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Regulated transcription of the immediate-early gene Zif268: Mechanisms and gene dosage-dependent function in synaptic plasticity and memory formation
Abstract
The immediate-early gene Zif268 is a member of the Egr family of inducible transcription factors. Data from gene expression studies have suggested that this gene may play a critical role in initial triggering of the genetic machinery that has long been considered a necessary mechanism for maintenance of the later phases of LTP and also for the consolidation or stabilization of long-lasting memories. Until recently, however, the data supporting this assumption have been based primarily on circumstantial evidence, with no direct evidence to suggest that Zif268 is required for long-lasting synaptic plasticity and memory. In this report, we review our own data using Zif268 mutant mice; we show that although the early phase of dentate gyrus LTP is normal in these mice, the later phases are not present, and the ability of the mice to maintain learned information over a 24-h period is deficient. In addition, we present new information showing a task-dependent gene dosage effect in Zif268 heterozygous mice. We show that spatial learning is particularly sensitive to reduced levels of Zif268, as one-half of the complement of Zif268 in heterozygous mice is insufficient to maintain spatial long-term memories.
... The synthesis of c-Fos ( Figure 1) follows the accumulation of its gene m RNA and could be detected by immunohistochemistry (IHC) between 20 and 90 min after stimulation, although it has also been observed up to 24 h after stimulation (in the cell nucleus). This protein has a half-life of approximately 2 h [3,[14][15][16]18,20,24,[30][31][32][33][34][35][36]. c-Fos activation is regulated by interactions with various transcriptional regulators, including Ca 2+ , cyclic adenosine monophosphate (cAMP or cAMP), or mitogen-activated protein kinases (MAPKs) [35,36]. ...
... Oxygen free radicals suppress c-fos expression, although the mechanism has not yet been fully elucidated. On the other hand, for c-Fos, the aging process increases expression in mice and dogs at the SC level due to neuronal distribution and natural neuronal damage/death caused by old age [4,14]. ...
For years, the biochemical processes that are triggered by harmful and non-harmful stimuli at the central nervous system level have been extensively studied by the scientific community through numerous techniques and animal models. For example, one of these techniques is the use of immediate expression genes, which is a useful, accessible, and reliable method for observing and quantifying cell activation. It has been shown that both the c-fos gene and its protein c-Fos have rapid activation after stimulus, with the length of time that they remain active depending on the type of stimulus and the activation time depending on the stimulus and the structure studied. Fos requires the participation of other genes (such as c-jun) for its expression (during hetero-dimer forming). c-Fos dimerizes with c-Jun protein to form factor AP-1, which promotes the transcription of various genes. The production and removal of c-Fos is part of cellular homeostasis, but its overexpression results in increased cell proliferation. Although Fos has been used as a marker of cellular activity since the 1990s, which molecular mechanism participates in the regulation of the expression of this protein is still unknown because the gene and the protein are not specific to neurons or glial cells. For these reasons, this work has the objective of gathering information about this protein and its use in neuroscience.
... Memory performance of Msk1-, Msk2-KO mice and control littermates were evaluated in object and object-place recognition memory tasks using standardized procedures as previously described Bozon et al., 2002). These memory tasks are based on the spontaneous preference of rodents for novelty and their ability to remember previously encountered objects and their location. ...
... In the DG, MAPK kinase pathways , CREB (Bozon et al., 2003) and several plasticity-related IEGs, including EGR1, are activated and required for the formation of long-term recognition memory Bozon et al., 2002;Soule et al., 2008). Here we found that loss of MSK1 was associated with a decrease in the basal levels of CREB and pCREB (29% and 49%, respectively), leading to a 25% reduction of pCREB/CREB ratio in the DG. ...
The mitogen-activated protein kinases (MAPK) are major signaling components of intracellular pathways required for memory consolidation. Mitogen- and stress-activated protein kinases 1 and 2 (MSK1 and MSK2) mediate signal transduction downstream of MAPK. MSKs are activated by Extracellular-signal Regulated Kinase 1/2 (ERK1/2) and p38 MAPK. In turn, they can activate cyclic AMP-response-element-binding protein (CREB), thereby modulating the expression of immediate early genes crucial for the formation of long-term memories. While MSK1 has been previously implicated in certain forms of learning and memory, little is known concerning MSK2. Our goal was to explore the respective contribution of MSK1 and MSK2 in hippocampal synaptic transmission and plasticity and hippocampal-dependent recognition memory. In Msk1- and Msk2-knockout mice, we evaluated object and object-place recognition memory, basal synaptic transmission, paired-pulse facilitation and inhibition, and the capacity to induce and sustain long-term potentiation (LTP) in vivo. We also assessed the level of two proteins downstream in the MAPK/ERK1/2 pathway crucial for long-term memory, CREB and the immediate early gene EGR1. Loss of Msk1, but not of Msk2, affected excitatory synaptic transmission at perforant path-to-dentate granule cell synapses, altered short-term presynaptic plasticity, impaired selectively long-term spatial recognition memory, and decreased basal levels of CREB and its activated form. LTP in vivo and LTP-induced CREB phosphorylation and EGR1 expression were unchanged after Msk1 or Msk2 deletion. Our findings demonstrate a dissimilar contribution of MSKs proteins in cognitive processes and suggest that Msk1 loss-of-function only has a deleterious impact on neuronal activity and hippocampal-dependent memory consolidation.
... We have begun to investigate the activation patterns of the immediate-early gene (IEG) early growth response gene 1 (Egr-1, Krox 24, NGFI-A, Zif268, TIS8) following the preexposure and training phases of the CPFE in developing rats (Asok, Schreiber, Jablonski, Rosen, & Stanton, 2013;Robinson-Drummer, Chakaraborty, Heroux, Rosen, & Stanton, 2018;Schreiber, Asok, Jablonski, Rosen, & Stanton, 2014). Egr-1 is an inducible regulatory transcription factor linked to neural plasticity and is broadly associated with learning and memory (for reviews, see Alberini, 2009;Bozon, Davis, & Laroche, 2002;Bozon et al., 2003;Rosen & Donley, 2006;Valenzuela, Morton, Diaz, & Topper, 2012). In the rat, Egr-1 mRNA is evident by the 2nd and 3rd postnatal weeks (Herms, Zurmöhle, Schlingensiepen, Brysch, & Schlingensiepen, 1994;Sanders, Happe, Bylund, & Murrin, 2008). ...
... These findings indicate that ethanol exposure during development may lead to persistent deficits in mPFC neuronal plasticity which could be partly responsible for the long-lasting dysregulation in neuronal circuits and behavioral function associated with human FASDs (El Shawa, Abbott, & Huffman, 2013). Egr-1 is regulated by the MAPK/ERK signaling pathway, specifically by the SRE and/or the CRE binding sites, via the transcription factors Elk-1 and CREB, respectively (Bozon et al., 2002;Davis et al., 2003). Targeting components of these pathways in the mPFC may be beneficial for reversing deficits in synaptic plasticity and improving behavioral outcomes, providing a potential mechanism of intervention for FASDs. ...
The context preexposure facilitation effect (CPFE) is a variant of contextual fear conditioning in which learning about the context (preexposure) and associating the context with a shock (training) occur on separate occasions. The CPFE is sensitive to a range of neonatal alcohol doses (Murawski & Stanton, 2011). The current study examined the impact of neonatal alcohol on Egr-1 mRNA expression in the infralimbic (IL) and prelimbic (PL) subregions of the mPFC, the CA1 of dorsal hippocampus (dHPC), and the lateral nucleus of the amygdala (LA), following the preexposure and training phases of the CPFE. Rat pups were exposed to a 5.25 g/kg/day single binge-like dose of alcohol (Group EtOH) or were sham intubated (SI; Group SI) over postnatal days (PD) 7–9. In behaviorally tested rats, alcohol administration disrupted freezing. Following context preexposure, Egr-1 mRNA was elevated in both EtOH and SI groups compared with baseline control animals in all regions analyzed. Following both preexposure and training, Group EtOH displayed a significant decrease in mPFC Egr-1 mRNA expression compared with Group SI. However, this decrease was greatest after training. Training day decreases in Egr-1 expression were not found in LA or CA1 in Group EtOH compared with Group SI. A second experiment confirmed that the EtOH-induced training-day deficits in mPFC Egr-1 mRNA expression were specific to groups which learned contextual fear (vs. nonassociative controls). Thus, memory processes that engage the mPFC during the context-shock association may be most susceptible to the teratogenic effects of neonatal alcohol.
... Several studies also indicate that IEGs are likely to be involved in stabilizing LTP mechanisms (Abraham et al. 1993;Dragunow 1996). Accordingly, several studies observed an involvement of the IEG egr-1 in LTP and in different forms of learning associated with selected regions within the telencephalon including the hippocampus of mice and rats (Abraham et al. 1993;Bozon et al. 2002Bozon et al. , 2003aFrankland and Bontempi 2005;Guzowski et al. 2001;Jones et al. 2001;Knapska and Kaczmarek 2004;Poirier et al. 2008). Knock-out mice lacking the Egr-1 gene were found to have an intact short-term memory but an impaired ability to establish hippocampal late long-term potentiation (LTP) in regard to spatial navigation, conditioned taste aversion or olfactory discriminations (Jones et al. 2001;Renaudineau et al. 2009). ...
... Similarly, an improved memory and support of its resistance to extinction has recently been described in transgenic mice overexpressing Egr-1 in the forebrain (Baumgärtel et al. 2008). Beyond hippocampus involvement, Egr-1 also plays a key role in the consolidation and/or reconsolidation of many memory tasks in conjunction with the mammalian isocortex and the amygdala (Bozon et al. 2002(Bozon et al. , 2003aDavis et al. 2003;Frankland and Bontempi 2005;Jones et al. 2001;Lee et al. 2004;Malkani et al. 2004;Poirier et al. 2008). ...
Using the expression of the immediate early gene (IEG) egr-1 as a neuronal activity marker, brain regions potentially involved in learning and long-term memory functions in the grey bamboo shark were assessed with respect to selected visual discrimination abilities. Immunocytochemistry revealed a significant up-regulation of egr-1 expression levels in a small region of the telencephalon of all trained sharks (i.e., ‘early’ and ‘late learners’, ‘recallers’) when compared to three control groups (i.e., ‘controls’, ‘undisturbed swimmers’, ‘constant movers’). There was also a well-defined difference in egr-1 expression patterns between the three control groups. Additionally, some staining was observed in diencephalic and mesencephalic sections; however, staining here was weak and occurred only irregularly within and between groups. Therefore, it could have either resulted from unintentional cognitive or non-cognitive inducements (i.e., relating to the mental processes of perception, learning, memory, and judgment, as contrasted with emotional and volitional processes) rather than being a training effect. Present findings emphasize a relationship between the training conditions and the corresponding egr-1 expression levels found in the telencephalon of Chiloscyllium griseum. Results suggest important similarities in the neuronal plasticity and activity-dependent IEG expression of the elasmobranch brain with other vertebrate groups. The presence of the egr-1 gene seems to be evolutionarily conserved and may therefore be particularly useful for identifying functional neural responses within this group.
... RC3 is involved in synaptic plasticity and memory via the modulation of Ca 2+ /calmodulin-dependent signaling [20,21]. This signaling pathway interacts with the MAPK/ERK-CREB pathway, also involved in synaptic plasticity and hippocampal functions [22][23][24]. Our recent studies showed that 5-week treatment with the antithyroid molecule, propylthiouracyl (PTU), induces hypothyroidism in adult rats. ...
... The MAPK/ERK-CREB and Ca 2+ /Calmodulin cascades are two major cell-signaling pathways involved in hippocampal synaptic plasticity and memory formation [47][48][49][50]. These pathways control activity-dependent regulation of gene programs via the regulated expression of inducible transcriptions factors such as EGR1/ZIF268, which is required for the expression of synaptic plasticity and hippocampal-dependent memories [22,51,52]. Our results in memory-deficient PTU-treated rats show a reduced expression of major components of both MAPK/ERK-CREB (pERK42, pCREB) and Ca 2+ /calmodulin (RC3, pRC3, CaM, pCaMKII, CaMKIV, and CaN protein levels) signaling pathways, associated with a decrease in EGR1/ZIF268 expression in the hippocampus. ...
Hypothyroidism is a condition that becomes more prevalent with age. Patients with untreated hypothyroidism have consistently reported symptoms of severe cognitive impairments. In patients suffering hypothyroidism, thyroid hormone supplementation offers the prospect to alleviate the cognitive consequences of hypothyroidism; however, the therapeutic value of TH supplementation remains at present uncertain and the link between cellular modifications associated with hypothyroidism and neurodegeneration remains to be elucidated. In the present study, we therefore evaluated the molecular and behavioral consequences of T3 hormone replacement in an animal model of hypothyroidism. We have previously reported that the antithyroid molecule propylthiouracil (PTU) given in the drinking water favors cerebral atrophy, brain neuroinflammation, Aβ production, Tau hyperphosphorylation, and altered plasticity-related cell-signaling pathways in the hippocampus in association with hippocampal-dependent spatial memory deficits. In the present study, our aim was to explore, in this model, the effect of hippocampal T3 signaling normalization on various molecular mechanisms involved in learning and memory that goes awry under conditions of hypothyroidism and to evaluate its potential for recovery of hippocampal-dependent memory deficits. We report that T3 supplementation can alleviate hippocampal-dependent memory impairments displayed by hypothyroid rats and normalize key markers of thyroid status in the hippocampus, of neuroinflammation, Aβ production, and of cell-signaling pathways known to be involved in synaptic plasticity and memory function. Together, these findings suggest that normalization of hippocampal T3 signaling is sufficient to reverse molecular and cognitive dysfunctions associated with hypothyroidism.
... Most of the mechanisms underlying such plasticity are mostly dependent on a genetic program in neurons, that drive de novo synthesis of proteins, as demonstrated from the extensive works that have shown that pharmacological inhibition of protein synthesis, impair long-term memory, leaving short-term memory intact (Davis and Squire, 1984; Meiri and Rosenblum, 1998). These results have allowed the proposition that the regulation of transcription of specific genes during learning is required for the establishment of a permanent memory trace (Bozon et al., 2002). Several evidences have demonstrated that LTP and long-lasting forms of synaptic plasticity depend on gene transcription , as well as on the novo protein synthesis (Otani and Abraham, 1989; Frey and Moris, 1997). ...
... Several evidences have demonstrated that LTP and long-lasting forms of synaptic plasticity depend on gene transcription , as well as on the novo protein synthesis (Otani and Abraham, 1989; Frey and Moris, 1997). In addition, several studies have demonstrated that both LTP and learning induce transcription of a variety of genes in specific areas in the brain of mammals (Bozon et al., 2002). Thus, these evidences support the idea that LTP is an important mechanism for long-term information storage in the brain (Bozon et al., 2002). ...
Extensive cellular and behavioral studies have led to the postulation that memories are encoded by changes in synaptic strength between neurons, as demonstrated by the correlation between the long-term changes in animal's behavior and long-term changes in neuronal connections underlying a specific behavior in invertebrate animals, or even in vertebrate animals, where cellular models of synaptic plasticity using genetic approaches, such as Long-Term Potentiation (LTP) and Long-Term Depression (LTD), have been shown to depend on long-term changes in synaptic activity implicated in behavioral learning and memory. Long-term memory (LTM) is crucial for animal's survival and thus represents a mechanism that underlies fundamental neurobiological events in the nervous system of vertebrate and non-vertebrate species including the human. Long-term changes in synaptic connectivity as well as long-term behavioral changes (both activities that underlie several of the properties of LTM and used as a parameter to explain the long-lasting enhancement of neuronal function after a stimulus) have been demonstrated to rely on signals that initially occur in the cell body. LTP is a form of synaptic plasticity widely accepted as a cellular model for stabilization of synapses in neurobiological phenomena such as development and learning and memory. Much of the experimental work concerning LTP in learning has been focused on the NMDA receptor dependent forms of LTP. But several questions have arisen regarding if LTP equals memory. If LTP has a real role in memory, a more appropriate hypothesis should be stated by postulating that activity-dependent synaptic plasticity and multiple forms of memory known to exist, share a common core; that is, the synaptic plasticity and memory, hypothesis states that activity dependent synaptic plasticity is induced at appropriate synapses during memory formation. Synaptic plasticity is a physiological phenomenon that induces specific patterns of neural activity sustained by chemical and molecular mechanisms, that gives rise to changes in synaptic efficacy and neural excitability which long outlast the events that trigger them. Based on the several properties of synaptic plasticity discovered, LTP may be proposed as a suitable neuronal mechanism for the development of several memory systems, including initial encoding and storage of memory traces and initial phases of trace consolidation over time. Such memory processing made up by LTP or LTD most probably occur as a network specific process, making LTP a universal mechanism for encoding and storage of memory traces and what gets encoded is part of a network property rather than mechanisms working at individual synapses. For example, the type of information processed at the hippocampus is quite different from the information processed by the amygdala, and such information should remain if the mechanisms of plasticity operating in each brain area are conserved. Decades of research have demonstrated that LTP in the hippocampus is induced by synaptic activity and that cytoplasmic membrane-bound molecule(s) are required to transduce extracellular signals mediated by receptor-activation into activation of intracellular signaling processes. Most of these processes depend on intracellular calcium activity, and thereby on calcium-dependent mechanisms that are recruited for LTP induction and expression. For instance, NMDA receptors have been shown to be essential for initiation of LTP, but expression of this phenomenon in brought primarily by AMPA receptors. Induction of LTP in CA1 hippocampal region has been shown to depend on increases of intracellular calcium and activation of specific calcium-dependent molecules such as the calcium/calmodulin-dependent protein kinase (CaMKII), whose cell expression is confined predominantly at postsynaptic densities. Moreover, long-term expression of LTP requires protein synthesis, where transient signals will be linked to activation of specific genes that ultimately will determine growth and remodeling of potential active synapses. Different types of synapses may express and use a different set of molecules mediating activation of intracellular signaling pathways for initiating and maintaining synaptic plasticity. Several studies have demonstrated that neuronal modifications of neurotransmitter receptors or membrane-receptor subunits at postsynaptic densities, represent one of the neuronal mechanisms by which neurons regulate their synaptic strength. For instance, it has been demonstrated that neuronal dendrites are able to regulate their own transmembrane receptor synthesis in response to external stimuli (i.e., GluR2 subunit of AMPA receptor) and such molecular mechanisms, posed important implications in the understanding of how individual synapses are selectively strengthened. In addition, recent experiments have demonstrated that specific intracellular signaling molecules (i.e., neuronal Synaptic GTPase-activating protein or SynGAP) are selectively expressed and enriched at excitatory synapses. Interestingly enough are the evidences that demonstrate that different subsets of protein kinases (MAPKs, SAPKs, MAPKAKs, p38MAPK, etc.) and intracellular signaling pathways activate transcription factors (AP-1 complex, CREB) that regulate the expression of different immediate early genes (IEG) which are crucial for neuronal development, glutamate receptor trafficking to specific synapses and for LTP induction. Much of the neurochemical and molecular changes that occurr in synaptic plasticity may be well associated with dynamic morphological changes in spine synapses as suggested to participate in the development and consolidation of LTP. In addition, glial cells, known to participate in the excitatory neurotransmission in the CNS besides their conceptualized cellular function, as elements for structural support and homeostasis, may play an important role in synaptic plasticity and thereby may regulate the information processed in the brain. As hippocampal LTP has been the target of intensive molecular genetic analysis, several studies have demonstrated that LTP is altered when particular single genes are knocked out or overexpressed in null mutant mice or transgenic mice. Such studies have led to the amazing observation that variations in LTP exist within natural inbred mouse strains.
... These processes could be mediated by phosphorylation of CREB (Miller and Kaplan, 2003). Although the mechanism of action of quercetinis is unclear, the involvement of CREB in the regulation of neurogenesis, dendrite formation, Zif268 expression, and formation of dendritic spines underscores its importance in neuroplasticity and neuroprotective processes (Murphy and Segal, 1997;Bozon et al., 2002;Tully et al., 2003). Zif268 (EGR-1), may play a critical role in consolidation or stabilization of long-lasting memories, long-lasting synaptic plasticity, and spatial memory. ...
... Zif268 (EGR-1), may play a critical role in consolidation or stabilization of long-lasting memories, long-lasting synaptic plasticity, and spatial memory. This gene is expressed primarily after synaptic activation and therefore serves as a marker of synaptic functionality, and it has been shown to be necessary for the transition from short-to long-term synaptic plasticity and formation of different forms of long-term memory (Steiner and Kitai, 2000;Jones et al., 2001;Bozon et al., 2002;Guzowski, 2002;Jessberger and Kempermann, 2003). ...
... EGR-1 expression exhibits a distinct pattern in the brain compared to c-Fos [68][69][70][71][72][73][74][75] . It plays a crucial role in mediating the expression of multiple genes involved in neural processes, ranging from growth control to changes in synaptic plasticity [76][77][78] . EGR-1 is relatively highly expressed during neural activity 72,79 , particularly in the hippocampus 3,80 and the dentate gyrus 7 . ...
The immediate early gene exhibits activation markers in the nervous system consisting of ARC, EGR-1, and c-Fos and is related to synaptic plasticity, especially in the hippocampus. Immediate early gene expression is affected by physical exercise, which induces direct ARC, EGR-1, and c-Fos expression.
Objective
To assess the impact of exercise, we conducted a literature study to determine the expression levels of immediate early genes (ARC, c-Fos, and EGR-1).
Methods
The databases accessed for online literature included PubMed-Medline, Scopus, and ScienceDirect. The original English articles were selected using the following keywords in the title: (Exercise OR physical activity) AND (c-Fos) AND (Hippocampus), (Exercise OR physical activity) AND (ARC) AND (Hippocampus), (Exercise OR physical activity) AND (EGR-1 OR zif268) AND (Hippocampus).
Results
Physical exercise can affect the expression of EGR-1, c-Fos, and ARC in the hippocampus, an important part of the brain involved in learning and memory. High-intensity physical exercise can increase c-Fos expression, indicating neural activation. Furthermore, the expression of the ARC gene also increases due to physical exercise. ARC is a gene that plays a role in synaptic plasticity and regulation of learning and memory, changes in synaptic structure and increased synaptic connections, while EGR-1 also plays a role in synaptic plasticity, a genetic change that affects learning and memory. Overall, exercise or regular physical exercise can increase the expression of ARC, c-Fos, and EGR-1 in the hippocampus. This reflects the changes in neuroplasticity and synaptic plasticity that occur in response to physical activity. These changes can improve cognitive function, learning, and memory.
Conclusion
c-Fos, EGR-1, and ARC expression increases in hippocampal neurons after exercise, enhancing synaptic plasticity and neurogenesis associated with learning and memory.
... LTP involves the mobilization of immediate-early genes activated by both the cAMP and calcium pathways. Fos up-regulation can be either calcium-or cAMP-dependent, while that of other immediate early genes, such as egr1, are cAMP-dependent [5,32,[62][63][64][65][66][67][68][69]. Fos induction initiated either by cAMP or calcium has been attributed variously to a PKA-dependent, Rap1-independent activation of ERK, leading to MSK-1 activation, and phosphorylation/activation of the transcription factor CREB, or to a CREB-dependent mechanism due to CREB phosphorylation directly by PKA [41,[70][71][72]. ...
The MAP kinase ERK is important for neuronal plasticity underlying associative learning, yet specific molecular pathways for neuronal ERK activation are undetermined. RapGEF2 is a neuron-specific cAMP sensor that mediates ERK activation. We investigated whether it is required for cAMP-dependent ERK activation leading to other downstream neuronal signaling events occurring during associative learning, and if RapGEF2-dependent signaling impairments affect learned behavior. Camk2α-cre +/-::RapGEF2 fl/fl mice with depletion of RapGEF2 in hippocampus and amygdala exhibit impairments in context-and cue-dependent fear conditioning linked to corresponding impairment in Egr1 induction in these two brain regions. Camk2α-cre +/-::RapGEF2 fl/fl mice show decreased RapGEF2 expression in CA1 and dentate gyrus associated with abolition of pERK and Egr1, but not of c-Fos induction, following fear conditioning, impaired freezing to context after fear conditioning, and impaired cAMP-dependent long-term potentiation at perforant pathway and Schaffer collateral synapses in hippocampal slices ex vivo. RapGEF2 expression is largely eliminated in basolateral amygdala, also involved in fear memory, in Camk2α-cre +/-::RapGEF2 fl/fl mice. Neither Egr1 nor c-fos induction in BLA after fear conditioning, nor cue-dependent fear learning, are affected by ablation of RapGEF2 in BLA. However, Egr1 induction (but not that of c-fos) in BLA is reduced after restraint stress-augmented fear conditioning, as is freezing to cue after restraint stress-augmented fear conditioning, in Camk2α-cre +/-::RapGEF2 fl/fl mice. Cyclic AMP-dependent GEFs have been genetically associated as risk factors for schizophrenia, a disorder associated with cognitive deficits. Here we show a functional link between one of them, RapGEF2, and cognitive processes involved in associative learning in amygdala and hippocampus.
... The genetic disruption of Arc gene in mice has been shown to impair both hippocampal LTP and LTD, thus causing the loss of long-lasting memories for implicit and explicit learning tasks [51,52]. EGR1 belongs to EGR family of inducible transcription factors which have three cysteine2-histidine2 (C 2 H 2 ) zinc fingers DNA-binding domain [53][54][55]. Following neuronal stimulation, EGR1 allows a second wave of transcriptional regulation to maintain the LTP, and thus, it plays essential role in consolidation and retrieval of memory [53,56]. ...
Amnesia is the inability to store new information and recall old memories. After the postulation of cholinergic hypothesis of geriatric memory dysfunction, the cholinergic signaling became a popular target to understand the underlying molecular mechanism of amnesia and its recovery. Scopolamine is a non-selective cholinergic receptor antagonist and induces amnesia through downregulation of synaptic plasticity genes including immediate early genes (IEGs). Scopolamine-induced amnesic mouse model is widely used to study the memory impairment that mimics the pathophysiology of aging, neurodegenerative, and neuropsychiatric disorders. However, a detailed understanding of cholinergic signaling-mediated regulation of plasticity-related gene expression remains elusive. Therefore, we have investigated the role of muscarinic acetylcholine receptors (mAChRs) and their downstream mediator protein kinase C (PKC) in the regulation of IEGs expression in amnesic mice hippocampus. Pilocarpine, a mAChRs agonist, was used to activate the cholinergic signaling in scopolamine-induced amnesia. Further, a PKC activator bryostatin 1 was used to understand the sole involvement of PKC as a downstream mediator of mAChRs-mediated signaling. Pilocarpine treatment significantly restored the scopolamine-induced impaired recognition memory and downregulated hippocampal IEGs expression and phosphorylation of ERK1/2 (extracellular signal-regulated kinase 1/2) and CREB (cAMP response element-binding protein). On the other hand, the bryostatin 1-mediated activation of PKC in scopolamine-induced amnesia selectively restored the hippocampal IEGs expression, recognition memory, and phosphorylation of ERK1/2 and CREB. Taken together, our findings suggest that mAChRs and their downstream mediator PKC regulate the hippocampal IEGs expression and ERK1/2-mediated CREB phosphorylation in scopolamine-induced amnesic mice.
... LTP involves the mobilization of several immediateearly genes activated by both the cAMP and calcium pathways. Fos up-regulation can be either calcium-or cAMP-dependent, and that of other immediate early genes, such as Egr-1/Zif268, are cAMP-dependent Ginty et al., 1992;Greenberg et al., 1992;Tai et al., 2001;Bozon et al., 2002;Bozon et al., 2003;Ravni et al., 2008;Gangarossa et al., 2011;Jiang et al., 2021). More controversial is the mode of regulation of fos initiated either by cAMP or calcium: this has been attributed both to a PKA-dependent, Rap1-independent activation of ERK, leading to MSK-1 activation and phosphorylation/activation of the transcription factor CREB, or to a CREB-dependent mechanism due CREB phosphorylation directly by PKA (Wong et al., 1999;Poser and Storm, 2001;Sindreu et al., 2007;Xia and Storm, 2012). ...
The MAP kinase ERK is important for neuronal plasticity underlying associative learning, yet specific molecular pathways for ERK activation in hippocampus are still largely undetermined. RapGEF2 has emerged as a neuron-specific cAMP sensor that mediates ERK activation. We investigated whether RapGEF2 might also be required for cAMP-dependent ERK activation leading to synaptic potentiation, and how this involvement might be penetrant to hippocampus-dependent learned behavior. We demonstrate that conditional knockout of Rapgef2 in forebrain neurons, specifically in dentate gyrus and CA1 of the hippocampus, leads to an attenuation of context-dependent fear conditioning, but not of cue-dependent fear conditioning, in mice. RapGEF2 knockout is associated with a reduction in cAMP-dependent synaptic potentiation at two central hippocampal synapses-the entorhinal cortex-granule cell synapse and the CA3-CA1 synapse. Furthermore, cAMP-induced postsynaptic potentiation requires both RapGEF2 and activation of ERK. Induction of Egr-1/Zif268 (and pERK), but not of c-Fos, immediately following fear conditioning, was abolished in CA1 and detate gyrus, in the absence of RapGEF2 expression in these hippocampal regions, thus revealing a link between learning (conditioning) and molecular pathways activated during conditioned fear memory formation. Hence, we suggest that contextual fear conditioning is mediated via RapGEF2-dependent ERK activation and downstream induction of Egr-1, via an underlying mechanism of cAMP-dependent long-term potentiation at hippocampal synapses. Cyclic AMP-dependent GEFs have been genetically associated as risk factors for schizophrenia, a disorder associated with cognitive deficits. This study provides a functional link between one of these cAMP-dependent GEFs, RapGEF2, and cognitive processes involved in associative learning.
... The important role of plasticity in courtship behavior was also evident upon blocking NMDA (N-methyl-D-aspartate) receptors, which play a critical role in learning-induced synaptic plasticity, while significantly affecting female preference behavior . Learning-induced synaptic plasticity can be observed also in various vertebrate taxa in various social (e.g., Sockman, 2007;Wang et al., 2014;Cummings, 2015;Cummings and Ramsey, 2015;Delclos et al., 2020) contexts and (non-social) learning tasks (e.g., Alcock, 2001;Bozon et al., 2002;Davis et al., 2003;Fuss and Schluessel, 2018). ...
Across taxa, mate choice is a highly selective process involving both intra- and intersexual selection processes aiming to pass on one’s genes, making mate choice a pivotal tool of sexual selection. Individuals adapt mate choice behavior dynamically in response to environmental and social changes. These changes are perceived sensorily and integrated on a neuronal level, which ultimately leads to an adequate behavioral response. Along with perception and prior to an appropriate behavioral response, the choosing sex has (1) to recognize and discriminate between the prospective mates and (2) to be able to assess and compare their performance in order to make an informed decision. To do so, cognitive processes allow for the simultaneous processing of multiple information from the (in-) animate environment as well as from a variety of both sexual and social (but non-sexual) conspecific cues. Although many behavioral aspects of cognition on one side and of mate choice displays on the other are well understood, the interplay of neuronal mechanisms governing both determinants, i.e., governing cognitive mate choice have been described only vaguely. This review aimed to throw a spotlight on neuronal prerequisites, networks and processes supporting the interaction between mate choice, sex roles and sexual cognition, hence, supporting cognitive mate choice. How does neuronal activity differ between males and females regarding social cognition? Does sex or the respective sex role within the prevailing mating system mirror at a neuronal level? How does cognitive competence affect mate choice? Conversely, how does mate choice affect the cognitive abilities of both sexes? Benefitting from studies using different neuroanatomical techniques such as neuronal activity markers, differential coexpression or candidate gene analyses, modulatory effects of neurotransmitters and hormones, or imaging techniques such as fMRI, there is ample evidence pointing to a reflection of sex and the respective sex role at the neuronal level, at least in individual brain regions. Moreover, this review aims to summarize evidence for cognitive abilities influencing mate choice and vice versa. At the same time, new questions arise centering the complex relationship between neurobiology, cognition and mate choice, which we will perhaps be able to answer with new experimental techniques.
... Although many transcription factors have been reported to regulate Wnt5a expression [25,26], we confirmed that Egr-1 regulated Wnt5a expression in human oral fibroblasts. Interestingly, Egr-1 is a typical immediate early gene (IEG) [38]. IEGs are genes which are activated transiently and rapidly in response to a wide variety of cellular stimuli. ...
Background
Arecoline is an alkaloid natural product found in the areca nut that can induce oral submucous fibrosis and subsequent development of cancer. However, numerous studies have shown that arecoline may inhibit fibroblast proliferation and prevent collagen synthesis.
Results
High doses of arecoline (> 32 μg/ml) could inhibit human oral fibroblast proliferation, while low doses of arecoline (< 16 μg/ml) could promote the proliferation of human oral fibroblasts. Wnt5a was found to be both sufficient and necessary for the promotion of fibroblast proliferation. Egr-1 could mediate the expression of Wnt5a in fibroblasts, while NF-κB, FOXO1, Smad2, and Smad3 did not. Treatment with siRNAs specific to Egr-1, Egr inhibitors, or Wnt5a antibody treatment could all inhibit arecoline-induced Wnt5a upregulation and fibroblast proliferation.
Conclusions
Egr-1 mediates the effect of low dose arecoline treatment on human oral mucosa fibroblast proliferation by transactivating the expression of Wnt5a. Therefore, Egr inhibitors and Wnt5a antibodies are potential therapies for treatment of oral submucosal fibrosis and oral cancer.
... Indeed, zif268 (EGR1) has been demonstrated to regulate the promotor activity of the GAD67 gene in developing neurons (Luo et al., 2008;Szabó et al., 1996). Zif268 is an immediate-early gene required for maintenance of the later phases of LTP and the consolidation or stabilization of long-lasting memories (Bozon et al., 2002) and has been shown to be active in early critical period of cats (Kaplan et al., 1995) and monkey visual cortex (Kaczmarek et al., 1999). DR has been reported to lower the number of zif268+ neurons in visual cortex of rats (Yamada et al., 1999) and cats (Mower & Kaplan, 2002) but leads to a stronger increase in labeled neurons with subsequent light exposure (Mower & Kaplan, 2002). ...
Early critical period of visual cortex is characterized by enhanced activity-driven neuronal plasticity establishing the specificity of neuronal connections required for optimal processing of sensory signals. Deprivation from visual input by dark rearing (DR) during this period leads to a lasting impairment of visual performance. Previously, we demonstrated that repetitive transcranial magnetic stimulation (rTMS) applied with intermittent theta-burst (iTBS) pattern during the critical period improved the visual performance of the DR rats. In this study, we describe that the excitability of the binocular part of the visual cortex (V1b), as measured in acute brain slices by input-output ratios of field excitatory synaptic potentials (fEPSPs), is lowered in DR rats compared to normal controls. Verum rTMS applied with the iTBS pattern during DR reversed this DR effect, while no rTMS effect was evident in the non-DR (nDR) rats. In addition, verum rTMS reduced the number of neurons expressing the 67 kD isoform of glutamic acid decarboxylase (GAD67), the calcium-binding protein calbindin (CB) and the zinc-finger transcription factor zif268/EGR1, as determined via immunohistochemistry, only in DR rats but not in nDR rats. Moreover, rTMS reduced the number of neurons expressing the calcium-binding protein parvalbumin (PV) only in nDR rats which showed more PV+ neurons compared to DR rats. This study confirms that iTBS-rTMS may be able to prevent or reverse the effects of DR on visual cortex physiology, likely through a modulation of the activity of inhibitory interneurons.
... Although many transcription factors have been reported to regulate Wnt5a expression [21,22], we nally con rmed that Egr-1 regulated Wnt5a expression. Interestingly, Egr-1 is a typical immediate early gene (IEG) [38]. IEGs are genes which are activated transiently and rapidly in response to a wide variety of cellular stimuli. ...
Background
Arecoline is the main carcinogens in Areca nut that induce oral submucous fibrosis to develop into cancer. However, many previous studies have showed that Arecoline may inhibit proliferation and prevent collagen synthesis of fibroblasts.
Results
High dose Arecoline (> 32 µg/ml) could inhibit but low dose Arecoline (< 16 µg/ml) could promote the proliferation of human oral fibroblasts. Wnt5a was both sufficient and necessary for the promotion of fibrobasts proliferation. Egr-1, but not NF-κB, FOXO1, Smad2 or Smad3, mediated the expression of Wnt5a in fibrobasts. The specific siRNAs of Egr-1, Egr inhibitors or Wnt5a antibodies treatment blocked Arecoline induced Wnt5a upregulation and fibroblasts proliferation.
Conclusions
Egr-1 mediates low dose Arecoline induced human oral mucosa fibroblasts proliferation by transaction the expression of Wnt5a, and Egr inhibitors or Wnt5a antibodies are potential therapeutic drugs of oral submucosal fibrosis and oral cancer.
... In the brain, Zif268 is transiently expressed in response to several stimuli [12][13][14] . In particular, hippocampal Zif268 increases in an NMDAR-dependent manner after LTP induction [15][16][17] and has been repeatedly linked to memory processing [18][19][20] . However, despite the fact that many consider Zif268 a non-declarative memory reconsolidation marker [20][21][22][23][24] , it was recently suggested that, on the contrary, Zif268 restricts the extinction of such memories 25 . ...
Object recognition memory (ORM) serves to distinguish familiar items from novel ones. Reconsolidation is the process by which active memories are updated. The hippocampus is engaged in ORM reconsolidation through a mechanism involving induction of long-term potentiation (LTP). The transcription factor Zif268 is essential for hippocampal LTP maintenance and has been frequently associated with memory processes. However, its possible involvement in ORM reconsolidation has not been determined conclusively. Using Zif268 antisense oligonucleotides in combination with behavioural, biochemical and electrophysiological tools in rats, we found that hippocampal Zif268 is necessary to update ORM through reconsolidation but not to retrieve it or keep it stored. Our results also suggest that knocking down hippocampal Zif268 during ORM reconsolidation deletes the active recognition memory trace.
... Dicho gen forma heterodímeros con los factores de transcripción c-Jun y JunB, produciendo un incremento en la expresión del primero y una inhibición del segundo. Se ha observado la expresión de zif268 después de la inducción de la LTP (potenciación a largo plazo, por sus siglas en inglés) y en varias etapas de la memoria (Bozon, Davis y Laroche, 2002). ...
... Activation of hippocampal GR increases Egr-1 expression through enhancing enzymatic activity in the MAPK signaling pathway including phosphorylation of CREB [30]. Since Egr-1 is required for late LTP and long-term memory [31], Egr-1-knockout mice showed impairment of long-term memory [32]. In this study, treadmill exercise enhanced Egr-1 expression in the hippocampus of both TG mice and wildtype mice (Fig. 4B), which is consistent with increased expressions of BDNF, p-CREB, and p-Erk1/2 (Figs. ...
Purpose:
Circadian rhythm affects learning process, memory consolidation, and long-term memory. In this study, the alleviating effect of exercise on circadian rhythm disruption-induced memory deficits was investigated.
Methods:
BMAL1 knockdown transgenic mice (BMAL1 TG) were used as the BMAL1-TG group and the BMAL1-TG with treadmill exercise group. Female C57BL/6J mice of the same age were used as the wildtype group and the wildtype with treadmill exercise group. The mice in the treadmill exercise groups performed running on a motorized treadmill under the dark-dark conditions for 8 weeks. Short-term memory, nonspatial object memory, and spatial learning memory were determined using stepdown avoidance test, novel object-recognition test, and radial 8-arm maze test. Immunohistochemistry for doublecortin and 5-bromo-2'-deoxyuridine was conducted for the determination of hippocampal neurogenesis. Using the western blot analysis, we determined the expressions of glucocorticoid receptor (GR) and factors related to the neurogenesis and memory consolidation, such as brain-derived neurotrophic factor, tyrosine kinase B, p44/42 mitogen-activated protein kinase, cyclic AMP-responsive element binding protein, phosphatidylinositol 3-kinase, protein kinas B, protein kinase C alpha, early-growth-response gene 1.
Results:
Circadian rhythm disruption impaired memory function through inhibiting the expressions of GR and the factors related to neurogenesis and memory consolidation. Treadmill exercise improved memory function via enhancing the expressions of GR and above-mentioned factors.
Conclusion:
Treadmill exercise acts as the zeitgeber that improves memory function under the circadian rhythm disrupted conditions.
... We also performed immunohistochemistry for ZENK because it has been related directly to learning, and its patterns of induction have been previously correlated with behavioral measures of habituation in the same Trained-Silence, Trained-Familiar, and Trained-Novel paradigm we used here Tischmeyer and Grimm 1999;Bozon et al. 2002;Dong and Clayton 2008;London and Clayton 2008). Further, despite the cross-talk between ERK and mTOR signaling cascades, Rapamycin and SC79 do not directly affect ERK activation (Hietman et al. 1991;Mendoza et al. 2011;Jo et al. 2012). ...
Nonassociative learning is considered simple because it depends on presentation of a single stimulus, but it likely reflects complex molecular signaling. To advance understanding of the molecular mechanisms of one form of nonassociative learning, habituation, for ethologically relevant signals we examined song recognition learning in adult zebra finches. These colonial songbirds learn the unique song of individuals, which helps establish and maintain mate and other social bonds, and informs appropriate behavioral interactions with specific birds. We leveraged prior work demonstrating behavioral habituation for individual songs, and extended the molecular framework correlated with this behavior by investigating the mechanistic Target of Rapamycin (mTOR) signaling cascade. We hypothesized that mTOR may contribute to habituation because it integrates a variety of upstream signals and enhances associative learning, and it crosstalks with another cascade previously associated with habituation, ERK/ZENK. To begin probing for a possible role for mTOR in song recognition learning, we used a combination of song playback paradigms and bidirectional dysregulation of mTORC1 activation. We found that mTOR demonstrates the molecular signatures of a habituation mechanism, and that its manipulation reveals the complexity of processes that may be invoked during nonassociative learning. These results thus expand the molecular targets for habituation studies and raise new questions about neural processing of complex natural signals.
... Further, the current review focuses on those IEG's which, based on the memory-specific deficits that occur following inhibition or knockout, have been shown to be important to memory-related plasticity. These include Arc (Guzowski et al., 2000;McIntyre et al., 2005;Plath et al., 2006), Zif268/Egr-1 (Bozon et al., 2002(Bozon et al., , 2003Jones et al., 2001) and c-fos (de Hoz et al., 2017;Fleischmann et al., 2003;Gandolfi et al., 2017;Kemp et al., 2013;Lamprecht & Dudai, 1996;Mileusnic et al., 1996;Swank et al., 1996). These studies suggest that, despite their limitation, IEG's provide useful information regarding learning and memory. ...
Context is an ever-present combination of discrete environmental elements capable of influencing many psychological processes. When context is associated with an aversive stimulus, a permanent contextual fear memory is formed. Context is hypothesized to greatly influence the treatability of various fear-based pathologies, in particular, post-traumatic stress disorder (PTSD). In order to understand how contextual fear memories are encoded and impact underlying fear pathology, delineation of the underlying neural circuitry of contextual fear memory consolidation and maintenance is essential. Past understandings of contextual fear suggest that the hippocampus only creates a unitary, or single, representation of context. This representation is sent to the amygdala, which creates the associative contextual fear memory. In contrast, here we review new evidence from the literature showing contextual fear memories to be consolidated and maintained by both amygdala and hippocampus. Based on this evidence, we revise the current model of contextual fear memory consolidation, highlighting a larger role for hippocampus. This new model may better explain the role of the hippocampus in PTSD.
... Historically, a number of studies supported the view that proposed that Egr1 expression is sensitive to information gained after the exposure to novelty or learning associated environments (Tischmeyer and Grimm, 1999;Bozon et al., 2002;Guzowski, 2002;Davis et al., 2003Davis et al., , 2006Knapska and Kaczmarek, 2004). This idea came from the increments in the expression of Egr1 mRNA or protein after different learning paradigms. ...
Many psychiatric disorders, despite their specific characteristics, share deficits in the cognitive domain including executive functions, emotional control and memory. However, memory deficits have been in many cases undervalued compared with other characteristics. The expression of Immediate Early Genes (IEGs) such as, c-fos, Egr1 and arc are selectively and promptly upregulated in learning and memory among neuronal subpopulations in regions associated with these processes. Changes in expression in these genes have been observed in recognition, working and fear related memories across the brain. Despite the enormous amount of data supporting changes in their expression during learning and memory and the importance of those cognitive processes in psychiatric conditions, there are very few studies analyzing the direct implication of the IEGs in mental illnesses. In this review, we discuss the role of some of the most relevant IEGs in relation with memory processes affected in psychiatric conditions.
... C-fos is necessary for consolidation of non-spatial hippocampal-dependent memory [56]. Genetic studies in mice have supported that zif268 is critical for memory consolidation and long-lasting memory stabilization [57]. Zif268 knockout mice show impaired long term memories but intact short-term retention [55]. ...
Mandarin fish refuse dead prey fish or artificial diets and can be trained to transform their inborn feeding habit. To investigate the effect of memory on feeding habit transformation, we compared the reaction time to dead prey fish and the success rate of feeding habit transformation to dead prey fish with training of mandarin fish in the 1st experimental group (trained once) and the 2nd experimental group (trained twice). The mandarin fish in the 2nd group had higher success rate of feeding habit transformation (100%) than those in the 1st group (67%), and shorter reaction time to dead prey fish (<1 s) than those in the 1st group (>1 s). Gene expression of cAMP responsive element binding protein I (Creb I), brain-derived neurotrophic factor (Bdnf), CCAAT enhancer binding protein delta (C/EBPD), fos-related antigen 2 (Fra2), and proto-oncogenes c-fos (c-fos) involved in long-term memory formation were significantly increased in the 2nd group after repeated training, and taste 1 receptor member 1 (T1R1), involved in feeding habit formation, was significantly increased in brains of the 2nd group after repeated training. DNA methylation levels at five candidate CpG (cytosine–guanine) sites contained in the predicted CpG island in the 5′-flanking region of T1R1 were significantly decreased in brains of the 2nd group compared with that of the 1st group. These results indicated that the repeated training can improve the feeding habit transformation through the memory formation of accepting dead prey fish. DNA methylation of the T1R1 might be a regulatory factor for feeding habit transformation from live prey fish to dead prey fish in mandarin fish.
... We focus on the Egr family of immediate early genes since they are activated in response to changes in the environment (Senba and Ueyama, 1997;Martinez et al., 2002), and regulate fundamental processes in the nervous system that are known to be dysfunctional in schizophrenia. These include myelination, vascularization, learning and memory, and synaptic plasticity (Paulsen et al., 1995;Guzowski et al., 2001;Nagarajan et al., 2001;Bozon et al., 2002Bozon et al., , 2003Flynn et al., 2003;Crabtree and Gogos, 2014). In addition, Egrs are activated downstream of N-methyl-D-aspartate receptors (NMDARs; Cole et al., 1989) and growth factors (Schulze et al., 2008;Shin et al., 2010), dysfunction of which have each been hypothesized to contribute to schizophrenia susceptibility (Olney et al., 1999;Moises et al., 2002;Calabrese et al., 2016). ...
While the causes of myriad medical and infectious illnesses have been identified, the etiologies of neuropsychiatric illnesses remain elusive. This is due to two major obstacles. First, the risk for neuropsychiatric disorders, such as schizophrenia, is determined by both genetic and environmental factors. Second, numerous genes influence susceptibility for these illnesses. Genome-wide association studies have identified at least 108 genomic loci for schizophrenia, and more are expected to be published shortly. In addition, numerous biological processes contribute to the neuropathology underlying schizophrenia. These include immune dysfunction, synaptic and myelination deficits, vascular abnormalities, growth factor disruption, and N-methyl-D-aspartate receptor (NMDAR) hypofunction. However, the field of psychiatric genetics lacks a unifying model to explain how environment may interact with numerous genes to influence these various biological processes and cause schizophrenia. Here we describe a biological cascade of proteins that are activated in response to environmental stimuli such as stress, a schizophrenia risk factor. The central proteins in this pathway are critical mediators of memory formation and a particular form of hippocampal synaptic plasticity, long-term depression (LTD). Each of these proteins is also implicated in schizophrenia risk. In fact, the pathway includes four genes that map to the 108 loci associated with schizophrenia: GRIN2A, nuclear factor of activated T-cells (NFATc3), early growth response 1 (EGR1) and NGFI-A Binding Protein 2 (NAB2); each of which contains the “Index single nucleotide polymorphism (SNP)” (most SNP) at its respective locus. Environmental stimuli activate this biological pathway in neurons, resulting in induction of EGR immediate early genes: EGR1, EGR3 and NAB2. We hypothesize that dysfunction in any of the genes in this pathway disrupts the normal activation of Egrs in response to stress. This may result in insufficient electrophysiologic, immunologic, and neuroprotective, processes that these genes normally mediate. Continued adverse environmental experiences, over time, may thereby result in neuropathology that gives rise to the symptoms of schizophrenia. By combining multiple genes associated with schizophrenia susceptibility, in a functional cascade triggered by neuronal activity, the proposed biological pathway provides an explanation for both the polygenic and environmental influences that determine the complex etiology of this mental illness.
... Egr1 and Fos are tightly linked to neuronal plasticity and memory [45,46]. Egr1 is implicated in the maintenance of synaptic plasticity and is necessary for the persistence of LTP [47] and the consolidation of different forms of long-term memory as well as during the transition from short-to long-term memories [48,49]. Interestingly, sleep is significantly involved in the regulation of brain plasticity and cognition (see [50] for review). ...
... Interestingly, we observed strong expression of Zif268 in the striatum and other brain regions after application of 6′-GNTI, but we cannot provide an underlying mechanism for this effect. Regulation of Zif268 expression potentially involves activation of PKA, PKC or CaMK signalling, involving either MAPK/ERK or CREB-dependent mechanisms (seeBozon et al., 2002). 6′-GNTI was described as a partial agonist for G-protein activation and inhibition of adenylate cyclase, with hardly any activation of β-arrestin compared with full agonists such as U-50488H and EKC, in HEK293T cells transfected with human κ receptors (Rives et al., 2012). ...
... The IEG Zif268 (also known as Krox24, NGF-I-A, Egr-1, TZs8, and Zenk) is a member of the Egr (early growth response) family of inducible transcription factors. Zif268 transcription is rapidly initiated following LTP and learning (Abraham et al., 1991;Demmer et al., 1993;Bozon et al., 2002). Initial studies on Zif268 transcription suggested that LTP triggers increased mRNA and protein levels of Zif268 mostly in the granule cells of stimulated dentate gyrus (Wisden et al., 1990). ...
Transcription is one of the most critical steps required for long-term memory formation. Over the last 20 years, research has explored the transcription factors that respond following neuronal activity and regulate gene expression linked to memory consolidation. Recently, the regulation of transcription factors through epigenetic modifications has changed our understanding of the neural mechanisms underlying learning and memory. In this chapter, we will introduce some of the molecular players required for memory formation and then discuss the role of epigenetics in transcription regulation during memory formation with a focus on histone acetylation and DNA methylation. We conclude with the possible sites of memory consolidation.
... Histogram represents fold change of acetylation level with respect to young as mean ± SEM. '*' and '#' denote significant differences (p < 0.05) as compared to young and adult, respectively Novel object recognition test in control, NaB, and antisense-treated old mice: novel object recognition test showing percentage of time spent with object and discrimination index in a saline and NaB group, b sham and antisense group. Data represent mean ± SEM. '*' denotes significant differences (p < 0.05) as compared to saline or sham group [41][42][43][44][45][46][47][48][49]. They were downregulated during amnesia, aging, neurodegeneration, and stress-induced cognitive decline [13-15, 27, 34, 50-52]. ...
The brain undergoes several anatomical, biochemical, and molecular changes during aging, which subsequently result in downregulation of synaptic plasticity genes and decline of memory. However, the regulation of these genes during aging is not clearly understood. Previously, we reported that the expression of histone deacetylase (HDAC)2 was upregulated in the hippocampus of old mice and negatively correlated with the decline in recognition memory. As HDAC2 regulates key synaptic plasticity neuronal immediate early genes (IEGs), we have examined their expression and epigenetic regulation. We noted that the expression of neuronal IEGs decreased both at mRNA and protein level in the hippocampus of old mice. To explore the underlying regulation, we analyzed the binding of HDAC2 and level of histone acetylation at the promoter of neuronal IEGs. While the binding of HDAC2 was higher, H3K9 and H3K14 acetylation level was lower at the promoter of these genes in old as compared to young and adult mice. Further, we inhibited HDAC2 non-specifically by sodium butyrate and specifically by antisense oligonucleotide to recover epigenetic modification, expression of neuronal IEGs, and memory in old mice. Inhibition of HDAC2 increased histone H3K9 and H3K14 acetylation level at the promoter of neuronal IEGs, their expression, and recognition memory in old mice as compared to control. Thus, inhibition of HDAC2 can be used as a therapeutic target to recover decline in memory due to aging and associated neurological disorders.
... Conversely, EGR1 overexpression in the forebrain enhances LTP in the mouse dentate gyrus (Penke et al., 2014). As hippocampal LTP is considered a molecular hallmark of spatial memory formation (Sweatt, 2016), the role of EGR1 in learning and memory paradigms has been extensively studied and recently reviewed elsewhere (Bozon et al., 2002;Knapska and Kaczmarek, 2004;Veyrac et al., 2014). This will thus not be discussed in detail here. ...
It is now clearly established that complex interactions between genes and environment are involved in multiple aspects of neuropsychiatric disorders, from determining an individual’s vulnerability to onset, to influencing its response to therapeutic intervention. In this perspective, it appears crucial to better understand how the organism reacts to environmental stimuli and provide a coordinated and adapted response. In the central nervous system, neuronal plasticity and neurotransmission are among the major processes integrating such complex interactions between genes and environmental stimuli. In particular, immediate early genes (IEGs) are critical components of these interactions as they provide the molecular framework for a rapid and dynamic response to neuronal activity while opening the possibility for a lasting and sustained adaptation through regulation of the expression of a wide range of genes. As a result, IEGs have been tightly associated with neuronal activity as well as a variety of higher order processes within the central nervous system such as learning, memory and sensitivity to reward. The immediate early gene and transcription factor early growth response 1 (EGR1) has thus been revealed as a major mediator and regulator of synaptic plasticity and neuronal activity in both physiological and pathological conditions. In this review article, we will focus on the role of EGR1 in the central nervous system. First, we will summarize the different factors influencing its activity. Then, we will analyze the amount of data, including genome-wide, that has emerged in the recent years describing the wide variety of genes, pathways and biological functions regulated directly or indirectly by EGR1. We will thus be able to gain better insights into the mechanisms underlying EGR1’s functions in physiological neuronal activity. Finally, we will discuss and illustrate the role of EGR1 in pathological states with a particular interest in cognitive functions and neuropsychiatric disorders.
... Each point represents one transcript that was both neuron-enriched and DE by TRAP-seq. (Bozon et al., 2002;Barbosa et al., 2008), and NFAT1 and MZF1 (Tables S9A,B), transcription factors that have not been reported to be involved in neuronal plasticity but that are expressed in hippocampal tissue as determined by our RNA-seq data. ...
The persistence of long-lasting changes in synaptic connectivity that underlie long-term memory require new RNA and protein synthesis. To elucidate the temporal pattern of gene expression that gives rise to long-lasting neuronal plasticity, we analyzed differentially-expressed (DE) RNAs in mouse hippocampal slices following induction of late phase long-term potentiation (L-LTP) specifically within pyramidal excitatory neurons using Translating Ribosome Affinity Purification RNA sequencing (TRAP-seq). We detected time-dependent changes in up- and down-regulated ribosome-associated mRNAs over 2 h following L-LTP induction, with minimal overlap of DE transcripts between time points. TRAP-seq revealed greater numbers of DE transcripts and magnitudes of LTP-induced changes than RNA-seq of all cell types in the hippocampus. Neuron-enriched transcripts had greater changes at the ribosome-loading level than the total RNA level, while RNA-seq identified many non-neuronal DE mRNAs. Our results highlight the importance of considering both time course and cell-type specificity in activity-dependent gene expression during memory formation.
... The c-fos is the most commonly used markers for neuronal plasticity. While a study on the c-fos gene provides information about the neural plasticity, Zif-268 has been implicated in long-term memory consolidation process [58], [59]. The c-fos promoter contains several critical regulatory elements, including the serum response element (SRE) that mediate transcription induced by glutamate in neurons. ...
Vitamins, especially the water-soluble complex of vitamins B, are highlighted in the daily clinical practice. Numerous studies emphasize the need for supplementation, mainly in groups with deficiency of these vitamins, such as the elderly, pregnant women, children and patients with diseases associates with cognitive disorder. Thiamine (B1), a vitamin of the diet, is an important cofactor for the three key enzymes involved in the citric acid cycle and the pentose phosphate cycle. Pyridoxine (B6) and cobalamin (B12) act in the CNS as a cofactor in the metabolism reactions of homocysteine. Deficiency of some neurotransmitter precursors can also cause symptoms of attention deficit hyperactivity disorder in children, especially amino acid and vitamin B deficiency. Inhibitory and excitatory neurotransmitters regulate diverse behavioral processes, including sleep, learning, memory and sensation of pain. They are also implicated in many pathological processes, such as epilepsy and neurotoxicity. Studies suggest that the excitatory amino acids may play a role in learning and memory. The binding of glutamate to its receptor triggers molecular and cellular events associated with numerous physiological and pathophysiological pathways, including the development of an increased sensation of pain (hyperalgesia), brain neurotoxicity or synaptic alterations involved in certain types of memory formation. Between the two major classes of neuroactive amino acids, γ-aminobutyric acid (GABA) is the major inhibitory amino acid. It is known that GABA plays a fundamental role in encoding information and behavioral control, in the regulation of motor function and in motor learning. The inter-relationships between diet, the brain and behavior are complex. However, micronutrients are known to have a direct influence on cognitive function through their involvement in the energy metabolism of neurons and glia cells, the synthesis of neurotransmitters, receptor binding and the maintenance of membrane ion pumps.
... We also examined two regions that directly and indirectly connect to the song system: the ventral tegmental area (VTA) and the medial preoptic nucleus (POM). The immediate early gene, ZENK, has been predominantly used to measure song-related activation of cells, because ZENK binds to promoter regions of many genes (Mello, 2002) and is important for late long-term potentiation and long-term memories (Bozon, Davis, & Laroche, 2002;Jones et al., 2001;Mello, 2002). Therefore, we chose ZENK as our immediate early gene of interest. ...
The nonapeptides oxytocin and vasopressin have been implicated in a variety of social behaviors. In zebra finches, oxytocin antagonists decrease pairing in both sexes, and pairing, in turn, increases expression of both mesotocin (the avian homologue of oxytocin) and vasotocin (the avian homologue of vasopressin). Increases in mesotocin and vasotocin mRNA are correlated with the amount of directed singing by males. Thus, in the present study, we examined the hypothesis that activation of cells containing nonapeptide receptors in song-related regions (ventral tegmental area, lateral septum, and medial preoptic nucleus) would also be correlated with directed singing in males. To rule out the possibility that these regions are involved in general pairing motivation, we also included females as subjects. In the ventral tegmental area, males had higher ZENK and V1aR than females and paired animals (regardless of sex) had higher ZENK and V1aR than did unpaired animals. In the medial preoptic nucleus, paired animals had higher ZENK than did unpaired animals, and there were no sex or pairing effects in the lateral septum. Only ZENK + V1aR in the medial preoptic nucleus was correlated with singing in males. These findings suggest that pairing is associated activation of nonapeptide receptors in the ventral tegmental area and the medial preoptic nucleus, but there is only partial evidence that courtship singing accounts for these findings. (PsycINFO Database Record
... One of the key signaling pathways under epigenetic control is that involving brain-derived neurotrophic factor (BDNF)-TrkB signaling (Martinowich et al. 2003;Bredy et al. 2007;Tsankova et al. 2007;Yasuda et al. 2009). In turn, one of the transcriptional targets of BDNF-TrkB signaling that has also been shown to be under epigenetic control is the regulatory immediate-early gene (IEG) Egr1 (Zif268) (Bozon et al. 2002;Knapska, Kaczmarek 2004;Nott et al. 2008;Guan et al. 2009). Egr1 encodes a zinc finger transcription factor and its mRNA expression is known to be upregulated in the hippocampus by associative learning (Knapska, Kaczmarek 2004). ...
Advancing our understanding of neuroplasticity and the development of novel therapeutics based upon this knowledge is critical in order to improve the treatment and prevention of a myriad of nervous system disorders. Epigenetic mechanisms of neuroplasticity involve the post-translational modification of chromatin and the recruitment or loss of macromolecular complexes that control neuronal activity-dependent gene expression. While over a century after Ramón y Cajal first described nuclear subcompartments and foci that we now know correspond to sites of active transcription with acetylated histones that are under epigenetic control, the rate and extent to which epigenetic processes act in a dynamic and combinatorial fashion to shape experience-dependent phenotypic and behavioral plasticity in response to various types of neuronal stimuli over a range of time scales is only now coming into focus. With growing recognition that a subset of human diseases involving cognitive dysfunction can be classified as ‘chromatinopathies’, in which aberrant chromatin-mediated neuroplasticity plays a causal role in the underlying disease pathophysiology, understanding the molecular nature of epigenetic mechanisms in the nervous system may provide important new avenues for the development of novel therapeutics. In this review, we discuss the chemistry and neurobiology of the histone deacetylase (HDAC) family of chromatin-modifying enzymes, outline the role of HDACs in the epigenetic control of neuronal function, and discuss the potential relevance of these epigenetic mechanisms to the development of therapeutics aiming to enhance memory and neuroplasticity. Finally, open questions, challenges, and critical needs for the field of ‘neuroepigenetics’ in the years to come will be summarized.
... Interestingly, we observed strong expression of Zif268 in the striatum and other brain regions after application of 6′-GNTI, but we cannot provide an underlying mechanism for this effect. Regulation of Zif268 expression potentially involves activation of PKA, PKC or CaMK signalling, involving either MAPK/ERK or CREB-dependent mechanisms (see Bozon et al., 2002). 6′-GNTI was described as a partial agonist for G-protein activation and inhibition of adenylate cyclase, with hardly any activation of β-arrestin compared with full agonists such as U-50488H and EKC, in HEK293T cells transfected with human κ receptors (Rives et al., 2012). ...
Background:
With a prevalence of 1-2%, epilepsies belong to the most frequent neurological diseases worldwide. Although antiepileptic drugs are available since several decades, the rate of patients refractory to medication is still over 30 %. Antiepileptic effects of kappa opioid receptor (κ receptor) agonists were suggested since the 1980s. However, their clinical use was hampered by dysphoric side effects. Recently, G-protein biased κ receptor agonists, were developed suggesting reduced aversive effects.
Experimental approach:
We investigated the effects of the κ receptor agonist U-50488H and the G-protein biased partial κ receptor agonist 6'-GNTI in models of acute seizures and drug resistant temporal lobe epilepsy and in the conditioned place avoidance test. Moreover, we performed slice electrophysiology to understand the functional mechanisms of 6'-GNTI.
Key results:
Like previously shown for U-50488H, 6'-GNTI markedly increased the threshold for pentylenetetrazole induced seizures. All treated mice displayed reduced paroxysmal activity in response to U-50488H (20 mg / kg) or 6'-GNTI (10 - 30 nmoles) treatment in the mouse model of intra-hippocampal injection of kainic acid. Single cell recordings on hippocampal pyramidal cells revealed enhanced inhibitory signalling as potential mechanisms causing the reduction of paroxysmal activity. Effects of 6'-GNTI were blocked in both seizure models by the κ receptor antagonist 5'-GNTI. Moreover, 6'-GNTI did not induce conditioned place avoidance, a measure of aversive effects, while U-50488H did.
... To better understand these molecular mechanism, we examined the effect of general anesthesia with isoflurane on behavioral training in rats and the corresponding transcription of plasticity-related immediateearly genes (IEGs; Alkire et al., 2007;Alkire and Guzowski, 2008). Synaptic plasticity is the physical basis of memory, and the expression of certain plasticity-related IEGs, including Arc and Zif268, is required to form long-term memory (Dragunow, 1996;Bozon et al., 2002Bozon et al., , 2003Davis et al., 2003;Plath et al., 2006;Bramham et al., 2008Bramham et al., , 2010Nakayama et al., 2015). In a neurobiological context, learning is the formation of longterm memory and amnesia is the loss of previously acquired memory (retrograde amnesia) or the failure to form new longterm memories (anterograde amnesia). ...
Background:
Anterograde amnesia is a hallmark effect of volatile anesthetics. Isoflurane is known to affect both the translation and transcription of plasticity-associated genes required for normal memory formation in many brain regions. What is not known is whether isoflurane anesthesia prevents the initiation of transcription or whether it halts transcription already in progress. We tested the hypothesis that general anesthesia with isoflurane prevents learning-induced initiation of transcription of several memory-associated immediate-early genes (IEGs) correlated with amnesia; we also assessed whether it stops transcription initiated prior to anesthetic administration.
Methods:
Using a Tone Fear Conditioning paradigm, rats were trained to associate a tone with foot-shock. Animals received either no anesthesia, anesthesia immediately after training, or anesthesia before, during, and after training. Animals were either sacrificed after training or tested 24 h later for long-term memory. Using Cellular Compartment Analysis of Temporal Activity by Fluorescence in situ Hybridization (catFISH), we examined the percentage of neurons expressing the IEGs Arc/Arg3.1 and Zif268/Egr1/Ngfi-A/Krox-24 in the dorsal hippocampus, primary somatosensory cortex, and primary auditory cortex.
Results:
On a cellular level, isoflurane administered at high doses (general anesthesia) prevented initiation of transcription, but did not stop transcription of Arc and Zif268 mRNA initiated prior to anesthesia. On a behavioral level, the same level of isoflurane anesthesia produced anterograde amnesia for fear conditioning when administered before and during training, but did not produce retrograde amnesia when administered immediately after training.
Conclusion:
General anesthesia with isoflurane prevents initiation of learning-related transcription but does not stop ongoing transcription of two plasticity-related IEGs, Arc and Zif268, a pattern of disruption that parallels the effects of isoflurane on memory formation. Combined with published research on the effects of volatile anesthetics on memory in behaving animals, our data suggests that different levels of anesthesia affect memory via different mechanisms: general anesthesia prevents elevation of mRNA levels of Arc and Zif268 which are necessary for normal memory formation, while anesthesia at lower doses affects the strength of memory by affecting levels of plasticity-related proteins.
... Zif268 has a high level of basal expression in many brain areas as compared to c-fos that has a low basal expression level in most neural systems [23,24]. As biological markers, Zif268 complements c-Fos as it is also largely distributed in many brain regions and, like c-Fos, has been linked to learning and memory [25][26][27]. Using these biomarkers, we showed that the processing of interference in WM might require a specific and negative control of the dentate gyrus (DG) of the dorsal hippocampus materialized by an absence of activation of the expression of both Zif268 and c-Fos. ...
How does the brain discriminate essential information aimed to be stored permanently from information required only temporarily, and that needs to be cleared away for not saturating our precious memory space? Reference Memory (RM) refers to the long-term storage of invariable information whereas Working Memory (WM) depends on the short-term storage of trial-unique information. Previous work has revealed that WM tasks are very sensitive to proactive interference. In order to prevent such interference, irrelevant old memories must be forgotten to give new ones the opportunity to be stabilized. However, unlike memory, physiological processes underlying this adaptive form of forgetting are still poorly understood. Here, we precisely ask what specific brain structure(s) could be responsible for such process to occur. To answer this question, we trained rats in a radial maze using three paradigms, a RM task and two WM tasks involving or not the processing of interference but strictly identical in terms of locomotion or motivation. We showed that an inhibition of the expression of Zif268 and c-Fos, two indirect markers of neuronal activity and synaptic plasticity, was observed in the dentate gyrus of the dorsal hippocampus when processing such interfering previously stored information. Conversely, we showed that inactivating the dentate gyrus impairs both RM and WM, but improves the processing of interference. Altogether, these results strongly suggest for the first time that the dentate gyrus could be a key structure involved in adaptive forgetting.
... To clarify how the altered histone acetylation leads to the laparotomy-induced cognitive deficits, we investigated the expression of proteins related to cognition performance in 16month old mice. Studies have linked the increased histone acetylation in the hippocampus to memory permissive for the transcription of learning-related plasticity genes (Ravi and Kannan, 2013) and attributed neuron damages and synaptic plasticity changes to the development of POCD (Bozon et al., 2002;Jungwirth et al., 2009;Cibelli et al., 2010;Lin and Zuo, 2011). We observed that neuroapoptosis-related proteins cleaved caspase-3 and iNOS were up-regulated, and synaptic plasticity-related proteins BDNF, synapsin 1, and PSD95 were down-regulated in the 16-month old mice after the laparotomy. ...
Postoperative cognitive dysfunction (POCD) is a recognized clinical entity characterized with cognitive deficits after anesthesia and surgery, especially in aged patients. Previous studies have shown that histone acetylation plays a key role in hippocampal synaptic plasticity and memory formation. However, its role in POCD remains to be determined. Here, we show that suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, attenuates POCD in aging Mice. After exposed to the laparotomy, a surgical procedure involving an incision into abdominal walls to examine the abdominal organs, 16- but not 3-month old male C57BL/6 mice developed obvious cognitive impairments in the test of long-term contextual fear conditioning. Intracerebroventricular (i.c.v.) injection of SAHA at the dose of (20 μg/2 μl) 3 h before and daily after the laparotomy restored the laparotomy-induced reduction of hippocampal acetyl-H3 and acetyl-H4 levels and significantly attenuated the hippocampus-dependent long-term memory (LTM) impairments in 16-month old mice. SAHA also reduced the expression of cleaved caspase-3, inducible nitric oxide synthase (iNOS) and N-methyl-D-aspartate (NMDA) receptor-calcium/calmodulin dependent kinase II (CaMKII) pathway, and increased the expression of brain-derived neurotrophic factor (BDNF), synapsin 1, and postsynaptic density 95 (PSD95). Taken together, our data suggest that the decrease of histone acetylation contributes to POCD and may serve as a target to improve the neurological outcome of POCD.
Transcription factors have a pivotal role in synaptic plasticity and the associated modification of neuronal networks required for memory formation and consolidation. The nuclear receptors subfamily 4 group A (Nr4a) have emerged as possible modulators of hippocampal synaptic plasticity and cognitive functions. However, the molecular and cellular mechanisms underlying Nr4a2-mediated hippocampal synaptic plasticity are not completely known. Here, we report that neuronal activity enhances Nr4a2 expression and function in cultured mouse hippocampal neurons (both sexes) by an ionotropic glutamate receptor/Ca ²⁺ /cAMP response element-binding protein/CREB-regulated transcription factor 1 (iGluR/Ca ²⁺ /CREB/CRTC1) pathway. Nr4a2 activation mediates Brain-derived neurotrophic factor (BDNF) production and increases expression of iGluRs, thereby affecting long-term depression (LTD) at CA3-CA1 synapses in acute mouse hippocampal slices (both sexes). Altogether, our results indicate that the iGluR/Ca 2+/ CREB/CRTC1 pathway mediates activity-dependent expression of Nr4a2 which is involved in glutamatergic synaptic plasticity by increasing BDNF and synaptic GluA1-AMPARs. Therefore, Nr4a2 activation could be a therapeutical approach for brain disorders associated with dysregulated synaptic plasticity.
SIGNIFICANT STATEMENT:
A major factor that regulates fast excitatory synaptic transmission and plasticity is the modulation of synaptic AMPA receptors. However, despite decades of research, the underlying mechanisms of this modulation remain poorly understood. Our study identified a molecular pathway that links neuronal activity with AMPA receptor modulation and hippocampal synaptic plasticity through the activation of Nr4a2, a member of the nuclear receptor subfamily 4. Since several compounds have been described to activate Nr4a2, our study not only provides mechanistic insights into the molecular pathways related to hippocampal synaptic plasticity and learning, but also identifies Nr4a2 as a potential therapeutic target for pathological conditions associated with dysregulation of glutamatergic synaptic function.
Previous studies have speculated that tidal rhythm of Onchidium reevesii was linked to its memory ability, which has not been well explained. Mapk1 and egr1 genes are closely related to memory formation in the MAPK signaling pathway that have been extensively studied in vertebrates. These two genes are involved in Long-term potentiation (LTP), which is generally regarded as one of the main molecular mechanisms underlying learning and memory. To investigate the relationship between tidal rhythm of O. reevesii and its memory ability, we studied the molecular mechanism of mapk1 and egr1 genes cooperating with tidal rhythm. The full-length cDNA sequence of mapk1 gene was cloned by RACE method and analyzed by bioinformatics, then qRT-PCR was used to analyze its expression levels in each tissue. A homology analysis and phylogenetic tree reconstruction revealed that O. reevesii is most closely related to the Biomphalaria glabrata. The qRT-PCR showed that mapk1 was expressed in all the tissues, but the highest expression was in the ganglion. We speculated that mapk1 is related to memory formation of O. reevesii. Then we used qRT-PCR to detect the expression of mapk1 and egr1 in ganglion of O. reevesi under tidal stimulation. The expression of mapk1 and egr1 at the rising tide points were significantly higher (P < 0.05) than that at the previous one lowest tide points on May 15–21, except the rising tide point on May 20 and May 21 of mapk1. The expression of mapk1 and egr1 genes in the ganglion sampled for 7-day were basically the same as that of the tide change of the same day. The expression of the two genes were upregulated at raising tide and downregulated at the lowest tide, it is presumed that the O. reevesii was affected by the tide for a long time and formed the tide memory.
This study investigated physical proximity and paracrine activity of neurotrophic
factor-secreting cells (NTF-SCs) on beta-amyloid treated cells. Mesenchymal stem cells
(MSCs) - to-NTF-SCs (Astrocyte –like cells) trans-differentiation was confirmed using
immunofluorescence staining of GFAP. BDNF and NGF levels were measured by
ELISA. To mimic AD-like condition, SH-SY5Y cells were exposed to 10 µM Aβ1-42. SHSY5Y cells were allocated into Control; and Aβ1–42-treated cells. Treated cells were
further classified into three subgroups including Aβ1–42 cells, Aβ1–42 cells + NTF-SCs
(CM) and Aβ1–42 cells + NTF-SCs co-culture. Cell viability was measured by MTT
assay. Anti-inflammatory and anti-tau hyperphosphorylation effects of NTF-SCs were
assessed via monitoring TNF-α and hyperphosphorylated Tau protein expression level
respectively. To explore the impact of NTF-SCs on synaptogenesis and synaptic
functionality, real-time PCR assay was performed to measure the expression of
synapsine 1, homer 1 and ZIF268. The level of synaptophysin was monitored via
immunofluorescence staining. Data showed MSCs potential in trans-differentiating
toward NTF-SCs indicated with enhanced GFAP expression (p<0.05). ELISA assay
confirmed the superiority of NTF-SCs in releasing NGF and BDNF compared to the
MSCs (p<0.05). Aβ significantly induced SH-SY5Y cells death while juxtacrine and
paracrine activity of NTF-SCs significantly blunted these conditions (p<0.05). Transdifferentiated cells had potential to reduce Tau hyperphosphorylation and TNF-α level
after treatment with Aβ through juxtacrine and paracrine mechanisms (p<0.05).
Moreover, NTF-SCs significantly increased the expression rate of synapsin 1, homer 1
and zif 268 genes in Aβ-treated cells compared to matched-control group coincided with
induction of synaptophysin at the protein level(p<0.05). NTF-SCs reversed AD-like
neuropathological alterations in SH-SY5Y cells via paracrine and juxtacrine
mechanisms.
Objective: Phosphatidylinositol (PI) has been suggested to have important functions in intracellular signal transduction and to be effective in improving brain function. However, its effects on memory and learning ability have not been elucidated. Therefore, in the present study, we aimed to clarify the effects of oral administration of 59% purified PI (PI 50) on memory and learning ability in rats.
Methods: Wistar male rats were divided into four groups: (1) distilled water administration group, (2) 30 mg/kg PI 50 administration group, (3) 100 mg/kg PI 50 administration group, and (4) 300 mg/kg PI 50 administration group. Effects of oral administration of PI 50 on memory and learning ability were investigated using behavioral and molecular biological techniques.
Result: In the learning and memory behavioral tests, the PI 50-administered group showed improvements in spatial memory and learning ability compared to the distilled water-administered group. Additionally, c-Fos- and BrdU-positive cells in the hippocampus increased significantly in the PI 50-administered group. The PI 50-administered group showed a significant increase in BDNF, PKC-α, and MAPK protein expression compared to the distilled water- administered group.
Conclusion: These results indicate that PI 50 intake stimulates nerve cell activation and growth factor secretion in the hippocampus by activating the PKC-α and MAPK signal pathways, thus, facilitating the development and proliferation of nerve cells and may affect the enhancement of learning and memory ability. This study provides evidence that PI 50 may affect the enhancement of learning and memory ability.
La maladie d’Alzheimer (MA) est une maladie multifactorielle et à ce jour aucune cause des formes sporadiques de la maladie, qui représente plus de 99% des cas, n’a été mise en évidence. Des données émergentes de la littérature suggèrent l’existence d’un lien entre les dysfonctionnements thyroïdiens et la MA. Dans ce contexte, l’objectif de cette étude était de préciser l’implication de l’hypothyroïdie dans les processus neuropathologiques de la MA. En utilisant un modèle de rats rendus hypothyroïdiens par un traitement au propylthiouracile (PTU), nous avons montré que l’hypothyroïdie favorise la mise en place de lésions caractéristiques de la MA dans l’hippocampe, structure du cerveau précocement altérée dans la maladie et qui joue un rôle crucial dans les processus de mémoire. Une étude d’IRM in vivo a révélé une diminution progressive du volume cérébral des rats hypothyroïdiens. Dans l’hippocampe, l’hypothyroïdie s’accompagne d’une augmentation de la production de peptides amyloïdes, d’une hyperphosphorylation de la protéine Tau et d’une augmentation de la libération de plusieurs cytokines pro-Inflammatoires. Ces lésions, caractéristiques de la MA, sont associées à des troubles de la mémoire spatiale à court et long terme et à une altération de deux voies de signalisation connues pour jouer un rôle important dans les processus de plasticité synaptique et de mémoire : la voie calcique et la voie ERK-MAPK. Afin d’évaluer le potentiel de restauration de ces lésions, une partie des rats hypothyroïdiens a reçu des injections intra-Péritonéales de triiodothyronine (T3), forme active des hormones thyroïdiennes. Nos résultats montrent que l’administration de T3 permet de restaurer les déficits de mémoire spatiale à court terme, mais pas à long terme. En outre, ce même traitement permet de restaurer les niveaux de cytokines pro-Inflammatoires, de peptides amyloïdes ainsi que les voies « calcique » et « ERK-MAPK ». Ces données renforcent l’existence d’un lien entre l’hypothyroïdie et la MA : elles suggèrent que l’hypothyroïdie pourrait représenter un facteur important pouvant impacter le risque de développer des formes sporadiques de la MA.
Schizophrenia is a multifactorial disorder caused by a combination of genetic variations and exposure to environmental insults. Sleep and circadian rhythm disturbances are a prominent and ubiquitous feature of many psychiatric disorders, including schizophrenia. There is growing interest in uncovering the mechanistic link between schizophrenia and circadian rhythms, which may directly affect disorder outcomes. In this review, we explore the interaction between schizophrenia and circadian rhythms from 2 complementary angles. First, we review evidence that sleep and circadian rhythm disturbances constitute a fundamental component of schizophrenia, as supported by both human studies and animal models with genetic mutations related to schizophrenia. Second, we discuss the idea that circadian rhythm disruption interacts with existing risk factors for schizophrenia to promote schizophrenia-relevant behavioral and neurobiological abnormalities. Understanding the mechanistic link between schizophrenia and circadian rhythms will have implications for mitigating risk to the disorder and informing the development of circadian-based therapies.
Learned vocal communication emerges from the coordination of sensory and motor learning, reflects the function of a distributed but integrated neural circuit, and unfolds across several timescales, often occurring in maturing animals. Because nearly all brain organization and function originates from patterns of genomic activation, it is crucial to understand principles of how the genome works in order to understand how learned vocal communication arises. In this chapter, the fact that genome functions have high evolutionary conservation will be leveraged to provide a conceptual guide for how research using a species of songbird, the zebra finch, can deepen and expand clinical findings from humans. Additionally, this chapter provides examples for how studies in the zebra finch can uncover fundamental processes of learned vocal communication that are of value for understanding human speech and language. Examples include the organization of specialized neural circuits, responses to social communication experiences, activation of motor plans, and consideration of how the age and sex of the individual intersect with vocal communication skills, all of which have potential to inform on vocal learning mechanisms in humans. Together, our current state of knowledge advances the idea that humans and songbirds do not simply share superficial parallels; rather, they share deep biological properties to accomplish the complex, multi-level processes required for learning and producing meaningful communication patterns.
The decline in neurogenesis is a very critical problem in Alzheimer disease. Different biological activities have been reported for medicinal application of quercetin. Herein, we investigated the neurogenesis potential of quercetin in a rat model of Alzheimer's disease induced by amyloid-beta injection. Rats were randomly divided into Control, Alzheimer + Saline and Alzheimer + Quercetin groups. Following the administration of Amyloid-beta, rats in the Alzheimer + Quercetin group received 40 mg/kg/day quercetin orally for one month. Our data demonstrated amyloid-β injection could impair learning and memory processing in rats indicated by passive avoidance test evaluation. We noted that one-month quercetin treatment alleviated the detrimental effects of amyloid-β on spatial learning and memory parameters using Morris water maze analysis. Quercetin was found to increase the number of proliferating neural stem/progenitor cells. Notably, quercetin increased the number of DCX-expressing cells, indicating the active dynamic growth of neural progenitor cells in the dentate gyrus of the hippocampus. We further observed that the quercetin improved the number of BrdU/NeuN positive cells contributed to enhanced adult neurogenesis. Based on our results, quercetin had the potential to promote the expression of BDNF, NGF, CREB, and EGR-1 genes involved in regulating neurogenesis. These data suggest that quercetin can play a valuable role in alleviating Alzheimer's disease symptoms by enhancing adult neurogenesis mechanism.
In humans, some males experience reductions in testosterone levels, as a natural consequence of aging or in the clinical condition termed hypogonadism, which are associated with impaired cognitive performance and mood disorder(s). Some of these behavioral deficits can be reversed by testosterone treatment. Our previous work in rats reported that sex differences in the expression of the transcription factor Zif268, a downstream target of testosterone, within the medial prefrontal cortex (mPFC) mediates sex differences in social interaction. In the present study, we aimed to examine the effects of gonadectomy (GNX) in male rats on mPFC Zif268 expression, mood and cognitive behaviors. We also examined whether reinstitution of Zif268 in GNX rats will correct some of the behavioral deficits observed following GNX. Our results show that GNX induced a downregulation of Zif268 protein in the mPFC, which was concomitant with impaired memory in the y-maze and spontaneous object recognition test, reduced social interaction time, and depression-like behaviors in the forced swim test. Reinstitution of mPFC Zif268, using a novel adeno-associated-viral (AAV) construct, abrogated GNX-induced working memory and long-term memory impairments, and reductions in social interaction time, but not GNX-induced depression-like behaviors. These findings suggest that mPFC Zif268 exerts beneficial effects on memory and social interaction, and could be a potential target for novel treatments for behavioral impairments observed in hypogonadal and aged men with declining levels of gonadal hormones.
Study objectives:
It is commonly accepted that sleep is beneficial to memory processes, but it is still unclear if this benefit originates from improved memory consolidation or enhanced information processing. It has thus been proposed that sleep may also promote forgetting of undesirable and non-essential memories, a process required for optimization of cognitive resources. We tested the hypothesis that non-rapid eye movement sleep (NREMS) promotes forgetting of irrelevant information, more specifically when processing information in working memory (WM), while REM sleep (REMS) facilitates the consolidation of important information.
Methods:
We recorded sleep patterns of rats trained in a radial maze in three different tasks engaging either the long-term or short-term storage of information, as well as a gradual level of interference.
Measurements and results:
We observed a transient increase in REMS amount on the day the animal learned the rule of a long-term/ reference memory task (RM), and, in contrast, a positive correlation between the performance of rats trained in a WM task involving an important processing of interference and the amount of NREMS or slow wave activity. Various oscillatory events were also differentially modulated by the type of training involved. Notably, NREMS spindles and REMS rapid theta increase with RM training, while sharp-wave ripples increase with all types of training.
Conclusions:
These results suggest that REMS, but also rapid oscillations occurring during NREMS would be specifically implicated in the long-term memory in RM, whereas NREMS and slow oscillations could be involved in the forgetting of irrelevant information required for WM.
Objective: To investigate the effects of ketamine on learning and memory function and expression of early growth responsive gene-1 (Egr-1) in hippocampus of minor rats. Methods: Twenty minor SD rats were randomly divided into normal saline control group (Ns group) and ketamine group (Ket group). Rats in Ket group were intraperitoneally injected with 50 mg/kg ketamine, and those in Ns group were administrated with same amount of normal saline for 6 d. Morris water maze test was adopted to explore the learning and memory function at 24 h after the final injection. The sections of hippocampus were obtained at 1 h of the final Morris water maze test, and the expression of Egr-1 mRNA and protein was determined by Real-Time PCR and Western blotting respectively. Results: In Morris water maze test, the latency in place navigation in Ket group was significantly longer than that in Ns group (P<0.05), and the times of crossing the platform and total swimming distance in Ket group were significantly decreased compared with Ns group (P<0.05). It was revealed that the expression of Egr-1 mRNA and protein in Ket group was significantly lower than that in Ns group (P<0.05). Conclusion: Application of ketamine in minor rats may result in learning and memory impairment, and the mechanism may be associated with the down-regulation of expression of Egr-1 in hippocampus.
Discoveries related to genes, genetic code, gene sequencing, genetic techniques, and RNA sequencing have allowed the manipulation of genes involved in memory and provided a better understanding of the role of genes expression to form memories. Memories are represented in cells as long-term changes in the function of proteins translated from mRNA, transcribed from genes. The first protein identified as being associated with the neurobiology of learning and memory was cyclic AMP (cAMP), which affects memory processes by initiating a signaling cascade leading to the phosphorylation of cyclic AMP responsive element binding protein. Neuronal growth-associated proteins (nGAPs) such as SCGIO and GAP-43, and immediate early genes (lEGs) such as c-fos, arc, and zif/268, are also mediated by learning and memory tasks. Experience-dependent gene expression increases in synaptic strength underlie memory formation in networks of neurons and are known as Hebb's postulate and long-term potentiation (LTP). Genes associated with LTP are temporally classified as immediate early genes (lEGs) or late-responder genes There are two fundamental types of LTP: N-methyl-D-aspartate (NMDA) receptor dependent and NMDA receptor independent. Knockout mutant mice studies have revealed the importance of several gene products in LTP induction and learning of a hippocampally dependent learning task, including tissue plasminogen activator (tPA), c-fos, and CREB. The mRNA transcription that encodes proteins essential for strengthening the synapse occurs during late-phase LTP, but not during early-phase LT. It suggests that synapse strengthening during late-phase-LTP is protein synthesis dependent.
Mastering the Morris water maze (MWM) requires the animal to consolidate, retain and retrieve spatial localizations of relevant visual cues. However, it is necessary to investigate whether a reorganization of the neural networks takes place when part of the spatial information is removed. We conducted four experiments using the MWM. A classical reference memory procedure was performed over five training days, RM5 (n=7), and eight days, RM8 (n=7), with the whole room and all the spatial cues presented. Another group of animals were trained in the same protocol, but they received an additional day of training with only partial cues, PC (n=8). Finally, a third group of animals performed the classical task, followed by an overtraining with partial cues for four more days, OPC (n=8). After completing these tasks, cytochrome c-oxidase activity (CO) in several brain limbic system structures was compared between groups. In addition, c-Fos positive cells were measured in the RM5, RM8, PC and OPC groups. No significant differences were found among the four groups in escape latencies or time spent in the target quadrant. CO revealed involvement of the prefrontal and parietal cortices, dorsal and ventral striatum, CA1 and CA3 subfields of the dorsal hippocampus, basolateral and lateral amygdala, and mammillary nuclei in the PC group, compared to the RM group. In the OPC group, involvement of the ventral striatum and anteroventral thalamus and the absence of amygdala involvement were revealed, compared to the PC group. C-Fos results highlighted the role of the prefrontal cortex, dorsal striatum, anterodorsal thalamus and CA3 in the PC group, compared to the OPC, RM5 and RM8 groups. The animals were able to find the escape platform even when only a portion of the space where the cues were placed was available. Although the groups did not differ behaviorally, energetic brain metabolism and immediate early gene expression revealed the engagement of different neural structures in the groups that received more training without the entire surrounding space.
Copyright © 2015. Published by Elsevier B.V.
Rats were given to drink an unfamiliar taste solution under conditions that result in long-term memory of that taste. The insular cortex, which contains the taste cortex, was then removed and assayed for activation of mitogen-activated protein kinase (MAPK) cascades by using antibodies to the activated forms of various MAPKs. Extracellular responsive kinase 1–2 (ERK1–2) in the cortical homogenate was significantly activated within <30 min of drinking the taste solution, without alteration in the total level of the ERK1–2 proteins. The activity subsided to basal levels within <60 min. In contrast, ERK1–2 was not activated when the taste was made familiar. The effect of the unfamiliar taste was specific to the insular cortex. Jun N-terminal kinase 1–2 (JNK1–2) was activated by drinking the taste but with a delayed time course, whereas the activity of Akt kinase and p38MAPK remained unchanged. Elk-1, a member of the ternary complex factor and an ERK/JNK downstream substrate, was activated with a time course similar to that of ERK1–2. Microinjection of a reversible inhibitor of MAPK/ERK kinase into the insular cortex shortly before exposure to the novel taste in a conditioned taste aversion training paradigm attenuated long-term taste aversion memory without significantly affecting short-term memory or the sensory, motor, and motivational faculties required to express long-term taste aversion memory. It was concluded that ERK and JNK are specifically and differentially activated in the insular cortex after exposure to a novel taste, and that this activation is required for consolidation of long-term taste memory.
The Ets family of transcription factors includes nuclear phosphoproteins that are involved in cell proliferation, differentiation and oncogenic transformation. The family is defined by a conserved DNA-binding domain (the ETS-DBD), which forms a highly conserved, winged, helix-turn-helix structural motif. As targets of the Ras–MAPK signaling pathway, Ets proteins function as critical nuclear integrators of ubiquitous signaling cascades. To direct signals to specific target genes, Ets proteins interact with (other) transcription factors that promote the binding of Ets proteins to composite Ras-responsive elements.
It has long been proposed that the cellular and molecular mechanisms responsible for LTP may well involve the mechanisms that lead to the type of synaptic modification that occurs during learning. However, it is also known that a single memory trace is encoded in spatially distributed networks; implying that alterations of synaptic strength occur at multiple sites along circuits of connected cells. Recent evidence suggests that regulation of the gene encoding syntaxin 1B, a presynaptic protein involved in exocytosis, plays an important role in the mediation of trans-synaptic LTP, a candidate mechanism for the propagation of plasticity in neural circuits during learning. Using in situ hybridization to measure the mRNA levels at different time points after learning a spatial working or reference memory task, we show that expression of the gene encoding this protein in the hippocampal and corticoprefrontal circuits increases linearly with performance at a critical window of learning when rats are reaching between 75% and 100% of their maximal performance. No changes were observed during the early phases of learning or when rats where overtrained. The correlational analysis indicates that coordinated increases in syntaxin 1B expression occurs in hippocampal circuits during working memory and in more widespread hippocampocortical circuits during reference memory. These results suggest that a form of trans-synaptic plasticity mediated in part by regulation of the expression of syntaxin 1B may play an active role in configuring specific spatially distributed circuits during the laying down of memories.
We investigated the participation of genomic regulatory events in the response of the songbird brain to a natural auditory stimulus of known physiological and behavioral relevance, birdsong. Using in situ hybridization, we detected a rapid increase in forebrain mRNA levels of an immediate-early gene encoding a transcriptional regulator (ZENK; also known as zif-268, egr-1, NGFI-A, or Krox-24) following presentation of tape-recorded songs to canaries (Serinus canaria) and zebra finches (Taeniopygia guttata). ZENK induction is most marked in a forebrain region believed to participate in auditory processing and is greatest when birds hear the song of their own species. A significantly lower level of induction occurs when birds hear the song of a different species and no induction is seen after exposure to tone bursts. Cellular analysis indicates that the level of induction reflects the proportion of neurons recruited to express the gene. These results suggest a role for genomic responses in neural processes linked to song pattern recognition, discrimination, or the formation of auditory associations.
Recent studies in invertebrates indicate that a rapid genomic response to neuronal stimulation has a critical role in long-term changes in synaptic efficacy. Because several of the genes (immediately early genes; IEGs) that respond rapidly to growth factor stimulation of vertebrate cells in vitro are also activated by neuronal stimulation in vivo, attention has focused on the possibility that they play a part in synaptic plasticity in vertebrate nervous systems. Four IEGs thought to encode transcription factors, zif/268 (also termed Egr-1, NGFI-A, Krox 24), c-fos, c-jun, and jun-B are rapidly induced in the brain by seizure activity, and we have now studied the induction of these genes in a well-characterized model of synaptic plasticity in the vertebrate brain--long-term potentiation (LTP) of the perforant pathgranule cell (pp-gc) synapse in vivo. We found that high-frequency (but not low-frequency) stimulation of the pp-gc synapse markedly increases zif/268 messenger RNA (mRNA) levels in the ipsilateral granule cell neurons; mRNA of c-fos, c-jun and jun-B is less consistently increased. The stimulus frequency and intensity required to increase zif/268 mRNA levels are similar to those required to induce LTP, which is also seen only ipsilaterally, and both responses are blocked by NMDA-receptor antagonists as well as by convergent synaptic inhibitory inputs already known to block LTP. Accordingly, zif/268 mRNA levels and LTP seem to be regulated by similar synaptic mechanisms.
We previously reported the identification of a mouse gene, Krox-20, encoding a protein with three "zinc fingers" (DNA-binding domains with coordinated zinc ions) whose expression is regulated during G0/G1 transition (cell-cycle reentry). We now have isolated cDNAs corresponding to a related gene, Krox-24. Krox-24 encodes a protein with zinc fingers nearly identical to those encoded by Krox-20 and similar to those of transcription factor Sp1. Similarity between Krox-20 and Krox-24 proteins also extends to several blocks of amino acid sequence located upstream of the finger region. Like Krox-20, Krox-24 is transiently activated in quiescent cells after treatment with fetal bovine serum or purified growth factors. The kinetics of activation are similar to those of the protooncogene c-fos. The induction does not require de novo protein synthesis, and cycloheximide treatment of the cells leads to superinduction due, at least in part, to mRNA stabilization. In the mouse, the two genes are expressed in a tissue-specific manner, with slightly different patterns. These properties suggest that Krox-20 and Krox-24 may encode transcription factors with identical DNA target sequences and that these factors may be involved in the modulation of cell proliferation and differentiation.
Reviews studies that have used protein synthesis inhibitors to test the hypothesis that memory in part depends on brain protein synthesis. Evidence from learning curves, examination of short-term retention, and posttraining drug injection indicate that initial acquisition is not dependent on such synthesis, but it appears that protein synthesis, during or shortly after training, is an essential step in the formation of long-term memory. Possible side effects of protein synthesis inhibitors are considered in terms of locomotor activity, abnormal cerebral electrical activity, conditioned aversion, and catecholamine biosynthesis. Stages of memory formation are discussed, and the possibility that kindling, drug tolerance, and enzyme induction are dependent on protein synthesis is considered. (8 p ref)
NGFI-A (also known as EGR-1, zif/268, and Krox-24) is a zinc finger transcription factor induced in many cell types by a variety
of growth and differentiation stimuli. To determine if NGFI-A plays a requisite role in these processes, we used homologous
recombination to mutate both alleles of NGFI-A in embryonic stem (ES) cells and examined its effect on growth and differentiation.
We find that ES cells lacking NGFI-A exhibit similar growth rates and serum-induced gene expression profiles compared to wild-type
parental cells. They are capable of differentiating into neurons, cardiac myocytes, chondrocytes, and squamous epithelium.
Chimeric mice were generated from targeted ES cells, and their progeny were crossed to produce homozygous mutant mice. Growth
and histological analyses of mice lacking NGFI-A confirm the finding in ES cells that NGFI-A is not required for many of the
processes associated with its expression and suggest that the function of NGFI-A is either more subtle in vivo or masked by redundant expression provided by other gene family members such as NGFI-C, Krox-20, or EGR3.
The zinc finger gene Krox-20 is transcribed in two alternate segments (rhombomeres) of the developing hindbrain. To investigate its function, we have used homologous recombination to generate mice carrying an in-frame insertion of the E. coli lacZ gene within Krox-20. Analysis of the beta-galactosidase pattern in heterozygous embryos confirmed the known profile with expression restricted to rhombomeres (r) 3 and 5. Mice homozygous for the mutation die during the first two weeks after birth. Anatomical analysis of the hindbrain and of the cranial nerves during embryogenesis, combined with the determination of the expression patterns of rhombomere-specific genes, demonstrated that Krox-20 inactivation results in a marked reduction or elimination of r3 and r5. We conclude that Krox-20, although not required for the initial delimitation of r3 and r5, plays an important role in the process of segmentation governing hindbrain development.
The molecular mechanisms controlling the process of myelination by Schwann cells remain elusive, despite recent progress in the identification and characterization of genes encoding myelin components (reviewed in ref. 1). We have created a null allele in the mouse Krox-20 gene, which encodes a zinc-finger transcription factor, by in-frame insertion of the Escherichia coli lacZ gene, and have shown that hindbrain segmentation is affected in Krox-20-/- embryos. We demonstrate here that Krox-20 is also activated in Schwann cells before the onset of myelination and that its disruption blocks Schwann cells at an early stage in their differentiation, thus preventing myelination in the peripheral nervous system. In Krox-20-/- mice, Schwann cells wrap their cytoplasmic processes only one and a half turns around the axon, and although they express the early myelin marker, myelin-associated glycoprotein, late myelin gene products are absent, including those for protein zero and myelin basic protein. Therefore Krox-20 is likely to control a set of genes required for completion of myelination in the peripheral nervous system.
We have investigated the early in vivo signaling events triggered by serum that lead to activation of the c-fos proto-oncogene in HeLa cells. Both RAF-1 and MEK kinase activities are fully induced within 3 min of serum treatment and quickly decrease thereafter, slightly preceding the activation and inactivation of p42MAPK/ERK2. ERK2 activity correlates tightly with a transient phosphatase-sensitive modification of ternary complex factor (TCF), manifested by the slower electrophoretic mobility of TCF-containing protein-DNA complexes. These induced complexes in turn correlate with the activity of the c-fos, egr-1, and junB promoters. Phorbol ester treatment induces the same events but with slower and prolonged kinetics. Inhibition of serine/threonine phosphatase activities by okadaic acid treatment reverses the repression of the c-fos promoter either after induction or without induction. This corresponds to the presence of the induced complexes and of ERK2 activity, as well as to the activation of a number of other kinases. Inhibition of tyrosine phosphatase activities by sodium vanadate treatment delays but does not block ERK2 inactivation, TCF dephosphorylation, and c-fos repression. The tight linkage in vivo between the activity of MAP kinase, TCF phosphorylation, and immediate-early gene promoter activity is consistent with the notion that a stable ternary complex over the serum response element is a direct target for the MAP kinase signaling cascade. Furthermore, serine/threonine phosphatases are implicated in regulating the kinase cascade, as well as the state of TCF modification and c-fos promoter activity, in vivo.
Repeated high-frequency trains of stimuli induce long-term potentiation (LTP) in the CA1 region that persists for up to 8
hours in hippocampal slices and for days in intact animals. This long time course has made LTP an attractive model for certain
forms of long-term memory in the mammalian brain. A hallmark of long-term memory in the intact animal is a requirement for
transcription, and thus whether the late phase of LTP (L-LTP) requires transcription was investigated here. With the use of
different inhibitors, it was found in rat hippocampal slices that the induction of L-LTP [produced either by tetanic stimulation
or by application of the cyclic adenosine monophosphate (cAMP) analog Sp-cAMPS (Sp-cyclic adenosine 3',5'-monophosphorothioate)]
was selectively prevented when transcription was blocked immediately after tetanization or during application of cAMP. As
with behavioral memory, this requirement for transcription had a critical time window. Thus, the late phase of LTP in the
CA1 region requires transcription during a critical period, perhaps because cAMP-inducible genes must be expressed during
this period.
Recent studies suggest a role for rapid induction of transcription factors in stimulus-induced neuronal plasticity in the mammalian brain. Synaptic activation of transcription factors has been analyzed in the hippocampus using the long-term potentiation or enhancement (LTP/LTE) paradigm. Using this approach, several studies have identified transcription factors that are induced in hippocampal granule cells by NMDA receptor-dependent mechanisms; however, the link between long-term plasticity and activation of these genes has been called into question by reports suggesting that the thresholds for LTE and gene activation differ. To address this issue, we have used a chronic in vivo recording technique to monitor mRNA responses of several transcription factor genes to two different patterns of LTE-inducing electrical stimulation of entorhinal cortical afferents to hippocampus. One pattern consisted of 10 repetitions of a 20 or 25 msec train of pulses at 400 Hz (80 or 100 pulses total). This "10-train" pattern has been used in previous studies of LTE and produces robust synaptic enhancement lasting at least 3 d (Barnes, 1979). The other stimulation pattern consisted of 50 repetitions of a 20 msec train delivered at 400 Hz (400 pulses total), which is similar to parameters used in other studies reporting induction of c-fos in association with LTE (Dragunow et al., 1989; Jeffery et al., 1990; Abraham et al., 1992). Our results indicate that expression of zif268, monitored by in situ hybridization and immunostaining, is strongly induced by the 10-train stimulus pattern to levels similar to those induced by seizure activity. JunB mRNA levels are also modestly increased by the 10-train stimulus pattern; however, increases in JunB immunostaining were not detected. Neither c-fos nor c-jun mRNA were detectably induced by this stimulus. In contrast, the 50-train stimulus pattern resulted in a robust induction of c-fos and c-jun mRNA, in addition to zif268 and junB. Transcription factor responses to either stimulus pattern were blocked by the noncompetitive NMDA receptor antagonist MK-801. Identical transcription factor responses were observed in adult (6-12-month-old) and aged (23-26-month-old) rats, suggesting that synaptic mechanisms involved in these responses are preserved in aged animals. Analysis of LTE following either the 10- or 50-train stimulus patterns revealed identical magnitudes of initial induction and decay kinetics (approximately 3 d) and indicates that the 10-train stimulus pattern is sufficient to produce maximal synaptic enhancement.(ABSTRACT TRUNCATED AT 400 WORDS)
Endochondral ossification is the prevalent mode of vertebrate skeleton formation; it starts during embryogenesis when cartilage models of long bones develop central regions of hypertrophy which are replaced by bony trabeculae and bone marrow. Although several transcription factors have been implicated in pattern formation in the limbs and axial skeleton, little is known about the transcriptional regulations involved in bone formation. We have created a null allele in the mouse Krox-20 gene, which encodes a zinc finger transcription factor, by in frame insertion of the E. coli lacZ gene and shown that hindbrain segmentation and peripheral nerve myelination are affected in Krox-20-/- embryos. We report here that Krox-20 is also activated in a subpopulation of growth plate hypertrophic chondrocytes and in differentiating osteoblasts and that its disruption severely affects endochondral ossification. Krox-20-/- mice develop skeletal abnormalities including a reduced length and thickness of newly formed bones, a drastic reduction of calcified trabeculae and severe porosity. The periosteal component to bone formation and calcification does not appear to be affected in the homozygous mutant suggesting that the major role for Krox-20 is to be found in the control of the hypertrophic chondrocyte-osteoblast interactions leading to endosteal bone formation.
Mitogen-activated protein kinase (MAPK) is an integral component of cellular signaling during mitogenesis and differentiation of mitotic cells. Recently MAPK activation in post-mitotic cells has been implicated in hippocampal long-term potentiation (LTP), a potential cellular mechanism of learning and memory. Here we investigate the involvement of MAPK in learning and memory in behaving animals. MAPK activation increased in the rat hippocampus after an associative learning task, contextual fear conditioning. Two other protein kinases known to be activated during hippocampal LTP, protein kinase C and α-calcium/calmodulin protein kinase II, also were activated in the hippocampus after learning. Inhibition of the specific upstream activator of MAPK, MAPK kinase (MEK), blocked fear conditioning. Thus, classical conditioning in mammals activates MAPK, which is necessary for consolidation of the resultant learning.
Recent studies suggest that the CREB-CRE transcriptional pathway is pivotal in the formation of some types of long-term memory. However, it has not been demonstrated that stimuli that induce learning and memory activate CRE-mediated gene expression. To address this issue, we used a mouse strain transgenic for a CRE-lac Z reporter to examine the effects of hippocampus-dependent learning on CRE-mediated gene expression in the brain. Training for contextual conditioning or passive avoidance led to significant increases in CRE-dependent gene expression in areas CA1 and CA3 of the hippocampus. Auditory cue fear-conditioning, which is amygdala dependent, was associated with increased CRE-mediated gene expression in the amygdala, but not the hippocampus. These data demonstrate that learning in response to behavioral conditioning activates the CRE transcriptional pathway in specific areas of brain.
The robust translocation of MAPK during synaptic plasticity (11xMartin, K.C, Michael, D, Rose, J.C, Barad, M, Casadio, A, Zhu, H, and Kandel, E.R. Neuron. 1997; 18: 899–912Abstract | Full Text | Full Text PDF | PubMed | Scopus (411)See all References, 9xImpey, S, Obrietan, K, Wong, S.T, Poser, S, Yano, S, Wayman, G, Deloulme, J.C, Chan, G, and Storm, D.R. Neuron. 1998; 21: 869–883Abstract | Full Text | Full Text PDF | PubMed | Scopus (644)See all References, 15xSgambato, V, Pages, C, Rogard, M, Besson, M.J, and Caboche, J. J. Neurosci. 1998; 18: 8814–8825PubMedSee all References) indicates that there are likely additional nuclear targets of MAPK signaling other than CREB. For example, several recent reports suggest that the transcription factor Elk1 is a major nuclear target of MAPK during synaptic plasticity and memory consolidation (2xBerman, D.E, Hazvi, S, Rosenblum, K, Seger, R, and Dudai, Y. J. Neurosci. 1998; 18: 10037–10044PubMedSee all References, 15xSgambato, V, Pages, C, Rogard, M, Besson, M.J, and Caboche, J. J. Neurosci. 1998; 18: 8814–8825PubMedSee all References).The prominent dendritic localization of activated MAPK following synaptic activity (Impey et al. 1998xImpey, S, Obrietan, K, Wong, S.T, Poser, S, Yano, S, Wayman, G, Deloulme, J.C, Chan, G, and Storm, D.R. Neuron. 1998; 21: 869–883Abstract | Full Text | Full Text PDF | PubMed | Scopus (644)See all ReferencesImpey et al. 1998) suggests that it may also have important cytosolic targets. The best example of such a target is the Aplysia cell adhesion molecule ApCAM. MAPK activity is required for the downregulation and internalization of ApCAM, a key step in the induction of LTF. This is an important observation because the Drosophila (Fas II) and murine (NCAM) homologs of ApCAM have also been implicated in neuronal plasticity.Collectively, these studies indicate that the MAPK pathway is a fundamental component of LTM formation in invertebrates and vertebrates. Thus, the MAPK cascade joins the cAMP/PKA pathway and the CREB transcriptional pathway as an evolutionarily conserved regulator of LTM consolidation (Figure 1Figure 1). Work showing that MAPK is a major activator of plasticity-associated CREB-dependent gene expression also strongly suggests that MAPK signaling facilitates memory consolidation and L-LTP by promoting de novo CREB-regulated gene expression. There are a number of unanswered questions regarding the role of MAPK in neuronal plasticity and memory formation. Is CREB a target of Ras/MAPK signaling during memory consolidation? How is MAPK activated during adaptive neuronal plasticity and memory consolidation? What are the cytosolic and nuclear targets of MAPK that facilitate memory formation and modulate synaptic efficacy? Additional research using temporally and spatially restricted transgenic technologies should help clarify and confirm the role of Ras/MAPK signaling in LTM.*To whom correspondence should be addressed (e-mail: dstorm@u.washington.edu).
The cAMP-responsive element-binding protein (CREB) has been implicated in the activation of protein synthesis required for long-term facilitation, a cellular model of memory in Aplysia. Our studies with fear conditioning and with the water maze show that mice with a targeted disruption of the alpha and delta isoforms of CREB are profoundly deficient in long-term memory. In contrast, short-term memory, lasting between 30 and 60 min, is normal. Consistent with models claiming a role for long-term potentiation (LTP) in memory, LTP in hippocampal slices from CREB mutants decayed to baseline 90 min after tetanic stimulation. However, paired-pulse facilitation and posttetanic potentiation are normal. These results implicate CREB-dependent transcription in mammalian long-term memory.
Although protein synthesis inhibition has been shown to affect long-term memory in a wide variety of animal species, cases have been reported in which protein synthesis inhibition failed to affect memory consolidation [S. Wittstock, R. Menzel, Color learning and memory in honey bees are not affected by protein synthesis inhibition, Behav. Neural Biol., 62 (1994) 224–229.]. Most findings argue that the critical time for protein synthesis is during or immediately after training. However, other reports show a second time window, hours after training, where protein synthesis inhibition can cause amnesia [F.M. Freeman, S.P.R. Rose, A.B. Scholey, Two time windows of anisomycin-induced amnesia for passive avoidance training in the day-old chick, Neurobiol. Learn. Mem., 63 (1995) 291–295.][G. Grecksch, H. Matthies, Two sensitive periods for the amnesic effect of anisomycin, Pharmacol. Biochem. Behav., 12 (1980) 663–665.]. In this study, we addressed two questions: (1) Is protein synthesis essential for spatial memory? and (2) At what injection time window(s) will protein synthesis inhibition cause spatial memory amnesia? We report that bilateral intraventricular microinjection of anisomycin (Ani) impairs consolidation of long-term memory, in the hippocampal-dependent Morris water maze spatial memory task. Memory was impaired in a dose-dependent manner without affecting short-term memory. Spatial memory was affected only if Ani was injected 20 min before performing the task and not in any other time window before or after the behavioral test. The inhibition did not affect pre-existing memories or the capability to memorize once the effect of the inhibition diminished.
Prolonged changes in synaptic strength, such as those that occur in LTP and LTD, are thought to contribute to learning and memory processes. These complex phenomena occur in diverse brain structures and use multiple, temporally staged and spatially resolved mechanisms, such as changes in neurotransmitter release, modulation of transmitter receptors, alterations in synaptic structure, and regulation of gene expression and protein synthesis. In the CA1 region of the hippocampus, the combined activation of SRC family tyrosine kinases, protein kinase A, protein kinase C and, in particular, Ca2+/calmodulin-dependent protein kinase II results in phosphorylation of glutamate-receptor-gated ion channels and the enhancement of subsequent postsynaptic current. Crosstalk between these complex biochemical pathways can account for most characteristics of early-phase LTP in this region.
Expression of the immediate early gene zif/268 (also termed NGFI-A, Krox 24, TIS8 and Egr-1) was investigated in awake rats following various long-term potentiation (LTP) induction protocols. zif/268 mRNA (Northern blots) and protein (immunohistochemistry) levels sharply increased following LTP, and followed a time course characteristic of other immediate early genes. When measured across 3 tetanization protocols known to produce differing degrees of LTP persistence, zif/268 induction was found to be more highly correlated with LTP duration than with the magnitude of initial LTP. These data support the hypothesis that the immediate early gene zif/268 plays a role as a third messenger in the cascade of cellular and nuclear events that govern the persistence of LTP.
C-fos nuclear protooncogene encodes a regulatory protein (Fos), able to directly influence both expression of itself and other genes. It has been repeatedly shown that c-fos expression coincides with different forms of cell activation, probably being functionally involved in the coupling of extracellular ligands to long-term cellular responses. In this study it has been found that c-fos mRNA accumulation in rat brain, as measured by northern blotting coincides with increase of performance level of learned behavior of a two-way active avoidance task. We have previously reported (Nikolaev et al., Brain Res. Bull., in press) that a single training session of two-way active avoidance strongly induces c-fos mRNA accumulation but that after long-term training up to the asymptotic level of performance no c-fos expression was detectable. In this paper we show that c-fos still remains inducible even after long-term, asymptotic training to darkness as conditioned stimulus (CS), provided that a novel stimulus, wide band noise, which elevated performance level, was given together with darkness as compound CS.
Immediate early genes (IEGs) are a class of genes that show rapid and transient but protein synthesis-independent increases in expression to extracellular signals such as growth factors and neurotransmitters. Many IEGs code for transcription factors that have been suggested to govern the growth and differentiation of many cell types by regulating the expression of other genes. IEGs are expressed in adult neurons both constitutively and in response to afferent activity, and it has been suggested that during learning, IEGs may play a role in the signal cascade, resulting in the expression of genes critical for the consolidation of long-term memory. Long-term potentiation (LTP) is a persistent, activity-dependent form of synaptic plasticity that stands as a good candidate for the mechanism of associative memory. A number of IEGs coding for transcription factors have been shown to transiently increase transcription in the dentate gyrus of rats following LTP-inducing afferent stimulation. These include zif/268 (also termed NGFI-A, Krox-24, TIS-8, and egr-l), c-fos-related genes, c-jun, junB, and junD. Of these, zif/268 appears to be the most specifically related to LTP since it is evoked under virtually all LTP-inducing situations and shows a remarkably high correlation with the duration of LTP. There are a number of outstanding questions regarding the role of zif/268 and other IEGs in LTP, including which second messenger systems are important for activating them, which "late effector" genes are regulated by them, and the exact role these genes play, if any, in the stabilization and maintenance of LTP.
A key event in the response of cells to proliferative signals is the rapid, transient induction of the c-fos proto-oncogene, which is mediated through the serum response element (SRE) in the fos promoter. Genomic footprinting and transfection experiments suggest that this activation occurs through a ternary complex that includes the serum response factor (SRF) and the ternary complex factor p62. Interaction of p62TCF with the SRF-SRE binary complex requires a CAGGA tract immediately upstream of the SRE. Proteins of the ets proto-oncogene family bind to similar sequences and we have found that a member of this family, Elk-1, forms SRF-dependent ternary complexes with the SRE. Elk-1 and p62TCF have the same DNA sequence requirements and antibodies against Elk-1 block the binding of both proteins. Furthermore, we show that like p62TCF, Elk-1 forms complexes with the yeast SRF-homologue MCM1 but not with yeast ARG80. But ARG80 mutants that convey interaction with p62TCF can also form complexes with Elk-1. The similarity, or even identity, between Elk-1 and p62TCF suggests a novel regulatory role for Ets proteins that is effected through interaction with other proteins, such as SRF. Furthermore, the possible involvement of an Ets protein in the control of c-fos has interesting implications for proto-oncogene cooperation in cellular growth control.
Transcription factors are rapidly and transiently induced in brain by excitatory stimuli and may be important in coordinating changes in gene expression underlying neuronal plasticity. In contrast to their transient induction after stimulation, certain transcription factors display stable, relatively high basal levels of expression in brain. Here we demonstrate that this "constitutive" expression of the transcription factor zif268 in cortex is driven by natural synaptic activity. Blockade of afferent visual activity with intraocular injections of tetrodotoxin results in rapid, dramatic reductions of Zif268 mRNA and immunoreactivity in visual cortex. Moreover, dark-adaptation for several days lowers zif268 expression in visual cortex, and expression rapidly returns to control levels upon subsequent light exposure. Several other transcription factors, which are induced in cortical neurons by excitatory stimuli, appear less responsive to changes in natural sensory input. These studies suggest that transcription factors play a role not only in responses to artificial stimuli but also in the normal maintenance of cortical physiology. Anatomic markers for zif268 may be useful in mapping normal cortical activity in brain.
The localization of zif/268 gene expression in adult rat and mouse brain was studied with in the situ hybridization, using 32P-labeled 30 mer oligonucleotide probes. Basal expression without intentional neuronal stimulation was examined. Distribution of zif/268 mRNA was highly differential. In the neocortex a layer specific expression was seen with high levels in layers IV and VI, most prominent in the visual and somatosensory cortices. In the hippocampus labeling was strongest in CA1 but very low in the dentate gyrus. Strong expression was also seen in the primary olfactory cortex, the corpus striatum, the nuclei amygdaloidei, the nucleus accumbens and the cerebellar cortex.
NGFI-A and NGFI-B are two genes that are induced in PC12 cells treated with nerve growth factor and encode zinc-finger proteins that may act as transcription factors. To study the function of these genes in vivo, their expression in rat embryos and postnatally developing tissues was examined. Both genes are expressed during embryogenesis from E12 to E18. In postnatally developing animals, these genes are induced in cortex, midbrain and cerebellum, suggesting that they may function in one or many of the changes that occur during postnatal maturation of the brain. Both genes are expressed at a low, constitutive level in a subset of other non-neuronal tissues, but also demonstrate developmental changes in expression in many other organs. To examine expression of these genes in ganglia of the peripheral nervous system, a sensitive and quantitative assay employing reverse transcription and polymerase chain reaction was developed. Results obtained with this assay demonstrated that expression of both NGFI-A and NGFI-B is modulated in the peripheral nervous system during postnatal maturation, but not in a pattern concordant with previously characterized NGF receptor expression. These studies suggest that NGFI-A and NGFI-B serve multiple, tissue-specific functions and are under complex regulation in vivo.
We have demonstrated that immediate early genes can be differentially activated within the central nervous system. We examined the effects of tetanic stimulation in the hippocampus and of noxious sensory stimulation of the spinal cord on the expression of eight immediate early genes. Induction of long-term potentiation (LTP) in the dentate gyrus resulted in an increase in mRNA and protein for NGFI-A (also termed Zif/268, Egr-1, or Krox 24), and less consistently for jun-B mRNA. No increase was seen for c-fos, NGFI-B, c-jun, jun-D, SRF, or PC4 mRNAs. Blockade of the NMDA receptor prevented the induction of both LTP and NGFI-A mRNA in the dentate gyrus. However, commissural stimulation, which prevented the induction of LTP, resulted in bilateral activation of all the genes examined, including NGFI-A. No change was seen in animals trained in a water maze. These results suggest that no simple relationship exists between LTP, spatial learning, and immediate early gene induction. Stimulation of sensory fibers resulted in an increase in mRNA for NGFI-A, c-fos, SRF, NGFI-B, and c-jun in spinal cord neurons. Blockade of the NMDA receptor had no effect on immediate early gene induction in the spinal cord.
Antibodies are used to localize the NGFI-A protein in the rat brain. The protein is found in a wide variety of neurons. However, not all neurons are stained. The protein is either absent or present at undetectable levels in glial cells. Neuronal nuclei stain intensely, cytoplasmic staining is lighter. Seizures cause a detectable increase in the intensity of staining.
Zif268, a zinc finger protein whose mRNA is rapidly activated in cells exposed to growth factors or other signaling agents, is thought to play a role in regulating the genetic program induced by extracellular ligands. We report that Zif268 has one of the characteristics of a transcriptional regulator, namely, sequence-specific binding to DNA. Zif268 synthesized in Escherichia coli bound to two sites upstream of the zif268 gene and to sites in the promoter regions of other genes. The nucleotide sequences responsible for binding were defined by DNase I footprinting, by methylation interference experiments, and by use of synthetic oligonucleotides. From these results we derived the following consensus sequence for a Zif268 high-affinity binding site: GCGTGGGGCG.
The structure of Krox-24, a mouse zinc-finger-encoding gene that is transiently activated during G0/G1 transition, has been established. Krox-24 is located on mouse chromosome 18, bands C-D. The gene product, as anticipated for a putative DNA-binding protein, is localized within the cell nucleus. The Krox-24 5'-flanking region contains a series of serum response elements (SREs) similar to the SRE observed upstream of the c-fos proto-oncogene. These elements can substitute for the c-fos SRE, their effect is cumulative and they bind the same cellular factor, the serum response factor (SRF), as the c-fos SRE. This suggests that the SRE and its cognate protein are likely to be involved in the regulation of Krox-24 and presumably of other immediate-early serum response genes. SRE and SRF therefore constitute key components in the regulatory pathway leading from mitogenic stimulation to cellular proliferation.
We examined whether the critical protein synthesis for maintenance of perforant path long-term potentiation (LTP) takes place in the dentate gyrus or the entorhinal cortex. Field potential recordings were made of responses in the dentate gyrus to stimulation of the perforant path in urethane-anaesthetized rats. Anisomycin (10 micrograms) injected into the dentate gyrus, but not the entorhinal cortex, 1 h prior to tetanization led to nearly complete decay of perforant path LTP of the excitatory postsynaptic potential (EPSP) within 3 h. Intra-dentate injection of neither actinomycin D (a mRNA synthesis inhibitor) nor boiled anisomycin affected LTP maintenance over 6 h. These results suggest that the proteins necessary for the maintenance of LTP over 6 h are synthesized in the dentate gyrus from already existing mRNA without involving protein synthesis in the cell bodies of the afferent fibres.
Egr-1 is an early growth response gene that displays fos-like induction kinetics in fibroblasts, epithelial cells, and lymphocytes following mitogenic stimulation. Sequence analysis of murine Egr-1 cDNA predicts a protein with three DNA binding zinc fingers. The human EGR1 gene maps to chromosome 5 (bands 5q23-31). Egr-1 mRNA increases dramatically during cardiac and neural cell differentiation, and following membrane depolarization both in vitro and in vivo. Thus, Egr-1 and c-fos are often coregulated with strikingly similar kinetics. These results, in conjunction with the Egr-1 primary structure, suggest that Egr-1 may function as a transcriptional regulator in diverse biological processes.
We have recently identified by cDNA cloning a set of genes that are rapidly activated in mouse 3T3 cells by serum or purified growth factors. Here we report that the cDNA (clone 268) derived from one of these immediate early genes (zif/268) encodes a protein with three tandem "zinc finger" sequences typical of a class of eukaryotic transcription factors. The mRNA of zif/268 is present in many organs and tissues of the mouse and is especially abundant in the brain and thymus tissue. The 5' genomic flanking sequence of zif/268 has sequences related to binding sites for known regulatory proteins, including four sequences that resemble the core of the serum response elements (SREs) upstream of the c-fos and actin genes. The SRE-like sequences could be responsible for the coordinate activation of zif/268 and fos after serum stimulation of 3T3 cells.
Nerve growth factor (NGF) is a trophic agent that promotes the outgrowth of nerve fibers from sympathetic and sensory ganglia. The neuronal differentiation stimulated by this hormone was examined in the NGF-responsive cell line PC12. Differential hybridization was used to screen a complementary DNA library constructed from PC12 cells treated with NGF and cycloheximide. One of the complementary DNA clones that was rapidly induced by NGF was found to have a nucleotide sequence that predicts a 54-kilodalton protein with homology to transcriptional regulatory proteins. This clone, NGFI-A, contains three tandemly repeated copies of the 28- to 30-amino acid "zinc finger" domain present in Xenopus laevis TFIIIA and other DNA-binding proteins. It also contains another highly conserved unit of eight amino acids that is repeated at least 11 times. The NGFI-A gene is expressed at relatively high levels in the brain, lung, and superior cervical ganglion of the adult rat.
1. The after‐effects of repetitive stimulation of the perforant path fibres to the dentate area of the hippocampal formation have been examined with extracellular micro‐electrodes in rabbits anaesthetized with urethane.
2. In fifteen out of eighteen rabbits the population response recorded from granule cells in the dentate area to single perforant path volleys was potentiated for periods ranging from 30 min to 10 hr after one or more conditioning trains at 10–20/sec for 10–15 sec, or 100/sec for 3–4 sec.
3. The population response was analysed in terms of three parameters: the amplitude of the population excitatory post‐synaptic potential (e.p.s.p.), signalling the depolarization of the granule cells, and the amplitude and latency of the population spike, signalling the discharge of the granule cells.
4. All three parameters were potentiated in 29% of the experiments; in other experiments in which long term changes occurred, potentiation was confined to one or two of the three parameters. A reduction in the latency of the population spike was the commonest sign of potentiation, occurring in 57% of all experiments. The amplitude of the population e.p.s.p. was increased in 43%, and of the population spike in 40%, of all experiments.
5. During conditioning at 10–20/sec there was massive potentiation of the population spike (‘frequency potentiation’). The spike was suppressed during stimulation at 100/sec. Both frequencies produced long‐term potentiation.
6. The results suggest that two independent mechanisms are responsible for long‐lasting potentiation: ( a ) an increase in the efficiency of synaptic transmission at the perforant path synapses; ( b ) an increase in the excitability of the granule cell population.
Long-term potentiation-inducing stimulation of the perforant path was followed in dentate gyrus granule cells by a dramatic increase of mRNA and protein for Krox20, a zinc-finger-containing transcription factor. Induction of Krox20 required stimulation sufficient to induce LTP and was prevented by NMDA antagonists CPP and MK-801, which block LTP induction. Krox20 protein increased within 20 min of tetanization, was maximal between 1 and 8 h, and was still significantly elevated at 24 h after LTP induction. This prolonged appearance is in striking contrast with the more transient induction of the related molecule, Krox24. The elevation in the mRNA for Krox20 and Krox24 was of similar duration, suggesting that the Krox20 protein has a greater stability and may play a key role in the stabilization of long-term potentiation.
It is now well established that environmental signals mediated via neurotransmitters and hormones can induce responses in cells which involve a cascade of receptors, G proteins, and second messengers. These in turn can induce transcription factors which regulate long-term changes in gene expression. It has been proposed that the stimulus-transcription coupling properties of these DNA-binding proteins can be exploited to visualize activated neurons by way of immunostaining. We have used standard immunohistochemical techniques to detect the expression of one specific transcription factor, Zif268, in the visual cortex (area 17, V1) of vervet monkeys (Cercopithecus aethiops). Immunopositive neurons were present in large numbers throughout the visual cortex of the normal animal, being concentrated in layers 2/3 and 6 and at moderate levels in 4C beta and 5. To determine if Zif268 expression was affected by visual stimulation in the monkey, we restricted light input to one eye with the aim of revealing ocular-dominance columns in striate cortex. We found that short-term monocular deprivation induced either by enucleation, intravitreal TTX injection, or eyelid suturing resulted in dramatic changes in Zif268 levels, revealing vertically oriented columns of reduced Zif268 staining interdigitated with columns of normal expression. Furthermore, these columns were discernible after just 2 h of monocular blockade. A comparison of the ocular-dominance pattern obtained with Zif268 immunostaining and cytochrome oxidase histochemistry in long-term monocularly deprived animals showed a coincident reduction of both markers along columns that were precisely aligned in adjacent sections, indicating that Zif268 expression is restricted to cortical regions of high metabolic activity. Simultaneous immunostaining for Zif268 and the calcium-binding proteins calbindin and parvalbumin showed a negative correlation, suggesting that the Zif268 protein may be expressed selectively within excitatory neurons. A similar approach with immunostaining for neurofilament and microtubule-associated proteins (SMI-32 and MAP2) revealed pyramidal neurons which were regularly found to contain a Zif268-positive nucleus. Furthermore, confocal images of lucifer yellow filled neurons possessing Zif268-positive nuclei all showed pyramidal morphology. Taken together, these results point to activity-dependent expression of Zif268 within a subset of excitatory neurons.
This chapter discusses the early growth response protein (Egr) gene family and its most extensively characterized member, Egr-1, first identified as an immediate-early gene responsive to growth factors and various differentiation cues, later confirmed to be a transcriptional regulatory protein. During development, a single Egr-1 transcript is predominantly expressed in cortex, midbrain, and cerebellum; in bone, cartilage, and muscle; and at several sites of epithelial-mesenchymal interactions. In addition, Egr-1 is expressed in diverse physiological contexts in particular cell types. The broad spectrum of extracellular stimuli that induce Egr-1 can be roughly subgrouped into four categories: (1) mitogens, (2) developmental or differentiation cues, (3) tissue or radiation injury, and (4) signals that cause neuronal excitation. Strong evidence for a role for Egr-1 in proliferation also comes, from the studies, with mouse skeletal muscle So18 cells. Although Egr-1 message was induced in response to mitogenic stimuli, differentiative stimuli (insulin), and other agents that caused neither proliferation nor differentiation, Egr-1 protein could be detected only in response to the mitogenic cues.
NGFI-A is the prototypic member of a family of immediate-early gene-encoded transcription factors which includes NGFI-C, Egr3, and Krox20. These proteins possess highly homologous DNA-binding domains, composed of three Cys2-His2 zinc fingers, and all bind to and activate transcription from the sequence GCGGGGGCG. We used a PCR-mediated random site selection protocol to determine whether other sites could be bound by these proteins and the extent to which their binding site preferences are similar or different. The high-affinity consensus sites generated from the selection data are similar, and the combined consensus sequence is T-G-C-G-T/g-G/A-G-G-C/a/t-G-G/T (lowercase letters indicate bases selected less frequently). Using gel shift assays, we found that sequences that diverge from the consensus were bound by NGFI-A, confirming that there is greater variability in binding sites than has generally been acknowledged. We also provide evidence that protein-DNA interactions not noted, or whose importance was not apparent from the X-ray cocrystal structure of the NGFI-A zinc fingers complexed with DNA, contribute significantly to the binding energy of these proteins and confirm that an optimal site is at least 10 instead of 9 nucleotides in length. In contrast to the similarities in binding specificity among these proteins we found that while NGFI-A, Egr3, and Krox20 have comparable DNA binding affinities and kinetics of dissociation, the affinity of NGFI-C is more than threefold lower. This could result in differential regulation of target genes in cells where NGFI-C and the other proteins are coexpressed. Furthermore, we show that this affinity difference is a property not of the zinc fingers themselves but rather of the protein context of the DNA-binding domain.
Following LTP induction in freely moving rats, in situ hybridization revealed discrete changes in the expression of one isoform in each of four families of serine/threonine kinases constitutively expressed in the dentate gyrus of the hippocampus. Expression of the alpha isoform of CaMKII showed a transient increase over the soma and a more persistent increase over the dendritic field of dentate granule cells. Of the PKC isoforms, only gamma PKC was up-regulated substantially 2 hr after LTP induction, declining to control levels 48 hr later. An increase in the expression of mRNA for ERK2 and raf-B was seen at 24 hr only. These results show that, during the maintenance phase of LTP in the hippocampus, there are selective increases in the expression of serine/threonine kinases and that these increases have specific and characteristic temporal and spatial profiles.
The cAMP-responsive element-binding protein (CREB) has been implicated in the activation of protein synthesis required for long-term facilitation, a cellular model of memory in Aplysia. Our studies with fear conditioning and with the water maze show that mice with a targeted disruption of the alpha and delta isoforms of CREB are profoundly deficient in long-term memory. In contrast, short-term memory, lasting between 30 and 60 min, is normal. Consistent with models claiming a role for long-term potentiation (LTP) in memory, LTP in hippocampal slices from CREB mutants decayed to baseline 90 min after tetanic stimulation. However, paired-pulse facilitation and posttetanic potentiation are normal. These results implicate CREB-dependent transcription in mammalian long-term memory.
Changes in the distribution pattern of mRNA encoding the zif268 transcription factor (also referred to as NGFI-A, Krox-24 or EGR-1) were investigated by in situ hybridization histochemistry during postnatal rat brain development. Marked changes in zif268 expression patterns were seen in particular in the cerebral cortex and the hippocampal formation during the first 3 wk. In the 1st postnatal week, zif268 mRNA levels were highest in the corpus striatum and the piriform cortex. In the neocortex, expression rose sharply in the sensorymotor area between postnatal days (PNDs) 10 and 12. In the frontal and occipital cortex, in contrast, an increase in zif268 mRNA levels was first seen on PND 14. After PND 17, levels decreased in the sensorymotor and the frontal cortex but remained high in the occipital and the piriform cortex. In the hippocampus, an initially uniform increase in expression during the 2nd week was followed by a marked dissociation in expression levels between CA1, with continuously high expression levels on the one hand, and CA3, CA4 and the dentate gyrus, with a strong decline of expression during the 3rd week, on the other hand. Our results indicate that zif268 expression displays a highly dynamic expression pattern during plastic adaptations of different cerebral subregions during postnatal development, suggesting a possible involvement in gene regulatory processes during these phases.
Krox20 is a zinc finger gene expressed in rhombomeres 3 and 5 during hindbrain development in vertebrates. Mice homozygous for a targeted mutation that deletes the majority of the Krox20 genes, including the zinc finger DNA-binding domain, died shortly after birth. The primary phenotype of the homozygous mutant animals was the loss of rhombomeres 3 and 5. This resulted in fusions of the trigeminal ganglion with the facial and vestibular ganglia, and of the superior ganglia of the glossopharyngeal and vagus nerves. These fusions resulted in a disorganization of the nerve roots of these ganglia as they entered the brain stem. These data demonstrate that Krox20 plays an essential role during development of the hindbrain and associated cranial sensory ganglia in mice.
The duration of long-term potentiation in the dentate gyrus of awake rats was examined following systematic manipulation of the number of stimulus trains delivered. This was correlated with the induction of immediate early genes in separate groups of animals given identical stimulus regimes. Following 10 trains of stimulation, long-term potentiation decayed with a time constant of up to several days (long-term potentiation 2), and this correlated with the appearance of an increase in the messenger RNA and protein levels of zif/268. Increasing the number of stimulus trains resulted in a greater probability of eliciting long-term potentiation with a time constant of several weeks (long-term potentiation 3), as well as increasing the induction of zif/268, c-Jun, Jun-B, Jun-D and Fos-related proteins. When 10 trains were delivered repeatedly on up to five consecutive days, only the zif/268 protein levels showed associated changes. These data provide support for the hypothesis that long-term potentiation 3 involves mechanisms additional to those for long-term potentiation 2. One possible mechanism is altered gene expression, initiated by immediate early gene transcription factors such as zif/268 and possibly homo- or heterodimers of Fos and Jun family members, that then contributes to the stabilization or maintenance of long-term potentiation 3.
The Elk-1 and SRF transcription factors form a ternary complex at the c-fos serum response element (SRE). Growth factor stimulation rapidly induces a reversible change in the electrophoretic mobility of the ternary complex, accompanied by increased phosphorylation of the Elk-1 C-terminal region and by the activation of a 42 kd cellular Elk-1 kinase. Phosphorylation of Elk-1 in vitro by partially purified p42/p44 MAP kinase induces a similar reduction in ternary complex mobility but has little effect on the efficiency of its formation. In vitro, MAP kinase phosphorylates the Elk-1 C-terminal region at multiple sites, which are also phosphorylated following growth factor stimulation in vivo. The Elk-1 C-terminal region functions as a regulated transcriptional activation domain whose activity in vivo is dependent on the integrity of the MAP kinase sites. These findings directly link transcriptional activation by the SRE to the growth factor-regulated phosphorylation of an SRE-binding protein.
Gene expression regulated by the cAMP response element (CRE) has been implicated in synaptic plasticity and long-term memory. It has been proposed that CRE-mediated gene expression is stimulated by signals that induce long-term potentiation (LTP). To test this hypothesis, we made mice transgenic for a CRE-regulated reporter construct. We focused on long-lasting long-term potentiation (L-LTP), because it depends on cAMP-dependent protein kinase activity (PKA) and de novo gene expression. CRE-mediated gene expression was markedly increased after L-LTP, but not after decremental UP (D-LTP). Furthermore, inhibitors of PKA blocked L-LTP and associated increases in CRE-mediated gene expression. These data demonstrate that the signaling required for the generation of L-LTP but not D-LTP is sufficient to stimulate CRE-mediated transcription in the hippocampus.
The immediate-early transcription factor NGFI-A (also called Egr-1, zif/268, or Krox-24) is thought to couple extracellular signals to changes in gene expression. Although activins and inhibins regulate follicle-stimulating hormone (FSH) synthesis, no factor has been identified that exclusively regulates luteinizing hormone (LH) synthesis. An analysis of NGFI-A-deficient mice derived from embryonic stem cells demonstrated female infertility that was secondary to LH-beta deficiency. Ovariectomy led to increased amounts of FSH-beta but not LH-beta messenger RNA, which suggested a pituitary defect. A conserved, canonical NGFI-A site in the LH-beta promoter was required for synergistic activation by NGFI-A and steroidogenic factor-1 (SF-1). NGFI-A apparently influences female reproductive capacity through its regulation of LH-beta transcription.