Article

Maturation of neurons in neocortical slice cultures. A light and electron microscopic study on in situ and in vitro material

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Abstract

Using light and electron microscopic methods, we investigated the development and morphology of neurons in neocortical slice cultures. Slices taken from the visual cortex of 6-day-old rats and cultivated for 14 or 20 days were compared with in situ material of corresponding age (P 20 and P 26). Maturation and differentiation of pyramidal and non-pyramidal cells kept in vitro were found to have progressed considerably. In the light microscope the neurons exhibited a morphological appearance strikingly similar to that of the neurons of the neocortex in situ at the same age. The fine structure of the tissue in vitro also had a mature appearance, corresponding in most respects to the material in situ. Synapses and dendritic spines were well-developed. Sometimes a spine apparatus was contained in the sections and occasionally a myelinated fiber could be seen. GABA-immunoreactive cells making symmetric synaptic contacts were also present. Despite these similarities, some quantitative differences could be observed. In slice cultures, only 52% of the synapses were located on spines (78% in situ). In vitro, a larger proportion of synapses (30%) showed a postsynaptically concave curvature than was the case in situ (12%). The areal density of synapses in vitro reached only about 70% of that in situ. This was probably a side-effect of the larger size of dendritic and axonal profiles on electron micrographs of in vitro-material. The most striking difference was that large synapses and synapses containing a large amount of synaptic vesicles were considerably more frequent in vitro than in situ.

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... In this study, we performed experiments on the organotypic cultures of rat cortex, which have been widely used in neuroscience as a biological model of neuronal activity (50)(51)(52)(53)(54) and in MRI studies (11,28,55). Organotypic cortical cultures largely maintain the in vivo cortical cytoarchitecture including cortical layers and cortical cell types, which can be grown and recorded for several weeks in the incubator (51,52). ...
... In this study, we performed experiments on the organotypic cultures of rat cortex, which have been widely used in neuroscience as a biological model of neuronal activity (50)(51)(52)(53)(54) and in MRI studies (11,28,55). Organotypic cortical cultures largely maintain the in vivo cortical cytoarchitecture including cortical layers and cortical cell types, which can be grown and recorded for several weeks in the incubator (51,52). More importantly, organotypic cultures in vitro display bursts of spontaneous neuronal activity, so-called up-and down-states, that is similar to in vivo nervous tissue (53,56,57). ...
... The use of organotypic cortical cultures as a biological model of neuronal activity eliminates any possible hemodynamic contributions to the MR signal. The organotypic cultures possess healthy neurons similar to those in the in vivo cortex with high cell densities and extracellular matrix (51). Moreover, spontaneous activity in organotypic cortex cultures organizes as neuronal avalanches (53), a common dynamic mode of ongoing activity also observed in vivo in humans and nonhuman primates (56,57). ...
Article
Recently, several new functional (f)MRI contrast mechanisms including diffusion, phase imaging, proton density, etc. have been proposed to measure neuronal activity more directly and accurately than blood-oxygen-level dependent (BOLD) fMRI. However, these approaches have proved difficult to reproduce, mainly because of the dearth of reliable and robust test systems to vet and validate them. Here we describe the development and testing of such a test bed for non-BOLD fMRI. Organotypic cortical cultures were used as a stable and reproducible biological model of neuronal activity that shows spontaneous activity similar to that of in vivo brain cortex without any hemodynamic confounds. An open-access, single-sided magnetic resonance (MR) "profiler" consisting of four permanent magnets with magnetic field of 0.32 T was used in this study to perform MR acquisition. A fluorescence microscope with long working distance objective was mounted on the top of a custom-designed chamber that keeps the organotypic culture vital, and the MR system was mounted on the bottom of the chamber to achieve real-time simultaneous calcium fluorescence optical imaging and MR acquisition on the same specimen. In this study, the reliability and performance of the proposed test bed were demonstrated by a conventional CPMG MR sequence acquired simultaneously with calcium imaging, which is a well-characterized measurement of neuronal activity. This experimental design will make it possible to correlate directly the other candidate functional MR signals to the optical indicia of neuronal activity in the future. Copyright © 2015 John Wiley & Sons, Ltd.
... Cortex slices taken into culture at early postnatal age develop the main features of cortical organization that includes all major classes of pyramidal neurons (Cäser, Bonhoeffer et al., 1989;Cäser and Schüz, 1992), interneurons (Götz and Bolz, 1989;Plenz and Aertsen, 1996a;Plenz and Aertsen, 1996b;Gorba, Klostermann et al., 1999;Klostermann and Wahle, 1999), and cortical layers (Götz and Bolz, 1992). Organotypic slice cultures therefore to date represent the most intact culture system for studying cortex function in isolation over the course of many weeks. ...
... While the spatial resolution of the LFP recordings is too poor to clearly identify layer-specific activity, our analysis revealed that nLFPs were located towards the upper half of the cortex cultures, which corresponds to the developing superficial cortical layers (Cäser and Schüz, 1992;Götz and Bolz, 1992;Porter, Rizzo et al., 1999). Furthermore, cortical slice cultures have been demonstrated to maintain their cortical layers for many months in culture (Plenz and Kitai, 1996;Plenz and Aertsen, 1996a;Klostermann and Wahle, 1999). ...
Article
Cortical networks in vivo and in vitro are spontaneously active in the absence of inputs, generating highly variable bursts of neuronal activity separated by up to seconds of quiescence. Previous measurements in adult rat cortex revealed an intriguing underlying organization of these dynamics, termed neuronal avalanches, which is indicative of a critical network state. Here we demonstrate that neuronal avalanches persist throughout development in cortical slice cultures from newborn rats. More specifically, we find that in spite of large variations of average rate in activity, spontaneous bursts occur with power-law distributed sizes (exponent -1.5) and a critical branching parameter close to 1. Our findings suggest that cortical networks homeostatically regulate a critical state during postnatal maturation.
... In a number of studies it has been shown that many of the unique properties of neocortex are maintained in organotypic cultures of this tissue. Pyramidal and non-pyramidal neurons from OTCs of rat neocortex and hippocampus display differentiation of morphology and transmitter phenotypes as well as connectivity and physiological properties comparable to the in vivo situation (Caesar et al., 1989; Caesar and Schuz, 1992; Finsen et al., 1992; Gotz and Bolz, 1994). Here we add the finding that basic properties of NKlR expressing neurons, namely the non-pyramidal shape and the co-expression of GABA, are maintained in OTCs. ...
... This suggestion is based on the observation that the layer specific phenotype restriction of NPYmRNA expressing cells occurring postnatally in the rat visual cortex occurs in co-cultures of cortex with cortex or thalamus but not in monocultures (Obst and Wahle, 1996). Others have reported changes in distribution and size of synapses in OTCs of rat neocortex; for example, in pyramidal neurons the proportion of axospinous over axodendritic synapses was higher in vivo than in OTCs (Caesar and Schuz, 1992). Another interesting finding was that of considerable sprouting of cut axons in the OTCs which would increase the probability of making contacts to neighbouring cells. ...
Article
The morphology and the distribution of neurons expressing the NK1-receptor (NK1R) and the co-expression of gamma-aminobutyric acid (GABA) in these neurons were studied in the rat occipital cortex and in organotypic cultures (OTCs) derived from this structure. By employing immunohistochemistry, we demonstrate that the NK1R-expressing neurons are non-pyramidal neurons and co-express GABA. Some differences were noted between in vivo and OTCs. NK1R-expressing neurons in OTCs had larger somata and longer dendrites and the proportion stained with an anti-GABA-antibody (approximately 50%) was smaller than in vivo (90%). The preferential location of NK1R-expressing neurons in layers II/III and VI, seen in vivo is not present in OTCs where these neurons distribute rather homogeneously through layers II-VI. Our findings imply that in contrast to the cat and monkey, in the rat occipital cortex the effects of substance P are almost exclusively mediated via inhibitory interneurons.
... Organotypic slice cultures provide a useful model for addressing developmental questions, because the cytoarchitectonics and intrinsic connectivity are preserved to a large extent. Furthermore, organotypic cultures are well suited for determining whether certain characteristics and developmental events are determined intrinsically (Frotscher et al., 1990(Frotscher et al., , 1995Caeser and Schütz, 1992;Frotscher and Heimrich, 1993;Gillies and Price, 1994;Rennie et al., 1994;Tuttle et al., 1995). To gain more information on the developmental roles of CR cells and on the mechanisms responsible for their degeneration, we have studied here the morphological features, survival properties, and fate of CR cells in single organotypic slice cultures from the somatosensory cortex and hippocampus, deprived of their extrinsic afferent inputs. ...
... Single organotypic slice cultures. Hippocampal and neocortical slice cultures were prepared from day of birth (P0) or P1 mouse pups (NMRI; Charles River Wiga, Sulzfeld, Germany) essentially as described (Caeser and Schütz, 1992;Frotscher and Heimrich, 1993). Animals were anesthetized by hypothermia, their brains were aseptically removed, and the hippocampus and prospective parietal cortex were dissected out under microscopic control. ...
Article
Full-text available
Cajal-Retzius (CR) cells are transient, pioneer neurons of layer I of the cortex that are believed to play essential roles in corticogenesis, e.g., in neuronal migration and synaptogenesis. Here we have used calretinin immunostaining to study the characteristics, survival, and fate of CR cells in single organotypic slice cultures of mouse neocortex and hippocampus deprived of their extrinsic afferents. In neocortical explants, CR cells were observed after 1-3 d in vitro (DIV), but they disappeared after 5-7 DIV, which is similar to their time of degeneration in vivo. The disappearance of CR cells in neocortical slices was prevented by incubation with tetrodotoxin and the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3,-dione but not by 2-amino-5-phosphonopentanoic acid, suggesting that neuronal activity and non-NMDA glutamate receptors may trigger CR cell death in the neocortex. In contrast to the situation in vivo, in which many hippocampal CR cells disappear at approximately the third postnatal week, CR cells survived in single hippocampal cultures after long incubation times (31 DIV), with their morphology essentially unaltered. In contrast, fewer CR cells were found when hippocampal slices were cocultured with explants from the entorhinal cortex. Because CR cells are transient synaptic targets for entorhinohippocampal afferents, these findings suggest a role for entorhinal afferents in the degeneration of CR cells in the hippocampus. In conclusion, this study shows different survival properties of CR cells in organotypic slice cultures of hippocampus and neocortex, and it suggests that different mechanisms are involved in the regulation of the process of naturally occurring CR cell death in the two cortical regions.
... The organotypic cortex culture is a well-characterized biological model of neuronal activity free of hemodynamic, respiratory, and other physiological confounds. Not only is the in vivo cortical cytoarchitecture preserved (including cortical layers and cortical cell types), but neuronal activity in the culture also displays bursts of spontaneous neuronal avalanches grouped into so-called up-states and separated by periods of low activity (22)(23)(24)(25), resembling resting neuronal activity in vivo (26)(27)(28). Specifically, fluorometric calcium (Ca 2+ ) imaging is used to detect intracellular Ca 2+ concentration changes that closely follow action potentials in neurons under Significance Diffusion functional MRI has been proposed as a noninvasive neuroimaging method to detect neuronal activity more directly than current blood-oxygen-level-dependent functional MRI, yet initial findings have proven difficult to interpret and reproduce. ...
... Organotypic cultures represent an in vitro model in which a slab of cortical tissue can be kept alive and active for many weeks. Anatomical studies have shown that these cultures maintain all major cell types and cortical layer characteristics as in vivo (22,23). Importantly, the neuronal activity in this in vitro system is found to organize into active 0.5-to 3-s-long periods (up-states) separated by up to 10 s of very low activity (down-states). ...
Article
Full-text available
Functional MRI (fMRI) is widely used to study brain function in the neurosciences. Unfortunately, conventional fMRI only indirectly assesses neuronal activity via hemodynamic coupling. Diffusion fMRI was proposed as a more direct and accurate fMRI method to detect neuronal activity, yet confirmative findings have proven difficult to obtain. Given that the underlying relation between tissue water diffusion changes and neuronal activity remains unclear, the rationale for using diffusion MRI to monitor neuronal activity has yet to be clearly established. Here, we studied the correlation between water diffusion and neuronal activity in vitro by simultaneous calcium fluorescence imaging and diffusion MR acquisition. We used organotypic cortical cultures from rat brains as a biological model system, in which spontaneous neuronal activity robustly emerges free of hemodynamic and other artifacts. Simultaneous fluorescent calcium images of neuronal activity are then directly correlated with diffusion MR signals now free of confounds typically encountered in vivo. Although a simultaneous increase of diffusion-weighted MR signals was observed together with the prolonged depolarization of neurons induced by pharmacological manipulations (in which cell swelling was demonstrated to play an important role), no evidence was found that diffusion MR signals directly correlate with normal spontaneous neuronal activity. These results suggest that, whereas current diffusion MR methods could monitor pathological conditions such as hyperexcitability, e.g., those seen in epilepsy, they do not appear to be sensitive or specific enough to detect or follow normal neuronal activity.
... For organotypic cortex cultures, glia cells have been demonstrated to protect the neuronal tissue from mechanical damage (Schultz-Süchting and Wolburg, 1994;Schmidt-Kastner and Humpel, 2002). Organotypic cortex cultures also show clear differences compared to in vivo cortex such as an overall reduced connectivity due to a reduction in the 3 rd dimension when preparing a slice (Cäser and Schüz, 1992) or change in glia protein expression (Staal et al., 2011). Organotypic cultures are typically prepared from newborn animals. ...
Preprint
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Self-organized criticality (SOC) refers to the ability of complex systems to evolve towards a second-order phase transition at which interactions between system components lead to scale-invariant events beneficial for system performance. For the last two decades, considerable experimental evidence has accumulated that the mammalian cortex with its diversity in cell types, interconnectivity, and plasticity might exhibit SOC. Here we review experimental findings of isolated, layered cortex preparations to self-organize towards the four dynamical motifs of up-states, oscillations, neuronal avalanches, and coherence potentials. During up-states, the synchronization observed for nested theta/gamma-oscillations embeds scale-invariant neuronal avalanches identified by robust power law scaling in avalanche sizes with a slope of -3/2. This precise dynamical coordination can be tracked in negative transients of the local field potential (nLFP), emerges autonomously in superficial layers of organotypic cortex cultures, is homeostatically regulated, exhibits separation of time scales, and reveals unique size vs. quiet time dependencies. A subclass of avalanches, the coherence potential, exhibits precise maintenance of temporal precision in propagated local synchrony. Avalanches emerge in superficial cortex layers under conditions of strong external driving. The balance of excitation and inhibition (E/I) and neuromodulators such as dopamine establish powerful control parameters for avalanche dynamics. This rich dynamical repertoire is not observed in dissociated cortex cultures, which lack the differentiation into layered components, suggesting that SOC in cortex requires essential components found in superficial layers of cortex. The precise interaction between up-states, nested oscillations and avalanches in layered cortex provides compelling evidence for SOC in the brain.
... Medium was changed every 3–5 d. Mature slice cultures prepared from rat cortex at P1 and grown for 4 weeks have been shown to have anatomical features that closely match those found in vivo (Caeser and Schuz, 1989; Gotz and Bolz, 1992; Plenz and Aertsen, 1996a,b; Plenz and Kitai, 1996 ) and develop precise efferent connections with target structures (for review, see Bolz, 1994). Because the current project is part of a larger study to analyze corticostriatal processing, cortex slices were cocultured with striatum and substantia nigra (Plenz and Kitai, 1998 ). ...
Article
Full-text available
A major goal of neuroscience is to elucidate mechanisms of cortical information processing and storage. Previous work from our laboratory (Beggs and Plenz, 2003) revealed that propagation of local field potentials (LFPs) in cortical circuits could be described by the same equations that govern avalanches. Whereas modeling studies suggested that these "neuronal avalanches" were optimal for information transmission, it was not clear what role they could play in information storage. Work from numerous other laboratories has shown that cortical structures can generate reproducible spatiotemporal patterns of activity that could be used as a substrate for memory. Here, we show that although neuronal avalanches lasted only a few milliseconds, their spatiotemporal patterns were also stable and significantly repeatable even many hours later. To investigate these issues, we cultured coronal slices of rat cortex for 4 weeks on 60-channel microelectrode arrays and recorded spontaneous extracellular LFPs continuously for 10 hr. Using correlation-based clustering and a global contrast function, we found that each cortical culture spontaneously produced 4736 +/- 2769 (mean +/- SD) neuronal avalanches per hour that clustered into 30 +/- 14 statistically significant families of spatiotemporal patterns. In 10 hr of recording, over 98% of the mutual information shared by these avalanche patterns were retained. Additionally, jittering analysis revealed that the correlations between avalanches were temporally precise to within +/-4 msec. The long-term stability, diversity, and temporal precision of these avalanches indicate that they fulfill many of the requirements expected of a substrate for memory and suggest that they play a central role in both information transmission and storage within cortical networks.
... At 10 DIV, the explants were briefly rinsed in 0.9% NaCl to remove excess growth medium and fixed for 48 h in 3.5% potassium dichromate–5% glutaraldehyde solution at 4 °C (Caeser & Aertsen, 1991; Caeser & Schuz, 1992). The explants were then rinsed with distilled water. ...
Article
The present study was undertaken to determine the effects of neurotrophin-3 (NT3) and spontaneous bioelectric activity (SBA) on dendritic elongation and branching in long-term isolated organotypic explants of rat neocortex. Viral vector-directed expression of NT3 was used as an effective means to ensure a continuous, local production of the neurotrophic factor. Quantitative light microscopic measurement of dendritic branching patterns was carried out on Golgi-stained materials. Explants were exposed to an adenoviral vector encoding the genetic sequence for neurotrophin-3 (Ad-NT3), or to exogenous additions of the neuropeptide NT3. In order to test for activity-dependent growth effects under control and experimental conditions, explants were exposed to glutamatergic blockade using a cocktail of APV and DNQX. Both Ad-NT3 and NT3 peptide potently promoted apical and basal dendritic growth (elongation and branching) in pyramidal neurons. This growth was observed to be significant in layers II-IV and V. These growth effects were also not activity dependent, inasmuch as they were elicited from explants in which spontaneous bioelectric activity had been suppressed. Non-pyramidal neurons, throughout the neocortical slice, showed no significant dendritic responses to the prolonged presence of NT3. These findings show that pyramidal dendritic growth in long-term neocortical explants responds to at least one neurotrophic growth factor, NT3, and is independent of intrinsic bioelectric activity. The use of viral vectors in delivering a continuous high level of neurotrophic factor within developing neural tissues demonstrates its potential application to in vivo tissues during development, or in the stimulation of neuritogenesis and neuroregeneration following injuries.
... Similarly, it is possible that our inability to detect a difference between mutant and wild-type animals can be attributed to the presence or absence of some factor in the culture medium. Although neurons in organotypic cultures mature roughly comparably to those in the intact brain (Caesar et al., 1989;Caeser and Schuz, 1992;Muller et al., 1993), a more subtle effect remains a possibility. Notably, a recent study in cultured hippocampal neurons failed to detect differences in spine length between mutant and wild-type animals, and instead found morphologic and electrophysiological evidence for presynaptic defects (Braun and Segal, 2000). ...
Article
Fragile X syndrome is caused by a mutation in the FMR1 gene leading to absence of the fragile X mental retardation protein (FMRP). Reports that patients and adult FMR1 knock-out mice have abnormally long dendritic spines of increased density suggested that the disorder might involve abnormal spine development. Because spine length, density, and motility change dramatically in the first postnatal weeks, we analyzed these properties in mutant mice and littermate controls at 1, 2, and 4 weeks of age. To label neurons, a viral vector carrying the enhanced green fluorescent protein gene was injected into the barrel cortex. Layer V neurons were imaged on a two-photon laser scanning microscope in fixed tissue sections. Analysis of >16,000 spines showed clear developmental patterns. Between 1 and 4 weeks of age, spine density increased 2.5-fold, and mean spine length decreased by 17% in normal animals. Early during cortical synaptogenesis, pyramidal cells in mutant mice had longer spines than controls. At 1 week, spine length was 28% greater in mutants than in controls. At 2 weeks, this difference was 10%, and at 4 weeks only 3%. Similarly, spine density was 33% greater in mutants than in controls at 1 week of age. At 2 or 4 weeks of age, differences were not detectable. The spine abnormality was not detected in neocortical organotypic cultures. The transient nature of the spine abnormality in the intact animal suggests that FMRP might play a role in the normal process of dendritic spine growth in coordination with the experience-dependent development of cortical circuits.
... For organotypic cortex cultures, glial cells have been demonstrated to protect the neuronal tissue from mechanical damage [29,30]. Also, organotypic cortex cultures show clear differences compared to the in vivo cortex, such as an overall reduced connectivity due to a reduction in the third dimension when preparing the brain slice taken into culture [31] or a change in glial protein expression [32]. Organotypic cultures are typically prepared from newborn animals. ...
Article
Full-text available
Self-organized criticality (SOC) refers to the ability of complex systems to evolve toward a second-order phase transition at which interactions between system components lead to scale-invariant events that are beneficial for system performance. For the last two decades, considerable experimental evidence has accumulated that the mammalian cortex with its diversity in cell types, interconnectivity, and plasticity might exhibit SOC. Here, we review the experimental findings of isolated, layered cortex preparations to self-organize toward four dynamical motifs presently identified in the intact cortex in vivo: up-states, oscillations, neuronal avalanches, and coherence potentials. During up-states, the synchronization observed for nested theta/gamma oscillations embeds scale-invariant neuronal avalanches, which can be identified by robust power law scaling in avalanche sizes with a slope of −3/2 and a critical branching parameter of 1. This precise dynamical coordination, tracked in the negative transients of the local field potential (nLFP) and spiking activity of pyramidal neurons using two-photon imaging, emerges autonomously in superficial layers of organotypic cortex cultures and acute cortex slices, is homeostatically regulated, exhibits separation of time scales, and reveals unique size vs. quiet time dependencies. A subclass of avalanches, the coherence potentials, exhibits precise maintenance of the time course in propagated local synchrony. Avalanches emerge in superficial layers of the cortex under conditions of strong external drive. The balance of excitation and inhibition (E/I), as well as neuromodulators such as dopamine, establishes powerful control parameters for avalanche dynamics. This rich dynamical repertoire is not observed in dissociated cortex cultures, which lack the differentiation into cortical layers and exhibit a dynamical phenotype expected for a first-order phase transition. The precise interactions between up-states, nested oscillations, and avalanches in superficial layers of the cortex provide compelling evidence for SOC in the brain.
... These neuroblasts proliferate and differentiate before migrating towards the developing cortical plate [61]. The rapid growth spurt of radial glial fibres, which guide migrating neuroblasts to their final destination, commences early and is completed by 20 weeks gestation [93,94]. Proliferation ceases at 18 weeks gestation and the last neuroblasts formed within the neural tube move to their final destinations at this time. ...
Article
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While many children with brain conditions present with cognitive, behavioural, emotional, academic and social impairments, other children recover with seemingly few impairments. Animal studies and preliminary child studies have identified timing of brain lesion as a key predictor in determining functional outcome following early brain lesions. This research suggests that knowledge of healthy developmental processes in brain structure and function is essential for better understanding functional recovery and outcome in children with brain lesions. This review paper aims to equip researchers with current knowledge of key principles of developmental processes in brain structure and function. Timetables for development of the prefrontal cortex (PFC), a brain region particularly vulnerable to lesions due to its protracted developmental course, are examined. In addition, timetables for development of executive skills, which emerge in childhood and have a prolonged developmental course that parallels development of the PFC, are also discussed. Equipped with this knowledge, researchers are now in a better position to understand functional recovery and outcome in children with brain conditions.
... Acute slices from rat cortex are commonly taken at PND 0 -1 and cultured for many weeks on the MEA. Early studies have clearly demonstrated that single cortex slice cultures, after several weeks in vitro, maintain a layered structure with identifiable cell types that can be easily compared to in vivo cell classes 18,[18][19][20][21] . The layered organization in this in vitro system has been conveniently used to study thalamic innervation of cortex during development [22][23][24] , as well as for driving subcortical structures such as the striatum 25,26 . ...
Article
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The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing. In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).
... Owing to the regeneration and development of synaptic connections in vitro, the proportion of interconnected neurons is higher in cultures than in acute slices [31]. This high degree of interconnectivity is causal of notable rates of spontaneous action potential activity, particularly in brain areas in which neuronal activity hinges on synaptic inputs [49,50]. ...
Article
The development of neuroactive drugs is a time consuming procedure. Candidate drugs must be run through a battery of tests, including receptor studies and behavioural tests on animals. As a rule, numerous substances with promising properties as assessed in receptor studies must be eliminated from the development pipeline in advanced test phases because of unforeseen problems like intolerable side-effects or unsatisfactory performance in the whole organism. Clearly, test systems of intermediate complexity would alleviate this inefficiency. In this review, we propose cultured organotypic brain slices as model systems that could bridge the 'interpolation gap' between receptors and the brain, with a focus on the development of new general anaesthetics with lesser side effects. General anaesthesia is based on the modulation of neurotransmitter receptors and other conductances located on neurons in diverse brain regions, including cerebral cortex and spinal cord. It is well known that different components of general anaesthesia, e.g. hypnosis and immobility, are produced by the depression of neuronal activity in distinct brain regions. The ventral horn of the spinal cord is an important structure for the induction of immobility. Thus, the potentially immobilizing effects of a newly designed drug can be estimated from its depressant effect on neuronal network activity in cultured spinal slices. A drug's sedative and hypnotic potential can be examined in cortical cultures. Combined with genetically engineered mice, this approach can point to receptor subtypes most relevant to the drug's intended net effect and in return can help in the design of more selective drugs. In conclusion, the use of organotypic cultures permits predictions of neuroactive properties of newly designed drugs on an intermediate level, and should therefore open up avenues for a more creative and economic drug development process.
... Organotypic slice cultures were used for electrophysiological recordings after 15 -35 days in vitro. As the changes in the reversal potential of GABA-evoked currents occur between postnatal day 5 and 12, all cultures used in the present study had developed into an adult status, which is also indicated by the morphological differentiation of individual cell types (Caeser and Schüz, 1992;Di Cristo et al., 2004). ...
Article
Neocortical neurons mediate the sedative and anticonvulsant properties of benzodiazepines. These agents enhance synaptic inhibition via positive modulation of γ-aminobutyric acid (GABA(A)) receptors harbouring α1-, α2-, α3- or α5-protein subunits. Benzodiazepine-sensitive GABA(A) receptors containing the α5-subunit are abundant in the neocortex, but their impact in controlling neuronal firing patterns is unknown. Here we studied how the discharge rates of cortical neurons are modified by a positive (SH-053-2'F-R-CH3) and a negative (L 655,708) α5-subunit-preferring allosteric modulator in comparison to diazepam, the classical non-selective benzodiazepine. Drug actions were characterized in slice cultures from wild-type and α5(H105R) knock-in mice by performing extracellular multi-unit-recordings. In knock-in mice, receptors containing the α5 subunit are insensitive to benzodiazepines. The non-selective positive allosteric modulator diazepam decreased the discharge rates of neocortical neurons during episodes of ongoing neuronal activity (up states). In contrast to diazepam, the α5-preferring positive modulator SH-053-2'F-R-CH3 accelerated action potential firing during up states. This promoting action was absent in slices from α5(H105R) mice, confirming that it is mediated by the α5-subunit. Consistent with these observations, the negative α5-selective modulator L 655,708 inhibited up state action potential activity in slices from wild-type mice. The opposing actions of diazepam and SH-053-2'F-R-CH3, which both enhance GABA(A) receptor function but differ in subtype-selectivity, uncovers contrasting roles of GABA(A) receptor subtypes in controlling the firing rates of cortical neurons. These findings may have important implications for the design of novel anaesthetic and anticonvulsant benzodiazepines displaying an improved efficacy and fewer side effects.
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Present noninvasive neuroimaging methods measure neuronal activity indirectly, via either cerebrovascular changes or extracranial measurements of electrical/magnetic signals. Recent studies have shown evidence that MRI may be used to directly and noninvasively map electrical activity associated with human brain activation, but results are inconclusive. Here, we show that MRI can detect cortical electrical activity directly. We use organotypic rat-brain cultures in vitro that are spontaneously active in the absence of a cerebrovascular system. Single-voxel magnetic resonance (MR) measurements obtained at 7 T were highly correlated with multisite extracellular local field potential recordings of the same cultures before and after blockade of neuronal activity with tetrodotoxin. Similarly, for MR images obtained at 3 T, the MR signal changed solely in voxels containing the culture, thus allowing the spatial localization of the active neuronal tissue. • cell culture • functional MRI • neuronal current imaging • brain
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General anaesthetics cause sedation, amnesia and hypnosis. Although these clinically desired actions are indicative of an impairment of neocortical information processing, it is widely held that they are to a large part mediated by subcortical neural networks. Anaesthetic action on brain stem, basal forebrain and thalamus, all of which are known to modulate cortical excitability, would thus ultimately converge on neocortex, perturbing and reducing action potential activity therein. However, as neocortex harbours molecular targets of anaesthetics in high densities, notably GABA(A) receptors, neocortex itself should be very sensitive to anaesthetics. Here, we performed experiments to reveal the extent to which neocortex proper is a relevant target of the low concentrations of volatile anaesthetics causing sedation and hypnosis. We compared the effects of isoflurane, enflurane and halothane on spontaneous action potential activity of rat neocortical neurons in vivo and in isolated cortical networks in vitro, i.e. in the presence and absence of subcortical arousal systems. We observed that the anaesthetics decreased spontaneous firing of neurons via intracortical mechanisms; concentrations inducing hypnosis in humans reduced discharge rates both in vivo and in vitro to the same extent, approximately 50%. This decrease in neuronal activity was paralleled by a significant enhancement of neocortical GABA(A) receptor-mediated inhibition. These findings challenge the notion of predominantly subcortical effects of volatile anaesthetics and suggest that intracortical targets, among them neocortical GABA(A) receptors, mediate the sedative and hypnotic properties of volatile anaesthetics.
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Fragile X syndrome is caused by a mutation in the FMR1 gene leading to absence of the fragile X mental retardation protein (FMRP). Reports that patients and adult FMR1 knock-out mice have abnormally long dendritic spines of increased density suggested that the disorder might involve abnormal spine development. Because spine length, density, and motility change dramatically in the first postnatal weeks, we analyzed these properties in mutant mice and littermate controls at 1, 2, and 4 weeks of age. To label neurons, a viral vector carrying the enhanced green fluorescent protein gene was injected into the barrel cortex. Layer V neurons were imaged on a two-photon laser scanning microscope in fixed tissue sections. Analysis of >16,000 spines showed clear developmental patterns. Between 1 and 4 weeks of age, spine density increased 2.5-fold, and mean spine length decreased by 17% in normal animals. Early during cortical synaptogenesis, pyramidal cells in mutant mice had longer spines than controls. At 1 week, spine length was 28% greater in mutants than in controls. At 2 weeks, this difference was 10%, and at 4 weeks only 3%. Similarly, spine density was 33% greater in mutants than in controls at 1 week of age. At 2 or 4 weeks of age, differences were not detectable. The spine abnormality was not detected in neocortical organotypic cultures. The transient nature of the spine abnormality in the intact animal suggests that FMRP might play a role in the normal process of dendritic spine growth in coordination with the experience-dependent development of cortical circuits.
Article
The reestablishment in vitro of the corticopontine projection was studied in organotypic co-cultures of cortex and pons of rats 0 (day of birth) - 3 days old. After 2-3 weeks in vitro, application of the lipophilic tracer DiI in the pontine explant retrogradely stained in layer V of the cortical explant pyramidal neurons which were characterized by large somata and spiny dendrites with an apical dendrite that reached upper cortical layers II/III. The projection developed only in co-cultures from rats 0-2 days old when the pontine explant was placed in close vicinity either to the white matter or the pial surface of the cortical explant. Control experiments demonstrated the specificity of the corticopontine projection in vitro by showing that the projections of the layer V pyramidal cells to the pons did not reflect a non-directed outgrowth pattern with subsequent survival of axons contacting the target explant. Our findings demonstrate that morphology and laminar position of corticopontine projection neurons in vitro are similar to those in vivo and thus support the "organotypic" nature of the explant co-culture system.
Article
An inhibitor of cathepsins B and L was used to test if lysosomal dysfunction in cultured slices of rat frontal cortex induces pathological features that develop in the human cortex during aging and Alzheimer's disease (AD). Incubation for 6 days with N-CBZ-L-phenylalanyl-L-alanine-diazomethylketone (ZPAD) resulted in a massive proliferation of endosomes-lysosomes in all cortical layers. Slices additionally exposed to a washout of 4 days had numerous meganeurites, blister-like structures in the region of the axon hillock, in layer III but not in other cortical laminae. Meganeurites are a characteristic feature of the human frontal cortex after age 50 and are largely restricted to layer III. Tests for apoptosis were carried out at two intervals following meganeurite formation. TUNEL-labeled neurons were confined to layers II/III on the surface of the slices but there was no evidence for a ZPAD effect. In all, 6 days of lysosomal dysfunction reproduces characteristic effects of normal aging in neocortex without generating some key features of AD.
Article
An important general question in neurobiology concerns the development and expression of the rich context of neuronal phenotypes, especially in relation to the diverse patterns of connectivity. Organotypic cultures of brain slices may offer distinct advantages for such studies if such a preparation survives, maintains a wide diverstiy of neuronal phenotypes and displays appropriate synaptic connections between regions. To address these requirements, we utilized long-term organotypic cultures of intact horizontal slices of rat forebrain and midbrain and assessed a variety of markers of phenotype in combination with functional tests of connectivity. This explant preparation displayed a distinct viability requirement such that the greatest explant survival was seen in slices taken from pups of less than postnatal day 7 and was independent of N-methyl-d-aspartate channel blockade. The anatomical features of the major brain regions (e.g., neocortex, striatum, septum, hippocampus, diencephalon and midbrain) were observed in their normal boundaries. The presence of cholinergic and catecholaminergic neurons was demonstrated with acetylcholinesterase histochemistry and tyrosine hydroxylase immunohistochemistry. Labelled neurons displayed multiple, regionally-appropriate cytoarchitectures and, in some cases, could be seen to project to brain regions in a manner quite similar to that seen in vivo. Finally, the direct demonstration of spontaneous and evoked interregional excitatory synaptic transmission was made using whole-cell patch-clamp recordings from striatal neurons which revealed an intact glutamate-using corticostriatal pathway.
Article
Volatile general anesthetics depress neuronal activity in the mammalian central nervous system and enhance inhibitory Cl- currents flowing across the gamma-aminobutyric acid A (GABA(A)) receptor-ion channel complex. The extent to which an increase in GABA(A)-mediated synaptic inhibition contributes to the decrease in neuronal firing must be determined, because many further effects of these agents have been reported on the molecular level. The actions of halothane, isoflurane, and enflurane on the firing patterns of single neurons were investigated by extracellular recordings in organotypic slice cultures derived from the rat neocortex. Volatile anesthetics depressed spontaneous action potential firing of neocortical neurons in a concentration-dependent manner. The estimated median effective concentration (EC50) values were about one half the EC50 values for general anesthesia. In the presence of the GABA(A) antagonist bicuculline (20 microM), the effectiveness of halothane, isoflurane, and enflurane in reducing the discharge rates were diminished by 48-65%, indicating that these drugs act via the GABA(A) receptor. Together with recent investigations, our results provide evidence that halothane, isoflurane, and enflurane reduced spontaneous action potential firing of neocortical neurons in cultured brain slices mainly by increasing GABA(A)-mediated synaptic inhibition. At concentrations, approximately one half the EC50 for general anesthesia, volatile anesthetics increased overall GABA(A)-mediated synaptic inhibition about twofold, thus decreasing spontaneous action potential firing by half.
Article
Electron microscopy has been employed to analyze the normal maturational sequence that characterizes the postnatal development of synaptic circuits in the ventrobasal (VB) and reticular (Rt) thalamic nuclei of rats at different ages (from birth to the end of the third postnatal week). Throughout the first postnatal week, similar signs of immaturity are observed in both nuclei, mainly consisting in scarcity of cytoplasmic organelles, presence of wide extracellular spaces, and absence of myelinated fibers. Several synaptic terminals are however present from birth, thus indicating that some of the afferents have already reached and contacted their thalamic target during embryonic life. Most of the terminals are small and contain only a few round, clear vesicles, and therefore their cytological features do not allow the identification of their origin. In particular, in both nuclei, terminals with flat vesicles and symmetric specialization are only rarely observed, and in VB the ascending terminals are not distinguishable from terminals of other sources as they are in adults. During the second postnatal week, progressive maturational changes in VB and Rt lead to neurons having well-developed cytoplasmic organelles and to an elaborate neuropil containing myelinated fibers and synaptic terminals that are morphologically heterogeneous and resemble the adult ones. The permanence of growth cone-like profiles and of numerous somatic and dendritic protrusions, often contacted by synaptic terminals, indicates that a certain degree of reorganization is still taking place in both nuclei. By the end of the third postnatal week the synaptic organization of VB and Rt is indistinguishable from that observed in adults. This ultrastructural study shows that the appearance of the neuropil of VB and Rt and the morphological complexity of the synaptic arrangements characteristic of the adult rat are not present in neonates, but are gradually acquired during the first three postnatal weeks, and that they result from progressive modifications in circuit organization involving both pre- and postsynaptic elements.
Article
Dendritic/axonal growth has been examined in long-term organotypic neocortical explants taken from neonatal rat pups and grown either as isolated slices or as co-cultures. The quantitative light microscopic measurement of dendritic and axonal branching patterns within both types of explants was carried out on Golgi-stained materials. Spontaneous bioelectric activity (SBA) was blocked within both types of explants using a combination of APV and DNQX, NMDA and non-NMDA receptor antagonists, respectively. No extracellularly measurable SBA was observed to occur in the silenced explants in the presence of both antagonists but reappeared following wash-out with control medium. In both control and silenced explants, the overall cellular organization of the slice was maintained throughout the culturing period, with distinguishable pyramidal and non-pyramidal neurons located within the same layers and with the same orientations as observed in situ. The major findings of the present study show the following. (i) Pyramidal neurones chronically exposed to APV/DNQX exhibited no basal dendritic growth in co-cultured explants, while growth of apical dendritic lengths was similar to control values in the absence of SBA. (ii) Pyramidal neurones, nonetheless, exhibited significant terminal segment growth under SBA blockade which was correlated with a concomitant decrease in number of basal dendrites. (iii) Axonal growth in co-cultures was not sustained in silenced pyramidal neurones. (iv) Non-pyramidal neurones showed significant total dendritic and axonal growth in co-cultures following APV/DNQX treatment. (v) Non-pyramidal cells in co-cultures experienced an increase in terminal segment length at 2 weeks which declined in the third week. This increase-decrease was correlated with a decrease-increase in the total number of dendritic segments during the second and third weeks, respectively. (vi) In isolated explants the only departure from control growth curves was a significant increase in terminal segment length which was offset by a similar decrease in number of dendritic segments under APV/DNQX growth conditions. Thus the chronic loss of glutamate-mediated SBA differentially effected pyramidal and non-pyramidal neurones in isolated and co-cultured explants, with pyramidal neurones experiencing the more pronounced effects. We conclude that SBA effects the dynamics of neuritic elongation and branching and that these changes are most dramatically seen in co-cultures which cross-innervate one another, presumably via pyramidal axons. We hypothesize that the activity-dependent changes associated with reduction in pyramidal dendritic and axonal growth may be associated with neurotrophin receptor production/maturation.
Article
Purpose fMRI is widely used to study brain activity. Unfortunately, conventional fMRI methods assess neuronal activity only indirectly, through hemodynamic coupling. Here, we show that active, steady‐state transmembrane water cycling (AWC) could serve as a basis for a potential fMRI mechanism for direct neuronal activity detection. Methods AWC and neuronal actitivity in rat organotypic cortical cultures were simultaneously measured with a hybrid MR‐fluorescence system. Perfusion with a paramagnetic MRI contrast agent, Gadoteridol, allows NMR determination of the kinetics of transcytolemmal water exchange. Changes in intracellular calcium concentration, [Cai²⁺] were used as a proxy of neuronal activity and were monitored by fluorescence imaging. Results When we alter neuronal activity by titrating with extracellular [K⁺] near the normal value, we see an AWC response resembling Na⁺‐K⁺‐ATPase (NKA) Michaelis‐Menten behavior. When we treat with the voltage‐gated sodium channel inhibitor, or with an excitatory postsynaptic inhibitor cocktail, we see AWC decrease by up to 71%. AWC was found also to be positively correlated with the basal level of spontaneous activity, which varies in different cultures. Conclusions These results suggest that AWC is associated with neuronal activity and NKA activity is a major contributor in coupling AWC to neuronal activity. Although AWC comprises steady‐state, homeostatic transmembrane water exchange, our analysis also yields a simultaneous measure of the average cell volume, which reports any slower net transmembrane water transport.
Article
Patients suffering from organophosphorus intoxication are compromised by generalised seizures and respiratory insufficiency, either being potentially lethal. In these patients induction of general anaesthesia to allow artificial ventilation is an important therapeutic option. Previously, it has been demonstrated that cholinergic overstimulation impaired network depressing effects of etomidate and sevoflurane. In this study we tested the impact of cholinergic overstimulation on inhibitory effects of diazepam in organotypic slice cultures of cerebrocortical neurons. Effects of clinically relevant concentrations of diazepam on spontaneous action potential activity were assessed by extracellular action potential recordings under basal cholinergic tone as well as in the presence of acetylcholine (1 μM). Diazepam at anaesthetic concentrations (25-500 μM) impeded spontaneous network activity in a concentration dependent manner (EC₅₀ 80.5±8.0 μM). In the presence of 1 μM acetylcholine the potency of diazepam was not significantly altered (EC₅₀ 83.6±8.4 μM). The results demonstrate that the potency of diazepam to depress neocortical network-excitability is not significantly impaired by cholinergic overstimulation. Diazepam thereby differs from other anaesthetics like etomidate or sevoflurane whose potencies and efficacies were severely attenuated. Hence diazepam might be preferable for induction and maintenance of general anaesthesia in patients suffering from nerve agent intoxication.
Article
An in vitro system was established to analyse corticostriatal processing. Cortical and striatal slices taken at postnatal days 0-2 were co-cultured for three to six weeks. The anatomy of the organotypic co-cultures was determined using immunohistochemistry. In the cortex parvalbumin-positive and calbindin-positive cells, which resembled those seen in vivo, had laminar distributions. In the striatum, strongly stained parvalbumin-positive cells resembling striatal GABAergic interneurons and cholinergic interneurons were scattered throughout the tissue. The soma area of these interneuron classes was larger than the average striatal soma area, thus enabling visual selection of cells by class before recording. Cortical neurons with projections to the striatum showed similar morphological features to corticostriatal projection neurons in vivo. No projections from the striatum to the cortex were found. Intracellular recordings were obtained from 94 neurons. These were first classified on the basis of electrophysiological characteristics and the morphologies of cells in each class were reconstructed. Two types of striatal secondary neurons with unique electrophysiological dynamics were identified: GABAergic interneurons (n = 17) and large aspiny, probably cholinergic, interneurons (n = 15). The electrophysiological and morphological characteristics of cortical pyramidal cells (n = 27), cortical interneurons (n = 1), as well as striatal principal neurons (n = 34), were identical to those reported for similar ages in vivo. Organotypic cortex-striatum co-cultures are therefore suitable as an in vitro system in which to analyse corticostriatal processing. The network dynamics, which developed spontaneously in that system, are examined in the companion paper.
Article
Calretinin, a calcium-binding protein, is expressed in a specific set of interneurons in the adult rat cortex. Although its role in development is not known with any degree of certainty, evidence in support of a neuroprotective function has been forthcoming. To test this hypothesis, we submitted organotypic cultures (interphase technique) of 4- to 6-day-old rat brain slices to nutritive stress by serum deprivation for 1-3 weeks. Cultures were immunolabelled either with an antiserum against calretinin or with an antibody against MAP2 (the latter being used to assess neuronal cell number). In control (serum-enriched) cultures, the pattern of development of calretinin immunoreactivity mimicked that evinced in vivo with respect to layer- and cell-type specificity, but the maturation process was retarded by about 1 week. In the experimental group, cultures were incubated for 1 week in the presence of serum and then transferred to serum-free medium for an additional 2 weeks. Tissue was characterized by necrotic foci, a marked decrease in neuronal cell number and a further retardation in the course of development of the calretinin immunoreactivity pattern. The proportion of calretinin-immunoreactive cells to total number of viable neurons was 16% in serum-free cultures as against 9% in serum-enriched ones, suggesting that cells expressing the calcium-binding protein exhibit a greater tenacity for survival under conditions of nutritive stress, and thereby supporting the contention that calretinin acts in a neuroprotective capacity.
Article
The forebrain area medio-rostral neostriatum/hyperstriatum ventrale, a presumed analogue to the mammalian prefrontal cortex, displays a variety of synaptic changes during auditory filial imprinting. In order to study the underlying basic mechanisms of this synaptic plasticity we developed slice cultures of the medio-rostral neostriatum/hyperstriatum ventrale from newly hatched chicks. As a prerequisite for these investigations and in order to test the suitability of this system for future studies, we performed a thorough characterization of the in vitro tissue, of its cellular components and some of their biochemical features in comparison with in situ tissue. Since in situ the medio-rostral neostriatum/hyperstriatum ventrale has been previously shown to contain three distinct neuron populations characterized by the activity-regulated Ca(2+)-binding proteins parvalbumin, calbindin D28K and calretinin, we used these proteins as neuronal markers to study the survival and preservation of the morphological features of medio-rostral neostriatum/hyperstriatum ventrale neurons in vitro. In agreement with in vivo studies the three Ca(2+)-binding proteins are confined to neuronal cells and they are not colocalized, i.e. they appear to characterize three different neuron populations. The immunoreactive neurons in medio-rostral neostriatum/hyperstriatum ventrale cultures to a certain extent appear to form synaptic contacts with each other, shown by the double immuncytochemical experiments. One difference between cells in vivo and in vitro is their soma size, which is much larger in vitro than in vivo. This and our previous study on neuronal morphology demonstrates that morphologically and biochemically intact neurons can be maintained in medio-rostral neostriatum/hyperstriatum ventrale slice cultures, which may thus provide a suitable in vitro system for further studies of neuronal and synaptic plasticity in vitro.
Article
Dendritic growth has been studied in long-term organotypic neonatal rat occipital neocortex grown either apart as isolated explants or in tandem as cocultures. Quantitative light microscopic measurement of dendritic and axonal branching patterns within the cortical slice was accomplished using rapid Golgi stained materials. In both isolates and cocultures the overall cellular organization of the slice was maintained over 4 weeks in vitro with morphologically distinguishable pyramidal and nonpyramidal neurons located within the same layers and with the same orientations as observed in situ. Long-term increases in the total length of basal dendrites, apical dendrite and axons were observed only in cocultures and were similar to growth patterns reported for in situ materials. Dendritic growth was mainly due to elongation of terminal dendritic segments. Surprisingly, isolated explants showed no long-term increases in total (basal) dendrites, apical dendrites or axons with time in vitro. A transient decrease in the number of basal dendritic segments and increase in terminal segment lengths at the end of the first week in vitro, however, was observed in nonpyramidal neurons. It is hypothesized that (i) afferent inputs and/or efferent targets develop only in cocultures and provide a crucial conditions for the continued growth of dendritic/axonal arborization for neocortical neurons in vitro, (ii) intrinsic interconnectivity within isolated explants is not sufficient to maintain long-term growth of neuritic arbors, and (iii) remodelling of dendritic arbors within isolated explants occurs at the same time as these explants are showing noticeable increases in the level of spontaneous bioelectric activity, which suggests that dendritic growth and network formation may be function dependent.
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