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Glutamate release during LTD at cerebellar climbing fiber-Purkinje cell synapses
Abstract
It is widely thought that persistent, use-dependent alterations in synaptic strength such as long-term synaptic potentiation (LTP) and depression (LTD) underlie at least a portion of memory traces in the brain, but the exact cellular locus of expression for these alterations remains to be determined. They could be expressed presynaptically as a decrease in transmitter release, postsynaptically as a decrease in the synaptic current evoked by a fixed delivery of transmitter, as an increase in the number of functional synaptic contacts, or by a combination of these mechanisms. Here we report that LTD at the climbing fiber-Purkinje cell synapse in rat cerebellum was not associated with changes in a synaptic cleft glutamate transient, indicating that this type of LTD is most likely expressed postsynaptically.
... Climbing fibers can express postsynaptic LTD, and similarly to postsynaptic PF-LTD, CF-LTD requires post-synaptic Ca 2+ influx and activation of mGluR1 and PKC (Hansel and Linden, 2000; Ohtsuki et al., 2009; Shen et al., 2002), with both expressed as a reduced number of AMPA receptors at the postsynaptic membrane (Shen et al., 2002 ). The consequences of CF-LTD are a reduction in the slow component of the complex spike (Hansel and Linden, 2000; Weber et al., 2003), a reduction in the complex spike afterhyperpolarization (Schmolesky et al., 2005), and decreased complex spike-evoked dendritic Ca 2+ transients (Weber et al., 2003), all of which reduce the probability of subsequent induction of parallel fiber plasticity requiring a high calcium threshold (Ohtsuki et al., 2009; Weber et al., 2003 ). ...
... Climbing fibers can express postsynaptic LTD, and similarly to postsynaptic PF-LTD, CF-LTD requires post-synaptic Ca 2+ influx and activation of mGluR1 and PKC (Hansel and Linden, 2000; Ohtsuki et al., 2009; Shen et al., 2002), with both expressed as a reduced number of AMPA receptors at the postsynaptic membrane (Shen et al., 2002 ). The consequences of CF-LTD are a reduction in the slow component of the complex spike (Hansel and Linden, 2000; Weber et al., 2003), a reduction in the complex spike afterhyperpolarization (Schmolesky et al., 2005), and decreased complex spike-evoked dendritic Ca 2+ transients (Weber et al., 2003), all of which reduce the probability of subsequent induction of parallel fiber plasticity requiring a high calcium threshold (Ohtsuki et al., 2009; Weber et al., 2003 ). ...
... The consequences of CF-LTD are a reduction in the slow component of the complex spike (Hansel and Linden, 2000; Weber et al., 2003), a reduction in the complex spike afterhyperpolarization (Schmolesky et al., 2005), and decreased complex spike-evoked dendritic Ca 2+ transients (Weber et al., 2003), all of which reduce the probability of subsequent induction of parallel fiber plasticity requiring a high calcium threshold (Ohtsuki et al., 2009; Weber et al., 2003 ). In addition, CF-LTD is not associated with changes in the synaptic glutamate transient (Shen et al., 2002). The observed decrease in complex spike-evoked Ca 2+ transients (Weber et al., 2003) has a significant effect on the probability for PF-LTD induction (Coesmans et al., 2004 ), could have a neuroprotective function (Coesmans et al., 2004) and/or could provide a critical component of cerebellar learning (Ohtsuki et al., 2009). ...
... Furthermore, the importance of PF–LTD for the formation of several types of motor memory, such as adaptation of the vestibuloocular reflex and associative eyeblink conditioning, has been convincingly demonstrated (De Zeeuw et al., 1998; Koekkoek et al., 2003). In contrast to the wealth of information currently available about PF–LTD, we have only recently discovered and begun to characterize long term depression at the climbing fiber–PC synapse (Hansel and Linden, 2000; Schmolesky et al., 2002; Shen et al., 2002; Weber et al., 2003). Although each PC is innervated by only one climbing fiber, this input is disproportionately strong as it forms more than 1500 high-probability release synapses that are near-simultaneously active (Dittman and Regehr, 1998; Silver et al., 1998; Strata and Rossi, 1998; Foster et al., 2002). ...
... This form of synaptic plasticity is expressed as a $ 20% decrease in the excitatory postsynaptic current (EPSC) amplitude. As in the case of PF–LTD, expression of CF–LTD is postsynaptic (Shen et al., 2002 ) and is dependent upon a rise in the postsynaptic calcium levels, activation of type 1 metabotropic glutamate receptors (mGluR1), and activation of PKC. However, CF–LTD is unique in that its expression is not restricted to the reduction of the AMPA-R generated EPSC. ...
... The evidence for this serial determinant model is manifold. CF–LTD at the EPSC level is postsynaptic (Hansel and Linden, 2000; Shen et al., 2002) and at least three of the key molecular requirements are identical for the induction of PF-and CF-LTD at the EPSC level. These facts suggest that AMPA-R internalization occurs in CF-LTD as has been proven for PF–LTD (Wang and Linden, 2000). ...
Purkinje cells (PC) receive two types of excitatory input—the parallel fibers rising from the granule cells of the cerebellar cortex and the climbing fibers (CF) rising from neurons of the inferior olive nucleus found in the brainstem. This chapter describes the long term plasticity at the CF–PC synapse and pays particular attention to the slow afterhyperpolarization (AHP). Climbing fibres–long term depression (CF–LTD) results in a LTD of CF-evoked Ca²⁺ transients in PC dendrites, as a result, CF–LTD could also prove significant for Ca²⁺-sensitive developmental processes, neuroprotection, and/or shifting the probability of plasticity at other synapses onto the PC. The chapter presents a critical replication of the CF–LTD induction experiments characterized at the complex spike level and also shows that CF–LTD induction is accompanied by a substantial reduction in the slow AHP and that this AHP is Ca²⁺-dependent. This study provides a better understanding of plasticity in one of the most important synapses in the cerebellum.
... First, we used conventional methods, including measuring the coefficient of variation (CV), the paired-pulse ratio (PPR), and NMDAR activity, to assess potential changes in presynaptic transmitter release probability. Then we probed the LTD expression locus further using a low-affinity, competitive AMPAR antagonist, ␥-D-glutamyl-glycine (␥-DGG), to detect potential changes in transmitter profile in the cleft (Liu et al., 1999;Wadiche and Jahr, 2001;Shen et al., 2002). Finally, inde-pendent confirmation of these results came from experiments demonstrating that NMDAR-dependent LTD was occluded by postsynaptic infusion of an N-ethylmaleimide-sensitive fusion protein (NSF)-AP2-clathrin adaptor protein inhibitory peptide, pep2m (KRMKVAKNAQ) (Lüscher et al., 1999;Lüthi et al., 1999;Lee et al., 2002), suggesting a mechanism involving postsynaptic AMPAR trafficking at CI-AMPAR synapses. ...
... Next, we explored the nature of transmitter release at mossy fiber-interneuron synapses. Taking advantage of the low-affinity, competitive AMPAR antagonist ␥-DGG, we probed for variations in transmitter glutamate concentration during paired-pulse paradigms and after LTD expression at both synapse types (Liu et al., 1999;Wadiche and Jahr, 2001;Shen et al., 2002). These experiments are based on the observation that a certain fraction of synaptic receptors continually exposed to ␥-DGG and then subjected to the rapid synaptic glutamate transient will replace bound ␥-DGG for glutamate because of the low affinity of ␥-DGG. ...
... These experiments are based on the observation that a certain fraction of synaptic receptors continually exposed to ␥-DGG and then subjected to the rapid synaptic glutamate transient will replace bound ␥-DGG for glutamate because of the low affinity of ␥-DGG. Consequently, the degree of ␥-DGG inhibition can be used to assess changes in the glutamate transient concentration in the synaptic cleft during paired-pulse protocols (Wadiche and Jahr, 2001) and after induction of LTD (Shen et al., 2002). ...
Two distinct forms of long-term depression (LTD) exist at mossy fiber synapses between dentate gyrus granule cells and hippocampal CA3 stratum lucidum interneurons. Although induction of each form of LTD requires an elevation of postsynaptic intracellular Ca2+, at Ca2+-impermeable AMPA receptor (CI-AMPAR) synapses, induction is NMDA receptor (NMDAR) dependent, whereas LTD at Ca2+-permeable AMPA receptor (CP-AMPAR) synapses is NMDAR independent. However, the expression locus of either form of LTD is not known. Using a number of criteria, including the coefficient of variation, paired-pulse ratio, AMPA-NMDA receptor activity, and the low-affinity AMPAR antagonist gamma-D-glutamyl-glycine, we demonstrate that LTD expression at CP-AMPAR synapses is presynaptic and results from reduced transmitter release, whereas LTD expression at CI-AMPAR synapses is postsynaptic. The N-ethylmaleimide-sensitive fusion protein-AP2-clathrin adaptor protein 2 inhibitory peptide pep2m occluded LTD expression at CI-AMPAR synapses but not at CP-AMPAR synapses, confirming that CI-AMPAR LTD involves postsynaptic AMPAR trafficking. Thus, mossy fiber innervation of CA3 stratum lucidum interneurons occurs via two parallel systems targeted to either Ca2+-permeable or Ca2+-impermeable AMPA receptors, each with a distinct expression locus for long-term synaptic plasticity.
... Our results showed that CF stimulation at 1 Hz produced robust LTD in cerebellar slices from WT rats, but no induction of LTD in slices from MUT rats. Climbing fiber stimulation alone can induce postsynaptic LTD, which requires post-synaptic activation of mGluR1, protein kinase Cɣ (PKCɣ), and Ca 2+ influx [55,67,68], and in turn leads to reduced numbers of GluA2containing AMPA receptors in the postsynaptic membrane [68] and decreased amplitude of the synaptic response. The elimination of LTD by the W246G ELOVL4 mutation is strikingly similar to the elimination of LTD reported in a number of mouse strains with knockout or loss-of-function mutations in postsynaptic mGluR1, its downstream signaling molecules PKCɣ, phospholipase C-β4, PICK1, or the GluA2-containing AMPA receptor [45,[69][70][71][72][73]. ...
... Our results showed that CF stimulation at 1 Hz produced robust LTD in cerebellar slices from WT rats, but no induction of LTD in slices from MUT rats. Climbing fiber stimulation alone can induce postsynaptic LTD, which requires post-synaptic activation of mGluR1, protein kinase Cɣ (PKCɣ), and Ca 2+ influx [55,67,68], and in turn leads to reduced numbers of GluA2containing AMPA receptors in the postsynaptic membrane [68] and decreased amplitude of the synaptic response. The elimination of LTD by the W246G ELOVL4 mutation is strikingly similar to the elimination of LTD reported in a number of mouse strains with knockout or loss-of-function mutations in postsynaptic mGluR1, its downstream signaling molecules PKCɣ, phospholipase C-β4, PICK1, or the GluA2-containing AMPA receptor [45,[69][70][71][72][73]. ...
Spinocerebellar ataxia (SCA) is a neurodegenerative disorder characterized by ataxia and cerebellar atrophy. A number of different mutations gives rise to different types of SCA with characteristic ages of onset, symptomatology, and rates of progression. SCA type 34 (SCA34) is caused by mutations in ELOVL4 (ELOngation of Very Long-chain fatty acids 4), a fatty acid elongase essential for biosynthesis of Very Long Chain Saturated and Polyunsaturated Fatty Acids (VLC-SFA and VLC-PUFA, resp., ≥28 carbons), which have important functions in the brain, skin, retina, Meibomian glands, testes, and sperm. We generated a rat model of SCA34 by knock-in of the SCA34-causing 736T>G (p.W246G) ELOVL4 mutation. Rats carrying the mutation developed impaired motor deficits by 2 months of age. To understand the mechanism of these motor deficits, we performed electrophysiological studies using cerebellar slices from rats homozygous for W246G mutant ELOVL4 and found marked reduction of long-term potentiation at parallel fiber synapses and long-term depression at climbing fiber synapses onto Purkinje cells. Neuroanatomical analysis of the cerebellum showed normal cytoarchitectural organization with no evidence of degeneration out to 6 months of age. These results point to ELOVL4 as essential for motor function and cerebellar synaptic plasticity. The results further suggest that ataxia in SCA34 patients may arise from a primary impairment of synaptic plasticity and cerebellar network desynchronization before onset of neurodegeneration and progression of the disease at a later age.
... Climbing ber stimulation alone can induce postsynaptic LTD, which requires post-synaptic activation of mGluR1, Protein Kinase C (PKC ), and Ca 2+ in ux [53,65,66], and in turn leads to reduced numbers of GluA2-containing AMPA receptors in the postsynaptic membrane [66] and decreased amplitude of the synaptic response. The elimination of LTD by the W246G ELOVL4 mutation is strikingly similar to the elimination of LTD reported in a number of mouse strains with knockout or loss-of-function mutations in postsynaptic mGluR1, its downstream signaling molecules (PKC , Phospholipase C-β4), PICK1, or the GluA2-containing AMPA receptor [45,[67][68][69][70][71]. ...
... Climbing ber stimulation alone can induce postsynaptic LTD, which requires post-synaptic activation of mGluR1, Protein Kinase C (PKC ), and Ca 2+ in ux [53,65,66], and in turn leads to reduced numbers of GluA2-containing AMPA receptors in the postsynaptic membrane [66] and decreased amplitude of the synaptic response. The elimination of LTD by the W246G ELOVL4 mutation is strikingly similar to the elimination of LTD reported in a number of mouse strains with knockout or loss-of-function mutations in postsynaptic mGluR1, its downstream signaling molecules (PKC , Phospholipase C-β4), PICK1, or the GluA2-containing AMPA receptor [45,[67][68][69][70][71]. ...
Spinocerebellar ataxias (SCA) are a group of neurodegenerative disorders caused by a number of different mutations the leads to loss of motor coordination with characteristic ages of onset, symptomatology, and rates of progression. SCA type 34 (SCA34) is an age-related cerebellar neurodegenerative disorder caused by mutations in the Fatty Acid Elongase-4 ( ELOVL4 ). The ELOVL4 is an essential enzyme that mediates biosynthesis of V ery L ong C hain S aturated and P oly u nsaturated F atty A cids (VLC-SFA and VLC-PUFA, resp., ≥28 carbons) that are critical for the normal function of brain, skin, retina, Meibomian glands, and testes in which ELOVL4 is expressed. Global deletion or homozygous expression of truncated mutant ELOVL4 that lack VLC-SFA and VLC-PUFA biosynthesis cause severe skin disorders, seizures and neonatal mortality in rodents and humans. To understand the consequences of ELOVL4 mutations in pathogenesis of SCA34, we generated a rat model of SCA34 by knock-in of the SCA34-causing 736T>G (p.W246G) ELOVL4 mutation. We show that heterozygous and homozygous rats carrying the W246G mutation developed impaired motor deficits by two months of age. Our electrophysiological studies using cerebellar slices found marked reduction of long-term potentiation at parallel fiber synapses and long-term depression at climbing fiber synapses onto Purkinje cells in the homozygous W246G mutant rats. Our results further point to ELOVL4 products as being essential for motor function and cerebellar synaptic plasticity. These results suggest that in SCA34 patients, ataxia likely arises from primary impairment of synaptic plasticity and cerebellar network desynchronization that precedes cerebellar degeneration and loss of motor coordination with aging.
... Our results showed that CF stimulation at 1 Hz produced robust LTD in cerebellar slices from WT rats, but no induction of LTD in slices from MUT rats. Climbing fiber stimulation alone can induce postsynaptic LTD, which requires post-synaptic activation of mGluR1, Protein Kinase Cɣ (PKCɣ), and Ca 2+ influx [53,65,66], and in turn leads to reduced numbers of GluA2-containing AMPA receptors in the postsynaptic membrane [66] and decreased amplitude of the synaptic response. The elimination of LTD by the W246G ELOVL4 mutation is strikingly similar to the elimination of LTD reported in a number of mouse strains with knockout or loss-of-function mutations in postsynaptic mGluR1, its downstream signaling molecules (PKCɣ, Phospholipase C-β4), PICK1, or the GluA2-containing AMPA receptor [45,[67][68][69][70][71]. ...
... Our results showed that CF stimulation at 1 Hz produced robust LTD in cerebellar slices from WT rats, but no induction of LTD in slices from MUT rats. Climbing fiber stimulation alone can induce postsynaptic LTD, which requires post-synaptic activation of mGluR1, Protein Kinase Cɣ (PKCɣ), and Ca 2+ influx [53,65,66], and in turn leads to reduced numbers of GluA2-containing AMPA receptors in the postsynaptic membrane [66] and decreased amplitude of the synaptic response. The elimination of LTD by the W246G ELOVL4 mutation is strikingly similar to the elimination of LTD reported in a number of mouse strains with knockout or loss-of-function mutations in postsynaptic mGluR1, its downstream signaling molecules (PKCɣ, Phospholipase C-β4), PICK1, or the GluA2-containing AMPA receptor [45,[67][68][69][70][71]. ...
Background: Spinocerebellar ataxias (SCA) are a group of neurodegenerative disorders characterized by neuronal degeneration leading to loss of motor coordination. A number of different mutations gives rise to different types of SCA with characteristic ages of onset, symptomatology, and rates of progression. SCA type 34 (SCA34) is an age-related cerebellar neurodegenerative disorder caused by mutations in the fatty acid elongase-4 (ELOVL4). The ELOVL4 is an essential enzyme that mediates biosynthesis of Very Long Chain Saturated and Polyunsaturated Fatty Acids (VLC-SFA and VLC-PUFA, resp., ≥28 carbons) that are critical for the normal function of brain, skin, retina, Meibomian glands, and testes in which ELOVL4 is expressed. Global deletion or homozygous expression of truncated mutant ELOVL4 that lack VLC-SFA and VLC-PUFA biosynthesis cause severe skin disorders, seizures and neonatal mortality in rodents and humans.
Methods: To understand role of ELOVL4 and its products in neuronal function and to evaluate the consequences of ELOVL4 mutations in pathogenesis of age-related SCA34, we generated a rat model of SCA34 by knock-in of the SCA34-causing 736T>G (p.W246G) ELOVL4 mutation. We performed biochemical, neuroanatomical and behavioral analyses by rotorod to measure motor function. We used electrophysiological recordings from cerebellar slices to determine the impact of the W246G mutation on neuronal function. Results were analyzed using GraphPad Prism Statistical software.
Results: Heterozygous and homozygous rats carrying the W246G mutation developed impaired motor deficits by two months of age. To understand the mechanism of these motor deficits, we performed electrophysiological studies using cerebellar slices from rats homozygous for W246G mutant ELOVL4 and found marked reduction of long-term potentiation at parallel fiber synapses and long-term depression at climbing fiber synapses onto Purkinje cells. Neuroanatomical analysis of the cerebellum up 6 months of age showed normal cytoarchitectural organization despite the early-onset motor deficits and defects in synaptic plasticity.
Conclusions: Our results point to ELOVL4 and its products being essential for motor function and cerebellar synaptic plasticity. The results further suggest that in SCA34 patients, ataxia arises from primary impairment of synaptic plasticity and cerebellar network desynchronization that precedes cerebellar degeneration and loss of motor coordination with aging.
... Cerebellar long-term depression (LTD) is the most extensively studied form of long-term synaptic plasticity in the cerebellum (Ito 2001). Cerebellar LTD is defined as a persistent depression of input efficacy at the synapse of PFs with PCs mediated by a downregulation of postsynaptic AMPA receptor function (Shen et al 2002). There is strong evidence that this downregulation is the result of a decrease in the number of functional synaptic AMPA receptors by clathrin-mediated endocytosis (Matsuda et al 2000, Wang & Linden 2000. ...
... There is strong evidence that this downregulation is the result of a decrease in the number of functional synaptic AMPA receptors by clathrin-mediated endocytosis (Matsuda et al 2000, Wang & Linden 2000. LTD induction occurs via repeated association of inputs from PFs and climbing fibres, and is postsynaptic in origin (Crepel and Jaillard 1991, Crepel and Krupa 1988, Shen et al 2002. The climbing fibre inputs contribute to induction of LTD via calcium influx through voltage-gated channels which occurs during the complex spike (Hansel et al 2001). ...
... If anything, we observed a trend toward faster block after TTX— implying a greater Pr or higher glutamate concentration in the synaptic cleft, which would be associated with greater depression rather than the reduced depression in TTX (Figure S6Cii) (Zucker and Regehr, 2002). This was confirmed by using the low-affinity, competitive AMPAR antagonist g-DGG, which suppresses AMPAR responses more effectively under reduced glutamate concentrations (Lei and McBain, 2004; Shen et al., 2002; Wadiche and Jahr, 2001). Again, this assay showed no significant difference between the two conditions, but pointed to a trend-wise increase in synaptic glutamate after TTX (as g- DGG was less effective in suppressing AMPAR responses) (Fig- ure S6Ci). ...
... i. The low affinity competitive AMPAR antagonist γ-DGG was used to investigate the synaptic glutamate transient (Lei and McBain, 2004; Shen et al., 2002; Wadiche and Jahr, 2001). To provide evidence that the synaptic glutamate transient (as a measure of release probability) is unaltered following TTX-treatment we measured the degree of EPSP reduction after washing in γ-DGG (0.5 mM). ...
... If anything, we observed a trend toward faster block after TTX— implying a greater Pr or higher glutamate concentration in the synaptic cleft, which would be associated with greater depression rather than the reduced depression in TTX (Figure S6Cii) (Zucker and Regehr, 2002). This was confirmed by using the low-affinity, competitive AMPAR antagonist g-DGG, which suppresses AMPAR responses more effectively under reduced glutamate concentrations (Lei and McBain, 2004; Shen et al., 2002; Wadiche and Jahr, 2001). Again, this assay showed no significant difference between the two conditions, but pointed to a trend-wise increase in synaptic glutamate after TTX (as g- DGG was less effective in suppressing AMPAR responses) (Fig- ure S6Ci). ...
... i. The low affinity competitive AMPAR antagonist γ-DGG was used to investigate the synaptic glutamate transient (Lei and McBain, 2004; Shen et al., 2002; Wadiche and Jahr, 2001). To provide evidence that the synaptic glutamate transient (as a measure of release probability) is unaltered following TTX-treatment we measured the degree of EPSP reduction after washing in γ-DGG (0.5 mM). ...
The AMPA-type glutamate receptor (AMPAR) subunit composition shapes synaptic transmission and varies throughout development and in response to different input patterns. Here, we show that chronic activity deprivation gives rise to synaptic AMPAR responses with enhanced fidelity. Extrasynaptic AMPARs exhibited changes in kinetics and pharmacology associated with splicing of the alternative flip/flop exons. AMPAR mRNA indeed exhibited reprogramming of the flip/flop exons for GluA1 and GluA2 subunits in response to activity, selectively in the CA1 subfield. However, the functional changes did not directly correlate with the mRNA expression profiles but result from altered assembly of GluA1/GluA2 subunit splice variants, uncovering an additional regulatory role for flip/flop splicing in excitatory signaling. Our results suggest that activity-dependent AMPAR remodeling underlies changes in short-term synaptic plasticity and provides a mechanism for neuronal homeostasis.
... We hypothesized that synaptic plasticity may be implicated in the maturation and refinement process of the CF projection pattern and therefore examined the plasticity at CF-PN synapses in immature mice. Previous studies reported postsynaptic long-term depression (LTD) at the synapses (Hansel & Linden, 2000;Shen et al., 2002) and recently postsynaptic long-term potentiation (LTP) was also reported (Bosman et al., 2008). Here, we show that a 5 Hz conditioning stimulation of CFs induced either LTP or LTD between postnatal days (P)5 and 9 in mice. ...
... The fact that the asynchronous EPSC amplitudes, following the test CF stimulation in the Sr 2+ -containing solution, were not changed significantly by the conditioning stimulation, supports the concept of presynaptic expression of LTP and LTD. The postsynaptic LTD at CF-PN synapses was reported in rat cerebellum at P14-22 (Hansel & Linden, 2000;Shen et al., 2002). The expression site of LTD might change during postnatal development. ...
Climbing fibers provide one of the two major excitatory inputs to the cerebellar cortex. In an immature animal, several climbing fibers form synapses with one Purkinje neuron. During postnatal development most climbing fiber innervations with a Purkinje neuron are eliminated and only one strong fiber remains. Previous studies suggested that this pruning of surplus climbing fiber innervations depends on the neuronal activity. We hypothesized that synaptic plasticity might play a role in the maturation and refinement of such a climbing fiber projection pattern, and examined the plasticity properties of synapses between postnatal days 5 and 9 in mice. We found that a 5 Hz conditioning stimulation of climbing fibers forming relatively strong synapses with a Purkinje neuron induced long-term potentiation of the transmission accompanied by a decrease in the paired-pulse ratio of excitatory postsynaptic current amplitudes. This was suggestive of an increased probability of presynaptic release. However, the conditioning stimulation of climbing fibers forming relatively weak synapses induced long-term depression and tended to increase the paired-pulse ratio. Thus, the direction of plasticity appears to be determined by the strength of synaptic connection. Long-term depression occurred only in the conditioned climbing fiber, whereas long-term potentiation spread to unconditioned climbing fibers. A postsynaptic increase in the intracellular Ca(2+) concentration was required for long-term potentiation but not for long-term depression. These results reveal the existence of novel presynaptic plasticity at immature climbing fiber-Purkinje cell synapses, which may contribute to the maturation and refinement of the climbing fiber projection pattern.
... First, the LTD of AMPA-EPSCs proflashes produced inward currents of peak amplitude duced by climbing fiber tetanization, bath application 77.3 ± 6.6pA (recorded with V hold = −70 mV; n = 5), of PDA, or postsynaptic application of (−)-indolacwhich had much slower kinetics compared to similar tam-V is not associated with changes in paired-pulse currents evoked in Purkinje cells (10%–90% rise: ratio, consistent with previous reports (Hansel and Lin- 26.9 ± 2.8 ms; 50% decay: 135.6 ± 10.2 ms). Application den, 2000; Shen et al., 2002 ...
... at t = 19 min; n = 5) or kinetics (data not shown). The antagonist γ-D-glutamylglycine (Shen et al., 2002). ...
Persistent, use-dependent modulation of synaptic strength has been demonstrated for fast synaptic transmission mediated by glutamate and has been hypothesized to underlie persistent behavioral changes ranging from memory to addiction. Glutamate released at synapses is sequestered by the action of excitatory amino acid transporters (EAATs) in glia and postsynaptic neurons. So, the efficacy of glutamate transporter function is crucial for regulating glutamate spillover to adjacent presynaptic and postsynaptic receptors and the consequent induction of plastic or excitotoxic processes. Here, we report that tetanic stimulation of cerebellar climbing fiber-Purkinje cell synapses results in long-term potentiation (LTP) of a climbing fiber-evoked glutamate transporter current recorded in Purkinje cells. This LTP is postsynaptically expressed and requires activation of an mGluR1/PKC cascade. Together with a simultaneously induced long-term depression (LTD) of postsynaptic AMPA receptors, this might reflect an integrated antiexcitotoxic cellular response to strong climbing fiber synaptic activation, as occurs following an ischemic episode.
... Kynurenic acid (0.5 mM) reduced the amplitude of the initial EPSC in rod-driven horizontal cells to 41 ± 3% of control (Fig. 5A, inset) and significantly increased PPD when compared with control trials conducted in the same cell pairs (Fig. 5A). The more efficacious block of the second EPSC by kynurenic acid is consistent with a lower concentration of glutamate being attained in the synaptic cleft (Hashimoto and Kano, 1998; Wadiche and Jahr, 2001; Shen et al., 2002), but these results can also be interpreted as a relief of postsynaptic receptor saturation during the first EPSC (Foster et al., 2002; Harrison and Jahr, 2003). These two possible interpretations are considered in the Discussion in greater detail. ...
... Greater PPD in the presence of a low-affinity, competitive glutamate antagonist can be interpreted in two ways: (1) greater PPD can be produced by a more efficacious block of the second EPSC by kynurenic acid. If so, this would suggest that a lower concentration of glutamate is attained in the synaptic cleft during the second EPSC (Hashimoto and Kano, 1998; Wadiche and Jahr, 2001; Shen et al., 2002). Such a situation can occur if there is multivesicular release (Wadiche and Jahr, 2001), as found at the bipolar cell ribbon synapse (Singer et al., 2004). ...
Synaptic depression produced by repetitive stimulation is likely to be particularly important in shaping responses of second-order retinal neurons at the tonically active photoreceptor synapse. We analyzed the time course and mechanisms of synaptic depression at rod and cone synapses using paired-pulse protocols involving two complementary measurements of exocytosis: (1) paired whole-cell recordings of the postsynaptic current (PSC) in second-order retinal neurons and (2) capacitance measurements of vesicular membrane fusion in rods and cones. PSCs in ON bipolar, OFF bipolar, and horizontal cells evoked by stimulation of either rods or cones recovered from paired-pulse depression (PPD) at rates similar to the recovery of exocytotic capacitance changes in rods and cones. Correlation between presynaptic and postsynaptic measures of recovery from PPD suggests that 80-90% of the depression at these synapses is presynaptic in origin. Consistent with a predominantly presynaptic mechanism, inhibiting desensitization of postsynaptic glutamate receptors had little effect on PPD. The depression of exocytotic capacitance changes exceeded depression of the presynaptic calcium current, suggesting that it is primarily caused by a depletion of synaptic vesicles. In support of this idea, limiting Ca2+ influx by using weaker depolarizing stimuli promoted faster recovery from PPD. Although cones exhibit much faster exocytotic kinetics than rods, exocytotic capacitance changes recovered from PPD at similar rates in both cell types. Thus, depression of release is not likely to contribute to differences in the kinetics of transmission from rods and cones.
... The increase of postsynaptic [Ca 2+ ]i is very important in long-term synaptic plasticity including LTP and LTD at PF synapse (Ekerot & Kano, 1985;Ito & Kano, 1982;Kakizawa et al., 2012;Konnerth et al., 1992;Lev-Ram et al., 1992;Sakurai, 1987), and LTD at CF synapse (Hansel & Linden, 2000;Shen et al., 2002;Weber et al., 2003). The distinct synaptic plasticity in Purkinje cells is governed by the complex calcium signalling that arise from different Ca 2+ sources including Ca 2+ influx via the VGCCs and Ca 2+ release from intracellular stores mediated by the mGluRs signalling pathways (Finch & Augustine, 1998;Inoue et al., 1998;Konnerth et al., 1992;Takechi et al., 1998). ...
Duchenne muscular dystrophy (DMD) is a rapidly progressive X-linked recessive disease affecting about 1 in 3500 live male births. It is caused by mutations in the dystrophin gene, which result in the loss of dystrophin or expression of a non-functional truncated protein product. Full-length dystrophin is mainly expressed in muscles and the central nervous system. In addition to the degeneration of skeletal musculature, about one-third of patients with DMD display various degrees of intellectual impairment, commonly found with intelligence quotient (IQ) scores of one standard deviation below (IQ of 85) the normal population mean (IQ of 100). However, the mechanism underlying the cognitive deficits in DMD remains unclear and no effective treatment is available to reverse this condition in the affected individual. Recent studies showed that the life span of DMD patients today has increased from teens to their fourth decades. With longer survival, the quality of life becomes increasing important. Therefore, research on the cognitive aspect of DMD is as important as research on the muscular aspects because improvements in cognitive function will enhance the quality of life for the growing population of adult DMD patients. The aim of this thesis was to investigate the role of dystrophin in the central nervous system of the mdx mouse, a widely accepted murine model for DMD. This study employed the use of animal with different age groups, corresponding to young (3-4 months), adult (11-12 months), and aged (23-26 months). Adult and aged mdx mice are the focus in this study with findings from the older mouse especially valuable as, disease progression in aged mice closely resembling that of DMD. As numerous evidence has shown a high similarity between the specific cognitive dysfunctions seen in DMD (i.e. impaired verbal intelligence) and in patients with cerebellar lesions (i.e. language disorders), this study examined the function of cerebellar Purkinje cells in mdx mice using electrophysiological recording and calcium imaging. Overall, the data presented in this thesis provides new insights into the role of dystrophin in cerebellar Purkinje neurons. The findings suggest that dystrophin is important for normal inhibitory synaptic function, intrinsic electrophysiological properties, and calcium handling of the mature cerebellar Purkinje cells. The consequences of the absence of dystrophin including the altered GABAA receptor clustering and reduced peak amplitude of mIPSCs could be ameliorated when dystrophin was successfully rescued with Pip6f-PMO in an organotypic mdx cerebellar culture. If mdx mice and DMD patients share similar neuropathogenesis, the development of drugs targeting the altered functions in mdx Purkinje cells may serve as a potential therapy in alleviating the cognitive impairments seen in DMD.
... The addition of a Ca ++ chelator (BAPTA), mGluR1 antagonist (AIDA) or a PKC inhibitor (chelerythrine) completely blocks CF-LTD (Hansel and Linden, 2000), suggesting that a rise of Ca ++ , the activation of mGluR1 and the activation of PKC are necessary factors in the induction of this form of homosynaptic long-term plasticity. CF-LTD has been proposed to be a postsynaptic downregulation of AMPAR, because it is not associated with an alteration in the transient synaptic glutamate (Shen et al., 2002). CF/PC synapse LTP is required to potentiate the wiring of the wining CF during the developmental homosynaptic competition. ...
The establishment and maturation of functional synapses underlies the proper function of the Central Nervous System. A large number of secreted and transmembrane molecules are needed to control the mechanisms ensuring synaptic development, maturation and plasticity during the life of the organism. Sushi domain-containing proteins are evolutionary conserved in synapses and mutations in synaptic Sushi domain proteins have been related to neurological and psychiatric disorders in humans. I used the olivocerebellar network as a model to study the potential role of several Sushi domain-containing proteins in excitatory synapse formation and function. Based on previous data from our laboratory, I decided to study two genes, one coding the transmembrane Sushi domain containing protein 4 (SUSD4) and another gene coding four isoforms of Masp1/3 (Mannan-binding lectin serine protease 1/3). First, I identified that the third isoform of Masp1/3, that produces the protein MAP44 (Mannose-binding lectin-associated protein of 44 kDa) lacking the serine protease domain, is the most abundantly expressed isoform in the cerebellum and in the inferior olive. The data obtained by analyzing the Masp1/3 knockout mice suggest that the cytoarchitecture of the cerebellum and excitatory synaptogenesis on cerebellar Purkinje cells is not grossly affected in the absence of Masp1/3. Second, I found that Susd4 is also expressed in the olivocerebellar network during postnatal development and in the adult. Using Susd4 knock-out mice, I found that the absence of SUSD4 leads to deficits in motor learning, lack of climbing fiber-dependent parallel fiber long-term depression and facilitation of parallel fiber long-term potentiation. Climbing fiber transmission is increased transiently during maturation and is associated with misregulation of AMPA receptors content at synapses. Affinity purification followed by mass spectrometry revealed that E3 ubiquitin ligases of the HECT family bind to the cytoplasmic tail of SUSD4. Finally, I provide evidence for a regulation of AMPA receptor degradation by the SUSD4/HECT ligase complex. While molecular mechanisms controlling synaptic incorporation of glutamate receptors are well described, what controls their specific removal from synapses remains to be found. SUSD4 function could be a general mechanism allowing precise spatiotemporal control of the turnover of specific target proteins in cells by HECT ubiquitin ligases. These results provide novel insights into the role of Sushi domain proteins in the molecular mechanisms controlling synaptic plasticity and function in the CNS.
... However, LTD has also been described at CF synapses in P14-30 rats (Fig. 4) 94,95 . CF LTD results from brief CF activation at 5 Hz and is, just like PF LTD, postsynaptically induced and expressed 96 . While CF LTD has not been characterized in as much molecular detail as its counterpart at PF synapses, it is known that CF LTD requires a rise in calcium transients for its induction as well as the activation of mGluR1 receptors and PKC (Fig. 4) 94 . ...
In long-term depression (LTD) at synapses in the adult brain, synaptic strength is reduced in an experience-dependent manner. LTD thus provides a cellular mechanism for information storage in some forms of learning. A similar activity-dependent reduction in synaptic strength also occurs in the developing brain and there provides an essential step in synaptic pruning and the postnatal development of neural circuits. Here we review evidence suggesting that LTD and synaptic pruning share components of their underlying molecular machinery and may thus represent two developmental stages of the same type of synaptic modulation that serve different, but related, functions in neural circuit plasticity. We also assess the relationship between LTD and synaptic pruning in the context of recent findings of LTD dysregulation in several mouse models of autism spectrum disorder (ASD) and discuss whether LTD deficits can indicate impaired pruning processes that are required for proper brain development.
... i. The low affinity competitive AMPAR antagonist γ-DGG was used to investigate the synaptic glutamate transient (Lei and McBain, 2004;Shen et al., 2002;Wadiche and Jahr, 2001). To provide evidence that the synaptic glutamate transient (as a measure of release probability) is unaltered following TTX-treatment we measured the degree of EPSP reduction after washing in γ-DGG (0.5 mM). ...
... CF-induced complex spikes in PCs are an important source of intracellular calcium that can determine the direction of plasticity at the PF-PC synapse. Indeed, CF-PC LTD [53] was shown to affect the probability of postsynaptic LTP and LTD induction at the PF-PC synapses [45]. ...
The cerebellum is involved in learning and memory of sensory motor skills. However, the way this process takes place in local microcircuits is still unclear. The initial proposal, casted into the Motor Learning Theory, suggested that learning had to occur at the parallel fiber-Purkinje cell synapse under supervision of climbing fibers. However, the uniqueness of this mechanism has been questioned, and multiple forms of long-term plasticity have been revealed at various locations in the cerebellar circuit, including synapses and neurons in the granular layer, molecular layer and deep-cerebellar nuclei. At present, more than 15 forms of plasticity have been reported. There has been a long debate on which plasticity is more relevant to specific aspects of learning, but this question turned out to be hard to answer using physiological analysis alone. Recent experiments and models making use of closed-loop robotic simulations are revealing a radically new view: one single form of plasticity is insufficient, while altogether, the different forms of plasticity can explain the multiplicity of properties characterizing cerebellar learning. These include multi-rate acquisition and extinction, reversibility, self-scalability, and generalization. Moreover, when the circuit embeds multiple forms of plasticity, it can easily cope with multiple behaviors endowing therefore the cerebellum with the properties needed to operate as an effective generalized forward controller.
... In addition to CF induction of LTD at the parallel fibre-PC synapse, CFs also exhibit homosynaptic LTD. This means that tetanic stimulation (>5 Hz) of the CF alone can lead to LTD at the CF-PC synapse (Hansel and Linden 2000;Carta et al., 2006) via a postsynaptic mechanism (Shen et al., 2002) which is not associated with changes in PPD. ...
... However, low-frequency (5 Hz, 30 s) CF stimulation may induce LTD of PC responses (Hansel and Linden, 2000;Carta et al., 2006). The CF-LTD is postsynaptically induced and expressed (Shen et al., 2002) and it is associated with an alteration in the complex spike waveform (Hansel and Linden, 2000), a reduction in the complex spike afterhyperpolarization (Schmolesky et al., 2005), and a depression of CF-evoked dendritic Ca 2+ transient (Weber et al., 2003). The CF-LTD has a significant effect on the probability of induction of postsynaptic LTD and LTP at PF-PC synapses (Coesmans et al., 2004). ...
The way long-term potentiation (LTP) and depression (LTD) are integrated within the different synapses of brain neuronal circuits is poorly understood. In order to progress beyond the identification of specific molecular mechanisms, a system in which multiple forms of plasticity can be correlated with large-scale neural processing is required. In this paper we take as an example the cerebellar network, in which extensive investigations have revealed LTP and LTD at several excitatory and inhibitory synapses. Cerebellar LTP and LTD occur in all three main cerebellar subcircuits (granular layer, molecular layer, deep cerebellar nuclei) and correspondingly regulate the function of their three main neurons: granule cells (GrCs), Purkinje cells (PCs) and deep cerebellar nuclear (DCN) cells. All these neurons, in addition to be excited, are reached by feed-forward and feed-back inhibitory connections, in which LTP and LTD may either operate synergistically or homeostatically in order to control information flow through the circuit. Although the investigation of individual synaptic plasticities in vitro is essential to prove their existence and mechanisms, it is insufficient to generate a coherent view of their impact on network functioning in vivo. Recent computational models and cell-specific genetic mutations in mice are shedding light on how plasticity at multiple excitatory and inhibitory synapses might regulate neuronal activities in the cerebellar circuit and contribute to learning and memory and behavioral control.
... CF synapses also express long-term depression (CF-LTD) (Hansel & Linden, 2000;Shen, Hansel, & Linden, 2002;Weber, De Zeeuw, Linden, & Hansel, 2003). CF-LTD is induced by repetitive activation of CF (5 Hz for 30 s) and is postsynaptically expressed in a Ca 2+ -dependent manner (Hansel & Linden, 2000). ...
The Purkinje cells in the cerebellum are unique neurons that generate local and global Ca(2+) signals in response to two types of excitatory inputs, parallel fiber and climbing fiber, respectively. The spatiotemporal distribution and interaction of these synaptic inputs produce complex patterns of Ca(2+) dynamics in the Purkinje cell dendrites. The Ca(2+) signals originate from Ca(2+) influx through voltage-gated Ca(2+) channels and Ca(2+) release from intracellular stores that are mediated by the metabotropic glutamate receptor signaling pathway. These Ca(2+) signals are essential for the induction of various forms of synaptic plasticity and for controlling the input-output relationship of Purkinje cells. In this article we review Ca(2+) signaling in Purkinje cell dendrites.
... CF-LTD is expressed as a 20% reduction in the CFevoked EPSC, dendritic calcium transient, complex spike second component and AHP (Hansel & Linden, 2000; Weber et al., 2003; Schmolesky et al., 2005). CF-LTD is a postsynaptic phenomenon (Shen et al., 2002), and its induction is dependent upon activation of group I metabotropic glutamate receptors (mGluRs), a rise in cytosolic Ca 2+ and protein kinase C (PKC) activation (Hansel & Linden, 2000). In this study we recorded from Purkinje cells in the in vitro cerebellar slice preparation to test the hypothesis that CRF plays a role in regulating activity and ⁄ or plasticity at the CF-PC synapse. ...
The climbing fibre (CF) input controls cerebellar Purkinje cell (PC) activity as well as synaptic plasticity at parallel fibre (PF)-PC synapses. Under high activity conditions, CFs release not only glutamate, but also the neuropeptide corticotropin-releasing factor (CRF). Brief periods of such high CF activity can lead to the induction of long-term depression (LTD) at CF-PC synapses. Thus, we have examined for the first time the role of CRF in regulating excitatory postsynaptic currents (EPSCs) and long-term plasticity at this synapse. Exogenous application of CRF alone transiently mimicked three aspects of CF-LTD, causing reductions in the CF-evoked excitatory postsynaptic current, complex spike second component and complex spike afterhyperpolarization. The complex spike first component is unaffected by CF-LTD induction and was similarly unaffected by CRF. Application of a CRF receptor antagonist reduced the expression amplitude and induction probability of CF-LTD monitored at the EPSC level. Collectively, these results suggest that under particular sensorimotor conditions, co-release of CRF from climbing fibres could down-regulate excitatory transmission and facilitate LTD induction at CF-PC synapses. Inhibition of either protein kinase C (PKC) or protein kinase A (PKA) attenuated the effects of CRF upon CF-EPSCs. We have previously shown that CF-LTD induction is PKC-dependent, and here demonstrate PKA-dependence as well. These results suggest that both the acute effects of CRF on CF-EPSCs as well as the facilitating effect of CRF on CF-LTD induction can be explained by a CRF-mediated recruitment of PKC and PKA.
... These investigators showed that repetitive stimulation of climbing fibers (5 Hz for 30 s) produces LTD of climbing fiber responses in slices from juvenile rats. Similar to parallel fiber LTD, the mechanism of climbing fiber LTD was found to require an mGluR1-dependent increase in postsynaptic [Ca 2þ ] i and activation of PKC, with no change in glutamate release (Hansel and Linden, 2000;Shen et al., 2002) (Fig. 5). In addition, climbing fiber LTD requires activation of PKA mediated by corticotropin-releasing factor (CRF), which is likely released from climbing fibers in an activity-dependent manner-it is noteworthy that CRF release from climbing fibers may also play a role in parallel fiber LTD ( Schmolesky et al., 2007;reviewed in Ohtsuki et al., 2009). ...
The cerebellum controls balance, posture, motor coordination, and cognition, and studies suggest that ethanol impairs these cerebellar functions. However, the mechanisms through which ethanol produces these effects are not fully understood. Here, we review evidence suggesting that ethanol acts, in part, by impairing synaptic plasticity mechanisms at cerebellar Purkinje neurons. We will primarily focus on recent experiments indicating that long-term depression at both parallel fiber- and climbing fiber-Purkinje cell synapses is inhibited by acute ethanol exposure. We will also discuss experimental evidence showing that chronic prenatal ethanol exposure converts long-term depression into long-term potentiation at parallel fiber-Purkinje cell synapses.
... In addition, induction of presynaptically expressed long-term depression (LTD) can also decrease MVR (Lei and McBain, 2004). At PFs synapses, LTD is primarily expressed through postsynaptic mechanisms (Hartell, 2002;Shen et al., 2002), but it was recently found that block of presynaptic LTP reveals a form of presynaptically-expressed LTD (Qiu and Knopfel, 2009). At this and other synapses where presynaptic LTD decreases P R (Lovinger, 2008), LTD may be expressed in part by a decrease in MVR. ...
At a number of synapses, long-term potentiation (LTP) can be expressed by an increase in presynaptic strength, but it is unknown whether presynaptic LTP is expressed solely through an increase in the probability that a single vesicle is released or whether it can increase multivesicular release (MVR). Here, we show that presynaptic LTP decreases inhibition of AMPA receptor EPSCs by a low-affinity antagonist at parallel fiber-molecular layer interneuron (PF-MLI) synapses. This indicates that LTP induction results in larger glutamate concentration transients in the synaptic cleft, a result indicative of MVR, and suggests that MVR can be modified by long-term plasticity. A similar decrease in inhibition was observed when release probability (PR) was increased by forskolin, elevated extracellular Ca2+, and paired-pulse facilitation. Furthermore, we show that MVR may occur under baseline physiological conditions, as inhibition increased when P(R) was lowered by reducing extracellular Ca2+ or by activating presynaptic adenosine receptors. These results suggest that at PF-MLI synapses, MVR occurs under control conditions and is increased when PR is elevated by both short- and long-term plasticity mechanisms.
... In current-clamp mode, LTD is associated with an alteration in the complex spike waveform (Hansel and Linden, 2000), a reduction in the complex spike afterhyperpolarization (Schmolesky et al., 2005 ), and a long-term depression of climbing fi ber evoked calcium transients (Weber et al., 2003).Figure 3 illustrates two crucial aspects of climbing fi ber LTD: climbing fi ber tetanization leads to a reduction in the amplitude of the slow spikelets that make up the late component of a complex spike (Figure 3A), and the associated calcium transients (Figure 3B). Climbing fi ber LTD is postsynaptically induced and expressed (Shen et al., 2002). The biochemical cascade for the induction of climbing fi ber LTD shares elements with the LTD induction cascade at parallel fi ber synapses: at both types of synapses, a postsynaptic calcium surge, activation of mGluR1 receptors, and activation of protein kinase C (PKC) are required for LTD induction (Hansel and Linden, 2000). ...
The physiology of climbing fiber signals in cerebellar Purkinje cells has been studied since the early days of electrophysiology. Both the climbing fiber-evoked complex spike and the role of climbing fiber activity in the induction of long-term depression (LTD) at parallel fiber-Purkinje cell synapses have become hallmark features of cerebellar physiology. However, the key role of climbing fiber signaling in cerebellar motor learning has been challenged by recent reports of forms of synaptic and non-synaptic plasticity in the cerebellar cortex that do not involve climbing fiber activity, but might well play a role in cerebellar learning. Moreover, cerebellar LTD does not seem to strictly require climbing fiber activity. These observations make it necessary to re-evaluate the role of climbing fiber signaling in cerebellar function. Here, we argue that climbing fiber signaling is about adjusting relative probabilities for the induction of LTD and long-term potentiation (LTP) at parallel fiber synapses. Complex spike-associated, dendritic calcium transients control postsynaptic LTD and LTP induction. High calcium transients, provided by complex spike activity, do not only favor postsynaptic LTD induction, but simultaneously trigger retrograde cannabinoid signaling, which blocks the induction of presynaptic LTP. Plasticity of the climbing fiber input itself provides additional means to fine-tune complex spike associated calcium signaling and thus to adjust the gain of heterosynaptic climbing fiber control. In addition to dendritic calcium transients, climbing fiber activity leads to the release of the neuropeptide corticotropin-releasing factor (CRF), which facilitates LTD induction at both parallel fiber and climbing fiber synapses.
... It has been shown that variations in the degree of EPSC blockade by the low-affinity competitive AMPA receptor-antagonist ␥-D-glutamyl-glycine (␥-DGG) can be used to detect changes in the synaptic glutamate transient (Wadiche and Jahr, 2001;Shen et al., 2002;Lei and McBain, 2004). To provide further evidence for a presynaptic expression of LTP in bursting cells, we therefore analyzed glutamate transients in the synaptic cleft before and after expression of forskolin-induced LTP. ...
cAMP is a critical second messenger involved in synaptic transmission and synaptic plasticity. Here, we show that activation of the adenylyl cyclase by forskolin and application of the cAMP-analog Sp-5,6-DCl-cBIMPS both mimicked and occluded tetanus-induced long-term potentiation (LTP) in subicular bursting neurons, but not in subicular regular firing cells. Furthermore, LTP in bursting cells was inhibited by protein kinase A (PKA) inhibitors Rp-8-CPT-cAMP and H-89. Variations in the degree of EPSC blockade by the low-affinity competitive AMPA receptor-antagonist gamma-d-glutamyl-glycine (gamma-DGG), analysis of the coefficient of variance as well as changes in short-term potentiation suggest an increase of glutamate concentration in the synaptic cleft after expression of LTP. We conclude that presynaptic LTP in bursting cells requires activation of PKA by a calcium-dependent adenylyl cyclase while LTP in regular firing cells is independent of elevated cAMP levels. Our results provide evidence for a differential role of cAMP in LTP at hippocampal output synapses.
... Taken together, these experiments argue that LTD of the CF EPSC, a process that is PKC-dependent (27) and postsynaptically expressed (29), results in LTD of both the slow spike and the CF-evoked dendritic Ca 2 transient. The LTD of the slow spike and dendritic Ca 2 transients are likely to be a simple consequence of the reduction in CF-EPSC amplitude because (i) application of a submaximal dose of an AMPA-receptor antagonist to mimic LTD of EPSCs produced LTD of the slow spike as well as the associated Ca 2 transient and (ii) LTD of all three measures was blocked by a PKC inhibitor. ...
In recent years much has been learned about the molecular requirements for inducing long-term synaptic depression (LTD) in various brain regions. However, very little is known about the consequences of LTD induction for subsequent signaling events in postsynaptic neurons. We have addressed this issue by examining homosynaptic LTD at the cerebellar climbing fiber (CF)-Purkinje cell (PC) synapse. This synapse is built for reliable and massive excitation: Activation of a single axon produces an unusually large alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor-mediated synaptic current, the depolarization of which drives a regenerative complex spike producing a large, widespread Ca(2+) transient in PC dendrites. Here we test whether CF LTD has an impact on dendritic, complex spike-evoked Ca(2+) signals by simultaneously performing long-term recordings of complex spikes and microfluorimetric Ca(2+) measurements in PC dendrites in rat cerebellar slices. Our data show that LTD of the CF excitatory postsynaptic current produces a reduction in both slow components of the complex spike waveform and complex spike-evoked dendritic Ca(2+) transients. This LTD of dendritic Ca(2+) signals may provide a neuroprotective mechanism and/or constitute "heterosynaptic metaplasticity" by reducing the probability for subsequent induction of those forms of use-dependent plasticity, which require CF-evoked Ca(2+) signals such as parallel fiber-PC LTD and interneuron-PC LTP.
... For these 'saturated' synapses, only unidirectional plasticity-synaptic depression can be induced readily. The mechanisms for longterm depression (LTD) are associated almost exclusively with changes to the number and properties of postsynaptic receptors, as in cerebellar climbing fiber LTD [53]. However, changes in cleft transmitter concentration might still occur during short-term synaptic plasticity. ...
Although the strength of quantal synaptic transmission is jointly controlled by pre- and post-synaptic mechanisms, the presynaptic mechanisms remain substantially less well characterized. Recent studies reveal that a single package of neurotransmitter is generally insufficient to activate all available postsynaptic receptors, whereas the sum of transmitter from multiple vesicles can result in receptor saturation. Thus, depending upon the number of vesicles released, a given synaptic pathway might be either 'reliable' or 'unreliable'. A lack of receptor saturation in turn makes it possible to modify quantal size by altering the flux of transmitter through the synaptic cleft. Studies are now illuminating several new mechanisms behind the regulation of this transmitter flux--characteristics that control how transmitter is loaded into vesicles, how it is released and the manner by which it interacts with postsynaptic receptors.
... CFs, like ON, express LTD (Hansel and Linden, 2000). CF-Purkinje cell LTD is also mGluR dependent, but, in contrast to ON-MC LTD, it is most likely mediated by postsynaptic changes (Hansel and Linden, 2000;Shen et al., 2002). Al- had no effect on test fEPSPs but depressed the expression of ON-MC LTD compared with control (n ϭ 14). ...
The synapses formed by the olfactory nerve (ON) convey sensory information to olfactory glomeruli, the first stage of central odor processing. Morphological and behavioral studies suggest that glomerular odor processing is plastic in neonate rodents. However, long-term synaptic plasticity, a cellular correlate of functional and structural plasticity, has not yet been demonstrated in this system. Here, we report that ON-->mitral cell (MC) synapses of 5- to 8-d-old mice express long-term depression (LTD) after brief low-frequency ON stimulation. Pharmacological techniques and imaging of presynaptic calcium signals demonstrate that ON-MC LTD is expressed presynaptically and requires the activation of metabotropic glutamate receptors but does not require fast synaptic transmission. LTD at the ON--> MC synapse is potentially relevant for the establishment, maintenance, and experience-dependent refinement of odor maps in the olfactory bulb.
... Thus, it could be that the rhythmic activity is controlled within the olivocerebello-nuclear loop in normal conditions. LTD at climbing fiber-Purkinje cell synapses [35][36][37] is a good candidate for mediating such control, because it would decrease corticonuclear inhibition due to synchronous olivary activation, as in cooling or ablation of cerebellar cortex. ...
The role of the climbing fiber input to the cerebellum has been enigmatic, with recent studies focusing on its temporal and spatial firing patterns. Debate remains as to whether climbing fibers provide a periodic clock for coordinating movements or lead to long-term modification of Purkinje cell activity as the basis of motor learning. Rhythmic and synchronous activity of climbing fibers can cause movements at the same frequency in some preparations, suggesting a role in motor timing. However, in awake monkeys climbing fiber signals have been reported to occur at random, presenting a problem for clock theories. Yet synchronous patterns of discharge are consistently observed among several Purkinje cells within a narrow parasagittal longitudinal band. Here, we review recent experimental and theoretical studies and attempt to provide a coherent account of the interplay between rhythmicity, randomness and synchrony in climbing fiber activity, with a particular reference to studies in chaos.
... CRH is but one of at least four transmitters (glutamate, aspartate, CRH, and calcitonin gene related peptide) released at climbing fiber synapses ( Palkovits et al., 1987;Bishop, 1990;Yuzaki et al., 1996;Beitz and Saxon, 2004). Long-term depression (LTD) at the climbing fiberPurkinje cell synapse is most likely expressed post-synaptically ( Shen et al., 2002) and LTD at the parallel fiber-Purkinje cell synapse may require CRH released by climbing fibers (Miyata et al., 1999). Furthermore, climbing fiber LTD requires post-synaptic calcium elevation, activation of group 1 metabotropic glutamate receptors, and protein kinase C (Hansel and Linden, 2000). ...
The genetically dystonic (dt) rat, an autosomal recessive model of generalized dystonia, harbors an insertional mutation in Atcay. As a result, dt rats are deficient in Atcay transcript and the neuronally-restricted protein caytaxin. Previous electrophysiological and biochemical studies have defined olivocerebellar pathways, particularly the climbing fiber projection to Purkinje cells, as sites of significant functional abnormality in dt rats. In normal rats, Atcay transcript is abundantly expressed in the granular and Purkinje cell layers of cerebellar cortex. To better understand the consequences of caytaxin deficiency in cerebellar cortex, differential gene expression was examined in dt rats and their normal littermates. Data from oligonucleotide microarrays and quantitative real-time reverse transcriptase-PCR (QRT-PCR) identified phosphatidylinositol signaling pathways, calcium homeostasis, and extracellular matrix interactions as domains of cellular dysfunction in dt rats. In dt rats, genes encoding the corticotropin-releasing hormone receptor 1 (CRH-R1, Crhr1) and plasma membrane calcium-dependent ATPase 4 (PMCA4, Atp2b4) showed the greatest up-regulation with QRT-PCR. Immunocytochemical experiments demonstrated that CRH-R1, CRH, and PMCA4 were up-regulated in cerebellar cortex of mutant rats. Along with previous electrophysiological and pharmacological studies, our data indicate that caytaxin plays a critical role in the molecular response of Purkinje cells to climbing fiber input. Caytaxin may also contribute to maturational events in cerebellar cortex.
... For completeness, it should be mentioned that an equivalent form of LTD at the climbing fibre-Purkinje cell synapse (Figure 1, 2) has been described that is characteristic of pfLTD. Climbing fibre LTD is Ca 2+ -dependent, requires mGluR, AMPA receptor and PKC activation, and is expressed postsynaptically (Hansel and Linden, 2000;Shen et al., 2002). ...
A major goal of learning and memory research is to correlate the function of molecules with the behaviour of organisms. The beautiful laminar structure of the cerebellar cortex lends itself to the study of synaptic plasticity, because its clearly defined patterns of neurons and their synapses form circuits that have been implicated in simple motor behaviour paradigms. The best understood in terms of molecular mechanism is the parallel fibre-Purkinje cell synapse, where presynaptic long-term potentiation and postsynaptic long-term depression and potentiation finely tune cerebellar output. Our understanding of these forms of plasticity has mostly come from the electrophysiological and behavioural analysis of knockout mutant mice, but more recently the knock-in of synaptic molecules with mutated phosphorylation sites and binding domains has provided more detailed insights into the signalling events. The present review details the major forms of plasticity in the cerebellar cortex, with particular attention to the membrane trafficking and intracellular signalling responsible. This overview of the current literature suggests it will not be long before the involvement of the cerebellum in certain motor behaviours is fully explained in molecular terms.
Since 1990s, long-term depression of (LTD) at parallel fiber–Purkinje cell synapses has been regarded as a cellular phenomenon for motor learning. However, parallel fiber LTD by itself cannot account for motor learning. Here, we review a rich variety of use-dependent plasticity in the cerebellar cortex and nuclei, including long-term potentiation (LTP) and LTD at excitatory and inhibitory synapses, and persistent modulation of intrinsic excitability. Prevailing studies demonstrated that intrinsic and extrinsic factors, including neuronal excitation, specific molecular mechanisms, theta oscillation, and external neuromodulators, are essential to different forms of plasticity in the cerebellum.KeywordsLong-term depressionLong-term potentiationPurkinje cellGranule cellParallel fiberClimbing fiber
This chapter describes how plasticity works at the cerebellar cortex, and how the different mechanisms may interact to cause learning. The occurrence of long-term depression (LTD) with stimulation of climbing and parallel fibers is actually quite surprising. LTD requires some back-and-forth communication between neurons, analogous to a conversation. This conversation is mediated by at least four substances in the extracellular space: glutamate, gamma-amino-butyric acid (GABA), one or more endocannabinoids, and the gas nitric oxide (NO). LTD requires a complex sequence of molecular signals. Low calcium concentration in the vicinity of the synapse (possibly within the spine) means that no plasticity will occur. More calcium enables LTP, and more still, LTD. If the LTP threshold is not reached, no learning occurs. Two forms of metaplasticity are brought about by repeated activation of the climbing fiber alone, and both make parallel-fiber LTD less likely to happen in the future.
Since 1990s, long-term depression of (LTD) at parallel fi ber-Purkinje cell synapses has been regarded as a cellular phenomenon for motor learning. However, parallel fi ber LTD by itself cannot account for motor learning. Here, I review a rich variety of use-dependent plasticity in the cerebellar cortex and nuclei, including long-term potentiation (LTP) and LTD at excitatory and inhibitory synapses, and persistent modulation of intrinsic excitability. Intrinsic and extrinsic factors, including neuronal excitation, specifi c molecular mechanisms and theta oscillation, and external neuromodulators, are essential to different forms of plasticity.
In neural networks, information can be stored by altering the efficacy of synaptic transmission. Synaptic plasticity therefore provides a candidate mechanism underlying learning and memory. Since its initial discovery in 1973, long-term potentiation (LTP) has been in the spotlight as a cellular correlate of various forms of learning, particularly those mediated by the hippocampus. Later, it was discovered that synapses can also undergo long-term depression (LTD), often in an activity-dependent way. In this article, we describe the molecular and cellular mechanisms of LTD induction and expression at (1) hippocampal Schaffer collateral-CA1 pyramidal cell synapses and (2) parallel fiber and climbing fiber synapses, respectively, onto cerebellar Purkinje cells. Using these types of synapses as examples, we characterize the different roles of LTD and compare bidirectional plasticity mechanisms at hippocampal and cerebellar synapses.
N-methyl-D-aspartate receptors (NMDA-R), that are key players of neuronal function, are widely expressed by most of integrative neurons. Regarding this rule, adult cerebellar Purkinje cells have long been considered as an exception by lacking NMDA-R and were therefore used as peculiar model of plastic integrative neurons. This view has largely emerged from electrophysiological studies performed on rats or mice aged of two or three weeks, that, however, are not yet adult. Furthermore, immunohistochemistry or in situ hybridization studies accorded on the presence of NR1 subunits in adult Purkinje cells, while NR2 subunits were detected only by some authors, suggesting possible species differences. Thus the question of functional NMDA-Rs expression by adult Purkinje cells deserved clarification. Using mouse older than 2 months, we have shown that adult Purkinje cells express functional NMDA receptors. In patch-clamp whole-cell recordings, exogenous applications of NMDA induce currents in Purkinje cells. Moreover, NMDA receptors participate to the excitatory postsynaptic current induced by the stimulation of the climbing fiber (CF-EPSC). This NMDA-mediated CF-EPSC is blocked by D-APV and external magnesium ions. Its I/V curve is typical of postsynaptic NMDA receptors and is blocked by intracellular MK801, showing that NMDA receptors are postsynaptic. We also detected NR2-A/B subunits by immunohistochemistry. We show that the NMDA-mediated CF-EPSC, hardly detectable before 3 postnatal weeks, appears in all Purkinje cells by 21 postnatal days but is still very small at this age. Then, its amplitude increases until 12 weeks after birth. Thus, there is actually a "gap" in the developmental expression of NMDA-Rs in Purkinje cells, during the second and the third postnatal weeks. In mature Purkinje cells, we demonstrate that NMDA-Rs contribute to the depolarizing plateau of complex spikes and modulate the spikelets latency and number. According to these observations and to their calcium permeability, our preliminary data indicate that these receptors favor long-term depression of parallel fiber synapses. Thus, in adult mice, the NMDA-Rs are located at climbing fiber synapses and, probably by shaping the complex spike, favor LTD induction.
Despite its various limitations, for many decades the experimental autoimmune encephalomyelitis (EAE) has been indispensable for understanding the pathology of multiple sclerosis (MS) and for establishing widely used MS therapeutics. We tested whether synaptic plasticity is a suitable measure for EAE and whether it can detect detrimental effects on supra-spinal structures that are too subtle to be captured by the motor score. Our data show functional synaptic deficits in the EAE that were beyond the measurable EAE score: long-term depression responses were strongly weakened in superior colliculus and cerebellum resulting from impaired postsynaptic transmission. In addition to further insight into neuronal deficits associated with the autoimmune disease, quantification of synaptic transmission may serve as a complementary method of EAE evaluation.
Cerebellar parallel fiber (PF)-Purkinje cell (PC) synapses can undergo postsynaptically expressed long-term depression (LTD) or long-term potentiation (LTP) depending on whether or not the climbing fiber (CF) input is coactivated during tetanization. Here, we show that modifications of the postsynaptic calcium load using the calcium chelator BAPTA or photolytic calcium uncaging result in a reversal of the expected polarity of synaptic gain change. At higher concentrations, BAPTA blocks PF-LTP. These data indicate that PF-LTD requires a higher calcium threshold amplitude than PF-LTP induction and suggest that CF activity acts as a polarity switch by providing dendritic calcium transients. Moreover, previous CF-LTD induction changes the relative PF-LTD versus -LTP induction probability. These findings suggest that bidirectional cerebellar learning is governed by a calcium threshold rule operating "inverse" to the mechanism previously described at other glutamatergic synapses (BCM rule) and that the LTD/LTP induction probability is under heterosynaptic climbing fiber control.
Le cervelet est une structure cérébrale nécessaire au maintien de la posture, à la coordination motrice ainsi qu'aux apprentissages moteurs. Son implication dans des fonctions cognitives ou émotionnelles, ainsi que dans des pathologies, comme l'autisme ou la schizophrénie, est également de plus en plus avancée. C'est enfin un modèle de choix pour comprendre le fonctionnement des neurones.
La cellule de Purkinje est un élément clef de la fonction cérébelleuse. Elle reçoit 2 grands types d'afférences glutamatergiques: les fibres parallèles (FP), et une fibre grimpante (FG), unique à partir du 21e jour postnatal chez la souris. La nature et le rôle des différents récepteurs du glutamate exprimés à ces synapses ont été largement étudiés et sont désormais assez bien connus. De manière surprenante, le récepteur N-methyl-D-aspartate (R-NMDA), qui joue un rôle clef dans la plupart des neurones intégrateurs du cerveau, en agissant comme détecteur de coïncidence et en permettant une signalisation calcique cruciale dans de nombreux processus cellulaires, a gardé quant à lui une fonction méconnue dans les cellules de Purkinje néonatales, et sa présence est demeurée quasiment inconnue chez les cellules adultes. Le but de cette thèse était donc, d'une part, d'étudier le rôle de la dépolarisation, via notamment les R-NMDA, dans les cellules de Purkinje néonatales, et d'autre part de clarifier la présence, puis le rôle des R-NMDA chez l'adulte.
Par le biais d'une étude menée en collaboration, nous pouvons proposer un rôle des R-NMDA dans l'effet neuroprotecteur de la dépolarisation, lors de la mort cellulaire développementale intervenant aux alentours du 3e jour post-natal. Dans des co-cultures olive-cervelet, la survie des cellules de Purkinje de cet âge est significativement favorisée par le voisinage de FGs. La synaptogénèse avec ces fibres aurait donc un effet neuroprotecteur sur les cellules de Purkinje et ce, via leurs R-NMDA juvéniles. Ces récepteurs, en détectant le glutamate libéré par les FGs voisines, pourraient ainsi favoriser la survie des seules cellules contactées par ces afférences à cette étape critique du développement.
Parce que les R-NMDA ne sont pas détectés dans les cellules de Purkinje entre la 2e et la 3e semaine postnatales, ces cellules étaient considérées comme un rare exemple de neurone intégrateur n'exprimant pas de R-NMDA chez l'adulte. Cependant, nous avons montré qu'après cette période transitoire d'absence, un nouveau type de R-NMDA apparaît aux synapses avec les FGs et reste exprimé à l'âge adulte. Nous avons mis en évidence la participation des R-NMDA dans la transmission excitatrice de FG, ce qui suggère leur rôle dans de nombreux mécanismes cellulaires au sein de la cellule de Purkinje, notamment dans la plasticité synaptique. En effet, nos résultats préliminaires présentés dans cette thèse sont en faveur d'un rôle des R-NMDA dans l'aiguillage de la plasticité à long terme des synapses FPs - cellules de Purkinje.
Nous avons également étudié la possibilité d'une compétition entre ces R-NMDA et un autre type de récepteur du glutamate spécifiquement exprimé dans les cellules de Purkinje, le récepteur GluRdelta2. Ce dernier joue un rôle essentiel dans la régression des FGs surnuméraires et la limitation de leur territoire dendritique d'innervation. Bien que nos résultats ne mettent pas en évidence une interaction entre ces récepteurs, ils montrent que chez la souris adulte n'exprimant pas GluRdelta2, lorsque l'innervation des cellules de Purkinje par de multiples FGs persiste, les R-NMDA ne participent qu'à la transmission synaptique de la plus forte FG. Ceci suggère également un rôle des R-NMDA dans le choix et la stabilisation d'une seule FG lors de la maturation finale de ces afférences.
The subiculum is the principal target of CA1 pyramidal cells. It functions as a mediator of hippocampal-cortical interaction and has been proposed to play an important role in the encoding and retrieval of long-term memory. The cellular mechanisms of memory formation are thought to include long-term potentiation (LTP) and depression (LTD) of synaptic strength. This review summarizes the contemporary knowledge of LTP and LTD at CA1-subiculum synapses. The observation that the underlying mechanisms of LTP and LTD at CA1-subiculum synapses correlate with the discharge properties of subicular pyramidal cell reveals a novel and intriguing mechanism of cell-specific consolidation of hippocampal output.
The acid-sensing ion channels (ASICs) are members of the DEG/ENaC superfamily of Na+ channels. Acid-gated cation currents have been detected in neurons from multiple regions of the brain including the cerebellum, but little is known about their molecular identity and function. Recently, one of ASICs (ASIC1a) was implicated in synaptic plasticity. In this study we examined the subcellular distribution of ASIC2a in rat cerebellum by immunostaining and confocal microscopy. Monoclonal antibodies for labeling of defined brain structures, for example, astroglia, Purkinje cell dendrites, nuclei, and presynaptic terminals were used for colocalization analyses. In the gray matter, the anti-ASIC2a antibody intensively stained dendrite branches of Purkinje cells evenly distributed throughout the entire molecular layer (ML). In the granule cell layer (GL), anti-ASIC2a antibody stained synaptic glomeruli. Neuronal localization of ASIC2a was confirmed by lack of co-staining with glial fibrillary acidic protein. Anti-ASIC2a staining in the ML colocalized with metabotropic glutamate receptor 1alpha (mGluR1alpha) in Purkinje cell dendrites and dendritic spines. Both proteins, mGluR1alpha and ASIC2a, were enriched in a crude synaptic membrane fraction prepared from cerebellum, suggesting synaptic expression of these proteins. Dual staining with anti-syntaxin 1A and anti-ASIC2a antibodies demonstrates characteristic complementary distribution of two proteins in both ML and GL. Because syntaxin 1A localized in presynaptic membranes and synaptic vesicles, complementary distribution with ASIC2a suggests postsynaptic localization of ASIC2a in these structures. This study shows specific localization of ASIC2a in both Purkinje and granule cell dendrites of the cerebellum and enrichment of ASIC2a in a crude cerebellar synaptic membrane fraction. The study is the first report of synaptic localization of ASIC2a in the CNS. The synaptic localization of ASIC2a in the cerebellum makes this channel a candidate for a role in motor coordination and learning.
The genetically dystonic rat (SD-dt:JFL) is an autosomal recessive model of generalized dystonia. Without cerebellectomy, the dt rat dies prior to Postnatal Day 40. The dt locus was mapped to a 4.2 Mb region on Chr 7q11 and candidate genes were screened with semi-quantitative RT-PCR. Then, Southern blotting and genomic DNA sequencing identified the 3'-long terminal repeat portion of an intracisternal A particle element inserted into Intron 1 of Atcay, the gene which encodes caytaxin. Northern and Western blotting and quantitative real-time RT-PCR defined the Atcay allele in dt rats (Atcay(dt)) as hypomorphic. To establish a framework for functional studies of caytaxin, the developmental expression of rat Atcay transcript was analyzed with Northern blotting, relative quantitative multiplex real-time RT-PCR (QRT-PCR) and in situ hybridization. With a multiple tissue Northern blot, three Atcay transcripts were identified in brain but none were present in heart, spleen, lung, liver, muscle, kidney or testis. With a multiple time-point Northern blot, the same three transcripts were present in cerebellum at Embryonic Day (E15), Postnatal Day 1 (P1), P7, P14, P36 and 8 months. During early development (E15 to P14), the relative proportion of the smallest transcript was increased. QRT-PCR was performed with total RNA from cerebral cortex, striatum, thalamus, hippocampus and cerebellum. Transcript levels peaked at P7 in hippocampus, increased linearly from P1 to P36 in cerebellum, and showed minimal developmental regulation in cerebral cortex. Radioactive in situ hybridization localized Atcay transcript to seemingly all neuronal populations in brain. In cerebellum, Atcay transcript was present in the molecular, Purkinje and granular layers; transcript density in the molecular layer peaked at P14. In the background of previous biochemical, behavioral and electrophysiological studies in the dt rat, our data are compatible with a vital role for caytaxin in the development and neurophysiology of cerebellar cortex.
Glutamatergic synaptic transmission is a dynamic process determined by the amount of glutamate released by presynaptic sites, the clearance of glutamate in the synaptic cleft, and the properties of postsynaptic glutamate receptors. Clearance of glutamate in the synaptic cleft depends on passive diffusion and active uptake by glutamate transporters. In this study, we examined the role of glial glutamate transporter 1 (GLT-1) in spinal sensory processing. Excitatory postsynaptic currents (EPSCs) of substantia gelatinosa neurons recorded from spinal slices of young adult rats were analyzed before and after GLT-1 was pharmacologically blocked by dihydrokainic acid. Inhibition of GLT-1 prolonged the EPSC duration and the EPSC decay phase. The EPSC amplitudes were increased in neurons with weak synaptic input but decreased in neurons with strong synaptic input upon inhibition of GLT-1. We suggest that presynaptic inhibition, desensitization of postsynaptic AMPA receptors, and glutamate "spillover" contributed to the kinetic change of EPSCs induced by the blockade of GLT-1. Thus, GLT-1 is a key component in maintaining the spatial and temporal coding in signal transmission at the glutamatergic synapse in substantia gelatinosa neurons.
The cerebellum has a parasagittal modular structure, in which Zebrin (Aldolase) positive and negative bands expressed in Purkinje cell layers alternate, and is involved in amphetamine psychosis. Administration of SKF38393, a D1 receptor agonist, reversed the behavioral sensitization of methamphetamine. In the vermis, there were the binding sites of SKF38393. In methamphetamine-sensitized rats the expression of the Aldolase mRNA positive bands move laterally in the rat vermis. We provide here the evidence that the D1 agonist injections also reversed the expression pattern of both the Aldolase mRNA in Purkinje cells and Ube (ubiquitin activating enzyme) 1x mRNA in Golgi interneurons of the sensitized rats. Thus the reverse changes in gene expression pattern in the vermis may be involved in the mechanisms of the behavioral plasticity and suggests the new treatment of drug abuse.
To understand the elementary unit of synaptic communication between CNS neurons, one must know what causes the variability of quantal postsynaptic currents and whether unitary packets of transmitter saturate postsynaptic receptors. We studied single excitatory synapses between hippocampal neurons in culture. Focal glutamate application at individual postsynaptic sites evoked currents (I(glu)) with little variability compared with quantal excitatory postsynaptic currents (EPSCs). The maximal I(glu) was >2-fold larger than the median EPSC. Thus, variations in [glu]cleft are the main source of variability in EPSC size, and glutamate receptors are generally far from saturation during quantal transmission. This conclusion was verified by molecular antagonism experiments in hippocampal cultures and slices. The general lack of glutamate receptor saturation leaves room for increases in [glu]cleft as a mechanism for synaptic plasticity.
In classic Marr-Albus-Ito models of cerebellar function, coactivation of the climbing fiber (CF) synapse, which provides massive, invariant excitation of Purkinje neurons (coding the unconditioned stimulus), together with a graded parallel fiber synaptic array (coding the conditioned stimulus) leads to long-term depression (LTD) of parallel fiber-Purkinje neuron synapses, underlying production of a conditioned response. Here, we show that the supposedly invariant CF synapse can also express LTD. Brief 5 Hz stimulation of the CF resulted in a sustained depression of CF EPSCs that did not spread to neighboring parallel fiber synapses. Like parallel fiber LTD, CF LTD required postsynaptic Ca2+ elevation, activation of group 1 mGluRs, and activation of PKC. CF LTD is potentially relevant for models of cerebellar motor control and learning and the developmental conversion from multiple to single CF innervation of Purkinje neurons.
Cerebellar long-term depression (LTD) is thought to play an important role in certain types of motor learning. However, the molecular mechanisms underlying this event have not been clarified. Here, using cultured Purkinje cells, we show that stimulations inducing cerebellar LTD cause phosphorylation of Ser880 in the intracellular C-terminal domain of the AMPA receptor subunit GluR2. This phosphorylation is accompanied by both a reduction in the affinity of GluR2 to glutamate receptor interacting protein (GRIP), a molecule known to be critical for AMPA receptor clustering, and a significant disruption of postsynaptic GluR2 clusters. Moreover, GluR2 protein released from GRIP is shown to be internalized. These results suggest that the dissociation of postsynaptic GluR2 clusters and subsequent internalization of the receptor protein, initiated by the phosphorylation of Ser880, are the mechanisms underlying the induction of cerebellar LTD.
Cerebellar LTD requires activation of PKC and is expressed, at least in part, as postsynaptic AMPA receptor internalization. Recently, it was shown that AMPA receptor internalization requires clathrin-mediated endocytosis and depends upon the carboxy-terminal region of GluR2/3. Phosphorylation of Ser-880 in this region by PKC differentially regulates the binding of the PDZ domain-containing proteins GRIP/ABP and PICK1. Peptides, corresponding to the phosphorylated and dephosphorylated GluR2 carboxy-terminal PDZ binding motif, were perfused in cerebellar Purkinje cells grown in culture. Both the dephospho form (which blocks binding of GRIP/ABP and PICK1) and the phospho form (which selectively blocks PICK1) attenuated LTD induction by glutamate/depolarization pairing, as did antibodies directed against the PDZ domain of PICK1. These findings indicate that expression of cerebellar LTD requires PKC-regulated interactions between the carboxy-terminal of GluR2/3 and PDZ domain-containing proteins.
The adult olivocerebellar axons and their terminal arbours, the climbing fibres, are capable of remarkable structural plasticity, regulated through their interaction with Purkinje cells. When these cells are deleted, terminal climbing fibre branches retract. In contrast, there is a vigorous outgrowth of entire terminal arbours when extra postsynaptic neurones are available. The new connections lead to a functional, highly specific pattern of innervation at the single Purkinje cell level and are topographically organized according to the principles of the original projection map. A reversible climbing fibre retraction occurs following depression of electrical activity of the cerebellar cortex. These remarkable plastic properties, together with the fact that these neurones express several growth-associated genes constitutively, suggest that the climbing fibre synapses might be adjusted dynamically to participate in physiological plasticity.
1. Climbing fibre-mediated excitatory postsynaptic potentials (CF-EPSPs) or currents (CF-EPSCs) were recorded from Purkinje cells in rat cerebellar slices using the whole-cell recording technique. 2. Climbing fibre responses displayed prominent paired-pulse depression (PPD). In the current-clamp recording mode, PPD resulted in a decreased number of spikelets in the second complex spike of the pair, and depression of the after-depolarization and after-hyperpolarization. 3. The mechanism of PPD was examined under voltage clamp. Manipulations that reduce transmitter release significantly affected PPD. These included lowering extracellular Ca2+ concentration and bath application of baclofen or adenosine. 4. Changing the number of stimulated climbing fibres, equivalent to changing the number of release sites, had no effect on PPD. 5. Selective manipulations of postsynaptic responsiveness had no effect on PPD. These included partial blockade of CF-EPSCs by a non-NMDA receptor antagonist, 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), and changing the holding potential. 6. A rapidly dissociating AMPA receptor antagonist, 2,3-cis-piperidine dicarboxylic acid, inhibited the second CF-EPSC of the pair proportionately more than the first, suggesting that presynaptic release by the second pulse is decreased. 7. PPD at interstimulus intervals of 50 ms or longer (up to 3000 ms) was not significantly affected by manipulations that change postsynaptic glutamate receptor desensitization. 8. Blockade of metabotropic glutamate, GABAB and adenosine receptors had no effect on PPD, suggesting that presynaptic autoreceptors do not contribute to PPD. 9. These results indicate that decreased transmitter release is a major cause of PPD at cerebellar climbing fibre-Purkinje cell synapses.
EPSCs were recorded under whole‐cell voltage clamp at room temperature from Purkinje cells in slices of cerebellum from 12‐ to 14‐day‐old rats. EPSCs from individual climbing fibre (CF) inputs were identified on the basis of their large size, paired‐pulse depression and all‐or‐none appearance in response to a graded stimulus.
Synaptic transmission was investigated over a wide range of experimentally imposed release probabilities by analysing fluctuations in the peak of the EPSC. Release probability was manipulated by altering the extracellular [Ca ²⁺ ] and [Mg ²⁺ ]. Quantal parameters were estimated from plots of coefficient of variation ( CV ) or variance against mean conductance by fitting a multinomial model that incorporated both spatial variation in quantal size and non‐uniform release probability. This ‘multiple‐probability fluctuation’ (MPF) analysis gave an estimate of 510 ± 50 for the number of functional release sites ( N ) and a quantal size ( q ) of 0.5 ± 0.03 nS ( n = 6 ).
Control experiments, and simulations examining the effects of non‐uniform release probability, indicate that MPF analysis provides a reliable estimate of quantal parameters. Direct measurement of quantal amplitudes in the presence of 5 mM Sr ²⁺ , which gave asynchronous release, yielded distributions with a mean quantal size of 0.55 ± 0.01 nS and a CV of 0.37 ± 0.01 ( n = 4 ). Similar estimates of q were obtained in 2 mM Ca ²⁺ when release probability was lowered with the calcium channel blocker Cd ²⁺ . The non‐NMDA receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 1 μM) reduced both the evoked current and the quantal size (estimated with MPF analysis) to a similar degree, but did not affect the estimate of N .
We used MPF analysis to identify those quantal parameters that change during frequency‐dependent depression at climbing fibre‐Purkinje cell synaptic connections. At low stimulation frequencies, the mean release probability (P̄ r ) was unusually high (0.90 ± 0.03 at 0.033 Hz, n = 5), but as the frequency of stimulation was increased, p r fell dramatically (0.02 ± 0.01 at 10 Hz, n = 4) with no apparent change in either q or N . This indicates that the observed 50‐fold depression in EPSC amplitude is presynaptic in origin.
Presynaptic frequency‐dependent depression was investigated with double‐pulse and multiple‐pulse protocols. EPSC recovery, following simultaneous release at practically all sites, was slow, being well fitted by the sum of two exponential functions (time constants of 0.35 ± 0.09 and 3.2 ± 0.4 s, n = 5). EPSC recovery following sustained stimulation was even slower. We propose that presynaptic depression at CF synapses reflects a slow recovery of release probability following release of each quantum of transmitter.
The large number of functional release sites, relatively large quantal size, and unusual dynamics of transmitter release at the CF synapse appear specialized to ensure highly reliable olivocerebellar transmission at low frequencies but to limit transmission at higher frequencies.
Short-term depression is a widespread form of use-dependent plasticity found in the peripheral and central nervous systems of invertebrates and vertebrates. The mechanism behind this transient decrease in synaptic strength is thought to be primarily the result of presynaptic "depletion" of a readily releasable neurotransmitter pool, which typically recovers with a time constant of a few seconds. We studied the mechanism and dynamics of recovery from depression at the climbing fiber to Purkinje cell synapse, where marked presynaptic depression has been described previously. Climbing fibers are well suited to studies of recovery from depression because they display little, if any, facilitation (even under conditions of low-release probability), which can obscure rapid recovery from depression for hundreds of milliseconds after release. We found that recovery from depression occurred in three kinetic phases. The fast and intermediate components could be approximated by exponentials with time constants of 100 msec and 3 sec at 24 degrees C. A much slower recovery phase was also present, but it was only prominent during prolonged stimulus trains. The fast component was enhanced by raising extracellular calcium and was eliminated by lowering presynaptic calcium, suggesting that, on short time scales, recovery from depression is driven by residual calcium. During regular and Poisson stimulus trains, recovery from depression was dramatically accelerated by accumulation of presynaptic residual calcium, maintaining synaptic efficacy under conditions that would otherwise deplete the available transmitter pool. This represents a novel form of presynaptic plasticity in that high levels of activity modulate the rate of recovery as well as the magnitude of depression.
Cerebellar long-term depression (LTD) is a cellular model system of information storage that may underlie certain forms of motor learning. While cerebellar LTD is expressed as a selective modification of postsynaptic AMPA receptors, this might involve changes in receptor number/distribution, unitary conductance, kinetics, or glutamate affinity. The observation that GluR2-containing synaptic AMPA receptors could be internalized by regulated clathrin-mediated endocytosis suggested that this process could underlie LTD expression. To test this hypothesis, we postsynaptically applied dynamin and amphiphysin peptides that interfere with the clathrin endocytotic complex and found that they blocked LTD expression in cultured Purkinje neurons. In addition, induction of LTD and attenuation of AMPA responses by stimulation of clathrin-mediated endocytosis occluded each other. These findings suggest that the expression of cerebellar LTD requires clathrin-mediated internalization of postsynaptic AMPA receptors.
In recent years, it has become clear that motor learning, as revealed by associative eyelid conditioning and adaptation of the vestibulo-ocular reflex, contributes to the well-established cerebellar functions of sensorimotor integration and control. Long-term depression of the parallel fiber-Purkinje cell synapse (which is often called 'cerebellar LTD') is a cellular phenomenon that has been suggested to underlie these forms of learning. However, it is clear that parallel fiber LTD, by itself, cannot account for all the properties of cerebellar motor learning. Here we review recent electrophysiological experiments that have described a rich variety of use-dependent plasticity in cerebellum, including long-term potentiation (LTP) and LTD of excitatory and inhibitory synapses, and persistent modulation of intrinsic neuronal excitability. Finally, using associative eyelid conditioning as an example, we propose some ideas about how these cellular phenomena might function and interact to endow the cerebellar circuit with particular computational and mnemonic properties.
Synapses driven by action potentials are thought to release transmitter in an all-or-none fashion; either one synaptic vesicle undergoes exocytosis, or there is no release. We have estimated the glutamate concentration transient at climbing fiber synapses on Purkinje cells by measuring the inhibition of excitatory postsynaptic currents (EPSCs) produced by a low-affinity competitive antagonist of AMPA receptors, gamma-DGG. The results, together with simulations using a kinetic model of the AMPA receptor, suggest that the peak glutamate concentration at this synapse is dependent on release probability but is not affected by pooling of transmitter released from neighboring synapses. We propose that the mechanism responsible for the elevated glutamate concentration at this synapse is the simultaneous release of multiple vesicles per site.
Cerebellar long-term synaptic depression (LTD) is a model system of neuronal information storage that is expressed postsynaptically as a functional down-regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. What properties of postsynaptic AMPA receptors are changed? Several lines of evidence argue against changes in AMPA-receptor kinetics. Neither LTD evoked in cultured granule-cell Purkinje cell (PC) pairs nor an LTD-like phenomenon evoked by phorbol ester application was associated with alterations in evoked AMPA receptor-mediated excitatory post-synaptic current (EPSC) or mEPSC kinetics. LTD produced by pairing glutamate pulses with depolarization was not altered by prior application of the desensitization-reducing compound cyclothiazide. Finally, rapid application of glutamate to lifted PCs revealed no significant alterations in AMPA-receptor kinetic properties after LTD induction. When this system was used to apply varying concentrations of glutamate, no alteration in AMPA-receptor glutamate affinity was seen after LTD induction. Finally, peak-scaled nonstationary fluctuation analysis was applied to estimate AMPA-receptor unitary conductance before and after LTD induction in a cultured cell pair, and this analysis too revealed no significant change. These results suggest that cerebellar LTD may be expressed solely as a reduction in the number of functional AMPA receptors in the postsynaptic density [Wang, Y.-T. & Linden, D. J. (2000) Neuron 25, 635-664].
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