Granule Cell Synapses Research Articles

Desensitization Properties of AMPA Receptors at the Cerebellar Mossy Fiber–Granule Cell Synapse

Native AMPA receptors (AMPARs) exhibit rapid and profound desensitization in the sustained presence of glutamate. Desensitization therefore contributes to short-term depression at synapses in which glutamate accumulates. At synapses that do not exhibit desensitization-dependent depression, AMPARs are thought to be protected against prolonged or repetitive exposure to synaptically released glutamate. At the cerebellar mossy fiber to granule cell (GC) synapse, in which high release probability and glutamate spillover produce a substantial buildup of glutamate concentration in the cleft ([Glut]cleft) during high-frequency transmission, only moderate desensitization of the phasic AMPAR EPSC occurs. To investigate how such currents are produced, we examined the kinetic properties of synaptic AMPARs in GCs using glutamate uncaging. Photolysis of 4-methoxy-7-nitroindolinyl-caged L-glutamate with large illumination spots produced step-like increases in [Glut]cleft that could be used to systematically probe AMPAR kinetics. At low levels of activation, synaptic AMPARs exhibited little desensitization. With larger activations, the desensitization time course became faster, but the level of desensitization was only weakly dependent on receptor occupancy. Indeed, a substantial desensitization-resistant current component remained (17%) in saturating glutamate. Photolysis with small illumination spots produced brief [Glut]cleft waveforms and transient AMPAR activations, similar to the EPSC current components. Paired-pulse uncaging with such spots revealed little desensitization after spillover-like activations and modest depression after activations that mimicked quantal and spillover components together. Our results show that GC AMPARs exhibit a resistance to desensitization at low occupancies and that this property is crucial for sustaining high-frequency transmission at a synapse in which glutamate accumulates.

Contribution of NMDA receptor NR2B subunit to synaptic plasticity during associative learning in behaving rats

The difference in the amounts of NR2 subunits contained in NMDA receptors of the hippocampus has been related to their different involvement in activity-dependent synaptic plasticity. Here, we show that Ro 25-6981, a high-affinity and selective blocker of NMDA receptors containing NR2B subunits, is able to block the acquisition of a trace conditioning paradigm in adult rats, a task that requires the active participation of hippocampal circuits. Reconditioning with the same trace paradigm was also prevented by Ro 25-6981. In addition, we show that the slope of monosynaptic field excitatory postsynaptic potentials evoked at the dentate gyrus by single pulses presented to the medial perforant pathway increases significantly across conditioning sessions and during reconditioning, in a linear relationship with the increase in the number of classically conditioned eyelid responses. Administration of Ro 25-6981 prevented these learning-related changes in synaptic strength at the perforant pathway-dentate granule cell synapse. The present results suggest the involvement of NR2B-containing NMDA receptors in hippocampal functions related to both associative learning and activity-dependent synaptic plasticity.

Synaptic fatigue at the naive perforant path–dentate granule cell synapse in the rat

Synaptic activation at low frequency is often used to probe synaptic function and synaptic plasticity, but little is known about how such low-frequency activation itself affects synaptic transmission. In the present study, we have examined how the perforant path-dentate granule cell (PP-GC) synapse adapts to low-frequency activation from a previously non-activated (naive) state. Stimulation at 0.2 Hz in acute slices from developing rats (7-12 days old) caused a gradual depression of the AMPA EPSC (at -80 mV) to about half within 50 stimuli. This synaptic fatigue was unaffected by the NMDA and metabotropic glutamate (mGlu) receptor antagonists d-AP5 and LY-341495. A smaller component of this synaptic fatigue was readily reversible when switching to very low-frequency stimulation (0.033-0.017 Hz) and is attributed to a reversible decrease in release probability, which is probably due to depletion of readily releasable vesicles. Thus, it was expressed to the same extent by AMPA and NMDA EPSCs, and was associated with a decrease in quantal content (measured as 1/CV(2)) with no change in the paired-pulse ratio. The larger component of the synaptic fatigue was not readily reversible, was selective for AMPA EPSCs and was associated with a decrease in 1/CV(2), thus probably representing silencing of AMPA signalling in a subset of synapses. In adult rats (> 30 days old), the AMPA silencing had disappeared while the low-frequency depression remained unaltered. The present study has thus identified two forms of synaptic plasticity that contribute to fatigue of synaptic transmission at low frequencies at the developing PP-GC synapse; AMPA silencing and a low-frequency depression of release probability.

Hippocampal Synaptic Metaplasticity Requires Inhibitory Autophosphorylation of Ca2+/Calmodulin-Dependent Kinase II

Virtually all CNS synapses display the potential for activity-dependent long-term potentiation (LTP) and/or long-term depression (LTD). Intriguingly, the potential to exhibit LTP or LTD at many central synapses itself is powerfully modulated by previous synaptic activity. This higher-order form of plasticity has been termed metaplasticity. Here, we show that inhibitory autophosphorylation of Ca2+/calmodulin-dependent kinase II (CaMKII) is required for hippocampal metaplasticity at the lateral perforant path-dentate granule cell synapse. Brief 10 Hz priming, which does not affect basal synaptic transmission, caused a dramatic, pathway-specific and long-lasting (up to 18 h) reduction in subsequently evoked LTP at lateral perforant path synapses. In contrast, LTD was unaffected by priming. The induction of lateral perforant path metaplasticity required the activation of NMDA receptors during priming. In addition, metaplasticity was absent in knock-in mice expressing alphaCaMKII that cannot undergo inhibitory phosphorylation, indicating that inhibitory autophosphorylation of alphaCaMKII at threonines 305/306 is required for metaplasticity. Metaplasticity was not observed in the medial perforant pathway, consistent with the observation that CaMKII activity was not required for the induction of LTP at this synapse. Thus, modulation of alphaCaMKII activity via autophosphorylation at Thr305/Thr306 is a key mechanism for metaplasticity that may be of importance in the integration of temporally separated episodes of activity.

Decreased expression and impaired function of muscarinic acetylcholine receptors in the rat hippocampus following transient forebrain ischemia

In this study, we investigated whether transient cerebral ischemia affects the function and molecular expression of specific muscarinic cholinergic receptors. Our results show that in contrast to the GABA-B and A1 adenosine receptor systems, the ability of muscarinic receptors to attenuate evoked excitatory responses at vulnerable CA1 synapses is significantly attenuated by 18 h following reperfusion. This attenuation in efficacy was restricted to the vulnerable CA1 subfield, as no significant change in muscarinic receptor-mediated attenuation of evoked responsiveness was observed within post-ischemic dentate granule cell synapses. Expression analysis revealed that the mRNA and immunoreactive protein levels for individual types of muscarinic receptors respond differently and uniquely to transient cerebral ischemia insult. Of particular interest is the m4 subtype of receptor, whose mRNA and protein expression levels were significantly diminished within the hippocampus by 12 and 24 h following reperfusion, respectively. As the m4 muscarinic receptor localizes to presynaptic terminals within the hippocampus, a decrease in its expression could account for the impaired functional responsiveness of the muscarinic receptor system following ischemic insult. Taken together, these results demonstrate that transient forebrain ischemia leads to dynamic alterations in the gene expression, protein prevalence, and functionality of muscarinic receptors in the post-ischemic hippocampus at times preceding the degeneration of the vulnerable neurons.

Short‐term synaptic plasticity during development of rat mossy fibre to granule cell synapses

Changes occur during the postnatal development of the rat glutamatergic mossy fibre to granule cell synapse: to the morphology of synapses, glutamate transporter expression, AMPA receptor expression and the kinetics of AMPA receptor-mediated synaptic transmission. For example, both the rise and decay times of AMPA receptor-mediated excitatory postsynaptic currents significantly shorten. To further define the development of mossy fibre to granule cell synaptic transmission, the properties and mechanisms of short-term plasticity have been described. The characterization of short-term plasticity will aid our understanding of the mechanisms that define the parameters of synaptic transmission during development and furthermore short-term plasticity may play an important role in determining information transfer between mossy fibres and granule cells. In response to pairs of stimuli (2-100-ms interval), depression (second excitatory postsynaptic current amplitude smaller than the first) was observed at both mature (older than 40 postnatal days) and immature (between 8 and 12 postnatal days) synapses. The degree of depression was similar at both stages of development, although recovery from depression was slower at mature synapses (tau 22 vs 12.5 ms). Several experimental approaches (coefficient of variation, low-affinity antagonists and cyclothiazide) suggest that depression at immature synapses results from multiple mechanisms. At mature synapses, postsynaptic receptor desensitization appears to be the major cause of depression.

Developmental expression of methyl-CpG binding protein 2 is dynamically regulated in the rodent brain

The gene encoding methyl-CpG binding protein 2 (MeCP2) is mutated in the large majority of girls that have Rett Syndrome (RTT), an X-linked neurodevelopmental disorder. To better understand the developmental role of MeCP2, we studied the ontogeny of MeCP2 expression in rat brain using MeCP2 immunostaining and Western blots. MeCP2 positive neurons were present throughout the brain at all ages examined, although expression varied by region and age. At early postnatal ages, regions having neurons that were generated early and more mature had the strongest MeCP2 expression. Late developing structures including cortex, hippocampus and cerebellum exhibited the most significant changes in MeCP2 expression. Of these regions, the cerebellum showed the most striking cell-specific changes in MeCP2 expression. For example, the early-generated Purkinje cells became MeCP2 positive by P6, while the late-generated granule cells did not express MeCP2 until the fourth postnatal week. The timing of MeCP2 expression in the granule cell layer is coincident with the onset of granule cell synapse formation. Although more subtle, the degree of MeCP2 expression in cortex and hippocampus was most closely correlated with synaptogenesis in both regions. Our finding that MeCP2 expression is correlated with synaptogenesis is consistent with the hypothesis that Rett Syndrome is caused by defects in the formation or maintenance of synapses.

Developmental expression of methyl-CpG binding protein 2 is dynamically regulated in the rodent brain

The gene encoding methyl-CpG binding protein 2 (MeCP2) is mutated in the large majority of girls that have Rett Syndrome (RTT), an X-linked neurodevelopmental disorder. To better understand the developmental role of MeCP2, we studied the ontogeny of MeCP2 expression in rat brain using MeCP2 immunostaining and Western blots. MeCP2 positive neurons were present throughout the brain at all ages examined, although expression varied by region and age. At early postnatal ages, regions having neurons that were generated early and more mature had the strongest MeCP2 expression. Late developing structures including cortex, hippocampus and cerebellum exhibited the most significant changes in MeCP2 expression. Of these regions, the cerebellum showed the most striking cell-specific changes in MeCP2 expression. For example, the early-generated Purkinje cells became MeCP2 positive by P6, while the late-generated granule cells did not express MeCP2 until the fourth postnatal week. The timing of MeCP2 expression in the granule cell layer is coincident with the onset of granule cell synapse formation. Although more subtle, the degree of MeCP2 expression in cortex and hippocampus was most closely correlated with synaptogenesis in both regions. Our finding that MeCP2 expression is correlated with synaptogenesis is consistent with the hypothesis that Rett Syndrome is caused by defects in the formation or maintenance of synapses.

An NMDAR-independent LTP mediated by group II metabotropic glutamate receptors and p42/44 MAP kinase in the dentate gyrus in vitro

The induction of long-term potentiation (LTP) under conditions of blockade of the N-methyl- d-aspartate receptor (NMDAR) was studied in the medial perforant path to granule cell synapse in the dentate gyrus. A small amplitude NMDAR-independent potentiation was induced by a single brief high frequency stimulation (HFS), and a summated larger LTP was induced by repeated spaced HFS. The NMDAR-independent LTP was mediated by activation of group II mGluR as it was inhibited by the group II antagonists EGLU and also low concentrations of LY341495, but not the group I mGluR antagonist MPEP. Perfusion of the group II mGluR agonist DCG-IV induced NMDAR-independent LTP in media containing an NMDAR antagonist. The NMDAR-independent LTP induced by HFS was mediated via activation of p42/44 MAP kinase as it was blocked by the selective inhibitor PD98059.

Ethanol effects on dentate granule cell LTP.

In previous studies we identified a lateral hypothalamic area (LHA) sensitive to ethanol, < 5.0 mM, when the perifornical region of the area is perfused with different concentrations of ethanol. Some of these perifornical neurons contain angiotensin (Ang) and project directly to the dentate gyrus where angiotensin is released and inhibits LTP in medial perforant path-dentate granule cell synapses. The AT1 subtype receptor is involved because pretreatment with losartan, an AT1 antagonist, prevents Ang II, diazepam, and ethanol impairment of LTP as well as their effects on behavior. There is a possibility that these effects were not specific to the LHA; but might be attributable to direct effects of ethanol on postsynaptic granule cells due to diffusion of the ethanol in the extracellular space or by the circulatory system. The purpose of the present study was to determine a dose effect of ethanol on LTP in these same synapses when the dentate gyrus was perfused with several different concentrations of ethanol under the same conditions in urethane anesthetized rats. Ethanol was administered directly into the dentate gyrus by means of a fine stainless steel cannula attached approximately 1.0 mm from the tip of the glass capillary recording electrode. Results show that the threshold for ethanol in the dentate is higher by a factor of ten, > 30 mM and < 50 mM; and that at higher doses ethanol can have a direct effect on the LHA; and possibly toxic due to increasing ethanol in the blood circulatory system.

Brain-Derived Neurotrophic Factor Triggers Transcription-Dependent, Late Phase Long-Term PotentiationIn Vivo

Acute intrahippocampal infusion of brain-derived neurotrophic factor (BDNF) leads to long-term potentiation (BDNF-LTP) of synaptic transmission at medial perforant path-->granule cell synapses in the rat dentate gyrus. Endogenous BDNF is implicated in the maintenance of high-frequency stimulation-induced LTP (HFS-LTP). However, the relationship between exogenous BDNF-LTP and HFS-LTP is unclear. First, we found that BDNF-LTP, like HFS-LTP, is associated with enhancement in both synaptic strength and granule cell excitability (EPSP-spike coupling). Second, treatment with a competitive NMDA receptor (NMDAR) antagonist blocked HFS-LTP but had no effect on the development or magnitude of BDNF-LTP. Thus, NMDAR activation is not required for the induction or expression of BDNF-LTP. Formation of stable, late phase HFS-LTP requires mRNA synthesis and is coupled to upregulation of the immediate early gene activity-regulated cytoskeleton-associated protein (Arc). Local infusion of the transcription inhibitor actinomycin D (ACD) 1 hr before or immediately before BDNF infusion inhibited BDNF-LTP and upregulation of Arc protein expression. ACD applied 2 hr after BDNF infusion had no effect, defining a critical time window of transcription-dependent synaptic strengthening. Finally, the functional role of BDNF-LTP was assessed in occlusion experiments with HFS-LTP. HFS-LTP was induced, and BDNF was infused at time points corresponding to early phase (1 hr) or late phase (4 hr) HFS-LTP. BDNF applied during the early phase led to normal BDNF-LTP. In contrast, BDNF-LTP was completely occluded during the late phase. The results strongly support a role for BDNF in triggering transcription-dependent, late phase LTP in the intact adult brain.

A Simplified Model of Long-Term Plasticity in Cerebellar Mossy Fiber–Granule Cell Synapses

We simulated synaptic transmission and modified a simple model of long-term potentiation and long-term depression in order to describe the long-term plasticity-related changes in cerebellar mossy fiber–granule cell synapses. In our model, protein autophosphorylation, leading to the maintenance of long-term plasticity, is controlled by Ca2+ entry through the NMDA receptor channels. The observed nonlinearity in the development of long-term changes of EPSP in granule cells is explained by the difference in the rate constants of two independent autocatalytic processes.

Evidence for nicotinic acetylcholine receptor activation in rat cerebellar slices

Neuronal nicotinic ACh receptor (nAChR) activation is known to enhance glutamate and GABA release in different brain areas. Moreover, nAChRs play an important role in neuronal differentiation. By using the patch-clamp technique, we have investigated the presence of nAChRs in cerebellar granule cells in slices from P5–P14 rats. Application of ACh (1 mM) could elicit a variety of effects. Some cells did not respond at all. In other cells, a somatic current was activated. In a proportion of cells, postsynaptic currents (PSCs), with or without somatic current, were elicited. Somatic nAChRs are likely to be of the α 4β 2 subtype, but the presence of other subunit combinations (α 7- or β 4-containing receptors) cannot be ruled out. The ACh-induced PSCs were glutamatergic in nature. Thus, in a reasonable proportion of cells, nicotinic receptors are present presynaptically. They are likely to be α 7 receptors whose activation elicits Glu release via a TTX-sensitive mechanism. Our experiments are the first electrophysiological evidence showing, in a native cerebellar preparation, the presence of nicotinic receptors at the mossy fibre–granule cell synapse at early developmental stages.

Acute and chronic intracerebroventricular morphine infusions affect long-term potentiation differently in the lateral perforant path

Experiments were performed to investigate the effects of acute and chronic intracerebroventricular (icv) morphine infusions via osmotic minipumps on long-term potentiation (LTP) in the lateral perforant path (LPP)-granule cell synapse of the rat dentate gyrus. Although significant antinociceptive activity was observed when morphine was infused (25 nmol/μl/h) for 30 min or 1 h, the activity was not observed in rats receiving morphine chronically for 72 h, and the tail-flick latency of this group was comparable to that of rats receiving saline. LTP induction was significantly attenuated after acute morphine infusion (1 h) in LPP-granule cell synapses of the dentate gyrus. In contrast, LTP induction was augmented after chronic morphine infusion for 72 h. Bath-perfused morphine augmented the baseline population spike (PS) amplitude in rats treated with saline, whereas it attenuated the LTP induced by chronic morphine infusion. Returning the LTP to the level of saline infusion after in vitro morphine perfusion suggests that enhancement of the LTP is a withdrawal-like phenomenon. These results suggest a difference between the effects of acute and chronic intracerebroventricular morphine infusions on synaptic plasticity in the LPP-granule cell synapses of the dentate gyrus.

NMDA receptor subunits GluRepsilon1, GluRepsilon3 and GluRzeta1 are enriched at the mossy fibre-granule cell synapse in the adult mouse cerebellum.

Cerebellar N-methyl-D-aspartate (NMDA) receptors are concentrated in the granular layer and are involved in motor coordination and the induction of long-term potentiation at mossy fibre-granule cell synapses. In the present study, we used immunohistochemistry to examine the distribution of NMDA receptor subunits in the adult mouse cerebellum. We found that appropriate pepsin pretreatment of sections greatly enhanced the sensitivity and specificity of immunohistochemical detection. As a result, intense immunolabelling for GluRepsilon1 (NR2A), GluRepsilon3 (NR2C), and GluRzeta1 (NR1) all appeared in synaptic glomeruli of the granular layer. Double immunofluorescence showed that these subunits were colocalized in individual synaptic glomeruli. Within the glomerulus, NMDA receptor subunits were located between centrally-located huge mossy fibre terminals and peripherally-located tiny Golgi axon terminals. By immunoelectron microscopy, all three subunits were detected at the postsynaptic junction in granule cell dendrites, forming synapses with mossy fibre terminals. Consistent with the known functional localization, GluRepsilon1, GluRepsilon3, and GluRzeta1 are, thus, anatomically concentrated at the mossy fibre-granule cell synapse. By contrast, immunohistochemical signals were very low in Purkinje cell somata and dendrites in the molecular layer. The lack of GluRzeta1 immunolabelling in Purkinje cells was unexpected because the cells express GluRzeta1 mRNA at high levels and high levels of GluRzeta1 protein in the molecular layer were revealed by immunoblot. As Purkinje cells are exceptionally lacking GluRepsilon expression, the discrepant result may provide in vivo evidence suggesting the importance of accompanying GluRepsilon subunits in synaptic localization of GluRzeta1.

Unsupervised learning of granule cell sparse codes enhances cerebellar adaptive control.

Marr [J. Physiol. (1969) 202, 437-470] and Albus [Math. Biosci. (1971) 10, 25-61] hypothesized that cerebellar learning is facilitated by a granule cell sparse code, i.e. a neural code in which the fraction of active neurons is low at any one time. In this paper, we re-examine this hypothesis in light of recent experimental and theoretical findings. We argue that cerebellar motor learning is enhanced by a sparse code that simultaneously maximizes information transfer between mossy fibers and granule cells, minimizes redundancies between granule cell discharges, and re-codes the mossy fiber inputs with an adaptive resolution such that inputs corresponding to large errors are finely encoded. We then propose that a set of biologically plausible unsupervised learning rules can produce such a code. To maintain a low mean firing rate compatible with a sparse code, an activity-dependent homeostatic mechanism sets the cells' thresholds. Then, to maximize information transfer, the mossy fiber--granule cell synapses are adjusted by a Hebbian rule. Furthermore, to minimize redundancies between granule cell discharges, the inhibitory Golgi cell--granule cell synapses are tuned by an anti-Hebbian rule. Finally, to allow adaptive resolution, a performance-based neuromodulator-like signal gates these three plastic processes. We integrate these gated learning rules into a simplified model of the cerebellum for arm movement control, and show that unsupervised learning of granule cell sparse codes greatly improves cerebellar adaptive motor control in comparison to a "fixed" Marr--Albus-type model. Until recently, activity-dependent cerebellar plasticity was thought to be largely confined to the granule cell--Purkinje cell synapses. This static view of the cerebellum is, however, quickly being replaced by an extremely dynamic view in which plasticity is omnipresent. The present theoretical study shows how several forms of plasticity in the granular layer of the cerebellum can produce fast, accurate and stable cerebellar learning.

The supramammillary nucleus contributes to associative EPSP-spike potentiation in the rat dentate gyrus in vivo.

The supramammillary nucleus (SUM) of the hypothalamus sends neural projections to the hippocampus and is supposed to be involved in learning and memory. To test the possibility that SUM afferents modulate hippocampal functions, we investigated the effect of electrical stimulation of the SUM on the induction of long-term potentiation (LTP) at medial perforant path (PP)--granule cell synapses in the dentate gyrus (DG) of anaesthetized rats. High-frequency stimulation of the SUM (100 pulses at 100 Hz) alone did not change PP--DG field potentials. However, when the SUM stimulation was applied simultaneously with weak tetanic stimulation of the PP (20 pulses at 20 Hz) which alone did not induce any potentiation, it produced a long-lasting potentiation of the population spike, without an accompanying increase in the population excitatory postsynaptic potential (EPSP). The EPSP-spike (E-S) potentiation induced by pairing SUM and PP stimulation was abolished by lesions of the fimbria--fornix, a major pathway of SUM afferents. SUM stimulation applied 1 s before or after PP stimulation failed to produce E-S potentiation, and SUM stimulation augmented PP--DG field potentials during tetanic stimulation. Furthermore, the E-S potentiation was abolished by blocking GABAergic neurotransmission with picrotoxin. These results suggest that coactivation of SUM and PP inputs produces a long-lasting increase of granule cell excitability by modulating GABAergic inhibition. SUM afferents may contribute to associative memory processing by modulating hippocampal excitability.

LTP induction threshold change in old rats at the perforant path–granule cell synapse

Old, memory-deficient rats do not show a change in the threshold for long-term potentiation (LTP) induction in hippocampal region CA1. This observation suggests that defective NMDA receptor mechanisms at the Schaffer collateral—CA1 pyramidal cell synapse cannot explain age-related LTP induction deficits that are observed under some stimulation protocols. The effects of aging on functional electrophysiology are not, however, identical between hippocampal subregions. In fact, at the perforant path—granule cell synapse of rats NMDA receptor-mediated responses are reduced, suggesting a possible change in the threshold for LTP induction at this synapse. This hypothesis was tested in the present experiment. We found that when weak orthodromic stimulation of medial perforant path fibers is paired with intracellular current injection of granule cells, the threshold for LTP induction is elevated in aged, spatial memory-impaired rats compared to middle-aged and young controls. Thus, in addition to there being fewer total medial perforant path synaptic contacts in old rats, greater depolarization and input convergence is required before durable modification of synaptic strength can be induced.

Long-Term Potentiation of Intrinsic Excitability at the Mossy Fiber–Granule Cell Synapse of Rat Cerebellum

Synaptic activity can induce persistent modifications in the way a neuron reacts to subsequent inputs by changing either synaptic efficacy or intrinsic excitability. After high-frequency synaptic stimulation, long-term potentiation (LTP) of synaptic efficacy is commonly observed at hippocampal synapses (Bliss and Collingridge, 1993), and potentiation of intrinsic excitability has recently been reported in cerebellar deep nuclear neurons (Aizenmann and Linden, 2000). However, the potential coexistence of these two aspects of plasticity remained unclear. In this paper we have investigated the effect of high-frequency stimulation on synaptic transmission and intrinsic excitability at the mossy fiber-granule cell relay of the cerebellum. High-frequency stimulation, in addition to increasing synaptic conductance (D'Angelo et al., 1999), increased granule cell input resistance and decreased spike threshold. These changes depended on postsynaptic depolarization and NMDA receptor activation and were prevented by inhibitory synaptic activity. Potentiation of intrinsic excitability was induced by relatively weaker inputs than potentiation of synaptic efficacy, whereas with stronger inputs the two aspect of potentiation combined to enhance EPSPs and spike generation. Potentiation of intrinsic excitability may extend the computational capability of the cerebellar mossy fiber-granule cell relay.

Critical Period for Activity-Dependent Synapse Elimination in Developing Cerebellum

Synapse elimination is considered to be the final step in neural circuit formation, by causing refinement of redundant connections formed at earlier developmental stages. The developmental loss of climbing fiber innervation from cerebellar Purkinje cells is an example of such synapse elimination. It has been suggested that NMDA receptors are involved in the elimination of climbing fiber synapses. In the present study, we probed the NMDA receptor-dependent period of climbing fiber synapse elimination by using daily intraperitoneal injections of the NMDA receptor antagonist MK-801. We found that blockade of NMDA receptors during postnatal day 15 (P15) and P16, but not before or after this period, resulted in a higher incidence of multiple climbing fiber innervation and caused a mild but persistent loss of motor coordination. Neither basic synaptic functions nor cerebellar morphology were affected by this manipulation. Chronic local application of MK-801 to the cerebellum during P15 and P16 also yielded a higher incidence of multiple climbing fiber innervation. During P15-P16, large NMDA receptor-mediated EPSCs were detected at the mossy fiber-granule cell synapse, but not at the parallel fiber-Purkinje cell or climbing fiber-Purkinje cell synapse. It is therefore likely that the NMDA receptors located at the mossy fiber-granule cell synapse mediate signals leading to the elimination of surplus climbing fibers. These results suggest that an NMDA receptor-dependent phase of climbing fiber synapse elimination lasts 2 d at most. During this phase, the final refinement of climbing fiber synapses occurs, and disruption of this process leads to permanent impairment of cerebellar function.