Mossy Fiber-granule Cell Research Articles

Hippocampal mossy fiber sprouting induced by chronic electroconvulsive seizures

Stress, which can precipitate and exacerbate depression, causes atrophy and in severe cases death of hippocampal neurons. Atrophy of the hippocampus has also been observed in patients suffering from recurrent major depression. The present study examines the influence of electroconvulsive seizures, one of the most effective treatments for depression, on the morphology and survival of hippocampal neurons. The results demonstrate that chronic administration of electroconvulsive seizures induces sprouting of the granule cell mossy fiber pathway in the hippocampus. This sprouting is dependent on repeated administration of electroconvulsive seizures, reaches a maximum 12 days after the last treatment and is long lasting (i.e. up to six months). Electroconvulsive seizure-induced sprouting occurs in the absence of neuronal loss, indicating that sprouting is not a compensatory response to cell death. This is different from the sprouting induced by kindling or excitotoxin treatment, which induce cell death along with recurrent seizures. Electroconvulsive seizure-induced sprouting is significantly diminished in brain-derived neurotrophic factor heterozygote knockout mice, indicating that this neurotrophic factor contributes to mossy fiber sprouting. However, infusion of brain-derived neurotrophic factor into the hippocampus does not induce sprouting of the mossy fiber pathway. The results demonstrate that chronic administration of electroconvulsive seizures induces mossy fiber sprouting and suggest that increased expression of brain-derived neurotrophic factor is necessary, but not sufficient for the induction on this sprouting. Although the functional consequences remain unclear, sprouting of the mossy fiber pathway would appear to oppose the actions of stress and could thereby contribute to the therapeutic actions of electroconvulsive seizure therapy.

Evidence for NMDA and mGlu receptor-dependent long-term potentiation of mossy fiber-granule cell transmission in rat cerebellum.

Long-term potentiation (LTP) is a form of synaptic plasticity that can be revealed at numerous hippocampal and neocortical synapses following high-frequency activation of N-methyl--aspartate (NMDA) receptors. However, it was not known whether LTP could be induced at the mossy fiber-granule cell relay of cerebellum. This is a particularly interesting issue because theories of the cerebellum do not consider or even explicitly negate the existence of mossy fiber-granule cell synaptic plasticity. Here we show that high-frequency mossy fiber stimulation paired with granule cell membrane depolarization (-40 mV) leads to LTP of granule cell excitatory postsynaptic currents (EPSCs). Pairing with a relatively hyperpolarized potential (-60 mV) or in the presence of NMDA receptor blockers [5-amino--phosphonovaleric acid (APV) and 7-chloro-kynurenic acid (7-Cl-Kyn)] prevented LTP, suggesting that the induction process involves a voltage-dependent NMDA receptor activation. Metabotropic glutamate receptors were also involved because blocking them with (+)-alpha-methyl-4-carboxyphenyl-glycine (MCPG) prevented potentiation. At the cytoplasmic level, EPSC potentiation required a Ca2+ increase and protein kinase C (PKC) activation. Potentiation was expressed through an increase in both the NMDA and non-NMDA receptor-mediated current and by an NMDA current slowdown, suggesting that complex mechanisms control synaptic efficacy during LTP. LTP at the mossy fiber-granule cell synapse provides the cerebellar network with a large reservoir for memory storage, which may be needed to optimize pattern recognition and, ultimately, cerebellar learning and computation.

Development of the quantal properties of evoked and spontaneous synaptic currents at a brain synapse.

In many studies of central synaptic transmission, the quantal properties of miniature synaptic events do not match those derived from synaptic events evoked by action potentials. Here we show that at mossy fiber-granule cell (MF-gc) synapses of mature cerebellum, evoked excitatory postsynaptic currents (EPSCs) are multiquantal, and their amplitudes vary in discrete steps, whereas miniature (m)EPSCs are monoquantal or multiquantal with quantal parameters identical to those of the EPSCs. In contrast, at immature MF-gc synapses, EPSCs are multiquantal, but their amplitudes do not vary in discrete steps, whereas most mEPSCs seem to be monoquantal with a broad and skewed amplitude distribution. The results demonstrate that quantal variance decreases during synaptic development. They also directly confirm the quantal hypothesis of neurotransmission at a mature brain synapse.

Vestibular ganglion neurons survive the loss of their cerebellar targets

Neuronal survival during mammalian development crucially depends on target-derived neurotrophic factors. Target loss removes this trophic support and leads in most cases to the transsynaptic retrograde degeneration of the respective afferents. Primary vestibular afferents (PVA) originating from bipolar neurons in the vestibular ganglion (VG) are the first mossy fibers that enter the cerebellum, but little is known about the survival requirements of VG neurons. In the present study the influence of the differential granule cell (GC) target loss on the survival of VG neurons was studied quantitatively using unbiased stereological methods in the cerebellar mutants Purkinje cell degeneration (pcd/pcd), Lurcher (Lc/+), and Weaver (wv/wv). Neither the secondary GC loss in the Purkinje cell deficient mutants pcd/pcd and Lc/+, nor the primary loss of GCs in wv/wv produced any significant reduction in the total number of bipolar neurons in the VG compared to controls. So, PVA neurons are highly resistant to cerebellar target deprivation and survive in the absence of cerebellar granule and Purkinje cells, regardless of whether the target loss occurs before (in wv/wv), during (in Lc/+) or after (in pcd/pcd) the mossy fiber-granule cell synaptogenesis.

Biosynthesis and metabolism of native and oxidized neuropeptide Y in the hippocampal mossy fiber system.

Neuropeptide Y (NPY) gene expression is known to be modulated in the mossy fiber projection of hippocampal granule cells following seizure. We investigated NPY biosynthesis and metabolism in an attempt to characterize NPY biochemically as a neurotransmitter in the granule cell mossy fiber projection. NPY biosynthesis was compared in normal control animals and in animals that had experienced a single pentylenetetrazole-induced seizure. In situ hybridization analysis established the postseizure time course of preproNPY mRNA expression in the hippocampal formation, localizing the majority of increased preproNPY mRNA content to the hilus of the dentate gyrus. Radioimmunoassay analysis of the CA3/mossy fiber terminal subfield confirmed a subsequent increase in NPY peptide content. Biosynthesis of NPY peptide by granule cells and transport to the CA3/mossy fiber subfield was demonstrated by in vivo radiolabel infusion to the dentate gyrus/hilus followed by sequential HPLC purification of identified radiolabeled peptide from the CA3/mossy fiber terminal subfield. Additional in vivo radiolabeling studies revealed a postseizure increase in an unidentified NPY-like immunoreactive (NPY-LI) species. HPLC/radioimmunoassay analyses of CA3 subfield tissue extracts comparing normal control animals and pentylenetetrazole-treated animals confirmed the increased total NPY-LI, and demonstrated that the increased NPY-LI was comprised of a minor increase in native NPY and a major increase in the unknown NPY-LI. Data from subsequent and separate analyses incorporating immunoprecipitation with anti-C-terminal flanking peptide of NPY, further HPLC purification, and matrix-assisted laser desorption/ionization mass spectrometry support the conclusion that the unknown NPY-LI is methionine sulfoxide NPY. NPY and NPY-sulfoxide displayed differential calcium sensitivity for release from mossy fiber synaptosomes. Similar to NPY, NPY sulfoxide displayed high-affinity binding to each of the cloned Y1, Y2, Y4, and Y5 receptor subtypes. Postrelease inactivation of NPY was demonstrated in a mossy fiber synaptosomal preparation. Thus, the present study in combination with previously reported electrophysiological activity of NPY in the CA3 subfield demonstrates that NPY fulfills the classical criteria for a neurotransmitter in the hippocampal granule cell mossy fiber projection, and reveals the presence of two molecular forms of NPY that display differential mechanisms of release while maintaining similar receptor potencies.

Integrated regulation of signal coding and plasticity by NMDA receptors at a central synapse.

The role of NMDA and non-NMDA glutamate receptors in long-term potentiation has been intensely investigated, yet recent evidence on the dynamics of synaptic depolarization suggests that the original view should be extended. NMDA receptor-mediated currents, apart from their Ca2+ permeability, show a marked voltage dependence, consisting of current increase and slowdown during membrane depolarization. During highfrequency synaptic transmission, NMDA current increase and slowdown are primed by non-NMDA receptor-dependent depolarization and proceed regeneratively. Thus, NMDA receptors make a decisive contribution to membrane depolarization and spike-firing. From the data obtained at the mossy fiber- granule cell synapse of the cerebellum, we propose that the electrogenic role of NMDA receptors is functional to LTP induction. Moreover, during LTP, both NMDA and non- NMDA receptor currents are potentiated, thus establishing a feed-forward mechanism that ultimately enhances spike firing. Thus, NMDA receptors exert an integrated control on signal coding and plasticity. This mechanism may have important implications for information processing at the cerebellar mossy fibergranule cell relay.

Effect of cerebellar granule cell depletion on learning of the equilibrium behaviour: study in postnatally X-irradiated rats.

To assess the role of the mossy fibre-granule cell pathway in learning, the cerebellum of young DA/HAN strain rats was irradiated to make the cortex completely or partially agranular. The X-rays were delivered according to two different schedules, between 5-14 postnatal days (early group) and between 10-14 postnatal days (late group). Histological controls at 35 days showed a mean loss of granule cells of 96 +/- 1% in the early group and of 61 +/- 3% in the late group. The irradiated animals were subjected, from day 23 to day 35, to daily sensorimotor training on a rotorod. The scores and the strategy used (walking or hanging) by the rats were noted. The results demonstrate that a partial loss of granule cells due to a late X-irradiation schedule induced mild motor disabilities but no learning deficit, the only problem being difficulty in elaborating rapidly an efficient strategy to solve a novel problem. A sub-total loss of the granule cells, due to an early X-irradiation schedule, induced gross motor disabilities and the animals used hanging > 90% of the time. Due to the discrepancy between the learning abilities, which were preserved at least in part, and the gross motor impairments, the animals elaborated a novel strategy (jumping from the beam), allowing them to escape the experimental situation. This avoidance behaviour may be due to a decrease of anxiety, a lack of behavioural inhibition and/or attentional deficits that have been already observed in several other examples of cerebellar abnormalities.

Delayed Decrease of Calbindin Immunoreactivity in the Granule Cell-Mossy Fibers after Kainic Acid-Induced Seizures

Kainic acid (KA) administration induces an abnormal excitation and spontaneous recurrent seizures. Alterations of granule cell properties may be potential mechanisms. In this study, dynamic alterations of calbindin, a calcium binding protein particularly abundant in the granule cells, have been investigated immunocytochemically in the rat hippocampus after the KA-induced seizures. The calbindin immunoreactivity decreased slightly in the CA1/CA2 fields already after 1 and 3 days, and was lost partly or completely in the pyramidal layer after 10 days. From day 21, the calbindin immunoreactivity decreased in dendrites and soma of the granule cells and mossy fibers. The alterations remained at least to day 90, while no evident neuronal loss occurred in the granule cells. This may reflect a disturbance of calcium homostasis in the granule cells after seizures. The delayed decrease of calbindin has a time course similar to the occurrence of spontaneous recurrent seizures, suggesting a possible correlation between the two events.

Motor Discoordination Results from Combined Gene Disruption of the NMDA Receptor NR2A and NR2C Subunits, But Not from Single Disruption of the NR2A or NR2C Subunit

NMDA receptors consist of two distinct classes of subunits. The NR1 subunit possesses all properties of the NMDA receptor-channel complex, whereas four NR2 subunits (NR2A-2D) potentiate and differentiate NMDA receptor responses by heteromeric assemblies with NR1. The mRNAs for the five NMDA receptor subunits are expressed in the cerebellum in a distinct temporospatial manner. To study functions of the NMDA receptors in the cerebellum, we generated knockout mice deficient in either NR2A or NR2C or both of these subunits. All three mutant mice developed normally and showed normal overall morphology of the cerebellum. The NMDA receptor-mediated components of EPSCs in granule cells, as assessed by whole-cell recordings of cerebellar slices, were reduced in NR2A- and NR2C-deficient mice and nearly abolished in mice lacking both NR2A and NR2C. The NR2A- and NR2C-deficient granule cells were different in the current-voltage relationship and time course of NMDA receptor responses. The NR2A and NR2C subunits thus contribute to distinct NMDA receptor-mediated excitatory transmission in mossy fiber-granule cell synapses in the mature cerebellum. Both NR2A- and NR2C-deficient mice showed no impaired movements in the motor coordination tasks tested. The mutant mice deficient in both NR2A and NR2C could also manage simple coordinated tasks, such as staying on a stationary or a slowly rotating rod, but failed more challenging tasks such as staying on a quickly rotating rod. These data demonstrate that the NMDA receptors play an active role in motor coordination.

The pathogenic and progressive features of chronic human hippocampal epilepsy.

To design useful experimental models of epilepsy, it is necessary to clearly understand the known clinical-pathologic features of the disease process. Studies of mesial temporal lobe epilepsy (MTLE) patients have identified several distinctive clinical and pathophysiologic characteristics and many of these can be analyzed in experimental models. For example, patients with typical MTLE have medical histories that often contain an initial precipitating injury (IPI), are likely to have hippocampal sclerosis in the surgical specimen, and have better seizure outcomes than patients with typical idiopathic temporal seizures (i.e. cryptogenic). Hippocampal from children as young as age 1 year with IPI histories also demonstrate neuron damage similar to adults with hippocampal sclerosis. Compared to IPI patients without seizures (i.e. trauma, hypoxia, etc.), IPI cases with severe seizures showed younger ages at the IPI, shorter latent periods, and longer durations of habitual MTLE. Hippocampal damage is often bilateral, however, the epileptogenic side shows hippocampal sclerosis and the opposite side usually shows only mild neuron losses. Moreover, MTLE patients show declines in hippocampal neuron densities with very long histories of habitual seizures (15 to 20 years), however, the additional neuron loss adds to the template of hippocampal sclerosis and occurs in limited subfields (granule cells, CA1 and prosubiculum). Hippocampal axon and synaptic reorganization is another pathologic feature of MTLE, and involves granule cell mossy fibers and axons immunoreactive for neuropeptide upsilon, somatostatin, and glutamate decarboxylase (which synthesizes GABA). Finally, MTLE patients with hippocampal sclerosis show increased granule cell mRNA levels for brain derived neurotropic factor, nerve growth factor, and neurotrophin-3 that correlate with mossy fiber sprouting or with declines in Ammon's horn neuron densities. Taken together, our data support the following concepts: (1) The pathogenesis of MTLE is associated with IPI histories that probably injure the hippocampus at some time prior to habitual seizure onsets, (2) most of the damage seems to occur with the IPI, (3) there can be additional neuron loss associated with long histories, (4) another pathologic feature of MTLE is axon reorganization of surviving fascia dentata and hippocampal neurons, and (5) reorganized axon circuits probably contribute to seizure or propagation.

Plasticity of granule cell–mossy fiber system following kainic acid induced seizures: an immunocytochemical study on neurofilament proteins

Plasticity of granule cell–mossy fiber system following kainic acid induced seizures: an immunocytochemical study on neurofilament proteins

Plasticity of granule cell-mossy fiber system following kainic acid induced seizures: an immunocytochemical study on neurofilament proteins

Abnormal reestablishment of mossy fibers with the CA3 pyramidal cells and granule cells is an important aspect of postlesional plasticity in epilepsy. However, basis for the structural reorganisation and functional consequences of the event remain uncertain. Therefore we have investigated alterations of neurofilaments, major cytoskeletal components of neurons, in the rat hippocampus after the kainic acid (KA) administration, an experimental model for the temporal lobe epilepsy. The immunoreactivity for phosphorylated heavy weight neurofilament (pNFH) and non-phosphorylated heavy weight neurofilament (npNFH), in particular the pNFH, decreased in the CA1 field and inner molecular layer of the dentate gyrus during 3 and 10 days after the KA administration. After 10 days, npNFH immunoreactivity appeared in the mossy fibers, in which it is normally absent, meanwhile the pNFH staining in the mossy fibers did not decrease. From day 21, the immunoreactivity of pNFH and npNFH was normal or above normal in the CA1 stratum lacunosum-moleculare, mossy fibers, hilus and inner molecular layer of the dentate gyrus. These alterations in the later phase remained at least to day 90. The reappearance and increase of the neurofilament immunoreactivity in the inner molecular layer of the dentate gyrus probably reflects a collateral extension of the granule cell axons known as mossy fiber sprouting. The results suggest that neurofilament changes in the granule cell-mossy fiber system may be a morphological basis for the structural reconstruction of granule cell axons, and neurofilaments are involved in the plasticity after the KA induced seizures.

Partial ablation of the neonatal external granular layer disrupts mossy fiber topography in the adult rat cerebellum.

The spinocerebellar projection in the rat is compartmentalized in an array of parasagittal bands of mossy fiber terminals. These bands align reproducibly with bands of Purkinje cells that differentially express zebrin II. To investigate whether this alignment is obligatory, Purkinje cell and mossy fiber compartmentation has been compared in the rat cerebellum where the cytoarchitecture was contorted by neonatal administration of methylazoxymethanol. Methylazoxymethanol ablates many granule cell precursors, leaving a much reduced external granular layer, and adult rats that received a single methylazoxymethanol injection at birth showed varying degrees of abnormal cerebellar foliation. Zebrin II immunocytochemistry nevertheless revealed no fundamental abnormality in the Purkinje cell compartments. However, despite the normal Purkinje cell compartmentation being retained, the spinocerebellar mossy fiber-Purkinje cell topography is disrupted by methylazoxymethanol treatment. The normal precise array of parasagittal mossy fiber terminal fields becomes blurred across the lobule, and the normal clear banding is difficult to follow. These data suggest that, despite the early topography being dependent on the Purkinje cells, the granule cell-mossy fiber interactions also regulate the topography of the spinocerebellar projection.

Partial ablation of the neonatal external granular layer disrupts mossy fiber topography in the adult rat cerebellum

The spinocerebellar projection in the rat is compartmentalized in an array of parasagittal bands of mossy fiber terminals. These bands align reproducibly with bands of Purkinje cells that differentially express zebrin II. To investigate whether this alignment is obligatory, Purkinje cell and mossy fiber compartmentation has been compared in the rat cerebellum where the cytoarchitecture was contorted by neonatal administration of methylazoxymethanol. Methylazoxymethanol ablates many granule cell precursors, leaving a much reduced external granular layer, and adult rats that received a single methylazoxymethanol injection at birth showed varying degrees of abnormal cerebellar foliation. Zebrin II immunocytochemistry nevertheless revealed no fundamental abnormality in the Purkinje cell compartments. However, despite the normal Purkinje cell compartmentation being retained, the spinocerebellar mossy fiber-Purkinje cell topography is disrupted by methylazoxymethanol treatment. The normal precise array of parasagittal mossy fiber terminal fields becomes blurred across the lobule, and the normal clear banding is difficult to follow. These data suggest that, despite the early topography being dependent on the Purkinje cells, the granule cell-mossy fiber interactions also regulate the topography of the spinocerebellar projection. © 1996 Wiley-Liss, Inc.

Non-NMDA glutamate receptor occupancy and open probability at a rat cerebellar synapse with single and multiple release sites.

1. Excitatory postsynaptic currents (EPSCs) were recorded under whole-cell voltage clamp from granule cells in slices of rat cerebellum. EPSCs from individual mossy fibre inputs were identified by their all-or-none appearance in response to a graded stimulus. Excitatory synaptic transmission was investigated at room temperature (approximately 24 degrees C) and at near-physiological temperature (approximately 34 degrees C) by analysing current fluctuations in the peak and decay of the non-N-methyl-D-aspartate (non-NMDA) component of EPSCs. 2. In a subset of synapses the mean EPSC amplitude remained unchanged as the probability of transmitter release was substantially lowered by raising the extracellular [Mg2+] and lowering [Ca2+]. These synapses were considered to have only one functional release site. Single-site synapses had small EPSCs (139 +/- 16 pS, n = 5, at 24 degrees C) with a large coefficient of variation (c.v. = 0.23 +/- 0.02, n = 5) and an amplitude distribution that was well fitted by a Gaussian distribution in four out of five cases. The EPSC latency had a unimodal distribution and its standard deviation had a temperature dependence with a temperature coefficient (Q10; range, 24-35 degrees C) of 2.4 +/- 0.4 (n = 4). 3. Peak-scaled non-stationary fluctuation analysis of single-site EPSCs indicated that the mean conductance of the underlying non-NMDA channels was 12 +/- 2 pS (n = 4) at 35 degrees C. Upper and lower limits for mean channel open probability (Po), calculated from fluctuations in the EPSC peak amplitude, were 0.51 and 0.38, respectively. These estimates, together with the open probability of the channel when bound by transmitter, suggest that only about 50% of the non-NMDA channels were occupied following the release of a quantum of transmitter. 4. At some multi-site synapses EPSCs had a low c.v. (0.4 +/- 0.01, n = 5) at 34 degrees C and non-stationary fluctuation analysis gave a parabolic variance-mean current relationship. This suggests that practically all of the non-NMDA receptors were occupied by glutamate at the peak of EPSC. The channel open probability (Po = 0.84 +/- 0.03, n = 5) at these 'saturated' multi-site synapses will therefore equal the open probability of the channel when bound by transmitter (Po,max). 5. Non-stationary fluctuation analysis of EPSCs from 'saturating' multi-site synapses indicated that 170 +/- 40 postsynaptic non-NMDA channels were exposed to transmitter at the peak of the EPSC. The mean conductance of the synaptic channels was 10 +/- 2 pS (n = 5) at 34 degrees C. 6. At synapses with multiple release sites the EPSC decay time became faster when release probability was lowered (by reducing the external [Ca2+]/[Mg2+] ratio), indicating that the transmitter concentration profile depended on release probability. No such speeding of the EPSC decay was observed at single-site synapses. 7. Our results suggest that release of a packet of transmitter from a single release site does not saturate postsynaptic non-NMDA receptors at cerebellar mossy fibre-granule cell synapses. However, at multi-site synapses transmitter released from neighbouring sites can overlap, changing the transmitter concentration profile in the synaptic cleft. We conclude that the level of postsynaptic receptor occupancy can depend on the probability of transmitter release at individual multi-site synapses.

Frequency dependent activation of a slow N-methyl-D-aspartate-dependent excitatory postsynaptic potential in turtle cerebellum by mossy fibre afferents.

The synaptic responses of turtle cerebellar Purkinje cells to stimulation of localized mossy fibre systems have been studied by use of intrasomatic and intradendritic recordings in a brainstem-cerebellum preparation in vitro. Activation of mossy fibre inputs from the spinocerebellar pathway evoked fast, disynaptic postsynaptic potentials which were graded in amplitude with stimulus intensity and elicited at latencies consistent with those reported for peripheral nerve stimulation. Repetitive activation (50-100 Hz, 2-10 stimuli) of both spinocerebellar and trigeminocerebellar pathways evoked a slow, long-lasting excitatory postsynaptic potential regardless of whether single stimuli resulted in excitatory, inhibitory, or no postsynaptic responses. This slow potential was capable of triggering dendritic pacemaker discharges in recorded Purkinje cells in addition to volleys of simple spikes when activated at or near resting membrane potential. The fast excitatory synaptic potentials evoked by spinocerebellar stimulation were blocked by the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, consistent with the hypothesis that they are mediated by activation of ionotropic glutamate receptors of the alpha-amino-3-hydroxy-5-methylisox-azole-4-proprionic acid subtype at the mossy fibre-granule cell synapse and the subsequent parallel fibre-Purkinje cell synapse. The slow excitatory synaptic potential evoked by repetitive stimulation of either the spinocerebellar tract or trigeminal nerve was blocked by DL-2-amino-5-phosphonvalerate, indicating that this potential is primarily dependent upon N-methyl-D-aspartate receptors at the mossy fibre-granule cell synapse for its expression. This slow potential was reversibly potentiated by L-2-amino-4-phosphonobutyrate and bicuculline; the metabotropic glutamate antagonist (+)-alpha-methyl-4-carboxyphenylglycine did not block this potentiation. The ability of mossy fibre inputs to drive long, slow excitatory events in Purkinje cells adds another dimension to the mechanisms by which various sensory modalities can be processed interactively in the cerebellar cortex. The ability of incoming systems to access a second, longer duration response of the cerebellar output neuron may be of significant consequence to our understanding of the manner in which this neural centre integrates sensory information from multiple sources.

Synaptic excitation mediated by AMPA receptors in rat cerebellar slices is selectively enhanced by aniracetam and cyclothiazide

AMPA receptors mediate fast, glutamatergic synaptic transmission in the central nervous system. The time-course of the associated postsynaptic current has been suggested to be determined principally by the kinetics of glutamate binding and receptor desensitization. Aniracetam and cyclothiazide are drugs capable of selectively preventing desensitization of the AMPA receptor. To investigate the relevance of desensitization to fast synaptic transmission in the cerebellum we have tested these compounds against AMPA-induced depolarizations and postsynaptic potentials using the grease-gap recording technique. Aniracetam (1 microM-5 mM) and cyclothiazide (1 microM-500 microM) both enhanced the depolarising action of AMPA (1 microM) on Purkinje cells in a concentration-dependent manner. At the highest concentrations tested, the increases over controls were approximately 600% and 800% respectively. Aniracetam also increased, in a concentration-dependent manner, the amplitude of the evoked synaptic potentials of both parallel fibre-Purkinje cell and mossy fibre-granule cell pathways, with the highest concentrations tested enhancing the potentials by approximately 60% and 75% respectively. These data suggest that, at two different synapses in the cerebellum, AMPA receptor desensitization occurs physiologically and is likely to contribute to the shape of fast synaptic currents.

Estimated conductance of glutamate receptor channels activated during EPSCs at the cerebellar mossy fiber-granule cell synapse

We have analyzed the variance associated with the decay of the non-NMDA receptor component of synaptic currents, recorded from mossy fiber-granule cell synapses in cerebellar slices, to obtain a conductance estimate for the synaptic channel. Current fluctuations arising from the random channel gating properties were separated from those arising from the fluctuations in the population of channels by subtracting the mean excitatory postsynaptic current (EPSC) waveform scaled to the EPSC peak amplitude. A weighted mean single-channel conductance of approximately 20 pS was determined from the relationship between the mean current and the variance around the mean during the decay of evoked and spontaneous synaptic currents. This result suggests that high conductance non-NMDA channels, such as the 10–30 pS glutamate receptor channel previously characterized in granule cells, carry the majority of the fast component of the EPSC at this synapse. In addition, our data are consistent with the activation of surprisingly few (approximately 10) non-NMDA channels by a single packet of transmitter.

Granule cells and the development of synapses with mossy fibres in the cerebellum of the jaundiced Gunn rat.

The development of granule cells and the formation of synapses with mossy fibres were investigated in cerebella from jaundiced (j/j) Gunn rats with hereditary hyperbilirubinemia. The external granular layer was thinner than that in cerebella from heterozygous (+/j) Gunn rats without hyperbilirubinemia on days 12 and 15 after birth, and it had disappeared in places by day 18. Migrating granule cells decreased in number from days 12 to 18. In the internal granular layer, the paucity of granule cells became apparent on day 12 and a reduction in the number of cells was prominent from day 18 onward. More degenerating cells were found in j/j cerebella than in +/j cerebella on days 12 and 15. Mossy fibre-granule cell synapses (glomeruli) were immature in appearance in j/j and +/j cerebella from days 7 to 12; small, roundish mossy fibre terminals were in contact with a few dendrites of granule cells. After day 18, the glomeruli were mature in terms of shape in +/j cerebella. By contrast, the mossy terminals remained small and roundish, and the granule cell dendrites that surrounded the mossy terminals did not increase in number nor develop digitiform branches in j/j cerebella. The mossy terminals were partially surrounded by glial processes and were occasionally surrounded by large dendrites of Golgi cells, which made multiple synaptic contacts. Some degenerating mossy terminals were present from days 18 to 30. These anomalous glomeruli may be caused by insufficient synaptic contacts with granule cells.

Induction of F1/GAP-43 gene: expression in hippocampal granule cells after seizures

In the adult rat hippocampus mRNA of F1/GAP-43, an axonal growth-associated protein, is highly expressed in pyramidal cells, but is absent in granule cells. To determine whether granule cells can be induced to express mRNA of F1/GAP-43, transcript levels were studied after limbic seizures, which can induce sprouting of granule cell mossy fibers. Seizure-inducing electrolytic lesions were made in the dentate gyrus hilus with stainless-steel electrodes and mRNA levels were measured in contralateral hippocampus by quantitative in situ hybridization. Induction of F1/GAP-43 mRNA expression was observed in granule cells at 24 h, but not at 6 or 12 h, after the hilar lesion. When equivalent sized hilar lesions were made with platinum electrodes, which do not induce seizures, no hybridization was apparent over the granule cells. Hybridization over granule cells had declined by 48 h post-lesion, but even at 10 days it was still slightly higher than in control rats. F1/GAP-43 mRNA expression was also increased 2-fold in CA1 pyramidal cells with peak expression at 48 h post-lesion. These are the first data to our knowledge that demonstrate that F1/GAP-43 gene expression can be altered in neurons located within the adult brain. Induction of F1/GAP-43 mRNA expression in the granule cells may be important for the sprouting of mossy fibers and could be triggered by the elevated levels of brain-derived neurotrophic factor in CA3 cells which precede the increased F1/GAP-3 gene expression in granule cells.