Granule Cell Activity Research Articles

Granule cell activity in the cerebellum during delay eyelid conditioning

Although granule cell activity is crucial in defining the information processing performed by the cerebellum, in vivo single unit recordings of granule cells are scarce. Granule cells, which make more than half of all neurons in the brain, are not currently amenable to in vivo recordings due to their small size. To compensate for this lack of experimental data we performed an optimization analysis that predicts the dynamics of granule cell activity during delay eyelid conditioning. We used a simplified version of a model developed by Mauk and Donegan to optimize granule cell activity given that we have available eyelid conditioned responses for interstimulus intervals ranging from 100 to 750 ms. The solutions found by the optimization algorithm converge on three important aspects of stimulus evoked activity of the granule cells: (a) during stimulus presentation different granule cells become active at different times, (b) for the majority of granule cells the duration of the stimulus evoked responses is not dependent on the duration of the stimulus and (c) peaks of granule cell activity are preceded and/or followed by inhibition. While the first feature has been suggested to be the byproduct of interactions between granule and Golgi cells, the latter two predictions are novel. The utility of these predictions is supported by tests in a detailed simulation of the cerebellum.

Stimulus specific activity patterns in the granule cell networks of mice

It has been speculated that the olfactory bulb encodes information in the form of stimulus specific activity patterns. One of the key features of this population activity is the emergence of odor-specific spatial patterns of mitral cell spiking over a time course of 200–800 ms following stimulus (-odor) onset. We are interested in understanding the mechanisms involved in establishing and maintaining these spatial patterns. Here, we investigated the temporal response characteristics of granule cell activity by imaging activity in populations of olfactory bulb cells following bulk loading of calcium dye in olfactory bulb slices. We found that granule cells show varied (ranging from 0–900 ms) but reliable activation latencies (std. dev. = 50 ms). Moreover, we found that these activity patterns played a significant role in the generation and the maintenance of reliable spike patterns in mitral cells. Experiments in which multiple glomeruli were stimulated showed that the latency of granule cell activity is input specific and that individual granule cells respond most reliably to specific temporal patterns of stimulation. These data suggest that glomerular (~stimulus) identity is encoded in the form of latencies of granule cells activity, which in turn results in distinct stimulus specific changes in the pattern of mitral cell activity.

Enigmas of the Dentate Gyrus

We are rapidly approaching a better understanding of the mechanisms that allow our brains to form distinct representations for similar events or episodes. McHugh et al. have brought that goal one step closer by showing that NMDA receptor-dependent synaptic plasticity in the dentate gyrus is necessary for immediate differentiation between environments with similar features.

Changes in Biochemical Processes in Cerebellar Granule Cells of Mice Exposed To Methylmercury

At postnatal day 34, male and female C57BL/6J mice were exposed orally once a day to a total of five doses totaling 1.0 or 5.0 mg/kg of methylmercuric chloride or sterile deionized water in moistened rodent chow. Eleven days after the last dose cerebellar granule cells were acutely isolated to measure reactive oxygen species (ROS) levels and mitochondrial membrane potential using CM-H(2)DCFDA and TMRM dyes, respectively. For visualizing intracellular calcium ion distribution using transmission electron microscopy, mice were perfused 11 days after the last dose of methylmercury (MeHg) using the oxalate-pyroantimonate method. Cytosolic and mitochondrial protein fractions from acutely isolated granule cells were analyzed for cytochrome c content using Western blot analysis. Histochemistry (Fluoro-Jade dye) and immunohistochemistry (activated caspase 3) was performed on frozen serial cerebellar sections to label granule cell death and activation of caspase 3, respectively. Granule cells isolated from MeHg-treated mice showed elevated ROS levels and decreased mitochondrial membrane potential when compared to granule cells from control mice. Electron photomicrographs of MeHg-treated granule cells showed altered intracellular calcium ion homeostasis ([Ca(2+)](i)) when compared to control granule cells. However, in spite of these subcellular changes and moderate relocalization of cytochrome c into the cytosol, the concentrations of MeHg used in this study did not produce significant neuronal cell death/apoptosis at the time point examined, as evidenced by Fluoro-Jade and activated caspase 3 immunostaining, respectively. These results demonstrate that short-term in vivo exposure to total doses of 1.0 and 5.0 mg/kg MeHg through the most common exposure route (oral) can result in significant subcellular changes that are not accompanied by overt neuronal cell death.

Theta phase precession emerges from a hybrid computational model of a CA3 place cell

The origins and functional significance of theta phase precession in the hippocampus remain obscure, in part, because of the difficulty of reproducing hippocampal place cell firing in experimental settings where the biophysical underpinnings can be examined in detail. The present study concerns a neurobiologically based computational model of the emergence of theta phase precession in which the responses of a single model CA3 pyramidal cell are examined in the context of stimulation by realistic afferent spike trains including those of place cells in entorhinal cortex, dentate gyrus, and other CA3 pyramidal cells. Spike-timing dependent plasticity in the model CA3 pyramidal cell leads to a spatially correlated associational synaptic drive that subsequently creates a spatially asymmetric expansion of the model cell's place field. Following an initial training period, theta phase precession can be seen in the firing patterns of the model CA3 pyramidal cell. Through selective manipulations of the model it is possible to decompose theta phase precession in CA3 into the separate contributing factors of inheritance from upstream afferents in the dentate gyrus and entorhinal cortex, the interaction of synaptically controlled increasing afferent drive with phasic inhibition, and the theta phase difference between dentate gyrus granule cell and CA3 pyramidal cell activity. In the context of a single CA3 pyramidal cell, the model shows that each of these factors plays a role in theta phase precession within CA3 and suggests that no one single factor offers a complete explanation of the phenomenon. The model also shows parallels between theta phase encoding and pattern completion within the CA3 autoassociative network.

Group I Metabotropic Glutamate Receptors Are Differentially Expressed by Two Populations of Olfactory Bulb Granule Cells

In the main olfactory bulb, several populations of granule cells (GCs) can be distinguished based on the soma location either superficially, interspersed with mitral cells within the mitral cell layer (MCL), or deeper, within the GC layer (GCL). Little is known about the physiological properties of superficial GCs (sGCs) versus deep GCs (dGCs). Here, we used patch-clamp recording methods to explore the role of Group I metabotropic glutamate receptors (mGluRs) in regulating the activity of GCs in slices from wildtype and mGluR-/- mutant mice. In wildtype mice, bath application of the selective Group I mGluR agonist DHPG depolarized and increased the firing rate of both GC subtypes. In the presence of blockers of fast synaptic transmission (APV, CNQX, gabazine), DHPG directly depolarized both GC subtypes, although the two GC subtypes responded differentially to DHPG in mGluR1-/- and mGluR5-/- mice. DHPG depolarized sGCs in slices from mGluR5-/- mice, although it had no effect on sGCs in slices from mGluR1-/- mice. By contrast, DHPG depolarized dGCs in slices from mGluR1-/- mice but had no effect on dGCs in slices from mGluR5-/- mice. Previous studies showed that mitral cells express mGluR1 but not mGluR5. The present results therefore suggest that sGCs are more similar to mitral cells than dGCs in terms of mGluR expression.

Group I metabotropic glutamate receptors are differentially expressed by two populations of olfactory bulb granule cells

In the main olfactory bulb, several populations of granule cells (GCs) can be distinguished based on the soma location either superficially, interspersed with mitral cells within the mitral cell layer (MCL), or deeper, within the GC layer (GCL). Little is known about the physiological properties of superficial GCs (sGCs) vs. deep GCs (dGCs). Here, we used patch-clamp recording methods to explore the role of Group I metabotropic glutamate receptors (mGluRs) in regulating the activity of GCs in slices from wildtype and mGluR −/− mutant mice. In wildtype mice, bath application of the selective Group I mGluR agonist DHPG depolarized and increased the firing rate of both GC subtypes. In the presence of blockers of fast synaptic transmission (APV, CNQX, gabazine), DHPG directly depolarized both GC subtypes. The two GC subtypes responded differentially to DHPG in mGluR1−/− and mGluR5−/− mice, however. DHPG depolarized sGCs in slices from mGluR5−/− mice, but it had no effect on sGCs in slices from mGluR1−/− mice. By contrast, DHPG depolarized dGCs in slices from mGluR1−/− mice, but it had no effect on dGCs in slices from mGluR5−/− mice. Previous studies have shown that mitral cells express mGluR1, but not mGluR5. The results suggest that sGCs are more similar to mitral cells than dGCs in terms of mGluR expression. Support: Whitehall Foundation, Howard Univ. New Faculty Research Program, Biomedical Research Foundation of Northwest Louisiana, and U.S. PHS grants 2-S06-GM08016-36 (NIGMS/NIH) and DC03195.

AMPA/Kainate Receptors Drive Rapid Output and Precise Synchrony in Olfactory Bulb Granule Cells

Gamma frequency (30-70 Hz) synchronized oscillatory activity in the olfactory bulb is widely believed to be important for odor detection and discrimination. As in other circuits with "gamma activity," the activity in the bulb is driven by GABAergic interneurons, specifically a class of axonless cells called granule cells. However, bulb granule cells appear to lack some key mechanistic features that promote rapid synchrony in other circuits, including direct electrical interconnections and dominant actions for fast neurotransmitter receptors. At least under "static" stimulus conditions, granule cells are driven by kinetically slow NMDA receptors. Here, I used patch-clamp recordings in rat olfactory bulb slices to better understand mechanisms that shape granule cell activity under "dynamic" stimulus conditions that mimic a natural odor stimulus. During a 4 Hz patterned stimulation of olfactory nerve afferents, activation of single granule cells was primarily controlled by two classes of AMPA/kainate receptor-mediated synaptic inputs derived from output mitral cells. The rapid kinetics of these receptors, together with inactivation of A-type potassium channels, ensured that granule cells had short spike-response times. Studies in cell pairs, moreover, indicated that excitatory inputs could synchronize granule cells on a rapid time scale (2-5 ms), in turn resulting in phase-locked GABA release onto mitral cells. The precision of granule cell synchrony was controlled by the same biophysical mechanisms that promoted rapid single-cell spiking. These studies demonstrate the mechanistic underpinnings that transform a circuit with slow, uncoupled activity under static conditions into a fast, dynamic circuit operating with high precision under physiological conditions.

The Linear Computational Algorithm of Cerebellar Purkinje Cells

The orchestration of simple motor tasks by the cerebellum results in coordinated movement and the maintenance of balance. The cerebellum integrates sensory and cortical information to generate the signals required for the coordinated execution of simple motor tasks. These signals originate in the firing rate of Purkinje cells, each of which integrates sensory and cortical information conveyed by granule cell synaptic inputs. Given the importance of the granule cell input-Purkinje cell output function for cerebellar computation, this algorithm was determined. Using several stimulation paradigms, including those that mimicked patterns of granule cell activity similar to those observed in vivo, we quantified the poststimulus maximum firing rate and number of extra spikes in response to granule cell synaptic input. Both of these parameters linearly encoded the strength of synaptic input when inhibitory synaptic transmission was blocked. This linear algorithm was independent of the location or temporal pattern of synaptic input. With inhibitory synaptic transmission intact, the maximum firing rate, but not the number of extra spikes, encoded the strength of granule cell synaptic input. Furthermore, the maximum firing rate of Purkinje cells linearly encoded the strength of synaptic input whether or not the activation of granule cells resulted in a pause in Purkinje cell firing. On the basis of the data presented, we propose that Purkinje cells encode the strength of granule cell synaptic input in their maximum firing rate with a linear algorithm.

Synaptic organization of the mouse cerebellar cortex in organotypic slice cultures

The cellular and synaptic organization of new born mouse cerebellum maintained in organotypic slice cultures was investigated using immunohistochemical and patch-clamp recording approaches. The histological organization of the cultures shared many features with that observed in situ. Purkinje cells were generally arranged in a monolayer surrounded by a molecular-like neuropil made of Purkinje cell dendritic arborizations. Purkinje cell axons ran between clusters of small round cells identified as granule cells by Kv3.1b potassium channel immunolabelling. The terminal varicosities of the Purkinje cells axons enwrapped presumptive neurons of the cerebellar nuclei whereas their recurrent collaterals were in contact with Purkinje cells and other neurons. Granule cell axons established contacts with Purkinje cell somata and dendrites. Parvalbumin and glutamine acid decarboxylase (GAD) immunohistochemistry revealed the presence of presumptive interneurons throughout the culture. The endings of granule cell axons were observed to be in contact with these interneurons. Similarly, interneurons endings were seen close to Purkinje cells and granule cells. Whole cell recordings from Purkinje cell somata showed AMPA receptor-mediated spontaneous excitatory post-synaptic currents (sEPSCs) and GABAA receptor-mediated spontaneous inhibitory post-synaptic currents (sIPSCs). Similar events were recorded from granule cell somata except that in this neuronal type EPSPs have both a NMDA component and an AMPA component. In addition, pharmacological experiments demonstrated a GABAergic control of granule cell activity and a glutamatergic control of GABAergic neurons by granule cells. This study shows that a functional neuronal network is established in such organotypic cultures even in the absence of the two normal excitatory afferents, the mossy fibers and the climbing fibers.

Sex steroids modulate the signals from volatile female odors in the accessory olfactory bulb of male mice

We previously reported that male mice detect volatile female odors via the accessory olfactory system, and that these odors activate granule cells in the accessory olfactory bulb (AOB) with a characteristic pattern. We also reported that sex steroids modulate the attraction of male mice to volatile female odors. The present study investigated hormonal modulation of signals from volatile female odors in the AOB with c-Fos immunostaining. After intact male mice were exposed to volatile female odors, there were more c-Fos positive cells in the caudal granule cell layer (GCL) than in the rostral GCL of the AOB. This effect was observed 3 days but not 7 days after castration, suggesting that hormonal deficiency causes the reorganization of the AOB after 3 days. There was no difference in the number of c-Fos positive cells between the rostral and caudal GCL of castrated male mice treated with 17 beta-estradiol (E). In contrast, there were more c-Fos positive cells in the caudal GCL than in the rostral GCL of castrated male mice treated with dihydrotestosterone (DHT). In both DHT- and E-treated castrated male mice, there was no difference in the number of c-Fos positive cells between the rostral GCL and caudal GCL. This finding suggests that E disrupts the effect of DHT, and that androgen is required for maintaining the intact neuronal network of the AOB. The present study suggests that sex steroids modulate the signals from volatile female odors in the AOB of male mice.

Metabotropic Glutamate Receptors in the Main Olfactory Bulb Drive Granule Cell-Mediated Inhibition

Main olfactory bulb (MOB) granule cells (GCs) express high levels of the group I metabotropic glutamate receptor (mGluR), mGluR5. We investigated the role of mGluRs in regulating GC activity in rodent MOB slices using whole cell patch-clamp electrophysiology. The group I/II mGluR agonist (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD) or the selective group I agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) depolarized ( approximately 20 mV) and increased the firing rate of GCs. In the presence of ionotropic glutamate and GABA receptor antagonists, DHPG evoked a more modest depolarization ( approximately 8 mV). In voltage clamp, DHPG, but not group II [(2S,2'R,3)-2-(2',3'-dicarboxycyclopropyl)glycine, DCG-IV] or group III [L(+)-2-amino-4-phosphonobutyric acid, L-AP4] mGluR agonists, induced an inward current. The inward current reversed polarity near the potassium equilibrium potential, suggesting mediation by closure of potassium channels. The DHPG-evoked inward current was unaffected by the mGluR1 antagonist (S)-(+)-alpha-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385), was blocked by the group I/II mGluR antagonist (alphaS)-alpha-amino-alpha-[(1S,2S)-2-carboxycyclopropyl]-9H-xanthine-9-propanoic acid (LY341495), and was absent in GCs from mGluR5 knockout mice. LY341495 also attenuated mitral cell-evoked voltage-sensitive dye signals in the external plexiform layer and mitral cell-evoked spikes in GCs. These results suggest that activation of mGluR5 increases GC excitability, an effect that should increase GC-mediated GABAergic inhibition of mitral cells. In support of this: DHPG increased the frequency of spontaneous GABAergic inhibitory postsynaptic currents in mitral cells and LY341495 attenuated the feedback GABAergic postsynaptic potential elicited by intracellular depolarization of mitral cells. Our results suggest that activation of mGluR5 participates in feedforward and/or feedback inhibition at mitral cell to GC dendrodendritic synapses, possibly to modulate lateral inhibition and contrast in the MOB.

Complementary Postsynaptic Activity Patterns Elicited in Olfactory Bulb by Stimulation of Mitral/Tufted and Centrifugal Fiber Inputs to Granule Cells

Main olfactory bulb (MOB) granule cells receive spatially segregated glutamatergic synaptic inputs from the dendrites of mitral/tufted cells as well as from the axons of centrifugal fibers (CFFs) originating in olfactory cortical areas. Dendrodendritic synapses from mitral/tufted cells occur on granule cell distal dendrites in the external plexiform layer (EPL), whereas CFFs preferentially target the somata/proximal dendrites of granule cells in the granule cell layer (GCL). In the present study, tract tracing, and recordings of field potentials and voltage-sensitive dye optical signals were used to map activity patterns elicited by activation of these two inputs to granule cells in mouse olfactory bulb slices. Stimulation of the lateral olfactory tract (LOT) produced a negative field potential in the EPL and a positivity in the GCL. CFF stimulation produced field potentials of opposite polarity in the EPL and GCL to those elicited by LOT. LOT-evoked optical signals appeared in the EPL and spread subsequently to deeper layers, whereas CFF-evoked responses appeared in the GCL and then spread superficially. Evoked responses were reduced by N-methyl-d-aspartate (NMDA) receptor antagonists and completely suppressed by AMPA receptor antagonists. Reduction of extracellular Mg(2+) enhanced the strength and spatiotemporal extent of the evoked responses. These and additional findings indicate that LOT- and CFF-evoked field potentials and optical signals reflect postsynaptic activity in granule cells, with moderate NMDA and dominant AMPA receptor components. Taken together, these results demonstrate that LOT and CFF stimulation in MOB slices selectively activate glutamatergic inputs to the distal dendrites versus somata/proximal dendrites of granule cells.

Plasticity of synaptopodin and the spine apparatus organelle in the rat fascia dentata following entorhinal cortex lesion

Synaptopodin is an actin-associated molecule essential for the formation of a spine apparatus in telencephalic spines. To study whether synaptopodin and the spine apparatus organelle are regulated under conditions of lesion-induced plasticity, synaptopodin and the spine apparatus were analyzed in granule cells of the rat fascia dentata following entorhinal denervation. Confocal microscopy was employed to quantify layer-specific changes in synaptopodin-immunoreactive puncta densities. Electron microscopy was used to quantify layer-specific changes in spine apparatus organelles. Within the denervated middle and outer molecular layers, the layers of deafferentation-induced spine loss, synaptogenesis, and spinogenesis, the density of synaptopodin puncta and the number of spine apparatuses decreased by 4 days postlesion and slowly recovered in parallel with spinogenesis by 180 days postlesion. Within the nondenervated inner molecular layer, the zone without deafferentation-induced spine loss, a rapid loss of synaptopodin puncta and spine apparatuses was also observed. In this layer, spine apparatus densities recovered by 14 days postlesion, in parallel with plastic remodeling at the synaptic level and the postlesional recovery of granule cell activity. These data demonstrate layer-specific changes in the distribution of synaptopodin and the spine apparatus organelle following partial denervation of granule cells: in the layer of spine loss, spine apparatus densities follow spine densities; in the layer of spine maintenance, however, spine apparatus densities appear to be regulated by other signals.

Effect of hypoxia on the transcription pattern of subunit isoforms and the kinetics of cytochrome c oxidase in cortical astrocytes and cerebellar neurons

Brain energy metabolism essentially depends on the availability of oxygen representing the energetic substrate for cytochrome c oxidase (COX). The catalytic activity of mammalian COX is regulated by binding of ATP to the N-terminus of subunit IV. This causes an allosteric inhibition of the enzyme at a high energy level and thus plays an important role in adjusting energy production to cellular energy requirements. We have studied COX activity in cortical astrocytes and cerebellar granule cells after normoxia and hypoxia treatment. Differences in the kinetic behaviour of COX from these two brain cell types can be addressed to a differential, but cell type-specific, expression of the COX subunit IV-2 isoform. Besides COX isoform IV-1, which is ubiquitously transcribed in all mammalian tissues, we also detected low levels of COX isoform IV-2 in cerebellar neurons, but not in cortical astrocytes. Under conditions of oxygen deprivation, transcription of COX IV-2 is induced in astrocytes and further up-regulated in cerebellar granule cells. Elevated transcription levels of the COX IV-2 isoform are accompanied by an abolition of the allosteric inhibition of COX by ATP. We conclude that the presence of the COX isoform IV-2 suppresses the sensitivity of COX to its allosteric regulator ATP and overrules the regulation of COX by the cellular energy level. This suggests a pivotal role of COX as an oxygen sensor for brain function.

Activation of GIRK Channels by Muscarinic Receptors and Group II Metabotropic Glutamate Receptors Suppresses Golgi Cell Activity in the Cochlear Nucleus of Mice

Granule cells and parallel fiber circuits in the dorsal cochlear nucleus (DCN) play a role in integration of multimodal sensory with auditory inputs. The activity of granule cells is regulated through inhibitory connections made by Golgi cells. Golgi cells in turn probably receive parallel fiber inputs and regulate activity of the DCN. We have investigated the electrophysiological properties of Golgi cells using the whole cell patch-clamp method in slices made from transgenic mice that express green fluorescent protein driven by the promotor of metabotropic glutamate receptor subtype 2. Stimulation of auditory nerve fibers (ANFs) and of parallel fibers evoked glutamatergic excitatory postsynaptic currents (EPSC) through AMPA receptors. The strengths and latencies of these inputs differed, however. ANF stimulation evoked EPSCs after 4.7 +/- 0.4 ms, whereas parallel fiber stimulation evoked EPSCs after 1.4 +/- 0.2 ms that were on average 2.5 times as large. The multiple peaks and prolonged activity suggest the presence of polysynaptic connections between ANFs and Golgi cells. Agonists for group II metabotropic glutamate receptors (mGluRs) and for muscarinic receptors induced membrane hyperpolarization and suppressed the firing of Golgi cells by activating G-protein-coupled inward rectifier K(+) (GIRK) channels. These results strongly suggest that Golgi cells were regulated through the combined activities of glutamatergic and cholinergic synapses, which presumably regulated the temporal firing patterns of granule cells and through them the activity of principal cells of the DCN.

N-Methyl-DD-aspartate Blocks Activation of JNK and Mitochondrial Apoptotic Pathway Induced by Potassium Deprivation in Cerebellar Granule Cells

During the postnatal development of cerebellum, lack of excitatory innervation from the mossy fibers results in cerebellar granule cell (CGC) apoptosis during the migration of the cells toward the internal granule cell layer. Accordingly, CGCs die by apoptosis when cultured in physiological KCl concentrations (5 mm; K5), and they survive in the presence of depolarizing conditions such as high KCl concentration (25 mm; K25) or N-methyl-D-aspartate (NMDA). We have recently shown that NMDA is able to exert a long lasting neuroprotective effect when added to immature (2 days in vitro) CGC cultures by inhibition of caspase-3 activity. Here we show that NMDA- and K25-mediated neuroprotection is associated with an increase in the levels of Bcl-2, an inhibition of K5-mediated increase in Bax, and the inhibition of the release of apoptogenic factors from mitochondria such as Smac/DIABLO and cytochrome c. Moreover, we have shown that similar effects are observed when c-Jun N-terminal kinases (JNKs) are inhibited and that treatment of CGC cultures with NMDA blocks K5-mediated JNK activation. These results allow us to postulate that the inhibition of JNK-mediated release of apoptogenic factors from mitochondria is involved in the NMDA protection from K5-mediated apoptosis of CGCs.

A role for mixed lineage kinases in granule cell apoptosis induced by cytoskeletal disruption

Microtubule disruption by colchicine induces apoptosis in selected neuronal populations. However, little is known about the upstream death signalling events mediating the neurotoxicity. We investigated first whether colchicine-induced granule cell apoptosis activates the c-Jun N-terminal kinase (JNK) pathway. Cultured murine cerebellar granule cells were exposed to 1 microm colchicine for 24 h. Activation of the JNK pathway was detected by western blotting as well as immunocytochemistry using antibodies against phospho-c-Jun (p-c-Jun). Next, adult male rats were injected intracerebroventricularly with colchicine (10 microg), and JNK pathway activation in dentate granule cells (DGCs) was detected by antibodies against p-c-Jun. The second part of the study tested the involvement of mixed lineage kinases (MLK) as upstream activators of the JNK pathway in colchicine toxicity, using CEP-1347, a potent MLK inhibitor. In vitro, significant inhibition of the JNK pathway, activated by colchicine, was achieved by 100-300 nm CEP-1347, which blocked both activation of cell death proteases and apoptosis. Moreover, CEP-1347 markedly delayed neurite fragmentation and cell degeneration. In vivo, CEP-1347 (1 mg/kg) significantly prevented p-c-jun increase following injection of colchicine, and enhanced survival of DGCs. We conclude that colchicine-induced neuronal apoptosis involves the JNK/MLK pathway, and that protection of granule cells can be achieved by MLK inhibition.

The Hyperinhibition Hypothesis in Epileptogenesis: An Assessment of the Evidence

Hippocampal Granule Cell Activity and c-fos Expression During Spontaneous Seizures in Awake, Chronically Epileptic, Pilocarpine-treated Rats: Implications for Hippocampal Epileptogenesis Harvey BD, Sloviter RS J Comp Neurol 2005;488(4):442–463 The process of postinjury hippocampal epileptogenesis may involve gradually developing dentate granule cell hyperexcitability caused by neuron loss and synaptic reorganization. We tested this hypothesis by repeatedly assessing granule cell excitability after pilocarpine-induced status epilepticus (SE) and monitoring granule cell behavior during 235 spontaneous seizures in awake, chronically implanted rats. During the first week post-SE, granule cells exhibited diminished paired-pulse suppression and decreased seizure discharge thresholds in response to afferent stimulation. Spontaneous seizures often began during the first week after SE, recruited granule cell discharges that followed behavioral seizure onsets, and evoked c-fos expression in all hippocampal neurons. Paired-pulse suppression and epileptiform discharge thresholds increased gradually after SE, eventually becoming abnormally elevated. In the chronic epileptic state, interictal granule cell hyperinhibition extended to the ictal state; granule cells did not discharge synchronously before any of 191 chronic seizures. Instead, granule cells generated only low-frequency voltage fluctuations (presumed “field excitatory postsynaptic potentials”) during 89% of chronic seizures. Granule cell epileptiform discharges were recruited during 11% of spontaneous seizures, but these occurred only at the end of each behavioral seizure. Hippocampal c-fos after chronic seizures was expressed primarily by inhibitory interneurons. Thus, granule cells became progressively less excitable, rather than hyperexcitable, as mossy fiber sprouting progressed and did not initiate the spontaneous behavioral seizures. These findings raise doubts about dentate granule cells as a source of spontaneous seizures in rats subjected to prolonged SE and suggest that dentate gyrus neuron loss and mossy fiber sprouting are not primary epileptogenic mechanisms in this animal model.

Temporal patterns of fos expression in the dentate gyrus after spontaneous seizures in a mouse model of temporal lobe epilepsy.

Identifying the brain regions and neuronal cell types that become active at the time of spontaneous seizures remains an important challenge for epilepsy research, and the involvement of dentate granule cells in early seizure events continues to be debated. Although Fos expression is commonly used to evaluate patterns of neuronal activation, there have been few studies of Fos localization after spontaneous seizures. Thus, in a pilocarpine model of recurrent seizures in C57BL/6 mice, Fos expression was examined at multiple time points after spontaneous seizures to follow the temporal and spatial patterns of Fos activation. By 15 min after the beginning of a spontaneous behavioral seizure, Fos labeling was evident in dentate granule cells. This labeling was particularly striking because of its wide extent and relatively uniform appearance in the granule cell layer. At later time points, from 30 min to 4 h after a spontaneous seizure, Fos labeling was also detected in interneurons within the dentate gyrus and in widespread regions of the temporal lobe. Interestingly, the timing of Fos activation appeared to differ among different types of GABAergic interneurons in the dentate gyrus, with labeling of parvalbumin neurons along the base of the granule cell layer preceding that of GABA neurons in the molecular layer. The findings in this mouse model are consistent with previous suggestions that spontaneous seizures in temporal lobe epilepsy may result from a periodic breakdown of the normal filter functions of the dentate gyrus and a resulting increase in hypersynchronous activity of dentate granule cells.