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At least 3 months after systemic treatment with pilocarpine to induce status epilepticus, Long-Evans and Sprague-Dawley rats were video-EEG monitored for seizures continuously for 1 month. Rats were then perfused, hippocampi were processed for Nissl staining, and hilar neurons were quantified. Seizure frequency in Long-Evans rats was 1/10th of that in Sprague-Dawley rats, and more variable. Hilar neuron loss was also less severe in Long-Evans rats. However, there was no correlation between hilar neuron loss and seizure frequency in either strain. The low and variable seizure frequency suggests limited usefulness of pilocarpine-treated Long-Evans rats for some epilepsy experiments., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The goal of this study was to test different combinations of acoustic pressure and doses of quinolinic acid (QA) for producing a focal neuronal lesion in the murine hippocampus without causing unwanted damage to adjacent brain structures. Sixty male CD-1 mice were divided into 12 groups that underwent magnetic resonance-guided focused ultrasound at high (0.67 MPa), medium (0.5 MPa) and low (0.33 MPa) acoustic peak negative pressures and received QA at high (0.012 mmol), medium (0.006 mmol) and low (0.003 mmol) dosages. Neuronal loss occurred only when magnetic resonance-guided focused ultrasound with adequate acoustic power (0.67 or 0.5 MPa) was combined with QA. The animals subjected to the highest acoustic power had larger lesions than those treated with medium acoustic power, but two mice had evidence of bleeding. When the intermediate acoustic power was used, medium and high dosages of QA produced lesions larger than those produced by the low dosage., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Temporal lobe epilepsy is a common and challenging clinical problem, and its pathophysiological mechanisms remain unclear. One possibility is insufficient inhibition in the hippocampal formation where seizures tend to initiate. Normally, hippocampal basket cells provide strong and reliable synaptic inhibition at principal cell somata. In a rat model of temporal lobe epilepsy, basket cell-to-granule cell (BC→GC) synaptic transmission is more likely to fail, but the underlying cause is unknown. At some synapses, probability of release correlates with bouton size, active zone area, and number of docked vesicles. The present study tested the hypothesis that impaired GABAergic transmission at BC→GC synapses is attributable to ultrastructural changes. Boutons making axosomatic symmetric synapses in the granule cell layer were reconstructed from serial electron micrographs. BC→GC boutons were predicted to be smaller in volume, have fewer and smaller active zones, and contain fewer vesicles, including fewer docked vesicles. Results revealed the opposite. Compared with controls, epileptic pilocarpine-treated rats displayed boutons with over twice the average volume, active zone area, total vesicles, and docked vesicles and with more vesicles closer to active zones. Larger active zones in epileptic rats are consistent with previous reports of larger amplitude miniature IPSCs and larger BC→GC quantal size. Results of this study indicate that transmission failures at BC→GC synapses in epileptic pilocarpine-treated rats are not attributable to smaller boutons or fewer docked vesicles. Instead, processes following vesicle docking, including priming, Ca(2+) entry, or Ca(2+) coupling with exocytosis, might be responsible., Significance Statement: One in 26 people develops epilepsy, and temporal lobe epilepsy is a common form. Up to one-third of patients are resistant to currently available treatments. This study tested a potential underlying mechanism for previously reported impaired inhibition in epileptic animals at basket cell-to-granule cell (BC→GC) synapses, which normally are reliable and strong. Electron microscopy was used to evaluate 3D ultrastructure of BC→GC synapses in a rat model of temporal lobe epilepsy. The hypothesis was that impaired synaptic transmission is attributable to smaller boutons, smaller synapses, and abnormally low numbers of synaptic vesicles. Results revealed the opposite. These findings suggest that impaired transmission at BC→GC synapses in epileptic rats is attributable to later steps in exocytosis following vesicle docking., (Copyright © 2016 the authors 0270-6474/16/363295-14$15.00/0.)

Previous studies suggest that spontaneous seizures in patients with temporal lobe epilepsy might be preceded by increased action potential firing of hippocampal neurons. Preictal activity is potentially important because it might provide new opportunities for predicting when a seizure is about to occur and insight into how spontaneous seizures are generated. We evaluated local field potentials and unit activity of single, putative excitatory neurons in the subiculum, CA1, CA3, and dentate gyrus of the dorsal hippocampus in epileptic pilocarpine-treated rats as they experienced spontaneous seizures. Average action potential firing rates of neurons in the subiculum, CA1, and dentate gyrus, but not CA3, increased significantly and progressively beginning 2-4 min before locally recorded spontaneous seizures. In the subiculum, CA1, and dentate gyrus, but not CA3, 41-57% of neurons displayed increased preictal activity with significant consistency across multiple seizures. Much of the increased preictal firing of neurons in the subiculum and CA1 correlated with preictal theta activity, whereas preictal firing of neurons in the dentate gyrus was independent of theta. In addition, some CA1 and dentate gyrus neurons displayed reduced firing rates preictally. These results reveal that different hippocampal subregions exhibit differences in the extent and potential underlying mechanisms of preictal activity. The finding of robust and significantly consistent preictal activity of subicular, CA1, and dentate neurons in the dorsal hippocampus, despite the likelihood that many seizures initiated in other brain regions, suggests the existence of a broader neuronal network whose activity changes minutes before spontaneous seizures initiate., (Copyright © 2014 the authors 0270-6474/14/3416671-17$15.00/0.)

Purpose: The role of granule cell axon (mossy fiber) sprouting in temporal lobe epileptogenesis is unclear and controversial. Rapamycin suppresses mossy fiber sprouting, but its reported effects on seizure frequency are mixed. The present study used high-dose rapamycin to more completely block mossy fiber sprouting and to measure the effect on seizure frequency., Methods: Mice were treated with pilocarpine to induce status epilepticus. Beginning 24 h later and continuing for 2 months, vehicle or rapamycin (10 mg/kg/day) was administered. Starting 1 month after status epilepticus, mice were monitored by video 9 h per day, every day, for 1 month to measure the frequency of spontaneous motor seizures. At the end of seizure monitoring, a subset of mice was prepared for anatomic analysis. Mossy fiber sprouting was measured as the proportion of the granule cell layer and molecular layer that displayed black labeling in Timm-stained sections., Key Findings: Extensive mossy fiber sprouting developed in mice that experienced status epilepticus and were treated with vehicle. In rapamycin-treated mice, mossy fiber sprouting was blocked almost to the level of naive controls. Seizure frequency was similar in vehicle-treated and rapamycin-treated mice., Significance: These findings suggest that mossy fiber sprouting is not necessary for epileptogenesis in the mouse pilocarpine model. They also reveal that rapamycin does not have antiseizure or antiepileptogenic effects in this model., (Wiley Periodicals, Inc. © 2013 International League Against Epilepsy.)

Mossy cells are likely to contribute to normal hippocampal function and to the pathogenesis of neurologic disorders that involve the hippocampus, including epilepsy. Mossy cells are the least well-characterized excitatory neurons in the hippocampus. Their somatic and dendritic morphology has been described qualitatively but not quantitatively. In the present study rat mossy cells were labeled intracellularly with biocytin in vivo. Somatic and dendritic structure was reconstructed three-dimensionally. For comparison, granule cells, CA3 pyramidal cells, and CA1 pyramidal cells were labeled and analyzed using the same approach. Among the four types of hippocampal neurons, granule cells had the smallest somata, fewest primary dendrites and dendritic branches, and shortest total dendritic length. CA1 pyramidal cells had the most dendritic branches and longest total dendritic length. Mossy cells and CA3 pyramidal cells both had large somata and similar total dendritic lengths. However, mossy cell dendrites branched less than CA3 pyramidal cells, especially close to the soma. These findings suggest that mossy cells have dendritic features that are not identical to any other type of hippocampal neuron. Therefore, electrotonic properties that depend on soma-dendritic structure are likely to be distinct in mossy cells compared to other neurons., (Wiley Periodicals, Inc. © 2012 International League Against Epilepsy.)

Neurons encode information by firing spikes in isolation or bursts and propagate information by spike-triggered neurotransmitter release that initiates synaptic transmission. Isolated spikes trigger neurotransmitter release unreliably but with high temporal precision. In contrast, bursts of spikes trigger neurotransmission reliably (i.e., boost transmission fidelity), but the resulting synaptic responses are temporally imprecise. However, the relative physiological importance of different spike-firing modes remains unclear. Here, we show that knockdown of synaptotagmin-1, the major Ca(2+) sensor for neurotransmitter release, abrogated neurotransmission evoked by isolated spikes but only delayed, without abolishing, neurotransmission evoked by bursts of spikes. Nevertheless, knockdown of synaptotagmin-1 in the hippocampal CA1 region did not impede acquisition of recent contextual fear memories, although it did impair the precision of such memories. In contrast, knockdown of synaptotagmin-1 in the prefrontal cortex impaired all remote fear memories. These results indicate that different brain circuits and types of memory employ distinct spike-coding schemes to encode and transmit information., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Purpose: Dentate granule cell axon (mossy fiber) sprouting creates an aberrant positive-feedback circuit that might be epileptogenic. Presumably, mossy fiber sprouting is initiated by molecular signals, but it is unclear whether they are expressed transiently or persistently. If transient, there might be a critical period when short preventative treatments could permanently block mossy fiber sprouting. Alternatively, if signals persist, continuous treatment would be necessary. The present study tested whether temporary treatment with rapamycin has long-term effects on mossy fiber sprouting., Methods: Mice were treated daily with 1.5 mg/kg rapamycin or vehicle (i.p.) beginning 24 h after pilocarpine-induced status epilepticus. Mice were perfused for anatomic evaluation immediately after 2 months of treatment ("0 delay") or after an additional 6 months without treatment ("6-month delay"). One series of sections was Timm-stained, and an adjacent series was Nissl-stained. Stereologic methods were used to measure the volume of the granule cell layer plus molecular layer and the Timm-positive fraction. Numbers of Nissl-stained hilar neurons were estimated using the optical fractionator method., Key Findings: At 0 delay, rapamycin-treated mice had significantly less black Timm staining in the granule cell layer plus molecular layer than vehicle-treated animals. However, by 6-month delay, Timm staining had increased significantly in mice that had been treated with rapamycin. Percentages of the granule cell layer plus molecular layer that were Timm-positive were high and similar in 0 delay vehicle-treated, 6-month delay vehicle-treated, and 6-month delay rapamycin-treated mice. Extent of hilar neuron loss was similar among all groups that experienced status epilepticus and, therefore, was not a confounding factor. Compared to naive controls, average volume of the granule cell layer plus molecular layer was larger in 0 delay vehicle-treated mice. The hypertrophy was partially suppressed in 0 delay rapamycin-treated mice. However, 6-month delay vehicle- and 6-month delay rapamycin-treated animals had similar average volumes of the granule cell layer plus molecular layer that were significantly larger than those of all other groups., Significance: Status epilepticus-induced mossy fiber sprouting and dentate gyrus hypertrophy were suppressed by systemic treatment with rapamycin but resumed after treatment ceased. These findings suggest that molecular signals that drive mossy fiber sprouting and dentate gyrus hypertrophy might persist for >2 months after status epilepticus in mice. Therefore, prolonged or continuous treatment might be required to permanently suppress mossy fiber sprouting., (Wiley Periodicals, Inc. © 2011 International League Against Epilepsy.)

Many patients with temporal lobe epilepsy display neuron loss in the dentate gyrus. One potential epileptogenic mechanism is loss of GABAergic interneurons and inhibitory synapses with granule cells. Stereological techniques were used to estimate numbers of gephyrin-positive punctae in the dentate gyrus, which were reduced short-term (5 days after pilocarpine-induced status epilepticus) but later rebounded beyond controls in epileptic rats. Stereological techniques were used to estimate numbers of synapses in electron micrographs of serial sections processed for postembedding GABA-immunoreactivity. Adjacent sections were used to estimate numbers of granule cells and glutamic acid decarboxylase-positive neurons per dentate gyrus. GABAergic neurons were reduced to 70% of control levels short-term, where they remained in epileptic rats. Integrating synapse and cell counts yielded average numbers of GABAergic synapses per granule cell, which decreased short-term and rebounded in epileptic animals beyond control levels. Axo-shaft and axo-spinous GABAergic synapse numbers in the outer molecular layer changed most. These findings suggest interneuron loss initially reduces numbers of GABAergic synapses with granule cells, but later, synaptogenesis by surviving interneurons overshoots control levels. In contrast, the average number of excitatory synapses per granule cell decreased short-term but recovered only toward control levels, although in epileptic rats excitatory synapses in the inner molecular layer were larger than in controls. These findings reveal a relative excess of GABAergic synapses and suggest that reports of reduced functional inhibitory synaptic input to granule cells in epilepsy might be attributable not to fewer but instead to abundant but dysfunctional GABAergic synapses., (2009 Wiley-Liss, Inc.)

In temporal lobe epilepsy, seizures initiate in or near the hippocampus, which frequently displays loss of neurons, including inhibitory interneurons. It is unclear whether surviving interneurons function normally, are impaired, or develop compensatory mechanisms. We evaluated GABAergic interneurons in the hilus of the dentate gyrus of epileptic pilocarpine-treated GIN mice, specifically a subpopulation of somatostatin interneurons that expresses enhanced green fluorescence protein (GFP). GFP-immunocytochemistry and stereological analyses revealed substantial loss of GFP-positive hilar neurons (GPHNs) but increased GFP-positive axon length per dentate gyrus in epileptic mice. Individual biocytin-labeled GPHNs in hippocampal slices from epileptic mice also had larger somata, more axon in the molecular layer, and longer dendrites than controls. Dual whole-cell patch recording was used to test for monosynaptic connections from hilar GPHNs to granule cells. Unitary IPSCs (uIPSCs) recorded in control and epileptic mice had similar average rise times, amplitudes, charge transfers, and decay times. However, the probability of finding monosynaptically connected pairs and evoking uIPSCs was 2.6 times higher in epileptic mice compared to controls. Together, these findings suggest that surviving hilar somatostatin interneurons enlarge, extend dendrites, sprout axon collaterals in the molecular layer, and form new synapses with granule cells. These epilepsy-related changes in cellular morphology and connectivity may be mechanisms for surviving hilar interneurons to inhibit more granule cells and compensate for the loss of vulnerable interneurons.

Dentate granule cell axon (mossy fiber) sprouting is a common abnormality in patients with temporal lobe epilepsy. Mossy fiber sprouting creates an aberrant positive-feedback network among granule cells that does not normally exist. Its role in epileptogenesis is unclear and controversial. If it were possible to block mossy fiber sprouting from developing after epileptogenic treatments, its potential role in the pathogenesis of epilepsy could be tested. Previous attempts to block mossy fiber sprouting have been unsuccessful. The present study targeted the mammalian target of rapamycin (mTOR) signaling pathway, which regulates cell growth and is blocked by rapamycin. Rapamycin was focally, continuously, and unilaterally infused into the dorsal hippocampus for prolonged periods beginning within hours after rats sustained pilocarpine-induced status epilepticus. Infusion for 1 month reduced aberrant Timm staining (a marker of mossy fibers) in the granule cell layer and molecular layer. Infusion for 2 months inhibited mossy fiber sprouting more. However, after rapamycin infusion ceased, aberrant Timm staining developed and approached untreated levels. When onset of infusion began after mossy fiber sprouting had developed for 2 months, rapamycin did not reverse aberrant Timm staining. These findings suggest that inhibition of the mTOR signaling pathway suppressed development of mossy fiber sprouting. However, suppression required continual treatment, and rapamycin treatment did not reverse already established axon reorganization.

Temporal lobe epilepsy is common and difficult to treat. Reduced inhibition of dentate granule cells may contribute. Basket cells are important inhibitors of granule cells. Excitatory synaptic input to basket cells and unitary IPSCs (uIPSCs) from basket cells to granule cells were evaluated in hippocampal slices from a rat model of temporal lobe epilepsy. Basket cells were identified by electrophysiological and morphological criteria. Excitatory synaptic drive to basket cells, measured by mean charge transfer and frequency of miniature EPSCs, was significantly reduced after pilocarpine-induced status epilepticus and remained low in epileptic rats, despite mossy fiber sprouting. Paired recordings revealed higher failure rates and a trend toward lower amplitude uIPSCs at basket cell-to-granule cell synapses in epileptic rats. Higher failure rates were not attributable to excessive presynaptic inhibition of GABA release by activation of muscarinic acetylcholine or GABA(B) receptors. High-frequency trains of action potentials in basket cells generated uIPSCs in granule cells to evaluate readily releasable pool (RRP) size and resupply rate of recycling vesicles. Recycling rate was similar in control and epileptic rats. However, quantal size at basket cell-to-granule cell synapses was larger and RRP size smaller in epileptic rats. Therefore, in epileptic animals, basket cells receive less excitatory synaptic drive, their pools of readily releasable vesicles are smaller, and transmission failure at basket cell-to-granule cell synapses is increased. These findings suggest dysfunction of the dentate basket cell circuit could contribute to hyperexcitability and seizures.

In patients with temporal lobe epilepsy some dentate granule cells develop basal dendrites. The extent of excitatory synaptic input to basal dendrites is unclear, nor is it known whether basal dendrites receive inhibitory synapses. We used biocytin to intracellularly label individual granule cells with basal dendrites in epileptic pilocarpine-treated rats. An average basal dendrite had 3.9 branches, was 612 microm long, and accounted for 16% of a cell's total dendritic length. In vivo intracellular labeling and postembedding GABA-immunocytochemistry were used to evaluate synapses with basal dendrites reconstructed from serial electron micrographs. An average of 7% of 1,802 putative synapses were formed by GABA-positive axon terminals, indicating synaptogenesis by interneurons. Ninety-three percent of the identified synapses were GABA-negative. Most GABA-negative synapses were with spines, but at least 10% were with dendritic shafts. Multiplying basal dendrite length/cell and synapse density yielded an estimate of 180 inhibitory and 2,140 excitatory synapses per granule cell basal dendrite. Based on previous estimates of synaptic input to granule cells in control rats, these findings suggest an average basal dendrite receives approximately 14% of the total inhibitory and 19% of excitatory synapses of a cell. These findings reveal that basal dendrites are a novel source of inhibitory input, but they primarily receive excitatory synapses., ((c) 2008 Wiley-Liss, Inc.)

Although much is known about persistent molecular, cellular, and circuit changes associated with temporal lobe epilepsy, mechanisms of seizure onset remain unclear. The dentate gyrus displays many persistent epilepsy-related abnormalities and is in the mesial temporal lobe where seizures initiate in patients. However, little is known about seizure-related activity of individual neurons in the dentate gyrus. We used tetrodes to record action potentials of multiple, single granule cells before and during spontaneous seizures in epileptic pilocarpine-treated rats. Subsets of granule cells displayed four distinct activity patterns: increased firing before seizure onset, decreased firing before seizure onset, increased firing only after seizure onset, and unchanged firing rates despite electrographic seizure activity in the immediate vicinity. No cells decreased firing rate immediately after seizure onset. During baseline periods between seizures, action potential waveforms and firing rates were similar among the four subsets of granule cells in epileptic rats and in granule cells of control rats. The mean normalized firing rate of granule cells whose firing rates increased before seizure onset deviated from baseline earliest, beginning 4 min before dentate gyrus electrographic seizure onset, and increased progressively, more than doubling by seizure onset. It is generally assumed that neuronal firing rates increase abruptly and synchronously only when electrographic seizures begin. However, these findings show heterogeneous and gradually building changes in activity of individual granule cells minutes before spontaneous seizures.

NeuN (Neuronal Nuclei), the neuron-specific marker of nuclear protein is used extensively in histological procedures to identify major cell-types in adult vertebrate nervous systems of a variety of species including rodents and humans. Some notable exceptions (i.e., NeuN-negative neurons) include Purkinje cells in cerebellum, mitral cells in olfactory bulb, and photoreceptors in retina. Here we report that neurons in gerbil (Meriones unguiculatus) substantia nigra pars reticulata (SNr), whose "neuronal" phenotype was confirmed via electrophysiology, biocytin-labeling, histology, and in situ hybridization, are also devoid of NeuN-immunoreactivity as assayed with the widely used monoclonal antibody A60. Immunohistochemistry of rat SNr using the same antibody yielded robust staining. These data suggest lack of NeuN-immonoreactivity observed in certain cell-types and brain regions can be species-specific.

Temporal lobe epilepsy is often associated with pathological changes in the dentate gyrus, and such changes may be more common in humans than in some nonprimate species. To examine species-specific characteristics that might predispose the dentate gyrus to epileptogenic damage, we evaluated recurrent excitation of granule cells with and without basal dendrites in macaque monkeys, measured miniature inhibitory postsynaptic currents (mIPSCs) of granule cells in macaque monkeys and compared them to rats, and estimated the granule cell-to-interneuron ratio in macaque monkeys and rats. In hippocampal slices from monkeys, whole-cell patch recording revealed antidromically evoked excitatory PSCs that were four times larger and inhibitory PSCs that were over two times larger in granule cells with basal dendrites than without. These findings suggest that granule cells with basal dendrites receive more recurrent excitation and, to a lesser degree, more recurrent inhibition. Miniature IPSC amplitude was slightly larger in monkey granule cells with basal dendrites than in those without, but mIPSC frequency was similar and only 26% of that reported for rats. In situ hybridization for glutamic acid decarboxylase and immunocytochemistry for somatostatin, parvalbumin, and neuronal nuclei revealed interneuron proportions and distributions in monkeys that were similar to those reported for rats. However, the interneuron-to-granule cell ratio was lower in monkeys (1:28) than in rats (1:11). These findings suggest that in the primate dentate gyrus, recurrent excitation is enhanced and inhibition is reduced compared with rodents. These primate characteristics may contribute to the susceptibility of the human dentate gyrus to epileptogenic injuries.

Temporal lobe epilepsy is the most common type of epilepsy in adults, and its underlying mechanisms are unclear. To investigate how the medial entorhinal cortex might contribute to temporal lobe epilepsy, we evaluated the histology and electrophysiology of slices from rats 3-7 d after an epileptogenic injury (pilocarpine-induced status epilepticus). Nissl staining, NeuN immunocytochemistry, and in situ hybridization for GAD65 mRNA were used to verify the preferential loss of glutamatergic neurons and the relative sparing of GABAergic interneurons in layer III. From slices adjacent to those that were used for anatomy, we obtained whole-cell patch recordings from layer II medial entorhinal cortical neurons. Recordings under current-clamp conditions revealed similar intrinsic electrophysiological properties (resting membrane potential, input resistance, single spike, and repetitive firing properties) to those of controls. Spontaneous IPSCs were less frequent (68% of controls), smaller in amplitude (57%), and transferred less charge (51%) than in controls. However, the frequency, amplitude, and rise time of miniature IPSCs were normal. These findings suggest that after epileptogenic injuries the layer II entorhinal cortical neurons receive less GABA(A) receptor-mediated synaptic input because presynaptic inhibitory interneurons become less active. To investigate the possible consequences of reduced spontaneous inhibitory input to layer II neurons, we recorded field potentials in the dentate gyrus, their major synaptic target. At 5 d after pilocarpine-induced status epilepticus the spontaneous field potentials recorded in vivo were over three times more frequent than in controls. These findings suggest that an epileptogenic injury reduces inhibition of layer II neurons and results in excessive synaptic input to the dentate gyrus.

Patients and models of temporal lobe epilepsy have fewer inhibitory interneurons in the dentate gyrus than controls, but it is unclear whether granule cell inhibition is reduced. We report the loss of GABAergic inhibition of granule cells in the temporal dentate gyrus of pilocarpine-induced epileptic rats. In situ hybridization for GAD65 mRNA and immunocytochemistry for parvalbumin and somatostatin confirmed the loss of inhibitory interneurons. In epileptic rats, granule cells had prolonged EPSPs, and they discharged more action potentials than controls. Although the conductances of evoked IPSPs recorded in normal ACSF were not significantly reduced and paired-pulse responses showed enhanced inhibition of granule cells from epileptic rats, more direct measures of granule cell inhibition revealed significant deficiencies. In granule cells from epileptic rats, evoked monosynaptic IPSP conductances were <40% of controls, and the frequency of GABA(A) receptor-mediated spontaneous and miniature IPSCs (mIPSCs) was <50% of controls. Within 3-7 d after pilocarpine-induced status epilepticus, miniature IPSC frequency had decreased, and it remained low, without functional evidence of compensatory synaptogenesis by GABAergic axons in chronically epileptic rats. Both parvalbumin- and somatostatin-immunoreactive interneuron numbers and the frequency of both fast- and slow-rising GABA(A) receptor-mediated mIPSCs were reduced, suggesting that loss of inhibitory synaptic input to granule cells occurred at both proximal/somatic and distal/dendritic sites. Reduced granule cell inhibition in the temporal dentate gyrus preceded the onset of spontaneous recurrent seizures by days to weeks, so it may contribute, but is insufficient, to cause epilepsy.

Little is known about the morphological characteristics and intracellular electrophysiological properties of neurons in the primate hippocampus and dentate gyrus. We have therefore begun a program of studies using intracellular recording and biocytin labeling in hippocampal slices from macaque monkeys. In the current study, we investigated mossy cells and proximal CA3 pyramidal cells. As in rats, macaque mossy cells display fundamentally different traits than proximal CA3 pyramidal cells. Interestingly, macaque mossy cells and CA3 pyramidal neurons display some morphological differences from those in rats. Macaque monkey mossy cells extend more dendrites into the molecular layer of the dentate gyrus, have more elaborate thorny excrescences on their proximal dendrites, and project more axon collaterals into the CA3 region. In macaques, three types of proximal CA3 pyramidal cells are found: classical pyramidal cells, neurons with their dendrites confined to the CA3 pyramidal cell layer, and a previously undescribed cell type, the "dentate" CA3 pyramidal cell, whose apical dendrites extend into and ramify within the hilus, granule cell layer, and molecular layer of the dentate gyrus. The basic electrophysiological properties of mossy cells and proximal CA3 cells are similar to those reported for the rodent. Mossy cells have a higher frequency of large amplitude spontaneous depolarizing postsynaptic potentials, and proximal CA3 pyramidal cells are more likely to discharge bursts of action potentials. Although mossy cells and CA3 pyramidal cells in macaque monkeys display many morphological and electrophysiological features described in rodents, these findings highlight significant species differences, with more heterogeneity and the potential for richer interconnections in the primate hippocampus., (Copyright 2001 Wiley-Liss, Inc.)

Patients with temporal lobe epilepsy display neuron loss in the hilus of the dentate gyrus. This has been proposed to be epileptogenic by a variety of different mechanisms. The present study examines the specificity and extent of neuron loss in the dentate gyrus of kainate-treated rats, a model of temporal lobe epilepsy. Kainate-treated rats lose an average of 52% of their GAD-negative hilar neurons (putative mossy cells) and 13% of their GAD-positive cells (GABAergic interneurons) in the dentate gyrus. Interneuron loss is remarkably specific; 83% of the missing GAD-positive neurons are somatostatin-immunoreactive. Of the total neuron loss in the hilus, 97% is attributed to two cell types-mossy cells and somatostatinergic interneurons. The retrograde tracer wheat germ agglutinin (WGA)-apoHRP-gold was used to identify neurons with appropriate axon projections for generating lateral inhibition. Previously, it was shown that lateral inhibition between regions separated by 1 mm persists in the dentate gyrus of kainate-treated rats with hilar neuron loss. Retrogradely labeled GABAergic interneurons are found consistently in sections extending 1 mm septotemporally from the tracer injection site in control and kainate-treated rats. Retrogradely labeled putative mossy cells are found up to 4 mm from the injection site, but kainate-treated rats have fewer than controls, and in several kainate-treated rats virtually all of these cells are missing. These findings support hypotheses of temporal lobe epileptogenesis that involve mossy cell and somatostatinergic neuron loss and suggest that lateral inhibition in the dentate gyrus does not require mossy cells but, instead, may be generated directly by GABAergic interneurons.

The pathophysiological bases of cognitive, motor, and behavioral abnormalities in patients infected with the human immunodeficiency virus (HIV-1) remain largely unknown. To test the possibility that changes in hippocampal neuronal structure may contribute to these neurologic abnormalities, we examined the brains of cats infected with the feline immunodeficiency virus (FIV), an animal model of HIV-1 infection. We evaluated the dentate gyrus by using Timm's staining to estimate the extent of granule cell axon reorganization and by using Nissl staining, immunocytochemistry, and the optical fractionator method to estimate changes in the number of different neuronal subtypes. FIV-infected cats had abnormally high amounts of Timm's staining in the inner molecular layer and granule cell layer and loss of Nissl-stained, somatostatin-immunoreactive, and parvalbumin-immunoreactive neurons in the hilus. An inverse correlation existed between hilar neuron numbers and extent of aberrant Timm's staining. Increased Timm's staining and hilar neuron loss occurred throughout the septotemporal axis of the hippocampus. This type of neuronal loss and synaptic reorganization may provide an anatomic basis for some of the neurologic symptoms found in FIV-infected cats and HIV-infected humans., (Copyright 1999 Wiley-Liss, Inc.)

In vivo intracellular recording and labeling in kainate-induced epileptic rats was used to address questions about granule cell axon reorganization in temporal lobe epilepsy. Individually labeled granule cells were reconstructed three dimensionally and in their entirety. Compared with controls, granule cells in epileptic rats had longer average axon length per cell; the difference was significant in all strata of the dentate gyrus including the hilus. In epileptic rats, at least one-third of the granule cells extended an aberrant axon collateral into the molecular layer. Axon projections into the molecular layer had an average summed length of 1 mm per cell and spanned 600 microm of the septotemporal axis of the hippocampus-a distance within the normal span of granule cell axon collaterals. These findings in vivo confirm results from previous in vitro studies. Surprisingly, 12% of the granule cells in epileptic rats, and none in controls, extended a basal dendrite into the hilus, providing another route for recurrent excitation. Consistent with recurrent excitation, many granule cells (56%) in epileptic rats displayed a long-latency depolarization superimposed on a normal inhibitory postsynaptic potential. These findings demonstrate changes, occurring at the single-cell level after an epileptogenic hippocampal injury, that could result in novel, local, recurrent circuits.

The hippocampi of species commonly used for in vitro physiologic studies were examined to determine if there were species-specific and regional differences in somatostatin immunoreactivity. The distributions of somatostatin-immunoreactive somata and fiber plexuses were determined, and the concentration of somatostatin along the septotemporal axis of the hippocampus was measured using a radioimmunoassay. There are many similarities in the patterns of somatostatin immunoreactivity in the hippocampi of mice, rats, guinea pigs, and rabbits. All species had a relatively even distribution of somatostatin-positive perikarya across three fields of the hippocampus (dentate gyrus, CA3, and CA1-2), a similar distribution of somatostatin-immunoreactive perikarya across the strata of the CA1-2 field and the dentate gyrus; and more somatostatin-positive cells in temporal than in septal hippocampus. However, there are species-specific differences in the distribution of somatostatin-immunoreactive perikarya across the strata of CA3. In addition, unlike the other species examined, mice appeared not to have a somatostatin-immunoreactive fiber plexus in the molecular layer of the dentate gyrus. The functional significance of these differences remains to be determined.

The ipsilateral associational pathway connects different septotemporal levels of the dentate gyrus. Neurons of the dentate hilus project hundreds of micrometers from the cells of origin to the inner molecular layer. The authors hypothesized that mossy cells, the major cell type of the hilus, also project locally to the inner molecular layer. Within a 400 microns slice, mossy cells were (1) recorded intracellularly while the inner molecular layer was stimulated to test for antidromic responses, and (2) labeled with biocytin and examined with light and electron microscopy for axonal projections into the molecular layer. The authors found that mossy cells can be antidromically activated by inner molecular layer stimulation and that axonal projections to the molecular layer can be visualized within a 400 microns hippocampal slice. In 13 of 19 intracellularly labeled and electrophysiologically characterized mossy cells, collaterals could be traced into the molecular layer. These results suggest that mossy cells contribute to the ipsilateral associational pathway and also participate in local recurrent circuitry to influence granule cell activity.