Seizure frequency correlates with loss of dentate gyrus GABAergic neurons in a mouse model of temporal lobe epilepsy.

Epilepsy occurs in one of 26 people. Temporal lobe epilepsy is common and can be difficult to treat effectively. It can develop after brain injuries that damage the hippocampus. Multiple pathophysiological mechanisms involving the hippocampal dentate gyrus have been proposed. This study evaluated a mouse model of temporal lobe epilepsy to test which pathological changes in the dentate gyrus correlate with seizure frequency and help prioritize potential mechanisms for further study. FVB mice (n = 127) that had experienced status epilepticus after systemic treatment with pilocarpine 31-61 days earlier were video-monitored for spontaneous, convulsive seizures 9 hr/day every day for 24-36 days. Over 4,060 seizures were observed. Seizure frequency ranged from an average of one every 3.6 days to one every 2.1 hr. Hippocampal sections were processed for Nissl stain, Prox1-immunocytochemistry, GluR2-immunocytochemistry, Timm stain, glial fibrillary acidic protein-immunocytochemistry, glutamic acid decarboxylase in situ hybridization, and parvalbumin-immunocytochemistry. Stereological methods were used to measure hilar ectopic granule cells, mossy cells, mossy fiber sprouting, astrogliosis, and GABAergic interneurons. Seizure frequency was not significantly correlated with the generation of hilar ectopic granule cells, the number of mossy cells, the extent of mossy fiber sprouting, the extent of astrogliosis, or the number of GABAergic interneurons in the molecular layer or hilus. Seizure frequency significantly correlated with the loss of GABAergic interneurons in or adjacent to the granule cell layer, but not with the loss of parvalbumin-positive interneurons. These findings prioritize the loss of granule cell layer interneurons for further testing as a potential cause of temporal lobe epilepsy.

Hippocampal neuropathology of domoic acid-induced epilepsy in California sea lions (Zalophus californianus).

California sea lions (Zalophus californianus) are abundant human-sized carnivores with large gyrencephalic brains. They develop epilepsy after experiencing status epilepticus when naturally exposed to domoic acid. We tested whether sea lions previously exposed to DA (chronic DA sea lions) display hippocampal neuropathology similar to that of human patients with temporal lobe epilepsy. Hippocampi were obtained from control and chronic DA sea lions. Stereology was used to estimate numbers of Nissl-stained neurons per hippocampus in the granule cell layer, hilus, and pyramidal cell layer of CA3, CA2, and CA1 subfields. Adjacent sections were processed for somatostatin immunoreactivity or Timm-stained, and the extent of mossy fiber sprouting was measured stereologically. Chronic DA sea lions displayed hippocampal neuron loss in patterns and extents similar but not identical to those reported previously for human patients with temporal lobe epilepsy. Similar to human patients, hippocampal sclerosis in sea lions was unilateral in 79% of cases, mossy fiber sprouting was a common neuropathological abnormality, and somatostatin-immunoreactive axons were exuberant in the dentate gyrus despite loss of immunopositive hilar neurons. Thus, hippocampal neuropathology of chronic DA sea lions is similar to that of human patients with temporal lobe epilepsy.

Rapamycin suppresses axon sprouting by somatostatin interneurons in a mouse model of temporal lobe epilepsy.

PURPOSE: In temporal lobe epilepsy many somatostatin interneurons in the dentate gyrus die. However, some survive and sprout axon collaterals that form new synapses with granule cells. The functional consequences of γ-aminobutyric acid (GABA)ergic synaptic reorganization are unclear. Development of new methods to suppress epilepsy-related interneuron axon sprouting might be useful experimentally. METHODS: Status epilepticus was induced by systemic pilocarpine treatment in green fluorescent protein (GFP)-expressing inhibitory nerurons (GIN) mice in which a subset of somatostatin interneurons expresses GFP. Beginning 24 h later, mice were treated with vehicle or rapamycin (3 mg/kg intraperitoneally) every day for 2 months. Stereologic methods were then used to estimate numbers of GFP-positive hilar neurons per dentate gyrus and total length of GFP-positive axon in the molecular layer plus granule cell layer. GFP-positive axon density was calculated. The number of GFP-positive axon crossings of the granule cell layer was measured. Regression analyses were performed to test for correlations between GFP-positive axon length versus number of granule cells and dentate gyrus volume. KEY FINDINGS: After pilocarpine-induced status epilepticus, rapamycin- and vehicle-treated mice had approximately half as many GFP-positive hilar neurons as did control animals. Despite neuron loss, vehicle-treated mice had over twice the GFP-positive axon length per dentate gyrus as controls, consistent with GABAergic axon sprouting. In contrast, total GFP-positive axon length was similar in rapamycin-treated mice and controls. GFP-positive axon length correlated most closely with dentate gyrus volume. SIGNIFICANCE: These findings suggest that rapamycin suppressed axon sprouting by surviving somatostatin/GFP-positive interneurons after pilocarpine-induced status epilepticus in GIN mice. It is unclear whether the effect of rapamycin on axon length was on interneurons directly or secondary, for example, by suppressing growth of granule cell dendrites, which are synaptic targets of interneuron axons. The mammalian target of rapamycin (mTOR) signaling pathway might be a useful drug target for influencing GABAergic synaptic reorganization after epileptogenic treatments, but additional side effects of rapamycin treatment must be considered carefully.

Initial loss but later excess of GABAergic synapses with dentate granule cells in a rat model of temporal lobe 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.

Surviving hilar somatostatin interneurons enlarge, sprout axons, and form new synapses with granule cells in a mouse model of temporal lobe epilepsy.

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.

Inhibition of the mammalian target of rapamycin signaling pathway suppresses dentate granule cell axon sprouting in a rodent model of temporal lobe epilepsy.

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.

Prolonged infusion of inhibitors of calcineurin or L-type calcium channels does not block mossy fiber sprouting in a model of temporal lobe epilepsy.

PURPOSE: It would be useful to selectively block granule cell axon (mossy fiber) sprouting to test its functional role in temporal lobe epileptogenesis. Targeting axonal growth cones may be an effective strategy to block mossy fiber sprouting. L-type calcium channels and calcineurin, a calcium-activated phosphatase, are critical for normal growth cone function. Previous studies have provided encouraging evidence that blocking L-type calcium channels or inhibiting calcineurin during epileptogenic treatments suppresses mossy fiber sprouting. METHODS: Rats were treated systemically with pilocarpine to induce status epilepticus, which lasted at least 2 h. Then, osmotic pumps and cannulae were implanted to infuse calcineurin inhibitors (FK506 or cyclosporin A) or an L-type calcium channel blocker (nicardipine) into the dorsal dentate gyrus. After 28 days of continuous infusion, extent of mossy fiber sprouting was evaluated with Timm staining and stereological methods. RESULTS: Percentages of volumes of the granule cell layer plus molecular layer that were Timm-positive were similar in infused and noninfused hippocampi. CONCLUSIONS: These findings suggest inhibiting calcineurin or L-type calcium channels does not block mossy fiber sprouting in the pilocarpine-treated rat model of temporal lobe epilepsy.

GABAA receptor-mediated IPSCs and alpha1 subunit expression are not reduced in the substantia nigra pars reticulata of gerbils with inherited epilepsy.

Domestic Mongolian gerbils, a model of inherited epilepsy, begin having spontaneous seizures at approximately 1.5 mo of age, making it possible to evaluate them during epileptic and pre-epileptic stages. Previous studies have shown that GABA binding is reduced in the substantia nigra pars reticulata (SNr) of both epileptic and pre-epileptic gerbils compared with controls, suggesting that reduced expression of GABAA receptors in SNr might be epileptogenic in this model. To test this hypothesis, we measured the expression of the GABAA receptor alpha1 subunit, the dominant alpha subunit expressed in the SNr, and evaluated GABAA receptor-mediated postsynaptic currents in SNr neurons. GABA(A) alpha1 subunit mRNA levels in substantia nigra-rich tissue from pre-epileptic animals were similar to controls, and immunocytochemistry for the alpha1 subunit showed similar strong expression in the SNr in both groups. Western analysis confirmed that expression of the alpha1 subunit protein was similar in substantia nigra-rich tissue from pre-epileptic and control gerbils. The frequency and amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) and the frequency of miniature (m)IPSCs in SNr neurons of pre-epileptic gerbil were similar to those of controls. The amplitude of mIPSCs in the pre-epileptics was significantly larger than controls. Zolpidem, an alpha1 subunit-specific modulator of the GABAA receptor, was equally efficacious in prolonging the decay time of mIPSCs in both groups. Hence, contrary to the predictions of the hypothesis, mRNA and protein expression levels of the major GABAA receptor alpha subunit were normal, and neurons of the SNr in pre-epileptic gerbils displayed normal or enhanced IPSC frequencies and amplitudes. Therefore reduced expression of GABAA receptors in SNr is not likely to be an epileptogenic mechanism in this model.

Reduced inhibition and increased output of layer II neurons in the medial entorhinal cortex in a model of temporal lobe epilepsy.

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.