Spontaneous recurrent seizures after pilocarpine-induced status epilepticus activate calbindin-immunoreactive hilar cells of the rat dentate gyrus.

Although it is now established that neurogenesis of dentate gyrus granule cells increases after experimental seizures, little is currently known about the function of the new granule cells. One question is whether they become integrated into the network around them. Recent experiments that focused on the newly born granule cells in the hilus showed that indeed the new cells appear to become synchronized with host hippocampal neurons [Scharfman et al. (2000) J. Neurosci. 20, 6144-6158]. To address this issue further, we asked whether the new hilar granule cells were active during spontaneous limbic seizures that follow status epilepticus induced by pilocarpine injection. Thus, we perfused rats after spontaneous seizures and stained sections using antibodies to c-fos, a marker of neural activity, and calbindin, a marker of the newly born hilar granule cells [Scharfman et al. (2000) J. Neurosci. 20, 6144-6158]. We asked whether calbindin-immunoreactive hilar neurons were also c-fos-immunoreactive.C-fos was highly expressed in calbindin-immunoreactive hilar neurons. Approximately 23% of hilar cells that expressed c-fos were double-labeled for calbindin. In addition, other types of hilar neurons, i.e. those expressing parvalbumin or neuropeptide Y, also expressed c-fos. Yet other hippocampal neurons, including granule cells and pyramidal cells, had weak expression of c-fos at the latency after the seizure that hilar neuron expression occurred. In controls, there was very little c-fos or calbindin expression in the hilus.These results indicate that calbindin-immunoreactive hilar cells are activated by spontaneous seizures. Based on the evidence that many of these cells are likely to be newly born, the data indicate that new cells can become functionally integrated into limbic circuits involved in recurrent seizure generation. Furthermore, they appear to do so in a manner similar to many neighboring hilar neurons, apparently assimilating into the local environment. Finally, the results show that a number of hilar cell types are activated during chronic recurrent seizures in the pilocarpine model, a surprising result given that many hilar neurons are thought to be damaged soon after pilocarpine-induced status epilepticus.

Survival of dentate hilar mossy cells after pilocarpine-induced seizures and their synchronized burst discharges with area CA3 pyramidal cells.

The clinical and basic literature suggest that hilar cells of the dentate gyrus are damaged after seizures, particularly prolonged and repetitive seizures. Of the cell types within the hilus, it appears that the mossy cell is one of the most vulnerable. Nevertheless, hilar neurons which resemble mossy cells appear in some published reports of animal models of epilepsy, and in some cases of human temporal lobe epilepsy. Therefore, mossy cells may not always be killed after severe, repeated seizures. However, mossy cell survival in these studies was not completely clear because the methods did allow discrimination between mossy cells and other hilar cell types. Furthermore, whether surviving mossy cells might have altered physiology after seizures was not examined. Therefore, intracellular recording and intracellular dye injection were used to characterize hilar cells in hippocampal slices from pilocarpine-treated rats that had status epilepticus and recurrent seizures ('epileptic' rats). For comparison, mossy cells were also recorded from age-matched, saline-injected controls, and pilocarpine-treated rats that failed to develop status epilepticus. Numerous hilar cells with the morphology, axon projection, and membrane properties of mossy cells were recorded in all three experimental groups. Thus, mossy cells can survive severe seizures, and those that survive retain many of their normal characteristics. However, mossy cells from epileptic tissue were distinct from mossy cells of control rats in that they generated spontaneous and evoked epileptiform burst discharges. Area CA3 pyramidal cells also exhibited spontaneous and evoked bursts. Simultaneous intracellular recordings from mossy cells and pyramidal cells demonstrated that their burst discharges were synchronized, with pyramidal cell discharges typically beginning first. From these data we suggest that hilar mossy cells can survive status epilepticus and chronic seizures. The fact that mossy cells have epileptiform bursts, and that they are synchronized with area CA3, suggest a previously unappreciated substrate for hyperexcitability in this animal model.

Granule-like neurons at the hilar/CA3 border after status epilepticus and their synchrony with area CA3 pyramidal cells: functional implications of seizure-induced neurogenesis.

A group of neurons with the characteristics of dentate gyrus granule cells was found at the hilar/CA3 border several weeks after pilocarpine- or kainic acid-induced status epilepticus. Intracellular recordings from pilocarpine-treated rats showed that these "granule-like" neurons were similar to normal granule cells (i. e., those in the granule cell layer) in membrane properties, firing behavior, morphology, and their mossy fiber axon. However, in contrast to normal granule cells, they were synchronized with spontaneous, rhythmic bursts of area CA3 pyramidal cells that survived status epilepticus. Saline-treated controls lacked the population of granule-like cells at the hilar/CA3 border and CA3 bursts. In rats that were injected after status epilepticus with bromodeoxyuridine (BrdU) to label newly born cells, and also labeled for calbindin D(28K) (because it normally stains granule cells), many double-labeled neurons were located at the hilar/CA3 border. Many BrdU-labeled cells at the hilar/CA3 border also were double-labeled with a neuronal marker (NeuN). Taken together with the recent evidence that granule cells that are born after seizures can migrate into the hilus, the results suggest that some newly born granule cells migrate as far as the CA3 cell layer, where they become integrated abnormally into the CA3 network, yet they retain granule cell intrinsic properties. The results provide insight into the physiological properties of newly born granule cells in the adult brain and suggest that relatively rigid developmental programs set the membrane properties of newly born cells, but substantial plasticity is present to influence their place in pre-existing circuitry.

Actions of brain-derived neurotrophic factor in slices from rats with spontaneous seizures and mossy fiber sprouting in the dentate gyrus.

This study examined the acute actions of brain-derived neurotrophic factor (BDNF) in the rat dentate gyrus after seizures, because previous studies have shown that BDNF has acute effects on dentate granule cell synaptic transmission, and other studies have demonstrated that BDNF expression increases in granule cells after seizures. Pilocarpine-treated rats were studied because they not only have seizures and increased BDNF expression in granule cells, but they also have reorganization of granule cell "mossy fiber" axons. This reorganization, referred to as "sprouting," involves collaterals that grow into novel areas, i.e., the inner molecular layer, where granule cell and interneuron dendrites are located. Thus, this animal model allowed us to address the effects of BDNF in the dentate gyrus after seizures, as well as the actions of BDNF on mossy fiber transmission after reorganization. In slices with sprouting, BDNF bath application enhanced responses recorded in the inner molecular layer to mossy fiber stimulation. Spontaneous bursts of granule cells occurred, and these were apparently generated at the site of the sprouted axon plexus. These effects were not accompanied by major changes in perforant path-evoked responses or paired-pulse inhibition, occurred only after prolonged (30-60 min) exposure to BDNF, and were blocked by K252a. The results suggest a preferential action of BDNF at mossy fiber synapses, even after substantial changes in the dentate gyrus network. Moreover, the results suggest that activation of trkB receptors could contribute to the hyperexcitability observed in animals with sprouting. Because human granule cells also express increased BDNF mRNA after seizures, and sprouting can occur in temporal lobe epileptics, the results may have implications for understanding temporal lobe epilepsy.

Basal expression and induction of glutamate decarboxylase and GABA in excitatory granule cells of the rat and monkey hippocampal dentate gyrus.

The excitatory, glutamatergic granule cells of the hippocampal dentate gyrus are presumed to play central roles in normal learning and memory, and in the genesis of spontaneous seizure discharges that originate within the temporal lobe. In localizing the two GABA-producing forms of glutamate decarboxylase (GAD65 and GAD67) in the normal hippocampus as a prelude to experimental epilepsy studies, we unexpectedly discovered that, in addition to its presence in hippocampal nonprincipal cells, GAD67-like immunoreactivity (LI) was present in the excitatory axons (the mossy fibers) of normal dentate granule cells of rats, mice, and the monkey Macaca nemestrina. Using improved immunocytochemical methods, we were also able to detect GABA-LI in normal granule cell somata and processes. Conversely, GAD65-LI was undetectable in normal granule cells. Perforant pathway stimulation for 24 hours, which evoked population spikes and epileptiform discharges in both dentate granule cells and hippocampal pyramidal neurons, induced GAD65-, GAD67-, and GABA-LI only in granule cells. Despite prolonged excitation, normally GAD- and GABA-negative dentate hilar neurons and hippocampal pyramidal cells remained immunonegative. Induced granule cell GAD65-, GAD67-, and GABA-LI remained elevated above control immunoreactivity for at least 4 days after the end of stimulation. Pre-embedding immunocytochemical electron microscopy confirmed that GAD67- and GABA-LI were induced selectively within granule cells; granule cell layer glia and endothelial cells were GAD- and GABA-immunonegative. In situ hybridization after stimulation revealed a similarly selective induction of GAD65 and GAD67 mRNA in dentate granule cells. Neurochemical analysis of the microdissected dentate gyrus and area CA1 determined whether changes in GAD- and GABA-LI reflect changes in the concentrations of chemically identified GAD and GABA. Stimulation for 24 hours increased GAD67 and GABA concentrations sixfold in the dentate gyrus, and decreased the concentrations of the GABA precursors glutamate and glutamine. No significant change in GAD65 concentration was detected in the microdissected dentate gyrus despite the induction of GAD65-LI. The concentrations of GAD65, GAD67, GABA, glutamate and glutamine in area CA1 were not significantly different from control concentrations. These results indicate that dentate granule cells normally contain two "fast-acting" amino acid neurotransmitters, one excitatory and one inhibitory, and may therefore produce both excitatory and inhibitory effects. Although the physiological role of granule cell GABA is unknown, the discovery of both basal and activity-dependent GAD and GABA expression in glutamatergic dentate granule cells may have fundamental implications for physiological plasticity presumed to underlie normal learning and memory. Furthermore, the induction of granule cell GAD and GABA by afferent excitation may constitute a mechanism by which epileptic seizures trigger compensatory interictal network inhibition or GABA-mediated neurotrophic effects.

Apoptosis and necrosis induced in different hippocampal neuron populations by repetitive perforant path stimulation in the rat.

Patients experiencing spontaneous seizures of temporal lobe origin often exhibit a shrunken hippocampus, which results from the loss of dentate granule cells, hilar neurons, and hippocampal pyramidal cells. Although experimental attempts to replicate the human pattern of hippocampal sclerosis in animals indicate that prolonged seizures cause prominent injury to dentate hilar neurons and hippocampal pyramidal cells, dentate granule cells of animals are generally regarded as relatively resistant to seizure-induced injury. By evaluating pathology shortly after hippocampal seizure discharges were induced electrically, we discovered that some granule cells are highly vulnerable to prolonged excitation and that they exhibit acute degenerative features distinct from those of other vulnerable cell populations. Intermittent perforant path stimulation for 24 hours induced acute degeneration of dentate granule cells, dentate hilar neurons, and hippocampal pyramidal cells. However, stimulation for 8 hours, which was insufficient to injure hilar neurons and hippocampal pyramidal cells, was nonetheless sufficient to induce bilateral granule cell degeneration. Degenerating granule cells were consistently more numerous in the infrapyramidal than the suprapyramidal blade, and were consistently more numerous in the rostral than caudal dentate gyrus. Depending on the nature of the insult, acutely degenerating neurons exhibit distinct morphological features that are classifiable as either apoptosis or necrosis, although the degree of possible overlap is unknown. Light and electron microscopic analysis of the acute pathology caused by prolonged afferent stimulation revealed that degenerating hilar neurons and pyramidal cells exhibited the morphological features of necrosis, which is characterized in part by early cytoplasmic vacuolization before nuclear changes occur. However, acutely degenerating granule cells exhibited the clearly distinct morphological features of apoptosis, which include an early coalescence of nuclear chromatin into multiple nuclear bodies, compaction of the cytoplasm, cell shrinkage, and budding-off of 'apoptotic bodies' that are engulfed by glia. Whereas pyramidal cell debris persisted for months, granule cell debris disappeared rapidly. This observation may explain why significant granule cell vulnerability has not been described previously. These data document for the first time that dentate granule cells are among the cell types most vulnerable to seizure-induced injury, and demonstrate that whereas hilar neurons and pyramidal cells undergo a typically necrotic degenerative process, granule cells simultaneously exhibit morphological features that more closely resemble the degenerative process of apoptosis. This finding implies that the type of cell death induced by excessive excitation may be determined postsynaptically by the way in which different target cells 'interpret' an excitatory insult. This experimental model may be useful for identifying the biochemical mechanisms that initiate and mediate neuronal apoptosis and necrosis, and for developing strategies to prevent or induce these presumably distinct forms of neuronal death.

Hippocampal dentate granule cell degeneration after adrenalectomy in the rat is not reversed by dexamethasone.

Although adrenalectomy has been reported to induce a selective and sometimes nearly complete degeneration of hippocampal dentate granule cells, Azmitia and colleagues recently reported (Mol. Brain Res., 19 (1993) 328-332) that normal hippocampal structure can nonetheless be restored within a matter of days by dexamethasone in the drinking water. We have attempted to confirm this remarkable finding. Four months after adrenalectomy, rats were given vehicle or dexamethasone for 5 days and then sacrificed. Histological analysis revealed that vehicle-treated adrenalectomized rats exhibited a full spectrum of granule cell loss, which spanned mild to nearly complete cell loss. Dexamethasone-treated adrenalectomized rats did not differ from vehicle-treated adrenalectomized rats and, in fact, exhibited a virtually identical spectrum of granule cell loss. These results confirm that adrenalectomy reliably induces hippocampal granule cell degeneration in a majority of animals and indicate that dexamethasone does not restore normal hippocampal structure once granule cell loss has occurred.

Adrenalectomy-induced granule cell degeneration in the rat hippocampal dentate gyrus: characterization of an in vivo model of controlled neuronal death.

The recent discovery that adrenalectomy results in hippocampal granule cell loss suggested that this phenomenon might be useful as a model of selective, experimentally controlled neuronal death possibly relevant to neurodegenerative disorders. This study was designed to provide a detailed qualitative anatomical description of the phenomenon and to determine whether adrenalectomy-induced dentate granule cell degeneration constitutes a reliable model of selective neuronal death. Silver impregnation staining revealed that granule cell degeneration begins immediately after adrenalectomy and continues for months in both sexes, in young and older adults, and in all strains tested. In one group of 77 adrenalectomized rats, 82% exhibited silver-impregnated granule cells. This phenomenon is extraordinarily selective in that no neurons other than dentate granule cells degenerated after adrenalectomy. There was considerable variability among animals in the number of cells degenerating at a given time-point or in the degree of ultimate cell loss. In the most extreme cases, virtually complete granule cell loss was present throughout approximately 80% of the dentate gyrus. Nissl staining revealed that degenerating granule cells exhibited coalescing of nuclear chromatin into multiple nuclear bodies and pyknosis without accompanying glial swelling. This morphology is distinct from the necrosis caused by other neurotoxic insults and is the subject of the ultrastructural companion paper identifying this type of cell death as apoptosis. Taken together, these results indicate that adrenalectomy reliably initiates an immediate, highly selective, and long-continuing process of hippocampal granule cell degeneration that exhibits morphological features characteristic of apoptosis, rather than necrosis. The possibility that this apoptotic cell death involves a biochemical cascade relevant to programmed cell death and/or neurodegenerative diseases suggests that this model may be valuable for studies of neuronal death and its prevention. Some practical guidelines for use of this model are described.

Calbindin-D28k immunoreactivity and selective vulnerability to ischemia in the dentate gyrus of the developing rat.

Hippocampal dentate granule cells normally express the calcium-binding protein calbindin-D28k and, in the adult, are the hippocampal neurons least vulnerable to an ischemic insult. We evaluated hippocampal structure 2-3 days after hypoxic/ischemic insult at postnatal day 7-10, and discovered that, unlike adult granule cells, developing granule cells were irreversibly injured. Localization of calbindin-D28k-like immunoreactivity (LI) revealed that the vulnerable cells were the immature granule cells at the base of the cell layer that were not yet calbindin-immunoreactive. Adjacent granule cells that did not die in response to the hypoxic/ischemic insult were calbindin-immunoreactive. Whether the lack of calbindin-LI in immature granule cells is causally related to their vulnerability, or is a coincidental reflection of cellular immaturity, remains to be determined.

Calcium-binding protein (calbindin-D28K) and parvalbumin immunocytochemistry in the normal and epileptic human hippocampus.

The calcium-binding proteins calbindin-D28K (CaBP) and parvalbumin (PV) were localized in the "normal" and "epileptic" human hippocampus to address the possible relationship between the expression of these constitutive cytosolic calcium-binding proteins and the resistance or selective vulnerability of different hippocampal neuron populations in temporal lobe epilepsy. Compared to rodents and a baboon (Papio papio), the pattern of CaBP-like immunoreactivity (LI) in the "normal" human hippocampus is unique. CaBP-LI is present in the dentate granule cells, neurons of the "resistant zone" (area CA2), and presumed interneurons of all regions. Unlike rodent and baboon CA1 pyramidal cells, human CA1 pyramidal cells appear to be devoid of CaBP-LI. Thus, the relatively resistant dentate granule cells and CA2 pyramidal cells are the only human hippocampal principal cells that contain CaBP-LI normally. As in lower mammals, PV-LI is present exclusively in interneurons of all human hippocampal subregions. CaBP- and PV-LI were localized in hippocampi surgically removed in the treatment of intractable temporal lobe epilepsy to determine whether surviving hippocampal cells were those that express these calcium-binding proteins. Hippocampi removed from patients with tumors or arteriovenous malformations that were associated with complex partial seizures arising from this region appeared relatively normal histologically. CaBP- and PV-LI in this patient group appeared similar to that seen in autopsy controls. Conversely, "cryptogenic" epileptics, who exhibit hippocampal sclerosis as the only lesion associated with the epilepsy, exhibited a preferential survival of hippocampal cells that were CaBP- or PV-immunoreactive. In the dentate hilus, which normally contains few CaBP-LI neurons, most of the few surviving hilar neurons were CaBP-immunoreactive. Their number and darkness of staining suggests that CaBP synthesis may be increased in cells that survive. Despite an obvious decrease of PV-LI specifically in the damaged parts of the sclerotic hippocampi, PV-immunoreactive interneurons were often among the few surviving cells. Nevertheless, large expanses of the surviving granule cell layer appeared to have lost the PV-immunoreactive axosomatic fiber plexus. These results reveal a unique and striking correlation between the human hippocampal cells that normally express these calcium-binding proteins and those that survive in the sclerotic epileptic hippocampus.

Selective loss of hippocampal granule cells in the mature rat brain after adrenalectomy.

Adrenalectomy of adult male rats resulted in a nearly complete loss of hippocampal granule cells 3 to 4 months after surgery. Nissl and immunocytochemical staining of hippocampal neurons revealed that the granule cell loss was selective; there was no apparent loss of hippocampal pyramidal cells or of gamma-amino butyric acid (GABA)-, somatostatin-, neuropeptide Y-, calcium binding protein-, or parvalbumin-containing hippocampal interneurons. The hippocampal CA1 pyramidal cells of adrenalectomized animals exhibited normal electrophysiological responses to afferent stimulation, whereas responses evoked in the dentate gyrus were severely attenuated. Corticosterone replacement prevented both the adrenalectomy-induced granule cell loss and the attenuated physiological response. Thus, the adrenal glands play a role in maintaining the structural integrity of the normal adult brain.