The gene encoding proline dehydrogenase modulates sensorimotor gating in mice.

Hemizygous cryptic deletions of the q11 band of human chromosome 22 have been associated with a number of psychiatric and behavioural phenotypes, including schizophrenia. Here we report the isolation and characterization of PRODH, a human homologue of Drosophila melanogaster sluggish-A (slgA), which encodes proline dehydrogenase responsible for the behavioural phenotype of the slgA mutant. PRODH is localized at chromosome 22q11 in a region deleted in some psychiatric patients. We also isolated the mouse homologue of slgA (Prodh), identified a mutation in this gene in the Pro/Re hyperprolinaemic mouse strain and found that these mice have a deficit in sensorimotor gating accompanied by regional neurochemical alterations in the brain. Sensorimotor gating is a neural filtering process that allows attention to be focused on a given stimulus, and is affected in patients with neuropsychiatric disorders. Furthermore, several lines of evidence suggest that proline may serve as a modulator of synaptic transmission in the mammalian brain. Our observations, in conjunction with the chromosomal location of PRODH, suggest a potential involvement of this gene in the 22q11-associated psychiatric and behavioural phenotypes.

Neuropeptide Y regulates recurrent mossy fiber synaptic transmission less effectively in mice than in rats: Correlation with Y2 receptor plasticity.

A unique feature of temporal lobe epilepsy is the formation of recurrent excitatory connections among granule cells of the dentate gyrus as a result of mossy fiber sprouting. This novel circuit contributes to a reduced threshold for granule cell synchronization. In the rat, activity of the recurrent mossy fiber pathway is restrained by the neoexpression and spontaneous release of neuropeptide Y (NPY). NPY inhibits glutamate release tonically through activation of presynaptic Y2 receptors. In the present study, the effects of endogenous and applied NPY were investigated in C57Bl/6 mice that had experienced pilocarpine-induced status epilepticus and subsequently developed a robust recurrent mossy fiber pathway. Whole cell patch clamp recordings made from dentate granule cells in hippocampal slices demonstrated that, as in rats, applied NPY inhibits recurrent mossy fiber synaptic transmission, the Y2 receptor antagonist (S)-N2-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6H)-oxodibenz[b,e]azepin-11-yl]-1-piperazinyl]-2-oxoethyl]cyclopentyl]acetyl]-N-[2-[1,2-dihydro-3,5(4H)-dioxo-1,2-diphenyl-3H-1,2,4-triazol-4-yl]ethyl]-argininamide (BIIE0246) blocks its action and BIIE0246 enhances synaptic transmission when applied by itself. Y5 receptor agonists had no significant effect. Thus spontaneous release of NPY tonically inhibits synaptic transmission in mice and its effects are mediated by Y2 receptor activation. However, both NPY and BIIE0246 were much less effective in mice than in rats, despite apparently equivalent expression of NPY in the recurrent mossy fibers. Immunohistochemistry indicated greater expression of Y2 receptors in the mossy fiber pathway of normal mice than of normal rats. Pilocarpine-induced status epilepticus markedly reduced the immunoreactivity of mouse mossy fibers, but increased the immunoreactivity of rat mossy fibers. Mossy fiber growth into the inner portion of the dentate molecular layer was associated with increased Y2 receptor immunoreactivity in rat, but not in mouse. These contrasting receptor changes can explain the quantitatively different effects of endogenously released and applied NPY on recurrent mossy fiber transmission in mice and rats.

Regionally selective effects of NMDA receptor antagonists against ischemic brain damage in the gerbil.

This study compared the ability of three N-methyl-D-aspartate (NMDA) receptor antagonists to prevent neuronal degeneration in an animal model of global cerebral ischemia. The model employed is characterized by damage to the striatum, hippocampus, and neocortex. Antagonists were administered to gerbils either before or after a 5-min bilateral carotid occlusion. The intraischemic rectal temperature was either maintained at 36-37 degrees C or allowed to fall passively to 28-32 degrees C. Antagonists and doses tested were 1 and 10 mg/kg of MK-801 (pre- or postischemia), 30 mg/kg of CGS 19755 preischemia, four 25 mg/kg doses of CGS 19755 administered between 0.5 and 6.5 h postischemia, and 40 mg/kg of MDL 27,266 (pre- or postischemia). All three NMDA receptor antagonists exhibited some degree of neuroprotective activity when the carotid occlusion was performed under normothermic conditions. Most of the treatments with antagonist markedly reduced striatal damage. CA1 hippocampal and neocortical pyramidal cells were spared by only three of the treatments, however, and the extent of neuroprotection varied widely from case to case. Toxic doses of antagonist were required to protect CA1 pyramidal cells from ischemic damage. Ischemic damage to hippocampal areas CA2-CA3a and CA4 appeared to be resistant to all of these treatments. Most CA1 pyramidal cells that were protected from degeneration by an NMDA receptor antagonist were histologically abnormal. The neuroprotective effects of MK-801 and intraischemic hypothermia appeared to be additive. MK-801 (10 mg/kg) consistently reduced the postischemic brain temperature, but only the magnitude of hypothermia produced soon after reperfusion correlated with its neuroprotective action. These results suggest that NMDA receptor antagonists are relatively poor neuroprotective agents against a moderately severe ischemic insult.

Hippocampal mossy fiber sprouting and synapse formation after status epilepticus in rats: visualization after retrograde transport of biocytin.

In complex partial epilepsy and in animal models of epilepsy, hippocampal mossy fibers appear to develop recurrent collaterals that invade the dentate molecular layer. Mossy fiber collaterals have been proposed to subserve recurrent excitation by forming granule cell-granule cell synapses. This hypothesis was tested by visualizing dentate granule cells and their mossy fibers after terminal uptake and retrograde transport of biocytin. Labeling studies were performed with transverse slices of the caudal rat hippocampal formation prepared 2.6-70.0 weeks after pilocarpine-induced or kainic acid-induced status epilepticus. Light microscopy demonstrated the progressive growth of recurrent mossy fibers into the molecular layer; the densest innervation was observed in slices from pilocarpine-treated rats that had survived 10 weeks or longer after status epilepticus. Thin mossy fiber collaterals originated predominantly from deep within the hilar region, crossed the granule cell body layer, and formed an axonal plexus oriented parallel to the cell body layer within the inner one-third of the molecular layer. When sprouting was most robust, some recurrent mossy fibers at the apex of the dentate gyrus reached the outer two-thirds of the molecular layer. The distribution and density of mossy fiber-like Timm staining correlated with the biocytin labeling. When viewed with the electron microscope, the inner one-third of the dentate molecular layer contained numerous mossy fiber boutons. In some instances, biocytin-labeled mossy fiber boutons were engaged in synaptic contact with biocytin-labeled granule cell dendrites. Granule cell dendrites did not develop large complex spines ("thorny excrescences") at the site of synapse formation, and they did not appear to have been permanently damaged by seizure activity. These results establish the validity of Timm staining as a marker for mossy fiber sprouting and support the view that status epilepticus provokes the formation of a novel recurrent excitatory circuit in the dentate gyrus. Retrograde labeling with biocytin showed that the recurrent mossy fiber projection often occupies a considerably greater fraction of the dendritic region than previous studies had suggested.

Degeneration of hippocampal CA3 pyramidal cells induced by intraventricular kainic acid.

Degeneration of hippocampal CA3 pyramidal cells was investigated by light and electron microscopy after intraventricular injection of the potent convulsant, kainic acid. Electron microscopy revealed evidence of pyramidal cell degeneration within one hour. The earliest degenerative changes were confined to the cell body and proximal dendritic shafts. These included an increased incidence of lysosomal structures, deformation of the perikaryal and nuclear outlines, some increase in background electron density, and dilation of the cisternae of the endoplasmic reticulum accompanied by detachment of polyribosomes. Within the next few hours the pyramidal cells atrophied and became electron dense. Then these cells became electron lucent once more as ribosomes disappeared and their membranes and organelles broke up and disintegrated. Light microscopic changes correlated with these ultrastructural observations. The dendritic spines and the initial portion of the dendritic shaft became electron dense within four hours and degenerated rapidly, whereas the intermediate segment of the dendrites swelled moderately and became more electron lucent. No degenerative changes were evident in pyramidal cell axons and boutons until one day after kainic acid treatment. Less than one hour after kainic acid administration, astrocytes in the CA3 area swelled, initially in the vicinity of the cell body and mossy fiber layers. It is suggested that the paroxysmal discharges initiated in CA3 pyramidal cells by kainic acid served as the stimulus for this response. Phagocytosis commenced between one and three days after kainic acid administration, but remained incomplete at survival times of 6-8 weeks. Astrocytes, microglia, and probably oligodendroglia phagocytized the degenerating material. These results point to the pyramidal cell body and possibly also the dendritic spines as primary targets of kainic acid neurotoxicity. In conjunction with other data, they support the view that lesions made by intraventricular kainic acid can serve as models of epileptic brain damage.

Fate of the hippocampal mossy fiber projection after destruction of its postsynaptic targets with intraventricular kainic acid.

Intraventricular injections of kainic acid were used to create a model of selective cell death in order to study the fate of afferent projections that are deprived of their postsynaptic targets. This treatment rapidly destroyed hippocampal CA3 pyramidal cells, but not those neurons that give rise to their mossy fiber and entorhinal afferents. Light microscopic studies with the Timm's sulfide silver stain indicated that half or more of the mossy fiber boutons in area CA3b were lost within the first 1-3 days after kainic acid administration. This finding was confirmed by electron microscopy. Electron-dense, usually vacuolated mossy fiber boutons accounted for about 10-20% of the total population present at a 4-hour survival time, but were not encountered in control rats nor at survival times longer than 1 day. Other mossy fiber boutons remained electron lucent, but enlarged, became more rounded in shape, and suffered an apparent loss of synaptic vesicles. It is suggested that degeneration of some mossy fiber boutons and resorption of others into the axon may have accounted for the precipitous decline in their number. The dendritic excrescences contacted by these boutons were nearly all undergoing electron-dense degeneration 4 hours after kainic acid administration. In rats that survived 6-8 weeks mossy fiber boutons remained somewhat scarce, individual boutons appeared relatively small, and only one-third the normal percentage were observed to be engaged in more than one synaptic contact within a single cross section. A qualitative electron microscopic study of the entorhinal projection to area CA3 suggested a response to kainic acid treatment similar to that of the mossy fiber projection, except that no entorhinal boutons were seen to become electron dense. These findings suggest that presynaptic fibers in the mature hippocampus adjust the size of their terminal arborizations and number of synaptic contacts to accommodate a reduction in the target cell population. The rapid loss of mossy fiber boutons may be attributable to an unusual fragility of these structures when they are deprived of the mechanical support normally provided by the pyramidal cell. Finally, the ability of kainic acid administration to alter the number and distribution of presynaptic elements must be taken into account whenever this toxin is used to make selective lesions of postsynaptic cells.

Histochemical evidence of altered development of cholinergic fibers in the rat dentate gyrus following lesions. I. Time course after complete unilateral entorhinal lesion at various ages.

The entorhinal cortex of rats was removed at various times during development, and the reaction of the cholinergic septohippocampal input to the dentate gyrus was examined by use of acetylcholinesterase histochemistry. When the ipsilateral entorhinal cortex is completely removed, the outer 70-75% of the molecular layer of the dentate gyrus is almost completely denervated. After such a lesion at 5 to 33 days of age, the acetylcholinesterase staining initially intensified throughout the denervated area, indicating that the septohippocampal fibers branched or elongated. This reaction could be detected within one day after a lesion at 11 days of age and within three or five days after lesions at earlier or later times. Whereas the initial response of the septohippocampal fibers was independent of the age at which the lesion was made, their final localization depended on the developmental state of the animal. After lesions at the age of 5 or 11 days, the reactive septohippocampal fibers became restricted to the outer one-sixth to one-third of the molecular layer within two days after appearance of their initial reaction. A similar concentration of reactive fibers was demonstrable after lesions at 16, 18 or 21 days of age, but some reaction persisted in the middle third of the molecular layer. Finally, after lesions at 26 or 33 days of age the proliferating cholinergic fibers ultimately were uniformly distributed throughout the outer 60% of the molecular layer. These results suggest that septohippocampal fibers initially extend or sprout throughout the denervated area to replace the lost perforant path fibers. However, the reactive fiber population becomes restricted to the outer edge of the molecular layer if the entorhinal lesion is made before the period of cholinergic synaptogenesis and concentrates in this same zone if it is made while cholinergic synapses are forming. We suggest that either the proliferative reaction continues in the outer part of the molecular layer and subsides in other parts of the denervated area or septohippocampal fibers move outward through the molecular layer to assume a more superficial location. After entorhinal lesions at 16 days of age or later the pale-staining zone (containing fibers that originate in hippocampus regio inferior) immediately deep to the denervated area widened. If the lesion was made earlier, this zone never developed at most septotemporal levels of the dentate gyrus. These results are probably related to the extension of regio inferior fibers into the denervated area.

Histochemical evidence of altered development of cholinergic fibers in the rat dentate gyrus following lesions. II. Effects of partial entorhinal and simultaneous multiple lesions.

It has been concluded previously that the septohippocampal fibers which project to the rat dentate gyrus extend or branch in the denervated area of the molecular layer following a complete ipsilateral entorhinal lesion. The septohippocampal fibers thus appear to replace some of the perforant fibers which degenerate as a result of the lesion. The reactive fibers eventually become localized to a much smaller and more superficial area after lesions of immature rats than after lesions made in adulthood. To determine whether this difference in the response results from a selective reaction to loss of the lateral perforant path in the immature rat, various portions of the entorhinal cortex were removed at the age of 11 days, and the cholinergic septohippocampal fibers were visualized by acetylcholinesterase histochemistry. An alternative possibility, that the difference between immature and adult rats is attributable to an interaction with other reactive afferents, was tested by removing other sources of input (the contralateral entorhinal cortex, contralateral hippocampal formation or both) along with the ipsilateral entorhinal cortex at the age of 11 days and then demonstrating the septohippocampal fibers histochemically. Lesions of the lateral part of the ipsilateral entorhinal cortex (source of the lateral perforant path) at 11 days of age evoked a septohippocampal reaction along the outer edge of the molecular layer, where the lateral perforant path fibers normally terminate. This result matched that produced by a complete entorhinal lesion. Lesions of the medial entorhinal cortex evoked no obvious reaction. In contrast, the septohippocampal fibers in adult rats proliferated in the denervated area of the molecular layer after lesions of either part of the entorhinal cortex. Combining lesions of other sources of innervation to the dentate gyrus with an ipsilateral entorhinal lesion at 11 days of age did not alter the response of septohippocampal fibers, as determined histochemically. Neither did the septohippocampal fibers react to removal of commissural afferents alone. The response at any age was unaffected by prior or subsequent removal of the contralateral entorhinal cortex. These results indicate that in immature rats the septohippocampal fibers respond only to loss of the lateral perforant path, but these same fibers can later react to loss of any part of the perforant path. They are regarded as support for the hypothesis that the reactive septohippocampal fibers preferentially interact with dendritic growth cones. Our results do not support explanations based on a hypothetical attraction between septohippocampal and crossed perforant path fibers (which react in the same area) or on competition with commissural fibers (which reinnervate an adjacent area). We suggest further that proximity to the degenerating elements does not in itself determine the pattern of reinnervation after lesions of the central nervous system.

Status epilepticus-induced hilar basal dendrites on rodent granule cells contribute to recurrent excitatory circuitry.

Mossy fiber sprouting into the inner molecular layer of the dentate gyrus is an important neuroplastic change found in animal models of temporal lobe epilepsy and in humans with this type of epilepsy. Recently, we reported in the perforant path stimulation model another neuroplastic change for dentate granule cells following seizures: hilar basal dendrites (HBDs). The present study determined whether status epilepticus-induced HBDs on dentate granule cells occur in the pilocarpine model of temporal lobe epilepsy and whether these dendrites are targeted by mossy fibers. Retrograde transport of biocytin following its ejection into stratum lucidum of CA3 was used to label granule cells for both light and electron microscopy. Granule cells with a heterogeneous morphology, including recurrent basal dendrites, and locations outside the granule cell layer were observed in control preparations. Preparations from both pilocarpine and kainate models of temporal lobe epilepsy also showed granule cells with HBDs. These dendrites branched and extended into the hilus of the dentate gyrus and were shown to be present on 5% of the granule cells in pilocarpine-treated rats with status epilepticus, whereas control rats had virtually none. Electron microscopy was used to determine whether HBDs were postsynaptic to axon terminals in the hilus, a site where mossy fiber collaterals are prevalent. Labeled granule cell axon terminals were found to form asymmetric synapses with labeled HBDs. Also, unlabeled, large mossy fiber boutons were presynaptic to HBDs of granule cells. These results indicate that HBDs are present in the pilocarpine model of temporal lobe epilepsy, confirm the presence of HBDs in the kainate model, and show that HBDs are postsynaptic to mossy fibers. These new mossy fiber synapses with HBDs may contribute to additional recurrent excitatory circuitry for granule cells.

Kindling reduces sensitivity of CA3 hippocampal pyramidal cells to competitive NMDA receptor antagonists.

Kindling is a form of experimental epilepsy in which periodic electrical stimulation of a brain pathway induces a permanently hyperexcitable state. A previous study demonstrated that kindling enhances the sensitivity of hippocampal CA3 pyramidal cells to NMDA (N-methyl-D-aspartate), consistent with a greater expression of NMDA receptors. We have tested the possibility that kindling also changes the sensitivity of these neurons to competitive NMDA receptor antagonists. When depolarizing responses to NMDA were studied with a grease-gap preparation 1-5 months after the last evoked seizure, higher concentrations of competitive antagonist were required to reduce response amplitudes. Schild analysis yielded higher KD values for all three antagonists tested. This finding suggests that kindling provokes the expression by CA3 pyramidal cells of NMDA receptors with reduced affinity for competitive antagonists.

Protective effects of mossy fiber lesions against kainic acid-induced seizures and neuronal degeneration.

The effects of a hippocampal mossy fiber lesion have been determined on neuronal degeneration and limbic seizures provoked by the subsequent intracerebroventricular administration of kainic acid to unanesthetized rats. Mossy fiber lesions were made either by transecting this pathway unilaterally or by destroying the dentate granule cells unilaterally or bilaterally with colchicine. All control rats eventually developed status epilepticus and each temporally discrete seizure that preceded status epilepticus was recorded from the hippocampus ipsilateral to the kainic acid infusion before the contralateral hippocampus. A mossy fiber lesion of the ipsilateral hippocampus prevented the development of status epilepticus in 26% of subjects and in 52% of subjects seizures were recorded from the contralateral hippocampus before the ipsilateral hippocampus. Unlike electrographic records from other treatment groups, those from rats which had received a bilateral colchicine lesion exhibited no consistent pattern indicative of seizure propagation from one limbic region to another. A bilateral, but not a unilateral, mossy fiber lesion also dramatically attenuated the behavioral expression of the seizures. Regardless of its effects on kainic acid-induced electrographic and behavioral seizures, a mossy fiber lesion always substantially reduced or completely prevented the degeneration of ipsilateral hippocampal CA3-CA4 neurons. This protective effect was specific for those hippocampal neurons deprived of mossy fiber innervation. Neurons in other regions of the brain were protected from degeneration only when the mossy fiber lesion also prevented the development of electrographic status epilepticus. These results suggest that the hippocampal mossy fibers constitute an important, though probably not an obligatory, link in the circuit responsible for the spread of kainic acid seizures. Degeneration of CA3-CA4 neurons appears to depend upon (1) the duration of hippocampal seizure activity and (2) an as yet undefined influence of or interaction with the mossy fiber projection which enhances the neurodegenerative effect of the seizures.

Aspartate release from rat hippocampal synaptosomes.

Certain excitatory pathways in the rat hippocampus can release aspartate along with glutamate. This study utilized rat hippocampal synaptosomes to characterize the mechanism of aspartate release and to compare it with glutamate release. Releases of aspartate and glutamate from the same tissue samples were quantitated simultaneously. Both amino acids were released by 25 mM K(+), 300 microM 4-aminopyridine (4-AP) and 0.5 and 1 microM ionomycin in a predominantly Ca(2+)-dependent manner. For a roughly equivalent quantity of glutamate released, aspartate release was significantly greater during exposure to elevated [K(+)] than to 4-AP and during exposure to 0.5 than to 1 microM ionomycin. Aspartate release was inefficiently coupled to P/Q-type voltage-dependent Ca(2+) channels and was reduced by KB-R7943, an inhibitor of reversed Na(+)/Ca(2+) exchange. In contrast, glutamate release depended primarily on Ca(2+) influx through P/Q-type channels and was not significantly affected by KB-R7943. Pretreatment of the synaptosomes with tetanus toxin and botulinum neurotoxins C and F reduced glutamate release, but not aspartate release. Aspartate release was also resistant to bafilomycin A(1), an inhibitor of vacuolar H(+)-ATPase, whereas glutamate release was markedly reduced. (+/-) -Threo-3-methylglutamate, a non-transportable competitive inhibitor of excitatory amino acid transport, did not reduce aspartate release. Niflumic acid, a blocker of Ca(2+)-dependent anion channels, did not alter the release of either amino acid. Exogenous aspartate and aspartate recently synthesized from glutamate accessed the releasable pool of aspartate as readily as exogenous glutamate and glutamate recently synthesized from aspartate accessed the releasable glutamate pool. These results are compatible with release of aspartate from either a vesicular pool by a "non-classical" form of exocytosis or directly from the cytoplasm by an as-yet-undescribed Ca(2+)-dependent mechanism. In either case, they suggest aspartate is released mainly outside the presynaptic active zones and may therefore serve as the predominant agonist for extrasynaptic N-methyl-D-aspartate receptors.

Ontogenetic changes in laminar distribution of ornithine decarboxylase during development of cerebellar cortex: autoradiographic localization with [3H]alpha-difluoromethylornithine.

Ornithine decarboxylase is the first enzyme in the biosynthesis of the polyamines, which control macromolecule synthesis during cellular development. Polyamines appear to play a critical role in the development of the rat cerebellar cortex, since postnatal treatment with the specific irreversible ornithine decarboxylase inhibitor, alpha-difluoromethylornithine, arrests cell division and migration in this region. To determine whether the distribution of ornithine decarboxylase within the developing cerebellar cortex correlates with specific maturational events, [3H]alpha-difluoromethylornithine, a specific marker for ornithine decarboxylase activity, was localized autoradiographically in 3-13-day-old rats. The density of autoradiographic grains within the cerebellar cortex as a whole paralleled the postnatal rise and fall of biochemically determined ornithine decarboxylase activity. Superimposed on this pattern was a selective laminar distribution of label which indicated specific association of ornithine decarboxylase with cell replication, as shown by preferential labeling of the superficial (mitotic) zone of the external granule cell layer. In addition, ornithine decarboxylase activity was temporally associated with regions in which post-mitotic cells were undergoing migration, axonogenesis and dendritic arborization, as shown by the patterns obtained in deeper layers. In contrast, there was no evidence for an association between ornithine decarboxylase activity and synaptogenesis, gliogenesis or myelination. These results, in combination with previous biochemical and morphological data, support the view that the ornithine decarboxylase/polyamine system plays an important role in both mitotic and post-mitotic events within the nervous system.

Autoradiographic localization of ornithine decarboxylase in cerebellar cortex of the developing rat with [3H]alpha-difluoromethylornithine.

Ornithine decarboxylase was autoradiographically localized in the developing rat cerebellar cortex after intracisternal injection of [3H]alpha-difluoromethylornithine, a specific, irreversible inhibitor of the enzyme. At nine days of age, when cerebellar ornithine decarboxylase activity is maximal, autoradiographic grains were distributed over all layers of the cerebellar cortex and throughout the brain stem. Within cerebellar folia, the highest grain density was associated with the molecular layer, whereas the internal and external granule cell layers were less densely labeled. Enhancement of ornithine decarboxylase activity by intracisternally-administered isoproterenol correspondingly increased the autoradiographic grain density over each layer. Thus much of the polyamine biosynthetic capability needed to support neuronal and/or glial differentiation appears to be associated with the developing cell processes. The combination of [3H]alpha-difluoromethylornithine autoradiography with localized injection techniques provides a potentially powerful tool for the study of the involvement of polyamine biosynthesis in brain development.

Selective neocortical and thalamic cell death in the gerbil after transient ischemia.

In animal models of transients ischemia, selective vulnerability and delayed neuronal death in the hippocampus have been extensively described. However, little is known about selective damage in the neocortex and the thalamus, even though deficits in sensorimotor function are common in humans surviving hypoxic/ischemic episodes. This study investigated the neurodegenerative effects of transient ischemia in the gerbil neocortex and thalamus with use of Cresyl Violet and silver impregnation staining methods. In addition, immunohistochemistry of an astrocyte-associated protein, glial fibrillary acidic protein, was used to assess the astrocytic response to ischemia. Pyramidal cells in layers 3 and 6 of somatosensory and auditory cortex were exceptionally sensitive to ischemia, whereas the neurons in layers 2, 4 and 5 were more resistant to ischemia. More pyramidal cells were killed in layer 3 than in layer 6. This bilaminar pattern of neuronal death developed after periods of ischemia ranging from 3 to 10 min and was identifiable at post-ischemic survival times of 6 h to one month. Somatodendritic argyrophilia in the neocortex was identified as early as 6-12 h after 5 min of ischemia. The greatest number of degenerating cortical neurons were stained two to four days after ischemia. With 10 min of ischemia, argyrophilic neurites and neurons were also found as early as 8 h after the occlusion. The most extensive damage was noted in the ventroposterior nucleus, the medial geniculate nucleus, and the intralaminar nuclei two to four days after ischemia. Thus, selective vulnerability and delayed neuronal death are evident in both the neocortex and the thalamus after transient ischemia. These regions need to be examined when considering the efficacy of potential neuroprotective drugs.

Kainate and quisqualate receptor autoradiography in rat brain after angular bundle kindling.

The kainate and quisqualate types of excitatory amino acid receptor were visualized autoradiographically in brain sections from rats kindled by stimulating the angular bundle. Kainate receptors were labeled with [3H]kainate and quisqualate receptors with L-[3H]glutamate. When assayed one day after the last evoked seizure, kainate receptor binding had declined by 24-29% in stratum lucidum of hippocampal area CA3 and by 12-14% in the inner third of the dentate molecular layer, but was unchanged in the neocortex and basolateral amygdala. Saturation binding curves revealed that, under the conditions of these experiments, [3H]kainate labeled a single class of binding sites with a KD of 33-36 nM. In stratum lucidum of area CA3, kindling reduced the density of kainate receptors without altering their affinity for kainate. At the same time, quisqualate receptor binding had declined by 20-35% in many layers of the hippocampal formation and neocortex, but remained unchanged in the basolateral amygdala. Repeated stimulation or repeated seizures were required to produce these effects, since both kainate and quisqualate receptor binding were unchanged one day after a single afterdischarge. These receptor changes largely or completely reversed during a 28-day period without further stimulation. Thus maintenance of the kindled state probably cannot be explained by a long-lasting change in the expression of kainate or quisqualate receptors. The transient, regionally-selective down-regulation of these receptors may represent a compensatory response of forebrain neurons to repeated stimulation or seizures.

Impaired development of cerebellar cortex in rats treated postnatally with alpha-difluoromethylornithine.

alpha-Difluoromethylornithine specifically and irreversibly inhibits the enzyme ornithine decarboxylase. Ornithine decarboxylase catalyses the initial step in the synthesis of polyamines, which are thought to play an essential role in growth and development of mammalian tissues. The current study examined the effects of alpha-difluoromethylornithine on the ontogenic development of the rat cerebellar cortex. Animals injected daily with alpha-difluoromethylornithine on postnatal days 1-21 suffered a deficit in the number of granule cells and many of the remaining granule cells became trapped in the molecular layer during migration. Purkinje cells were also scattered throughout the molecular layer and their mean diameter was 38% smaller than in controls. In general, the cerebellar cortex of alpha-difluoromethylornithine-treated rats failed to progress much beyond the stage of development reached in control rats during the first postnatal week. These effects of alpha-difluoromethylornithine were already clearly visible at 10-15 days of age. The final size of the cerebellum as a whole and of individual folia was markedly subnormal. These data indicate that polyamines play an obligatory role in cerebellar neurogenesis and histogenesis.

Selective neuronal death after transient forebrain ischemia in the Mongolian gerbil: a silver impregnation study.

An important feature of ischemic brain damage is the exceptional vulnerability of specific neuronal populations and the relative resistance of others. Silver impregnation was used to delineate the extent and time-course of neuronal degeneration produced by 5 min of complete forebrain ischemia in the Mongolian gerbil. Lesions were confined to four brain regions: (1) hippocampal areas CA1, CA2-CA3a and CA4; (2) the dorsomedial portion of the lateral septal nucleus; (3) the dorsolateral portion of the striatum; and (4) the somatosensory neocortex. The ischemic lesion evolved with time in all four regions, but at different rates. Somatic argyrophilia developed rapidly in the striatum and hippocampal area CA4 (maximal in 24 h or less), at intermediate rates in the somatosensory neocortex, hippocampal areas CA1a and CA2-CA3a and the lateral septal nucleus (maximal in 2 days), and slowly in hippocampal area CA1b (maximal in 3 days). These results emphasize that the extent and rate of neuronal degeneration can vary even within a presumably homogeneous neuronal population, as evidenced by the different results in areas CA1a and CA1b. Similar results were obtained from analysis of brain sections stained with Cresyl Violet, hematoxylin-eosin or hematoxylin-eosin/Luxol Fast Blue. Terminal-like silver granules were observed in the projection fields of degenerated neurons. They also appeared, however, in the perforant path terminal zone of the hippocampal dentate molecular layer 1-2 days after transient ischemia and in stratum oriens and stratum radiatum of area CA1b prior to somatic degeneration. These granular deposits could not be clearly related to the degeneration of neuronal somata. Novel findings of this study include the degeneration of some dentate basket cells and lateral septal neurons and the appearance of terminal-like argyrophilia in the hippocampal formation without any obvious relation to somatic degeneration. Some of our results lend support to the hypothesis that ischemic neuronal cell death constitutes an excitotoxic process. Other results, however, suggest that the selective vulnerability of neurons to transient ischemia must involve factors beyond excitotoxicity.

Benzodiazepines protect hippocampal neurons from degeneration after transient cerebral ischemia: an ultrastructural study.

The ability of full and partial benzodiazepine receptor agonists to prevent DNA fragmentation and neuronal death after transient cerebral ischemia was investigated in the Mongolian gerbil. Diazepam (10mg/kg, i.p.) or the partial agonist imidazenil (3mg/kg, i.p.) was administered 30 and 90min after transient forebrain ischemia produced by occlusion of the carotid arteries for 5min. Treatment with diazepam completely protected CA1b hippocampal pyramidal neurons in 94% of the animals and partially protected pyramidal neurons in 6% of the animals, as assessed with a standard Nissl stain three and four days after ischemia. DNA fragmentation was examined by the terminal dUTP nick-end labeling (TUNEL) reaction. Prior to cell death, there were no TUNEL-positive neurons in area CA1b. By three days after ischemia, when neuronal degeneration was nearly complete, 14 out of 16 gerbils exhibited a positive TUNEL reaction throughout area CA1b stratum pyramidale. In 13 out of 14 gerbils treated with diazepam, no TUNEL-positive neurons were observed in this region. Imidazenil was less effective than diazepam with respect to both neuroprotection and prevention of DNA fragmentation. Three days after ischemia, six out of eight gerbils treated with imidazenil showed partial to complete neuroprotection. Imidazenil completely prevented DNA fragmentation in only one of the animals; varying degrees of TUNEL reaction persisted in the remainder. To determine whether the neurons protected by diazepam had a normal ultrastructure, gerbils were killed two to 30 days after ischemia and the hippocampal neurons in area CA1b were examined by electron microscopy. Within the first 48h after ischemia, early cytoplasmic changes of varying degrees (e.g., vacuolation, rough endoplasmic reticulum stacking, swollen mitochondria) and electron-dense dendrites were observed in gerbils not treated with diazepam. Degeneration was nearly complete by three days after ischemia. In contrast, pyramidal neuron ultrastructure appeared normal in gerbils that exhibited complete area CA1b neuroprotection (defined at the light microscope level) by diazepam when studied two, seven or 30 days after ischemia. In gerbils with partial protection of area CA1b, most of the remaining neurons exhibited varying degrees of necrosis when studied 30 days after ischemia. No apoptotic bodies were observed. We conclude that: (i) diazepam can fully protect CA1 pyramidal cells from the toxic effects of transient cerebral ischemia; (ii) when diazepam affords only partial neuroprotection, the residual CA1 pyramidal cells exhibit ultrastructural abnormalities consistent with necrotic damage; and (iii) diazepam is a more efficacious neuroprotectant than the partial benzodiazepine receptor agonist, imidazenil.

Effects of baclofen on synaptically-induced cell firing in the rat hippocampal slice.

The effects of baclofen on the synaptically-induced firing of pyramidal and granule cell populations were tested in the rat hippocampal slice. Population spikes were evoked by stimulating excitatory pathways in the presence and absence of bath-applied drug. (+/-)-Baclofen (20 microM) completely blocked the firing of CA1 or CA3 hippocampal pyramidal cells subsequent to stimulation of projections that originate in area CA3. In contrast, the firing of dentate granule cells evoked by stimulation of the perforant path fibres was depressed by only 46% and baclofen did not affect the monosynaptic firing of CA3 pyramidal cells evoked by mossy fibre stimulation. These results are consistent with the effects of baclofen on the corresponding extracellularly-recorded excitatory postsynaptic potentials (e.p.s.ps). The Schaffer collateral-commissural population spike in area CA1 was depressed by (-)-baclofen (EC50 = 2.8 microM), GABA (EC50 = 2.2 mM) and 3-aminopropanesulphonic acid (3-APS) (EC50 = 0.34 mM). (-)-Baclofen was 180 times as potent as (+)-baclofen. Bicuculline methiodide (100 microM) did not reverse the depressant action of (-)-baclofen. GABA-induced depressions were antagonized to only a small degree, whilst the effect of 3-APS was readily reversed. Raising the concentration of bicuculline from 100 microM to 500 microM did not further reverse the action of GABA. The effects of (-)-baclofen and 3-APS on the relationship between extracellular e.p.s.p. and population spike were tested by stimulation of the Schaffer collateral-commissural fibres in area CA1. (-)-Baclofen shifted the 'input/output' curve to the right at a concentration of 1 microM, but less or not at all at 3 microM. In contrast, increasing the concentration of 3-APS shifted this curve farther to the right.