J.V. Nadler

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.

Comparative toxicity of kainic acid and other acidic amino acids toward rat hippocampal neurons

Abstract Acidic amino acids were tested by histological criteria for their ability to destroy rat hippocampal neurons. When injected directly into the hippocampal formation, kainic acid preferentially destroyed CA3-CA4 pyramidal cells and was least toxic toward dentate granule cells and pyramidal cells of the h2 area. All other acidic amino acids tested destroyed hippocampal neurons non-selectively, the vulnerability of these neurons depending simply on their proximity to the injection site. At sufficient doses, kainic acid destroyed neurons in distant limbic regions, but the other acidic amino acids did not. All amino acid excitants tested were neurotoxic, the order of potency being:kainate>ibotenate>N-methyl- dl -aspartate>dihydrokainate> dl -homocysteate> l -cysteate> l -aspartate> l -glutamate. Most excitatory amino acid antagonists were only very weakly neurotoxic, but the phosphonate analogues of glutamate and aspartate were about as potent as the strong excitants, dl -homocysteate and l -cysteate. Intraventricular injections similarly revealed a distinction between selective hippocampal lesions made by kainic acid and non-selective hippocampal lesions made by high doses of other acidic amino acids. These results generally support previous correlations between neuro-excitatory and neurotoxic effects of acidic amino acids. However, the considerable neurotoxic potency of the phosphonate antagonists is difficult to reconcile with the conventional view that the ability of acidic amino acids to destroy neurons is determined solely by their ability to depolarize them. Several mechanisms are discussed to account for the different patterns of neuronal cell death observed after injection of kainic acid and other acidic amino acids. Finally,N-methyl- dl -aspartate is suggested as a useful agent for making localized axonsparing lesions.

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.

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.

Impaired development of cerebellar cortex in rats treated postnatally with α-difluorometh ylornithine

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.

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.

Regulation of glutamate and aspartate release from the Schaffer collaterals and other projections of CA3 hippocampal pyramidal cells.

Excitatory synaptic transmission in the CNS can be modulated by endogenous substances and metabolic states that alter release of the transmitter, usually glutamate and/or aspartate. To explore this issue, we have studied the release of endogenous glutamate and aspartate from synaptic terminals of the CA3-derived Schaffer collateral, commissural and ipsilateral associational fibers in slices of hippocampal area CA1. These terminals release glutamate and aspartate in about a 5:1 ratio. The release process is modulated by adenosine, by the transmitters themselves and by nerve terminal metabolism. Adenosine inhibits the release of both amino acids by acting upon an A1 receptor. The transmitters, once released, can regulate their further release by acting upon both an NMDA and a non-NMDA (quisqualate/kainate) receptor. Activation of the NMDA receptor enhances the release of both glutamate and aspartate, whereas activation of the non-NMDA receptor depresses the release of aspartate only. Superfusion of CA1 slices with a glucose-deficient medium increases the release of both amino acids and reduces the glutamate/aspartate ratio. These results have implications for the regulation of excitatory synaptic transmission in the CA1 area and for the mechanism of hypoglycemic damage to CA1 pyramidal cells.

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.

NMDA Receptor Plasticity in the Kindling Model

The N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptor serves a critical role in the development and stabilization of synapses in the developing nervous system (Cline et al., 1987) and in plasticity of the adult nervous system, particularly with respect to formation of some forms of learning and memory (Morris et al., 1986; Mondadori et al., 1989) Its role in these processes almost certainly derives from two unique features of this ionotropic neurotransmitter receptor: 1) its regulation by magnesium which results in its sensitivity to membrane voltage, thereby endowing it with associative properties (MacDonald et al., 1982; Flatman et al., 1983; Nowak et al., 1984; Mayer et al., 1984); and 2) its permeability to calcium (MacDermott et al., 1986), a second messenger capable of controlling a host of calcium sensitive enzymes.

Autoradiographic localization of ornithine decar☐ylase in cerebellar cortex of the developing rat with [3H]α-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.