H. Pollard

Kainate-induced apoptotic cell death in hippocampal neurons

We have examined the role apoptosis plays in epileptic brain damage using intra-amygdaloid injection of kainate. With the silver staining technique of Gallyas, argyrophylic (dying) neurons were observed, a few hours after the injection, in the amygdala and in the vulnerable pyramidal neurons of the hippocampal CA3 region. In both areas, cell death has apoptotic features, including: (i) nuclear chromatin condensation and marginalization with light and electron microscopy; (ii) DNA fragmentation with a typical ladder pattern on agarose gel electrophoresis; (iii) positive nuclear labelling with a selective in situ DNA fragmentation staining method. Combined in situ DNA labelling and silver staining showed that the DNA fragmentation occurred in dying neurons. CA1 or granule cells which do not degenerate following intra-amygdaloid injection of kainate were not stained with the in situ DNA labelling or the argyrophylic technique. Administration of diazepam blocked the kainate-induced seizures and prevented DNA fragmentation in CA3 but not in the amygdala. Therefore, apoptosis contributes to the local and distant damage induced by kainate.

Histamine as a Neurotransmitter in Mammalian Brain: Neurochemical Evidence

For a long time the history of histamine (HA) has been confounded with that of autacoids, i.e., endogenous substances with potent biological effects but whose physiological function is only a matter of speculation. However, starting from about 1970, sufficient data, mostly of a biochemical nature, have been collected to justify its present classification in most reviews or textbooks at the top of the list of putative neurotransmitters in the brain. The main landmarks in this evolution are probably the development of neurochemical methods allowing for assessment of the possibilities that: synthesis and storage of HA occurs in neuronal pools, in which it is in a dynamic state; that selective lesions of neuronal tracts are followed by changes in HA and L-histidine decarboxylase (HD); and that HA receptors can be detected in brain by various approaches. Recently the view that HA might have a neurotransmitter role in mammalian brain has been indirectly reinforced by the identification of histaminergic neurons in the CNS of the marine mollusk Aplysia californica (for a review, see Weinreich, 1977). The short review will consider the. most significant neurochemical data obtained concerning the metabolism of the amine, the localisation of putative histaminergic pathways and the characterization of receptors. Other aspects not covered here can be found in recent reviews: behavioral and pharmacological aspects (Green et al . , 1978; Schwartz et al., 198Oa), neuroendocrinological aspects (Weiner and Ganong, 1978), and neurovegetative aspects (Owen, 1977).

A detailed autoradiographic mapping of histamine H3 receptors in rat brain areas.

[3H](R)alpha-methylhistamine, a selective histamine H3-receptor ligand, was used to perform binding studies with membranes and generate light microscopic autoradiograms in sections of the rat brain. High densities of H3 receptors were found in membranes from the anterior part of the cerebral cortex, the accumbens nucleus, the striatum, the olfactory tubercles and the substantia nigra. Autoradiography of sagittal and frontal sections evidenced specific labelling in a number of gray matter areas over a very low background, as determined using thioperamide, a selective H3-receptor antagonist, as competing drug. Labelled areas were identified by comparison with adjacent Nissl-stained sections and their labelling was rated visually. H3 receptors are heterogeneously distributed among areas known to receive histaminergic projections. In the cerebral cortex, H3 receptors are present in all areas and layers, with a rostrocaudal gradient and a higher density in deep layers (laminae IV-VI). In the hippocampal formation, H3 receptors are the most abundant in the dentate gyrus and the subiculum. In the amygdaloid complex, the highest densities are found in the central, lateral and basolateral groups of nuclei. In the basal forebrain, the accumbens nucleus, the striatum, the olfactory tubercles and the globus pallidus are highly labelled. In the thalamus in which histaminergic fibres are scarce, H3 receptors are present in a rather high density, particularly in the midline, median and intralaminar groups of nuclei. In the hypothalamus, where the densest network of histaminergic fibres is found, H3 receptors occur in moderate density, being slightly more abundant in the anterior and medial part. They are also present at the level of the tuberomammillary nuclei where they may reside on histaminergic perikarya. In mesencephalon and lower brainstem, H3 receptors are abundant in the reticular part of the substantia nigra and central gray. They are present in low density in areas of noradrenergic and serotoninergic perikarya and in the spinal cord, where a faint specific labelling is detected in the gray matter, particularly in the external layers of the dorsal horn. In the cerebellum and pituitary gland, H3 receptors are scarce. Kainic acid infusions into the striatum were followed by marked local decreases in H3 receptors evidenced in both membrane binding and autoradiographic studies. Unilateral interruption of the ascending histaminergic pathways via electrocoagulation of the lateral hypothalamic area was followed by ipsilateral increase in striatal [3H](R)alpha-methylhistamine binding, a process consistent with denervation up regulation of postsynaptic H3 receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Regional variability in DNA fragmentation after global ischemia evidenced by combined histological and gel electrophoresis observations in the rat brain

Abstract: We have studied whether the delayed cell death induced by transient forebrain ischemia is associated with an inter‐nucleosomal cleavage of DNA into oligonucleosome‐sized fragments. The integrity of genomic DNA in various brain regions after a 20‐min four‐vessel ischemia was examined using gel elec‐trophoresis. We found typical ladders of oligonucleosomal DNA fragments in the striatum and in the Ammons horn. In the latter we also often found a random DNA degradation as a smear pattern. These findings were reinforced by a specific in situ labeling of DNA breaks in tissue sections. A dark staining of nuclei was observed in the cell bodies of neurons—in particular in the head of the caudate and in the vulnerable CAl hippocampal area. With biochemical and histological approaches, there was no evidence of DNA degradation in regions that are resistant to the injury. We conclude that the association of multiple mechanisms of cell damage may occur after a global ischemia. The regional variability in DNA fragmentation stresses the importance of using histological approaches in parallel with gel electrophoresis.

Involvement of GABAA receptors in the outgrowth of cultured hippocampal neurons.

Whereas GABA is a major inhibitory neurotransmitter in the adult central nervous system, recent experiments performed in our laboratory have shown that the activation of GABAA receptors in the hippocampus leads to excitatory effects during the early post-natal period. The possible consequence of a depolarizing effect of GABA was assessed on the neuritic outgrowth of embryonic hippocampal neurons in culture. No morphological alterations were observed when hippocampal neurons were cultured for three days in the presence of muscimol, a GABAA receptor agonist. In contrast, the neuritic outgrowth of cultured hippocampal neurons was profoundly affected by the presence of bicuculline in the culture medium. In the presence of this GABAA receptor antagonist neurons displayed a reduction in the number of primary neurites and branching points, resulting in a concomitant decrease of the total neuritic length. Thus, this study suggests that GABA, acting on GABAA subtype of receptors, is able to affect the development of the hippocampus.

Three histamine receptors (H1, H2 and H3) visualized in the brain of human and non-human primates

The distribution of histamine H1, H2 and H3 receptors in postmortem human and rhesus monkey brain was examined using receptor autoradiography. [125I]Iodobolpyramine, [125I]iodoaminopotentine and [3H](R) alpha-methylhistamine were used as ligands to label H1, H2 and H3 receptors respectively. The 3 receptor subtypes were identified in the human and monkey brains. Each receptor presented comparable distribution in the two primate brains. H1 and H2 receptors were particularly enriched in the caudate and putamen and observed in other brain areas such as the neocortex and hippocampus. H3-receptors were found to predominate in the basal ganglia where the highest densities were localized in the two segments of the globus pallidus. They were also observed in the hippocampus and cortical areas. The distribution of these 3 histamine receptors in the primate brain suggests the involvement of histaminergic mechanism in the functions of many brain areas. In particular, H2 and H3 receptors could play a role in the regulation of the basal ganglia functions in primates.

Alterations of the GluR-B AMPA receptor subunit flip/flop expression in kainate-induced epilepsy and ischemia

In the hippocampus, glutamatergic pathways are altered following seizure activity or transient global ischemia, both pathological conditions leading to selective neuronal degeneration. Glutamatergic receptors, and notably alpha-amino-3-hydroxy-5-methyl-4-isoxazolopropionate (AMPA) receptors, a family of glutamate receptors involved in fast synaptic transmission and in the maintenance of synaptic potentiation may play an important role in the pathological outcome. AMPA receptors are assembled from GluR-A, GluR-B, GluR-C and GluR-D polypeptides which exist in flop and flip variants, the latter allowing larger glutamate responses. Using in situ hybridization techniques, we show that kainate-induced epilepsy provokes a rapid but transient increase (50%) of GluR-B flip mRNA levels in all subregions of the hippocampus (CA1, CA3, dentate gyrus). This early phase is followed by a second, persistent GluR-B flip increase in regions in which neurons are known to be seizure-resistant (i.e. CA1 an dentate gyrus) while a 35% decrease is observed in the vulnerable CA3 area. Following global ischemia, the levels of GluR-B flip and flop variants are dramatically reduced (90-100%), well before any morphological signs of cell death, in the subiculum and CA1, two areas known to be particularly sensitive to ischemic insult. In keeping with the properties of GluR flip variants, it is suggested that altered subunit stoichiometry may lead to long-lasting enhanced efficiency of fast synaptic transmission in the epileptic hippocampus. Since GluR-B containing receptors are Ca2+ impermeable, our results also suggest altered Ca2+ permeability in the vulnerable pyramidal neurons of areas CA3 and CA1 in the epileptic and ischemic hippocampi, respectively.

Catabolism of 3H-histamine in the rat brain after intracisternal administration☆

The fate of 3H-histamine of high specific activity has been studied after its administration into the cisterna magna or into the lateral ventricle. 3H-histamine metabolites were analyzed by a combination of methods involving ion-exchange and thin-layer chromatography, solvent extraction and isotope dilution. Histamine catabolism occurred mainly, if not solely, through ring-methylation followed by deamination into methylimidazole acetic acid which was found to be the main catabolite. This last step was inhibited in tranylcypromine-treated rats, resulting in a marked rise in brain 3H-methylhistamine. Partial inhibition of histamine methylation was achieved by administration of methylhistamine in high doses and resulted in a slower disappearance of 3H-histamine; it also induced a prompt increase in the level of endogenous histamine, suggesting a high turnover rate for the brain amine. Deamination of 3H-methylhistamine occurred more slowly in the newborn than in the adult rat brain, probably in relation with incomplete development of monoamine oxidase activity. If present, direct oxidative deamination of histamine was a minor pathway, as evidenced by the low 3H-imidazole acetic formation as well as by the slight effects of aminoguanidine treatment, both on 3H-histamine catabolism and on endogenous amine level. The efflux of 3H-deaminated metabolites from brain was not modified by treatment with probenecid.

RADIOIMMUNOASSAY OF METHIONINE‐AND LEUCINE‐ENKEPHALINS IN REGIONS OF RAT BRAIN AND COMPARISON WITH ENDORPHINS ESTIMATED BY A RADIORECEPTOR ASSAY

Abstract— Radioimmunoassays (RIAs) selective for methionine‐enkephalin (Met‐ENK) and leucineenkephalin (Leu‐ENK) have been developed using competition towards binding of 10 pM 125I‐enkephalins to antibodies raised in rabbits against ENKs coupled to ovalbumin with carbodiimide. The high sensitivity of the RIAs (IC50 0.57 nm and 0.55 nm for Met‐ and Leu‐ENK, respectively) allowed estimation of the enkephalin content in extracts of all rat brain regions. Regional levels are compared with those determined on the same extracts by a radioreceptor assay (RRA) using competition towards binding of 5 nm [3H]Leu‐ENK to rat striatal membranes. Optimal conditions for killing the animals and extracting the endorphins have been carefully investigated: killing by rapid microwave irradiation was not found necessary as long as brain regions were homogenized into 0.1 n‐HCl before deproteinization.

Opiate receptors on mesolimbic dopaminergic neurons

Abstract Following electrocoagulation of dopaminergic cell bodies of the mesolimbic system, [ 3 H]naloxone binding decreases significantly in septum and nucleus accumbens. The latter effect is also observed after local injection of 6-hydroxydopamine. These findings indicate that opiate receptors are localized presynaptically on dopaminergic neurons from limbic areas. They could mediate presynaptic inhibitions of dopamine release which would account for a large number of behavioral effects of morphine in single or repeated administrations.