NeuroAiD: properties for neuroprotection and neurorepair.

BACKGROUND: Treatments for stroke and other brain injuries are limited. NeuroAiD has been shown to be beneficial in clinical studies. We reviewed the pharmacological effects of NeuroAiD on the normal and ischemic brain and neurons. METHODS: In vivo and in vitro experiments using mouse model of stroke (focal ischemia), rat model of cardiac arrest (global ischemia) and cortical neurons in culture were reviewed and summarized. RESULTS: NeuroAiD improved survival, attenuated infarct size, improved functional recovery in the model of focal ischemia, and protected neurons against glutamate-induced injury. Furthermore, it enhanced cognitive recovery by reducing hippocampal CA1 cell degeneration, DNA fragmentation, Bax expression and ma-londialdehyde release in the model of global ischemia. Activation of the Akt survival pathway and opening of KATP channels may contribute to the neuroprotective properties of NeuroAiD. NeuroAiD increased BDNF expression and induced proliferation of cells which differentiate and mature into neurons. It enhanced rosette formation of human embryonic stem cells. NeuroAiD-treated embryonic cortical neurons developed into neurons with longer neurites, denser outgrowths and networks, and more synaptic release sites. CONCLUSIONS: NeuroAiD demonstrated both neuroprotective and neuroregenerative properties in rodent models of focal and global ischemia and in cortical cell cultures. These properties would be important for developing a treatment strategy in reducing the long-term disability of stroke, cardiac arrest and other brain injuries.

Specific caspase pathways are activated in the two stages of cerebral infarction.

Necrosis and apoptosis have been initially identified as two exclusive pathways for cell death. In acute brain lesions, such as focal ischemia, this binary scheme is challenged by demonstrations of mixed morphological and biochemical characteristics of both apoptosis and necrosis in single cells. The resulting difficulty in defining the nature of cell death that is triggered by severe insults has dramatically impeded the development of therapeutic strategies. We show that in the early stages of cerebral infarction, neurons of the so-called "necrotic" core display a number of morphological, physiological, and biochemical features of early apoptosis, which include cytoplasmic and nuclear condensations and specific caspase activation cascades. Early activation cascades involve the death receptor pathway linked to caspase-8 and the caspase-1 pathway. They are not associated with alterations of mitochondrial respiration or activation of caspase-9. In contrast, pathways that are activated during the secondary expansion of the lesion in the penumbral area include caspase-9. In agreement with its downstream position in both mitochondria-dependent and -independent pathways, activation of caspase-3 displays a biphasic time course. We suggest that apoptosis is the first commitment to death after acute cerebral ischemia and that the final morphological features observed results from abortion of the process because of severe energy depletion in the core. In contrast, energy-dependent caspase activation cascades are observed in the penumbra in which apoptosis can fully develop because of residual blood supply.

Comparative study of the glial fibrillary acidic protein in vertebrates by PAP immunohistochemistry.

Glial fibrillary acidic protein (GFA) has been visualized by direct peroxidase antiperoxidase (APA) immunohistochemistry in various vertebrates (cyclostomes, teleosts, amphibians, reptiles, birds, and several placental mammals). In this study GFA-immunoreactivity (GFA-I) was observed in all species examined except in cyclostomes and amphibians. Two types of immunoreactive elements were observed: astrocytes and long processes without visible somata. Astrocytic cells with GFA-I were first found in the snake, and more cells were in birds where the pattern of distribution was similar to that of mammals. Within mammals, few differences in the manner of localization were observed among different species, except in the corpus callosum and the ependymal and subependymal layers. Long straight processes were observed in the lower submammalians--the lamprey, carp, and turtle. They radiated through the neuropil from the ventricular wall and followed nerve fiber bundles in the white matter. An uncommon feature was observed in the turtle brain, which possessed very intense GFA-I within the ependymal layer. The presence of GFA-containing profiles in the ependyma of adult animals is discussed in relation to GFA-positive structures seen in the human brain during ontogeny or under certain pathological conditions.

Distribution of gamma-aminobutyric acid-immunoreactive neurons in the septal region of the rat brain.

The distribution of gamma-aminobutyric acid-immunoreactive (GABA-I) elements was examined in the septal region of the rat brain. The indirect peroxidase-antiperoxidase technique was used with anti-GABA antibodies in normal and colchicine-pretreated rats, with or without use of detergent in the incubation medium. Intraventricular injection of colchicine did not result in any change in the staining of neuronal perikarya. Intraseptal injections increased the intensity of labelling of GABA-I cell bodies in the lateral septal nucleus and increased the number of labelled cells in the medial septal nucleus and diagonal band of Broca (dbB). Triton X-100 added to the incubation media decreased the intensity of staining and number of GABA-I somata in all septal nuclei with a concentration-dependent effect. No change was observed concerning GABA-I varicosities. The septal area, including the lateral, medial, and triangular septal nuclei; the anterior rudiment of the hippocampus; the island of Calleja magna; the septofimbrial nucleus; the bed nucleus of the stria terminalis; and the dbB showed a strong reaction to anti-GABA antibodies with regard to GABA-containing surrounding structures. GABA-I axonal varicosities were observed in all the regions with an uneven distribution. The highest density was found in the dorsal and ventral parts of the lateral septal nucleus and in a band situated between the dbB and the nucleus accumbens. Labelled varicosities were frequently observed surrounding GABA-I and nonimmunoreactive cell bodies. GABA-I somata ranged from 10 to 30 micron in diameter. Small neurons were present in great number at the ventricular border and in the zona limitans. Medium-size and large neurons were mostly observed in the medial part of the dorsal lateral nucleus and in the intermediate lateral nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution and role of aspartate in the nervous system of the chaetognath Sagitta.

Cholinergic control of locomotory muscles in chaetognaths is monitored by diffuse transmitter release through layers of collagen fibers that form the connective stratum of the hydroskeleton. Despite the lack of morphologically defined synaptic junctions, the control of locomotor activity in chaetognaths is highly specific and allows complex behavioral patterns. This complexity suggests the existence of neuromediators acting to modulate the effects of the main motor neurotransmitter, acetylcholine, on muscular contraction. Immunocytochemical investigations performed in Sagitta friderici by using antibodies directed against L-aspartate revealed the presence of the amino acid within abundant fiber networks regularly distributed in the head, trunk, and tail and within discrete groups of cell bodies. In addition to known components of the sensory and motor nervous systems, L-aspartate immunoreactivity revealed previously undescribed intraepidermal networks of axonal profiles. With the exception of two giant anterior fibers radiating from the ventral ganglion, L-aspartate-immunoreactive processes were usually thin and varicose, occasionally making an anastomosis. As indicated by electron microscopy, L-aspartate-immunoreactive varicosities apposed to the connective stratum were filled with synaptic-like vesicles but displayed no synaptic differentiation. Physiologic investigations suggested a potent inhibitory effect of L-aspartate on acetylcholine-induced muscle contraction. The wide distribution pattern of immunoreactive profiles suggests an important role of L-aspartate in motor and sensory functions in chaetognaths. Although classified among excitatory amino acids in vertebrates, aspartate may function as an inhibitory modulator of acetylcholine-induced muscle contraction in these enterocoelous gastroneuralians.

Hypothalamic galanin-immunoreactive neurons projecting to the posterior lobe of the rat pituitary: a combined retrograde tracing and immunohistochemical study.

UNLABELLED: To identify the galanin-immunoreactive neurons projecting to the posterior lobe of the pituitary in the rat hypothalamus, a retrograde tracer (complex of wheat germ agglutinin-enzymatically inactive horseradish peroxidase-colloidal gold) was injected into the posterior lobe of the pituitary. Sections of the hypothalamus were treated with a combination of silver enhancement of retrogradely transported tracer and immunohistochemistry of galanin. Of the total number of hypothalamic cells doubly labeled with retrograde tracing and galanin-immunostaining, 56-60% were found in the supraoptic nucleus, 18-23% in the retrochiasmatic nucleus, 8-10% in the lateral magnocellular portion of the paraventricular nucleus. The ratio of (number of doubly labeled cells/number of galanin-immunoreactive cells) in each of the above regions was similar to the ratio of (number of retrogradely labeled cells/number of Nissl-stained cells) in the supraoptic nucleus. Of all retrogradely labeled cells in the hypothalamus, 51-56% also contained galaninlike immunoreactivity. IN CONCLUSION: (1) galanin-immunoreactive fibers in the posterior lobe of the pituitary originate mainly in the supraoptic nucleus, retrochiasmatic nucleus, and lateral magnocellular portion of the paraventricular nucleus, (2) most of galanin-immunoreactive cells in these regions project to the posterior lobe of the pituitary, and (3) about half the neurons constituting the hypothalamo-neurohypophyseal system contain galaninlike immunoreactivity.

Differential regulation of NGF receptors in primary sensory neurons by adjuvant-induced arthritis in the rat.

In the adult brain, neurotrophins play a key role in adaptive processes linked to increased neuronal activity. A growing body of evidence suggests that chronic pain results from long-term plasticity of central pathways involved in nociception. We have investigated the involvement of nerve growth factor (NGF) in adaptive responses of primary sensory neurons during the course of a long-lasting inflammatory pain model. The amount and distribution of the NGF receptors p75(NTR) and TrkA were measured in the dorsal horn and dorsal root ganglia (DRG) of animals subjected to Freund's adjuvant-induced arthritis (AIA). We observed an increased immunoreactivity of both receptors in the central terminals of primary sensory neurons in the arthritic state. The increases were seen in the same population of afferent terminals in deep dorsal horn laminae. These changes paralleled the variations of clinical and behavioral parameters that characterize the course of the disease. They occurred in NGF-sensitive, but not GDNF-sensitive, nerve terminals. However, p75(NTR) and TrkA protein levels in the DRG (in the cell body of these neurons) showed different response patterns. An immediate rise of p75(NTR) was seen in parallel with the initial inflammation that developed after administration of Freund's adjuvant in hindpaws. In contrast, increases of the mature (gp140(trk)) form of TrkA occurred later and seemed to be linked to the development of the long-lasting inflammatory response. The changes in receptor expression were observed exclusively at lumbar levels, L3-L5, somatotopically appropriate for the inflammation. Together, these results implicate NGF in long-term mechanisms accompanying chronic inflammatory pain, via the up-regulation of its high affinity receptor, and offer additional evidence for differential processes underlying short- versus long-lasting inflammatory pain.

The expression pattern of somatostatin and calretinin by postnatal hippocampal interneurons is regulated by activity-dependent and -independent determinants.

Hippocampal interneurons form distinct populations identified on the basis of their projection pattern and neurochemical characteristics, which includes the expression of specific neuropeptides and/or calcium-binding proteins. The neurochemical maturation of hippocampal interneurons is largely a postnatal event, and factors which govern this maturation are presently unknown. Using slice cultures, we have investigated the role of neuronal activity in regulating the expression of somatostatin and calretinin during the postnatal maturation of hippocampal interneurons. Blocking inhibitory activity with bicuculline, or excitatory activity with 6,7-dinitroquinoxaline-2,3-dione, for 14 days in slice cultures from seven-day-old rat increased and decreased, respectively, the number of somatostatin-immunoreactive neurons. Withdrawal of the blocking agents resulted in a reversal of the effects on somatostatin immunoreactivity. In addition, bicuculline slightly increased the number of calretinin-positive neurons, while 6,7-dinitroquinoxaline-2,3-dione exerted no effect. However, bicuculline and 6,7-dinitroquinoxaline-2,3-dione markedly increased and decreased, respectively, the number of calretinin-labelled axons. Despite activity-linked modifications of immunoreactivity levels, no change in the organotypic location of somatostatin-labelled neurons was observed, whatever the treatment. Double labelling studies demonstrated that somatostatin and calretinin were expressed by different neurons, even when the number of labelled cells was highly increased. These results show that the levels of expression of somatostatin and calretinin in maturing hippocampal interneurons are tuned to the endogenous balance of excitatory and inhibitory activity. In contrast, the neurochemical specificity of each subtype of interneurons does not depend upon variations in neuronal activity.

Ultrastructural immunocytochemical study of the dopaminergic innervation of the rat lateral septum with anti-dopamine antibodies.

The dopaminergic innervation of the rat lateral septum has been investigated at ultrastructural level by immunocytochemistry using the unlabelled peroxidase-anti-peroxidase method with anti-dopamine antibodies. The specificity of the reaction has been carefully checked by immunological and histochemical controls. A strong immunoreaction was observed in fibres of the lateral septum as well as in their cells of origin in the ventral tegmental area. In the lateral septum, dopamine-immunoreactive fibres were localized in two distinct areas. A first area, located ventrally in the anterior part of the septum was characterized by a high density of immunoreactive varicosities with barely visible intervaricose segments. A more dorsal area, extending throughout the anteroposterior region of the septum, was characterized by immunoreactive fibres in pericellular arrangements. Electron microscopic observations revealed no difference in the ultrastructure of dopamine-immunoreactive profiles in the different areas. Reaction product was found in vesicles, linked to microtubules and in the cytoplasm. Three types of vesicles were seen: (i) small vesicles (30-50 nm) with varying intensity of immunoreaction, filling up the varicosities; (ii) rare large clear vesicles (50-80 nm) with no internal immunoreaction; (iii) very rare large dense vesicles (50-100 nm) with a strong dopamine immunoreactivity. Labelled profiles were observed in clearly defined asymmetrical synaptic contacts with somata and dendrites. Due to the lack of previous work dealing with the use of anti-dopamine antibodies for electron microscope immunocytochemistry, our observations are compared to previous data obtained by more indirect labelling techniques.

Host dopaminergic afferents affect the development of DARPP-32 immunoreactivity in transplanted embryonic striatal neurons.

Homotopic transplantation provides an interesting way to observe the relationships between developing cells and ingrowing host afferents. We have performed a complete and selective elimination of the mesostriatal dopaminergic system in adult rats to observe the influence of its absence on the development and chemical differentiation of embryonic striatal cells. Cell suspensions from striatal primordia of 14-15-day-old embryos were transplanted into host striata that were (i) neuron-depleted by kainic acid (control group) or (ii) deprived of dopamine by 6-hydroxydopamine prior to the neuronal depletion by kainic acid (experimental group). The expression of dopamine- and adenosine 3',5'-monophosphate-regulated phosphoprotein (DARPP-32) by transplanted cells was observed in correlation with their innervation by host dopaminergic afferents which in turn were identified by tyrosine hydroxylase immunohistochemistry. Observations were made between four days and three months after transplantation. Four days after transplantation, no immunoreactivity for DARPP-32 was observed in transplants of control animals despite the presence of tyrosine hydroxylase-immunopositive fibers growing from the host to discrete cell clusters in the transplant. DARPP-32-labeled cells appeared soon afterwards. Six days after transplantation they displayed varying intensities of immunoreaction, ranging from just detectable to normal levels and were specifically targeted by developing tyrosine hydroxylase-immunopositive fibers. The number of DARPP-32-labeled cells increased rapidly and they formed increasingly compact clusters. Fourteen days after transplantation and afterwards, all the DARPP-32-labeled cells displayed an intensity of immunoreaction and a distribution comparable to that observed in long-term transplants. Transplants in the experimental hosts displayed the same organization and developmental features as the control transplants with the exception of DARPP-32 labeling which was not detected before eight days after transplantation. Ten days after transplantation, the distribution and intensity of DARPP-32 labeling was similar to that observed at six days in the control group. The evolution of DARPP-32 labeling after 10 days in the experimental group paralleled that observed six days post-transplantation and beyond in the control group. Dopaminergic mesostriatal host afferents are able to provide developing cells in grafted striatal tissues with normal innervation very rapidly. Despite this rapidity, the innervation does not seem to have any trophic influence on the general development of the transplant but does affect the onset time of the expression of neurochemical markers that are directly related to its synaptic function.(ABSTRACT TRUNCATED AT 400 WORDS)

Anatomical distribution and ultrastructural organization of the GABAergic system in the rat spinal cord. An immunocytochemical study using anti-GABA antibodies.

gamma-Aminobutyric acid (GABA)-containing elements have been studied by light and electron microscopy in the rat spinal cord, using immunocytochemistry with anti-GABA antibodies. Light microscopy showed immunoreactive somata localized principally in laminae I-III, and occasionally in the deeper laminae of the dorsal horn and in the ventral horn. Small somata were also observed around the central canal. Punctate GABA-immunoreactive profiles were particularly concentrated in laminae I-III, and moderately abundant in the deeper laminae and in the ventral horn where they were observed surrounding the unlabelled motoneurons. At the ultrastructural level, the punctate profiles corresponded to GABA-containing axonal varicosities or small dendrites. GABA-immunoreactive varicosities were presynaptic to labelled or unlabelled dendrites and cell bodies. Some unlabelled terminals presynaptic to unlabelled dendrites received symmetrical synaptic contacts from GABA-immunoreactive terminals. These results confirm data obtained with L-glutamate decarboxylase immunocytochemistry, and support the role of GABA in pre- and postsynaptic inhibition in the spinal cord, respectively via axoaxonal and axosomatic or axodendritic synapses.

Dissimilar responses of adult thalamic monoaminergic and somatosensory afferent fibers to implantation of thalamic fetal cells.

It is generally accepted that transplanted fetal neurons can, after several weeks to months, establish connections with the host CNS. Host afferent systems seem, however, to show different types of responses to the presence of grafted fetal neurons. The present study is a preliminary step to identify mechanisms involved in the reactions of adult axons to transplanted fetal neurons. The right ventrobasal thalamus of adult rats was depleted of neurons by in-situ injection of kainic acid and cell suspensions from homotopic thalamic embryonic primordia which were injected into the lesioned area. After various post-implantation delays, ranging from five to 30 days, two types of experiments were performed: (i) noradrenaline and serotonin immunohistochemistry with specific antibodies on alternate sections; and (ii) anterograde tracing using wheat germ agglutinin conjugated to horseradish peroxidase from the dorsal column nuclei and the principal sensory trigeminal nucleus. Five days after transplantation, host monoaminergic fibers (either noradrenergic or serotoninergic) had already grown into the transplants. Ingrowing fibers were thin and poorly varicose, exhibiting endings morphologically similar to the growth cones observed during axogenesis. Seven days after grafting, growth cones were no longer visible and monoaminergic fibers exhibited either normal-sized or very large varicosities. Large varicosities progressively decreased in number and, after three weeks, the fibers displayed a normal adult morphology, forming a dense network all over the transplants. In contrast, host somatosensory afferents, labeled by anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase, did not grow into the transplants. Intermingling of somatosensory afferents and transplanted cells was observed only after 10 days, when grafted neurons extended outside the original transplantation site into the neuron-depleted area containing the somatosensory afferents. The present results demonstrate that adult monoaminergic and somatosensory afferents, when deprived of their usual target, do not react in a similar way to the addition of fetal neurons. It is proposed that adult monaminergic fibers have the ability to regain morphological (and probably functional) immature forms which were considered to be restricted to the period of axogenesis or to lesion-induced regeneration. In contrast, fetal transplants do not seem to induce, by themselves, a similar alteration of genetic expression in adult somatosensory neurons. It has been proposed that "diffuse" and "point-to-point" axonal systems may be differentiated in the CNS on anatomical bases. The present results add to the identification of two different systems by demonstrating that, in the thalamus, they present dissimilar responses to the implantation of fetal cells.

Correlated long-term increase of brain-derived neurotrophic factor and Trk B proteins in enlarged granule cells of mouse hippocampus after kainic acid injection.

Our previous studies have shown that a single injection of kainic acid into the dorsal hippocampus of adult mice resulted in hypertrophy of the dentate gyrus granule cells. This hypertrophy was correlated with a long-lasting increase of brain-derived neurotrophic factor messenger RNA, and prevented by anti-sense brain-derived neurotrophic factor oligonucleotide treatment. These results suggest that an increase of brain-derived neurotrophic factor messenger RNA may be a major trigger of granule cells enlargement. However, the level of messenger RNA of Trk B, the high-affinity receptor of brain-derived neurotrophic factor, was not increased significantly, raising the question of whether increased brain-derived neurotrophic factor messenger RNA level leads actually to an increased protein production. The objective of the present study was to examine this; changes in contents of brain-derived neurotrophic factor and TrkB protein were monitored by immunohistochemistry during kainic acid-induced hypertrophy. Results show that immunoreactivities of brain-derived neurotrophic factor and Trk B were present in enlarged granule cells. These immunoreactivities increased from two to 16 weeks after kainic acid injection and were maintained up to 12 months. Simultaneous increases of brain-derived neurotrophic factor messenger RNA and protein, and of TrkB protein were coupled tightly to the chronology of granule cell enlargement, suggesting that the action of brain-derived neurotrophic factor in the induction and maintenance of kainic acid-induced granule cells enlargement is likely to be mediated by TrkB. The discrepancy between the previously described lack of increase of TrkB messenger RNA and the herein observed increase of the protein further reveals the existence of translational regulations of the receptor messenger RNA.

Short increase of BDNF messenger RNA triggers kainic acid-induced neuronal hypertrophy in adult mice.

Neurotrophin gene expression in adult brain varies according to physiological activity and following brain injury, suggesting a role in neuronal maintenance and plasticity. However, the exact roles and mechanisms of action of neurotrophins in the adult brain are still poorly understood. We have recently demonstrated that neurons of the adult mouse dentate gyrus can develop a conspicuous morphogenetic response to intrahippocampal injection of kainic acid. This response is correlated with long-lasting overexpression of the brain-derived neurotrophic factor gene, suggesting a causal relationship between molecular and structural changes. To test this hypothesis, brain-derived neurotrophic factor messenger RNA were sequestered in vivo by administration of antisense oligodeoxynucleotides. When administered before 20 h post-kainate, antisense oligodeoxynucleotides totally prevented the kainate-induced neuronal hypertrophy, while sense or missense sequences had no effect. On the other hand, the hypertrophic response was observed when antisense administration was begun 24 h post-kainate, indicating an involvement of brain-derived neurotrophic factor messenger RNA in the initiation of structural changes, but not in their evolution. The hypertrophy was blocked by inhibition of tyrosine kinase activities by K252a, suggesting an involvement of Trk high affinity receptors. Administration of human recombinant brain-derived neurotrophic factor without previous treatment by kainate failed to induce any morphogenetic response. These results show that a short activation of the brain-derived neurotrophic factor gene can, in association with neuronal activation by kainate, trigger dramatic and long-lasting morphological changes in adult neurons. A physiological role of brain-derived neurotrophic factor in adult brain could therefore be to link, by autocrine/paracrine action, activation of glutamate receptors and neuronal morphological adaptive responses.

Decreased choline acetyltransferase activity in nerve growth factor-transgenic mice during brain development.

Activity of the synthetic enzyme for acetylcholine, choline acetyltransferase was investigated during development and in adult nerve growth factor-transgenic mice. A conspicuous reduction of choline acetyltransferase activity was observed in the anterior brain of nerve growth factor-transgenic embryos from embryonic days 13 to 16 (E13 to E16). Choline acetyltransferase activity levels subsequently resumed to normal levels, with the exception of a 15% increase in the adult hippocampus. Nerve growth factor contents followed a similar time-course and regional distribution in normal and nerve growth factor-transgenic animals and displayed significantly higher values from E14 to the early postnatal period. Nerve growth factor contents were normal in the adult brain. In vitro experiments confirmed the involvement of nerve growth factor in the decrease of choline acetyltransferase activity levels observed in transgenic neurons during development. These results suggest a role for nerve growth factor in the initial phase of the phenotypic differentiation of cholinergic neurons. They show that nerve growth factor may, under specific development conditions, lead to a paradoxical down-regulation of choline acetyltransferase activity.

Long-term increase of GluR2 alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor subunit in the dispersed dentate gyrus after intrahippocampal kainate injection in the mouse.

Intrahippocampal injection of a subtoxic dose of kainate in mice has been shown to induce a dispersion of granule cells of the dentate gyrus, which is a characteristic morphological change often seen in human hippocampal sclerosis. In addition, it has been shown recently that such injections lead to recurrent hippocampal seizures and changes in glucose metabolism, which are reminiscent of temporal lobe epilepsy. Previous reports on human hippocampal sclerosis have shown an increase of the expression of the GluR2 alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate subunits in the dispersed granule cell somata. However, no such changes have been observed so far in animal models of epilepsy with hippocampal sclerosis. In this study, the expression of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor subunits was examined by immunohistochemistry following intrahippocampal injection of kainate in mice and rats. In mice, such injection induced a persistent increase of GluR2 immunoreactivity in the granule cells for up to 180 days. By contrast, GluR1 immunoreactivity was transiently increased during the first four days after the injection and progressively decreased thereafter. By contrast, intrahippocampal injection of kainate in rats did not result in granule cell dispersion and no changes in GluR1 immunoreactivity or GluR2 immunoreactivity were observed. These results show that, in addition to morphological, clinical and metabolical similarities, intrahippocampal injection of kainate results in a persistent increase of GluR2 associated with granule cell dispersion, as in human hippocampal sclerosis. These data suggest the existence of common mechanisms between granule cell dispersion and regulation of GluR2 subunits associated with hippocampal sclerosis.

Morphogenetic effect of kainate on adult hippocampal neurons associated with a prolonged expression of brain-derived neurotrophic factor.

Intraperitoneal or intrahippocampal injections of kainate induce both hippocampal cell death and axonal remodeling of the dentate gyrus granular neurons. We report here that injection of kainate into the dorsal hippocampus of adult mice may also trigger a conspicuous and long-lasting global trophic response of granule cells. Morphological changes include somatic and dendritic growth and increased nuclear volume with ultrastructural features characteristic of neuronal development. The trophic response is correlated with a specific overexpression of brain-derived neurotrophic factor that is maintained for at least six months. This shows that plasticity in adult neurons can, in addition to axonal remodeling, extend to generalized cell growth. Our results further suggest that brain-derived neurotrophic factor could be involved in the activation and/or maintenance of this phenomenon.

Inter- and intracellular relationship of substance P-containing neurons with serotonin and GABA in the dorsal raphe nucleus: combination of autoradiographic and immunocytochemical techniques.

Double-labeling experiments were performed at the electron microscopic level in the dorsal raphe nucleus of rat, in order to study the inter- and intracellular relationship of substance P with gamma-aminobutyric acid (GABA) and serotonin. Autoradiography for either [3H]serotonin or [3H]GABA was coupled, on the same tissue section, with peroxidase-antiperoxidase immunocytochemistry for substance P in colchicine-treated animals. Intercellular relationships were represented by synaptic contacts made by [3H]serotonin-labeled terminals on substance P-containing somata and dendrites, and by substance P-containing terminals on [3H]GABA-labeled cells. Intracellular relationships were suggested by the occurrence of the peptide within [3H]serotonin-containing and [3H]GABA-containing cell bodies and fibers. Doubly labeled varicosities of the two kinds were also observed in the supraependymal plexus adjacent to the dorsal raphe nucleus. The results demonstrated that, in addition to reciprocal synaptic interactions made by substance P with serotonin and GABA, the dorsal raphe nucleus is the site of intracellular relationships between the peptide and either the amine or the amino acid.

Target-deprived CNS neurons express the NGF gene while reactive glia around their axonal terminals contain low and high affinity NGF receptors.

Reactive gliosis is part of the response of central nervous system to injury and neurodegeneration. Cellular components of the reactive gliosis have the capability to synthesize neurotrophic factors, and thus are capable of affecting the fate of neuronal populations in the injured tissue. In this study, we explored the putative involvement of reactive glia-derived neurotrophins in sustaining the axonal projections of target-deprived neurons. Neuronal targets of the dorsal column nuclei neurons were suppressed through excitotoxic lesion of the ventrobasal complex of the rat thalamus (VB). Despite the development of reactive gliosis, neither up-regulation of NGF, nor BDNF or NT3 mRNA could be detected by solution hybridization in the lesioned site at all times tested. In contrast, expression of the LNGFR gene increased progressively up to 90 days post-lesion. Immunocytochemical studies localized the LNGFR protein in a subset of small cells with ramified processes resembling microglia at 7 and 20 days post-lesion. At longer times, double immunolabelling studies revealed that a substantial part of LNGFR-immunoreactive cells filling the area of neuronal loss were neither microglial cells nor astrocytes although presence of LNGFR in a subset of microglial cells could not be excluded. Previous ultrastructural studies of the kainate-lesioned VB suggest that these LNGFR-immunoreactive cells correspond to oligodendrocytes and/or Schwann cells. At 2 months post-lesion, when LNGFR expression was maximal, increased levels of trkA mRNA were detected in the lesioned site. Immunocytochemical studies revealed the presence of numerous trkA-immunoreactive astrocytes. TrkB mRNA, encoding the full-length high-affinity receptor for BDNF, remained undetectable by non-isotopic in situ hybridization. In contrast to the lack of neurotrophin gene expression by glial components of the lesioned VB, dorsal column nuclei neurons contained NGF mRNA as revealed by in situ hybridization studies at 10 days--prior to enhanced LNGFR expression in the lesion--and 2 months post-lesion. In addition, the number and the staining intensity of NGF mRNA-positive neurons was increased in the target-deprived neurons, as compared with the contra-lateral nucleus projecting to intact targets. These results show that glial cells present in a reactive gliosis which develops in the kainic acid-lesioned thalamus, do not synthesize neurotrophins but instead produce high levels of both low- and high-affinity NGF receptors, LNGFR by Schwann cells/oligodendrocytes and possibly a subset of microglial cells, and trkA by reactive astrocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Reduction of cortical infarction and impairment of apoptosis in NGF-transgenic mice subjected to permanent focal ischemia.

The neuroprotective potential of the nerve growth factor (NGF) against permanent ischemic brain damage has been investigated in vivo using NGF-transgenic (tg) mice. The expression of the transgene is driven by part of the promoter of the proto-oncogene c-fos, which belongs to the first set of genes activated after brain ischemic insult. Wild-type (wt) mice and tg mice were subjected to permanent focal ischemia induced by electrocoagulation of the middle cerebral artery. Twenty four hours (h) after the ischemic shock, when compared to wt, tg mice displayed a 40% reduction of the infarcted area, which lasted up to 1 week. However, infarcted brain areas were similar in wt and tg mice within the first hours post-occlusion, indicating that NGF acted to block the progression of neuronal damage. Kinetics of NGF synthesis assessed by ELISA was in good agreement with the observed neuroprotective effect, since NGF content peaked 6 h post-ischemia. This was further correlated with the time-course of c-Fos immunoreactivity, detectable only from 6 h post-ischemia. The neuroprotective effect of NGF involved the impairment of apoptotic cell death, as evidenced by a marked decrease of the number of apoptotic profiles inside the ischemic zone in tg mice. These results underline the potential of c-fos-NGF-tg mice to study in vivo the molecular and cellular mechanisms of the NGF-induced neuroprotective effect against ischemic damage.