Resveratrol and quercetin, two natural polyphenols, reduce apoptotic neuronal cell death induced by neuroinflammation.

Parkinson's disease (PD) is a movement disorder characterized by a progressive loss of nigrostriatal dopaminergic neurons. Microglia activation and neuroinflammation have been associated with the pathogenesis of PD. Indeed, cytokines have been proposed as candidates that mediate the apoptotic cell death of dopaminergic neurons seen in PD. In this study, we investigated the effect of two natural polyphenols, resveratrol and quercetin, on neuroinflammation. For glial cells, we observed that lipopolysaccharide (LPS)-induced mRNA levels of two proinflammatory genes, interleukin 1-alpha and tumor necrosis factor-alpha, are strongly decreased by treatments with resveratrol or quercetin. We also undertook microglial-neuronal coculture to examine the influence of resveratrol and quercetin on dopaminergic neuronal cell death evoked by LPS-activated microglia. Cytotoxicity assays were performed to evaluate the percentage of cell death, with apoptotic cells identified by both the TdT-mediated dUTP nick end labeling technique and the detection of cleaved caspase-3. We report that treatment of N9 microglial cells with resveratrol or quercetin successfully reduced the inflammation-mediated apoptotic death of neuronal cells in our coculture system. Altogether our results demonstrate that resveratrol and quercetin diminished apoptotic neuronal cell death induced by microglial activation and suggest that these two phytoestrogens may be potent antiinflammatory compounds.

Effect of oxidative stress on stability and structure of neurofilament proteins.

Neurofilament proteins are highly phosphorylated molecules in the axonal compartment of the adult nervous system. We report the structural analysis of neurofilament proteins after oxidative damage. SDS-PAGE, immunoblotting, circular dichroism, and Fourier transform infrared spectroscopy were used to investigate the relative sensitivity of neurofilaments to oxidative stress and to identify changes in their molecular organization. An ascorbate-Fe+3-O2 buffer system as well as catechols were used to generate free radicals on a substrate of phosphorylated and dephosphorylated neurofilaments. By Fourier Transform Infrared spectroscopy and circular dichroism, we established that the neurofilament secondary structure is mainly composed of alpha-helices and that after free radical damage of the peptide backbone of neurofilaments, those helices are partly modified into beta-sheet and random coil structures. These characteristic reorganizations of the neurofilament structure after oxidative exposure suggest that free radical activity might play an important role in the biogenesis of the cytoplasmic inclusions found in several neurodegenerative diseases.

AMPA receptor properties in adult rat hippocampus following environmental enrichment.

In adult rats, environmental enrichment has been shown to selectively increase -AMPA binding in the hippocampus but the molecular mechanisms underlying this effect remain unknown. We used in situ hybridization with antisense oligonucleotides to determine possible changes in the hippocampal expression of messenger RNAs for different subunits of AMPA receptors in adult rats following exposure to an enriched environment. Quantitative analysis revealed that mRNA levels for three subtypes of AMPA glutamate receptors (GluR1-3; Flip and Flop variants) were not modified in any hippocampal region after environmental enrichment. In addition, no differences were detected in the levels of GluR1 and GluR2/3 proteins in Western blots of hippocampal membranes from enriched rats. Nevertheless, quantitative ligand binding autoradiography indicated that environmental enrichment evoked a significant and uniform decrease in the capacity of calcium or phosphatidylserine (PS) to up-regulate -AMPA binding in various hippocampal regions but not in the cerebral cortex. These findings support previous observations suggesting that post-translational changes in AMPA receptor properties, as a result of the activation of calcium-dependent processes, may represent an important mechanism underlying long-term modifications of synaptic efficacy in the rat hippocampus.

Binding properties of glutamate receptors in streptozotocin-induced diabetes in rats.

The biochemical mechanisms by which diabetes modulates cognitive function are not well established. Here, we determined the effects of streptozotocin (STZ) administration on the binding properties of alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) subtypes of glutamate receptors in rats, using quantitative autoradiographic analysis of (3)H-AMPA and [(3)H]glutamate binding on brain tissue sections. The STZ injection (70 mg/kg intraperitoneally) produced a reduction of (3)H-AMPA binding in various brain regions, an effect that is due to a decrease in receptor affinity. The STZ-induced reduction of (3)H-AMPA binding varied in different brain structures, being more pronounced in the striatum, cerebral cortex, and hippocampus and almost absent in the cerebellum. Western blots performed on hippocampal membranes revealed that the decrease in (3)H-AMPA binding is possibly associated with changes in immunologic properties for one glutamate receptor subunit (GluR1). Finally, the effect of STZ-induced diabetes appeared to be specific to the AMPA subtype of glutamate receptors, as the same treatment did not modify [(3)H]glutamate binding to NMDA receptors. These changes in AMPA receptor properties may have important implications for understanding the biochemical mechanisms underlying cognitive impairment in diabetes.

Apolipoprotein E genotype in schizophrenia: frequency, age of onset, and neuropathologic features.

Apolipoprotein E (ApoE) genotype has been found to affect the expression of a variety of neuropsychiatric disorders. We determined ApoE genotype frequencies and their relationship to clinical and pathological features in a diverse cohort of individuals with schizophrenia. There were no differences in ApoE genotype frequencies between schizophrenics and controls. However, the ApoE epsilon 4 genotype was associated with a younger age of onset of schizophrenia, and in an elderly subsample, individuals with the epsilon 4 allele more frequently exhibited co-existent dementia and had more neurofibrillary pathology (although none of the cases met criteria for Alzheimer's disease). This examination of ApoE in relation to clinical and neurobiological features of schizophrenia suggests that it modifies the phenotypic expression of the disease.

Melatonin receptors are present in non-optic regions of the brain of a deep-sea fish living in the absence of solar light.

Pineal melatonin hormonally transduces photoperiod to influence daily and seasonal cycles in most vertebrates (1, 2). Evidence of melatonin receptors throughout the brain of several fish species (3-5), particularly in retinorecipient structures, also indicates a role in visual processing. Despite the absence of solar light many deepsea organisms show seasonality (6-8). The presence of central melatonin receptors was investigated by quantitative in vitro autoradiography in the deep-sea fish Coryphaenoides (Nematonurus) armatus. Specific, time-dependent, saturable, high affinity and guanine nucleotide sensitive, 2-[125I]iodomelatonin binding was found over the mid-brain tegmentum and hindbrain. Competing ligand potency was iodomelatonin > melatonin >> 5-HT. Although C.(N.) armatus has well developed eyes no 2-[125I]iodomelatonin binding occurred in optic tectum, cerebellum or hypothalamus. Thus melatonin involvement in processing of visual information and control of seasonal physiology via hypothalamic areas appears to be absent in this species. The presence of central G-protein coupled receptors indicates a function for melatonin unrelated to solar light.

Alterations of intermediate filaments in various histopathological conditions.

Intermediate filament proteins belong to a multigene family and constitute an important cytoskeletal component of most vertebrate cells. Their pattern of expression is tissue specific and is highly controlled during embryonic development. Numerous pathologies are known to be associated with modifications of intermediate filament organisation, although their precise role has not yet been elucidated. The present review focuses on the most recent data concerning the possible causes of intermediate filaments disorganization in specific pathologic conditions affecting the epidermis, the liver, and the nervous system. We discuss the formation of abnormal intermediate filament networks that arise as a consequence of mutations that directly affect intermediate filament structure or are induced by multifactorial causes such as modifications of post-translational processes and changes in the levels of expression.

Association of apolipoprotein epsilon 4 allele and neuropathologic findings in patients with dementia.

Apolipoprotein E (APOE) is a lipoprotein expressed in liver and brain as one of three isoforms (APOE 2, APOE 3 and APOE 4). Recent findings suggest that the presence of APOE 4 is associated with an increased risk for both familial Alzheimer's disease and late-onset Alzheimer's disease. We extended these observations by determining the frequency of APOE alleles in patients with pathologically confirmed Alzheimer's Disease (AD), Parkinson's disease (PD), diffuse Lewy Body disease (DLBD), AD with concomitant PD pathology, demented PD patients without or with concomitant AD pathology and in schizophrenics with a progressive dementia (SCHIZ+DEM). The APOE genotype was determined by restriction digestion of polymerase chain reaction-amplified DNA isolated from frozen brain samples. The frequency of the APOE epsilon 4 allele was highest among sporadic AD and DLBD patients (0.30 and 0.38, respectively) and lowest in the SCHIZ+DEM and non-demented PD patients (0.06 and 0.1, respectively). Thus, the APOE epsilon 4 allele is over-represented selectively in patients with dementias associated with plaques and tangles and/or cortical Lewy bodies, but not in demented schizophrenics or non-demented PD patients.

Variants of the heavy neurofilament subunit are associated with the development of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder primarily affecting motor neurons. The etiology of the majority of cases remains unknown. Recent findings from several laboratories suggest a role for neurofilaments in the development of motor neuron disorders. The C-terminal region of the human neurofilament heavy subunit (NEFH) contains a unique functional domain consisting of 43 repeat motifs of the amino acids Lys-Ser-Pro (KSP). This C-terminal region of NEFH forms the sidearm projections which cross-link the neurofilaments. Previously, we have demonstrated polymorphism in the C-terminal region of the human NEFH: an allelic variant of a slightly larger molecular size, containing an additional KSP phosphorylation motif. Novel mutations in this region were found in five ALS patients. We propose that changes in the KSP-repeat domain may affect the cross-linking properties of the heavy neurofilament subunit and perhaps contribute to the development of neurofilamentous swellings in motor neurons, a hallmark of ALS.

A new orphan member of the nuclear hormone receptor superfamily that interacts with a subset of retinoic acid response elements.

We have identified and characterized a new orphan member of the nuclear hormone receptor superfamily, called MB67, which is predominantly expressed in liver. MB67 binds and transactivates the retinoic acid response elements that control expression of the retinoic acid receptor beta 2 and alcohol dehydrogenase 3 genes, both of which consist of a direct repeat hexamers related to the consensus AGGTCA, separated by 5 bp. MB67 binds these elements as a heterodimer with the 9-cis-retinoic acid receptor, RXR. However, MB67 does not bind or activate other retinoic acid response elements with alternative hexamer arrangements or any of several other wild-type and synthetic hormone response elements examined. The transactivation of retinoic acid response elements by MB67 is weaker than that conferred by the retinoic acid receptors but does not require the presence of all-trans retinoic acid, 9-cis-retinoic acid, or any exogenously added ligand. We propose that MB67 plays an important role in the complex network of proteins that govern response to retinoic acid and its metabolites.

Isolation of IFAPa-400 cDNAs: evidence for a transient cytostructural gene activity common to the precursor cells of the myogenic and the neurogenic cell lineages.

Differentiation of neural and muscle cells is characterized by a switch in the expression of the type of intermediate filament protein subunit. In these lineages, vimentin is transiently expressed in the initial stages of development and is gradually replaced by a tissue specific protein. We have identified a giant developmentally regulated antigen (IFAPa-400) which colocalizes with vimentin in the precursor cells of the neurogenic and myogenic lineages of the chick embryo [Chabot and Vincent (1990) Dev. Brain Res. 54, 195-204; Cossette and Vincent (1991) J. Cell Sci. 98, 251-260]. Based on the expression of this protein during neurogenesis and myogenesis, we hypothesize that IFAPa-400 and vimentin define a special intermediate filament network, common to the non-differentiated cells derived from the neuroectoderm and those of the myogenic tissues. We report here the isolation and sequence of partial cDNAs encoding more than 400 amino acids of the carboxy-terminus of this protein. RNA blot analysis and in situ hybridization indicate that IFAPa-400 represents a bona fide developmentally regulated gene product. These results further confirm that IFAPa-400 mRNA transcripts are limited to the early precursor cells of both neurogenic and myogenic lineages.

Ontogeny of Ha-ras and c-myc mRNA levels in rabbit embryo and extraembryonic tissues by quantitative in situ hybridization.

A large variety of proto-oncogenes are known to be of key importance in cellular growth and differentiation during embryonic development. Using quantitative in situ hybridization, we studied in detail the levels of the proto-oncogenes Ha-ras and c-myc mRNA in embryos and extraembryonic tissues (maternal and embryonic placentas, trophoblast, and endometrial epithelium) during prenatal life of rabbit. cDNA probes encoding for Ha-ras (fragment Kpn 1-BstE II of 883 bp) and c-myc (fragment Pst 1-Pst 1 of 490 bp) were used to detect specific transcripts in fixed cryostat sections. High levels of Ha-ras and c-myc mRNA were detected in the rabbit embryo as well as in the decidua and in the trophoblast as early as day 9 of gestation. At 12 and 15 days of gestation, Ha-ras and c-myc mRNA levels decreased in both embryonic and maternal placenta while in the embryo a significant increase of Ha-ras and c-myc expression was detected with particular evidence in the central nervous system. Finally, at 25 days of gestation the expression of the two proto-oncogenes, Ha-ras and c-myc, was greatly decreased in both the embryo and extraembryonic tissues, and was undetectable by 30 days of gestation. These results show that in rabbit the expression of the two proto-oncogenes Ha-ras and c-myc is localized in the same tissues with similar intensity and follows an unparallel temporal modulation in the embryo and in the extraembryonic tissues during prenatal development.

Growth hormone and somatostatin gene expression in adult and aging rats as measured by quantitative in situ hybridization.

We have investigated the variations in growth hormone (GH) and somatostatin (SOM) mRNAs in adult, middle-aged and aging rats of both sexes using quantitative in situ hybridization. cDNA probes complementary to GH mRNA and SOM mRNA were used on fixed pituitary and hypothalamic sections, respectively. A clear sexual dimorphism in GH gene expression was observed in all age groups examined, mRNA levels always being higher in males than in females. In male rats, GH mRNA levels reached a maximum at 7 months of age and then dramatically decreased in middle-aged and aging animals. In female animals, the highest expression of GH was observed at 9 months of age followed by a gradual and constant decline during the aging period. SOM mRNA levels in the periventricular nucleus of the hypothalamus also showed a sexual dimorphism in adult rats, being higher in males than females. A gradual decline in SOM mRNA was observed in middle-aged and aging rats of both sexes. These results suggest that middle age and aging are critical periods for the control of GH and SOM gene expression and that the decrease in GH mRNA levels observed during aging is probably not a consequence of an increase of SOM activity.

Dihydrotestosterone (DHT) regulation of insulin-like growth factor II mRNA in neonatal rats.

Insulin-like growth factors (IGFs) are well known as peptide mitogens and important growth factors in fetal as well as in early postnatal development. In particular, IGF II is strongly expressed during fetal life and in neonatal animals. Very little is known about the regulation of IGF II gene expression. In order to study in detail the regulation of IGF II mRNA levels in the liver by the potent nonaromatizable androgen dihydrotestosterone (DHT), we have used quantitative in situ hybridization to detect the mRNA encoding for this growth factor. Pups were separated into 4 groups and injected twice a day immediately after birth with 3 different doses of DHT: 0.1 mg DHT/day, 0.25 mg DHT/day, 0.5 mg DHT/day for 4 and 7 days, and the control groups were injected with the vehicle alone. Animals were perfused with 4% paraformaldehyde and sections from the liver, heart, kidneys and brain were cut with a cryostat. A [35S]-labeled cDNA probe was used to detect IGF II mRNA levels. After hybridization, sections were autoradiographed with X-ray films and then coated with liquid photographic emulsion. Densitometric measurement revealed that, at 4 days of age, IGF II mRNA levels were lower in DHT-treated rats than in control animals. No statistically significant differences in IGF II mRNA levels were observed among the three groups treated with the different doses of DHT, thus revealing that even the lowest dose of DHT (0.1 mg/day) used was sufficient to inhibit IGF II gene expression in neonatal rats. Moreover, at 7 days of age, DHT-treated rats showed the same levels of IGF II mRNA as those observed in rats treated with DHT for 4 days. These results suggest that DHT may play an important role in the regulation of IGF II gene expression in the rat liver during the neonatal period.

Regional changes of striatal dopamine receptors following denervation by 6-hydroxydopamine and fetal mesencephalic grafts in the rat.

Young adult female rats received a 6-hydroxydopamine lesion in the left substantia nigra and, 3 weeks later, some of them were grafted with a cell suspension from the ventral mesencephalon of rat embryos (14-15 days old). Six months after transplantation, some grafted rats, following injection of amphetamine, had switched to turning only toward the intact side (type 1), whereas others turned toward the intact side only during the first half of the test (type 2). Levels of dopamine, dihydroxyphenylacetic acid and homovanillic acid were, respectively, 2%, 15% and 35% of the intact side in the denervated striatum of 6-hydroxydopamine rats. Dopamine concentrations were restored to 13% and 10% of the intact side in the grafted striatum of type 1 and type 2 animals, respectively. Levels of homovanillic acid were unchanged following grafts whereas those of dihydroxyphenylacetic acid increased by 209% and 247% in the grafted striatum of type 1 and type 2 animals, respectively. The ratios of dihydroxyphenylacetic acid/dopamine as well as homovanillic acid/dopamine were low in the intact striatum whereas they increased in the denervated striatum with or without graft. The tyrosine hydroxylase immunoreactivity decreased by about 80% in the denervated striatum of 6-hydroxydopamine rats. In type 1 rats, tyrosine hydroxylase immunoreactivity revealed that the graft was localized in the dorsomedial part of the denervated striatum, whereas in type 2 animals, it was also in the medial striatum but it overlapped the dorsal and ventral parts of it equally. D1 as well as D2 dopamine receptors were measured throughout the striatum (9.0-7.6 rostral-caudal coordinates), by autoradiography, using [3H]SCH 23390 (D1 antagonist) and [3H]spiperone (D2 antagonist) binding. Supersensitive D2 receptors were normalized in the dorso- and ventromedial parts of the grafted striatum. D2 receptor density was higher in type 2 than in type 1 rats, more specifically at 8.6-8.2 rostral-caudal coordinates, where the graft was. D1 receptor supersensitivity was modest compared to D2 receptors in the striatum of 6-hydroxydopamine rats and decreased following grafts. DA receptors changes in the striatum, following fetal mesencephalic grafts, may explain the behavioral recovery seen in grafted rats.

The ontogeny of central melatonin binding sites in the rat.

The maternal transfer of circadian rhythmicity and photoperiodic information to the fetus has been clearly demonstrated in several species, as has the importance of the pineal hormone, melatonin, in conveying this information. Maternal melatonin is known to readily cross the placenta and be taken up by the fetal brain. Specific melatonin receptors have been demonstrated in the rodent brain and pituitary from the 21st day of gestation. To better understand the mechanisms by which melatonin brings about the transfer of information to the fetus and to define the receptivity of the fetus to the melatonin signal, we have followed the ontogeny of central melatonin binding sites in the rat from the 13th day of gestation to 5 days after birth. The use of in vitro autoradiography allows for the precise localization of binding sites as well as their quantification. In the present study melatonin binding sites were first found on the 15th day of gestation, at which time specific binding was limited to the pituitary. At birth it was possible to identify a strong label in the pars tuberalis of the pituitary, whereas the pars distalis appeared to be less intensely and more unevenly labeled. Neuronal melatonin binding sites became apparent from day 17 of gestation in an area of the dorsal brain that in older fetuses and in neonatal rats appeared to be the paraventricular nucleus of the thalamus. Moreover, melatonin binding sites are identifiable over the suprachiasmatic nuclei from day 18 of gestation. These data show that the fetal pituitary may have the potential to respond to the maternal melatonin signal as early as the 15th day of gestation, and that the brain may attain that potential at the 17th or 18th day of gestation.

Effects of triiodothyronine, dexamethasone and estradiol-17 beta on GH mRNA in rat pituitary cells in culture as revealed by in situ hybridization.

The GH lines of rat pituitary tumour cells have been largely used to study the regulation of GH mRNA. In order to investigate the role of T3, dexamethasone and estradiol-17 beta on GH expression in non-tumoural pituitary cells, we have used in situ hybridization techniques performed on rat anterior pituitary cells in monolayer culture. The amounts of mRNA encoding for GH, as evaluated by counting the number of grains per somatotrope, were markedly reduced after 4 days of culture in a steroid-free medium supplemented with an hypothyroid calf serum. Addition of T3 or dexamethasone for 3 days increased GH mRNA levels. The concomitant administration of the two hormones produced a synergistic effect on GH mRNA levels which became higher than those observed after T3 or dexamethasone administration alone. However, this effect did not restore GH mRNA levels to those measured in monolayer pituitary cells grown in medium containing 10% fetal calf serum. Moreover GH mRNA levels appeared higher in male than in female pituitary cells. The administration of E2 to pituitary cell cultures from both male and female rats produced an increase by 15, and 12.8% in GH mRNA levels in male and female, respectively. This stimulatory effect of E2 in cell culture was competitively blocked by simultaneous incubation with the antiestrogen LY156758 (Keoxifene). These results demonstrate that T3, dexamethasone as well as E2 act directly on somatotropic cells to regulate GH gene expression.

Distribution of central melatonin binding sites in the goldfish (Carassius auratus).

In fish, as well as in birds and mammals, the pineal hormone melatonin acts as a neuroendocrine transducer of photoperiod and as such has a profound influence over the timing of seasonal behavior, especially reproduction. With the aim of better understanding how melatonin may exert its influence in the fish, we have studied the distribution and characteristics of the melatonin binding sites in the brain and pituitary of the goldfish (Carassius auratus) using the ligand [2-(125)I]iodomelatonin and a combination of in vitro autoradiography and membrane homogenate receptor assays. Specific binding of the ligand was defined as that displaced by 1 muM melatonin. High-affinity melatonin binding sites were found to have a widespread distribution within the brain of the goldfish and measurements of the relative optical densities on X-ray films using image analysis showed that the highest concentrations were present in the visual system (optic tract, pretectal area, optic tectum), the molecular layer of the cerebellum, and the hypothalamus. No specific binding could be detected in either the pituitary gland or the olfactory bulbs. Displacement of binding by increasing concentrations of melatonin measured by comparative optical density readings on serial sections revealed an ED(50) for melatonin of 10(-9)M. No displacement of binding by increasing concentrations of serotonin up to a concentration of 10(-6) M could be detected. Binding was shown to be time dependent with maximal binding occurring after 1 h at room temperature (22 degrees C). Addition of 1 muM melatonin after 2 h resulted in a time-dependent dissociation of ligand binding. Saturation studies carried out on crude membrane homogenates of whole brains gave rise to a straight line Scatchard plot with a K(D) of 69 pM and a B(max) of 14 fmol/mg protein. The widespread distribution of high-affinity melatonin binding sites in the brain of the goldfish suggests that melatonin may influence circadian and seasonal behavior in fish via numerous sites involving sensory and neuroendocrine systems but does not have a direct action on the pituitary.

Ontogenesis and Sexual Dimorphism of Rat Growth Hormone Messenger Ribonucleic Acid as Studied by in situ Hybridization.

Abstract We have investigated the ontogeny of growth hormone (GH) mRNA in the developing rat foetus and also from birth to adulthood. Using quantitative in situ hybridization, we studied the variations in the levels of GH mRNA during foetal and postnatal life in the pituitary of both male and female rats. A cDNA probe to GH mRNA was used to detect GH transcripts on fixed pituitary sections at different stages of development. Few labelled cells were observed in the lateral wings of the anterior pituitary from the 17th to 19th day of gestation in both sexes. The amounts of GH mRNA significantly increased in both male and female rats from neonatal to adult life, reaching the highest levels after puberty. A clear sexual dimorphism was observed at the 20th day of foetal life, GH mRNA levels being higher in male than in female rats. After birth, no significant differences of GH mRNA levels could be observed between male and female rats until 30 days of age (prepubertal period) when male rats exhibited GH mRNA levels higher than females. This sexual difference in GH mRNA levels remained constant throughout adult life. Moreover, gonadectomy performed at neonatal, prepubertal and adult periods in both male and female rats did not modify GH mRNA levels in either sex. These results indicate that an early synthesis of GH mRNA occurs in foetal pituitary and that the sexual dimorphism of GH mRNA observed from 30 days of age is not related to sex steroids.

Distribution of GABA-immunoreactive neurons in the forebrain of the goldfish, Carassius auratus.

The distribution of gamma-aminobutyric acid (GABA) immunoreactivity was studied in the forebrain (tel- and diencephalon) of the goldfish by means of immunocytochemistry on Vibratome sections using antibodies against GABA. Positive perikarya were detected in the olfactory bulbs and in all divisions of the telencephalon, the highest density being found along the midline. In the diencephalon, GABA-containing cell bodies were found in the hypothalamus, in particular in the preoptic and tuberal regions. The inferior lobes, the nucleus recessus lateralis, and more laterodorsal regions, such as the nucleus glomerulosus and surrounding structures, also exhibited numerous GABA-positive perikarya. Cell bodies were also noted in the thalamus, in particular in the dorsomedial, dorsolateral and ventromedial nuclei. The relative density of immunoreactive fibers was evaluated for each brain nucleus and classified into five categories. This ubiquitous distribution indicates that, as in higher vertebrates, GABA most probably represents one of the major neurotransmitters in the brain of teleosts.