The Absence of Caspase-8 in the Dopaminergic System Leads to Mild Autism-like Behavior.

In the last decade, new non-apoptotic roles have been ascribed to apoptotic caspases. This family of proteins plays an important role in the sculpting of the brain in the early stages of development by eliminating excessive and nonfunctional synapses and extra cells. Consequently, impairments in this process can underlie many neurological and mental illnesses. This view is particularly relevant to dopamine because it plays a pleiotropic role in motor control, motivation, and reward processing. In this study, we analyze the effects of the elimination of caspase-8 (CASP8) on the development of catecholaminergic neurons using neurochemical, ultrastructural, and behavioral tests. To do this, we selectively delete the CASP8 gene in cells that express tyrosine hydroxylase with the help of recombination through the Cre-loxP system. Our results show that the number of dopaminergic neurons increases in the substantia nigra. In the striatum, the basal extracellular level of dopamine and potassium-evoked dopamine release decreased significantly in mice lacking CASP8, clearly showing the low dopamine functioning in tissues innervated by this neurotransmitter. This view is supported by electron microscopy analysis of striatal synapses. Interestingly, behavioral analysis demonstrates that mice lacking CASP8 show changes reminiscent of autism spectrum disorders (ASD). Our research reactivates the possible role of dopamine transmission in the pathogenesis of ASD and provides a mild model of autism.

Apoptosis-inducing factor mediates dopaminergic cell death in response to LPS-induced inflammatory stimulus: evidence in Parkinson's disease patients.

We show that intranigral lipopolysaccharide (LPS) injection, which provokes specific degeneration of DA neurons, induced caspase-3 activation in the rat ventral mesencephalon, which was mostly associated with glial cells. In contrast, nigral DA neurons exhibited AIF nuclear translocation in response to LPS. A significant decrease of the Bcl-2/Bax ratio in nigral tissue after LPS injection was observed. We next developed an in vitro co-culture system with the microglial BV2 and the DA neuronal MN9D murine cell lines. The silencing of caspase-3 or AIF by small interfering RNAs exclusively in the DA MN9D cells demonstrated the key role of AIF in the LPS-induced death of DA cells. In vivo chemical inhibition of caspases and poly(ADP-ribose)polymerase-1, an upstream regulator of AIF release and calpain, proved the central role of the AIF-dependent pathway in LPS-induced nigral DA cell death. We also observed nuclear translocation of AIF in the ventral mesencephalon of Parkinson's disease subjects.

Fatal levofloxacin failure in treatment of a bacteremic patient infected with Streptococcus pneumoniae with a preexisting parC mutation.

The fatal outcome of levofloxacin treatment in a patient with bacteremic pneumonia caused by Streptococcus pneumoniae with a preexisting parC mutation is reported. Failure was due to the emergence of a gyrA mutation after 4 days of therapy. Problems encountered in detecting first-step mutation isolates are discussed.

Regional-specific regulation of BDNF and trkB correlates with nigral dopaminergic cell sprouting following unilateral nigrostriatal axotomy.

Axotomy is a powerful stimulus of axon growth and plastic changes. We investigated the potential role of BDNF/trkB signaling in the sprouting of dopaminergic nigral axons in response to axotomy of the medial forebrain bundle. Tyrosine hydroxylase immunohistochemistry revealed the existence of sprouting mechanisms in the axotomized substantia nigra (SN). Time-course changes of trkB mRNA expression demonstrated a robust increase in an area projecting from the rostral tip of the SN to the glial scar, which coincided with evidence of nigral dopaminergic sprouting. In addition, we found an early loss of this messenger in areas related to the knife cut, which recovered by 7 days postlesion. TrkB down-regulation appeared to be associated to the lesion-induced local damage, as it was restricted to an area showing Fluoro-Jade B- and TUNEL positive cells. In trkB-depleted areas, an inverse correlation between mRNA expressions of BDNF and trkB was apparent. Specific induction of BDNF mRNA was mostly seen in border of areas devoid of trkB mRNA. In contrast, in the areas exhibiting trkB mRNA expression, no BDNF mRNA was detected. We suggest that trkB levels could be a determinant element in regulating BDNF expression. Finally, the search for molecules involved in either promoting or inhibiting axonal growth, demonstrated up-regulation of GAP-43 and Nogo-A mRNA at sites close to the knife transections as early as 1 day postlesion. However, overall, Nogo-A induction was more robust than that seen for GAP-43.

Stress increases vulnerability to inflammation in the rat prefrontal cortex.

Inflammation could be involved in some neurodegenerative disorders that accompany signs of inflammation. However, because sensitivity to inflammation is not equal in all brain structures, a direct relationship is not clear. Our aim was to test whether some physiological circumstances, such as stress, could enhance susceptibility to inflammation in the prefrontal cortex (PFC), which shows a relative resistance to inflammation. PFC is important in many brain functions and is a target for some neurodegenerative diseases. We induced an inflammatory process by a single intracortical injection of 2 microg of lipopolysaccharide (LPS), a potent proinflammogen, in nonstressed and stressed rats. We evaluated the effect of our treatment on inflammatory markers, neuronal populations, BDNF expression, and behavior of several mitogen-activated protein (MAP) kinases and the transcription factor cAMP response element-binding protein. Stress strengthens the changes induced by LPS injection: microglial activation and proliferation with an increase in the levels of the proinflammatory cytokine tumor necrosis factor-alpha; loss of cells such as astroglia, seen as loss of glial fibrillary acidic protein immunoreactivity, and neurons, studied by neuronal-specific nuclear protein immunohistochemistry and GAD67 and NMDA receptor 1A mRNAs expression by in situ hybridization. A significant increase in the BDNF mRNA expression and modifications in the levels of MAP kinase phosphorylation were also found. In addition, we observed a protective effect from RU486 [mifepristone (11beta-[p-(dimethylamino)phenyl]-17beta-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one)], a potent inhibitor of the glucocorticoid receptor activation. All of these data show a synergistic effect between inflammation and stress, which could explain the relationship described between stress and some neurodegenerative pathologies.

Aquaporins in the central nervous system.

In this review, we have tried to summarize most available data dealing with the aquaporin (AQP) family of water channels in the CNS. Two aquaporins have been identified so far in the CNS, AQP1 and AQP4. AQP1 is restricted to the choroid plexus of the lateral ventricles, which raises a role for this aquaporin in cerebrospinal fluid formation. AQP4 is the predominant water channel in the brain and it is more widely distributed than originally believed, with a marked prevalence over periventricular areas. In the first part of this review, we examine the complete distribution pattern of AQP4 in the CNS including its rostro-caudal localization to end with its subcellular location. After discussing scarce data dealing with regulation of aquaporins in the CNS, we focus in potential roles for aquaporins. Novel recent data highlights very important roles for this aquaporin in the normal and pathological brain including, among others, role in potassium buffering, body fluid homeostasis, central osmoreception and development and restoration of brain edema.

Chronic inhibition of the high-affinity dopamine uptake system increases oxidative damage to proteins in the aged rat substantia nigra.

The effect of chronic treatment of aged rats with nomifensine has been studied in the rat nigrostriatal dopaminergic system. The rat substantia nigra suffers an oxidative damage during aging that results in both an increase in carbonyl groups of its total proteins and the oxidative inactivation of tyrosine hydroxylase (TH) enzyme, which are partially reversed by chronic treatment with deprenyl. Different mechanisms may account for this effect, including inhibition of the high-affinity dopamine uptake system. We treated aged rats chronically with nomifensine for 2 months and found some significant effects. Nomifensine treatment significantly increased TH enzyme amount in substantia nigra (39.2%), which was accompanied by a significant increase in TH enzyme activity (47.8%). However, these effects were not observed in the terminal field (striatum). As a further step we quantified the oxidative level of proteins by measuring the number of carbonyl groups coupled either to total proteins or specifically to TH enzyme. The proteins of aged rat substantia nigra showed a significant increase of carbonyl groups following nomifensine treatment. The number of carbonyl groups coupled to nigral TH enzyme also increased in the nomifensine-treated animals. However, this increase was lower than that found in the total homogenate proteins. All these results show that the oxidative damage produced during aging in tyrosine hydroxylase enzyme and total proteins is not reduced by nomifensine treatment. On the contrary, the nomifensine treatment increased the oxidative damage to proteins. These results suggest the capability of deprenyl to induce TH enzyme could be due to inhibition of the high-affinity dopamine uptake system, but its ability to protect against oxidative damage is not produced by this mechanism.

Oxidative inactivation of tyrosine hydroxylase in substantia nigra of aged rat.

Study of the tyrosine hydroxylase enzyme from substantia nigra and striatum during the aging period of the rat has discovered a significant decrease (55%) of TH activity in substantia nigra between 12 and 24 mo of age. The amount of TH in substantia nigra also decreased (30%) during aging. This loss in TH activity of substantia nigra appears to be produced by the decrease in TH content along with an inactivation process. Our finding showed a significant increase of carbonyl groups in the proteins of rat substantia nigra with aging. A statistically significant increase of carbonyl groups in TH enzyme was found in aged rat brain substantia nigra, indicating that oxidative damage could be the inactivation process that explains the decrease in TH activity found during aging. This hypothesis was corroborated by the fact that when rat striatal homogenate was incubated with hydrogen peroxide, there was a time-dependent decrease in TH activity, which highly correlated with measurements of carbonyl groups content of TH enzyme. The importance of these results may be in their relationship, considering that substantia nigra is preferentially affected in many neurodegenerative disorders.

Effect of ageing on monoamine turnover in the prefrontal cortex of rats.

Turnover of dopamine (DA), serotonin (5-hydroxytryptamine) (5-HT), noradrenaline (NA) and their metabolites has been measured in control and aged rats. In addition, tyrosine hydroxylase (TH) activity has been studied. After pargyline treatment, the turnover rates of DA, NA, 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT), 5-HT and 5-hydroxy-3-indolacetic acid (5-HIAA) and TH activity increased in aged rats with respect to controls. At the same time the DA and 3-MT turnover increase are consistent with the hypothesis that enhanced release of DA may participate in some degenerative processes in ageing. After probenecid treatment, the turnover of homovanillic acid (HVA) was lower in aged rats than in controls. However, DOPAC turnover was higher in the aged rats. The DOPAC increase seems to indicate a reinforcement of this pathway in aged rats.

Determination of levels of biogenic amines and their metabolites and both forms of monoamine oxidase in prefrontal cortex of aged rats.

Levels of dopamine, noradrenaline and serotonin and their metabolites and the activities of monoamine oxidase A and B, have been determined in the prefrontal cortex of the rat during ageing. Serotonin turnover rate has been measured as 5-hydroxytryptophan accumulation rate after central decarboxylase inhibition. The major changes were an increase of dopamine and noradrenaline levels and a decrease of serotonin in aged animals compared with control animals. A decrease of the MAO-A to MAO-B ratio was found in aged rats.

Changes in monoamines and their metabolite levels in substantia nigra of aged rats.

A decrease in the monoamine and monoamine metabolite contents in the substantia nigra of aged rats vs. controls has been found. Hence, it can be speculated that this decrease may play a role in the multiple alterations in dopaminergic functions observed in aged rats.

Retrograde transport of nerve growth factor from hippocampus and amygdala to trkA messenger RNA expressing neurons in paraventricular and reuniens nuclei of the thalamus.

We previously reported the presence of trkA messenger RNA expressing non-cholinergic neurons in the paraventricular anterior and reuniens nuclei, which are located in the thalamic midline. In the present study, retrograde labeling with iodinated (125I) nerve growth factor was used to identify the innervation target of these cells. Neurons in the paraventricular anterior and reuniens nuclei were labeled following injection of iodinated nerve growth factor into amygdala and hippocampus, but not into nucleus accumbens and entorhinal cortex, the two other main areas receiving strong innervation from the thalamic midline. Target ablation of hippocampus or amygdala failed to down-regulate trkA messenger RNA expression in the two thalamic nuclei, thus suggesting a role for nerve growth factor different from a critical survival factor. The thalamic paraventricular anterior and reuniens nuclei are part of the reticular formation which plays a role in general cortical activation, behavioral arousal and control of awareness. Retrograde transport of nerve growth factor by trkA messenger RNA expressing neurons in these nuclei suggests a physiological role of this trophic factor in the function of these cells.

Intrastriatal infusion of nerve growth factor after quinolinic acid prevents reduction of cellular expression of choline acetyltransferase messenger RNA and trkA messenger RNA, but not glutamate decarboxylase messenger RNA.

Excitotoxic striatal lesions induced by quinolinic acid, a model for Huntington's disease, were used to test for neuroprotective actions of nerve growth factor on striatal cholinergic and GABAergic neurons. Expressions of the trkA receptor for nerve growth factor, choline acetyltransferase and glutamate decarboxylase were analysed by messenger RNA in situ hybridization in adult rats following quinolinic acid lesion (150 nmol) and daily striatal administration of nerve growth factor (1 microgram) or control protein (cytochrome C) for one week. One week after toxin administration, the numbers of cells expressing trkA or choline acetyltransferase messenger RNAs were decreased when compared with unlesioned animals. Moreover, the surviving cells showed a strong down-regulation of these messenger RNAs as deduced from grain count analysis of sections processed for emulsion autoradiography. Daily intrastriatal nerve growth factor administration for one week completely prevented the reduction in the number of cells expressing either of the two markers. Nerve growth factor treatment increased the cellular expression of choline acetyltransferase messenger RNA three times above control levels and restored the levels of trk A messenger RNA expression to control levels. In contrast to the protective effects on cholinergic cells, nerve growth factor treatment failed to attenuate the quinolinic acid-induced decrease in glutamate decarboxylase messenger RNA levels. Optical density measurements of the entire striatum on autoradiographs of brain sections from quinolinic acid-lesioned animals revealed a reduction of the glutamate decarboxylase messenger RNA-specific hybridization signal, which was unaltered by infusion of nerve growth factor or control protein. Our findings strongly suggest that in both the intact and the quinolinic acid-lesioned adult rat striatum, nerve growth factor action is confined to trk A-expressing cholinergic neurons. Striatal glutamate decarboxylase messenger RNA-expressing GABAergic neurons which degenerate in Huntington's disease are not responsive to nerve growth factor.

Protective effects of neurotrophin-4/5 and transforming growth factor-alpha on striatal neuronal phenotypic degeneration after excitotoxic lesioning with quinolinic acid.

Lesioning of the mammalian striatum with the excitotoxin quinolinic acid results in a pattern of neuropathology that resembles that of post mortem Huntington's disease brain. Certain neurotrophic factors can rescue degenerating cells in a variety of lesion types, including those produced by neurotoxins. Several neurotrophic factors promote the survival of striatal neurons and/or are localized within the striatum. Of these factors, neurotrophin-4/5 and transforming growth factor-alpha were chosen for administration to rats lesioned with quinolinic acid. Adult rats received a single unilateral intrastriatal injection of quinolinic acid (120 nmol) and either trophic factors or the control protein cytochrome c for seven days thereafter. The pattern of phenotypic degeneration was assessed by immunocytochemical labeling of various striatal neuronal populations at five rostrocaudal levels. Quinolinic acid produced a preferential loss in the number of cells immunoreactive for glutamate decarboxylase, with a relative sparing of the number of choline acetyltransferase-immunoreactive cells and, to a lesser degree, calretinin-immunoreactive cells. None of these phenotypic populations was protected by either neurotrophin-4/5 or transforming growth factor-alpha. In contrast, when glutamate decarboxylase cells were alternatively identified by calbindin immunolabeling, both factors were found to have partially reversed the loss in the number of calbindin-positive cells induced by excitolesioning. In addition, the loss in the number of parvalbumin-immunopositive cells due to quinolinic acid was partially reversed by neurotrophin-4/5, while the loss in the number of NADPH-diaphorase-stained cells was partially reversed by transforming growth factor-alpha. These findings reveal a new population of striatal cells, calretinin neurons, that are relatively resistant to quinolinic acid toxicity and that neurotrophin-4/5 and transforming growth factor-alpha partially protect against the phenotypic degeneration of striatal cell populations in an in vivo animal model of Huntington's disease.

Expression of neurotrophin and trk receptor genes in adult rats with fimbria transections: effect of intraventricular nerve growth factor and brain-derived neurotrophic factor administration.

The expression of the specific trk receptors for nerve growth factor and brain-derived neurotrophic factor (trkA and trkB) has been assayed by messenger RNA in situ hybridization in adult rats with partial fimbrial transections along with intraventricular treatment of nerve growth factor or brain-derived neurotrophic factor. In the forebrain, specific hybridization labeling for trkA messenger RNA showed an identical pattern to that of choline acetyltransferase messenger RNA, supporting the view that trkA expression is confined to the cholinergic population in the basal forebrain and the cholinergic interneurons in the striatum. After partial unilateral transections of the fimbria there was a progressive loss of choline acetyltransferase and trkA messenger RNA expression in the septal region ipsilateral to the lesion. Daily intraventricular administration of brain-derived neurotrophic factor or nerve growth factor partially prevented the lesion-induced decrease in the levels of both messengers, the latter being more effective than the former. Grain count analysis of individual cells was used to test whether the two factors upregulated choline acetyltransferase or trkA expression in individual cells surviving the lesion. Brain-derived neurotrophic factor treatment failed to induce any change in the levels of both messengers per neuron in the septal area. In contrast, daily intraventricular administration of nerve growth factor upregulated both choline acetyltransferase and trkA messenger RNA expression in individual neurons. This upregulation was evident on ipsilateral and contralateral sides, suggesting that nerve growth factor is able to upregulate these markers in intact and injured cholinergic cells in the basal forebrain. Similar to the situation in the septum, brain-derived neurotrophic factor did not upregulate choline acetyltransferase or trkA expression in the striatum. However, nerve growth factor administration strongly upregulated choline acetyltransferase messenger RNA expression by individual cholinergic neurons of the striatum. A medial to lateral gradient decrease in this upregulation was detected in the striatum ipsilateral to the side of administration, suggesting a limited diffusion of the nerve growth factor protein from the ventricle into brain parenchyma. In contrast to the strong effect on choline acetyltransferase expression, nerve growth factor treatment was ineffective in altering trkA messenger RNA in the striatum. The contrasting findings between septum and striatum suggest different regulatory mechanisms for trkA messenger RNA expression in the two cholinergic populations. Since nerve growth factor was found to upregulate the expression of its trkA receptor, we tested whether brain-derived neurotrophic factor administration had similar effects on the regulation of its trkB receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Serotonin hyperinnervation in the adult rat ventral mesencephalon following unilateral transection of the medial forebrain bundle. Correlation with reactive microglial and astroglial populations.

We have previously studied changes in the serotoninergic and dopaminergic nigrostriatal systems following transection of the medial forebrain bundle and found a long-term axotomy-induced increase in the levels of serotonin and its main metabolite, 5-hydroxyindolacetic acid in substantia nigra [Venero et al. (1997) J. Neurochem. 68, 2458-2468]. In an attempt to find a rationale for this effect, we have performed an immunohistochemical study. Transection of the medial forebrain bundle of the rat interrupted most of the ascending serotoninergic pathways from the raphe nuclei as revealed by serotonin immunoreactivity. While serotonin immunostaining was almost absent in striatum, it doubled in the ventral mesencephalon at 21 days postlesion. This axotomy-induced increase was accompanied by an increased density of the serotonin nerve terminal network in the ipsilateral substantia nigra and ventral tegmental area. The increase in serotonin immunoreactivity was in line with the measured levels of serotonin and 5-hydroxyindolacetic acid in substantia nigra. In addition, the distribution pattern of glial fibrillary acidic protein-immunoreactive astrocytes and OX42-immunoreactive microglia correlated highly with the location of increased serotonin fibre density in the ventral mesencephalon, especially in ventral tegmental area and in the most medial part of substantia nigra. We suggest that a pruning effect may underly the axotomy-induced increase in serotonin immunoreactivity in the ventral mesencephalon, and further, that activated astroglia and microglia may play a role in directing serotoninergic axonal regeneration following axotomy.

Delayed apoptotic pyramidal cell death in CA4 and CA1 hippocampal subfields after a single intraseptal injection of kainate.

We have performed a detailed time-course analysis of cell death in the hippocampal formation, basal forebrain and amygdala following a single intraseptal injection of kainate in adult rats. Acetylcholinesterase histochemistry revealed a profound loss of staining in the medial septum but not in the diagonal band, and cholinergic fiber density was highly reduced in the hippocampus and amygdala at 10 days postinjection. Terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphatebiotin nick end labeling (TUNEL) histochemistry was performed for precise location of apoptotic cells. Both the medial septum and amygdala exhibited numerous TUNEL-positive nuclei after the intraseptal injection of kainate, while the lateral septum exhibited a lower but significant incidence in terms of apoptotic cells. In the medial septum, the presence of apoptotic cells was at a location displaying acetylcholinesterase staining. TUNEL histochemistry revealed a time-dependent sequential apoptotic cell death in hippocampal pyramidal cells. During the first two days postinjection, apoptosis in the hippocampus was only evident in the CA3 region. At five days postinjection, the entire CA4 region became apoptotic. At 10 days postinjection, the whole extent of the CA1 pyramidal cell layer exhibited numerous TUNEL-positive nuclei. The time-course of kainate-induced apoptosis in Ammons's horn correlated with the disappearance of hippocampal pyramidal neurons as detected by Nissl staining, which is suggestive of a prominent apoptotic death for these cells. The temporal delayed distant damage to CA4 and CA1 hippocampal subfields after a single intraseptal kainate injection is not seen in other models employing kainate and may be a valuable tool for exploring the cellular mechanisms leading to cell death in conditions of status epilepticus.

Long-lasting induction of brain-derived neurotrophic factor is restricted to resistant cell populations in an animal model of status epilepticus.

We have recently characterized an animal model of status epilepticus induced by a single intraseptal injection of kainate. Under these conditions, there is a delayed expanding apoptotic hippocampal and amygdalar cell death. In order to further characterize this animal model, we have performed a detailed time-course analysis of the appearance of cell death, brain-derived neurotrophic factor messenger RNA expression and astroglial and microglial response in different brain areas related to the limbic system. We found a long-lasting delayed apoptotic cell death in the hippocampal formation, amygdala, medial thalamus, dorsal endopiriform nucleus and multiple cortical areas from two to 21 days post-injection. There was a spatiotemporal correlation between the appearance of cell death and induction of brain-derived neurotrophic factor messenger RNA expression in the areas studied, and interestingly this induction was found in non-degenerating cells. We conclude that our animal model of status epilepticus exhibits remarkable features of recurrent seizure activity and provides evidence for a neuroprotective role of brain-derived neurotrophic factor against seizure-induced apoptotic cell death.

Less induced 1-methyl-4-phenylpyridinium ion neurotoxicity on striatal slices from guinea-pigs fed with a vitamin C-deficient diet.

The effect of ascorbic acid depletion on the 1-methyl-4-phenylpyridinium ion (MPP+)-induced neurotoxicity in the dopaminergic system has been tested in guinea-pig striatal slices. Guinea-pigs were divided into three groups and fed on a control diet, ascorbic acid-free diet and ascorbic acid-supplemented diet, respectively. Diets were maintained during 30 days. Striatal slices from ascorbic acid-deficient animals showed the highest levels of dopamine following 25 microM MPP+ treatment; the results from animals under this treatment condition were statistically different from both control and ascorbic acid-supplemented animals under identical experimental conditions. In addition, neurochemical analysis demonstrated that the levels of ascorbic acid and dehydroascorbic acid were highly reduced in striatal tissue from ascorbic acid-deficient animals, thus proving scorbutic conditions in our experimental animals. In view of the higher resistance of the ascorbic acid-deficient animals to the neurotoxicity elicited by MPP+, additional dopaminergic parameters were also measured in striatal tissue from ascorbic acid-deficient animals in the absence of MPP+, including levels of dopamine and its metabolites, tyrosine hydroxylase activity and dopamine uptake, with the aim of finding an explanation for this unexpected result. While dopamine levels and tyrosine hydroxylase activity remained close to control levels, dopamine uptake was significantly reduced in striatal synaptosomes from ascorbic acid-deficient animals as compared with control animals. Since MPP+ is actively accumulated into dopaminergic nerve terminals via the high-affinity dopamine uptake system, this finding could explain the higher resistance of ascorbic acid-deficient animals to the dopamine-depleting effect induced by MPP+ toxicity assayed in striatal slices.

Detailed localization of aquaporin-4 messenger RNA in the CNS: preferential expression in periventricular organs.

We have performed a detailed in situ hybridization study of the distribution of aquaporin-4 messenger RNA in the CNS. Contrary to expectation, we demonstrate that aquaporin-4 is ubiquitously expressed in the CNS. Strong hybridization labeling was detected in multiple olfactory areas, cortical cells, medial habenular nucleus, bed nucleus of the stria terminalis, tenia tecta, pial surface, pontine nucleus, hippocampal formation and multiple thalamic and hypothalamic areas. A low but significant hybridization signal was found, among others, in the choroid plexus of the lateral ventricles, ependymal cells, dorsal raphe and cerebellum. Overall, a preferential distribution of aquaporin-4 messenger RNA-expressing cells was evident in numerous periventricular organs. From the distribution study, the presence of aquaporin-4 messenger RNA-expressing cells in neuronal layers was evident in neuronal layers including the CA1 -CA3 hippocampal pyramidal cells, granular dentate cells and cortical cells. Further evidence of neuronal expression comes from the semicircular arrangement of aquaporin-4 messenger RNA-expressing cells in the bed nucleus of the stria terminalis and medial habenular nucleus exhibiting Nissl-stained morphological features typical of neurons. Combined glial fibrillary acidic protein immunohistochemistry and aquaporin-4 messenger RNA in situ hybridization demonstrated that aquaporin-4 messenger RNA is expressed by glial fibrillary acidic protein-lacking cells. We conclude that aquaporin-4 messenger RNA is present in a collection of structures typically involved in the regulation of water and sodium intake and that aquaporin-4 water channels could be the osmosensor mechanism responsible for detecting changes in cell volume by these cells.