Cannabinoid type 2 receptors in dopamine neurons inhibits psychomotor behaviors, alters anxiety, depression and alcohol preference.

Cannabinoid CB2 receptors (CB2Rs) are expressed in mouse brain dopamine (DA) neurons and are involved in several DA-related disorders. However, the cell type-specific mechanisms are unclear since the CB2R gene knockout mice are constitutive gene knockout. Therefore, we generated Cnr2-floxed mice that were crossed with DAT-Cre mice, in which Cre- recombinase expression is under dopamine transporter gene (DAT) promoter control to ablate Cnr2 gene in midbrain DA neurons of DAT-Cnr2 conditional knockout (cKO) mice. Using a novel sensitive RNAscope in situ hybridization, we detected CB2R mRNA expression in VTA DA neurons in wildtype and DAT-Cnr2 cKO heterozygous but not in the homozygous DAT-Cnr2 cKO mice. Here we report that the deletion of CB2Rs in dopamine neurons enhances motor activities, modulates anxiety and depression-like behaviors and reduces the rewarding properties of alcohol. Our data reveals that CB2Rs are involved in the tetrad assay induced by cannabinoids which had been associated with CB1R agonism. GWAS studies indicates that the CNR2 gene is associated with Parkinson's disease and substance use disorders. These results suggest that CB2Rs in dopaminergic neurons may play important roles in the modulation of psychomotor behaviors, anxiety, depression, and pain sensation and in the rewarding effects of alcohol and cocaine.

Retrograde Axonal Degeneration in Parkinson Disease.

In spite of tremendous research efforts we have not yet achieved two of our principal therapeutic goals in the treatment of Parkinson's disease (PD), to prevent its onward progression and to provide restoration of systems that have already been damaged by the time of diagnosis. There are many possible reasons for our inability to make progress. One possibility is that our efforts thus far may not have been directed towards the appropriate cellular compartments. Up until now research has been largely focused on the loss of neurons in the disease. Thus, neuroprotection approaches have been largely aimed at blocking mechanisms that lead to destruction of the neuronal cell body. Attempts to provide neurorestoration have been almost entirely focused on replacement of neurons. We herein review the evidence that the axonal component of diseased neuronal systems merit more of our attention. Evidence from imaging studies, from postmortem neurochemical studies, and from genetic animal models suggests that the axons of the dopaminergic system are involved predominantly and early in PD. Since the mechanisms of axonal destruction are distinct from those of neuron cell body degeneration, a focus on axonal neurobiology will offer new opportunities for preventing their degeneration. At present these mechanisms remain largely obscure. However, defining them is likely to offer new opportunities for neuroprotection. In relation to neurorestoration, while it has been classically believed that neurons of the adult central nervous system are incapable of new axon growth, recent evidence shows that this is not true for the dopaminergic projection. In conclusion, the neurobiology of axons is likely to offer many new approaches to protective and restorative therapeutics.

An early axonopathy in a hLRRK2(R1441G) transgenic model of Parkinson disease.

Mutations in the gene for LRRK2 are the most common cause of familial Parkinson's disease (PD) and patients with these mutations manifest clinical features that are indistinguishable from those of the more common sporadic form. Thus, investigations of disease mechanisms based on disease-causing LRRK2 mutations can be expected to shed light on the more common sporadic form as well as the inherited form. We have shown that as human BAC transgenic hLRRK2(R1441G) mice age, they exhibit two abnormalities in the nigrostriatal dopaminergic system: an axonopathy and a diminished number of dendrites in the substantia nigra (SN). To better understand disease mechanisms it is useful to determine where in the affected neural system the pathology first begins. We therefore examined the nigrostriatal dopaminergic system in young mice to determine the initial site of pathology. Brains from hLRRK2(R1441G) and littermate control mice at 2-4months of age were examined by immunohistochemistry, anterograde fluorescent axon labeling and ultrastructural analysis. SN neurons, their projecting axons and the striatal terminal fields were assessed. The first identifiable abnormality in this system is an axonopathy characterized by giant polymorphic axon spheroids, the presence of intra-axonal autophagic vacuoles and intra-axonal myelin invagination. An initial involvement of axons has also been reported for other genetic models of PD. These observations support the concept that axons are involved early in the course of the disease. We suggest that effective neuroprotective approaches will be aimed at preventing axonal degeneration.

Neurotrophic effects of serum- and glucocorticoid-inducible kinase on adult murine mesencephalic dopamine neurons.

Mesencephalic dopamine neurons are central to many aspects of human cognition, motivational, and motor behavior, and they are uniquely vulnerable to degenerative neurologic disorders such as Parkinson's disease. There is growing evidence that in the mature brain these neurons not only remain responsive to neurotrophic support, but are dependent on it for viability and function. Little is known of the cellular signaling pathways that mediate this support, although some evidence suggests that protein kinase Akt/PKB may play such a role. Another candidate for such a role is serum- and glucocorticoid-inducible kinase (SGK), a member of the AGC kinase family that is closely related to Akt. We have herein examined the responsiveness of adult mouse dopamine neurons in vivo to overexpression of wild-type and a constitutively active form of SGK by use of viral vector transfer in normal mice and both before and after 6-OHDA lesion. We find that SGK induces a broad spectrum of neurotrophic effects on these neurons, including induction of neuronal hypertrophy, protection from both neuron death and neurotoxin-induced retrograde axonal degeneration, and axon regeneration. Given the diverse and robust effects of SGK on these neurons, and its abundant expression in them, we suggest that SGK, like closely related Akt, may play a role in their responsiveness to neurotrophic factors and in adult maintenance. It therefore offers a novel target for therapeutic development.

Distribution of CB2 cannabinoid receptor in adult rat retina.

Cannabinoid effects are mediated through two receptors, CB1 and CB2. In the retina CB1 has been reported in bipolar cells, gabaergic amacrine cells, horizontal cells, and inner plexiform layer. CB2 receptor mRNA localization was shown in photoreceptors, inner nuclear layer, and ganglion cell layer by using RT-PCR. The aim of this work was to localize CB2 receptor in the rat retina by using immunocytochemistry. Our results showed that CB2 receptor was localized in retinal pigmentary epithelium, inner photoreceptor segments, horizontal and amacrine cells, cells localized in ganglion cell layer, and in fibers of inner plexiform layer. These results are in agreement with studies using RT-PCR and provide some additional information about the distribution of CB2 receptor. Further studies are needed to clarify the role of this cannabinoid receptor in the retina.

Functional expression of brain neuronal CB2 cannabinoid receptors are involved in the effects of drugs of abuse and in depression.

Major depression and addiction are mental health problems associated with stressful events in life with high relapse and recurrence even after treatment. Many laboratories were not able to detect the presence of CB2 cannabinoid receptors (CB2-Rs) in healthy brains, but CB2-R expression has been demonstrated in rat microglial cells and other brain-associated cells during inflammation. Thus, neuronal expression of CB2-Rs has been ambiguous and controversial, and its role in depression and substance abuse is unknown. In this study we tested the hypothesis that genetic variants of the CB2 gene might be associated with depression in a human population and that alteration in CB2 gene expression may be involved in the effects of abused substances, including opiates, cocaine, and ethanol, in rodents. Here we demonstrate that a high incidence of Q63R but not H316Y polymorphism in the CB2 gene was found in Japanese depressed subjects. CB2-Rs and their gene transcripts are expressed in the brains of naïve mice and are modulated after exposure to stressors and administration of abused drugs. Mice that developed an alcohol preference had reduced CB2 gene expression, and chronic treatment with JWH015 a putative CB2-R agonist, enhanced alcohol consumption in stressed but not in control mice. The direct intracerebroventricular microinjection of CB2 antisense oligonucleotide into the mouse brain reduced mouse aversions in the plus-maze test, indicating the functional presence of CB2-Rs in the brain that modifies behavior. Using electron microscopy we report the subcellular localization of CB2-Rs that are mainly on postsynaptic elements in rodent brain. Our data demonstrate the functional expression of CB2-Rs in the brain that may provide novel targets for the effects of cannabinoids in depression and substance abuse disorders beyond neuroimmunocannabinoid activity.

Postsynaptic localization of CB2 cannabinoid receptors in the rat hippocampus.

The expression of CB2 cannabinoid receptors (CB2-Rs) in the brain and their neuronal function has now attracted research interest, since we and others have demonstrated the presence of CB2-Rs in neuronal and glial cells in the brain. In this study, we show the subcellular distribution of CB2-Rs in neuronal, glial, and endothelial cells in the rat hippocampus using immunohistochemical electron microscopy. Brain sections from the hippocampus were immunolabeled for CB2-R, visualized, and analyzed by electron microscopy. We found that in neurons, CB2-R immunoreactivity is present in the cell body as well as in large and medium-sized dendrites. In the soma, the CB2-R labeling is associated with the rough endoplasmic reticulum and Golgi apparatus demonstrating that CB2-Rs are synthesized by hippocampal neurons. CB2-R labeling in dendrites was observed in the cytoplasm and associated with the plasma membrane near the area of synaptic contact with axon terminals indicating a postsynaptic distribution of these receptors. In CB2-R immunoreactive glial and endothelial cells, the labeling was also found to be associated with the plasma membrane. These results provide the first ultrastructural evidence that CB2-Rs are mainly postsynaptic in the rat hippocampus.

Brain neuronal CB2 cannabinoid receptors in drug abuse and depression: from mice to human subjects.

BACKGROUND: Addiction and major depression are mental health problems associated with stressful events in life with high relapse and reoccurrence even after treatment. Many laboratories were not able to detect the presence of cannabinoid CB2 receptors (CB2-Rs) in healthy brains, but there has been demonstration of CB2-R expression in rat microglial cells and other brain associated cells during inflammation. Therefore, neuronal expression of CB2-Rs had been ambiguous and controversial and its role in depression and substance abuse is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study we tested the hypothesis that genetic variants of CB2 gene might be associated with depression in a human population and that alteration in CB2 gene expression may be involved in the effects of abused substances including opiates, cocaine and ethanol in rodents. Here we demonstrate that a high incidence of (Q63R) but not (H316Y) polymorphism in the CB2 gene was found in Japanese depressed subjects. CB2-Rs and their gene transcripts are expressed in the brains of naïve mice and are modulated following exposure to stressors and administration of abused drugs. Mice that developed alcohol preference had reduced CB2 gene expression and chronic treatment with JWH015 a putative CB2-R agonist, enhanced alcohol consumption in stressed but not in control mice. The direct intracerebroventricular microinjection of CB2 anti-sense oligonucleotide into the mouse brain reduced mouse aversions in the plus-maze test, indicating the functional presence of CB2-Rs in the brain that modifies behavior. We report for the using electron microscopy the sub cellular localization of CB2-Rs that are mainly on post-synaptic elements in rodent brain. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate the functional expression of CB2-Rs in brain that may provide novel targets for the effects of cannabinoids in depression and substance abuse disorders beyond neuro-immunocannabinoid activity.

Synapses between corticotropin-releasing factor-containing axon terminals and dopaminergic neurons in the ventral tegmental area are predominantly glutamatergic.

Interactions between stress and the mesocorticolimbic dopamine (DA) system have been suggested from behavioral and electrophysiological studies. Because corticotropin-releasing factor (CRF) plays a role in stress responses, we investigated possible interactions between neurons containing CRF and those producing DA in the ventral tegmental area (VTA). We first investigated the cellular distribution of CRF in the VTA by immunolabeling VTA sections with anti-CRF antibodies and analyzing these sections by electron microscopy. We found CRF immunoreactivity present mostly in axon terminals establishing either symmetric or asymmetric synapses with VTA dendrites. We established that nearly all CRF asymmetric synapses are glutamatergic, insofar as the CRF-immunolabeled axon terminals in these synapses coexpressed the vesicular glutamate transporter 2, and that the majority of CRF symmetric synapses are GABAergic, insofar as the CRF-immunolabeled axon terminals in these synapses coexpressed glutamic acid decarboxylase, findings that are of functional importance. We then looked for synaptic interactions between CRF- and DA-containing neurons, by using antibodies against CRF and tyrosine hydroxylase (TH; a marker for DA neurons). We found that most synapses between CRF-immunoreactive axon terminals and TH neurons are asymmetric (in the majority likely to be glutamatergic) and suggest that glutamatergic neurons containing CRF may be part of the neuronal circuitry that mediates stress responses involving the mesocorticolimbic DA system. The presence of CRF synapses in the VTA offers a mechanism for interactions between the stress-associated neuropeptide CRF and the mesocorticolimbic DA system.

Discovery of the presence and functional expression of cannabinoid CB2 receptors in brain.

Two well-characterized cannabinoid receptors (CBrs), CB1 and CB2, mediate the effects of cannabinoids and marijuana use, with functional evidence for other CBrs. CB1 receptors are expressed primarily in brain and peripheral tissues. For over a decade several laboratories were unable to detect CB2 receptors in brain and were known to be intensely expressed in peripheral and immune tissues and have traditionally been referred to as peripheral CB2 CBrs. We have reported the discovery and functional presence of CB2 cannabinoid receptors in mammalian brain that may be involved in depression and drug abuse and this was supported by reports of identification of neuronal CB2 receptors that are involved in emesis. We used RT-PCR, immunoblotting, hippocampal cultures, immunohistochemistry, transmission electron microscopy, and stereotaxic techniques with behavioral assays to determine the functional expression of CB2 CBrs in rat brain and mice brain exposed to chronic mild stress (CMS) or those treated with abused drugs. RT-PCR analyses supported the expression of brain CB2 receptor transcripts at levels much lower than those of CB1 receptors. In situ hybridization revealed CB2 mRNA in cerebellar neurons of wild-type but not of CB2 knockout mice. Abundant CB2 receptor immunoreactivity (iCB2) in neuronal and glial processes was detected in brain and CB2 expression was detected in neuron-specific enolase (NSE) positive hippocampal cell cultures. The effect of direct CB2 antisense oligonucleotide injection into the brain and treatment with JWH015 in motor function and plus-maze tests also demonstrated the functional presence of CB2 cannabinoid receptors in the central nervous system (CNS). Thus, contrary to the prevailing view that CB2 CBrs are restricted to peripheral tissues and predominantly in immune cells, we demonstrated that CB2 CBrs and their gene transcripts are widely distributed in the brain. This multifocal expression of CB2 immunoreactivity in brain suggests that CB2 receptors may play broader roles in the brain than previously anticipated and may be exploited as new targets in the treatment of depression and substance abuse.

A low chronic ethanol exposure induces morphological changes in the adolescent rat brain that are not fully recovered even after a long abstinence: an immunohistochemical study.

Little is known about the morphological effects of alcoholism on the developing adolescent brain and its consequences into adulthood. We studied here the relationship between two neurotransmitter systems (the serotoninergic and nitrergic) and the astrocytic and neuronal cytoskeleton immediately and long after drinking cessation of a chronic, but low, ethanol administration. Adolescent male Wistar rats were exposed to ethanol 6.6% (v/v) in drinking water for 6 weeks and studied after ending exposure or after a 10-week recovery period drinking water. Control animals received water. Brain sections were processed by immunohistochemistry using antibodies to serotonin (5-HT); glial fibrillary acidic protein (GFAP); astroglial S-100b protein; microtubule associated protein-2 (MAP-2); 200 kDa neurofilaments (Nf-200); and neuronal nitric oxide synthase (nNOS). The mesencephalic dorsal and median raphe nucleus (DRN; MRN) and three prosencephalic areas closely related to cognitive abilities (CA1 hippocampal area, striatum and frontal cortex) were studied by digital image analysis. 5-HT immunoreactivity (-ir) decreased in the DRN and recovered after abstinence and was not changed in the MRN. In the three prosencephalic areas, astrocytes' cell area (GFAP-ir cells) increased after EtOH exposure and tended to return to normality after abstinence, while cytoplasmic astroglial S100b protein-ir, relative area of MAP-2-ir and Nf-200-ir fibers decreased, and later partially recovered. In the striatum and frontal cortex, nNOS-ir decreased only after abstinence. In conclusion, in the adolescent brain, drinking cessation can partially ameliorate the ethanol-induced morphological changes on neurons and astrocytes but cannot fully return it to the basal state.

Morphometric study on cytoskeletal components of neuronal and astroglial cells after chronic CB1 agonist treatment.

One of the major goals for the use of digital image analysis systems in neuroanatomy is to visualize structures, cells, or other tissue components in order to compare various populations. In addition, digital image analysis allows semi-quantification of cell labeling because it is capable of measuring simultaneously the staining intensity, location, size, and shape of labeled profiles. In the present work, the morphological changes in the CB1 hippocampal area and corpus striatum induced by chronic treatment with the synthetic CB1-receptor agonist WIN55,212-2 were analyzed as an example of digital image analysis application. Twice-daily treatment for 14 d with the CB1-receptor agonist demonstrated significant changes in the expression of neuronal cytoskeletal proteins and in neuronal morphology, as evidenced by immunocytochemical and digital analysis studies. However, changes in the expression of astroglial cytoskeletal proteins were not found.

Methods to study the behavioral effects and expression of CB2 cannabinoid receptor and its gene transcripts in the chronic mild stress model of depression.

Behavioral and molecular methods were used to study and determine whether there is a link between depression that may be a factor in drug/alcohol addiction, and the endocannabinoid hypothesis of substance abuse. Depression is a lack of interest in the pleasurable things of life (termed anhedonia) and depressed mood. It is unknown whether CB2 cannabinoid receptors are expressed in the brain and whether they are involved in depression and substance abuse. Therefore, mice were subjected daily for 4 wk to chronic mild stress (CMS), and anhedonia was measured by the consumption of 2% sucrose solution. Behavioral and rewarding effects of abused substances were determined in the CMS and control animals. The expression of CB2 receptors and their gene transcripts was compared in the brains of CMS and control animals by Western blotting using CB2 receptor antibody and reverse transcriptase-polymerase chain reaction (RT-PCR). Furthermore, the expression and immunocytochemical identification of CB2 cannabinoid receptor in the rat brain were determined. CMS induced gender-specific aversions, which were blocked by WIN55,212-2, a nonspecific CB1 and CB2 cannabinoid receptor agonist. Direct CB2 antisense oligonucleotide microinjection into the mouse brain induced anxiolysis, indicating that CB2 or CB2-like receptors are present in the brain and may influence behavior. The major finding from these studies was the expression of CB2 receptor and its gene transcript in the mouse brain, which was enhanced by CMS. These preliminary results, if confirmed, suggest that the CB2 receptors are expressed in the mammalian brain and may be involved in depression and substance abuse.

Neuronal cytoskeleton and synaptic densities are altered after a chronic treatment with the cannabinoid receptor agonist WIN 55,212-2.

Cannabinoid CB1 receptors are the most abundant G-protein-coupled receptors in the brain. Its presynaptic location suggests a role for cannabinoids in modulating the release of neurotransmitters from axon terminals by retrograde signaling. The neuroprotective effects of cannabinoid agonists in animal models of ischemia, seizures, hypoxia, Multiple Sclerosis, Huntington and Parkinson disease have been demonstrated in several reports. The proposed mechanism for the neuroprotection ranges from antioxidant effects, reduction of microglial activation and anti-inflammatory reaction to receptor-mediated reduction of glutamate release. In the present work, we analyzed the morphological changes induced by a chronic treatment with the synthetic cannabinoid receptor agonist, WIN 55,212-2, in four brain regions where the CB1 cannabinoid receptor is present in high density: the CA1 hippocampal area, corpus striatum, cerebellum and frontal cortex. After a twice-daily treatment for 14 days with the cannabinoid receptor agonist (3 mg/kg sc, each dose) to male Wistar rats (150-170 g), the expression of neurofilaments (Nf-160 and Nf-200), microtubule-associated protein-2 (MAP-2), synaptophysin (Syn) and glial fibrillary acidic protein (GFAP) was studied by immunohistochemistry and digital image analysis. Ultrastructural study of the synapses was done using electron microscopy. After the treatment, a significant increase in the expression of neuronal cytoskeletal proteins (Nf-160, Nf-200, MAP-2) was observed, but we did not find changes in the expression of GFAP, the main astroglial cytoskeletal protein. In cerebellum, there was an increase in Syn expression and in the number of synaptic vesicles, while, in the hippocampus, an increase in the Syn expression and in the thickness of the postsynaptic densities was observed. The results obtained from these studies provide evidences on the absence of astroglial reaction and a sprouting phenomena induced by the WIN treatment that might be a key contributor to the long-term neuroprotective effects observed after cannabinoid treatments in different models of central nervous system (CNS) injury reported in the literature.

Cannabinoid CB2 receptors: immunohistochemical localization in rat brain.

Brain expression of CB2 cannabinoid receptors has been much less well established and characterized in comparison to the expression of brain CB1 receptors. Since CB2 receptors are intensely expressed in peripheral and immune tissues, expression in brain microglia has been anticipated. Nevertheless, we now describe expression of CB2-receptor-like immunoreactivity in brain in neuronal patterns that support broader CNS roles for this receptor. Two anti-CB2 affinity purified polyclonal antibodies were raised in rabbits immunized with peptide conjugates that corresponded to amino acids 1-33 and 20-33. Western blot analyses revealed specific bands that were identified using these sera and were absent when the sera were preadsorbed with 8.3 mug/ml of the immunizing peptides. These studies, and initial RT-PCR analyses of brain CB1 and CB2 mRNAs, also support the expression of brain CB2 receptor transcripts at levels much lower than those of CB1 receptors. CB2 cannabinoid receptor mRNA was clearly expressed in the cerebellum of wild type but not in CB2 knockout mice. CB2 immunostaining was detected in the interpolar part of spinal 5th nucleus of wild type but not in CB2 knockout mice, using a mouse C-terminal CB2 receptor antibody. Immunohistochemical analyses revealed abundant immunostaining for CB2 receptors in apparent neuronal and glial processes in a number of rat brain areas. Cerebellar Purkinje cells and hippocampal pyramidal cells revealed substantial immunoreactivity that was absent when sections were stained with preadsorbed sera. CB2 immunoreactivity was also observed in olfactory tubercle, islands of Calleja, cerebral cortex, striatum, thalamic nuclei, hippocampus, amygdala, substantia nigra, periaqueductal gray, paratrochlear nucleus, paralemniscal nucleus, red nucleus, pontine nuclei, inferior colliculus and the parvocellular portion of the medial vestibular nucleus. In-vitro, CB2 immunoreactivity was also present in hippocampal cell cultures. The multifocal expression of CB2 immunoreactivity in glial and neuronal patterns in a number of brain regions suggests reevaluation of the possible roles that CB2 receptors may play in the brain.

Changes in the postnatal development on nitric oxide system induced by serotonin depletion.

Serotonin (5-HT) is expressed early during central nervous system (CNS) development and plays an important role during this period. Nitric oxide (NO) is also involved in neuronal development. Morphological and functional relationships between NO and 5-HT, demonstrated as alterations of the nitrergic system, were observed after a 5-HT depletion. It has been hypothesized that NO may be related to the neuronal damage induced by some 5-HT neurotoxins. A parachloroamphetamine (PCA) treatment is able to damage ascending 5-HT fibers proceeding from the dorsal raphe nucleus (DRN) and depletes 5-HT storage in neuronal somata. In order to study the effects of a 5-HT depletion on the nitrergic system during postnatal development, Wistar rat pups were injected subcutaneously twice, on postnatal day (PND) 3 and PND4 with PCA. Neuronal nitric oxide synthase (nNOS) immunoreactivity and NADPH diaphorase reactivity were performed on brain sections from PND5, 7, 12, 19, 29 and 62 animals. After the treatment, we found an increased NADPH-d staining and nNOS immunoreactivity in striatum, frontal cortex and hippocampus along the different studied time periods. Interestingly, the expression of both NO markers was higher when 5-HT depletion was more evident, suggesting a very close relationship between 5-HT and NO systems during postnatal development.

Altered neuron-glia interactions in a low, chronic prenatal ethanol exposure.

Serotoninergic neurons, astrocytes and nitrergic system play an important role in central nervous system (CNS) development. These systems are altered in prenatal ethanol exposure (PEE) but ethanol (EtOH) effects may be very diverse under different conditions. In this study, we analyzed morphologically two serotoninergic mesencephalic nuclei and three prosencephalic areas of serotoninergic innervation in a model of pre- and postnatal low-ethanol exposure. Female Wistar rats were orally exposed to EtOH 6.6% (v/v), ad libitum, for 6 weeks before mating and during gestation and lactation while control group received water ad libitum. Twenty-day-old offspring (P21) brains were processed and immunoreactivity (IR) using antibodies against tryptophan hydroxylase (TPH), 5-HT, 5-HT transporter (5HTT), glial fibrillary acidic protein (GFAP), S-100B protein, 200-kDa neurofilaments (Nf-200) and neuronal nitric oxide synthase (nNOS) was evaluated. Dorsal and median raphe nucleus (DRN and MRN), hippocampus (Hipp), striatum (Strt) and frontal cortex (FCx) were studied by computer-assisted image analysis. Relative optical density (ROD) of TPH-IR, 5-HT-IR and nNOS-IR neurons; cell area of GFAP-IR astrocytes; relative area of 5HTT-IR fibers and Nf-200-IR were evaluated. TPH-IR was increased in DRN and MRN and 5-HT-IR was increased only in MRN. 5-HTT-IR fibers and ROD of S-100B-IR astrocytes were increased in the three prosencephalic areas while GFAP-IR astrocytes were hypertrophied only in Hipp and FCx. Nf-200 expression was increased in Hipp and Strt and morphologically altered in the FCx. ROD of nNOS-IR neurons was increased in Strt and FCx but was not detected in Hipp. We have also detected morphological changes resembling accelerated development and maturation, and early aging. Considering the evidences of a close 5-HT-astroglial-NO relationship during CNS development the differential response of the studied regions is an interesting result that could be due to different gradients of development in the studied areas and/or different responses of those areas to the effects of a low pre- and postnatal ethanol exposure.

Neuronal and inducible nitric oxide synthase immunoreactivity following serotonin depletion.

Serotonin (5HT) modulates the development and plasticity of its innervation areas in the central nervous system (CNS). Astrocytic 5HT(1A) receptors are involved in the plastic phenomena by releasing the astroglial-derived neurotrophic factor S-100beta. Several facts have demonstrated that nitric oxide (NO) and the nitric oxide synthase enzyme (NOS) may also be involved in this neuroglial interaction: (i) NO, S-100beta and 5HT are involved in CNS plasticity; (ii) micromolar S-100beta concentration stimulates inducible-NOS (iNOS) expression; (iii) neuronal NOS (nNOS) immunoreactive neurons are functionally and morphologically related to the serotoninergic neurons; (iv) monoamines level, including 5HT, can be modulated by NO release. We have already shown that 5HT depletion increases astroglial S-100beta immunoreactivity, induces neuronal cytoskeletal alterations and produces an astroglial reaction, while once 5HT level is recovered, a sprouting phenomenon occurs [Brain Res. 883 (2000) 1-14]. To further characterize the relationship among nNOS, iNOS and 5HT we have analyzed nNOS and iNOS expression in the CNS after 5HT depletion induced by parachlorophenylalanine (PCPA) treatment. Studies were performed immediately after ending the PCPA treatment and during a recovery period of 35 days. Areas densely innervated by 5HT fibers were studied by means of nNOS and iNOS immunoreactivity as well as NADPH diaphorase (NADPHd) staining. All parameters were quantified by computer-assisted image analysis. Increased nNOS immunoreactivity in striatum and hippocampus as well as increased NADPHd reactivity in the striatum, hippocampus and parietal cortex were found after PCPA treatment. The iNOS immunoreactivity in the corpus callosum increased 14 and 35 days after the end of PCPA treatment. These findings showed that nNOS immunoreactivity and NADPHd activity increased immediately after 5HT depletion evidencing a close functional interaction between nitrergic and serotoninergic systems. However, iNOS immunoreactivity increased when 5HT levels were normalized, which could indicate one of the biological responses to S-100beta release.

Distribution of NADPH-diaphorase in rat mesencephalon: a light and electron microscopical study.

NADPH-diaphorase is a useful technique to reveal NO producing neurons at light microscopic level (LM). A modification of the technique using the tetrazolium salt BSPT as substrate, is useful to study the ultrastructure of NO neurons. The aim of this work was to perform a detailed analysis of NADPH-diaphorase reactive neurons in rat mesencephalon both at light and electron microscopic levels. NADPH-diaphorase reactive neurons were observed in superior colliculus, in central gray matter, in dorsal and medial raphe and in the pedunculopontine tegmental nucleus using two histochemical techniques at LM. Electron microscopy showed deposits on membranes of the endoplasmic reticulum, Golgi apparatus and nuclear envelope of dorsal raphe neurons. Presynaptic and postsynaptic terminals showed deposits on membranous elements but postsynaptic terminals also showed deposits on the inner surface of their membranes. Further physiological studies are needed to clarify the meaning of the ultrastructural findings such as the putative interaction of NOS with postsynaptic proteins, receptors or membranous channels.

Distribution of NADPH-diaphorase in rat mesencephalon: a light and electron microscopical study

NADPH-diaphorase is a useful technique to reveal NO producing neurons at light microscopic level (LM). A modification of the technique using the tetrazolium salt BSPT as substrate, is useful to study the ultrastructure of NO neurons. The aim of this work was to perform a detailed analysis of NADPH-diaphorase reactive neurons in rat mesencephalon both at light and electron microscopic levels. NADPH-diaphorase reactive neurons were observed in superior colliculus, in central gray matter, in dorsal and medial raphe and in the pedunculopontine tegmental nucleus using two histochemical techniques at LM. Electron microscopy showed deposits on membranes of the endoplasmic reticulum, Golgi apparatus and nuclear envelope of dorsal raphe neurons. Presynaptic and postsynaptic terminals showed deposits on membranous elements but postsynaptic terminals also showed deposits on the inner surface of their membranes. Further physiological studies are needed to clarify the meaning of the ultrastructural findings such as the putative interaction of NOS with postsynaptic proteins, receptors or membranous channels.