Functional Interplay of Type-2 Corticotrophin Releasing Factor and Dopamine Receptors in the Basolateral Amygdala-Medial Prefrontal Cortex Circuitry.

BACKGROUND: Basolateral amygdala (BLA) excitatory projections to medial prefrontal cortex (PFC) play a key role controlling stress behavior, pain, and fear. Indeed, stressful events block synaptic plasticity at the BLA-PFC circuit. The stress responses involve the action of corticotrophin releasing factor (CRF) through type 1 and type 2 CRF receptors (CRF1 and CRF2). Interestingly, it has been described that dopamine receptor 1 (D1R) and CRF peptide have a modulatory role of BLA-PFC transmission. However, the participation of CRF1 and CRF2 receptors in BLA-PFC synaptic transmission still is unclear. METHODS: We used in vivo microdialysis to determine dopamine and glutamate (GLU) extracellular levels in PFC after BLA stimulation. Immunofluorescence anatomical studies in rat PFC synaptosomes devoid of postsynaptic elements were performed to determine the presence of D1R and CRF2 receptors in synaptical nerve endings. RESULTS: Here, we provide direct evidence of the opposite role that CRF receptors exert over dopamine extracellular levels in the PFC. We also show that D1R colocalizes with CRF2 receptors in PFC nerve terminals. Intra-PFC infusion of antisauvagine-30, a CRF2 receptor antagonist, increased PFC GLU extracellular levels induced by BLA activation. Interestingly, the increase in GLU release observed in the presence of antisauvagine-30 was significantly reduced by incubation with SCH23390, a D1R antagonist. CONCLUSION: PFC CRF2 receptor unmasks D1R effect over glutamatergic transmission of the BLA-PFC circuit. Overall, CRF2 receptor emerges as a new modulator of BLA to PFC glutamatergic transmission, thus playing a potential role in emotional disorders.

Dopamine D1 and corticotrophin-releasing hormone type-2α receptors assemble into functionally interacting complexes in living cells.

BACKGROUND AND PURPOSE: Dopamine and corticotrophin-releasing hormone (CRH; also known as corticotrophin-releasing factor) are key neurotransmitters in the interaction between stress and addiction. Repeated treatment with cocaine potentiates glutamatergic transmission in the rat basolateral amygdala/cortex pathway through a synergistic action of D1 -like dopamine receptors and CRH type-2α receptors (CRF2 α receptors). We hypothesized that this observed synergism could be instrumented by heteromers containing the dopamine D1 receptor and CRF2 α receptor. EXPERIMENTAL APPROACH: D1 /CRF2 α receptor heteromerization was demonstrated in HEK293T cells using co-immunoprecipitation, BRET and FRET assays, and by using the heteromer mobilization strategy. The ability of D1 receptors to signal through calcium, when singly expressed or co-expressed with CRF2 α receptors, was evaluated by the calcium mobilization assay. KEY RESULTS: D1 /CRF2 α receptor heteromers were observed in HEK293T cells. When singly expressed, D1 receptors were mostly located at the cell surface whereas CRF2 α receptors accumulated intracellularly. Interestingly, co-expression of both receptors promoted D1 receptor intracellular and CRF2 α receptor cell surface targeting. The heteromerization of D1 /CRF2 α receptors maintained the signalling through cAMP of both receptors but switched D1 receptor signalling properties, as the heteromeric D1 receptor was able to mobilize intracellular calcium upon stimulation with a D1 receptor agonist. CONCLUSIONS AND IMPLICATIONS: D1 and CRF2 α receptors are capable of heterodimerization in living cells. D1 /CRF2 α receptor heteromerization might account, at least in part, for the complex physiological interactions established between dopamine and CRH in normal and pathological conditions such as addiction, representing a new potential pharmacological target.

Intron retention as an alternative splice variant of the rat urocortin 1 gene.

Urocortin 1, highly conserved metazoan gene of the corticotropin-releasing hormone family, is a simple gene structured in two exons and the corresponding intron. The urocortin 1 prepropeptide is entirely coded in the second exon. Preliminary non-isotopic in situ hybridization experiments with an oligonucleotide complementary to an intron sequence of the urocortin 1 gene showed a significant cytoplasmic-like staining, suggesting the occurrence of an intron-retained urocortin 1 transcript. This observation prompted us to study whether the urocortin 1 gene presents alternative splicing by intron retention event. Confocal fluorescent in situ hybridization for urocortin 1 RNA and the use of the specific DNA dye TOPRO-3 allowed us to show significant expression of the intron-retained urocortin 1 transcript that did not colocalize with TOPRO-3 staining indicating a cytoplasmic localization for the intron-retained urocortin 1 transcript. The natural occurrence of a polyadenylated intron-retained urocortin 1 RNA was further documented by reverse transcriptase polymerase chain reaction (PCR), primed with oligo(dT), of total RNA extracted from three brain regions, a midbrain region containing the Edinger-Westphal nucleus, cerebellum and prefrontal cortex. In the three brain regions studied, it was possible to amplify both intron-less as well as intron-retained urocortin 1 transcripts. The use of PCR primers that simultaneously amplify both urocortin 1 transcripts allowed us to show that the expression of both urocortin 1 transcripts differs among the brain regions analyzed, suggesting a tissue specific regulation of this alternative splicing. In silico analysis of the five known mammalian urocortin 1 genomic sequences showed high conservation of the urocortin 1 intron sequence. Further studies should investigate the regulation of this intron retention event and its consequence for the functionality of the urocortin 1 gene.

8-oxoguanine DNA glycosylase, but not Kin17 protein, is translocated and differentially regulated by estrogens in rat brain cells.

8-oxoguanine DNA glycosylase and Kin17 are proteins widely distributed and phylogenetically conserved in the CNS. 8-oxoguanine DNA glycosylase is a DNA repair enzyme that excises 7,8-dihydro-8-oxoguanine present in DNA damaged by oxidative stress. Kin17 protein is involved in DNA repair and illegitimate recombination in eukaryotic cells. The present study evaluates the effect of ovarian hormones on the expression of both proteins in the magnocellular paraventricular nucleus of the hypothalamus and the bed nucleus of the stria terminalis in female and male rat brains. In the paraventricular nucleus, ovariectomy induced a significant decrease in the number of 8-oxoguanine DNA glycosylase-positive nuclei as well as in their relative fluorescent intensity as compared with ovariectomized-estradiol treated and proestrous groups. Confocal microscopy observation demonstrated that oxoguanine DNA glycosylase protein is located in the Hoechst-dyed nuclei and cytoplasm in male and ovariectomized rats. Surprisingly, following estradiol administration to ovariectomized and proestrous rats, the 8-oxoguanine DNA glycosylase immunolabeling was observed in the nucleolus, the cytoplasm and the dendrites of cells, while Kin17 protein was always localized in the cell nuclei. In the bed nucleus of the stria terminalis, the number of 8-oxoguanine DNA glycosylase-positive nuclei during proestrous was significantly lower than the number obtained in males and ovariectomized rats and similar to the number of ovariectomized-estradiol-treated groups. In contrast to these observations, no significant differences were observed in the expression of kin17 protein. Our results suggest that estrogens differentially regulate the expression of 8-oxoguanine DNA glycosylase, but not that of Kin17 protein, in specific regions of the rat brain and that estradiol can translocate the 8-oxoguanine DNA glycosylase protein within nuclei and to other subcellular compartments.

Chronic morphine treatment and withdrawal increase extracellular levels of norepinephrine in the rat bed nucleus of the stria terminalis.

Extracellular levels of norepinephrine (NE) and glutamate (Glu) in the ventral bed nucleus of the stria terminalis (vBNST) of saline- and chronic morphine-treated rats, with or without withdrawal, were studied by means of the in vivo microdialysis technique in anesthetized rats. In addition, the tissue concentration of NE was studied at different rostrocaudal levels of the vBNST. Chronic morphine treatment significantly increased extracellular levels of NE, but not Glu, in vBNST. At 48 h after naloxone-induced morphine withdrawal there was a further significant increase in the extracellular levels of NE, but not Glu, in vBNST. The presence of UK 14304, an alpha(2)-adrenergic agonist, induced a significant decrease in NE extracellular levels in all experimental groups. In contrast, UK 14304 induced a significant decrease in Glu extracellular levels only in saline-treated rats. The results also show that the vBNST presents a rostrocaudal gradient of NE and contains 9.4% of total brain NE. The increase in NE extracellular levels in vBNST induced by chronic morphine treatment and the further increase in NE levels 48 h after naloxone-induced morphine withdrawal suggest that NE in vBNST may be involved in the pharmacological effects of chronic morphine and withdrawal.

Medullary noradrenergic neurons projecting to the bed nucleus of the stria terminalis express mRNA for the NMDA-NR1 receptor.

The bed nucleus of the stria terminalis pars ventralis (vBNST) receives dense noradrenergic terminals and contains the highest concentration of noradrenaline (NA) in the brain. We used autoradiography following retrograde axonal transport of [(3)H]-NA to identify selectively whether noradrenergic neurons innervating the vBNST originate in the medulla oblongata and/or the locus coeruleus. In combination with this technique, non-isotopic in situ hybridization for the NMDA-NR1 receptor subunit mRNA was used to examine, on the same brain sections, its expression in noradrenergic neurons that innervate the vBNST. The results showed that 60 +/- 6% and 35 +/- 7% of the total number of radiolabeled cells detected after injection of [(3)H]-NA in the vBNST were located in brainstems A1 and A2 noradrenergic cell groups, respectively. In addition, 18.5 +/- 4.2% of radiolabeled cells in A1 and 15.7 +/- 5% in A2 also expressed the mRNA for the NMDA-NR1 receptor subunit. In contrast, only 4 +/- 3% of the radiolabeled cells were present in the locus coeruleus, and none of these cells was positive to NMDA-NR1 receptor subunit mRNA. The present results provide evidence that BNST noradrenergic fibers and terminals originate predominantly from A1 and A2 noradrenergic cell groups, and that a significant number of these noradrenergic neurons also express the mRNA for the NMDA-NR1 receptor subunit. The observation that brainstem noradrenergic neurons innervating the vBNST express NMDA receptor mRNA gives anatomical support to the regulation of NA release by NMDA presynaptic receptors.

Raphe serotonergic neurons projecting to the olfactory bulb contain galanin or somatostatin but not neurotensin.

Retrograde axonal transport with [3H]5-HT has been developed as a specific tracing technique to identify serotonergic projections. This method, in combination with immunocytochemistry, offers considerable advantage of specificity and sensitivity to study pathways of multitransmitter-containing neurons. In this work, we studied the presence of galanin, somatostatin, and neurotensin in serotonergic neurons of dorsal and median raphe, which project to the olfactory bulbs. After [3H]5-HT injections into the rat olfactory bulbs, double galanin-immunoreactive and [3H]5-HT radiolabelled cells were located in the dorsal, lateral, and ventral region of dorsal raphe, but they were never seen in the median raphe. In the dorsal raphe, galanin-radiolabelled neurons represented 28% of the total number of radiolabelled cells. Double somatostatin-immunoreactive and radiolabelled neurons were located in the dorsal and median raphe. In the dorsal raphe, double somatostatin-radiolabelled neurons represented only 11% of the radiolabelled cells and they were mainly located ventral to the aqueduct. In the median raphe, 15% of radiolabelled cells were also immunopositive for somatostatin. In contrast, neurotensin-immunoreactive cells in the dorsal and median raphe were distributed among [3H]5-HT radiolabelled neurons but they were never radiolabelled. Our results show subpopulations of serotonergic raphe-olfactory bulb projection neurons containing either galanin or somatostatin, but not neurotensin.

Noradrenaline inhibits glutamate release in the rat bed nucleus of the stria terminalis: in vivo microdialysis studies.

The microdialysis technique was used to simultaneously study the in vivo extracellular levels of noradrenaline, glutamate, and gamma aminobutyric acid (GABA) in the bed nucleus of the stria terminalis in order to assess the regulation that noradrenaline may exert upon the release of amino acid neurotransmitters. Perfusion through the probe with UK14304, a selective alpha2-adrenergic agonist, produced a significant decrease of noradrenaline and glutamate extracellular levels. Perfusion through the probe with RX821002, a selective alpha2-adrenergic antagonist, produced a significant increase of noradrenaline and glutamate basal extracellular levels. Perfusion with prazosine, a selective alpha1-adrenergic antagonist, produced a significant decrease of noradrenaline basal extracellular levels without affecting glutamate levels. Under the same conditions, GABA basal extracellular levels were not changed in the presence of any of the alpha-adrenergic ligands studied. The perfusion of high potassium through the probe induced a significant Ca++dependent release of the three neurotransmitters; however, extracellular noradrenaline returned to normal levels even though potassium was still present. In addition, it was observed that alpha-adrenergic receptor ligands exerted differential effects upon K+-induced release of noradrenaline and glutamate. Perfusion with the nonselective alpha-adrenergic antagonist, phenoxybenzamine, presented a biphasic effect upon K+-induced release of noradrenaline; a significant decrease during the first 5 min of stimulation followed by a significant increase in the next 5 min of stimulation. Perfusion with RX821002 produced a significant increase in K+-induced release of noradrenaline that returned to normal basal values before the end of the stimulation period. In contrast, local perfusion with prazosine caused a significant decrease of K+-induced noradrenaline release. In the case of glutamate, perfusion through the probe with phenoxybenzamine produced a significant increase in K+-induced release of glutamate. In addition, RX821002 and prazosine produced a significant increase in K+-induced release of glutamate. Perfusion through the probe with UK14304 produced a significant decrease of both noradrenaline and glutamate K+-induced release. The present results show that noradrenaline in the bed nucleus of stria terminalis exerts a significant inhibition over its own release through alpha2-adrenergic receptors and over glutamate release mainly through alpha2-adrenergic receptors. Thus, the results suggest that noradrenaline in the bed nucleus of the stria terminalis maintains an inhibitory tone over the information flow mediated by glutamate.

Differential regulation of dopamine release by N-methyl-D-aspartate receptors in rat striatum after partial and extreme lesions of the nigro-striatal pathway.

The participation of N-methyl-d-aspartate (NMDA) receptors on dopamine (DA) efflux in the striatum of anaesthetized rats, which had their DA nigrostriatal pathway previously lesioned with different doses of 6-hydroxydopamine (6-OH-DA), was assessed by in vivo microdialysis methodology. In addition, the in vivo basal DA and dihydroxy-phenyl-acetic acid (DOPAC) effluxes and the effect of local K+-depolarization on DA release were also evaluated in the striatum of these 6-OH-DA treated rats. Lesioned rats were divided in three groups corresponding to animals with 25-75%, 75-95% and >95% of striatum tissue DA depletion, respectively. Striatal DA tissue depletion between 25-75% occurred in parallel with a 30% reduction in DA extracellular levels, with a moderate 10% increase in basal fractional DA efflux, and with no statistical changes in the fractional DA efflux induced by NMDA (500 microM) receptor stimulation by reverse dialysis. Rats with higher DA tissue depletion (between 75-95%) exhibited a 60% reduction in DA extracellular levels in the striatum and this reduction occurred in parallel with a modest rise in basal fractional DA efflux, but with a striking decrease in the NMDA-induced fractional DA efflux. In rats with extreme or >95% of striatal DA tissue depletion, basal fractional DA efflux in the striatum increased quite substantially along with a recovery in the ability of NMDA receptor stimulation to induce fractional DA release. The >95% striatal DA-depleted rats also exhibited a significant decrease in tissue and extracellular DOPAC/DA ratio when compared to sham and partially DA-depleted rats. In contrast to the previous results, fractional DA efflux induced by reverse dialysis with K+ (40 mM) remained the same in the striatum of sham and all groups of DA-tissue depleted rats. The present findings suggest the existence of at least three features associated to the regulation of basal and NMDA-induced extracellular levels of DA in the striatum of rats as a function of striatal tissue DA depletion produced by 6-OH-DA. They also support the view that a differential regulation of basal and NMDA-induced DA extracellular levels occur in partial and extreme DA-depleted striatum after 6-OH-DA treatment. Such findings may have implications as regard to the participation of the NMDA receptor in the compensatory mechanisms associated to the progress of Parkinson's disease, as well as in the therapeutic treatment of this neurological disorder.

Regulation of norepinephrine release from the rat bed nucleus of the stria terminalis: in vivo microdialysis studies.

The microdialysis technique was used to study the in vivo extracellular levels of norepinephrine in the bed nucleus of the stria terminalis. A basal level of 2.34 +/-0.25 fmol/microl of norepinephrine was observed. Desipramine (2 and 10 microM), a norepinephrine uptake blocker, significantly increased extracellular levels of norepinephrine. Reversed perfusion with high potassium in the presence of 2 microM desipramine induced the release of norepinephrine. Instead, in the presence of 10 microM desipramine, a significant decrease in the induced release of norepinephrine was observed. Clonidine, an alpha2-adrenergic agonist, significantly decreased basal extracellular levels of norepinephrine and the K+-induced release of norepinephrine. In contrast, yohimbine and RX821002, two alpha2-adrenergic antagonists, significantly increased basal extracellular levels of norepinephrine but not the release of norepinephrine induced by 70 mM K+. Perfusion of tetrodotoxin through the probe located in the bed nucleus of the stria terminalis significantly decreased both the basal extracellular level and the K+-induced release of norepinephrine. Furthermore, perfusion of tetrodotoxin through a microdialysis probe implanted in the medial forebrain bundle also decreased basal extracellular levels of norepinephrine in the bed nucleus of the stria terminalis. The results show that in vivo there is a significant noradrenergic tonic activity in the bed nucleus of the stria terminalis. This tonic activity depends on the impulse flow through medial forebrain bundle nerve fibers. Under these conditions, extracellular levels of norepinephrine in the bed nucleus of the stria terminalis are regulated by the magnitude of norepinephrine uptake and by presynaptic alpha2-adrenergic receptors.

NMDA-NR1 receptor subunit mRNA expression in rat brain after 6-OH-dopamine induced lesions: a non-isotopic in situ hybridization study.

Antisense digoxigenin-labeled deoxyoligonucleotides probes and non-isotopic in situ hybridization (HIS) techniques have been used to explore the NMDA-NR1 receptor subunit mRNA distribution in different brain areas of rats which had their dopaminergic nigrostriatal pathway previously lesioned with intracerebral administration of 6-OH-dopamine (6-OH-DA). Intense and significant hybridization signals for NR1 mRNA were found in dentate gyrus and regions CA1-CA2-CA3 of the hippocampus, in layers II-III and V-VI of the cerebral cortex, and in the cerebellum of sham-treated rats. Basal ganglia structures such as the striatum exhibited few NR1 mRNA hybridization signals as compared to the hippocampus and cerebral cortex. In contrast, both zona compacta and reticulata of substantia nigra (SN) showed a reduced number of cells with nevertheless intense NR1 mRNA HIS signals. The NR1 mRNA distribution in the brain was affected in a brain regional selective manner by 6-OH-DA induced lesions of DA neuronal systems. A striking increase in NR1 mRNA HIS signals was observed in both striata after unilateral lesioning with 6-OH-DA. Instead, in SN compacta but not in reticulata, a moderate but significant bilateral reduction of NR1 mRNA was observed after unilateral 6-OH-DA injection. No significant changes in NR1 mRNA were detected in cerebral cortex and other brain regions after 6-OH-DA treatment. These studies, and others reported in the literature, support the view that extensive lesions of nigrostriatal DA-containing neurons in the brain may trigger compensatory or adaptative responses in basal ganglia structures such as striatum and substantia nigra which involve glutamateric neurons and the genic expression of NMDA receptors.

Regulation of endogenous noradrenaline release from the bed nucleus of stria terminalis.

The bed nucleus of stria terminalis (BNST) contains the highest concentration of noradrenaline (NA) in the brain. Minislices of the ventral portion of the bed nucleus of stria terminalis (vBNST) were used to study the release of endogenous NA. High K+ induced a Ca(2+)-dependent and reserpine-sensitive release of NA. Clonidine (1 microM), an alpha 2-noradrenergic receptor agonist, significantly decreased K(+)-induced release of NA, whereas yohimbine (1 microM), an alpha 2-noradrenergic antagonist, increased this release. N-Methyl-D-aspartate (NMDA), a specific agonist of NMDA-type glutamate receptors, evoked the release of NA from vBNST minislices. In the presence of D-serine (10 microM), an agonist at the glycine site associated with the NMDA receptor, the NMDA effect was significantly higher. Glycine (1 microM) also increased NA release evoked by NMDA. However, glycine exhibited a significant effect by itself, suggesting the existence of strychnine-sensitive glycine receptors in vBNST. Endogenous NA release induced by 40 mM K+ and NMDA was not additive. Thus, vBNST minislices seem to be a good model to study the release of endogenous NA in the CNS. Such NA release in the vBNST is regulated by alpha 2-noradrenergic receptors and by glutamate through NMDA receptors.

Effects of in vitro ethanol and chronic ethanol consumption on the release of excitatory amino acids in the rat hippocampus.

In CA1-CA3 hippocampal slices, in vitro ethanol (EtOH) (10-100 mM) evoked, as a function of EtOH concentration, a differential release of aspartate (Asp) and glutamate (Glu). Omission of Ca2+ ions from the superfusion media completely abolished the EtOH-induced release of Asp but not that of Glu. In addition, at 20 mM, EtOH enhanced K(+)-evoked release only of Asp. Finally, delayed changes were observed on NMDA-evoked release of [3H]noradrenaline (NA) in the dentate gyrus (DG) after withdrawal from EtOH for 30 days.

Changes in extracellular levels of glutamate and aspartate in rat substantia nigra induced by dopamine receptor ligands: in vivo microdialysis studies.

The microdialysis technique was utilized to study the local effects of D1 and D2 family type dopamine (DA) receptor (R) ligands on the in vivo release of endogenous glutamate (GLU) and aspartate (ASP) from rat substantia nigra (SN). Addition to the dialysis perfusion solution of either D1-R and D2-R agonists, such as SKF-38393 (50 and 100 microM) and Quinpirole (5 and 10 microM), resulted in dose-dependent increases in extracellular concentrations of GLU and ASP, respectively. The SKF-38393 and Quinpirole-induced effects were reduced by SCH-23390 (0.5 microM), a D1-R antagonist, and by Spiperone (1.0 microM), a D2-R antagonist, respectively. However, SCH-23390 and Spiperone did increase GLU and ASP extracellular concentrations. Local infusion with Tetrodotoxin (TTX) (1.0 microM), a blocker of voltage-dependent Na+ channels, increased basal extracellular levels of GLU. In addition, co-infusion of TTX and SKF-38393 evoked increases in extracellular GLU levels higher than those observed after SKF-38393 alone. Finally, chemical lesions of nigral DA cells with 6-OH-DA increased the basal extracellular levels of GLU. It is proposed that the release of GLU and ASP from SN may be regulated by D1- and D2-receptors present in this basal ganglia structure. In addition, part of the D1 receptors present in SN might be located presynaptically on GLU-containing nerve endings.

Release of endogenous catecholamines from the striatum and bed nucleus of stria terminalis evoked by potassium and N-methyl-D-aspartate: in vitro microdialysis studies.

Induced release of endogenous dopamine and noradrenaline from coronal slices containing the striatum and the bed nucleus of the stria terminalis, respectively, was studied by means of in vitro microdialysis. A Ca(+2)-dependent and reserpine-sensitive K(+)-induced release of catecholamines was detected in both nuclei. We confirmed that N-methyl-D-aspartate (2.5 and 5.0 mM in the dialysis perfusion solution) induces the release of dopamine from the striatum, and this effect was blocked by prior dialysis perfusion with 500 microM MK-801, a noncompetitive N-methyl-D-aspartate receptor antagonist. Infusion of N-methyl-D-aspartate (1-10 mM) or glutamate through the dialysis probe did not produce any detectable modification in the extracellular levels of noradrenaline in the bed nucleus of the stria terminalis. In addition, perfusion with D-serine (100 microM) alone or in the presence of desipramine (10 microM), resulted in a slight increase in extracellular noradrenaline in the bed nucleus of the stria terminalis. However, N-methyl-D-aspartate in the presence of D-serine and desipramine produced a marked increase in extracellular noradrenaline from the bed nucleus of the stria terminalis. These results indicate that N-methyl-D-aspartate receptors might regulate the release of noradrenaline from the bed nucleus of the stria terminalis as is the case of dopamine release in the striatum. The in vitro microdialysis seems to be a suitable complement to the in vivo microdialysis for the study of catecholamine release in discrete regions of the central nervous system and its local regulation by excitatory amino acid receptors.

Some noradrenergic neurons of locus ceruleus-olfactory pathways contain neuropeptide-Y.

We report a specific tracing technique for studying projections of noradrenergic neurons that contain other transmitters. Autoradiography after retrograde axonal transport of tritiated-noradrenaline ([3H]NA) was combined with immunocytochemical detection of endogenous NA or neuropeptide Y (NPY). The specificity of [3H]NA retrograde labeling was dependent on the concentration of [3H]NA injected at the terminal region. At 2 x 10(-3) M, the specificity of [3H]NA retrograde labeling was confirmed by immunodetection of endogenous NA in radiolabeled cell bodies of the locus ceruleus. Combination of autoradiography of [3H]NA retrograde labeling with immunodetection of NPY demonstrated that a some scattered locus ceruleus noradrenergic neurons (about 26%) projecting to the olfactory bulb do contain NPY.

Regulation of [3H]norepinephrine release by N-methyl-D-aspartate receptors in minislices from the dentate gyrus and the CA1-CA3 area of the rat hippocampus.

It has been reported previously that N-methyl-D-aspartic acid induces a significant release of [3H]norepinephrine preaccumulated in slices from the hippocampus. In the present study, we investigated whether there are regional differences in the hippocampus regarding this N-methyl-D-aspartate effect. In the absence of Mg2+, N-methyl-D-aspartate (10-200 microM) induced the release of [3H]norepinephrine from superfused minislices containing the dentate gyrus area or the CA1-CA3 region of the hippocampus. Such N-methyl-D-aspartate effects on [3H]norepinephrine release were significantly higher in the dentate gyrus than in the CA1-CA3 area. The N-methyl-D-aspartate effects in both hippocampal areas were also reduced significantly by D-2-amino-5-phosphonovaleric acid (50 microM), an antagonist of the N-methyl-D-aspartate receptor, and by tetrodotoxin, a blocker of the voltage-dependent Na+ channels. The extent of this reduction was the same in the dentate gyrus and the CA1-CA3 area. Further experiments, conducted in the presence of Mg2+, demonstrated that N-methyl-D-aspartic acid increased K(+)-induced release of [3H]norepinephrine from dentate gyrus minislices but not from the CA1-CA3 area. The results are consistent with the existence of a higher density and/or different subtypes of N-methyl-D-aspartate receptors modulating [3H]norepinephrine release in the dentate gyrus as compared with the CA1-CA3 hippocampal area.

Studies of cholecystokinin in the rat bed nucleus of stria terminalis.

The release of cholecystokinin from the dorsal and ventral region of the rat bed nucleus of stria terminalis was studied. Minislices from both regions were superfused with Krebs-Ringer-phosphate, and the cholecystokinin released into the physiological medium was concentrated previous to radioimmunoassay determination. For this purpose, cholecystokinin was adsorbed onto a C18 reverse-phase column and eluted with acetonitrile. Cholecystokinin standards (10-50 pg) were subjected to the above procedure, which allowed a 20- to 50-fold concentration of the peptide with an 80% recovery. Potassium-induced release of cholecystokinin from minislices of dorsal and ventral regions of the bed nucleus of stria terminalis was measured successfully using the above procedure to concentrate the peptide. Lesion of the stria terminalis, a fiber tract originating in the amygdala, provoked a significant decrease in cholecystokinin levels in the ventral region of the bed nucleus of strial terminalis. Thus, cholecystokinin released from minislices of the ventral region of the stria terminalis may be of amygdaloid origin.

Differential effects of haloperidol on negative symptoms in drug-naive schizophrenic patients: effects on plasma homovanillic acid.

After 5 weeks of haloperidol, positive symptoms in drug-naive schizophrenic patients substantially subsided. Negative symptoms, although with a different temporal pattern, decreased after the fifth week of haloperidol treatment; specifically, a decrease was seen in anhedonia and affective flattening, whereas avolition-apathy and attentional impairment presented no changes. Alogia showed a decrease during the third week and a trend to return to placebo scores during weeks 4 and 5. Changes in affective flattening, alogia and attentional impairment correlated with changes in positive symptoms. During placebo, plasma homovanillic acid (HVA) correlated with negative symptoms and with changes presented by negative symptoms between the first and the fifth treatment week. These data show that negative symptoms respond differentially to neuroleptics and suggest that avolition-apathy may represent a different behavioral component of the schizophrenia process.