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Several findings have suggested the existence in the subventricular zone (SVZ) from sagittal sections of adult rat brain of a trophic mechanism, mediated by fibroblast growth factor-2 (FGF-2) and its multiple high-affinity FGF receptors (FGFRs), regulating neurogenesis mainly by controlling precursor cell proliferation. However, no clear data are available on the expression of FGF-2 and FGFRs in proliferating precursor cells of the SVZ. To address these questions we examined FGF-2 mRNA and its FGFR mRNA expression in proliferating precursor cells of the SVZ by using a double labeling procedure, combining in situ hybridization for FGF-2 and its FGFR mRNA with immunohistochemistry for bromodeoxyuridine (BrdU), a marker for proliferating cells. The results showed that FGFR1 and FGFR2 mRNAs were expressed in BrdU-labeled proliferating precursor cells, whereas FGFR3 and FGF-2 mRNAs were not, suggesting that in the SVZ the proliferating precursor cells express FGFR1 or FGFR2 and they may respond to FGF-2 released by non-proliferating cells. The FGFR4 mRNA was not found expressed in the SVZ. In the future, by identifying the cell types expressing FGFRs, it will be possible to gain insight into the functional activity of FGF2 within the SVZ.

The intercalated islands are intra-amigdaloid clusters of D1 receptor rich GABAergic neurons, which control impulse traffic between the basolateral complex and the central nucleus of the amygdala. As dopaminergic transmission within the amygdala may play a role in anxiety, the effect of the D1 antagonist SCH23390 microinjected mainly close to the rostral intercalated islands in rats was studied, using the White and Black Box test. SCH23390 reduced anxiety by an increase in the latency of the first entry into the black compartment and by an increase in the total time spent in the white compartment of the White and Black Box test, while there was no significant modification of locomotion. It is suggested that blockade of D1 receptors in the rostral intercalated islands may reduce anxiety through a reduction of GABA-mediated dishinibition of the central amygdaloid nucleus.

To understand the morphological substrate of the nicotine effect on nigral FGF-2 expression, a stereological analysis of FGF-2 immunoreactive neuronal and glial profiles has been performed in the substantia nigra of the rat after acute intermittent nicotine treatment. The major finding of this paper is the demonstration that this type of nicotine treatment produces a significant reduction in the total number of nuclear FGF-2 immunoreactive astroglial profiles in the substantia nigra. A parallel analysis of nigral FGF-1 and FGF-5 immunoreactivities showed no effect of this type of nicotine treatment. The results may be explained by an inhibition of FGF-2 synthesis in a subpopulation of nigral astroglia by nicotinic receptor activation, favouring a reduction of astroglial neurotrophism in the substantia nigra.

The kinetics of L-(-)-[N-propyl-(3)H(N)]-norapomorphine ([(3)H]NPA) interactions with rat striatal membranes were studied. The analysis revealed that in addition to specific dopaminergic binding a substantial part of the radioligand was bound irreversibly to heterogeneous populations of non-specific binding sites of these membranes. The specific binding of [(3)H]NPA with dopamine receptors, determined from the differences of kinetic curves of total and non-specific binding, was fast, reversible, and revealed high affinity. The irreversible component was heterogeneous and seems to be related to oxidative degradation of the radioligand, as the rate of this process was substantially reduced by antioxidants like ascorbic acid and dithiothreitol.

Interactions between subtypes of dopamine, glutamate and adenosine receptors seem to play an important integrative role in the function of striatal gamma-aminobutyric acid (GABA)ergic efferent neurons. Recent behavioral and biochemical studies suggest the existence of specific interactions between adenosine A2A receptors (A(2A)R), dopamine D2 receptors (D2R) and the group I metabotropic mGlu5 receptors (mGlu5R) in the dorsal striatum. The dual-probe approach in vivo microdialysis technique in freely moving rats was used to study the role of mGlu5R/A2AR/D2R interactions in the modulation of the ventral striopallidal GABA pathway. Perfusion of a selective mGlu5R agonist (CHPG) in the nucleus accumbens facilitated GABA release in the ipsilateral ventral pallidum. This effect was strongly potentiated by co-perfusion with the A2AR agonist CGS 21680. Co-perfusion with the D2R agonist quinpirole counteracted the increase in pallidal GABA levels induced by CGS 21680 and by CGS 21680 plus CHPG. These results demonstrate that mGlu5R/A2AR/D2R interactions play an important modulatory role in the function of the ventral striopallidal GABA pathway, which might have implications for the treatment of schizophrenia and drug addiction.

The Flinders Sensitive Line rats showed an increased immobility response by 104% (P<0.01) in the forced swim test with respect to the Flinders Resistant Line rats (control). The Flinders Sensitive Line rats also had an increase of [(125)I]galanin (porcine) binding in the dorsal raphe (55+/-3.3 fmol/mg protein) with respect to the Flinders Resistant Line rats (38+/-3.6 fmol/mg protein) (42%, P<0.05) without changes in galanin receptor affinity and with a significant reduction (32.3%, P<0.05) of galanin-like immunoreactivity in the dorsal raphe. The results indicate that enhancement of galanin receptor function in the dorsal raphe rich in 5-HT neurons could be a mechanism involved in the production of depressive-like activity in this animal model of depression.

The cross regulation of agonist binding to D2 dopamine receptors and guanosine nucleotide binding to G proteins was studied using membranes of Chinese hamster ovary cells expressing rat D2short dopamine receptors. All guanosine nucleotides studied caused a concentration-dependent loss of high-affinity agonist binding sites of D2 receptors with potencies corresponding to their affinity to bind to G proteins in these membranes. On the other hand, the dopaminergic agonists, but not antagonists, decreased the affinities of guanosine diphosphate and guanosine monophosphate, but not of guanosine 5'-(gamma-thiotriphosphate). The cross regulation of ligand binding to D2 dopamine receptors and G proteins suggests the existence of several conformational states of these proteins during the signal transduction and that the high-affinity state of agonist binding is a transient state of the agonist-receptor-G protein complex, where no nucleotides are bound.

Galanin (3 nmol/rat), 2 h after its intracerebroventricular (i.c.v.) administration to male rats, attenuated the passive avoidance (PA) retention deficit induced by the 5-hydroxytryptamine (HT)(1A) receptor agonist 8-hydroxy-2-(di-N-propylamino)tetraline (8-OH-DPAT) (0.2 mg/kg) The reduction in the postjunctional 5-HT(1A) receptor-mediated response after i.c.v. galanin was not associated with changes in the mRNA levels and agonist binding properties of cortical limbic 5-HT(1A) receptors, believed to be the target receptors mediating the PA deficit caused by 8-OH-DPAT. These results suggest that acute increases of galanin transmission in vivo even after 2 h can counteract limbic 5-HT(1A) receptor-mediated responses of relevance for affective disorders without significantly affecting gene expression and binding characteristics of cortical limbic 5-HT(1A) receptors.

Rat dopamine D(2short) expressed in Chinese hamster ovary (CHO) cells were characterized by means of activation of [(35)S]-guanosine 5'-O-(gamma-thiotriphosphate) ([(35)S]GTPgammaS) binding and inhibition of [(3)H]raclopride binding. Among 18 dopaminergic ligands studied dopamine, NPA, apomorphine and quinpirole were full agonists in activation of [(35)S]GTPgammaS binding, while seven ligands were partial agonists with efficacies from 16 to 69% of the effect of dopamine and seven ligands were antagonists having no effect on the basal level of [(35)S]GTPgammaS binding, but inhibited dopamine-dependent activation in a dose-response manner. Despite the different efficacies, the potencies of all 18 ligands to modulate [(35)S]GTPgammaS binding revealed a good correlation with their potencies to inhibit [(3)H]raclopride binding in the CHO cell membranes. This indicates that the binding of the ligand to the receptor determines its potency, but has no direct correlation with its intrinsic activity.

The effect of an acute administration of the vigilance-promoting drug modafinil ((+/-)(diphenyl-methyl)-sulfinyl-2 acetamide; Modiodal) on the nigrostriatal dopamine system was studied after damage induced by MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) by means of immunohistochemistry for tyrosine hydroxylase (TH) and a stereological method. MPTP (40 mg/kg) reduced from 24,380 +/- 902 to 13,501 +/- 522 and from 37,868 +/- 3300 to 20,568 +/- 1270, respectively, the number of TH immunoreactive (IR) and non-TH IR nigral neurons. Co-administration of Modafinil restored to normal the number of these neuronal populations. MPTP treatment induced also a reduction in the volume of TH IR neurons, which was counteracted by Modafinil administration. The data provide morphological evidence, based on unbiased stereological analysis, for a potential neuroprotective role of Modafinil, not only in dopaminergic neurons, but also with a similar magnitude in the non-DA nerve cell population of the substantia nigra after MPTP lesion. These results suggest that Modafinil has a neuroprotective role in the substantia nigra via a still undefined mechanism in which a crucial role of DA uptake blockade should be excluded. Modafinil may therefore have a therapeutic potential in neurodegenerative processes such as those occurring in Parkinson's disease.

The effect of a subchronic (4-week) exposure to low concentrations of toluene (40 or 80 parts per million, ppm) on the brain dopaminergic system has been examined in a rat model of Parkinson's disease. A unilateral lesion of the substantia nigra (SN) dopamine (DA) nerve cells was performed by injection of a low dose of 6-hydroxydopamine (6-OH DA). The peak activity of contralateral rotational behavior induced by apomorphine was significantly decreased after exposure to 80 ppm toluene. Analysis of the neostriatum and SN ipsilateral to the lesion revealed that toluene (80 ppm, but not 40 ppm) counteracted the 6-OH DA-induced reductions of DA tissue levels both within the SN and the neostriatum. Also the lesion-induced reduction of immunoreactivity for tyrosine hydroxylase (TH IR) in the neostriatum was partly counteracted by the toluene exposure (80 ppm). In conclusion, a subchronic exposure to low doses of toluene (80 ppm) leads to signs of reduced dysfunction of the nigrostriatal dopaminergic system after the neurotoxic treatment.

Previously, we observed specific alterations of neuropeptide Y (NPY) and Y1 receptor mRNA expression in discrete regions of the Flinders Sensitive Line rats (FSL), an animal model of depression. In order to clarify the correlation between mRNA expression and protein content, radioimmunoassay and receptor autoradiography were currently performed. In the FSL rats, NPY-like immunoreactivity (NPY-LI) was decreased in the hippocampal CA region, while Y1 binding sites were increased; NPY-LI was increased in the arcuate nucleus. Fluoxetine treatment elevated NPY-LI in the arcuate and anterior cingulate cortex and increased Y1 binding sites in the medial amygdala and occipital cortex in both strains. No differences were found regarding the Y2 binding sites. The results demonstrate a good correlation between NPY peptide and mRNA expression, and sustain the possible involvement of NPY and Y1 receptors in depression.

The effects of the vigilance promoting drug modafil were studied ex vivo (100 mg/kg; i.p.) and in vitro (10-1000 microM modafinil) on the synthesis of [3H]gamma-aminobutyric acid ([3H]GABA) and [3H]glutamate from [3H]glutamine within the rat hypothalamus. No effects of modafinil were observed on the overall synthesis of these neurotransmitters nor, in vitro (1-33 microM modafinil) on other parameters related to the compartmentalization of their synthesis (glutamate decarboxylase and phosphate-activated glutaminase activities, and [3H]glutamine uptake). It is suggested on these grounds, that the modafinil-induced reductions and increases in regional GABA and glutamate extracellular levels respectively using in vivo microdialysis may be a consequence of an indirect effect of modafinil on these neurons.

The effects of the anti-narcoleptic drug modafinil (30-300 mg/kg i.p.) on GABA and glutamate release were evaluated in the basal ganglia of the conscious rat, by using the microdialysis technique. Modafinil (100 mg/kg) inhibited striatal (85+/-4% of basal values) and pallidal (85+/-2%) GABA release without influencing local glutamate release. At the highest dose (300 mg/kg), modafinil induced a further reduction of pallidal (75+/-2%) but not striatal (82+/-7%) GABA release and increased striatal (134+/-11%) but not pallidal glutamate release. On the contrary, in the substantia nigra modafinil reduced GABA release only at the 300 mg/kg dose (59+/-5%) without affecting glutamate release. The preferential reduction in striato-pallidal GABA release at the 100 mg/kg dose of modafinil suggests that modafinil may be useful in the treatment of Parkinsonian diseases.

The motor effects of selective adenosine A1 and A2A receptor antagonists were tested in young (2 months) and aged (24 months) Wistar rats. In young rats, both the selective A2A receptor antagonist 5-amino-7-(2-phenylethyl)-2-2(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazo++ + lo[1,5-c]pyrimidine (SCH 58261, minimal effective dose 2 mg/kg intraperitoneally (i.p.)) and the selective A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT, minimal effective dose 1.2 mg/kg i.p.) stimulated motor activity. In old rats, both compounds induced significant motor activation starting from doses lower than those required in young animals. Specifically, the minimal effective doses of SCH 58261 and CPT in aged rats were 1 and 0.6 mg/kg i.p, respectively. The results indicate that both adenosine A1 and A2A receptors play a functional role in the control of motor activity, and, therefore, the blockade of both receptor subtypes is involved in the motor stimulating properties of methylxanthines. Also the evidence indicates, for the first time, that in aged animals the motor inhibitory adenosinergic tone seems to be increased with respect to young animals.

Alteration of ligand binding to dopamine D2 receptors through activation of adenosine A2A receptors in rat striatal membranes has been studied by means of kinetic analysis. The binding of dopaminergic agonist [3H]quinpirole to rat striatal membranes was characterized by the constants Kd = 1.50+/-0.09 nM and Bmax = 115+/-2 fmol/mg of protein. The kinetic analyses revealed that the binding had at least two consecutive and kinetically distinguishable steps, the fast equilibrium of complex formation between receptor and agonist (KA = 5.9+/-1.7 nM), followed by a slow isomerization equilibrium (Ki = 0.06). Activation of adenosine A2A receptors by CGS 21680 caused enhancement of the rate [3H]quinpirole binding, altering mainly the formation of the receptor-ligand complexes (KA) as well as the isomerization rate of this complexes (ki), while the deisomerization rate (k[-i]) and the apparent dissociation rate remained unchanged.

Difference in striatal dopamine D2 receptors and in spontaneous and theophylline-induced motor activity in rats withdrawn (for 72 h) from prolonged treatment with haloperidol or clozapine were studied. Haloperidol but not clozapine pretreatment increased the binding of the dopamine D2 antagonist [3H]raclopride in striatal membrane preparations, due to an increased number of striatal dopamine D2 receptors (Bmax) without changes in affinity (Kd). Haloperidol induced a significant increase (in motility and locomotion) and clozapine a decrease (in locomotion) of the spontaneous motor activity. Haloperidol but not clozapine pretreatment was associated with a stronger theophylline-induced motor activation. These results suggest that prolonged treatment with typical but not with atypical antipsychotics are associated with adaptive changes at both dopamine and adenosine receptors.

A G(i)-protein antibody AS/7 at 1:10 dilution significantly increased the K(d) values of the D2 agonist [3H]N-propylnorapomorphine (NPA) binding sites in the rat striatal membranes, and coincubation with sulphated cholecystokinin octapeptide (CCK-8; 1 nM) did not further increase the K(d) values. A GTP analogue guanylyl-imidodiphosphate (GMP-PNP) at 100 microM markedly increased the K(d) values of the [3H]NPA binding sites in the rat forebrain sections, and coincubation with CCK-8 (1 nM) again did not produce a further increase in the K(d) values. The present results indicate that abnormal activity of G-proteins abolished the ability of CCK-8 to reduce the D2 receptor affinity in the brain.

Glucocorticoids and dopamine (DA) may affect brain development and permanently programme central nervous system (CNS) responses. The ontogeny of the striatal glucocorticoid receptor (GR) mRNA and of DA D1, D2 and D3 receptor subtype mRNAs were, therefore, studied by means of in situ hybridization techniques. The expression of GR and the dopamine D3 receptor mRNAs but not of DA D1 and D2 receptor subtype mRNAs were observed in the striatal neuroepithelium during all prenatal stages studied (E14.5-E20.5). These results suggest that GR may directly influence striatal developmental processes, possibly involving the dopamine D3 receptor.

Double immunolabelling immunohistochemistry in the arcuate nucleus of the rat demonstrates that neuropeptide Y (NPY) Y1 receptor like immunoreactivity is strongly present in a subpopulation of beta-endorphin immunoreactive nerve cell bodies, while the small NPY immunoreactive nerve cell bodies located medially lack NPY Y1 receptor like immunoreactivity. The NPY Y1 like immunoreactive nerve cell bodies lie in an arcuate area rich in NPY immunoreactive nerve terminals forming an uniform plexus. It is postulated that NPY Y1 receptors in beta-endorphin neurons may mediate some actions of NPY on motivational processes and pain control as well as on hypophyseal hormone secretion, involving at the least in part a regulation of the tubero-infundibular DA neurons.

The effect of modafinil on endogenous gamma-aminobutyric acid (GABA) release in the medial preoptic area (MPA) and posterior hypothalamus (PH) and the role of local 5-HT3 receptors in this effect was investigated in the awake rat using in vivo microdialysis. Modafinil (30-100 mg/kg i.p.) dose-dependently decreased GABA release from the MPA, while only the 100 mg/kg dose markedly reduced GABA release in the PH. The modafinil (100 mg/kg) induced inhibition of GABA release in the MPA and the PH was partially counteracted by the 5-HT3 receptor antagonist MDL72222 (1 microM) when perfused locally alone or together with the non-selective 5-HT receptor antagonist methysergide (1 microM). Thus, the reduction of GABA transmission induced by modafinil in the MPA and in the PH, at least in part, involves local 5-HT3 receptors. The GABA release inhibition by modafinil in the above areas may be relevant for its vigilance promoting action.

An antagonistic interaction between adenosine A1 and dopamine D1 receptors has previously been found in the basal ganglia. However, direct evidence for a selective adenosine A1 antagonist-induced potentiation of dopamine D1-mediated motor activation is lacking. The systemic administration of the adenosine A1 antagonist 8-cyclopentyl-1,3-dimethylxanthine significantly potentiated the motor activating properties of the systemically administered dopamine D1 agonist SKF 38393 in both reserpinized mice and unilaterally 6-hydroxy-dopamine-lesioned rats. However, 8-cyclopentyl-1, 3-dimethylxanthine did not modify the motor effects of the dopamine D2 agonist quinpirole. The present work shows that an antagonistic interaction between adenosine A1 and dopamine D1 receptors may be involved in the motor activating effects of adenosine antagonists, like caffeine.

The potential neurotoxicity of the solvent toluene to the nigrostriatal dopaminergic system was assessed in a rat model of Parkinson's disease. Rats, 1 day after a unilateral injection of 6-hydroxydopamine (6-OH DA) into the substantia nigra, inhaled air or different concentrations of toluene (80, 300 or 1000 ppm), 6 h/day for 3 days. The animals were sacrificed 2 days after the last exposure and biochemical measurements of catecholamines and 3,4-dihydroxyphenylacetic acid (DOPAC) were performed in the neostriatum and substantia nigra. Toluene at 80 and 1000 ppm significantly enhanced the depletion of striatal DOPAC levels induced by the lesion and produced at 80 and 300 ppm a trend for intensifying the 6-OH DA-induced depletion of striatal DA stores. The alterations induced after the combined challenge to the dopaminergic nigrostriatal system may reflect endangering actions of toluene.

Excitotoxic lesion of the medial septum with ibotenic acid leading to partial disappearance of the septal cholinergic nerve cells was used to investigate the role of cholinergic mechanisms in the control of trophic factors for hippocampal plasticity, namely brain-derived neurotrophic factor (BDNF) and glucocorticoid receptor (GR). Their mRNA levels were tested by in situ hybridization 13 days after the lesion. A persistent and widespread increase of BDNF mRNA was found in all parts of the dorsal hippocampal formation that was not accompanied by a significant modification in GR expression. The present data suggest that subcortical excitotoxic lesions at the septal level have long-term consequences for the adaptive trophic responses occurring in the dorsal hippocampus.

Antagonistic interactions between adenosine A2a and dopamine D2 receptors and between adenosine A1 and dopamine D1 receptors have been previously found in the basal ganglia. Those interactions have been proposed to be key mechanisms of action responsible for the motor depressant effects of adenosine agonists and the motor activating effects of adenosine antagonists, like caffeine. By using quantitative receptor autoradiography, the selective adenosine A1 receptor agonist N6-cyclopentyladenosine was found to decrease the affinity of dopamine D1 receptors for the specific D1 antagonist [(125)I]SCH 23982 in both the nucleus accumbens and the medial prefrontal cortex of the rat brain. The present results suggest that dopamine neurotransmission, through an A1-D1 interaction, might also he involved in the behavioural effects of adenosine agonists and antagonists not related to motor activity, like the sedative-hypnogenic properties of adenosine analogues and the psychostimulant effects of caffeine.

Presynaptic receptors may reduce transmitter release with different mechanisms. Both the alpha 2-agonist, clonidine and the Y2-agonist, neuropeptide Y fragment 13-36 (NPY 13-36), induce a concentration-dependent inhibition of the 4-aminopyridine (4-AP)-evoked [3H]noradrenaline ([3H]NA) release from hypothalamic synaptosomes. Changes in alpha 2- and Y2-modulation of noradrenaline (NA) release were observed by lowering the calcium influx with the use of omega-conotoxin (omega-CgTx), a calcium-channel blocking agent. In these experimental conditions, clonidine was less active, whereas NPY 13-36 preserved its efficacy. It therefore seems possible that presynaptic alpha 2-adrenoceptors can primarily inhibit NA release by reducing calcium influx via voltage-sensitive calcium channels (VSCC), while Y2-receptors may inhibit the intracellular release process with a mechanism independent of the calcium entry.

The effect of striatal perfusion with the intestinal peptide PEC-60 on endogenous dopamine (DA) and gamma-aminobutyric acid (GABA) release in the dorsolateral striatum and GABA release in the globus pallidus was monitored using in vivo microdialysis in the halothane anaesthetized rat. The results show that PEC-60 (100 nM) increases DA release in the dorsolateral striatum without influencing GABA release in the dorsolateral striatum or in the globus pallidus. In addition, PEC-60 failed to influence the extracellular striatal 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels. The PEC-60 induced increase in striatal DA was abolished by the addition of tetrodotoxin (1 microM) to the perfusion medium. These data suggest that PEC-60 plays a role in modulating striatal DA release but not DA metabolism and that this effect is primarily targeted on the presynaptic DA terminals of the nigrostriatal DA pathway rather than on the postsynaptic striatopallidal GABA projection neurons in the dorsolateral striatum.

The effects of acute and repeated treatments with modafinil (30 mg/kg, s.c.) alone or after i.c.v. 6-hydroxy-dopamine injection were studied on cortical GABA release as well as on cortical/striatal catecholamine levels in awake freely moving guinea pig. The results show that repeated daily modafinil treatment produces a similar but short-lasting reduction of GABA outflow compared with acute administration. A significant reduction of cortical basal GABA outflow was observed in animals treated with 6-hydroxy-dopamine, which was maintained also after modafinil treatments. Furthermore, after the 6-hydroxy-dopamine treatment, modafinil fails to inhibit cortical GABA release. The depleting action of the toxin, 17% reduction of neostriatal dopamine levels and 35% of noradrenaline levels in the parietal cortex, was not influenced by repeated modafinil treatment. The catecholaminergic telencephalic networks therefore seem essential for the elicitation of the inhibitory effects of modafinil on GABA release.

The cardiovascular effects of adrenaline microinjected alone or together with neuropeptide Y (NPY) receptor agonists into the nucleus tractus solitarius (Sol) of the anaesthetized rat have been investigated in order to evaluate NPY/adrenergic receptor interactions. In the dose range 0.05-20 nmol, adrenaline microinjected unilaterally into the Sol produced significant dose-related reductions in mean arterial blood pressure and heart rate. The vasodepressor action of a close to ED50 dose of adrenaline (0.5 nmol) was significantly counteracted by a threshold dose of NPY (1-36) (1 pmol) and of the NPY Y1 receptor agonist [Leu31,Pro34]NPY (2.5 pmol) microinjected into the Sol, but not by a threshold dose of NPY(13-36)(50 fmol), a selective Y2 receptor agonist. The present study provides evidence for an antagonistic NPY Y1/adrenergic receptor interaction in the Sol of the rat, involved in cardiovascular regulation.

Using solution and in situ hybridization techniques we have studied the effects of adrenalectomy with or without restitution therapy with corticosterone on galanin mRNA levels in discrete regions of the male rat brain. Galanin peptide levels were also measured using a radioimmunoassay. The solution hybridization showed a two-fold increase in galanin mRNA 7 days, but not 14 days, after adrenalectomy in the preoptic area including the hypothalamic paraventricular nucleus (PVN). No changes were observed in the mediobasal hypothalamus including the arcuate nucleus. In situ hybridization showed that the increase in galanin mRNA was localized to the PVN and that the arcuate nucleus was not affected. The changes observed could be fully counteracted by corticosterone treatment. Radioimmunoassay showed decreased galanin levels in the median eminence 14 days, but not 7 days, after adrenalectomy and increased levels in the anterior pituitary and neurointermediate lobe. The results give evidence for a regional regulation of galanin gene expression and galanin peptide synthesis by adrenocortical steroids.

By means of a highly specific hyaluronic acid-recognizing protein the localization and regional distribution of hyaluronic acid was demonstrated in the tel- and diencephalon and in the midbrain of the adult rat nervous system. Histochemistry revealed labeling associated with the plasma membrane in highly discrete nerve cell bodies of the frontoparietal cortex, the red nucleus, the zona reticulata of the substantia nigra, the oculomotor nucleus and the reticular thalamic nucleus. A strong labeling without association with perikarya was demonstrated in the subgranular zone of the dentate gyrus of the hippocampal formation. The present results open up the possibility that the hyaluronic acid found in high concentrations associated with some perikarya may have a special role in plasticity responses in these discrete nerve cell populations.

Using angiotensin II (ANG II) to compete with (3-[125I]iodotyrosyl-4, Sar1, Ile8)ANG-II ([125I]Sar1, Ile8)ANG II) for its binding sites in the nucleus of the solitary tract (nTS) and the paraventricular hypothalamic nucleus (PV) bradykinin (10 nM) reduced the IC50 value (48 nM) of ANG II, an action blocked by the bradykinin B2 antagonist HOE-140 (100 nM). In contrast, when analysing the high-affinity site (Kd 3.1 nM) for [125I]ANG II in the nTS bradykinin (10 nM) increased the Kd value. Thus, a central bradykinin/ANG II receptor interaction may exist involving a differential regulation of the high- and low-affinity ANG II receptors in the nTS. This regulation by bradykinin of angiotensin receptors in the nTS may help to explain the central vasopressor effect of bradykinin.

By means of two colour immunofluorescence procedures it has been possible to demonstrate in the rat brain the coexistence of TH and bFGF immunoreactivities (IRs) in the perikarya of large numbers of noradrenaline (NA) nerve cells of the locus coeruleus and of the NA cell groups A1, A5 and A7 and in many perikarya of the adrenaline (A) cell groups C1, C2 and C3. The coexistence of 5-hydroxytryptamine (5-HT) and bFGF IRs was demonstrated in the perikarya of large numbers of 5-HT nerve cells of practically all raphe nuclei. These results open up the possibility that bFGF may have a trophic role in the NA, A and 5-HT cell groups of the rat brain.

In the concentration range of 1-10 nM, neuromedin N produced a significant concentration-related increase in the Kd values of [3H]L-(-)-N-propylnorapomorphine binding sites in rat neostriatal membranes with a peak action at 10 nM (36% increase versus the control group mean value). The Bmax values were not affected by neuromedin N. Neurotensin at 10 nM induced an increase in the Kd values, which was not affected by a threshold concentration of neuromedin N (0.1 nM). In view of the higher potency of neuromedin N versus neurotensin to modulate neostriatal D2 receptors in contrast to the higher potency of neurotensin versus neuromedin N to bind to the cloned neurotensin receptors, it seems possible that the neuromedin N activated neostriatal neurotensin receptors controlling the D2 receptors represent a distinct subtype of neurotensin receptors.

The effects of NPY(13-36) on cardiorespiratory responses elicited by microinjections of L-glutamate into the nucleus tractus solitarius (NTS) have been studied in anaesthetized (alpha-chloralose + urethane) Sprague-Dawley rats. NPY(13-36) in doses ranging from 50 to 500 fmol produces a significant increase in mean arterial pressure (MAP) without significant effects on heart rate (HR) and respiratory rate (RR). The ED50 dose (100 fmol) of NPY(13-36) counteracts significantly the decrease in MAP and HR elicited by L-glutamate (1500 pmol) microinjected into NTS. Similar results are obtained using a threshold dose of the peptide (50 fmol) and an ED50 dose of L-glutamate (300 pmol). These results indicate that the Y2 receptors in NTS can mediate vasopressor responses to femtomolar amounts of NPY(13-36) and counteract cardiovascular responses to L-glutamate.

The cellular localization of angiotensin type 1 receptor (AT 1) and angiotensinogen mRNA expression in the subfornical organ (SFO) of the rat brain has been studied by means of non-radioactive in situ hybridization combined with immunocytochemistry for glial fibrillary acidic protein (GFAP) and Neutral red staining. The AT 1 receptor mRNA expression is shown to be within putative nerve cells without any association with the glial fibrillary acidic protein (GFAP)-immunoreactive (IR) cells. In contrast the angiotensinogen cRNA expression is associated predominantly with GFAP-IR cells. The results demonstrate that a neuronal AT 1 receptor mediates the actions of circulating angiotensin II on the SFO and that the angiotensinogen mRNA is predominantly expressed in the SFO astroglial cells.

By means of triple immunofluorescence procedures the codistribution and coexistence of glucocorticoid receptor (GR), c-Fos and corticotropin-releasing factor (CRF) immunoreactive (IR) nerve cell profiles have been analyzed within the paraventricular hypothalamic nucleus at various rostrocaudal levels following acute immobilization stress (2 h) in the male rat. One population of CRF IR neurons is shown to contain both nuclear GR and c-Fos immunoreactivities following the stress, while another contains GR immunoreactivity alone. These results indicate heterogeneities among parvocellular CRF paraventricular hypothalamic nerve cell populations in the response to acute immobilization stress.

The present study demonstrates the existence and regional distribution of angiotensin II AT1 receptor subtype mRNA expression in the rat brain by the use of in situ hybridization and RNase protection assay. Substantial expression levels in the brain have only been detected in certain distinct areas, such as the subfornical organ, the parvocellular part of the paraventricular hypothalamic nucleus, and the median preoptic nucleus. The results give further evidence for the involvement of the angiotensin II AT1 receptor subtype in the classical functions of central angiotensin II, like blood pressure control, body fluid homeostasis and in corticotropin-releasing factor (CRF) secretion.

The effects of intracerebroventricular (i.v.t.) injections of pertussis toxin (PTX) (10 micrograms/30 microliters, 48 h) were studied on the cardiovascular actions of i.v.t. administered neuropeptide Y(13-36) (NPY(13-36)) as evaluated in the awake unrestrained male rat. The vasopressor action of NPY(13-36) in the peak dose of 3000 pmol per rat was significantly inhibited by pretreatment by PTX. Pertussis toxin treatment alone significantly reduced the baroreceptor reflex elicited by L-phenylephrine. The results are compatible with the view that G-proteins mediate the central vasopressor actions of NPY(13-36) and thus probably are involved in NPY Y2-receptor transduction in cardiovascular areas of the brainstem.

Spontaneous and electrically evoked endogenous acetylcholine release and [3H]-choline efflux from slices of guinea pig nucleus basalis magnocellularis (nbM) were studied. Tetrodotoxin reduced the spontaneous endogenous release by 55%, while the Ca(2+)-free medium reduced it by about 30%. Evoked [3H]-choline efflux was Na+ and Ca2+ dependent and frequency related. Physostigmine, 30 microM, nearly halved the stimulation-evoked efflux; atropine, 0.15 microM, not only antagonized, but even reversed this effect into facilitation. Pirenzepine, 1 microM, and AFDX 116, 1 microM, were less effective than atropine, and reversed the inhibitory effect of physostigmine only when applied together. 4-DAMP, 0.01 microM, was ineffective. These findings indicate that acetylcholine release in guinea pig nbM slices is inhibited by the cooperation of muscarinic autoreceptors, possibly belonging to the M1 and M2 subclasses.

The effects of glucocorticoids on monosodium glutamate-induced neurotoxicity in the neonatal basal hypothalamus were studied by means of semiquantitative immunocytochemistry for tyrosine hydroxylase, growth hormone releasing factor and luteinizing hormone releasing hormone. Neonatal monosodium glutamate treatment induced a marked decrease in tyrosine hydroxylase immunoreactive neurons in the arcuate nucleus and growth hormone releasing factor immunoreactive nerve terminals in the median eminence. These effects were significantly antagonized by the coadministration of corticosterone. Corticosterone alone had no effect on the parameters studied. No significant change in luteinizing hormone releasing hormone immunoreactivity in the median eminence was detected after any treatment. These results demonstrate that corticosterone, possibly acting via type II corticosterone receptors which are highly enriched in the arcuate neurons, can exert a protective action on glutamate-induced neurotoxicity.

The central administration of the adenosine A2 agonist CGS 21680 induced catalepsy in the rat. This effect was counteracted by the previous systemic administration of the adenosine antagonist theophylline or the D2 agonist BHT-920. These results are in agreement with the view that adenosine A2 receptors regulate central dopamine D2 transmission and underline the potential antipsychotic activity of A2 agonists.

Using a rabbit endothelin-1 (ET-1) antiserum together with a goat-anticholineacetylase antiserum or a mouse anti-somatostatin antiserum it was possible by means of double immunolabelling procedures to demonstrate ET-like immunoreactivity in striatal nerve cell bodies of the rat, which were shown to contain either cholineacetylase or somatostatin immunoreactivity. Absorption studies with ET-3, ET-1 or big ET-1 indicated that the ET-like immunoreactivity was ET-3 like. In agreement the radioimmunoassay showed that ET-3-like immunoreactivity was present in higher concentrations than ET-1-like immunoreactivity in the neostriatum and other brain areas. Characterization by reversed phase HPLC revealed, however, that a major portion of the neostriatal ET-3-like immunoreactivity was not identical to ET-3. The nature of neuronal ET in the rat may thus be more complicated than hitherto assumed.

The effects of galanin (GAL) have been evaluated on the depolarization evoked release of [3H]5-HT (serotonin or 5-hydroxytryptamine) from rat hypothalamic synaptosomal preparations, using low concentrations of potassium (15 mM). In the same preparation effects of GAL were also evaluated on [3H]5-HT uptake, using kinetic analysis to determine effects on Vmax values and Km values. GAL concentrations of 0.1-10 nM caused a concentration-related increase of the depolarization-evoked release of [3H]5-HT without influencing [3H]5-HT uptake. The results indicate the existence of high affinity GAL receptors on the hypothalamic 5-HT nerve terminals, exerting a facilitatory influence on depolarization-evoked [3H]5-HT release.

The effect of alpha-difluoromethylornithine (alpha-DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC) the rate limiting enzyme of polyamine biosynthesis, was studied on the astroglial reaction in a model of mechanical brain injury. alpha-DFMO markedly decreased the astroglial activation induced by the microdialysis probe implantation in the striatum of the male rat, as studied by glial fibrillary acidic protein (GFAP) immunocytochemistry. This response was restored by putrescine (20 nmol/ml) administered via the microdialysis probe. These results suggest that the astroglial reaction and the polyamine biosynthesis activation induced by a localized mechanical lesion are causally linked phenomena.

Intracerebroventricular injections (140-4300 pmol) of a 60-residue polypeptide (now designated PEC-60) isolated from pig intestine produced a dose-dependent increase (peak action 21% +/- 2; ED50 value of 1.7 nmol for the peak effect) in mean arterial blood pressure (MAP) in the awake, unrestrained male rat. The heart rate (HR) was significantly reduced with the highest doses used (1400 and 4300 pmol). The pancreatic secretory trypsin inhibitor (PSTI), with a high degree of sequence similarity to PEC-60, did not significantly change MAP and HR (4300 pmol). These results may indicate a biological role for the putative PEC-60-like peptide demonstrated within central catecholamine cardiovascular neurons.

Intraventricular injections of the Y2 neuropeptide Y (NPY) receptor agonist porcine NPY (13-36) (pNPY (13-36); 25-3000 pmol) produced a dose-dependent increase (up to 14%; ED50 value of 0.3 nmol for overall effects and 0.97 nmol for the peak effects) in mean arterial blood pressure in the awake, unrestrained male rat without affecting heart rate. Furthermore, a subthreshold dose of pNPY (13-36) (25 pmol) counteracted the vasodepressor action of the parent compound pNPY (1-36) (75 pmol), which also acts at NPY receptors of the Y1 type. These results suggest that NPY receptors of the Y1 and Y2 type have opposing actions in central cardiovascular regulation.

By using double immunolabelling techniques the possible coexistence of tyrosine hydroxylase (TH)- and angiotensin II (ANG II)-like immunoreactivities (IR) has been studied within the noradrenaline A1 and adrenaline C1 areas of the ventral medulla of the rat. In the A1 area 20-30% of the TH IR nerve cell bodies were ANG II IR and in the C1 area 5-40% of the TH IR nerve cell bodies displayed ANG II IR. These results suggest the existence of ANG II/NA and ANG II/A costoring neurons in the A1 and C1 group, respectively, opening new possibilities for ANG II-catecholamine interactions in cardiovascular and neuroendocrine regulation.

The cellular localization and the distribution of the mas proto-oncogene/angiotensin receptor mRNA have been studied in the male rat brain using in situ hybridization with radiolabelled mas cRNA probes. Neuronal cell populations in the forebrain were selectively labelled. A strong specific labelling was demonstrated in the dentate gyrus, the CA3 and CA4 areas of the hippocampus, the olfactory tubercle (medical part), the piriform cortex and the olfactory bulb, while a weak to moderate labelling was present all over the neocortex and especially in the frontal lobe.

The effects of neurotensin in vitro were investigated on the binding characteristics of N-[3H]propylnorapomorphine [( 3H]NPA) binding sites in crude membrane preparations from post mortem human caudate-putamen, obtained within 10 h after death from 3 patients without reported neurological or psychiatric diseases. Neurotensin (0.1-30 nM) was found to increase the KD value of [3H]NPA binding sites with a maximal increase of about 50% (420 +/- 45 pM) at 3 nM of neurotensin, without significantly affecting the Bmax value (123 +/- 20 pmol/g protein). These results indicate the existence of an intramembrane regulation by neurotensin receptors of D2 receptors in the human caudate-putamen.