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The dopaminergic system integrated by cell groups distributed in several brain regions exerts a modulatory role in brain. Particularly important for this task are the mesencephalic dopamine neurons, which from the substantia nigra and ventral tegmental area project to the dorsal striatum and the cortical/subcortical limbic systems, respectively. Dopamine released from these neurons operates mainly via the short distance extrasynaptic volume transmission and activates five different dopaminergic receptor subtypes modulating synaptic GABA and glutamate transmission. To accomplish this task dopaminergic neurons keep mutual modulating interactions with neurons of other neurotransmitter systems, including allosteric receptor-receptor interactions in heteroreceptor complexes. As a result of its modulatory role dopaminergic mechanisms are involved in either the etiology or physiopathology of many brain diseases such as Parkinsońs disease and schizophrenia. The aim of this work is to review some novel and conventional approaches that either have been used or are currently employed to treat these diseases. Particular attention is paid to the approaches derived from the knowledge recently acquired in the realm of receptor-receptor interactions taking place through multiple dopamine heteroreceptor complexes in the plasma membrane. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries., (Copyright © 2019. Published by Elsevier Ltd.)

Preclinical data indicate that ethanol produces behavioral effects that can be regulated by many neurotransmitters and neuromodulators like adenosine (A). The most important receptors with respect to the rewarding effects of ethanol seem to be the A 2A receptors. This study used a transgenic strategy, specifically rats overexpressing the A 2A receptor, to characterize the neurobiological mechanisms of ethanol consumption as measured by intermittent access to 20% ethanol in a two-bottle choice paradigm. In this model, no change in ethanol consumption was observed in transgenic animals compared to wild type controls during the acquisition/maintenance phase. Following alcohol deprivation, only transgenic rats overexpressing the A 2A receptor exhibited escalation of ethanol consumption and drank more (by ca. 90%), but not significantly, ethanol than did the wild type rats. During ethanol withdrawal, the immobility time of rats overexpressing the A 2A receptor in the forced swim test was lower than that of wild type rats. Moreover, transgenic rats withdrawn from ethanol, compared to the drug-naive transgenic animals, exhibited an increase above 70% in locomotion. The results indicated that the overexpression of A 2A receptors may be a risk factor for the escalation of ethanol consumption despite the reduction in depression-like signs of ethanol withdrawal., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

Several clinical observations have demonstrated a link between heart rate and anxiety or panic disorders. In these patients, β-adrenergic receptor function was altered. This prompted us to investigate whether the β-adrenergic receptor agonist isoproterenol, at a dose that stimulates peripheral β-adrenergic system but has no effects at the central nervous system, can induce anxiety-like behavior in rats. Moreover, some possible messengers involved in the peripheral to brain communication were investigated. Our results showed that isoproterenol (5 mg kg(-1) i.p.) increased heart rate, evoked anxiety-like behavior, did not result in motor impairments and increased extracellular vesicle content in the blood. Plasma corticosterone level was unmodified as well as vesicular Hsp70 content. Vesicular miR-208 was also unmodified indicating a source of increased extracellular vesicles different from cardiomyocytes. We can hypothesize that peripheral extracellular vesicles might contribute to the β-adrenergic receptor-evoked anxiety-like behavior, acting as peripheral signals in modulating the mental state., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The anterior and rostral paracapsular intercalated islands (AIC and PIC, respectively) were studied in the context of the amygdaloid modulation of fear/anxiety using horizontal sections. The structural analysis carried out using silver-impregnated specimens revealed that the AIC is composed of tightly packed, medium-sized spiny neurons with distinct dendritic and axonal patterns that send projecting axons to the central nucleus of the amygdala. The AIC occupies a strategic position between the basolateral amygdaloid complex and the caudal limb of the anterior commissure from which it receives fibers en passage and axon terminals. Electron microscopic observation of terminal (i.e., synaptic) degeneration 72 h after the surgical interruption of the anterior commissure, confirms the synaptic interaction between the latter and the AIC neurons. These observations suggest that these islands may gate the activity of neurons from the contralateral basal forebrain and synchronize the anxiogenic output of both amygdalae. Immunohistochemical analysis indicated that, within the AIC and rostral PIC, the distance between tyrosine hydroxylase-immunoreactive terminals and the punctate dopamine D(1) receptor immunoreactivity, was in the micrometer range. These results indicate a short distance and a rapid extrasynaptic form of dopamine volume transmission mediated via D(1) receptors in the AIC and PIC which may enhance fear and anxiety by suppressing feed-forward inhibition in the basolateral and central amygdaloid nuclei. The strong suggestion for a commissural axon projection to the AIC documented here, coupled with the previous evidences indicting an isocortical and amygdalar contributions to the anterior commissure, opens the possibility that the AIC may be involved in decoding nerve impulses arising from both the ipsi- and contra-lateral forebrain to, in turn, modulate the homolateral amygdala., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Amygdaloid dopamine D(2) receptors play an important role in the modulation of fear/anxiety. Their topographical distribution within the amygdala is however unclear, and their role in unconditioned fear/anxiety remains largely unknown. The aim of this paper was to study the intra-amygdaloid distribution of D(2) receptors and to ascertain their role in unconditioned anxiety. Chemical anatomical studies in the rat, using D(2) and D(3)in situ hybridization, quantitative receptor autoradiography with either [(3)H]raclopride or [(125)I]sulpiride, and D(2)-like immunocytochemistry showed that the highest density of dopamine D(2) receptors is present in the central amygdaloid nucleus, particularly within its latero-capsular division, in which a D(2) but not a D(3) mRNA signal was observed. However, although at considerably reduced densities dopamine D(2) receptors were also found in other locations within the amygdala, including the basolateral nucleus. Behaviorally, the infusion of raclopride (0.75-4 μg/side) in the area of the central amygdaloid nucleus resulted at low doses in the appearance of anxiogenic-like effects in the Shock-Probe Burying test, whereas no effects of raclopride treatment were found at any dose in the Elevated Plus-Maze and the Open-Field test. Our results indicate that amygdaloid dopamine D(2)-like receptors have a topographically differentiated distribution within the rat amygdala, the major location being in the central amygdaloid nucleus. D(2)-like receptors play a role in the modulation of anxiety responses involving a potential differential function of D(2)-like receptors in the central amygdaloid nucleus versus the basolateral amygdaloid nucleus., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

The recently cloned histamine H(4) receptor is expressed predominantly in haematopoietic cells and has been found to modulate the function of mast cells, eosinophils, dendritic cells and T lymphocytes. It represents an attractive target for pharmacological interventions against a number of inflammatory and autoimmune disorders. In the present work we used two-electrode voltage-clamp to demonstrate histamine H(4) receptor modulation of G protein-coupled inward rectifier potassium (GIRK) channels heterologously expressed in Xenopus oocytes. In accordance with earlier findings in other effector systems, full agonism by histamine and (R)-alpha-methylhistamine, partial agonism by clobenpropit and inverse agonism by thioperamide were observed. Furthermore, in oocytes injected with low amounts of receptor cRNA, clobenpropit apparently acted as a neutral antagonist. We also used the high temporal resolution afforded by this system to study the differential time courses of response deactivation upon ligand washout for clobenpropit and (R)-alpha-methylhistamine. GIRK channels represent a novel effector system for histamine H(4) receptor modulation, which may be of physiological relevance and prove useful in the development of compounds targeting this receptor.

The role of the dopamine D(4) receptor in cognitive processes and its association with several neuropsychiatric disorders have been related to its preferential localization in the cerebral cortex. In the present work we have studied in detail the regional and cellular localization of the dopamine D(4) receptor immunoreactivity (IR) in the rat cerebral cortex and its relationship to the dopaminergic and noradrenergic nerve terminal networks, since both dopamine and noradrenaline have a high affinity for this receptor. High levels of D(4) IR were found in motor, somatosensory, visual, auditory, temporal association, cingulate, retrosplenial and granular insular cortices, whereas agranular insular, piriform, perirhinal and entorhinal cortices showed low levels. D(4) IR was present in both pyramidal and non-pyramidal like neurons, with the receptor being mainly concentrated to layers II/III. Layer I was observed to be exclusively enriched in D(4) IR branches of apical dendrites. Finally, mismatches were observed between D(4) IR and tyrosine hydroxylase and dopamine beta-hydroxylase IR nerve terminal plexuses, indicating that these receptors may be activated at least in part by dopamine and noradrenaline operating as volume transmission signals. The present findings support a major role of the dopamine D(4) receptor in mediating the transmission of cortical dopamine and noradrenaline nerve terminal plexuses.

The dopamine D1 and D2 receptors are major receptors in the regulation of striatal function and striatal adenosine A1 and A2A receptors are major modulators of their signaling. The evidence suggests the existence of antagonistic A1-D1 heteromeric receptor complexes in the basal ganglia and prefrontal cortex and especially in the direct striatonigral-striatoentopeduncular GABA pathways. The neurochemical and behavioral findings showing antagonistic A1-D1 receptor interactions can be explained by the existence of such A1-D1 heteromeric receptor complexes and of antagonistic interactions at the level of the second messengers. In contrast, A2A-D2 receptor heteromers may exist in the dorsal and ventral striato-pallidal GABA pathways, where activation of A2A receptors reduces D2 receptor recognition, coupling and signaling. As a result of the A2A receptor-induced reduction of D2 receptor signaling, the activity of these GABA neurons is increased resulting in reduced motor and reward functions mediated via the indirect pathway, causing a reduced glutamate drive to the prefrontal and motor areas of the cerebral cortex. Thus, A2A receptor antagonists and A2A receptor agonists, respectively, may offer novel treatments of Parkinson's disease (reduced D2 receptor signaling) and of schizophrenia and drug addiction (increased D2 receptor signaling).

The mammalian corticospinal tract is known to contain axons that travel from the cerebral cortex to various levels of the spinal cord and its main function is thought to be the mediation of voluntary movement. The current study describes neuroanatomy related to the corticospinal tract of the giraffe. This animal presents a specific morphology that may present challenges to this neural pathway in terms of the metabolism required for correct functioning and maintenance of potentially very long axons. The spinal cord of the giraffe can be up to 2.6 m long and forms the conus medullaris at the level of the sacral vertebrae. Primary motor cortex was found in a location typical of that of other ungulates, and the cytoarchitectonic appearance of this cortical area was similar to that previously reported for sheep, despite the potential distance that the axons emanating from the layer 5 gigantopyramidal neurons must travel. A typically mammalian dorsal striatopallidal complex was transected by a strongly coalesced internal capsule passing through to the pons and forming clearly identifiable but somewhat flattened (in a dorsoventral plane) pyramidal tracts. These tracts terminated in a spinal cord that exhibited no unique anatomical features related to its length. Our results, at least at the level of organization investigated herein, show that the corticospinal tract of the giraffe resembled that of a typical ungulate.

The histamine H(3) receptor mediates inhibitory responses in the nervous system. Here, we demonstrate the coupling of the human histamine H(3) receptor to G protein-coupled inward rectifier potassium (GIRK) channels in Xenopus oocytes, using voltage-clamp. The histamine H(3) receptor agonist (R)-alpha-methylhistamine increased GIRK currents with an EC(50) of 2.5 nM. The response to (R)-alpha-methylhistamine was inhibited by the specific antagonist/inverse agonist clobenpropit. GIRK channels represent a novel effector pathway for the histamine H(3) receptor, also suggesting the use of electrophysiology assays in histamine H(3) receptor drug screening, allowing for the resolution of G protein activation kinetics.

Over the past years, evidence has accumulated that stem cells are present in the adult brain, and generate neurons and/or glia from two active germinal zones: the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus of the hippocampus. This study shows that acute intermittent nicotine treatment significantly enhances neuronal precursor cell proliferation in the SVZ of adult rat brain, but not in the SGZ of the hippocampus, and pre-treatment with mecamylamine, a nonselective nAChR antagonist, blocks the enhanced precursor proliferation by nicotine. This effect is supported by up-regulation of fibroblast growth factor-2 (FGF-2) mRNA in the SVZ and the expression of its receptor FGFR-1 in cells of SVZ showing precursor cells profile. It is also demonstrated that the nicotine effect on neuronal precursor proliferation is mediated by FGF-2 via fibroblast growth factor receptor 1 (FGFR-1) activation by showing that i.c.v. pre-treatment with anti-FGF-2 antibodies or with FGFR-1 inhibitor 3-[(3-(2-carboxyethyl)-4-methylpyrrol-2-yl)methylene]-2-indolinone (SU5402) blocks nicotine-induced precursor cell proliferation. This nicotine enhancement of neuronal precursor cell proliferation was not accompanied by an increase in the number of apoptotic cells. Taken together the present findings revealed the existence in the SVZ of the adult rat brain of a trophic mechanism mediated by FGF-2 and its receptor and regulated by nAchR activation. This possibility of in vivo regulation of neurogenesis in the adult brain by exogenous factors may aid to develop treatments stimulating neurogenesis with potential therapeutic implications.

Mismatches between dopamine innervation and dopamine D1 receptor (D1) distribution have previously been demonstrated in the intercalated cell masses of the rat amygdala. Here the distribution of enkephalin and beta-endorphin immunoreactive (IR) nerve terminals with respect to their mu-opioid receptors is examined in the intercalated cell masses, along with a further immunohistochemical analysis of the dopamine/D1 mismatches. A similar analysis is also made within the extended amygdala. A spatial mismatch in distribution patterns was found between the mu-opioid receptor-1 immunoreactivity and enkephalin IR in the main intercalated island of the amygdala. Discrete cell patches of dopamine D1 receptor and mu-opioid receptor-1 IR were also identified in a distinct region of the extended amygdala, the interstitial nucleus of the posterior limb of the anterior commissure, medial division (IPACM), which displayed sparse tyrosine hydroxylase or enkephalin/beta-endorphin IR nerve terminals. Furthermore, distinct regions of the main intercalated island that showed dopamine/D1 receptor matches (the rostral and rostrolateral parts) were associated with strong dopamine and cyclic AMP regulated phosphoprotein, 32 kDa-IR in several D1 IR neuronal cell bodies and dendrites, whereas this was not the case for the dopamine/D1 mismatch areas (the rostromedial and caudal parts) of the main intercalated island. The lack of correlation between the terminal/receptor distribution patterns suggests a role for volume transmission for mu-opioid receptor- and dopamine D1 receptor-mediated transmission in distinct regions of the amygdala and extended amygdala. This may have implications for amygdaloid function, where slow long lasting responses may develop as a result of volume transmission operating in opioid peptide and dopaminergic communication.

Uncoupling proteins in the inner mitochondrial membrane uncouples oxidative phosphorylation from ATP synthesis. It has been suggested that these proteins are involved in thermogenesis as well as in the regulation of reactive oxygen species production in the mitochondria. The present work was conducted to investigate the localization of the uncoupling protein 2-like immunoreactivity (uncoupling protein 2/3 immunoreactivity) in the main catecholaminergic projection fields in the rat brain as well as in the areas of the dopaminergic and noradrenergic nerve cell groups. In particular, the relationships of tyrosine hydroxylase, dopamine beta-hydroxylase and uncoupling protein 2/3 immunoreactivity were assessed by double immunolabeling and confocal laser microscopy analysis associated with computer-assisted image analysis. Uncoupling protein 2/3 immunoreactivity was observed in discrete dopaminergic terminals in the nucleus accumbens and in the cerebral cortex whereas it was found in scattered noradrenergic terminals in the caudate putamen and Islands of Calleja Magna. One interesting finding was that uncoupling protein 2/3 immunoreactivity together with tyrosine hydroxylase immunoreactivity in the shell of nucleus accumbens was observed surrounding the previously characterized D1 receptor rich nerve cell column system characterized by a relative lack of tyrosine hydroxylase immunoreactivity. Moreover, in animal models of dopaminergic pathway degeneration, plastic changes in uncoupling protein 2/3 terminals have been shown in the cerebral cortex and striatum as seen from the increased size and intensity of uncoupling protein 2/3 immunoreactivity of their varicosities. Taken together, these findings open up the possibility that uncoupling protein 2/3 could play an important role modulating the dopaminergic and noradrenergic neurotransmission within discrete brain regions.

Several studies in different in vitro and in vivo models have demonstrated neuroprotective effects of nicotinic receptor agonists and indirect trophic actions of nicotine on brain are suggested from observations describing nicotine as a cognitive enhancer by increasing vigilance and improving learning and memory. While an increasing number of studies have given evidence of neuroprotective and neurotrophic effects of nicotine treatment, the molecular mechanism mediating the neurotrophic effects of nicotine are not fully understood. Previously in an analysis of several neurotrophic factors as possible mediators of nicotine-induced neuroprotection and/or neurotrophic effects we could reveal that an acute intermittent nicotine treatment increases fibroblast growth factor-2 mRNA and protein in several brain regions of rat brain. Even if other studies have demonstrated in different paradigms that nicotine administration modulates expression level of a variety of genes, there is still a lack of indication which candidate genes, involved in neuroprotective responses are modulated by nicotine. In the present work we have used a microarray assay to further find and characterize new genes responsive to acute intermittent nicotine treatment and linked to neuroprotection. Therefore, we used Rat Genome U34A Affymetrix GeneChip arrays containing about 8800 probe sets to characterize transcriptional responses in the rat parietal cortex after acute intermittent nicotine treatment. We focused our attention to expression of transcription factors and several of them were up- or down-regulated by nicotine, among these Nr4a1 (Nurr77), Egr-1 and Egr-2. In situ hybridization was used to corroborate the microarray data and to reveal further spatial and temporal patterns of these nicotine induced genes. Taken together the present results identified several novel candidate genes modified by acute intermittent nicotine exposure and as such potentially involved in neuroprotective-neurotrophic actions.

Adenosine A2A receptors are a new target for drug development in Parkinson's disease. Some experimental and clinical data suggest that A2A receptor antagonists can provide symptomatic improvement by potentiating the effects of L-DOPA as well as a decrease in secondary effects such as L-DOPA-induced dyskinesia. L-DOPA-induced behavioral sensitization in unilateral 6-hydroxydopamine-lesioned rats is frequently used as an experimental model of L-DOPA-induced dyskinesia. In the present work this model was used to evaluate the effect of the A2A receptor agonist CGS 21680 and the A2A receptor antagonist MSX-3 on L-DOPA-induced behavioral sensitization and 6-hydroxydopamine-induced striatal dopamine denervation. L-DOPA-induced behavioral sensitization was determined as an increase in L-DOPA-induced abnormal involuntary movements and enhancement of apomorphine-induced turning behavior. Striatal dopamine innervation was determined by measuring tyrosine-hydroxylase immunoreactivity. Chronic administration of MSX-3 was not found to be effective at counteracting L-DOPA-induced behavioral sensitization. On the other hand, CGS 21680 completely avoided the development of L-DOPA-induced behavioral sensitization. The analysis of the striatal dopamine innervation showed that L-DOPA-CGS 21680 co-treatment conferred neuroprotection to the toxic effects of 6-hydroxydopamine. This neuroprotective effect was dependent on A2A and D2 receptor stimulation, since it was counteracted by MSX-3 and by the D2 receptor antagonist haloperidol. These results open new therapeutic avenues in early events in Parkinson's disease.

Emerging evidence shows that G protein-coupled receptors can form homo- and heteromers. These include adenosine A(2A) receptor-dopamine D(2) receptor heteromers, which are most probably localized in the dendritic spines of the striatopallidal GABAergic neurons, where they are in a position to modulate glutamatergic neurotransmission. The discovery of A(2A) receptor-dopamine D(2) receptor heteromers gives a frame for the well-known antagonistic interaction between both receptors, which is the bases for a new therapeutic approach for neuro-psychiatric disorders, such as Parkinson's disease and schizoprenia. The present review deals mainly with the biochemical and molecular aspects of A(2A) receptor-dopamine D(2) receptor interactions. Recent results at the molecular level show that A(2A) receptor-dopamine D(2) receptor heteromers represent the first example of epitope-epitope electrostatic interaction underlying receptor heteromerization. Most probably A(2A) receptor-D(2) receptor heteromerization is not static, but subject to a dynamic regulation, related to the phosphorylation dependence of the A(2A) receptor epitope and to the ability of the D(2) receptor epitope to bind different partners. Finding out the mechanisms involved in this dynamic regulation can have important implications for the treatment of basal ganglia disorders, schizophrenia and drug addiction.

In this brief comment, we emphasize the importance of circadian rhythms, sleep stages and especially REM sleep for motor and procedural learning which needs to be taken into account when studying striatal plasticity. Mode and timing of application could also play a crucial role for long-term dopaminergic therapies and behavioural and other changes. We further propose a model where the brain, during REM sleep/dreaming, by random recombinations of small pieces of past experiences, tries to anticipate situations not yet experienced and to prepare it-self, in an 'off' situation, for adequate new motor procedural responses.

Behavioral and microdialysis studies have been performed on antagonistic A(2A)/D(2) interactions in animal models of Parkinson's Disease. The behavioral analysis involved studies on locomotor activity in reserpinized mice, haloperidol-induced catalepsy in rats and rotational behavior in rats with unilateral 6-OHDA lesions of the ascending DA pathways (Ungerstedt model). Dual probe microdialysis studies were indirectly performed on the striatopallidal GABA neurons by studying extracellular glutamate levels in the striatum and globus pallidus of the awake freely moving rat. The striatum was perfused with A(2A) and/or D(2) agonists via reverse microdialysis. The results show that the A(2A) antagonists SCH58261 and KF17837 can increase locomotor activity in reserpinized mice and produce contralateral rotational behavior only after administration of subthreshold doses of l-DOPA or the D(2) like agonist quinpirole. Furthermore, antagonizing the A(2A) receptor (R) reduced haloperidol induced catalepsy. The behavioral results underline the view that A(2A) antagonists act by blocking A(2A) R in A(2A)/D(2) heterodimers where A(2A) R inhibits the D(2) R transduction and D(2) inhibits the adenylate cyclase (AC) activated by A(2A) R. The microdialysis studies show that the A(2A) agonist CGS21680 striatally coperfused with the D(2) agonist quinpirole more potently counteract the D(2) agonist (quinpirole) induced reduction of pallidal glutamate levels in the DA denervated vs the control striatum indicating an enhancement of the inhibitory A(2A)/D(2) interaction. In the DA denervated but not in the control striatum the A(2A) agonist CGS21680 could strongly increase striatal glutamate levels, indicating an increased receptor signaling in the A(2A) R located on the striatal glutamate terminals, where also D(2) like R exist, here probably as D(4). Thus, the signaling of this A(2A) R may be set free by the loss of D(4) tone on the AC activated by A(2A) in this postulated A(2A)/D(4) heteromer on the glutamate terminals. Taken together, the results indicate that the antiparkinsonian actions of A(2A) antagonists probably are produced by blockade of A(2A) R in the A(2A)/D(2) heterodimers mainly located in the striatopallidal GABA neurons.

The intercalated cell masses are GABAergic neurons interposed between the major input and output structures of the amygdala. Dopaminergic projections to the main and paracapsular intercalated islands were examined by determining the relationship of the dopamine nerve-terminal networks to the D1-receptor immunoreactive staining of cells within the intercalated islands, using double-fluorescence immunolabelling procedures in combination with confocal laser microscopy. The relationship of terminals positive for both tyrosine hydroxylase and dopamine beta-hydroxylase (noradrenaline and/or adrenaline) to terminals positive for tyrosine hydroxylase but negative for dopamine beta-hydroxylase (dopamine terminals) was studied in relation to the D1-receptor immunoreactivity in adjacent sections at various rostrocaudal levels. The microscopy and image analysis revealed that there was only a minor dopaminergic innervation of the D1 receptor-immunoreactive cells in the rostromedial and caudal component of the main intercalated island, suggesting volume transmission as the main communication mode for dopamine in these regions. In contrast, the D1 receptor-immunoreactive areas in the rostrolateral part of the main island and also the paracapsular intercalated islands showed a high degree of dopaminergic innervation, indicating that synaptic and perisynaptic dopamine transmission plays a dominant role in these regions. It is known that amygdala neurons are involved in the elicitation and learning of fear-related behaviors. We suggest that slow dopaminergic volume transmission in the rostromedial and caudal parts of the main intercalated island may have a role in tonic excitatory modulation in these parts of the main island, allowing GABAergic activity to develop in the central amygdaloid nucleus and thereby contributing to inhibition of fear-related behavioral and autonomic responses. In contrast, a faster synaptic and perisynaptic dopaminergic transmission in the rostrolateral part of the main intercalated island and in the paracapsular intercalated islands may have a role in allowing a more rapid elicitation of fear-related behaviors.

The role of adenosine deaminase in the interactions between adenosine A(1) and dopamine D(1) receptors was studied in a mouse fibroblast cell line stably cotransfected with human D(1) receptor and A(1) receptor cDNAs (A(1)D(1) cells). Confocal laser microscopy analysis showed a high degree of adenosine deaminase immunoreactivity on the membrane of the A(1)D(1) cells but not of the D(1) cells (only cotransfected with human D(1) receptor cDNAs). In double immunolabelling experiments in A(1)D(1) cells and cortical neurons a marked overlap in the distribution of the A(1) receptor and adenosine deaminase immunoreactivities and of the D(1) receptor and adenosine deaminase immunoreactivities was found. Quantitative analysis of A(1)D(1) cells showed that adenosine deaminase immunoreactivity to a large extent colocalizes with A(1) and D(1) receptor immunoreactivity, respectively. The A(1) receptor agonist caused in A(1)D(1) cells and in cortical neurons coaggregation of A(1) receptors and adenosine deaminase, and of D(1) receptors and adenosine deaminase. The A(1) receptor agonist-induced aggregation was blocked by R-deoxycoformycin, an irreversible adenosine deaminase inhibitor. The competitive binding experiments with the D(1) receptor antagonist [(3)H]SCH-23390 showed that the D(1) receptors had a better fit for two binding sites for dopamine, and treatment with the A(1) receptor agonist produced a disappearance of the high-affinity site for dopamine at the D(1) receptor. R-Deoxycoformycin treatment, which has previously been shown to block the interaction between adenosine deaminase and A(1) receptors, and which is crucial for the high-affinity state of the A(1) receptor, also blocked the A(1) receptor agonist-induced loss of high-affinity D(1) receptor binding. The conclusion of the present studies is that the high-affinity state of the A(1) receptor is essential for the A(1) receptor-mediated antagonistic modulation of D(1) receptors and for the A(1) receptor-induced coaggregates of A(1) and adenosine deaminase, and of D(1) and adenosine deaminase. Thus, the confocal experiments indicate that both A(1) and D(1) receptors form agonist-regulated clusters with adenosine deaminase, where the presence of a structurally intact adenosine deaminase bound to A(1) receptors is important for the A(1)-D(1) receptor-receptor interaction at the level of the D(1) receptor recognition.

Evidence for a role of dopaminergic neurotransmission in the motor effects of adenosine antagonists, such as caffeine, is reviewed, based on the existence of specific antagonistic interactions between specific subtypes of adenosine and dopamine receptors in the striatum. Both adenosine A(1) and adenosine A(2A) receptor antagonists induce motor activation in rodents. At least a certain degree of dopaminergic activity is required to obtain adenosine antagonist-induced motor activation, with adenosine A(1) antagonists being the most sensitive and non-selective adenosine antagonists the most resistant to striatal dopamine depletion. When considering long-term treatment with adenosine antagonists concomitant administration of dopamine agonists might be required in order to obtain strong motor effects (cross-sensitization) and to avoid the development of telerance.

Dual-probe microdialysis in the awake rat was employed to investigate the effects of intranigral perfusion with the tridecapeptide neurotensin on local dialysate glutamate and GABA levels in the substantia nigra pars reticulata and on dialysate GABA levels in the ventral thalamus. Intranigral neurotensin (10-300nM, 60min) dose-dependently increased (+29+/-3% and +46+/-3% vs basal for the 100 and 300nM concentrations, respectively) local dialysate glutamate levels, while the highest 300nM concentration of the peptide exerted a long-lasting and prolonged reduction in both local and ventral thalamic (-20+/-4% and -22+/-2%, respectively) GABA levels. Intranigral perfusion with the inactive neurotensin fragment neurotensin(1-7) (10-300nM, 60min) was without effect. Furthermore, the non-peptide neurotensin receptor antagonist SR 48692 (0.2mg/kg) and tetrodotoxin (1microM) fully counteracted the intranigral neurotensin (300nM)-induced increase in local glutamate. SR 48692 (0.2mg/kg) also counteracted the decreases in nigral and ventral thalamic GABA release induced by the peptide. In addition, intranigral perfusion with the dopamine D(2) receptor antagonist raclopride (1microM) fully antagonized the neurotensin (300nM)-induced decreases in nigral and ventral thalamic GABA levels. The ability of nigral neurotensin receptor activation to differently influence glutamate and GABA levels, whereby it increases nigral glutamate and decreases both nigral and ventral thalamic GABA levels, suggests the involvement of neurotensin receptor in the regulation of basal ganglia output at the level of the nigra.

It has been shown that galanin plays a role in central cardiovascular regulation. Galanin administered centrally induces an increase of heart rate and a weak vasodepressor response, whereas the N-terminal galanin fragment (1-15) elicits vasopressor effects and tachycardia. Furthermore, it has been shown that galanin-(1-15), but not galanin-(1-29), decreases the baroreceptor reflex sensitivity. Since these data demonstrate that both galanin and its N-terminal fragment (1-15) exert a different modulation on central cardiovascular control, the aim of this work has been to study if the specific galanin receptor antagonist Galanin-(1-12)-Pro-(Ala-Leu)(2)-Ala]-amide (M40) could modulate their cardiovascular actions. Urethane anaesthetized rats were injected intracisternally and the changes in mean arterial pressure and heart rate were monitored. Two doses of M40 alone have been tested for their cardiovascular effects. With the dose of 1.0 nmol, a significant tachycardia was observed (P<0.001), but 0.1 nmol was ineffective. This suggests a possible agonistic effect for the higher doses of M40. The galanin receptor antagonist M40 at the dose of 0.1 nmol failed to modify the weak vasodepressor effects and tachycardia induced by 3.0 nmol of galanin-(1-29). However, the same dose completely blocked the vasopressor and tachycardic responses elicited by 3.0 nmol of galanin-(1-15). These data show that M40 differentially counteracts the central cardiovascular responses of the galanin fragment and give a functional support for the existence of galanin receptor subtypes within the brainstem. Therefore, the present findings can be explained on the basis that the cardiovascular actions of galanin-(1-29) could be mediated by one type of galanin receptor, whereas a galanin receptor subtype that recognizes N-terminal fragments of galanin may mediate the actions of galanin-(1-15).

We have examined the motor responses to the dopamine D(3) receptor-preferring agonist, S(+)-(4aR,10bR)-3,4,4a, 10b-tetrahydro-4-propyl-2H,5H-1-benzopyranol[4,3-b]-1,4-oxazin+ ++-9-ol ((+)-PD128,907), by non-habituated male rats during postnatal development. (+)-PD128,907 (0.025 and 0.1 mg/kg) increased motor activity (rearing, motility and locomotion) in 14-day-old rats without inducing oral stereotypies. However, in 21-, 28- and 70-day-old rats, (+)-PD128,907 caused a significant reduction in motor activity. This reduction was most pronounced in 70-day-old rats. In addition, the stimulatory effects of (+)-PD128,907 in 14-day-old rats were fully blocked by the dopamine D(3) receptor antagonist 5,6-dimethoxy-2-(di-u-propylamino) indan (U99194A). These results suggest that the motor inhibition mediated by the activation of the dopamine D(3) receptors develops between the second and the third postnatal weeks.

Human adenosine A2A and rat dopamine D2 receptors (A2A and D2 receptors) were co-transfected in Chinese hamster ovary (CHO) cells to study the interactions between two receptors that are co-localized in striatopallidal gamma-aminobutyric acid-(GABA)ergic neurons. Membranes from transfected cells showed a high density of D2 (3.6 pmol per mg protein) and A2A receptors (0.56 pmol per mg protein). The D2 receptors were functional: an agonist, quinpirole, could stimulate GTPgammaS binding and reduce stimulated adenylyl cyclase activity. The A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) decreased high-affinity binding of the agonist dopamine at D2 receptors. Activation of adenosine A2A receptors shifted the dose-response curve for quinpirole on adenosine 3',5'-cyclic monophosphate (cAMP) to the right. However, CGS 21680 did not affect dopamine D2 receptor-induced GTPgammaS binding, but did cause a concentration-dependent increase in cAMP accumulation. The maximal cAMP response was decreased by the D2 agonist quinpirole in a concentration-dependent manner, but there was no change in EC50 and no effect in cells transfected only with adenosine A2A receptors. A2A receptor activation also increased phosphorylation of cAMP response element-binding protein and expression of c-fos mRNA. These effects were also strongly counteracted by quinpirole. These results show that the antagonistic actions between adenosine A2A and dopamine D2 receptors noted previously in vivo can also be observed in CHO cells where the two receptors are co-transfected. Thus, no brain cell-specific factors are required for such interactions. Furthermore, the interaction at the second messenger level and beyond may be quantitatively more important than A2A receptor-mediated inhibition of high affinity D2 agonist binding to the receptor.

The distribution of dopamine D1 and D2 receptor immunoreactivities in the nucleus accumbens and the olfactory tubercle of adult and postnatal male rats were compared with the distribution of tyrosine hydroxylase and dopamine transporter immunoreactivities. An overall co-distribution of D1 and D2 receptor immunoreactivities with tyrosine hydroxylase immunoreactivity was found in the nucleus accumbens and the olfactory tubercle. However, the major finding in this study was, following a more detailed analysis in coronal sections of the shell part of the nucleus accumbens, the existence of nerve cell patches of strong D1 receptor immunoreactivity associated with low D2 receptor, dopamine transporter and tyrosine hydroxylase immunoreactivities. These patches were mainly surrounded by areas of strong D2 receptor, tyrosine hydroxylase and dopamine transporter immunoreactivities and could be found also in the olfactory tubercle. Similar observations were made in postnatal rats. Serial reconstructions of the patches of strong D1 receptor immunoreactivity in the rostrocaudal direction were made. The patches formed a continuous tubular nerve cell system in the shell part of the nucleus accumbens. Since this nerve cell system was found to be surrounded by a high density of dopamine terminals, it may represent a compartment where dopamine transmission mainly acts on D1 receptors via local diffusion (i.e. via volume transmission). However, it must be noted that the D1 receptor rich patches constitute only a small fraction of the nucleus accumbens and the overall density of tyrosine hydroxylase immunoreactive terminals correlates with the density of both D1 and D2 receptors in the nucleus accumbens. In conclusion, the present paper gives new aspects on the chemical microarchitecture of the nucleus accumbens.

Functional activation of dopamine receptors in the crude membranes from rat striatum was studied by a [35S]-guanosine 5'-O-(gamma-thiotriphosphate) ([35S]GTPgammaS) binding assay. Binding of [35S]GTPgammaS could be characterized with a dissociation constant (Kd) = 14.6+/-0.8 nM and this did not depend on the presence of dopamine. The displacement of [35S]GTPgammaS binding by GDP could be characterized with an inhibition constant (K(i)) = 78+/-15 microM in the presence of 10 microM of butaclamol, while the presence of 100 microM of dopamine decreased it to a K(i) = 0.13+/-0.02 mM. Dopamine increased the association rate of [35S]GTPgammaS binding in the presence of GDP in a dose-dependent manner with an EC50 = 1.45+/-0.48 microM. Other dopamine receptor agonists studied displayed a potency to stimulate the [35S]GTPgammaS binding in the order R(-)-10,11dihydroxy-N-n-propylnorapomorphine (NPA) > pergolide > or = apomorphine > dopamine approximately quinpirole > R(+)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride (SKF-38393) > S(+)(4aR,10bR)-3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1]be nzopyrano[4,3-b]-1,4-oxazin-9-ol hydrocholoride (PD 128,907). The dopamine-induced stimulation of [35S]GTPgammaS binding was inhibited by different dopamine receptor antagonists in the potency order: (+)butaclamol > haloperidol approximately clorpromazine > or = raclopride > (-)-sulpride > remoxipride > 5,6-dimethoxy-2-(dipropylamine)indan (U 991944A) > R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzaz epine (SCH-23390). Comparison of the obtained data with the dissociation constants of these ligands to different subtypes of dopamine receptors gave a good correlation only with constants for the D2 subtype, supporting the idea that this subtype is most likely responsible for the dopaminergic activation of [35S]GTPgammaS binding in rat striatal membranes.

Volume transmission in the brain is mediated by the diffusion of neurotransmitters, modulators and other neuroactive substances in the extracellular space. The effects of nitric oxide synthase inhibition on extracellular space diffusion properties were studied using two different approaches, the histological dextran method and the real-time iontophoretic tetramethylammonium method. The spread of biotinylated dextran (mol. wt 3000) in the extracellular space was measured morphometrically following microinjection into the neostriatum of male rats. Two parameters were used to describe the spread of biotinylated dextran in brain tissue, namely, total volume of spread and the mean grey value. The nonspecific nitric oxide synthase inhibitors NG-nitro-L-arginine methyl ester (10-100 mg/kg) and NG-monomethyl-L-arginine acetate (30-200 mg/kg) decreased the total volume of spread of dextran in a dose-dependent manner. 7-Nitroindazole monosodium salt (50-100 mg/kg), a specific neuronal nitric oxide synthase inhibitor, did not change the total volume of spread of dextran. Using the tetramethylammonium method, the extracellular space diffusion properties can be described by the volume fraction (alpha = extracellular space volume/total tissue volume), tortuosity lambda (lambda2 = free diffusion coefficient/apparent diffusion coefficient in tissue), and non-specific uptake kappa' [Nicholson C. and Syková E. (1998) Trends Neurosci. 21, 207-215]. Nitric oxide synthase inhibition by NG-nitro-L-arginine methyl ester (50 mg/kg) had relatively little effect on volume fraction and tortuosity, and no changes were observed after NG-monomethyl-L-arginine acetate (20 mg/kg) or 7-nitroindazole monosodium salt (100 mg/kg) treatment. A substantial increase was found only in non-specific uptake, by 13% after NG-nitro-L-arginine methyl ester and by 16% after NG-monomethyl-L-arginine acetate, which correlates with the decreased total volume of spread of dextran observed with the dextran method. NG-Nitro-L-arginine methyl ester treatment (100 mg/kg) decreased striatal blood flow and increased mean arterial blood pressure. The changes in dextran spread and non-specific uptake can be explained by an increased capillary clearance following the inhibition of endothelial nitric oxide synthase, as neuronal nitric oxide synthase inhibition had no effect. The observed changes after non-specific nitric oxide synthase inhibition may affect the extracellular space concentration of neurotransmitters and modulators, and influence volume transmission pathways in the central nervous system by increased capillary and/or cellular clearance rather than by changes in extracellular space diffusion.

Neuropeptide Y is the most abundant peptide in the mammalian central nervous system and exhibits a variety of potent neurobiological functions. In the present study, double immunolabelling histochemistry was performed, using previously characterized antibodies against neuropeptide Y and the neuropeptide Y Y1 receptor subtype, to clarify the cellular distribution of Y1 receptors in the rat brain in relation to the neuropeptide Y-immunoreactive systems. Based on fluorescence and confocal laser microscopy analysis, morphological evidence is presented that the perikaryal and dendritic Y1 receptor-like immunoreactivity demonstrated in discrete regions of the tel-, diencephalon and of the lower brain stem, shown to be cytoplasmic and membrane associated, in many brain regions is not co-distributed with the neuropeptide Y-immunoreactive terminal network. These findings may partly be explained by the existence of volume transmission in Y1 receptor-mediated neuropeptide Y transmission involving short to long distance diffusion and/or convection of neuropeptide Y from its site of release to the neuronal target cells, containing the high-affinity Y1 receptors. Furthermore, neuropeptide Y and Y1 receptor-like immunoreactivities were in no case co-localized in the same nerve cell, suggesting that, in the rat brain, the Y1 receptor subtype may not be a neuropeptide Y autoreceptor.

The neuropeptide Y Y2 receptor is one of six receptor subtypes mediating the multiform physiological actions of neuropeptide Y. The Y2 receptor has been demonstrated to be the most predominant receptor subtype in the human brain and appears to be involved in many neuropeptide Y actions, such as the regulation of locomotor activity, cardiovascular functions, memory processing, circadian rhythms and release of other neurotransmitters. We have recently demonstrated the widespread and abundant distribution of neuropeptide Y Y1 receptor messenger RNA in the human cerebral cortex (different laminar patterns within distinct cortical regions), hippocampal dentate gyrus and striatum. To assess a possible differential distribution of Y1 and Y2 receptor messenger RNAs, the regional expression of neuropeptide Y Y2 messenger RNA-containing cells in the human brain was analysed, in particular within the cerebral cortex and striatum. In situ hybridization experiments revealed the localization of the Y2 messenger RNA signal throughout all cortical regions, with the highest intensity per cell apparent in lamina IV, with the exception of the striate cortex, which showed an intense labelling primarily in layer VI. The striatum expressed low to undetectable levels of the Y2 receptor messenger RNA. The dentate gyrus and the CA2 region presented the highest hybridization signals, while a very weak Y2 messenger RNA expression was found in the CA1 region and subiculum. Positive Y2 messenger RNA hybridization signals were also detected in the lateral geniculate nucleus, amygdala, substantia nigra, hypothalamus, cerebellum and choroid plexus. These results demonstrate the widespread distribution of neuropeptide Y Y2 receptor messenger RNA in the human brain, with a pattern of expression distinct from the Y1 subtype, suggesting that these two receptor subtypes may mediate different neuropeptide Y functions in the human brain, mainly through actions on different neuronal systems.

The effects of the systemic (i.p.) administration of the selective adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) and the selective adenosine A2A receptor agonist sodium 2-p-carboxyethyl)phenylamino-5'-N-carboxamidoadenosine (CGS 21680) on different dopamine receptor agonist-induced behaviours were studied in the male rat. CGS 21680 (1 micromol/kg), but not CPA, was found to counteract the stereotypies induced by the non-selective dopamine receptor agonist apomorphine (0.25 mg/kg s.c.). Low doses of CGS 21680 (0.1 micromol/kg) and high doses of CPA (3 micromol/kg) counteracted yawning induced by the dopamine D2 selective agonist quinpirole (0.05 mg/kg). On the other hand, low doses of CPA (0.3 micromol/kg) antagonized grooming induced by the selective dopamine D1 receptor-selective agonist SKF 38393 (10 mg/kg i.p.), while CGS 21680 was ineffective. These results are consistent with the proposed existence of a selective antagonistic modulation of dopamine D1 and D2 receptors by adenosine A1 and A2A receptors, respectively. The ability of CGS 21680 to counteract apomorphine-induced stereotypies is weaker compared to its previously reported antagonistic effect of amphetamine-induced motor activity. This supports the hypothesis that adenosine A2A receptor agonists may be potential antipsychotic drugs with a low potential for extrapyramidal side effects.

Several findings show a neuroprotective effect of nicotine treatment in different experimental models, and a negative correlation has been observed between cigarette smoking and the incidence of Parkinson's disease. It seems possible that nicotine may in part exert its neuroprotective actions by favouring the synthesis of neurotrophic factors. The aim of this study was to determine whether the nicotine treatment could be associated with the induction of a neurotrophic factor in brain regions with nicotinic receptors. Thus, we analysed by in situ hybridization and RNAse protection assay the effects of (-)nicotine on basic fibroblast growth factor messenger RNA and by immunocytochemistry fibroblast growth factor-2 protein in the tel- and diencephalon of rats following single or acute intermittent (-)nicotine treatment. The present results showed that acute intermittent (-)nicotine treatment (four i.p. injections at intervals of 30 min), but not single injections, lead to a substantial and dose-related (0.1-2 mg/kg) up-regulation of fibroblast growth factor-2 messenger RNA levels in the cerebral cortex, in the hippocampus, in the striatum and ventral midbrain. This induction of fibroblast growth factor-2 expression peaked 4 h after the first injection and returned to normal levels within 24 h. The change of fibroblast growth factor-2 messenger RNA levels was associated with increased fibroblast growth factor-2 immunoreactivity mainly localized to nerve cells. The treatment was effective also when repeated in the same animals three or five days after the first injection. The pre-treatment with the non-competitive (-)nicotine receptor antagonist mecamylamine blocked the (-)nicotine effects on fibroblast growth factor-2 messenger RNA levels. In the above areas, no changes were observed in the fibroblast growth factor-1, 2 and 3 receptor messenger RNA levels nor in brain-derived neurotrophic factor messenger RNA levels. The present data indicate an ability of intermittent (-)nicotine to increase fibroblast growth factor-2 in many tel- and diencephalic areas. In view of the trophic function of fibroblast growth factor-2, the previously observed neuroprotective effects of (-)nicotine may at least in part involve an activation of the neuronal fibroblast growth factor-2 signalling, and open up new avenues for treatment of Parkinson's disease and Alzheimer's disease based on the existence of nicotinic receptor subtypes enhancing fibroblast growth factor-2 signalling in many regions of the tel- and diencephalon.

The potential influence of glucocorticoids on fetal brain development was investigated after corticosterone administration via pellets to pregnant rats during the last trimester of gestation. We examined both spontaneous motor activity and dopamine-mediated motor responses to apomorphine, a D1, D2 and D3 receptor agonist, given at a postsynaptic dose (1 mg/kg, s.c.) to both prepubertal and adult male and female offspring. Prenatal corticosterone was found to produce the following alterations in the offspring. (1) Prepubertal stage: Male offspring: a statistically significant (P < 0.05) increase was observed in spontaneous rearing, motility and locomotion (activity measured during the first 30 min) without changes in apomorphine-induced motor responses. Female offspring: a reduction (P < 0.05) only in spontaneous rearing activity was observed during the exploratory phase (activity measured during the first 10 min) without significant changes in apomorphine-induced motor responses. (2) Adult stage: Male offspring: the exploratory activity to the novel environment was increased (P < 0.05) without significant changes in apomorphine-induced motor activity. Female offspring: an increase (P < 0.05) in spontaneous locomotion was observed during the first 30 min of testing without significant changes in exploratory activity to the novel environment. However, the apomorphine-induced motility and locomotion were reduced (P < 0.05) during the first 30 min. These observations indicate that prenatal corticosterone induces both short-term and long-term changes in spontaneous motor activity as well as long-lasting alterations in dopamine receptor response in the motor network mechanisms controlled by DA receptors. These changes are in part age and sex-dependent. The possible relationship between prenatal programming of the mesolimbic and nigrostriatal dopaminergic pathways by corticosterone and the observed changes in motor function is discussed.

There exist at least two distinct subtypes of angiotensin II receptors in the brain, namely the AT1 and AT2 subtypes. The high density of angiotensin II AT1 receptors is present in the medulla oblongata. The AT1 subtype of angiotensin II receptors mainly mediates central cardiovascular events. In the present study a polyclonal antibody against the angiotensin II AT1 receptor and a monoclonal antibody against tyrosine hydroxylase were employed to evaluate the possible presence of angiotensin II AT1 receptor-like immunoreactivity in the catecholaminergic neurons of the rat medulla oblongata by means of the double colour immunofluorescence technique. A weak, diffuse cytoplasmic angiotensin II AT1 receptor-like immunoreactivity was observed in almost all the catecholaminergic cell bodies of the A2, C1, C2 and C3 cell groups, except those of the A1 cell group containing moderately intense, diffuse cytoplasmic angiotensin II AT1 receptor-like immunoreactivity, occasionally found in the noradrenergic dendrites of the A1 cell group. There was a higher density of the angiotensin II AT1 receptor-like immunoreactive profiles in the A2 cell group area than in other catecholaminergic cell group areas. In addition, the angiotensin II AT1 receptor-like immunoreactivity was seen in non-catecholaminergic neurons. The present results provide evidence for the existence of the specific angiotensin II AT1 receptor-like immunoreactivity in the noradrenergic and adrenergic neurons of the rat medulla oblongata known to have a cardiovascular role. Thus, the findings support the view that angiotensin II AT1 receptors in the medulla oblongata participate in cardiovascular control and indicate a cellular substrate for the documented interaction between the angiotensin II and adrenergic transmission lines in cardiovascular function at the level of the nucleus tractus solitarii.

The regulation by neuropeptide Y of alpha2-adrenoceptors in the nucleus tractus solitarii was evaluated in the adult normotensive Wistar Kyoto rat and the adult spontaneously hypertensive rat. The microinjection of a submaximal dose of l-noradrenaline (800 pmol in 50 nl) alone into the nucleus tractus solitarii produced a significant reduction in the mean arterial blood pressure in either strain. The threshold dose (1 pmol in 50 nl) of neuropeptide Y(1-36) for the vasodepressor response in the Wistar Kyoto rat was five times higher than that (0.2 pmol in 50 nl) in the spontaneously hypertensive rat. Furthermore, neuropeptide Y(1-36) at 0.2 pmol in 50 nl could significantly counteract the vasodepressor response to l-noradrenaline (800 pmol in 50 nl) in the spontaneously hypertensive rat, but not in the Wistar Kyoto rat, in which 1 pmol in 50 nl of neuropeptide Y(1-36) must be employed to counteract the vasodepressor response to l-noradrenaline (800 pmol in 50 nl), although the vasodepressor responses are of a similar magnitude. The in situ hybridization and quantitative receptor autoradiographical experiments showed that the alpha2A-adrenoceptor messenger RNA levels and the B(max) value of the alpha2-adrenoceptor agonist [3H]p-aminoclonidine binding sites measured in the nucleus tractus solitarii of the spontaneously hypertensive rat were substantially lower than those in the Wistar Kyoto rat. The quantitative receptor autoradiographical results were consistent with the cardiovascular results and showed that in the spontaneously hypertensive rat, neuropeptide Y(1-36) at 1 nM led to a significant increase in the K(d) value of [3H]p-aminoclonidine binding sites. In the Wistar Kyoto rat, neuropeptide Y(1-36) produced this effect only at 10 nM. The present study provides evidence for an increase of the potency of neuropeptide Y(1-36) to antagonistically modulate alpha2-adrenoceptors in the nucleus tractus solitarii of the spontaneously hypertensive rat. This enhanced antagonistic action may partly be related to a reduction in the number of alpha2A-adrenoceptors in the nucleus tractus solitarii of the spontaneously hypertensive rat, since a decrease has been observed in the alpha2A-adrenoceptor messenger RNA levels and the alpha2-adrenoceptor binding sites in the spontaneously hypertensive rat. This increased potency of neuropeptide Y(1-36) to antagonize alpha2-adrenoceptor function in the nucleus tractus solitarii of the spontaneously hypertensive rat may contribute to the development of high blood pressure in this hypertensive strain.

In membrane preparations from rat striatum, where adenosine A2A and dopamine D2 receptors are coexpressed, stimulation of adenosine A2A receptors was found to decrease the affinity of dopamine D2 receptors for dopamine agonists. We now demonstrate the existence of this antagonistic interaction in a fibroblast cell line (Ltk-) stably transfected with the human dopamine D2 (long-form) receptor and the dog adenosine A2A receptor cDNAs (A2A-D2 cells). In A2A-D2 cells, but not in control cells only containing dopamine D2 receptors (D2 cells), the selective adenosine A2A agonist 2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethyl-carboxamido adenosine (CGS 21680) induced a 2-3-fold decrease in the affinity of dopamine D2 receptors for dopamine, as shown in competition experiments with dopamine versus the selective dopamine D2 antagonist [3H]raclopride. By contrast, activation of the constitutively expressed adenosine A2B receptors with 5'-N-ethyl-carboxamidoadenosine (NECA) did not modify dopamine D2 receptor binding. In A2A-D2 cells CGS 21680 failed to induce or induced only a small increase in adenosine-3',5'-cyclic-monophosphate (cAMP) accumulation. In D2 cells NECA- or forskolin-induced adenylyl cyclase activation was not associated with any change in dopamine D2 receptor binding. These results indicate that adenylyl cyclase activation is not involved in the adenosine A2A receptor-mediated modulation of the binding characteristics of the dopamine D2 (long-form) receptor.

In the present study we characterized the cholecystokinin receptor regulation of (i) the dopamine D2 agonist binding sites in striatal sections including the nucleus accumbens and (ii) GABA and dopamine release in the central part of the rat nucleus accumbens, by combining the in vitro filter wipe-off and the in vivo microdialysis techniques. In the binding study we demonstrate that sulphated cholecystokinin octapeptide (1 nM) increased (219 +/- 30%) the KD value of the D2 agonist [3H]N-propylnorapomorphine binding sites in sections from the striatum including the accumbens. This effect was counteracted by the cholecystokinin-B antagonist PD134308 (50 nM). In a parallel study using microdialysis in the central nucleus accumbens, we found that local perfusion with sulphated cholecystokinin octapeptide (1 microM) induced an increase in GABA (135 +/- 7%) and dopamine (146 +/- 8%) release which was unaffected by the cholecystokinin-A antagonist L-364,718 (10 nM). In contrast, when the cholecystokinin-B antagonist PD134308 (10 nM) was co-perfused with the peptide it prevented the increase in dopamine and decreased GABA release (-24 +/- 2%). This reduction was counteracted by the addition to the perfusate medium of the cholecystokinin-A antagonist or the cholinergic muscarinic M2 receptor antagonist AF-DX 116 (0.1 microM). Taken together, these data demonstrate that the facilitation by sulphated cholecystokinin octapeptide of GABA and dopamine release in the central accumbens probably reflects an inhibitory effect of the peptide on both pre- and postsynaptic D2 receptors, mediated via cholecystokinin-B receptor activation. In addition, for the first time we provide evidence for a differential cholecystokinin-A and -B receptor-mediated regulation of GABA transmission in the central accumbens, where the cholecystokinin-B receptor exerts a dominant excitatory influence while the cholecystokinin-A receptor mediates an inhibition of GABA release via a local muscarinic M2 receptor.

Neuropeptide Y receptors in the medulla oblongata participate in central cardiovascular control. The neuropeptide Y1 receptor subtype gene and amino acid sequence have been identified by molecular cloning studies. In this study, a C-terminal peptide representing amino acids 355-382 of the neuropeptide Y1 receptor was synthesized and cross-linked to thyroglobulin to produce an antibody against a partial sequence of the neuropeptide Y1 receptor, used to localize neuropeptide Y1 receptor-like immunoreactivity in the catecholaminergic neurons of the medulla oblongata. The double colour immunofluorescence technique with a polyclonal antibody against the neuropeptide Y1 receptor and a monoclonal antibody against tyrosine hydroxylase revealed that in the rat medulla oblongata, a weak (the C3 cell group) to moderately intense (the A1, A2, C1 and C2 cell groups), diffuse cytoplasmic neuropeptide Y1 receptor-like immunoreactivity was distributed primarily in the noradrenergic and adrenergic cell bodies and occasionally seen in the noradrenergic and adrenergic cell processes. Almost all tyrosine hydroxylase-like immunoreactive cell bodies in the A1, A2, C1, C2 and C3 cell groups showed neuropeptide Y1 receptor-like immunoreactivity. The neuropeptide Y1 receptor-like immunoreactivity in the A2 cell group was somewhat stronger. The present findings show localization of specific neuropeptide Y1 receptor-like immunoreactivity in the vast majority of the noradrenergic and adrenergic cell bodies of the A1, A2, C1, C2 and C3 cell groups, which are putative cardiovascular regions. The results support the view that neuropeptide Y1 receptors in the medulla oblongata are involved in central cardiovascular control and may coexist with another important receptor, the alpha 2A-adrenoceptor, also involved in central, cardiovascular regulation, since the alpha 2A-adrenoceptor-like immunoreactivity has been shown to exist in almost all noradrenergic and adrenergic cell bodies in the brainstem. In conclusion, centrally administered neuropeptide Y may act in part via neuropeptide Y1 receptors located on the soma and dendrites of noradrenergic and adrenergic neurons, where it may interact with alpha 2-adrenoceptors at least in the noradrenergic A2 neurons. This noradrenaline system may be involved in at least part of the vasodepressor actions of neuropeptide Y, noradrenaline and adrenaline in the nucleus tractus solitarii in view of the present findings.

The present in vivo microdialysis study demonstrated that the subcutaneous injection of modafinil (diphenyl-methyl-sulfinyl-2-acetamide) in doses of 30-300 mg/kg dose dependently increased dopamine release from the intermediate level of the nucleus accumbens along the rostro-caudal axis of the halothane anaesthetized rat. The effect of modafinil in a dose of 100 mg/kg was counteracted by the local perfusion in the nucleus accumbens with the GABAB receptor antagonist phaclofen (beta-p-chlorophenyl-gamma-aminopropyl-phosphonic acid) (50 microM), the GABAA agonist muscimol (3-hydroxy-5-aminomethyl-isoxazolol) (10 microM) and the neuronal GABA reuptake inhibitor SKF89976A (4,4-diphenyl-3-butenyl-nipecotic acid) (0.1 microM), whereas it was increased by the GABAB receptor agonist (-)-baclofen [beta-(p-chlorophenyl-gamma-aminobutyric acid)] (10 microM). In addition, the modafinil-induced increase of dopamine release was associated with a significant reduction of accumbens GABA release. These results suggest that the dopamine releasing action of modafinil in the rat nucleus accumbens is secondary to its ability to reduce local GABAergic transmission, which leads to a reduction of GABAA receptor signaling on the dopamine terminals.

The influence of adenosine A1 (N6-cyclopentyladenosine, CPA) and A2 (2-[4-(2-carboxylethyl)phenethylamino]-5'-N-ethylcarboxamido -adenosine hydrochloride, CGS 21680) receptor agonists on SKF 38393-induced electroencephalographic (EEG) arousal was studied in rabbits. While CPA (0.1 mg/kg i.v.) significantly prevented the EEG effects of SKF 38393, CGS 21680 (0.2 mg/kg i.v.) did not affect them. These results demonstrate that adenosine A1 receptors can modulate dopamine D1 receptor-induced EEG arousal and show, for the first time, that adenosine-dopamine interactions are involved in brain functions other than motor activity.

In the present study we used the dual probe approach to investigate striatal N-methyl-D-aspartate receptor regulation of GABA release from the substantia nigra pars reticulata of the awake, freely moving rat. One microdialysis probe of concentric design was implanted in the dorsolateral striatum and another in the ipsilateral substantia nigra pars reticulata. Perfusion with N-methyl-D-aspartate (100 microM) in the dorsolateral striatum decreased local dopamine release (-25%) and increased both glutamate (+40%) and GABA (+35%) release. Moreover, perfusion with N-methyl-D-aspartate (100 microM) in the dorsolateral striatum increased GABA release (+20%) in the substantia nigra pars reticulata. Perfusion with the lower (10 microM) N-methyl-D-aspartate concentration in the dorsolateral striatum did not affect striatal dopamine, glutamate and GABA release or nigral GABA release. Intrastriatal perfusion with the N-methyl-D-aspartate receptor antagonist dizocilpine maleate (10 microM), at a dose which by itself did not affect basal striatal or nigral neurotransmitter levels, prevented the effects of striatal perfusion with N-methyl-D-aspartate on both striatal and nigral neurotransmitter release. Intrastriatal dizocilpine maleate was also perfused concurrently with intranigral tetrodotoxin (10 microM) (see accompanying paper). Intrastriatal perfusion with dizocilpine maleate prevented the tetrodotoxin-induced rise in both striatal and nigral GABA levels and profoundly reduced the tetrodotoxin-induced contralateral turning. In addition, intrastriatal dizocilpine maleate delayed the increase in striatal glutamate release evoked by intranigral tetrodotoxin without affecting the associated decrease in striatal dopamine release. The present study demonstrates that N-methyl-D-aspartate receptors in the dorsolateral striatum regulate GABA release in the substantia nigra pars reticulata of the awake rat and provides evidence that this regulation plays a key role in motor function.

In the present study we employed the dual probe approach to investigate functional interactions between the nigrostriatal dopaminergic and striatonigral GABAergic pathways in the awake, freely moving rat and their role in motor function. One microdialysis probe of concentric design was implanted in the substantia nigra pars reticulata and another in the ipsilateral dorsolateral striatum. Perfusion with a low-Ca2+ (0.1 mM) medium and with the voltage-dependent Na(+)-channel blocker tetrodotoxin (10 microM) was alternatively performed in both brain regions and the dialysate dopamine, glutamate and GABA levels were simultaneously measured in the dorsolateral striatum, whereas GABA levels alone were monitored in the substantia nigra. Perfusion with a low-Ca2+ medium in the substantia nigra pars reticulata did not affect local GABA levels, but transiently increased striatal dopamine release (+40%) without modifying striatal glutamate and GABA levels. Conversely, intranigral perfusion with tetrodotoxin transiently increased local GABA levels (+40%), while it decreased striatal dopamine (-60%) and increased glutamate (+70%) and GABA (+50%) levels. Perfusion with a low-Ca2+ medium in the dorsolateral striatum reversibly decreased local dopamine (-70%), glutamate (-20%) and GABA (-20%) levels, while local perfusion with tetrodotoxin decreased dopamine (-70%), increased glutamate (+30%) but did not affect dialysate GABA levels in this brain area. Neither of these intrastriatal treatments significantly affected GABA levels in the substantia nigra. Intranigral but not intrastriatal perfusion with tetrodotoxin was also associated with an increase in spontaneous locomotor activity as expressed by contralateral turning. Intranigral and intrastriatal perfusion with low-Ca2+ medium did not influence locomotor activity. On the basis of these neurochemical and behavioural findings, we propose a new dynamic model for the study of motor behaviour as mediated by basal ganglia circuitry.

A negative correlation has been found between smoking and Parkinson's disease. There is evidence to suggest that this correlation appears to be associated with a neuroprotective role of nicotine for dopamine neurons at least in relation to mechanical injury. However, 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP) neurotoxicity to dopamine neurons is enhanced by chronic continuous (-)nicotine. More recently, basic fibroblast growth factor has been found to possess neurotrophic activities for many nerve cells including the dopamine cells in vivo and in vitro. Therefore, it is of interest to explore a possible effect of nicotine on basic fibroblast growth factor expression in the ventral midbrain of intact and 6-hydroxydopamine-lesioned rats and how treatment with nicotine can alter the 6-hydroxydopamine-induced injury of the nigral dopamine nerve cells as evaluated by dopamine biochemistry. In the present paper, an analysis of the effects of chronic continuous infusion of (-)nicotine via minipumps was carried out on basic fibroblast growth factor expression in the vental midbrain of the intact male rat and of the 6-hydroxydopamine lesioned rat. A quantitative messenger RNA protection assay analysis was used as well as an immunocytochemical analysis in the substantia nigra. Our findings give evidence that a two-week continuous infusion with (-)nicotine in the intact rat leads to substantial and dose-related (0.03-0.3 mg/kg per h) reductions of basic fibroblast growth factor messenger RNA levels in the ventral midbrain. These changes are not associated with changes in neuronal and glial basic fibroblast growth factor immunoreactivity in this region with the antibodies used. However, a one-week continuous infusion with (-)nicotine (0.125 mg/kg per h) failed to significantly alter the basic fibroblast growth factor messenger RNA levels in the ventral midbrain of solvent and 6-hydroxydopamine-injected rats and thus also the 6-hydroxydopamine-induced increase of basic fibroblast growth factor messenger RNA levels in the ventral midbrain of the lesioned side observed at this time-interval and known to be of astroglial origin [Chadi G. et al. (1994) Neuroscience 61, 891-910]. In agreement, the 6-hydroxydopamine-induced depletion of dopamine in the neostriatum was unaltered by the nicotine treatment (0.125 mg/kg per h). Thus, chronic continuous (-)nicotine treatment may lead to a reduced basic fibroblast growth factor trophic tone in the ventral midbrain of the intact but not of the 6-hydroxydopamine-lesioned rat neither on the lesioned nor on the unlesioned side of the ventral midbrain. It seems possible that chronic nicotine treatment mainly reduces basic fibroblast growth factor messenger RNA levels of neuronal origin, since the astroglial messenger RNA levels dominate after the 6-hydroxydopamine-induced lesions.

During the past two decades several revisions of the concepts underlying interneuronal communication in the central nervous system have been advanced. We propose here to classify communicational phenomena between cells of the central neural tissue under two general frames: "wiring" and "volume" transmission. "Wiring" transmission is defined as intercellular communication occurring through a well-defined connecting structure. Thus, wiring transmission is characterized by the presence of physically identifiable communication channels within the neuronal and/or glial cell network. It includes synaptic transmission but also other types of intercellular communication through a connecting structure (e.g., gap junctions). "Volume" transmission is characterized by signal diffusion in a three-dimensional fashion within the brain extracellular fluid. Thus, multiple, structurally often not well characterized extracellular pathways connect intercommunicating cells. Volume transmission includes short- (but larger than synaptic cleft, i.e. about 20 nm) and long-distance diffusion of signals through the extracellular and cerebrospinal fluid. It must be underlined that the definitions of wiring and volume transmission focus on the modality of transmission and are neutral with respect to the source and target of the transmission, as well as type of informational substance transmitted. Therefore, any cell present in the neural tissue (neurons, astroglia, microglia, ependyma, tanycytes, etc.) can be a source or a target of wiring and volume transmission. In this paper we discuss the basic definitions and some distinctive characteristics of the two types of transmission. In addition, we review the evidence for different types of intercellular communication besides synaptic transmission in the central nervous system during phylogeny, and in vertebrates in physiological and pathological conditions.

The mode of coupling between neurotransmitter receptors and G proteins was investigated by agonist-induced high-affinity GTPase activity in rat striatal membranes. There was a simple additive relationship among dopamine-, carbachol-, and gamma-aminobutyric acid (GABA)-sensitive high-affinity GTPase activity in any combination, indicating that the respective receptors stimulated by these agonists (i.e., dopamine D2, pirenzepine-insensitive muscarinic, and GABAB receptors) interact independently with distinct pools of G proteins. Unexpectedly non-additivity was observed between dopamine- and norepinephrine-stimulation. This lack of additivity was apparently due to stimulation of the same dopamine D2 receptors by both dopamine and norepinephrine, since norepinephrine-stimulated high-affinity GTPase activity could be inhibited by dopaminergic but not adrenergic antagonists. The same non-additivity as seen in rat striatum was confirmed in the membranes prepared from cultured mouse fibroblast cells co-transfected with dopamine D2 and adenosine A2A receptors. The implication of the (non-)additivity between receptor-mediated high-affinity GTPase activity was discussed with a consideration of the possible underlying molecular mechanism.

A stably co-transfected mouse fibroblast cell line, which expresses the long form of the human dopamine D2 receptor and the dog adenosine A2a receptor, was used to analyse the mechanism underlying changes in the cytosolic free calcium concentration ([Ca2+]i) induced by activation of D2 (long-form) receptors and its modulation by the A2a receptor agonist CGS 21680 by means of fura-2 imaging. Quinpirole (1-1000 nM), a D2 receptor agonist, caused a concentration-dependent increase in [Ca2+]i. Haloperidol, a preferential D2 receptor antagonist, completely blocked this [Ca2+]i response to quinpirole. Preincubation with Ca(2+)-free medium containing 2 mM EGTA or a medium containing 320 mM ethanol, an inositol 1,4,5-triphosphate receptor antagonist, substantially diminished the increase in [Ca2+]i evoked by quinpirole. Furthermore, quinpirole totally failed to elevate [Ca2+]i in a medium containing both 2 mM EGTA and 320 mM ethanol. CGS 21680 (1-500 nM) did not, by itself, exert any significant effect on [Ca2+]i. However, 100 nM of CGS 21680 substantially counteracted the [Ca2+]i responses to quinpirole (10-1000 nM). Moreover, this counteraction still occurred after blocking Ca2+ mobilization from intracellular stores with ethanol, but disappeared when the cells were pretreated with the Ca(2+)-free medium containing 2 mM EGTA. Our findings imply that the D2 (long-form) receptors in the present fibroblast cell line can raise [Ca2+]i both via Ca2+ influx from the extracellular medium and Ca2+ release from intracellular stores via activation of inositol 1,4,5-triphosphate receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Agonist-induced high-affinity GTPase activity was investigated using the crude membrane preparation from rat striatum. High-affinity GTPase activity was stimulated by dopamine and carbachol in a Mg(2+)-dependent manner and with possible optimum NaCl concentrations of 50-100 mM to detect the percent increase induced by each agonist. Dopamine and selective (as well as non-selective) D2 receptor agonists, but not selective D1 receptor agonists, stimulated activity in a concentration-dependent manner, with affinities which were significantly correlated with those for adenylate cyclase inhibition as previously reported in the literature. Maximal percent stimulation above basal high-affinity GTPase activity was 9.8 +/- 0.6% and 4.4-7.6% for dopamine and other synthetic dopamine D2 receptor agonists, respectively. Dopamine-stimulated activity was inhibited by several dopamine receptor antagonists with the following rank order of potency: (+)-butaclamol > spiperone > raclopride > S(-)-sulpiride; but not by (-)-butaclamol or SCH 23390. High-affinity GTPase activity was also stimulated by carbachol and acetylcholine through the pirenzepine-insensitive muscarinic receptors. Preincubation of the membranes with AS/7, a specific antiserum to Gi1 and Gi2, appeared to attenuate dopamine-sensitive activity, suggesting that Gi1 and/or Gi2 may be at least partially involved. These results indicate that high-affinity GTPase activity in rat striatal membranes is activated through dopamine D2-like receptors and pirenzepine-insensitive muscarinic receptors, both of which are negatively coupled to adenylate cyclase via Gi proteins.

Recently, both glucocorticoid receptor immunoreactivity and glucocorticoid receptor messenger RNA levels were found in multiple brain areas, especially in the neuroepithelium during the late prenatal development of the rat brain. To better understand the potential influence of stress on fetal brain development by release of maternal adrenocortical steroids, we have investigated the effects of corticosterone administration to pregnant rats on the locomotor activity of their prepubertal offspring. On day 16 of pregnancy female rats were implanted with either placebo or corticosterone pellets (release of 2.4 mg/day for seven days). After birth their offspring were nursed by foster mothers to avoid any postnatal effects of the corticosterone pellets. At three weeks of age, the offspring were tested for spontaneous motor behaviours. Both male and female offspring from corticosterone treated mothers showed significantly increased spontaneous ambulation, motility and rearing compared to placebo treated groups. No significant sex differences were found in locomotor activity between male and female offspring from placebo groups. Following d-amphetamine (1.5 mg/kg) treatment, a preferential dopamine releasing agent, we observed a significant increase in ambulation, motility and rearing activity in the male offspring treated with corticosterone. In the female offspring, only the rearing activity was significantly higher after d-amphetamine treatment in the prenatal corticosterone group compared with the placebo treated group. Basal dopamine metabolism (dihydroxyphenylacetic acid/dopamine ratio) was increased in the dorsal striatum and ventral striatum of male and female offspring from corticosterone-treated dams. In the male offspring, corticosterone treatment was associated with a disappearance of the right side dominance of dopamine metabolism in the dorsal striatum.(ABSTRACT TRUNCATED AT 250 WORDS)

To investigate the long-term effects of excitatory amino acid microinjections into the basal forebrain and its correlation with a possible Ca2+ imbalance associated with the excitotoxic process, ibotenic acid, mainly an N-methyl-D-aspartate receptor agonist, and quisqualic acid, an agonist of non-N-methyl-D-aspartate receptors, were injected into two regions rich in cholinergic neurons, namely the medial septal nucleus and the ventral globus pallidus. Within the globus pallidus but not within the medial septal nucleus, 13 days and one year postlesion, nerve cell death was associated with the appearance of calcium deposits within the large putative GABAergic pallidal neurons, being more pronounced in ibotenic acid than quisqualic acid-lesioned rats. An intermediate two month post-lesion study with alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and ibotenic acid microinjections in globus pallidus demonstrated that the AMPA subtype of glutamate receptor may also be involved in this Ca2+ imbalance, together with the N-methyl-D-aspartate and metabotropic subtype receptors. Quisqualic acid lesions in globus pallidus and medial septum were associated with a substantial disappearance of cholinergic cell bodies and their nerve terminal networks within the cerebral cortex and hippocampal formation respectively, as assessed by choline acetyltransferase and acetylcholine esterase immunocytochemistry. Ibotenic acid lesions resulted in a lower reduction of cholinergic markers. One year after septal lesions induced either by ibotenic or quisqualic acid, a marked atrophy of the entire dorsolateral septal nucleus was observed. Our results support the hypothesis that brief and intense glutamate exposure can induce long-term neurodegenerative processes and give evidence that long-term excitotoxic lesions of the two areas studied result in marked differences in neuronal damage, including intracellular calcium deposits which do not correlate with the cholinergic deficits produced by multiple glutamate receptor subtypes.