Search

Glucagon-like peptide 1 (GLP-1) and serotonin play critical roles in energy balance regulation. Both systems are exploited clinically as antiobesity strategies. Surprisingly, whether they interact in order to regulate energy balance is poorly understood. Here we investigated mechanisms by which GLP-1 and serotonin interact at the level of the central nervous system. Serotonin depletion impaired the ability of exendin-4, a clinically used GLP-1 analog, to reduce body weight in rats, suggesting that serotonin is a critical mediator of the energy balance impact of GLP-1 receptor (GLP-1R) activation. Serotonin turnover and expression of 5-hydroxytryptamine (5-HT) 2A (5-HT2A) and 5-HT2C serotonin receptors in the hypothalamus were altered by GLP-1R activation. We demonstrate that the 5-HT2A, but surprisingly not the 5-HT2C, receptor is critical for weight loss, anorexia, and fat mass reduction induced by central GLP-1R activation. Importantly, central 5-HT2A receptors are also required for peripherally injected liraglutide to reduce feeding and weight. Dorsal raphe (DR) harbors cell bodies of serotonin-producing neurons that supply serotonin to the hypothalamic nuclei. We show that GLP-1R stimulation in DR is sufficient to induce hypophagia and increase the electrical activity of the DR serotonin neurons. Finally, our results disassociate brain metabolic and emotionality pathways impacted by GLP-1R activation. This study identifies serotonin as a new critical neural substrate for GLP-1 impact on energy homeostasis and expands the current map of brain areas impacted by GLP-1R activation.

Summary: Variations in the human FTO gene have been linked to obesity and altered connectivity of the dopaminergic neurocircuitry. Here, we report that fat mass and obesity-associated protein (FTO) in dopamine D2 receptor-expressing medium spiny neurons (D2 MSNs) of mice regulate the excitability of these cells and control their striatopallidal globus pallidus external (GPe) projections. Lack of FTO in D2 MSNs translates into increased locomotor activity to novelty, associated with altered timing behavior, without impairing the ability to control actions or affecting reward-driven and conditioned behavior. Pharmacological manipulations of dopamine D1 receptor (D1R)- or D2R-dependent pathways in these animals reveal altered responses to D1- and D2-MSN-mediated control of motor output. These findings reveal a critical role for FTO to control D2 MSN excitability, their projections to the GPe, and behavioral responses to novelty. : Ruud et al. find that FTO, an obesity-risk gene, regulates physical activity via dopamine D2 receptor-expressing medium spiny neurons in the brain. Although FTO regulates the firing of and limits locomotion through those neurons, these changes do not predispose mice to weight gain or altered food reward.

Testosterone deficiency in men is associated with increased risk for autoimmunity and increased B cell numbers through unknown mechanisms. Here we show that testosterone regulates the cytokine BAFF, an essential survival factor for B cells. Male mice lacking the androgen receptor have increased splenic B cell numbers, serum BAFF levels and splenic Baff mRNA. Testosterone deficiency by castration causes expansion of BAFF-producing fibroblastic reticular cells (FRCs) in spleen, which may be coupled to lower splenic noradrenaline levels in castrated males, as an α-adrenergic agonist decreases splenic FRC number in vitro. Antibody-mediated blockade of the BAFF receptor or treatment with the neurotoxin 6-hydroxydopamine revert the increased splenic B cell numbers induced by castration. Among healthy men, serum BAFF levels are higher in men with low testosterone. Our study uncovers a previously unrecognized regulation of BAFF by testosterone and raises important questions about BAFF in testosterone-mediated protection against autoimmunity., Testosterone deficiency is associated with autoimmunity and increased B cell numbers, but the underlying mechanism is unclear. Here the authors show that testosterone may modulate the production of B cell survival factor BAFF by fibroblastic reticular cells via regulation of splenic neurotransmitter levels.

Impulsivity, defined as impaired decision making, is associated with many psychiatric and behavioral disorders, such as attention-deficit/hyperactivity disorder as well as eating disorders. Recent data indicate that there is a strong positive correlation between food reward behavior and impulsivity, but the mechanisms behind this relationship remain unknown. Here we hypothesize that ghrelin, an orexigenic hormone produced by the stomach and known to increase food reward behavior, also increases impulsivity. In order to assess the impact of ghrelin on impulsivity, rats were trained in three complementary tests of impulsive behavior and choice: differential reinforcement of low rate (DRL), go/no-go, and delay discounting. Ghrelin injection into the lateral ventricle increased impulsive behavior, as indicated by reduced efficiency of performance in the DRL test, and increased lever pressing during the no-go periods of the go/no-go test. Central ghrelin stimulation also increased impulsive choice, as evidenced by the reduced choice for large rewards when delivered with a delay in the delay discounting test. In order to determine whether signaling at the central ghrelin receptors is necessary for maintenance of normal levels of impulsive behavior, DRL performance was assessed following ghrelin receptor blockade with central infusion of a ghrelin receptor antagonist. Central ghrelin receptor blockade reduced impulsive behavior, as reflected by increased efficiency of performance in the DRL task. To further investigate the neurobiological substrate underlying the impulsivity effect of ghrelin, we microinjected ghrelin into the ventral tegmental area, an area harboring dopaminergic cell bodies. Ghrelin receptor stimulation within the VTA was sufficient to increase impulsive behavior. We further evaluated the impact of ghrelin on dopamine-related gene expression and dopamine turnover in brain areas key in impulsive behavior control. This study provides the first demonstration that the stomach-produced hormone ghrelin increases impulsivity and also indicates that ghrelin can change two major components of impulsivity-motor and choice impulsivity.

The glucagon-like peptide 1 (GLP-1) system is a recently established target for type 2 diabetes treatment. In addition to regulating glucose homeostasis, GLP-1 also reduces food intake. Previous studies demonstrate that the anorexigenic effects of GLP-1 can be mediated through hypothalamic and brainstem circuits which regulate homeostatic feeding. Here, we demonstrate an entirely novel neurobiological mechanism for GLP-1-induced anorexia in rats, involving direct effects of a GLP-1 agonist, Exendin-4 (EX4) on food reward that are exerted at the level of the mesolimbic reward system. We assessed the impact of peripheral, central, and intramesolimbic EX4 on two models of food reward: conditioned place preference (CPP) and progressive ratio operant-conditioning. Food-reward behavior was reduced in the CPP test by EX4, as rats no longer preferred an environment previously paired to chocolate pellets. EX4 also decreased motivated behavior for sucrose in a progressive ratio operant-conditioning paradigm when administered peripherally. We show that this effect is mediated centrally, via GLP-1 receptors (GLP-1Rs). GLP-1Rs are expressed in several key nodes of the mesolimbic reward system; however, their function remains unexplored. Thus we sought to determine the neurobiological substrates underlying the food-reward effect. We found that the EX4-mediated inhibition of food reward could be driven from two key mesolimbic structures—ventral tegmental area and nucleus accumbens—without inducing concurrent malaise or locomotor impairment. The current findings, that activation of central GLP-1Rs strikingly suppresses food reward/motivation by interacting with the mesolimbic system, indicate an entirely novel mechanism by which the GLP-1R stimulation affects feeding-oriented behavior.

6-Hydroxydopamine (6-OHDA) lesions are being used in the mouse for basic research on Parkinson's disease and L-DOPA-induced dyskinesia. We set out to compare unilateral lesion models produced by intrastriatal or intramesencephalic injections of a fixed 6-OHDA concentration (3.2 μg/μl) in C57BL/6 mice. In the first experiment, toxin injections were performed either at two striatal coordinates (1 or 2 μl per site, termed "striatum(2 × 1 μl)" and "striatum(2 × 2 μl)" models), in the medial forebrain bundle (MFB), or in the substantia nigra pars compacta (SN) (1 μl per site). All the four lesion models produced significant forelimb use asymmetry, but spontaneous turning asymmetry only occurred in the MFB and striatum(2 × 2 μl) models. After the behavioral studies, the induction of phosphorylated extracellular signal-regulated kinases 1 and 2 (pERK1/2) by acute L-DOPA (30 mg/kg) was used as a marker of post-synaptic supersensitivity. Striatal pERK1/2 expression was sparse in the SN and striatum(2 × 1 μl) groups, but pronounced in the striatum(2 × 2 μl) and MFB-lesioned mice. In further experiments, mice with MFB and striatal(2 × 2 μl) lesions were used to compare behavioral and molecular responses to chronic L-DOPA treatment (12 days at 3 and 6 mg/kg/day). Maximally severe abnormal involuntary movements (AIMs) occurred in all MFB-lesioned mice, whereas only 35% of the mice with striatal lesions developed dyskinesia. Striatal tissue levels of dopamine were significantly lower in the dyskinetic animals (both MFB and striatum(2 × 2 μl) groups) in comparison with the non-dyskinetic ones. Noradrenaline levels were significantly reduced only in MFB lesioned animals and did not differ among the dyskinetic and non-dyskinetic cases with striatal lesions. In all groups, the L-DOPA-induced AIM scores correlated closely with the number of cells immunoreactive for tyrosine hydroxylase or FosB/∆FosB in the striatum. In conclusion, among the four lesion procedures examined here, only the MFB and striatum(2 × 2 μl) models yielded a degree of dopamine denervation sufficient to produce spontaneous postural asymmetry and molecular supersensitivity to L-DOPA. Both lesion models are suitable to reproduce L-DOPA-induced dyskinesia, although only MFB lesions yield a pronounced and widespread expression of post-synaptic supersensitivity markers in the striatum.

L-DOPA-induced dyskinesia in Parkinson's disease is associated with large increases in brain dopamine (DA) levels following drug dosing, but the precise significance of this phenomenon is not understood. Here we compare DA efflux and metabolism in the striatum and the substantia nigra in dyskinetic and non-dyskinetic animals following a standard dose of L-DOPA. Rats with 6-hydroxydopamine lesions were treated chronically with L-DOPA, monitored on the abnormal involuntary movements scale, and then subjected to intracerebral microdialysis under freely-moving conditions. Following s.c. L-DOPA injection, peak extracellular DA levels in both striatum and substantia nigra were about twice as large in dyskinetic animals compared to non-dyskinetic rats. This effect was not attributable to differences in DOPA levels or DA metabolism. The larger DA efflux in dyskinetic animals was blunted by 5-HT1A/5-HT1B receptor agonists and tetrodotoxin infusion, reflecting release from serotonin neurons. Striatal levels of serotonin and its main metabolite, 5-hydroxyindolacetic acid were indeed elevated in dyskinetic animals compared to non-dyskinetic rats, indicating a larger serotonergic innervation density in the former group. High DA release was, however, not sufficient to explain dyskinesia. The 'abnormal involuntary movements output' per unit concentration of striatal extracellular DA was indeed much larger in dyskinetic animals compared to non-dyskinetic cases at most time points examined. The present results indicate that both a high DA release post-L-DOPA administration and an increased responsiveness to DA must coexist for a full expression of dyskinesia.

Glucagon-like peptide 1 (GLP-1), produced in the intestine and hindbrain, is known for its glucoregulatory and appetite suppressing effects. GLP-1 agonists are in clinical use for treatment of type 2 diabetes and obesity. GLP-1, however, may also affect brain areas associated with emotionality regulation. Here we aimed to characterize acute and chronic impact of GLP-1 on anxiety and depression-like behavior. Rats were subjected to anxiety and depression behavior tests following acute or chronic intracerebroventricular or intra-dorsal raphe (DR) application of GLP-1 receptor agonists. Serotonin or serotonin-related genes were also measured in the amygdala, DR and the hippocampus. We demonstrate that both GLP-1 and its long lasting analog, Exendin-4, induce anxiety-like behavior in three rodent tests of this behavior: black and white box, elevated plus maze and open field test when acutely administered intraperitoneally, into the lateral ventricle, or directly into the DR. Acute central GLP-1 receptor stimulation also altered serotonin signaling in the amygdala. In contrast, chronic central administration of Exendin-4 did not alter anxiety-like behavior but significantly reduced depression-like behavior in the forced swim test. Importantly, this positive effect of Exendin-4 was not due to significant body weight loss and reduced food intake, since rats pair-fed to Exendin-4 rats did not show altered mood. Collectively we show a striking impact of central GLP-1 on emotionality and the amygdala serotonin signaling that is divergent under acute versus chronic GLP-1 activation conditions. We also find a novel role for the DR GLP-1 receptors in regulation of behavior. These results may have direct relevance to the clinic, and indicate that Exendin-4 may be especially useful for obese patients manifesting with comorbid depression.

Background: The anxiety-reducing effect of long-term administration of serotonin reuptake inhibitors is usually seen only in subjects with anxiety disorders, and such patients are also abnormally inclined to experience a paradoxical anxiety-enhancing effect of acute serotonin reuptake inhibition. These unique responses to serotonin reuptake inhibitors in anxiety-prone subjects suggest, as do genetic association studies, that inter-individual differences in anxiety may be associated with differences in serotonergic transmission. Methods: The one-third of the animals within a batch of Wistar rats most inclined to spend time on open arms in the elevated plus maze were compared with the one-third most inclined to avoid them with respect to indices of brain serotonergic transmission and how their behavior was influenced by serotonin-modulating drugs. Results: “Anxious” rats displayed higher expression of the tryptophan hydroxylase-2 gene and higher levels of the tryptophan hydroxylase-2 protein in raphe and also higher levels of serotonin in amygdala. Supporting these differences to be important for the behavioral differences, serotonin depletion obtained by the tryptophan hydroxylase-2 inhibitor p-chlorophenylalanine eliminated them by reducing anxiety in “anxious” but not “non-anxious” rats. Acute administration of a serotonin reuptake inhibitor, paroxetine, exerted an anxiety-enhancing effect in “anxious” but not “non-anxious” rats, which was eliminated by long-term pretreatment with another serotonin reuptake inhibitor, escitalopram. Conclusions: Differences in an anxiogenic impact of serotonin, which is enhanced by acute serotonin reuptake inhibitor administration, may contribute to differences in anxiety-like behavior amongst Wistar rats.

Previous data indicate that the release of somatodendritic dopamine in substantia nigra influences motor activity and coordination, but the relative importance of somatodendritic dopamine release vs. terminal striatal dopamine release remains to be determined. We utilized simultaneous measurement of dopamine neurotransmission by microdialysis and motor performance assessment by rotarod test to investigate the effects of local dopamine depletion in rats. The vesicular monoamine transporter inhibitor tetrabenazine (100 microm) was administered locally in substantia nigra as well as in striatum. Nigral tetrabenazine administration decreased nigral dopamine dialysate concentrations to 7% of baseline and whole-tissue dopamine content by 60%. Nigral dopamine depletion was associated with a reduction in motor performance to 73 +/- 6% of pretreatment value, but did not alter dialysate dopamine concentrations in the ipsilateral striatum. Striatal tetrabenazine administration decreased striatal dopamine dialysate concentrations to 5% of baseline and doubled the somatodendritic dopamine response to motor activity, but it was not associated with changes in motor performance or dopamine content in striatal tissue. Simultaneous treatment of substantia nigra and striatum reduced motor performance to 58 +/- 5% of the pretreatment value. The results of this study indicate that partial depletion of nigral dopamine stores can significantly impair motor functions, and that increased nigral dopamine release can counteract minor impairments of striatal dopamine transmission.

Although dendritic release was first proposed in the 1970s, the mechanism of release is still subject to debate. We have used in vivo microdialysis to study the acute effects of botulinum toxin A, B and tetanus toxin injected in the substantia nigra or striatum of freely moving rats. Spontaneous and evoked dopamine release decreased in both regions after treatment with the SNAP-25 (synaptosome-associated protein of 25 kDa) cleaving protease botulinum toxin A (1000 mouse lethal doses, MLD). Tetanus toxin (4000 MLD) did not significantly change spontaneous or evoked dopamine release in striatum or in the substantia nigra. Another synaptobrevin cleaving protease, botulinum toxin B, inhibited release in the striatum by 55% but did not affect dopamine release when injected in the substantia nigra. The results indicate that both terminal and somatodendritic dopamine release need intact SNAP-25 to occur, but somatodendritic dopamine release in contrast to terminal release depends on a botulinum toxin B resistant pathway.

The sigma-1 receptor, an endoplasmic reticulum-associated molecular chaperone, is attracting great interest as a potential target for neuroprotective treatments. We provide the first evidence that pharmacological modulation of this protein produces functional neurorestoration in experimental parkinsonism. Mice with intrastriatal 6-hydroxydopamine lesions were treated daily with the selective sigma-1 receptor agonist, PRE-084, for 5 weeks. At the dose of 0.3 mg/kg/day, PRE-084 produced a gradual and significant improvement of spontaneous forelimb use. The behavioural recovery was paralleled by an increased density of dopaminergic fibres in the most denervated striatal regions, by a modest recovery of dopamine levels, and by an upregulation of neurotrophic factors (BDNF and GDNF) and their downstream effector pathways (extracellular signal regulated kinases 1/2 and Akt). No treatment-induced behavioural-histological restoration occurred in sigma-1 receptor knockout mice subjected to 6-hydroxydopamine lesions and treated with PRE-084. Immunoreactivity for the sigma-1 receptor protein was evident in both astrocytes and neurons in the substantia nigra and the striatum, and its intracellular distribution was modulated by PRE-084 (the treatment resulted in a wider intracellular distribution of the protein). Our results suggest that sigma-1 receptor regulates endogenous defence and plasticity mechanisms in experimental parkinsonism. Boosting the activity of this protein may have disease-modifying effects in Parkinson's disease.

Intravenous administration of m-chloro-phenylpiperazine (mCPP) (0.25 or 2.5 mg/kg) induced a marked and dose-related increase in extracellular concentrations of serotonin in hippocampus (300-1,400% of baseline) as measured using in vivo microdialysis in awake male Wistar rats of the spontaneously hypertensive (SH) strain. Indicating that the effect of mCPP was caused by a reversal of the serotonin transporter, it was antagonized by pretreatment with the serotonin re-uptake inhibitor citalopram (10 mg/kg) but was unaffected by local administration of the sodium channel blocker tetrodotoxin (TTX; 1 microns). mCPP was also shown to induce an increase in extracellular concentrations of dopamine in the nucleus accumbens and the striatum of SH rats and in the nucleus accumbens of rats of the Sprague-Dawley (SD) strain; this effect of mCPP was, however, much weaker (125-170% of baseline) than the effect on serotonin; moreover, it seems to be TTX-sensitive. In anesthetized SD rats, mCPP induced a moderate reduction of nigral dopamine cell firing rate; supporting the assumption that this effect is secondary to the observed increase in dopamine release, it was blocked by pretreatment either with the dopamine synthesis inhibitor alpha-methyl-para-tyrosine or with the dopamine D2 receptor antagonist haloperidol. In conclusion, the results suggest that mCPP induces a marked, TTX-insensitive increase in serotonin release in rat brain, but only a modest and TTX-sensitive increase in the extracellular levels of dopamine.

A 15-mer, all-phosphorothioate-modified antisense oligodeoxynucleotide (ASO) targeted against rat dopamine D3 receptor mRNA (4 microM, 5 days) significantly reduced (28%) the amount of binding sites labelled with [3H]spiperone in monolayer cultured Chinese hamster ovary (CHO) cells transfected with the complementary desoxyribonucleic acid (cDNA) for the rat D3 receptor. In contrast, D3-ASO treatment did not reduce the amount of bound [3H]spiperone in CHO cells transfected with D2 receptor cDNA. Intracerebroventricular infusion of D3-ASO (osmotic minipump, 10 microg/microl/h, 7 days) influenced dopamine receptor density in the limbic forebrain such that the upper part of the dopamine/[3H]spiperone displacement curve--tentatively representing the D3 receptor--was altered significantly. Spontaneous locomotor activity of non-habituated rats was increased significantly in D3-ASO-treated animals; in addition, in vivo microdialysis revealed a moderate increase in dopamine release in the nucleus accumbens in these animals. In all experiments, an oligodeoxynucleotide comprising the same nucleotides as the antisense sequence, but in random order, was used as control. It is concluded that the antisense strategy is useful for investigating the functional role of dopamine D3 receptors and that the dopamine D3 receptor is involved in rat locomotor behaviour.

Previous studies have shown that administration of gamma-hydroxybutyric acid (GHBA) or the GABA(B) receptor agonist baclofen are associated with a decrease in firing rate, a regularisation of firing pattern and a decrease in burst activity of midbrain dopamine (DA) neurons in the substantia nigra (SN). In the present study we compared the ability of the novel GABA(B) receptor antagonist SCH 50911 and the selective antagonist of GHBA binding sites, NCS-382, to antagonise the effects of baclofen or GHBA, respectively, on the neuronal activity of DA neurons in anaesthetised rats. SCH 50911 (75 mg/kg, i.v.) was found to antagonise the decrease in firing rate, the regularisation of firing rhythm and the decrease of burst activity in DA cells, induced by baclofen (1-32 mg/kg, i.v.) or GHBA (12.5-1600 mg/kg, i.v.). NCS-382 (100 mg/kg, i.v.) did not affect the baclofen-induced changes in neuronal activity. Neither was the drug able to influence the GHBA-induced alterations in firing rate or in burst activity, although NCS-382 to some extent antagonised the regularisation of the firing pattern observed following low doses of GHBA (or =100 mg/kg). The results of the present study give further support for the notion that the GHBA-induced changes in neuronal activity of nigral dopamine neurons are mediated by stimulation of GABA(B) receptors.

The importance of voltage-dependent sodium channels and different types of voltage-sensitive calcium channels for depolarisation-induced release of endogenous dopamine from dendrites and cell bodies in superfused guinea pig substantia nigra slices was investigated. The stimulatory effect of veratridine (10 microM) on dopamine release was only marginally attenuated in Ca(2+)-free medium but was completely blocked by tetrodotoxin (1 microM) and by the dopamine reuptake inhibitor GBR 12909 (10 microM). Low extracellular concentration of Na+ stimulated the dopamine release. Potassium-evoked dopamine release was completely Ca(2+)-dependent, not blocked by GBR 12909 and partially blocked by tetrodotoxin. Nifedipine (20 microM), omega-conotoxin GVIA (0.5 microM), penfluridol (5 microM), and Ni2+ (20 microM) had no effect, amiloride (1 mM) attenuated and neomycin (350 microM), and omega-agatoxin IVA (1 microM) almost totally blocked the potassium-induced dopamine release. The results suggest that veratridine released dopamine mostly by reversing the dopamine transporter. High concentrations of potassium induced release of nigral dopamine by opening of voltage-sensitive calcium channels of P/Q type but not L-type, N-type and probably not T-type. The depolarisation evoked by high concentrations of potassium seems to open voltage-sensitive calcium channels both by the depolarisation induced by potassium per se and by the secondary depolarisation induced by opening of voltage-dependent sodium channels.

In the present study the effect of inhibition of the re-uptake of dopamine (DA) was analysed with respect to DA release and to the firing pattern of DA neurons in the substantia nigra (SN). Intravenous administration of GBR 12909 (0.5-8 mg/kg), a specific and potent inhibitor of DA re-uptake, was found to dose-dependently increase the DA concentration both in the SN and in the striatum, as measured by microdialysis. However, the drug failed to significantly affect the firing rate of the nigral DA neurons. In contrast, GBR 12909 dose-dependently induced a regularisation of the firing pattern, concomitant with a reduction in burst activity. An acute hemisection of the brain, which by itself produced a slight regularisation of the firing pattern of the nigral DA neurons without changing the firing rate of the ability of the DA neurons to fire in bursts, was found to prevent the regulatory action of GBR 12909. Pretreatment with the selective GABAB-receptor antagonist CGP 35348 (200 mg/kg, i.v., 5 min) did not significantly affect the firing rate, the regularity of the DA neurons, or their ability to fire in bursts. However, CGP 35348 markedly antagonised the ability of GBR 12909 to induce pacemaker-like firing or a decrease in burst activity of the nigral DA neurons. The results of the present study suggest that a striatonigral feedback projection may serve to control the activity of nigral DA neurons not primarily by regulating the firing rate, but, preferably, by modulating the firing pattern of the neurons. In this regard, activation of somatodendritic GABAB-receptors may form the final link in this feedback inhibitory control system.

Mesolimbic dopamine plays a critical role in food-related reward processing and learning. The literature focuses primarily on the nucleus accumbens as the key dopaminergic target in which enhanced dopamine signaling is associated with reward. Here, we demonstrate a novel neurobiological mechanism by which dopamine transmission in the amygdala regulates food intake and reward. We show that food intake was associated with increased dopamine turnover in the amygdala. Next, we assess the impact of direct intra-amygdala D1 and D2 receptor activation on food intake and sucrose-driven progressive ratio operant conditioning in rats. Amygdala D2 receptor activation reduced food intake and operant behavior for sucrose, whereas D2 receptor blockade increased food intake but surprisingly reduced operant behavior. In contrast, D1 receptor stimulation or blockade did not alter feeding or operant conditioning for food. The glucagon-like peptide 1 (GLP-1) system, a target for type 2 diabetes treatment, in addition to regulating glucose homeostasis, also reduces food intake. We found that central GLP-1R receptor activation is associated with elevated dopamine turnover in the amygdala, and that part of the anorexic effect of GLP-1 is mediated by D2 receptor signaling in the amygdala. Our findings indicate that amygdala dopamine signaling is activated by both food intake and the anorexic brain-gut peptide GLP-1 and that amygdala D2 receptor activation is necessary and sufficient to change feeding behavior. Collectively these studies indicate a novel mechanism by which the dopamine system affects feeding-oriented behavior at the level of the amygdala.

Previous studies have shown that administration of γ-hydroxybutyric acid (GHBA) or baclofen is associated with a decrease in locomotor activity as well as an increase of dopamine (DA) in brain. In the present study we analyse whether these actions are related to activation of GABA B -receptors utilising a GABA B -receptor antagonist, CGP 35348. Administration of GHBA (200 or 800 mg/kg, i.p.) or baclofen (4 or 16 mg/kg, i.p.) induced a marked and dose-dependent decrease in locomotor activity in mice, that was antagonised by pretreatment with CGP 35348 (400 mg/kg, i.p.). Treatment with the highest doses of GHBA and baclofen produced clear-cut increases in forebrain DA concentration. Also these effects were effectively antagonised by pretreatment with CGP 35348. Treatment with the GABA B -receptor antagonist alone did not influence the locomotor activity or brain DA concentration. These results indicate that the behaviourally depressive and DA increasing effects of GHBA and baclofen are mediated by activation of GABA B -receptors.

Background The vestibular system is connected to spinal, cerebellar and cerebral motor control structures and can be selectively activated with external electrodes. The resulting sensation of disturbed balance can be avoided by using stochastic stimulation patterns. Adding noise to the nervous system sometimes improves function. Small clinical trials suggest that stochastic vestibular stimulation (SVS) may improve symptoms in Parkinson's disease. We have investigated this claim and possible mechanisms using the 6-hydroxydopamine (6-OHDA) hemilesion model of Parkinson's disease. Methodology/Principal Findings Animals were tested in the accelerating rod test and the Montoya staircase test of skilled forelimb use. In 6-OHDA hemilesioned animals, SVS improved rod performance by 56±11 s. At group level L-DOPA treatment had no effect, but positive responders improved time on rod by 60±19 s. Skilled forelimb use was not altered by SVS. To investigate how SVS may influence basal ganglia network activity, intracerebral microdialysis was employed in four regions of interest during and after SVS. In presence of the γ-amino buturic acid (GABA) transporter inhibitor NNC 711, SVS induced an increase in GABA to 150±15% of baseline in the substantia nigra (SN) of unlesioned animals, but had no effect in the pedunculopontine nucleus (PPN), the striatum or the ventromedial thalamus (VM). Dopamine release remained stable in all areas, as did GABA and amine concentrations in the SN of unstimulated controls. Following SVS, a sustained increase in GABA concentrations was observed in the ipsilesional, but not in the contralesional SN of 6-OHDA hemilesioned rats. In contrast, L-DOPA treatment produced a similar increase of GABA in the ipsi- and contra-lesional SN. Conclusions/Significance SVS improves rod performance in a rat model of Parkinson's disease, possibly by increasing nigral GABA release in a dopamine independent way. We propose that SVS could be useful for treating symptoms of Parkinson's disease.

Results obtained previously indicate that the firing pattern of midbrain dopamine (DA) neurons is of importance for the terminal DA release. In the present combined electrophysiological and microdialysis study, the influence of the firing pattern on striatal DA release was studied by using the previously observed ability of low doses of β-hydroxybutyrate (GHBA) to profoundly regularise the firing pattern of rat DA neurons in the substantia nigra (SN). Administration of GHBA (200 mg/kg, i.v.) did not significantly reduce the firing rate of any of the DA neurons recorded from, but rather caused a slight transient excitation. However, this dose of the drug caused a profound regularisation of the firing pattern and abolished burst activity of the DA neurons. The DA concentration in the dialysate obtained from the striatum (10 min sampling periods) decreased with the lowest value (67% of predrug value) observed at the sampling period 20-30 min after the GHBA administration. As a complement to microdialysis, the 3-methoxytyramine (3-MT) accumulation in striatal tissue following monoamine oxidase inhibition was determined as an indirect measure of DA release in vivo. The 3-MT concentrations in the striatum decreased to 84% of controls following 200 mg/kg of GHBA. To exclude an effect on DA release conceivably mediated by GHBA locally in the striatum, GHBA (10−7-10−3 M) was given locally in the dialysis probe and was found to increase DA in the dialysate (maximally to 140% of controls). The present results are in line with previous electrophysiological studies which have demonstrated that artificially induced burst firing by electrical stimulation is associated with an increased extracellular level of striatal DA (as determined by in vivo voltammetric techniques or microdialysis) and support the idea that firing pattern may be an important physiological modulatory mechanism for the release of terminal neurotransmitter. © 1994 Wiley-Liss, Inc.

Previous electrophysiological experiments have emphasized the importance of the firing pattern for the functioning of midbrain dopamine (DA) neurons. In this regard, excitatory amino acid receptors appear to constitute an important modulatory control mechanism. In the present study, extracellular recording techniques were used to investigate the significance of GABAB-receptor activation for the firing properties of DA neurons in the substantia nigra (SN) in the rat. Intravenous administration of the GABAB-receptor agonist baclofen (1-16 mg/kg) was associated with a dose-dependent regularization of the firing pattern, concomitant with a reduction in burst firing. At higher doses (16-32 mg/kg), the firing rate of the DA neurons was dose-dependently decreased. Also, microiontophoretic application of baclofen regularized the firing pattern of nigral DA neurons, including a reduction of burst firing. Both the regularization of the firing pattern and inhibition of firing rate produced by systemic baclofen administration was antagonized by the GABAB-receptor antagonist CGP 35348 (200 mg/kg, i.v.). The GABAA-receptor agonist muscimol produced effects on the firing properties of DA neurons that were opposite to those observed following baclofen, i.e., an increase in firing rate accompanied by a decreased regularity. The NMDA receptor antagonist MK 801 (0.4-3.2 mg/kg, i.v.) produced a moderate, dose-dependent increase in the firing rate of the nigral DA neurons as well as a slightly regularized firing pattern. Pretreatment with MK 801 (3.2 mg/kg, i.v., 3-10 min) did neither promote nor prevent the regularization of the firing pattern or inhibition of firing rate on the nigral DA neurons produced by baclofen. The present results clearly show that GABAB-receptors can alter the firing pattern of nigral DA neurons, hereby counterbalancing the previously described ability of glutamate to induce burst firing activity on these neurons.

Nigro-striatal neurons release dopamine not only from their axon terminals in the striatum, but also from somata and dendrites in the substantia nigra. Somatodendritic dopamine release in the substantia nigra can facilitate motor function by mechanisms that may act independently of axon terminal dopamine release in the striatum. The dopamine neurons in the substantia nigra receive a cholinergic input from the pedunculopontine nucleus. Despite recent efforts to introduce this nucleus as a potential target for deep brain stimulation to treat motor symptoms in Parkinson's disease; and the well-known antiparkinsonian effects of anticholinergic drugs; the cholinergic influence on somatodendritic dopamine release is not well understood. The aim of this study was to investigate the possible regulation of locomotor-induced dopamine release in the substantia nigra by endogenous acetylcholine release. In intact and 6-OHDA hemi-lesioned animals alike, the muscarinic antagonist scopolamine, when perfused in the substantia nigra, amplified the locomotor-induced somatodendritic dopamine release to approximately 200% of baseline, compared to 120-130% of baseline in vehicle-treated animals. A functional importance of nigral muscarinic receptor activation was demonstrated in hemi-lesioned animals, where motor performance was significantly improved by scopolamine to 82% of pre-lesion performance, as compared to 56% in vehicle-treated controls. The results indicate that muscarinic activity in the substantia nigra is of functional importance in an animal Parkinson's disease model, and strengthen the notion that nigral dopaminergic regulation of motor activity/performance is independent of striatal dopamine release.

Cytochrome P450 2E1 (CYP2E1) has been detected in brain regions which are of relevance for the pathophysiology of Parkinson's disease, such as the substantia nigra (SN). Furthermore, CYP2E1 is known to generate reactive oxygen species (ROS), toxic molecules which have been implicated in the pathogenesis of the disease. We have previously reported that CYP2E1 inhibition increases extracellular dopamine (DA) in the SN. The aims of the present study were by using in vivo microdialysis in rat, to elucidate the mechanisms responsible for the increase in extracellular DA induced by CYP2E1 inhibition and to explore whether ROS is produced in the SN, both with and without the presence of an exogenous CYP2E1 substrate. The effect of inhibition of CYP2E1 by phenylethyl isothiocyanate (100 mg/kg) on extracellular DA in the SN was unaltered following pretreatment with gamma-butyrolactone and GBR-12909, drugs that inhibit firing of DA neurons and DA re-uptake, respectively. Preadministration of tetrodotoxin or reserpine, however, abolished the effect of CYP2E1 inhibition. Administration of isoflurane, an anesthetic which is metabolized by CYP2E1, increased the production of *OH in the SN, as measured by the transformation of 4-hydroxybenzoic acid to 3,4-dihydroxybenzoic acid during local perfusion compared with animals given other anesthetics. The results support the notion that CYP2E1 is located near or in the same compartment in the SN as stored DA, tentatively the endoplasmatic reticulum, and that the enzyme activity might modulate the amount of DA that is available for release. Furthermore, our findings indicate that the production of ROS can be stimulated by CYP2E1 substrates.

The release of dopamine from soma and dendrites of dopaminergic neurons in substantia nigra has been reported to be calcium-dependent, but it remains to be determined which calcium channels mediate this effect. We have used in vivo microdialysis in rat substantia nigra and striatum to investigate the effect of Ca(v)3.1-3.3 (T-type) and Ca(v)2.3 (R-type) calcium channel antagonists on somatodendritic and terminal dopamine release. Local reverse dialysis administration of 0.1-10 microM of the Ca(v)2.3 inhibitor SNX-482, or 100 microM of mibefradil, decreased the concentrations of dopamine and its metabolites in dialysate from substantia nigra, whereas 1 microM mibefradil or 40-80 microM nickel(II) induced an increase in nigral dialysate dopamine concentrations. Dopamine concentrations in striatal dialysates were decreased only by 10 microM of SNX-482 or 100 microM of mibefradil. Nickel(II) induced an increase in striatal dialysate dopamine concentration similar to that in substantia nigra. The results indicate a role for Ca(v)2.3 (R-type) voltage sensitive calcium channels in the calcium dependency of somatodendritic dopamine release, but argue against a calcium dependency mediated substantially by Ca(v)3.1-3.3 (T-type) channels.

Although the major routes of dopamine metabolism seem to be established, at least in terminal regions such as the striatum, it is important to search for previously unknown metabolites and to investigate the relevance of previously suggested minor alternative pathways. An urgent issue is to verify and quantify the transformation of dopamine to putative toxic species, another is to further explore metabolism of dopamine located in cell bodies/dendrites, e.g. in the substantia nigra. We have developed a new method in order to widen the search for alternative metabolites of dopamine. The method is based on systemic injection of tritiated L-DOPA to rats in vivo . Brain tissue was homogenised and centrifuged and the resulting supernatant fractioned following passage through a liquid chromatography system. The radioactivity of each fraction was measured using a scintillation system. By identifying fractions containing major catecholamines and metabolites, according to a standard solution, novel metabolites can be searched for in the remaining fractions. It was possible to obtain sufficient radioactivity in separate fractions of supernatant of homogenised tissue, even from such a small brain nucleus as substantia nigra. Radioactivity was obtained in those fractions that contained the major catecholamines and their metabolites, as well as in other fractions where it may represent previously unknown metabolites of L-DOPA/dopamine. The method was used to evaluate the possibility that cytochrome P450 2E1 is involved in the metabolism of dopamine in the substantia nigra. Significant changes in the radioactivity pattern were induced by inhibition of the enzyme but conclusions about whether cytochrome P450 2E1 is involved in the metabolism of dopamine or not requires further studies. The method can be used to study the metabolism of dopamine and can be extended, by using other radiolabelled precursors, also to evaluate metabolism of other transmitters, e.g. serotonin.

The physiological role of somatodendritic dopamine release in the rat substantia nigra was evaluated with a combination of dual probe microdialysis and simultaneous motor performance tests on an accelerating rod. Three main findings support a modulating influence of somatodendritic dopamine release on motor coordination. (1) The rod performance tests were associated with an increase in extracellular dopamine but not 5-hydroxytryptamine concentrations in substantia nigra and with increases in both dopamine and 5-hydroxytryptamine concentrations in the striatum. (2) Nigral application of dopamine antagonists without intrinsic activity resulted in changed performances on the accelerating rod. The response to nigral perfusion with low concentrations (0.1, 1.0 microM) of the D(2)/D(3)-antagonist raclopride consisted of an impairment in rod performance to 63% of the pre-perfusion performance. Higher concentrations (10, 100 microM), however, were not associated with impaired rod performance, but with increased striatal dopamine concentrations. Perfusion of the substantia nigra with 1, 10 and 100 microM of the D(1)/D(5)-antagonist SCH 23390 dose-dependently impaired rod performance. SCH 23390 consistently increased dopamine and 5-hydroxytryptamine concentrations in substantia nigra but did not change the dialysate in the striatum. (3) In unilaterally 6-hydroxydopamine-lesioned rats, a dose-dependent improvement in rod performance was observed during perfusion of the substantia nigra with the non-selective dopamine agonist apomorphine.

We presented data previously on dopamine (DA) synthesis and catabolism in the rat substantia nigra (SN) suggesting that a substantial part of the synthesized DA in this brain part is metabolized by unknown nonclassical metabolic pathways. On the basis of that a relatively high density of cytochrome P450 2E1 (CYP 2E1) has been detected in rat SN the aim of the present study was to investigate the possibility that this enzyme is involved in the metabolism of DA. Systemic administration of either phenylethyl isothiocyanate (100 mg/kg ip), diethyldithiocarbamate (500 mg/kg, ip) or diallyl sulfide (200 mg/kg, sc or ip), three different inhibitors of cytochrome P450 2E1, induced an increase of the extracellular DA concentration in the SN, measured with microdialysis in awake rats, by 130%, 90%, and 35%, respectively. A tendency to increased concentrations of the classical DA metabolites in the dialysate from the SN was also observed in some experiments. In the striatum, no profound effects were induced by the drugs on the concentrations of DA or its metabolites. The results show that CYP 2E1 activity affects dopaminergic neurotransmission in the SN, possibly by participating in DA metabolism. Other mechanisms, such as the influence on the DA transporter or the release process cannot, however, be ruled out.

Several serotonergic drugs are effective for anxiety disorders, but underlying mechanisms are unclear, and findings in experimental animals are difficult to reconcile with human data. It has been proposed that differential effects of serotonin within specific anatomical systems may account for these difficulties, and the amygdala has been suggested as one of the structures involved. To examine this hypothesis, the neurotoxin 5,7-dihydroxytryptamine was administered locally in rat amygdala. Within the amygdala, serotonin was depleted by approximately 80%, with other transmitters unaffected, and serotonin transporter labelling was decreased by approximately 85%. Cortical areas near the lesion site were also affected, although to a lesser degree. Other forebrain areas were unaffected. Lesions resulted in a specific anti-conflict effect in a punished drinking test, but did not influence elevated plus-maze behavior (under baseline conditions and after restraint stress), locomotor activity or ethanol intake. These data suggest that the punished drinking test and the elevated plus-maze may activate different components of fear circuitry, and that the serotonergic input to the amygdala specifically participates in fear-related behavioral suppression mediated by this structure. © 2001 American College of Neuropsychopharmacology.

Previous studies have shown that systemic as well as local administration of the GABA(B)-receptor agonist baclofen is associated with a decrease in firing rate, a regularisation of firing rhythm and a decrease in burst firing activity of dopamine (DA) containing midbrain neurons. In the present electrophysiological study we have utilised the novel, selective and potent GABA(B)-receptor antagonist SCH 50911 in order to further analyse the importance of GABA(B)-receptors for the overall activity of rat nigral DA neurons. SCH 50911 given intravenously (1-64 mg/kg) or locally, by microiontophoretic techniques, was found to increase firing rate and to increase the burst firing activity of DA neurons. The present data suggest that the GABA(B)-receptor antagonist blocks somatodendritic receptors on nigral DA neurons. This GABA-receptor input appears to be of a tonic nature. It is proposed that the activation of nigral DA neurons may underlie the beneficial effects of GABA(B)-receptor antagonists in the modulation of cognition and that GABA(B)-receptor antagonists may be of therapeutic value in the treatment of Parkinson's disease.

The pivotal role for voltage-sensitive calcium channels in initiating synaptic transmitter release is undisputed, but it is only partly known to what extent the different subtypes contribute in vivo. Their importance for the dendritic release of dopamine has not been investigated in vivo previously. To evaluate comprehensively the relative importance of different voltage-sensitive calcium channel subtypes for striatal dopamine release, and to further investigate the mechanism of dendritic dopamine release in the reticulate part of substantia nigra, dopamine was measured by in vivo microdialysis in the striatum or substantia nigra of awake rats. The calcium channel blockers nimodipine, omega-conotoxin-GVIA, omega-agatoxin-IVA, and neomycin were administered locally through the dialysis probes and compared with calcium-free perfusion. Results indicate that dopamine release in the striatum is mainly dependent on N- and P/Q-type channels, but the dendritic dopamine release in the substantia nigra is mediated mainly by some other calcium-dependent mechanism, for example, calcium mobilization through T-, O-, or R-type calcium channels. A portion of the dendritic release is calcium independent but can be inhibited partially by neomycin, which might suggest a role for inositol 4,5-bisphosphate breakdown products.

This study was undertaken, using microdialysis, to compare the extracellular concentration of 3-methoxytyramine and dopamine in dialysate from the striatum and substantia nigra, after pargyline (75 mg/kg), after pargyline plus amphetamine (3 mg/kg), and after pargyline plus reserpine (5 mg/kg) administration. Treatment with pargyline alone increased the extracellular dopamine concentration by 70% in the striatum and by 140% in the substantia nigra and induced in both regions a time-dependent accumulation of 3-methoxytyramine. The addition of d-amphetamine to pargyline increased the extracellular dopamine concentration, compared with pargyline-treated controls, to the same extent in both the substantia nigra (maximally by 360%) and the striatum (maximally by 400%), but the concomitant increase of 3-methoxytyramine accumulation in the dialysate was relatively smaller in the substantia nigra compared with the striatum. Reserpine treatment decreased the extracellular dopamine concentration in both regions below the detection level (

Intracerebroventricular infusion of an all-phosphorothioate antisense oligodeoxynucleotide targeted at the rat dopamine D3 receptor mRNA (10 micrograms h-1, 5 days) resulted in a significant reduction (19%) of the binding of the [D2 + D3] ligand [3H]spiperone in the limbic forebrain, where D3 receptors are relatively abundant, but not in caudate-putamen, where D3 receptors are sparse. In nucleus accumbens antisense treatment also caused an increase in dopamine synthesis; in contrast, antisense administration did not counteract the effect of apomorphine on dopamine formation. No effect of antisense administration on dopamine synthesis was observed in caudate-putamen. The results support the usefulness of the antisense strategy for the study of D3 receptors and suggest that D3 receptors may influence dopamine synthesis.

It has been suggested that ethanol may interact with the central nicotinic acetylcholine receptor, thus providing a basis for the often observed high consumption of both ethanol and nicotine. In the present in vivo microdialysis study, ethanol (2.5 g/kg) moderately increased dopamine overflow in the rat nucleus accumbens. The central nicotinic acetylcholine receptor antagonist mecamylamine totally counteracted this effect in a dose (1.0 mg/kg) that did not alter dopamine overflow per se. Ethanol also increased the overflow of dihydroxyphenylacetic acid and homovanillic acid, but this effect was not altered by mecamylamine (1.0 mg/kg). Furthermore, the ethanol-induced enhancement of 3,4-dihydroxyphenylalanine accumulation in the mesolimbic dopamine terminal area after NSD 1015 (an inhibitor of l-aromatic amino acid decarboxylase) was completely antagonized by mecamylamine in doses (3.0 and 6.0 mg/kg) that exerted no effects per se. Neither ethanol nor mecamylamine changed the catecholamine synthesis rate in the striatum or the cerebral cortex. These results provide further evidence that ethanol-induced activation of the mesolimbic dopamine system (increased dopamine synthesis and release) may be mediated via stimulation of central nicotinic acetylcholine receptors. It is suggested that antagonists of central nicotinic acetylcholine receptors may be useful in the treatment of alcoholism.

In the present study the actions of gamma-hydroxybutyric acid (GHBA) on dopaminergic neurons in the rat substantia nigra (SN) were pharmacologically analysed utilising extracellular single unit recording techniques. Intravenous administration of GHBA was associated with several effects on the neuronal activity of nigral dopamine (DA) neurons. Low doses (200 mg/kg) of GHBA produced a slight excitation of the neurons, concomitant with a regularized firing rhythm and lack of burst activity. In higher doses GHBA produced an even higher degree of regularization but, in contrast to low doses, an inhibition of firing rate. Administration of the GABAB-receptor agonist baclofen, in all essential respects, mimicked the effect of GHBA on the firing of nigral DA neurons. Both the regularization of the firing pattern and inhibition of firing rate produced by systemic administration of GHBA were antagonised by the GABAB-receptor antagonist CGP 35348 (200 mg/kg, i.v.). Our observations show that GHBA affects the firing pattern of nigral DA neurons in doses considerably lower than those required to inhibit the firing rate of the neurons. This action, as well as the decreased firing rate observed after high doses of GHBA, are mediated via activation of GABAB-receptors.

Dopamine (DA) is synthesized and released not only from the terminals of the nigrostriatal dopaminergic neuronal pathway but also from cell bodies and dendrites in the substantia nigra (SN). In most of the in vitro studies on DA release in the SN, release of exogenously applied 3H-DA has been determined either from slices or from synaptosomes. However 3H-DA has some disadvantages; 3H-DA is taken up and released not only from dopaminergic cells but also from other neuronal elements and radiolabelled DA may not be evenly distributed with the releasable endogenous pool of the amine. Therefore we have developed a method for determination of the release of endogenous DA from superfused guinea pig SN slices. We have used a superfusion system in which 6 slices are placed into a microchamber. Samples of superfusate were collected every 10 min for up to 3 h and biochemical analyses were performed by electrochemical detection preceded by adsorbtion of DA on alumina and chromatography on a cation exchange column. Expected increases of the release of endogenous DA were obtained following d -amphetamine and potassium administration. The data indicate that it is possible to measure endogenous release of DA from guinea pig SN slices with standard HPLC technique and follow the release for a relatively long time.

The effects of serotoninergic drugs on dopaminergic neurotransmission in the substantia nigra, the striatum and the limbic forebrain of rat have been investigated. The accumulation of 3-methoxytyramine (3-MT) following inhibition of monoamine oxidase with pargyline was used as an indirect measure of dopamine (DA) activity in vivo. The effects of the following serotoninergic drugs were tested: the 5-HT1A receptor agonist 8-OH-DPAT, the 5-HT1B receptor agonist trifluoromethyl-phenylpiperazine (TFMPP), CGS 12066B and RU 24969, the 5-HT1A/1B antagonist (±)pindolol, the 5-HT2/1C receptor antagonist ritanserin, the 5-HT2/1C receptor agonist DL-1-(2,5-dimethoxy-4-iodo-phenyl)-2-aminopropane (DOI), the 5-HT3 receptor antagonist BRL 43 694, the unselective 5-HT1 receptor antagonist methiothepin, and carbidopa+L-5-hydroxytryptophan (L5-HTP) to achieve a general, unselective stimulation of multiple 5-HT receptors. In the substantia nigra, carbidopa + 5-HTP treatment increased the 3-MT accumulation by 26% and decreased the DA concentration to 67% of controls, tentatively suggesting a 5-HTP-induced displacement of nigral DA. A minor, non dose-related reduction in nigral 3-MT was seen after the 5-HT1A receptor agonist 8-OH-DPAT. None of the other serotonin receptor acting drugs induced any pronounced effect on the nigral 3-MT accumulation. Taken together, the findings provide little support for the idea that one single 5-HT1 receptor subtype serves a modulatory function on DA activity in the substantia nigra. In the striatum and the limbic forebrain, trifluoromethylphenylpiperazine dose-dependently increased the 3-MT accumulation to maximally 200%–220% of controls. In the limbic forebrain also the highest dose of RU 24 969 (15 mg/kg) increased the 3-MT accumulation (78%), whereas in the striatum the lowest does of the drug (1.5 mg/kg) decreased it by 30%. The trifluoromethylphenylpiperazine-induced stimulation of 3-MT accumulation was not blocked by ritanserin. In the limbic forebrain, also DL-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane and carbidopa+Lr5-HTP treatment increased the 3-MT concentrations to 120% and 150% of controls, respectively. Paradoxically, methiothepin also induced an increase of the 3-MT accumulation in these brain regions, probably due to its DA receptor antagonism. None of the other serotoninergic drugs induced any pronounced effects on the 3-MT accumulation in these brain parts. The results may overall support the hypothesis that 5-HT1 does modulate the DA activity in the striatum and limbic forebrain, tentatively via 5-HT1B receptors in the striatum and 5-HT1B and 5-HT2 or 5-HT1C receptors in the limbic forebrain. It may be speculated therefore, that clinical application of 5-HT1 receptor-modulating drugs to influence central dopaminergic activity might be of therapeutical benefit, for example, in motor disorders like Parkinson's disease.

In order to investigate the physiological importance of the membrane pump in eliminating released dopamine (DA) we have studied the effects of the putative selective dopamine re-uptake inhibitor, GBR 12909, on synthesis and metabolism of monoamines in the rat striatum, limbic forebrain, cortical hemispheres and substantia nigra (SN). The effects of the drug on the firing rate of catecholamine containing neurons in the SN and locus coeruleus (LC) were also investigated. For comparison we have investigated the effects of desipramine and maprotiline. As a measure of the synthesis of noradrenaline (NA), DA and 5-hydroxytryptamine (5-HT) we determined the 3,4-dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan (5-HTP) accumulation after inhibition of aromatic L-amino acid decarboxylase by 3-hydroxy-benzylhydrazine (NSD 1015). As indirect measurements of DA and NA release in vivo, we have assessed pargyline-induced 3-methoxytyramine (3-MT) and normetanephrine (NM) accumulation and disappearance rates of DA and NA after inhibition of their synthesis by alpha-methyl-p-tyrosine (alpha-MT). Administration of GBR 12909 (2.5, 5, 10, 20 or 40 mg/kg) decreased the NSD 1015-induced DOPA accumulation in the striatum and in the limbic forebrain. In contrast, only minor effects of the drug were seen on the DOPA accumulation in the cortical hemisphere and on the cerebral 5-HTP accumulation. GBR 12909 increased the 3-MT accumulation in the striatum, limbic forebrain and the cortical hemispheres, an effect that was even more pronounced in haloperidol-pretreated animals. However, GBR 12909 did not alter the 3-MT accumulation in the SN either when given alone or when given to haloperidol-pretreated rats. In haloperidol-pretreated rats GBR 12909 markedly enhanced the DA disappearance in the striatum and in the limbic forebrain, but not in the SN. Furthermore, GBR 12909 did not significantly affect the firing rate of dopaminergic neurons in the SN or that of noradrenergic neurons in the LC. Taken together, our results support the notion that GBR 12909 is a specific DA uptake inhibitor without a transmitter releasing action. In addition, our findings indicate that DA re-uptake is of physiological importance in the elimination of DA from the synaptic cleft in the striatum, limbic forebrain and cortical hemispheres, but not in the SN. Furthermore, a large part of the DA taken up by the dopaminergic terminals in the striatum and in the limbic forebrain seems to be re-incorporated into the storage vesicles.

Dopamine (DA) is synthesized and released not only from the terminals of the nigrostriatal dopaminergic neuronal pathway, but also from the dendrites in the substantia nigra. We have investigated the regulation of the DA turnover, the DA synthesis rate, and the DA release in the substantia nigra pars compacts (SNpc) and pars reticulata (SNpr) in vivo. As a measure of DA turnover, we have assessed the concentrations of 3,4-dihydroxyphenylacetic acid and homovanillic acid. As a measure of the DA synthesis rate, we have determined the 3,4-dihydroxyphenylalanine accumulation after inhibition of aromatic L-amino acid decarboxylase by 3-hydroxybenzylhydrazine. As a measure of DA release, we have investigated the disappearance rate of DA after inhibition of its synthesis by alpha-methyl-p-tyrosine and the 3-methoxytyramine accumulation following monoamine oxidase inhibition by pargyline. Both the DA turnover and the DA synthesis rate increased following treatment with the DA receptor antagonist haloperidol and decreased following treatment with the DA receptor agonist apomorphine in the SNpc and in the SNpr, but the effects of the drugs were less pronounced than in the striatum. gamma-Butyrolactone treatment, which suppresses the firing of the dopaminergic neurons, increased the DA synthesis rate in the striatum (165%), but had no such effect in the SNpc or SNpr. Haloperidol, apomorphine, and gamma-butyrolactone increased, decreased, and abolished, respectively, the DA release in the striatum, but the drugs had no or only slight effects on the alpha-methyl-p-tyrosine-induced DA disappearance and on the pargyline-induced 3-methoxytyramine accumulation in the SNpc or SNpr. Taken together, these results indicate that the DA synthesis rate, but not the DA release, are influenced by DA receptor activity and neuronal firing in the SNpc and SNpr. This is in contrast to the situation in the striatum, where both the DA synthesis rate and the DA release are under such control.

The central ghrelin signaling system engages key pathways of importance for feeding control, recently shown to include those engaged in anxiety-like behavior in rodents. Here we sought to determine whether ghrelin impacts on the central serotonin system, which has an important role in anxiety. We focused on two brain areas, the amygdala (of importance for the mediation of fear and anxiety) and the dorsal raphe (i.e. the site of origin of major afferent serotonin pathways, including those that project to the amygdala). In these brain areas, we measured serotonergic turnover (using HPLC) and the mRNA expression of a number of serotonin-related genes (using real-time PCR). We found that acute central administration of ghrelin to mice increased the serotonergic turnover in the amygdala. It also increased the mRNA expression of a number of serotonin receptors, both in the amygdala and in the dorsal raphe. Studies in ghrelin receptor (GHS-R1A) knock-out mice showed a decreased mRNA expression of serotonergic receptors in both the amygdala and the dorsal raphe, relative to their wild-type littermates. We conclude that the central serotonin system is a target for ghrelin, providing a candidate neurochemical substrate of importance for ghrelin's effects on mood.

Thermoregulatory and neurochemical effects of capsaicin microinjection into the substantia nigra (SN) or caudatus putamen (CPu) were studied in rats. Administration of capsaicin into these brain structures induced a peripheral vasodilatation which was associated with a decrease in body temperature. Pretreatment of the rats with capsaicin either as adults or neonates abolished the thermolytic response to the drug, indicating that the effect is executed specifically upon capsaicin sensitive structures. Analyses of the levels of monoamines and their metabolites in the striatum following injection of capsaicin into the SN or CPu revealed that dopaminergic neurons are not primarily involved in this effect. This view is also supported by our findings that neurochemical lesion of unilateral nigrostriatal dopaminergic neurons did not influence the vasodilatatory response. Since the pharmacological effect of intranigral capsaicin was not abolished by unilateral axotomy (hemisection) we presume a capsaicin-sensitive, non-dopaminergic descending vasodilatatory pathway from the SN.

In rat substantia nigra a biphasic disappearance curve of dopamine (DA) was seen after tyrosine hydroxylase inhibition by α-methyl-p-tyrosine (α-MT): the initial phase had a fast turnover and a half life of 0ṁ5 h and the later phase had an extremely slow turnover. In contrast to the effects in striatum, neither haloperidol nor apomorphine influenced α-MT-induced DA disappearance in the substantia nigra. Furthermore, inhibition of impulse flow by γ-butyrolactone prevented DA disappearance in striatum but not in the substantia nigra. Measurements of DA and 3-methoxytyramine following treatment with inhibitors of monoamine oxidase (pargyline) and catechol-O-methyl transferase (tropolone) indicated that O-methylation is a more important metabolic pathway in the substantia nigra than in the striatum. The data are interpreted to indicate that the release and metabolism of DA in the substantia nigra are largely beyond the control of nerve impulses and DA receptors. It is suggested that such an arrangement forms an important feature of autoreceptor-mediated feedback control of DA nerve cell activity.

The accumulation rates of 3,4′-dihydroxyphenyl-alanine (DOPA) and 5-hydroxytrypthophan (5-HTP) after inhibition of aromatic amino acid decarboxylase (AADC) by 3-hydroxybenzylhydrazine (NSD 1015) or 1-(dl-seryl)-2(2,3,4-trihydroxybenzyl)hydrazine (Ro 4-4602) have widely been used as measurements of the in vivo synthesis rates of monoamines. However, the values of dopamine (DA) turnover in rat striatum obtained using these drugs are much lower than values obtained by other methods. This discrepancy prompted us to further investigate the AADC inhibitor 1-(3-hydroxybenzyl)-1-methylhydrazine (NSD 1034) which earlier has been shown to give a DOPA accumulation rate in the striatum of the same magnitude as other measures of DA turnover. NSD 1034 was found to give a more than twofold higher DOPA accumulation rate than NSD 1015, NSD 1024, NSD 1039, NSD 1055 and Ro 4-4602 in the striatum. Also, in the limbic region and the hemispheres, but not in the substantia nigra, the DOPA accumulation was higher after NSD 1034 than after NSD 1015, but the difference was less pronounced. There was, however, no difference in 5-HTP accumulation between the drugs in any of the brain parts investigated. Although the DOPA accumulation rates are higher after NSD 1034 than after NSD 1015, the NSD 1015-induced DOPA accumulation seems to be more sensitive to changes in dopamine receptor occupancy. The different DOPA accumulation rates obtained with NSD 1015 and NSD 1034 are not due to differences in MAO inhibition, to interference with classical DA receptors, or to different degrees of AADC inhibition, but to an ability of NSD 1034 to stimulate DA synthesis. In addition, under certain conditions NSD 1034 also has a DA releasing action, like amphetamine. It is proposed that NSD 1034 and amphetamine stimulate DA synthesis and release by a common mechanism. The low value of DA synthesis rate, obtained when measured as DOPA accumulation after NSD 1015, is due to a substantial efflux of DOPA from the brain. The efflux of DOPA is equally large after NSD 1034 but the loss is compensated for by an increase in DOPA synthesis.