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The cerebellum, a primary brain structure involved in the control of sensorimotor tasks, also contributes to higher cognitive functions including reward, emotion and social interaction. Although the regulation of these behaviors has been largely ascribed to the monoaminergic system in limbic regions, the contribution of cerebellar dopamine signaling in the modulation of these functions remains largely unknown. By combining cell-type-specific transcriptomics, histological analyses, three-dimensional imaging and patch-clamp recordings, we demonstrate that cerebellar dopamine D2 receptors (D2Rs) in mice are preferentially expressed in Purkinje cells (PCs) and regulate synaptic efficacy onto PCs. Moreover, we found that changes in D2R levels in PCs of male mice during adulthood alter sociability and preference for social novelty without affecting motor functions. Altogether, these findings demonstrate novel roles for D2R in PC function and causally link cerebellar D2R levels of expression to social behaviors.

The dorsal striatum has been linked to decision-making under conflict, but the mechanism by which striatal neurons contribute to approach-avoidance conflicts remains unclear. We hypothesized that striatopallidal dopamine D2 receptor (D2R)-expressing neurons promote avoidance, and tested this hypothesis in two exploratory approach-avoidance conflict paradigms in mice: the elevated zero maze and open field. Genetic elimination of D2Rs on striatopallidal neurons (iMSNs), but not other neural populations, increased avoidance of the open areas in both tasks, in a manner that was dissociable from global changes in movement. Population calcium activity of dorsomedial iMSNs was disrupted in mice lacking D2Rs on iMSNs, suggesting that disrupted output of iMSNs contributes to heightened avoidance behavior. Consistently, artificial disruption of iMSN output with optogenetic stimulation heightened avoidance of open areas of these tasks, while inhibition of iMSN output reduced avoidance. We conclude that dorsomedial striatal iMSNs control approach-avoidance conflicts in exploratory tasks, and highlight this neural population as a potential target for reducing avoidance in anxiety disorders.

A recent clinical trial found that pharmacological blockade of V1b receptors reduces alcohol relapse in alcohol‐dependent patients. SSR149415 is a selective V1b receptor antagonist that has potential for development as an alcohol dependency treatment. In this study, we investigated whether SSR149415 alone or in combination with the mu‐opioid receptor (MOP‐r) antagonist naltrexone (NTN) would alter excessive alcohol drinking in mice. Both sexes of C57BL/6J (B6) mice were subjected to a chronic intermittent access (IA) drinking paradigm (2‐bottle choice, 24‐hour access every other day) for 3 weeks. Sucrose and saccharin drinking were used as controls for alcohol‐specific drug effects. Neuronal proopiomelanocortin (POMC) enhancer (nPE) knockout mice with hypothalamic‐specific loss of POMC(including beta‐endorphin, the main endogenous ligand of MOP‐r) were used as a genetic control for the effects of NTN. Acute administration of SSR149415 (1 to 30 mg/kg) reduced alcohol intake and preference in a dose‐dependent manner in both male and female B6 mice after IA. To investigate potential synergistic effects between NTNand SSR149415, we tested 6 different combination doses of SSR149415 and NTN, and found that a combination of SSR149415 (3 mg/kg) and NTN(1 mg/kg) reduced alcohol intake profoundly at doses lower than the individual effective doses in both sexes of B6 mice. We confirmed the effect of SSR149415 on reducing alcohol intake in nPE−/− male mice, consistent with independent mechanisms by which SSR149415 and NTNdecrease alcohol drinking. The combination of V1b antagonist SSR149415 with NTNat individual subthreshold doses shows potential in alcoholism treatment, possibly with less adverse effects. Pharmacological blockade of V1b receptors reduces alcohol relapse in alcohol‐dependent patients. We tested combination doses of selective V1b antagonist SSR149415 and naltrexone (NTN) and found that combinations of the two human compounds reduced alcohol intake profoundly at doses lower than individual effective doses in both sexes of mice. The combination of SSR149415 with NTN shows potential in alcoholism treatment, possibly with less adverse‐effect. Figure shows dose responses of SSR149415 with NTN on reducing excessive alcohol intake (A) and preference (B).

Wireless mesh networks are emerging as a promising solution for ubiquitous Internet access with mobility support. In such networks, user mobility may lead to Internet gateway changes and consequently, impact the performance of continuous media applications. In this article, we investigate the impact of user mobility on the performance of peer-to-peer video applications over wireless mesh networks. Peer-to-peer video streaming applications rely on the collaborative behavior of peers to assist the source in delivering multimedia content, reduce costs, and increase the scalability of video distribution. We identify practical issues related to mobility for P2P video streaming implementation in WMNs, such as addressing and forwarding strategies. Furthermore, we evaluate the performance of different P2P streaming applications as the user walks in our WMN testbed. Results indicate mobile users benefit from the frequent shortlived connections established in modern P2P video sessions.

Recently, the importance of the dopaminergic D2 receptor (D2R) subtype in normal body growth and neonatal GH secretion has been highlighted. Disruption of D2R alters the GHRH–GH–IGF-I axis and impairs body growth in adult male mice. The D2R knockout (KO) dwarf mouse has not been well characterized; we therefore sought to determine somatotrope function in the adult pituitary. Using immunohistochemistry and confocal microscopy, we found a significant decrease in the somatotrope population in pituitaries from KO mice (P=0.043), which was paralleled by a decreased GH output from pituitary cells cultured in vitro.In cells from adult mice the response amplitude to GHRH differed between genotypes (lower in KO), but this difference was less dramatic after taking into account the lower basal release and hormone content in the KO cells. Furthermore, there were no significant differences in cAMP generation in response to GHRH between genotypes. By Western blot, GHRH-receptor in pituitary membranes from KO mice was reduced to 46% of the level found in wildtype (WT) mice (P=0.016). Somatostatin induced a concentration-dependent decrease in GH and prolactin (PRL) secretion in both genotypes, and 1×10−7M ghrelin released GH in cells from both genotypes (P=0.017) in a proportionate manner to basal levels. These results suggest that KO somatotropes maintain a regulated secretory function. Finally, we tested the direct effect of dopamine on GH and PRL secretion in cells from both genotypes at 20 days and 6 months of life. As expected, we found that dopamine could reduce PRL levels at both ages in WT mice but not in KO mice, but there was no consistent effect of the neurotransmitter on GH release in either genotype at the ages studied. The present study demonstrates that in the adult male D2R KO mouse, there is a reduction in pituitary GH content and secretory activity. Our results point to an involvement of D2R signaling at the hypothalamic level as dopamine did not release GH acting at the pituitary level either in 1-month-old or adult mice. The similarity of the pituitary defect in the D2R KO mouse to that of GHRH-deficient models suggests a probable mechanism. A loss of dopamine signaling via hypothalamic D2Rs at a critical age causes the reduced release of GHRH from hypophyseotropic neurons leading to inadequate clonal expansion of the somatotrope population. Our data also reveal that somatotrope cell number is much more sensitive to changes in neonatal GHRH input than their capacity to develop properly regulated GH-secretory function.

It is now established that the hypothalamus is essential in coordinating endocrine, autonomic, and behavioral responses to changes in energy availability. However, the interaction of key peptides, neuropeptides, and neurotransmitters systems within the hypothalamus has yet to be delineated. Recently, we investigated the mechanisms through which central serotonergic (5-hydroxytryptamine, 5-HT) systems recruit leptin-responsive hypothalamic pathways, such as the melanocortin systems, to affect energy balance. Through a combination of functional neuroanatomy, feeding, and electrophysiology studies in rodents, we found that 5-HT drugs require functional melanocortin pathways to exert their effects on food intake. Specifically, we observed that anorectic 5-HT drugs activate pro-opiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (Arc). We provide evidence that the serotonin 2C receptor (5-HT2CR) is expressed on POMC neurons and contributes to this effect. Finally, we found that 5-HT drug-induced hypophagia is attenuated by pharmacological or genetic blockade of downstream melanocortin 3 and 4 receptors. We review candidate brain regions expressing melanocortin 3 and 4 receptors that play a role in energy balance. A model is presented in which activation of the melanocortin system is downstream of 5-HT and is necessary to produce the complete anorectic effect of 5-HT drugs. The data reviewed in this paper incorporate the central 5-HT system to the growing list of metabolic signals that converge on melanocortin neurons in the hypothalamus.

Feeding behavior can be divided into appetitive and consummatory phases, differing in neural substrates and effects of deprivation. Opioids play an important role in the appetitive aspects of feeding, but they also have acute stimulatory effects on food consumption. Because the opioid peptide ?-endorphin is co-synthesized and released with melanocortins from proopiomelanocortin (POMC) neuronal terminals, we examined the physiological role of ?-endorphin in feeding and energy homeostasis using a strain of mutant mice with a selective deficiency of ?-endorphin. Male ?-endorphin-deficient mice unexpectedly became obese with ad libitum access to rodent chow. Total body weight increased by 15 with a 50-100 increase in the mass of white fat. The mice were hyperphagic with a normal metabolic rate. Despite the absence of endogenous ?-endorphin, the mutant mice did not differ from wild-type mice in their acute feeding responses to ?-endorphin or neuropeptide Y administered intracerebroventricularly or naloxone administered intraperitoneally. Additional mice were studied using an operant behavioral paradigm to examine their acquisition of food reinforcers under increasing work demands. Food-deprived, ?-endorphin-deficient male mice emitted the same number of lever presses under a progressive ratio schedule compared to wild-type mice. However, the mutant mice worked significantly less than did the wild-type mice for food reinforcers under nondeprived conditions. Controls for nonspecific effects on acquisition of conditioned learning, activity, satiety, and resistance to extinction revealed no genotype differences, supporting our interpretation that ?-endorphin selectively affects a motivational component of reward behavior under nondeprived conditions. Therefore, we propose that ?-endorphin may function in at least two primary modes to modulate feeding. In the appetitive phase, ?-endorphin release increases the incentive value of food as a primary reinforcer. In contrast, it appears that endogenous ?-endorphin may inhibit food consumption in parallel with melanocortins and that the orexigenic properties previously ascribed to it may actually be due to other classes of endogenous opioid peptides.

We used β-endorphin-deficient mice as a novel approach to confirm the physiological role that opioid peptides play in the development or regulation of the immune system. We found that mice lacking β-endorphin possessed an enhanced immune response, measured in terms of splenocyte proliferation and interleukin (IL)-2 mRNA levels, in vitro production of the splenic macrophage inflammatory cytokines IL-6 and Tumor Necrosis Factor (TNF)-α and plasma IL-6 following lipopolysaccharide (LPS) administration. β-Endorphin-deficient mice had attenuated increases of plasma ACTH and corticosterone levels in response to LPS. These results are consistent with a postulated inhibitory role of endogenous β-endorphin on the immune system at multiple levels.

Phenotypes were assessed topographically in mice lacking functional D2dopamine receptors [‘knockouts’], using an ethologically based approach to assess all behaviours in the natural repertoire. D2-null mice evidenced an ethogram characterised initially by modest reductions in locomotion and shifts in rearing topographies. Subsequently, topographies of behaviour habituated similarly for wildtypes and ‘knockouts’. Following challenge with the D2-like agonist RU 24213, both inhibition of rearing at a lower dose and induction of stereotyped sniffing and ponderous locomotion at higher doses were essentially absent in D2-null mice. Following challenge with the D1-like agonist A 68930, vacuous chewing was released in D2-null mice. This topographical approach to phenotypic characterisation implicates: (i) the D2receptor in these D2-like agonist effects and in oppositional D1-like: D2-like interactions; and (ii) the operation of material compensatory processes consequent to the developmental absence of D2receptors which are able to maintain ethological function under tonic, ‘naturalistic’ conditions but not under ‘phasic’ challenge.

1Sigma receptors bind a diverse group of chemically unrelated ligands, including pentazocine, apomorphine (a dopamine receptor agonist) and haloperidol (a dopamine receptor antagonist). Although sigma binding sites are widely distributed, their physiological roles are poorly understood. Here, the whole‐terminal patch‐clamp technique was used to demonstrate that sigma receptors modulate K+channels in rodent neurohypophysis.2Previous work suggested that dopamine type 4 (D4) receptors modulate neurohypophysial K+current, so this study initially tested the role of dopamine receptors. Experiments using transgenic mice lacking D2, D3or D4receptors indicated that the reduction of K+current by PPHT and U101958 (ligands thought to be selective for dopamine receptors) is not mediated by dopamine receptors. The sensitivity of the response to U101958 (a drug that binds to D4receptors) was the same in both wild‐type and D4receptor‐deficient mice.3Experiments with other ligands revealed a pharmacological signature inconsistent with any known dopamine receptor. Furthermore, dopamine itself (at 100 μm) had no effect. Thus, despite the activity of a number of putative dopamine receptor ligands, dopamine receptors play no role in the modulation of neurohypophysial K+channels.4Because of the negative results regarding dopamine receptors, and because some of the dopamine receptors ligands used here are known to bind also to sigma receptors, experiments were conducted to test for the involvement of sigma receptors. In rat neurohypophysis the sigma receptor ligands SKF10047, pentazocine, and ditolylguanidine all reversibly inhibited K+current in a concentration‐dependent fashion, as did haloperidol and apomorphine (ligands that bind to both dopamine and sigma receptors). The activity of these and other ligands tested here matches the reported binding specificity for sigma receptors.5Fifteen candidate endogenous sigma receptor ligands, including biogenic amines (e.g dopamine and serotonin), steroids (e.g. progesterone), and peptides (e.g. neuropeptide Y), were screened for activity at the sigma receptor. All were without effect.6Haloperidol reduced K+current proportionally at all voltages without shifting the voltage dependence of activation and inactivation. Sigma receptor ligands inhibited current through two distinct K+channels, the A‐channel and the Ca2+‐dependent K+channel. In rat, all drugs reduced current through both channels proportionally, suggesting that both channels are modulated by a single population of sigma receptors. In contrast, mouse peptidergic nerve terminals either have two receptors which are sensitive to these drugs, or a single receptor that is differentially coupled to ion channel function.7The inhibition of voltage‐activated K+current by sigma receptors would be expected to enhance the secretion of oxytocin and vasopressin from the neurohypophysis.

TO study the specific contribution of the D3dopamine receptor in the generation of locomotor activity, total or partially dopamine-depleted rats were pretreated with an antisense oligodeoxynucleotide for the D3receptor (D3R-as) and locomotor activity induced by apomorphine was measured. A 35.7 increase in locomotor activity was seen in the totally dopaminedepleted rats pretreated with the D3R-as, whereas the same antisense, caused a significantly greater increase in the locomotor response (95) in the partially dopamine-depleted rats compared with control groups (pretreated with a control oligodeoxynucleotide or vehicle). In situautoradiography for D3receptors showed a 27 fall in the density of D3receptors in the islands of Calleja compared with control animals. Our results seem to confirm that D3receptors exert an inhibitory effect on locomotor activity, through the stimulation of both pre- and postsynaptic components.

CONTRALATERAL rotations induced by the D1-like agonist SKF 38393 in unilaterally 6-hydroxydopamine-lesioned rats were completely prevented by the administration of the D1-like antagonist SCH 23390. A similar result was obtained after intracerebroventricular administration of an antisense oligodeoxynucleotide for the D1 receptor (D1R-as). Contrariwise, administration of a D5R-as potentiated the effects of SKF 38393, showing a 60 increase in the rotational scores. Both effects were reversible upon cessation of D1R-as or D5R-as treatment and were also specific since rotational scores in rats treated with vehicle or with a randomly designed oligodeoxynucleotide were not modified. These results suggest that whereas D1 receptors play a facilitatory role in locomotion, D5 receptors exert an inhibitory effect.

Abstract: We measured the minimum amount of endogenous dopamine (EDA), necessary for the expression of rotational behavior induced by D2 receptor stimulation in striatal or medial forebrain bundle (MFB) lesioned rats. We correlated these results with the minimum dose of D1 receptor agonists needed to substitute EDA in its permissive role for D2 motor effects to take place. Rats with unilateral quinolinic acid (QA) striatal or 6-hydroxydopamine (6-OHDA) MFB lesions were given increasing doses of the tyrosine hydroxylase inhibitor alpha-methyl-para-tyrosine (AMPT) in combination with a fixed dose of the D2 receptor agonist quinpirole (trans-(–)-4aR-4,4a,5,6,7,8,8a,9-Octahydro-5-propyl-1H-pyrazolo(3,4-g)quinoline hydrochloride) and tested for rotational behavior. The animals were later sacrificed and striata removed; EDA was measured by high performance liquid chromatography (HPLC). Rotational responses were abolished by increasing doses of AMPT inducing a stepwise depletion of EDA. EDA content and rotational behavior to D2 stimulation showed a high degree of correlation. There was an abrupt reduction in rotational behavior at dopamine levels of 50–60% of controls in both animal models. In addition, striatal or MFB lesioned rats which were maximally depleted of dopamine by AMPT pretreatment received a fixed dose of quinpirole and then challenged with increasing doses of a D1 receptor agonist SKF 38393 ((±)-1-Phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride). Rotational behavior was restored by SKF 38393 in both animal models in a dose-dependent fashion. Our results confirm the need for simultaneous D1/D2 stimulation in the generation of rotational behavior in both animal models. Moreover, they demonstrate the existence of a threshold level of D1 stimulation necessary to exert its permissive role on D2 mediated responses.

A correction to this paper has been published and can be accessed via a link at the top of the paper.

Alcohol produces both stimulant and sedative effects in humans and rodents. In humans, alcohol abuse disorder is associated with a higher stimulant and lower sedative responses to alcohol. Here, we show that this association is conserved in mice and demonstrate a causal link with another liability factor: low expression of striatal dopamine D2 receptors (D2Rs). Using transgenic mouse lines, we find that the selective loss of D2Rs on striatal medium spiny neurons enhances sensitivity to ethanol stimulation and generates resilience to ethanol sedation. These mice also display higher preference and escalation of ethanol drinking, which continues despite adverse outcomes. We find that striatal D1R activation is required for ethanol stimulation and that this signaling is enhanced in mice with low striatal D2Rs. These data demonstrate a link between two vulnerability factors for alcohol abuse and offer evidence for a mechanism in which low striatal D2Rs trigger D1R hypersensitivity, ultimately leading to compulsive-like drinking.

Dopamine receptors play an important role in systemic blood pressure (BP) regulation. Recent studies have shown that disruption of D1, D2, D3, or D5 dopamine receptors leads to hypertension in mice, via specific central and peripheral mechanisms. D4 receptors are expressed in the heart, renal collecting ducts, juxtaglomerular cells, and in brain nuclei (e.g., nucleus tractus solitarius (NTS), dorsal motor nucleus of the vagus) known to affect BP. In pentobarbital-anesthetized mice, directly measured systolic (S), diastolic, and mean BPs were elevated in D4-/- mice (SBP = 128 ± 1 mm Hg, n=27) compared to wild type mice (SBP = 104 ± 1, n=18). Heart weight (% body weight) was greater in D4-/- (0.50 ± 0.02%) than in D4+/+ (0.43 ± 0.01%) mice. An acute saline load (5% body weight) did not increase BP and was excreted to a similar extent in D4-/- and D4+/+ mice. Whereas the V1 receptor antagonist, cyclopentamethylene proprionic acid-2-(O-methyl) tyrosine-Arg-vasopressin (10 µg/kg), had a minimal effect on BP in either D4+/+ or D-/- mice (n=6/strain), after 8 min, it slightly increased BP in the D4-/- mice (10 ± 2%). Neither ETA (BQ610, 100 µg/kg/min X 10 min) nor ETB (BQ788, 6.6 µg/kg/min X 10 min) receptor blockade had any significant effect on BP. Although a-adrenergic blockade with phentolamine (5 ng/kg/min X 30min) decreased BP to a similar extent in both mouse strains (n=6/strain/study), AT1 receptor blockade with Losartan (3 mg/kg IV) significantly decreased BP to a greater and longer extent in D4-/- than in D4+/+ mice such that 45 min after Losartan, SBP was decreased by 12 ± 3% in D4+/+ mice (n=6) and by 46 ± 3% in D4-/- mice (n=5). Because renal and plasma renin concentrations were similar in the two mouse strains, the dependence of the hypertension displayed by the D4-/- mice on the AT1 receptors was probably of central origin. Given that D4 receptors are co-expressed with depressor V1 receptors and pressor AT1 receptors in some neurons of the NTS, we suggest that D4 receptors may normally act to inhibit the firing of these cells, thereby diminishing the effects of activating AT1 and V1 receptors. Consequently, in the absence of D4 receptors, the pressor action of AT1 receptors becomes more pronounced while at the same time a weak depressor action of V1 receptors is unmasked.

The introduction of small mutations instead of null alleles into the mouse genome has broad applications to the study of protein structure-function relationships and the creation of animal models of human genetic diseases. To test a simple mutational strategy we designed a targeting vector for the mouse pro-opiomelanocortin (POMC) gene containing a single nucleotide insertion that converts the initial tyrosine codon of β-endorphin 1-31 to a premature translational termination codon and introduces a unique HpaI endonuclease restriction site. The targeting vector also contains a neo cassette immediately 3′ to the last POMC exon and a herpes simplex virus thymidine kinase cassette to allow positive and negative selection. Homologous recombination occurred at a frequency of 1/30 clones of electroporated embryonic stem cells selected in G418 and gancyclovir. 10/11 clones identified initially by a polymerase chain reaction (PCR) strategy had the predicted structure without evidence of concatemer formation by Southern blot analysis. We used a combination of Hpa I digestion of PCR amplified fragments and direct nucleotide sequencing to further confirm that the point mutation was retained in 9/10 clones. The POMC gene was transcriptionally silent in embryonic stem cells and the targeted allele was not activated by the downstream phosphoglycerate kinase-1 promoter that transcribed the neo gene. Under the electroporation conditions used, we have demonstrated that a point mutation can be introduced with high efficiency and precision into the POMC gene using a replacement type vector containing a retained selectable marker without affecting expression of the allele in the embryonic stem cells. A similar strategy may be useful for a wide range of genes.