John F. Neumaier

5-HT1B mrna regulation in two animal models of altered stress reactivity

BACKGROUNDnAcute stress has profound effects on serotonergic activity, but it is not known whether alterations in the serotonin system can predispose individuals to exaggerated stress responses. We examined the regulation of 5-HT(1B) and 5-HT(1A) mRNA in two rodent models of differential sensitivity to stress: congenital learned helplessness (cLH) and handling and maternal separation (HMS).nnnMETHODSn5-HT(1B) and 5-HT(1A) mRNAs in brain tissue sections were quantitated by in situ hybridization from control, stress-sensitive, and stress-resistant male rats in the HMS model and stress-sensitive and stress-resistant rats (both males and females) in the cLH model. Dorsal raphe nucleus, striatum, and hippocampus were examined.nnnRESULTSnThe main result was that dorsal raphe 5-HT(1B) mRNA was substantially elevated (63-73%) in male rats in the stress-resistant group of both models compared with stress-sensitive animals. 5-HT(1B) mRNA in female rats did not differ between groups in the cLH model. There were no differences in 5-HT(1A) mRNA between HMS groups.nnnCONCLUSIONSnThese findings suggest that 5-HT(1B) autoreceptor regulation is altered in animals with diminished stress reactivity. These results suggest that 5-HT(1B) autoreceptors in unstressed and acutely stressed animals differ, indicating the importance of state versus trait changes in serotonin function in animal models of anxiety and depression.

BGC20-761, a novel tryptamine analog, enhances memory consolidation and reverses scopolamine-induced memory deficit in social and visuospatial memory tasks through a 5-HT6 receptor-mediated mechanism.

Inhibition of 5-HT(6) receptors has been shown to improve memory consolidation, thus we tested whether a novel tryptamine analog with high affinity for 5-HT(6) receptors, BGC20-761 (5-methoxy-2-phenyl-N,N-dimethyltryptamine, PMDT), can enhance long-term memory. BGC20-761 (10 mg/kg i.p.) alone had no effect on social recognition in young rats, however, at doses of 5 mg/kg and 10 mg/kg i.p, BGC20-761 dose-dependently reversed a deficit of social recognition induced by scopolamine (0.4 mg/kg i.p.), an anticholinergic drug that impairs memory. BGC20-761 (10 mg/kg i.p.), scopolamine (0.2 mg/kg i.p.) or BGC20-761 + scopolamine had no effects on novel object discrimination in young rats (2 months). In mature rats (6 months), recognition of the novel object was improved following administration of BGC20-761. Scopolamine had no effect in object recognition. However, the addition of scopolamine disrupted the memory-enhancing effect of BGC20-761. Based on the high affinity of BGC20-761 for 5-HT(6) receptors, these cognitive enhancing effects are most likely mediated by 5-HT(6) receptor inhibition. The difference in effects of BGC20-761 in young vs. mature rats may reflect the status of memory consolidation in these different age ranges.

DREADDing the lateral habenula: A review of methodological approaches for studying lateral habenula function

The lateral habenula (LHb) is part of the habenular complex in the dorsal diencephalon. The LHb is an important regulator of several neurotransmitter systems in the midbrain; disturbances in this regulation may contribute to mood disorders, abnormalities in cognition, drive, and addiction. Owing to the critical role this nucleus plays in modulating activity of midbrain nuclei, there has been a rapid increase in studies targeting the LHb in the recent years. In this review, we describe studies using traditional approaches to elucidate the function of this brain region, such as lesion, electrical and chemical stimulation, electrophysiology and in vivo microdialysis. We have selected a variety of illustrative studies to discuss each of these methods. Next, we describe studies using methods that are based upon recent advances in molecular biology techniques including recent results from our laboratory using the Designer Receptor Exclusively Activated by Designer Drug (DREADD) technology. Using a Gi/o-coupled DREADD, we found that inhibition of the LHb reduces depression-like behavior in the forced swim test in a manner that suggests enhanced serotonergic activity. The emerging picture reveals that the LHb is likely to be a critical node in the network of subcortical nuclei that regulate aversive learning, motivation, stress responses, etc. We describe how recently developed methods have advanced the study of the LHb and are leading research of this brain region in promising new directions. This article is part of a Special Issue entitled Optogenetics (7th BRES).

Increased expression of 5-HT1B receptor in dorsal raphe nucleus decreases fear-potentiated startle in a stress dependent manner

5-HT(1B) autoreceptors regulate serotonin release from terminals of dorsal raphe nucleus (DRN) projections. Due to postsynaptic 5-HT(1B) receptors in DRN terminal fields, it has not previously been possible to manipulate 5-HT(1B) autoreceptor activity without also changing 5-HT(1B) heteroreceptor activity. We have developed a viral gene transfer strategy to express epitope-tagged 5-HT(1B) and green fluorescent protein in vivo, allowing us to increase 5-HT(1B) expression in DRN neurons. We have shown that increased 5-HT(1B) autoreceptor expression reduced anxiety in unstressed animals but increased anxiety following inescapable stress. These findings suggest that effects of increased 5-HT(1B) autoreceptor expression are dependent on stress context. To better understand the mechanisms underlying these observations, we have used fear-potentiated startle (FPS). FPS is especially sensitive to the activity of the amygdala, which shares reciprocal connections with DRN. In the absence of an inescapable stressor, increased 5-HT(1B) autoreceptor expression attenuated FPS response compared with animals injected with a virus expressing only green fluorescent protein. Administration of the 5-HT(1B) antagonist SB224289 (5 mg/kg i.p.) before startle testing blocked the effects of increased 5-HT(1B) autoreceptor expression. Since SB224289 had no effect on FPS in the absence of viral gene transfer, these results suggest that the antagonist reversed the behavioral effects of increased 5-HT(1B) autoreceptor expression through blockade of transgenic receptors. When tested 24 h following water-restraint stress, animals with increased 5-HT(1B) autoreceptors demonstrated restoration of robust FPS response. These results extend our previous studies and suggest explanations for the complex relationship between 5-HT(1B) autoreceptor expression, stress, and anxiety behavior.

5-HT6 receptor antagonist reversal of emotional learning and prepulse inhibition deficits induced by apomorphine or scopolamine

5-HT6 receptors have been implicated in consolidation of visuospatial and reward-based learning tasks. Since 5-HT6 receptors may be important in modulation of sensory gating which is often affected in schizophrenic patients, we tested whether Ro 4368554, a 5-HT6 selective antagonist at a dose of 10 mg/kg, could reverse the loss of prepulse inhibition from apomorphine or scopolamine. In addition, we also tested whether Ro 4368554 altered fear conditioning using fear potentiated startle, a model for emotional learning. Prepulse inhibition of startle was disrupted by apomorphine (0.5 mg/kg) when prepulse emissions were 5 dB above background but not above 15 dB, while scopolamine (0.5 mg/kg) caused disruption at both prepulse levels. Scopolamine-mediated disruption was not reversed by Ro 4368854 but apomorphine-mediated disruption was significantly ameliorated by 5-HT6 inhibition. For fear potentiated startle, scopolamine and/or Ro 4368554 were administered before two daily fear conditioning sessions; rats were tested on the following day. Rats that received scopolamine displayed no fear potentiated startle but Ro 4368554 reversed this scopolamine deficit. Additionally, we mapped Fos induction in rats treated with scopolamine and/or Ro 4368554; scopolamine increased Fos expression in the central nucleus of the amygdala and this was attenuated by Ro 4368554. In summary, we have demonstrated the efficacy of 5-HT6 antagonists in modulating sensory gating and fear conditioning, and thus may be of therapeutic use for schizophrenia-related disorders.

Clozapine downregulates 5-hydroxytryptamine6 (5-HT6) and upregulates 5-HT7 receptors in HeLa cells.

Clozapine is an atypical antipsychotic with high affinity for several serotonin receptors. This drug causes paradoxical downregulation of 5-hydroxytryptamine(2A) (5-HT)(2A) receptors, but its modulation of other serotonin receptors has not been studied. We examined the effects of clozapine and several other drugs on the regulation of rat 5-HT(6) and 5-HT(7) receptors individually expressed in transfected HeLa cells. Both 5-HT(6) and 5-HT(7) receptor densities (B(max)) were reduced by 5-carboxamidotryptamine, an agonist, and methiothepin, an inverse agonist. Clozapine reduced 5-HT(6) B(max). This suggests that 5-HT(6) receptors are also paradoxically downregulated by the antagonist clozapine. 5-Hydroxytryptamine(7) receptor B(max), on the other hand, was increased by clozapine. Clozapines modulation of the 5-HT(6) and 5-HT(7) receptor levels may be important in the action of this atypical antipsychotic.

Cocaine increases 5-HT1B mRNA in rat nucleus accumbens shell neurons.

Serotonin 5-HT(1B) receptors modulate behavioral responses to cocaine, but the effects of cocaine on endogenous 5-HT(1B) receptor expression are not known. Therefore, we examined the effect of binge cocaine administration on 5-HT1B mRNA expression in rat brain. We found that chronic, but not acute, binge cocaine exposure increased 5-HT(1B) mRNA by approximately 80% in nucleus accumbens shell and dorsal striatum. Surprisingly, 5-HT(1B) mRNA was increased in nucleus accumbens shell after chronic vehicle treatment as well, but this effect was driven by animals that were housed with cocaine-treated animals. Thus, 5-HT(1B) mRNA is upregulated by repeated exposure to cocaine and perhaps by social stress as well; both of these factors are relevant to the risk for relapse in cocaine addiction.

The effects of SB 224289 on anxiety and cocaine-related behaviors in a novel object task.

Cocaine facilitates dopamine transmission from ventral tegmental area (VTA) neurons that project to nucleus accumbens (NAcc), and previous experiments suggest that serotonin-1B (5-HT1B) receptors are involved in this effect. Specifically, activation of 5-HT1B receptors in VTA during cocaine exposure increases dopamine release in NAcc and enhances cocaine-induced locomotor activity, reward, and reinforcement. Thus, it is reasonable to hypothesize that blocking 5-HT1B activity may have the opposite effect. To investigate this hypothesis, SB 224289, a highly selective 5-HT1B antagonist, was used to block this receptor. In an open field/novel object exploration test, SB 224289 reduced cocaine-induced locomotion. However, SB 224289 also increased anxiety-like behavior, both alone and in combination with cocaine. This experiment gives evidence that 5-HT1B antagonists may reduce some of the behavioral effects of cocaine, but may have negative effects on anxiety as well.

5-HT1B receptor mRNA levels in dorsal raphe nucleus: inverse association with anxiety behavior in the elevated plus maze.

Serotonergic neurons in the dorsal raphe nucleus, the major source of forebrain serotonin projections, synthesize a terminal autoreceptor that inhibits serotonin release-the 5-HT(1B) autoreceptor. Overexpression of this autoreceptor is hypothesized to contribute to anxiety. Antidepressants decrease (while learned helplessness increases) 5-HT(1B) mRNA in dorsal raphe neurons, and viral-mediated overexpression of 5-HT(1B) here increases anxiety behavior after stress. However, 5-HT(1B) mRNA levels in dorsal raphe are substantially elevated in unstressed rats in two models of stress resistance. Thus, the role of dorsal raphe 5-HT(1B) autoreceptors in anxiety is complex. Therefore, we tested whether different stressors differentially affect dorsal raphe 5-HT(1B) mRNA [via in situ hybridization histochemistry] and anxiety behavior (using the elevated plus maze). Rats were assigned to a stressor (either forced swim, water restraint, dry restraint, or electric tail shock) or a control condition, then were tested and sacrificed 24 h later. Overall, controls exhibited less anxiety than stressed rats as indicated by a higher ratio of open arm to total arm entries (OTR). The stressors did not differentially affect the OTR, nor did any alter dorsal raphe 5-HT(1B) mRNA levels. There was, however, a significant positive correlation between the OTR and 5HT(1B) mRNA intensity in controls (r=.64; P=.006), but not in stressed rats (r=.16, P=.36), providing further evidence that elevated dorsal raphe 5-HT(1B) levels are associated with reduced anxiety in animals that have not been exposed to stress.

Overexpression of 5-HT1B mRNA in nucleus accumbens shell projection neurons differentially affects microarchitecture of initiation and maintenance of ethanol consumption

Serotonin 1B (5-HT(1B)) heteroreceptors on nucleus accumbens shell (NAcSh) projection neurons have been shown to enhance the voluntary consumption of alcohol by rats, presumably by modulating the activity of the mesolimbic reward pathway. The present study examined whether increasing 5-HT(1B) receptors expressed on NAcSh projection neurons by means of virus-mediated gene transfer enhances ethanol consumption during the initiation or maintenance phase of drinking and alters the temporal pattern of drinking behavior. Animals received stereotaxic injections of viral vectors expressing either 5-HT(1B) receptor and green fluorescent protein (GFP) or GFP alone. Home cages equipped with a three-bottle (water and 6 and 12% ethanol) lickometer system recorded animals drinking behaviors continuously, capturing either initiation or maintenance of drinking behavior patterns. Overexpression of 5-HT(1B) receptors during initiation increased consumption of 12% ethanol during both forced-access and free-choice consumption. There was a shift in drinking pattern for 6% ethanol with an increase in number of drinking bouts per day, although the total number of drinking bouts for 12% ethanol was not different. Finally, increased 5-HT(1B) expression induced more bouts with very high-frequency licking from the ethanol bottle sippers. During the maintenance phase of drinking, there were no differences between groups in total volume of ethanol consumed; however, there was a shift toward drinking bouts of longer duration, especially for 12% ethanol. This suggests that during maintenance drinking, increased 5-HT(1B) receptors facilitate longer drinking bouts of more modest volumes. Taken together, these results indicate that 5-HT(1B) receptors expressed on NAcSh projection neurons facilitate ethanol drinking, with different effects during initiation and maintenance of ethanol-drinking behavior.