Roh-Yu Shen

A Role for Hypocretin (Orexin) in Male Sexual Behavior

The role of hypocretin (orexin; hcrt/orx) neurons in regulation of arousal is well established. Recently, hcrt/orx has been implicated in food reward and drug-seeking behavior. We report here that in male rats, Fos immunoreactivity (ir) in hcrt/orx neurons increases markedly during copulation, whereas castration produces decreases in hcrt/orx neuron cell counts and protein levels in a time course consistent with postcastration impairments in copulatory behavior. This effect was reversed by estradiol replacement. Immunolabeling for androgen (AR) and estrogen (ERα) receptors revealed no colocalization of hcrt/orx with AR and few hcrt/orx neurons expressing ERα, suggesting that hormonal regulation of hcrt/orx expression is via afferents from neurons containing those receptors. We also demonstrate that systemic administration of the orexin-1 receptor antagonist SB 334867 [N-(2-methyl-6-benzoxazolyl)-N″-1,5-naphthyridin-4-yl urea] impairs copulatory behavior. One locus for the prosexual effects of hcrt/orx may be the ventral tegmental area (VTA). We show here that hcrt-1/orx-A produces dose-dependent increases in firing rate and population activity of VTA dopamine (DA) neurons in vivo. Activation of hcrt/orx during copulation, and in turn, excitation of VTA DA neurons by hcrt/orx, may contribute to the robust increases in nucleus accumbens DA previously observed during male sexual behavior. Subsequent triple immunolabeling in anterior VTA showed that Fos-ir in tyrosine hydroxylase-positive neurons apposed to hcrt/orx fibers increases during copulation. Together, these data support the view that hcrt/orx peptides may act in a steroid-sensitive manner to facilitate the energized pursuit of natural rewards like sex via activation of the mesolimbic DA system.

The Wake-Promoting Peptide Orexin-B Inhibits Glutamatergic Transmission to Dorsal Raphe Nucleus Serotonin Neurons through Retrograde Endocannabinoid Signaling

The wake-promoting neuropeptides orexins (hypocretins) play a crucial role in controlling neuronal excitability and synaptic transmission in the CNS. In this study, using whole-cell patch-clamp recordings in an acute dorsal raphe nucleus (DRN) slice preparation, we report that orexin B (Orx-B) depresses the evoked glutamate-mediated synaptic currents in DRN 5-HT neurons. The Orx-B-induced depression is accompanied by an increase in the paired-pulse ratio and the coefficient of variance, suggesting a presynaptic site of action. Orx-B also reduces the frequency but not the amplitude of miniature EPSCs, indicating that depression of glutamatergic transmission is mediated by a decrease in glutamate release. Surprisingly, the Orx-B-induced inhibition of glutamatergic transmission is abolished by postsynaptic inhibition of G-protein signaling with GDPβS, suggesting that this effect is signaled by postsynaptic orexin receptors and expressed presynaptically, presumably through a retrograde messenger. Interestingly, the Orx-B-induced depression of glutamate release is mimicked and occluded by the cannabinoid receptor agonist WIN 55,212-2, and is abolished by the CB1 cannabinoid receptor antagonist AM 251. These results imply that the Orx-B-induced depression of glutamatergic transmission to DRN 5-HT neurons is mediated by retrograde endocannabinoid release. Examination of downstream signaling pathways involved in this response indicates that the effect of Orx-B requires the activation of phospholipase C and DAG lipase enzymatic pathways but not a rise in postsynaptic intracellular calcium. Therefore, our findings reveal a previously unsuspected mechanism by which postsynaptic orexin receptors can modulate glutamatergic synaptic transmission to DRN 5-HT neurons.

D2-Like Dopamine Receptors Depolarize Dorsal Raphe Serotonin Neurons through the Activation of Nonselective Cationic Conductance

The dorsal raphe (DR) receives a prominent dopamine (DA) input that has been suggested to play a key role in the regulation of central serotoninergic transmission. DA is known to directly depolarize DR serotonin neurons, but the underlying mechanisms are not well understood. Here, we show that activation of D2-like dopamine receptors on DR 5-HT neurons elicits a membrane depolarization and an inward current associated with an increase in membrane conductance. The DA-induced inward current (IDA) exhibits a linear I-V relationship and reverses polarity at around –15 mV, suggesting the involvement of a mixed cationic conductance. Consistent with this notion, lowering the extracellular concentration of sodium reduces the amplitude of IDA and induces a negative shift of its reversal potential to approximately –45 mV. This current is abolished by inhibiting G-protein function with GDPβS. Examination of the downstream signaling mechanisms reveals that activation of the nonselective cation current requires the stimulation of phospholipase C but not an increase in intracellular calcium. Thus, pharmacological inhibition of phospholipase C reduces the amplitude of IDA. In contrast, buffering intracellular calcium has no effect on the amplitude of IDA. Bath application of transient receptor potential (TRP) channels blockers, 2-aminoethoxydiphenyl borate and SKF96365 [1-(β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole], strongly inhibits IDA amplitude, suggesting the involvement of TRP-like conductance. These results reveal previously unsuspected mechanism by which D2-like DA receptors induce membrane depolarization and enhance the excitability of DR 5-HT neurons.

Prenatal alcohol exposure causes attention deficits in male rats

Children with fetal alcohol spectrum disorder (FASD) are often diagnosed with attention-deficit/ hyperactivity disorder (ADHD). These children show increases in reaction time (RT) variability and false alarms on choice reaction time (CRT) tasks. In this study, adult rats prenatally exposed to ethanol were trained to perform a CRT task. An analysis of the distribution of RTs obtained from the CRT task found that rats with a history of prenatal ethanol exposure had more variable RT distributions, possibly because of lapses of attention. In addition, it was found that, similar to children with FASD, the ethanol-exposed rats had more false alarms. Thus, rats with prenatal ethanol exposure show attention deficits that are similar to those of children with FASD and ADHD.

Endocannabinoids Suppress Excitatory Synaptic Transmission to Dorsal Raphe Serotonin Neurons through the Activation of Presynaptic CB1 Receptors

Endocannabinoid signaling in the dorsal raphe (DR) has recently been implicated in the regulation of anxiety and depression. However, the cellular mechanisms by which endocannabinoids (eCBs) regulate the excitability of DR 5-hydroxytryptamine (serotonin; 5-HT) neurons remain poorly understood. In the present study, using whole-cell recording from DR 5-HT neurons, we examined the effects of eCBs on glutamatergic synapses in the DR. We found that the eCB anandamide decreased the amplitude of evoked excitatory postsynaptic currents (eEPSCs). This effect was blocked by CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM 251) and mimicked by (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN 55,212-2), a CB1 receptor agonist. The inhibition of eEPSC amplitude was associated with an increase in the paired-pulse ratio and coefficient of variance. Activation of CB1 receptors also reduced the frequency, but not the amplitude, of miniature excitatory postsynaptic currents, indicating that eCBs inhibit glutamate release in the DR. In addition, we found that depolarization of DR 5-HT neurons induced a transient inhibition of the amplitude of eEPSCs, termed depolarization-induced suppression of excitation (DSE). The induction of DSE required an increase in postsynaptic intracellular calcium and was due to a decrease in glutamate release. Furthermore, pharmacological studies showed that blockade of CB1 receptors with AM 251 abolished the DSE. In contrast, activation of CB1 receptors with WIN 55,212-2 mimicked and occluded the DSE, indicating that depolarization of DR 5-HT neurons triggers eCB release, which in turn mediates the DSE. Together, these results indicate that eCBs play a role in modulating glutamatergic synaptic transmission to DR 5-HT neurons.

Ethanol Withdrawal Reduces the Number of Spontaneously Active Ventral Tegmental Area Dopamine Neurons in Conscious Animals

Withdrawal from chronic ethanol treatment leads to a reduction in the electrical activity in dopamine (DA) neurons in the ventral tegmental area (VTA). However, there is disagreement on how the electrical activity is reduced (i.e., in the number of spontaneously active DA neurons or their firing rates and burst firing activity) and on the underlying mechanisms. The use of general anesthesia has been suggested to cause this discrepancy. In the present study, we demonstrate that ethanol withdrawal, in conscious animals, causes a reduction in the number of spontaneously active VTA DA neurons, but not in their firing rate or burst firing activity. Similar results were obtained in a previous study using anesthetized preparation, showing that general anesthesia does not cause this difference. Ethanol withdrawal-induced reduction in a number of spontaneously active VTA DA neurons could be mediated by depolarization inactivation because this effect was reversed by systemic administration of amphetamine, which inhibits VTA DA neurons by hyperpolarization. In addition, the withdrawal effect was normalized by acute ethanol administration, suggesting that the decrease in the number of spontaneously active VTA DA neurons represents an adaptational change to chronic ethanol treatment. Because the electrical activity of DA neurons controls the release of DA, it is possible that the decreased DA release during ethanol withdrawal observed in previous studies is caused by the reduction in the electrical activity of VTA DA neurons.

Prenatal ethanol exposure alters the postnatal development of the spontaneous electrical activity of dopamine neurons in the ventral tegmental area.

Prenatal ethanol exposure causes a persistent reduction in the spontaneous electrical activity of dopamine (DA) neurons in the ventral tegmental area (VTA) in adult animals. Because DA neuron activity matures into adult pattern during postnatal development, it is possible that reduced activity in VTA DA neurons after prenatal ethanol exposure is caused by impaired postnatal development. This possibility was investigated in the present study using the in vivo extracellular single-unit recording and brain stimulation techniques. The results show an age-dependent decrease in the number of spontaneously active VTA DA neurons from 2 to 4 weeks of age in both the control and prenatal ethanol-exposed animals. In ethanol-exposed animals, the age-dependent decrease was more prominent after 3 weeks of age, resulting in lower numbers of spontaneously active VTA DA neurons in 4-week-old and adult animals. In both the control and ethanol-exposed animals, there were age-dependent increases in the firing rates and burst firing activity of VTA DA neurons after 2 weeks of age. Ethanol exposure led to slightly lower firing rates in 4-week-old and adult animals and did not impact the burst firing pattern in any age groups. There were no changes in axon conduction velocity and antidromic spike characteristics of VTA DA neurons. These results indicate that reduced activity of VTA DA neurons during adulthood after prenatal ethanol exposure does not begin prenatally. Instead, it is a result of impaired postnatal development manifested only when animals reach 4 weeks of age. These results suggest that early intervention may be an effective treatment strategy for attention deficit/hyperactivity disorder, a behavioral dysfunction related to the abnormalities of DA systems and often observed in children with fetal alcohol spectrum disorder.

Modulation of the serotonin system by endocannabinoid signaling

The cannabinoid CB(1) receptors and their endogenous agonists, endocannabinoids (eCBs), are ubiquitously distributed throughout the central nervous system (CNS), where they play a key role in the regulation of neuronal excitability. As such, CB signaling has been implicated in the regulation of a myriad of physiological functions ranging from feeding homoeostasis to emotional and motivational processes. Ample evidence from behavioral studies also suggests that eCBs are important regulators of stress responses and a deficit in eCB signaling contributes to stress-related disorders such as anxiety and depression. The eCB-induced modulation of stress-related behaviors appears to be mediated, at least in part, through the regulation of the serotoninergic system. In this article, we review the role of eCB signaling in the regulation of the serotoninergic system with special emphasis on the cellular mechanisms by which cannabinoid CB(1) receptors modulate the excitability of dorsal raphe serotonin neurons.

Long-Term Reduction in Ventral Tegmental Area Dopamine Neuron Population Activity Following Repeated Stimulant or Ethanol Treatment

BACKGROUND Drugs of abuse exert profound effects on the mesolimbic/mesocortical dopaminergic (DA) systems. Few studies have investigated the long-term adaptations in ventral tegmental area (VTA) DA neuron activity after repeated exposure to drugs of abuse. We investigated changes in the electrical activity of VTA DA neurons after cessation from treatment with several stimulants and ethanol. METHODS Adult rats were treated with stimulants (amphetamine: 2 mg/kg per day, 5 days/week, 2 weeks; cocaine: 15 mg/kg per day, 5 days/week, 2 weeks; nicotine: .5 mg/kg per day, 5 days; ethanol: 10 g/kg per day, 3 weeks) and the single-unit activity of VTA DA neurons was studied in vivo 3 to 6 weeks later. RESULTS Stimulant and ethanol treatment decreased basal VTA DA neuron population activity but not firing rate or firing pattern. This effect was reversed by acute apomorphine, suggesting that the underlying mechanism for reduced population activity was depolarization inactivation. Anesthesia did not confound this result, as similar effects were observed in amphetamine-treated rats recorded in a conscious preparation. CONCLUSIONS Reduced basal VTA DA neuron population activity presumably due to depolarization inactivation is a common and long-term neuroadaptation to repeated treatment with stimulants and ethanol. This change in VTA DA neuron activity could underlie the persistent nature of addiction-associated behaviors.

Regulation of plasticity of glutamate synapses by endocannabinoids and the cyclic‐AMP/protein kinase A pathway in midbrain dopamine neurons

Endocannabinoids (eCBs) are lipid signalling molecules which play a key role in the regulation of synaptic transmission and plasticity in the central nervous system. Previous studies have reported that eCBs are released ‘on demand’ in the ventral tegmental area (VTA), a brain region critical for reward learning. However, their role in modulating the long‐term plasticity of glutamate synapses of VTA dopamine (DA) neurons remains unknown. In the present study, we showed that low frequency afferent stimulation paired with moderate postsynaptic depolarization elicited an N‐methyl‐d‐aspartate (NMDA) receptor‐independent long‐term depression (LTD) at glutamate synapses of VTA DA neurons. This form of LTD was caused by a decrease in the probability of glutamate release. Examination of the mechanisms underlying this form of LTD revealed that it was mediated by retrograde eCB signalling. In addition, we found that inhibition of 2‐arachidonoyl glycerol biosynthesis blocked LTD induction, suggesting that 2‐arachidonoyl glycerol is the most likely retrograde eCB messenger mediating LTD. The eCB‐LTD induced at glutamate synapses of VTA DA neurons also required the inhibition of the presynaptic cAMP/PKA pathway. Taken together, these results reveal a critical role of eCBs in controlling the long‐term plasticity of glutamate synapses in VTA DA neurons.