The rostromedial tegmental (RMTg) "brake" on dopamine and behavior: A decade of progress but also much unfinished work.

Between 2005 and 2009, several research groups identified a strikingly dense inhibitory input to midbrain dopamine neurons arising from a previously uncharted region posterior to the ventral tegmental area (VTA). This region is now denoted as either the rostromedial tegmental nucleus (RMTg) or the "tail of the VTA" (tVTA), and is recognized to express distinct genetic markers, encode negative "prediction errors" (inverse to dopamine neurons), and play critical roles in behavioral inhibition and punishment learning. RMTg neurons are also influenced by many categories of abused drugs, and may drive some aversive responses to such drugs, particularly cocaine and alcohol. However, despite much progress, many important questions remain about RMTg molecular/genetic properties, diversity of projection targets, and applications to addiction, depression, and other neuropsychiatric disorders. This article is part of the special Issue on 'Neurocircuitry Modulating Drug and Alcohol Abuse'.

Prenatal exposure to nicotine in mice is associated with alterations in development and cellular and synaptic effects of alcohol in a brainstem arousal nucleus.

Using drugs of abuse while pregnant has tremendous negative consequences for the offspring, including an enhanced risk for substance use disorder (SUD). This vulnerability suggests that gestational exposure to drugs alters the developmental trajectory of neurons important in SUD processes, which could lead to later life changes in responsiveness to motivationally salient stimuli. The laterodorsal tegmentum (LDT) gates the behaviorally relevant firing pattern signaling stimuli saliency in mesoaccumbal circuits. Accordingly, any alterations in LDT functionality could alter output, and play a role in negative outcomes on motivated behavior associated with early-life nicotine exposure. Therefore, we investigated whether prenatal exposure to nicotine (PNE), which is a known teratogen, altered responsiveness of LDT neurons to alcohol by conducting electrophysiology in brain slices. Alcohol induced an outward current in control LDT cells, which was not seen in PNE LDT neurons. The frequency of mEPSCs was significantly decreased by alcohol in LDT PNE cells and accompanied by a decrease in action potential frequency, which were actions not seen in controls. Changes in baseline activity of PNE LDT cells were also observed. In summary, PNE LDT neurons showed alterations in baseline activity and membrane and synaptic responses to postnatal exposures to alcohol. The differences in PNE baseline activity and alcohol responses likely lead to differential output from the LDT to mesoaccumbal targets that could play a role in biasing coding of relevant stimuli, which could participate in the enhanced proclivity for development of SUD in those exposed during gestation to nicotine.

Synaptic Adaptations at the Rostromedial Tegmental Nucleus Underlie Individual Differences in Cocaine Avoidance Behavior.

Although cocaine is powerfully rewarding, not all individuals are equally prone to abusing this drug. We postulate that these differences arise in part because some individuals exhibit stronger aversive responses to cocaine that protect them from cocaine seeking. Indeed, using conditioned place preference (CPP) and a runway operant cocaine self-administration task, we demonstrate that avoidance responses to cocaine vary greatly between individual high cocaine-avoider and low cocaine-avoider rats. These behavioral differences correlated with cocaine-induced activation of the rostromedial tegmental nucleus (RMTg), measured using both in vivo firing and c-fos, whereas slice electrophysiological recordings from ventral tegmental area (VTA)-projecting RMTg neurons showed that relative to low avoiders, high avoiders exhibited greater intrinsic excitability, greater transmission via calcium-permeable AMPA receptors (CP-AMPARs), and higher presynaptic glutamate release. In behaving animals, blocking CP-AMPARs in the RMTg with NASPM reduced cocaine avoidance. Hence, cocaine addiction vulnerability may be linked to multiple coordinated synaptic differences in VTA-projecting RMTg neurons.SIGNIFICANCE STATEMENT Although cocaine is highly addictive, not all individuals exposed to cocaine progress to chronic use for reasons that remain unclear. We find that cocaine's aversive effects, although less widely studied than its rewarding effects, show more individual variability, are predictive of subsequent propensity to seek cocaine, and are driven by variations in RMTg in response to cocaine that arise from distinct alterations in intrinsic excitability and glutamate transmission onto VTA-projecting RMTg neurons.

Repeated but Not Single Administration of Ketamine Prolongs Increases of the Firing Activity of Norepinephrine and Dopamine Neurons.

BACKGROUND: Clinical studies have shown that the rapid antidepressant effect of the glutamate N-methyl-D-aspartate receptor antagonist ketamine generally disappears within 1 week but can be maintained by repeated administration. Preclinical studies showed that a single ketamine injection immediately increases the firing and burst activity of norepinephrine (NE) neurons, but not that of serotonin (5-HT) neurons. It also enhances the population activity of dopamine (DA) neurons. In the present study, we investigated whether such alterations of monoamine neuronal firing are still present 1 day after a single injection, and whether they can be maintained by repeated injections. METHODS: Rats received a single ketamine injection or 6 over 2 weeks and the firing activity of dorsal raphe nucleus 5-HT, locus coeruleus NE, and ventral tegmental area DA neurons was assessed. RESULTS: One day following a single injection of ketamine, there was no change in the firing activity of 5-HT, NE, or DA neurons. One day after repeated ketamine administration, however, there was a robust increase of the firing activity of NE neurons and an enhancement of burst and population activities of DA neurons, but still no change in firing parameters of 5-HT neurons. The increased activity of NE neurons was no longer present 3 days after the last injection, whereas that of DA neurons was still present. DA neurons were firing normally 7 days after repeated injections. CONCLUSION: These results imply that the enhanced activity of NE and DA neurons may play a significant role in the maintenance of the antidepressant action of ketamine.

Stress-related endogenous neuropeptides induce neuronal excitation in the Laterodorsal Tegmentum.

Stress is a physiological response that promotes maintenance of balance against harmful stimuli. Unfortunately, chronic activation of stress systems facilitates the development of psychiatric disorders. A stress-mediated hypercholinergic state could underlie this facilitation, as cholinergic mechanisms have been suggested to play a role in anxiety, depression, and substance use disorder (SUD). Stimulation by stress hormones, urocortin (Ucn1) or corticotropin-releasing factor (CRF), of the CRF receptor type 1 (CRFR1) of acetylcholine-containing neurons of the laterodorsal tegmental nucleus (LDT) could be involved in modulation of cholinergic transmission during periods of stress hormone activation, which could play a role in psychiatric disorders as cholinergic LDT neurons project to, and control activity in, mood-, arousal- and SUD-controlling regions. The present study investigated for the first time the membrane effects and intracellular outcomes of CRFR1 activation by endogenous stress hormones on LDT neurons. Patch clamp recordings of immunohistochemically-identified cholinergic and non-cholinergic LDT neurons with concurrent calcium imaging were used to monitor cellular responses to CRFR1 stimulation with Ucn1 and CRF. Postsynaptically-mediated excitatory currents were elicited in LDT cholinergic neurons, accompanied by an enhancement in synaptic events. In addition, CRFR1 activation resulted in rises in intracellular calcium levels. CRFR1 stimulation recruited MAPK/ERK and SERCA-ATPase involved pathways. The data presented here provide the first evidence that Ucn1 and CRF exert pre and postsynaptic excitatory membrane actions on LDT cholinergic neurons that could underlie the hypercholinergic state associated with stress which could play a role in the heightened risk of psychiatric disorders associated with a chronic stress state.

The rostromedial tegmental nucleus: a key modulator of pain and opioid analgesia.

A recently defined structure, the rostromedial tegmental nucleus (RMTg; aka tail of the ventral tegmental area [VTA]), has been proposed as an inhibitory control center for dopaminergic activity of the VTA. This region is composed of GABAergic cells that send afferent projections to the ventral midbrain and synapse onto dopaminergic cells in the VTA and substantia nigra. These cells exhibit µ-opioid receptor immunoreactivity, and in vivo, ex vivo, and optogenetic/electrophysiological approaches demonstrate that morphine excites dopamine neurons by targeting receptors on GABAergic neurons localized in the RMTg. This suggests that the RMTg may be a key modulator of opioid effects and a major brake regulating VTA dopamine systems. However, no study has directly manipulated RMTg GABAergic neurons in vivo and assessed the effect on nociception or opioid analgesia. In this study, multiplexing of GABAergic neurons in the RMTg was achieved using stimulatory Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) and inhibitory kappa-opioid receptor DREADDs (KORD). Our data show that locally infused RMTg morphine or selective RMTg GABAergic neuron inhibition produces 87% of the maximal antinociceptive effect of systemic morphine, and RMTg GABAergic neurons modulate dopamine release in the nucleus accumbens. In addition, chemoactivation of VTA dopamine neurons significantly reduced pain behaviors both in resting and facilitated pain states and reduced by 75% the dose of systemic morphine required to produce maximal antinociception. These results provide compelling evidence that RMTg GABAergic neurons are involved in processing of nociceptive information and are important mediators of opioid analgesia.

Blockade of calcium-permeable AMPA receptors in the lateral habenula produces increased antidepressant-like effects in unilateral 6-hydroxydopamine-lesioned rats compared to sham-lesioned rats.

At present, role of the lateral habenula (LHb) calcium-permeable AMPA receptors (CP-AMPARs) in depression is not understood, particularly in Parkinson's disease-related depression. Here we found that lesions of the substantia nigra pars compacta (SNc) in rats induced depressive-like behaviors, and intra-LHb injection of CP-AMPAR antagonist Naspm produced antidepressant-like effects in SNc sham-lesioned and SNc-lesioned rats, however, the doses inducing these effects in SNc-lesioned rats were lower than that of SNc sham-lesioned rats. Blockade of LHb CP-AMPARs decreased the firing rate of the neurons and increased release of dopamine and serotonin in the medial prefrontal cortex (mPFC) in both groups, but the duration of Naspm action on the firing rate and release of the transmitters were prolonged in SNc-lesioned rats. These changes in SNc-lesioned rats were involved in increased expression of ßCaMKII and p-GluR1-S831 in the LHb. Intra-LHb injection of Naspm inhibited dopaminergic neurons in the anterior ventral tegmental area and serotonergic neurons in the dorsal raphe nucleus and excited dopaminergic neurons in the posterior ventral tegmental area (pVTA) and serotonergic neurons in the median raphe nucleus (MRN), and lesioning the GABAergic rostromedial tegmental nucleus (RMTg) decreased the percentages of excited pVTA dopaminergic neurons and MRN serotonergic neurons. Our findings indicate that blockade of LHb CP-AMPARs produces antidepressant-like effects, which attribute to decreased firing activity of LHb neurons and increased levels of dopamine and serotonin in the mPFC, and provide further evidence that LHb CP-AMPARs regulate the firing activity of pVTA dopaminergic neurons and MRN serotonergic neurons indirectly via the RMTg.

Alcohol withdrawal drives depressive behaviors by activating neurons in the rostromedial tegmental nucleus.

Rostromedial tegmental nucleus (RMTg) GABA neurons exert a primary inhibitory drive onto midbrain dopamine neurons and are excited by a variety of aversive stimuli. There is, however, little evidence that the RMTg-ventral tegmental area (VTA)-nucleus accumbens shell (Acb) circuit plays a role in the aversive consequences of alcohol withdrawal. This study was performed in adult male Long-Evans rats at 48-h withdrawal from chronic alcohol drinking in the intermittent schedule. These rats displayed clear anhedonia and depression-like behaviors, as measured with the sucrose preference, and forced swimming tests. These aberrant behaviors were accompanied by a substantial increase in cFos expression in the VTA-projecting RMTg neurons, identified by a combination of immunohistochemistry and retrograde-tracing techniques. Pharmacological or chemogenetic inhibition of RMTg neurons mitigated the anhedonia and depression-like behaviors. Ex vivo electrophysiological data showed that chemogenetic inactivation of RMTg neurons reduced GABA release and accelerated spontaneous firings of VTA dopamine neurons. Finally, using a functional hemispheric disconnection procedure, we demonstrated that inhibition of unilateral RMTg, when combined with activation of D1 and D2 dopamine receptors in the contralateral (but not ipsilateral) Acb, mitigated the anhedonia and depression-like behaviors in alcohol-withdrawal rats. These data show that the integrity in the RMTg-VTA-Acb pathway in a single hemisphere is sufficient to elicit depression-like behavior during ethanol-withdrawal. Overall, the present results reveal that the RMTg-VTA-Acb pathway plays a crucial role in the depression-like behavior in animals undergoing alcohol withdrawal, further advocating the RMTg as a potential therapeutic target for alcoholism.

Stress augments the rewarding memory of cocaine via the activation of brainstem-reward circuitry.

Effects of stress on the reward system are well established in the literature. Although previous studies have revealed that stress can reinstate extinguished addictive behaviors related to cocaine, the effects of stress on the rewarding memory of cocaine are not fully understood. Here, we provide evidence that stress potentiates the expression of rewarding memory of cocaine via the activation of brainstem-reward circuitry using a cocaine-induced conditioned place preference (CPP) paradigm combined with restraint stress in rats. The rats exposed to 30-minute restraint stress immediately before posttest exhibited significantly larger CPP scores compared with non-stressed rats. Intra-laterodorsal tegmental nucleus (LDT) microinjection of a ß or α2 adrenoceptor antagonist attenuated the stress-induced enhancement of cocaine CPP. Consistent with this observation, intra-LDT microinjection of a ß or α2 adrenoceptor agonist before posttest increased cocaine CPP. Additionally, intra-ventral tegmental area (VTA) microinjection of antagonists for the muscarinic acetylcholine, nicotinic acetylcholine or glutamate receptors attenuated the stress-induced enhancement of cocaine CPP. Finally, intra-medial prefrontal cortex (mPFC) microinjection of a D1 receptor antagonist also reduced the stress-induced enhancement of cocaine CPP. These findings suggest a mechanism wherein the LDT is activated by noradrenergic input from the locus coeruleus, leading to the activation of VTA dopamine neurons via both cholinergic and glutamatergic transmission and the subsequent excitation of the mPFC to enhance the memory of cocaine-induced reward value.

Pedunculopontine tegmentum cholinergic loss leads to a progressive decline in motor abilities and neuropathological changes resembling progressive supranuclear palsy.

Progressive supranuclear palsy (PSP) is the most common atypical Parkinsonism. Although PSP shares some symptomology with Parkinson's disease (PD), PSP has a different underlying pathology characterized by tau aggregation. Furthermore, PSP sufferers respond poorly to PD medications and there are no effective alternative therapeutics. The development of both palliative and disease altering therapeutics has been hampered by the lack of an animal model that displays relevant PSP-like pathology and behavioral deficits. Previously, our lab found that in rats the selective removal of cholinergic pedunculopontine neurons (whose axonal projections overlap with areas of PSP pathology), mimics the extensive loss of cholinergic pedunculopontine neurons seen in PSP, and produces a unique PSP-like combination of deficits in: startle reflex, attention, and motor function. The present study extends those findings by allowing the lesion to incubate for over a year and compares behavioral and post-mortem pathology of pedunculopontine-cholinergic-lesioned and sham-lesioned rats. There was an early startle reflex deficit which did not improve over time. Progressive declines in motor function developed over the course of the year, including an increase in the number of "slips" while navigating various beams and poorly coordinated transitions from an elevated platform into homecages. Histological analysis discovered that the loss off cholinergic pedunculopontine neurons precipitated a significant loss of substantia nigra tyrosine hydroxylase-positive neurons and a significant enlargement of the lateral ventricles. The latter is a distinguishing feature between PSP and PD. This preclinical animal model of PSP has the potential to further our understanding of PSP and aid in the testing of potential therapeutic agents.

Nicotine aversion is mediated by GABAergic interpeduncular nucleus inputs to laterodorsal tegmentum.

Nicotine use can lead to dependence through complex processes that are regulated by both its rewarding and aversive effects. Recent studies show that aversive nicotine doses activate excitatory inputs to the interpeduncular nucleus (IPN) from the medial habenula (MHb), but the downstream targets of the IPN that mediate aversion are unknown. Here we show that IPN projections to the laterodorsal tegmentum (LDTg) are GABAergic using optogenetics in tissue slices from mouse brain. Selective stimulation of these IPN axon terminals in LDTg in vivo elicits avoidance behavior, suggesting that these projections contribute to aversion. Nicotine modulates these synapses in a concentration-dependent manner, with strong enhancement only seen at higher concentrations that elicit aversive responses in behavioral tests. Optogenetic inhibition of the IPN-LDTg connection blocks nicotine conditioned place aversion, suggesting that the IPN-LDTg connection is a critical part of the circuitry that mediates the aversive effects of nicotine.

Prenatal nicotine exposure alters postsynaptic AMPA receptors and glutamate neurotransmission within the laterodorsal tegmentum (LDT) of juvenile mice.

Despite dissemination of information regarding the harm on fetal development of smoking while pregnant, the number of pregnancies associated with nicotine exposure appears to have stagnated. Presence of nicotine during neural formulation is associated with a higher susceptibility of drug dependence, suggesting an altered development of neurons in circuits involved in saliency and motivation. The laterodorsal tegmental nucleus (LDT) plays a role in coding stimuli valence via afferents to mesolimbic nuclei. Accordingly, alterations in development of neural mechanisms in the LDT could be involved in vulnerability to drug dependency. Therefore, we examined the effect of prenatal nicotine exposure (PNE) on glutamatergic functioning of LDT neurons in mouse brain slices using whole-cell, patch clamp concurrent with fluorescence-based calcium imaging. PNE was associated with larger amplitudes of AMPA-induced currents, and greater AMPA-mediated rises in intracellular calcium. AMPA/NMDA ratios and the AMPA-current rectification index were lower and higher, respectively, consistent with changes in the functionality of AMPA receptors in the PNE, which was substantiated by a greater inhibition of evoked and spontaneous glutamatergic synaptic events by a selective inhibitor of GluA2-lacking AMPA receptors. Paired pulse ratios showed a decreased probability of glutamate release from presynaptic inputs, and fluorescent imaging indicated a decreased action potential-dependent calcium increase associated with PNE. When taken together, our data suggest that PNE alters LDT glutamatergic functioning, which could alter output to mesolimbic targets. Such an alteration could play a role in altered coding of relevancy of drug stimuli that could enhance risk for development of drug dependency.

Pharmacological profile of vestibular inhibitory inputs to superior oblique motoneurons.

Vestibulo-ocular reflexes (VOR) are mediated by three-neuronal brainstem pathways that transform semicircular canal and otolith sensory signals into motor commands for the contraction of spatially specific sets of eye muscles. The vestibular excitation and inhibition of extraocular motoneurons underlying this reflex is reciprocally organized and allows coordinated activation of particular eye muscles and concurrent relaxation of their antagonistic counterparts. Here, we demonstrate in isolated preparations of Xenopus laevis tadpoles that the discharge modulation of superior oblique motoneurons during cyclic head motion derives from an alternating excitation and inhibition. The latter component is mediated exclusively by GABA, at variance with the glycinergic inhibitory component in lateral rectus motoneurons. The different pharmacological profile of the inhibition correlates with rhombomere-specific origins of vestibulo-ocular projection neurons and the complementary segmental abundance of GABAergic and glycinergic vestibular neurons. The evolutionary conserved rhombomeric topography of vestibulo-ocular projections makes it likely that a similar pharmacological organization of inhibitory VOR neurons as reported here for anurans is also implemented in mammalian species including humans.

Glucagon-Like Peptide-1 Receptor Signaling in the Lateral Dorsal Tegmental Nucleus Regulates Energy Balance.

The neurobiological substrates that mediate the anorectic effects of both endogenous glucagon-like peptide-1 (GLP-1) and exogenous GLP-1 receptor (GLP-1R) agonists are an active area of investigation. As the lateral dorsal tegmental nucleus (LDTg) expresses the GLP-1R and represents a potential neuroanatomical hub connecting the nucleus tractus solitarius (NTS), the major central source of GLP-1, with the other nuclei in the midbrain and forebrain, we tested the hypothesis that GLP-1R signaling in the LDTg controls food intake. Direct activation of LDTg GLP-1R suppresses food intake through a reduction in average meal size and independent of nausea/malaise. Immunohistochemical data show that GLP-1-producing neurons in the NTS project to the LDTg, providing anatomical evidence of endogenous central GLP-1 in the LDTg. Pharmacological blockade of LDTg GLP-1Rs with exendin-(9-39) dose-dependently increases food intake and attenuates the hypophagic effects of gastric distension. As GLP-1 mimetics are administered systemically in humans, we evaluated whether peripherally administered GLP-1R agonists access the LDTg to affect feeding. Immunohistochemical data show that a systemically administered fluorescent GLP-1R agonist accesses the LDTg and is juxtaposed with neurons. Additionally, blockade of LDTg GLP-1Rs attenuates the hypophagic effects of a systemic GLP-1R agonist. Together, these data indicate that LDTg GLP-1R signaling controls energy balance and underscores the role of the LDTg in integrating energy balance-relevant signals to modulate feeding.

Enhanced wakefulness following lesions of a mesopontine locus essential for the induction of general anesthesia.

The induction of general anesthesia shares many features with the transition from wakefulness to non-rapid eye movement (NREM) sleep, suggesting that the two types of brain-state transition are orchestrated by a common neuronal mechanism. Previous studies revealed a brainstem locus, the mesopontine tegmental anesthesia area (MPTA), that is of singular importance for anesthetic induction. Microinjection of GABAergic anesthetics there induces rapid loss-of-consciousness and lesions render the animal relatively insensitive to anesthetics administered systemically. Here we show that MPTA lesions also alter the natural sleep-wake rhythm by increasing overall wake time at the expense of time asleep (NREM and REM sleep equally), with nearly all of the change occurring during the dark hours of the light-dark cycle. The effect was proportional to the extent of the lesion and was not seen after lesions just outside of the MPTA, or following sham lesions. Thus, MPTA neurons appear to play a role in natural bistable brain-state switching (sleep-wake) as well as in loss and recovery of consciousness induced pharmacologically.

Cellular and Molecular Mechanisms of REM Sleep Homeostatic Drive: A Plausible Component for Behavioral Plasticity.

Homeostatic regulation of REM sleep drive, as measured by an increase in the number of REM sleep transitions, plays a key role in neuronal and behavioral plasticity (i.e., learning and memory). Deficits in REM sleep homeostatic drive (RSHD) are implicated in the development of many neuropsychiatric disorders. Yet, the cellular and molecular mechanisms underlying this RSHD remain to be incomplete. To further our understanding of this mechanism, the current study was performed on freely moving rats to test a hypothesis that a positive interaction between extracellular-signal-regulated kinase 1 and 2 (ERK1/2) activity and brain-derived neurotrophic factor (BDNF) signaling in the pedunculopontine tegmentum (PPT) is a causal factor for the development of RSHD. Behavioral results of this study demonstrated that a short period (<90 min) of selective REM sleep restriction (RSR) exhibited a strong RSHD. Molecular analyses revealed that this increased RSHD increased phosphorylation and activation of ERK1/2 and BDNF expression in the PPT. Additionally, pharmacological results demonstrated that the application of the ERK1/2 activation inhibitor U0126 into the PPT prevented RSHD and suppressed BDNF expression in the PPT. These results, for the first time, suggest that the positive interaction between ERK1/2 and BDNF in the PPT is a casual factor for the development of RSHD. These findings provide a novel direction in understanding how RSHD-associated specific molecular changes can facilitate neuronal plasticity and memory processing.

Nitric oxide in the medial prefrontal cortex contributes to the acquisition of cocaine place preference and synaptic plasticity in the laterodorsal tegmental nucleus.

Nitric oxide (NO), a gaseous neurotransmitter, is involved in a variety of brain functions, including drug addiction. Although previous studies have suggested that NO plays an important role in the development of cocaine addiction, the brain region(s) in which NO acts and how it contributes to cocaine addiction remain unclear. In this study, we examined these issues using a cocaine-induced conditioned place preference (CPP) paradigm and ex vivo electrophysiological recordings in rats. Specifically, we focused on the medial prefrontal cortex (mPFC) and laterodorsal tegmental nucleus (LDT), brain regions associated with cocaine CPP development and cocaine-induced plasticity. Intra-mPFC injection of the non-selective NO synthase (NOS) inhibitor L-NAME or the neuronal NOS (nNOS) selective inhibitor L-NPA during the conditioning phase disrupted cocaine CPP. Additionally, intra-mPFC injection of L-NPA prior to each cocaine injection prevented the induction of presynaptic plasticity, induced by repeated cocaine administration, in LDT cholinergic neurons. These findings indicate that NO generated in the mPFC contributes to the acquisition of cocaine CPP and the induction of neuroplasticity in LDT cholinergic neurons. Together with previous studies showing that NO induces membrane plasticity in mPFC neurons, that mPFC neurons project to the LDT, and that LDT activity is critical for the acquisition of cocaine CPP, the present findings suggest that NO-mediated neuroplasticity induced in the mPFC-LDT circuitry is critical for the development of cocaine addiction.

Neuropeptide S (NPS) is a neuropeptide with cellular actions in arousal and anxiety-related nuclei: Functional implications for effects of NPS on wakefulness and mood.

Neuropeptide S (NPS) is a peptide recently recognized to be present in the CNS, and believed to play a role in vigilance and mood control, as behavioral studies have shown it promotes arousal and has an anxiolytic effect. Although NPS precursor is found in very few neurons, NPS positive fibers are present throughout the brain stem. Given the behavioral actions of this peptide and the wide innervation pattern, we examined the cellular effects of NPS within two brain stem nuclei known to play a critical role in anxiety and arousal: the dorsal raphe (DR) and laterodorsal tegmentum (LDT). In mouse brain slices, NPS increased cytoplasmic levels of calcium in DR and LDT cells. Calcium rises were independent of action potential generation, reduced by low extracellular levels of calcium, attenuated by IP3 - and ryanodine (RyR)-dependent intracellular calcium store depletion, and eliminated by the receptor (NPSR) selective antagonist, SHA 68. NPS also exerted an effect on the membrane of DR and LDT cells inducing inward and outward currents, which were driven by an increase in conductance, and eliminated by SHA 68. Membrane actions of NPS were found to be dependent on store-mediated calcium as depletion of IP3 and RyR stores eliminated NPS-induced currents. Finally, NPS also had actions on synaptic events, suggesting facilitation of glutamatergic and GABAergic presynaptic transmission. When taken together, actions of NPS influenced the excitability of DR and LDT neurons, which could play a role in the anxiolytic and arousal-promoting effects of this peptide.

Effects of early-life exposure to THIP on brainstem neuronal excitability in the Mecp2-null mouse model of Rett syndrome before and after drug withdrawal.

Rett syndrome (RTT) is mostly caused by mutations of the X-linked MECP2 gene. Although the causal neuronal mechanisms are still unclear, accumulating experimental evidence obtained from Mecp2-/Y mice suggests that imbalanced excitation/inhibition in central neurons plays a major role. Several approaches may help to rebalance the excitation/inhibition, including agonists of GABAA receptors (GABAAR). Indeed, our previous studies have shown that early-life exposure of Mecp2-null mice to the extrasynaptic GABAAR agonist THIP alleviates several RTT-like symptoms including breathing disorders, motor dysfunction, social behaviors, and lifespan. However, how the chronic THIP affects the Mecp2-/Y mice at the cellular level remains elusive. Here, we show that the THIP exposure in early lives markedly alleviated hyperexcitability of two types of brainstem neurons in Mecp2-/Y mice. In neurons of the locus coeruleus (LC), known to be involved in breathing regulation, the hyperexcitability showed clear age-dependence, which was associated with age-dependent deterioration of the RTT-like breathing irregularities. Both the neuronal hyperexcitability and the breathing disorders were relieved with early THIP treatment. In neurons of the mesencephalic trigeminal nucleus (Me5), both the neuronal hyperexcitability and the changes in intrinsic membrane properties were alleviated with the THIP treatment in Mecp2-null mice. The effects of THIP on both LC and Me5 neuronal excitability remained 1 week after withdrawal. Persistent alleviation of breathing abnormalities in Mecp2-/Y mice was also observed a week after THIP withdrawal. These results suggest that early-life exposure to THIP, a potential therapeutic medicine, appears capable of controlling neuronal hyperexcitability in Mecp2-/Y mice, which occurs in the absence of THIP in the recording solution, lasts at least 1 week after withdrawal, and may contribute to the RTT-like symptom mitigation.

Chronic cocaine exposure induces noradrenergic modulation of inhibitory synaptic transmission to cholinergic neurons of the laterodorsal tegmental nucleus.

The laterodorsal tegmental nucleus (LDT), which sends cholinergic efferent connections to dopaminergic (DA) neurons in the ventral tegmental area (VTA), plays a critical role in the development of addictive behavior and the reinstatement of cocaine-seeking behavior. Although repeated cocaine exposure elicits plastic changes in excitatory synaptic transmission and intrinsic membrane excitability in LDT cholinergic neurons, it remains unclear whether inhibitory synaptic transmission is modulated by cocaine exposure. The LDT receives fibers containing noradrenaline (NA), a neurotransmitter whose extracellular levels increase with cocaine exposure. Therefore, it is hypothesized that repeated cocaine exposure induces plastic changes in LDT cholinergic neurons via NA. Ex vivo electrophysiological recordings in LDT cholinergic neurons were obtained from rats repeatedly exposed to cocaine. Bath-application of NA induced similar levels of hyperpolarization in both saline- and cocaine-treated neurons. However, NA attenuated the amplitude of inhibitory postsynaptic currents (IPSCs) in cocaine- but not saline-treated neurons through α2 adrenoceptors. This NA-induced IPSC attenuation was observed in the presence of strychnine, but not gabazine, indicating that NA modulated GABAergic but not glycinergic neurotransmission. NA increased the paired-pulse ratios of evoked IPSCs and decreased the frequencies of miniature IPSCs (mIPSCs) without affecting their amplitudes, suggesting a presynaptic mechanism. These findings suggest that repeated cocaine exposure induces neuroplasticity in GABAergic synaptic transmission onto LDT cholinergic neurons by probably modulating presynaptic α2 adrenoceptors. This potentially increases the activity of LDT cholinergic neurons, which might contribute to the development of addictive behavior by enhancing VTA DA neuronal activity.