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 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.

Muscarinic control of rostromedial tegmental nucleus GABA neurons and morphine-induced locomotion.

Opioids induce rewarding and locomotor effects by inhibiting rostromedial tegmental GABA neurons that express µ-opioid and nociceptin receptors. These GABA neurons then strongly inhibit dopamine neurons. Opioid-induced reward, locomotion and dopamine release also depend on pedunculopontine and laterodorsal tegmental cholinergic and glutamate neurons, many of which project to and activate ventral tegmental area dopamine neurons. Here we show that laterodorsal tegmental and pedunculopontine cholinergic neurons project to both rostromedial tegmental nucleus and ventral tegmental area, and that M4 muscarinic receptors are co-localized with µ-opioid receptors associated with rostromedial tegmental GABA neurons. To inhibit or excite rostromedial tegmental GABA neurons, we utilized adeno-associated viral vectors and DREADDs to express designed muscarinic receptors (M4D or M3D respectively) in GAD2::Cre mice. In M4D-expressing mice, clozapine-N-oxide increased morphine-induced, but not vehicle-induced, locomotion. In M3D-expressing mice, clozapine-N-oxide blocked morphine-induced, but not vehicle-induced, locomotion. We propose that cholinergic inhibition of rostromedial tegmental GABA neurons via M4 muscarinic receptors facilitates opioid inhibition of the same neurons. This model explains how mesopontine cholinergic systems and muscarinic receptors in the rostromedial tegmental nucleus and ventral tegmental area are important for dopamine-dependent and dopamine-independent opioid-induced rewards and locomotion.

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.

Role for the Rostromedial Tegmental Nucleus in Signaling the Aversive Properties of Alcohol.

BACKGROUND: While the rewarding effects of alcohol contribute significantly to its addictive potential, it is becoming increasingly appreciated that alcohol's aversive properties also play an important role in the propensity to drink. Despite this, the neurobiological mechanism for alcohol's aversive actions is not well understood. The rostromedial tegmental nucleus (RMTg) was recently characterized for its involvement in aversive signaling and has been shown to encode the aversive properties of cocaine, yet its involvement in alcohol's aversive actions have not been elucidated. METHODS: Adult male and female Long-Evans rats underwent conditioned taste aversion (CTA) procedures where exposure to a novel saccharin solution was paired with intraperitoneal administration of saline, lithium chloride (LiCl), or ethanol (EtOH). Control rats underwent the same paradigm except that drug and saccharin exposure were explicitly unpaired. Saccharin consumption was measured on test day in the absence of drug administration, and rats were sacrificed 90 to 105 minutes following access to saccharin. Brains were subsequently harvested and processed for cFos immunohistochemistry. The number of cFos-labeled neurons was counted in the RMTg and the lateral habenula (LHb)-a region that sends prominent glutamatergic input to the RMTg. RESULTS: In rats that received paired drug and saccharin exposure, EtOH and LiCl induced significant CTA compared to saline to a similar degree in males and females. Both EtOH- and LiCl-induced CTA significantly enhanced cFos expression in the RMTg and LHb but not the hippocampus. Similar to behavioral measures, no significant effect of sex on CTA-induced cFos expression was observed. cFos expression in both the RMTg and LHb was significantly correlated with CTA magnitude with greater cFos being associated with more pronounced CTA. In addition, cFos expression in the RMTg was positively correlated with LHb cFos. CONCLUSIONS: These data suggest that the RMTg and LHb are involved in the expression of CTA and are consistent with previous work implicating the RMTg in aversive signaling. Furthermore, increased cFos expression in the RMTg following EtOH-induced CTA suggests that this region plays a role in signaling alcohol's aversive properties.

Age-related changes in functional postsynaptic nicotinic acetylcholine receptor subunits in neurons of the laterodorsal tegmental nucleus, a nucleus important in drug addiction.

The earlier an individual initiates cigarette smoking, the higher the likelihood of development of dependency to nicotine, the addictive ingredient in cigarettes. One possible mechanism underlying this higher addiction liability is an ontogenetically differential cellular response induced by nicotine in neurons mediating the reinforcing or euphoric effects of this drug, which could arise from age-related differences in the composition of nicotinic acetylcholine receptor (nAChR) subunits. In the current study, we examined whether the subunit composition of nAChRs differed between neurons within the laterodorsal tegmentum (LDT), a nucleus importantly involved in drug addiction associated behaviours, across two periods of ontogeny in which nicotine-mediated excitatory responses were shown to depend on age. To this end, whole-cell patch-clamp recordings in mouse brain slices from identified LDT neurons, in combination with nAChR subunit-specific receptor antagonists, were conducted. Comparison of the contribution of different nAChR subunits to acetylcholine (ACh)-induced inward currents indicated that the contributions of the ß2 and/or ß4 and α7 nAChR subunits alter across age. Taken together, we conclude that across a limited ontogenetic period, there is plasticity in the subunit composition of nAChRs in LDT neurons. In addition, our data indicate, for the first time, functional presence of α6 nAChR subunits in LDT neurons within the age ranges studied. Changes in subunit composition of nAChRs across ontogeny could contribute to the age-related differential excitability induced by nicotine. Differences in the subunit composition of nAChRs within the LDT would be expected to contribute to ontogenetic-dependent outflow from the LDT to target regions, which include reward-related circuitry.

A role for the lateral dorsal tegmentum in memory and decision neural circuitry.

A role for the hippocampus in memory is clear, although the mechanism for its contribution remains a matter of debate. Converging evidence suggests that hippocampus evaluates the extent to which context-defining features of events occur as expected. The consequence of mismatches, or prediction error, signals from hippocampus is discussed in terms of its impact on neural circuitry that evaluates the significance of prediction errors: Ventral tegmental area (VTA) dopamine cells burst fire to rewards or cues that predict rewards (Schultz, Dayan, & Montague, 1997). Although the lateral dorsal tegmentum (LDTg) importantly controls dopamine cell burst firing (Lodge & Grace, 2006) the behavioral significance of the LDTg control is not known. Therefore, we evaluated LDTg functional activity as rats performed a spatial memory task that generates task-dependent reward codes in VTA (Jo, Lee, & Mizumori, 2013; Puryear, Kim, & Mizumori, 2010) and another VTA afferent, the pedunculopontine nucleus (PPTg, Norton, Jo, Clark, Taylor, & Mizumori, 2011). Reversible inactivation of the LDTg significantly impaired choice accuracy. LDTg neurons coded primarily egocentric information in the form of movement velocity, turning behaviors, and behaviors leading up to expected reward locations. A subset of the velocity-tuned LDTg cells also showed high frequency bursts shortly before or after reward encounters, after which they showed tonic elevated firing during consumption of small, but not large, rewards. Cells that fired before reward encounters showed stronger correlations with velocity as rats moved toward, rather than away from, rewarded sites. LDTg neural activity was more strongly regulated by egocentric behaviors than that observed for PPTg or VTA cells that were recorded by Puryear et al. and Norton et al. While PPTg activity was uniquely sensitive to ongoing sensory input, all three regions encoded reward magnitude (although in different ways), reward expectation, and reward encounters. Only VTA encoded reward prediction errors. LDTg may inform VTA about learned goal-directed movement that reflects the current motivational state, and this in turn may guide VTA determination of expected subjective goal values. When combined it is clear the LDTg and PPTg provide only a portion of the information that dopamine cells need to assess the value of prediction errors, a process that is essential to future adaptive decisions and switches of cognitive (i.e. memorial) strategies and behavioral responses.

The effects of systemic administration and local microinjection into the central nervous system of the selective serotonin 5-HT2C receptor agonist RO-600175 on sleep and wakefulness in the rat.

The effects of RO-600175, a selective 5-HT2C receptor agonist, were studied in adult rats implanted for chronic sleep recordings. Intraperitoneal administration of RO-600175 (4 mg/kg) during the light phase of the light-dark cycle significantly increased wakefulness and reduced slow wave sleep and rapid-eye-movement sleep during the first 2 h of the recording period. Direct infusion of RO-600175 into the dorsal raphe nucleus (4 mmol/l), laterodorsal tegmental nucleus (4 mmol/l), or horizontal limb of the diagonal band of Broca (4 mmol/l) also decreased rapid-eye-movement sleep. It is proposed that the activation of γ-aminobutyric acid-ergic cells located in the dorsal raphe nucleus, laterodorsal tegmental nucleus, and horizontal limb of the diagonal band of Broca is responsible, at least in part, for the effects of RO-600175 on rapid-eye-movement sleep. It is suggested that the increased wakefulness observed after systemic injection of the 5-HT2C receptor ligand could be partly related to the increased release of acetylcholine in the frontal cortex and hippocampus. However, additional studies are required to characterize the neurotransmitter systems responsible for the increase in wakefulness.

Action controls dopaminergic enhancement of reward representations.

Dopamine is widely observed to signal anticipation of future rewards and thus thought to be a key contributor to affectively charged decision making. However, the experiments supporting this view have not dissociated rewards from the actions that lead to, or are occasioned by, them. Here, we manipulated dopamine pharmacologically and examined the effect on a task that explicitly dissociates action and reward value. We show that dopamine enhanced the neural representation of rewarding actions, without significantly affecting the representation of reward value as such. Thus, increasing dopamine levels with levodopa selectively boosted striatal and substantia nigra/ventral tegmental representations associated with actions leading to reward, but not with actions leading to the avoidance of punishment. These findings highlight a key role for dopamine in the generation of appetitively motivated actions.

Cocaine exposure enhances excitatory synaptic drive to cholinergic neurons in the laterodorsal tegmental nucleus.

Accumulating evidence indicates that the laterodorsal tegmental nucleus (LDT) is associated with reward processing and addiction. The cholinergic projection from the LDT to the ventral tegmental area is essential for a large dopamine release in the nucleus accumbens, which is critically involved in the reinforcing effects of addictive drugs, including cocaine. In contrast to the large number of studies on plasticity induced after cocaine exposure in the mesocorticolimbic dopaminergic system, it remains unknown whether LDT cholinergic neurons exhibit plastic changes following cocaine administration. To address this issue, we performed ex vivo whole-cell recordings in LDT cholinergic neurons obtained from rats following cocaine administration. Neurons obtained from 1 day after 5-day cocaine-treated rats showed significantly smaller paired-pulse ratios of evoked EPSCs and higher miniature EPSC frequencies than those from saline-treated rats, indicating an induction of presynaptic plasticity of increased glutamate release. This plasticity seemed to recover after a 5-day withdrawal from repeated cocaine exposure, and required NMDA receptor stimulation and nitric oxide production. Additionally, pharmacological suppression of activity of the medial prefrontal cortex inhibited the presynaptic plasticity in the LDT. On the other hand, AMPA/NMDA ratios were not different between saline- and cocaine-treated groups, revealing an absence of postsynaptic plasticity. These findings provide the first direct evidence of cocaine-induced synaptic plasticity in LDT cholinergic neurons and suggest that the presynaptic plasticity enhances the activity of LDT cholinergic neurons, contributing to the expression of cocaine-induced addictive behaviors through the dysregulation of the mesocorticolimbic system.

Opioid-sensitive GABA inputs from rostromedial tegmental nucleus synapse onto midbrain dopamine neurons.

Opioids increase dopamine release in the brain through inhibition of GABA-A IPSCs onto dopamine cells. Immunolabeling indicates that GABA neurons in the rostromedial tegmental nucleus (RMTg), also known as the tail of the ventral tegmental area, send a dense projection to midbrain dopamine neurons stain for µ-opioid receptors. There is however, little functional evidence that these neurons play a role in the opioid-dependent increase in dopamine neuron activity. The present study used retrograde tracers injected into the ventral tegmental area and substantia nigra (VTA/SN) to identify RMTg neurons that project to the VTA/SN. Whole-cell current-clamp and cell-attached recordings from labeled RMTg neurons were performed in sagittal slices from rat. The rhythmic spontaneous firing rate of RMTg neurons was decreased and the membrane potential was hyperpolarized in response to application of µ-opioid agonist DAMGO. Agonists that act at κ- and δ-opioid receptors (U69593 and DPDPE) failed to hyperpolarize RMTg neurons. Whole-cell recordings made in dopamine neurons revealed rhythmic, large amplitude spontaneous IPSCs that had a similar frequency, pattern and opioid sensitivity to the firing of RMTg neurons. In addition, electrical and channelrhodopsin-2 stimulation within the RMTg evoked GABA-A IPSCs in dopamine neurons that were inhibited by µ-opioid agonists DAMGO, but not κ- and δ-opioid agonists. Thus, this study demonstrates functional connection from the RMTg to the VTA/SN mediated by a dense, opioid-sensitive GABA innervation, and that the RMTg is a key structure in the µ-opioid receptor-dependent regulation of dopamine neurons.

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.

Nicotinic receptors in the habenulo-interpeduncular system are necessary for nicotine withdrawal in mice.

In humans, tobacco withdrawal produces symptoms that contribute to the difficulty associated with smoking cessation. Nicotine withdrawal symptoms can also be observed in rodents. A major standing question is which nicotinic receptor subtypes and which areas of the brain are necessary for nicotine withdrawal to occur. Using knock-out mice, we previously showed that the beta4, but not the beta2 subunit of nicotinic acetylcholine receptors, is necessary for the somatic manifestations of nicotine withdrawal. Since the beta4 subunit is highly expressed in the medial habenula, we focused our studies on the medial habenula and its primary target, the interpeduncular nucleus. In particular, we studied nicotine withdrawal in mice lacking the alpha2 or the alpha5 nicotinic receptor subunits, which are highly expressed in the interpeduncular nucleus. We precipitated withdrawal by systemically injecting the nicotinic antagonist mecamylamine in mice chronically treated with nicotine. Both the alpha2 and the alpha5 null mutations abolished the somatic manifestations of nicotine withdrawal. In addition, in wild-type mice chronically treated with nicotine, mecamylamine precipitated withdrawal when microinjected into the habenula or the interpeduncular nucleus, but not into the cortex, ventral tegmental area or hippocampus. Our results demonstrate a major role for the habenulo-interpeduncular system and the nicotinic receptor subunits expressed therein, in nicotine withdrawal symptoms. Our data suggest that the efforts to develop new smoking cessation therapies should concentrate on these areas and receptor types.

Activity profiles of cholinergic and intermingled GABAergic and putative glutamatergic neurons in the pontomesencephalic tegmentum of urethane-anesthetized rats.

Cholinergic neurons in the pontomesencephalic tegmentum form part of the ascending activating system and are thought to participate in stimulating cortical activation. Yet in the laterodorsal tegmental and pedunculopontine tegmental nuclei (LDT and PPT), they lie intermingled with GABAergic and glutamatergic neurons, which could also modulate cortical activity and sleep-wake state. To characterize the discharge of these cell types in relation to cortical activity, we recorded neurons in urethane-anesthetized rats during spontaneous slow wave and somatosensory evoked fast electroencephalographic (EEG) activity, then labeled the cells by juxtacellular technique with Neurobiotin (Nb) and dual-immunostained them for vesicular acetylcholine transporter (VAChT) and glutamic acid decarboxylase (GAD). All cholinergic cells discharged minimally during prestimulation (approximately 0.5 Hz) and moderately in a tonic manner (approximately 4 Hz) during stimulation. Being heterogeneous, some GABAergic, called "On," cells (approximately 48%) increased their discharge (from approximately 4 to 7 Hz), whereas others, called "Off" cells (approximately 38%), decreased or ceased firing during stimulation. Similarly, some noncholinergic/non-GABAergic On cells increased (from approximately 2 to 6 Hz, approximately 49%), whereas other Off cells decreased firing ( approximately 35%) during stimulation. Putative glutamatergic On together with GABAergic On neurons could thus act in parallel with cholinergic cells to stimulate cortical activation. Possibly influenced by cholinergic On and glutamatergic Off cells, whose change in discharge precedes theirs, the GABAergic Off cells could oppose neighboring neurons such as noradrenergic cells, which discharge during waking and cease firing during sleep. By concerted activity, these heterogeneous cell groups can modulate cortical activity and behavioral state across the sleep-waking cycle.

Interleukin-1 inhibits putative cholinergic neurons in vitro and REM sleep when microinjected into the rat laterodorsal tegmental nucleus.

STUDY OBJECTIVES: REM sleep is suppressed during infection, an effect mimicked by the administration of cytokines such as interleukin-1 (IL-1). In spite of this observation, brain sites and neurochemical systems mediating IL-1-induced suppression of REM sleep have not been identified. Cholinergic neurons in the brainstem laterodorsal tegmental nucleus (LDT) are part of the neuronal circuitry responsible for REM sleep generation. Since IL-1 inhibits acetylcholine synthesis and release, the aim of this study was to test the two different, but related hypotheses. We hypothesized that IL-1 inhibits LDT cholinergic neurons, and that, as a result of this inhibition, IL-1 suppresses REM sleep. DESIGN, MEASUREMENT, AND RESULTS: To test these hypotheses, the electrophysiological activity of putative cholinergic LDT neurons was recorded in a rat brainstem slice preparation. Interleukin-1 significantly inhibited the firing rate of 76% of recorded putative cholinergic LDT neurons and reduced the amplitude of glutamatergic evoked potentials in 60% of recorded neurons. When IL-1 (1 ng) was microinjected into the LDT of freely behaving rats, REM sleep was reduced by about 50% (from 12.7% +/- 1.5% of recording time [after vehicle] to 6.1% +/- 1.4% following IL-1 administration) during post-injection hours 3-4. CONCLUSIONS: Results of this study support the hypothesis that IL-1 can suppress REM sleep by acting at the level of the LDT nucleus. Furthermore this effect may result from the inhibition of evoked glutamatergic responses and of spontaneous firing of putative cholinergic LDT neurons.

Muscarinic receptor blockade in the ventral tegmental area attenuates cocaine enhancement of laterodorsal tegmentum stimulation-evoked accumbens dopamine efflux in the mouse.

The reinforcing properties of cocaine have been related to increased extracellular concentrations of dopamine in the nucleus accumbens (NAc). M5 muscarinic acetylcholine receptors (mAChRs) on dopamine cells in the ventral tegmental area (VTA) facilitate mesoaccumbens dopamine transmission and are critically involved in mediating natural and drug reinforcement. We investigated the effects of pharmacological blockade of mAChRs in the VTA on cocaine's ability to enhance electrically evoked NAc dopamine efflux. Using fixed potential amperometry together with carbon fiber recording microelectrodes positioned in the NAc core, we quantified dopamine oxidation currents (dopamine efflux) evoked by brief stimulation (15 monophasic pulses at 50 Hz every 30 s) of the laterodorsal tegmentum (LDT) in urethane (1.5 g/kg, i.p.) anesthetized mice. Compared to predrug baseline responses, cocaine (5 or 10 mg/kg, i.p.) dose-dependently enhanced LDT stimulation-evoked NAc dopamine efflux, whereas the nonsubtype selective mAChR antagonist scopolamine (10 microg/0.5 microl) microinfused into the VTA diminished LDT-evoked NAc dopamine efflux. Preinfusion of scopolamine into the VTA diminished the facilitatory actions of cocaine on LDT stimulation-evoked NAc dopamine efflux, and when infused at the peak effect of cocaine attenuated LDT-evoked dopamine efflux to below predrug baseline levels. These findings suggest that LDT cholinergic inputs to dopamine neurons in the VTA, via activation of mAChRs (probably of the M5 subtype), are involved in modulating the facilitatory effects of cocaine on NAc dopamine neurotransmission. They also suggest that the development of antagonists aimed at selectively disrupting M5 receptor function may be valuable in reducing abuse liability of psychostimulants.

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.