Effects of pedunculopontine nucleus cholinergic lesion on gait and dyskinesia in hemiparkinsonian rats.

Pedunculopontine nucleus (PPN) cholinergic neurons are implicated in freezing of gait in Parkinson's disease (PD) and motor stereotypy in normal animals, but the causal role of these neurons on specific gait parameters and treatment-induced dyskinesia remains speculative. Therefore, we examined whether selective cholinergic lesion of the rostral PPN affects PD motor and gait deficits, L-DOPA-induced dyskinesia and motor improvement, and DA-agonist-induced dyskinesia. Sprague-Dawley rats were assigned to one unilaterally lesioned group: Sham lesion, PPN cholinergic lesion with diphtheria urotensin II fusion toxin, medial forebrain bundle dopamine lesion with 6-hydroxydopamine, or dual acetylcholine and dopamine lesion. We used gait analysis and forepaw adjusting steps to examine PD gait and motor deficits. Forepaw adjusting steps were also used to assess motor improvement with L-DOPA treatment. The abnormal involuntary movements scale measured L-DOPA and dopamine D1- and D2-receptor agonist-induced dyskinesia. Lesions, verified via tyrosine hydroxylase and choline acetyltransferase immunohistochemistry reduced an average of 95% of nigral dopamine neurons and 80% of PPN cholinergic neurons, respectively. Rats receiving acetylcholine and dual lesion demonstrated enhanced freezing, and acetylcholine lesioned rats exhibited increased print area and stand index. Dopamine and dual lesion produced similar forepaw adjusting steps task on and off L-DOPA. Relative to DA lesioned rats, dual lesioned rats displayed reduced L-DOPA and DA agonist-induced dyskinesia at specific time points. Our results indicate that PPN cholinergic neurons affect gait parameters related to postural stability. Therefore, therapeutically targeting PPN cholinergic neurons could reduce intractable postural instability in PD without affecting motor benefits or side effects of L-DOPA treatment.

Human wildtype tau expression in cholinergic pedunculopontine tegmental neurons is sufficient to produce PSP-like behavioural deficits and neuropathology.

Progressive Supranuclear Palsy (PSP) is the most common atypical parkinsonism and exhibits hallmark symptomology including motor function impairment and dysexecutive dementia. In contrast to Parkinson's disease, the underlying pathology displays aggregation of the protein tau, which is also seen in disorders such as Alzheimer's disease. Currently, there are no pharmacological treatments for PSP, and drug discovery efforts are hindered by the lack of an animal model specific to PSP. Based on previous results and clinical pathology, it was hypothesized that viral deposition of tau in cholinergic neurons within the hindbrain would produce a tauopathy along neural connections to produce PSP-like symptomology and pathology. By using a combination of ChAT-CRE rats and CRE-dependent AAV vectors, wildtype human tau (the PSP-relevant 1N4R isoform; hTau) was expressed in hindbrain cholinergic neurons. Compared to control subjects (GFP), rats with tau expression displayed deficits in a variety of behavioural paradigms: acoustic startle reflex, marble burying, horizontal ladder and hindlimb motor reflex. Postmortem, the hTau rats had significantly reduced number of cholinergic pedunculopontine tegmentum and dopaminergic substantia nigra neurons, as well as abnormal tau deposits. This preclinical model has multiple points of convergence with the clinical features of PSP, some of which distinguish between PSP and Parkinson's disease.

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.

Divergent effects of ERα and ERß on fluid intake by female rats are not dependent on concomitant changes in AT1R expression or body weight.

Estradiol (E2) decreases both water and saline intakes by female rats. The ERα and ERß subtypes are expressed in areas of the brain that control fluid intake; however, the role that these receptors play in E2's antidipsogenic and antinatriorexigenic effects have not been examined. Accordingly, we tested the hypothesis that activation of ERα and ERß decreases water and saline intakes by female rats. We found a divergence in E2's inhibitory effect on intake: activation of ERα decreased water intake, whereas activation of ERß decreased saline intake. E2 decreases expression of the angiotensin II type 1 receptor (AT1R), a receptor with known relevance to water and salt intakes, in multiple areas of the brain where ERα and ERß are differentially expressed. Therefore, we tested for agonist-induced changes in AT1R mRNA expression by RT-PCR and protein expression by analyzing receptor binding to test the hypothesis that the divergent effects of these ER subtypes are mediated by region-specific changes in AT1R expression. Although we found no changes in AT1R mRNA or binding in areas of the brain known to control fluid intake associated with agonist treatment, the experimental results replicate and extend previous findings that body weight changes mediate alterations in AT1R expression in distinct brain regions. Together, the results reveal selective effects of ER subtypes on ingestive behaviors, advancing our understanding of E2's inhibitory role in the controls of fluid intake by female rats.

Sleep disorders in Parkinsonian macaques: effects of L-dopa treatment and pedunculopontine nucleus lesion.

Patients with Parkinson's disease (PD) display significant sleep disturbances and daytime sleepiness. Dopaminergic treatment dramatically improves PD motor symptoms, but its action on sleep remains controversial, suggesting a causal role of nondopaminergic lesions in these symptoms. Because the pedunculopontine nucleus (PPN) regulates sleep and arousal, and in view of the loss of its cholinergic neurons in PD, the PPN could be involved in these sleep disorders. The aims of this study were as follows: (1) to characterize sleep disorders in a monkey model of PD; (2) to investigate whether l-dopa treatment alleviates sleep disorders; and (3) to determine whether a cholinergic PPN lesion would add specific sleep alterations. To this end, long-term continuous electroencephalographic monitoring of vigilance states was performed in macaques, using an implanted miniaturized telemetry device. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment induced sleep disorders that comprised sleep episodes during daytime and sleep fragmentation and a reduction of sleep efficiency at nighttime. It also induced a reduction in time spent in rapid eye movement (REM) sleep and slow-wave sleep and an increase in muscle tone during REM and non-REM sleep episodes and in the number of awakenings and movements. l-Dopa treatment resulted in a partial but significant improvement of almost all sleep parameters. PPN lesion induced a transient decrease in REM sleep and in slow-wave sleep followed by a slight improvement of sleep quality. Our data demonstrate the efficacy of l-dopa treatment in improving sleep disorders in parkinsonian monkeys, and that adding a cholinergic PPN lesion improves sleep quality after transient sleep impairment.

Assessment of sensorimotor gating following selective lesions of cholinergic pedunculopontine neurons.

Sensorimotor gating is the state-dependent transfer of sensory information into a motor system. When this occurs at an early stage of the processing stream it enables stimuli to be filtered out or partially ignored, thereby reducing the demands placed on advanced systems. Prepulse inhibition (PPI) of the acoustic startle reflex (ASR) is the standard measure of sensorimotor gating. A brainstem-midbrain circuitry is widely viewed as mediating both PPI and ASR. In this circuitry, the pedunculopontine tegmental nucleus (PPTg) integrates sensory input and cortico-basal ganglia output and, via presumed cholinergic signaling, inhibits ASR-generating neurons within the reticular formation. Non-selective damage to all neuronal types within PPTg reduces PPI. We assessed whether this effect originates in the loss of cholinergic signaling by examining ASR and PPI in rats bearing non-selective (excitotoxic) or selective cholinergic (Dtx-UII) lesions of PPTg. Excitotoxic lesions had no effect on ASR but reduced PPI at all prepulse levels tested. In contrast, selective depletion of cholinergic neurons reduced ASR to the extent that PPI was not measurable with standard (10-20 s) inter-trial intervals. Subsequent testing revealed appreciable ASRs could be generated when the inter-trial interval was increased (180 s). Under these conditions, PPI was assessed and no deficits were found after lesions of cholinergic PPTg neurons. These results show that cholinergic output from PPTg is essential for rapidly regenerating the ASR, but has no influence on PPI. Results are discussed in terms of sensorimotor integration circuitry and psychiatric disorders that feature disrupted ASR and PPI.

Deficits in motor performance after pedunculopontine lesions in rats--impairment depends on demands of task.

Anatomically and functionally located between basal ganglia and brainstem circuitry, the pedunculopontine tegmental nucleus (PPTg) is in a pivotal position to contribute to motor behavior. Studies in primates have reported akinesia and postural instability following destruction of the PPTg. In humans, the PPTg partially degenerates in Parkinson's disease and stimulation of this region is under investigation as a possible therapeutic. Studies in rats report no crude motor impairment following PPTg lesion, although a detailed assessment of the role of the PPTg in rat motor function has not been reported. Our studies applied motor tests generally used in rodent models of Parkinson's disease to rats bearing either excitotoxic damage to all neuronal populations within PPTg, or selective destruction of the cholinergic subpopulation created with the toxin Dtx-UII. Neither lesion type altered baseline locomotion. On the rotarod, excitotoxic lesions produced a persistent impairment on the accelerating, but not fixed speed, conditions. In the vermicelli handling task (a quantitative measure of fine motor control and effective behavioral sequencing) excitotoxic lesions produced no single impairment, but globally increased the number of normal and abnormal behaviors. In contrast, depletion of cholinergic PPTg neurons produced impairment on the accelerating rotarod but no changes in vermicelli handling. Together, these results show that while PPTg lesions produce no impairment in the execution of individual motor actions, impairments emerge when the demands of the task increase. Results are discussed in terms of PPTg acting as part of a rapid action selection system, which integrates sensory information into motor output.

Properly timed exposure to central ANG II prevents behavioral sensitization and changes in angiotensin receptor expression.

Previous studies show that the angiotensin type 1 receptor (AT1R) is susceptible to rapid desensitization, but that more chronic treatments that stimulate ANG II lead to sensitization of several responses. It is unclear, however, if the processes of desensitization and sensitization interact. To test for differences in AT1R expression associated with single or repeated injections of ANG II, we measured AT1R mRNA in nuclei that control fluid intake of rats given ANG II either in a single injection or divided into three injections spaced 20 min apart. Rats given a single injection of ANG II had more AT1R mRNA in the subfornical organ (SFO) and the periventricular tissue surrounding the anteroventral third ventricle (AV3V) than did controls. The effect was not observed, however, when the same cumulative dose of ANG II was divided into multiple injections. Behavioral tests found that single daily injections of ANG II sensitized the dipsogenic response to ANG II, but a daily regimen of four injections did not cause sensitization. Analysis of (125)I-Sar(1)-ANG II binding revealed a paradoxical decrease in binding in the caudal AV3V and dorsal median preoptic nucleus after 5 days of single daily injections of ANG II; however, this effect was absent in rats treated for 5 days with four daily ANG II injections. Taken together, these data suggest that a desensitizing treatment regimen prevents behavior- and receptor-level effects of repeated daily ANG II.

RTI-263, a biased neuropeptide S receptor agonist that retains an anxiolytic effect, attenuates cocaine-seeking behavior in rats.

Neuropeptide S (NPS) is a neuromodulatory peptide that acts via a G protein-coupled receptor. Centrally administered NPS suppresses anxiety-like behaviors in rodents while producing a paradoxical increase in arousal. In addition, NPS increases drug-seeking behavior when administered during cue-induced reinstatement. Conversely, an NPS receptor (NPSR) antagonist, RTI-118, decreases cocaine-seeking behavior. A biased NPSR ligand, RTI-263, produces anxiolytic-like effects and has memory-enhancing effects similar to those of NPS but without the increase in arousal. In the present study, we show that RTI-263 decreased cocaine seeking by both male and female rats during cue-induced reinstatement. However, RTI-263 did not modulate the animals' behaviors during natural reward paradigms, such as palatable food intake, feeding during a fasting state, and cue-induced reinstatement of sucrose seeking. Therefore, NPSR biased agonists are a potential pharmacotherapy for substance use disorder because of the combined benefits of decreased drug seeking and the suppression of anxiety.

A cluster of neuropeptide S neurons regulates breathing and arousal.

Neuropeptide S (NPS) is a highly conserved peptide found in all tetrapods that functions in the brain to promote heightened arousal; however, the subpopulations mediating these phenomena remain unknown. We generated mice expressing Cre recombinase from the Nps gene locus (NpsCre) and examined populations of NPS+ neurons in the lateral parabrachial area (LPBA), the peri-locus coeruleus (peri-LC) region of the pons, and the dorsomedial thalamus (DMT). We performed brain-wide mapping of input and output regions of NPS+ clusters and characterized expression patterns of the NPS receptor 1 (NPSR1). While the activity of all three NPS+ subpopulations tracked with vigilance state, only NPS+ neurons of the LPBA exhibited both increased activity prior to wakefulness and decreased activity during REM sleep, similar to the behavioral phenotype observed upon NPSR1 activation. Accordingly, we found that activation of the LPBA but not the peri-LC NPS+ neurons increased wake and reduced REM sleep. Furthermore, given the extended role of the LPBA in respiration and the link between behavioral arousal and breathing rate, we demonstrated that the LPBA but not the peri-LC NPS+ neuronal activation increased respiratory rate. Together, our data suggest that NPS+ neurons of the LPBA represent an unexplored subpopulation regulating breathing, and they are sufficient to recapitulate the sleep/wake phenotypes observed with broad NPS system activation.

Identification of a Novel Neuropeptide S Receptor Antagonist Scaffold Based on the SHA-68 Core.

Activation of the neuropeptide S receptor (NPSR) system has been shown to produce anxiolytic-like actions, arousal, and enhance memory consolidation, whereas blockade of the NPSR has been shown to reduce relapse to substances of abuse and duration of anesthetics. We report here the discovery of a novel core scaffold (+) N-benzyl-3-(2-methylpropyl)-1-oxo-3-phenyl-1H,3H,4H,5H,6H,7H-furo[3,4-c]pyridine-5-carboxamide with potent NPSR antagonist activity in vitro. Pharmacokinetic parameters demonstrate that 14b reaches pharmacologically relevant levels in plasma and the brain following intraperitoneal (i.p.) administration, but is cleared rapidly from plasma. Compound 14b was able to block NPS (0.3 nmol)-stimulated locomotor activity in C57/Bl6 mice at 3 mg/kg (i.p.), indicating potent in vivo activity for the structural class. This suggests that 14b can serve as a useful tool for continued mapping of the pharmacological functions of the NPS receptor system.

Neuropeptide S stimulates dopaminergic neurotransmission in the medial prefrontal cortex.

Neuropeptide S (NPS) is known to produce anxiolytic-like effects and facilitate extinction of conditioned fear. Catecholaminergic neurotransmission in the medial prefrontal cortex (mPFC) has been suggested to be crucially involved in these brain functions. In the current study, we investigated the effect of NPS on the release of dopamine and serotonin in the mPFC by in vivo microdialysis in rats. Central administration of NPS dose-dependently enhanced extracellular levels of dopamine and its major metabolite 3,4-dihydroxyphenylacetic acid, with maximal effects lasting up to 120 min. In contrast, no effect on serotonergic neurotransmission was detected. Dopamine release in the mPFC has been previously linked to modulation of anxiety states and fear extinction. The present results may thus provide a physiological and anatomical basis for the reported effects of NPS on these behaviors.

Behavioral assessment of rimonabant under acute and chronic conditions.

Cannabinoid subtype 1 receptor (CB1R) antagonists were originally developed as anti-obesity agents. Unfortunately, SR1417116A (rimonabant), the first marketed inverse agonist of CB1R, produced CNS-related adverse effects including depression and suicidal ideation, and thus it was withdrawn from the market. These effects of rimonabant became evident in patients following chronic dosing. Standard preclinical toxicity studies failed to detect these adverse effects. The goal of these studies was to perform an integrated battery of behavioral assays to better understand the behavioral effects of rimonabant following both acute and chronic administration. In the present study, acute dosing with rimonabant in rats significantly decreased food consumption; decreased measures of locomotor activity; increased scratching, grooming and wet-dog shakes; and increased defecation. Subsequently, animals were tested using a chronic dosing regimen but prior to drug administration for that day. The highest dose of rimonabant tested significantly decreased marble burying behavior, presumably anxiolysis. There were also significant effects in social interaction after chronic dosing. Our results did not reveal significant rimonabant-induced anxiogenic behaviors. Future studies to characterize behavioral screens for anxiogenic effects of CB1 antagonists in rodents should further explore social interaction paradigms and potential comorbid factors of rimonabant dosing such as sex, age, and obesity.

Microinjection of urotensin II into the pedunculopontine tegmentum leads to an increase in the consumption of sweet tastants.

The pedunculopontine tegmentum (PPTg) plays a role in processing multiple sensory inputs and innervates brain regions associated with reward-related behaviors. The urotensin II receptor, activated by the urotensin II peptide (UII), is selectively expressed by the cholinergic neurons of the PPTg. Although the exact function of cholinergic neurons of the PPTg is unknown, they are thought to contribute to the perception of reward magnitude or salience detection. We hypothesized that the activation of PPTg cholinergic neurons would alter sensory processing across multiple modalities (ex. taste and hearing). Here we had three aims: first, determine if cholinergic activation is involved in consumption behavior of palatable solutions (sucrose). Second, if so, distinguish the impact of the caloric value by using saccharin, a zero calorie sweetener. Lastly, we tested the UII-mediated effects on perception of acoustic stimuli by measuring acoustic startle reflex (ASR). Male Sprague-Dawley rats were bilaterally cannulated into the PPTg, then placed under food restriction lasting the entire consumption experiment (water ad lib.). Treatment consisted of a microinjection of either 1 µL of aCSF or 1 µL of 10 µM UII into the PPTg, and the rats were immediately given access to either sucrose or saccharin. For the remaining five days, rats were allowed one hour access per day to the same sweet solution without any further treatments. During the saccharin experiment rats were tested in a contact lickometer which recorded each individual lick to give insight into the microstructure of the consumption behavior. ASR testing consisted of a baseline (no treatment), treatment day, and two additional days (no treatment). Immediately following the microinjection of UII, consumption of both saccharin and sucrose increased compared to controls. This significant increase persisted for days after the single administration of UII, but there was no generalized arousal or increase in water consumption between testing sessions. The effects on ASR were not significant. Activating cholinergic PPTg neurons may lead to a miscalculation of the salience of external stimuli, implicating the importance of cholinergic input in modulating a variety of behaviors. The long-lasting effects seen after UII treatment support further research into the role of sensory processing on reward related-behaviors at the level of the PPTg cholinergic neurons.

Neuropeptide S: a neuropeptide promoting arousal and anxiolytic-like effects.

Arousal and anxiety are behavioral responses that involve complex neurocircuitries and multiple neurochemical components. Here, we report that a neuropeptide, neuropeptide S (NPS), potently modulates wakefulness and could also regulate anxiety. NPS acts by activating its cognate receptor (NPSR) and inducing mobilization of intracellular Ca2+. The NPSR mRNA is widely distributed in the brain, including the amygdala and the midline thalamic nuclei. Central administration of NPS increases locomotor activity in mice and decreases paradoxical (REM) sleep and slow wave sleep in rats. NPS was further shown to produce anxiolytic-like effects in mice exposed to four different stressful paradigms. Interestingly, NPS is expressed in a previously undefined cluster of cells located between the locus coeruleus (LC) and Barrington's nucleus. These results indicate that NPS could be a new modulator of arousal and anxiety. They also show that the LC region encompasses distinct nuclei expressing different arousal-promoting neurotransmitters.

Identification of the first biased NPS receptor agonist that retains anxiolytic and memory promoting effects with reduced levels of locomotor stimulation.

The neuropeptide S system has been implicated in a number of centrally mediated behaviors including memory consolidation, anxiolysis, and increased locomotor activity. Characterization of these behaviors has been primarily accomplished using the endogenous 20AA peptide (NPS) that demonstrates relatively equal potency for the calcium mobilization and cAMP second messenger pathways at human and rodent NPS receptors. This study is the first to demonstrate that truncations of the NPS peptide provides small fragments that retain significant potency only at one of two single polymorphism variants known to alter NPSR function (NPSR-107I), yet demonstrate a strong level of bias for the calcium mobilization pathway over the cAMP pathway. We have also determined that the length of the truncated peptide correlates with the degree of bias for the calcium mobilization pathway. A modified tetrapeptide analog (4) has greatly attenuated hyperlocomotor stimulation in vivo but retains activity in assays that correlate with memory consolidation and anxiolytic activity. Analog 4 also has a bias for the calcium mobilization pathway, at the human and mouse receptor. This suggests that future agonist ligands for the NPS receptor having a bias for calcium mobilization over cAMP production will function as non-stimulatory anxiolytics that augment memory formation.

Urotensin II modulates rapid eye movement sleep through activation of brainstem cholinergic neurons.

Urotensin II (UII) is a cyclic neuropeptide with strong vasoconstrictive activity in the peripheral vasculature. UII receptor mRNA is also expressed in the CNS, in particular in cholinergic neurons located in the mesopontine tegmental area, including the pedunculopontine tegmental (PPT) and lateral dorsal tegmental nuclei. This distribution suggests that the UII system is involved in functions regulated by acetylcholine, such as the sleep-wake cycle. Here, we tested the hypothesis that UII influences cholinergic PPT neuron activity and alters rapid eye movement (REM) sleep patterns in rats. Local administration of UII into the PPT nucleus increases REM sleep without inducing changes in the cortical blood flow. Intracerebroventricular injection of UII enhances both REM sleep and wakefulness and reduces slow-wave sleep 2. Intracerebroventricular, but not local, administration of UII increases cortical blood flow. Moreover, whole-cell recordings from rat-brain slices show that UII selectively excites cholinergic PPT neurons via an inward current and membrane depolarization that were accompanied by membrane conductance decreases. This effect does not depend on action potential generation or fast synaptic transmission because it persisted in the presence of TTX and antagonists of ionotropic glutamate, GABA, and glycine receptors. Collectively, these results suggest that UII plays a role in the regulation of REM sleep independently of its cerebrovascular actions by directly activating cholinergic brainstem neurons.

Role of pedunculopontine cholinergic neurons in the vulnerability of nigral dopaminergic neurons in Parkinson's disease.

Pedunculopontine nucleus (PPN) cholinergic neurons, which exert excitatory nicotinic control over substantia nigra dopaminergic neurons, degenerate in Parkinson's disease (PD). This finding and other studies showing that nicotine, the preferential agonist of nicotinic acetylcholine receptors, is neuroprotective in experimental models of PD suggest that a deficit in PPN excitatory cholinergic inputs might contribute to the death of nigral dopaminergic neurons in PD. To explore this possibility, we used lesion paradigms of dopaminergic and/or cholinergic systems in rats and monkeys. Consistent with our hypothesis, we observed that stereotaxic lesioning of PPN cholinergic neurons with diphtheria toxin coupled to urotensin II resulted in a significant loss of nigral dopaminergic neurons in rats and induced morphological changes in these neurons in macaques. Unexpectedly, a lesion of dopaminergic neurons induced by unilateral striatal injection of 6-hydroxydopamine (6-OHDA) in rats, or by repeated systemic injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in macaques, led to a 29% and 7% loss of PPN cholinergic neurons, respectively. Lastly, when the PPN cholinergic lesion was performed in rats in which the dopaminergic lesion induced by 6-OHDA was in progress, loss of cholinergic neurons was more drastic than when each neurotransmitter system was lesioned separately. Thus, our results suggest that strong PPN cholinergic and dopaminergic interactions may be an important mechanism in the pathophysiology of PD.

Clozapine N-Oxide Administration Produces Behavioral Effects in Long-Evans Rats: Implications for Designing DREADD Experiments.

Clozapine N-oxide (CNO) is a ligand for a powerful chemogenetic system that can selectively inhibit or activate neurons; the so-called Designer Receptors Exclusively Activated by Designer Drugs (DREADD) system. This system consists of synthetic G-protein-coupled receptors, which are not believed to be activated by any endogenous ligand, but are activated by the otherwise inert CNO. However, it has previously been shown that the administration of CNO in humans and rats leads to detectable levels of the bioactive compounds clozapine and N-desmethylclozapine (N-Des). As a follow-up, experiments were conducted to investigate the effects of CNO in male Long-Evans rats. It was found that 1 mg/kg CNO reduced the acoustic startle reflex but had no effect on prepulse inhibition (PPI; a measure of sensorimotor gating). CNO (2 and 5 mg/kg) had no effect on the disruption to PPI induced by the NMDA antagonist phencyclidine or the muscarinic antagonist scopolamine. In locomotor studies, CNO alone (at 1, 2, and 5 mg/kg) had no effect on spontaneous locomotion, but 5 mg/kg CNO pretreatment significantly attenuated d-amphetamine-induced hyperlocomotion. In line with the behavioral results, fast-scan cyclic voltammetry found that 5 mg/kg CNO significantly attenuated the d-amphetamine-induced increase in evoked dopamine. However, the effects seen after CNO administration cannot be definitively ascribed to CNO because biologically relevant levels of clozapine and N-Des were found in plasma after CNO injection. Our results show that CNO has multiple dose-dependent effects in vivo and is converted to clozapine and N-Des emphasizing the need for a CNO-only DREADD-free control group when designing DREADD-based experiments.

Urotensin II acts as a modulator of mesopontine cholinergic neurons.

Urotensin II (UII) is a vasomodulatory peptide that was not predicted to elicit CNS activity. However, because we have recently shown that the urotensin II receptor (UII-R) is selectively expressed in rat mesopontine cholinergic (MPCh) neurons, we hypothesize that UII may have a central function. The present study demonstrates that the UII system is able to modulate MPCh neuron activity. Brain slice experiments demonstrate that UII excites MPCh neurons of the mouse laterodorsal tegmentum (LDTg) by activating a slow inward current. Furthermore, microinfusion of UII into the ventral tegmental area produces a sustained increase in dopamine efflux in the nucleus accumbens, as measured by in vivo chronoamperometry. In agreement with UII activation of MPCh neurons, intracerebroventricular injections of UII significantly modulate ambulatory movements in both rats and mice but do not significantly affect startle habituation or prepulse inhibition. The present study establishes that UII is a neuromodulator that may be exploited to target disorders involving MPCh dysfunction.