Mouse strain-specific nicotinic acetylcholine receptor expression by inhibitory interneurons and astrocytes in the dorsal hippocampus

The response by individuals to nicotine is likely to reflect the interaction of this compound with target nAChRs. However, resolving how different genetic backgrounds contribute to unique mouse strain-specific responses to this compound remains an important and unresolved issue. To examine this question in detail, expression of the nicotine acetylcholine receptor (nAChR) subunits alpha3, alpha4, alpha5, alpha7, beta2, and beta4 was measured in the dorsal hippocampus using immunohistochemistry in mouse strains or lines BALB/c, C3H/J, C57BL/6, CBA/J, DBA/2, Long Sleep (LS), Short Sleep (SS), and CF1. The nAChRs in all mice colocalized with glutamic acid decarboxylase (GAD)-positive interneurons that were subclassified into at least four groups based on nAChR subunit heterogeneity. A notable difference between mouse strains was the expression of nAChRs by astrocyte subpopulations in CA1 subregions whose numbers vary inversely with nAChR-immunostained neurons. This novel relationship also correlated with published parameters of strain sensitivity to nicotine. Attempts to identify the origin of this significant difference in nAChR expression among strains included comparison of the entire nAChRalpha4 gene sequence. Although multiple polymorphisms were identified, including two that changed nAChRalpha4 amino acid coding, none of these clearly correlate with strain-related differences in cell type-specific nAChR expression. These findings suggest that mouse strain-specific behavioral and physiological responses to nicotine are likely to be a reflection of a complex interplay between genetic factors that shape differences in expression and cellular architecture of this modulatory neurotransmitter system in the mammalian nervous system.     

Cholinergic control of striatal neurons to modulate L‐dopa‐induced dyskinesias

L‐dopa induced dyskinesias (LIDs) are a disabling motor complication of L‐dopa therapy for Parkinson's disease (PD) management. Treatment options remain limited and the underlying network mechanisms remain unclear due to a complex pathophysiology. What is well‐known, however, is that aberrant striatal signaling plays a key role in LIDs development. Here, we discuss the specific contribution of striatal cholinergic interneurons (ChIs) and GABAergic medium spiny projection neurons (MSNs) with a particular focus on how cholinergic signaling may integrate multiple striatal systems to modulate LIDs expression. Enhanced ChI transmission, altered MSN activity and the associated abnormal downstream signaling responses that arise with nigrostriatal damage are well known to contribute to LIDs development. In fact, enhancing M4 muscarinic receptor activity, a receptor favorably expressed on D1 dopamine receptor‐expressing MSNs dampens their activity to attenuate LIDs. Likewise, ChI activation via thalamostriatal neurons is shown to interrupt cortical signaling to enhance D2 dopamine receptor‐expressing MSN activity via M1 muscarinic receptors, which may interrupt ongoing motor activity. Notably, numerous preclinical studies also show that reducing nicotinic cholinergic receptor activity decreases LIDs. Taken together, these studies indicate the importance of cholinergic control of striatal neuronal activity and point to muscarinic and nicotinic receptors as significant pharmacological targets for alleviating LIDs in PD patients.     

Synchronous firing of inhibitory interneurons results in saturation of fast GABAA IPSC magnitude but not saturation of fast inhibitory efficacy in rat neocortical pyramidal cells

The kinetic properties of evoked fast inhibitory postsynaptic currents were examined to elucidate factors underlying the limit on the magnitude of fast inhibition in neocortex. Using whole-cell voltage-clamp recordings from layer V pyramidal neurons in slices of rat somatosensory cortex, fast γ-aminobutyric acid-A (GABA_A)ergic inhibitory postsynaptic currents were selectively recorded by holding cells at potentials equal to excitatory postsynaptic current reversal (∼0 mV). As stimulus intensity was increased, the magnitude and duration of the fast inhibitory postsynaptic current increased. Over the range of stimuli applied (2–10 V), fast GABA_A-mediated inhibitory postsynaptic currents reached a maximum peak conductance of 25.9 ± 4.2 nS (range 10.5–41.2 nS) at intensities approximately 2-times threshold. As stimulus intensities were increased beyond this point of maximal conductance, the time constant of current decay increased as function of stimulus strength, while rise time remained unaffected. Exposure to nominally magnesium-free solutions did not affect maximal peak conductances of fast inhibitory postsynaptic currents, but did cause an increase in the time constants of current decay by 66.3 ± 23.6%, resulting in an 85.6 ± 24.6% increase in the total charge flux carried by single inhibitory postsynaptic currents. This effect may be due to prolonged activation of postsynaptic GABA_A receptors by excess GABA released in response to increased excitation. Exposure to the GABA uptake blocker, nipecotic acid, similarly prolonged current decay without affecting the maximal peak conductance. Our findings suggest that the limit on recruitment of evoked fast inhibition in neocortical layer V pyramidal cells arises from the saturation of postsynaptic GABA_A receptors. However, while there is a limit to the peak fast inhibitory postsynaptic conductance which can be recruited with increasing excitation, inhibitory strength may still be modulated by increasing charge flux through the prolongation of fast inhibition. Synapse 28:91–102, 1998. © 1998 Wiley-Liss, Inc.     

Activity‐induced large amplitude postsynaptic mPSPs at soma–soma synapses between Lymnaea neurons

Spontaneous transmitter release has been observed at various synapses that permit analysis at a sufficient resolution as a miniature postsynaptic potential (mPSP). However, the precise mechanisms that regulate spontaneous transmitter release have not yet been fully defined. Activity and ligand‐mediated modulation of large amplitude, spontaneous events significantly enhances postsynaptic excitation in the absence of action potential activity suggesting a more complicated role for this mode of transmitter release, and thus warrants further analysis. Here, we used Lymnaea soma–soma synaptic connections to demonstrate that a transient increase in both the frequency and amplitude of spontaneous events (mPSPs) occurs following a short burst of action potentials in the presynaptic cell. These events were of presynaptic origin and the increase in mPSP amplitude could also be achieved with a stimulatory concentration of ryanodine. Ryanodine also occluded the activity‐induced increase in mPSP amplitude implicating calcium release from these channels in the production of large amplitude spontaneous transmitter release events. This suggests that presynaptic activity triggers ryanodine receptor‐mediated large amplitude minis, indicating that although these events are action potential‐independent, they are nevertheless responsive to the prior activity of the synapse. Synapse 63:117–125, 2009. ©2008 Wiley‐Liss, Inc.     

The involvement of GABA-C receptors in paired-pulse depression of inhibitory postsynaptic currents in rat hippocampal CA1 pyramidal neurons

In rat hippocampal CA1 pyramidal neurons, γ-aminobutyric acid (GABA) A receptor-mediated inhibitory postsynaptic currents (IPSCs) undergo a paired-pulse depression (PPD) by the second of two pulses, with inter-pulse intervals of 100-2000 ms, applied to the stratum radiatum. While GABA-C receptors are described in the CA1 area, their functional significance is unknown. In this study, the involvement of GABA-C receptors in PPD was examined using an in vitro hippocampal slice preparation. IPSCs evoked by stimulations in stratum radiatum were recorded with patch pipettes from CA1 pyramidal cells. PPD, when induced in the above fashion, was blocked by the GABA-C receptor antagonist (1,2,5,6-Tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA, 10 μM, applied in the superfusing medium). GABA-A and GABA-B receptor-mediated IPSCs, as well as the baclofen-induced suppression of the GABA-A receptor mediated IPSC, were not antagonized by TPMPA (10-20 μM). These results indicate that PPD of the IPSC is mediated by the activation of GABA-C receptors.     

Differential release of β‐NAD+ and ATP upon activation of enteric motor neurons in primate and murine colons

Background The purinergic component of enteric inhibitory neurotransmission is important for normal motility in the gastrointestinal (GI) tract. Controversies exist about the purine(s) responsible for inhibitory responses in GI muscles: ATP has been assumed to be the purinergic neurotransmitter released from enteric inhibitory motor neurons; however, recent studies demonstrate that β‐nicotinamide adenine dinucleotide (β‐NAD⁺) and ADP‐ribose mimic the inhibitory neurotransmitter better than ATP in primate and murine colons. The study was designed to clarify the sources of purines in colons of Cynomolgus monkeys and C57BL/6 mice. Methods High‐performance liquid chromatography with fluorescence detection was used to analyze purines released by stimulation of nicotinic acetylcholine receptors (nAChR) and serotonergic 5‐HT₃ receptors (5‐HT₃R), known to be present on cell bodies and dendrites of neurons within the myenteric plexus. Key Results Nicotinic acetylcholine receptor or 5‐HT₃R agonists increased overflow of ATP and β‐NAD⁺ from tunica muscularis of monkey and murine colon. The agonists did not release purines from circular muscles of monkey colon lacking myenteric ganglia. Agonist‐evoked overflow of β‐NAD⁺, but not ATP, was inhibited by tetrodotoxin (0.5 μmol L^?1) or ω‐conotoxin GVIA (50 nmol L^?1), suggesting that β‐NAD⁺ release requires nerve action potentials and junctional mechanisms known to be critical for neurotransmission. ATP was likely released from nerve cell bodies in myenteric ganglia and not from nerve terminals of motor neurons. Conclusions & Inferences These results support the conclusion that ATP is not a motor neurotransmitter in the colon and are consistent with the hypothesis that β‐NAD⁺, or its metabolites, serve as the purinergic inhibitory neurotransmitter.     

Matrix metalloproteinase‐9 reversibly affects the time course of NMDA‐induced currents in cultured rat hippocampal neurons

Matrix Metalloproteinase 9 (MMP‐9) has been demonstrated to play a crucial role in maintenance of NMDA receptor‐dependent LTP and in lateral mobility of these receptors. However, the effect of MMP‐9 on NMDA receptor (NMDAR) functional properties is unknown. For this purpose we have investigated the impact of recombinant MMP‐9 on the whole‐cell NMDAR‐mediated current responses in cultured hippocampal neurons. Treatment with MMP‐9 induced a reversible acceleration of desensitization and deactivation kinetics but had no effect on current amplitude. Interestingly, phorbol ester, a PKC activator known to enhance NMDAR lateral mobility, induced kinetic changes of currents similar to those produced by MMP‐9. In conclusion, our results show that MMP‐9 reversibly modulates the NMDAR kinetics and raise a possibility that this modulation could be related to the lateral mobility of these receptors. © 2009 Wiley‐Liss, Inc.     

Dopamine receptor mechanisms mediate corticotropin‐releasing factor‐induced long‐term potentiation in the rat amygdala following cocaine withdrawal

Corticotropin‐releasing factor (CRF) in the amygdala is involved in stress responses. Moreover, dopaminergic neurotransmission in the brain reward system including the amygdala plays a significant role in the pathology of cocaine addiction. The present study analysed CRF‐induced synaptic plasticity, its pharmacological sensitivity and interactions with the dopamine (DA) system in the basolateral to lateral capsula central amygdala (lcCeA) pathway after a 2‐week withdrawal from repeated cocaine administration. A physiologically relevant CRF concentration (25 nm ) induced long‐term potentiation (LTP) that was enhanced after cocaine withdrawal. In saline‐treated rats, CRF‐induced LTP was mediated through N‐methyl‐d ‐aspartate (NMDA) receptors, L‐type voltage‐gated calcium channels (L‐VGCCs) and CRF₁ receptors. However, in cocaine‐withdrawn animals, activation of CRF₁ and CRF₂ receptors was found to enhance LTP. This enhanced CRF‐induced LTP after cocaine withdrawal was mediated through endogenous activation of both D1‐ and D2‐like receptors. Furthermore, expression of the D1 receptor (D1R) but not the D2R, D3R, D4R or D5R was significantly increased after cocaine withdrawal. CRF₁ but not CRF₂ protein expression was increased, suggesting that elevated levels of these proteins contributed to the enhancement of CRF‐induced LTP during cocaine withdrawal. CRF interactions with the DA system in the amygdala may represent a fundamental neurochemical and cellular mechanism linking stress to cocaine‐induced neuronal plasticity.     

Calcium permeable AMPA receptor‐dependent long lasting plasticity of intrinsic excitability in fast spiking interneurons of the dentate gyrus decreases inhibition in the granule cell layer

The local fast‐spiking interneurons (FSINs) are considered to be crucial for the generation, maintenance, and modulation of neuronal network oscillations especially in the gamma frequency band. Gamma frequency oscillations have been associated with different aspects of behavior. But the prolonged effects of gamma frequency synaptic activity on the FSINs remain elusive. Using whole cell current clamp patch recordings, we observed a sustained decrease of intrinsic excitability in the FSINs of the dentate gyrus (DG) following repetitive stimulations of the mossy fibers at 30 Hz (gamma bursts). Surprisingly, the granule cells (GCs) did not express intrinsic plastic changes upon similar synaptic excitation of their apical dendritic inputs. Interestingly, pairing the gamma bursts with membrane hyperpolarization accentuated the plasticity in FSINs following the induction protocol, while the plasticity attenuated following gamma bursts paired with membrane depolarization. Paired pulse ratio measurement of the synaptic responses did not show significant changes during the experiments. However, the induction protocols were accompanied with postsynaptic calcium rise in FSINs. Interestingly, the maximum and the minimum increase occurred during gamma bursts with membrane hyperpolarization and depolarization respectively. Including a selective blocker of calcium‐permeable AMPA receptors (CP‐AMPARs) in the bath; significantly attenuated the calcium rise and blocked the membrane potential dependence of the calcium rise in the FSINs, suggesting their involvement in the observed phenomenon. Chelation of intracellular calcium, blocking HCN channel conductance or blocking CP‐AMPARs during the experiment forbade the long lasting expression of the plasticity. Simultaneous dual patch recordings from FSINs and synaptically connected putative GCs confirmed the decreased inhibition in the GCs accompanying the decreased intrinsic excitability in the FSINs. Experimentally constrained network simulations using NEURON predicted increased spiking in the GC owing to decreased input resistance in the FSIN. We hypothesize that the selective plasticity in the FSINs induced by local network activity may serve to increase information throughput into the downstream hippocampal subfields besides providing neuroprotection to the FSINs. © 2014 Wiley Periodicals, Inc.     

Different output properties of perisomatic region‐targeting interneurons in the basal amygdala

The perisomatic region of principal neurons in cortical regions is innervated by three types of GABAergic interneuron, including parvalbumin‐containing basket cells (PVBCs) and axo‐axonic cells (AACs), as well as cholecystokinin and type 1 cannabinoid receptor‐expressing basket cells (CCK/CB1BCs). These perisomatic inhibitory cell types can also be found in the basal nucleus of the amygdala, however, their output properties are largely unknown. Here, we performed whole‐cell recordings in morphologically identified interneurons in slices prepared from transgenic mice, in which the GABAergic cells could be selectively targeted. Investigating the passive and active membrane properties of interneurons located within the basal amygdala revealed that the three interneuron types have distinct single‐cell properties. For instance, the input resistance, spike rate, accommodation in discharge rate, or after‐hyperpolarization width at the half maximal amplitude separated the three interneuron types. Furthermore, we performed paired recordings from interneurons and principal neurons to uncover the basic features of unitary inhibitory postsynaptic currents (uIPSCs). Although we found no difference in the magnitude of responses measured in the principal neurons, the uIPSCs originating from the distinct interneuron types differed in rise time, failure rate, latency, and short‐term dynamics. Moreover, the asynchronous transmitter release induced by a train of action potentials was typical for the output synapses of CCK/CB1BCs. Our results suggest that, despite the similar uIPSC magnitudes originating from the three perisomatic inhibitory cell types, their distinct release properties together with the marked differences in their spiking characteristics may contribute to accomplish specific functions in amygdala network operation.     

Septohippocampal GABAergic neurons mediate the altered behaviors induced by n‐methyl‐D‐aspartate receptor antagonists

We hypothesize that selective lesion of the septohippocampal GABAergic neurons suppresses the altered behaviors induced by an N‐methyl‐D ‐aspartate (NMDA) receptor antagonist, ketamine or MK‐801. In addition, we hypothesize that septohippocampal GABAergic neurons generate an atropine‐resistant theta rhythm that coexists with an atropine‐sensitive theta rhythm in the hippocampus. Infusion of orexin‐saporin (ore‐SAP) into the medial septal area decreased parvalbumin‐immunoreactive (GABAergic) neurons by ～80%, without significantly affecting choline‐acetyltransferase‐immunoreactive (cholinergic) neurons. The theta rhythm during walking, or the immobility‐associated theta induced by pilocarpine, was not different between ore‐SAP and sham‐lesion rats. Walking theta was, however, more disrupted by atropine sulfate in ore‐SAP than in sham‐lesion rats. MK‐801 (0.5 mg/kg i.p.) induced hyperlocomotion associated with an increase in frequency, but not power, of the hippocampal theta in both ore‐SAP and sham‐lesion rats. However, MK‐801 induced an increase in 71–100 Hz gamma waves in sham‐lesion but not ore‐SAP lesion rats. In sham‐lesion rats, MK‐801 induced an increase in locomotion and an impairment of prepulse inhibition (PPI), and ketamine (3 mg/kg s.c.) induced a loss of gating of hippocampal auditory evoked potentials. MK‐801‐induced behavioral hyperlocomotion and PPI impairment, and ketamine‐induced auditory gating deficit were reduced in ore‐SAP rats as compared to sham‐lesion rats. During baseline without drugs, locomotion and auditory gating were not different between ore‐SAP and sham‐lesion rats, and PPI was slightly but significantly increased in ore‐SAP as compared with sham lesion rats. It is concluded that septohippocampal GABAergic neurons are important for the expression of hyperactive and psychotic symptoms an enhanced hippocampal gamma activity induced by ketamine and MK‐801, and for generating an atropine‐resistant theta. Selective suppression of septohippocampal GABAergic activity is suggested to be an effective treatment of some symptoms of schizophrenia. © 2012 Wiley Periodicals, Inc.     

Volatile anesthetic sevoflurane pretreatment alleviates hypoxia‐induced potentiation of excitatory inputs to striatal medium spiny neurons of mice

Sevoflurane, a commonly used anesthetic in surgery, has drawn attention because of its preconditioning effects in hypoxic conditions. To investigate the preconditioning effects in the striatum, a common site for ischemic stroke, we collected whole‐cell current‐clamp recordings from striatal medium spiny neurons. In our in vitro brain slice experiments, deprivation of oxygen and glucose depolarized the striatal neurons to subthreshold potentials, and the pre‐administration of sevoflurane (4%, 15 min) prolonged the time to depolarization. Furthermore, transient hypoxia induced the potentiation of excitatory postsynaptic potentials, which play a part in post‐ischemic excitotoxicity. Glibenclamide, a K_ATP channel inhibitor, reversed the prolonged time to depolarization and the prevention of the pathological potentiation of excitatory responses, indicating that the short exposure to sevoflurane likely participates in neuroprotection against hypoxia via activation of K_ATP channels. A monocarboxylate transporter blocker, 4‐CIN, also depolarized striatal neurons. Interestingly, the blockade of monocarboxylate transporters that supply lactate to neurons caused the pathological potentiation, even in the presence of enough oxygen and glucose. In this case, sevoflurane could not prevent the pathological potentiation, suggesting the involvement of monocarboxylate transporters in the sevoflurane‐mediated effects. These results indicate that sevoflurane protects striatal neurons from hypoxic damage and alleviates the pathological potentiation. Under these conditions, sevoflurane may become an effective intervention for patients undergoing surgery.     

Effect of optogenetic manipulation of accumbal medium spiny neurons expressing dopamine D2 receptors in cocaine‐induced behavioral sensitization

Repetitive exposure to addictive drugs causes synaptic modification in the mesocorticolimbic dopamine (DA) system. Dopamine D1 receptors (D1R) or D2 receptors (D2R) expressed in the medium spiny neurons (MSNs) of the nucleus accumbens (NAc) play critical roles in the control of addictive behaviors. Optogenetic activation of D2R‐expressing MSNs (D2R‐MSNs) in the NAc previously demonstrated that these neurons play a key role in withdrawal‐induced plasticity. Here, we examined the effect of optogenetic inhibition of D2R‐MSNs in the NAc on cocaine‐induced behavioral sensitization. Adeno‐associated viral vectors encoding archaerhodopsin (ArchT) were delivered into the NAc of D2‐Cre transgenic mice. Activation of ArchT produced photoinhibition of D2R‐MSNs and caused disinhibition of neighboring MSNs in the NAc. However, such optogenetic silencing of D2R‐MSNs in the NAc in vivo affected neither the initiation nor the expression of cocaine‐induced behavioral sensitization. Similarly, photoinhibition of NAc D2R‐MSNs in the NAc during the drug withdrawal period did not affect the expression of cocaine‐induced behavioral sensitization. More detailed analysis of the effects of optogenetic activation of D2R‐MSNs suggests that D2R‐MSNs in the NAc exert important modulatory effects on neighboring MSN neurons, which may control the balanced output of NAc MSNs to control addictive behaviors.     

Serotonergic‐postsynaptic receptors modulate gripping‐induced immobility episodes in male taiep rats

The Taiep rat is a myelin mutant with a motor syndrome characterized by tremor, ataxia, immobility, epilepsy, and paralysis. The rat shows a hypomyelination followed by a progressive demyelination. During immobilities taiep rats show a REM‐like sleep pattern and a disorganized sleep‐wake pattern suggesting taiep rats as a model of narcolepsy‐cataplexy. Our study analyzed the role of postsynaptic serotonin receptors in the expression of gripping‐induced immobility episodes (IEs) in 8‐month‐old male taiep rats. The specific postsynaptic serotonin agonist ±1‐(2,5‐dimethoxy‐4‐iodoamphetamine hydrochloride (±DOI) decreased the frequency of gripping‐induced IEs, but that was not the case with α‐methyl‐serotonin maleate (α‐methyl‐5HT), a nonspecific postsynaptic agonist. Although the serotonin antagonists, ketanserine and metergoline, produced a biphasic effect, first a decrease followed by an increase with higher doses, similar effects were obtained with a mean duration of gripping‐induced IEs. These findings correlate with the pharmacological observations in narcoleptic dogs and humans in which serotonin‐reuptake inhibitors improve cataplexy, particularly in long‐term treatment that could change the serotonin receptor levels. Polysomnographic recordings showed an increase in the awakening time and a decrease in the slow wave and rapid eye movement sleep concomitant with a decrease in immobilities after use of ±DOI, this being stronger with the highest dose. Taken together, our results show that postsynaptic serotonin receptors are involved in the modulation in gripping‐induced IEs caused by the changes in the organization of the sleep‐wake cycle in taiep rats. It is possible that specific agonists, without side effects, could be a useful treatment in human narcoleptic patients. Synapse 63:737–744, 2009. © 2009 Wiley‐Liss, Inc.     

Protein kinase C regulates tonic GABAA receptor‐mediated inhibition in the hippocampus and thalamus

Tonic inhibition mediated by extrasynaptic GABA_A receptors (GABA_ARs) is an important regulator of neuronal excitability. Phosphorylation by protein kinase C (PKC) provides a key mode of regulation for synaptic GABA_ARs underlying phasic inhibition; however, less attention has been focused on the plasticity of tonic inhibition and whether this can also be modulated by receptor phosphorylation. To address this issue, we used whole‐cell patch clamp recording in acute murine brain slices at both room and physiological temperatures to examine the effects of PKC‐mediated phosphorylation on tonic inhibition. Recordings from dentate gyrus granule cells in the hippocampus and dorsal lateral geniculate relay neurons in the thalamus demonstrated that PKC activation caused downregulation of tonic GABA_AR‐mediated inhibition. Conversely, inhibition of PKC resulted in an increase in tonic GABA_AR activity. These findings were corroborated by experiments on human embryonic kidney 293 cells expressing recombinant α4β2δ GABA_ARs, which represent a key extrasynaptic GABA_AR isoform in the hippocampus and thalamus. Using bath application of low GABA concentrations to mimic activation by ambient neurotransmitter, we demonstrated a similar inhibition of receptor function following PKC activation at physiological temperature. Live cell imaging revealed that this was correlated with a loss of cell surface GABA_ARs. The inhibitory effects of PKC activation on α4β2δ GABA_AR activity appeared to be mediated by direct phosphorylation at a previously identified site on the β2 subunit, serine 410. These results indicate that PKC‐mediated phosphorylation can be an important physiological regulator of tonic GABA_AR‐mediated inhibition.     

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.