Opioid-induced rewards, locomotion, and dopamine activation: A proposed model for control by mesopontine and rostromedial tegmental neurons.

Opioids, such as morphine or heroin, increase forebrain dopamine (DA) release and locomotion, and support the acquisition of conditioned place preference (CPP) or self-administration. The most sensitive sites for these opioid effects in rodents are in the ventral tegmental area (VTA) and rostromedial tegmental nucleus (RMTg). Opioid inhibition of GABA neurons in these sites is hypothesized to lead to arousing and rewarding effects through disinhibition of VTA DA neurons. We review findings that the laterodorsal tegmental (LDTg) and pedunculopontine tegmental (PPTg) nuclei, which each contain cholinergic, GABAergic, and glutamatergic cells, are important for these effects. LDTg and/or PPTg cholinergic inputs to VTA mediate opioid-induced locomotion and DA activation via VTA M5 muscarinic receptors. LDTg and/or PPTg cholinergic inputs to RMTg also modulate opioid-induced locomotion. Lesions or inhibition of LDTg or PPTg neurons reduce morphine-induced increases in forebrain DA release, acquisition of morphine CPP or self-administration. We propose a circuit model that links VTA and RMTg GABA with LDTg and PPTg neurons critical for DA-dependent opioid effects in drug-naïve rodents.

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.

Disruption of Src Is Associated with Phenotypes Related to Williams-Beuren Syndrome and Altered Cellular Localization of TFII-I

Src is a nonreceptor protein tyrosine kinase that is expressed widely throughout the central nervous system and is involved in diverse biological functions. Mice homozygous for a spontaneous mutation in Src (Src (thl/thl) ) exhibited hypersociability and hyperactivity along with impairments in visuospatial, amygdala-dependent, and motor learning as well as an increased startle response to loud tones. The phenotype of Src (thl/thl) mice showed significant overlap with Williams-Beuren syndrome (WBS), a disorder caused by the deletion of several genes, including General Transcription Factor 2-I (GTF2I). Src phosphorylation regulates the movement of GTF2I protein (TFII-I) between the nucleus, where it is a transcriptional activator, and the cytoplasm, where it regulates trafficking of transient receptor potential cation channel, subfamily C, member 3 (TRPC3) subunits to the plasma membrane. Here, we demonstrate altered cellular localization of both TFII-I and TRPC3 in the Src mutants, suggesting that disruption of Src can phenocopy behavioral phenotypes observed in WBS through its regulation of TFII-I.

Nicotine receptors mediating sensorimotor gating and its enhancement by systemic nicotine.

Prepulse inhibition (PPI) of startle occurs when intensity stimuli precede stronger startle-inducing stimuli by 10-1000 ms. PPI deficits are found in individuals with schizophrenia and other psychiatric disorders, and they correlate with other cognitive impairments. Animal research and clinical studies have demonstrated that both PPI and cognitive function can be enhanced by nicotine. PPI has been shown to be mediated, at least in part, by mesopontine cholinergic neurons that project to pontine startle neurons and activate muscarinic and potentially nicotine receptors (nAChRs). The subtypes and anatomical location of nAChRs involved in mediating and modulating PPI remain unresolved. We tested the hypothesis that nAChRs that are expressed by pontine startle neurons contribute to PPI. We also explored whether or not these pontine receptors are responsible for the nicotine enhancement of PPI. While systemic administration of nAChR antagonists had limited effects on PPI, PnC microinfusions of the non-α7nAChR preferring antagonist TMPH, but not of the α7nAChR antagonist MLA, into the PnC significantly reduced PPI. Electrophysiological recordings from startle-mediating PnC neurons confirmed that nicotine affects excitability of PnC neurons, which could be antagonized by TMPH, but not by MLA, indicating the expression of non-α7nAChR. In contrast, systemic nicotine enhancement of PPI was only reversed by systemic MLA and not by TMPH or local microinfusions of MLA into the PnC. In summary, our data indicate that non-α7nAChRs in the PnC contribute to PPI at stimulus intervals of 100 ms or less, whereas activation of α7nAChRs in other brain areas is responsible for the systemic nicotine enhancement of PPI. This is important knowledge for the correct interpretation of behavioral, preclinical, and clinical data as well as for developing drugs for the amelioration of PPI deficits and the enhancement of cognitive function.

Parvalbumin and GAD65 interneuron inhibition in the ventral hippocampus induces distinct behavioral deficits relevant to schizophrenia.

Hyperactivity within the ventral hippocampus (vHPC) has been linked to both psychosis in humans and behavioral deficits in animal models of schizophrenia. A local decrease in GABA-mediated inhibition, particularly involving parvalbumin (PV)-expressing GABA neurons, has been proposed as a key mechanism underlying this hyperactive state. However, direct evidence is lacking for a causal role of vHPC GABA neurons in behaviors associated with schizophrenia. Here, we probed the behavioral function of two different but overlapping populations of vHPC GABA neurons that express either PV or GAD65 by selectively inhibiting these neurons with the pharmacogenetic neuromodulator hM4D. We show that acute inhibition of vHPC GABA neurons in adult mice results in behavioral changes relevant to schizophrenia. Inhibiting either PV or GAD65 neurons produced distinct behavioral deficits. Inhibition of PV neurons, affecting ∼80% of the PV neuron population, robustly impaired prepulse inhibition of the acoustic startle reflex (PPI), startle reactivity, and spontaneous alternation, but did not affect locomotor activity. In contrast, inhibiting a heterogeneous population of GAD65 neurons, affecting ∼40% of PV neurons and 65% of cholecystokinin neurons, increased spontaneous and amphetamine-induced locomotor activity and reduced spontaneous alternation, but did not alter PPI. Inhibition of PV or GAD65 neurons also produced distinct changes in network oscillatory activity in the vHPC in vivo. Together, these findings establish a causal role for vHPC GABA neurons in controlling behaviors relevant to schizophrenia and suggest a functional dissociation between the GABAergic mechanisms involved in hippocampal modulation of sensorimotor processes.

Cholinergic control of morphine-induced locomotion in rostromedial tegmental nucleus versus ventral tegmental area sites.

M5 muscarinic acetylcholine receptors expressed on ventral tegmental dopamine (DA) neurons are needed for opioid activation of DA outputs. Here, the M5 receptor gene was bilaterally transfected into neurons in the ventral tegmental area (VTA) or the adjacent rostromedial tegmental nucleus (RMTg) in mice by means of a Herpes simplex viral vector (HSV) to increase the effect of endogenous acetylcholine. Three days after HSV-M5 gene infusion in VTA sites, morphine-induced locomotion more than doubled at two doses, while saline-induced locomotion was unaffected. When the HSV-M5 gene was infused into the adjacent RMTg, morphine-induced locomotion was strongly inhibited. The sharp boundary between these opposing effects was found where tyrosine hydroxylase (TH) and cholinesterase labelling decreases (-4.00 mm posterior to bregma). The same HSV-M5 gene transfections in M5 knockout mice induced even stronger inhibitory behavioural effects in RMTg but more variability in VTA sites due to stereotypy. The VTA sites where HSV-M5 increased morphine-induced locomotion receive direct inputs from many RMTg GAD-positive neurons, and from pontine ChAT-positive neurons, as shown by cholera-toxin B retrograde tracing. Therefore, morphine-induced locomotion was decreased by M5 receptor gene expression in RMTg GABA neurons that directly inhibit VTA DA neurons. Conversely, enhancing M5 receptor gene expression on VTA DA neurons increased morphine-induced locomotion via cholinergic inputs.

Acute food deprivation reverses morphine-induced locomotion deficits in M5 muscarinic receptor knockout mice.

Lesions of the pedunculopontine tegmental nucleus (PPT), one of two sources of cholinergic input to the ventral tegmental area (VTA), block conditioned place preference (CPP) for morphine in drug-naïve rats. M5 muscarinic cholinergic receptors, expressed by midbrain dopamine neurons, are critical for the ability of morphine to increase nucleus accumbens dopamine levels and locomotion, and for morphine CPP. This suggests that M5-mediated PPT cholinergic inputs to VTA dopamine neurons critically contribute to morphine-induced dopamine activation, reward and locomotion. In the current study we tested whether food deprivation, which reduces PPT contribution to morphine CPP in rats, could also reduce M5 contributions to morphine-induced locomotion in mice. Acute 18-h food deprivation reversed the phenotypic differences usually seen between non-deprived wild-type and M5 knockout mice. That is, food deprivation increased morphine-induced locomotion in M5 knockout mice but reduced morphine-induced locomotion in wild-type mice. Food deprivation increased saline-induced locomotion equally in wild-type and M5 knockout mice. Based on these findings, we suggest that food deprivation reduces the contribution of M5-mediated PPT cholinergic inputs to the VTA in morphine-induced locomotion and increases the contribution of a PPT-independent pathway. The contributions of cholinergic, dopaminergic and GABAergic neurons to the effects of acute food deprivation are discussed.

Duplication of GTF2I results in separation anxiety in mice and humans.

Duplication (dup7q11.23) and deletion (Williams syndrome) of chromosomal region 7q11.23 cause neurodevelopmental disorders with contrasting anxiety phenotypes. We found that 30% of 4- to 12-year-olds with dup7q11.23 but fewer than 5% of children with WS or in the general population met diagnostic criteria for a separation-anxiety disorder. To address the role of one commonly duplicated or deleted gene in separation anxiety, we compared mice that had varying numbers of Gtf2i copies. Relative to mouse pups with one or two Gtf2i copies, pups with additional Gtf2i copies showed significantly increased maternal separation-induced anxiety as measured by ultrasonic vocalizations. This study links the copy number of a single gene from 7q11.23 to separation anxiety in both mice and humans, highlighting the utility of mouse models in dissecting specific gene functions for genomic disorders that span many genes. This study also offers insight into molecular separation-anxiety pathways that might enable the development of targeted therapeutics.

Muscarinic receptors in brain stem and mesopontine cholinergic arousal functions.

All five muscarinic receptor subtypes and mRNAs are found widely in the brain stem, with M2 muscarinic receptors most concentrated in the hindbrain. Three cholinergic cell groups, Ch5: pedunculopontine (PPT); Ch6: laterodorsal tegmental (LDT); Ch8: parabigeminal (PBG), are found in the tegmentum. Ch5,6 neurons are activated by arousing and reward-activating stimuli, and inhibited via M2-like autoreceptors. Ch5,6 ascending projections activate many forebrain regions, including thalamus, basal forebrain, and orexin/hypocretin neurons (via M3 receptors) for waking arousal and attention. Ch5,6 activation of dopamine neurons of the ventral tegmental area and substantia nigra (via M5 receptors) increases reward-seeking and energizes motor functions. M5 receptors on dopamine neurons facilitate brain-stimulation reward, opiate rewards and locomotion, and male ultrasonic vocalizations during mating in rodents. Ch5 cholinergic activation of superior colliculus intermediate layers facilitates fast saccades and approach turns, accompanied by nicotinic and muscarinic inhibition of the startle reflex in pons. Ch8 PBG neurons project to the outer layers of the superior colliculus only, where M2 receptors are associated with retinotectal terminals. Ch5,6 descending projections to dorsal pontine reticular formation contribute to M2-dependent REM sleep.

M5 muscarinic receptors mediate striatal dopamine activation by ventral tegmental morphine and pedunculopontine stimulation in mice.

Opiates, like other addictive drugs, elevate forebrain dopamine levels and are thought to do so mainly by inhibiting GABA neurons near the ventral tegmental area (VTA), in turn leading to a disinhibition of dopamine neurons. However, cholinergic inputs from the laterodorsal (LDT) and pedunculopontine (PPT) tegmental nucleus to the VTA and substantia nigra (SN) importantly contribute, as either LDT or PPT lesions strongly attenuate morphine-induced forebrain dopamine elevations. Pharmacological blockade of muscarinic acetylcholine receptors in the VTA or SN has similar effects. M5 muscarinic receptors are the only muscarinic receptor subtype associated with VTA and SN dopamine neurons. Here we tested the contribution of M5 muscarinic receptors to morphine-induced dopamine elevations by measuring nucleus accumbens dopamine efflux in response to intra-VTA morphine infusion using in vivo chronoamperometry. Intra-VTA morphine increased nucleus accumbens dopamine efflux in urethane-anesthetized wildtype mice starting at 10 min after infusion. These increases were absent in M5 knockout mice and were similarly blocked by pre-treatment with VTA scopolamine in wildtype mice. Furthermore, in wildtype mice electrical stimulation of the PPT evoked an initial, short-lasting increase in striatal dopamine efflux, followed 5 min later by a second prolonged increase in dopamine efflux. In M5 knockout mice, or following systemic pre-treatment with scopolamine in wildtype mice, the prolonged increase in striatal dopamine efflux was absent. The time course of increased accumbal dopamine efflux in wildtype mice following VTA morphine was consistent with both the prolonged M5-mediated excitation of striatal dopamine efflux following PPT electrical stimulation and accumbal dopamine efflux following LDT electrical stimulation. Therefore, M5 receptors appear critical for prolonged PPT excitation of dopamine efflux and for dopamine efflux induced by intra-VTA morphine.

GABA receptors and prepulse inhibition of acoustic startle in mice and rats.

The acoustic startle reflex is strongly inhibited by a moderate-intensity acoustic stimulus that precedes the startling stimulus by roughly 10-1000 ms (prepulse inhibition, PPI). At long interstimulus intervals (ISIs) of 100-1000 ms, PPI in rats is reduced by the muscarinic receptor antagonist scopolamine. Here, we studied the role of GABA receptors in PPI at full ISI ranges in both mice and rats. In B6 mice, PPI begins and ends at shorter ISIs (4 and 1000 ms, respectively) than in Wistar rats (8 and 5000 ms). The GABA(A) antagonist bicuculline (1 mg/kg i.p.) reduced PPI at ISIs near the peak of PPI in both rats and mice. The GABA(B) antagonist phaclofen (10 or 30 mg/kg i.p. in rats or mice, respectively) reduced PPI only at long ISIs, similar to the effects of the muscarinic antagonist scopolamine (1 mg/kg i.p.). The effects of phaclofen and scopolamine were additive in rats, suggesting independent effects of GABA(B) and muscarinic receptors. Patch-clamp recordings of startle-mediating PnC (nucleus reticularis pontis caudalis) giant neurons in rat slices show that EPSCs evoked by either trigeminal or auditory fiber stimulation were inhibited by the GABA(A/C) agonist muscimol or the GABA(B) agonist baclofen via postsynaptic mechanisms. Hyperpolarization of PnC neurons by muscimol was reversed with bicuculline, indicating that postsynaptic GABA(A) receptors strongly inhibit PnC giant neurons needed for startle. Therefore, GABA receptors on PnC giant neurons mediate a substantial part of PPI, with GABA(A) receptors contributing at the peak of PPI, and GABA(B) receptors adding to muscarinic effects on PPI at long ISIs.

CBP histone acetyltransferase activity regulates embryonic neural differentiation in the normal and Rubinstein-Taybi syndrome brain.

Increasing evidence indicates that epigenetic changes regulate cell genesis. Here, we ask about neural precursors, focusing on CREB binding protein (CBP), a histone acetyltransferase that, when haploinsufficient, causes Rubinstein-Taybi syndrome (RTS), a genetic disorder with cognitive dysfunction. We show that neonatal cbp(+/-) mice are behaviorally impaired, displaying perturbed vocalization behavior. cbp haploinsufficiency or genetic knockdown with siRNAs inhibited differentiation of embryonic cortical precursors into all three neural lineages, coincident with decreased CBP binding and histone acetylation at promoters of neuronal and glial genes. Inhibition of histone deacetylation rescued these deficits. Moreover, CBP phosphorylation by atypical protein kinase C zeta was necessary for histone acetylation at neural gene promoters and appropriate differentiation. These data support a model in which environmental cues regulate CBP activity and histone acetylation to control neural precursor competency to differentiate, and indicate that cbp haploinsufficiency disrupts this mechanism, thereby likely causing cognitive dysfunction in RTS.

M5 muscarinic receptor knockout mice show reduced morphine-induced locomotion but increased locomotion after cholinergic antagonism in the ventral tegmental area.

M(5) muscarinic receptors are the only muscarinic receptor subtype expressed by mesencephalic dopamine neurons and provide an important excitatory input to mesolimbic and nigrostriatal dopamine systems. Here, we studied locomotion induced by systemic morphine (3, 10, and 30 mg/kg i.p.) in M(5) knockout mice of the C57BL/6 (B6) and CD1 x 129SvJ background strains. M(5) knockout mice of both strains showed reduced locomotion in response to 30 mg/kg morphine. B6 M(5) knockout mice were less sensitive to naltrexone in either the antagonism of morphine-induced locomotion or in the reduction of locomotion by naltrexone alone. This suggests that M(5) knockout mice are less sensitive to the effects of either exogenous or endogenous opiates on locomotion and that spontaneous locomotion in B6 mice is sustained by endogenous opiates. In B6 wild-type mice, ventral tegmental area (VTA) pretreatment with the muscarinic receptor antagonist atropine (3 microg bilateral), but not the nicotinic receptor antagonist mecamylamine (5 microg bilateral), reduced locomotion in response to 30 mg/kg morphine to a similar extent as systemic M(5) knockout, suggesting that reduced morphine-induced locomotion in M(5) knockout mice is due to the loss of M(5) receptors on VTA dopamine neurons. In contrast, in M(5) knockout mice, but not in wild-type mice, either intra-VTA atropine or mecamylamine alone increased locomotion by almost 3 times relative to saline and potentiated morphine-induced locomotion. Therefore, in M(5) knockout mice, blockade of either VTA muscarinic or nicotinic receptors increased locomotion, suggesting that in the absence of VTA M(5) receptors, VTA cholinergic inputs inhibit locomotion.

Ultrasonic vocalizations induced by sex and amphetamine in M2, M4, M5 muscarinic and D2 dopamine receptor knockout mice.

Adult mice communicate by emitting ultrasonic vocalizations (USVs) during the appetitive phases of sexual behavior. However, little is known about the genes important in controlling call production. Here, we study the induction and regulation of USVs in muscarinic and dopaminergic receptor knockout (KO) mice as well as wild-type controls during sexual behavior. Female mouse urine, but not female rat or human urine, induced USVs in male mice, whereas male urine did not induce USVs in females. Direct contact of males with females is required for eliciting high level of USVs in males. USVs (25 to120 kHz) were emitted only by males, suggesting positive state; however human-audible squeaks were produced only by females, implying negative state during male-female pairing. USVs were divided into flat and frequency-modulated calls. Male USVs often changed from continuous to broken frequency-modulated calls after initiation of mounting. In M2 KO mice, USVs were lost in about 70-80% of the mice, correlating with a loss of sexual interaction. In M5 KO mice, mean USVs were reduced by almost 80% even though sexual interaction was vigorous. In D2 KOs, the duration of USVs was extended by 20%. In M4 KOs, no significant differences were observed. Amphetamine dose-dependently induced USVs in wild-type males (most at 0.5 mg/kg i.p.), but did not elicit USVs in M5 KO or female mice. These studies suggest that M2 and M5 muscarinic receptors are needed for male USV production during male-female interactions, likely via their roles in dopamine activation. These findings are important for the understanding of the neural substrates for positive affect.

Carbachol injections into the intergeniculate leaflet induce nonphotic phase shifts.

Nonphotic phase shifts of the circadian clock in mammals are mediated by the intergeniculate leaflet (IGL) of the thalamus via a geniculohypothalamic projection to the suprachiasmatic nucleus. These shifts can be induced by arousing stimuli, such as wheel running, brain stimulation reward and foot shock. Because mesopontine cholinergic neurons are also activated by arousing stimuli, we tested the hypothesis that cholinergic input to the IGL mediates nonphotic phase shifts. Carbachol injected into the IGL of hamsters in their subjective day (CT8) induced phase advances similar to shifts that are induced by arousal at the same circadian time. Control injections of saline at CT8 did not advance phase similarly. Carbachol injections outside the IGL produced smaller shifts. Pre-injections of the muscarinic antagonist, atropine, reduced carbachol-induced phase advances relative to saline pre-injections. The results indicate that muscarinic input to the IGL can induce nonphotic phase shifts.

Casein kinase I epsilon gene transfer into the suprachiasmatic nucleus via electroporation lengthens circadian periods of tau mutant hamsters.

Circadian activity rhythms in mammals are controlled by the expression and transcriptional regulation of clock genes in the suprachiasmatic nucleus (SCN). The circadian cycle length in hamsters is regulated in part by casein kinase I epsilon (CKIepsilon). A semidominant mutation (C-->T, R178C, CKIepsilon(tau)) appears to act as a dominant-negative allele to shorten the period of circadian rhythms. We tested this hypothesis in vivo by expressing wild-type CKIepsilon gene in homozygous tau mutant hamsters. High-level CKIepsilon(+/+) gene transfer and expression (as indicated by green fluorescent protein) were obtained by injecting CKIepsilon-containing plasmids bilaterally near the SCN, followed by in vivo electroporation. Rhythmicity reappeared 5-7 days after electroporation, with a gradual increase in circadian period over the next 10 days. The circadian period returned to the baseline over the next 20 days. For the five hamsters with clearest gene expression in the SCN, the mean lengthening time was 39.6 min. Period change was not observed in either control tau mutant hamsters electroporated with plasmids lacking the CKIepsilon gene or in wild-type hamsters with plasmids containing the wild-type CKIepsilon gene. Therefore, normal periodicity in homozygous CKIepsilon(tau) hamsters was partially rescued by expression of the wild-type CKIepsilon gene in the SCN, supporting a competitive and dominant-negative action of the mutant allele. This study shows that electroporation of wild-type CKIepsilon gene into the SCN is sufficient for lengthening the shorter circadian period of tau mutant hamsters in a time-dependent way and supports the conclusion that CKIepsilon(tau) is the cause of the shorter period.

Opposite effects of tetanic stimulation of the auditory thalamus or auditory cortex on the acoustic startle reflex in awake rats.

The amygdala mediates both emotional learning and fear potentiation of startle. The lateral amygdala nucleus (LA) receives auditory inputs from both the auditory thalamus (medial geniculate nucleus; MGN) and auditory association cortex (AAC), and is critical for auditory fear conditioning. The central amygdala nucleus, which has intra-amygdaloid connections with LA, enhances startle magnitude via midbrain connections to the startle circuits. Tetanic stimulation of either MGN or AAC in vitro or in vivo can induce long-term potentiation in LA. In the present study, behavioural consequences of tetanization of these auditory afferents were investigated in awake rats. The acoustic startle reflex of rats was enhanced by tetanic stimulation of MGN, but suppressed by that of AAC. All the tetanization-induced changes of startle diminished within 24 h. Blockade of GABAB receptors in the LA area reversed the suppressive effect of tetanic stimulation of AAC on startle but did not change the enhancing effect of tetanic stimulation of MGN. Moreover, transient electrical stimulation of MGN enhanced the acoustic startle reflex when it lagged behind acoustic stimulation, but inhibited the acoustic startle reflex when it preceded acoustic stimulation. The results of the present study indicate that MGN and AAC afferents to LA play different roles in emotional modulation of startle, and AAC afferents are more influenced by inhibitory GABAB transmission in LA.

M3-like muscarinic receptors mediate Ca2+ influx in rat mesencephalic GABAergic neurones through a protein kinase C-dependent mechanism.

GABAergic neurones in the mesencephalon are important regulators of dopamine neurones. Cholinergic projections from mesopontine nuclei preferentially synapse onto these GABAergic neurones, thus suggesting that ACh can regulate dopamine neurones indirectly by modulating GABAergic interneurones. Muscarinic receptors mediate excitation of these interneurones through a Ca(2+)-dependent mechanism. Using a mesencephalic primary culture model, we show here that muscarine (10 microM) increases intracellular Ca2+ concentrations ([Ca2+]i) in GABAergic interneurones. Compatible with previous anatomical data, our pharmacological studies further suggest that the M3 receptor is the primary mediator of this increase. The rise in [Ca2+]i induced by muscarine was not activity-dependent but required influx of Ca2+ from the extracellular medium. Consistent with the known coupling of the M3 receptor to PKC, the effect of muscarine was blocked by bisindolylmaleimide, a selective PKC antagonist. The effect of muscarine was inhibited by SKF 96365 and verapamil, drugs known to block non-selective cationic channels such as those formed by transient receptor potential (TRPC) proteins. Finally, GABAergic neurones were found to be immunopositive for TRPC1, 3, 5 and 6. Taken together, these results suggest that the Ca(2+)-dependent regulation of mesencephalic GABAergic neurones by muscarinic receptors requires activation of some receptor-operated Ca2+ channels through a PKC-dependent mechanism.

Linkage of M5 muscarinic and alpha7-nicotinic receptor genes on 15q13 to schizophrenia.

Most antipsychotic drugs act on the forebrain by blocking dopamine receptors. In rodents, the M5 muscarinic receptor (CHRM5) is important for prolonged dopamine release. We typed polymorphisms in CHRM5 and alpha7-nicotinic receptor (CHRNA7) genes on 15q13 in 82 Canadian families having at least 1 schizophrenic patient. Using the Family-Based Association Test, we performed haplotype analysis of the 2 loci and found biased transmission in schizophrenia (z = -2.651, p = 0.008). In the families tested, the 2 cholinergic genes interacted to affect schizophrenia in combination, while neither was sufficiently alone to confer susceptibility. Our present study provided the first line of direct evidence suggesting that the CHRM5 gene combined with the CHRNA7 gene may be linked to schizophrenia.

Decreased amphetamine-induced locomotion and improved latent inhibition in mice mutant for the M5 muscarinic receptor gene found in the human 15q schizophrenia region.

M5 muscarinic receptors are coexpressed with D2 dopamine receptors in the ventral tegmentum and striatum, and are important for reward in rodents. Previously, we reported that disruption of the M5 receptor gene in mice reduced dopamine release in the nucleus accumbens. In this study, we established a polymerase chain reaction (PCR) genotyping method for M5 mutant mice, and, using RT-PCR, found that M5 mRNA expression was highest in the ventral tegmentum, striatum, and thalamus in wild-type mice. In the M5 mutant mice, D2 mRNA expression was increased in several brain structures, including the striatum. Genome mapping studies showed the M5 gene is localized to chromosome 2E4 in mice, and to 15q13 in humans in the region that has been linked to schizophrenia. Amphetamine-induced locomotion, but not baseline locomotion or motor functions, decreased in M5 mutant mice, consistent with lower accumbal dopamine release. Previous reports found latent inhibition improvement in rats following nucleus accumbens lesions, or blockade of dopamine D2 receptors with neuroleptic drugs. Here, latent inhibition was significantly increased in M5 mutant mice as compared with controls, consistent with reduced dopamine function in the nucleus accumbens. In summary, our results showed that M5 gene disruption in mice decreased amphetamine-induced locomotion and increased latent inhibition, suggesting that increased M5 mesolimbic function may be relevant to schizophrenia.