Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 96 (18), 10483-8

Enhancement of D1 Dopamine Receptor-Mediated Locomotor Stimulation in M(4) Muscarinic Acetylcholine Receptor Knockout Mice

Affiliations

Enhancement of D1 Dopamine Receptor-Mediated Locomotor Stimulation in M(4) Muscarinic Acetylcholine Receptor Knockout Mice

J Gomeza et al. Proc Natl Acad Sci U S A.

Abstract

Muscarinic acetylcholine receptors (M(1)-M(5)) regulate many key functions of the central and peripheral nervous system. Primarily because of the lack of receptor subtype-selective ligands, the precise physiological roles of the individual muscarinic receptor subtypes remain to be elucidated. Interestingly, the M(4) receptor subtype is expressed abundantly in the striatum and various other forebrain regions. To study its potential role in the regulation of locomotor activity and other central functions, we used gene-targeting technology to create mice that lack functional M(4) receptors. Pharmacologic analysis of M(4) receptor-deficient mice indicated that M(4) receptors are not required for muscarinic receptor-mediated analgesia, tremor, hypothermia, and salivation. Strikingly, M(4) receptor-deficient mice showed an increase in basal locomotor activity and greatly enhanced locomotor responses (as compared with their wild-type littermates) after activation of D1 dopamine receptors. These results indicate that M(4) receptors exert inhibitory control on D1 receptor-mediated locomotor stimulation, probably at the level of striatal projection neurons where the two receptors are coexpressed at high levels. Our findings offer new perspectives for the treatment of Parkinson's disease and other movement disorders that are characterized by an imbalance between muscarinic cholinergic and dopaminergic neurotransmission.

Figures

Figure 1
Figure 1
Disruption of the mouse M4 muscarinic receptor gene. (a) Knockout strategy showing restriction maps of wild-type receptor locus, targeting construct, and targeted allele. Only relevant restriction sites are shown. The receptor coding region is represented by a solid bar. The probes used for Southern analysis and the sizes of the restriction fragments detected with these probes are indicated. Bg, BglII; H, HindIII; Ns, NsiI; K, KpnI; X, XhoI. (b) Genotyping of F2 offspring via Southern blot analysis of NsiI-digested mouse tail DNA (probe 2). The 5.5- and 6.7-kb bands represent the wild-type and mutant M4 receptor alleles, respectively.
Figure 2
Figure 2
In situ hybridization analysis of M4 receptor mRNA expression in wild-type and M4 receptor mutant mice. Horizontal brain sections obtained from mice of the indicated genotypes were analyzed. A 35S-labeled ribonucleotide antisense probe corresponding in sequence to the genomic fragment that was deleted during the construction of the M4 targeting vector (see Fig. 1a) was used as a probe. CPu, caudate-putamen; Cx, cerebral cortex; Hc, hippocampus; OB, olfactory bulb.
Figure 3
Figure 3
Immunoprecipitation analysis of muscarinic receptor expression. Immunoprecipitation studies were carried out as described in Materials and Methods, using [3H]QNB-labeled receptors solubilized from the indicated brain regions of wild-type and M4 receptor mutant mice. M2 or M4 muscarinic receptors were immunoprecipitated with subtype-specific anti-M2 or anti-M4 rabbit antisera, respectively. Cx, cerebral cortex; Hc, hippocampus; Str, striatum; OB, olfactory bulb; Cer, cerebellum; BS; brain stem. Data are given as means ± SD (n = 3–4 for each dose and genotype). ∗, P < 0.001 (Student’s t test).
Figure 4
Figure 4
OXO-induced antinociceptive responses in M4 receptor mutant mice. (a) Tail-flick test. (b) Hot-plate assay. Mice of the indicated genotypes were injected s.c. with vehicle (Veh) or increasing doses of the nonselective muscarinic agonist, OXO. Analgesia measurements were carried out as described in Materials and Methods. Data are presented as means ± SEM (n = 18–20 for each dose and genotype) and are expressed as the percentage of MPE. Analgesic responses were not significantly different among the various genotypes (ANOVA).
Figure 5
Figure 5
Basal locomotor activity of M4−/− receptor mutant mice and their wild-type littermates. (a) Number of total photobeam breaks during a 1-hr observation period (n = 115) studied with vehicle-injected mice. (b) Time course of basal locomotor activity studied with vehicle-injected mice (n > 100 for each data point). Number of total photobeam breaks were measured in 10-min intervals. Locomotor activity measurements were carried out as described in Materials and Methods. Data are given as means ± SEM. ∗, P < 0.05 (Newman–Keuls post hoc comparison).
Figure 6
Figure 6
Effect of dopamine receptor agonists on the locomotor activity of M4−/− receptor mutant mice and their wild-type littermates. Mice of the indicated genotypes were injected s.c. with vehicle (Veh) or increasing doses of the following dopamine receptor agonists: apomorphine (nonselective), SKF 38393 (D1-type selective), and quinpirole (D2-type selective). Locomotor activity measurements were carried out as described in Materials and Methods. Data (number of total photobeam breaks during a 1-hr observation period) are given as means ± SEM (n ≥ 14 for each dose and genotype). ∗, P < 0.05 (Newman–Keuls post hoc comparison).
Figure 7
Figure 7
Effect of dopamine receptor antagonists on the locomotor activity of M4−/− receptor mutant mice and their wild-type littermates. Mice of the indicated genotypes were injected s.c. with vehicle (Veh) or increasing doses of SCH 23390, a D1-type receptor antagonist, or haloperidol, a D2-type receptor antagonist. Locomotor activity measurements were carried out as described in Materials and Methods. Data (number of total photobeam breaks during a 1-hr observation period) are given as means ± SEM (n = 13–16 for each dose and genotype). ∗, P < 0.05 (Newman–Keuls post hoc comparison).

Comment in

Similar articles

See all similar articles

Cited by 95 PubMed Central articles

See all "Cited by" articles

MeSH terms

Feedback