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M₄ muscarinic receptors regulate the dynamics of cholinergic and dopaminergic neurotransmission: relevance to the pathophysiology and treatment of related CNS pathologies

Eli Lilly and Co., Indianapolis, Indiana, USA; and
^* Laboratory Bioorganic Chemistry, National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, Maryland, USA

¹ Correspondence: Lilly Corporate Center, Indianapolis, IN, 46240-0510, USA. E-mail: gnomikos{at}lilly.com

SPECIFIC AIMS

Dopaminergic dysfunction is an important pathogenetic factor for brain pathologies such as Parkinson’s disease, ADHD, schizophrenia, and addiction as well as for metabolic disorders and anorexia. Dopaminergic neurons projecting from the midbrain to forebrain regions, such as the nucleus accumbens and the prefrontal cortex, regulate motor and cognitive functions and coordinate the patterned response of the organism to sensory, affective, and rewarding stimuli. The aim of this study was to determine the role of the inhibitory metabotropic cholinergic receptors (M₂ and M₄ receptors) in regulating dopaminergic and cholinergic neurotransmission in the whole animal, and thus to examine the potential of these receptors as therapeutic molecular targets for diseases in which the dynamic balance of the two neurotransmitter systems is purportedly disrupted.

PRINCIPAL FINDINGS

1. Hyperdopaminergia in the nucleus accumbens of M₄ knockout mice
To overcome the lack of selective muscarinic ligands that has impeded understanding of the role of muscarinic receptors in regulating CNS functions, we used a powerful molecular tool, genetically targeted mice, specifically invalidated for M₂ or M₄ receptors [mixed 129J1 x CF-1 hybrids for M₂ knockout (KO) and corresponding wild-type (WT) mice or 129SvEv x CF-1 hybrids for M₄ KOs and corresponding WT mice].

Basal values of dopamine and its metabolites DOPAC and HVA, as well as of the serotonin metabolite 5-HIAA, were determined in the nucleus accumbens of M₄ and M₂ KO mice and their corresponding WT controls by in vivo microdialysis. Basal dopamine efflux was elevated by 2-fold in M₄ KO mice compared with their WT controls. Dopamine levels in pmol/mL dialysate were 2.76 ± 0.86 for M₄ WT and 5.15 ± 0.91 for M₄ KO mice (statistically significant difference at P<0.05). Basal levels of DOPAC and HVA were elevated in M₄ KO mice (301±38 and 243±28 pmol/mL for DOPAC and HVA in M₄ KOs vs.175±34 and 127±28 in M₄ WTs; P<0.05). There was no difference in the levels of 5-HIAA between M₄ KO (139±12 pmol/mL) and WT (109±23 pmol/mL) animals, suggesting that M₄ receptor deletion results in a selective increase in basal dopamine levels in the nucleus accumbens by affecting synthesis/release of the neurotransmitter. There was no difference in dopamine, DOPAC, HVA, or 5-HIAA levels between M₂ KO and M₂ WT mice.

2. Enhanced dopaminergic sensitivity to psychostimulants in the nucleus accumbens of M₄ KO mice
The elevated dopaminergic tone in M₄ KO mice could be indicative of a generalized hyper-responsiveness of the mesolimbic dopaminergic system, possibly leading to enhanced responses to psychostimulants in these animals, as has been suggested from behavioral data. Thus, we assessed dopamine efflux in the nucleus accumbens of M₂ and M₄ WT and KO mice in response to acute administration of the psychostimulants d-amphetamine (1 and 3 mg/kg i.p.) or PCP (10 mg/kg i.p.). Administration of 1 and 3 mg/kg d-amphetamine resulted in a significant increase in dopamine efflux in the nucleus accumbens in all mouse genotypes compared with saline administration. Dopamine release was significantly (P<0.05) enhanced in M₄ KO mice compared with their WT controls but not significantly different between M₂ WT and M₂ KO mice (Fig. 1 ). Similar results were obtained with PCP.

View larger version (34K): [in this window] [in a new window]   Figure 1. Time course of DA efflux in the nAcc in response to d-amphetamine in M4 and M2 KO mice and their corresponding WT controls. d-Amphetamine at 1 and 3 mg/kg (i.p.) significantly increased DA efflux in all different genotype groups. a, b) This response was significantly enhanced in M4 but not in (c, d) M2 KO mice compared with their WT controls. Arrows indicate injection of 1 mg/kg (a, c) and 3 mg/kg (b, d) d-amphetamine or vehicle. Time points correspond to 30 min samples acquired before or after the injection. Data are expressed as multifold change over baseline and represent means ±SE of 5–7 mice per group. *P<0.05 vs. WT controls.

3. Increased basal acetylcholine efflux in the midbrain of M₄-KO mice
Cholinergic projections of the Ch5/Ch6 cell groups to the midbrain (VTA/SN) monosynaptically activate dopaminergic afferents to the nucleus accumbens. Thus, M₄ muscarinic receptor dysfunction could affect basal and evoked dopamine release in the nucleus accumbens indirectly by altering acetylcholine (ACh) content and muscarinic receptor activation in the midbrain. To test this hypothesis, we assessed basal ACh levels in the midbrain of M₄ and M₂ KO mice and their corresponding WT controls. Basal ACh levels were significantly (P<0.05) elevated in M₄ KO mice vs. WT controls (96±10 and 49±9 fmol/15 min dialysate sample, respectively). In contrast, there was no difference in baseline ACh values between M₂ WT and M₂ KO mice.

4. M₄ receptor dysfunction results in impaired homeostatic control of cholinergic activity that leads to increased basal acetylcholine efflux in the midbrain
In some brain regions, inhibitory M₂ and/or M₄ receptors operate as autoreceptors that regulate ACh homeostasis. To determine the possible autoreceptor role of M₄ and M₂ muscarinic receptors in regulating ACh dynamics in the midbrain, we assessed release of ACh in response to a local challenge with scopolamine in M₂ and M₄ WT and KO mice. Scopolamine at 1 or 10 µM infused through the microdialysis probe in the midbrain for 45 min induced a marked increase in ACh efflux in this region in M₄ and M₂ WT mice. Midbrain ACh efflux enhancement in response to 1 or 10 µM scopolamine infusions was significantly (P<0.05) reduced (by 50%) in M₄-KO compared with their corresponding WT controls (Fig. 2 ). There was no significant difference between M₂ KO and M₂ WT animals.

View larger version (27K): [in this window] [in a new window]   Figure 2. ACh efflux in the midbrain of M4 and M2 WT and KO mice in response to scopolamine. Scopolamine infused at both concentrations significantly increased ACh efflux in all different genotype groups. a) This response was significantly attenuated in M4 but not in b) M2 KO mice compared with their WT controls. Scopolamine (1 or 10µM) was infused locally through the microdialysis probe for 45 min. Data are expressed as average multifold change over baseline for the entire 45 min infusion period, and represent means ±SE of 4–6 mice per group. **P<0.01, ***P<0.001 filled stars vs. vehicle-treated mice; open stars vs. WT mice.

CONCLUSIONS AND SIGNIFICANCE

In this study we show that dopaminergic neurotransmission in the forebrain is highly dependent on M₄ cholinergic muscarinic receptor function in the midbrain. Using in vivo microdialysis in M₄ KO mice, we found elevated dopamine (DA) basal values and enhanced DA response to psychostimulants in the nucleus accumbens of these animals. Earlier studies predicted that cholinergic/muscarinic dysfunction might lead to abnormal dopaminergic neurotransmission and dysregulated dopamine-related behavioral and physiologic responses. Because of the lack of specific muscarinic ligands, it has not been possible to distinguish which one among the five muscarinic receptors cloned and characterized up to now—in particular, between the two inhibitory cholinergic receptors M₂ and M₄—is important in the regulation of dopaminergic dynamics. An M₂ implication was often suggested on the basis of the high levels of expression of M₂ receptors within the CNS, but no proof for this involvement was given. Contrary to this widespread assumption, our results suggest that the M₂ receptor plays only a secondary role in regulating dopaminergic neurotransmission and related behaviors.

The importance of M₄ receptors as regulators of dopaminergic activity has been largely underestimated because most studies were conducted with in vitro techniques that were limited in targeting responses occurring exclusively at the level of the dopaminergic terminal field. Using an in vivo functional approach, we show here that M₄ receptor activation is critical for the tonic and phasic modulation of dopaminergic responsiveness in the whole animal. Our findings suggest that M₄ receptors regulate dopaminergic homeostasis indirectly by affecting cholinergic activity in the midbrain.

M₄ receptor expression has been detected in midbrain cholinergic afferents (originating from the Ch5/Ch6 cell complex, i.e., the pendunculopontine tegmental nucleus and the laterodorsal tegmental nucleus, which monosynaptically contact and excite dopaminergic neurons. We hypothesized that on these cholinergic neurons M₄ receptors could function as presynaptic inhibitory autoreceptors to regulate ACh from the cell body and the terminal region. Indeed, we show that M₄ receptor dysfunction results in impaired homeostatic control of cholinergic activity, which leads to increased basal ACh efflux in the midbrain. Loss of M₄ muscarinic receptor control of cholinergic function effectuates a state of dopaminergic hyperexcitability (Fig. 3 ). This may be responsible for pathological conditions in which appetitive motivation as well as affective and cognitive processing is impaired.

View larger version (18K): [in this window] [in a new window]   Figure 3. Schematic diagram. Model of dysregulated afferent modulation of mesolimbic DA neurotransmission in M4 KO mice. M4 receptors present on cholinergic interneurons and on medium spiny GABAergic neurons in the nucleus accumbens are not depicted for reasons of clarity. In the midbrain, M4 receptors are localized on GABAergic afferents that also express D1 receptors; this projection indirectly, possibly through an interposed GABA collateral, activates DA firing (red dashed arrow). M4 receptors are also localized to cholinergic laterodorsal tegmental (LDT) and pendunculopontine (PPT) afferents that monosynaptically stimulate DA activity (red solid arrow). In WT animals (a), ACh released from LDT terminals activates M4 receptors (blue arrows) that act as inhibitory hetero- or autoreceptors to reduce net excitatory input to DA neurons. In M4 KO animals (b), loss of M4 receptors results in impeded autoinhibition of cholinergic projections, increased midbrain ACh efflux, enhanced stimulation of mesolimbic DA efferents, and increased DA in the nucleus accumbens (n.Acc.).

Preliminary clinical studies have shown the efficacy of M₄ muscarinic ligands in patients with Alzheimer’s disease or affective and psychotic symptoms. However lack of proof of concept for these ligands has hindered progress in the field. The evidence we provide here permits us to consider the use of M₄ receptor ligands in the pharmacotherapy of disorders associated with hyperdopaminergia. According to our scheme, activation of M₄ receptors on midbrain afferents is predicted to sequentially reduce excitatory cholinergic input to dopaminergic neurons, normalize aberrant dopamine firing patterns and mesolimbic dopamine release, and restore goal-oriented patterns of dopamine-related behaviors.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1575fje; doi: 10.1096/fj.04-1575fje

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