Reduced striatal acetylcholine efflux in the R6/2 mouse model of Huntington's disease: An examination of the role of altered inhibitory and excitatory mechanisms

Abstract

Huntington's disease (HD) is a genetic neurodegenerative disorder that is characterized by the progressive onset of cognitive, psychiatric, and motor symptoms. In parallel, the neuropathology of HD is characterized by progressive loss of projection neurons in cortex and striatum; striatal cholinergic interneurons are relatively spared. Nonetheless, there is evidence that striatal acetylcholine (ACh) function is altered in HD. The present study is the first to examine striatal ACh function in awake, behaving animals, using the R6/2 mouse model of HD, which is transgenic for exon 1 of the mutant huntingtin gene. Physiological levels of extracellular striatal ACh were monitored in R6/2 mice and wild type controls using in vivo microdialysis. Results indicate that spontaneous ACh release is reduced in R6/2 mice relative to controls. Intrastriatal application of the GABA_A antagonist bicuculline methiodide (10.0 μM) significantly elevated ACh levels in both R6/2 mice and wild type controls, while overall ACh levels were reduced in the R6/2 mice compared to the wild type group. In contrast, systemic administration of the D₁ dopamine receptor partial agonist, SKF-38393 (10.0 mg/kg, IP), elevated ACh levels in control animals, but not R6/2 mice. Taken together, the present results suggest that GABA-mediated inhibition of striatal ACh release is intact in R6/2 mice, further demonstrating that cholinergic interneurons are capable of increased ACh release, whereas D₁ receptor-dependent activation of excitatory inputs to striatal cholinergic interneurons is dysfunctional in R6/2 mice. Reduced levels of extracellular striatal ACh in HD may reflect abnormalities in the excitatory innervation of cholinergic interneurons, which may have implications ACh-dependent processes that are altered in HD, including corticostriatal plasticity.

Introduction

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder that is caused by an unstable expansion of CAG repeats in exon 1 on the huntingtin (Htt) gene (The Huntington's Disease Collaborative Research Group, 1993). The hallmark symptoms of HD include cognitive, psychiatric, and motor disturbances. The onset of HD symptoms is progressive, with psychiatric and cognitive symptoms appearing early, followed by the occurrence of gross motor dysfunction in later stages of the disease.

The neuropathology of HD is characterized by progressive loss of neurons in cortex and striatum, with medium spiny striatopallidal projection neurons being the most vulnerable early in the disease, while giant aspiny cholinergic striatal interneurons are relatively spared (Deng et al., 2004, Ferrante et al., 1985, Ferrante et al., 1987, Reiner et al., 1988). The process governing the differential vulnerability of neuronal subtypes remains largely unknown. Despite the fact that cholinergic interneurons are resistant to degeneration, there is evidence that striatal acetylcholine (ACh) function is altered in HD. Several indices of cholinergic function have been shown to be reduced in both HD mutant mice and in post-mortem striatal tissue from HD patients, including reduced levels of vesicular ACh transporter and the synthetic enzyme, choline-acetyl transferase (Smith et al., 2006), as well as reduced expression of muscarinic ACh receptors (Cha et al., 1998). To date, one study, which employed in vitro superfusion, found that electrically stimulated striatal ACh release was reduced in striatal slice preparations from HD mutant mice (Vetter et al., 2003), although the precise mechanism was not clear. Critically, dysfunction of striatal cholinergic interneurons has profound implications for ACh-dependent processes, including the regulation of corticostriatal plasticity (Calabresi et al., 1999, Wang et al., 2006) and striatal information processing (Calabresi et al., 2000), which may underlie aspects of motor control and cognition that are disrupted in HD.

Several genetic mouse models of HD have been developed that reflect many aspects of the human disease. One of the best-characterized models is the R6/2 mouse line, which is transgenic for exon 1 of the mutant huntingtin gene and expresses approximately 150 CAG repeats (Mangiarini et al., 1996). R6/2 mice display subtle motor and cognitive deficits as early as 4 weeks of age, that progressively worsen until spontaneous death, which generally occurs by 14 weeks of age. Additionally, R6/2 mice show several neuropathological alterations, including the presence of nuclear inclusions, morphological changes in cortical pyramidal and striatal medium spiny neurons, as well as altered electrophysiological properties of the corticostriatal pathway (see Cepeda et al., 2007, Levine et al., 2004, for review). Despite the considerable work describing abnormalities in corticostriatal circuitry in mouse models of HD, relatively little is known about the afferent regulation of cholinergic interneurons.

The aim of the present study was to examine the release dynamics of striatal ACh, as well as the functional integrity of inhibitory and excitatory regulation of striatal cholinergic interneurons in the R6/2 transgenic mouse model of HD. R6/2 mice were selected for microdialysis experiments using a “behavioral clamping” design, wherein microdialysis experiments commenced upon observing a moderate deficit on the fixed speed Rotarod treadmill task. The functional status of cholinergic inhibitory afferents was evaluated by monitoring changes in ACh release in response to local administration of the GABA_A receptor antagonist, bicuculline, in R6/2 mice and wild type controls. Excitatory cholinergic afferents were similarly assessed by monitoring ACh efflux in response to systemic administration of the D₁ receptor partial agonist, SKF-38393, thereby activating a circuit which results in increased excitatory drive of striatal cholinergic interneurons (Abercrombie and DeBoer, 1997). It was hypothesized that treatment with both bicuculline and SKF-39383 would result in increased ACh release. Differential effects of either bicuculline or SKF-38393 treatment between R6/2 and wild type mice would thus reflect abnormalities in the inhibitory or excitatory regulation of striatal cholinergic interneurons, respectively.