Changes in Purkinje cell firing and gene expression precede behavioral pathology in a mouse model of SCA2.

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominantly inherited disorder, which is caused by a pathological expansion of a polyglutamine (polyQ) tract in the coding region of the ATXN2 gene. Like other ataxias, SCA2 most overtly affects Purkinje cells (PCs) in the cerebellum. Using a transgenic mouse model expressing a full-length ATXN2(Q127)-complementary DNA under control of the Pcp2 promoter (a PC-specific promoter), we examined the time course of behavioral, morphologic, biochemical and physiological changes with particular attention to PC firing in the cerebellar slice. Although motor performance began to deteriorate at 8 weeks of age, reductions in PC number were not seen until after 12 weeks. Decreases in the PC firing frequency first showed at 6 weeks and paralleled deterioration of motor performance with progression of disease. Transcription changes in several PC-specific genes such as Calb1 and Pcp2 mirrored the time course of changes in PC physiology with calbindin-28 K changes showing the first small, but significant decreases at 4 weeks. These results emphasize that in this model of SCA2, physiological and behavioral phenotypes precede morphological changes by several weeks and provide a rationale for future studies examining the effects of restoration of firing frequency on motor function and prevention of future loss of PCs.

Response to ethanol induced ataxia between C57BL/6J and 129X1/SvJ mouse strains using a treadmill based assay.

More sensitive assays of mouse motor ataxia may provide a better understanding of the pathological profile. Treadmill gait analysis using ventral imaging allows for unhindered access to the ambulating mouse. In contrast to genetic mutations or exogenous brain injury, ethanol (EtOH) allows for the detection of dose dependent changes in motor behavior, which can be used to assess an assay's detection sensitivity. EtOH induced ataxia was assessed in C57BL/6J (B6) and 129X1/SvJ (129) mice using the DigiGait imaging system. Gait was analyzed across EtOH dosage (1.75, 2.25 and 2.75 g/kg) in each strain using a linear mixed effects model. Overall, 129 mice displayed greater susceptibility to EtOH ataxia than their B6 counterparts. In both strains, hind paws exhibited greater sensitivity to EtOH dosage than fore paws. Across most variables analyzed, only a modest EtOH-induced change in motor behavior was observed in each strain with the 1.75 g/kg EtOH doses failing to elicit significant change. These data indicate the ability to detect motor differences between strains, yet only moderate ability to detect change across EtOH dosage using the automated treadmill. Rotarod assays, however, were able to detect motor impairment at lower doses of EtOH. The significant, but opposite changes in paw placement with increasing EtOH doses highlight strain-specific differences in biophysical adaptations in response to acute EtOH intoxication.

ETS1 regulates the expression of ATXN2.

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant disorder caused by the expansion of a CAG tract in the ATXN2 gene. The SCA2 phenotype is characterized by cerebellar ataxia, neuropathy and slow saccades. SCA2 foreshortens life span and is currently without symptomatic or disease-modifying treatments. Identifying function-specific therapeutics for SCA2 is problematic due to the limited knowledge of ATXN2 function. As SCA2 is likely caused by a gain-of-toxic or gain-of-normal function like other polyglutamine disorders, targeting ATXN2 expression may represent a valid therapeutic approach. This study characterized aspects of ATXN2 expression control using an ATXN2 promoter-luciferase (luc) reporter construct. We verified the fidelity of construct expression by generating transgenic mice expressing the reporter construct. High reporter expression was seen in the cerebellum and olfactory bulb in vivo but there was relatively low expression in other tissues, similar to the expression of endogenous ataxin-2. We verified the second of two possible start codons as the functional start codon in ATXN2. By evaluating deletions in the ATXN2 promoter, we identified an E-twenty six (ETS)-binding site required for ATXN2 expression. We verified that endogenous ETS1 interacted with the ATXN2 promoter by an electromobility supershift assay and chromatin immunoprecipitation polymerase chain reaction. ETS1 overexpression increased ATXN2-luc (ATXN2-luciferase) as well as endogenous ATXN2 expression. Deletion of the putative ETS1-binding site abrogated the effects on the expression of ATXN2-luc. A dominant negative ETS1 and an ETS1 short-hairpin RNA both reduced ATXN2-luc expression. Our study broadens the understanding on the transcriptional control of ATXN2 and reveals specific regulatory features of the ATXN2 promoter that can be exploited therapeutically.

Cholinergic modulation of mesolimbic dopamine function and reward.

The substantial health risk posed by obesity and compulsive drug use has compelled a serious research effort to identify the neurobiological substrates that underlie the development these pathological conditions. Despite substantial progress, an understanding of the neurochemical systems that mediate the motivational aspects of drug-seeking and craving remains incomplete. Important work from the laboratory of Bart Hoebel has provided key information on neurochemical systems that interact with dopamine (DA) as potentially important components in both the development of addiction and the expression of compulsive behaviors such as binge eating. One such modulatory system appears to be cholinergic pathways that interact with DA systems at all levels of the reward circuit. Cholinergic cells in the pons project to DA-rich cell body regions in the ventral tegmental area (VTA) and substantial nigra (SN) where they modulate the activity of dopaminergic neurons and reward processing. The DA terminal region of the nucleus accumbens (NAc) contains a small but particularly important group of cholinergic interneurons, which have extensive dendritic arbors that make synapses with a vast majority of NAc neurons and afferents. Together with acetylcholine (ACh) input onto DA cell bodies, cholinergic systems could serve a vital role in gating information flow concerning the motivational value of stimuli through the mesolimbic system. In this report we highlight evidence that CNS cholinergic systems play a pivotal role in behaviors that are motivated by both natural and drug rewards. We argue that the search for underlying neurochemical substrates of compulsive behaviors, as well as attempts to identify potential pharmacotherapeutic targets to combat them, must include a consideration of central cholinergic systems.

Injection of CART (cocaine- and amphetamine-regulated transcript) peptide into the nucleus accumbens reduces cocaine self-administration in rats.

Cocaine- and amphetamine-regulated transcript (CART) peptides appear to modulate various effects of psychostimulant drugs. Injections of CART peptide into the nucleus accumbens (NAcc) inhibit locomotion produced by systemic injections of the psychostimulants cocaine and amphetamine. Intra-NAcc injections of CART peptide also inhibit locomotion produced by microinfusions of dopamine into the NAcc, suggesting that the effects of CART peptides may be due to an interaction with the dopaminergic system in the NAcc. We sought to determine if this inhibitory effect of CART peptide generalizes to other measures of dopaminergic function such as reward/reinforcement by testing the effect of bilateral intra-NAcc CART infusions (0, 0.25, 1.0 and 2.5 microg per side) on cocaine and food self-administration. One group of rats self-administered cocaine (0.75 mg/kg per 140 microl IV infusion) on a progressive ratio schedule. A separate group received 45 mg food pellets on the same progressive ratio schedule. Bilateral intra-NAcc injections of CART peptide dose-dependently decreased the number of cocaine infusions, the breakpoint of cocaine self-administration, and the total number of bar presses on the cocaine-associated lever. There were no effects of CART injections on the breakpoint for food reward. Thus, we conclude that injections of CART into the NAcc appear to functionally antagonize a major site of action for cocaine self-administration in rats.

The nicotinic acetylcholine receptor antagonist mecamylamine prevents escalation of cocaine self-administration in rats with extended daily access.

RATIONALE: Escalation from moderate to excessive drug intake is a hallmark of human addiction that can be modeled in rats by giving them longer daily access time to self-administer cocaine. Nicotine and cocaine are commonly coabused drugs in humans and recent work in animals suggests that activation of nicotinic acetylcholine receptors (nAChR) can increase cocaine self-administration. OBJECTIVES: Determine the role of nAChR in the escalation of cocaine self-administration. METHODS: Control rats self-administered cocaine (0.75 mg/kg/infusion) for either 1 or 6 h per day. Experimental groups had the nAChR antagonist mecamylamine (MEC) added to the cocaine solution for 5 days after the transition from short (1 h per day) to long access (6 h per day) for cocaine self-administration. After 5 days, MEC was removed from the cocaine solution. RESULTS: Control rats and rats that received a low dose of MEC (7 microg/infusion) with cocaine increased their average hourly intake over 5 days of 6 h per day cocaine access. Rats that received a higher dose of MEC (70 microg/infusion) did not increase their intake of cocaine during 6 h access but continued to self-administer cocaine. When MEC was removed, this group showed an escalation in cocaine self-administration. MEC did not alter cocaine intake in a group that had continuous 1 h access. CONCLUSIONS: Antagonism of nAChRs during the initial exposure to extended cocaine self-administration access time prevented escalation of, but did not eliminate, drug intake. These findings indicate that MEC-sensitive nAChRs are critical for determining cocaine intake as a function of longer access time.

Operant self-administration of ethanol in C57BL/6 mice lacking beta-endorphin and enkephalin.

To test whether endogenous opioid peptides are necessary for the rewarding effects of ethanol, we examined operant oral self-administration of ethanol in mice congenic to the C57BL/6J strain but lacking expression of beta-endorphin, enkephalin or both peptides. The influences of prandial state, schedule interval and type, and ethanol concentration were all examined. Food-restricted subjects were tested in postprandial and preprandial states and subsequently at normal body weight when feeding ad libitum (ad lib). Operant studies were conducted using fixed ratio (FR) intervals of 2 and 8 as well as a progressive ratio (PR) interval of 2. The main significant effect relevant to our hypothesis was increased responding by female mice lacking beta-endorphin under ad lib feeding conditions and only for lower ethanol concentrations (3% and 6%). Importantly, all subjects including those lacking both beta-endorphin and enkephalins learned to self-administer ethanol similarly to wild-type mice and maintained responding for ethanol under a variety of procedural variables. Consequently, the two opioid peptides believed to be the endogenous ligands for the micro-opioid receptor (MOR) were not necessary to shape or perpetuate ethanol-reinforced operant responding. These results suggest that the rewarding effects of ethanol do not require beta-endorphin or enkephalin signaling.