Kamran Khodakhah - US grants

DESCRIPTION (provided by applicant): The mesolimbic system, particularly the dopaminergic neurons of the ventral tegmental area (VTA) play an essential role in endogenous reward. Activation of the reward pathway is associated with increased dopamine release brought about by an increase in the activity of VTA dopaminergic neurons. The reinforcing actions of drugs of abuse such as opioids and psychostimulants are mediated by increasing the activity of dopamine neurons. A grasp of the mechanisms that regulate the excitability of these cells, therefore, is essential for a better understanding of the cellular mechanisms involved in endogenous reward and in drug addiction. Recently, it has been shown that stimulation of glutametergic synaptic inputs result in a slow hyperpolarization of VTA dopamine cells that requires the activation of glutamate metabotropic receptors (mGluRs). It has been proposed that these inhibitory postsynaptic potentials (IPSPs) are produced by activation of small conductance calcium-dependent potassium channels (SK) by calcium release from intracellular stores. Both of the two calcium release channels, the inositol triphosphate (InsP3) and ryanodine receptors, have been implicated in this process. The main goal of this proposal is to test the hypothesis that InsP3-evoked calcium release, augmented by calcium-induced calcium release (CICR), mediates the mGluR-dependent IPSPs in VTA dopaminergic cells. These experiments will be done by combining whole-cell recordings from VTA dopaminergic neurons in acutely prepared midbrain slices with calcium imaging. To mobilize calcium, all second messenger cascades will be bypassed and InsP3 will be photoreleased into the cytosol by flash photolysis of caged InsP3. The extent to which CICR contributes to InsP3-evoked calcium transients will be evaluated by inhibition of ryanodine receptors with selective antagonists. In addition, the extent to which, and the mechanism by which, prior neuronal activity modulates the mGluR-mediated IPSPs will be delineated.

DESCRIPTION (provided by applicant): Several forms of hereditary ataxia such as episodic ataxia type 2 (EA2) and spinocerebellar ataxia type 6 (SCA6) are caused by mutations in P/Q-type voltage-gated calcium channels. The main objective of this grant is to test a new hypothesis regarding the cause of ataxia in patients suffering from these movement disorders. Malfunction of the cerebellum, either because of pathological damage or alterations in the physiological function of its neurons, results in uncoordinated movement (ataxia). In humans and mice several forms of hereditary ataxia have been identified. Many of these are the consequence of mutations in the P/Q-type voltage-gated calcium channels which are present throughout the central nervous system (CNS). P/Q-type calcium channels are expressed throughout the CNS and trigger neurotransmitter release at many nerve terminals. The accepted hypothesis suggests that in P/Q-channel related ataxias poor motor coordination is the consequence of impaired synaptic transmission in the cerebellum. Reported changes in synaptic transmission, however, do not fully account for the extent of ataxia. This grant examines whether additional cerebellar defects contribute to ataxia in these disorders. In the cerebellum P/Q channels are expressed at a high density in the soma and dendrites of Purkinje cells. Purkinje cells form the core of the computational units of the cerebellum and their dysfunction causes ataxia. In Purkinje cells of the ataxic P/Q-channel mutants the P/Q calcium current is significantly reduced. Further, in different mutant mice, the severity of ataxia correlates well with the extent of reduction in the P/Q current in Purkinje cells. We have previously shown that in Purkinje cells P/Q-type calcium channels regulate firing by activating calcium-activated potassium channels. Here we propose that P/Q channel mutations that cause ataxia alter the intrinsic, spontaneous, firing of Purkinje cells. Because Purkinje cells provide the sole output of the cerebellar cortex large alterations in their intrinsic firing will impair their function. Such dysfunction is likely to make a significant contribution to poor motor coordination. We will examine whether the intrinsic firing of Purkinje cells is altered in the P/Q-channel mutant mice, and if so, we will explore whether agents that make the firing of P/Q mutant Purkinje cells regular reduce ataxia.

DESCRIPTION (provided by applicant): A major function of the cerebellum is motor coordination and maintenance of balance. Dysfunction of the cerebellum, either as a consequence of a hereditary disorder or acquired deficiency, results in uncoordinated movement (ataxia). There is presently no cure for any of the hereditary ataxias, and there are few effective therapeutic options for ataxia in general. However, during the last two decades serotonergic agonists have been shown to be therapeutically effective in reducing the symptoms of a wide variety of cerebellar ataxias. The mechanism of action of serotonergic drugs is not understood. Given the therapeutic utility of serotonin it is important to delineate its mechanism of action in the cerebellum as a first step towards improving its therapeutic use in ataxia. The goal of this proposal is to delineate the effects of serotonergic drugs on cerebellar Purkinje cells. In particular, we will test the hypothesis that serotonin regulates the trafficking of AMPA/Kainate receptors in these neurons. PUBLIC HEALTH RELEVANCE: A major function of the cerebellum is motor coordination and maintenance of balance. Dysfunction of the cerebellum, either as a consequence of a hereditary disorder or acquired deficiency, results in uncoordinated movement (ataxia). There is presently no cure for any of the hereditary ataxias, and there are few effective therapeutic options for ataxia in general. However, during the last two decades serotonergic agonists have been shown to be therapeutically effective in reducing the symptoms of a wide variety of cerebellar ataxias. The mechanism of action of serotonergic drugs is not understood. Given the therapeutic utility of serotonin it is important to delineate its mechanism of action in the cerebellum as a first step towards improving its therapeutic use in ataxia. The goal of this proposal is to delineate the effects of serotonergic drugs on cerebellar Purkinje cells.

Abstract There is broad agreement that the cerebellum does more than just coordinate movement, with clear indications that it participates in a number of cognitive functions, and that its dysfunction may contribute to mental health disorders such as schizophrenia, autism, and addiction. Yet, compared to studies aimed at understanding the contribution of the cerebellum to motor coordination, there is little research focused on elucidating the non-motor functions of the cerebellum, and our understanding of its cognitive functions is rudimentary at best. The literature provides compelling evidence consistent with the idea that the cerebellum contributes to addiction and drug-seeking behavior both in experimental animals and in humans. However, the nature of this contribution has remained, by and large, unexplored. Our pilot data reveal a potential substrate for this effect: a previously little-appreciated direct projection from the cerebellum to the ventral tegmental area (VTA). Because the VTA is the seat of the mesolimbic dopamine projection that is critically important in addiction and reward, we propose the overarching hypothesis that the direct cerebellum to VTA projection (Cb?VTA) is a critical element of the neural circuitry underlying drug- seeking and natural reward-seeking behavior, as well as drug and natural reinforcement. A primary aim of the current proposal is to delineate, using state-of-the-art anatomical and physiological approaches, the pathways by which the cerebellum can affect the activity of neurons in the VTA, and also those in the prefrontal cortex and nucleus accumbens (two additional brain regions which are target of the VTA projections and are intimately associated with addictive behavior). To complement and expand upon the anatomical and physiological studies, an additional goal is to directly examine the potential utility of cerebellar projections to the VTA in acquisition and expression of addictive behavior using behavioral experiments during which the relevant cerebellar pathways are optogenetically or chemogenetically manipulated. Successful completion of the proposed aims would not only advance our understanding of the non- motor functions of the cerebellum, but has the potential to substantiate a number of mechanistic hypotheses on acquisition and extinction of addictive behaviors. Such knowledge, while fundamental basic science in nature, in the future may contribute to new insights for treatment of drug abusers and prevention of relapse after treatment.

Abstract DYT1 is a debilitating movement disorder caused by loss-of-function mutations in torsinA. How these mutations cause dystonia remains unknown. Mouse models which have embryonically targeted torsinA have failed to recapitulate the dystonia seen in patients, possibly due to differential developmental compensation between rodents and humans. To address this issue, we have developed a new mouse model where torsinA is acutely knocked down in select brain regions of adult mice using shRNAs. We have found that torsinA knockdown in the cerebellum, but not in the basal ganglia, is sufficient to induce dystonia. Abnormal motor symptoms in dystonic animals were associated with irregular cerebellar output caused by changes in the intrinsic activity of both Purkinje cells and neurons of the deep cerebellar nuclei. This proposal capitalizes on this dystonic model of DYT1 to explore at circuit, neuronal, and molecular levels how loss of torsinA causes dystonia. The proposal is based on three specific aims. In the first specific aim we will test the hypothesis that in DYT1 abnormal cerebellar output causes dystonia by altering the activity of the basal ganglia via the thalamic disynaptic Cb-BG pathway that we have characterized. Specific Aim 2 will test the hypothesis that selective knock down of torsinA in cerebellar Purkinje cells and/or DCN neurons causes cerebellar dysfunction and dystonia. And lastly the third specific Aim tests the hypothesis that knock down of torsinA alters the intrinsic pacemaking of Purkinje cells and DCN neurons by altering the expression or function of select conductances. Successful accomplishment of the aims set will significantly advance our understanding of DYT1 dystonia, and may provide valuable potential therapeutic targets for its treatment.