Isabelle M. Mintz - US grants

Although it generally is appreciated that Ca channels play crucial roles in the shaping of action potentials and firing patterns, as well as in the control of transmitter release at nerve terminals, the study of these roles has been limited by the availability of selective tools for blocking Ca channels. The recent isolation of omega-Aga-IVA (from spider venom) constitutes a significant advance since this toxin may be used to target a unique population of Ca channels whose roles were difficult to isolate previously. Funds are requested to continue these studies in three different ways: 1) patch-clamp recordings from tissue slices or freshly isolated neurons to investigate whether the toxin blocks more than one population of Ca channels (as suggested recently by R. W. Tsien and collaborators); 2) photometric studies of presynaptic Ca transients to determine the regulation of synaptic strength by transmitters that affect presynaptic Ca channels differentially; and 3) patch-clamp recordings of Purkinje cells in combination with photometric measurements of Ca to study the link between P-type Ca channels and internal Ca release, and their contribution to the processing of synaptic inputs. Since Ca channels sensitive to omega-Aga-IVA are ubiquitous in the CNS, there is no question that these studies will provide greater insight into the many cellular functions controlled by calcium (e.g. promotion of neuronal growth, regulation of genes and enzymes, etc.) as well as into the pathological situations that arise when Ca homeostatic mechanisms are disrupted, as is thought to occur with epilepsy, excitotoxicity, and neuronal death.

Axon terminals are the conventional structures in the brain that release neurotransmitters and ensure communication between neurons. Dendrites, which are the typical postsynaptic component of the synapse, are nevertheless able to release neurotransmitters. In particular, dendrites in the substantia nigra pars compacta (SNc) release dopamine in a manner regulated by incoming neuronal activity and neuromodulators. The goal of this proposal is to investigate the mechanisms of dopamine dendritic release, when triggered by a physiological input from the subthalamus. A combination of techniques will be used which include patch-clamp recordings of neurons, maintained in slices or freshly isolated, and tyrosine hydroxylase immunostaining. The specific aims are i) to establish the existence of monosynaptic connections between subthalamic neurons and dopaminergic SNc neurons, ii) to demonstrate that the dendritic release of dopamine occurs upon stimulation of subthalamic afferents, iii) to clarify the role of the dopamine transporter in mediating such release, and finally iv) to investigate how dopamine controls subthalamic neuron excitability and enhances the potential excitotoxicity of their connection with SN dopaminergic neurons. Subthalamic neurons are hyperactive in Parkinson's patients and their lesion dramatically improves the symptoms of the disease. There is little doubt that the proposed studies focus on mechanisms likely to be involved in the onset and progression of Parkinson's disease.

DESCRIPTION (provided by applicant): Neurotransmitter transporters constitute an important class of related proteins, whose major function is the re-uptake of molecules released upon fusion of synaptic vesicles, thus aiding the rapid clearance of the neurotransmitter from the synaptic cleft. In dopaminergic neurons, the dopamine transporter (DAT) has long been implicated in the effects of numerous psychostimulants and substances of abuse, such as amphetamines and cocaine, which induce an accumulation of dopamine (DA) in the extracellular space. The normal transport cycle of the DAT is electrogenic and coupled to the stoichiometric movement of Na and CI ions, whose electrochemical potential can fuel the translocation of DA against its concentration gradient. This process is reversible. Recent observations have demonstrated that, in the substantia nigra (SN), activation of neural inputs can lead to the release of DA through reverse transport. Such mechanisms could enable the release of DA from non-synaptic regions, a concept of far-reaching implications. This proposal will exploit a new model system, developed in native dopaminergic neurons, which affords a great degree of experimental control together with the high resolution needed for single-cell detection of DA release. The goals are to identify the physiological determinants for DAT reversal during neuronal activity (aim 1) or exposure to amphetamines (aim 2). Using amperometric and patch-clamp recordings of isolated dopaminergic neurons from the SN, we shall first identify the variables (membrane potential, ionic currents, local Na gradients...) affected by the release paradigms and, conversely, measure DA release while manipulating these variables, in order to determine their individual contribution to DAT reversal. Should the observations depart from the predicted behavior of ion-coupled DAT cycling (as suggested for amphetamine by preliminary studies), we shall examine whether neuronal activity and amphetamine recruit distinct modes for DAT reverse operation. Finally, to link these studies to the releasing function of DAT in a physiological context, we shall use multiphoton imaging of fluorescent Na indicators, in dopaminergic neurons maintained in slices, to determine how neural excitation and amphetamines affect local Na gradients (aim 3). These experiments may identify new mechanisms by which mid-brain dopaminergic systems are utilized for ceil-cell communication and are targeted by psychostimulant drugs.