Lloyd D. Partridge - US grants

Calcium plays a crucial role in coupling excitation to many functions of neurons. One important example is the activation of ion channel gating. A recently discovered class of Ca++.activated channels is the non.specific cation (CAN) channel. Dr. Partridge intends to establish that these channels, found in a wide diversity of tissues, constitute a unique channel class. Dr. Partridge has shown previously that CAN channels exist in burster neurons. Although the characteristics of CAN channels can be surmised from piecemeal data from numerous tissues, a solid understanding of their role in cell function and a detailed study of channel characteristics in a single preparation are lacking. The proposed research will utilize cell.attached and excised membrane patches from burster neurons to measure CAN channel selectivity, kinetics, and links to cell metabolism. The project will accomplish two important goals. (1) The physiological role of CAN currents has been established in burster neurons, thus a detailed description of CAN channels will provide an important link between the cellular phenomenon of bursting and the action of a specific class of membrane channels; and (2) Focusing descriptive information about CAN channels on one preparation will provide strong evidence to support the hypothesis that CAN channels represent a unique class of membrane.

Calcium-activated non-specific cation (CAN) channels play a crucial role in the generation and modulation of bursting activity in neurons. Bursting, the grouping of action potentials in high periodic frequency discharges, is an important neuronal property characteristic of such phenomena as pacemaking and neurosecretion. Because CAN channels have a central function in burst generation, their characteristics are significant determinants in neuron encoding. The goal of this research project is to investigate the selectivity and voltage dependence of CAN channels. Dr. Partridge will study these channels by using the patch clamp technique, an experimental technique which allows for the study of single channels in isolation. Studies on how CAN channel activity is modulated by phosphorylation, the modification of protein structure induced by the attachment of phosphate groups, will be performed. This investigation explores basic neuronal properties important to the signalling function in the nervous system.

DESCRIPTION (provided by applicant): Neurosteroids have important roles in mental health-related disorders and these compounds have great potential for neuropsychopharmacological applications. Neurosteroids affect ligand-gated ion channels and metabotropic receptors by nongenomic mechanisms, but their mechanism of action is not completely understood. We have convincing preliminary evidence of a novel effect of the neurosteroids. We found that these compounds act at a presynaptic site to dramatically enhance glutamatergic paired-pulse facilitation, which is a model of short-term presynaptic plasticity. Additionally we observed an increase spontaneous miniature glutamatergic excitatory postsynaptic currents. We thus propose the following hypothesis: HYPOTHESIS: Neurosteroid actions in the CNS are due in part to enhancement of glutamate release in the hippocampus. To test this hypothesis we will address the following two specific aims: Specific Aim #1. Investigate the modulation of glutamate release by neurosteroids. We will use hippocampal slices and sharp-electrode electrophysiological techniques to study effect of two physiologically important neurosteroids, pregnenolone sulfate and dehydroapiandrosterone sulfate, on paired pulse facilitation. We will also examine effects of these compounds on spontaneous miniature glutamatergic excitatory postsynaptic currents. We will use hippocampal slices and whole-cell patch-clamp electrophysiological techniques for these studies. Specific Aim #2. Investigate the mechanisms by which neurosteroids modulate glutamate release. We will consider the role of presynaptic metabotropic receptors and ion channels in the modulation of glutamate release by neurosteroids. Specifically we will test a role of sigma receptors, voltage-gated calcium channels and potassium channels. The proposed studies will increase our understanding of the mechanism of action of these compounds in the brain.

DESCRIPTION (provided by applicant): Neurosteroids are produced in the brain independently of peripheral endocrine glands and act on neurons and glia. They have important roles in mental health-related disorders with great potential for neuropsychopharmacological applications. We have recently demonstrated a novel presynaptic effect of neurosteroids that could help explain their mechanism of action. We found that pregnenolone sulfate (PREGS) enhances facilitated glutamate release in mature neurons and increases tonic glutamate release in immature neurons. We thus propose the following hypothesis: The modulation of glutamate release by neurosteroids is developmentally regulated. In the neonatal brain, neurosteroids enhance maturation of hippocampal circuits while, in the adult brain, neurosteroids modulate signal filtering and short-term plasticity in these same circuits. We will test this hypothesis with two specific aims. (1) To characterize the mechanism of the PREGS-induced enhancement of tonic glutamate release in the neonatal hippocampus and to determine its impact. We have shown that PREGS increases mEPSC frequency in cultured hippocampal neurons and hippocampal slices from neonatal rats. Our goal here is to show that unique characteristics of immature neurons allow the same endogenous neurosteroids to subserve very different functions in neonatal synaptic terminals from those that they will ultimately play in adult neurons. Specifically, we will characterize the effect on PREGS on glutamate release, investigate its effect on silent synapses, and determine its role in modulating synchronous potentials. (2) Investigate the mechanisms by which PREGS enhances short term facilitation of presynaptic glutamate release in the adult hippocampus. We have shown that PREGS causes an enhancement of the bandpass filtering characteristics of the Schaffer to CA1 synapses and also produces a very dramatic frequency-dependent effect on signal transmission through the perforant path to dentate synapses. These effects must contribute significantly to the documented modulation that neurosteroids exert on the processing of spatial/relational information by the hippocampus. Our goal here will be to determine the effect of PREGS on presynaptic Ca2+ dynamics, focus on effects in the CAS field, and determine the functional consequences of PREGS acting at presynaptic glutamatergic terminals in the adult hippocampus. Finally we will confirm that our findings in the in vitro slice also apply to the in vivo hippocampus.