William E. Armstrong - US grants

We propose to utilize a model system, the rat hypothalamo-neurohypophysial explant in vitro, to examine with electrophysiological, immunocytochemical and radioimmunoassay techniques the organization of magnocellular neurosecretory neurons inthe supraoptic nucleus (SON). Our primary goals will be to 1) determine the relationship between electrical activity in SON neurons and vasopressin release; 2) determine the osmosensitivity of SON neurons, its dependence on synaptic input and its relationship to the anteroventral third ventricle; and 3) examine neuronal, ionic and humoral factors influencing phasic, bursting activity, which in vivo is correlated with vasopressin release. Extracellular unit recordings will be made with electrodes filled with horseradish peroxidase (HRP) while changing the composition of the medium and sampling the effluent for correlative changes in vasopressin release. Stimulating electrodes placed on the neurohypophysial stalk will allow the antidromic identification of some of the cells. Media compositions will be varied in the osmolality, drug concentrations and Ca++ and Mg++ proportions to test the role of putative transmitters and synaptic transmission per se in controlling vasopressin cell activity and release. The recording site will be marked with HRP and related tothe distribution of immunoreactive vasopressin and oxytocin neurons. Our long range objective is to understand the mechanisms governing vasopressin release. This hormone is present in all mammals, including man, and dysfunctions in its normal control lead to many clinical syndromes associated with irregularities in sal and water balance, including diabetes insipidus and chronic hypernatremia.

The long-term objective of this research is to understand the neural mechanisms underlying the release of oxytocin during lactation. The experiments in the present proposal are aimed specifically at determining how norepinephrine and excitatory amino acids, such as glutamate, affect the electrophysiological characteristics of identified oxytocin neurons in the lactating rat using intracellular current and voltage clamp recordings in the hypothalamo-neurohypophysial explant or in acutely isolated neurons from the lactating rat. The present proposal combines the characterization of the actions of excitatory amino acids and norepinephrine with the identification of the recorded neurons as oxytocinergic or vasopressinergic using immunocytochemical or immunoblotting techniques. The broad hypothesis of this project is that norepinephrine and excitatory amino acids interact to shape the firing behavior of oxytocin neurons during lactation. The Specific Aims of this project are: 1) to examine the effects of norepinephrine and alpha-1 agonists on the membrane properties of oxytocin neurons in lactating female rats; 2) to characterize the action of excitatory amino acids on oxytocin neurons and in particular to identify whether both AMPA and NMDA receptors are present; and 3) to determine the level at which excitatory amino acids and norepinephrine act synergistically on oxytocin neurons. For each aim the effects on the firing pattern of oxytocin neurons as well as the effects on specific membrane potential characteristics and ion channels will be addressed. In addition, the relevance of any effects to oxytocin release will be tested using transmitter-modulated spike trains to drive the electrical stimulation of hormone release from isolated neural lobes. Oxytocin release is essential to normal lactation in all mammals, yet virtually nothing is known about the specific membrane properties of these neurons during lactation.

DESCRIPTION (applicant's abstract): Neurosecretory neurons have a large capacity for morphological and physiological plasticity as a function of endocrine state during adulthood. Of principal interest in this project is the formation of new synapses in the supraoptic and paraventricular nuclei of the hypothalamo-neurohypophysial system during lactation, when the demand for oxytocin (OT) release is high. This morphological plasticity is accompanied by significant increases in the activity of glutamate-releasing synapses which are selective for OT neurons, These changes include a doubling in miniature excitatory postsynaptic currents and an increase in the probability of glutamate release. A similar up-regulation has been found for GABAergic synapses. Profound changes in gonadal hormone secretion during late pregnancy are thought to program these changes, anticipating the increased activity of OT neurons. Furthermore, local OT release feeds back on both types of synapse through autoreceptors on OT neurons and/or their presynaptic terminals. Central OT release is critical for the morphological plasticity, and for the normal expression of OT neuronal firing during lactation. In this project, the mechanisms and consequence of this increased synaptic activity will be investigated. There are four Specific Aims: Aim 1) A) To test whether glutamate release at AMPA/Kainate receptors on OT neurons is enhanced during lactation by a change in the presynaptic regulation of its own release; B) To determine whether NMDA receptors participate in this plasticity; Aim 2) To test the combined and differential contribution of GABAergic and glutamatergic activity to spike patterning in OT neurons during lactation; Aim 3) To test whether OT's direct and presynaptic effects on OT neurons are state-dependent, and whether OT can alter the firing pattern of OT neurons during lactation; Aim 4) To test the time-dependence of the increase in glutamatergic activity during pregnancy, and whether it is controlled by gonadal steroid hormones. These studies are important for understanding how the central control of OT neurons adapts to the demands of increased hormone secretion during pregnancy and lactation.

Mammalian neurosecretory neurons are excellent models for understandingthe relationship between spike discharge patterns and hormone/transmitter release in the central nervous system. Neurosecretory neurons adopt a bursting pattern that promotes maximally efficient stimulus-secretion coupling at the nerve terminal. The phasic bursting activity in vasopressin neurons is exemplary of this pattern, and relatively unique in ihat it can be studied in vitro, as it results largely from intrinsic membrane properties. The global hypothesis of this proposal is that phasic burstingper se can be explained largely by the understanding of one primary current, its modulation by Ca++, and its autoregulation by dynorphin through Kopiate receptors. The Specific Aims are: Aim 1) Determine the ion species, underlyingconductance change, and Ca++-dependence of the current underlying the DAP (IDAP)- Experimentally, we will test several predictions from a powerful computational model simulating phasic activity developed during the past grant period. We predict that the plateau potential underlying bursts results from a Ca++ dependent inhibition of a K+ leak current, such that this current attains voltage-dependence in elevated [Ca++].. Aim 2) Determine the mechanism of the autoregulatory inhibition of VP neurons via Kopiate receptors. We predict that dynorphin, released locally during a burst from vasopressin neurons, shifts the Ca++ sensitivity of a K+ leak current rightward, raising its threshold, and terminating the burst. Aim 3) Determine the role of autoregulation in the variability of phasic bursting expression in vitro. We predict that phasic bursting is correlated with the ability of dynorphin to regulate the DAP and burst length and complimentarily, that deficits in phasic bursting activity are due to deficits in autoregulation. A corollary is that phasic bursting may be related to the degree of dendritic arbor present. Understanding the origin of phasic bursting is critical to understanding how VP release is controlled in both physiological and pathophysiological conditions. VP release is critical to normal water balance and cardiovascular regulation. PERFORMANCE. SITE(S) (organization, city, state) University of Tennessee Health Science Center Department of Anatomy and Neurobiology 855 Monroe Avenue Memphis, TN 38163 KEY PERSONNEL. See instructions. Use continuation pages as neededto provide the required information in the format shown below. Start with Principal Investigator. List all other key personnel in alphabetical order, last name first. Name Organization Role on Project William E. Armstrong, Ph.D. Universityof Tennessee, Memphis Principal Investigator Ryoichi Teruyama, Ph.D. University of Tennessee, Memphis Co-Investigator Jay Callaway, Ph.D. University of Tennessee, Memphis Co-Investigator Angela Cantrell, Ph.D. Universityof Tennessee, Memphis Co-Investigator Chunyun Li Universityof Tennessee, Memphis Postdoc To be named Universityof Tennessee, Memphis Research Technician Peter Roper, Ph.D. NIH, NIDDK Collaborator/consultant Disclosure Permission Statement. Applicable to SBIR/STTR Only. Seeinstructions. L~] Yes TTNO PHS 398 (Rev. 05/01) Page 2 Number pages consecutively at the bottom throughout Form Page 2 the application. Do not use suffixes such as 2a, 2b. Principal Investigator/Program Director (Last, First, Middle): The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page 1 Description,

DESCRIPTION (provided by applicant): The proposal is to purchase a modern confocal laser confocal scanning microscope (Zeiss LSM 710) with wide spectral scanning capability, high resolution, and high sensitivity for a Neuroscience Imaging Center at the University of Tennessee Health Science Center. The LSM will be mounted on a Zeiss Axio Observer inverted microscope, providing great flexibility in the type of material examined. The microscope stage will have several adaptors such that live neurons may be imaged in a warmed, oxygenated, humidified environment in a variety of incubation wells, or by switching adapters, fixed brain slices may be imaged with specific structures and/or molecules labeled with fluorescent probes. The LSM comes equipped with six laser lines, and three photomultipliers (PMTs) capable of scanning 34 channels, and is much more sensitive than previous LSMs. This sensitivity means that less laser power, which is damaging to live cells and which bleaches the probes used in these studies, is required. These fluorescent probes can span a variety of emitted wavelengths, and up to 10 probes may be simultaneously viewed and characterized. The spectral scanning sensitivity is such that probes with fluorescent emission profiles 10 nm or even less may be separated, graphically marked, and analyzed. Thus, neuroscientists will have great flexibility to determine the precise location, and biochemical nature, of neurons, their extensive processes, and their synaptic connections. The spatial resolution of this LSM (6144 x 6144) combined with Zeiss objectives and confocal optics, means that structures well below the limit of conventional light microscopes (~0.10 5m) may be viewed, measured and counted. Neuroscientists here will be studying a broad array of problems, from how neurons develop appropriate connections, to understanding which ion channels sculpt their electrical behavior necessary for communication. This information is critical to understanding how neurodegenerative or neuropathic diseases such Parkinson's, Huntington's, and dystonia arise and progress - information critical to understanding how we might cure these conditions. Brain proteins abnormally expressed in a variety of genetic conditions, such as those related to some forms of autism, also will be examined with this microscope. Live imaging capability means that drugs may be tested for their efficacy in fighting brain tumor cells, with real-time results. The basic structures of many brain areas, such as those serving olfaction, nervous system control of pituitary hormones, sensory-motor integration, coordination of movement and cognition will be examined, as will normal brain development, and the deleterious effects of ethanol.

DESCRIPTION (provided by applicant): Oxytocin (OT) functions as a hormone in labor and lactation and as a neuromodulator in the brain. OT receptors are present on OT neurons, which they autoregulate during somatodendritic OT release. During pregnancy and lactation, OT release is pulsatile, not continuous, and this pattern is critical to avoid OT receptor desensitization and insure proper hormone function at target smooth muscle in uterus and mammary gland. Neurons in the supraoptic (SON) and paraventricular nuclei (PVN) undergo remarkable plasticity during late pregnancy and lactation, including a glial-neuronal rearrangement and increases in synaptic activity. We have found enhanced spike afterhyperpolarizations (AHPs) during both periods. AHPs are critical in sculpting the short (~4 sec) high frequency (~50 Hz) bursts from OT neurons underlying this pulsatility, bursts that produce a bolus release of OT into the bloodstream to maximally contract uterine smooth muscle or mammary gland myoepithelium. Reproductive-associated plasticity in the SON and PVN depends on the pattern of ovarian steroid release during pregnancy and on the somatodendritic release of OT. We present the first in vivo evidence showing that central OT receptors are critical for the enhancement of the calcium-dependent AHPs normally manifest at late pregnancy. A specific OT antagonist administered chronically to the third ventricle during late pregnancy blocked this form of plasticity with no effect on VP neurons. In addition, we mimicked this plasticity by applying OT to hypothalamic slices from pregnant (E18-19) rats. The goal of this proposal is to understand the mechanisms of this adaptation made by OT neurons, and how it shapes OT neuronal firing to maximize hormone release and thereby insure normal parturition and lactation for infant development. Understanding the mechanisms of central OT receptor transduction at the OT neuron will have important applications for understanding the wider roles of OTRs in brain function. There are Three Specific Aims: Specific Aim 1. To determine if the enhancement of AHPs in OT neurons by OT is specific to OT neurons and OT receptor activation, whether it is specific to pregnant animals, and whether it is due to a change in the Ca2+ sensitivity of the underlying currents. Specific Aim 2. To test whether OT's effects on AHPs are mediated through a pERK 1/2-MAPK cascade, and whether it is associated with an increase expression of the AHP channels and/or the enzymes CK2 and PP2a, known to modulate AHP Ca2+ sensitivity. Specific Aim 3. To determine how enhanced AHPs modulate OT neuronal bursts, and to determine whether the reinsertion of missing excitatory activity differentially alters OT firing pattern in lactating vs. virgin rats, in an OT-dependent manner.