Matthew Ennis - US grants

The long term goal of this research is to elucidate the pathways and mechanisms by which the midbrain periaqueductal gray (PAG) regulates autonomic functions. The proposed studies use multidisciplinary approaches to test specific hypotheses, suggested by out new pilot data and previous studies, of the role of PAG in cardiovascular regulation. Most investigations of PAG have focused on the role of this structure in analgesia, the "defense and escape reactions", and sexual behavior. Our new results suggests that PAG exerts remarkably potent, selective, bidirectional actions on blood pressure. Pressor and depressor responses elicited by electrical or chemical microstimulation of PAG are obtained from two discrete, longitudinally organized columns of neurons along the rostrocaudal axis of PAG. PAG neurons in the vicinity of these pressor- depressor columns heavily and selectively innervate the rostral ventrolateral medulla (RVM), a major sympathoexcitatory region, and nucleus ambiguous (NA), the source of preganglionic parasympathetic neurons that project to the heart. Additional new results demonstrate that PAG receives robust, highly organized inputs from several forebrain areas specifically implicated in cardiovascular regulation. Our findings show that these forebrain areas focally innervate discrete subregions of PAG, forming distinct longitudinal input columns that span the entire rostrocaudal extent of PAG. These longitudinal input columns from forebrain pressor- depressor areas may selectively target corresponding pressor-depressor columns in PAG, including neurons projecting to known cardiovascular regulatory sites in the medulla. Taken together, our new results suggest that PAG pressor=depressor columns selectively target and functionally engage sympathoexcitatory and vagal cardioinhibitory neurons in RVM and NA. We further hypothesize that forebrain pressor-depressor sites focally target longitudinal columns of PAG pressor-depressor neurons that project to these medullary vasomotor sites. These forebrain-PAG-medullary circuits may function to provide coordinated cardiovascular adjustments during emotional reactions. We will test these hypotheses by a set of highly coordinate anatomical and neurophysiological experiments. At the present time, there is only fragmentary information about the circuits and physiological mechanisms mediating PAG's actions on the nervous system. The proposed research, by closing this gap, will advance our understanding of CNS regulation of the autonomic nervous system. In addition, this research may provide new insights into disturbances in cardiovascular tone (e.g. hypertension) associated with stress and emotional disturbances.

DESCRIPTION: The present proposal seeks to answer fundamental questions about mammalian olfactory nerve synapses. Despite advances in our understanding of the circuitry and synaptic physiology in the main olfactory bulb (MOB), many questions about neurotransmission at the first synapse in the olfactory system have not been answered. Primary olfactory neurons in the epithelium terminate in the glomerular layer of MOB where they synapse on the dendrites of mitral cells (MC). Activation of the olfactory nerve (ON) directly activates MCs, the primary relay cell in the olfactory system. However the transmitter(s) and receptors(s) mediating synaptic transmission between the ON and MCs in the mammal are not known. Our new findings in rat MOB slices demonstrate that ON stimulation elicits a rapid, short latency and a delayed excitation of MCs that are differentially blocked by non-NMDA and NMDA receptor antagonists. We propose a set of neurophysiological studies to rigorously rest the hypothesis the glutamate release from ON terminals monosynaptically activates MCs via actions at NMDA and non-NMDA receptors. Our new findings indicate that the NMDA response undergoes long-term potentiation (LTP) after a burst of high frequency ON stimulation in vitro. Rodents have an olfactory recognition memory located at the first relay in this sensory system. Odor memories are acquired with one-trial learning and last for weeks. Our finding of LTP at the ON->MC synapse discloses a mechanism that may underlie the capacity of the olfactory system to form rapid odor-specific memories. A major goal of this proposal, therefore, is to test the hypothesis that high frequency ON activation induces selective LTP of NMDA receptor-mediated responses in vitro and in vivo. The peptide carnosine, and the metal ion zinc, are present in the mammalian ON but have no known physiological function. Our preliminary studies show that zinc selectively attenuates the NMDA receptor-mediated component of MC response to ON simulation. We will test the hypothesis the endogenously released zinc modulates ON-evoked excitation of MCs by inhibiting glutamates actions at the NMDA receptor. Carnosine is co-localized with glutamate in ON terminals. Carnosine is a potent chelator of zinc. We hypothesize that carnosine modulates (i.e.) potentiates the actions of glutamate at the NMDA receptor by binding zinc. We will test this hypothesis. Peptides are preferentially released by high frequency activity. We hypothesize that carnosine release during high frequency ON activity plays a key neuromodulatory role that facilitates the induction of NMDA-receptor-dependent LTP.

DESCRIPTION (adapted from the applicant's abstract): Studies by Blass and colleagues demonstrate profound antinociceptive and calming effects of oral ingestion of sucrose, milk and fat in newborn rats and humans. Sucrose-induced analgesia in rats and humans develops within seconds, lasts for minutes and is prevented by opiate receptor antagonists. Thus, the mechanisms appear to be remarkably well conserved phylogenetically. The neural circuits by which taste stimuli engage central analgesic mechanisms are not known. Taste buds responsive to sweet-tasting stimuli are located mostly on the palate in the rat. They project via the superficial petrosal nerve and synapse in discrete target zones in the rostral, gustatory pathways to the parabrachial (PBC), ventrobasal thalamus, central nucleus of the amygdala (CNA), lateral hypothalamic area (LHA), and insular cortex (IC). Although analgesia can be elicited from a constellation of sites ranging from the frontal lobe to the brainstem, there is consensus that two major nodal points are involved in circuitry that produces opiate-mediated analgesia: the midbrain periaqeductal grey (PAG) and nucleus raphe magnus (NRM). Based on the finding that opiate receptor antagonists prevent sucrose-induced analgesia, we hypothesize that sucrose engages opiate analgesia via activation of PAG or NRM descending output neurons. Recent work in our laboratory suggests several potential sites of interaction between gustatory and central analgesia circuits. Ascending gustatory pathways synapse in PBC and ultimately terminate in IC and CNA. We have shown that PBC, IC, LHA, and CNA send dense projections to PAG. Stimulation of PBC, LHA and CNA elicits analgesia. However, we do not know if gustatory responsive neurons in these areas project to PAG, or if other gustatory nuclei project to PAG or NRM. The goal of this project is to identify the anatomical substrates underlying the antinociceptive effect of oral sucrose. A coordinate set of Fos mapping, tract tracing and lesion studies will be used to pinpoint the sites(s) of linkage between gustatory nuclei and descending pain inhibitory circuits and to verify that these links are necessary for the production of sucrose-induced analgesia. This work will lead to a better understanding of neural circuits controlling taste and pain and how these circuits regulate analgesia in newborns.

This subproject is focused on the organization of glomerular inputs to the piriform cortex and the intracortical circuitry that is related to the glomerular inputs. The studies use an in vitro slice preparation, developed by the investigators, that includes, intact, the OB in connection with the PC. The preparation contains intracortical connections and centrifugal PC to OB connections as well. There are two aims. Aim 1 is to test the hypothesis that mitral/tufted cells whose dendrites coalesce in an individual glomerulus have axons that terminate with topographic specificity in the PC. This will be accomplished with focal glomerular applications of anterograde anatomical tracers and with field potential and voltage sensitive dye techniques. to determine whether circuits within the PC form topographically arranged functional units, and whether the patterns of those units are related to patterns of afferent inputs from the glomeruli. This aim utilizes coordinated tract-tracing and functional (field potential, voltage sensitive dye) approaches. Neuronal recording in the PC will determine how synaptic interactions between glomerular inputs zones are organized in the PC. Intracellular staining will map axon collaterals from recorded cortical cells in relation to glomerular inputs zones optically imaged in the same experiment. The overall goal is the elucidation of the circuit diagram involving PC pyramidal cells, glomerular input zones, and intracortical pyramidal axon connections. Physical resources, space and equipment are all adequate for the performance of this subproject.

DESCRIPTION (provided by applicant): Glutamate plays a dominant role in neurotransmission at number of synapses in the mammalian main olfactory bulb (MOB). Work from our laboratory as well as by others over the last 5 years has demonstrated that (1) sensory transmission from olfactory nerve (ON) terminals to mitral/tufted cells (M/TCs) and juxtaglomerular cells, and (2) transmission from MiTCs to granule cells (GCs), is mediated by glutamate acting at AMPA and NMDA receptors. Additionally, glutamate released by M!TCs can activate AMPA and NMDA receptors on other MrrCs, providing a source of autoexcitation that enhances responses to ON input. Receptor localization studies suggest that another class of glutamate receptors, metabotropic glutamate receptors (mGluRs), are densely expressed at several glutamatergic synapses in the MOB. In paiticular, the density of mGluRs on MTFCs and GCs are higher than most other regions of the brain. However, the role(s) of mGluRs in synaptic processing in the MOB are unknown. The goal of the present proposal is to close this gap. Preliminary data show that the operation of the MOB network, and the excitability of MCs and GCs, are potently modulated by activation and inactivation of mGluRs. Direct activation of mGluRs on MCs increases their excitability, and in turn, increases their excitatory drive on GCs. Activation of mGluRs also directly increases GC excitability and GABA release. Inactivation of mGluRs, by contrast, potently attenuates MC - GC excitatory transmission. Based on these results and other preliminary data, we hypothesize that activation of mGluRs by synaptically-released glutamate positively modulates lateral inhibition and increases contrast in the MOB network. In agreement with this hypothesis, preliminary studies using optical imaging of voltage-sensitive dyes show that mGluR antagonists dramatically decrease the amplitude, spatial spread and duration of postsynaptic activity in the external plexiform layer evoked by focal glomerular stimulation. This suggests that endogenous activation of mGluRs amplifies lateral inhibition. This hypothesis will be tested at the cellular and circuit levels using patch clamp electrophysiology and functional imaging approaches in mammalian MOB slices from rats, as well as slices from mice with targeted gene deletions of the major mGluRs expressed by MCs and GCs. The overarching goal of this proposal is to elucidate the roles of mGluRs in the operation of the MOB network and odor coding.

Metabotropic glutamate receptors (mGluRs) are densely expressed by the major cell types of the main olfactory bulb (MOB), yet their functional role(s) in olfactory processing remain poorly understood. During the previous funding cycle, studies from our lab demonstrated that activation of mGluRs directly enhances the excitability of mitral cell and granule cells. New preliminary data suggest that specific mGluR subtypes (mGluRI and mGluRS) are differentially expressed by morphologically discrete subpopulations of GABAergic granule cells. Based on these and other new findings, we hypothesize that activation of mGluRI and mGluRS differentially modulates short vs. long-range lateral inhibition in the MOB via the mitral cell-to granule cell dendrodendritic circuit. This hypothesis will be tested using patch clamp electrophysiology in mice MOB slices. The role of mGluRs in intraglomerular processing is unknown. New preliminary studies demonstrate that activation and inactivation mGluRs potently modifies the spontaneous and sensory evoked excitability of external tufted (ET) cells in the glomerulus. Activation of mGluRs also enhances electrotonic coupling between ET cells and leads to the emergence of slow rhythmic activity in the glomerular layer. Based on these and other new findings, we hypothesize that activation of mGluRs on ET cells enhances synchrony in the glomerular network and leads to the emergence of slow rhythmic oscillations in mitral cells that are linked to respiratory-driven sensory input to the MOB. Additional findings demonstrate that mGluRs also enhance intraglomerular GABAergic inhibition of ET cells. These hypotheses will be tested at the cellular and circuit level using patch clamp electrophysiology in MOB slices from wildtype mice, as well as slices from mice with targeted gene deletions of the major mGluR expressed by MOB neurons. The overarching goal of this proposal is to elucidate the roles of mGluRs in the operation of the MOB network and odor coding. It is hoiped that a better understanding of the normal operation of the olfactory system will aid in the treatment or prevention of olfactory impairments that occur with aging and in certain neurodegenerative disorders.