Mirjana Randic - US grants

Available evidence provides a support for the idea that substance P, gramma-amino-butyric acid and somatostatin are involved in synaptic transmission processes in the superficial layers of the spinal dorsal horn. However, the identity of the neural elements responsive to these agents, and the ionic mechanisms underlying atheir synaptic actions are not as yet clear. The intent of this proposal is to identify the elements these putative transmitters act upon, determine their membrane effects and try to explain the mechanisms of their actions. Conventional approach will be to use cat spinal cord in situ and apply GABA, somatostatin and substance P iontophoretically or by micropressure injections into the laminae I-III of the dorsal horn, while performing extra - and intracellular recordings from the large diameter primary afferents or dorsal root ganglion cells. Ionic mechanisms of presynaptic and postsynaptic actions of GABA, somatostatin and substance P will be investigated in virtro by using the rat spinal cord slice preparation. Morphological identification of physiologically and pharmacologically characterized dorsal horn interneurons and primary afferent neurons will be done by intracellular labeling with horseradish peroxidase.

The primary aim of this proposal is to study ionic and molecular mechanisms of action of several neuropeptides and the functional role of putative second messenger systems in peptide actions, and in transmission and regulation of sensory information at afferent synapses in the dorsal horn of the immature rat spinal cord. Substance P (SP), neurokinin A (NKA) and calcitonin gene-related peptide (CGRP), have been found to excite dorsal horn neurons, although the ionic and molecular mechanisms by which the peptide signals produce cellular responses have not been completely understood. In addition, the cellular mechanisms of the slow excitatory synaptic potentials have not been adequately tested. Therefore, we propose to utilize intracellular recording methodology in the rat dorsal horn slice preparation, and the isolated dorsal horn neurons, to learn more about possible ionic and molecular mechanisms underlying the peptide actions and the fast and slow excitatory transmission in the dorsal horn of the spinal cord. The following topics will be investigated: 1. Voltage-clamp analysis of the slow excitatory transmission in the rat spinal dorsal horn slice preparation: possible mediation by tachykinins (SP, NKA). 2. Possible molecular mechanisms underlying the SP, NKA and CGRP actions and the fast and slow excitatory synaptic transmission in the rat spinal dorsal horn. The three second messenger systems that may be linked to peptide receptors and which will be examined for their role in the control of neuronal excitability in the rat spinal dorsal horn, are as follows: 1) The adenylate cyclase and cyclic AMP-dependent protein kinase system, 2) The guanylate cyclase and cyclic GMP-dependent protein kinase system, and 3) The inositol triphosphate/diacylglycerol-protein kinase C system. 3. Mechanisms of modulation of calcium conductances by neuropeptides (SP, NKA, and CGRP) in acutely isolated rat dorsal horn neurons using patch-clamp technique.

Intercellular communication mediated by bioactive substances occurs in virtually all multicellular systems. Chemical neurotransmission in the vertebrate nervous system represents a form of this type of signalling. Fast chemical signalling (within milliseconds), in which the neurotransmitter is released at specialized neuronal junctions, called synapses, stimulates the opening of receptor-controlled ion channels in the post- synaptic cell. There is evidence that the amino acids, glutamate and aspartate, may represent the principal fast transmitters used by many central neurons. However, the actions of some chemical mediators, for instance neuropeptides, are slow (acting over seconds or even minutes) and they are functionally important as modulators of synaptic transmission. Since the chemical nature of excitatory synaptic transmitters in the mammalian spinal cord is still unknown, this research project will analyze cellular mechanisms underlying the rat spinal dorsal horn in vitro. The experiments will be directed at the role of excitatory amino acid synaptic mediators (l-glutamate and its analogues), receptors for excitatory amino acids, the role of tachykinins (substance P and neurokinin A), and the activation of intracellular second messenger systems (phoshoinositides) by excitatory amino acids and the tachykinins. Specific objectives to be examined are: 1) the pharmacological properties and ionic mechanisms of fast and slow excitatory postsynaptic potentials in the rate spinal cord slice preparation. The emphasis will be on NMDA receptors, since information about their role in primary afferent transmission is lacking; 2) the possibility of the involvement of second messenger systems (phosphoinositides and cyclic nucleotides) in the regulation of actions of excitatory amino acids and tachykinins at afferent synapses in the rat spinal dorsal horn; and 3) modulation of voltage-dependent calcium conductance of acutely-isolated rat dorsal horn neurons by excitatory amino acids, tachykinins and second messengers using whole-cell voltage clamp technique with a patch microelectrode. These studies will provide for a better understanding of synaptic signalling via chemical mediators within the nervous system.

The perception of pain requires excitatory synaptic transmission from primary afferent sensory fibers to secondary projection neurons in the dorsal horn of the spinal cord. Two groups of neurotransmitter candidates are thought to mediate this initial excitatory step in the pain pathway: one being excitatory amino acids (EAA: glutamate, asparate), and another are peptides (including tachykinins such as substance P and neurokinin A). These peptides may be colocalized with an excitatory amino acid(s) in the same neurons. Chemical signal transfer via such neurons presents new aspects and complexities of presynaptic (synaptic efficacy) and postsynaptic (membrane excitability) regulation which have not previously been considered and may have important implications for the performance of the somatosensory, especially of pain pathways. Dr. Randic has recently found that substance P and excitatory amino acids interact to produce neurophysiological signs of hyperalgesia i.e. a prolonged enhancement of responses to excitatory manipulation. However, the sites and the molecular mechanisms by which the peptide signals produce enhanced EAA responses have yet to be elucidated. Dr. Randic will examine the hypothesis that the activation of distinct neurokinin receptors causes modulation of the responses of freshly isolated spinal dorsal horn neurons to EAAs, especially to N-methyl-D-aspartic- acid (NMDA). To gain understanding of molecular mechanism(s) underlying interactions between EAAs, and tachykinins possible involvement of glycine allosteric site of NMDA receptor channel complex, guanine nucleotide-binding proteins (G-proteins) or change in Ca++-sensitive second messenger systems, will be investigated. Whole-cell voltage-clamp recording of EAA responses will be utilized in this study. Delineating the cellular mechanism(s) of peptide actions on dorsal horn neurons is an important step toward understanding anatomical and neurochemical organization of the spinal dorsal horn.***//

Primary afferent terminals in the dorsal horn (DH) of the spinal cord release besides glutamate (GLU) and aspartate (ASP) also peptides that may be involved in the slow excitatory synaptic transmission and the central processing of pain information. However, the sites and the molecular mechanisms by which the peptide signals produce cellular responses have yet to be elucidated. The co-existence of tachykinins and GLU, has been reported in small primary sensory neurons. Chemical signal transfer via such neurons presents new aspects and complexities of presynaptic (synaptic efficacy) and postsynaptic (membrane excitability) regulation which have not previously been considered and may have important implications for the performance of the somatosensory system. This proposal focuses on the functional postsynaptic aspects of the co-release of tachykinins and excitatory amino acids (EAAs) in the rat spinal DH and especially on the analysis of their direct excitatory interactions and underlying cellular mechanisms. We propose to examine the hypothesis that modulation of the responses of rat spinal DH neurons to EAA (glutamate, aspartate, N-methyl- D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid, kainate and quisqualate) by tachykinins (e.g. substance P, neurokinin A) and opioid peptides (acting at mu, delta and kappa receptors) occurs and might involve the distinct subtypes of glutamate and the peptide receptors. Possible involvement of G proteins, changes in the concentration of intracellular Ca2+ and second messenger mechanisms in the regulation of the chemosensitivity of DH neurons to NMDA and tachykinins will also be investigated. Whole-cell voltage-clamp and nystatin- perforated patch-clamp techniques and monitoring of the levels of intracellular free Ca2+ concentration using fura-2 based microfluorimetry in freshly isolated DH neurons will be used to obtain information about the molecular mechanisms underlying the interactions between NMDA and tachykinins. Delineating the cellular mechanisms of tachykinin actions on DH neurons is an important step toward understanding anatomical and neurochemical organization of the spinal DH. Such knowledge may provide the possibility of selective pharmacological manipulation of sensory perception mechanisms with important therapeutic implications, especially for pain.

Primary afferent terminals in the dorsal horn (DH) of the spinal cord release besides glutamate (GLU) and aspartate (ASP) also peptides that may be involved in the slow excitatory synaptic transmission and the central processing of pain information. However, the sites and the molecular mechanisms by which the peptide signals produce cellular responses have yet to be elucidated. The co-existence of tachykinins and GLU, has been reported in small primary sensory neurons. Chemical signal transfer via such neurons presents new aspects and complexities of presynaptic (synaptic efficacy) and postsynaptic (membrane excitability) regulation which have not previously been considered and may have important implications for the performance of the somatosensory system. This proposal focuses on the functional postsynaptic aspects of the co-release of tachykinins and excitatory amino acids (EAAs) in the rat spinal DH and especially on the analysis of their direct excitatory interactions and underlying cellular mechanisms. We propose to examine the hypothesis that modulation of the responses of rat spinal DH neurons to EAA (glutamate, aspartate, N-methyl- D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid, kainate and quisqualate) by tachykinins (e.g. substance P, neurokinin A) and opioid peptides (acting at mu, delta and kappa receptors) occurs and might involve the distinct subtypes of glutamate and the peptide receptors. Possible involvement of G proteins, changes in the concentration of intracellular Ca2+ and second messenger mechanisms in the regulation of the chemosensitivity of DH neurons to NMDA and tachykinins will also be investigated. Whole-cell voltage-clamp and nystatin- perforated patch-clamp techniques and monitoring of the levels of intracellular free Ca2+ concentration using fura-2 based microfluorimetry in freshly isolated DH neurons will be used to obtain information about the molecular mechanisms underlying the interactions between NMDA and tachykinins. Delineating the cellular mechanisms of tachykinin actions on DH neurons is an important step toward understanding anatomical and neurochemical organization of the spinal DH. Such knowledge may provide the possibility of selective pharmacological manipulation of sensory perception mechanisms with important therapeutic implications, especially for pain.

INVOLVEMENT OF GLUTAMATE METABOTROPIC RECEPTORS IN SYNAPTIC PLASTICITY IN THE SPINAL CORD

It is now well established that the primary afferent fibers use glutamate as a principal fast excitatory transmitter in the superficial dorsal horn (SDH) of the spinal cord, the first modulatory site in the relay of sensory information from the peripheral receptors to the brain. Glutamate acts through two broad classes of receptors, ion-channel-linked (ionotropic) receptors (AMPA/kainate and NMDA receptors) and metabotropic receptors (mGluRs), which couple via G-proteins to the intracellular second messenger cascades. Group I mGluRs (mGluR1 and 5 subtypes) are expressed by neurons in the SDH, but their roles in synaptic function, and contribution to spinal somatosensory transmission and synaptic plasticity, have yet to be elucidated. The recent development of ligands that bind specifically to these receptors, and availability of mutant mice lacking mGluR1 or mGluR5 receptors, has provided means of characterizing the important roles they may play in tuning of fast and slow excitatory synaptic transmission, including the induction of long-term changes in synaptic efficacy, as suggested by our recent preliminary evidence.
Two major objectives in the proposal are: 1) to study involvement of Group I mGluRs via ICAN (non-selective Ca2+-dependent cation channels) in the generation of primary afferent-evoked slow excitatory synaptic potential (sEPSP) in the SDH region; and 2) to study regulation of AMPA and NMDA receptor-mediated sensory transmission by activation of Group I mGluRs. A combination of intracellular and whole-cell patch-clamp recordings, Ca2+ imaging, and pharmacological techniques will be employed to study the involvement of Group I mGluR activation in synaptic transmission and plasticity in SDH neurons within spinal cord slices prepared from young rats and mutant mice lacking mGlu1 or 5 receptors.
Delineating the physiological roles of the Group I mGluRs in the SDH region, and cellular and molecular mechanisms underlying their actions, is an important step toward understanding of implications of glutamate-mediated transmission in the spinal cord DH synaptic function, but in particular for synaptic plasticity and nociception.