Amy B. MacDermott - US grants

The primary goal of these studies is to understand the regulation of intracellular calcium in mammalian central neurons following activation by excitatory amino acids, especially through the NMDA receptor. Elevation of calcium via glutamate receptor activation has been implicated in regulation of long term potentiation in mammalian hippocampal pyramidal neurons, generation of rythmic pacemaker-like activity which may control pattern generation in efferent motor systems, as well as the excitotoxic action of some excitatory amino acid agonists. The three sources of calcium that may contribute to the elevation of intracellular calcium following activation are through voltage- dependent calcium channels, glutamate-gated channels, or glutamate activation of intracellular release of calcium. By combining the use of electrophysiological techniques and optical recording of intracellular calcium through the use of calcium- sensitive dyes, it will be possible to determine the conditions under which each source contributes optimally to elevation of intracellular calcium. It will also be possible to measure and manipulate the kinetics of recovery from agonist-induced calcium loads in order to help identify the mechanisms which contribute to calcium clearance. Finally, the physiological consequences of glutamate-induced elevation of intracellular calcium will also be studied including possible activation of calcium-dependent potassium, chloride, and other cation channels. The experiments in this proposal will provide new, direct information on a previously incompletely described component of the response to glutamate; elevation of intracellular calcium.

The dorsal horn of the spinal cord is an important site of afferent sensory transmission to central neurons. Interactions between the synaptic receptors and voltage-gated calcium channels (VGCCs), and their combined effects on Ca2+ entry, have not been well studied in dorsal horn neurons or elsewhere in the central nervous system. Yet the physiological effects of [Ca2+]i and calcium-driven changes in membrane potentials could have profound influence on the sensation of peripheral stimuli in both normal and pathological states. Fast synaptic transmission between primary afferents and their dorsal horn targets primarily involves an excitatory transmitter, glutamate, acting on one or more of a family of synaptic excitatory amino acid (EAA) receptors known as the NMDA, and non-NMDA or kainate and quisqualate receptors. EAAs will depolarize the cells by activation of these receptors and indirectly activate VGCCs causing [Ca2+]i to elevate and membrane potential to become more depolarized. This form of interaction implies synaptic activation of Ca2+-dependent potentials with highly nonlinear boosting of both the change in membrane potential and [Ca2+]i. Recent experiments have also suggested that kainate-activated receptor operated channels (ROCs) permeable to Ca2+ exist. If these are in dorsal horn neurons, it could completely change the localization, kinetics and voltage dependence of Ca2+ entry during synaptic activation. Finally, the possibility of a non-NMDA receptor that is metabolically coupled having a role in synaptic transmission in dorsal horn neurons is untested. Therefore, the regulation of Ca2+ entry will be studied following non-NMDA receptor activation to determine 1) if Ca2+ entry through the non-NMDA receptors occurs, 2) the contribution of VGCC properties to determining the amplitude and time course of the evoked [Ca2+]i transient and 3) the role of Ca2+ release in non-NMDA receptor mediated changes in [Ca2+]i. Simultaneous measurement of [Ca2+]i and membrane potential changes should be particularly informative about whether Ca2+ entry is due to Ca2+- dependent action potentials or sustained opening of VGCCs. Dendritic receptor and channel properties will be studied using low light level video imaging for good spatial resolution and photomultiplier tubes for good time resolution. Such studies will improve our understanding of the complex interactions that normally occur among synaptic inputs and VGCCs in the dorsal horn. Furthermore changes in [Ca2+]i have been implicated in processes of activity dependent facilitation of synaptic transmission such as might underlie pathological pain syndromes like allodynia. Finally, since sustained EAA-mediated elevation of [Ca2+]i in spinal cord neurons is associated with secondary cell death following traumatic spinal cord injury, these experiments may improve the basic understanding of the mechanisms associated with cell death.

The dorsal horn of the spinal cord is an important site of afferent sensory transmission to central neurons. Interactions between the synaptic receptors and voltage-gated calcium channels (VGCCs), and their combined effects on Ca2+ entry, have not been well studied in dorsal horn neurons or elsewhere in the central nervous system. Yet the physiological effects of [Ca2+]i and calcium-driven changes in membrane potentials could have profound influence on the sensation of peripheral stimuli in both normal and pathological states. Fast synaptic transmission between primary afferents and their dorsal horn targets primarily involves an excitatory transmitter, glutamate, acting on one or more of a family of synaptic excitatory amino acid (EAA) receptors known as the NMDA, and non-NMDA or kainate and quisqualate receptors. EAAs will depolarize the cells by activation of these receptors and indirectly activate VGCCs causing [Ca2+]i to elevate and membrane potential to become more depolarized. This form of interaction implies synaptic activation of Ca2+-dependent potentials with highly nonlinear boosting of both the change in membrane potential and [Ca2+]i. Recent experiments have also suggested that kainate-activated receptor operated channels (ROCs) permeable to Ca2+ exist. If these are in dorsal horn neurons, it could completely change the localization, kinetics and voltage dependence of Ca2+ entry during synaptic activation. Finally, the possibility of a non-NMDA receptor that is metabolically coupled having a role in synaptic transmission in dorsal horn neurons is untested. Therefore, the regulation of Ca2+ entry will be studied following non-NMDA receptor activation to determine 1) if Ca2+ entry through the non-NMDA receptors occurs, 2) the contribution of VGCC properties to determining the amplitude and time course of the evoked [Ca2+]i transient and 3) the role of Ca2+ release in non-NMDA receptor mediated changes in [Ca2+]i. Simultaneous measurement of [Ca2+]i and membrane potential changes should be particularly informative about whether Ca2+ entry is due to Ca2+- dependent action potentials or sustained opening of VGCCs. Dendritic receptor and channel properties will be studied using low light level video imaging for good spatial resolution and photomultiplier tubes for good time resolution. Such studies will improve our understanding of the complex interactions that normally occur among synaptic inputs and VGCCs in the dorsal horn. Furthermore changes in [Ca2+]i have been implicated in processes of activity dependent facilitation of synaptic transmission such as might underlie pathological pain syndromes like allodynia. Finally, since sustained EAA-mediated elevation of [Ca2+]i in spinal cord neurons is associated with secondary cell death following traumatic spinal cord injury, these experiments may improve the basic understanding of the mechanisms associated with cell death.

Macdermott 9514720 The functional contacts between nerve cells are called synapses, and the development of the nervous system involves elaboration and specialization of those synaptic inputs. A fundamental aspect of development of the mammalian spinal cord is the representation of sensory input. While the sensory nerves have grown into the spinal cord by birth and already are synapsing with some of their target neurons, the quality of these sensory signals is immature. For example, the response to stimuli may be prolonged in time, and the area of the body, called the "receptive field", from which a central neuron receives input may be considerably broader than in the adult animal. This project uses pharmacology and physiology to study the role of a particular molecule, the NMDA- receptor that acts at the synapse to bind the NMDA transmitter molecule. This component of the nerve cell surface is proposed to have two different tasks during early postnatal development of the spinal cord. One is the processing of sensory pain signals in the spinal cord, and the other is the active fine tuning of synaptic connections between neurons involved in sensory transmission.Results from this work will not only help understand the maturation of sensory pain perception, but will have a broader impact on neurophysiology and developmental biology. The results will clarify the role of an important receptor molecule in development, and will help understand the developmental mechanisms by which sensory stimuli are represented in the central nervous system.

AMPA receptors mediate pain transmission and have been implicated as critical components in initiating some forms of central hyperalgesia and allodynia. Ca2+ is a second messenger often involved in plasticity at central synapses and some of the AMPA receptors are directly permeable to Ca2+. However the role of these Ca2+ permeable receptors in synaptic plasticity, even their role in synaptic transmission in the spinal cord (but see Preliminary studies), is unknown. If Ca2+ entry through synaptic AMPA receptors is responsible for regulating synaptic strength in the dorsal horn pain pathway, then pharmacological intervention with those receptors would provide an entirely new way to control conditions leading to pathological pain states. Joro spider toxin (JSTX), a polyamine purified from the spider Nephila clavata, selectively blocks Ca2+ permeable AMPA receptors without blocking Ca2+ impermeable AMPA receptors, providing a good example of such selective drug action. Furthermore, due to the unique properties of the Ca2+ -permeable AMPA receptors compared to NMDA receptors, synaptically evoked Ca2+ entry through the AMPA receptors should be enhanced by co-incident inhibitory synaptic activation (rather than inhibited as is the case for the NMDA receptors) making the Ca2+- permeable AMPA receptors a novel type of co-incidence detector. Using synthetic JSTX-3, we have evidence establishing that the Ca2+- permeable AMPA receptors are expressed postsynaptically on dorsal horn neurons. We now intend to clarify the role of Ca2+-permeable AMPA receptors in the central transmission and modulation of pain information. We plan to determine the role of Ca2+-permeable AMPA receptors in the modification of neighboring synaptic receptors in a manner dependent on Ca2+ entry through the AMPA channels themselves. We have already demonstrated that Ca2+ entry through the Ca2+-permeable AMPA receptors causes desensitization of the NMDA receptors (Kyrozis et al, 1995) and now we will determine if this happens during synaptic transmission. We will measure the Ca2+ accumulation following entry through Ca2+-permeable AMPA receptors and determine its dependence on membrane potential. We will contrast these observations with those made on synaptically gated Ca2+ accumulation through the NMDA receptors in the presence of Mg2+ as a way of determining if Ca2+-permeable AMPA receptors could function as co- incidence detectors for inhibitory input. Finally, we will identify which subpopulation of neurons in laminae I and II of the dorsal horn pain pathway most prominently express Ca2+-permeable AMPA receptors at synaptic sites which will give us insight into the major role these receptors play in pain transmission.

DESCRIPTION: Glutamate and substance P are released by primary afferent fibers of the dorsal root ganglion (DRG) nerve terminals to transmit nociceptive signals to the central nervous system and to modulate pain signaling. Activation of presynaptic receptors on the DRG nerve terminal has been postulated to account for one critical level of control of synaptic coupling between the afferent DRG neuron and its target in the dorsal horn. The best known of these presynaptic regulatory receptors is of the metabotropic type. Activation of opiate and GABAB receptors, for example, has been suggested to inhibit release of substance P and glutamate from the DRG nerve terminal. However, ionotropic receptors are just beginning to be appreciated as important and powerful regulators of transmitter release as well. Recent evidence shows that ATP P2X receptors potently influence glutamate release from the DRG nerve terminal. The glutamate release is detected as large, synchronous release of multiple quanta of glutamate driven by regenerative action potential activity or as an increase in the frequency of mEPSC activity or quantal glutamate release. These studies suggest that elevation of ATP near the DRG nerve terminals, by a mechanism including Ca2+ dependent ATP release or release following spinal cord jury, will enhance signal output by increasing glutamate release during synaptic transmission. It may also evoke spurious signaling causing pain signals that are actually initiated in the spinal cord itself. The investigator plan's to address two main hypotheses that follow from these initial observations using electro-physiological and Ca2 imagining techniques to study synaptic transmission between DRG and dorsal horn neurons in co-culture and in spinal cord slices. The first hypothesis to be tested is that the presynaptic ATP P2X receptors expressed on DRG nerve terminals represent an important mechanism for regulating the release of the transmitters glutamate and substance P and that P2X receptor desensitization is a major determinant of the activity dependence of the P2X receptor presynaptic regulation. Activation of presynaptic ATP P2X receptors on DRG nerve terminals evokes a large synchronous release of multiple quanta of glutamate when action potential activity is allowed. ATP induces release of single quanta of glutamate when all action potential activity is suppressed. The second hypothesis we will test is whether ATP is able to depolarize the DRG nerve terminals by activation of Ca2+ permeable P2X receptors, that the depolarization initiates action potential activity at the DRG nerve terminal, and that these action potentials require the presence of TTX-resistant Na+ channels. The result of the studies in this proposal will be to provide insight on the role of presynaptic P2X receptors in sensory information processing as well as elucidate important pharmacological targets for the development of new pain therapies.

DESCRIPTION (adapted from applicant's abstract): Cutaneous nociceptors are a heterogeneous group of peripheral sensory neurons that sense noxious, damage-causing stimuli in the periphery and transmit that signal to the central nervous system. They extend their processes peripherally and centrally from cell bodies located in the dorsal root ganglia (DRG). The primary site of termination of the central projection of these unmyelinated and lightly myelinated fibers is predominantly in the superficial laminae (I and II) of the spinal cord dorsal horn. There are multiple subpopulations of nociceptors with cells bodies in the dorsal root ganglion (DRG). They can be grouped by sensory modality, by intracellular markers such as peptides, or by surface markers such as those developed and characterized by Dodd and Jessell (1985). We will use immunocytochemistry to identify DRG nociceptor subpopulations. We will test individual populations of nociceptors for expression of several nociceptive properties including low and high threshold heat and capsaicin sensitivity, using Ca2+ imaging and electrophysiology to measure responses. Kainate and AMPA receptors are expressed on subpopulations of DRG neurons, including functionally identified nociceptors, although the subpopulations of nociceptors have not yet been identified. Kainate and AMPA receptors expressed by nociceptors have been suggested to contribute to detection of damaging stimuli in the skin (Carlton and Coggeshall, 1995; Jackson and Hargreaves, 1995). There is immunocytochemical evidence for peripheral kainate and AMPA receptor expression (Coggeshall and Carlton, 1998) that could underlie such a transduction mechanism. There is physiological evidence that DRG neurites central to the ganglion itself respond to kainate, suggesting kainate or AMPA receptors may have a function at the central terminals of nociceptors, as well (Agrawal and Evans, 1986). Using the spinal cord slice preparation with attached dorsal root and co-cultures of identified nociceptors and dorsal horn neurons, we will investigate where AMPA and kainate receptors are expressed central to the DRG and specifically whether they are expressed on the nociceptor nerve terminals. We will determine the impact of receptor activation on glutamate release from the primary afferent terminals.

DESCRIPTION: (Applicant's Abstract) Cutaneous nociceptive signaling is carried into the CNS by high threshold primary afferent fibers, or nociceptors, that form glutamatergic/peptidergic synapses with neurons in the superficial dorsal horn. Glutamate released from these terminals acts on multiple receptor types including the ionotropic N-methyl-D-aspartate (NMDA) and non-NMDA receptors. The two main types of Ca2+ permeable glutamate receptors expressed by dorsal horn neurons, the NMDA and Ca2+ permeable AMPA receptors, have different voltage dependent properties making Ca2+ entry through these receptors have different activity dependence. Both of these receptors have been implicated in forms of central sensitization association with hyperalgesia and allodynia. In addition to glutamatergic synaptic transmission, GABA and glycine are also importantly involved in regulating nociception (Willis and Coggeshall, 1991; Dickenson et al, 1997). It is not known whether the critical importance of non-NMDA receptors in the regulation of nociception is due to their action to enhance inhibition by mediating excitation of GABA/glycinergic neurons at postsynaptic sites or even at the nerve terminals of these inhibitory neurons. Understanding the role of Ca2+ permeable glutamate receptors in activation of projection neurons and modulating excitation of inhibitory neurons is key to understanding regulation of information flow through the superficial dorsal horn. We plan to use postnatal rat spinal cord slices to study the role of pre and postsynaptic AMPA receptors in regulating synaptic transmission in the spinal cord dorsal horn. Using electrophysiology, Ca2+ and general fluorescent imaging, and immunocytochemistry, we will clarify the location and function of the Ca2+ permeable non-NMDA receptors in the dorsal horn. We will use co-cultures of nociceptors and dorsal horn neurons grown on microislands to determine the rules for sorting of Ca2+ permeable AMPA receptors and NMDA receptors at individual synaptic sites and how that impacts on the activity dependence of each synapse studied. In addition to developing our understanding of regulation of nociceptive information transmission in the dorsal horn, these studies may also contribute to the development of new drugs for alleviating pain that are targeted to the unexplored cell specific AMPA receptors we propose to study.

[unreadable] DESCRIPTION (provided by applicant): In the spinal cord dorsal horn, lamina I and II are the primary sites of synaptic innervation by high threshold pain and temperature sensory afferent fibers whereas low threshold (touch) afferent fibers generally terminate in deeper lamina. The rostrally rising projection neurons that carry pain and temperature signals to higher brain centers arise primarily in lamina I, III and V. Because more than 80% of the lamina I and III projection neurons express receptor for substance P, called the NK1 receptor (NK1R), it has been possible to selectively ablate them and thus demonstrate their key role in expression of hyperalgesia and allodynia (Mantyh et al, 1997; Nichols et al, 1999; Mantyh and Hunt, 2004). In this grant, we propose to investigate 3 aspects of regulation of synaptic transmission onto these critical lamina I neurons expressing the NK1R. To do this, we take advantage of our ability to label NK1R expressing (NK1R+) neurons in live rat spinal cord slices using fluorescently labeled substance P. Because acute intrathecal administration of strychnine and bicuculline produces tactile allodynia (Yaksh, 1989), we hypothesize that in the absence of local inhibition, low threshold input will become prominent while high threshold input onto NK1R+ neurons will be enhanced. To test this hypothesis, we will characterize which primary afferent fibers, defined by threshold and conduction velocity, synapse onto the NK1R+ neurons in lamina I. Then we will assess alterations in afferent input to NK1R+ neurons in the absence of inhibition. If this disinhibition reveals enhanced excitatory drive onto NK1R+ lamina I neurons, we will determine whether the NMDA receptors play a critical role in mediating this activity. To investigate the synaptic basis for such control, we will estimate the power with which NMDA receptors control excitatory synaptic activity onto lamina I NK1R+ neurons. Taking advantage of a transgenic mouse with EGFP expressing GABAergic neurons, we will identify the inhibitory neurons that directly regulate synaptic excitation of NK1R+ neurons in lamina I. Furthermore, we will test the hypotheses that fast synaptic inhibition of NK1R+ neurons is predominantly glycinergic and that inhibitory transmitter release onto NK1R+ neurons is regulated through ionotropic glutamate receptor activation. Identifying and characterizing the excitatory and inhibitory synapses impinging on the NK1R+ lamina I neurons is an undertaking that goes to the heart of understanding the synaptic drive contributing to allodynia and hyperalgesia and therefore is key to development of targeted drugs for chronic pain. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The peripheral sensory nerves specialized for detection of noxious stimuli, temperature and itch have their peripheral terminals in the skin, muscle, bone and viscera, their cell bodies in the dorsal root ganglion (DRG), and their central terminals in the spinal dorsal horn. The stimuli are carried by AD and C-fibers of small diameter sensory neurons, many of which express the noxious heat-sensitive receptor, TRPV1, a Ca2+ permeable cation channel. TRPV1 in the peripheral sensory terminals of DRG neurons serves as a major transducer of multiple noxious stimuli, including noxious thermal stimuli above 43oC. Despite the well- established presence of TRPV1 on the central synaptic terminals of DRG neurons in the dorsal horn, its physiological relevance there has not been clearly established. Recent findings imply that under certain conditions, TRPV1 can be expected to become active at core body temperature (~37oC), well below TRPVI's normal 43oC temperature threshold for activation. The first condition involves the interaction between temperature and voltage. At resting membrane potentials, TRPV1 shows measurable, tonic activation at 37oC. Subsequent depolarization leads to substantial increases in TRPV1 activation (Voets et al, 2004). However, most investigations of TRPV1 function in central terminals have been carried out at room temperature rather than body temperature. Second, activation of PLC-coupled receptors leads to a dramatic reduction in TRPVI's temperature threshold of activation leading to its activation even at room temperature (Chuang et al, 2001). We propose to test the hypothesis that at physiological or body temperatures, and under the conditions of either receptor mediated PLC activation or physiological membrane depolarization mediated by action potentials and ionotropic receptor transmitters, TRPV1 activation in central sensory terminals is substantial and that this activation will lead to modulation of synaptic transmission at DRG-spinal cord synapses. These studies focus on a critical part of the pain pathway; the intersection of the peripheral sensory fiber with the central nervous systems and they will provide insight into new regulatory mechanisms in that pathway. [unreadable] [unreadable]

Tactile allodynia refers to a painful sensation induced by a simple touch. It is a serious and often debilitating medical problem associated with nerve injury and inflammation. In experiments supported by this grant over the last funding period, we have demonstrated the presence of a polysynaptic excitatory pathway, normally under powerful inhibitory control mediated by GABAA and glycine receptor activation that connects low threshold afferent input [unreadable]:touch[unreadable];to nociceptive output neurons [unreadable]:pain[unreadable];within the spinal cord dorsal horn. This means that there is built in circuitry within the spinal cord dorsal horn that permits the mixing of sensory modalities, allowing input generated by light touch to drive the pain projection neurons sending axons to higher brain centers. That is, there is an existing circuitry that is likely to contribute to allodynia. This pathway becomes revealed with pharmacological disinhibition [unreadable]:Torsney and MacDermott, 2006[unreadable];or by disinhibition associated with peripheral nerve injury and tactile allodynia[unreadable]:Keller et al., 2007[unreadable];. Over the next two years, we propose to investigate the inhibitory control mechanisms that may keep the polysynaptic excitatory pathway suppressed under normal conditions. In Specific Aim one, we will test whether inhibitory neurons in lamina I through III receive low threshold mono or polysynaptic afferent drive and how the strength of that afferent drive varies with stimulation frequency. In Specific Aim two, we will investigate tonic inhibition of excitatory and inhibitory neurons in lamina I-III to provide insight into how the inhibitory GABA and glycine receptors contribute to suppression of the polysynaptic low threshold pathway in the spinal cord slice preparation. In Specific Aim 3, we will investigate is presynaptic inhibition of low threshold afferents. The model we will test is whether the disinhibition allowing low threshold drive of lamina I pain neurons, which can be mimicked with GABAA and glycine receptor antagonists, works, at least in part by relief of endogenous presynaptic inhibition of low threshold afferents.

DESCRIPTION (provided by applicant): Twenty-first Century pre-college science teachers face unprecedented challenges in preparing U.S. students for, and interesting them in, careers in science and technology. Neuroscience poses special challenges for these teachers since few of them study neuroscience in college. Yet, advances in our understanding of the human nervous system have practical implications for their teaching practices, and are of importance to their students'education. To elevate teachers'neuroscience knowledge, Professor Amy MacDermott (P.I.) has joined forces with Professor Samuel Silverstein, Founder of Columbia University's Summer Research Program for Science Teachers (SRP), to expand opportunities allowing New York metropolitan area K-12 science teachers to participate in high quality, focused and sustained research experiences and professional development in the neurosciences. Teachers selected for support through NIH's Summer Research Experience Programs solicitation (PAR-11-050, CFDA 93.853) will perform hands-on research for eight weeks in each of two consecutive summers under the mentorship of NINDS-supported Columbia University neuroscientists (86 Columbia faculty currently receive NINDS support). Teachers will be full members of their laboratory's research team. In addition, they will participate one full day/week in Columbia University Summer Research Program's demonstrably successful professional development activities. Designed specifically for science teachers, these special pedagogical sessions include science seminars led by faculty from Columbia and other science-rich institutions, workshops in molecular modeling, data-driven instruction, lesson plan development, peer coaching and practice teaching. The program provides breakfast and lunch on professional development days, thereby encouraging informal interactions between participants. By the end of each summer the group has coalesced into an effective Professional Learning Community. Columbia's SRP is a world leader in documenting program effectiveness in promoting teacher retention, in stimulating teachers to implement more hands-on classroom and laboratory exercises, and in improving student achievement in science (Science 326:440- 442, 2009). In the academic years following teacher participation in the program, 10.1% more of their students (a 22.9% increase) passed a New York State Regents science exam than in the academic year preceding their entry into it, or than students of non-participating teachers in the same school in the same years. PUBLIC HEALTH RELEVANCE: To elevate pre-college science teachers'neuroscience knowledge, and assist them in providing current health sciences instruction to their students, Columbia University's Summer Research Program for Science Teachers will provide the teachers with high quality, focused and sustained research experiences and professional development in the neurosciences. The teachers will perform hands-on research under the mentorship University neuroscientists.