Edgar T. Walters, Ph.D. - US grants

The objective of the proposed research is to identify cellular mechanisms underlying associative learning and to relate these mechanisms to general principles of information processing by sensory systems. Building on the recent demonstration of activity-dependent associative plasticity in individual sensory neurons mediating afferent input for the tail withdrawal reflex in Aplysia, the proposed experiments will test the hypothesis that this associative plasticity is a mechanism for classical conditioning of tail withdrawal. Differential classical conditioning of tail withdrawal will be examined in a semi-intact preparation in which behavioral and cellular alterations in identified neurons within the reflex circuit can be measured simultaneously. Alterations both in the monosynaptic connections to identified tail motor neurons and in the electro-physiological properties of the sensory neuron soma will be measured. Both classes of assciative modification will be compared to associative plasticity produced by pairing intracellular activation of sensory neurons with application of the presumed reinforcing neuromodulator (serotonin) in both the semi-intact preparation and the isolated sensory neuron soma. The possible interactions of associatively specific activity-dependent neuromodulation with changes in membrane potential and lateral inhibition will be examined in an attempt to identify sensory processes that may contribute to more complex features of associative learning such as sequence specificity, overshadowing, and blocking. By precisely defining conditioning and test stimuli in the semi-intact preparation and relating the effects of conditioning with these stimuli to the details of cellular associations measured under analogous conditions in the isolated soma, the groundwork for an eventual quantitative model of associative processing in this sensory system will be laid. Since learning is one of the fundamental capabilities of most if not all nervous systems, these studies may shed light on general mechanisms involved in both the normal function and some of the dysfunctions of the human brain.

The long-term objective of the proposed research is to identify cellular mechanisms underlying associative learning and to relate these mechanisms to general principles of information processing by sensory systems. Building on the recent demonstration of activity-dependent associative plasticity in individual sensory neurons mediating afferent input for the tail withdrawal reflex in Aplysia, the proposed experiments will test the hypothesis that this associative plasticity is a mechanism for classical conditioning. Initially differential classical conditioning of tail withdrawal will be examined in a semi-intact preparation in which behavioral and cellular alterations in identified neurons within the reflex circuit can be measured simultaneously. An analog of the conditioning process will then be applied to the isolated sensory cell soma by pairing intracellular stimulation with bath application of the presumed neuromodulator mediating the unconditioned effects. Specific hypotheses concerning the interaction of lateral inhibition with cellular mechanisms of associative plasticity will be tested in an attempt to identify sensory processes that may contribute to complex features of associative learing. By precisely defining conditioning and test stimuli in the simi-intact preparation and relating the effects of conditioning with these stimuli to the details of cellular associations measured under analogous conditions in the isolated soma, quantitative models of the functional interactions underlying associative processing in this sensory system will be developed. If basic principles relating specific neuronal properties and patterns of organization to associative modifiability emerge, these principles will be tested in collaborative studies on a suitable vertebrate preparation. Since learning is one of the fundamental capabilities of most if not all nervous systems, these studies may shed light on general mechanisms involved in both the normal function and some of the dysfunctions of the human brain.

The long-term objective is to analyze the cellular mechanisms underlying two important capabilities of the nervous system: (1) the ability to associate a given stimulus with a novel motor response (stimulus-response or S-R learning), and (2) long-term sensory modifiability. This objective requires the development of preparations, involving well-defined behavioral alterations and identified neuronal networks, that permit direct analysis of physiological mechanisms. Building on preliminary studies using the siphon, tail, head, and parapodia of the marine gastropod, Aplysia, intact and semi-intact preparations will be developed that show the acquisition of novel siphon responses after pairing parapodial stimulation with head or tail stimulation. Electrophysiological correlates of this S-R conditioning will be examined in identified siphon motor neurons and interneurons. Two hypotheses for the development of novel S-R connections will be tested using intracellular recording, voltage clamp, and quantal analysis techniques. Long-term sensory memory will be investigated in the central and peripheral processes of parapodial sensory neurons, which offer special advantages for sensory analysis. The general hypothesis that associative information storage in sensory systems makes use of mechanisms evolved for sensory compensation after injury will be tested. The contribution of a specific cellular associative mechanism - activity-dependent extrinsic modulation (ADEM) - to sensory modifiability will be tested. Several potential ADEM-related enhancements of signaling effectiveness produced by associative conditioning and by injury of the receptive field will be examined: synaptic facilitation, increased central and/or peripheral excitability, and sprouting of peripheral and/or central processes. These studies should provide basic information on general mechanisms of learning, sensory compensation, and neuronal regeneration that may eventually contribute to an understanding of normal and abnormal physiological plasticity within the human nervous system.

Although identified clusters of mechanosensory neurons in gastropod molluscs have provided detailed information about cellular events correlated with behavioral modifications, little is known about the functions or evolutionary significance of these cellular mechanisms. In these studies, comparisons will focus on short-and long-term changes in the excitability of the sensory neuron's cell body within the central nervous system and changes in the sensory neuron's peripheral receptive field. These comparisons should illuminate the functions of mechanosensory plasticity in this group of animals, and may point to primitive mechanisms involved in memory traces of peripheral injury.

This project by Dr. Walters will compare cellular mechanisms within the same neurons contributing to 3 classes of long-lasting alteration. These alterations, which have generally been considered separately in different biological preparations by different laboratories are: 1) adaptive responses following injury of a neuron's axon, 2) hyperexcitability of nociceptive neurons (functionally equivalent to neurons in mammalian pain pathways) during persistent nociceptive sensitization ("hyperalgesia"), and 3) aversive learning and memory. Dr. Walters will be using electrophysiological, pharmacological, and morphological methods, to characterize functionally identified neurons in an invertebrate preparation to address the following questions: First, does axonal injury induce long-term changes in excitability, synaptic transmission, and morphology in functionally diverse neurons? Second, how do alterations induced by axonal injury compare to activity-dependent alterations that have previously been linked to learning and memory. In addition, how do injury-related and learning-related effects interact? Third, is long-term plasticity induced by axonal injury initially triggered by the interruption of retrograde axonal transport of trophic factors, or do the axonal triggers involve elevation within the axon of known signals for neuronal plasticity such as the second messengers calcium and cAMP? Fourth, do cAMP and calcium in or near the cell body of the neuron contribute to the triggering of long-term plasticity during both learning and cellular reactions to axonal injury? These investigations may reveal basic mechanisms of plasticity that appeared early in evolution and which may be involved in memory, regeneration, and chronic pain-like states.***//

DESCRIPTION: The general objective is to use a simple invertebrate model of traumatic neuropathic sensitization to probe fundamental cellular mechanisms that may be important substrates for neuropathic hyperalgesia--a common component of intractable and chronic pain in humans. Various observations, as well as evolutionary arguments, suggest that some basic mechanisms of neuropathic sensitization are likely to be widely conserved. Identified nociceptors and other neurons controlling the tail withdrawal reflex in the mollusc, Aplysia californica, provide a special opportunity to test the roles of specific cellular mediators in the induction, expression, and termination of neuropathic sensitization involving nerve injury. Using this system, a model of neuropathic sensitization involving crush injury of the tail will be developed that allows direct tests of hypotheses about mechanisms underlying expression and induction of persistent behavioral and sensory alterations lasting for months. Tests will be made of the contributions of peripheral changes, including sensitization of afferents, spontaneous discharge of injured axons, and background neuromodulator release. Tests of persistent central changes will examine hyperexcitability of nociceptor somata, interneurons and motor neurons, facilitation of central synapses, disinhibition, and background electrical and synaptic activity. Tests of induction mechanisms will examine contributions of fast, activity-dependent signals, neuromodulator release, long-term synaptic potentiation, and slow axoplasmic signals. A general hypothesis about the combined role of cAMP, calcium ions and slow axoplasmic injury signals in the induction of persistent nociceptor hyperexcitability and enhancement of nociceptor regeneration will be tested in a reduced preparation of ganglia and nerves, and in isolated nociceptors growing in cell culture. Other potential intracellular and extracellular signals for induction and maintenance of persistent nociceptor hyperexcitability will begin to be screened in individual cells.

Central memory of peripheral injury relies on mechanisms that are likely prototypes of other transcription-dependent forms of long-term memory, and which directly contribute to the clinical problems of persistent hyperalgesia and neuropathic pain. In addition, derangements of these fundamental plasticity mechanisms may contribute to problems of memory and learning in humans. The invertebrate, Aplysia, contains nociceptive sensory neurons with defined roles in defensive behavior and which display long-term hyperexcitability (LTH) of their central as well as peripheral components lasting for days to months after intense noxious stimulation. These sensory neurons are particularly favorable for investigating long-term memory mechanisms because they can be manipulated and tested individually before, during, and after memory induction -- in ways not possible with vertebrate neurons. The proposed studies will test a multiphase hypothesis about the cellular signaling pathways and transcription factors responsible for induction of LTH in these neurons, focusing on the phases that depend upon signals evoked by intense neural activity. A number of specific questions will be systematically addressed. What ionic mechanisms underlie the expression Of LTH? During LTH are there changes in the mRNA levels of any ion channels that have been cloned from Aplysia? How long do different phases of LTH induction last? How do they depend upon NO and cGMP signals? Do NO and PKG act through activation of MAPK? Is prolonged or repeated elevation of intracellular Ca2+ necessary or sufficient to induce LTH? Is prolonged PKA activation important for LTH induction? Is there simultaneous or sequential synergism between cAMP and either Ca2+ or cGMP signals during the induction of LTH? How does the activity of protein kinases potentially important for LTH (e.g. PKG, MAPK IAK-1, PKA, PKC, CaMK, SAPK) change during different phases of the induction and maintenance of LTH? Which transcription factors are required for the rapid induction of LTH? Are any phases of LTH prevented by injection of decoy oligonucleotides encoding response elements such as CRE, SRE and ERE? Can LTH be induced by injection of activated transcription factors? As is evident by the detailed nature of these questions, the proposed experiments directly probe specific pathways that are important for the induction of LTH. Our findings will provide significant insights into fundamental mechanisms important for both memory formation and persistent pain.

Description (provided by applicant): Summary Persistent neuropathic pain is produced by spinal cord injury (SCI) in a majority of patients. Like other forms of central neuropathic pain, SCI pain is often debilitating and quite resistant to clinical treatment. Most research on mechanisms of SCI pain has focused on increases in the responsiveness and spontaneous electrical activity of central neurons within pain pathways, especially second-order neurons in the dorsal horn near the injury site. Hyperexcitability of dorsal horn neurons after SCI appears to involve many plausible causes, but one that has received little attention is an enhancement of spontaneous activity (SA) and excitability in the sensory neurons (especially nociceptors) that normally excite dorsal horn neurons. Indeed, surprisingly little is known about how sensory neurons in the dorsal root ganglion (DRG) respond to SCI. The proposed studies are based on the novel hypothesis that SCI triggers a chronic hyperfunctional state in nociceptors which results in the generation of SA within their somata in DRGs, and that this continuing SA excites central pain pathways, driving spontaneous pain, allodynia, and hyperalgesia. This hypothesis will be tested by 1) examining the effects of SCI on SA and electrophysiological properties of DRG neurons that are subsequently dissociated and tested in depth, 2) examining SA in DRG neurons in vivo after SCI, and 3) by attempting to block SA (and associated SCI pain) by disconnecting the DRG from the spinal cord (dorsal rhizotomy prior to the contusion) or by knocking down a voltage-gated Na+ channel that is necessary for the generation of SA by nociceptors. SCI will be produced in a standard contusion injury model, with the impact at spinal level T10. Behavioral and electrophysiological tests will be conducted 3 days, 1 month, and 3 months after injury. The behavioral tests will assess motor loss and possible recovery, and (at 1 and 3 months) spontaneous pain, allodynia, and hyperalgesia at, above, and below the injury level. In vitro electrophysiological tests will be conducted with whole-cell current clamp methods on DRG neurons dissociated from T9, T11, and L4 levels. In addition to recording SA, a complex test protocol will define intrinsic passive and active membrane properties at resting membrane potential and at holding potentials of -80 mV (where little inactivation of voltage-gated sodium channels occurs) and -50 mV (where many of these channels are inactivated). In vivo electrophysiological tests will use extracellular recording from filaments teased from the dorsal root to see how much SA is present before and after disconnecting the DRG from the periphery. Four predictions of the hyperfunctional nociceptor hypothesis will be tested: first, SCI should enhance SA of putative nociceptive DRG neurons, initially at and below the level of injury, but later above the injury as well. Second, that enhanced SA in vitro and in vivo, and hyperexcitability in vitro, should be correlated with enhanced behavioral signs of pain, allodynia, and hyperalgesia. Third, if SCI pain depends in part upon SA in nociceptors, SCI pain should be reduced by selectively suppressing nociceptor SA in vivo. This will be tested by delivering antisense oligonucleotides intrathecally to knock down the expression of a Na+ channel, Nav1.8, that is expressed selectively in nociceptive sensory neurons and is necessary for generating SA in these neurons. Fourth, the assumption that retrograde signals to nociceptor somata from central processes of these neurons are necessary for triggering the SA will be tested by performing a dorsal rhizotomy immediately before the SCI. These exploratory studies will test a novel hypothesis about mechanisms important for SCI pain, define intrinsic electrophysiological alterations in DRG neurons linked to neuropathic pain, and begin to test an intervention that appears potentially useful for treating SCI pain. PUBLIC HEALTH RELEVANCE: Spinal cord injury patients often suffer debilitating pain that is highly resistant to clinical treatments. Although most investigations of this problem have focused on alterations in central neurons within pain pathways, preliminary data suggest that alterations of sensory neurons that normally convey pain information from peripheral tissues may play an important role. The proposed studies will test the hypothesis that chronic pain caused by spinal cord injury is produced in part by spontaneous electrical activity in sensory neurons, and that pain may be reduced by blocking this activity.

All animals, including humans, experience injury, and every species examined shows adaptive responses to help avoid injury or re-injury. Injury to humans causes pain, which can be long lasting (even permanent) and sometimes untreatable. Although much has been learned about other aspects of human biology through comparative studies of simpler animals, including selected invertebrates, systematic comparative studies of behavioral and neural responses to injury in invertebrates have been lacking. This project will compare behavioral and neural responses to peripheral injury in two invertebrates that, because of their giant neurons, have been sources of pioneering discoveries in neuroscience: the common Atlantic squid, Loligo, and a large marine snail, Aplysia. Both are molluscs, but they have very different life styles and cognitive capabilities. Hypotheses to be tested include: (1) In the peripheral sensory neurons of both species, injury-induced, long-lasting behavioral sensitization involves a similar long-term enhancement of responsiveness that resembles the effects reported in mammals, (2) these changes are induced by common chemical signals that also drive inflammatory pain in mammals, (3) the large-brained squid, but not small-brained snail display ongoing awareness of an injury, and (4) sensitization of defensive responses in the squid increases the chances of survival in a wounded state by decreasing the vulnerability to predatory attacks. The broader impacts of this research project will be the encouragement of the use of molluscs to explore the evolution of fundamental behavioral patterns involving core mechanisms that are widely conserved and may be important for human pain, and the education of future scientists (including undergraduates) interested in comparative and evolutionary approaches to behavioral neurobiology. The resulting scientific information will be published in scientific journals and disseminated to foster rational and humane treatment of molluscs and other invertebrates in research.

? DESCRIPTION (provided by applicant): Chronic pain caused by injury to the peripheral or central nervous system (neuropathic pain) is notoriously resistant to treatment. The mechanisms that maintain any type of neuropathic pain for months or longer are poorly understood. Chronic pain in a rat model of spinal cord injury (SCI) has recently been shown to depend upon hyperactivity in nociceptive sensory neurons (nociceptors), with much of the pain-initiating activity generated within the cell bodies. The continued expression of pain-linked nociceptor hyper excitability and spontaneous activity (SA) in vitro provides a special opportunity to link biochemical mechanisms directly to electrophysiological activity critical for maintaining chronic SCI pain. Preliminary results indicate that continuing signaling by complexes containing adenylyl cyclase (AC), protein kinase A (PKA), and A-kinase anchoring proteins (AKAPs), and possibly exchange protein activated by cAMP (EPAC) plays a vital role. While cAMP signaling has long been known to be important for acute pain lasting hours to days, a major role in maintaining pain lasting months is unexpected. Agents selectively inhibiting different steps along cAMP-dependent pathways blocked chronic SCI-induced SA, including inhibitors of AKAP5 (AKAP79/150)-anchored complexes. Biochemical studies of membranes from dorsal root ganglia revealed a change in AC regulation after SCI, suggesting the existence of a previously unknown mechanism at the level of AC function that contributes to chronic pain. These and related observations led to the hypothesis that chronic nociceptor SA and pain after SCI are maintained by 1) alterations in AC regulation and 2) AKAP5-scaffolded macromolecular complexes that facilitate cAMP-dependent PKA and EPAC regulation of ion channels. The proposed studies will exploit complementary strengths of the two PIs' laboratories by combining in vitro biochemistry, cell biology, and electrophysiology coordinated with in vivo tests of pain-related behavior after SCI. Experiments will take advantage of our findings that robust SCI-induced SA in numerous nociceptors below the spinal injury level is clearly linked to behaviorally expressed hypersensitivity and pain. This will allow the use of electrophysiological and molecular alterations in dissociated nociceptors as informative endpoints for studies evaluating pain-related functions of signaling molecules within the cAMP pathway. It will also allow pooling of multiple ganglia from SCI animals to facilitate biochemical and molecular studies. Predicted behavioral and cellular effects of interventions targeting macromolecular complexes disclosed in the in vitro studies will be tested in the whole animal using complementary approaches, including a novel viral vector for expression of disrupting peptides selectively in nociceptors, an knockdown and inhibitor methods targeting specific cAMP signaling components. Information gained from these studies may lead to major mechanistic discoveries that could guide future efforts to treat chronic pain by targeting the persistent intracellular signaling that maintains hyperactivity in nociceptors that promotes chronic pain.

Project Summary The long-term objective of this project is to discover novel, highly targeted approaches for treating ongoing pain by defining critical mechanisms of ongoing activity (OA) in primary nociceptors that drive this pain. Recent discoveries revealed that the OA generated spontaneously in probable nociceptors and linked to ongoing pain after spinal cord injury (SCI) is associated with all three electrophysiological alterations that, in principle, can promote OA. These are depolarization of resting membrane potential (RMP), reduced voltage threshold for action potentials (APs), and increased frequency of large, transient, depolarizing spontaneous fluctuations (DSFs). Two extrinsic mediators related to inflammation, serotonin (5-HT) and capsaicin (mimicking endogenous TRPV1 activators), also promote OA, in large part by enhancing DSFs. Virtually nothing is known about mechanisms underlying large DSFs. Three specific aims will test hypotheses about DSF generation and potentiation, employing whole cell patch recording, stimulation by Ca2+ uncaging, pharmacological and transgenic approaches, in vivo recording, and behavioral tests. Aim 1 will define ion conductance and cell signaling (Ca2+ and cAMP) contributions to the acute generation of large DSFs, taking advantage of the ability of 5-HT, forskolin, and capsaicin to rapidly stimulate large DSFs, using naïve rats and transgenic mice. The focus will include HCN channels, T-type Ca2+ channels, and Nav1.8 channels. Special attention will be paid to TRPC4/5 channels, which are important for OA and have unusual properties that account for unique features of large DSFs. Aim 2 will define ion conductances and cell signals that promote large DSF generation in chronic SCI and in a subacute peripheral inflammation model (hindpaw injection of complete Freund's adjuvant - CFA). The channels found in Aim 1 to be important for large DSFs will be tested for altered contributions and expression in each model. Alterations promoting OA are predicted to be shared in these models (and thus to potentially drive many forms of ongoing pain). Aim 3 will test the prediction that combined interventions selectively blocking large DSFs and elevating AP threshold will reduce ongoing pain. A novel analgesic strategy will be tested, which combines a drug that prevents large DSF generation (a TRPC4/5 blocker) with a drug that selectively elevates AP threshold in nociceptors (a Nav1.8 blocker). The combination should efficiently suppress nociceptor OA and consequent ongoing pain at doses lower than required to observe any effect on ongoing pain from either drug alone. This prediction will be tested in vivo both on C-fiber OA recorded from dorsal roots of anesthetized rats and on ongoing pain in SCI rats and in rat and mouse CFA models. This targeted approach could lay the foundation for new treatments for severe ongoing pain that have relatively few side effects and provide an alternative to opioids, with their attendant risks.