Shawn Hochman - US grants

DESCRIPTION:(adapted from applicant's abstract) The identity of the spinal interneurons that comprise the mammalian central pattern generator (CPG) for locomotion remains largely unknown. It is known that brainstem drive centers can activate the CPG, and the P.I. proposes that descending monoaminergic pathways remodel the spinal CPG for locomotion and may play a role in facilitating interactions within the CPG to allow it to generate rhythmic output. Four major aims are proposed, with increasing levels of analysis: 1) Identify neurons active during the locomotor program using the activity-dependent dye sulforhodamine, and analyze the modulatory actions of monoamines and their agonists on ligand- and voltage-gated conductances in these neurons. 2) Determine the effects of monoamines on the firing properties of locomotor-related interneurons, specifically looking for conditional bursting properties. 3) Identify the 5HT receptor subtypes responsible for activating the locomotor CPG, with specific emphasis on the hypothesis that 5HT-7 receptors are critical to locomotor rhythmogenesis. 4) Determine whether interneurons active during locomotion express 5HT-7 receptors, which could then serve as markers for CPG neurons. These studies will contribute to our understanding of the CPG for locomotion and could aid in recovery after spinal cord injury. Specifically, pharmacotherapeutic strategies to facilitate CPG activation may assist limited reconnections from the brain in activating locomotion. In addition, identification and characterization of the neurons in the CPG may be helpful in future studies involving transplantation of the neurons in the future.

An important objective in the treatment of the hyperreflexia that follows spinal cord injury is to understand the mechanisms that control plastic alterations in reflex gain. Recently, we have discovered that serotonin (5-HT) can potently and reproducibly facilitate spinal reflexes for hours. These actions resemble persistent flexion reflex responses seen following nociceptive stimuli, and suggest that both primary afferents and descending systems can modulate the gain of the spinal interneurons interposed in flexor reflex afferent (FRA) pathways. "Unfortunately, much less is known about the FRA interneurons than other interneurons". Hence, we propose to use the easily- inducible augmentation in FRA pathways by 5-HT to undertake a detailed analysis of the network, cellular, and molecular mechanisms controlling plasticity of this well known behavior, the flexor withdrawal reflex. 1 We propose to characterize the afferent and interneuronal populations involved in the 5-HT-induced reflex facilitation to test the hypothesis that the flexor reflex afferent (FRA) interneurons are preferentially facilitated by 5-HT. 2 Injury- inducing noxious stimuli lead to the activation of protein kinase C (PKC) and a long-lasting plasticity in spinal neurons. Because 5-HT2 receptors also activate PKC, we propose to test the hypothesis that 5-HT2 receptors mediate the 5-HT-induced long- lasting facilitation of spinal reflexes via activation of PKC. 3 We then propose to use cellular/molecular approaches to identify `molecular markers' selective to interneurons serving facilitated reflexes. We hypothesize that membrane translocation of the PKCgamma and/or PKCepsilon isozymes will identify the facilitated interneurons. Using similar techniques, we will also test the hypothesis that the hyperreflexia that follows chronic spinal cord injury is due to a novel expression of constitutively active PKC isozymes. In summary, this proposal will use a multidisciplinary approach to characterize a newly-discovered form of plasticity in spinal cord reflex pathways. Information derived from these studies should provide insights into controlling the hyperreflexia that follows spinal cord injury so that the flexion reflex other behaviors that utilize the same spinal circuitry (e.g. locomotion) are allowed to function within a normal range of sensori-motor gain.

DESCRIPTION (provided by applicant): Restless legs syndrome (RLS) is a CNS disorder involving abnormal muscle sensations that are reduced during motor activity, worsen at rest, and have a marked circadian pattern. Primary treatment involves providing drugs that increase CNS dopaminergic activity, particularly activation of D2-1ike receptors. The hypothalamus controls autonomic function and circadian rhythmicity. The dorso-posterior (A11) region contains the only dopaminergic (DA) projections to spinal cord. DA fibers terminate largely in the intermediolateral column (IML) housing preganglionic sympathetic neurons and in dorsal horn regions related to muscle afferent processing. We hypothesize: (i) That a deficit in hypothalamo-spinal DA activity results in an aberrant activation of muscle afferents; directly, by reducing tonic inhibition of afferent input, and; indirectly, via a disinhibition-induced increase in sympathetic drive to skeletal muscle afferents. (ii) That low-threshold afferent activity (e.g. during movement) presynaptically depresses high-threshold muscle afferents. (iii) That DA disinhibitory actions should peak at night, the nadir of hypothalamic circadian dopamine release. As the A11 region provides the only DA input, all spinal modulatory actions can be ascribed to its function. Hence, studies of DA modulatory actions in the in vitro spinal cord will characterize the complex cellular and network actions of hypothalamo-spinal dopamine function. First, we plan to characterize the dopamine receptor distribution in spinal cord using immunostaining and in situ hybridization techniques. We will then study 5-HT and dopamine modulation of IML neuronal excitability and whether increases in sympathetic drive facilitate muscle afferent activity and input to spinal neurons. Lastly, we will use A11 neurochemical lesioning and D3 receptor knockout mice to examine their effects on alterations in spinal cord function and relate these changes to changes in several movement-related behavioral parameters concomitant with recordings of EEG, neck & limb EMG, and EKG. The uniqueness of our proposal is the development of novel and testable hypotheses on putative spinal mechanisms causing RLS. It involves the first detailed study of DA modulation of spinal cord function and it is undertaken at behavioral, network, and cellular levels, including an attempt to develop an animal model of RLS.

Tyramine, octopamine, and tryptamine belong to a family of endogenous amines called trace amines (TAs) that have structural and metabolic similarities to the 'classical' neurotransmitters serotonin, dopamine, and nor-epinephrine. While receptors for TAs were recently identified, their physiological relevance remains elusive. This project seeks to reveal that these substances control leg movements by acting on the spinal cord circuits responsible for locomotion. TAs are applied directly to the spinal cord to characterize their modulation of neural circuit function. In addition, electrical recordings from limb muscles are coupled with video-based analyses of limb movements to allow quantitative study of locomotor cycle-based activity patterns, detailing the effects of TAs on motor performance. The advances made will spur investigations that impact our understanding of how the spinal cord is 'engineered' to control locomotion. Overall, this project establishes ground-breaking science and methodology that incorporates cross-disciplinary mentoring of underrepresented engineering students from the Georgia Institute of Technology with training in the biological sciences at Emory, and a broadening of their activities through participation in conferences.

DESCRIPTION (provided by applicant): Sensory neurotransmission is the fundamental first step in the central processing of sensory stimuli. It is controlled by pre- and post-synaptic inhibitory mechanisms. Presynaptic inhibition (PSI) is more powerful than postsynaptic inhibition in depressing the central excitatory actions of almost all primary afferent sensory fibers. A major mechanism producing afferent PSI is via a counterintuitive channel-mediated depolarization of their intraspinal terminals which, conveniently, can be recorded as a dorsal root potential. It is thought that, via a trisynaptic pathway, GABAergic inhibitory interneurons release GABA as the neurotransmitter to produce PSI of large-diameter (low threshold) muscle and cutaneous sensory afferents. However to this day there is little 'squeaky clean' evidence. We have heretical evidence suggesting instead that much of this afferent stimulation-evoked PSI is generated by more direct synaptic pathways that may be; (i) independent of classical GABAA receptors and (ii) independent of GABA. A mechanistic proof of these assertions require a coalescence of pioneering electrophysiological studies in the in vitro nerves-attached mouse spinal cord (P10-14) and includes transgenic lines that identify and knockout specific genes in larger- diameter sensory afferent subpopulation. Specifically, we will test the following two hypotheses: 1. GABA is not the only transmitter producing PSI. Alternates include acetylcholine, taurine and 2-alanine, and these are found in discrete afferent and interneuronal subpopulations. 2. GABAA receptor subunits are not the only subunits in activated receptors. Alternates are nicotinic and glycinergic subunits and these may be assembled in unique heteromeric compositions. If successful, a decades-old view of mechanisms producing PSI will require dramatic conceptual revision. This new perspective broadens our understanding of somatosensory information processing, and may introduce novel control strategies for sensory dysfunction.

Project Summary The present project explores a barely studied and poorly-understood area of vertebrate autonomic neuroscience: the recruitment properties of thoracic paravertebral sympathetic postganglionic neurons (tSPNs). The prominent role of thoracic paravertebral sympathetic chain ganglia is as the final neural control element regulating vasomotor tone. Given their strategic nodal site in autonomic signaling to body, any plasticity in tSPNs is likely to be of high significance. Unfortunately, tSPNs are largely inaccessible for in vivo study, so operational principles are inferred from studies in cervical and lumbar chain ganglia. Only 3 in vitro studies have revealed tSPN electrophysiological properties: none accurately measure cellular integrative properties or underlying recruitment principles due to electrode impalement injury. We undertook the first physiological studies on caudal thoracic chain ganglia in the adult mouse by developing an ex vivo preparation with intact segmental preganglionic and rostrocaudal interganglionic connections. We obtained the first whole- cell patch clamp recordings of tSPNs and observed fundamentally different integrative and firing properties are than previously observed. This reliable data set is a critical prerequisite to realistic computational simulation. We propose to interleave experimental testing with modeling to understand tSPN recruitment principles and their integrative properties. [SA1] We will test the hypothesis that tSPNs have heterogeneous synaptic, cellular, and network properties, and are active participants in input-output recruitment strategies. Higher thoracic spinal cord injuries (SCI) disrupt the brainstem pathways that regulate tSPN excitability via spinal preganglionic loops. Such disruption can lead to sudden life-threatening tSPN mediated hypertensive crises (autonomic dysreflexia). Whether paravertebral sympathetic chain ganglia dysfunction contributes to amplification in a vasomotor response is unknown. To fill this significant gap in knowledge, experimental studies will disclose plasticity in the cellular and synaptic organizational rules serving tSPN recruitment. [SA2] We will test the hypothesis that tSPNs increased their intrinsic excitability and convert from linear to non-linear gain amplifiers after SCI. Computational simulation will construct a database amenable to realistic modeling of recruitment principles of potential clinical relevance that could be transformative to the field. The relative simplicity of the organization makes discovery of principles through modeling more assured than in more complex systems. Realistic simulation of the neural bases of tSPN function and emergent dysfunction could catalyze predictive drug discovery-based high throughput simulations that normalize function for rapid preclinical testing. Significance: we aim to uncover the operational principles governing the final neural command pathways regulating vascular tone. As sympathetic hyperactivity is implicated in various autonomic disorders, a database amenable to realistic modeling studies will be of broad predictive use in preclinical and translational studies.

Abstract A dominant class of postganglionic neurons in paravertebral thoracic chain ganglia are vasoconstrictors innervating distributed organ systems including skin and muscle. For muscle, control of blood flow via vasoconstrictors operates over a narrow range. In comparison, dramatic changes in blood flow are controlled by the cutaneous vasoconstrictor system as part of a highly complex control system vitally important in homeostasis including thermoregulation. After spinal cord injuries (SCI) there is loss of cutaneous thermoregulatory control below the injury site. This can lead to detrimental effects on physiology including rendering individuals as functionally poikilothermic and prone to hypothermia and hyperthermia. This field of autonomic dysfunction after SCI is highly understudied. A completely unexplored area is whether selective neuromodulation stimulation-based control targeting of thoracic paravertebral sympathetic chain be used to control cutaneous vasoconstrictor function. Preclinical studies are required to assess targeted interventions at this site. Modulatory control of cutaneous vasoconstrictor function after SCI in innervation territories deprived of brainstem drive circuits may also be amenable for use in smart feedback-based control technologies for homeostatic control of vascular vasoconstrictor function. Presently we take advantage of the R21 exploratory research mechanism to assess whether thoracic chain ganglia can be used as a novel site for neuromodulation-based control of cutaneous vasoconstrictors. We recently developed an ex vivo adult mouse model that makes us rather uniquely positioned for comprehensive assessment of thoracic chain ganglia as a target site for therapeutic control via interfacing neuromodulation technologies. Here we leverage powerful optogenetic approaches to selectively recruit vasoconstrictors to guide optimization of electrical stimulation strategies that preferentially target activation of postganglionic over preganglionics axons. Due to their projections to prevertebral ganglia (e.g. celiac and mesenteric) recruitment of preganglionics is likely to have undesirable off target actions. Assessment of vasoconstrictor recruitment along the thoracic sympathetic chain is likely to offer ability for selective segmental control of vasomotor function. Once characterized we will compare recruitment results to those seen after high thoracic spinal cord injury at acute (first week) and more chronic stages (3 to 6 weeks). If successful, combined approaches will have determined that; (1) selective recruitment of thoracic chain paravertebral ganglia can be used to preferentially control cutaneous vasoconstrictor activity, (2) measured changes in skin temperature can be used as a feedback variable to increase cutaneous vasoconstrictor activity, and (3) whether targeting these ganglia could provide a substrate for translational neuromodulation-based approaches to control dysfunction in cutaneous thermoregulatory systems after SCI.

PROJECT SUMMARY Spinal cord sympathetic preganglionic neurons (SPNs) are found in the thoracic and lumbar spinal cord. They are the final arbiters of CNS sympathetic output. SPN axons branch to issue highly divergent multisegmental projections on paravertebral sympathetic chain ganglia postganglionic neurons (PNs). Divergence provides a mechanism for amplification of CNS sympathetic commands to the numerically much greater PNs. It is assumed that spike conduction is reliable across multisegmental branch points. This was based on ex vivo recordings from the sympathetic chain at room temperature (T°) where large increases in spike amplitude and width ensure a high safety factor for branch point conduction. As increasing T° promotes conduction failures, it is likely that SPN branch point conduction is also T°-sensitive. In somatosensory systems, branch points provide an important site for control of axonal conduction and extrasynaptic ?5-containing GABAA receptors (GABAARs) are implicated. SPNs also express ?5-GABAARs, and their expression at presynaptic axonal branch points may similarly control divergence and hence response amplification to PNs. We recorded from SPN axons diverging across ganglia in the adult mouse ex vivo thoracic paravertebral chain following stimulation of attached ventral roots. Initial experiments observed that conduction block was dependent on; number of traversed chain ganglia, T°, frequency, and GABAAR antagonists. Results support axonal divergence as a modifiable output stage in sympathetic gain control. [SA1] We hypothesize that preganglionic axonal conduction block across branch points is under neuromodulatory control by constitutively active, ?5-containing GABAAR. We undertake experiments to aess axonal recruitment changes following application of GABAAR agonists, antagonists and endogenous neurosteroid allosteric modulators and tested across the physiological range of firing frequencies. [SA2] Many spinal cord injured (SCI) individuals have thermoregulatory dysfunction. As even small elevations in body T° can compromise spike conduction, SPN axonal function in the SCI population may be particularly vulnerable. We hypothesize that conduction across branch points is sensitive to the broader changes in T°core and will test the effect of T° on conduction block over a range of T° consistent with hypo- or hyperthermia. [SA3] We hypothesize that SCI leads to changes that promote conduction across branch points, including (a) increased GABAAR activity and (b) spike width broadening. Results above will be compared to those seen after T2 thoracic SCI at early (1-3 days) and chronic (4-6 weeks) time points. Exploring mechanisms that promote or prevent conduction across branch points is critical to understanding whether SPN signal amplification is hard-wired or physiologically modifiable (e.g. behavioral state dependent) and whether plasticity after SCI alters function at this important sympathetic output stage.