Asaf Keller - US grants

The purpose of the proposed studies is to further the understanding of how the brain controls voluntary movements. Voluntary movements are controlled, in part, by the motor areas of the cerebral cortex. This control is accomplished by correlated activity of specific groups of brain cells, or neurons. Each group of neurons is responsible for the control of individual muscles. Therefore, an understanding of the mechanisms of motor control is contingent upon deciphering the circuits that link these neurons into functional groups. Furthermore, knowledge about the interactions among different functional groups is important. This information can be obtained by identifying the functional properties of single neurons, and then defining the specific connections that link them into neuronal groups. These studies require an interdisciplinary approach, combining electrophysiological recordings of neuronal activity, labeling of neuronal pathways, and electron-microscopical examination of connections between identified neurons. The importance of this research is that it contributes to fundamental knowledge about motor control.

The proposed research will investigate mechanisms that shape and modulate the response properties of cortical neurons. Key to understanding these mechanisms is the elucidation of both the physiological and morphological attributes of cortical neurons, and the synaptic interactions by which they form functional circuits. In addition, our recent findings suggest that the receptive fields of cortical neurons are dynamically regulated by patterns of sensory inputs. These studies focus on the barrel cortex, the region of the somatosensory cortex containing discrete representations of individual whiskers on rodents' snouts. The first aim is to test the hypothesis that the microcircuitry within individual barrels is sufficient for integration of inputs from multiple whiskers. Cortical neurons respond best to inputs from their principal whisker, and these responses are shaped principally by thalamocortical (TC) inputs. In contrast, there is controversy about the mechanisms that shape the responses to inputs from adjacent whiskers - the surround receptive field (SRF). One hypothesis states that SRFs are shaped entirely by intracortical interactions between barrels. The second hypothesis is that they are shaped entirely by synaptic interactions within an individual barrel. We will test these hypotheses with the use of functional imaging, whole-cell recordings, photostimulation, and neuroanatomical approaches, applied to in vitro slice preparations. The second aim is to test the hypothesis that corticothalamic (CT) neurons play a critical role in the initial stages of cortical processing. Anatomical considerations suggest that CT cells play a critical role in this process. In addition, CT cells are strategically placed to provide potent feed-forward inhibition to layer IV neurons. These postulates will be tested by determining the responses of CT neurons to TC inputs, and their postsynaptic influences on layer IV cells, in both in vivo and in vitro preparations. The third aim is to test the hypothesis that the receptive field properties of barrel cortex neurons are dynamically regulated by patterns of sensory inputs. The preceding aims address steady-state mechanisms involved in shaping receptive fields. Based on preliminary data we propose that SRFs are continually modulated by whisking frequency during exploratory behavior. We will test this hypothesis in vivo, and determine the mechanisms responsible for this dynamic regulation in vitro. The proposed studies will provide data pertinent to understanding the normal functions of the cerebral cortex and the processes underlying congenital or acquired neurological diseases resulting in sensory-motor deficits.

The aim of the research program, of which this application is a part, is to decipher the mechanisms by which restitution of motor function occurs following congenital or acquired neurological disorders. Although several brain structures are involved in this recovery process, one of the important structures is the motor cortex. Within the motor cortex there are somatotopic representations of the major body parts, and each of these contains multiple, non contiguous representations of different movement patterns. Movement representation zones can be modified in a use- dependent fashion, following sensory-motor perturbations and during the acquisition of motor skills. The pliability of motor representation maps is particularly dramatic during the postnatal developmental period, and is thought to be correlated with the acquisition of novel motor skills. Available data indicate that intrinsic connections within the motor cortex link neurons within this distributed network to perform coordinated, multi-jointed movements. It is likely that similar synaptic interactions are involved also in the use-dependent plasticity of the motor cortex. The proposed studies will test the hypothesis that plasticity in the functional organization of the motor cortex is dependent on changes in intrinsic synaptic pathways within the motor cortex. This hypothesis predicts that the development of movement representations is associated with corresponding changes in the patterns of intracortical connections. To test this prediction we will examine the normal developmental patterns of intrinsic circuits in the motor cortex. The development of movement representations in the rat will be revealed using microstimulation techniques. Neuroanatomical tract tracing techniques will be used to determine the development and refinement of intrinsic synaptic pathways among these representation zones. In addition, the development of intrinsic axons belonging to several classes of cortico-fugal neurons will be examined, using neuroanatomical and electrophysiological procedures. Particular emphasis will be placed on studying the development of intrinsic axon collaterals belonging to pyramidal tract neurons (PTNs) that provide output from the motor cortex to spinal motoneuron pools. Our working hypothesis also predicts that abnormal development of movement representations-induced by sensory-motor manipulations-is associated with abnormal development of intracortical circuits. To test this prediction we will examine the effects of experimental manipulations on the development of these intrinsic circuits. In these studies, the effects of clipping the mystacial vibrissae (whiskers) on the development of movement representation zones and of intrinsic connections will be examined. The electrophysiological and anatomical approaches described above will also be used for these experiments. This approach will serve as a model to study the effects of removal of an effector organ, at various developmental stages, on the development of the functional organization of the cerebral cortex. These data are necessary for the elucidation of the neurobiological mechanisms responsible for compensation from neurological disturbances and for restitution of behavioral functions, and are thus relevant for understanding the neuropathology of congenital motor disturbances such as cerebral palsy.

DESCRIPTION (provided by applicant): Whisking--the rhythmic whisker movements that rats perform while exploring their environment--is emerging as an exceptionally promising model for investigating mechanisms that control of voluntary movements. Exploitation of this model has been limited by lack of data on the pathways and mechanisms responsible for this important motor behavior. In the current funding period we made the exciting discovery that serotonergic pre-motoneurons are both necessary and sufficient to generate rhythmic firing in the facial motoneurons that control whisking. Thus, these serotonergic neurons are a critical component of a central pattern generator (CPG) for rhythmic whisking. We also showed that this serotonergic CPG receives dense inputs from the whisker representation of the motor cortex (wMCx). Finally, we demonstrated that--contrary to prevailing dogma--lesions of wMCx significantly affect whisking kinematics and rhythmicity. Our central hypothesis is based on these convergent findings. It states that wMCx modulates the activity of the serotonergic CPG that regulates the frequency of exploratory whisking, a function critical for sensorimotor integration and discrimination abilities. From this central hypothesis we formulate and test three novel hypotheses directed at a complete understanding of the neural mechanisms underlying this important motor behavior: Hypothesis I: Facial motoneurons that control whisking (whisking motoneurons) are synchronized by reciprocal electrical synapses. Hypothesis II: Serotonin released from the CPG dynamically regulates whisking frequency. Hypothesis III: wMCx modulates whisking frequency by its action on the serotonergic CPG.

DESCRIPTION (provided by applicant): The neocortex receives inputs from two classes of thalamic nuclei. First-order ("specific") nuclei relay to the neocortex sensory inputs from the periphery. Higher-order ("non-specific") thalamic nuclei receive and provide inputs to various cortical areas. Based on exciting new data we hypothesize that this dichotomy is overly simplistic, in that higher-order nuclei function also as important relays of sensory inputs from the periphery to cortical and subcortical structures. We will show that sensory processing by higher-order nuclei-specifically the posterior medial nucleus (POm), the higher-order nucleus of the somatosensory system-is regulated by a critical subthalamic nucleus - the zona incerta (ZI). We will further show that ZI's inhibitory regulation of POm is dynamically modulated by brainstem cholinergic inputs related to sleep-wake states, and by phasic motor cortex inputs related to voluntary movements. This State Dependent Gating (SDG) hypothesis is relevant to understanding the functions of all "higher-order" thalamic nuclei, as they are all subject to potent inhibition from ZI. Because both ZI and POm are implicated in various movement disorders, the anticipated findings may be relevant for understanding the pathogenesis of disorders such as Parkinson's disease.

Insults to the brain and spinal cord result not only in debilitating motor, sensory and cognitive deficits, but also in chronic, excruciating and relentless pain that is largely resistant to treatment. In most patients, pain starts weeks or months after the original insult, and includes increased pain with noxious stimulation (hyperalgesia), pain in response to previously innocuous stimuli (allodynia), and spontaneous pain. Spinal cord lesions typically produce particularly painful CPS symptoms, with unremitting pain that can be diffuse, bilateral, and may extend, "below-lesion", to locations caudal to the spinal injury. The delayed expression of CPS and the diffuse localization of painful symptoms suggest that the pathophysiology does not reflect only direct effects at the denervated spinal segments. Rather, these features of CPS strongly suggest the occurence of maladaptive plasticity in supraspinal structures at which inputs from various body parts converge, 'The ultimate goal of this application is to identify the factors that are causally responsible for this maladaptive plasticity following spinal cord injury. To this end, we adapted a rodent spinal cord injury model of CPS. We demonstrated that rats suffering from CPS have abnormally high neuronal activity in the posterior nucleus of the thalamus (PO). We also demonstrated that the activity of PO, and related thalamic nuclei, is tightly regulated by inhibitory inputs from the zona incerta and the anterior pretectal .nucleus. These findings suggest that CPS is associated with maladaptive plasticity in the incerto-thalamic pathway. Based on these exciting new findings, we propose that CPS can result from suppressed inhibition to thalamic.nuclei from zona incerta and the anterior pretectal nucleus. The proposed studies will use electrophysiological, behavioral and anatomical approached to test the validity of 4 key predictions that emerge from our overarching hypothesis.

DESCRIPTION (provided by applicant): The development of animal models of persistent pain has advanced our knowledge of the initiation of pain hypersensitivity due to sensitization of peripheral nociceptors and neurons in the medullary and spinal dorsal horns as well as other sites in the central nervous system (CNS). It is now commonly proposed that the initiation of this plasticity and resultant pain amplification is dependent upon activation of peripheral nociceptors. It is assumed that peripheral mechanisms are also responsible for the maintenance of the pain hypersensitivity, though the findings in this area are less compelling. Recent findings suggest that behavioral hyperalgesia following injury persists despite a reduction in peripheral neuronal activity suggesting that peripheral drive may be necessary but not sufficient for the maintenance of pain hypersensitivity after injury. The primary aim of this proposal is to study the mechanisms that underlie the maintenance of pain amplification after injury and to determine whether there are CNS mechanisms that participate in pain chronicity and how persistent pain emerges from the more acute stage after injury. Current animal models are not entirely adequate for the study of the chronicity and maintenance of pain hypersensitivity. We have developed two new models in the trigeminal system: a tendomyositis model of the masseter muscle and a neuropathic pain model of the infraorbital nerve. Both models exhibit long-term hyperalgesia/allodynia that is constant and lasts for months, and the inflammation due to the surgical procedure can be separated with the use of long-duration local anesthetics. Our major hypothesis is that tissue and nerve injury in the orofacial region can lead to the maintenance of secondary hyperalgesia that involves the activation CNS descending mechanisms and is less dependent on the peripheral drive associated with the injured target. The following Specific Aims will test these hypotheses using multidisciplinary approaches. #1: Test the hypothesis that the maintenance of long-term hyperalgesia after injury involves an attenuation of peripheral afferent drive and a transition to central mechanisms. #2: Test the hypothesis that the maintenance of long-term hyperalgesia is dependent upon an enhancement of descending facilitatory or a reduction of descending inhibitory inputs. #3: Test the hypothesis that the maintenance of long-term hyperalgesia is dependent upon an enhancement of descending facilitatory drive involving activation of trigeminal non-neural glial cells and their release of cytokines. #4: Test the hypothesis that the maintenance of long-term hyperalgesia is dependent upon an enhancement of descending facilitatory drive that involves shifts in the anionic reversal potential of trigeminal GABA-responsive neurons and a reduction in GABAA-induced inhibitory tone. #5: Test the hypothesis that the maintenance of long-term mechanical hyperalgesia is dependent upon rostral ventromedial medulla activation of a trigeminal brain stem interactive signaling cascade of 5-HT3, GABAA and NMDA receptors as well as glia and cytokines.

DESCRIPTION (provided by applicant): Classical theories of sensory processing view the brain as a passive, stimulus-driven device. More recent views see perception as an active and highly selective operation in which top-down influences strongly shape the bottom-up information flow. An important component of top-down regulation is the corticotrigeminal tract, which directly impacts the very first processing station for sensations from the head and neck. Despite its anatomical prominence very little is known about the functions of the corticotrigeminal pathway. Here we focus on its role in modulating noxious inputs. Based on strong preliminary findings, our central hypothesis is that corticotrigeminal inputs modulate pain perception by suppressing responses of neurons in the trigeminal nuclei. The caudal spinal trigeminal nucleus (SpVc), which plays a pivotal role in pain processing, receives cortical inputs primarily from primary (SI) and second (SII) somatosensory cortex, and the insular cortex. Aim I will use single unit recordings in anesthetized rats to: (1) determine whether corticotrigeminal inputs suppress the activity of SpVc projection neurons, and (2) compare the roles of inputs from SI, SII and insular cortex. Exciting preliminary findings indicate that SII strongly suppresses while SI excites SpVc projection neurons. Aim II will investigate the cellular bases of corticotrigeminal function using our recently developed optogenetic approach in which the light sensitive cation channel, channelrhodopsin, is expressed in corticotrigeminal neurons and their axon terminals. This novel approach allows selective activation of corticotrigeminal synapses in SpVc. Patch clamp recordings in in vitro slices will compare the properties of corticotrigeminal synaptic inputs to projection and local circuit neurons in SpVc. This will dissect the circuit and synaptic mechanisms by which excitatory corticotrigeminal inputs are transformed into potent feed- forward inhibition in SpVc. In Aim III we directly test the hypothesis that corticotrigeminal inputs regulate behavioral responses to nociceptive inputs. For this we have adapted a new operant behavioral paradigm that measures responses to thermal stimuli, and the effects of manipulating corticotrigeminal activity on these behavioral responses. These studies will disclose, for the first time, how corticotrigeminal inputs from each of the three cortical areas regulate pain perception. Unraveling the synaptic mechanisms of these modulatory influences will provide information needed to improve pharmacologic therapies for persistent pain. Finally, by pinpointing the roles of specific cortical regions in pain modulation, these results will advance current treatments that use cortical stimulation for pain relief.

DESCRIPTION (provided by applicant): This application requests support for years 15-20 of a highly successful training program in cellular and integrative neuroscience at the University of Maryland School of Medicine (UMSOM). Our goal is to provide multidisciplinary training to highly-qualified postdoctoral fellows based on the concept that neuroscientists require expertise in many facets of integrative neuroscience in order to participate effectively in advanced investigations of the nervous system. Cellular communication is central to the integrative properties of the nervous system and plasticity of communication is a broad fundamental principle; indeed, it is the organizing theme of the program. Six interactive interest groups constitute focal points for investigation and training. They are: 1) neural systems, 2) intercellular communication, 3) molecular factors in cell signaling, 4) developmental neuroscience, 5) glia, and 6) novel optical approaches in neuroscience. In keeping with our philosophy of preparing trainees for all aspects of a career in biomedical research, this application places increased emphasis on training in non-scientific aspects of the career. With support from the UMSOM administration, formal workshops and seminars in grant writing, lab management, mentoring skills, ethics in research, etc., are available to our trainees. The program steering committee, an interdepartmental group of five senior investigators, is responsible for the administration of the program. It evaluates applicants and accepts successful trainees, monitors trainee progress, and organizes an annual retreat. The steering committee and 16 other established neuroscientists with long track records of publications, successful postdoctoral training experience and extramural funding, constitute the primary faculty, which is responsible for supervision of trainees. Eight secondary faculty members collaborate with the primary faculty in research and training. Of the 24 program graduate since 2004, 23 remain in scientific careers (22 in research) in academia, government, research institutes or industry. This includes 10 Assistant Professors (9 at research universities), 9 of whom have won significant independent federal funding (2 R01's, 4 K01s, 2 R21s, and 1 R03). In view of this record, we are requesting continued support for seven trainees.

Project Summary/Abstract Chronic pain is the most common complaint of patients, affecting over 100 million Americans, and costing the nation more than $650 billion/year in medical treatment and lost productivity. Most chronic pain patients are resistant to pharmaceutical or surgical therapies, in large part because the underlying pathophysiology of their chronic pain condition is unknown. The ultimate goal of this research program is to rectify this deficiency. Most spinal cord pain-related afferents target the parabrachial nuclear complex (PB), which then projects to multiple pain-related cortical and subcortical targets. New preliminary data indicate that inhibitory inputs from the central nucleus of the amygdala (CeA) to PB are reduced in a rodent neuropathic pain model: chronic constriction of the infraorbital nerve (CCI). This reduced inhibition dramatically `amplifies' both spontaneous and evoked PB neural activity. As a consequence, there is increased PB excitation of several pain-related nuclei, including the rostral ventral medulla (RVM), a key node of the descending pain modulation system. Based on this exciting new evidence we hypothesize that chronic pain results from the development of a pathologic positive feedback network: Reduced inhibition from CeA to PB ?> amplified PB activity ?> increased activation of RVM neurons ?> increased activation of nociceptive neurons in the spinal cord. With the use of electrophysiological recordings from intact rodents and from brain slices, and taking advantage of behavioral approaches, optogenetics and pharmacogenetics, we will directly test this overarching hypothesis. Specifically, we will (1) Test the hypothesis CCI causes a progressive and significant reduction of inhibitory inputs to nociceptive PB neurons that project to RVM, and dramatically increases their firing; (2) Test the hypothesis that amplified PB activity is due to reduced inhibition from CeA.; (3) Test the hypothesis that reduced CeAI inhibition to PB is causally related to the development of CCI-Pain. The anticipated findings are expected to reveal novel mechanisms for the development of chronic pain, and may lead to development of novel therapies to ameliorate, and perhaps even prevent, this devastating condition.

Project Summary/Abstract Chronic pain is the most common complaint of patients. Most chronic pain patients are resistant to therapy, in large part because the underlying pathophysiology of their chronic pain condition is unknown. The ultimate goal of this research program is to fill this critical gap. Pain is strongly modulated by the rostroventral medulla (RVM) that directly regulates the activity of nociceptive dorsal horn neurons. Prominent in RVM are serotonin- containing neurons. However, the role of these neurons in chronic pain remains controversial, with evidence for both pathological increases and decreases in 5HT output. Our exciting preliminary findings?using a model of chronic pain after chronic constriction injury of the infraorbital nerve (CCI-Pain)?may resolve this important controversy. We show that, in CCI-Pain, RVM-5HT neuronal activity is amplified, resulting in abnormally high release of 5HT in the caudal dorsal horn ? trigeminal nucleus (SpVc). This causes SpVc neurons to produce a barrage of after-discharges (ADs) that far outlast nociceptive stimuli, and that are considered a manifestation of chronic pain. The increased 5HT release also potentiates the strength of nociceptive inputs to SpVc neurons. Coupled with our previous demonstration that reducing 5HT levels in RVM suppresses ADs and blocks pain sensitization, we hypothesize that increased serotonergic drive from RVM causes hyperexcitability of dorsal horn neurons, which results in chronic pain. Aim I tests the hypothesis that amplified activity of 5HT-RVM neurons results in increased release of 5HT in SpVc and the development of chronic pain. We test the prediction that the electrophysiological activity of optogenetically- identified 5HT RVM ?> SpVc projection neurons is amplified in CCI-Pain. We will also use in vivo fast scanning voltammetry, and quantitative mass spectrometry, to test the prediction that CCI-Pain is associated with increased 5HT release in SpVc. Aim II tests the hypothesis that increased 5HT release is causally related to the development of chronic pain. We will test the prediction that in vivo optogenetic release of 5HT from RVM terminals in SpVc results in signs of sensory and affective pain, and that these signs are exacerbated by repeated 5HT release. We will also test the converse prediction, that optogenetic inhibition of these 5HT terminals results in relief from CCI-Pain. Aim III tests the hypothesis that amplified 5HT activity produces chronic pain by inducing abnormal ADs in dorsal horn neurons. We will test the prediction that in vivo optogenetic release of 5HT induces ADs in SpVc neurons of uninjured animals, and that optogenetic inhibition of 5HT release will suppress ADs in CCI-Pain animals. Aim IV tests the hypothesis that amplified 5HT activity produces chronic pain by potentiating primary afferent inputs to dorsal horn neurons. We will test the prediction that optogenetic release of 5HT in vitro evokes potentiation of trigeminal inputs to SpVc neurons. The predicted findings have novel translational relevance for the development of new pharmaceuticals to treat chronic pain.