Marco Martina - US grants

Pain is the most common motive leading people to seek health care. When it becomes chronic, pain can produce several long term effects such as depression, loss of sleep, depressed immune function, decreased mobility, and other long-term deleterious consequences, several of which suggest the involvement of cortical areas implicated in higher cognitive functions. Although animal models advanced over the last 15 years have revolutionized our understanding of chronic pain mechanisms, the knowledge garnered in these models has concentrated primarily on mechanisms involving afferent inputs, spinal cord processes, and descending modulation. Little is known about supraspinal mechanisms, even less so about the interaction of pain and cortical processes. Recent human brain imaging studies in chronic back pain patients indicate medial prefrontal cortical hyperactivity, even in absence of nociceptive peripheral inputs. Other studies show impairment of decision making tasks in patients suffering of chronic pain and animal models show that blocking neuronal activity in the medial prefrontal cortex reversibly decreases neuropathic pain. Animal studies on inflammatory pain show functional consequences on glutamatergic synaptic transmission in the prefrontal cortex. All these observations suggest that functional and morphological changes may be present in the prefrontal cortex of animals with neuropathic pain. We will investigate this hypothesis in SNI rats, a highly reproducible model of neuropathic pain. Patch clamp recordings and morphological analysis of biocytin filled neurons will be performed to compare the functional and morphological properties of pyramidal neurons of the medial prefrontal cortex (mPFC) of SNI and sham-operated animals. We will compare the number and length of the dendrites and the dendritic spine density in SNI and sham-operated animals. Immunohistochemical analysis will be performed to investigate the expression of molecular markers of neuronal reorganization. Intrinsic electrophysiological properties as well as the pharmacological properties of glutamatergic synaptic transmission will also be investigated. Nucleated patch recordings and fast solution exchange will be used to perform a detailed study of the functional properties of the glutamate receptors expressed in mPFC pyramidal neurons of control and SNI rats. Our preliminary data show that, compared to sham-operated counterparts, mPFC neurons from SNI rats expressed higher levels of c-Fos, have larger dendritic trees, increased dendritic spine density and different molecular composition of glutamate receptors. Interestingly, several of these changes are correlated with the pain threshold in the injured paw. These observations support our hypothesis that neuropathic pain induces functional reorganization of the mPFC. Successful completion of our experiments could represent a leap forward in the study of the cellular mechanisms of neuropathic pain and open new fields of investigation.

DESCRIPTION (provided by applicant): Chemoreceptors sensitive to the levels of CO2 or pH in the central nervous system are critical to the regulation of cardiorespiratory homeostasis. Disturbances in their function contribute to the morbidity and mortality associated with a number of diseases such as congenital central hypoventilation syndrome, chronic obstructive pulmonary disease, as well as sleep apnea and sudden infant death syndrome (SIDS). Despite the importance of central chemoreceptors in cardiorespiratory function, the relevance of specific chemoreceptor sites to respiratory regulation, and even the specific cell types serving this function (eg neurons or glia) remain controversial. Among the best supported sites for central chemoreception are the nucleus of the solitary tract (NTS) and the retrotrapezoid nucleus (RTN). To define the relative contributions of these sites to central chemoreception and the conditions under which they are active we will address two essential questions: 1) What are the molecular/biophysical bases of chemoreception for the candidate brainstem neurons? and 2) What are the pathways by which they provide input to central circuits controlling breathing? The proposed project will employ complementary in vivo and in vitro electrophysiological approaches, combined with neuroanatomical and molecular methods to define the molecular/biophysical basis of chemosensitivity within NTS and RTN cells, as well as their direct or indirect connections with brainstem respiratory circuits. In vitro recordings will take advantage of isolated neurons as well as slice recordings. Two-photon calcium imaging in acute slices will provide information on the response profiles of chemosensitive neurons. Neurons in these brainstem areas will be dissociated to allow careful electrophysiological characterization of the chemosensitive response to extracellular acidification. Effects of intracellular acidification will be determined using dual pipette patch clamp recordings with intracellular perfusion. Chemosensitive cells recorded in vivo will be juxtacellularly labeled to define their somatodendritic organization and local axonal arborization. Their brainstem targets will be determined by retrograde labeling. Homology of the filled neurons recorded in vivo with chemosensitive cells identified in vitro will be determined by comparing their content of the relevant pH sensitive ion channels, cell morphology including axonal projection, and related neurochemical markers. The impact of specific chemosensitive neuron types on the response to hypercapnia will be assessed during pharmacological blockade/stimulation of the target ion channels (identified in vitro) where specific antagonist/agonists exist (eg for TRPV1 channels implicated in our preliminary data). These experiments will shed light on one central question of animal physiology and may suggest new pharmacological targets for drug therapy. PUBLIC HEALTH RELEVANCE: Breathing is finely tuned by the partial pressures of arterial and brain carbon dioxide and failure of this tuning may lead to dramatic consequences such as the sudden Infant Death Syndrome (SIDS) or congenital central hypoventilation syndrome (CCHS);yet, the mechanisms underlying this regulation are not well understood. In particular, the circuitries and molecular mechanisms of CNS chemoreception remain the subjects of major debate. This proposal marshals electrophysiological, molecular and histochemical techniques to identify the neuronal types involved in central chemoreception and dissect the underlying molecular mechanisms.

The Unipolar Brush Cells (UBCs) are interneurons situated in the cerebellar granular layer and in granule cell containing regions of the cochlear nuclear complex. In the cerebellar cortex, UBCs and granule cells share mossy fiber inputs and represent the only two excitatory glutamatergic neuron classes. In rodents, UBCs have a more restricted distribution than in other mammals and abound in vestibulo-cerebellar lobules, with minor contingents in other lobules of the midline vermis. Across mammalian species, UBCs are preferentially associated with sensory input systems, while they appear to eschew regions targeted primarily by the cortico-ponto-cerebellar pathway. The UBC has typically only one dendrite that terminates with a brush of dendrioles; these establish a giant excitatory synapse with the rosette-like terminal of a mossy fiber; UBC axons ramify among granule cells and form a strikingly unique, cortex-intrinsic system of mossy fiber-like branches. The UBC, with its one-to-one giant synapse, is thought to amplify the input of an individual fiber and synchronize the activity of hundreds of target granule cells, thus influencing the firing pattern of subsets of overlying Purkinje cells. While in cerebellum UBCs are highly enriched in the caudal vermal and lateral lobules densely innervated by primary and secondary vestibular fibers, in the cochlear nuclear complex their density is highest in the polysensory innervated dorsal nucleus. Consequently, the notion has been put forward that these unique neurons are important for regulating head position in space, influencing posture and eye movements and improving auditory performance. Previous evidence indicates the UBC population is chemically heterogeneous and consists of two main chemotypes, a subset expressing the calcium binding protein calretinin, and a calretinin-negative subset expressing the metabotropic glutamate receptor mGluR1a. In this competitive renewal, the P.I. and his collaborators propose to test individual facets of the hypotheses that the properties of UBCs subclasses are related to specific types of inputs, that additional subclass specific chemotypes exist, and that input qualities transmitted by the UBC axon affect the cerebellar network. Specific aims will analyze possible sublineage specific inputs of UBCs, investigate their developmental plasticity and electrophysiologic properties, search for novel UBC chemotypes and study the network impact of UBC excitation. The proposed research is based on multidisciplinary approaches and will be primarily centered on mice to take advantage of the availability of strains of mutant animals with genetically transmitted neurological defects as well as of bacterial artificial chromosome (BAC)-transgenic mice expressing enhanced green fluorescent protein (eGFP) under the control of a specific promoter.

? DESCRIPTION (provided by applicant): Febrile seizures affect up to 5% of children under the age of 5, and are the most common seizures in children of this age group. Current treatment of febrile seizures is limited and inadequate, often resulting in long uncontrolled seizures. Clinical observations suggest a possible etiologic link between early life prolonged febrile seizures and later development of chronic temporal lobe epilepsy. Despite increased risk of epilepsy in children with febrile seizures, these seizures are routinely not treated with anticonvulsants even if they are recurrent. In the past, phenobarbital had been used for over 25 years as prophylaxis in the treatment of recurrent febrile seizures, but anticonvulsant prophylaxis is no longer recommended because side effects appeared to outweigh the potential benefits. One of the main hurdles to the design of effective treatments is that, despite their obvious association with hyperthermia, the detailed molecular mechanisms of febrile seizures remain unclear. This proposal is based on our observation that a temperature dependent increase in intrinsic neuronal excitability driven by L-type voltage-gated calcium channels plays a critical role in the generation of febrile seizures in naive rodents. We will use patch clamp recordings and single-cell RT-PCR from acute slices to characterize the precise molecular identity of the channels involved and test the hypothesis that the same channels are also critical mediators of hyperthermic depolarization in neurons obtained from Scn1atm1Kea mice, an animal model of Dravet syndrome. Further experiments will test the hypothesis that nimodipine can prevent the development of seizures in slices obtained from the Dravet syndrome mice. We will investigate the effect of bath applied nimodipine on the temperature threshold, frequency and magnitude of the epileptic discharges. Finally, we will use behavioral analysis and EEG recordings to test the efficacy of nimodipine for the treatment of seizures in the rodent model of Dravet syndrome. If successful, these experiments may have immediate translational relevance because dihydropyridines have been used for decades in clinical context for the treatment of high blood pressure with negligible adverse effects. Therefore it is likely that the use of nimodipine for the treatment or prevention of febrile seizures would be devoid of negative side effects and that this compound could be used for the treatment of all types of febrile seizures, including those in Dravet patients for which novel pharmacological treatments are badly needed.

Project Summary Spinocerebellar ataxias (SCAs) are autosomal dominant neurodegenerative conditions characterized by cerebellar atrophy causing progressive motor incoordination (ataxia). Mechanistic insights into disease pathogenesis have been slow in coming, largely because many of the genes mutated encode for proteins whose function is still unknown. Consequently, no disease modifying therapy exists for these invalidating conditions. Recent data show that increased activity of one particular ion channel, TRPC3, which is highly enriched in the cerebellum and in cerebellar Purkinje cells in particular, is the cause of SCA41 and may also represent a shared mechanism in many SCAs. The TRPC3 gain-of-function in turn leads to impaired firing and neuronal toxicity. In this proposal we will take advantage of the ?moonwalker? mutant, a mouse model of SCA41 to test the therapeutic efficacy of TRPC3 blockade in slowing or stopping the course of the disease. The effectiveness of the treatment will be evaluated by quantification of behavioral, electrophysiological and pathological parameters.

Summary This proposal investigates the hypothesis that a heretofore unknown auditory pathway projects to unipolar brush cells (UBCs) of the cerebellar flocculus and paraflocculus (FL/PFL). This hypothesis is based upon three different and converging sets of preliminary data. The first line of evidence is provided by in vivo extracellular electrophysiological recordings from the gerbil flocculus showing a short latency, frequency-dependent response to acoustic stimulation. Interestingly, the firing pattern of the responder neuron closely resembles the peculiar firing pattern of UBCs. The second line of evidence shows that auditory stimulation leads to c-Fos expression in floccular neurons that have spatial distribution compatible with that of UBCs. The third evidence was obtained from injection of biotinylated dextran amine (BDA), an anterograde tracer dye, into the cochlear spiral ganglion. We found that BDA labeled fibers terminated in FL/PFL and contacted UBCs. Thus multiple sets of converging data strongly suggest that cerebellar UBCs receive auditory inputs. This finding is further supported by the fact that the only CNS region outside the cerebellar cortex where UBCs are expressed are the cochlear nuclei. The goal of this proposal is two-fold. We will perform further in-vivo electrophysiological recordings and examine auditory stimulation-induced c-Fos staining to confirm the existence and define the properties of the cerebellar responses to auditory stimulation (Specific Aim 1), and we will perform anatomical tracing experiments to determine the origin of the afferent cerebellar fibers carrying the auditory information (Specific Aim 2). Besides confirming this novel auditory pathway, our experiments will also determine the connectivity of cerebellar unipolar brush cells, which is still largely unknown. Cerebellar UBCs are classified into the less numerous type I and the more abundant type II neurons. Type I UBCs of the cerebellum are innervated by primary and secondary afferents from vestibular end organs and the vestibular nuclei, and, according to our preliminary finding, by primary auditory fibers. The afferents to type II UBCs are still completely unknown. Our experiments combining in vivo injections of anterograde and retrograde tracers, immuno-histochemical staining with cell-specific cell markers and electron microscopy will also determine the synaptic connectivity of these neurons. Successful completion of the proposed experiments will not only test the novel hypothesis that primary auditory fibers project to cerebellar UBCs, but will also help determine the connectivity of this still understudied cell type. Finally, because UBCs have the ability to generate action potentials in the absence of synaptic stimulation and their axons sprout and grow in response to cellular deafferentation, conclusive evidence that these UBCs are embedded in an auditory pathway would also suggest that they may originate electrophysiological activity that mediates phantom auditory perception after deafferentation. Therefore our studies may also open the way for new mechanistic studies of the generation of tinnitus.

Abstract - Rodent Behavior Core Our Center will have a Rodent Behavior Core to give scientific support to the animal research teams. This Core will provide the program team with services that are critical to the Center?s success. The Core will perform pain/control surgeries in rats and mice for all the laboratories involved in the Center. Most importantly the core will perform a whole set of behavioral tests to assess the multiple components of the neuropathic pain phenotype and its effect on drug seeking behavior. Having a common space to perform all the behavioral tests will allow higher reproducibility of these complex protocols. Along this line, the Core personnel will not only perform the behavioral assessments, but will also provide training and supervision to external personnel that will be designated by the individual laboratories part of this Center Grant to help with the tests. The Core will further aim to optimize and refine the behavioral protocols to ideally suit them to assess the complex behavioral interaction between pain and drug seeking phenotype. The results of such effort will be summarized in highly detailed standard operating procedures that will be posted online and will be available to the entire scientific community. Finally, the Core will allow laboratories currently not directly involved with pain and/ or drug addiction research to become involved in this type of research by providing them with preliminary data critical to apply for external funding. Thus, this Core will play a critical role in expanding the scientific community interested in this very important topic, which represents one of the most urgent priorities for NIH.

Summary The neurobiological basis of chronic pain is poorly understood and no scientifically validated therapies exist for such condition. Yet, chronic pain has an enormous socio-economic price, estimated to reach US$ 635 billion annually in healthcare costs and lost productivity. To make things worse, the majority of opioid abusers begin their addiction with prescription medications for chronic pain. Consequently, the search for new, non-opioid, pharmacological treatments for chronic pain constitutes one of the most urgent unmet medical needs. Beside its sensory symptoms, chronic pain is characterized by impairment of cognitive tasks such as attention and working memory, which depend on cholinergic modulation of medial prefrontal cortex (mPFC). Accordingly, mPFC deactivation was found to have a causal role for the neuropathic pain phenotype, and our preliminary data show that excitatory cholinergic modulation is severely impaired in mPFC pyramidal neurons of male rats. Yet, the precise mechanisms mediating the mPFC deactivation, how this deactivation influences pain perception and cognitive performance, and whether it similarly impacts males and females remain largely unknown. Our preliminary data show that a current mediated by the M1 receptor is critical for mPFC pyramidal cell excitability and is strongly reduced in neuropathic pain; our overarching hypothesis is that impaired cholinergic modulation of the mPFC represents a major mechanism of mPFC deactivation in neuropathic pain and mediates several of the sensory, cognitive and emotional symptoms. In particular, we hypothesize that in neuropathic pain: (1) cholinergic modulation of mPFC activity is disrupted and this critically contributes to the global mPFC deactivation in both sexes; (2) blockade of M1-mediated mPFC excitation is sufficient to mimic, at least in part, the neuropathic pain phenotype; (3) pharmacological manipulations that counterbalance the cholinergic disruption and restore mPFC output ameliorate cognitive and sensory symptoms of neuropathic pain. To test these hypotheses we will take advantage of the Spared-Nerve-Injury (SNI) model of neuropathic pain to pursue two specific aims. In Aim 1 we will combine optogenetic activation of individual cholinergic inputs, patch clamp recordings in acute slices and PCR analysis, to test the hypothesis that impaired cholinergic modulation contributes to the global mPFC deactivation, to determine the identity of the receptors involved, and to dissect the relative impact of cholinergic inputs from local interneurons and from the basal forebrain on mPFC activity in both females and males. In Aim 2 we will test the behavioral effects of impaired mPFC cholinergic modulation. We will use in-vivo chemogenetic and pharmacological modulation of the mPFC to reverse the SNI phenotype. We obtained preliminary data showing that enhancing mPFC excitability through pharmacological antagonism of the 5HT1a receptor has potent analgesic effects. Conversely, we will also investigate whether blockade of M1-mediated mPFC excitation in naive animals is sufficient to mimic the SNI phenotype. Our work will thus identify new potential targets for non-opioid neuropathic pain treatment.