Thomas M. Jessell - US grants

The aim of this project is to examine the cellular mechanisms responsible for the transmission of sensory information at afferent synapses in the dorsal horn of the spinal cord. Several classes of cutaneous afferent fibres have been defined physiologically and morphologically but the transmitters released from individual subclasses of sensory neurons are unknown. Biochemical, cytochemical and immunological techniques will be used to identify specific chemical markers for subpopulations of dorsal root ganglion (DRG) neurons. These markers will be used to correlate chemical and functional properties of DRG neurons and to provide information on the transmitters released by sensory neurons. The physiological action of potential sensory transmitters, will be examined by intracellular recording from dorsal horn neurons grown in dissociated cell culture. Co-cultures of DRG and dorsal horn neurons will be used to compare the response of dorsal horn neurons to sensory synaptic input and transmitter candidates. Attention will be focused on a class of DRG neurons that contain a 5'-nucleotide hydrolysing enzyme and the possibility that these, and perhaps other, DRG neurons release ATP as an excitatory transmitter. A subpopulation of dorsal horn neurons are selectively and potently excited by ATP and indirect evidence suggests that ATP may be a sensory transmitter. This possibility will be investigated using biochemical, immunological and physiological techniques. To provide new information on the properties of sensory afferents, monoclonal antibodies will be generated against both defined and novel antigens expressed on the surface and in the cytoplasm of DRG neurons. Defined antigens include the 5'-nucleotide hydrolysing enzyme and the ATP binding site. Somatic cell hybridization techniques will be used to generate hybrid cell lines that express differentiated properties of DRG neurons and provide large amounts of material for biochemical and immunological studies. These studies are intended to improve understanding of the way in which information is processed in the spinal cord.

The aim of this project is to examine the cellular mechanisms responsible for the transmission of sensory information at afferent synapses in the dorsal horn of the spinal cord. Several classes of cutaneous afferent fibres have been defined physiologically and morphologically but the transmitters released from individual subclasses of sensory neurons are unknown. Biochemical, cytochemical and immunological techniques will be used to identify specific chemical markers for subpopulations of dorsal root ganglion (DRG) neurons. These markers will be used to correlate chemical and functional properties of DRG neurons and to provide information on the transmitters released by sensory neurons. The physiological action of potential sensory transmitters, will be examined by intracellular recording from dorsal horn neurons grown in dissociated cell culture. Co-cultures of DRG and dorsal horn neurons will be used to compare the response of dorsal horn neurons to sensory synaptic input and transmitter candidates. Attention will be focused on a class of DRG neurons that contain a 5'-nucleotide hydrolysing enzyme and the possibility that these, and perhaps other, DRG neurons release ATP as an excitatory transmitter. A subpopulation of dorsal horn neurons are selectively and potently excited by ATP and indirect evidence suggests that ATP may be a sensory transmitter. This possibility will be investigated using biochemical, immunological and physiological techniques. To provide new information on the properties of sensory afferents, monoclonal antibodies will be generated against both defined and novel antigens expressed on the surface and in the cytoplasm of DRG neurons. Defined antigens include the 5'-nucleotide hydrolysing enzyme and the ATP binding site. Somatic cell hybridization techniques will be used to generate hybrid cell lines that express differentiated properties of DRG neurons and provide large amounts of material for biochemical and immunological studies. These studies are intended to improve understanding of the way in which information is processed in the spinal cord.

The aim of this proposal is to provide a clearer understanding of the cellular and molecular mechanisms underlying the transmission of nociceptive information at synapses between small diameter primary afferent fibers and neurons in the dorsal horn of the mammalian spinal cord. Studies over the past decade have identified acidic amino acids and neuropeptides as two major classes of excitatory neurotransmitters that appear to be release from primary afferent fibers. However the detailed mechanism of action and interaction of these substances and the sensory modalities that they transmit are poorly understood. These problems will be addressed using two new in-vitro preparations; I/ a transverse slice preparation of adult rat spinal cord with an attached dorsal root that permits intracellular recording from identified substantia gelatinosa (s.g.) neurons and activation of A-delta and C fiber synaptic inputs to these neurons. II/ a preparation of isolated neonatal rat dorsal horn neurons that permits the post-synaptic actions of i-glutamate and substance P to be examined by whole cell recording and by measurement of intracellular Ca++ levels. These preparations will be used to determined the chemical identify and physiological actions of transmitters that mediate fine fiber input to s.g. neurons. the post-synaptic actions of I-glutamate on s.g. neurons will be examined in detail to determine the role of different excitatory amino acid receptors in mediating I-glutamate actions and afferent evoked excitation. The possibility that some primary afferents use fast transmitters other than I-glutamate will also be examined. In particular, the role of ATP as an excitatory transmitter in the dorsal horn will be determined. Intracellular dye injection will be used to determine the morphology and anatomical classification of s.g. neurons that have been characterized physiologically. The role of substance P in sensory transmission will be examined in two ways; I/ by determining the actions of substance P on s.g., neurons in the slice preparation, with attention focussed on the actions of the peptide on after potentials in these neurons, II/ by monitoring the intracellular events triggered by substance P receptor activation using Ca++ sensitive dyes and whole cell recording. Two distinct classes of dorsal horn neuronal responses to substance P have been defined through the use of Ca++ sensitive dyes. the mechanisms of signalling evoked by substance P in these two classes of neurons will be examined in detail.

The floor plate is a transient epithelial cell group which begins to differentiate at the midline of the neural plate and later occupies the ventral midline of the neural tube and developing central nervous system (CNS). Recent studies indicate that cells of the floor plate have a central role in the early development of the vertebrate CNS. The floor plate, together with the underlying notochord, appears to control the pattern of cell differentiation along the dorsoventral axis of the neural tube, and may also contribute to the regionalization of the neural plate along its anteroposterior axis. Later, the floor plate appears to guide the axons of a subset of central neurons by releasing a diffusible chemoattractant which promotes the outgrowth and orientation of axons in vitro and in vivo. In addition, the floor plate appears to act as an intermediate target involved in the contact-dependent guidance of axons that cross the ventral midline of the CNS. The molecular mechanism that underlie these cellular functions of the floor plate remain poorly characterized. In an attempt to analyze the molecular basis of floor plate function we have begun to identify the novel floor plate-specific transcripts. Using a new subtractive hybridization strategy we have isolated several floor plate enriched or specific cDNAs and assessed their distribution by in situ hybridization. In this application we propose to perform a series of experiments to determine the identity and function of floor plate specific genes. One of the genes that has already been cloned and characterized, FP5, encodes on novel diffusible protein with striking homologies to proteins implicated in cell adhesion, chemotaxis and haptotaxis. We will examine the function of the FP5 protein, focussing on its potential roles in the control of neural cell differentiation and axon growth and guidance. In addition, we will complete the structural and functional characterization of two other floor plate-specific genes using similar assays. Finally, we will use refined subtractive hybridization methods to identify additional floor plate specific genes that may be involved in the developmental signalling properties of the floor plate. The information that derives from these studies should provide a clearer understanding of the cellular and molecular mechanisms that control the early development of the vertebrate central nervous system.

DESCRIPTION (provided by applicant): Hox proteins and their transcriptional co-factors have crucial roles in specifying motor neuron identity but their role in directing the assembly of sensory-motor circuits remains less clear. This proposal takes advantage of the discovery that the forkhead transcription factor FoxP1 gates the output of the entire motor neuron Hox repertoire, and determines the subtype identity of spinal motor neurons. We will use motor neuron-specific FoxP1 mutant mice to probe three aspects of the way in which motor neuron pool specification nucleates the organization and function of this prototypic mammalian sensory- motor circuit. First, we will explore the role of Hox/FoxP1 programming in the formation and maintenance of topographic motor projections to target limb muscles. Anatomical tracing methods will be used to define the intraspinal position of lumbar motor neurons that innervate specific limb muscle targets in the absence of FoxP1 function, asking whether motor neuron cell bodies that innervate a given muscle target are positioned randomly within the limb-innervating cohort. In addition, we will use physiological recording and kinematic analyses to assess muscle activity patterns and locomotor behavior in mice in which motor neuron Hox/FoxP1 programming has been abolished. Second, we will examine how the loss of motor pool identity influences patterns of proprioceptive sensory connectivity. Anatomical tracing and physiological recordings from spinal motor neurons will compare the profile of monosynaptic connections between antagonist pairs of sensory and motor neurons in control and motor neuron-specific FoxP1 mutant mice. In parallel, we will determine the impact of loss of motor pool identity on the molecular specification of subsets of proprioceptive sensory neuron that supply individual limb muscles. Third, we will examine how the loss of motor pool identity in FoxP1 mutants perturbs the selectivity of assembly of local inhibitory circuits. Physiological studies will determine how the pattern of connectivity of Ia inhibitory interneurons with motor neurons is altered in motor neuron specific FoxP1 mutants. Molecular markers will be used to identify Ia inhibitory interneurons and examine how the erosion of motor pool identity influences the output and connectivity of this set of interneurons. Together, these studies are intended to forge a link between molecular programs of motor circuit assembly and the behavioral output of these circuits. In the long term, these insights should aid in the design of more effective strategies for promoting functional recovery of spinal circuits after traumatic spinal cord injury, and in motor neuron degenerative diseases.

The study of motor neuron differentiation in the developing spinal cord has provided a model system for defining the cellular interactions and molecular nature of inducing factors that control neuronal identify in the vertebrate nervous system. The source and origin of signals that control the diversification of motor neuron subtypes however remains unclear. In this project we will focus on the control of motor neuron diversity in the lateral motor column (LMC), a class of motor neurons that innervates target muscles in the limb. In preliminary studies we have provided evidence that three distinct signaling molecules that have been implicated in cellular differentiation and transformation: retinoids, ETS domain transcription factors and receptor tyrosine kinases of the Eph family are expressed by subsets of motor neurons in the LMC. These studies suggest that the analysis of motor neuron subtype diversity in the LMC may provide novel information on the function of classes of molecules implicated more generally in cellular differentiation and oncogenesis. Using a combination of in vitro assays of motor neuron differentiation and molecular genetic manipulations of gene expression in motor neurons in mouse embryo we will address three main issues: 1. The role of retinoid signaling in the specification of motor neuron columnar subtype identity within the LMC. 2. The role of ETS transcription factors in specifying the identity of motor neuron pool identity within the LMC. 3. The role of Eph kinases and their Ephrin ligands in the organization of motor neurons in the LMC and in the projection of specific subsets of motor axons to their targets in the limb.

The floor plate is a transient epithelial cell group which begins to differentiate at the midline of the neural plate and later occupies the ventral midline of the neural tube and developing central nervous system (CNS). Recent studies indicate that cells of the floor plate have a central role in the early development of the vertebrate CNS. The floor plate, together with the underlying notochord, appears to control the pattern of cell differentiation along the dorsoventral axis of the neural tube, and may also contribute to the regionalization of the neural plate along its anteroposterior axis. Later, the floor plate appears to guide the axons of a subset of central neurons by releasing a diffusible chemoattractant which promotes the outgrowth and orientation of axons in vitro and in vivo. In addition, the floor plate appears to act as an intermediate target involved in the contact-dependent guidance of axons that cross the ventral midline of the CNS. The molecular mechanism that underlie these cellular functions of the floor plate remain poorly characterized. In an attempt to analyze the molecular basis of floor plate function we have begun to identify the novel floor plate-specific transcripts. Using a new subtractive hybridization strategy we have isolated several floor plate enriched or specific cDNAs and assessed their distribution by in situ hybridization. In this application we propose to perform a series of experiments to determine the identity and function of floor plate specific genes. One of the genes that has already been cloned and characterized, FP5, encodes on novel diffusible protein with striking homologies to proteins implicated in cell adhesion, chemotaxis and haptotaxis. We will examine the function of the FP5 protein, focussing on its potential roles in the control of neural cell differentiation and axon growth and guidance. In addition, we will complete the structural and functional characterization of two other floor plate-specific genes using similar assays. Finally, we will use refined subtractive hybridization methods to identify additional floor plate specific genes that may be involved in the developmental signalling properties of the floor plate. The information that derives from these studies should provide a clearer understanding of the cellular and molecular mechanisms that control the early development of the vertebrate central nervous system.

DESCRIPTION: The assembly of neurons into functional neural networks underlies all aspects of behavior-from simple sensory reflexes and motor responses to more complex cognitive functions. Within the developing vertebrate central nervous system, perhaps the simplest neural circuit is that coordinating sensory input with motor output-the monosynaptic stretch reflex circuit of the spinal cord. The function of this circuit depends on the selective interconnections of three major classes of neurons: motor neurons, primary sensory neurons, and a set of ventral interneurons that regulates the output of motor neurons. Understanding how this simple circuit is generated during development depends first on defining how the distinct identities of these specific neuronal subtypes if acquired. For it is the early acquisition of such subtype identities that provides neurons with the ability to form selective connections with specific neural targets. The major aim of this project is therefore to define the molecular mechanism that controls the differentiation of motor neurons and interneurons that underlie the formation and function of motor circuits in the developing spinal cord. The project will focus in particular on the key role of transcription factors in the specification of neuronal subtype identity in the ventral spinal cord. Our previous studies have begun to provide evidence that genes encoding two classes of homeodomain proteins, the Pax and Nhr genes have critical roles in defining motor neuron and ventral interneuron identity and eventually their connectivity. The present proposal will attempt to define in molecular detail the function of these transcription factors in directing the identity and assembly of neuronal subtypes in the developing mammalian spinal cord. These studies should therefore provide a first step towards the understanding of the molecular basis of neuronal circuit formation and its control of a simple motor behavior. We propose to define the mechanisms whereby distinct classes of motor neurons and interneurons are generated. To this end, we propose to carry out three specific sets of experiments. 1. Defining Pax6 Functions in Shh Signaling and Ventral Patterning. 2. Defining the Role of Nkx Genes in Shh Signaling and Ventral Patterning. 3. Analysis of Ventral Patterning Defects in Compound Nkx2.2 and Nkx2.9, Pax6, NKx2.2, and Pax6 x Nkx2.9 Mutants.

DESCRIPTION (provided by applicant): The pattern of monosynaptic sensory-motor connections in the mammalian spinal cord is thought to be hard-wired, but there has been slow progress in identifying surface recognition systems that mediate input selectivity. Classical cadherins are known to delineate motor neuron subtypes according to their pool identities, and gain and loss of function studies have demonstrated that cadherin activity is required for the sorting of motor neurons into pools. Cadherins are also expressed by proprioceptive sensory neurons, and there is evidence for coordination in the profiles of cadherin expression by functionally interconnected sensory and motor neuron subsets. These observations raise the possibility of a developmentally relevant cadherin 'matching code' in which the homophilic or heterophilic interactions of paired cadherins expressed by sensory afferents and motor neurons promote adhesive interactions that direct synaptic specificity. This proposal aims to clarify the role of cadherin recognition in sensory-motor connectivity through a molecular analysis of the impact of classical cadherin inactivation in motor neurons. The early lethality of existing mouse motor neuron ß?-cat and N-cad mutants has precluded analysis of an independent role in sensory-motor connectivity. To bypass this problem we will use anatomy and physiology to examine sensory-motor connectivity profiles in viable ß?-cat and cadherin mutants, generated through the use of more selective cre driver lines that permit inactivation of target genes in restricted subsets of spinal motor neurons. We will examine whether selective disruption of ß?-cat, N-cad or type II cadherins alone or in combination is sufficient to erode the fidelity of sensory motor connections. To probe further the mechanisms of motor neuron cadherin signaling we will use cell binding assays to explore the logic and specificity of interactions between N-cadherin and the many type II cadherins expressed by motor neurons. Together, these studies are intended to define the molecular basis of synaptic connectivity at sensory-motor synapses, a key early step in the establish ment of functional motor circuits.

DESCRIPTION (provided by applicant): Proprioceptive sensory neurons (pSNs) serve a key role in refining the output of the spinal motor system through the provision of feedback signals that convey the state of muscle activity to central and spinal motor neurons. Distinct pSN subtypes engage with select spinal circuits dedicated to specific musculoskeletal tasks (e.g. postural control, knee flexion, ankle extension etc). This precision in sensory-motor connectivity is presumed to rest in large part on the molecular distinctions between the various proprioceptor subtypes, yet surprisingly little is known of the way in which proprioceptor subtype identity is established. Challenging prevailing views, our recent studies suggest that certain aspects pSN subtype character are mediated by graded signaling by neurotrophin 3 (NT3) rather than by intrinsic transcriptional determinants. This idea is founded on several observations, most notably the finding that embryonic muscles exhibit muscle- by-muscle differences in NT3 expression levels at the time when pSNs establish their subtype identity. The hypothesis that variations in the strength of NT3 signaling direct pSN subtype character leads to two predictions. First, if graded NT3 signaling drives pSN subtype diversity, NT3 should elicit distinct molecular responses in pSNs that innervate muscle targets expressing different levels of NT3. Second, based on the notion that pSN identity is inherently linked to spinal connectivity patterns, changes in NT3 signaling levels should result in alterations in pSN connectivity patterns. The experiments in the proposal are designed to test these expectations. In agreement with our predictions, in preliminary studies, we identified several molecular markers that are differentiall expressed between pSN subsets that innervate NT3high -and those that innervate NT3low muscle targets. These molecular markers not only provide new insights into the various aspects of pSN subtype identity, but importantly, are powerful tools through which to assess the role of NT3 in regulating pSN diversity (Aim 1). In addition (Aim 2), we will take advantage of newly developed strategies - based on anterograde transsynaptic transfer of recombinant rabies virus - to construct an anatomical framework of the spinal connectivity patterns of defined NT3low and NT3high pSN subsets, and examine the role of NT3 signaling in establishing these patterns. Ultimately, these analysis' should lead to new insights in cardinal molecular and network features of pSN subtypes and may begin to reveal the organizational rules that underlie the formation of spinal sensory- motor circuits.

Abstract Motor cortex (M1) plays a crucial role in the control of voluntary movement but the neuronal mechanisms by which this region converges on downstream circuits remain obscure. To better understand how activity in M1 is translated into context-appropriate behaviors, we propose to characterize the role of specific cortical projection subtypes as defined by their spinal interneuron targets. A growing body of electrophysiological and behavioral evidence suggests that interneuron subsets in the spinal cord represent functional units that, when recruited, play a role in imposing a task-appropriate pattern of muscle activation. An example of this is seen in the alternating activity of opposing flexor and extensor muscles, which is in part regulated by defined spinal circuits. These circuits can be over-ridden to achieve simultaneous antagonist muscle coactivation. Two inhibitory spinal interneuron (IN) populations, presynaptic GABAergic (GABApre) and reciprocal inhibitory GABA/glycineric (Ia) neurons, play a role in this transition, undergoing activation or suppression of their activity, respectively. Using a recently developed CVS-N2C based rabies tracing strategy combined with genetic access to specific cardinal interneuron populations, we have identified that both classes receive direct corticospinal neuron (CSN) inputs. Moreover, this input has been suggested to play a role in the task-appropriate switching from alternating activation to co-activation of flexor-extensor muscle pairs. In order to investigate the nature of this role, we have developed a motor behavioral task in, which mice are trained to either alternate or co-contract opposing forelimb muscles in response to a visual cue. For the first time, we can now simultaneously monitor and manipulate the activity of defined subsets of CSNs during a motor-cortically dependent behavior.

Understanding the mechanisms that the nervous system uses to control movement is critical for understanding brain and behavior, and one of the fundamental questions in neuroscience. The control of movement emerges from the activity of different motor control centers, that converge onto output systems, mostly located in the spinal cord. While the spinal circuits that underlie different aspects of motor control have been relatively well characterized, the way by which these circuits are coordinated by supraspinal motor control centers remains elusive. In this project, we aim to understand the functional and computational logic of connectivity between a motor control centers, the motor cortex, and the spinal cord and muscle. We will anatomically and functionally characterize the role of projection-specific populations of corticospinal neurons during particular modes of motor control. Because even the simplest motor program requires the activation of many neuronal populations across multiple brain areas, we will also investigate the contribution of other cortical and subcortical areas to the output of the brain to the spinal cord, and to muscle activity. This understanding requires It also requires extracting the information that is carried between brain areas and neuronal cell types, and understanding the computations that are operated in the circuits in order to achieve specific patterns of muscle activation. We will extract computational principles governing the relation between brain activity and muscle activity that are conserved between rodents and , and will construct predictive models of . In order to achieve a mechanistic understanding of the brain circuits underlying motor control, we will dissect the contributions of activity in specific neural populations using closed-loop optogenetic manipulations. The level of understanding that we are seeking requires a dynamic back and forth between anatomical and functional mapping experiments, computational and conceptual models, and causal testing of predictions. We put together a a multidisciplinary team of PIs working in a tight network, sharing the latest technologies to measure and manipulate the brain through an Advanced Imaging and Instrumentation core, creating and refining circuit models based on data that generate testable predictions, and establishing real-time knowledge exchange between team members through a Data Science Core. Our U19BCP Motor Control team proposes a comprehensive and ambitious project to establish the computational and circuit mechanisms underlying classical modes of motor control based on cell-type specific connectivity between brain and spinal cord, novel technology to measure and manipulate functionally and genetically-defined neural populations, and state-of-the-art computational tools. primates multi-area dynamics during motor control