Ford F. Ebner - US grants

Both physiological mapping and anatomical studies have demonstrated major differences in somatic sensory and motor cortex of mammals. These differences are reflected in the spatial separateness of the sensory and motor representations, the enlarged or diminished representation of each part of the body, and the degree of development of association cortex. In rats, there are even differences within SI; SI and MI are amalgamated in the hindlimb representation, but they are separated in the face region. We propose to study cortical organization in the rat using a combination of physiological and pathway tracing techniques at light and electron microscopic levels. The rat will be used as an experimental model to explore several factors that are important to maintaining separate sensory and motor areas in rodents. We will compare the corticocortical circuitry of identified face and hindlimb SI cortex to determine whether the separate SI face area has more extensive interconnections than the hindlimb cortex. Each area thus identified as interconnected with SI will be studied in a similar way to determine its connections with the thalamus and with other areas of cortex. In other studies, laminar lesions of face area SI cortex will be made in newborn animals in an attempt to establish an SI-MI amalgam for the face region that is similar to the naturally occurring amalgam of hindlimb SI cortex. The anatomical reorganization expected is that the VB projections can be induced to overlap with the VL projections in face area as they do normally in hindlimb cortex. These studies will clarify the cellular mechanisms underlying the differentiation of neocortex into more specialized subdivisions.

Experiments are proposed to determine why thalampcortical axons do not respond to injury in a way that leads to regeneration of connections and return of cortical function. Four hypotheses will be tested in the somatic sensory thalamus and cortex of the BALB/c mouse to find an explanation for this inability to grow and reform these important connections. One possible explanation is that the thalamic neurons are unable to mount an axonal growth response because they die after a cortical lesion. A second hypothesis is that extrathalamic axons may out-compete the regeneration of the thalamic fibers, perhaps by preempting available synaptic space. Third, the thalamic axons may start to grow, but not find the substrate molecules in the immediate area to use for guidance. Finally, after the thalamic axons mature they may permanently lose the capacity to elongate and form new synapses. Thalamic fiber growth will be studied using a transplantation paradigm in which embryonic cortex is placed in a corresponding region of an adult brain. The design for the first three sets of experiments involves changing the age of the donor tissue at the time it is placed in an adult host, while the last experiment requires that the host age be progressively reduced from adult to newborn. The analyses are focused on labeling the thalamic axons for examination in the light and electorn microscope and then correlating the axon distribution with the location of immunocytochemically localized cell surface and matrix molecules in the transplants. These results may potentially lead to improved clinical techniques for restoring neural circuitry following injury to the brain and spinal cord.

The goal of the proposed experiments is to understand the mechanisms that control sensory information processing in the cerebral cortex of adults mammals. Previous results by Armstrong- James and Fox, included in this proposal, have quantified the receptive field of cortical neurons in the barrel field cortex representing the mystacial vibrissae by measuring their response to vibrissal deflections. An important conclusion from these studies is that layer IV neurons that responded with the highest probability (2 or more spikes per stimulus over 50 repetitions) to stimulation of their appropriate vibrissa also responded at short latencies (<10 msec). In addition to the best response (here called the Center Receptive Field or CRF), layer IV neurons also showed lower probability, longer latency to onset of discharge to an additional number of whiskers (2.6 on average in the Excitatory Surround or SRF). Two hypothesis will be tested by the proposed experiments. One is that the high probability (>2 spikes/stimulus) + short latency (<10 msec) responses of barrel field cells to vibrissae deflection are generated through the VB pathway to cortex, while the lower probability (1 or fewer spikes/stimulus) + longer latency (>10 msec) responses are generated either through the spinal V to POM pathway or through corticocortical connections; either through SII cortex or short intracortical connections within SI or both. The second hypothesis is based on the observation that some components within SI cell activity depend upon mechanisms that require activation of acidic amino acid receptors, specifically the NMDA receptors. NMDA receptors have been shown to be essential for the generation of spontaneous burst activity, but not for the activation of cortical neurons by sensory inputs. Based on this differential dependence on the NMDA receptors, we predict that these receptors will be necessary for generating the surround, but not the center component of the layer IV cortical cell receptive field responses. Evidence supporting these hypotheses would provide new insights into the neural circuitry underlying the receptive fields of cortical neurons and could identify important functional interactions between the specific and the nonspecific thalamocortical projection systems that may regulate the strength of synapses in sensory cortex.

Neurons in somatic sensory cortex receive sensory information from the periphery throughout postnatal life. The central hypothesis for the proposed studies is that simple positive biases in the level of ongoing activity from selected whiskers is enough to potentiate their targeted synapses in cortex through glutamate receptor mechansisms. The studies will be carried out in the rat whisker to barrel field cortex pathway because the discrete, non-overlapping receptors in each whisker follide maintain a dear topography to and induding layer Iv of barrel field cortex. In addition, we have demonstrated that the receptive fields of neurons in adult rat barrel field cortex are modified by simply trimming some, but not all, of the whiskers for periods as short as 1 day or up to 30 days. Trimming all but two adjacent whiskers significantly increased the response of cortical neurons to spared whiskers and dramatically diminished the response to cut whiskers. The changes induced by whisker pairing were multifaceted and depend upon cell location in the barrel field cortex, the barrel's relationship to each whisker in the receptive field and how long they were trimmed or spared. Four specific aims are identified to analyze the circuits and mechanisms that support these plastic responses in adult rat cortex: Aim #1 is to compare the rate of change of synapses in each cortical layer induced by whisker pairing. Preliminary results show that supragranular neurons change before cells in any other layer. Two specific sub-goals are to test whether whisker pairing plasticity in the deeper layers requires prior changes in the superficial layers of cortex and whether the plasticity depends on glutamate receptor mechanisms. Aim #2 is to compare the limits of response enhancement that can be generated in the VPM thalamocortical fiber synapses before and after whisker pairing. Aim #3 is to analyze the role of extraIemniscal sensory modulators to barrel field cortex in the spread of activity between non-adjacent barrels when induced by whisker pairing. We will trim all but two non-adjacent whiskers to determine the effect of SII and POm lesions on the distance-limit for modifying connections between cortical barrels of the two intact whiskers. Aim #4 is to show the effect of depletion of non-sensory neuromodulators in cortex on whisker pairing plasticity.

The restoration of functional circuitry in damaged adult mammalian cerebral cortex remains the goal of the proposed research. During the previous grant period we showed that solid grafts consisting of thousands of neurons can be induced to form functional circuits with the host brain, so that graft cells can be driven by activity in the adult host sensory pathways. However, despite the fact that neurons in the solid grafts develop extensive internal circuitry, they form only weak connections with the host brain. That is, graft cells do not show normal levels of spontaneous activity and host-graft synapses do not show normal synaptic drive. The hypothesis that arises from these previous results is that the maturation of embryonic neurons grafted into adult cerebral cortex must be assisted in specific ways during sequential phases of differentiation: namely 1) during initial cell survival and process outgrowth, 2) during synaptogenesis and onset of neural activity and 3) during the activity-dependent (normally postnatal) phase of excitatory synapse adjustment. The proposed experiments will institute different procedures to facilitate each of these phases, with the ultimate goal of optimizing NMDA-receptor mediated plasticity in the host-graft synapses. In Specific Aim 1 the effect of growth factors will be tested on the initial survival of the dissociated embryonic cells and on the elongation of their neurites. In Specific Aim 2 the effect of modulatory neurotransmitters (NE + Ach,) on the graft cells will be tested after process formation has occurred to determine the modulators effect on initial synaptic activity. In Specific Aim 3 graft cells will be stimulated in ways that synchronize afferent sensory fiber activity and depolarization of the grafted neurons. Stimulation of sensory inputs will allow us to assay the effect of activity on the development of synaptic properties in the grafted neurons and on the induction of NMDA receptor function. The main series of experiments will be carried out in vivo, with quantitative studies of growth factors, modulators and stimulation dose-response effects being quantified in cultures of dissociated neurons. In parallel with the cellular analysis, a final aim (4) will be to assay the ability of the graft to restore specific behavioral deficits caused by the cortical lesion. The results of these studies should lead to a clearer understanding of the optimal strategy for enhancing the development of plasticity mechanisms and for restoring function in damaged cortical circuits.

neurosciences; developmental neurobiology; biomedical facility; microscopy; histology; immunocytochemistry; imaging /visualization /scanning;

The restoration of functional circuitry in damaged adult mammalian cerebral cortex remains the goal of the proposed research. During the previous grant period we showed that solid grafts consisting of thousands of neurons can be induced to form functional circuits with the host brain, so that graft cells can be driven by activity in the adult host sensory pathways. However, despite the fact that neurons in the solid grafts develop extensive internal circuitry, they form only weak connections with the host brain. That is, graft cells do not show normal levels of spontaneous activity and host-graft synapses do not show normal synaptic drive. The hypothesis that arises from these previous results is that the maturation of embryonic neurons grafted into adult cerebral cortex must be assisted in specific ways during sequential phases of differentiation: namely 1) during initial cell survival and process outgrowth, 2) during synaptogenesis and onset of neural activity and 3) during the activity-dependent (normally postnatal) phase of excitatory synapse adjustment. The proposed experiments will institute different procedures to facilitate each of these phases, with the ultimate goal of optimizing NMDA-receptor mediated plasticity in the host-graft synapses. In Specific Aim 1 the effect of growth factors will be tested on the initial survival of the dissociated embryonic cells and on the elongation of their neurites. In Specific Aim 2 the effect of modulatory neurotransmitters (NE + Ach,) on the graft cells will be tested after process formation has occurred to determine the modulators effect on initial synaptic activity. In Specific Aim 3 graft cells will be stimulated in ways that synchronize afferent sensory fiber activity and depolarization of the grafted neurons. Stimulation of sensory inputs will allow us to assay the effect of activity on the development of synaptic properties in the grafted neurons and on the induction of NMDA receptor function. The main series of experiments will be carried out in vivo, with quantitative studies of growth factors, modulators and stimulation dose-response effects being quantified in cultures of dissociated neurons. In parallel with the cellular analysis, a final aim (4) will be to assay the ability of the graft to restore specific behavioral deficits caused by the cortical lesion. The results of these studies should lead to a clearer understanding of the optimal strategy for enhancing the development of plasticity mechanisms and for restoring function in damaged cortical circuits.

high performance liquid chromatography; histochemistry /cytochemistry; laboratory mouse; tissue /cell culture

Neurons in somatic sensory cortex receive sensory information from the periphery throughout postnatal life. The central hypothesis for the proposed studies is that simple positive biases in the level of ongoing activity from selected whiskers is enough to potentiate their targeted synapses in cortex through glutamate receptor mechansisms. The studies will be carried out in the rat whisker to barrel field cortex pathway because the discrete, non-overlapping receptors in each whisker follide maintain a dear topography to and induding layer Iv of barrel field cortex. In addition, we have demonstrated that the receptive fields of neurons in adult rat barrel field cortex are modified by simply trimming some, but not all, of the whiskers for periods as short as 1 day or up to 30 days. Trimming all but two adjacent whiskers significantly increased the response of cortical neurons to spared whiskers and dramatically diminished the response to cut whiskers. The changes induced by whisker pairing were multifaceted and depend upon cell location in the barrel field cortex, the barrel's relationship to each whisker in the receptive field and how long they were trimmed or spared. Four specific aims are identified to analyze the circuits and mechanisms that support these plastic responses in adult rat cortex: Aim #1 is to compare the rate of change of synapses in each cortical layer induced by whisker pairing. Preliminary results show that supragranular neurons change before cells in any other layer. Two specific sub-goals are to test whether whisker pairing plasticity in the deeper layers requires prior changes in the superficial layers of cortex and whether the plasticity depends on glutamate receptor mechanisms. Aim #2 is to compare the limits of response enhancement that can be generated in the VPM thalamocortical fiber synapses before and after whisker pairing. Aim #3 is to analyze the role of extraIemniscal sensory modulators to barrel field cortex in the spread of activity between non-adjacent barrels when induced by whisker pairing. We will trim all but two non-adjacent whiskers to determine the effect of SII and POm lesions on the distance-limit for modifying connections between cortical barrels of the two intact whiskers. Aim #4 is to show the effect of depletion of non-sensory neuromodulators in cortex on whisker pairing plasticity.

[unreadable] DESCRIPTION (provided by applicant): The goal of the proposed research is to understand how tactile experience with the whiskers modifies the receptive field (RF) of neurons in the septa around cortical barrels, and what role septal neurons play in sensory processing and cortical plasticity. Anatomically, layer IV barrel neurons project horizontally into the septa surrounding the barrels and vertically above the barrel to layers II+III in a cap-like pattern that overlaps around each barrel. In contrast to barrels, which are dominated by specific thalamic VPM inputs, septal neurons are the cortical target of less dense inputs from the thalamus and commissural inputs from the contralateral cortex. When two whiskers are stimulated nearly simultaneously, some cells in the septum show increased (facilitated) responses. If there is a delay of 20-50 ms between whisker stimuli, the response to the second whisker is inhibited in barrel cells, but not studied in septal cells in relation to their location. We propose that the septa are specialized regions for modifying cortical responses to inputs that are generated by several vibrissa. Our main hypothesis is that the topography in the septal regions provides a distributed cell matrix in which the strength of whisker inputs is compared and modified through use-dependent, intracortical connection plasticity. We propose that septal cells operate as sensory information modulators in that they integrate the activity of several barrels and feed back reciprocally to modify barrel RF's. We will test the notion that septal cells contribute strongly to experience-driven modifications of barrel cell responses using a test for plasticity that we developed, called "whisker pairing plasticity". Specifically, we wish to define the role of whisker experience in determining adult septal cell plasticity to show how use-dependent plasticity is developmentally regulated. Analysis of these characteristics of the septal cells will provide significant new information about sensory processing in the barrel field cortex. The results will generate a better understanding of how sensory activity modifies the RF of cortical neurons throughout life, and the experiments will specify integrative deficits that are produced in septal neurons by inadequate early sensory experience (sensory deprivation). [unreadable] [unreadable]