Joseph Travers - US grants

DESCRIPTION (Adapted From The Applicant's Abstract): The objectives of this proposal are to determine the neural substrates in the rodent hindbrain through which gustatory stimuli guide ingestive behavior. The regulation of ingestive behavior is exceedingly complex, involving widespread regions of the central nervous system, but the basic regulatory function of taste to determine palatable from unpalatable stimuli is complete in the caudal brainstem. Anatomical studies suggest that polysynaptic pathways in the reticular formation serve as an interface between the gustatory region of the nucleus of the solitary tract and the oromotor nuclei that produce the consummatory responses of ingestion and rejection. Delineating this local circuitry is prerequisite to understanding how descending forebrain pathways further influence consummatory function. Specific subdivisions of the reticular formation including the lateral parvocellular and intermediate zones are hypothesized to mediate specific components of sensorimotor coordination. The proposed experiments combine several techniques to further understand the connectivity, neurochemistry and function of these reticular regions by (1) using double-labeling immunohistochemistry for identification of neurotransmitter phenotypes with concomitant increases in the expression of the immediate early gene c-fos produced by gustatory stimulation, (2) assessing the connectivity and neurochemical phenotype of interneurons with projections to specific pools of lingual and masticatory motorneurons assayed with double labeling techniques that use the transynaptic properties of two genetically altered strains of pseudorabies virus, (3) determining the effects of reversible inactivation of small regions of the reticular formation following microinfusions of either lidocaine or antagonists to suspected neurotransmitters in the circuitry on ingestion and rejection elicited with intraoral stimulation and measured electromyographically from a subset of lingual, masticatory and pharyngeal muscles. Chronic diseases such as obesity, hypertension, and anorexia nervosa all involve ingestive dysfunction. Understanding the neurological basis of the fundamental biological decision to ingest or reject food will contribute to the eventual solution of these chronic debilitating disorders.

The objective of this research proposal is to determine the neural substrate in the rodent hindbrain through which gustatory stimuli modulate ingestive behavior. Although the regulation of ingestive behavior is exceedingly complex, involving many regions of the CNS, the basic regulatory function of taste to evoke either an ingestion or rejection response, is complete in the caudal brain stem. Chronic EMG recording experiments have characterized the behaviors of ingestion and rejection to sapid stimuli by determining the activation sequences of a subset of trigeminal, facial, ambiguus and hypoglossal innervated muscles. A rejection response to quinine monohydrochloride (bitter) is characterized by retraction of the tongue (styloglossus contraction) in conjunction with jaw opening (anterior digastric contraction) in contrast to an ingestion sequence in which tongue protrusion (genioglossus contraction) is simultaneous with jaw opening. These stereotypic motor components of ingestion and rejection are also evident in a restrained, anesthetized preparation in which central recording is possible. The neural substrate mediating these oro-motor responses is polysnaptic and anatomical experiments have delineated several regions of the brain stem reticular formation (RF) that project to all (or a subset) of the oral motor nuclei producing them. Thus, these RF regions are candidates for coordinating and sequencing the complex synergies of ingestion and rejection. Using neurophysiological techniques, neurons in these reticular regions will be studied for (1) Sensitivities to gustatory, intra-oral tactile and oral proprioceptive stimuli, (2) responsiveness during specific motor components of ingestion and rejection defined electromyographically, (3) connectivity with oro-motor nuclei using antidromic stimulation. Additional experiments using retrograde transport of horseradish peroxidase will determine if these reticular regions receive projections from gustatory nuclei in the brain stem or whether other interneurons are interposed. Ingestion and rejection represent the final behavioral decision required for replenishing energy, fluid, and electrolyte stores. Chronic disorders such as obesity, hypertension and anorexia nervosa are related to disordered ingestion. Understanding the neurological basis of this fundamental biological decision, to ingest or reject will contribute to the eventual solution of these chronic debilitating disorders.

biomedical equipment resource; biomedical equipment purchase;

DESCRIPTION (provided by applicant): We propose to establish microdialysis in the awake, behaving preparation as a technique for investigating amino acid transmitters and neuropeptides in the gustatory-responsive (rostral) nucleus of the solitary tract (rNST). Although microdialysis has been used in many regions of the CNS with great success, the potential of this technique has remained unexploited in the gustatory system. Salt deprivation, hunger, satiety and conditioned aversions have all been demonstrated to impact neural responsiveness in the gustatory system. The neural basis for these state-dependent changes is unknown, but, based on their role in other sensory systems, a predominant role for peptide neuromodulators, perhaps acting via interneurons utilizing excitatory or inhibitory amino acids, is a compelling hypothesis. Understanding the functional neurochemistry of gustatory processing would be greatly facilitated by the ability to monitor the release of multiple neuroactive agents in an animal actively engaged in gustatory-driven behavior. Microdialysis can meet these requirements. The present studies will provide a foundation for assessing this hypothesis by establishing the capabilities of microdialysis followed by HPLC and electrochemical detection in the first-order gustatory relay. The first experiments will monitor amino acid release in an anesthetized preparation, in which precisely controlled taste stimulation is possible. The results of these experiments will be critical in interpreting data obtained in a second series of studies which will apply microdialysis to monitor amino acid release in an awake preparation. A third series of studies will develop microdialysis for assessing the release of multiple neuropeptides, by adapting electrochemical detection techniques to this purpose. When the proposed studies are brought to fruition, future work will be directed at examining the neurochemical basis for fluctuations in gustatory sensitivity as a function of metabolic or nutritional status, and as a function of learning or task demands. The characterization of the dynamic neurochemistry associated with gustatory processing under varying homeostatic conditions offers therapeutic avenues to either increase food intake in response to cachexia or to limit intake for obesity.

DESCRIPTION (provided by applicant): This proposal describes a high risk, high impact project using an optogenetic approach to determine how visceral neurons associated with satiey modulate the taste system. We have created a novel viral vector (AAV-9, PRSx8-ChR2(h134r)-mCherry) that expresses channelrhodopsin-2 (Chr2) and mCherry under the control of a Phox2-selective promoter that labels catecholaminergic neurons containing norepinephrine and other non-GABAergic viscerosenory neurons. A second optogenetic approach will target catecholamine neurons more specifically using a transgenic mouse that expresses Cre under the control of a dopamine- ¿ hydroxylase promoter (D ¿HCre/0) and a cre-dependent virus driving ChR2 expression. Neurons in the caudal brainstem including catecholamine neurons are intimately associated with visceral signaling underlying satiey mechanisms and glucoprivic-induced feeding, but where and how they modulate taste signals is unknown. Although catecholamine neurons are clustered in groups, e.g. A1 & A2, these clusters are embedded in a more heterogeneous population of cells making it difficult to study specific neural pathways. Optogenetic techniques allow defined neuron types to be specifically activated. Neuronal DNA is modified by inserting a gene expressing a light-sensitive channel (protein) capable of depolarizing the neuron membrane. Following neuronal transduction with the genetic construct, optical stimulation with a laser or high-intensity LED tuned to the optimal frequency of the light-sensitive channel will subsequently stimulate and depolarize those neurons genetically defined by the promoter. Recent work suggests that significant interactions between caudal solitary nucleus neurons sensitive to visceral signals and feeding circuits occur through local brainstem pathways including intrasolitary pathways. The outcomes of our proposal will thus (1) provide a novel technical approach to determine how a specific group of visceral-sensitive neurons modulate gustatory neurons and (2) determine if a previously unexplored brainstem intrasolitary pathway contibutes to taste/visceral integration.