Margaret Wong-Riley - US grants

The brain receives most of its energy from oxidative metabolism, of which cytochrome oxidase is a key energy-deriving enzyme. There is tight coupling between neuronal activity and energy expenditure. Likewise, our previous studies have revealed the close correlation between the level of cytochrome oxidase and that of neuronal activity. The major goal of our proposed research is to understand the cellular basis of the dynamic metabolic adjustment of neurons to altered functional demand in the adult. That is, we wish to know if there are metabolically distinct classes of neurons within a single nuclear group, and whether they respond differently to varying levels of functional insult. The dorsal lateral geniculate nucleus of adult cat is chosen for this study, because it has well-defined physiological and morphological cell types and because experimental manipulations can be applied to the retina without physically invading the LGN itself. To begin with, we will analyze and quantify the metabolic characteristics of large, medium and small cells in the LGN at both the light and E.M. levels by means of cytochrome oxidase histo- and cyto-chemistry. We will attempt to correlate these metabolic indentities with the known sizes, distribution and physiological properties of X-, Y- and W-cells. We then wish to ask whether and how these cell types respond differently to varying degrees of trauma: (a) Sensory deprivation by means of monocular lid suture, which reduces but not eliminate the amount of natural stimulus (light) from entering the eye; (b) afferent impulse blockade by means of intravitreal injection of tetrodotoxin, at dosage that does not block axoplasmic transport; and (c) deafferentation my means of unilateral enucleation, which effectively eliminates both afferent impulses as well as presumed "trophic" factors. We wish to know if these procedures would bring about varying degrees of responses in the LGN neurons, whether all neurons respond similarly to each type of trauma, or whether a specific cell type suffer a greater or lesser degree of susceptibility. These differences will be quantified at the light and E.M. levels. We hope that these data will help us gain a better understanding of the dynamic aspect of cellular response to functional deprivation in the adult nervous system.

The primate visual cortex possesses a unique array of metabolically active zones that are rich in cytochrome oxidase activity. In area 17, these C.O.-rich supragranular puffs and the granular fourth layer appear to coincide in space with geniculocortical terminations, and are likely to be strongly influenced by subcortical sensory input. The major goal of our proposed research is to understand how the metabolic integrity of mature visual cortical neurons is governed by normal and altered retinal input. While there is a substantial amount of information on both the anatomical and functional adjustments in the developing visual system, little is known about such plasticity in the adult. In fact, the mature neuron has traditionally been regarded as refractory to the trauma of sensory deprivation. We propose to examine the effect of various forms of sensory insult on the level of cytochrome oxidase in the primate visual cortex. Since there is tight coupling between neuronal activity and energy metabolism, mainly via the oxidative pathway, the level of cytochrome oxidase, a key energy-deriving oxidative enzyme, can be used as an indicator of the level of neuronal activity. Three paradigms will be tested for differential cortical responsiveness: (a) Sensory deprivation by means of monocular lid suture, which reduces but not eliminate the amount of natural stimulus (light) from entering the eye; (b) afferent impulse blockade by means of intravitreal injections of tetrodotoxin, at a dosage that does not block axoplasmic transport; and (c) deafferentation by means of unilateral enucleation, which effectively eliminates both afferent impulses as well as presumed "trophic" factors. We wish to know if cortical neurons are seensitive to any or all of these treatments. Do all neurons respond uniformly to each type of trauma, or does a specific type of neuron suffer a grater or lesser degree of susceptibility? We also wish to know if the time course and degree of reaction differ between the puffs and lamina IV, between the magnorecipient IVC and the pavro-recipient IVC, and between the center and the periphery of cortical puffs. We hope that these data will help us gain a better understanding of the dynamic aspect of cellular response to functional deprivations in the adult visual system.

The brain receives most of its energy from oxidative metabolism, of which cytochrome oxidase is a key energy-deriving enzyme. There is tight coupling between neuronal activity and energy expenditure. Likewise, our previous studies have revealed the close correlation between the level of cytochrome oxidase and that of neuronal activity. The major goal of our proposed research is to understand the cellular basis of the dynamic metabolic adjustment of neurons to altered functional demand in the adult. That is, we wish to know if there are metabolically distinct classes of neurons within a single nuclear group, and whether they respond differently to varying levels of functional insult. The dorsal lateral geniculate nucleus of adult cat is chosen for this study, because it has well-defined physiological and morphological cell types and because experimental manipulations can be applied to the retina without physically invading the LGN itself. To begin with, we will analyze and quantify the metabolic characteristics of large, medium and small cells in the LGN at both the light and E.M. levels by means of cytochrome oxidase histo- and cyto-chemistry. We will attempt to correlate these metabolic indentities with the known sizes, distribution and physiological properties of X-, Y- and W-cells. We then wish to ask whether and how these cell types respond differently to varying degrees of trauma: (a) Sensory deprivation by means of monocular lid suture, which reduces but not eliminate the amount of natural stimulus (light) from entering the eye; (b) afferent impulse blockade by means of intravitreal injection of tetrodotoxin, at dosage that does not block axoplasmic transport; and (c) deafferentation my means of unilateral enucleation, which effectively eliminates both afferent impulses as well as presumed "trophic" factors. We wish to know if these procedures would bring about varying degrees of responses in the LGN neurons, whether all neurons respond similarly to each type of trauma, or whether a specific cell type suffer a greater or lesser degree of susceptibility. These differences will be quantified at the light and E.M. levels. We hope that these data will help us gain a better understanding of the dynamic aspect of cellular response to functional deprivation in the adult nervous system.

The primate visual cortex uniquely possesses a regular array of metabolically active, cytochrome oxidase (C.O.)-rich zones (blots or puffs) in the supragranular layers with distinct physiological properties, particularly those related to color processing. Our previous studies indicate that unilateral retinal impulse blockage in the adult severely affects the most metabolically active neurons and induces synaptic reorganization within the puffs. These findings suggest that the mature visual cortex is not static but, rather, responds dynamically to altered functional demands. Besides changes in puffs, our preliminary light microscopic analysis of surrounding C.O.-poor interpuff regions indicate compensatory increases in C.O. levels within zones related to the non-treated eye. Such dynamic changes in the adult are of obvious clinical and functional importance, consequently our initial ultrastructural and quantitative analyses of the puffs will be extended to the interpuff regions. Selective vulnerability of the most metabolically active neurons deserves further investigation, since it appears to implicate a specific neurotransmitter type, GABA. Whether all puff neurons with intense C.O. activity are GABAergic, or whether GABAergic neurons encompass a wide range of oxidative metabolic capacities, will be examined by means of combined C.O. histo- or immunohisto-chemistry and GABA-immunohistochemistry on the same histological section. To directly address the relationship of C.O. levels to physiological activity and visual processing, single neurons will be recorded extracellularly from C.O.-rich and poor zones in normal cortex and during periods of physiological modification: 1) following monocular retinal impulse blockage in the adult and 2) during the critical period of postnatal development, when the innately determined cortical organization undergoes further physiological maturation. To understand the anatomical basis of these changes during development and specifically to explore the dynamics of maturational plasticity, we will examine structural reorganization in developing visual cortical neurons following retinal blockade. In summary, our approach is to combine histochemical, immunohistochemical, ultrastructural, and physiological observations to yield an integrated understanding of metabolic adjustments to altered functional demands within developing and mature neurons of the primary visual cortex.

DESCRIPTION: This competing renewal consists of 5 specific aims. The first three aims will examine using postembedding immuno-EM combined with cytochrome oxidase histochemistry to examine the neurochemical composition of synapses and neurons in the cytochrome oxidase rich puff and cytochrome oxidase poor regions of layer 2/3 in monkey visual cortex. These same analyses will be done following monocular deprivation to assess how activity might differentially regulate the neurochemical nature of synaptic connections in puff and interpuff regions.

DESCRIPTION (Adapted from applicant's abstract): The primate striate cortex is parceled into functional modules and streams revealed partly by the distribution of the enzyme cytochrome oxidase (CO). CO is a sensitive indicator of neuronal functional integrity, not only under normal conditions but also in response to visual deprivation. Deprived cortical neurons do not response alike to the same functional insult; the metabolically most active ones are most vulnerable. Despite the wide use of CO, little is known about the molecular mechanism of its regulation, which is critical for understanding how visual cortical neurons regulate their activity-dependent energy metabolism. CO, a bigenomic enzyme, requires precise coordination between the nuclear and the mitochondrial genomes to form a functional holoenzyme. Two transcription factors, nuclear respiratory factors 1 and 2 (NRF-1 & NRF-2) may play a coordinating role. They are known to activate genes for some of the nuclear-encoded CO subunits, and a gene that indirectly regulates the production of mitochondrial-encoded CO subunits. The goal of the PI is to probe these transcription factors at the protein and mRNA levels in the visual cortex of normal and visual deprived monkeys. The distribution of these proteins will be compared to CO activity, and the density of the NRF-2 subunits, (and (, will be compared to the density of CO in distinct metabolic cell types within cortical puffs. If NRF-1 and NRF-2 are molecularly linked to CO expression in vivo, then their distributions should closely correlate with that of CO. Monocular deprivation will determine if the regulation of NRF-1 and NRF-2 is activity-dependent and whether this occurs by translational or transcriptional control. An in vitro model of primary cultures of rat visual cortical neurons will reveal how NRF-1 responds to depolarizing stimulation, and if the time course of the response is upstream of CO gene expression in the two genomes. The gene for a glutamate receptor, GluR2, was recently reported to be activated by NRF-1 in vitro. As glutamate is a major excitatory transmitter in visual neurons, the co-expression of GluR2 and NRF-1 may provide another link between neuronal activity and energy metabolism. NRF-1 knockout mice will be studied to see if visual cortical neurons are adversely affected, if levels of CO & GluR2 are reduced, and if NRF-2 is up regulated in compensation. Results from these studies are expected to provide insight into the molecular basis of metabolic responses of visual cortical neurons to changing functional demands.

DESCRIPTION (provided by applicant): Neuronal activity and energy metabolism are tightly coupled. Under normal conditions, neuronal activity controls energy metabolism and not vice versa. Visual neurons are among the metabolically most active cells in the nervous system, and their energy is derived almost exclusively from oxidative metabolism. Defective energy metabolism adversely affects visual neurons, often leading to blindness. Thus far, energy metabolism and neuronal activity have been assumed to be regulated at the molecular level by two independent mechanisms. The present proposal aims to test the hypothesis that a candidate transcription factor, nuclear respiratory factor 1 (NRF-1), co-regulates both energy metabolism and synaptic neurochemicals (glutamate AMPA receptor subunit 2 [GluR2] and/or neuronal nitric oxide synthase [nNOS]) in specific types of visual cortical neurons. NRF-1 is known to activate genes involved in mitochondrial function, including subunit genes of cytochrome oxidase (CO). Binding sites for NRF-1 have been reported on the promoters of GluR2 and nNOS genes, but it is not known if NRF-1 is necessary for their expression. Recently, NRF-1 was found to be present in mammalian visual cortical neurons and its expression was regulated by neuronal activity. Both GluR2 and nNOS are associated with glutamatergic synaptic neurotransmission and are present in mammalian visual cortex. Specific aim 1 is to establish baseline data on the normal distribution and levels of NRF-1, GluR2, and nNOS mRNAs and proteins in visual cortical neurons in vivo and in vitro, using rat as a model. Specific aim 2 is to suppress the expression of NRF-1 by means of small interference RNA silencing to determine if expressions of GluR2, nNOS, and CO will be severely affected in visual cortical neurons. NRF-1 knockdown neurons will be subjected to depolarizing stimulation to determine if increased activity differentially stresses GluR2-rich and nNOS-rich neurons, and if one or both of them will undergo cell death. Specific aim 3 is to overexpress NRF-1 to ascertain if expressions of GluR2, nNOS, and CO reduced by impulse blockade can be rescued. These studies will shed light on how a transcription factor can simultaneously regulate both energy metabolism and synaptic neurochemicals in visual cortical neurons, and how genetic perturbation of this factor can lead to impaired energy production, defective expression of neurochemicals, and possible death of visual cortical neurons.

[unreadable] DESCRIPTION (provided by applicant): Respiratory control is multifaceted and complex. In mammals, a network of brain stem nuclei is essential for rhythm generation, pattern formation, and respiratory modulation. The mediators of respiratory neural processing are diverse sets of excitatory and inhibitory neurotransmitters, neuromodulators, and their receptors. Respiratory control differs between different stages of development. The assumption is that growth is progressive and the maturation of respiratory control follows a straight path. This may prove not to be the case, as the incidence of Sudden Infant Death Syndrome suggests that the peak of susceptibility is not at birth, but between the 2nd and 4th months after birth. Physiological studies of responses to hypoxia, hypercapnia, and other insults also suggest critical periods of postnatal development in animals. If such periods exist, the neurochemical bases underlying them need to be explored. When the development of several key neurochemicals in the rat brain stem was analyzed daily from postnatal day (P) 0 to 21, we found that, despite general trends of increasing or decreasing expressions with age, there is a distinct fall in the expression of excitatory neurotransmitters and receptors and a prominent rise in the expression of inhibitory neurotransmitters and receptors at P12. These events suggest a transient period of neurochemical imbalance, during which the system is under stronger inhibitory than excitatory drive, concomitant with a sudden drop in metabolic enzyme activity at P12. Remarkably, an apparent switch in subunit dominance of GABAA receptors from oc3 to a1 also occurs around P12 in the rat pre-Botzinger complex, suggesting that the same neurotransmitter may have different physiological effects before and after the subunit switch. The goal of the present proposal is to test our hypothesis that receptor subunit switching is a common theme for a number of neurotransmitter receptors during the presumed critical period of development in rats. Specifically, we will investigate in several key brain stem nuclei by quantitative immunohistochemical assays the developmental expressions of: 1) GABAA receptor subunits a1, a2, and a3; 2) glutamate receptors, including NMDA receptor subunits 2A and 2B, and AMPA receptor subunits GluR1 and GluR2; and 3) serotonin receptors 5-HT1A, 1B, 2A, 2C, and 3, tryptophan hydroxylase (TPH) and serotonin transporter (SERT). Finally, 4) the possibility that switches occur at the mRNA level will be explored using isolated cDNA fragments and in situ hybridization of subunit-specific riboprobes for GABA, NMDA, and 5-HT receptors. These studies will provide a solid anatomical and neurochemical foundation regarding the temporal events during postnatal development of brain stem respiratory nuclei, so that future physiological and behavioral studies may extend such knowledge toward a more complete understanding of respiratory control in neonatal and early postnatal animals. [unreadable] [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): It is estimated that addiction and its associated costs: crime, domestic violence and child abuse, health care costs, and loss of employment and family structure, exceed half of a trillion dollars per year. A major barrier to successful treatment of a estimated 20 million Americans suffering from addiction is the lack of understanding of factors important for drug seeking, withdrawal and reinstatement (relapse). One such factor, brain-derived neurotrophic factor (BDNF) has long been recognized as a critical trophic factor for the growth, development and survival of developing neurons as well as for mediating synaptic transmission and synaptic plasticity. In recent years, however, BDNF has been strongly implicated in a number of neuropsychiatric disorders, including drug addiction, depression, bipolar disorders, and schizophrenia. The rat is the preferred rodent model in the field for understanding behavior and could serve as a powerful preclinical model for investigating the role of BDNF in these disorders, however, the tools for manipulating rat genes have historically been lacking. Our group has recently developed a number of tools to overcome this barrier, creating the world's first gene knockout rats and developing new and efficient transgenic techniques. We propose to adapt these innovative methods to generate transgenic rats where expression levels of BDNF, or other genes, can be fine-tuned in a spatial and temporal manner. These novel rat models will be the first animal models of their kind to address the role of BDNF in addiction behavior. An innovative aspect of the design of these transgenic animals will also allow easy exchange of genes of interest, promoters, and reporter genes to tailor the needs of many different NIDA investigators in the future. The current proposal has two specific aims: Aim 1 is to generate rats with flexible transgenes to allow conditional and inducible gene knockdown in rats via RNA interference, focusing on BDNF as our first example. These transgenic lines will be useful to many researchers investigating drug addiction, affective disorders, and other neurological diseases. Aim 2 is to characterize the effects of BDNF knockdown on addiction-related behavior in an established rat paradigm of cocaine addiction. Our hypothesis is that BDNF knockdown will selectively reduce BDNF expression in adult neurons, leading to abnormal neurochemical and behavioral responses to cocaine administration, withdrawal, and reinstatement. We have assembled a collaborative and productive team of investigators to deploy these tools and to determine the impact of reduced forebrain levels of BDNF on cocaine-induced reinstatement in transgenic rats. Once established, these rat models and methodologies will be made available to all interested researchers in the neuroscience community.