Yasuko Nakajima, Md, PhD - US grants

DESCRIPTION (provided by applicant): Ion channels are the major determinant for excitability changes of neurons. Our long-term objective is to elucidate the mechanism by which slow excitation and slow inhibition of brain neurons take place. These events occur on time scales of tens of seconds to a few minutes, and are mostly mediated by G proteins. Our immediate research will focus on clarifying the signal transduction mechanism by which G-protein-coupled inward rectifier Kv (GIRK) channels are modulated by substance P (SP), an excitatory peptide neurotransmitter. Native cultured noradrenergic neurons in the locus coeruleus (LC) from newborn rats and mice, as well ascloned GIRKs expressed in HEK293 cells will be used. Electrophysiological and molecular biological techniques will be employed for the investigation. The LC contains neurons that innervate and supply norepinephrine to a wide area of the brain, and plays a vital role in arousal and alertness. Furthermore, LC neurons often degenerate in Alzheimer's disease. LC neurons are dually regulated by opposing signals: inhibitory transmitters, such as somatostatin, activate GIRK channels, whereas excitatory transmitters, such as SP, inhibit the GIRK activity that is activated by somatostatin. The mechanism of the GIRK inhibition, however, is controversial and yet to be determined. The goal of the proposed project is to elucidate the signal transduction mechanism of the SP-induced GIRK channel inhibition. Three possible signal transduction pathways could be responsible for the SP-induced GIRK channel inhibition: the phosphatidyl inositol 4,5-bisphosphate (PIP2) pathway, the protein kinase C pathway, and a new pathway, which we designate as the "quick pathway." The existence of the quick pathway is hypothesized because the inhibition is too rapid to be accounted for by the other pathways. We intend to elucidate the mechanism of this pathway. We also intend to investigate the possible synergistic relation between the quick and the PIP2 pathways.

The modulation of neuronal function by various kinds of endogenous opioid peptides is one of the most important cellular events of brain function. However, not much is known about the cellular and membranous mechanisms of opioid actions. The objective of this grant proposal is to elucidate the ionic channel and second messenger mechanisms of two kinds of endogenous opioid peptides, beta-endorphin and dynorphin. Primary cultures from two brain regions of postnatal animals, the locus coeruleus and the substantia nigra will be used. Electrophysiological techniques including the whole cell version of patch clamp and intracellular microelectrode methods will be used. The proposal is divided into two projects. The first project deals with the effects of beta-endorphin on dissociated cultured noradrenergic neurons from the locus coeruleus. A preliminary study in the laboratory shows that beta- endorphin produces a slow hyperpolarization at the resting stare, resulting in an inhibition of neuronal activity. The preliminary study also shows that pertussis toxin inhibits this hyperpolarization, suggesting that inhibitory GTP-binding proteins may be involved as second messengers. To investigate further the precise ionic and second messenger mechanisms of this slow hyperpolarization is the aim of the first project. The second project consists of two parts. The first part is to make primary dissociated cell culture of the substantia nigra of postnatal rats and mice. Dopaminergic neurons will be identified by using immunohistochemical or cathecholamine histofluorescence methods. The second part is to investigate the effects of dynorphin on cultured neurons from the substantia nigra, which is known to be richly innervated with dynorphin-containing nerve fibers. The ionic and second messenger mechanisms of dynorphin effects will be investigated.

The modulation of neuronal function by various kinds of endogenous opioid peptides is one of the most important cellular events of brain function. However, not much is known about the cellular and membranous mechanisms of opioid actions. The objective of this grant proposal is to elucidate the ionic channel and second messenger mechanisms of two kinds of endogenous opioid peptides, beta-endorphin and dynorphin. Primary cultures from two brain regions of postnatal animals, the locus coeruleus and the substantia nigra will be used. Electrophysiological techniques including the whole cell version of patch clamp and intracellular microelectrode methods will be used. The proposal is divided into two projects. The first project deals with the effects of beta-endorphin on dissociated cultured noradrenergic neurons from the locus coeruleus. A preliminary study in the laboratory shows that beta- endorphin produces a slow hyperpolarization at the resting stare, resulting in an inhibition of neuronal activity. The preliminary study also shows that pertussis toxin inhibits this hyperpolarization, suggesting that inhibitory GTP-binding proteins may be involved as second messengers. To investigate further the precise ionic and second messenger mechanisms of this slow hyperpolarization is the aim of the first project. The second project consists of two parts. The first part is to make primary dissociated cell culture of the substantia nigra of postnatal rats and mice. Dopaminergic neurons will be identified by using immunohistochemical or cathecholamine histofluorescence methods. The second part is to investigate the effects of dynorphin on cultured neurons from the substantia nigra, which is known to be richly innervated with dynorphin-containing nerve fibers. The ionic and second messenger mechanisms of dynorphin effects will be investigated.

There are many transmitter substances which regulate the activity of nerve cells of the central nervous system. The objective of this proposal is to elucidate the mechanisms by which these transmitter substances activate intracellular messenger processes that lead to the modulation of excitability through their action on potassium channel conductance properties. Recent studies have shown that excitability of neurons is dependent on potassium channels which are, in turn, regulated by G-proteins called Gi and Go, of which there are several subtypes. The specific aim of this investigation is to identify which of the G-protein subtypes are related to neurotransmitter action (acetylcholine and somatostatin). Molecular biological techniques will be used to produce specific mutations to selectively inactivate G-proteins and thereby determine which of them is directly affected by the neurotransmitters to act on potassium conductance of the cell. For this purpose mutated alpha subunits will be inserted into AtT-20 cells, a pituitary tumor cell line which contain G-protein regulated potassium channels. The effect of the mutants can then be ascertained on potassium channel function.

This grant project investigates the properties of a non- selective ion channel (channels are functionally important proteins located in the cell membrane). The non-selective channel plays an important part in slow excitation of brain cells induced by brain chemicals. The overall summation of the excitatory state of each of the brain cells in each specialized nucleus (region) of the brain determines the tone of the brain. In spite of its widespread nature and its functional importance, the non-selective channel has long been ignored, and little is known about its properties. To investigate this channel in brain neurons has been difficult because of the lack of a suitable cell type. Recently, the laboratory of the principal investigator has found that cultured neurons from the ventral tegmental area (a brain region playing an important part in the pathogenesis of schizophrenia) are an excellent cell type for this purpose, since this channel is consistently activated by four excitayory chemicals: (1) neurotensin, (2) neurokinin B, (3) metabotropic glutamate agonist, (4) and muscarine Thus, it is now feasible to conduct a systematic investigation of this channel. The specific projects examine: (1) whether each one of these brain chemicals acts on its own receptor, (2) the permeability of the channel to various ions, (3) the signal transduction mechanism (details of the events leading to a slow excitation of the neuron). (4) the pharmacology of this channel, and (5) the single channel properties of this channel. Preliminary results suggest that this non- selective channel has unusual characteristics: (1) It is different from a Ca-activated non-specific cation channel. (2) The channel activiation is independent of G proteins (an important protein for many types of signal transduction). (3) Yet, it does not seem to be a ligand- gated channel (usual channel for very quick excitation), since the latency of activation is very long.

DESCRIPTION (provided by applicant): The main objective of this research proposal is to investigate neuropharmacologically the cellular mechanisms of arousal and sleep with a special emphasis on the pathogenesis of narcolepsy. Hypocretins/orexins, newly discovered peptide neurotransmitters, and their receptors will be studied. Hypocretins/orexins and their receptors play a role in narcolepsy and sleep disorders and in the regulations of food intake. Currently little is known about their physiological effects on brain neurons. Previous studies have shown that lack of excitation produced by hypocretins/orexins through hypocretin2/orexin2 receptors in the brain nuclei is the central component of narcolepsy. Proposed projects focus on further elucidating their actions at the cellular and molecular level by using dissociated primary cultures of rat and mouse brain nuclei that are involved in regulating arousal and sleep. The specific projects are: (1) to elucidate hypocretin/orexin effects on histaminergic neurons in the tuberomammillary nucleus which are rich in hypocretin2/orexin2 receptors, (2) to determine the transmitter effects on noradrenergic neurons in the locus coeruleus which are rich in hypocretin1/orexin 1 receptors, and (3) to investigate hypocretin/orexin effects on cholinergic neurons in the nucleus basalis of Meynert. In addition, a heterologous system (HEK293A) which is transfected with each type of hypocretin/orexin receptor will be used. All of these projects emphasize elucidating signal transduction mechanisms of hypocretins/orexins, and determining the identity and roles of G proteins. Electrophysiological techniques (patchclamp) and pharmacological techniques combined with molecular biological methods will be used. These projects are important for deepening the understanding of narcolepsy and other sleep disorders. The knowledge obtained is essential in developing more effective treatments for narcolepsy and other sleep disorders. This research on the sleep-arousal centers of the brain will contribute in helping those who suffer sleep disorders.