Bibie M. Chronwall - US grants

The turbero-hypophyseal axis is characterized by limited neuronal complexity and few trans-interactions with a homogenous target tissue which produces only one major prohormone (POMC). We have demonstrated that the biosynthetic activity of intermediate lobe (IL) melanotropes is regulated by dopaminergic inhibition. Dopamine mitochondria; POMC mRNA levels and mitotic rate. DA antagonists have the opposite effect. The objective of this project is to establish how axons containing the colocalized neurotransmitters DA and gamma- aminobutyric acid (GABA) regulate the melanotropes. This system provides an excellent vehicle to study the mechanisms of transsynaptic regulation by colocalized neurotransmitters of peptide biosynthesis, biosynthetic heterogeneity and cellular hyperplasia. The specific aims are: (1) To morphologically characterize IL neuronal terminals and to quantitatively described their distribution and GABA/DA content, using light- and electron microscopic immunohistochemistry and morphometry. (2) To quantitate both in vitro and in vivo the effects of GABA on melanotrope biosynthetic potential hormone secretion and mitotic rate. The methods to be employed are in situ hybridization histochemistry, using a pro-opiomelanocortin (POMC) probe, RIA and incorporation of [3H] thymidine resolved at the single cell level. (3) To evaluate modulatory interactions between GABA and DA on POMC mRNA, hormone secretion (by RIA) and mitotic rate in the IL. (4) To determine the time sequence of the appearance of GABA and DA and to evaluate differing roles of the neurotransmitters during ontogeny, (immunohistochemistry, morphometry, [3H] thymidine incorporation, in situ hybridization histochemistry using a POMC probe). Studies of the effects of colocalized transmitters are of great importance for the understanding of the wide diversity of modulatory possibilities inherent in neuronal regulation. Comprehension of the mechanism of cotransmitter action is a necessary step in understanding normal neuronal regulation; human neurological and psychological diseases; and subsequently, how to treat such disorder without adverse drug effects emanating from interference with colocalized neurotransmitters.

The GABA- and dopaminergic (DA) neurons of the tubero-hypothalamic area and their target cells, the pituitary intermediate lobe (IL) melanotropes, will be used as a model system for the ontogeny of the mechanisms for neuronal co-transmitter control of endocrine cells. The concept of co- localization of multiple neurotransmitters within one neuron has become established through recent research. Co-localization adds a level of increased complexity to synaptic communication which is important to study to better comprehend neuronal regulation. Ontogenic studies unfold the processes by which a developing system forms and can explain the basis of the complexity of the mature system. The overall objective of this project is to link pre- and postsynaptic events during the ontogeny of the tubero-hypophysial system. The specific aims are: (1) to establish the chronology of the development of the co- localization of GABA and DA in neurons of the tubero-hypothalamic area; (2) to define the time-sequence and spatial pattern of the GABA and DAergic innervation of the IL; (3) to establish the ontogeny of dopamine (d 2) and GABA receptors; and, (4) to study the impact of the event of innervation on IL melanotrope mitotic rate, pro-opiomelanocortin (POMC) gene expression, and secretory vesicle population. The techniques to be used include immunohistochemistry, immunocytochemistry, electron microscopic morphometry, in situ hybridization histochemistry, [3H]thymidine incorporation, and receptor binding assays. The significance of these studies relates to how co-localized neurotransmitters regulate their target cells. Understanding these regulatory mechanisms, which are unlikely to be unique to GABA/DA regulation of melanotropes, will allow the elucidation of more complex cellular communication systems. Comprehension of the mechanisms for co- transmitter action is a necessary step toward the design of therapeutic interventions to treat neurological disorders without adverse drug effects.

A new design for real-time low light fluorescence video microscopy for capturing kinetic images of up to 4 dyes contained in the same living cell will be constructed and tested. It will be used to determine intracellular calcium and pH and show how the measurements may be used to correct the intracellular calcium values. The dissociation values (Kd's) for the fluorescent probes of Calcium are strongly pH dependent and failure to correct the Kd for the prevailing pH can greatly bias the apparent calcium concentration, especially in acidic conditions. The method will be used to investigate the rapid kinetics of intracellular calcium and pH in cultured pituitary intermediate lobe melanotropes and to their relationship to hormone release. These cells respond to potassium ion induced depolarization by entry of calcium into the cell with a simultaneous acidification of the cell. The hypothesis will be tested that the rapid acidification is caused by the influx of calcium and is not its antecedent. The second hypothesis to be tested is that the hormone, dopamine, through its specific receptors (D2) regulated melantrope calcium and pH.

9321667 Chronwall The purpose of the Fifteenth Winter Neuropeptide Conference is to encourage experts and young scientists in the expanding field of peptide research to discuss recent findings related to "Models of Peptide Action." The topics range from behavioral aspects to basic mechanisms of action, passage of peptide across the brain blood barrier,cytokines, neurotrophism and development. In addition, a plenary presentation and a poster session will be included. It is anticipated that 80-120 participants from all over the US as well as Europe and Japan will attend. ***

The turbero-hypophyseal axis is characterized by limited neuronal complexity and few trans-interactions with a homogenous target tissue which produces only one major prohormone (POMC). We have demonstrated that the biosynthetic activity of intermediate lobe (IL) melanotropes is regulated by dopaminergic inhibition. Dopamine mitochondria; POMC mRNA levels and mitotic rate. DA antagonists have the opposite effect. The objective of this project is to establish how axons containing the colocalized neurotransmitters DA and gamma- aminobutyric acid (GABA) regulate the melanotropes. This system provides an excellent vehicle to study the mechanisms of transsynaptic regulation by colocalized neurotransmitters of peptide biosynthesis, biosynthetic heterogeneity and cellular hyperplasia. The specific aims are: (1) To morphologically characterize IL neuronal terminals and to quantitatively described their distribution and GABA/DA content, using light- and electron microscopic immunohistochemistry and morphometry. (2) To quantitate both in vitro and in vivo the effects of GABA on melanotrope biosynthetic potential hormone secretion and mitotic rate. The methods to be employed are in situ hybridization histochemistry, using a pro-opiomelanocortin (POMC) probe, RIA and incorporation of [3H] thymidine resolved at the single cell level. (3) To evaluate modulatory interactions between GABA and DA on POMC mRNA, hormone secretion (by RIA) and mitotic rate in the IL. (4) To determine the time sequence of the appearance of GABA and DA and to evaluate differing roles of the neurotransmitters during ontogeny, (immunohistochemistry, morphometry, [3H] thymidine incorporation, in situ hybridization histochemistry using a POMC probe). Studies of the effects of colocalized transmitters are of great importance for the understanding of the wide diversity of modulatory possibilities inherent in neuronal regulation. Comprehension of the mechanism of cotransmitter action is a necessary step in understanding normal neuronal regulation; human neurological and psychological diseases; and subsequently, how to treat such disorder without adverse drug effects emanating from interference with colocalized neurotransmitters.

Morris 9515226 All electrically excitable cells, such as those in the brain and other organs, are constantly receiving chemical signals in the form of neurotransmitters and hormones. These chemical messengers interact with specific proteins, called receptors, on the outer surface of their target cells and through changes brought about by the activation of these receptors, transmit their message into the workings of the cell. The studies proposed by Dr. Morris study one such message system, the dopamine D2 receptor system. Dopamine is a small molecule which is crucial for brain communication and organismal function, particularly movement. Dr. Morris and his colleagues are using sophisticated molecular biological techniques to develop an easily accessible model system in which to study the events that occur in a cell subsequent to dopamine binding to its cognizant receptor. This work will provide a detailed understanding of the basic molecular mechanisms activated by a neurotransmitter binding to its receptor and will have major implications on revealing how the brain encodes chemical information and its translation.