Carl M. Rovainen - US grants

The objective of this proposal is to determine the neural circuits and cellular mechanisms which produce a state of generalized enhanced excitability in the lamprey brain. This active state is proposed as a model for arousal in the vertebrate brain stem. The advantages of the lamprey brain stem for this study are its relative simplicity, similar principles of organization as in other vertebrates, the ability of the isolated preparation to produce activities corresponding to behavior and at the same time to be suitable for intracellular recordings and pharmacological modulation, and background studies by the P.I. on the subsystems which are involved in the active state. The lamprey brain in vitro and lampreys in vivo display resting and active states. Active episodes in vitro occur or in response to sensory stimuli and are expressed as increased activities in trigeminal and spinal motor pathways, enhanced rate and intensity of fictive breathing, and altered responsiveness to sensory stimuli. Corresponding behavioral changes in vivo are active attachment by the sucker, changes in breathing rate, body movements, and altered responsiveness to vibration. The strategy of this proposal is (a) to use the known organization of the trigeminal, respiratory, reticulospinal, and sensory systems in the brain stem to assay behavioral changes in vivo and neurophysiological changes in vitro during the active state, (b) to use lesions, extracellular recording, and focal stimulation to localize critical brain regions involved in the active state, (c) to use receptor agonists and antagonists, including cholinergic and aminergic drugs, to initiate or block components of the active state in vitro, (d) to use intracellular recordings from target neurons to determine changes in cellular properties and monosynaptic transmission during the active state, (e) to identify brain stem interneurons which are involved in the active state, and (f) to integrate this information as cellular and circuit-related models for neural modulation during an arousal-like state in the isolated brain stem of a simpler vertebrate.

The long term goal of this research is to understand how the growth of blood vessels in the brain is controlled. Endothelium is a vital cellular component of the nervous system, is the conduit for circulation and exchange of metabolites, is the major interface of the blood-brain barrier, and has important chemical and regulatory functions. The cellular and molecular mechanisms which regulate the growth of brain endothelium during normal angiogenesis are uncertain. These mechanisms relate to congenital arteriovenous malformations, which arise from errors during brain angiogenesis, and to brain hemangioblastomas, in which proliferation of endothelial-like cells is uncontrolled. In addition, insufficient capillary growth in the retina of premature infants may initiate retrolental fibroplasia and blindness. Finally, brain tumors depend upon angiogenesis from brain endothelium for their growth and survival and might be controlled by inhibiting these mechanisms. The model selected for brain angiogenesis is the transparent albino Xenopus laevis tadpole. The structure of individual capillaries on the surface of the optic tectum can be resolved by light microscopy in vivo. The first specific aim is to chart the growth of the network of blood vessels in individual tadpoles from stage 43 with only a few capillaries through the major 4-6 week period of angiogenesis as the optic tectum grows about 10-fold in surface area. Second, quantitative determinations will be made for density of capillary sprouts, intratectal vascular branches, and capillary lengths per surface area. Third, angiogenic growth factors and inhibitors will be injected into the blood, ventricular fluid, and pial spaces with micropipettes to alter the growth of tectal blood vessels. Fourth, antisera and monoclonal antibodies will be produced against Xenopus endothelium as developmental markers. A fifth aim is to culture capillary endothelium, macrophages, and pericytes from the same albino strain of Xenopus and to test angiogenic responses in vitro and interactions of marked cells with brain angiogenesis in vivo.

The proposed research addresses the ability of brain blood vessels to adapt their responsiveness, local blood flow and architecture to alterations in the cerebral cortex during cortical development, in response to neural activity, and after sensory deafferentation or deprivation. We use the well established model system, the whisker-- barrel cortex of the rat. This collaboration combines the complementary talents and resources of cerebrovascular laboratories in the Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences, St. Petersburg, Russian Republic, USSR, and in Washington University, St. Louis, Missouri, USA. This proposal will expand and enhance two research projects funded by NIH: (a) ROlHL41075 "Models for brain angiogenesis", and (b) R01 NS28781 "Imaging brain blood vessels during cortical activity". It will facilitate collaborative and correlative research on local cerebral blood flow, P02, evoked activity, angiogenesis, and vascular remodeling. Methods for videomicroscopy in vivo, fluorescent tracers, and histology of the barrel cortex of rodents are well established in St. Louis. The laboratory in St. Petersburg provides expertise in biophysical measurements of local cerebral blood flow (ICBF), extracellular fluid space (ECS), and tissue P02, using the same barrel cortex model. The expected results will include the correlation of physiological and videomicroscopic parameters in normal barrel cortex of rats and remodeling of cerebral blood vessels during development and after selective sensory deafferentation.