Project:      Central nervous mechanisms regulating mammalian neuroendocrine and cardiovascular function

Start Date:             July 01, 1998

Finish Date:           June 30, 2000

Scientific or Technological Objectives:    This project is in the category of Basic Research.  In mammals, the hypothalamic suprachiasmatic nucleus is regarded as the main biological clock that entrains various body functions, including neuroendocrine and cardiovascular functions, into a circadian rhythmicity.  Manifestations of this entrainment are the early morning peaks in circulating glucocorticoids (endocrine) and arterial blood pressure (cardiovascular).  However the cellular mechanisms involved in this entrainment remain to be identified.  Neurons in the hypothalamic paraventricular nucleus designated as regulators of neuroendocrine or autonomic functions are likely targets for suprachiasmatic entrainment.  On the hypothesis that suprachiasmatic neurons exert this control via their projections to paraventricular nucleus, the objective is to use an in vitro brain slice preparation of rat hypothalamus and patch clamp recording techniques to define the characteristics of neurotransmission between the suprachiasmatic nucleus and neurons in the paraventricular nucleus.  Since most suprachiasmatic nucleus neurons synthesize the inhibitory neurotransmitter GABA, we will seek evidence that electrical stimulation in suprachiasmatic nucleus evokes a monosynaptic inhibitory postsynaptic potential in specific paraventricular nucleus neurons, blockable with selective GABA-A receptor antagonists.  Such observations would be proof of a direct connection, and that suprachiasmatic outflow pathways do indeed affect (inhibit) the excitability of neurons in the target nucleus.  Target paraventricular neurons with a neuroendocrine role (i.e. secretion of posterior pituitary hormones) can be recognized by their known and unique intrinsic electrical properties, and large cell size when visualized after intracellular labeling.  Target neurons regulating cardiovascular functions can be recognized through retrogradely transported labels injected into cardiovascular sites in the brainstem medulla.

Scientific or Technological Advancement: In the broad field of Neuroscience, this research is classed as neurophysiology and neuropharmacology.  The current approaches to this area involve the use of in-vitro preparations and patch clamp recordings, so as to achieve a measure of experimental control not possible with in vivo whole animal recording.   The technological advancement is the achievement of a brain slice preparation that contains the actual connections to be studied between the source, the suprachiasmatic nucleus, and the putative target, the paraventricular nucleus.  The scientific advancement  would be indisputable evidence that the connections can actually be demonstrated in data that are indisputable, revealing direct pathways that use a neurotransmitter that is likely to be made by the suprachiasmatic neurons.

Scientific or  Technological Uncertainty:

1.     The existence of ‘direct’ connections from the suprachiasmatic nucleus to the various subtypes of neurons in the paraventricular nucleus has been at best controversial mainly because the most recent anatomical tracer studies have not been done at the level of resolution (electron microscopic) that would prove their existence.  While we lack the detailed anatomical proof, we interpret our data as functional indication for the existence of ‘direct’ connections between suprachiasmatic and paraventricular nucleus neurons.

2.     Use of electrical stimulation always raises concerns that an observed response is due to an artifact of current spread to neurons that are outside the area of interest, in the peri-suprachiasmatic region.  We attempt to replicate the results of electrical stimulation by alternative means, that is chemical (glutamate) microstimulation, which only activates cell bodies, not fibers of passage.

3.     The major technical concern is the variability in the slice preparation itself.  This arises in part because of the way the brain is sliced, resulting in variances in the data used to support the hypothesis.  We are attempting to capture the majority of the neural pathways we wish to study by cutting thicker brain slice preparations.  A drawback is that cells become less viable in thicker slices because of poorer penetration of the nutrient media.