Methods to identify different classes of neurons and decipher their properties represent the Rosetta stone of neuroscience research. These tools are essential in order to understand how to selectively manipulate the activity of defined neuronal circuits in nervous system disorders. Thus, our goal here is to validate a novel mouse model to investigate glutamatergic neurons in the basal forebrain (BF), an important brain region controlling activation of the cortex, attention and sleep. The BF is impaired in coma, sleep disorders and Alzheimer's disease. Thus, a better understanding of its component neurons and their properties may lead to novel treatments for these disorders. We will study BF neurons which use the vesicular glutamate transporter, type 2 (vGluT2), the major subtype of BF glutamate neurons, including those which are project to the cortex. Mice expressing the bacterial enzyme, Cre Recombinase (Cre), under the control of the vGluT2 gene promoter region will be crossed with a reporter mouse strain which expressing a red fluorescent protein tdTomato in the presence of Cre, to yield vGluT2-Tomato mice. These novel mice will allow online identification of BF vGluT2 neurons, and is an essential first step prior to in vivo studiesof the role of BF glutamate neurons in sleep-wake behavior. Use of tdTomato as a reporter will allow combinatorial approaches using pre-existing transgenic mice and viral vectors expressing green/yellow fluorescent proteins.
Aim1 will validate the selective expression of tdTomato in BF glutamatergic neurons. We will confirm that tdTomato is not expressed in the two other major BF neurotransmitter classes, cholinergic and GABAergic neurons, and test if a subset of putative cortically-projecting BF vGluT2 neurons expresses the calcium-binding protein, calbindin. Optogenetic techniques will validate the release of glutamate and reveal the targets of BF glutamate neurons.
Aim 2 will reveal the intrinsic electrical properties of BF glutamate neurons for the first time using whole-cell patch-clamp recordings from vGluT2-Tomato neurons. Excitingly, our preliminary data indicate that a subset of medium-large sized, putative cortically projecting neurons exhibits a unique pattern of burst firing, reminiscent of the firing patterns of putative glutamate neurons recorded in vivo. Injections of retrograde tracer in the prefrontal cortex will be used to definitively identify cortically-projecting BF glutamate neurons.
Aim3 will investigate the effects of a cholinergic receptor agonist and adenosine on BF glutamate neurons in vitro. BF cholinergic neurons are involved in cortical activation during wakefulness and REM sleep, and degenerate early in the course of Alzheimer's disease. Our preliminary data suggests that cortically-projecting BF glutamate neurons are hyperpolarized by cholinergic inputs, facilitating rhythmic firing during waking and REM sleep by de-inactivating a low-threshold calcium current. Adenosine is an important homeostatic sleep factor. We predict adenosine promotes sleep via postsynaptic inhibition of local BF glutamate interneurons and presynaptic inhibition of excitatory inputs to cortically-projecting BF glutamate neurons.

Public Health Relevance

Here we aim to validate a novel transgenic mouse model (vGluT2-Tomato mice) to investigate for the first time the properties of identified glutamatergic neurons in the basal forebrain (BF), an important brain region controlling cortical activation, attention and sleep. BF function is impaired in coma, sleep disorders and Alzheimer's disease. Thus, an understanding of BF glutamate neurons and their properties may lead to novel treatments for these disorders.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Exploratory/Developmental Grants (R21)
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Neuroendocrinology, Neuroimmunology, Rhythms and Sleep Study Section (NNRS)
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Stewart, Randall R
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Harvard Medical School
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