Allan T. Gulledge, Ph.D. - US grants

DESCRIPTION (provided by applicant): Acetylcholine (ACh) plays a critical role in cognition, and decreased cholinergic input to the cerebral cortex contributes to the cognitive deficits observed in Alzheimer's disease, dementia with Lewy Bodies, Parkinson's dementia, and other neurological diseases. However, a lack of knowledge regarding the pharmacology of cholinergic effects, or the mechanisms by which ACh influences neuronal activity, have hampered the development of therapies specific to these debilitating diseases. The data that do exist appear conflicting, and have been difficult to reconcile. Indeed, ACh paradoxically generates two opposing responses in the deep-layer pyramidal neurons that provide the majority of cortical output: a fast transient inhibition and a longer-lasting excitation. Although the mechanisms mediating inhibitory cholinergic responses in these neurons (M1-like muscarinic acetylcholine receptor activation, calcium- release from intracellular calcium stores, and subsequent activation of an SK-type calcium-activated potassium conductance) have been well described, the mechanisms mediating cholinergic excitation, and the functional relationship between excitatory and inhibitory cholinergic signaling, remain unknown. This project aims to determine the receptor subtypes, signaling cascades, and ionic mechanisms responsible for cholinergic excitation in cortical layer 5 pyramidal neurons, and to test the overarching hypothesis that excitatory actions of ACh reflect activation of a calcium-permeable non-selective cationic conductance that acts functionally to replenish the intracellular calcium stores that gate inhibitory cholinergic signaling. We propose to use electrophysiological and imaging approaches in a brain slice preparation to address the following three specific aims: 1. To identify the specific muscarinic receptor(s) mediating cholinergic excitation and inhibition in neocortical layer 5 pyramidal neurons. 2. To determine the signaling cascades and ionic mechanism responsible for cholinergic excitation of layer 5 neurons. 3. To test the unifying hypothesis that excitatory cholinergic conductances serve functionally to refill intracellular calcium stores depleted during inhibitory cholinergic signaling. Our results will provide a framework for understanding the biological basis for cholinergic facilitation of cognitive function. This new knowledge will increase our understanding of why dysfunction of cholinergic systems leads to the functional deficits observed in dementia and other disease states, and will provide new targets for therapeutic intervention. The Public Health Relevance: Acetylcholine is a brain chemical necessary for normal cognitive function, and loss of acetylcholine is associated with Alzheimer's disease and other disease states. This project will determine the biological mechanisms by which acetylcholine influences the activity of neurons in the normal cerebral cortex, with the aim of understanding why loss of acetylcholine during aging or disease leads to cognitive dysfunction.

DESCRIPTION (provided by applicant): Cortical microcircuits, comprising specialized neuron subpopulations and their selective synaptic connections, form functionally segregated output channels to other cortical and subcortical targets. The activity of cortical microcircuits is regulated by a number of modulatory neurotransmitters, such as serotonin (5-HT), that optimize circuit performance for specific cognitive tasks, and which are implicated in a wide spectrum of mental health disorders. For instance, disrupted 5-HT signaling contributes to schizophrenia, depression, and anxiety, while serotonergic drugs are widely used to treat these disorders. However, despite their functional and clinical importance, little is known about how modulatory transmitters selectively regulate the activity of cortical neuron subpopulations subserving specific functional roles within cortical microcircuits. Our long-term goal is to characterize the functional impact of modulatory neurotransmitters on cortical microcircuit function. The research proposed here represents an important first step toward this goal by identifying key cellular and synaptic components of cortical microcircuits differentially regulated by 5-HT. Serotonergic signaling in excitatory cortical neurons relies primarily on two G-protein coupled receptors, 5-HT1A (1A) and 5-HT2A (2A), that have opposing influences on neuron excitability. Based on recent results showing commissural/callosal (COM) projection neurons are selectively excited by 5-HT, our central hypothesis is that endogenous 5-HT acts to selectively enhance the activity of neurons participating in specific executive functions, while suppressing the bulk of cortical output to subcortical structures. Our first aim is to identify the cellular components of cortical circuits that are functionally excited or inhibited by endogenous 5-HT. This will be accomplished in a mouse model in which channelrhodopsin-2 is selectively expressed in 5-HT neurons, and using fluorescent retrograde tracers delivered in vivo. Our second aim is to characterize synaptic connectivity among populations of 5-HT-excited neurons to determine whether they form networks capable of the sustained activity associated with executive functions. Finally, our third aim will confirm in behaving animals preferential activatio of COM or other 5-HT-excited neuron populations by 2A agonists, fear conditioning, and extinction of conditioned fear. These aims are innovative in combining anatomical, physiological, optogenetic, and behavioral approaches to characterize selective modulation of cellular components and synaptic connections underlying functionally defined cortical output channels. The results will be significant in providing a framework for understanding the functional role of 5 HT in regulating the output of cortical circuits. The new knowledge gained will provide insight into how 5-HT facilitates normal cognition and behavior, and why dysregulation of serotonergic signaling in the cerebral cortex leads to the behavioral deficits observed in schizophrenia, depression, and other mental health disorders.

Project Summary Individuals with fear disorders such as post-traumatic stress disorder (PTSD) experience excessively strong and intrusive fear memories about stimuli that were encountered during a prior trauma, including individual sounds or visual stimuli (?cue-specific? fear memory) or the combination of stimuli that together define the place in which the event occurred (?contextual? fear memory). The memories may have been formed recently or long ago (?remote? memories), in which case they may plague a person for a substantial portion of his/her life. The development of effective therapies depends on a thorough understanding the neural mechanisms that underlie these different types of fear memories, as well as fear extinction, which is the basis for exposure- based therapy commonly used to reduce fear in humans. To date, a substantial body of research has identified discrete neural systems that support recent versus remote contextual memory, and other studies have identified the substrates of recently-acquired cue-specific memory and extinction, but very little work has focused on the brain mechanisms involved in remote cue- specific memory and extinction. This is important to resolve particularly with respect to PTSD since individuals often do not seek therapy until long after the traumatic event, especially in cases of combat trauma or sexual assault. To address this, the proposed research advances a new theoretical model of the neural circuits that underlie remote cue-specific fear memory and extinction. This model is based on new data from our laboratory and combines state-of-the art chemogenetic and optogentic-anatomical approaches to test the hypotheses that a) communication between the retrosplenial cortex and secondary sensory cortices is necessary for remote cue-specific fear memory, and b) the postrhinal cortex mediates the context-dependency of extinction of remote cue-specific fear.

ABSTRACT PTEN is a phosphatidylinositol phosphatase that antagonizes signaling downstream of growth factor receptors. Mutations in PTEN have repeatedly been identified in patients with autism spectrum disorder (ASD) and macrocephaly. Further, experimental deletion of Pten in the mouse brain causes macrocephaly and deficits in social behavior, suggesting a causative role in the development of ASD. In neurons, Pten knockout results in aberrant growth and increased excitatory synapse function. Thus, studying Pten fits with our long- term goal of understanding how synaptic connectivity and activity contribute to cognitive and emotional processes. My central hypothesis is that Pten dysfunction causes aberrant neuronal growth and excitability leading to altered synaptic circuit formation during development. Guided by this hypothesis, the specific aims of this proposal will strengthen our understanding of the molecular and neurophysiological basis of ASD. There is a lack of pharmacological therapies for ASDs because we are just beginning to identify the molecular mechanisms underlying these disorders. We have defined a set of robust and reproducible cellular phenotypes elicited by Pten knockout in developing neurons. Understanding the molecular mechanisms underlying cellular phenotypes could lead to new treatments for ASDs. Our first aim will test the hypothesis that cellular phenotypes are caused by deregulation of translation and cytoskeletal organization to alter the developmental elaboration of neurons. Defining cell-autonomous changes in neuronal development is a first step into understanding the emergent impact on network formation and function. Our second aim will test our central hypothesis by determining whether Pten knockout results in similar cellular phenotypes across neuronal types and contexts. Different genetic models of ASDs display disparate cellular changes. Some models display synaptic hyperconnectivity while others display hypoconnecectivity and there is variability in excitation/inhibition ratios. Activity-dependent sculpting of synaptic connectivity during development fundamentally shapes network activity allowing for appropriate responses to our environment. A common feature shared by models of ASD may be pathological activity-dependent sculpting of synaptic connectivity during development. For the third aim, we will test the hypothesis that Pten dysfunction alters the activity-dependent sculpting of neuronal connectivity during development. This proposal will use innovative genetic approaches to manipulate gene expression and control neuronal activity in vivo. We will test the consequences of these genetic manipulations through detailed neuronal morphological and electrophysiological analyses. The broad goal of this research is to define the molecular basis of how Pten dysfunction contributes to aberrant neuronal development and network function.