Kirstie A. Cummings - US grants

DESCRIPTION (provided by applicant): Glycinergic N-methyl-D-aspartate receptors (NRs) are a unique type of excitatory channels. They differ from the traditional glutamatergic NRs in that that they are activated by glycine alone, are insensitive to glutamate, and have a lower unitary conductance, Ca2+ permeability, and sensitivity to voltage-dependent Mg2+ block. They are tetramers of GluN1 (N1) and GluN3 (N3A or N3B) subunits. The N1 subunit has eight splice variants (N1-1a through N1-4b), each with specific spatiotemporal expression patterns in the brain. The N3A subunit is specifically expressed neonatally and is critical for spine development and synaptic plasticity. Altered expression of N3A has been linked to the negative symptoms of schizophrenia. Contrary to what has been observed in glutamatergic NRs (N1/N2), strikingly distinct macroscopic current is produced dependent on the N1 splice variant with which N3A assembles. I propose that these differences may arise in part from distinct gating kinetics conferred onto N1/N3A receptors by the C-terminal cassettes of N1 splice variants. To test this hypothesis, I will pursue the following three aims: 1) Fully characterize the high-activity N1-4a/N3 isoform using kinetic analyses and state modeling. 2) Identify the kinetic contributions of each C-terminal cassette by systematically comparing reaction mechanisms of selected N1/N3A isoforms. 3) Identify the elements on each cassette responsible for gating modulation by combining sequence-based mutagenesis and kinetic analyses. The results from this proposal will provide insights into the mechanism by which differential slicing of N1 subunits controls the functional output of N1/N3A receptors. Despite having been cloned almost two decades ago, very little is known about the functional mechanism of N1/N3A receptors and how they contribute to both physiological and pathological states. Insights from this proposal will spur rational hypotheses to address these gaps in knowledge and will afford a more comprehensive understanding of the underlying molecular mechanism of the pathophysiology of schizophrenia.

Project Summary Fear responses to environmental threats are critical for survival. However, enhanced fear responses are a hallmark of anxiety disorders, such as posttraumatic stress disorder (PTSD), due in part to increased activity in the dorsal anterior cingulate (dACC). Rodents are widely used for to study mechanisms of fear expression due to the numerous anatomical similarities that exist with humans. In rodents, the functional analog of dACC is the prelimbic (PL) subdivision of the medial prefrontal cortex which also exhibits increased activity after fear memory formation. However, a complete understanding of how PL participates in fear memory formation as well as the details of the circuit-based mechanisms supporting this function are lacking. PL exhibits a highly organized laminar structure composed of excitatory projection-specific output neurons as well as a prominent network of inhibitory interneurons. Both classes of neurons receive layer-specific inputs from another fear- related structure, the basolateral amygdala (BLA). However, it is unclear how BLA inputs and PL laminar organization participate in memory formation. Preliminary data suggest that a sparse PL neural ensemble is recruited during fear memory formation, that optogenetic ensemble reactivation induces fear- and anxiety-like behaviors, and that excitatory inputs onto specific excitatory and inhibitory neurons exhibit fear memory-related synaptic plasticity. Based on these data, I propose that a PL neural ensemble contributes to fear memory storage and is selectively recruited by joint plasticity of excitatory and inhibitory circuits after fear learning. To test this hypothesis, I will pursue the following two aims: 1) Characterize a fear-related PL neural ensemble using activity-dependent ensemble tagging, confocal imaging, and in vivo optogenetic ensemble manipulation. 2) Identify the plasticity of BLA inputs and PL microcircuits ensuring the selective recruitment of the fear memory ensemble by using intersectional- and optogenetics-assisted cell type-specific dual patch-clamp recordings in mouse brain slices. Results from this proposal will be the first to identify plasticity mechanisms underlying selective PL fear memory ensemble recruitment. Moreover, these findings will afford a more comprehensive understanding of PL-mediated fear expression and therefore mechanisms underlying pathologic fear expression in PTSD and will lay the foundation for interrogating mechanisms of ensemble recruitment in many diverse behaviors and brain regions.

Anxiety disorders such as post-traumatic stress disorder (PTSD) typically nucleate when individuals experience a highly traumatic event. One hallmark of PTSD is pronounced expression of fear and resistance to fear-suppressing behavioral therapies. The medial prefrontal cortex (mPFC) is important for mediating both the expression and inhibition of learned fear. Specifically, the human dorsal anterior cingulate and ventromedial (vmPFC) subdivisions of mPFC are generally believed to be responsible for mediating the expression and inhibition of fear, respectively. However, several studies have suggested that in addition to its fear-inhibiting role, activity in the vmPFC is associated with increased anxiety in non-human primates and humans. Moreover, vmPFC activity is elevated in a third of PTSD patients. More strikingly, damage to the vmPFC has been suggested to protect against the development of PTSD. Despite its clear relevance to pathological fear behaviors, the mechanisms underlying this functional dichotomy in vmPFC remain unclear. Rodents are routinely used to study the mechanisms of fear memory regulation due to their numerous circuit parallels with humans. Similar to the vmPFC in humans, the rodent vmPFC mediates the inhibition of fear and can be divided into two anatomically distinct subregions, including the infralimbic (IL) and dorsal peduncular (DP) cortices. While both vmPFC subregions are thought to mediate the inhibition of fear, all studies have centered on IL and there are no studies that explicitly examine the potential contributions of DP during the regulation of memory. In contrast to its hypothesized role in mediating fear inhibition, my preliminary data indicate that DP is engaged during the expression of conditioned fear and exhibits evidence of fear learning-dependent plasticity. Based on these data, I propose that DP encodes learned fear through learning-dependent excitatory and inhibitory plasticity. To test this hypothesis, I propose to pursue the following two aims: 1. Resolve the activity dynamics and long-range targets of fear-activated DP neurons by using Miniscope in vivo calcium imaging in DP, anterograde circuit tracing, and combining the use of in vivo optogenetic manipulation of fear-tagged DP neurons and fiber photometry in target brain regions. 2. Determine the circuit mechanisms leading to fear learning-dependent DP recruitment by combining in vivo optogenetic manipulation of DP-projecting populations and fiber photometry in DP. We will also employ ex vivo electrophysiological measurements of experience- dependent plasticity of long-range projections to DP in a cell type-specific manner. Results from this proposal will be the first to characterize the role of DP in the regulation of fear memory, to outline how it integrates into existing models of fear circuitry, and to delineate the circuit plasticity mechanisms ensuring is recruitment after fear learning.