Periodic Reporting for period 1 - PeptidesAndFear (Neuropeptidergic modulation of synaptic circuits in fear and anxiety)
Reporting period: 2022-05-01 to 2024-10-31
Fear and anxiety are essential for survival. Adequate reactions to anticipated and perceived threats strongly depend on the current state, context and previous experience. What are the neurobiological mechanisms that underlie these adaptive behaviors? While synaptic circuits are the backbone to process threat signals and transform them into behavioral responses, increasing evidence suggests that neuropeptides tune these synaptic circuits in a state- and context-dependent manner, thus allowing for temporal control of excitability and plasticity. Neuropeptide receptors are abundantly expressed in neuronal circuits that regulate fear and anxiety. In fact, recent evidence suggests that most neurons synthesize multiple neuropeptides and, in turn, are regulated by multiple neuropeptide receptors that are expressed in highly cell type-specific patterns. Indeed, genetic alterations in humans as well as pharmacological and genetic experiments in mice indicate important roles for multiple neuropeptides in regulating fear and anxiety and, if dysfunctional, increasing the risk for multiple neuropsychiatric disorders. However, due to the lack of specificity of existing tools, the circuit mechanisms underlying these effects are largely unknown.
In this proposal we aim to reveal cell type-specific neuropeptidergic signaling mechanisms for context-dependent regulation of cortical circuit activity, cellular excitability and synaptic plasticity. We are developing and using cutting-edge in vivo imaging and genetic manipulation tools to reveal how these neuropeptidergic signaling mechanisms regulate behavioral responses in the context of fear and anxiety.
In this proposal we aim to reveal cell type-specific neuropeptidergic signaling mechanisms for context-dependent regulation of cortical circuit activity, cellular excitability and synaptic plasticity. We are developing and using cutting-edge in vivo imaging and genetic manipulation tools to reveal how these neuropeptidergic signaling mechanisms regulate behavioral responses in the context of fear and anxiety.
The overarching goal of this project is to identify how cell type- and circuit-specific interactions of neuropeptides alter behavioral responses to threats and anxiogenic stimuli.
In the first two years of this project, we used in vitro labeling techniques, in vitro imaging and electrophysiological techniques to identify targets of neuropeptidergic communication in the medial prefrontal cortex of mice, an area that is implicated in the regulation of fear and anxiety. We used optogenetic stimulation and electrical recordings to reveal synaptic mechanisms through which neuropeptides alter circuit communication. Thus, we found neuropeptide-mediated modulation of inhibitory and excitatory neurotransmission within the cortex. Our current electrophysiological and optical recordings aim at deciphering in detail the contribution of presynaptic release machinery components and ion channels that are involved in the regulation of neuronal excitability and vesicle release.
Based on our detailed analysis of neuropeptidergic mechanisms, we developed CRISPR-Cas9 based genetic tools that allow us to ablate neuropeptidergic pathways in defined cell types and neuronal circuits. Our newly developed tools target neuropeptide receptors as well as intracellular signaling pathways that we identified as important modulators of prefrontal neuronal circuits in our in vitro assays. Using these ablation techniques in a cell type- and circuit-specific manner will allow us to dissect the neuropeptidergic functions and mechanisms in vivo and identify functional implications for fear and anxiety.
Moreover, we developed imaging techniques to observe neuronal activity and intracellular signaling within the medial prefrontal cortex during fear and anxiety. In combination with our genetic ablation tools, these imaging techniques will allow us to correlate behavioral changes with changes in neuronal signaling dependent on the presence or absence of neuropeptidergic signaling.
In the first two years of this project, we used in vitro labeling techniques, in vitro imaging and electrophysiological techniques to identify targets of neuropeptidergic communication in the medial prefrontal cortex of mice, an area that is implicated in the regulation of fear and anxiety. We used optogenetic stimulation and electrical recordings to reveal synaptic mechanisms through which neuropeptides alter circuit communication. Thus, we found neuropeptide-mediated modulation of inhibitory and excitatory neurotransmission within the cortex. Our current electrophysiological and optical recordings aim at deciphering in detail the contribution of presynaptic release machinery components and ion channels that are involved in the regulation of neuronal excitability and vesicle release.
Based on our detailed analysis of neuropeptidergic mechanisms, we developed CRISPR-Cas9 based genetic tools that allow us to ablate neuropeptidergic pathways in defined cell types and neuronal circuits. Our newly developed tools target neuropeptide receptors as well as intracellular signaling pathways that we identified as important modulators of prefrontal neuronal circuits in our in vitro assays. Using these ablation techniques in a cell type- and circuit-specific manner will allow us to dissect the neuropeptidergic functions and mechanisms in vivo and identify functional implications for fear and anxiety.
Moreover, we developed imaging techniques to observe neuronal activity and intracellular signaling within the medial prefrontal cortex during fear and anxiety. In combination with our genetic ablation tools, these imaging techniques will allow us to correlate behavioral changes with changes in neuronal signaling dependent on the presence or absence of neuropeptidergic signaling.
The identification of mechanisms that regulate fear and anxiety is of great interest not only for the basic understanding of the brain, but also for clinical applications. In the past, manipulations to neuropeptidergic signaling in the context of fear and anxiety were mostly performed globally (whole brain) or systemically. Increasing research on neuropeptide functions however reveal more sophisticated signaling mechanisms that suggest highly brain area- and cell type-specific effects. These can be easily overlooked by global manipulations that trigger compensatory and counteracting mechanisms. Therefore, cell type- and circuit-specific manipulation tools are quintessential for future investigation of neuropeptidergic functions and mechanisms. Our newly developed genetic ablation tools fill a technical gap that will allow us to reveal mechanisms of anxiety and fear in more detail than before. Moreoever, our new tools will allow also other researchers easy access to cell type- and circuit-specific manipulations of neuropeptidergic signaling mechanisms and thereby advance insights into many other functions of neuropeptides in the brain.