in-vivo-calcium-imaging-with-miniaturized-microscope-hypothalamus-for

<em>In Vivo</em> Calcium Imaging with a Miniaturized Microscope in the Hypothalamus for Understanding Social Behaviors in Mice

Social behavior in vertebrates is tightly regulated through hypothalamic circuits. Molecularly defined hypothalamic neuron types were recently identified ...

Harvard University

Although mGluR5-antagonists prevent fear and anxiety, little is known about how the same receptor in the amygdala gives rise to both. Combining in vitro and in vivo activation of mGluR5 in rats, we identify specific changes in intrinsic excitability and synaptic plasticity in basolateral amygdala neurons that give rise to temporally distinct and mutually exclusive effects on fear-related behaviors. The immediate impact of mGluR5 activation is to produce anxiety manifested as indiscriminate fear of both tone and context. Surprisingly, this state does not interfere with the proper encoding of tone-shock associations that eventually lead to enhanced cue-specific fear. These results provide a new framework for dissecting the functional impact of amygdalar mGluR-plasticity on fear versus anxiety in health and disease.

Activation of the same mGluR5 receptors in the amygdala causes divergent effects on specific versus indiscriminate fear.

Stress is known to exert its detrimental effects not only by enhancing fear, but also by impairing its extinction. However, in earlier studies stress exposure preceded both processes. Thus, compared to unstressed animals, stressed animals had to extinguish fear memories that were strengthened by prior exposure to stress. Here, we dissociate the two processes to examine if stress specifically impairs the acquisition and recall of fear extinction. Strikingly, when fear memories were formed before stress exposure, thereby allowing animals to initiate extinction from comparable levels of fear, recall of fear extinction was unaffected. Despite this, we observed a persistent increase in theta activity in the BLA. Theta activity in the mPFC, by contrast, was normal. Stress also disrupted mPFC-BLA theta-frequency synchrony and directional coupling. Thus, in the absence of the fear-enhancing effects of stress, the expression of fear during and after extinction reflects normal regulation of theta activity in the mPFC, not theta hyperactivity in the amygdala.

Extinction recall of fear memories formed before stress is not affected despite higher theta activity in the amygdala.

Fragile X syndrome (FXS), a commonly inherited form of autism and intellectual disability, is associated with emotional symptoms that implicate dysfunction of the amygdala. However, current understanding of the pathogenesis of the disease is based primarily on studies in the hippocampus and neocortex, where FXS defects have been corrected by inhibiting group I metabotropic glutamate receptors (mGluRs). Here, we observe that activation, rather than inhibition, of mGluRs in the basolateral amygdala reverses impairments in a rat model of FXS. FXS rats exhibit deficient recall of auditory conditioned fear, which is accompanied by a range of in vitro and in vivo deficits in synaptic transmission and plasticity. We find presynaptic mGluR5 in the amygdala, activation of which reverses deficient synaptic transmission and plasticity, thereby restoring normal fear learning in FXS rats. This highlights the importance of modifying the prevailing mGluR-based framework for therapeutic strategies to include circuit-specific differences in FXS pathophysiology.

Correction of amygdalar dysfunction in a rat model of fragile X syndrome.

Estimating the pose of multiple animals is a challenging computer vision problem: frequent interactions cause occlusions and complicate the association of detected keypoints to the correct individuals, as well as having highly similar looking animals that interact more closely than in typical multi-human scenarios. To take up this challenge, we build on DeepLabCut, an open-source pose estimation toolbox, and provide high-performance animal assembly and tracking-features required for multi-animal scenarios. Furthermore, we integrate the ability to predict an animal's identity to assist tracking (in case of occlusions). We illustrate the power of this framework with four datasets varying in complexity, which we release to serve as a benchmark for future algorithm development.

Multi-animal pose estimation, identification and tracking with DeepLabCut.

Parenting behavior comprises a variety of adult-infant and adult-adult interactions across multiple timescales. The state transition from nonparent to parent requires an extensive reorganization of individual priorities and physiology and is facilitated by combinatorial hormone action on specific cell types that are integrated throughout interconnected and brainwide neuronal circuits. In this review, we take a comprehensive approach to integrate historical and current literature on each of these topics across multiple species, with a focus on rodents. New and emerging molecular, circuit-based, and computational technologies have recently been used to address outstanding gaps in our current framework of knowledge on infant-directed behavior. This work is raising fundamental questions about the interplay between instinctive and learned components of parenting and the mutual regulation of affiliative versus agonistic infant-directed behaviors in health and disease. Whenever possible, we point to how these technologies have helped gain novel insights and opened new avenues of research into the neurobiology of parenting. We hope this review will serve as an introduction for those new to the field, a comprehensive resource for those already studying parenting, and a guidepost for designing future studies.

The neurobiology of parenting and infant-evoked aggression.

Multiplexed error-robust fluorescence in situ hybridization (MERFISH) allows genome-scale imaging of RNAs in individual cells in intact tissues. To date, MERFISH has been applied to image thin-tissue samples of ~10 µm thickness. Here, we present a thick-tissue three-dimensional (3D) MERFISH imaging method, which uses confocal microscopy for optical sectioning, deep learning for increasing imaging speed and quality, as well as sample preparation and imaging protocol optimized for thick samples. We demonstrated 3D MERFISH on mouse brain tissue sections of up to 200 µm thickness with high detection efficiency and accuracy. We anticipate that 3D thick-tissue MERFISH imaging will broaden the scope of questions that can be addressed by spatial genomics.

Article	Year
Activation of the same mGluR5 receptors in the amygdala causes divergent effects on specific versus indiscriminate fear. eLife\| PubMed ID: 28555566	2017
Extinction recall of fear memories formed before stress is not affected despite higher theta activity in the amygdala. eLife\| PubMed ID: 30102149	2018
Correction of amygdalar dysfunction in a rat model of fragile X syndrome. Cell reports\| PubMed ID: 34644573	2021
Multi-animal pose estimation, identification and tracking with DeepLabCut. Nature methods\| PubMed ID: 35414125	2022
The neurobiology of parenting and infant-evoked aggression. Physiological reviews\| PubMed ID: 39146250	2025
Three-dimensional single-cell transcriptome imaging of thick tissues. eLife\| PubMed ID: 39727221	2024

Mohammed Mostafizur Rahman

Biography

JoVE Journal Publications

Other Publications