Combining RNAscope and immunohistochemistry to visualize inflammatory gene products in neurons and microglia.

A challenge for central nervous system (CNS) tissue analysis in neuroscience research has been the difficulty to codetect and colocalize gene and protein expression in the same tissue. Given the importance of identifying gene expression relative to proteins of interest, for example, cell-type specific markers, we aimed to develop a protocol to optimize their codetection. RNAscope fluorescent in situ hybridization (FISH) combined with immunohistochemistry (IHC) in fixed (CNS) tissue sections allows for reliable quantification of gene transcripts of interest within IHC-labeled cells. This paper describes a new method for simultaneous visualization of FISH and IHC in thicker (14-µm), fixed tissue samples, using spinal cord sections. This method's effectiveness is shown by the cell-type-specific quantification of two genes, namely the proinflammatory cytokine interleukin-1beta (IL-1b) and the inflammasome NLR family pyrin domain containing 3 (NLRP3). These genes are challenging to measure accurately using immunohistochemistry (IHC) due to the nonspecificity of available antibodies and the hard-to-distinguish, dot-like visualizations of the labeled proteins within the tissue. These measurements were carried out in spinal cord sections after unilateral chronic constriction injury of the sciatic nerve to induce neuroinflammation in the spinal cord. RNAscope is used to label transcripts of genes of interest and IHC is used to label cell-type specific antigens (IBA1 for microglia, NeuN for neurons). This combination allowed for labeled RNA transcripts to be quantified within cell-type specific boundaries using confocal microscopy and standard image analysis methods. This method makes it easy to answer empirical questions that are intractable with standard IHC or in situ hybridization alone. The method, which has been optimized for spinal cord tissue and to minimize tissue preparation time and costs, is described in detail from tissue collection to image analysis. Further, the relative expression changes in inflammatory genes NLRP3 and IL-1b in spinal cord microglia vs. neurons of somatotopically relevant laminae are described for the first time.

Bed nucleus of the stria terminalis GABA neurons are necessary for changes in foraging behaviour following an innate threat.

Foraging is a universal behaviour that has co-evolved with predation pressure. We investigated the role of the bed nucleus of the stria terminalis (BNST) GABA neurons in robotic and live predator threat processing and their consequences in post-threat encounter foraging. Both robotic and live predator interactions increased BNST GABA neuron activity. Mice were trained to procure food in a laboratory-based foraging apparatus in which food pellets were placed at incrementally greater distances from a nest zone. After mice learned to forage, they were exposed to a robotic or live predator threat, while BNST GABA neurons were chemogenetically inhibited. Post-robotic threat encounter, mice spent more time in the nest zone, but other foraging parameters were unchanged compared with pre-encounter behaviour. Inhibition of BNST GABA neurons had no effect on foraging behaviour post-robotic threat encounter. Following live predator exposure, control mice spent significantly more time in the nest zone, increased their latency to successfully forage, and significantly altered their overall foraging performance. Inhibition of BNST GABA neurons during live predator exposure prevented changes in foraging behaviour from developing after a live predator threat. BNST GABA neuron inhibition did not alter foraging behaviour during robotic or live predator threats. We conclude that these results demonstrate that while both robotic and live predator encounters effectively intrude on foraging behaviour, the perceived risk and behavioural consequences of the threat are distinguishable. Additionally, BNST GABA neurons may play a role in the integration of prior innate predator threat experience that results in hypervigilance during post-encounter foraging behaviour.

Ventral tegmental area glutamate neurons establish a mu-opioid receptor gated circuit to mesolimbic dopamine neurons and regulate opioid-seeking behavior.

A two-neuron model of ventral tegmental area (VTA) opioid function classically involves VTA GABA neuron regulation of VTA dopamine neurons via a mu-opioid receptor dependent inhibitory circuit. However, this model predates the discovery of a third major type of neuron in the VTA: glutamatergic neurons. We found that about one-quarter of VTA neurons expressing the mu-opioid receptor are glutamate neurons without molecular markers of GABA co-release. Glutamate-Mu opioid receptor neurons are largely distributed in the anterior VTA. The majority of remaining VTA mu-opioid receptor neurons are GABAergic neurons that are mostly within the posterior VTA and do not express molecular markers of glutamate co-release. Optogenetic stimulation of VTA glutamate neurons resulted in excitatory currents recorded from VTA dopamine neurons that were reduced by presynaptic activation of the mu-opioid receptor ex vivo, establishing a local mu-opioid receptor dependent excitatory circuit from VTA glutamate neurons to VTA dopamine neurons. This VTA glutamate to VTA dopamine pathway regulated dopamine release to the nucleus accumbens through mu-opioid receptor activity in vivo. Behaviorally, VTA glutamate calcium-related neuronal activity increased following oral oxycodone consumption during self-administration and response-contingent oxycodone-associated cues during abstinent reinstatement of drug-seeking behavior. Further, chemogenetic inhibition of VTA glutamate neurons reduced abstinent oral oxycodone-seeking behavior in male but not female mice. These results establish 1) a three-neuron model of VTA opioid function involving a mu-opioid receptor gated VTA glutamate neuron pathway to VTA dopamine neurons that controls dopamine release within the nucleus accumbens, and 2) that VTA glutamate neurons participate in opioid-seeking behavior.

Monosynaptic inputs to ventral tegmental area glutamate and GABA co-transmitting neurons.

A unique population of ventral tegmental area (VTA) neurons co-transmits glutamate and GABA as well as functionally signals rewarding and aversive outcomes. However, the circuit inputs to VTA VGluT2+VGaT+ neurons are unknown, limiting our understanding of the functional capabilities of these neurons. To identify the inputs to VTA VGluT2+VGaT+ neurons, we coupled monosynaptic rabies tracing with intersectional genetic targeting of VTA VGluT2+VGaT+ neurons in mice. We found that VTA VGluT2+VGaT+ neurons received diverse brain-wide inputs. The largest numbers of monosynaptic inputs to VTA VGluT2+VGaT+ neurons were from superior colliculus, lateral hypothalamus, midbrain reticular nucleus, and periaqueductal gray, whereas the densest inputs relative to brain region volume were from dorsal raphe nucleus, lateral habenula, and ventral tegmental area. Based on these and prior data, we hypothesized that lateral hypothalamus and superior colliculus inputs were glutamatergic neurons. Optical activation of glutamatergic lateral hypothalamus neurons robustly activated VTA VGluT2+VGaT+ neurons regardless of stimulation frequency and resulted in flee-like ambulatory behavior. In contrast, optical activation of glutamatergic superior colliculus neurons activated VTA VGluT2+VGaT+ neurons for a brief period of time at high stimulation frequency and resulted in head rotation and arrested ambulatory behavior (freezing). For both pathways, behaviors induced by stimulation were uncorrelated with VTA VGluT2+VGaT+ neuron activity. However, stimulation of glutamatergic lateral hypothalamus neurons, but not glutamatergic superior colliculus neurons, was associated with VTA VGluT2+VGaT+ footshock-induced activity. We interpret these results such that inputs to VTA VGluT2+VGaT+ neurons may integrate diverse signals related to the detection and processing of motivationally-salient outcomes. Further, VTA VGluT2+VGaT+ neurons may signal threat-related outcomes, possibly via input from lateral hypothalamus glutamate neurons, but not threat-induced behavioral kinematics.

Bed Nucleus of the Stria Terminalis GABA neurons are necessary for changes in foraging behavior following an innate threat.

Foraging is a universal behavior that has co-evolved with predation pressure. We investigated the role of bed nucleus of the stria terminalis (BNST) GABA neurons in robotic and live predator threat processing and their consequences in post-threat encounter foraging. Mice were trained to procure food in a laboratory-based foraging apparatus in which food pellets were placed at discrete and incrementally greater distances from a nest zone. After mice learned to forage, they were exposed to either a robotic or live predator threat, while BNST GABA neurons were chemogenetically inhibited. Post-robotic threat encounter, mice spent more time in the nest zone, but other foraging parameters were unchanged compared to pre-encounter behavior. Inhibition of BNST GABA neurons had no effect on foraging behavior post-robotic threat encounter. Following live predator exposure, control mice spent significantly more time in the nest zone, increased their latency to successfully forage, and their overall foraging performance was significantly a ltered. I nhibition o f BNST GABA neurons during live predator exposure prevented changes in foraging behavior from developing after live predator threat. BNST GABA neuron inhibition did not alter foraging behavior during robotic or live predator threat. We conclude that while both robotic and live predator encounter effectively intrude on foraging behavior, the perceived risk and behavioral consequence of the threats are distinguishable. Additionally, BNST GABA neurons may play a role in the integration of prior innate predator threat experience that results in hypervigilance during post-encounter foraging behavior.

Ventral tegmental area glutamate neurons mediate nonassociative consequences of stress.

Exposure to trauma is a risk factor for the development of a number of mood disorders, and may enhance vulnerability to future adverse life events. Recent data demonstrate that ventral tegmental area (VTA) neurons expressing the vesicular glutamate transporter 2 (VGluT2) signal and causally contribute to behaviors that involve aversive or threatening stimuli. However, it is unknown whether VTA VGluT2 neurons regulate transsituational outcomes of stress and whether these neurons are sensitive to stressor controllability. This work adapted an operant mouse paradigm to examine the impact of stressor controllability on VTA VGluT2 neuron function as well as the role of VTA VGluT2 neurons in mediating transsituational stressor outcomes. Uncontrollable (inescapable) stress, but not physically identical controllable (escapable) stress, produced social avoidance and exaggerated fear in male mice. Uncontrollable stress in females led to exploratory avoidance of a novel brightly lit environment. Both controllable and uncontrollable stressors increased VTA VGluT2 neuronal activity, and chemogenetic silencing of VTA VGluT2 neurons prevented the behavioral sequelae of uncontrollable stress in male and female mice. Further, we show that stress activates multiple genetically-distinct subtypes of VTA VGluT2 neurons, especially those that are VGluT2+VGaT+, as well as lateral habenula neurons receiving synaptic input from VTA VGluT2 neurons. Our results provide causal evidence that mice can be used for identifying stressor controllability circuitry and that VTA VGluT2 neurons contribute to transsituational stressor outcomes, such as social avoidance, exaggerated fear, or anxiety-like behavior that are observed within trauma-related disorders.

A Semi-Automated Workflow for Brain Slice Histology Alignment, Registration, and Cell Quantification (SHARCQ).

Tools for refined cell-specific targeting have significantly contributed to understanding the characteristics and dynamics of distinct cellular populations by brain region. While advanced cell-labeling methods have accelerated the field of neuroscience, specifically in brain mapping, there remains a need to quantify and analyze the data. Here, by modifying a toolkit that localizes electrodes to brain regions (SHARP-Track; Slice Histology Alignment, Registration, and Probe-Track analysis), we introduce a post-imaging analysis tool to map histological images to established mouse brain atlases called SHARCQ (Slice Histology Alignment, Registration, and Cell Quantification). The program requires MATLAB, histological images, and either a manual or automatic cell count of the unprocessed images. SHARCQ simplifies the post-imaging analysis pipeline with a step-by-step GUI. We demonstrate that SHARCQ can be applied for a variety of mouse brain images, regardless of histology technique. In addition, SHARCQ rectifies discrepancies in mouse brain region borders between atlases by allowing the user to select between the Allen Brain Atlas or the digitized and modified Franklin-Paxinos Atlas for quantifying cell counts by region. SHARCQ produces quantitative and qualitative data, including counts of brain-wide region populations and a 3D model of registered cells within the atlas space. In summary, SHARCQ was designed as a neuroscience post-imaging analysis tool for cell-to-brain registration and quantification with a simple, accessible interface. All code is open-source and available for download (https://github.com/wildrootlab/SHARCQ).