Probes for GAD2

Mechanical allodynia (MA) represents one prevalent symptom of chronic pain. Previously we and others have identified spinal and brain circuits that transmit or modulate the initial establishment of MA. However, brain-derived descending pathways that control the laterality and duration of MA are still poorly understood. Here we report that the contralateral brain-to-spinal circuits, from Oprm1 neurons in the lateral parabrachial nucleus (lPBNOprm1), via Pdyn neurons in the dorsal medial regions of hypothalamus (dmHPdyn), to the spinal dorsal horn (SDH), act to prevent nerve injury from inducing contralateral MA and reduce the duration of bilateral MA induced by capsaicin. Ablating/silencing dmH-projecting lPBNOprm1 neurons or SDH-projecting dmHPdyn neurons, deleting Dyn peptide from dmH, or blocking spinal κ-opioid receptors all led to long-lasting bilateral MA. Conversely, activation of dmHPdyn neurons or their axonal terminals in SDH can suppress sustained bilateral MA induced by lPBN lesion.

As the discovery of cellular diversity in the brain accelerates, so does the need for functional tools that target cells based on multiple features, such as gene expression and projection target. By selectively driving recombinase expression in a feature-specific manner, one can utilize intersectional strategies to conditionally promote payload expression only where multiple features overlap. We developed Conditional Viral Expression by Ribozyme Guided Degradation (ConVERGD), a single-construct intersectional targeting strategy that combines a self-cleaving ribozyme with traditional FLEx switches. ConVERGD offers benefits over existing platforms, such as expanded intersectionality, the ability to accommodate larger and more complex payloads, and a vector design that is easily modified to better facilitate rapid toolkit expansion. To demonstrate its utility for interrogating neural circuitry, we employed ConVERGD to target an unexplored subpopulation of norepinephrine (NE)-producing neurons within the rodent locus coeruleus (LC) identified via single-cell transcriptomic profiling to co-express the stress-related endogenous opioid gene prodynorphin ( Pdyn ). These studies showcase ConVERGD as a versatile tool for targeting diverse cell types and reveal Pdyn -expressing NE + LC neurons as a small neuronal subpopulation capable of driving anxiogenic behavioral responses in rodents.

Sleep disturbances are strongly associated with cardiovascular diseases. Baroreflex, a basic cardiovascular regulation mechanism, is modulated by sleep-wake states. Here, we show that neurons at key stages of baroreflex pathways also promote sleep. Using activity-dependent genetic labeling, we tagged neurons in the nucleus of the solitary tract (NST) activated by blood pressure elevation and confirmed their barosensitivity with optrode recording and calcium imaging. Chemogenetic or optogenetic activation of these neurons promoted non-REM sleep in addition to decreasing blood pressure and heart rate. GABAergic neurons in the caudal ventrolateral medulla (CVLM)-a downstream target of the NST for vasomotor baroreflex-also promote non-REM sleep, partly by inhibiting the sympathoexcitatory and wake-promoting adrenergic neurons in the rostral ventrolateral medulla (RVLM). Cholinergic neurons in the nucleus ambiguous-a target of the NST for cardiac baroreflex-promoted non-REM sleep as well. Thus, key components of the cardiovascular baroreflex circuit are also integral to sleep-wake brain-state regulation.

Exposure to irregular lighting schedules leads to deficits in affective behaviors. The retino-recipient perihabenular nucleus (PHb) of the dorsal thalamus has been shown to mediate these effects in mice. However, the mechanisms of how light information is processed within the PHb remains unknown. Here, we show that the PHb contains a distinct cluster of GABAergic neurons that receive direct retinal input. These neurons are part of a larger inhibitory network composed of the thalamic reticular nucleus and zona incerta, known to modulate thalamocortical communication. In addition, PHbGABA neurons locally modulate excitatory-relay neurons, which project to limbic centers. Chronic exposure to irregular light-dark cycles alters photo-responsiveness and synaptic output of PHbGABA neurons, disrupting daily oscillations of genes associated with inhibitory and excitatory PHb signaling. Consequently, selective and chronic PHbGABA manipulation results in mood alterations that mimic those caused by irregular light exposure. Together, light-mediated disruption of PHb inhibitory networks underlies mood deficits.

Single-cell RNA sequencing data can unveil the molecular diversity of cell types. Cell type atlases of the mouse spinal cord have been published in recent years but have not been integrated together. Here, we generate an atlas of spinal cell types based on single-cell transcriptomic data, unifying the available datasets into a common reference framework. We report a hierarchical structure of postnatal cell type relationships, with location providing the highest level of organization, then neurotransmitter status, family, and finally, dozens of refined populations. We validate a combinatorial marker code for each neuronal cell type and map their spatial distributions in the adult spinal cord. We also show complex lineage relationships among postnatal cell types. Additionally, we develop an open-source cell type classifier, SeqSeek, to facilitate the standardization of cell type identification. This work provides an integrated view of spinal cell types, their gene expression signatures, and their molecular organization.