Neurokinin B-Expressing Neurons of the Central Extended Amygdala Mediate Inhibitory Synaptic Input onto Melanin-Concentrating Hormone Neuron Subpopulations

Abstract

The lateral hypothalamic area (LHA) is a highly conserved brain region critical for maintaining physiological homeostasis and goal-directed behavior. LHA neurons that express melanin-concentrating hormone (MCH) are key regulators of arousal, energy balance, and motivated behavior. However, cellular and functional diversity among LHAMCHneurons is not well understood. Previous anatomic and molecular data suggest that LHAMCHneurons may be parsed into at least two distinct subpopulations, one of which is enriched in neurokinin-3 receptor (NK3R), the receptor for neurokinin B (NKB), encoded by theTac2gene. This tachykininergic ligand-receptor system has been implicated in reproduction, fear memory, and stress in other brain regions, but NKB interactions with LHAMCHneurons are poorly understood. We first identified how LHAMCHsubpopulations may be distinguished anatomically and electrophysiologically. To dissect functional connectivity between NKB-expressing neurons and LHAMCHneurons, we used Cre-dependent retrograde and anterograde viral tracing in maleTac2-Cre mice and identifiedTac2/EYFP+ neurons in the bed nucleus of the stria terminalis and central nucleus of the amygdala, the central extended amygdala, as major sources of NKB input onto LHAMCHneurons. In addition to innervating the LHA, these limbic forebrain NKB neurons also project to midbrain and brainstem targets. Finally, using a dual-virus approach, we found that optogenetic activation of these inputs in slices evokes GABA release onto a subset of LHAMCHneurons but lacked specificity for the NK3R+ subpopulation. Overall, these data define parallel tachykininergic/GABAergic limbic forebrain projections that are positioned to modulate multiple nodes of homeostatic and behavioral control.SIGNIFICANCE STATEMENTThe LHA orchestrates fundamental behavioral states in the mammalian hypothalamus, including arousal, energy balance, memory, stress, and motivated behavior. The neuropeptide MCH defines one prominent population of LHA neurons, with multiple roles in the regulation of homeostatic behavior. Outstanding questions remain concerning the upstream inputs that control MCH neurons. We sought to define neurochemically distinct pathways in the mouse brain that may communicate with specific MCH neuron subpopulations using viral-based retrograde and anterograde neural pathway tracing and optogenetics in brain slices. Here, we identify a specific neuropeptide-defined forebrain circuit that makes functional synaptic connections with MCH neuron subpopulations. This work lays the foundation for further manipulating molecularly distinct neural circuits that modulate innate behavioral states.

Neuroanatomical, electrophysiological, and morphological characterization of melanin-concentrating hormone cells coexpressing cocaine- and amphetamine-regulated transcript

“…Our findings extended those by Fujita and colleagues (2021) who noted that Pmch cells may express at least two electrical fingerprints, but these two fingerprints were not exclusively correlated to Carpt and Tacr3 coexpression. Interestingly, we noted that Tacr3 expression may be higher than the expression of its corresponding protein NK3R.…”

“…Interestingly, NK3R expression defined another subset of MCH cells, as NK3R was almost exclusively seen in MCH/CART+ cells, and only a few MCH cells expressed NK3R in the absence of CART. This is consistent with single-cell RNAseq analysis (Mickelsen et al, 2019; Fujita et al, 2021) revealing that Pmch cells can be divided into two major subclusters: one that strongly expressed both Carpt and Tacr3 , the genes for CART and NK3R respectively, and one that was Cartpt - and Tacr3 -negative. Furthermore, Cartpt and Tacr3 have also been recognized as highly expressed genes in Pmch cells by other RNAseq datasets like HypoMap (Steuernagel et al, 2022).…”

“…About half of the hypothalamic MCH cell population expressed CART. The prominence of CART coexpression in half of MCH cells has also been independently reported by others (Broberger, 1999; Elias et al, 2001; Cvetkovic et al, 2004; Vrang, 2006; Croizier et al, 2010; Mickelsen et al, 2017) while Cartpt mRNA coexpression may be even higher (Mickelsen et al, 2019; Fujita et al, 2021). CART-positive MCH cells can also be spatially differentiated from CART-negative cells.…”

“…Finally, MCH/CART+ and MCH/CART− cells differentially express other neuropeptides. Notably, over two-thirds of MCH/CART+ cells coexpress NK3R, but this receptor is less abundant in MCH/CART− cells (Mickelsen et al, 2019;Fujita et al, 2021). Neurokinin B, the predominant ligand at NK3R (Steinhoff et al, 2014), is implicated in reproductive behaviours and physiology (Rance et al, 2010(Rance et al, , 2013, and MCH/CART/NK3R+ cells could represent a further subdivision of the MCH/CART+ population.…”

A molecularly distinct cell type in the midbrain regulates intermale aggression behaviors in mice

“…In the CeA, Tac2 -expressing neurons mainly modulate consolidation of fear memory (Andero et al ., 2014; Florido et al, 2021). A recent study showed that Tac2 -expressing neurons in the BNST and the CeA regulate behavioral states, i.e., emotional valence, by sending inhibitory inputs to the LHA neurons expressing melanin-concentrating hormone (Fujita et al, 2021). These studies suggest that Tac2 -expressing neurons are uniquely positioned in specific brain regions to modulate behavioral homeostasis.…”