Neurochemical Heterogeneity Among Lateral Hypothalamic Hypocretin/Orexin and Melanin-Concentrating Hormone Neurons Identified Through Single-Cell Gene Expression Analysis

Abstract

The lateral hypothalamic area (LHA) lies at the intersection of multiple neural and humoral systems and orchestrates fundamental aspects of behavior. Two neuronal cell types found in the LHA are defined by their expression of hypocretin/orexin (Hcrt/Ox) and melanin-concentrating hormone (MCH) and are both important regulators of arousal, feeding, and metabolism. Conflicting evidence suggests that these cell populations have a more complex signaling repertoire than previously appreciated, particularly in regard to their coexpression of other neuropeptides and the machinery for the synthesis and release of GABA and glutamate. Here, we undertook a single-cell expression profiling approach to decipher the neurochemical phenotype, and heterogeneity therein, of Hcrt/Ox and MCH neurons. In transgenic mouse lines, we used single-cell quantitative polymerase chain reaction (qPCR) to quantify the expression of 48 key genes, which include neuropeptides, fast neurotransmitter components, and other key markers, which revealed unexpected neurochemical diversity. We found that single MCH and Hcrt/Ox neurons express transcripts for multiple neuropeptides and markers of both excitatory and inhibitory fast neurotransmission. Virtually all MCH and approximately half of the Hcrt/Ox neurons sampled express both the machinery for glutamate release and GABA synthesis in the absence of a vesicular GABA release pathway. Furthermore, we found that this profile is characteristic of a subpopulation of LHA glutamatergic neurons but contrasts with a broad population of LHA GABAergic neurons. Identifying the neurochemical diversity of Hcrt/Ox and MCH neurons will further our understanding of how these populations modulate postsynaptic excitability through multiple signaling mechanisms and coordinate diverse behavioral outputs.

Melanin-concentrating hormone neurons promote rapid eye movement sleep independent of glutamate release

“…Importantly, most MCH neurons express only Vglut2, but not other vesicular glutamate transporters viz. Vglut1 or Vglut3 (only a very small percentage of MCH neurons express Vglut3) (Mickelsen et al 2017) and therefore loss of Vglut2 was expected to completely abolish glutamate release from these neurons.…”

“…In contrast, we recently found that MCH neurons may not contain vesicular GABA transporter (Vgat) necessary for packaging GABA into the vesicles for synaptic release (Chee et al 2015a). While vesicular monoamine transporters (VMAT 1 and 2) can mediate GABAergic neurotransmission, MCH neurons are also devoid of these transporters (Mickelsen et al 2017). Consistent with this, optogenetic stimulation of MCH neurons did not release GABA in the lateral septum, the brain region that receives the densest MCH projections (Chee et al 2015a).…”

“…In addition to these neuropeptides, almost all (>98%) MCH neurons contain vesicular glutamate transporter 2 (Vglut2), which is necessary for packaging glutamate into synaptic vesicles (Chee et al 2015a; Mickelsen et al 2017; Schneeberger et al 2018). In contrast, none of them express vesicular GABA transporter (Vgat) necessary for packaging GABA (Chee et al 2015a; Mickelsen et al 2017) although contrary evidences exist (Jego et al 2013). Consistent with this, in vitro activation of MCH neurons evokes the release of glutamate but not GABA in the lateral septum, which receives densest input from MCH neurons (Chee et al 2015a).…”

Mapping Molecular Datasets Back to the Brain Regions They are Extracted from: Remembering the Native Countries of Hypothalamic Expatriates and Refugees

“…Eberwine et al (1992) [98] performed gene expression analysis on individual, freshly dissociated hippocampal neurons. More recently, single-cell isolation has now been conducted using laser-capture microdissection (LCM) methods; e.g., [37, 47, 56, 239, 456], or cell sorting methods (e.g., [64, 69, 95; 160; 264; 278; 368]; also see [312, 351]). Thus, single cell isolation methods first used for the purposes of morphological and structural investigation evolved for use in biochemical, molecular and functional analyses.…”

“…The Jackson laboratory has recently reported single-cell transcriptomic data obtained from LHA H/O-expressing neurons and MCH-expressing neurons in mouse transgenic lines [278]. Importantly, in their study, they show specific delineations of the regions they dissected using atlas-based coordinates and drawings of the estimated areas they micropunched.…”