Research ArticleElectrophysiological Properties of Genetically Identified Histaminergic Neurons
Introduction
Histamine is a biogenic amine involved in multiple physiological functions, including the regulation of arousal, energy homeostasis, body temperature, and the immune response (Schwartz et al., 1991, Schneider et al., 2002, Haas et al., 2008). Within the central nervous system (CNS), histamine acts as a neurotransmitter, with histaminergic neuron somas being confined to the tuberomammillary nucleus (TMN) in the posterior hypothalamus (Panula et al., 1984, Takeda et al., 1984, Watanabe et al., 1984). Histaminergic neurons project extensively throughout the brain and spinal cord, with histamine exerting its physiological effects via activation of four G-protein coupled histamine receptors (H1–H4) (Ericson et al., 1987, Haas and Panula, 2003). The wide range of physiological functions influenced by histaminergic neurons, and their heavy innervation of the CNS, has led to concentrated efforts in understanding the regulation of histamine and histaminergic neurons in multiple research settings.
Identification of histaminergic neurons in brain slice preparations have traditionally been aided by their characteristic electrophysiological properties. Initial studies in rats reported that histaminergic neurons were spontaneously active, with a resting membrane potential of −50 mV, a distinctive A-like current (IA – outward rectification), hyperpolarization-activated time-dependent inward rectification (Ih), and a prominent after-hyperpolarization following action potentials (Haas and Reiner, 1988). Subsequent studies have exploited these characteristics to identify histaminergic neurons in both rats (Greene et al., 1990, Kamondi and Reiner, 1991, Schonrock et al., 1991, Eriksson et al., 2001a, Eriksson et al., 2001b, Parmentier et al., 2009, Yanovsky et al., 2011, Yin et al., 2019) and mice (Mochizuki et al., 2000, Schone et al., 2012).
Overall, limited comparisons have been made between the electrophysiological properties of histaminergic neurons from rats and mice, and it is currently unclear if species differences exist. In addition, the original characterization of histaminergic neurons was constrained to TMN neurons near the ventral surface of the brain (Haas and Reiner, 1988). The TMN is a diffuse nucleus, and is categorized into multiple subregions (Kohler et al., 1985, Ericson et al., 1987, Inagaki et al., 1990), which are more widespread in mice than rats (Karlstedt et al., 2001). Growing evidence suggests that histaminergic neurons are a functionally heterogeneous population (Arrang et al., 1991, Miklos and Kovacs, 2003, Giannoni et al., 2009, Blandina et al., 2012, Michael et al., 2020). Therefore, the identification of mouse histaminergic neurons based on electrophysiological properties reported from one subregion of the TMN in rats may not be entirely accurate.
The rapid increase and reliance on mouse genetics has allowed for the development of mouse models aiding in the targeting of specific neuronal populations. Histaminergic neurons are characterized by the expression of histidine decarboxylase (HDC), the sole enzyme required for histamine synthesis, which catalyzes the formation of histamine from the amino acid l-histidine (Taylor and Snyder, 1972, Green et al., 1987). Multiple HDC Cre-recombinase mouse models have been developed (GENSAT project: Gong et al., 2007, Yanovsky et al., 2012, Zecharia et al., 2012), allowing for identification of histaminergic neurons in brain slices when mice are bred to fluorescent reporter mice.
Fujita et al. (2017) utilized such transgenic mice and provided comprehensive electrophysiological data from genetically identified HDC neurons. Unfortunately, this report was also limited to HDC cells in the ventral TMN. The penetrance of the Cre recombination in this model was below 50%, further restricting the sample of HDC neurons used for characterization (Fujita et al., 2017). Therefore, the present study aimed to determine the electrophysiological properties of genetically identified histaminergic neurons from mice. In addition to contrasting these characteristics with that originally obtained by Haas and Reiner (1988) in rats, we also aimed to determine if HDC neurons are electrophysiologically similar across the subregions of the TMN, and between male and female mice.
Section snippets
Animals
To target genetically identified histaminergic neurons we used the Hdc-Cre mouse developed by Jeffrey Zigman, which has been previously validated (Yanovsky et al., 2012, Walker et al., 2013). The Hdc-Cre mouse was bred to a tdTomato reporter mouse (Ai14, JAX stock # 007914) obtained from The Jackson Laboratory (Maine), to allow for fluorescent labeling of Hdc-expressing cells. All mice were bred to have one copy of each transgene and were maintained on a C57BL/6J background.
Mice were maintained
Passive membrane properties and action potential firing in HDC neurons
Histaminergic neurons were identified via red fluorescence in Hdc-cre::tdTomato mice and were clearly discernable from surrounding neurons (Fig. 1A). HDC expressing neurons were targeted for electrophysiological recordings throughout the rostro-caudal axis of the TMN. Whole-cell patch-clamp recordings were performed on 141 HDC neurons from male mice. HDC neurons displayed an average resting membrane potential of −47.6 ± 0.5 mV (n = 141, Fig. 1B, C). Half (71/141) of the HDC neurons were silent.
Discussion
This study provides an assessment of the electrophysiological properties from over 200 genetically identified histaminergic neurons located throughout the TMN in mice. HDC neurons displayed a resting membrane potential of approximately −48 mV, a firing rate of 1.3 Hz in active cells, and pervasive expression of an A-like current (IA). Minimal differences were observed between HDC neurons from male versus female mice, or between different sub-regions of the TMN. Despite many similarities with
Conflict of interest statement
The authors declare no competing financial interests.
Acknowledgements
We thank Dr Alexandre Caron (UT Southwestern Medical Center) for critically reviewing the manuscript. We also thank the National Institutes of Health (NIH) United States for its support (R01 DK008423 and R01 DK118725 to JKE, R01 DK119169, R01 DK100699 and P01 DK119130 to KWW, as well as a gift from the David and Teresa Disiere Foundation to JMZ.
Author contributions
Conceptualization, NJM and JKE; Methodology, NJM and KWW; Formal Analysis NJM; Investigation, NJM; Writing - Original Draft, NJM; Writing - Review and Editing, NJM, KWW, and JKE; Funding Acquisition, JKE; Supervision, JKE and KWW; Resources, JKE, KWW, JMZ. All authors contributed to editing the manuscript and approved the manuscript for publication.
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