An ex vivo Multi-Electrode Approach to Evaluate Endogenous Hormones and Receptor Subtype Pharmacology on Evoked and Spontaneous Neuronal Activity within the Ventromedial Hypothalamus; Translation from Female Receptivity

Abstract

Introduction The ventromedial hypothalamus (VMH) controls female rodent copulatory behavior, which can be modulated by injection of various compounds into the VMH. Aim The aim was to determine whether evoked excitatory postsynaptic potentials (EPSPs) or single-unit activity within the VMH ex vivo is a better parameter to predict lordosis. Methods VMH slices were placed onto a 64 microelectrode chip and spontaneous single-unit activity was recorded or slices stimulated to evoke EPSPs. Main Outcome Measures The sodium channel blocker, tetrodotoxin and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX) inhibited EPSPs, confirming EPSPs were glutamatergic in origin. The GABAA antagonist bicuculline potentiated EPSPs implying endogenous GABA tone. Single-unit activity was abolished by tetrodotoxin but unaffected by DNQX or bicuculline. Results Glutamatergic neurotransmission was greatest during metestrous and following ovariectomization. The number of regions within the VMH eliciting single-unit activity was reduced following ovariectomy without changing spike frequency. Adrenergic agents increasing lordosis via the VMH in vivo, decreased glutamate neurotransmission but increased single-unit activity. Conversely, agents decreasing lordosis via the VMH increased glutamatergic neurotransmission and inhibited single-unit activity (8-OH-DPAT, [D-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin, corticotropin releasing factor, bicuculline). Melanocortin and pituitary adenylate cyclase-activating polypeptide agonists had no effect. Conclusions Here we present a novel, robust VMH in vitro technique that (i) is consistent with the hypothesis that glutamate via non-NMDA receptors inhibits lordosis; (ii) glutamate is under the endogenous tone of GABA and steroid hormones; (iii) inhibition of lordosis during metestrous and following ovariectomy potentiates glutamatergic neurotransmission; (iv) activation of Gq- and Gi-coupled receptors decreases and increases glutamate neurotransmission, respectively, with an inverse correlation on single-unit activity; (v) activation of Gs-coupled receptors has no direct effect on glutamate or single-unit activity; and (vi) potency, receptor subtypes and localization can be determined prior to in vivo studies.

RU486 facilitates or disrupts the sensitization of sexual behaviors by estradiol in the ovariectomized Long–Evans rat: Effect of timecourse

“…Moreover, we found that the attenuation was driven by the receipt of VCS (Jones et al, submitted), and mimicked by AMPA receptor infusions to the vlVMH in place of copulation (Jones et al, 2015). Together those findings show that glutamate signaling in the vlVMH opposes the sensitization of appetitive sexual behaviors by EB, and suggests that the induction of sensitization may involve the removal of inhibitory glutamate signaling potentially via an increase in GABAergic signaling in the vlVMH (Booth et al, 2010;Georgescu et al, 2009Georgescu et al, , 2014McCarthy et al, 1990). Repeated treatments of EB may potentiate neuroprogesterone synthesis which may, in turn, not only bind to estradiol-induced PR but also upregulate GABA-a receptor subunits (Arbo et al, 2014), and/or its metabolites may also bind to GABA-a receptors directly (Frye and Walf, 2008a,b), leading to a facilitation of sexual behaviors with time.…”

Changes in neuronal activity across the mouse ventromedial nucleus of the hypothalamus in response to low glucose: Evaluation using an extracellular multi‐electrode array approach

“…No synaptic blockers were used during recordings. Although the voltage‐gated sodium channel blocker TTX has been useful in patch clamp experiments to distinguish between intrinsic and extrinsic glucosensing capabilities of neurones, we could not use this approach during MEA recordings as bath application of TTX abolishes the extracellularly recorded spontaneous action potentials in VMN‐containing brain slices. However, at the end of a subset of experiments, slices were either treated with bath application of the glutamatergic NMDA receptor agonist, NMDA (20 µmol L ‐1 ), to confirm maintained cell responsiveness, and/or were followed by application of TTX (1 µmol L ‐1 ).…”

Repeated administration of estradiol promotes mechanisms of sexual excitation and inhibition: Glutamate signaling in the ventromedial hypothalamus attenuates excitation

“…Thus to extend on a potential role for to the PAG, it is hypothesized that if AMPA receptor activation within the vlVMH (following VCS) interacts with the PAG to inhibit lordosis and facilitate defensive behaviors, this pathway must be overridden in those females that are repeatedly treated with EB and allowed to copulate. Overall, these findings are important as they further demonstrate that glutamate signaling in the vlVMH via AMPA receptors, is inhibitory to sexual behavior, and induces a behavioral pattern reminiscent of estrous termination [6,15,16,[18][19][20].…”

“…That inhibitory effect was most robustly induced by AMPA [19]. Importantly, that endogenous glutamate within the VMH is inhibitory to lordosis and under the control of GABA and steroid hormones, has also been supported electrophysiologically in tissue slices examined across the natural estrous cycle [6]. The onset of estrous termination is accelerated by the receipt of VCS from penile intromissions or experimenter applied with a glass rod (e.g., [12,30,41]), however if the frequency of hormone priming is increased from every 28 days to every 14 days to every 4 days, the inhibitory effect of VCS becomes progressively less robust [41].…”