HINDBRAIN MEDULLA CATECHOLAMINE CELL GROUP INVOLVEMENT IN LACTATE-SENSITIVE HYPOGLYCEMIA-ASSOCIATED PATTERNS OF HYPOTHALAMIC NOREPINEPHRINE AND EPINEPHRINE ACTIVITY P. K. SHRESTHA, P. TAMRAKAR, B. A. IBRAHIM AND K. P. BRISKI * Department of Basic Pharmaceutical Sciences, School of Pharmacy, The University of Louisiana at Monroe, Monroe, LA 71201, United States Abstract—Cell-type compartmentation of glucose metabo- lism in the brain involves trafficking of the oxidizable glyco- lytic end product, L-lactate, by astrocytes to fuel neuronal mitochondrial aerobic respiration. Lactate availability within the hindbrain medulla is a monitored function that regulates systemic glucostasis as insulin-induced hypoglycemia (IIH) is exacerbated by lactate repletion of that brain region. A2 noradrenergic neurons are a plausible source of lactoprivic input to the neural gluco-regulatory circuit as caudal fourth ventricular (CV4) lactate infusion normalizes IIH-associated activation, e.g. phosphorylation of the high-sensitivity energy sensor, adenosine 5 0 -monophosphate-activated pro- tein kinase (AMPK), in these cells. Here, we investigated the hypothesis that A2 neurons are unique among medullary catecholamine cells in directly screening lactate-derived energy. Adult male rats were injected with insulin or vehicle following initiation of continuous L-lactate infusion into the CV4. Two hours after injections, A1, C1, A2, and C2 neurons were collected by laser-microdissection for Western blot analysis of AMPK a1/2 and phosphoAMPK a1/2 proteins . Results show that AMPK is expressed in each cell group, but only a subset, e.g. A1, C1, and A2 neurons, exhibit increased sensor activity in response to IIH. Moreover, hind- brain lactate repletion reversed hypoglycemic augmentation of pAMPK a1/2 content in A2 and C1 but not A1 cells, and nor- malized hypothalamic norepinephrine and epinephrine con- tent in a site-specific manner. The present evidence for discriminative reactivity of AMPK-expressing medullary catecholamine neurons to the screened energy substrate lactate implies that that lactoprivation is selectively signaled to the hypothalamus by A2 noradrenergic and C1 adrenergic cells. Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: A2 noradrenergic neurons, C1 adrenergic neurons, insulin-induced hypoglycemia, laser-catapult microdissection, Western blotting, pAMPK. INTRODUCTION The neural network that controls glucostasis is extensive, linking multiple populations of metabolic-sensory cells situated within and outside the brain to a hierarchy of integrative, premotor, and motor components situated at various levels of the central neuroaxis (Watts and Donovan, 2010). The need to characterize the neuro- chemical elements of this system and their connectivity is undisputed. Hindbrain catecholamine signaling is evi- dently requisite for optimal function of this circuitry as col- lective destruction of noradrenergic (A1, A2) and adrenergic (C1, C2) cells by toxin uptake at a common hypothalamic projection field prevents feeding and auto- nomic responses to systemic pharmacological glucopriva- tion (Ritter et al., 2011). There remains the need to distinguish the respective roles (or lack thereof) of these individual cell groups in brain reactivity to insulin-induced hypoglycemia (IIH), a recurring complication of insulin- dependent diabetes mellitus management (Cryer, 2008). In particular, there is a keenness to know if one or more of these populations function to detect hypoglycemia- associated reductions in cellular energy. Signals of nerve cell metabolic instability derive from a small number of brain sites where specialized neurons adjust synaptic fir- ing in response to diminished substrate fuel supply, including the dorsal vagal complex (DVC) of the dorsome- dial medulla, where A2 and C2 catecholamine cells reside (Mizuno and Oomura, 1984; Adachi et al., 1995; Balfour et al., 2006). The efficacy of anti-glycolytic drugs to induce hyperphagia and hyperglycemia upon delivery into the DVC or ventrolateral medulla (the location of A1 and C1 cells) demonstrates connectivity of metabolic sensors in these sites with the central glucoregulatory circuitry (Ritter et al., 2000). http://dx.doi.org/10.1016/j.neuroscience.2014.07.033 0306-4522/Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. * Corresponding author. Address: Department of Basic Pharmaceu- tical Sciences, School of Pharmacy, The University of Louisiana at Monroe, 356 Bienville Building, 1800 Bienville Drive, Monroe, LA 71201, United States. Tel: +1-318-342-3283; fax: +1-318-342- 1737. E-mail address: briski@ulm.edu (K. P. Briski). Abbreviations: aCSF, artificial cerebrospinal fluid; AMPK, adenosine 5 0 -monophosphate-activated protein kinase; ARH, arcuate hypothalamic nucleus; AVPV, anteroventral periventricular nucleus; BSA, bovine serum albumin; CORT, corticosterone; CV4, caudal fourth ventricular; DMH, dorsomedial hypothalamic nucleus; DVC, dorsal vagal complex; E, epinephrine; EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked immunosorbent assay; I, insulin; IgG, immunoglobulin G; IIH, insulin-induced hypoglycemia; LHA, lateral hypothalamic area; MPN, medial preoptic nucleus; NE, norepinephrine; O.D., optical densities; pAMPK, phosphoAMPK; PVH, paraventricular hypothalamic nucleus; TBS, tris-buffer saline; TH, tyrosine hydroxylase; TH-ir, TH-immunoreactive; V, vehicle; VMH, ventromedial hypothalamic nucleus. Neuroscience 278 (2014) 20–30 20