8578 • The Journal of Neuroscience, September 17, 2003 • 23(24):8578–8585 Behavioral/Systems/Cognitive Metabolic Regulation of Fertility through Presynaptic and Postsynaptic Signaling to Gonadotropin-Releasing Hormone Neurons Shannon D. Sullivan, R. Anthony DeFazio, and Suzanne M. Moenter 1Internal Medicine and Cell Biology, University of Virginia, Charlottesville, Virginia 22908 Gonadotropin-releasing hormone (GnRH) neurons form the final common pathway for the central regulation of reproduction and are inhibited by negative energy balance. In normal adults, these neurons maintain elevated intracellular chloride so that GABAA receptor activation is excitatory. We hypothesized that fasting alters homeostatic mechanisms to eliminate excitatory responses to GABA but rejected this hypothesis when brief, local GABA application elicited action currents in GnRH neurons from fed and fasted mice. This response was specific to GABAA receptors, because glycine elicited no response. We next found that fasting reduced the frequency of spontaneous GABAergic postsynaptic currents (PSCs) and that this was reversed by in vivo treatment with leptin during the fast. In the presence of tetrodotoxin to minimize presynaptic actions, leptin also potentiated the postsynaptic response of these cells to GABAA receptor activation. Postsynaptic effects of leptin on GABAergic miniature PSCs were eliminated by inhibiting JAK2/3 (Janus kinase), the tyrosine kinase through which leptin receptors signal. In all experiments, elimination of PSCs at ECl or by treatment with the GABAA receptor antagonist bicuculline confirmed that PSCs were specifically mediated by GABAA receptor chloride channels. These data dem- onstrate that fasting and leptin act presynaptically and postsynaptically to alter GABAergic drive to GnRH neurons, providing evidence for GABAergic communication of metabolic cues to GnRH neurons, and suggest the possibility for functional leptin receptors on GnRH neurons. They further demonstrate cytokine modulation of the postsynaptic response to GABA in mammals, which may be important to central neural regulation in both healthy and diseased states. Key words: GnRH; GABA; PSC; leptin; cytokine; fertility Introduction of these metabolic signals is only beginning to be understood, Darwin linked the fate of species to the outcome of the search for although, among potential candidates, the adipocyte hormone food and mates over one century ago (Darwin, 1859). Subsequent leptin has received considerable attention because it both inhibits experiments have shown that negative energy balance is a potent food intake (Rentsch et al., 1995; Weigle, 1995) and reverses inhibitor of fertility, as evidenced by numerous animal models fasting-induced reproductive suppression (Ahima et al., 1996; (Dyer et al., 1985; Ahima et al., 1996; Schneider and Zhou, 1999; Nagatani et al., 1998; Sullivan et al., 2002). No conclusive evi- Ohkura et al., 2000), and clinical syndromes such as anorexia dence exists to date for leptin receptor expression in GnRH neu- nervosa (Stoving et al., 1999). Information regarding energy ho- rons (Finn et al., 1998; Cunningham et al., 1999), suggesting that meostasis is relayed to the gonadotropin-releasing hormone presynaptic effects of this hormone may be important, although (GnRH) neurons of the hypothalamus to regulate reproductive direct action on GnRH neurons cannot be discounted. In the function. These cells are the final common pathway controlling following report, we attempt to elucidate some of the neurobio- fertility and produce a frequency-modulated endocrine signal logical mechanisms by which metabolic information is commu- that is conveyed to the anterior pituitary gland. Metabolic signals nicated to the GnRH neuronal axis to regulate fertility. affect reproduction at least in part by altering the frequency and The neurotransmitter GABA is one candidate mediator of met- amplitude of GnRH secretion (I’Anson et al., 2000). The nature abolic signals to GnRH neurons. GnRH neurons receive functional synaptic input via GABAA receptors (Sim et al., 2001). Furthermore, GABAergic neurons express leptin receptors, indicating that their Received Feb. 21, 2003; revised July 21, 2003; accepted July 22, 2003. activity may be under direct metabolic control (Ovesjo et al., 2001). This work was supported by National Science Foundation Grant IBN 98058023, Whitehall Foundation Grant The A subtype of the GABA receptor gates an intrinsic ion channel 2000-12-43A, and National Institute of Child Health and Human Development—National Institutes of Health Cooperative Agreement U54 HD28934. We thank Catherine Christian, Glenn Harris, and Chun Xu for editorial com- that passes mainly chloride; thus, GABAA receptor activation can ments and Dr. Xu-Zhi Xu for expert technical assistance. either inhibit or excite a cell depending on chloride levels (Kaila, Correspondence should be addressed to S. M. Moenter, Internal Medicine and Cell Biology, P.O. Box 800578, 1994; Rivera et al., 1999; Wagner et al., 2001). Unlike most mature Jefferson Park Avenue, University of Virginia, Charlottesville, VA 22908. E-mail: [email protected]. neurons, adult GnRH cells are depolarized by GABA receptor acti- R. A. DeFazio’s present address: University of Miami School of Medicine, Department of Physiology and Biophys- A ics, 1600 Northwest 10th Avenue, Room 4044, Miami, FL 33136. E-mail: [email protected]. vation sufficiently to induce action potential firing in ad libitum fed Copyright © 2003 Society for Neuroscience 0270-6474/03/238578-08$15.00/0 animals (DeFazio et al., 2002). Sullivan et al. • Presynaptic and Postsynaptic Signaling to GnRH Neurons J. Neurosci., September 17, 2003 • 23(24):8578–8585 • 8579 We examined effects of energy balance on three facets of amplitude fast current spikes were detected in voltage clamp at 0 mV with GABAA receptor-mediated inputs to GnRH neurons. First, we signals filtered at 10 kHz. Seal resistance was periodically monitored and asked whether negative energy balance sufficient to suppress re- remained Ͼ1G⍀. In addition, the response to pressure application of production alters GnRH neuron chloride homeostasis so that the glycine (1 mM in HEPES buffer) was tested (n ϭ 2 cells fed, 2 fasted, 3 fasted with in vivo leptin) to determine whether GnRH neurons respond response to GABAA receptor activation becomes inhibitory. Such a change in response has been proposed to underlie circadian to another transmitter that binds a receptor with an intrinsic chloride changes in the firing rate of neurons of the suprachiasmatic nu- channel. Recording postsynaptic currents. Electrodes (2–4M⍀) were filled with cleus of the hypothalamus (Wagner et al., 1997, 2001; De Jeu and the high-chloride pipette solution with the addition of 4 mM MgATP and Pennartz, 2002), as well as changes in neuronal excitability after 0.4 mM NaGTP before adjusting to pH 7.2 with NaOH. GFP-GnRH injury (Nabekura et al., 2002). Second, we examined the effects of neurons were identified, and the whole-cell recording configuration was negative energy balance and leptin on GABAergic drive to GnRH achieved. Membrane potential was clamped at Ϫ60 mV, and signals were neurons, a presynaptic effect. Finally, we tested for functional filtered at 7 kHz with gain set at 10 mV/pA for 180 sec recording periods. evidence of direct, postsynaptic leptin action on these cells via Liquid junction potential of 3 mV (Barry, 1994) was not corrected for. leptin receptors. Postsynaptic currents (PSCs) were stored as Event Tracker files using Pulse Control and IGOR PRO software. Input resistance (Rin), series resistance (Rs), and membrane capacitance (Cm) were continually mon- Materials and Methods itored as described previously (DeFazio et al., 2002). Only recordings Animals. Adult transgenic female mice in which green fluorescent pro- Ͼ ⍀ Ͻ ⍀ with Rin 500 M and Rs 20 M were included for analysis. Mean Rin, tein (GFP) is genetically targeted to GnRH neurons (Suter et al., 2000) Ͼ Rs, and Cm were not different ( p 0.05) among or within cells in which were used. Animals were housed in groups of three to five and were ␮ comparisons were made. Bicuculline (20 M), a GABAA receptor antag- maintained on standard rodent chow (Harlan 7012; Harlan Sprague onist, was bath applied during a subset of recordings to determine that Dawley, Bartonsville, IL) and water ad libitum. All animals were held on detected PSC events were GABAA receptor mediated. Individual cells a 14/10 light/dark cycle with lights on at 5:00 A.M. eastern standard time. were further examined to determine that changes in the PSC properties Female mice were placed in one of three treatment groups: (1) diestrous examined were not attributable to alterations in passive properties or R fed; (2) fasted for 48 hr during diestrus, during which only water ad s within the acceptable ranges defined above. In a sample of recorded cells, libitum was given; or (3) fasted for 48 hr during diestrus and treated with GnRH neuron phenotype was confirmed post hoc by immunocytochem- leptin (1 mg/kg, i.p., two times per day) during the fast. Estrous cycle ical recovery of biocytin (included in internal recording solution at 4 stage was determined by vaginal cytology; all animals were diestrous on mM) with streptavidin-Cy3 and immunodetection of GnRH peptide the day of the experiment. Importantly, and as shown previously (Ahima within the same cell as described previously (Suter et al., 2000). et al., 1996; Sullivan et al., 2002), fasted mice remained in diestrus