Classically, 17beta-estradiol (E2) is thought to control homeostatic functions such as reproduction, stress responses, feeding, sleep cycles, temperature regulation, and motivated behaviors through transcriptional events. Although it is increasingly evident that E2 can also rapidly activate kinase pathways to have multiple downstream actions in CNS neurons, the receptor(s) and the signal transduction pathways involved have not been identified. We discovered that E2 can alter mu-opioid and GABA neurotransmission rapidly through nontranscriptional events in hypothalamic GABA, proopiomelanocortin (POMC), and dopamine neurons. Therefore, we examined the effects of E2 in these neurons using whole-cell recording techniques in ovariectomized female guinea pigs. E2 reduced rapidly the potency of the GABAB receptor agonist baclofen to activate G-protein-coupled, inwardly rectifying K+ channels in hypothalamic neurons. These effects were mimicked by the membrane impermeant E2-BSA and selective estrogen receptor modulators, including a new diphenylacrylamide compound, STX, that does not bind to intracellular estrogen receptors alpha or beta, suggesting that E2 acts through a unique membrane receptor. We characterized the coupling of this estrogen receptor to a Galpha(q)-mediated activation of phospholipase C, leading to the upregulation of protein kinase Cdelta and protein kinase A activity in these neurons. Moreover, using single-cell reverse transcription-PCR, we identified the critical transcripts, PKCdelta and its downstream target adenylyl cyclase VII, for rapid, novel signaling of E2 in GABA, POMC, and dopamine neurons. Therefore, this unique Gq-coupled estrogen receptor may be involved in rapid signaling in hypothalamic neurons that are critical for normal homeostatic functions.

Estrogens are involved in the hypothalamic control of multiple homeostatic functions including reproduction, stress responses, energy metabolism, sleep cycles, temperature regulation, and motivated behaviors. The critical role of 17␤-estradiol (E 2 ) is evident in hypoestrogenic states (e.g., postmenopause) in which many of these functions go awry. The actions of E 2 in the brain have been attributed to the activation of estrogen receptors ␣ and ␤ through nuclear, cytoplasmic, or membrane actions. However, we have identified a putative membrane-associated estrogen receptor that is coupled to desensitization of GABA B and -opioid receptors in guinea pig and mouse hypothalamic proopiomelanocortin neurons. We have synthesized a new nonsteroidal compound, STX, which selectively targets the G␣q-coupled phospholipase C-protein kinase C-protein kinase A pathway, and have established that STX is more potent than E 2 in mediating this desensitization in an ICI 182, 780-sensitive manner in both guinea pig and mouse neurons. Both E 2 and STX were fully efficacious in estrogen receptor ␣,␤ knock-out mice. Moreover, in vivo treatment with STX, similar to E 2 , attenuated the weight gain in hypoestrogenic female guinea pigs. Therefore, this membrane-delimited signaling pathway plays a critical role in the control of energy homeostasis and may provide a novel therapeutic target for treatment of postmenopausal symptoms and eating disorders in females.

Hypothalamic proopiomelanocortin (POMC) neurons are critical for controlling homeostatic functions in the mammal. We used a transgenic mouse model in which the POMC neurons were labeled with enhanced green fluorescent protein to perform visualized, whole-cell patch recordings from prepubertal female hypothalamic slices. The mouse POMC-enhanced green fluorescent protein neurons expressed the same endogenous conductances (a transient outward K(+) current and a hyperpolarization-activated, cation current) that have been described for guinea pig POMC neurons. In addition, the selective micro -opioid receptor agonist DAMGO induced an outward current (maximum of 12.8 +/- 1.2 pA), which reversed at K(+) equilibrium potential (E(K+)), in the majority (85%) of POMC neurons with an EC(50) of 102 nM. This response was blocked by the opioid receptor antagonist naloxone with an inhibition constant of 3.1 nM. In addition, the gamma-aminobutyric acid(B) receptor agonist baclofen (40 micro M) caused an outward current (21.6 +/- 4.0 pA) that reversed at E(K+) in these same neurons. The ATP-sensitive potassium channel opener diazoxide also induced an outward K(+) current (maximum of 18.7 +/- 2.2 pA) in the majority (92%) of POMC neurons with an EC(50) of 61 micro M. The response to diazoxide was blocked by the sulfonylurea tolbutamide, indicating that the POMC neurons express both Kir6.2 and sulfonylurea receptor 1 channel subunits, which was verified using single cell RT-PCR. This pharmacological and molecular profile suggested that POMC neurons might be sensitive to metabolic inhibition, and indeed, we found that their firing rate varied with changes in glucose concentrations. Therefore, it appears that POMC neurons may function as an integrator of metabolic cues and synaptic input for controlling homeostasis in the mammal.

Leptin can exert its potent appetite-suppressing effects via activation of hypothalamic proopiomelanocortin (POMC) neurons. It depolarizes POMC neurons via activation of a yet unidentified nonselective cation current. Therefore, we sought to identify the conductance activated by leptin using whole-cell recording in EGFP-POMC neurons from transgenic mice. The TRPC channel blockers SKF96365 (1-[␤-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride), flufenamic acid, and 2-APB (2-aminoethyl diphenylborinate) potently inhibited the leptin-induced current. Also, lanthanum (La 3ϩ ) and intracellular Ca 2ϩ potentiated the effects of leptin. Moreover, the diacylglycerol-permeable analog OAG (2-acetyl-1-oleoyl-sn-glycerol) failed to activate any TRPC current. Using a Cs ϩ -gluconate-based internal solution, the leptin-activated current reversed near Ϫ20 mV. After replacement of external Na ϩ and K ϩ with Cs ϩ , the reversal shifted to near 0 mV, and the I/V curve exhibited a negative slope conductance at voltages more negative than Ϫ40 mV. Based on scRT-PCR, TRPC1 and TRPC4 -7 mRNA were expressed in POMC neurons, with TRPC5 being the most prevalent. The leptin-induced current was blocked by the Jak2 inhibitor AG490, the PI3 kinase inhibitor wortmannin, and the phospholipase C inhibitors, U73122 and ET-18-OCH3. Notably, we identified PLC␥1 transcripts in the majority of POMC neurons. Therefore, leptin through a Jak2-PI3 kinase-PLC␥ pathway activates TRPC channels, and TRPC1, 4, and 5 appear to be the key channels mediating the depolarizing effects of leptin in POMC neurons.