Sex differences in the cannabinoid modulation of an A-type K ϩ current in neurons of the mammalian hypothalamus. J Neurophysiol 94: 2983-2986, 2005. First published May 18, 2005 doi:10.1152/jn.01187.2004. Cannabinoids regulate biological processes governed by the hypothalamus including, but not limited to, energy homeostasis and reproduction. The present study sought to determine whether cannabinoids modulate A-type K ϩ currents (I A ) in neurons of the hypothalamic arcuate nucleus (ARC). Whole cell patch-clamp recordings were performed in slices through the ARC prepared from castrated female and male guinea pigs. Forty percent of guinea pig ARC neurons exhibited a transient outward current that was antagonized by high (mM) concentrations of 4-aminopyridine and (100 nM) rHeteropodatoxin-2. Five of these neurons also were immunopositive for both ␤-endorphin and the Kv4.2 channel subunit. Bath application of the CB1 receptor agonists WIN 55,212-2 (1 M) or ACEA (1 M) selectively induced a rightward shift in the inactivation curve for the I A , significantly increasing the half-maximal voltage without affecting the peak current magnitude, in neurons from female but not male animals. The CB1 receptor antagonist AM251 (1 M) reversed this action. Collectively, these data reveal that guinea pig ARC neurons, including proopiomelanocortin neurons, express a prominent I A that is positively modulated by cannabinoids in a sex-specific way by altering the voltage dependence of its inactivation. The resultant inhibitory effect on this neuronal population may shed some insight into the mechanism(s) by which cannabinoids influence hypothalamic function.

Summary This review highlights the progress made thus far in characterizing the behavioral and cellular mechanisms through which cannabinoids regulate energy homeostasis. We performed microstructural analysis of feeding behavior in gonadectomized guinea pigs and gonadally intact, transgenic CB1 receptor knockout mice to determine how cannabinoids affect circadian rhythms in food intake and meal pattern. We also implanted data loggers into the abdominal cavity to correlate the appetite-modulating properties of cannabinoids with changes in core body temperature. We then coupled the effects on feeding behavior and temperature regulation with synaptic changes in the hypothalamic feeding circuitry via whole-cell patch clamp electrophysiological recording from neurons in the arcuate nucleus (ARC), in order to gain a more global perspective on the cannabinoid modulation of energy homeostasis. We observed marked sex differences in cannabinoid effects on food intake and core body temperature — with male guinea pigs exhibiting a comparatively greater sensitivity to the hyperphagia and hypophagia, as well as the hypothermia and hyperthermia, produced by CB1 receptor agonists and antagonists, respectively. In addition, male but not female CB1 receptor knockout mice show a diminished nocturnal food intake and average daily body weight relative to their wildtype littermate controls. The disparity in the CB1 receptor-mediated hyperphagia is paralleled by sex differences in the cellular effects of cannabinoids at anorexigenic, guinea pig proopiomelanocortin (POMC) synapses. Postsynaptically, cannabinoids potentiate an A-type K+ current (IA) in POMC neurons from female guinea pigs, whereas in males the activation of an inwardly rectifying K+ current is observed. Presynaptically, while cannabinoids inhibit glutamatergic input onto POMC neurons in males and females to similar degrees, males are more refractory to the cannabinoid-induced inhibition of convergent GABAergic input than females. These data reveal pervasive sex differences in the cannabinoid regulation of energy homeostasis that are consistent with changes in the excitability of POMC neurons.

Cannabinoid signaling exerts profound influence over the hypothalamic control of homeostasis including but not limited to feeding. The hypothalamic feeding circuitry consists of several components. The stimulatory component of appetite includes the neuropeptide Y (NPY), orexin (hypocretin), ghrelin and melanin concentrating hormone (MCH) neurons. Inhibitory inputs are predominately from the proopiomelanocortin (POMC) neurons originating in the hypothalamic arcuate nucleus (ARC). POMC neurons synthesize and release anorexigenic peptides such as cocaine amphetamine related transcript (CART), α-melanocyte stimulating hormone (α-MSH), and β-endorphin. This study sought to determine whether cannabinoids modulate A-type K+ current (IA) in POMC neurons. To this end, whole-cell patch clamp recordings were performed in hypothalamic slices through the ARC prepared from castrated female and male guinea pigs. A robust IA that was blocked by high concentrations of K+ channel blocker 4-aminopyridine (3mM & 10mM) was found in 46% of male and 39% in female cells. We observed that the neuronal responsiveness to cannabinoids was sexually differentiated. In recordings of female neurons, bath application of the brain cannabinoid (CB1) receptor agonists WIN 55,212-2 (1μM) or arachidonyl-2′-chloroethylaminde (ACEA, 1μM) selectively induced a depolarizing rightward shift in the inactivation curve for the IA, significantly increasing the half-maximal voltage (V1/2) for inactivation. This effect was completely blocked by the CB1 receptor antagonist AM251 (1μM) and occurred without affecting the peak current magnitude (Imax). Post-hoc immunofluorescent labeling confirmed that these effects occurred in POMC cells. We observed no effect on the V1/2 or the Imax for the activation curve. In contrast, recordings from male neurons indicated no discernable effect of cannabinoids on the V1/2 or the Imax for either the inactivation or the activation of the IA. Collectively, these data reveal that POMC neurons express a prominent IA, and that cannabinoids positively modulate this current in a sex-specific way by altering the voltage dependence of its inactivation. The resultant inhibitory effect on this neuronal population may shed some insight into the mechanism(s) by which cannabinoids influence appetite.

We explored the recently described method of in vivo electroporation as an alternate means to introduce foreign DNA constructs into mouse retinas. Ultimately, we will use this method for future studies of wildtype and mutant rhodopsin synthesis and turnover in transfected photoreceptor cells. Such studies may help to better understand the effects of some of the rhodopsin mutations that are causal for autosomal dominant retinitis pigmentosa. Methods: To test in vivo electroporation technique, a DNA plasmid containing GFP driven by the ubiquitin promoter was generated. Under a dissecting microscope, DNA was injected into the subretinal space of newborn CD1 mice using a Hamilton syringe. After injection, tweezer-type electrodes soaked in PBS were placed on the sides of heads of pups and used to create five 80-V pulses. Electroporated retinae were harvested 21 days after injection, fixed, and cut on a cryostat in sections of 20 µm. Cryosections were then examined with a Zeiss Axioplan fluorescent microscope. To examine turnover of mutant rhodopsin in photoreceptors, constructs were made that use the rhodopsin promoter to drive expression of chimeric proteins containing timer fluorescence (dsRed-E5) and either wildtype or mutant rhodopsin. Results: When viewed under the fluorescent microscope, cryosections of retinae containing the ubiquitin-GFP construct demonstrated expression of GFP in outer nuclear layer of photoreceptor cells as well as cells in the inner nuclear layer. Plasmids containing mouse rhodopsin promoter driving expression of wildtype rhodopsin-dsRed-E5 and rhodopsin mutant-dsRed-E5 were constructed and verified by sequencing. Conclusions: Expression of GFP in the retinae of P21 mice after DNA injection demonstrates in vivo electroporation is a possible means to study mutant rhodopsin in mice. Future experiments will use in vivo electroporation to study the turnover of the mutant rhodopsin-dsRed-E5 constructs.