in a population of neurons in the arcuate nucleus (ARC) of the hypothalamus and stimulates food intake for up to 7 days if injected intracerebroventricularly. The prolonged food intake stimulation does not seem to depend on continued competition at the melanocortin-4 receptor (MC4R), because the relatively specific MC4R agonist MTII regains its ability to suppress food intake 24 h after AgRP injection. Intracerebroventricular AgRP also stimulates c-Fos expression 24 h after injection in several brain areas, so the neurons exhibiting delayed Fos expression might be particularly important in feeding behavior. Thus we aimed to identify the neurochemical phenotype of some of these neurons in select hypothalamic areas, using double-label immunohistochemistry. AgRP-injected rats ingested significantly more chow (10.2 Ϯ 0.6 g) vs. saline controls (3.4 Ϯ 0.7 g) in the first 9 h (light phase) after injection. In the lateral hypothalamus (particularly the perifornical area) 23 h after injection, AgRP induced significantly more Fos vs. saline in orexin-A (OXA) neurons (25.6 Ϯ 4.9 vs. 4.8 Ϯ 3.1%), but not in melaninconcentrating hormone (MCH) or cocaine-and amphetamineregulated transcript (CART) neurons. In the ARC, AgRP induced significantly more Fos in CART (40.6 Ϯ 5.9 vs. 13.4 Ϯ 1.8%) but not NPY neurons. In the paraventricular nucleus, there was no significant difference in Fos expression induced by AgRP vs. saline in oxytocin and CART neurons. We conclude that the long-lasting hyperphagia induced by AgRP is correlated with and possibly partially mediated by hyperactive OXA neurons in the lateral hypothalamus and CART neurons in the ARC, but not by NPY and MCH neurons. The substantial increase in light-phase food intake by AgRP supports a role for the arousing effects of OXA. Activation of CART neurons in the ARC (which likely coexpress proopiomelanocortin) could indicate attempts to activate counterregulatory decreases in food intake. food intake; cocaine-and amphetamine-regulated transcript; neuropeptide Y; oxytocin; lateral hypothalamus; paraventricular nucleus; arcuate nucleus AGOUTI-RELATED PROTEIN (AgRP) is coexpressed with neuropeptide Y (NPY) in a population of neurons in the medial aspects of the arcuate nucleus (ARC) of the hypothalamus (17, 30). AgRP/NPY neurons project locally, within the ARC (3, 19), to the paraventricular nucleus (PVN) (25, 36), dorsomedial nucleus (50), perifornical hypothalamus (13, 23), and several other brain areas (3).AgRP competes with ␣-melanocyte-stimulating hormone (␣-MSH) for the melanocortin-4 receptor (MC4R) and thus acts as an endogenous antagonist of MC4R (38,49,69). Stimulation of MC4R in the hypothalamus with the natural ligand ␣-MSH (63) or with pharmacological agonists such as MTII (46, 61) suppresses food intake. In contrast, intracerebroventricular injection of AgRP induces robust food intake (54) that lasts for several days (29). In addition, overexpression of AgRP (27) and chronic facilitation of AgRP receptor binding via overexpression of syndecan (51) lead to obesit...

In the vertebrate retina, multiple cell types express G protein-coupled receptors linked to the IP3 signaling pathway. The signaling engendered by activation of this pathway can involve activation of calcium permeable transient receptor potential (TRP) channels. To begin to understand the role of these channels in the retina, we undertake an immunocytochemical localization of two TRP channel subunits. Polyclonal antibodies raised against mammalian TRPC1 and TRPC4 are used to localize the expression of these proteins in sections of the adult chicken retina. Western blot analysis indicates that these antibodies recognize avian TRPC1 and TRPC4. TRPC1 labeling is almost completely confined to the inner plexiform layer (IPL) where it labels a subset of processes that ramify in three broad stripes. Occasionally, cell bodies are labeled. These can be found in the inner nuclear layer (INL) proximal to the IPL, the IPL, and the ganglion cell layer (GCL). Double-labeling experiments using a polyclonal antibody that recognizes brain nitric oxide synthase (bNOS) in the chicken indicate that many of the TRPC1-positive processes and cell bodies also express bNOS. Labeling with the TRPC4 antibody was much more widespread with some degree of labeling found in all layers of the retina. TRPC4 immunoreactivity was found in the photoreceptor layer, in the outer plexiform layer (OPL), in radially oriented cells in the INL, diffusely in the IPL, and in vertically oriented elements below the GCL. Double-labeling experiments with a monoclonal antibody raised against vimentin indicate that the TRPC4-positive structures in the INL and below the GCL are Müller cells. Thus, TRPC1 and TRPC4 subunits have unique expression patterns in the adult chicken retina. The distributions of these two subunits indicate that different retinal cell types express TRP channels containing different subunits.

Crousillac S, Colonna J, McMains E, Dewey JS, Gleason E. Sphingosine-1-phosphate elicits receptor-dependent calcium signaling in retinal amacrine cells. J Neurophysiol 102: 3295-3309, 2009. First published September 23, 2009 doi:10.1152/jn.00119.2009. Evidence is emerging indicating that sphingosine-1-phosphate (S1P) participates in signaling in the retina. To determine whether S1P might be involved in signaling in the inner retina specifically, we examine the effects of this sphingolipid on cultured retinal amacrine cells. Whole cell voltage-clamp recordings reveal that S1P activates a cation current that is dependent on signaling through G i and phospholipase C. These observations are consistent with the involvement of members of the S1P receptor family of G-proteincoupled receptors in the production of the current. Immunocytochemistry and PCR amplification provide evidence for the expression of S1P1R and S1P3R in amacrine cells. The receptor-mediated channel activity is shown to be highly sensitive to blockade by lanthanides consistent with the behavior of transient receptor potential canonical (TRPC) channels. PCR products amplified from amacrine cells reveal that TRPCs 1 and 3-7 channel subunits have the potential to be expressed. Because TRPC channels provide a Ca 2ϩ entry pathway, we asked whether S1P caused cytosolic Ca 2ϩ elevations in amacrine cells. We show that S1P-dependent Ca 2ϩ elevations do occur in these cells and that they might be mediated by S1P1R and S1P3R. The Ca 2ϩ elevations are partially due to release from internal stores, but the largest contribution is from influx across the plasma membrane. The effect of inhibition of sphingosine kinase suggests that the production of cytosolic S1P underlies the sustained nature of the Ca 2ϩ elevations. Elucidation of the downstream effects of these signals will provide clues to the role of S1P in regulating inner retinal function.

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