Molecular annotation of integrative feeding neural circuits.

The identity of higher-order neurons and circuits playing an associative role to control feeding is unknown. We injected pseudorabies virus, a retrograde tracer, into masseter muscle, salivary gland, and tongue of BAC-transgenic mice expressing GFP in specific neural populations and identified several CNS regions that project multisynaptically to the periphery. MCH and orexin neurons were identified in the lateral hypothalamus, and Nurr1 and Cnr1 in the amygdala and insular/rhinal cortices. Cholera toxin ß tracing showed that insular Nurr1(+) and Cnr1(+) neurons project to the amygdala or lateral hypothalamus, respectively. Finally, we show that cortical Cnr1(+) neurons show increased Cnr1 mRNA and c-Fos expression after fasting, consistent with a possible role for Cnr1(+) neurons in feeding. Overall, these studies define a general approach for identifying specific molecular markers for neurons in complex neural circuits. These markers now provide a means for functional studies of specific neuronal populations in feeding or other complex behaviors.

Identification of neuronal subpopulations that project from hypothalamus to both liver and adipose tissue polysynaptically.

The autonomic nervous system regulates fuel availability and energy storage in the liver, adipose tissue, and other organs; however, the molecular components of this neural circuit are poorly understood. We sought to identify neural populations that project from the CNS indirectly through multisynaptic pathways to liver and epididymal white fat in mice using pseudorabies virus strains expressing different reporters together with BAC transgenesis and immunohistochemistry. Neurons common to both circuits were identified in subpopulations of the paraventricular nucleus of the hypothalamus (PVH) by double labeling with markers expressed in viruses injected in both sites. The lateral hypothalamus and arcuate nucleus of the hypothalamus and brainstem regions (nucleus of the solitary tract and A5 region) also project to both tissues but are labeled at later times. Connections from these same sites to the PVH were evident after direct injection of virus into the PVH, suggesting that these regions lie upstream of the PVH in a common pathway to liver and adipose tissue (two metabolically active organs). These common populations of brainstem and hypothalamic neurons express neuropeptide Y and proopiomelanocortin in the arcuate nucleus, melanin-concentrating hormone, and orexin in the lateral hypothalamus and in the corticotrophin-releasing hormone and oxytocin in the PVH. The delineation of this circuitry will facilitate a functional analysis of the possible role of these potential command-like neurons to modulate autonomic outflow and coordinate metabolic responses in liver and adipose tissue.

Transgenic mice expressing green fluorescent protein under the control of the corticotropin-releasing hormone promoter.

CRH is widely expressed in the brain and is of broad functional relevance to a number of physiological processes, including stress response, parturition, immune response, and ingestive behavior. To delineate further the organization of the central CRH network, we generated mice expressing green fluorescent protein (GFP) under the control of the CRH promoter, using bacterial artificial chromosome technology. Here we validate CRH-GFP transgene expression within specific brain regions and confirm the distribution of central GFP-producing cells to faithfully recapitulate that of CRH-expressing cells. Furthermore, we confirm the functional integrity of a population of GFP-producing cells by demonstrating their opposite responsiveness to nutritional status. We anticipate that this transgenic model will lend itself as a highly tractable tool for the investigation of CRH expression and function in discrete brain regions.

Embryonic motor neuron dendrite growth is stunted by inhibition of nitric oxide-dependent activation of soluble guanylyl cyclase and protein kinase G.

We have examined the participation of a neuronal nitric oxide synthase (nNOS) signaling pathway in the elaboration of motor neuron dendrites during embryonic life. During chick embryogenesis, nNOS is expressed by interneurons that surround the motor neuron pools in the ventral horn. Pseudorabies virus tracing suggests that these cells, while juxtaposed to motor neurons are not synaptically connected to them. The downstream effectors, soluble guanylyl cyclase (sGC) and protein kinase G (PKG), are found in motor neurons as well as several other populations of spinal cord cells. To determine the functional significance of the nNOS/sGC/PKG signaling pathway, pharmacological inhibitors were applied to chick embryos and the effects on motor neuron dendrites monitored. Inhibition of nNOS activity led to a lasting reduction in the overall size and degree of branching of the dendritic tree. These alterations in dendritic architecture were also seen when the activity of sGC or PKG was blocked. Our results suggest that normal motor neuron dendrite elaboration depends, in part, on the activity-dependent generation of NO by ventral horn interneurons, which then activates sGC and PKG in motor neurons.

Definition of estrogen receptor pathway critical for estrogen positive feedback to gonadotropin-releasing hormone neurons and fertility.

The mechanisms through which estrogen regulates gonadotropin-releasing hormone (GnRH) neurons to control mammalian ovulation are unknown. We found that estrogen positive feedback to generate the preovulatory gonadotropin surge was normal in estrogen receptor beta knockout (ERbeta) mutant mice, but absent in ERalpha mutant mice. An ERalpha-selective compound was sufficient to generate positive feedback in wild-type mice. As GnRH neurons do not express ERalpha, estrogen positive feedback upon GnRH neurons must be indirect in nature. To establish the cell type responsible, we generated a neuron-specific ERalpha mutant mouse line. These mice failed to exhibit estrogen positive feedback, demonstrating that neurons expressing ERalpha are critical. We then used a GnRH neuron-specific Pseudorabies virus (PRV) tracing approach to show that the ERalpha-expressing neurons innervating GnRH neurons are located within rostral periventricular regions of the hypothalamus. These studies demonstrate that ovulation is driven by estrogen actions upon ERalpha-expressing neuronal afferents to GnRH neurons.

Trpm5 null mice respond to bitter, sweet, and umami compounds.

Trpm5 is a calcium-activated cation channel expressed selectively in taste receptor cells. A previous study reported that mice with an internal deletion of Trpm5, lacking exons 15-19 encoding transmembrane segments 1-5, showed no taste-mediated responses to bitter, sweet, and umami compounds. We independently generated knockout mice null for Trpm5 protein expression due to deletion of Trpm5's promoter region and exons 1-4 (including the translation start site). We examined the taste-mediated responses of Trpm5 null mice and wild-type (WT) mice using three procedures: gustatory nerve recording [chorda tympani (CT) and glossopharyngeal (NG) nerves], initial lick responses, and 24-h two-bottle preference tests. With bitter compounds, the Trpm5 null mice showed reduced, but not abolished, avoidance (as indicated by licking responses and preference ratios higher than those of WT), a normal CT response, and a greatly diminished NG response. With sweet compounds, Trpm5 null mice showed no licking response, a diminished preference ratio, and absent or greatly reduced nerve responses. With umami compounds, Trpm5 null mice showed no licking response, a diminished preference ratio, a normal NG response, and a greatly diminished CT response. Our results demonstrate that the consequences of eliminating Trmp5 expression vary depending upon the taste quality and the lingual taste field examined. Thus, while Trpm5 is an important factor in many taste responses, its absence does not eliminate all taste responses. We conclude that Trpm5-dependent and Trpm5-independent pathways underlie bitter, sweet, and umami tastes.

Contribution of vasoconstriction to the origin of atherosclerosis: a conceptual study.

Research during the past century has clearly shown that endothelial injury (EI) and/or endothelial dysfunction (ED) are among the major events determining the onset of atherosclerosis. Included in the events that may elicit endothelial damage, vasoconstriction (VC) has received relatively little attention. This conceptual review attempts to show that in elastic and conduit arteries, VC is not only capable of producing EI/ED, but is also closely associated with many recognized proatherogenic stimuli. Of related interest is the observation that a number of suspected antiatherogenic stimuli oppose VC by their vasodilatory effects, lending further support to this relationship. In addition, recent developments in the knowledge of the molecular basis of VC (including the role of specific inhibitors) are discussed, and their potential for preventing lesion formation and thus becoming novel therapeutic alternatives against the onset of atherosclerosis are highlighted.

Making sense with TRP channels: store-operated calcium entry and the ion channel Trpm5 in taste receptor cells.

The sense of taste plays a critical role in the life and nutritional status of organisms. During the last decade, several molecules involved in taste detection and transduction have been identified, providing a better understanding of the molecular physiology of taste receptor cells. However, a comprehensive catalogue of the taste receptor cell signaling machinery is still unavailable. We have recently described the occurrence of calcium signaling mechanisms in taste receptor cells via apparent store-operated channels and identified Trpm5, a novel candidate taste transduction element belonging to the mammalian family of transient receptor potential channels. Trpm5 is expressed in a tissue-restricted manner, with high levels in gustatory tissue. In taste cells, Trpm5 is co-expressed with taste-signaling molecules such as alpha-gustducin, Ggamma(13), phospholipase C beta(2) and inositol 1,4,5-trisphosphate receptor type III. Biophysical studies of Trpm5 heterologously expressed in Xenopus oocytes and mammalian CHO-K1 cells indicate that it functions as a store-operated channel that mediates capacitative calcium entry. The role of store-operated channels and Trpm5 in capacitative calcium entry in taste receptor cells in response to bitter compounds is discussed.

A transient receptor potential channel expressed in taste receptor cells.

We used differential screening of cDNAs from individual taste receptor cells to identify candidate taste transduction elements in mice. Among the differentially expressed clones, one encoded Trpm5, a member of the mammalian family of transient receptor potential (TRP) channels. We found Trpm5 to be expressed in a restricted manner, with particularly high levels in taste tissue. In taste cells, Trpm5 was coexpressed with taste-signaling molecules such as alpha-gustducin, Ggamma13, phospholipase C-beta2 (PLC-beta2) and inositol 1,4,5-trisphosphate receptor type III (IP3R3). Our heterologous expression studies of Trpm5 indicate that it functions as a cationic channel that is gated when internal calcium stores are depleted. Trpm5 may be responsible for capacitative calcium entry in taste receptor cells that respond to bitter and/or sweet compounds.