Acetylcholinesterase (AChE), an important component of cholinergic synapses, colocalizes with amyloid-beta peptide (A beta) deposits of Alzheimer's brain. We report here that bovine brain AChE, as well as the human and mouse recombinant enzyme, accelerates amyloid formation from wild-type A beta and a mutant A beta peptide, which alone produces few amyloid-like fibrils. The action of AChE was independent of the subunit array of the enzyme, was not affected by edrophonium, an active site inhibitor, but it was affected by propidium, a peripheral anionic binding site ligand. Butyrylcholinesterase, an enzyme that lacks the peripheral site, did not affect amyloid formation. Furthermore, AChE is a potent amyloid-promoting factor when compared with other A beta-associated proteins. Thus, in addition to its role in cholinergic synapses, AChE may function by accelerating A beta formation and could play a role during amyloid deposition in Alzheimer's brain.

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.

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.

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.

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 commandlike neurons to modulate autonomic outflow and coordinate metabolic responses in liver and adipose tissue.autonomic nervous system | neuronal tracing | pseudorabies virus T he autonomic nervous system plays a prominent role in modulating carbohydrate and lipid metabolism. The original theories of sympathetic activation (1) proposed a body-wide increase in sympathetic outflow. However, later studies suggested differential sympathetic activation on specific organs through distinct autonomic projections, which permits more finely tuned control of metabolism (2, 3).The liver and adipose tissue play important roles in fuel storage and release. These organs respond to altered energy availability with a set of homeostatic responses mediated by humoral factors and autonomic outflow. For example, activation of hepatic sympathetic aminergic and peptidergic innervation increases glucose output and modulates fatty acid transport. Conversely, parasympathetic activity decreases hepatic glucose output and increases carbohydrate storage (4). Likewise, the sympathetic innervation of white adipose tissue induces lipolysis (5) and alters glucose uptake. Thus, in general, parasympathetic activity favors fuel storage, whereas sympathetic activity increases the fuel available for immediate use.Many CNS regions regulate autonomic outflow to hepatic or adipose tissue to influence peripheral energy homeostasis. In particular, the ventromedial hypothalamus (VMH), dorsomedial hypothalamus (DMH), lateral hypothalamus (LH), and nucleus of the solitary tract (NTS) (6)...

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.

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