DREADDs, designer receptors exclusively activated by designer drugs, are engineered G protein-coupled receptors (GPCR) which can precisely control GPCR signaling pathways (for example, Gq, Gs and Gi). This chemogenetic technology for control of GPCR signaling has been successfully applied in a variety of in vivo studies, including in mice, to remotely control GPCR signaling, for example, in neurons, glia cells, pancreatic beta-cells, or cancer cells. In order to fully explore the in vivo applications of the DREADD technology we generated hM3Dq and hM4Di strains of mice which allow for Cre recombinase-mediated restricted expression of these pathway-selective DREADDs. With the many Cre driver lines now available, these DREADD lines will be applicable to studying a wide array of research and preclinical questions.

SUMMARYObjectives: Mutations in the ATP1a3 subunit of the neuronal Na + /K + -ATPase are thought to be responsible for seizures, hemiplegias, and other symptoms of alternating hemiplegia of childhood (AHC). However, the mechanisms through which ATP1A3 mutations mediate their pathophysiologic consequences are not yet understood. The following hypotheses were investigated: (1) Our novel knock-in mouse carrying the most common heterozygous mutation causing AHC (D801N) will exhibit the manifestations of the human condition and display predisposition to seizures; and (2) the underlying pathophysiology in this mouse model involves increased excitability in response to electrical stimulation of Schaffer collaterals and abnormal predisposition to spreading depression (SD). Methods: We generated the D801N mutant mouse (Mashlool, Mashl +/À ) and compared mutant and wild-type (WT) littermates. Behavioral tests, amygdala kindling, flurothyl-induced seizure threshold, spontaneous recurrent seizures (SRS), and other paroxysmal activities were compared between groups. In vitro electrophysiologic slice experiments on hippocampus were performed to assess predisposition to hyperexcitability and SD. Results: Mutant mice manifested a distinctive phenotype similar to that of humans with AHC. They had abnormal impulsivity, memory, gait, motor coordination, tremor, motor control, endogenous nociceptive response, paroxysmal hemiplegias, diplegias, dystonias, and SRS, as well as predisposition to kindling, to flurothyl-induced seizures, and to sudden unexpected death. Hippocampal slices of mutants, in contrast to WT animals, showed hyperexcitable responses to 1 Hz pulse-trains of electrical stimuli delivered to the Schaffer collaterals and had significantly longer duration of K + -induced SD responses. Significance: Our model reproduces the major characteristics of human AHC, and indicates that ATP1a3 dysfunction results in abnormal short-term plasticity with increased excitability (potential mechanism for seizures) and a predisposition to more

Leptin deficiency results in a complex obesity phenotype comprising both hyperphagia and lowered metabolism. The hyperphagia results, at least in part, from the absence of induction by leptin of melanocyte stimulating hormone (MSH) secretion in the hypothalamus; the MSH normally then binds to melanocortin-4 receptor expressing neurons and inhibits food intake. The basis for the reduced metabolic rate has been unknown. Here we show that leptin administered to leptin-deficient (ob͞ob) mice results in a large increase in peripheral MSH levels; further, peripheral administration of an MSH analogue results in a reversal of their abnormally low metabolic rate, in an acceleration of weight loss during a fast, in partial restoration of thermoregulation in a cold challenge, and in inducing serum free fatty acid levels. These results support an important peripheral role for MSH in the integration of metabolism with appetite in response to perceived fat stores indicated by leptin levels.R ecent research has outlined a pathway for control of body weight (1-4): leptin, the product of the ob gene in mouse, is produced by adipocytes (5). It circulates to the hypothalamus where it binds to cells expressing the leptin receptor, the product of the db gene in mouse (6-9). Proopiomelanocortin (POMC) neurons are among the hypothalamic neurons expressing the leptin receptor (10). This leptin binding leads to the secretion of melanocyte stimulating hormone (MSH), which in turn binds to neurons expressing the melanocortin-4 receptor (MC4-R) (11); these neurons then suppress appetite (12)(13)(14). This outline is based on the phenotypes of spontaneous and induced mouse mutants (5,9,13,(15)(16)(17)(18)(19) as well as on the phenotype of homologous mutations in humans (20-24). These interpretations are in agreement that leptin is the signal from the fat stores (adipocytes) to the center, and further that MSH regulates appetite. However, there are significant aspects of the mutant phenotypes that suggest both a greater complexity of body weight homeostasis, specifically the integration of appetite and metabolism, and a factor from the central nervous system (CNS) to the periphery mediating this integration.First, pomc͞pomc mutants that completely lack POMC peptides, including MSH, show a phenotype of altered lipid metabolism in addition to hyperphagia. As the fat content of the diet increases, the mice gain weight out of proportion to their food intake (17). This shows a particular inability to use dietary fat for sustaining metabolic rate. And when these pomc͞pomc mutants are treated by peripheral administration of an ␣-MSH analog the mice lose weight and eat less, but the weight loss is much greater than the decrease in appetite (17). Again, this result is consistent with a role for MSH in mobilizing peripheral fat stores.Second, leptin-deficient mice (ob͞ob) show decreased metabolic rate (increased metabolic efficiency; ref. 25), which precedes the onset of obesity. Notably these mutants show: (i) weight gain when pair-fed with normal c...

Down syndrome (DS) is the most common genetic cause of significant cognitive disability. We hypothesize that by identifying metabolic alterations associated with cognitive impairment, it may be possible to develop medical or dietary interventions to ameliorate cognitive disabilities in persons with DS. Evidence suggests that one-carbon/transsulfuration (1C-TS) metabolism is abnormal in persons with DS. Cystathionine beta-synthase (CBS) plays a critical role in this metabolic system. The gene for CBS is on human chromosome 21, and there is evidence of elevated CBS enzyme activity in tissues and cells from individuals with DS. To analyze the possible role of CBS in Down syndrome, we have produced several lines of transgenic mice expressing the human CBS gene. We describe the use of Florescence Situ Hybridization (FISH) analysis to characterize the transgene insertion site for each line. Our initial expression analysis of each transgenic line by RT-PCR shows that the tissue specificity of human CBS mRNA levels in these mice may differ from the tissue specificity of mouse CBS mRNA levels in the same animals. These mice will be invaluable for assessing the regulation of the CBS gene and the role of CBS in cognition. They can also be used to develop therapies that target abnormalities in 1C-TS metabolism to improve cognition in persons with DS.