Narcoleptic orexin receptor knockout mice express enhanced cholinergic properties in laterodorsal tegmental neurons.

Pharmacological studies of narcoleptic canines indicate that exaggerated pontine cholinergic transmission promotes cataplexy. As disruption of orexin (hypocretin) signaling is a primary defect in narcolepsy with cataplexy, we investigated whether markers of cholinergic synaptic transmission might be altered in mice constitutively lacking orexin receptors (double receptor knockout; DKO). mRNA for Choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT) and the high-affinity choline transporter (CHT1) but not acetylcholinesterase (AChE) was significantly higher in samples from DKO than wild-type (WT) mice. This was region-specific; levels were elevated in samples from the laterodorsal tegmental nucleus (LDT) and the fifth motor nucleus (Mo5) but not in whole brainstem samples. Consistent with region-specific changes, we were unable to detect significant differences in Western blots for ChAT and CHT1 in isolates from brainstem, thalamus and cortex or in ChAT enzymatic activity in the pons. However, using ChAT immunocytochemistry, we found that while the number of cholinergic neurons in the LDT and Mo5 were not different, the intensity of somatic ChAT immunostaining was significantly greater in the LDT, but not Mo5, from DKO than from WT mice. We also found that ChAT activity was significantly reduced in cortical samples from DKO compared with WT mice. Collectively, these findings suggest that the orexins can regulate neurotransmitter expression and that the constitutive absence of orexin signaling results in an up-regulation of the machinery necessary for cholinergic neurotransmission in a mesopontine population of neurons that have been associated with both normal rapid eye movement sleep and cataplexy.

Increased sensitivity to acute and persistent pain in neuron-specific endothelin-1 knockout mice.

Endothelin-1 (ET-1) exists in endothelial cells as well as a variety of other cell types. The presence of ET-1 and its receptors in neurons suggests its possible role as a neurotransmitter and/or neuromodulator. Studies utilizing exogenous ET-1 have suggested that ET-1 affects pain transmission. This study was designed to examine the possible role(s) of neuronal ET-1 in pain processing. We produced neuron-specific ET-1 knockout mice using the Cre/loxP system with a synapsin I promoter and examined the effects produced by the lack of neuronal ET-1 on pain behavior using common pain models and a model of stress-induced analgesia. In acute nociceptive pain models, paw withdrawal thresholds to radiant heat and mechanical stimuli applied with von Frey hairs were significantly lower in the knockout mice compared with control. This indicated that the absence of neuronal ET-1 leads to greater sensitivity to acute nociceptive stimuli. After inflammation was produced by intraplantar injection of carrageenan, there was a significantly greater degree of thermal hyperalgesia and mechanical allodynia in the knockout mice even after the difference in baseline values was compensated. Furthermore, in a neuropathic pain model produced by spinal nerve ligation, there was also a greater degree of mechanical allodynia in the knockout mice. Finally, in a swim-stress model, the magnitude of stress-induced analgesia was less in the knockout mice, indicating the involvement of neuronal ET-1 in stress-induced analgesia. The results suggest that there is a basal release of ET-1 from neurons that affect baseline pain thresholds as well as an additional release during persistent pain states that acts to suppress the pain. The involvement of neuronal ET-1 in stress-induced analgesia further suggests its role in endogenous pain inhibitory systems. To confirm that ET-1 is released in persistent pain states and to determine which part of the CNS is involved, we measured the concentrations of ET-1 before and after inducing peripheral inflammation in different parts of the CNS involved in endogenous pain inhibitory systems in normal mice. We found that ET-1 was increased in the hypothalamus while no significant increase was observed in the midbrain, medulla and spinal cord. The results of the present study suggest that neuronal ET-1 is involved in endogenous pain inhibitory control likely via pathways through the hypothalamus.

Endothelium-specific loss of murine thrombomodulin disrupts the protein C anticoagulant pathway and causes juvenile-onset thrombosis.

The thrombomodulin (TM) gene was ablated in mice in a cell type-restricted manner from vascular endothelium by Cre-recombinase-mediated excision controlled by the endothelial cell lineage-specific Tie2 promoter. Forty percent of mutant (TMLox-) mice display a distinct lethal embryonic phenotype not observed in completely TM-deficient embryos. The remaining 60% of TMLox mice survive beyond birth, but invariably succumb to a severe hypercoagulable state and massive thrombosis after 3 weeks, terminating in a lethal consumptive coagulopathy. The progression of thrombosis was age- and sex-dependent. Disruption of the TM/protein C pathway was not associated with a latent proinflammatory state. Disease onset and progression could be prevented by warfarin anticoagulation. These results show that in mice, loss of endothelial cell TM function causes spontaneous and fatal thrombosis in the arterial and venous circulation, resulting from unfettered activation of the coagulation system. The combination of complete disease penetrance, uniform disease onset at young age, large vessel thrombosis of the extremities and multiple organ systems, and consumptive coagulopathy as the disease end-point provides a unique mouse model of human thrombotic disease.

Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo.

Endocardial cells are thought to contribute at least in part to the formation of the endocardial cushion mesenchyme. Here, we created Tie2-Cre transgenic mice, in which expression of Cre recombinase is driven by an endothelial-specific promoter/enhancer. To analyze the lineage of Cre expressing cells, we used CAG-CAT-Z transgenic mice, in which expression of lacZ is activated only after Cre-mediated recombination. We detected pan-endothelial expression of the Cre transgene in Tie2-Cre;CAG-CAT-Z double-transgenic mice. This expression pattern is almost identical to Tie2-lacZ transgenic mice. However, interestingly, we observed strong and uniform lacZ expression in mesenchymal cells of the atrioventricular canal of Tie2-Cre;CAG-CAT-Z double-transgenic mice. We also detected lacZ expression in the mesenchymal cells in part of the proximal cardiac outflow tract, but not in the mesenchymal cells of the distal outflow tract and branchial arch arteries. LacZ staining in Tie2-Cre;CAG-CAT-Z embryos is consistent with endocardial-mesenchymal transformation in the atrioventricular canal and outflow tract regions. Our observations are consistent with previously reported results from Cx43-lacZ, Wnt1-Cre;R26R, and Pax3-Cre;R26R transgenic mice, in which lacZ expression in the cardiac outflow tract identified contributions in part from the cardiac neural crest. Tie2-Cre transgenic mice are a new genetic tool for the analyses of endothelial cell-lineage and endothelial cell-specific gene targeting.