20-HETE is a potent vasoconstrictor that is implicated in the regulation of blood pressure, cerebral blood flow and neuronal death following ischemia. Numerous human genetic studies have shown that inactivating variants in the cytochrome P450 enzymes that produce 20-HETE are associated with hypertension, stroke and cerebrovascular disease. However, little is known about the expression and cellular distribution of the cytochrome P450A enzymes (CYP4A) that produce 20-HETE or the newly discovered 20-HETE receptor (GPR75) in the brain. The present study examined the cell types and regions in the rat forebrain that express CYP4A and GPR75. Brain tissue slices from Sprague Dawley (SD), Dahl Salt-Sensitive (SS) and CYP4A1 transgenic rat strains, as well as cultured human cerebral pericytes and cerebral vascular smooth muscle cells, were analyzed by fluorescent immunostaining. Tissue homogenates from these strains and cultured cells were examined by Western blot. In the cerebral vasculature, CYP4A and GPR75 were expressed in endothelial cells, vascular smooth muscle cells and the glial limiting membrane of pial arteries and penetrating arterioles but not in the endothelium of capillaries. CYP4A, but not GPR75, was expressed in astrocytes. CYP4A and GPR75 were both expressed in a subpopulation of pericytes on capillaries. The diameters of capillaries were significantly decreased at the sites of first and second-order pericytes that expressed CYP4A. Capillary diameters were unaffected at the sites of other pericytes that did not express CYP4A. These findings implicate 20-HETE as a paracrine mediator in various components of the neurovascular unit and are consistent with 20-HETE's emerging role in the regulation of cerebral blood flow, blood-brain barrier integrity, the pathogenesis of stroke and the vascular contributions to cognitive impairment and dementia. Moreover, this study highlights GPR75 as a potential therapeutic target for the treatment of these devastating conditions.

Kisspeptin/Neurokinin B/Dynorphin (KNDy) neurons of the arcuate nucleus (ARC) play a key role in the regulation of fertility. The ability to detect features of KNDy neurons that are essential for fertility may require three-dimensional (3D) imaging of the complete population. Recently developed protocols for optical tissue clearing permits 3D imaging of neuronal populations in un-sectioned brains. However, these techniques have largely been described in the mouse brain. We report 3D imaging of the KNDy cell population in the whole rat brain and sheep hypothalamus using immunolabelling and modification of a solvent-based clearing protocol, iDISCO. This study expands the use of optical tissue clearing for multiple mammalian models and provides versatile analysis of KNDy neurons across species. Additionally, we detected a small population of previously unreported kisspeptin neurons in the lateral region of the ovine mediobasal hypothalamus, demonstrating the ability of this technique to detect novel features of the kisspeptin system.

Individual variance in vulnerability to develop addictions is influenced by social factors. Specifically, drug-taking in a sexual context appears to enhance further drug-seeking behavior in human users, as these users identify the effects of drugs to enhance sexual pleasure as a primary reason for continued drug use. Methamphetamine (Meth) is commonly used in this context. Similarly, male rats that self-administered Meth immediately followed by sexual behavior display enhanced drug-seeking behavior, including attenuation of extinction and increased reinstatement to seeking of Meth-associated cues. Hence, drug-taking in a sexual context enhances vulnerability for addiction. However, the neural mechanisms by which this occurs are unknown. Here the hypothesis was tested that medial prefrontal cortex is essential for this effect of Meth and sex when experienced concurrently. First it was shown that CaMKII neurons in the anterior cingulate area (ACA) were co-activated by both Meth and sex. Next, chemogenetic inactivation of ACA CaMKII cells using AAV5-CaMKIIa-hM4Di-mCherry was shown not to affect Meth-induced locomotor activity or sexual behavior. Subsequently, chemogenetic inactivation of ACA CaMKII neurons during Meth self-administration followed by sexual behavior was shown to prevent the effects of Meth and sex on enhanced reinstatement of Meth-seeking but did not affect enhanced drug-seeking during extinction tests. These results indicate that ACA CaMKII cell activation during exposure to Meth in a sexual context plays an essential role in the subsequent enhancement of drug-seeking during reinstatement tests.