(3α,5α)3-hydroxypregnan-20-one (3α,5α-THP) regulation of hypothalamic and extrahypothalamic corticotropin releasing factor (CRF): Sexual dimorphism and brain region specificity in Sprague Dawley rats.

CRF is the main activator of the hypothalamic-pituitary-adrenal (HPA) axis in response to stress. CRF neurons are found mainly in the hypothalamus, but CRF positive cells and CRF1 receptors are also found in extrahypothalamic structures, including amygdala (CeA), hippocampus, NAc and VTA. CRF release in the hypothalamus is regulated by inhibitory GABAergic interneurons and extrahypothalamic glutamatergic inputs, and disruption of this balance is found in stress-related disorders and addiction. (3α,5α)3-hydroxypregnan-20-one (3α,5α-THP), the most potent positive modulator of GABAA receptors, attenuates the stress response reducing hypothalamic CRF mRNA expression and ACTH and corticosterone serum levels. In this study, we explored 3α,5α-THP regulation of hypothalamic and extrahypothalamic CRF mRNA and peptide expression, in male and female Sprague Dawley rats, following vehicle or 3α,5α-THP administration (15 mg/kg). In the hypothalamus, we found sex differences in CRF mRNA expression (females +74%, p < 0.01) and CRF peptide levels (females -71%, p < 0.001). 3α,5α-THP administration reduced hypothalamic CRF mRNA expression only in males (-50%, p < 0.05) and did not alter CRF peptide expression in either sex. In hippocampus and CeA, 3α,5α-THP administration reduced CRF peptide concentrations only in the male (hippocampus -29%, p < 0.05; CeA -62%, p < 0.01). In contrast, 3α,5α-THP injection increased CRF peptide concentration in the VTA of both males (+32%, p < 0.01) and females (+26%, p < 0.01). The results show sex and region-specific regulation of CRF signals and the response to 3α,5α-THP administration. This data may be key to successful development of therapeutic approaches for stress-related disorders and addiction.

Phenotyping CCL2 Containing Central Amygdala Neurons Controlling Alcohol Withdrawal-Induced Anxiety.

Chemokines such as chemokine (C-C motif) ligand 2 (CCL2) play a role in several behaviors, including anxiety-like behavior, but whether neurons are an important source of CCL2 for behavior and how neuronal CCL2 may work to affect behavior are still debated. When a herpes simplex virus (HSV) vector was used to knockdown CCL2 mRNA in neurons of the central nucleus of the amygdala (CeA) in rats experiencing multiple withdrawals from low dose ethanol, anxiety-like behavior appeared in the social interaction task. To examine this finding further Fractalkine (CX3CL1), a chemokine that is often found to have an opposing function to CCL2 was measured in these rats. Both alcohol withdrawal and CCL2 knockdown increased the levels of the anti-inflammatory protein CX3CL1. The combination of alcohol withdrawal and CCL2 knockdown decreased CX3CL1 and may alter pro-inflammatory/anti-inflammatory balance, and thus highlights the potential importance of CCL2 and CCL2/CX3CL1 balance in anxiety. To find a mechanism by which neuronal chemokines like CCL2 could affect behavior, retrograde tracing with fluorescent nanobeads was done in two brain regions associated with anxiety the bed nucleus of the stria terminalis (BNST) and the ventral periaqueductal gray (VPAG). These studies identified CeA projection neurons to these brain regions that contain CCL2. To demonstrate that CCL2 can be transported via axons to downstream brain regions, the axonal transport blocker, colchicine, was given and 24 h later, the accumulation of CCL2 in CeA neuronal cell bodies was found. Finally, CCL2 in CeA neurons was localized to the synapse using confocal microscopy with enhanced resolution following deconvolution and electron microscopy, which along with the other evidence suggests that CCL2 may be transported down axons in CeA neurons and released from nerve terminals perhaps into brain regions like the BNST and VPAG to affect behaviors such as anxiety. These results suggest that neurons are an important target for chemokine research related to behavior.