Corticotrophin-releasing factor (CRF) is a 41 amino acid neuropeptide that coordinates adaptive responses to stress. CRF projections from neurons in the central nucleus of the amygdala (CeA) to the brainstem are of particular interest for their role in motivated behavior. To directly examine the anatomy and function of CRF neurons, we generated a BAC transgenic Crh-Cre rat in which bacterial Cre recombinase is expressed from the Crh promoter. Using Cre-dependent reporters, we found that Cre expressing neurons in these rats are immunoreactive for CRF and are clustered in the lateral CeA (CeL) and the oval nucleus of the BNST. We detected major projections from CeA CRF neurons to parabrachial nuclei and the locus coeruleus, dorsal and ventral BNST, and more minor projections to lateral portions of the substantia nigra, ventral tegmental area, and lateral hypothalamus. Optogenetic stimulation of CeA CRF neurons evoked GABA-ergic responses in 11% of non-CRF neurons in the medial CeA (CeM) and 44% of non-CRF neurons in the CeL. Chemogenetic stimulation of CeA CRF neurons induced Fos in a similar proportion of non-CRF CeM neurons but a smaller proportion of non-CRF CeL neurons. The CRF1 receptor antagonist R121919 reduced this Fos induction by two-thirds in these regions. These results indicate that CeL CRF neurons provide both local inhibitory GABA and excitatory CRF signals to other CeA neurons, and demonstrate the value of the Crh-Cre rat as a tool for studying circuit function and physiology of CRF neurons.

The central amygdala (CeA) is important for fear responses to discrete cues. Recent findings indicate that the CeA also contributes to states of sustained apprehension that characterize anxiety, although little is known about the neural circuitry involved. The stress neuropeptide corticotropin releasing factor (CRF) is anxiogenic and is produced by subpopulations of neurons in the lateral CeA and the dorsolateral bed nucleus of the stria terminalis (dlBST). Here we investigated the function of these CRF neurons in stress-induced anxiety using chemogenetics in male rats that express Cre recombinase from a Crh promoter. Anxiety-like behavior was mediated by CRF projections from the CeA to the dlBST and depended on activation of CRF1 receptors and CRF neurons within the dlBST. Our findings identify a CRF CeA ¡CRF dlBST circuit for generating anxiety-like behavior and provide mechanistic support for recent human and primate data suggesting that the CeA and BST act together to generate states of anxiety.

Graphical AbstractHighlights d Central amygdala CRF neurons release GABA and several neuropeptides d Preventing GABA release in these neurons suppresses basal anxiety-like behavior d Knockdown of CRF or dynorphin prevents anxiety evoked by chemogenetic activation d CRF or dynorphin knockdown reduces but neurotensin knockdown enhances fear learning SUMMARY Central amygdala (CeA) neurons that produce corticotropin-releasing factor (CRF) regulate anxiety and fear learning. These CeA CRF neurons release GABA and several neuropeptides predicted to play important yet opposing roles in these behaviors. We dissected the relative roles of GABA, CRF, dynorphin, and neurotensin in CeA CRF neurons in anxiety and fear learning by disrupting their expression using RNAi in male rats. GABA, but not CRF, dynorphin, or neurotensin, regulates baseline anxiety-like behavior. In contrast, chemogenetic stimulation of CeA CRF neurons evokes anxiety-like behavior dependent on CRF and dynorphin, but not neurotensin. Finally, knockdown of CRF and dynorphin impairs fear learning, whereas knockdown of neurotensin enhances it. Our results demonstrate distinct behavioral roles for GABA, CRF, dynorphin, and neurotensin in a subpopulation of CeA neurons. These results highlight the importance of considering the repertoire of signaling molecules released from a given neuronal population when studying the circuit basis of behavior.

GABA A receptors mediate the majority of inhibitory neurotransmission in the CNS. Genetic deletion of the ␣1 subunit of GABA A receptors results in a loss of ␣1-mediated fast inhibitory currents and a marked reduction in density of GABA A receptors. A grossly normal phenotype of ␣1-deficient mice suggests the presence of neuronal adaptation to these drastic changes at the GABA synapse. We used cDNA microarrays to identify transcriptional fingerprints of cellular plasticity in response to altered GABAergic inhibition in the cerebral cortex and cerebellum of ␣1 mutants. In silico analysis of 982 mutation-regulated transcripts highlighted genes and functional groups involved in regulation of neuronal excitability and synaptic transmission, suggesting an adaptive response of the brain to an altered inhibitory tone. Public gene expression databases permitted identification of subsets of transcripts enriched in excitatory and inhibitory neurons as well as some glial cells, providing evidence for cellular plasticity in individual cell types. Additional analysis linked some transcriptional changes to cellular phenotypes observed in the knock-out mice and suggested several genes, such as the early growth response 1 (Egr1), small GTP binding protein Rac1 (Rac1), neurogranin (Nrgn), sodium channel ␤4 subunit (Scn4b), and potassium voltage-gated Kv4.2 channel (Kcnd2) as cell type-specific markers of neuronal plasticity. Furthermore, transcriptional activation of genes enriched in Bergman glia suggests an active role of these astrocytes in synaptic plasticity. Overall, our results suggest that the loss of ␣1-mediated fast inhibition produces diverse transcriptional responses that act to regulate neuronal excitability of individual neurons and stabilize neuronal networks, which may account for the lack of severe abnormalities in ␣1 null mutants.

Previous studies have shown that ethanol enhanced [ 3 H]dopamine uptake in Xenopus oocytes expressing the dopamine transporter (DAT). This increase in DAT activity was mirrored by an increase in the number of transporters expressed at the cell surface. In the present study, ethanol potentiated the function of DAT expressed in HeLa cells but inhibited the function of the related norepinephrine transporter (NET). Chimeras generated between DAT and NET were examined for ethanol sensitivity and demonstrated that a 76-amino acid region spanning transmembrane domains (TMD) 2 and 3 was essential for ethanol potentiation of DAT function. The second intracellular loop between TMD 2 and 3 of DAT, which differs from that of NET by four amino acids, was explored for possible sites of ethanol action. Site-directed mutagenesis was used to replace each of these residues in DAT with the corresponding residue in NET, and the resulting cRNA were expressed in Xenopus oocytes. We found that mutations G130T or I137F abolished ethanol potentiation of DAT function, whereas the mutations F123Y and L138F had no significant effect. These results identify novel sites in the second intracellular loop that are important for ethanol modulation of DAT activity.