A Transgenic Rat for Investigating the Anatomy and Function of Corticotrophin Releasing Factor Circuits.

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

Lmo4 in the Basolateral Complex of the Amygdala Modulates Fear Learning

Pavlovian fear conditioning is an associative learning paradigm in which mice learn to associate a neutral conditioned stimulus with an aversive unconditioned stimulus. In this study, we demonstrate a novel role for the transcriptional regulator Lmo4 in fear learning. LMO4 is predominantly expressed in pyramidal projection neurons of the basolateral complex of the amygdala (BLC). Mice heterozygous for a genetrap insertion in the Lmo4 locus (Lmo4gt/+), which express 50% less Lmo4 than their wild type (WT) counterparts display enhanced freezing to both the context and the cue in which they received the aversive stimulus. Small-hairpin RNA-mediated knockdown of Lmo4 in the BLC, but not the dentate gyrus region of the hippocampus recapitulated this enhanced conditioning phenotype, suggesting an adult- and brain region-specific role for Lmo4 in fear learning. Immunohistochemical analyses revealed an increase in the number of c-Fos positive puncta in the BLC of Lmo4gt/+ mice in comparison to their WT counterparts after fear conditioning. Lastly, we measured anxiety-like behavior in Lmo4gt/+ mice and in mice with BLC-specific downregulation of Lmo4 using the elevated plus maze, open field, and light/dark box tests. Global or BLC-specific knockdown of Lmo4 did not significantly affect anxiety-like behavior. These results suggest a selective role for LMO4 in the BLC in modulating learned but not unlearned fear

Regulation of the dopamine transporter: a role for ethanol and protein interactions

The dopamine (DA) transporter (DAT) serves to clear released DA from the synaptic cleft and is an important part of the mesolimbic DA system, which mediates the rewarding and reinforcing effects of various drugs of abuse. Several studies suggest that the function of DAT is regulated by protein-protein interactions and signaling systems that alter cellular trafficking of DAT. Ethanol potentiates DAT function in Xenopus oocytes expressing DAT in a manner consistent with altered cellular trafficking. In contrast to ethanol’s effects on DAT, the function of the related norepinephrine transporter (NET) is inhibited by ethanol. To delineate mechanisms of ethanol action on DAT, chimeras were generated between DAT and NET. The results of these as well as site directed mutagenesis experiments revealed ethanol sensitive sites in the first intracellular loop of DAT. The absence of consensus phosphorylation sites in this loop led to the hypothesis that ethanol modulates the interaction between DAT and a putative regulatory protein important for ethanol-induced trafficking of DAT and that this interaction occurs at the first intracellular loop. To identify proteins and signaling pathways that might regulate DAT function, an interaction proteomics based approach was used to isolate and identify proteins associated with DAT. These studies revealed that DAT is part of a large multiprotein complex consisting of 21 proteins that can be classified as ion channels, trafficking proteins, extracellular matrix associated and cytoskeletal proteins. Finally, the effects of ethanol on DAT trafficking were ascertained by examining ethanol-induced changes in DAT function in several cell types. Studies on MDCK cells stably expressing GFP-DAT suggest that ethanol potentiates DAT function in this cell type. SH-SY5Y cells stably expressing DAT were also examined for ethanol effects on DAT function. Ethanol produced a 25% enhancement in DAT function in these cells, which was not statistically significant. The effects of ethanol on DAT trafficking in neuronal cells were observed by using a sindbis viral construct encoding GFP-DAT. The experiments outlined above have led to the identification of a novel role for DAT in ethanol-induced neuroadaptation and in the identification of several novel proteins that could modulate DAT function.Institute for Cellular and Molecular Biolog

Co-localization of LMO4 and CaMKII- α in the BLC.

<p>Top panels: Dual-channel immunofluorescence images show high levels of expression for both LMO4 (A) and CaMKII-α (A′) in the BLC. Scale bar: 200 µm. Bottom panels: strong co-localization of both markers is evident in numerous BLC neurons (arrows, B, B′, and B″), with the LMO4-postive nuclei and CaMKII-α-positive perikarya. Scale bar: 50 µm.</p

Reduction in <i>Lmo4</i> levels does not affect anxiety-like behavior.

<p>The effects of global (<i>Lmo4gt/+</i>) or adult-and brain region-specific knockdown of <i>Lmo4</i> in the BLC on anxiety-like behavior was examined. Mice were subjected to the elevated plus maze, light/dark box, or the open field test. Mean values ± SEM are depicted. N = 11–12 (WT) and 9–11 (<i>Lmo4gt/+</i>) mice and n = 9–10 for shLmo4 and 10 for ShScr injected mice.</p

<i>Lmo4gt/+</i> mice display increased neuronal activation after FC.

<p>Neuronal activation in the BLC was assessed 2 hours after FC in both WT and <i>Lmo4gt/+</i> mice by measuring c-Fos positive puncta in the BLC. Control mice were exposed to the FC chambers but not subjected to tone/shock pairings. A) Genotypic differences in c-Fos staining were not observed in the BLC under control conditions. After FC, <i>Lmo4gt/+</i> mice displayed significantly more c-Fos positive puncta than their WT counterparts. Scale bar: 200 µm. B) Quantification of results in panel A. Data are presented as mean ± SEM. *, p<0.05. n = 3–5/group.</p

Knockdown of <i>Lmo4</i> in the BLC leads to enhanced freezing to the context and the cue.

<p>A) Quantification of knockdown of <i>Lmo4</i> (RNA) expression in the BLC. <i>Lmo4</i> levels were quantified in laser captured tissue from sh<i>Lmo4</i> and shScr infected mice by QPCR. <i>Lmo4</i> levels were significantly decreased (by 46%) in sh<i>Lmo4</i> infected cells compared to the shScr control. *, p<0.05, n = 6 (shLmo4) and 10 (shscr). B) Immunohistochemical analyses revealed a robust decrease in LMO4 expression in GFP positive/infected cells (arrows point to infected neurons, right panel) from shLmo4-injected mice but not from shScr-injected mice (arrows point to infected neurons, left panel). Co-localization of LMO4 and GFP observed as yellow overlap in nuclei can be observed in shScr- but not shLmo4-infected neurons. Scale bar: 50 µm B) Knockdown of <i>Lmo4</i> expression in the BLC results in increased freezing to both the context and the cue. No differences in baseline freezing were observed between the two different shRNA's. Data are represented as mean ± SEM. n = 10–12/group. C) Left panel: Representative image of viral infection is shown. Viral infection was visualized by staining for GFP. Right panel: Serial reconstruction of injection sites is shown.</p

Tissue plasminogen activator modulates the cellular and behavioral response to cocaine

Cocaine exposure induces long-lasting molecular and structural adaptations in the brain. In this study, we show that tissue plasminogen activator (tPA), an extracellular protease involved in neuronal plasticity, modulates the biochemical and behavioral response to cocaine. When injected in the acute binge paradigm, cocaine enhanced tPA activity in the amygdala, which required activation of corticotropin-releasing factor type-1 (CRF-R1) receptors. Compared with WT mice, tPA−/− mice injected with cocaine displayed attenuated phosphorylation of ERK, cAMP response element binding protein (CREB), and dopamine and cAMP-regulated phosphoprotein 32 kDa (DARPP-32) and blunted induction of immediate early genes (IEGs) c-Fos, Egr-1, and Homer 1a in the amygdala and the nucleus accumbens (NAc). tPA−/− mice also displayed significantly higher basal preprodynorphin (ppDyn) mRNA levels in the NAc in comparison to WT mice, and cocaine decreased ppDyn mRNA levels in tPA−/− mice only. Cocaine-induced locomotor sensitization and conditioned place preference (CPP) were attenuated in tPA−/− mice. Cocaine exposure also had an anxiolytic effect in tPA−/− but not WT mice. These results identify tPA as an important and novel component of the signaling pathway that modulates cocaine-induced changes in neuroadaptation and behavior

Selective chemical genetic inhibition of protein kinase C epsilon reduces ethanol consumption in mice

Reducing expression or inhibiting translocation of protein kinase C epsilon (PKCε) prolongs ethanol intoxication and decreases ethanol consumption in mice. However, we do not know if this phenotype is due to reduced PKCε kinase activity or to impairment of kinase-independent functions. In this study, we used a chemical-genetic strategy to determine whether a potent and highly selective inhibitor of PKCε catalytic activity reduces ethanol consumption. We generated ATP analog-specific PKCε (AS-PKCε) knock-in mice harboring a point mutation in the ATP binding site of PKCε that renders the mutant kinase highly sensitive to inhibition by 1-tert-butyl-3-naphthalen-1-ylpyrazolo[3,4-d]pyrimidin-4-amine (1-NA-PP1). Systemically administered 1-NA-PP1 readily crossed the blood brain barrier and inhibited PKCε-mediated phosphorylation. 1-NA-PP1 reversibly reduced ethanol consumption by AS-PKCε mice but not by wild type mice lacking the AS-PKCε mutation. These results support the development of inhibitors of PKCε catalytic activity as a strategy to reduce ethanol consumption, and they demonstrate that the AS- PKCε mouse is a useful tool to study the role of PKCε in behavior