Cellular, circuit and transcriptional framework for modulation of itch in the central amygdala

Itch is an unpleasant sensation that elicits robust scratching and aversive experience. However, the identity of the cells and neural circuits that organize this information remains elusive. Here, we show the necessity and sufficiency of chloroquine-activated neurons in the central amygdala (CeA) for both itch sensation and associated aversion. Further, we show that chloroquine-activated CeA neurons play important roles in itch-related comorbidities, including anxiety-like behaviors, but not in some aversive and appetitive behaviors previously ascribed to CeA neurons. RNA-sequencing of chloroquine-activated CeA neurons identified several differentially expressed genes as well as potential key signaling pathways in regulating pruritis. Finally, viral tracing experiments demonstrate that these neurons send projections to the ventral periaqueductal gray that are critical in modulation of itch. These findings reveal a cellular and circuit signature of CeA neurons orchestrating behavioral and affective responses to pruritus in mice

New technologies for examining neuronal ensembles in drug addiction and fear

Correlational data suggest that learned associations are encoded within neuronal ensembles. However, it has been difficult to prove that neuronal ensembles mediate learned behaviours because traditional pharmacological and lesion methods, and even newer cell type-specific methods, affect both activated and non-activated neurons. Additionally, previous studies on synaptic and molecular alterations induced by learning did not distinguish between behaviourally activated and non-activated neurons. Here, we describe three new approaches—Daun02 inactivation, FACS sorting of activated neurons and c-fos-GFP transgenic rats — that have been used to selectively target and study activated neuronal ensembles in models of conditioned drug effects and relapse. We also describe two new tools — c-fos-tTA mice and inactivation of CREB-overexpressing neurons — that have been used to study the role of neuronal ensembles in conditioned fear

Conserved epigenomic signals in mice and humans reveal immune basis of Alzheimer’s disease

Alzheimer’s disease (AD) is a severe1 age-related neurodegenerative disorder characterized by accumulation of amyloid-β (Aβ) plaques and neurofibrillary tangles, synaptic and neuronal loss, and cognitive decline. Several genes have been implicated in AD, but chromatin state alterations during neurodegeneration remain uncharacterized. Here, we profile transcriptional and chromatin state dynamics across early and late pathology in the hippocampus of an inducible mouse model of AD-like neurodegeneration. We find a coordinated downregulation of synaptic plasticity genes and regulatory regions, and upregulation of immune response genes and regulatory regions, which are targeted by factors that belong to the ETS family of transcriptional regulators, including PU.1. Human regions orthologous to increasing-level enhancers show immune cell-specific enhancer signatures as well as immune cell expression quantitative trait loci (eQTL), while decreasing-level enhancer orthologs show fetal-brain-specific enhancer activity. Notably, AD-associated genetic variants are specifically enriched in increasing-level enhancer orthologs implicating immune processes in AD predisposition. Indeed, increasing enhancers overlap known AD loci lacking protein-altering variants and implicate additional loci that do not reach genome-wide significance. Our results reveal new insights into the mechanisms of neurodegeneration and establish the mouse as a useful model for functional studies of AD regulatory regions

Neuronal development is promoted by weakened intrinsic antioxidant defences due to epigenetic repression of Nrf2

Forebrain neurons have weak intrinsic antioxidant defences compared with astrocytes, but the molecular basis and purpose of this is poorly understood. We show that early in mouse cortical neuronal development in vitro and in vivo, expression of the master-regulator of antioxidant genes, transcription factor NF-E2-related-factor-2 (Nrf2), is repressed by epigenetic inactivation of its promoter. Consequently, in contrast to astrocytes or young neurons, maturing neurons possess negligible Nrf2-dependent antioxidant defences, and exhibit no transcriptional responses to Nrf2 activators, or to ablation of Nrf2’s inhibitor Keap1. Neuronal Nrf2 inactivation seems to be required for proper development: in maturing neurons, ectopic Nrf2 expression inhibits neurite outgrowth and aborization, and electrophysiological maturation, including synaptogenesis. These defects arise because Nrf2 activity buffers neuronal redox status, inhibiting maturation processes dependent on redox-sensitive JNK and Wnt pathways. Thus, developmental epigenetic Nrf2 repression weakens neuronal antioxidant defences but is necessary to create an environment that supports neuronal development

Identifying Molecular Neuroadaptations in Cocaine-activated Rat Striatal Neuronal Ensembles using Fluorescence Activated Cell Sorting (FACS)

Context-specific sensitization is due to a learned association between drug and stimuli in the administration environment. We had previously shown that this learned association is encoded by a pattern of sparsely distributed neurons called a neuronal ensemble. Until now, scientists have studied molecular neuroadaptations in brain homogenates without differentiating between activated neuronal ensembles and surrounding non-activated neurons. This likely obscures the changes seen only in the activated cells. Therefore, we developed a novel method for purifying activated neurons from rat striatal neuronal ensembles and assessing their unique set of cocaine-induced molecular neuroadaptations. We used c-fos-lacZ transgenic rats to identify cocaine-activated neuronal ensembles in striatum. Electrophysiological activation of these neurons induces β-galactosidase protein that can be labeled with a fluorescent antibody against β-galactosidase. We then separated these fluorescently labeled activated neurons from the majority of non-activated neurons using Fluorescence Activated Cell Sorting (FACS). Compared to non-activated neurons from the same cocaine-treated rats or all neurons from saline-treated rats, microarray analysis and quantitative PCR showed that activated neurons from cocaine-treated rats had much higher expression levels of many immediate early genes including arc, fos, fosB, and nr4a3. Because several of these genes are transcription factors, they can influence the expression of many downstream genes, and thus produce a vastly different gene expression profile in the activated neurons. The activated β-galactosidase-expressing neurons also had increased prodynorphin mRNA (a marker of D1-type neurons) and decreased dopamine D2 receptor mRNA; thus, the majority of neurons activated by cocaine are of the D1-type. Finally, the β-galactosidase-positive neurons likely have attenuated p38 MAPK signaling, because they expressed lower levels of the kinase that activates p38 (map2k6) and higher levels of the phosphatase that deactivates p38 (dusp1 or mkp1). Since p38 has been demonstrated to be involved in long term depression and to inhibit long term potentiation, these neurons may be primed for altered plasticity. We have shown that the neurons mediating context-specific locomotor sensitization to cocaine have a broadly different gene expression profile than the surrounding non-activated neurons. This represents the first multi-gene analysis of a neuronal ensemble encoding a learned behavior