Noradrenergic depletion causes sex specific alterations in the endocannabinoid system in the Murine prefrontal cortex.

Brain endocannabinoids (eCB), acting primarily via the cannabinoid type 1 receptor (CB1r), are involved in the regulation of many physiological processes, including behavioral responses to stress. A significant neural target of eCB action is the stress-responsive norepinephrine (NE) system, whose dysregulation is implicated in myriad psychiatric and neurodegenerative disorders. Using Western blot analysis, the protein expression levels of a key enzyme in the biosynthesis of the eCB 2-arachidonoylglycerol (2-AG), diacylglycerol lipase-α (DGL-α), and two eCB degrading enzymes monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH) were examined in a mouse model that lacks the NE-synthesizing enzyme, dopamine ß-hydroxylase (DßH-knockout, KO) and in rats treated with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4). In the prefrontal cortex (PFC), DGL-α protein expression was significantly increased in male and female DßH-KO mice (P < 0.05) compared to wild-type (WT) mice. DßH-KO male mice showed significant decreases in FAAH protein expression compared to WT male mice. Consistent with the DßH-KO results, DGL-α protein expression was significantly increased in male DSP-4-treated rats (P < 0.05) when compared to saline-treated controls. MGL and FAAH protein expression levels were significantly increased in male DSP-4 treated rats compared to male saline controls. Finally, we investigated the anatomical distribution of MGL and FAAH in the NE containing axon terminals of the PFC using immunoelectron microscopy. MGL was predominantly within presynaptic terminals while FAAH was localized to postsynaptic sites. These results suggest that the eCB system may be more responsive in males than females under conditions of NE perturbation, thus having potential implications for sex-specific treatment strategies of stress-related psychiatric disorders.

High resolution approaches for the identification of amyloid fragments in brain.

BACKGROUND: It is now widely recognized that endogenous, picomolar concentrations of the 42 amino acid long peptide, amyloid-ß (Aß42) is secreted under normal physiological conditions and exerts important functional activity throughout neuronal intracellular compartments. Transgenic animal models that overexpress Aß42 and its precursor, amyloid precursor protein (APP), have not provided predictive value in testing new treatments for Alzheimer's disease (AD), resulting in failed clinical trials. While these results are discouraging, they underscore the need to understand the physiological roles of Aß42 and APP under normal conditions as well as at early pre- symptomatic stages of AD. New method: We describe the use of acrolein-perfusion in immunoelectron microscopy in combination with novel antibodies directed against endogenous murine Aß42 and APP fragments to study abnormalities in the endolysosomal system at early stages of disease. The specific requirements, limitations and advantages of novel antibodies directed against human and murine Aß42, APP and APP fragments are discussed as well as parameters for ultrastructural analysis of endolysosomal compartments. RESULTS: Novel antibodies and a detailed protocol for immunoelectron microscopy using acrolein as a fixative are described. Acrolein is shown to preserve intraneuronal Aß42 species, as opposed to paraformaldehyde fixed tissue, which primarily preserves membrane bound species. Comparison with existing method(s): Technology sensitive enough to detect endogenous Aß42 under physiological conditions has not been widely available. We describe a number of novel and highly sensitive antibodies have recently been developed that may facilitate the analysis of endogenous Aß42. CONCLUSIONS: Using novel and highly specific antibodies in combination with electron microscopy may reveal important information about the timing of aberrant protein accumulation, as well as the progression of abnormalities in the endolysosomal systems that sort and clear these peptides.

Cortical adrenoceptor expression, function and adaptation under conditions of cannabinoid receptor deletion.

A neurochemical target at which cannabinoids interact to have global effects on behavior is brain noradrenergic circuitry. Acute and repeated administration of a cannabinoid receptor synthetic agonist is capable of increasing multiple indices of noradrenergic activity. This includes cannabinoid-induced 1) increases in norepinephrine (NE) release in the medial prefrontal cortex (mPFC); 2) desensitization of cortical α2-adrenoceptor-mediated effects; 3) activation of c-Fos in brainstem locus coeruleus (LC) noradrenergic neurons; and 4) increases in anxiety-like behaviors. In the present study, we sought to examine adaptations in adrenoceptor expression and function under conditions of cannabinoid receptor type 1 (CB1r) deletion using knockout (KO) mice and compare these to wild type (WT) controls. Electrophysiological analysis of α2-adrenoceptor-mediated responses in mPFC slices in WT mice showed a clonidine-induced α2-adrenoceptor-mediated increase in mPFC cell excitability coupled with an increase in input resistance. In contrast, CB1r KO mice showed an α2-adrenoceptor-mediated decrease in mPFC cell excitability. We then examined protein expression levels of α2- and ß1-adrenoceptor subtypes in the mPFC as well as TH expression in the locus coeruleus (LC) of mice deficient in CB1r. Both α2- and ß1-adrenoceptors exhibited a significant decrease in expression levels in CB1r KO mice when compared to WT in the mPFC, while a significant increase in TH was observed in the LC. To better define whether the same cortical neurons express α2A-adrenoceptor and CB1r in mPFC, we utilized high-resolution immunoelectron microscopy. We localized α2A-adrenoceptors in a knock-in mouse that expressed a hemoagglutinin (HA) tag downstream of the α2A-adrenoceptor promoter. Although the α2A-adrenoceptor was often identified pre-synaptically, we observed co-localization of CB1r with α2-adrenoceptors post-synaptically in the same mPFC neurons. Finally, using receptor binding, we confirmed prior results showing that α2A-adrenoceptor is unchanged in mPFC following acute or chronic exposure to the synthetic cannabinoid receptor agonist, WIN 55,212-2, but is increased, following chronic treatment followed by a period of abstinence. Taken together, these data provide convergent lines of evidence indicating cannabinoid regulation of the cortical adrenergic system.

Localization of the delta opioid receptor and corticotropin-releasing factor in the amygdalar complex: role in anxiety.

It is well established that central nervous system norepinephrine (NE) and corticotropin-releasing factor (CRF) systems are important mediators of behavioral responses to stressors. More recent studies have defined a role for delta opioid receptors (DOPR) in maintaining emotional valence including anxiety. The amygdala plays an important role in processing emotional stimuli, and has been implicated in the development of anxiety disorders. Activation of DOPR or inhibition of CRF in the amygdala reduces baseline and stress-induced anxiety-like responses. It is not known whether CRF- and DOPR-containing amygdalar neurons interact or whether they are regulated by NE afferents. Therefore, this study sought to better define interactions between the CRF, DOPR and NE systems in the basolateral (BLA) and central nucleus of the amygdala (CeA) of the male rat using anatomical and functional approaches. Irrespective of the amygdalar subregion, dual immunofluorescence microscopy showed that DOPR was present in CRF-containing neurons. Immunoelectron microscopy confirmed that DOPR was localized to both dendritic processes and axon terminals in the BLA and CeA. Semi-quantitative dual immunoelectron microscopy analysis of gold-silver labeling for DOPR and immunoperoxidase labeling for CRF revealed that 55 % of the CRF neurons analyzed contained DOPR in the BLA while 67 % of the CRF neurons analyzed contained DOPR in the CeA. Furthermore, approximately 41 % of DOPR-labeled axon terminals targeted BLA neurons that expressed CRF while 29 % of DOPR-labeled axon terminals targeted CeA neurons that expressed CRF. Triple label immunofluorescence microscopy revealed that DOPR and CRF were co-localized in common cellular profiles that were in close proximity to NE-containing fibers in both subregions. These anatomical results indicate significant interactions between DOPR and CRF in this critical limbic region and reveal that NE is poised to regulate these peptidergic systems in the amygdala. Functional studies were performed to determine if activation of DOPR could inhibit the anxiety produced by elevation of NE in the amygdala using the pharmacological stressor yohimbine. Administration of the DOPR agonist, SNC80, significantly attenuated elevated anxiogenic behaviors produced by yohimbine as measured in the rat on the elevated zero maze. Taken together, results from this study demonstrate the convergence of three important systems, NE, CRF, and DOPR, in the amygdala and provide insight into their functional role in modulating stress and anxiety responses.

Repeated administration of a synthetic cannabinoid receptor agonist differentially affects cortical and accumbal neuronal morphology in adolescent and adult rats.

Recent studies demonstrate a differential trajectory for cannabinoid receptor expression in cortical and sub-cortical brain areas across postnatal development. In the present study, we sought to investigate whether chronic systemic exposure to a synthetic cannabinoid receptor agonist causes morphological changes in the structure of dendrites and dendritic spines in adolescent and adult pyramidal neurons in the medial prefrontal cortex (mPFC) and medium spiny neurons (MSN) in the nucleus accumbens (Acb). Following systemic administration of WIN 55,212-2 in adolescent (PN 37-40) and adult (P55-60) male rats, the neuronal architecture of pyramidal neurons and MSN was assessed using Golgi-Cox staining. While no structural changes were observed in WIN 55,212-2-treated adolescent subjects compared to control, exposure to WIN 55,212-2 significantly increased dendritic length, spine density and the number of dendritic branches in pyramidal neurons in the mPFC of adult subjects when compared to control and adolescent subjects. In the Acb, WIN 55,212-2 exposure significantly decreased dendritic length and number of branches in adult rat subjects while no changes were observed in the adolescent groups. In contrast, spine density was significantly decreased in both the adult and adolescent groups in the Acb. To determine whether regional developmental morphological changes translated into behavioral differences, WIN 55,212-2-induced aversion was evaluated in both groups using a conditioned place preference paradigm. In adult rats, WIN 55,212-2 administration readily induced conditioned place aversion as previously described. In contrast, adolescent rats did not exhibit aversion following WIN 55,212-2 exposure in the behavioral paradigm. The present results show that synthetic cannabinoid administration differentially impacts cortical and sub-cortical neuronal morphology in adult compared to adolescent subjects. Such differences may underlie the disparate development effects of cannabinoids on behavior.

Alcohol-induced dysregulation of stress-related circuitry: The search for novel targets and implications for interventions across the sexes.

While the ability to process fermented fruits and alcohols was once an adaptive trait that improved nutrition and quality of life, the availability and prevalence of high potency alcoholic drinks has contributed to alcohol abuse disorders in a vulnerable portion of the population. Although the neural reward systems take part in the initial response to alcohol, negative reinforcement and stress, which are normally adaptive responses, can intersect to promote continued alcohol use at all stages of the addiction cycle. Eventually a point is reached where these once adaptive responses become dysregulated resulting in uncontrolled intake that constitutes a clinically important condition termed alcohol use disorder (AUD). Current research is targeted at both the behavioral and molecular adaptations in AUDs in an effort to better develop novel approaches to intervention. In this review, historical context is provided demonstrating the societal burden of alcohol use and abuse disorders. The importance of gender in the mechanism of action of alcohol is discussed. Finally, the impact of alcohol on stress-related circuitry, uncovered by preclinical research, is outlined to provide insight into potential novel pharmacological approaches to the treatment of AUD.

Ultrastructural evidence for synaptic contacts between cortical noradrenergic afferents and endocannabinoid-synthesizing post-synaptic neurons.

Endocannabinoids (eCBs) are involved in a myriad of physiological processes that are mediated through the activation of cannabinoid receptors, which are ubiquitously distributed within the nervous system. One neurochemical target at which cannabinoids interact to have global effects on behavior is brain noradrenergic circuitry. We, and others, have previously shown that CB type 1 receptors (CB1r) are positioned to pre-synaptically modulate norepinephrine (NE) release in the rat frontal cortex (FC). Diacylglycerol lipase (DGL) is a key enzyme in the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). While DGL-α is expressed in the FC in the rat brain, it is not known whether noradrenergic afferents target neurons expressing synthesizing enzymes for the endocannabinoid, 2-AG. In the present study, we employed high-resolution neuroanatomical approaches to better define cellular sites for interactions between noradrenergic afferents and FC neurons expressing DGL-α. Immunofluorescence microscopy showed close appositions between processes containing the norepinephrine transporter (NET) or dopamine-ß-hydroxylase (DßH) and cortical neurons expressing DGL-α-immunoreactivity. Ultrastructural analysis using immunogold-silver labeling for DGL-α and immunoperoxidase labeling for NET or DßH confirmed that NET-labeled axon terminals were directly apposed to FC somata and dendritic processes that exhibited DGL-α-immunoreactivity. Finally, tissue sections were processed for immunohistochemical detection of DGL-α, CB1r and DßH. Triple label immunofluorescence revealed that CB1r and DßH were co-localized in common cellular profiles and these were in close association with DGL-α. Taken together, these data provide anatomical evidence for direct synaptic associations between noradrenergic afferents and cortical neurons exhibiting endocannabinoid synthesizing machinery.

Amygdalar neuronal plasticity and the interactions of alcohol, sex, and stress.

Alcohol abuse and alcoholism are major medical problems affecting both men and women. Previous animal studies reported a difference in c-Fos neuronal activation after chronic alcohol exposure; however, females remain an understudied population. To model chronic alcohol exposure match-pair fed adult male and female rats were administered 14 days of a liquid ethanol containing diet. Analysis focused on the central nucleus of the amygdala (CeA), a region integral to stress sensitivity and substance abuse. Immunocytochemical approaches identified cells containing ΔFosB, a marker of sustained neuronal activation, and activity patterns within the CeA were mapped by subdivision and rostral-caudal extent. Significant interactions were present between all groups, with gender differences noted among control groups, and ethanol exposed animals having the greatest number of ΔFosB immunoreactive cells indicating baseline dysregulation. Compared with c-Fos, a marker of recent neuronal activation, male ethanol treated animals had similar activity to controls, indicating a neuronal habituation not seen in females. Next, a cohort of animals were exposed to the forced swim test (FST), and c-Fos was examined in addition to FST behavior. Neuronal activity was increased in ethanol exposed animals compared to controls, and control females compared to males, indicating a potentiated stress response. Further, a population of activated neurons were shown to contain either corticotropin releasing factor or enkephalin. The present data suggest that dysregulation in the CeA neuronal activity may underlie some of the negative sequelae of alcohol abuse, and may, in part, underlie the distinctive response seen between genders to alcohol use.

Chronic alcohol exposure differentially affects activation of female locus coeruleus neurons and the subcellular distribution of corticotropin releasing factor receptors.

Understanding the neurobiological bases for sex differences in alcohol dependence is needed to help guide the development of individualized therapies for alcohol abuse disorders. In the present study, alcohol-induced adaptations in (1) anxiety-like behavior, (2) patterns of c-Fos activation and (3) subcellular distribution of corticotropin releasing factor receptor in locus coeruleus (LC) neurons was investigated in male and female Sprague-Dawley rats that were chronically exposed to ethanol using a liquid diet. Results confirm and extend reports by others showing that chronic ethanol exposure produces an anxiogenic-like response in both male and female subjects. Ethanol-induced sex differences were observed with increased c-Fos expression in LC neurons of female ethanol-treated subjects compared to controls or male subjects. Results also reveal sex differences in the subcellular distribution of the CRFr in LC-noradrenergic neurons with female subjects exposed to ethanol exhibiting a higher frequency of plasmalemmal CRFrs. These adaptations have implications for LC neuronal activity and its neural targets across the sexes. Considering the important role of the LC in ethanol-induced activation of the hypothalamo-pituitary-adrenal (HPA) axis, the present results indicate important sex differences in feed-forward regulation of the HPA axis that may render alcohol dependent females more vulnerable to subsequent stress exposure.