E.J. Van Bockstaele

GABA-containing neurons in the ventral tegmental area project to the nucleus accumbens in rat brain.

The ventral tegmental area receives a gamma-aminobutyric acid (GABA) innervation from the nucleus accumbens and contains GABA immunoreactive neurons believed to be interneurons. We combined the immunocytochemical detection of retrogradely transported Fluoro-Gold (FG) from the nucleus accumbens (Acb) with the detection of GABA within the same section of tissue in the ventral tegmental area (VTA) of the rat brain to determine whether there might also be reciprocal GABAergic projections in the mesolimbic pathway. Immunoperoxidase labeling for FG and immunogold-silver labeling for GABA were most readily distinguished within perikarya and dendrites in sections examined by electron microscopy. Ultrastructural observations indicated that 36% (n = 110) of the FG-labeled perikarya and dendrites also contained GABA immunoreactivity. The present results provide the first evidence that GABA is contained in a subpopulation of neurons in the mesolimbic pathway from the VTA to the Acb. The reciprocity of this circuitry may provide an important feedback loop thus facilitating inhibition of motor activity.

Systemic administration of WIN 55,212-2 increases norepinephrine release in the rat frontal cortex.

Cannabinoid agonists modulate a variety of behavioral functions by activating cannabinoid receptors that are widely distributed throughout the central nervous system. In the present study, norepinephrine efflux was assessed in the frontal cortex of rats that received a systemic administration of the cannabinoid agonist, WIN 55,212-2. The synthetic cannabinoid agonist dose-dependently increased the release of norepinephrine in this brain region. Pretreatment with the cannabinoid receptor antagonist, SR 141716A, blocked the increase in norepinephrine release. To identify sites of cellular activation, immunocytochemical detection of c-Fos was combined with detection of the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH), in the brainstem nucleus locus coeruleus (LC), a region that is the sole source of norepinephrine to the frontal cortex. Systemic administration of WIN 55,212-2 significantly increased the number of c-Fos immunoreactive cells within TH-containing neurons in the LC compared to vehicle-treated rats. Pretreatment with SR 141716A inhibited the WIN 55,212-2 induced c-Fos expression, while the antagonist alone did not affect c-Fos expression. Taken together, these data indicate that systemically administered cannabinoid agonists stimulate norepinephrine release in the frontal cortex by activating noradrenergic neurons in the coeruleo-frontal cortex pathway. These effects may partially underlie changes in attention, arousal and anxiety observed following exposure to cannabis-based drugs.

The locus coeruleus: a key nucleus where stress and opioids intersect to mediate vulnerability to opiate abuse

The interaction between the stress axis and endogenous opioid systems has gained substantial clinical attention as it is increasingly recognized that stress predisposes to opiate abuse. For example, stress has been implicated as a risk factor in vulnerability to the initiation and maintenance of opiate abuse and is thought to play an important role in relapse in subjects with a history of abuse. Numerous reports indicating that stress alters individual sensitivity to opiates suggest that prior stress can influence the pharmacodynamics of opiates that are used in clinical settings. Conversely, the effects of opiates on different components of the stress axis can impact on individual responsivity to stressors and potentially predispose individuals to stress-related psychiatric disorders. One site at which opiates and stress substrates may interact to have global effects on behavior is within the locus coeruleus (LC), the major brain norepinephrine (NE)-containing nucleus. This review summarizes our current knowledge regarding the anatomical and neurochemical afferent regulation of the LC. It then presents physiological studies demonstrating opposing interactions between opioids and stress-related neuropeptides in the LC and summarizes results showing that chronic morphine exposure sensitizes the LC-NE system to corticotropin releasing factor and stress. Finally, new evidence for novel presynaptic actions of kappa-opioids on LC afferents is provided that adds another dimension to our model of how this central NE system is co-regulated by opioids and stress-related peptides.

Cannabinoid and opioid interactions: implications for opiate dependence and withdrawal

Withdrawal from opiates, such as heroin or oral narcotics, is characterized by a host of aversive physical and emotional symptoms. High rates of relapse and limited treatment success rates for opiate addiction have prompted a search for new approaches. For many opiate addicts, achieving abstinence may be further complicated by poly-drug use and co-morbid mental disorders. Research over the past decade has shed light on the influence of endocannabinoids (ECs) on the opioid system. Evidence from both animal and clinical studies point toward an interaction between these two systems, and suggest that targeting the EC system may provide novel interventions for managing opiate dependence and withdrawal. This review will summarize the literature surrounding the molecular effects of cannabinoids and opioids on the locus coeruleus-norepinephrine system, a key circuit implicated in the negative sequelae of opiate addiction. A consideration of the trends and effects of marijuana use in those seeking treatment to abstain from opiates in the clinical setting will also be presented. In summary, the present review details how cannabinoid-opioid interactions may inform novel interventions in the management of opiate dependence and withdrawal.

Repeated cannabinoid administration increases indices of noradrenergic activity in rats

The present study examined the impact of repeated administration of a synthetic cannabinoid agonist, WIN 55,212-2 on the coeruleo-cortical pathway, a circuit implicated in anxiety. Male Sprague-Dawley rats received repeated systemic injections of WIN 55,212-2 (3.0 mg/kg). A separate group of rats received repeated WIN 55,212-2 injections followed by a period of abstinence. Control animals received vehicle injections. Ninety minutes following the last injection on day 8, anxiety-related behavior was assessed using the elevated plus maze. The abstinent group was tested after another 8 days. Following behavioral testing, brain tissue was extracted from the locus coeruleus (LC) and probed for tyrosine hydroxylase (TH) expression. In a separate group of animals, in vivo microdialysis was used to monitor extracellular norepinephrine efflux in the frontal cortex following repeated WIN 55,212-2 administration and following a period of abstinence. Repeated administration of WIN 55,212-2 evoked an anxiogenic-like response that was accompanied by an increase in TH protein expression in the LC. A similar neurochemical profile was observed using in vivo microdialysis where an augmented increase in cortical norepinephrine efflux was identified in response to a systemic injection of WIN 55,212-2 on day 8. Anxiety-like behavior, catecholamine synthesizing enzyme levels and NE efflux returned to control values after 8 days of abstinence. The present findings indicate that repeated administration of a synthetic cannabinoid receptor agonist induces transient anxiety-like behaviors that correlate with increases in catecholamine synthesizing enzyme expression in the LC and augmented norepinephrine efflux in response to a challenge injection of WIN 55,212-2.

Corticotropin-releasing factor is preferentially colocalized with excitatory rather than inhibitory amino acids in axon terminals in the peri-locus coeruleus region.

Corticotropin-releasing factor(CRF)-immunoreactive terminals form synaptic specializations with locus coeruleus (LC) dendrites in rat brain. Within these terminals, CRF-immunoreactive dense core vesicles are colocalized with non-labeled dense core vesicles and clear vesicles, implicating other neuromodulators in the actions of CRF on LC neurons. Excitatory (glutamate) and inhibitory (GABA) amino acid afferents to the LC, have been identified which regulate noradrenergic responses to sensory stimuli. This study was designed to determine whether these amino acid neurotransmitters are colocalized with CRF in terminals within the LC/peri-LC region in the rat. Sections through the LC region that were dually labeled using immunohistochemical techniques to visualize either CRF and glutamate or CRF and GABA were examined using electron microscopy. Numerous terminals that contained immunolabeling for both CRF and glutamate (e.g. 30% of 106 CRF-immunoreactive terminals and 13% of 232 glutamate-immunolabeled terminals) were observed in the peri-LC. Additionally, single labeled CRF and glutamate terminals were often apposed to one another or found to converge on common dendritic targets. In contrast, relatively few terminals exhibited immunolabeling for both GABA and CRF (5% of 317 CRF-immunoreactive terminals). However, evidence for a postsynaptic effect of CRF on GABA-containing profiles included synapses between CRF axon terminals and GABA-labeled dendrites (10% of 317 CRF-labeled terminals), as well as appositions between CRF- and GABA-labeled terminals. These results indicate that CRF is preferentially colocalized with glutamate in the rostrolateral LC region and may impact on glutamate neurotransmission in the LC via presynaptic or postsynaptic actions. They argue against colocalization of CRF with GABA, although CRF may modulate GABA release via postsynaptic effects in the peri-LC region.

Dynorphin and stress related peptides in rat locus coeruleus: contribution of amygdalar efferents

The interaction between the stress axis and endogenous opioid systems has gained substantial attention, because it is increasingly recognized that stress alters individual sensitivity to opiates. One site at which opiates and stress substrates may interact to have global effects on behavior is within the locus coeruleus (LC). We have previously described interactions of several opioid peptides [e.g., proopiomelanocortin, enkephalin (ENK)] with the stress‐related peptide corticotropin‐releasing factor (CRF) in the LC. To examine further the interactions among dynorphin (DYN), ENK, and CRF in the LC, sections were processed for detection of DYN and CRF or DYN and ENK in rat brain. DYN‐ and CRF‐containing axon terminals overlapped noradrenergic dendrites in this region. Dual immunoelectron microscopy showed coexistence of DYN and CRF; 35% of axon terminals containing DYN were also immunoreactive for CRF. In contrast, few axon terminals contained both DYN and ENK. A potential DYN/CRF afferent is the central nucleus of the amygdala (CeA). Dual in situ hybridization showed that, in CeA neurons, 31% of DYN mRNA‐positive cells colocalized with CRF mRNA, whereas 53% of CRF mRNA‐containing cells colocalized with DYN mRNA. Finally, to determine whether limbic DYN afferents target the LC, the CeA was electrolytically lesioned. Light‐level densitometry of DYN labeling in the LC showed a significant decrease in immunoreactivity on the side of the lesion. Taken together, these data indicate that DYN‐ and CRF‐labeled axon terminals, most likely arising from amygdalar sources, are positioned dually to affect LC function, whereas DYN and ENK function in parallel. J. Comp. Neurol. 508:663–675, 2008.

Amygdalar peptidergic circuits regulating noradrenergic locus coeruleus neurons: linking limbic and arousal centers.

The endogenous opioid peptides, met- or leu-enkephalin, and corticotropin-releasing factor (CRF) regulate noradrenergic neurons in the locus coeruleus (LC) in a convergent manner via projections from distinct brain areas. In contrast, the opioid peptide dynorphin (DYN) has been shown to serve as a co-transmitter with CRF in afferents to the LC. To further define anatomical substrates targeting noradrenergic neurons by DYN afferents originating from limbic sources, anterograde tract-tracing of biotinylated dextran amine (BDA) from the central amygdaloid complex was combined with immunocytochemical detection of DYN and tyrosine hydroxylase (TH) in the same section of tissue. Triple labeling immunocytochemistry was combined with electron microscopy in the LC where BDA was identified using an immunoperoxidase marker, and DYN and TH were distinguished by the use of sequential immunogold labeling and silver enhancement to produce different sized gold particles. Results show direct evidence of a monosynaptic pathway linking amygdalar DYN afferents with LC neurons. To determine whether DYN-containing amygdalar LC-projecting neurons colocalize CRF, retrograde tract-tracing using fluorescent latex microspheres injected into the LC was combined with immunocytochemical detection of DYN and CRF in single sections in the central amygdala. Retrogradely labeled neurons from the LC were distributed throughout the rostro-caudal extent of the central nucleus of the amygdala (CeA) as previously described. Cell counts showed that approximately 42% of LC-projecting neurons in the CeA contained both DYN and CRF. Taken with our previous studies showing monosynaptic projections from amygdalar CRF neurons to noradrenergic LC cells, the present study extends this by showing that DYN and CRF are co-transmitters in monosynaptic projections to the LC and are poised to coordinately impact LC neuronal activity.

Local administration of a cannabinoid agonist alters norepinephrine efflux in the rat frontal cortex.

Delta(9)-tetrahydrocannabinol, the main psychoactive ingredient in marijuana, activates specific cannabinoid (CB) receptors to exert complex actions on modulatory neurotransmitters involved in attention and cognition. Previous research has demonstrated that systemic administration of the synthetic cannabinoid agonist, WIN 55,212-2, increases norepinephrine efflux in the frontal cortex. The distribution of CB1 receptors on noradrenergic fibers in the frontal cortex suggests this may be one potential site for the regulation of norepinephrine release. In the present study, we first examined the ability of a CB1 antagonist, applied locally in the frontal cortex of adult male Sprague-Dawley rats, to block the actions of systemic WIN 55,212-2. Pretreatment with SR 141716A (300 microM) significantly attenuated the excitatory effects of WIN 55,212-2 (15 mg/kg, i.p.). Next, the impact of direct perfusion of WIN 55,212-2 into the frontal cortex on extracellular norepinephrine efflux was measured. Direct application of WIN 55,212-2 (100 microM) into the frontal cortex elicited a significant increase in extracellular norepinephrine efflux suggesting that activation of cortical cannabinoid receptors contributes to alterations in norepinephrine levels in this brain region. Finally, local administration of SR 141716A followed by local administration of WIN 55,212-2 revealed a paradoxical inhibition of norepinephrine efflux.

Betaxolol, a selective β1-adrenergic receptor antagonist, diminishes anxiety-like behavior during early withdrawal from chronic cocaine administration in rats

BACKGROUND Anxiety has been indicated as one of the main symptoms of the cocaine withdrawal syndrome in human addicts and severe anxiety during withdrawal may potentially contribute to relapse. As alterations in noradrenergic transmission in limbic areas underlie withdrawal symptomatology for many drugs of abuse, the present study sought to determine the effect of cocaine withdrawal on beta-adrenergic receptor (beta(1) and beta(2)) expression in the amygdala. METHODS Male Sprague Dawley rats were administered intraperitoneal (i.p.) injections of cocaine (20 mg/kg) once daily for 14 days. Two days following the last cocaine injection, amygdala brain regions were micro-dissected and processed for Western blot analysis. Results showed that beta(1)-adrenergic receptor, but not beta(2)-adrenergic receptor expression was significantly increased in amygdala extracts of cocaine-withdrawn animals as compared to controls. This finding motivated further studies aimed at determining whether treatment with betaxolol, a highly selective beta(1)-adrenergic receptor antagonist, could ameliorate cocaine withdrawal-induced anxiety. In these studies, betaxolol (5 mg/kg via i.p. injection) was administered at 24 and then 44 h following the final chronic cocaine administration. Anxiety-like behavior was evaluated using the elevated plus maze test approximately 2 h following the last betaxolol injection. Following behavioral testing, betaxolol effects on beta(1)-adrenergic receptor protein expression were examined by Western blotting in amygdala extracts from rats undergoing cocaine withdrawal. RESULTS Animals treated with betaxolol during cocaine withdrawal exhibited a significant attenuation of anxiety-like behavior characterized by increased time spent in the open arms and increased entries into the open arms compared to animals treated with only saline during cocaine withdrawal. In contrast, betaxolol did not produce anxiolytic-like effects in control animals treated chronically with saline. Furthermore, treatment with betaxolol during early cocaine withdrawal significantly decreased beta(1)-adrenergic receptor protein expression in the amygdala to levels comparable to those of control animals. CONCLUSIONS The present findings suggest that the anxiolytic-like effect of betaxolol on cocaine-induced anxiety may be related to its effect on amygdalar beta(1)-adrenergic receptors that are up-regulated during early phases of drug withdrawal. These data support the efficacy of betaxolol as a potential effective pharmacotherapy in treating cocaine withdrawal-induced anxiety during early phases of abstinence.