Viktor Kharazia

RACK1 and Brain-Derived Neurotrophic Factor: A Homeostatic Pathway That Regulates Alcohol Addiction

Alcoholism is a devastating disease that manifests as uncontrolled drinking. Consumption of alcohol is regulated by neurochemical systems within specific neural circuits, but endogenous systems that may counteract and thus suppress the behavioral effects of ethanol intake are unknown. Here we demonstrate that BDNF plays a role in reducing the behavioral effects of ethanol, including consumption, in rodents. We found that decreasing the levels of BDNF leads to increased behavioral responses to ethanol, whereas increases in the levels of BDNF, mediated by the scaffolding protein RACK1, attenuate these behaviors. Interestingly, we found that acute exposure of neurons to ethanol leads to increased levels of BDNF mRNA via RACK1. Importantly, acute systemic administration of ethanol and voluntary ethanol consumption lead to increased levels of BDNF expression in the dorsal striatum. Taken together, these findings suggest that RACK1 and BDNF are part of a regulatory pathway that opposes adaptations that lead to the development of alcohol addiction.

Neurons in rat cerebral cortex that synthesize nitric oxide: NADPH diaphorase histochemistry, NOS immunocytochemistry, and colocalization with GABA

Neurons that stain for NADPH diaphorase, which colocalizes with nitric oxide synthase (NOS), are scattered uniformly across neocortex, and denser in entorhinal cortex. In the primary sensorimotor cortex, 0.5-2% of neurons contain NOS. These are most numerous in layers II-III, whereas NOS-positive fibers are concentrated in layers IV and VI. Most stained neurons are aspiny bipolar cells. Some in deep layers are multipolar; very few are pyramidal-shaped. In layer IV, NOS-positive neurons and their dendrites are confined to the septa between barrels. Retrograde tracing experiments demonstrate that NOS-positive cells are local circuit neurons. Double staining demonstrates that NOS-positive neurons also contain GABA.

Endogenous BDNF in the Dorsolateral Striatum Gates Alcohol Drinking

We previously found that brain-derived neurotrophic factor (BDNF)-haplodeficient mice exhibit greater ethanol-induced place preference and psychomotor sensitization, and greater ethanol consumption after deprivation, than control mice. We further observed that, in mice, voluntary ethanol intake increases BDNF expression in the dorsal striatum (DS). Here, we determined whether BDNF within the DS regulates ethanol self-administration in Long–Evans rats trained to self-administer a 10% ethanol solution. We observed a greater increase in BDNF expression after ethanol self-administration in the dorsolateral striatum (DLS) than in the dorsomedial striatum (DMS). We further found that downregulation of endogenous BDNF using viral-mediated siRNA in the DLS, but not in the DMS, significantly increased ethanol self-administration. Infusion of exogenous BDNF (0.25 μg/μl/side into the DMS; 0.25 and 0.75 μg/μl/side into the DLS) attenuated responding for ethanol when infused 3 h before the beginning of the self-administration session. Although the decrease in ethanol intake was similar in the DLS and DMS, BDNF infused in the DLS, but not in the DMS, induced an early termination of the drinking episode. Furthermore, the action of BDNF in the DLS was specific for ethanol, as infusion of the neurotrophic factor in the DMS, but not DLS, resulted in a reduction of sucrose intake. Together, these findings demonstrate that the BDNF pathway within the DLS controls the level of ethanol self-administration. Importantly, our results suggest that an endogenous signaling pathway within the same brain region that mediates drug-taking behavior also plays a critical role in gating the level of ethanol intake.

Neutrophil protein kinase Cδ as a mediator of stroke-reperfusion injury

Thrombolysis is widely used to intervene in acute ischemic stroke, but reestablishment of circulation may paradoxically initiate a reperfusion injury. Here we describe studies with mice lacking protein kinase Cδ (PKCδ) showing that absence of this enzyme markedly reduces reperfusion injury following transient ischemia. This was associated with reduced infiltration of peripheral blood neutrophils into infarcted tissue and with impaired neutrophil adhesion, migration, respiratory burst, and degranulation in vitro. Total body irradiation followed by transplantation with bone marrow from PKCδ-null mice donors reduced infarct size and improved neurological outcome in WT mice, whereas marrow transplantation from WT donors increased infarction and worsened neurological scores in PKCδ-null mice. These results indicate an important role for neutrophil PKCδ in reperfusion injury and strongly suggest that PKCδ inhibitors could prove useful in the treatment of stroke.

Ligand-induced down-regulation of the cannabinoid 1 receptor is mediated by the G-protein-coupled receptor-associated sorting protein GASP1

The cannabinoid 1 receptor (CB1R) is one of the most abundant seven transmembrane (7TM) spanning/G‐protein‐coupled receptors in the central nervous system and plays an important role in pain transmission, feeding, and the rewarding effects of cannabis. Tolerance to cannabinoids has been widely observed after long‐term use, with concomitant receptor desensitization and/or down‐regulation depending on the brain region studied. Several CB1R agonists promote receptor internalization after activation, but the postendocytic sorting of the receptor has not been studied in detail. Utilizing human embryonic kidney (HEK293) cells stably expressing the CB1R and primary cultured neurons expressing endogenous CB1R, we show that treatment with cannabinoid agonists results in CB1R degradation after endocytosis and that the G‐protein‐coupled receptor‐associated sorting protein GASP1 plays a major role in the postendocytic sorting process. Thus, these results may identify a molecular mechanism underlying tolerance and receptor down‐regulation after long‐term use of cannabinoids.—Martini, L., Waldhoer, M., Pusch, M., Kharazia, V., Fong, J., Lee, J. H., Freissmuth, C., Whistler, J. L. Ligand‐induced down‐regulation of the cannabinoid 1 receptor is mediated by the G‐protein‐coupled receptor‐associated sorting protein GASP1. FASEB J. 21, 802–811 (2007)

Type I nitric oxide synthase fully accounts for nadph-diaphorase in rat striatum, but not cortex

The novel gaseous neuromediator nitric oxide is thought to play an important role in development and plasticity. Despite this, gene-knockout mice lacking neuronal (Type I) nitric oxide synthase exhibit relatively normal brain development and behavior. The nervous system of these mice (especially the forebrain) retains some calcium-dependent nitric oxide synthesis, presumably reflecting other isozymes. Type I nitric oxide synthase has NADPH-dependent diaphorase activity. However, this stain also recognizes other isozymes, and it remains controversial whether all diaphorase-positive neurons contain Type I nitric oxide synthase. To assess whether neurons containing another isoform of nitric oxide synthase may be present in the forebrain of normal rodents, we studied co-localization of diaphorase staining with immunocytochemistry for Type I nitric oxide synthase. Co-localization was complete in the striatum, but some neurons deep in cortex were diaphorase-positive and immunonegative, and therefore may contain a splice variant or novel isozyme of nitric oxide synthase.

Disruption of alcohol-related memories by mTORC1 inhibition prevents relapse

Relapse to alcohol abuse is an important clinical issue that is frequently caused by cue-induced drug craving. Therefore, disruption of the memory for the cue-alcohol association is expected to prevent relapse. It is increasingly accepted that memories become labile and erasable soon after their reactivation through retrieval during a memory reconsolidation process that depends on protein synthesis. Here we show that reconsolidation of alcohol-related memories triggered by the sensory properties of alcohol itself (odor and taste) activates mammalian target of rapamycin complex 1 (mTORC1) in select amygdalar and cortical regions in rats, resulting in increased levels of several synaptic proteins. Furthermore, systemic or central amygdalar inhibition of mTORC1 during reconsolidation disrupts alcohol-associated memories, leading to a long-lasting suppression of relapse. Our findings provide evidence that the mTORC1 pathway and its downstream substrates are crucial in alcohol-related memory reconsolidation and highlight this pathway as a therapeutic target to prevent relapse.

Tangential synaptic distribution of NMDA and AMPA receptors in rat neocortex

We performed an electron microscopic study in layers II-III of S-1 in rats, using postembedding immunogold histochemistry to compare the synaptic distribution of N-methyl D-aspartate (NMDA) receptors (assessed with an antibody for the NMDAR1 subunit) with that of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors (assessed with an antibody for the GluR2/3 subunit). Labeling for each receptor was concentrated at active zones of asymmetric synapses. Analysis of the tangential position of gold particles along the postsynaptic active zone revealed that NMDA receptors were at highest concentration in the middle of the synaptic apposition, whereas AMPA receptors were concentrated in an annulus away from its center. These data support the view that the two types of receptors are anchored by distinct subsynaptic assemblies, and raise the possibility of independent synaptic microdomains.

EM colocalization of AMPA and NMDA receptor subunits at synapses in rat cerebral cortex

Electrophysiology and light microscopy suggest that a single excitatory synapse may use both amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors. Using immunogold electron microscopy, we here provide direct evidence for colocalization at individual synapses in sensorimotor cortex of adult rats. Colocalization was most commonly observed on dendritic spines; subunits of the two classes of receptors seemed to be independently distributed within the synaptic active zone.

Acute cocaine exposure alters spine density and long-term potentiation in the ventral tegmental area

Growing evidence indicates that the expression of synaptic plasticity in the central nervous system results in dendritic reorganization and spine remodeling. Although long‐term potentiation of glutamatergic synapses after cocaine exposure in the ventral tegmental area (VTA) has been proposed as a cellular mechanism underlying addictive behaviors, the relationship between long‐term potentiation and dendritic remodeling induced by cocaine on the dopaminergic neurons of the VTA has not been demonstrated. Here we report that rat VTA cells classified as type I and II showed distinct morphological responses to cocaine, as a single cocaine exposure significantly increased dendritic spine density in type I but not in type II cells. Further, only type I cells had a significant increase in the AMPA receptor : NMDA receptor ratio after a single cocaine exposure. Taken together, our data provide evidence that increased spine density and synaptic plasticity are coexpressed within the same VTA neuronal population and that only type I neurons are structurally and synaptically modified by cocaine.