RESUMO
Research has identified the large conductance voltage- and calcium-activated potassium channel (BK) as a key regulator of neuronal excitability genetically associated to behavioral alcohol tolerance. Sensitivity to ethanol at the molecular level is characterized by acute potentiation of channel activity. BK isoforms show variations in alcohol sensitivity and are differentially distributed on the plasma membrane surface in response to prolonged exposure. MicroRNA (MiRNA) targeting of alcohol-sensitive isoforms coupled with active internalization of BK channels in response to ethanol are believed to be key in establishing homeostatic adaptations that produce persistent changes within the plasma membrane of neurons. In fact, microRNA 9 (miR-9) upregulated expression is a key event in persistent alcohol tolerance mediating acute EtOH desensitization of BK channels. The exact nature of these interactions remains a current topic of discussion. To further study the effects of miR-9 on the expression and distribution of BK channel isoforms we designed an experimental model by transfecting human BK channel isoforms ZERO heterologous constructs in human embryonic kidney cells 293 (HEK293) cells respectively expressing 2.1 (miR-9 responsive), 2.2 (unresponsive) and control (no sequence) 3'untranslated region (3'UTR) miRNA recognition sites. We used imaging techniques to characterize the stably transfected monoclonal cell lines, and electrophysiology to validate channel activity. Finally, we used immunocytochemistry to validate isoform responsiveness to miR-9. Our findings suggest the cell lines were successfully transfected to express either the 2.1 or 2.2 version of ZERO. Patch clamp recordings confirm that these channels retain their functionality and immunohistochemistry shows differential responses to miR-9, making these cells viable for use in future alcohol dependence studies.
Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Alta , MicroRNAs , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Regiões 3' não Traduzidas/genética , Células HEK293 , Etanol/farmacologia , MicroRNAs/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Rim/metabolismo , Cálcio/metabolismoRESUMO
BACKGROUND: Recent research in humans and rodents has explored the use of deep brain stimulation (DBS) of the ventral capsule/ventral striatum (VS) as a possible treatment for drug addiction. However, the optimum electrode placement and optimum DBS parameters have not been thoroughly studied. Here we varied stimulation sites and frequencies to determine whether DBS of the VS could facilitate the extinction of morphine-induced conditioned place preference in rats. METHODS: Rats were implanted with DBS electrodes in the dorsal or ventral subregions of the VS and trained to the morphine conditioned place preference. Subsequently, rats received extinction sessions over 9 days, combined with 60 min of either high- (130 Hz) or low- (20 Hz) frequency DBS. To study circuit-wide activations after DBS of the VS, c-fos immunohistochemistry was performed in regions involved in the extinction of drug-seeking behaviors. RESULTS: High-frequency DBS of the dorsal-VS impaired both extinction training and extinction memory, whereas high-frequency DBS of the ventral-VS had no effect. In contrast, low-frequency DBS of the dorsal-VS strengthened extinction memory when tested 2 or 9 days after the cessation of stimulation. Both DBS frequencies increased c-fos expression in the infralimbic prefrontal cortex, but only low-frequency DBS increased c-fos expression in the basal amygdala and the medial portion of the central amygdala. CONCLUSIONS: Our results suggest that low-frequency (rather than high-frequency) DBS of the dorsal-VS strengthens extinction memory and may be a potential adjunct for extinction-based therapies for treatment-refractory opioid addiction.