BK ZERO isoform HEK293 stably transfected cell lines differing 3'UTRs to assess miR-9 regulation.

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.

Binge Alcohol Drinking Alters Synaptic Processing of Executive and Emotional Information in Core Nucleus Accumbens Medium Spiny Neurons.

The nucleus accumbens (NAc) is a forebrain region mediating the positive-reinforcing properties of drugs of abuse, including alcohol. It receives glutamatergic projections from multiple forebrain and limbic regions such as the prefrontal cortex (PFCx) and basolateral amygdala (BLA), respectively. However, it is unknown how NAc medium spiny neurons (MSNs) integrate PFCx and BLA inputs, and how this integration is affected by alcohol exposure. Because progress has been hampered by the inability to independently stimulate different pathways, we implemented a dual wavelength optogenetic approach to selectively and independently stimulate PFCx and BLA NAc inputs within the same brain slice. This approach functionally demonstrates that PFCx and BLA inputs synapse onto the same MSNs where they reciprocally inhibit each other pre-synaptically in a strict time-dependent manner. In alcohol-naïve mice, this temporal gating of BLA-inputs by PFCx afferents is stronger than the reverse, revealing that MSNs prioritize high-order executive processes information from the PFCx. Importantly, binge alcohol drinking alters this reciprocal inhibition by unilaterally strengthening BLA inhibition of PFCx inputs. In line with this observation, we demonstrate that in vivo optogenetic stimulation of the BLA, but not PFCx, blocks binge alcohol drinking escalation in mice. Overall, our results identify NAc MSNs as a key integrator of executive and emotional information and show that this integration is dysregulated during binge alcohol drinking.

Voltage-induced Ca2+ release by ryanodine receptors causes neuropeptide secretion from nerve terminals.

Depolarisation-secretion coupling is assumed to be dependent only on extracellular calcium ([Ca2+ ]o ). Ryanodine receptor (RyR)-sensitive stores in hypothalamic neurohypophysial system (HNS) terminals produce sparks of intracellular calcium ([Ca2+ ]i ) that are voltage-dependent. We hypothesised that voltage-elicited increases in intraterminal calcium are crucial for neuropeptide secretion from presynaptic terminals, whether from influx through voltage-gated calcium channels and/or from such voltage-sensitive ryanodine-mediated calcium stores. Increases in [Ca2+ ]i upon depolarisation in the presence of voltage-gated calcium channel blockers, or in the absence of [Ca2+ ]o , still give rise to neuropeptide secretion from HNS terminals. Even in 0 [Ca2+ ]o , there was nonetheless an increase in capacitance suggesting exocytosis upon depolarisation. This was blocked by antagonist concentrations of ryanodine, as was peptide secretion elicited by high K+ in 0 [Ca2+ ]o . Furthermore, such depolarisations lead to increases in [Ca2+ ]i . Pre-incubation with BAPTA-AM resulted in > 50% inhibition of peptide secretion elicited by high K+ in 0 [Ca2+ ]o . Nifedipine but not nicardipine inhibited both the high K+ response for neuropeptide secretion and intraterminal calcium, suggesting the involvement of CaV1.1 type channels as sensors in voltage-induced calcium release. Importantly, RyR antagonists also modulate neuropeptide release under normal physiological conditions. In conclusion, our results indicate that depolarisation-induced neuropeptide secretion is present in the absence of external calcium, and calcium release from ryanodine-sensitive internal stores is a significant physiological contributor to neuropeptide secretion from HNS terminals.

Alcohol Regulates BK Surface Expression via Wnt/ß-Catenin Signaling.

It has been suggested that drug tolerance represents a form of learning and memory, but this has not been experimentally established at the molecular level. We show that a component of alcohol molecular tolerance (channel internalization) from rat hippocampal neurons requires protein synthesis, in common with other forms of learning and memory. We identify ß-catenin as a primary necessary protein. Alcohol increases ß-catenin, and blocking accumulation of ß-catenin blocks alcohol-induced internalization in these neurons. In transfected HEK293 cells, suppression of Wnt/ß-catenin signaling blocks ethanol-induced internalization. Conversely, activation of Wnt/ß-catenin reduces BK current density. A point mutation in a putative glycogen synthase kinase phosophorylation site within the S10 region of BK blocks internalization, suggesting that Wnt/ß-catenin directly regulates alcohol-induced BK internalization via glycogen synthase kinase phosphorylation. These findings establish de novo protein synthesis and Wnt/ß-catenin signaling as critical in mediating a persistent form of BK molecular alcohol tolerance establishing a commonality with other forms of long-term plasticity. SIGNIFICANCE STATEMENT: Alcohol tolerance is a key step toward escalating alcohol consumption and subsequent dependence. Our research aims to make significant contributions toward novel, therapeutic approaches to prevent and treat alcohol misuse by understanding the molecular mechanisms of alcohol tolerance. In our current study, we identify the role of a key regulatory pathway in alcohol-induced persistent molecular changes within the hippocampus. The canonical Wnt/ß-catenin pathway regulates BK channel surface expression in a protein synthesis-dependent manner reminiscent of other forms of long-term hippocampal neuronal adaptations. This unique insight opens the possibility of using clinically tested drugs, targeting the Wnt/ß-catenin pathway, for the novel use of preventing and treating alcohol dependency.

Time-Dependent Effects of Ethanol on BK Channel Expression and Trafficking in Hippocampal Neurons.

BACKGROUND: The large conductance Ca(2+) - and voltage-activated K(+) channel (BK) is an important player in molecular and behavioral alcohol tolerance. Trafficking and surface expression of ion channels contribute to the development of addictive behaviors. We have previously reported that internalization of the BK channel is a component of molecular tolerance to ethanol (EtOH). METHODS: Using primary cultures of hippocampal neurons, we combine total internal reflection fluorescence microscopy, electrophysiology, and biochemical techniques to explore how exposure to EtOH affects the expression and subcellular localization of endogenous BK channels over time. RESULTS: Exposure to EtOH changed the expression of endogenous BK channels in a time-dependent manner at the perimembrane area (plasma membrane and/or the area adjacent to it), while total protein levels of BK remain unchanged. These results suggest a redistribution of the channel within the neurons rather than changes in synthesis or degradation rates. Our results showed a temporally nonlinear effect of EtOH on perimembrane expression of BK. First, there was an increase in BK perimembrane expression after 10 minutes of EtOH exposure that remained evident after 3 hours, although not correlated to increases in functional channel expression. In contrast, after 6 hours of EtOH exposure, we observed a significant decrease in both BK perimembrane expression and functional channel expression. Furthermore, after 24 hours of EtOH exposure, perimembrane levels of BK had returned to baseline. CONCLUSIONS: We report a complex time-dependent pattern in the effect of EtOH on BK channel trafficking, including successive increases and decreases in perimembrane expression and a reduction in active BK channels after 3 and 6 hours of EtOH exposure. Possible mechanisms underlying this multiphasic trafficking are discussed. As molecular tolerance necessarily underlies behavioral tolerance, the time-dependent alterations we see at the level of the channel may be relevant to the influence of drinking patterns on the development of behavioral tolerance.

Lipids modulate the increase of BK channel calcium sensitivity by the ß1 subunit.

Co-expression of the auxiliary ß1 subunit with the pore forming α subunit of BK dramatically alters apparent calcium sensitivity. Investigation of the mechanism underlying the increase in calcium sensitivity of BK in smooth muscle has concentrated on the energetic effect of ß1's interaction with α. We take a novel approach, exploring whether ß1 modification of calcium sensitivity reflects altered interaction between the channel protein and surrounding lipids. We reconstituted hSlo BK α and BK α+ß1 channels into two sets of bilayers. One set contained POPE with POPS, POPG, POPA and POPC, where the length of acyl chains is constant, but surface charge differs. The second set is a series of neutral bilayers formed from DOPE with phosphatidylcholines (PCs) of varying acyl chain lengths: C (14:1), C (18:1), C (22:1) and C (24:1), and with brain sphingomyelin (SPM), in which surface charge is constant, but bilayer thickness varies. The increase in calcium sensitivity caused by the ß1 subunit was preserved in negatively charged lipid bilayers but not in neutral bilayers, indicating that modification of apparent Ca(2+) sensitivity by ß1 is modulated by membrane lipids, requiring negatively charged lipids in the membrane. Moreover, the presence of ß1 reduces BK activity in thin bilayers of PC 14:1 and thick bilayers containing SPM, but has no significant effect on activity of BK in PC 18:1, PC 22:1 and PC 24:1 bilayers. These data suggest that auxiliary ß1 subunits fine-tune channel gating not only through direct subunit-subunit interactions but also by modulating lipid-protein interactions.

Large conductance voltage- and Ca2+-gated potassium (BK) channel ß4 subunit influences sensitivity and tolerance to alcohol by altering its response to kinases.

Tolerance is a well described component of alcohol abuse and addiction. The large conductance voltage- and Ca(2+)-gated potassium channel (BK) has been very useful for studying molecular tolerance. The influence of association with the ß4 subunit can be observed at the level of individual channels, action potentials in brain slices, and finally, drinking behavior in the mouse. Previously, we showed that 50 mm alcohol increases both α and αß4 BK channel open probability, but only α BK develops acute tolerance to this effect. Currently, we explore the possibility that the influence of the ß4 subunit on tolerance may result from a striking effect of ß4 on kinase modulation of the BK channel. We examine the influence of the ß4 subunit on PKA, CaMKII, and phosphatase modulation of channel activity, and on molecular tolerance to alcohol. We record from human BK channels heterologously expressed in HEK 293 cells composed of its core subunit, α alone (Insertless), or co-expressed with the ß4 BK auxiliary subunit, as well as, acutely dissociated nucleus accumbens neurons using the cell-attached patch clamp configuration. Our results indicate that BK channels are strongly modulated by activation of specific kinases (PKA and CaMKII) and phosphatases. The presence of the ß4 subunit greatly influences this modulation, allowing a variety of outcomes for BK channel activity in response to acute alcohol.

µ-Opioid inhibition of Ca2+ currents and secretion in isolated terminals of the neurohypophysis occurs via ryanodine-sensitive Ca2+ stores.

µ-Opioid agonists have no effect on calcium currents (I(Ca)) in neurohypophysial terminals when recorded using the classic whole-cell patch-clamp configuration. However, µ-opioid receptor (MOR)-mediated inhibition of I(Ca) is reliably demonstrated using the perforated-patch configuration. This suggests that the MOR-signaling pathway is sensitive to intraterminal dialysis and is therefore mediated by a readily diffusible second messenger. Using the perforated patch-clamp technique and ratio-calcium-imaging methods, we describe a diffusible second messenger pathway stimulated by the MOR that inhibits voltage-gated calcium channels in isolated terminals from the rat neurohypophysis (NH). Our results show a rise in basal intracellular calcium ([Ca(2+)]i) in response to application of [D-Ala(2)-N-Me-Phe(4),Gly5-ol]-Enkephalin (DAMGO), a MOR agonist, that is blocked by D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), a MOR antagonist. Buffering DAMGO-induced changes in [Ca(2+)]i with BAPTA-AM completely blocked the inhibition of both I(Ca) and high-K(+)-induced rises in [Ca(2+)]i due to MOR activation, but had no effect on κ-opioid receptor (KOR)-mediated inhibition. Given the presence of ryanodine-sensitive stores in isolated terminals, we tested 8-bromo-cyclic adenosine diphosphate ribose (8Br-cADPr), a competitive inhibitor of cyclic ADP-ribose (cADPr) signaling that partially relieves DAMGO inhibition of I(Ca) and completely relieves MOR-mediated inhibition of high-K(+)-induced and DAMGO-induced rises in [Ca(2+)]i. Furthermore, antagonist concentrations of ryanodine completely blocked MOR-induced increases in [Ca(2+)]i and inhibition of I(Ca) and high-K(+)-induced rises in [Ca(2+)]i while not affecting KOR-mediated inhibition. Antagonist concentrations of ryanodine also blocked MOR-mediated inhibition of electrically-evoked increases in capacitance. These results strongly suggest that a key diffusible second messenger mediating the MOR-signaling pathway in NH terminals is [Ca(2+)]i released by cADPr from ryanodine-sensitive stores.

Modulation/physiology of calcium channel sub-types in neurosecretory terminals.

The hypothalamic-neurohypophysial system (HNS) controls diuresis and parturition through the release of arginine-vasopressin (AVP) and oxytocin (OT). These neuropeptides are chiefly synthesized in hypothalamic magnocellular somata in the supraoptic and paraventricular nuclei and are released into the blood stream from terminals in the neurohypophysis. These HNS neurons develop specific electrical activity (bursts) in response to various physiological stimuli. The release of AVP and OT at the level of neurohypophysis is directly linked not only to their different burst patterns, but is also regulated by the activity of a number of voltage-dependent channels present in the HNS nerve terminals and by feedback modulators. We found that there is a different complement of voltage-gated Ca(2+) channels (VGCC) in the two types of HNS terminals: L, N, and Q in vasopressinergic terminals vs. L, N, and R in oxytocinergic terminals. These channels, however, do not have sufficiently distinct properties to explain the differences in release efficacy of the specific burst patterns. However, feedback by both opioids and ATP specifically modulate different types of VGCC and hence the amount of AVP and/or OT being released. Opioid receptors have been identified in both AVP and OT terminals. In OT terminals, µ-receptor agonists inhibit all VGCC (particularly R-type), whereas, they induce a limited block of L-, and P/Q-type channels, coupled to an unusual potentiation of the N-type Ca(2+) current in the AVP terminals. In contrast, the N-type Ca(2+) current can be inhibited by adenosine via A(1) receptors leading to the decreased release of both AVP and OT. Furthermore, ATP evokes an inactivating Ca(2+)/Na(+)-current in HNS terminals able to potentiate AVP release through the activation of P2X2, P2X3, P2X4 and P2X7 receptors. In OT terminals, however, only the latter receptor type is probably present. We conclude by proposing a model that can explain how purinergic and/or opioid feedback modulation during bursts can mediate differences in the control of neurohypophysial AVP vs. OT release.

The relationship between duration of initial alcohol exposure and persistence of molecular tolerance is markedly nonlinear.

The neuronal calcium- and voltage-activated BK potassium channel is modulated by ethanol, and plays a role in behavioral tolerance in vertebrates and invertebrates. We examine the influence of temporal parameters of alcohol exposure on the characteristics of BK molecular tolerance in the ventral striatum, an important component of brain reward circuitry. BK channels in striatal neurons of C57BL/6J mice exhibited molecular tolerance whose duration was a function of exposure time. After 6 h exposure to 20 mm (0.09 mg%) ethanol, alcohol sensitivity was suppressed beyond 24 h after withdrawal, while after a 1 or 3 h exposure, sensitivity had significantly recovered after 4 h. This temporally controlled transition to persistent molecular tolerance parallels changes in BK channel isoform profile. After withdrawal from 6 h, but not 3 h alcohol exposure, mRNA levels of the alcohol-insensitive STREX (stress axis-regulated exon) splice variant were increased. Moreover, the biophysical properties of BK channels during withdrawal from 6 h exposure were altered, and match the properties of STREX channels exogenously expressed in HEK 293 cells. Our results suggest a temporally triggered shift in BK isoform identity. Once activated, the transition does not require the continued presence of alcohol. We next determined whether the results obtained using cultured striatal neurons could be observed in acutely dissociated striatal neurons, after alcohol administration in the living mouse. The results were in remarkable agreement with the striatal culture data, showing persistent molecular tolerance after injections producing 6 h of intoxication, but not after injections producing only 3 h of intoxication.

Differential modulation of N-type calcium channels by micro-opioid receptors in oxytocinergic versus vasopressinergic neurohypophysial terminals.

Opioids modulate the electrical activity of magnocellular neurons (MCN) and inhibit neuropeptide release at their terminals in the neurohypophysis. We have previously shown that micro-opioid receptor (MOR) activation induces a stronger inhibition of oxytocin (OT) than vasopressin (AVP) release from isolated MCN terminals. This higher sensitivity of OT release is due, at least in part, to the selective targeting of R-type calcium channels. We now describe the underlying basis for AVP's weaker inhibition by MOR activation and provide a more complete explanation of the complicated effects on neuropeptide release. We found that N-type calcium channels in AVP terminals are differentially modulated by MOR; enhanced at lower concentrations but increasingly inhibited at higher concentrations of agonists. On the other hand, N-type calcium channels in OT terminals were always inhibited. The response pattern in co-labeled terminals was analogous to that observed in AVP-containing terminals. Changes in intracellular calcium concentration and neuropeptide release corroborated these results as they showed a similar pattern of enhancement and inhibition in AVP terminals contrasting with solely inhibitory responses in OT terminals to MOR agonists. We established that fast translocation of Ca(2+) channels to the plasma membrane was not mediating current increments and thus, changes in channel kinetic properties are most likely involved. Finally, we reveal a distinct Ca-channel beta-subunit expression between each type of nerve endings that could explain some of the differences in responses to MOR activation. These results help advance our understanding of the complex modulatory mechanisms utilized by MORs in regulating presynaptic neuropeptide release.

Voltage-dependent kappa-opioid modulation of action potential waveform-elicited calcium currents in neurohypophysial terminals.

Release of neurotransmitter is activated by the influx of calcium. Inhibition of Ca(2+) channels results in less calcium influx into the terminals and presumably a reduction in transmitter release. In the neurohypophysis (NH), Ca(2+) channel kinetics, and the associated Ca(2+) influx, is primarily controlled by membrane voltage and can be modulated, in a voltage-dependent manner, by G-protein subunits interacting with voltage-gated calcium channels (VGCCs). In this series of experiments we test whether the kappa- and micro-opioid inhibition of Ca(2+) currents in NH terminals is voltage-dependent. Voltage-dependent relief of G-protein inhibition of VGCC can be achieved with either a depolarizing square pre-pulse or by action potential waveforms. Both protocols were tested in the presence and absence of opioid agonists targeting the kappa- and micro-receptors in neurohypophysial terminals. The kappa-opioid VGCC inhibition is relieved by such pre-pulses, suggesting that this receptor is involved in a voltage-dependent membrane delimited pathway. In contrast, micro-opioid inhibition of VGCC is not relieved by such pre-pulses, indicating a voltage-independent diffusible second-messenger signaling pathway. Furthermore, relief of kappa-opioid inhibition during a physiologic action potential (AP) burst stimulation indicates the possibility of activity-dependent modulation in vivo. Differences in the facilitation of Ca(2+) channels due to specific G-protein modulation during a burst of APs may contribute to the fine-tuning of Ca(2+)-dependent neuropeptide release in other CNS terminals, as well.

ATP elicits inward currents in isolated vasopressinergic neurohypophysial terminals via P2X2 and P2X3 receptors.

Effects of extracellular adenosine tri-phosphate (ATP) on ionic currents were investigated using the perforated-patch whole-cell recording technique on isolated terminals of the Hypothalamic Neurohypophysial System (HNS). ATP induced a current response in 70% of these isolated terminals. This inwardly-rectifying, inactivating current had an apparent reversal near 0 mV and was dose-dependent on ATP with an EC50=9.6+/-1.0 microM. In addition, current amplitudes measured at maximal ATP concentrations and optimum holding potentials had a current density of 70.8 pA pF(-1) and were greatly inhibited by suramin and PPADS. Different purinergic receptor agonists were tested, with the following efficacy: ATP > or = 2-methylthioATP > ATP-gamma-S > Bz-Bz-ATP > alpha,beta-methylene-ATP > beta,gamma-methylene-ATP. However, UTP and ADP were ineffective. These data suggest the involvement of a P2X purinergic receptor in the ATP-induced responses. Immunocytochemical labeling in vasopressinergic terminals indicates the existence of P2X(2,3,4, and 7), but not P2X6 receptors. Additionally, P2X(2 and 3) were not found in terminals which labeled for oxytocin. In summary, the EC50, decay, inactivation, and pharmacology indicate that a functional mixture of P2X(2 and 3) homomeric receptors mediate the majority of the ATP responses in vasopressinergic HNS terminals. We speculate that the characteristics of these types of receptors reflect the function of co-released ATP in the terminal compartment of these and other CNS neurons.

Ca2+ syntillas, miniature Ca2+ release events in terminals of hypothalamic neurons, are increased in frequency by depolarization in the absence of Ca2+ influx.

Localized, brief Ca2+ transients (Ca2+ syntillas) caused by release from intracellular stores were found in isolated nerve terminals from magnocellular hypothalamic neurons and examined quantitatively using a signal mass approach to Ca2+ imaging. Ca2+ syntillas (scintilla, L., spark, from a synaptic structure, a nerve terminal) are caused by release of approximately 250,000 Ca ions on average by a Ca2+ flux lasting on the order of tens of milliseconds and occur spontaneously at a membrane potential of -80 mV. Syntillas are unaffected by removal of extracellular Ca2+, are mediated by ryanodine receptors (RyRs) and are increased in frequency, in the absence of extracellular Ca2+, by physiological levels of depolarization. This represents the first direct demonstration of mobilization of Ca2+ from intracellular stores in neurons by depolarization without Ca2+ influx. The regulation of syntillas by depolarization provides a new link between neuronal activity and cytosolic [Ca2+] in nerve terminals.