Commonly used anesthetics modify alcohol and (-)-trans-delta9-tetrahydrocannabinol in vivo effects on rat cerebral arterioles.

BACKGROUND: Ethyl alcohol and cannabis are widely used recreational substances with distinct effects on the brain. These drugs increase accidental injuries requiring treatment under anesthesia. Moreover, alcohol and cannabis are often used in anesthetized rodents for biomedical research. Here, we compared the influence of commonly used forms of anesthesia, injectable ketamine/xylazine (KX) versus inhalant isoflurane, on alcohol- and (-)-trans-delta9-tetrahydrocannabinol (THC) effects on cerebral arteriole diameter evaluated in vivo. METHODS: Studies were performed on male and female Sprague-Dawley rats subjected to intracarotid catheter placement for drug infusion, and cranial window surgery for monitoring pial arteriole diameter. Depth of anesthesia was monitored every 10-15 min by toe-pinch. Under KX, the number of toe-pinch responders was maximal after the first dose of anesthesia and diminished over time in both males and females. In contrast, the number of toe-pinch responders under isoflurane slowly raised over time, leading to increase in isoflurane percentage until deep anesthesia was re-established. Rectal temperature under KX remained stable in males while dropping in females. As expected for gaseous anesthesia, both males and females exhibited rectal temperature drops under isoflurane. RESULTS: Infusion of 50 mM alcohol (ethanol, EtOH) into the cerebral circulation rendered robust constriction in males under KX anesthesia, this alcohol action being significantly smaller, but still present under isoflurane anesthesia. In females, EtOH did not cause measurable changes in pial arteriole diameter regardless of the anesthetic. These findings indicate a strong sex bias with regards to EtOH induced vasoconstriction. Infusion of 42 nM THC in males and females under isoflurane tended to constrict cerebral arterioles in both males and females when compared to isovolumic infusion of THC vehicle (dimethyl sulfoxide in saline). Moreover, THC-driven changes in arteriole diameter significantly differed in magnitude depending on the anesthetic used. Simultaneous administration of 50 mM alcohol and 42 nM THC to males constricted cerebral arterioles regardless of the anesthetic used. In females, constriction by the combined drugs was also observed, with limited influence by anesthetic presence. CONCLUSIONS: We demonstrate that two commonly used anesthetic formulations differentially influence the level of vasoconstriction caused by alcohol and THC actions in cerebral arterioles.

Dual-color miniscope imaging of microvessels and neuronal activity in the hippocampus CA1 region of freely moving mice following alcohol administration.

Moderate-to-heavy episodic ("binge") drinking is the most common form of alcohol consumption in the United States. Alcohol at binge drinking concentrations reduces brain artery diameter in vivo and in vitro in many species including rats, mice, and humans. Despite the critical role played by brain vessels in maintaining neuronal function, there is a shortage of methodologies to simultaneously assess neuron and blood vessel function in deep brain regions. Here, we investigate cerebrovascular responses to ethanol by choosing a deep brain region that is implicated in alcohol disruption of brain function, the hippocampal CA1, and describe the process for obtaining simultaneous imaging of pyramidal neuron activity and diameter of nearby microvessels in freely moving mice via a dual-color miniscope. Recordings of neurovascular events were performed upon intraperitoneal injection of saline versus 3 g/kg ethanol in the same mouse. In male mice, ethanol mildly increased the amplitude of calcium signals while robustly decreasing their frequency. Simultaneously, ethanol decreased microvessel diameter. In females, ethanol did not change the amplitude or frequency of calcium signals from CA1 neurons but decreased microvessel diameter. A linear regression of ethanol-induced reduction in number of active neurons and microvessel constriction revealed a positive correlation (R = 0.981) in females. Together, these data demonstrate the feasibility of simultaneously evaluating neuronal and vascular components of alcohol actions in a deep brain area in freely moving mice, as well as the sexual dimorphism of hippocampal neurovascular responses to alcohol.

Differential Functional Contribution of BK Channel Subunits to Aldosterone-Induced Channel Activation in Vascular Smooth Muscle and Eventual Cerebral Artery Dilation.

Calcium/voltage-activated potassium channels (BK) control smooth muscle (SM) tone and cerebral artery diameter. They include channel-forming α and regulatory ß1 subunits, the latter being highly expressed in SM. Both subunits participate in steroid-induced modification of BK activity: ß1 provides recognition for estradiol and cholanes, resulting in BK potentiation, whereas α suffices for BK inhibition by cholesterol or pregnenolone. Aldosterone can modify cerebral artery function independently of its effects outside the brain, yet BK involvement in aldosterone's cerebrovascular action and identification of channel subunits, possibly involved in steroid action, remains uninvestigated. Using microscale thermophoresis, we demonstrated that each subunit type presents two recognition sites for aldosterone: at 0.3 and ≥10 µM for α and at 0.3-1 µM and ≥100 µM for ß1. Next, we probed aldosterone on SM BK activity and diameter of middle cerebral artery (MCA) isolated from ß1-/- vs. wt mice. Data showed that ß1 leftward-shifted aldosterone-induced BK activation, rendering EC50~3 µM and ECMAX ≥ 10 µM, at which BK activity increased by 20%. At similar concentrations, aldosterone mildly yet significantly dilated MCA independently of circulating and endothelial factors. Lastly, aldosterone-induced MCA dilation was lost in ß1-/- mice. Therefore, ß1 enables BK activation and MCA dilation by low µM aldosterone.

Interspecies and regional variability of alcohol action on large cerebral arteries: regulation by KCNMB1 proteins.

Alcohol intake leading to blood ethanol concentrations (BEC) ≥ legal intoxication modifies brain blood flow with increases in some regions and decreases in others. Brain regions receive blood from the Willis' circle branches: anterior, middle (MCA) and posterior cerebral (PCA), and basilar (BA) arteries. Rats and mice have been used to identify the targets mediating ethanol-induced effects on cerebral arteries, with conclusions being freely interchanged, albeit data were obtained in different species/arterial branches. We tested whether ethanol action on cerebral arteries differed between male rat and mouse and/or across different brain regions and identified the targets of alcohol action. In both species and all Willis' circle branches, ethanol evoked reversible and concentration-dependent constriction (EC50s ≈ 37-86 mM; below lethal BEC in alcohol-naïve humans). Although showing similar constriction to depolarization, both species displayed differential responses to ethanol: in mice, MCA constriction was highly sensitive to the presence/absence of the endothelium, whereas in rat PCA was significantly more sensitive to ethanol than its mouse counterpart. In the rat, but not the mouse, BA was more ethanol sensitive than other branches. Both interspecies and regional variability were ameliorated by endothelium. Selective large conductance (BK) channel block in de-endothelialized vessels demonstrated that these channels were the effectors of alcohol-induced cerebral artery constriction across regions and species. Variabilities in alcohol actions did not fully matched KCNMB1 expression across vessels. However, immunofluorescence data from KCNMB1-/- mouse arteries electroporated with KCNMB1-coding cDNA demonstrate that KCNMB1 proteins, which regulate smooth muscle (SM) BK channel function and vasodilation, regulate interspecies and regional variability of brain artery responses to alcohol.

Cerebrovascular Effects of Alcohol Combined with Tetrahydrocannabinol.

Introduction: Alcohol (ethanol) and cannabis are among the most widely used recreational drugs in the world. With increased efforts toward legalization of cannabis, there is an alarming trend toward the concomitant (including simultaneous) use of cannabis products with alcohol for recreational purpose. While each drug possesses a distinct effect on cerebral circulation, the consequences of their simultaneous use on cerebral artery diameter have never been studied. Thus, we set to address the effect of simultaneous application of alcohol and (-)-trans-Δ-9-tetrahydrocannabinol (THC) on cerebral artery diameter. Materials and Methods: We used Sprague-Dawley rats because rat cerebral circulation closely mimics morphology, ultrastructure, and function of cerebral circulation of humans. We focused on the middle cerebral artery (MCA) because it supplies blood to the largest brain territory when compared to any other cerebral artery stemming from the circle of Willis. Experiments were performed on pressurized MCA ex vivo, and in cranial windows in vivo. Ethanol and THC were probed at physiologically relevant concentrations. Researchers were "blind" to experimental group identity during data analysis to avoid bias. Results: In males, ethanol mixed with THC resulted in greater constriction of ex vivo pressurized MCA when compared to the effects exerted by separate application of each drug. In females, THC, ethanol, or their mixture failed to elicit measurable effect. Vasoconstriction by ethanol/THC mixture was ablated by either endothelium removal or pharmacological block of calcium- and voltage-gated potassium channels of large conductance (BK type) and cannabinoid receptors. Block of prostaglandin production and of endothelin receptors also blunted constriction by ethanol/THC. In males, the in vivo constriction of MCA by ethanol/THC did not differ from ethanol alone. In females, the in vivo constriction of this artery by ethanol was significantly smaller than in males. However, artery constriction by ethanol/THC did not differ from the constriction in males. Conclusions: Our data point at the complex nature of the cerebrovascular effects elicited by simultaneous use of ethanol and THC. These effects include both local and systemic components.

Celastrol Dilates and Counteracts Ethanol-Induced Constriction of Cerebral Arteries.

The increasing recognition of the role played by cerebral artery dysfunction in brain disorders has fueled the search for new cerebrovascular dilators. Celastrol, a natural triterpene undergoing clinical trials for treating obesity, exerts neuroprotection, which was linked to its antioxidant/anti-inflammatory activities. We previously showed that celastrol fit pharmacophore criteria for activating calcium- and voltage-gated potassium channels of large conductance (BK channels) made of subunits cloned from cerebrovascular smooth muscle (SM). These recombinant BK channels expressed in a heterologous system were activated by celastrol. Activation of native SM BK channels is well known to evoke cerebral artery dilation. Current data demonstrate that celastrol (1-100 µM) dilates de-endothelialized, ex vivo pressurized middle cerebral arteries (MCAs) from rats, with EC50 = 45 µM and maximal effective concentration (Emax)= 100 µM and with MCA diameter reaching a 10% increase over vehicle-containing, time-matched values (P < 0.05). A similar vasodilatory efficacy is achieved when celastrol is probed on MCA segments with intact endothelium. Selective BK blocking with 1 µM paxilline blunts celastrol vasodilation. Similar blunting is achieved with 0.8 mM 4-aminopirydine, which blocks voltage-gated K+ channels other than BK. Using an in vivo rat cranial window, we further demonstrate that intracarotid injections of 45 µM celastrol into pial arteries branching from MCA mimics celastrol ex vivo action. MCA constriction by ethanol concentrations reached in blood during moderate-heavy alcohol drinking (50 mM), which involves SM BK inhibition, is both prevented and reverted by celastrol. We conclude that celastrol could be an effective cerebrovascular dilator and antagonist of alcohol-induced cerebrovascular constriction, with its efficacy being uncompromised by conditions that disrupt endothelial and/or BK function. SIGNIFICANCE STATEMENT: Our study demonstrates for the first time that celastrol significantly dilates rat cerebral arteries both ex vivo and in vivo and both prevents and reverses ethanol-induced cerebral artery constriction. Celastrol actions are endothelium-independent but mediated through voltage-gated (KV) and calcium- and voltage-gated potassium channel of large conductance (BK) K+ channels. This makes celastrol an appealing new agent to evoke cerebrovascular dilation under conditions in which endothelial and/or BK channel function are impaired.