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Prenatal ethanol exposure (PE) causes Fetal Alcohol Spectrum Disorders (FASD), characterized by cognitive, behavioral, and emotional deficits, including anxiety and depression. PE-induced alteration in the function of dorsal raphe nucleus (DRN) serotonin (5-HT) neurons is thought to be major contributing factor for increased anxiety. However, the precise neuronal circuits involved are unknown. Using electrophysiology, optogenetics, chemogenetics, and behavioral approaches, we find that PE preferentially potentiates medial prefrontal cortex (mPFC) glutamatergic inputs, but not lateral habenula (LHb), to DRN 5-HT neurons projecting to mPFC. Additionally, PE also increases the strength of LHb but not mPFC excitatory inputs to DRN 5-HT neurons projecting to central amygdala (Ce). This input and target selective effect of PE was mediated by a circuit-specific increase in nitric oxide (NO) signaling. Importantly, chemogenetic inhibition of mPFC-DRN neuronal circuit blunted anxiety-like behaviors in PE rats. As such, our results unraveled the DRN neuronal circuitries affected by PE, which gate FASD-induced anxiety-like behaviors.

Prenatal ethanol exposure (PE) causes Fetal Alcohol Spectrum Disorders (FASD), characterized by cognitive, behavioral, and emotional deficits, including anxiety and depression. PE-induced alteration in the function of dorsal raphe nucleus (DRN) serotonin (5-HT) neurons is thought to be major contributing factor for increased anxiety. However, the precise neuronal circuits involved are unknown. Using electrophysiology, optogenetics, chemogenetics, and behavioral approaches, we find that PE preferentially potentiates medial prefrontal cortex (mPFC) glutamatergic inputs, but not lateral habenula (LHb), to DRN 5-HT neurons projecting to mPFC. Additionally, PE also increases the strength of LHb but not mPFC excitatory inputs to DRN 5-HT neurons projecting to central amygdala (Ce). This input and target selective effect of PE was mediated by a circuit-specific increase in nitric oxide (NO) signaling. Importantly, chemogenetic inhibition of mPFC-DRN neuronal circuit blunted anxiety-like behaviors in PE rats. As such, our results unraveled the DRN neuronal circuitries affected by PE, which gate FASD-induced anxiety-like behaviors.

The unclear mechanisms of ethanol metabolism in the brain highlight the need for a deeper understanding of its metabolic pathways. This study used in vivo microdialysis to simultaneously sample ethanol and its metabolites, acetaldehyde and acetate, in the rat striatum following self-administration of ethanol, emphasizing the natural oral exposure route. To enhance the self-administration, rats underwent two-bottle-choice and limited access training. Dialysate samples, collected every 10 min for 2.5 h, were analyzed using gas chromatography with flame ionization detection (GC-FID). The measured time courses of dialysate concentrations of ethanol, acetaldehyde, and acetate provided insights into dynamics of ethanol metabolism. Notably, in a subject with low ethanol consumption (0.29 g/kg), the concentration of acetaldehyde remained below the limit of detection throughout the experiment. However, the acetate concentration was clearly increased after ethanol consumption in this subject and was comparable to that of other rats with higher ethanol consumption. Compared with focusing only on peak values in the time-courses of concentrations of ethanol and its metabolites, calculating areas under curves provided better models of the relationships between ethanol intake and individual ethanol metabolites, as indicated by the R-square values for the linear regressions. This approach of using the area under the curve accounts for both the amplitude and duration of the concentration profiles, reducing the impact of variations in individual drinking patterns. In vivo microdialysis enables concurrent sampling of brain metabolites during oral ethanol administration, contributing insights into metabolite dynamics. To our knowledge, this paper is the first to report measurement of all three analytes in the brain following self-administration of ethanol. Future studies will explore regional variations and dynamics post-ethanol dependence, further advancing our understanding of ethanol metabolism in the brain.

The unclear mechanisms of ethanol metabolism in the brain highlight the need for a deeper understanding of its metabolic pathways. This study used in vivo microdialysis to simultaneously sample ethanol and its metabolites, acetaldehyde and acetate, in the rat striatum following self-administration of ethanol, emphasizing the natural oral exposure route. To enhance the self-administration, rats underwent two-bottle-choice and limited access training. Dialysate samples, collected every 10 min for 2.5 h, were analyzed using gas chromatography with flame ionization detection (GC-FID). The measured time courses of dialysate concentrations of ethanol, acetaldehyde, and acetate provided insights into dynamics of ethanol metabolism. Notably, in a subject with low ethanol consumption (0.29 g/kg), the concentration of acetaldehyde remained below the limit of detection throughout the experiment. However, the acetate concentration was clearly increased after ethanol consumption in this subject and was comparable to that of other rats with higher ethanol consumption. Compared with focusing only on peak values in the time-courses of concentrations of ethanol and its metabolites, calculating areas under curves provided better models of the relationships between ethanol intake and individual ethanol metabolites, as indicated by the R-square values for the linear regressions. This approach of using the area under the curve accounts for both the amplitude and duration of the concentration profiles, reducing the impact of variations in individual drinking patterns. In vivo microdialysis enables concurrent sampling of brain metabolites during oral ethanol administration, contributing insights into metabolite dynamics. To our knowledge, this paper is the first to report measurement of all three analytes in the brain following self-administration of ethanol. Future studies will explore regional variations and dynamics post-ethanol dependence, further advancing our understanding of ethanol metabolism in the brain.

Previous electrophysiology studies show that acute ethanol inhibits firing of orbitofrontal (OFC) cortex neurons while chronic intermittent ethanol (CIE) exposure increases firing accompanied by enhanced ethanol drinking. The acute ethanol inhibition of OFC neuronal firing is mediated by inhibitory glycine receptors and is reduced by expressing a plasma membrane calcium ATPase (PMCA) in OFC astrocytes. In this study, we tested the effects of astrocyte PMCA on CIE-induced increases in excitability and alcohol consumption and the physical interaction between OFC astrocytes and neurons. CIE increased neuronal firing in male mice as compared to Air controls while PMCA itself increased firing in Air control male mice. In contrast, PMCA reduced CIE-mediated hyperexcitability of firing in females. CIE did not affect OFC astrocyte size in control or PMCA male mice but increased astrocyte size in female mice. Similar to spiking, PMCA and CIE both increased the number of GluA1 containing synapses within the vicinity of virally labeled astrocytes in male mice but had differential effects in females. The astrocytic interaction with GluA1 labeled synapses was not affected by CIE treatment in male or female control mice, but there was a treatment-dependent effect of PMCA in male mice. CIE increased alcohol consumption in control but not PMCA male mice and had no effect on drinking in female mice. Lastly, OFC astrocyte PMCA expression had no effect on behavioral measures of locomotion, anxiety, spontaneous alternation, or spatial memory. These findings reveal important sex-dependent differences in the physiological, structural and behavioral actions of OFC astrocytes.

Previous electrophysiology studies show that acute ethanol inhibits firing of orbitofrontal (OFC) cortex neurons while chronic intermittent ethanol (CIE) exposure increases firing accompanied by enhanced ethanol drinking. The acute ethanol inhibition of OFC neuronal firing is mediated by inhibitory glycine receptors and is reduced by expressing a plasma membrane calcium ATPase (PMCA) in OFC astrocytes. In this study, we tested the effects of astrocyte PMCA on CIE-induced increases in excitability and alcohol consumption and the physical interaction between OFC astrocytes and neurons. CIE increased neuronal firing in male mice as compared to Air controls while PMCA itself increased firing in Air control male mice. In contrast, PMCA reduced CIE-mediated hyperexcitability of firing in females. CIE did not affect OFC astrocyte size in control or PMCA male mice but increased astrocyte size in female mice. Similar to spiking, PMCA and CIE both increased the number of GluA1 containing synapses within the vicinity of virally labeled astrocytes in male mice but had differential effects in females. The astrocytic interaction with GluA1 labeled synapses was not affected by CIE treatment in male or female control mice, but there was a treatment-dependent effect of PMCA in male mice. CIE increased alcohol consumption in control but not PMCA male mice and had no effect on drinking in female mice. Lastly, OFC astrocyte PMCA expression had no effect on behavioral measures of locomotion, anxiety, spontaneous alternation, or spatial memory. These findings reveal important sex-dependent differences in the physiological, structural and behavioral actions of OFC astrocytes.

Background: Acetylation of α-tubulin is an important post-translational modification that helps maintain microtubules’ stability and dynamics, including axonal transport, cell signaling, and overall neuronal integrity. This study investigates sex-based differences in alcohol-induced acetylation of α-tubulin in mouse cerebellum. Methods: Adult, 3-month-old male and female C57BL/6 mice were administered 20% ethanol intraperitoneally. The cerebellum was dissected at 30 min, 1 h, 2 h, and 4 h post-injection. Expression levels of cerebellar acetylation of α-tubulin and enzymes mediating acetylation/deacetylation were analyzed by Western blot. The downstream product of ethanol metabolism, acetyl-CoA, was quantified by HPLC. Results: In males, α-tubulin acetylation levels increased significantly as early as 30 min post-ethanol injection, whereas females exhibited increased acetylation at a later time point, after 1 h. These sex-specific changes coincided with alterations in acetyl-CoA levels that increased significantly at 15 min in males and 1 h in females following ethanol administration. Furthermore, the level of acetyltransferase that acetylates tubulin increased significantly at 30 min in males and 1 h in females. Notably, however, no significant changes were observed in the level of the tubulin deacetylating enzyme, HDAC6, in either sex. Conclusions: Our data demonstrate that these sex differences stem from variations in expression levels of tubulin acetyltransferase (αTAT1), and the rate of ethanol metabolism-related acetyl-CoA production between male and female animals.

Background: Acetylation of α-tubulin is an important post-translational modification that helps maintain microtubules’ stability and dynamics, including axonal transport, cell signaling, and overall neuronal integrity. This study investigates sex-based differences in alcohol-induced acetylation of α-tubulin in mouse cerebellum. Methods: Adult, 3-month-old male and female C57BL/6 mice were administered 20% ethanol intraperitoneally. The cerebellum was dissected at 30 min, 1 h, 2 h, and 4 h post-injection. Expression levels of cerebellar acetylation of α-tubulin and enzymes mediating acetylation/deacetylation were analyzed by Western blot. The downstream product of ethanol metabolism, acetyl-CoA, was quantified by HPLC. Results: In males, α-tubulin acetylation levels increased significantly as early as 30 min post-ethanol injection, whereas females exhibited increased acetylation at a later time point, after 1 h. These sex-specific changes coincided with alterations in acetyl-CoA levels that increased significantly at 15 min in males and 1 h in females following ethanol administration. Furthermore, the level of acetyltransferase that acetylates tubulin increased significantly at 30 min in males and 1 h in females. Notably, however, no significant changes were observed in the level of the tubulin deacetylating enzyme, HDAC6, in either sex. Conclusions: Our data demonstrate that these sex differences stem from variations in expression levels of tubulin acetyltransferase (αTAT1), and the rate of ethanol metabolism-related acetyl-CoA production between male and female animals.