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1.
Alcohol ; 2024 Sep 05.
Article in English | MEDLINE | ID: mdl-39243874

ABSTRACT

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 minutes for 2.5 hours, 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.

2.
J Appl Polym Sci ; 141(21)2024 Jun 05.
Article in English | MEDLINE | ID: mdl-39282595

ABSTRACT

Sterilization of devices is important in hospitals, operating theatres, and emergency rooms. Microdialysis allows in vivo sampling of small molecules and is used for clinical studies. Microdialysis probes are made of soft, flexible, porous polymeric membranes. They have been traditionally disinfected using either ethanol (which fails to eliminate all microbes and doesn't satisfy regulatory requirements) or ethylene oxide gas and gamma irradiation (that are expensive and resource-intensive). In this work, three methods for microdialysis probe-sterilization were studied - autoclave and two chemicals (commercially available sterilization solutions): Sporox II and MetriCide. Following sterilization, the regenerated cellulose membranes were characterized under scanning electron microscopy and by measuring the changes in pore characteristics using nitrogen sorption. To determine the effect of sterilization on analyte diffusion through the membrane, microdialysis probes were fabricated, sterilized and tested with two analytes; ethanol and dopamine. The autoclaved membranes suffered thermo-mechanical damage and were deemed unfit for further testing. Probes sterilized with the chemical solutions were subsequently characterized by in vitro microdialysis experiments performed under regulated mass flux conditions. It is concluded that autoclaving is not a suitable sterilization technique for the cellulose membranes, while both of the chemical sterilizers were found to be good candidates for sterilization.

3.
Anal Methods ; 16(26): 4322-4332, 2024 Jul 04.
Article in English | MEDLINE | ID: mdl-38888243

ABSTRACT

Microdialysis is an important technique for in vivo sampling of tissue's biochemical composition. Understanding the factors that affect the performance of the microdialysis probes and developing methods for sample analysis are crucial for obtaining reliable results. In this work, we used experimental and numerical procedures to study the performance of microdialysis probes having different configurations, membrane materials and dimensions. For alcohol research, it is important to understand the dynamics of ethanol metabolism, particularly in the brain and in other organs, and to simultaneously measure the concentrations of ethanol and its metabolites - acetaldehyde and acetate. Our work provides a comprehensive characterization of three microdialysis probes, in terms of recovery rates and backpressure, allowing for interpretation and optimization of experimental procedures. In vivo experiments were performed to measure the time course concentration of ethanol, acetaldehyde, and acetate in the rat brain dialysate. Additionally, the combination of in vitro experimental results with numerical simulations enabled us to calculate diffusion coefficients of molecules in the microdialysis membranes and study the extent of the depletion effect caused by continuous microdialysis sampling, thus providing additional insights for probe selection and data interpretation.


Subject(s)
Brain , Ethanol , Microdialysis , Microdialysis/methods , Ethanol/metabolism , Ethanol/analysis , Ethanol/pharmacokinetics , Animals , Rats , Brain/metabolism , Acetaldehyde/analysis , Acetaldehyde/metabolism , Male , Acetates/metabolism , Acetates/pharmacokinetics
4.
Article in English | MEDLINE | ID: mdl-37051259

ABSTRACT

Continuous measurement of the concentrations of ethanol and its metabolites, acetaldehyde and acetic acid, in vivo is important for the alcohol research community. Most studies only measure ethanol because accurate measurement of all three compounds is challenging. Measurement inside tissue/brain is done using a microdialysis technique, followed by off-line analysis using gas chromatography (GC). To realize simultaneous measurement of ethanol and its metabolites, one can take advantage of infrared (IR) spectroscopy as a rapid and reagent-free method. Here we report a feasibility study of using IR spectroscopy to simultaneously measure ethanol, acetaldehyde and acetic acid in aqueous solution. Different concentrations in transmission mode at different optical pathlengths and using attenuated total reflectance (ATR) were measured. In vitro microdialysis was performed on the mixture of the three compounds, and the collected sample was measured using IR to demonstrate the capability of quantifying the concentrations of the three analytes simultaneously. Lastly, to overcome the limitations of the microdialysis technique, direct measurement using evanescent-field IR spectroscopy can be a potential alternative. A hydrophobic polymer coating that adsorbs ethanol and excludes water, could improve sensitivity. Sorption kinetics in polymethyl methacrylate (PMMA) and polydimethylsiloxane (PDMS) coatings on an ATR crystal were measured. Both polymers demonstrate preferential adsorption of ethanol over water.

5.
Metallomics ; 15(4)2023 04 03.
Article in English | MEDLINE | ID: mdl-36990693

ABSTRACT

Loss-of-function mutations in SLC30A10 induce hereditary manganese (Mn)-induced neuromotor disease in humans. We previously identified SLC30A10 to be a critical Mn efflux transporter that controls physiological brain Mn levels by mediating hepatic and intestinal Mn excretion in adolescence/adulthood. Our studies also revealed that in adulthood, SLC30A10 in the brain regulates brain Mn levels when Mn excretion capacity is overwhelmed (e.g. after Mn exposure). But, the functional role of brain SLC30A10 under physiological conditions is unknown. We hypothesized that, under physiological conditions, brain SLC30A10 may modulate brain Mn levels and Mn neurotoxicity in early postnatal life because body Mn excretion capacity is reduced in this developmental stage. We discovered that Mn levels of pan-neuronal/glial Slc30a10 knockout mice were elevated in specific brain regions (thalamus) during specific stages of early postnatal development (postnatal day 21), but not in adulthood. Furthermore, adolescent or adult pan-neuronal/glial Slc30a10 knockouts exhibited neuromotor deficits. The neuromotor dysfunction of adult pan-neuronal/glial Slc30a10 knockouts was associated with a profound reduction in evoked striatal dopamine release without dopaminergic neurodegeneration or changes in striatal tissue dopamine levels. Put together, our results identify a critical physiological function of brain SLC30A10-SLC30A10 in the brain regulates Mn levels in specific brain regions and periods of early postnatal life, which protects against lasting deficits in neuromotor function and dopaminergic neurotransmission. These findings further suggest that a deficit in dopamine release may be a likely cause of early-life Mn-induced motor disease.


Subject(s)
Cation Transport Proteins , Manganese , Humans , Adult , Animals , Mice , Adolescent , Manganese/metabolism , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Zinc Transporter 8/genetics , Dopamine , Brain/metabolism , Mice, Knockout , Synaptic Transmission
6.
Biochem Biophys Res Commun ; 637: 136-143, 2022 12 31.
Article in English | MEDLINE | ID: mdl-36399799

ABSTRACT

Although microdialysis is a common in vivo sampling technique, a detailed characterization of the performance of a microdialysis probe used for sampling ethanol molecules has not been conducted. In this work, experimental and computational investigations were carried out to quantitatively study ethanol diffusion characteristics for home-made and commercially available probes. Probe efficiency, i.e. recovery rate (defined as the ethanol concentration in the dialysate to that in the external medium surrounding the probe) was used to characterize the performance. The recovery rate was measured at different perfusion flow rates (0.1, 0.2, 0.5, 1, 1.5, 2 µL/min) and external ethanol concentrations (1, 2.5, 5, 10, 20 mM) with controlled environmental conditions. Effect of temperature was also investigated at 19, 37 and 47 °C. The results show that reducing the flow rate from 2 to 0.1 µL/min at least triples the recovery rate for the home-made probes, and it remains nearly unchanged when varying external ethanol concentration. The performance for two commercial microdialysis probes with different membrane materials and configurations were also determined and have similar recovery rates. Through computational modeling, the diffusion coefficient of ethanol in the semipermeable membrane of the home-made probe was determined by fitting the experimental data, and it was found to be 9 × 1011 m2/s (R2 > 0.99). In addition, the depletion effect over time at different flow rates along with estimated in vivo ethanol clearance were simulated numerically, showing that the depletion region shrinks significantly when the flow rate is below 1 µL/min. The results provide better understanding of the diffusion characteristics of the microdialysis probe when used for sampling ethanol which can be used for better interpretation of in vivo measurements and for microdialysis probe optimization.


Subject(s)
Ethanol , Microdialysis , Perfusion , Computer Simulation , Diffusion
7.
Front Behav Neurosci ; 16: 954906, 2022.
Article in English | MEDLINE | ID: mdl-35967900

ABSTRACT

In this selective review article, we showcase our collaborations with our colleague, Dr. Nadia Chaudhri. Dr. Chaudhri was an esteemed colleague and researcher who contributed greatly to our understanding of Pavlovian alcohol conditioning. From 2014 to 2019, we collaborated with Nadia. Here, we reflect on our friendship, the work we did together, and the continued impact on the field.

8.
Biol Psychiatry ; 91(12): 1008-1018, 2022 06 15.
Article in English | MEDLINE | ID: mdl-35430085

ABSTRACT

BACKGROUND: Alcohol use disorder (AUD) is a leading preventable cause of death. The central amygdala (CeA) is a hub for stress and AUD, while dysfunction of the noradrenaline stress system is implicated in AUD relapse. METHODS: Here, we investigated whether alcohol (ethanol) dependence and protracted withdrawal alter noradrenergic regulation of the amygdala in rodents and humans. Male adult rats were housed under control conditions, subjected to chronic intermittent ethanol vapor exposure to induce dependence, or withdrawn from chronic intermittent ethanol vapor exposure for 2 weeks, and ex vivo electrophysiology, biochemistry (catecholamine quantification by high-performance liquid chromatography), in situ hybridization, and behavioral brain-site specific pharmacology studies were performed. We also used real-time quantitative polymerase chain reaction to assess gene expression of α1B, ß1, and ß2 adrenergic receptors in human postmortem brain tissue from men diagnosed with AUD and matched control subjects. RESULTS: We found that α1 receptors potentiate CeA GABAergic (gamma-aminobutyric acidergic) transmission and drive moderate alcohol intake in control rats. In dependent rats, ß receptors disinhibit a subpopulation of CeA neurons, contributing to their excessive drinking. Withdrawal produces CeA functional recovery with no change in local noradrenaline tissue concentrations, although there are some long-lasting differences in the cellular patterns of adrenergic receptor messenger RNA expression. In addition, postmortem brain analyses reveal increased α1B receptor messenger RNA in the amygdala of humans with AUD. CONCLUSIONS: CeA adrenergic receptors are key neural substrates of AUD. Identification of these novel mechanisms that drive alcohol drinking, particularly during the alcohol-dependent state, supports ongoing new medication development for AUD.


Subject(s)
Alcoholism , Central Amygdaloid Nucleus , Alcohol Drinking , Animals , Central Amygdaloid Nucleus/metabolism , Ethanol/pharmacology , Humans , Male , Norepinephrine , RNA, Messenger , Rats , Receptors, Adrenergic/metabolism
9.
Addict Biol ; 26(2): e12899, 2021 03.
Article in English | MEDLINE | ID: mdl-32255261

ABSTRACT

Although alcohol (i.e., ethanol) is a major drug of abuse, the acute functional effects of ethanol on the reward circuitry are not well defined in vivo. In freely moving rats, we examined the effect of intravenous ethanol administration on neuronal unit activity in the posterior ventral tegmental area (VTA), a central component of the mesolimbic reward system. VTA units were classified as putative dopamine (DA) neurons, fast-firing GABA neurons, and unidentified neurons based on a combination of electrophysiological properties and DA D2 receptor pharmacological responses. A gradual infusion of ethanol significantly altered the firing rate of DA neurons in a concentration-dependent manner. The majority of DA neurons were stimulated by ethanol and showed enhanced burst firing activity, but a minority was inhibited. Ethanol also increased the proportion of DA neurons that exhibited pacemaker-like firing patterns. In contrast, ethanol mediated a variety of effects in GABA and other unidentified neurons that were distinct from DA neurons, including a nonlinear increase in firing rate, delayed inhibition, and more biphasic activity. These results provide evidence of discrete electrophysiological effects of ethanol on DA neurons compared with other VTA cell types, suggesting a complex role of the VTA in alcohol-induced responses in freely moving animals.


Subject(s)
Action Potentials/drug effects , Dopaminergic Neurons/drug effects , Ethanol/pharmacology , GABAergic Neurons/drug effects , Ventral Tegmental Area/drug effects , Animals , Dose-Response Relationship, Drug , Male , Rats , Rats, Long-Evans , Reward
10.
Alcohol Clin Exp Res ; 44(8): 1529-1539, 2020 08.
Article in English | MEDLINE | ID: mdl-32573991

ABSTRACT

BACKGROUND: Norepinephrine has been suggested to regulate ethanol (EtOH)-related behaviors, but little is known about the effects of EtOH on norepinephrine release in mesocortical and mesolimbic brain areas that are targets of EtOH actions. METHODS: We used in vivo microdialysis to examine the effects of EtOH on extracellular norepinephrine concentrations in mesocorticolimbic brain regions of male Long Evans rats. We determined the effects of intravenous infusion of saline or EtOH in the medial prefrontal cortex (mPFC) and the basal forebrain. We also measured dialysate norepinephrine concentrations during operant self-administration of EtOH in the mPFC. RESULTS: Intravenous infusion (1 or 0.25 ml/min) of 1.0 g/kg EtOH stimulated an increase in dialysate norepinephrine in mPFC and in basal forebrain. In the basal forebrain, an infusion of 0.5 g/kg EtOH did not stimulate dialysate norepinephrine concentrations. In both regions, saline infusions did not increase dialysate norepinephrine concentrations. In the behavioral experiment, 1 week of experience with operant self-administration of sweetened EtOH resulted in an apparent reduction in basal dialysate norepinephrine concentrations in the mPFC relative to the sucrose control. Dialysate norepinephrine increased during the transfer from home cage to the operant chamber in all groups. CONCLUSIONS: We conclude that acute EtOH stimulates both the locus coeruleus (which projects to the mPFC) and the nucleus tractus solitarius (which projects to the basal forebrain) noradrenergic neurons. Additionally, limited EtOH self-administration experience alters dialysate norepinephrine in the mPFC in a manner consistent with a decrease in tonic norepinephrine release. Further studies are necessary to elucidate the mechanisms by which EtOH exerts these variable effects.


Subject(s)
Basal Forebrain/drug effects , Central Nervous System Depressants/pharmacology , Ethanol/pharmacology , Norepinephrine/metabolism , Prefrontal Cortex/drug effects , Administration, Intravenous , Animals , Basal Forebrain/metabolism , Conditioning, Operant , Extracellular Space/metabolism , Male , Microdialysis , Prefrontal Cortex/metabolism , Rats , Self Administration
11.
Pharmacol Ther ; 212: 107573, 2020 08.
Article in English | MEDLINE | ID: mdl-32437827

ABSTRACT

Alcohol use disorder has multiple characteristics including excessive ethanol consumption, impaired control over drinking behaviors, craving and withdrawal symptoms, compulsive seeking behaviors, and is considered a chronic condition. Relapse is common. Determining the neurobiological targets of ethanol and the adaptations induced by chronic ethanol exposure is critical to understanding the clinical manifestation of alcohol use disorders, the mechanisms underlying the various features of the disorder, and for informing medication development. In the present review, we discuss ethanol's interactions with a variety of neurotransmitter systems, summarizing findings from preclinical and translational studies to highlight recent progress in the field. We then describe animal models of ethanol self-administration, emphasizing the value, limitations, and validity of commonly used models. Lastly, we summarize the behavioral changes induced by chronic ethanol self-administration, with an emphasis on cue-elicited behavior, the role of ethanol-related memories, and the emergence of habitual ethanol seeking behavior.


Subject(s)
Ethanol/administration & dosage , Self Administration , Alcohol Drinking/psychology , Animals , Appetite/drug effects , Behavior, Animal/drug effects , Dopamine/physiology , Ethanol/pharmacology , Humans , Models, Animal , Neuroimmunomodulation/drug effects , Norepinephrine/physiology , Receptors, Opioid/physiology
12.
Alcohol ; 81: 1-9, 2019 12.
Article in English | MEDLINE | ID: mdl-31002878

ABSTRACT

The ability of environmental cues to trigger alcohol-seeking behaviors is believed to facilitate problematic alcohol use. We previously showed that the development of this cue-evoked alcohol approach reflects cue-alcohol learning and memory in the adult male rat; however, we do not know whether the same is true for similarly aged female rats. Consequently, adult Long-Evans female rats were allowed to drink unsweetened alcohol in the home cage (Monday, Wednesday, Friday; 24-h two-bottle choice; 5 weeks) and were subsequently split into two experimental groups: Paired and Unpaired. Groups were matched for ingested doses and alcohol bottle preference across the pre-conditioning home cage period. Both groups were trained in conditioning chambers using a Pavlovian procedure. For the Paired group, the chamber houselight was illuminated to signal access to an alcohol sipper. Houselight onset was yoked for the Unpaired group, but access to the alcohol sipper was scheduled to occur only during the intervening periods (in the absence of light). We found that in the Paired, but not Unpaired group, an alcohol approach reaction was conditioned to houselight illumination, and the level of cue-conditioned reactivity predicted drinking behavior within trials. Groups experienced equivalently low but non-negligible blood alcohol concentrations over the course of conditioning sessions. We conclude that cue-triggered alcohol-seeking behavior in adult female rats reflects associative learning about the relationship between alcohol availability and houselight illumination.


Subject(s)
Association Learning/drug effects , Ethanol/pharmacology , Alcohol Drinking , Animals , Conditioning, Classical/drug effects , Cues , Dose-Response Relationship, Drug , Female , Rats , Rats, Long-Evans
13.
Alcohol ; 76: 91-102, 2019 05.
Article in English | MEDLINE | ID: mdl-30612041

ABSTRACT

Alcohol self-administration produces brain and behavior adaptations that facilitate a progressive loss of control over drinking and contribute to relapse. One possible adaptation is the ability of antecedent environmental stimuli that are consistently paired with alcohol to trigger alcohol-seeking behaviors. We previously modeled this adaptation in rats using a Pavlovian conditioning procedure in which illumination of a houselight preceded the presentation of a sipper tube that produced unsweetened alcohol when licked. However, in our previous work we did not demonstrate whether this adaptation represented a consequence of repeated exposure to alcohol or the houselight, or whether it was the consequence of associative learning and memory. Thus, in the present study, we tested the associative basis of alcohol seeking in response to houselight illumination in our task using adult male rats that were not food- or water-deprived and were not dependent on alcohol. Separate groups of rats received houselight illumination that was explicitly paired or unpaired with presentation of the retractable sipper that provided access to unsweetened alcohol. Our primary dependent variable was appetitive alcohol-directed behavior: the frequency of movement toward and interaction with the hole in the wall of the chamber through which the sipper was presented during the period of houselight illumination trial before each sipper presentation. However, we also analyzed consummatory sipper licking behavior and blood ethanol concentration in the same rats. Finally, we explored the brain basis of cue-elicited alcohol seeking using c-Fos immunohistochemistry. Our findings confirmed the associative basis of cue-elicited alcohol seeking in our paradigm and mapped these onto the insular cortex, suggesting a role for this brain region in early stages of brain and behavior adaptation to regular alcohol use.


Subject(s)
Alcohol Drinking/psychology , Cerebral Cortex/physiology , Conditioning, Classical/physiology , Drug-Seeking Behavior/drug effects , Animals , Blood Alcohol Content , Cerebral Cortex/metabolism , Cues , Male , Proto-Oncogene Proteins c-fos/metabolism , Rats , Self Administration
14.
Psychopharmacology (Berl) ; 235(9): 2777, 2018 09.
Article in English | MEDLINE | ID: mdl-30094500

ABSTRACT

After publication of this paper, the authors determined an error in the calculation of the norepinephrine standard concentrations for the HPLC calibration curves.

15.
Alcohol ; 69: 41-49, 2018 06.
Article in English | MEDLINE | ID: mdl-29635111

ABSTRACT

Implicit learning about antecedent stimuli and the unconditional stimulus (US) properties of alcohol may facilitate the progressive loss of control over drinking. To model this learning, Cofresí et al. (2017) developed a procedure in which a discrete, visual conditional stimulus (houselight illumination; CS) predicted the availability of a retractable sipper that rats could lick to receive unsweetened alcohol [Alcoholism: Clinical and Experimental Research, 41, 608-617]. Here we investigated the possibility that houselight illumination, sipper presentation, and oral alcohol receipt might each exert control over alcohol seeking and drinking. We also determined the relationship between ingested dose and blood alcohol concentration, in order to validate the idea that the US is a post-ingestive action of alcohol. Finally, we tested a major prediction from the conditioning account of problematic drinking [Tomie, A., & Sharma, N. (2013). Current Drug Abuse Reviews, 6, 201-219], which is that once learned, responses elicited by a CS will promote drinking. We found that despite having constrained opportunities to drink alcohol during the conditioning procedure, ingested doses produced discriminable blood concentrations that supported cue conditioning. Based on our analysis of the dynamics of cue reactivity in well-trained rats, we found that houselight illumination triggered conditioned approach, sipper presentation evoked licking behavior, and alcohol receipt promoted drinking. Reactivity to these cues, which varied in terms of their temporal proximity to the alcohol US, persisted despite progressive intoxication or satiety. Additionally, rats with the greatest conditioned reactivity to the most distal alcohol cue were also the fastest to initiate drinking and drank the most. Our findings indicate that the post-ingestive effects of alcohol may condition multiple cues simultaneously in adult rats, and these multiple cues help to trigger alcohol seeking and drinking. Moreover, identification and characterization of these cues should be helpful for designing interventions that attenuate the power of these cues over behavior.


Subject(s)
Alcohol Drinking/psychology , Conditioning, Classical/drug effects , Cues , Drug-Seeking Behavior/drug effects , Ethanol/pharmacology , Alcohol Drinking/blood , Animals , Blood Alcohol Content , Dose-Response Relationship, Drug , Male , Rats , Time Factors
16.
Neurochem Res ; 43(2): 306-315, 2018 Feb.
Article in English | MEDLINE | ID: mdl-29127598

ABSTRACT

Inhibitory signaling in the ventral tegmental area (VTA) is involved in the mechanism of action for many drugs of abuse. Although drugs of abuse have been shown to alter extracellular γ-aminobutyric acid (GABA) concentration in the VTA, knowledge on how uptake mechanisms are regulated in vivo is limited. Quantitative (no-net-flux) microdialysis is commonly used to examine the extracellular concentration and clearance of monoamine neurotransmitters, however it is unclear whether this method is sensitive to changes in clearance for amino acid neurotransmitters such as GABA. The purpose of this study was to determine whether changes in GABA uptake are reflected by in vivo extraction fraction within the VTA. Using quantitative (no-net-flux) microdialysis adapted for transient conditions, we examined the effects of local perfusion with the GABA uptake inhibitor, nipecotic acid, in the VTA of Long Evans rats. Basal extracellular GABA concentration and in vivo extraction fraction were 44.4 ± 1.9 nM (x-intercepts from 4 baseline regressions using a total of 24 rats) and 0.19 ± 0.01 (slopes from 4 baseline regressions using a total of 24 rats), respectively. Nipecotic acid (50 µM) significantly increased extracellular GABA concentration to 170 ± 4 nM and reduced in vivo extraction fraction to 0.112 ± 0.003. Extraction fraction returned to baseline following removal of nipecotic acid from the perfusate. Conventional microdialysis substantially underestimated the increase of extracellular GABA concentration due to nipecotic acid perfusion compared with that obtained from the quantitative analysis. Together, these results show that inhibiting GABA uptake mechanisms within the VTA alters in vivo extraction fraction measured using microdialysis and that in vivo extraction fraction may be an indirect measure of GABA clearance.


Subject(s)
Microdialysis , Neurons/metabolism , Ventral Tegmental Area/metabolism , gamma-Aminobutyric Acid/metabolism , Amino Acids/metabolism , Animals , Dopamine/metabolism , Extracellular Space/metabolism , GABA Uptake Inhibitors/metabolism , Microdialysis/methods , Nipecotic Acids/metabolism , Nucleus Accumbens/drug effects , Rats, Long-Evans
18.
Article in English | MEDLINE | ID: mdl-28433865

ABSTRACT

Reversed-phase HPLC with derivatization using o-phthalaldehyde (OPA) and sulfite allows electrochemical detection of γ-aminobutyric acid (GABA) in microdialysis samples. However, OPA/sulfite derivatives have been reported to produce lower fluorescent yield than OPA derivatives using organic thiols as the nucleophile. To overcome this limitation we examined excitation and emission spectra, reaction time, pH, and concentration of reagents in the derivatization solution. Optimal detection parameters were determined as λex=220nm and λem=385nm for maximal fluorescence. The derivatization reaction occurred immediately and the product was stable up to 24 h [corrected]. A pH of 10.4 for the borate buffer used in the derivatization solution was significantly better than lower pH. Increasing the amount of sulfite combined with diluting the derivatization solution in borate buffer resulted in complete separation of the GABA peak from contaminants without any loss in signal. Controlling the temperature of the detector at 15°C significantly improved sensitivity with a detection limit of approximately 1nM. To validate this assay, we performed microdialysis in the dorsal striatum and ventral tegmental area (VTA) of adult Long Evans rats. GABA concentrations in dialysates were determined using external standards and standard additions, in order to further confirm interfering peaks were not present in biological samples. Within the dorsal striatum (n=4), basal GABA concentrations were 12.9±2.2 and 14.5±2.2nM (external and additions, respectively). Respective basal GABA concentrations in the VTA (n=3) were 4.6±1.1 and 5.1±0.6nM. Thus, we have developed a novel, sensitive fluorescence method to determine GABA in microdialysates using HPLC of an OPA/sulfite derivative.


Subject(s)
Chromatography, High Pressure Liquid/methods , Microdialysis/methods , gamma-Aminobutyric Acid/analysis , Animals , Corpus Striatum/chemistry , Fluorescence , Limit of Detection , Male , Rats, Long-Evans , Sulfites/chemistry , o-Phthalaldehyde/chemistry
19.
Alcohol Clin Exp Res ; 41(3): 608-617, 2017 03.
Article in English | MEDLINE | ID: mdl-28169439

ABSTRACT

BACKGROUND: Conditioned responses to alcohol-associated cues can hinder recovery from alcohol use disorder (AUD). Cue exposure (extinction) therapy (CET) can reduce reactivity to alcohol cues, but its efficacy is limited by phenomena such as spontaneous recovery and reinstatement that can cause a return of conditioned responding after extinction. Using a preclinical model of alcohol cue reactivity in rats, we evaluated whether the efficacy of alcohol CET could be improved by conducting CET during the memory reconsolidation window after retrieval of cue-alcohol associations. METHODS: Rats were provided with intermittent access to unsweetened alcohol. Rats were then trained to predict alcohol access based on a visual cue. Next, rats were treated with either standard extinction (n = 14) or postretrieval extinction (n = 13). Rats were then tested for long-term memory of extinction and susceptibility to spontaneous recovery and reinstatement. RESULTS: Despite equivalent extinction, rats treated with postretrieval extinction exhibited reduced spontaneous recovery and reinstatement relative to rats treated with standard extinction. CONCLUSIONS: Postretrieval CET shows promise for persistently attenuating the risk to relapse posed by alcohol cues in individuals with AUD.


Subject(s)
Alcohol Drinking/psychology , Cues , Ethanol/administration & dosage , Extinction, Psychological/physiology , Animals , Extinction, Psychological/drug effects , Male , Rats , Rats, Long-Evans
20.
Alcohol Clin Exp Res ; 40(12): 2528-2536, 2016 12.
Article in English | MEDLINE | ID: mdl-27785807

ABSTRACT

BACKGROUND: The objective of this study was to characterize the acute pharmacological effects of ethanol (EtOH) on extracellular dopamine in the dorsomedial and dorsolateral striata. This is the first study to quantify and directly compare the effects of acute EtOH on dopamine in these subregions. Therefore, we also tested the nucleus accumbens as a positive control. We hypothesized that while EtOH may increase extracellular dopamine in the dorsomedial striatum and dorsolateral striatum, the magnitude of this increase and the temporal profiles of extracellular dopamine concentrations would differ among the dorsomedial striatum, dorsolateral striatum, and nucleus accumbens. METHODS: We performed in vivo microdialysis in adult, male Long Evans rats as they received a single (experiment 1) or repeated (experiment 2) doses of EtOH. RESULTS: The results of our positive control study validate earlier work by our laboratory demonstrating that acute intravenous EtOH immediately and robustly increases extracellular dopamine in the nucleus accumbens (Howard et al., ). In contrast, a single 1-g/kg dose of intravenous EtOH did not significantly affect extracellular dopamine in the dorsomedial striatum or the dorsolateral striatum. However, following a cumulative EtOH dosing protocol, we observed a ramping up of tonic dopamine activity in both the dorsomedial striatum and dorsolateral striatum over the course of the experiment, but this effect was more robust in the dorsomedial striatum. CONCLUSIONS: These results suggest that distinct mechanisms underlie the stimulating effects of acute EtOH on extracellular dopamine in striatal subregions. Additionally, our findings suggest a role for the dorsomedial striatum and minimal-to-no role for the dorsolateral striatum in mediating the intoxicating effects of acute moderate to high doses of EtOH.


Subject(s)
Corpus Striatum/drug effects , Corpus Striatum/metabolism , Dopamine/metabolism , Ethanol/administration & dosage , Ethanol/pharmacology , Extracellular Fluid/metabolism , Administration, Intravenous , Animals , Male , Microdialysis , Nucleus Accumbens/metabolism , Rats
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