Acute taurine reduced alcohol intake and preference in alcohol-experienced, but not in alcohol-näive rats by central mechanisms.

Taurine is a non-essential ß sulfonated amino acid involved in a plethora of biological functions in the mammalian central nervous system. Taurine is easily accessible in energy drinks for human consumption. Previous preclinical and clinical reports suggest that acute systemic administration of taurine could inhibit some of the behavioral and metabolic effects of alcohol use disorder. Overall, both in rodent and human studies, acute taurine administration reduced voluntary alcohol intake. This study aimed to assess the pharmacological effects of taurine (intracerebroventricular; i.c.v.) on ethanol intake/preference of rats either control (i.e., alcohol naïve) or forced ethanol intake (since juvenile age with a chronic intermittent access model). In addition, to explore anxiety-like behavior (through defensive burying behavior test) as pharmacological control of taurine. We found that acute (i.c.v.) taurine reduced alcohol consumption, i.e., taurine significantly decreased both alcohol intake and preference in adult male Wistar rats. Moreover, taurine elicits an anxiolytic-like effect in all administered groups independently of previous alcohol exposure.

Pharmacologic hyperreactivity of kappa opioid receptors in periaqueductal gray matter during alcohol withdrawal syndrome in rats.

BACKGROUND: Periaqueductal gray matter (PAG) is a brain region rich in kappa-opioid receptors (KOR). KOR in PAG mediates behavioral responses related to pain integration, and panic response, among others. Its participation in the addiction phenomena has been poorly studied. Hence, this preliminary study explored the pharmacological effects of KOR stimulation/blockade in dorsal-PAG (D-PAG) during alcohol withdrawal on anxiety-type behaviors and alcohol intake/preference. METHODS: Juvenile male Wistar rats were unexposed (A-naïve group) or exposed to alcohol for 5 weeks and then restricted (A-withdrawal group). Posteriorly, animals received intra D-PAG injections of vehicle (10% DMSO), salvinorin A (SAL-A; a selective KOR agonist), or 2-Methyl-N-((2'-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine (PF-04455242; a highly selective KOR-antagonist). Subsequently, the defensive burying behavior (DBB) and alcohol intake/preference paradigms were evaluated. RESULTS: SAL-A markedly increased burying time, the height of bedding, and alcohol consumption/preference in A-withdrawal, while slightly increased the height of bedding in A-näive rats. PF-04455242 decreased both burying and immobility duration, whereas increases latency to burying, frequency of rearing, and the number of stretches attempts with no action on alcohol intake/preference in A-withdrawal rats. CONCLUSIONS: In general, stimulation/blockade of KOR in A-withdrawal animals exert higher responses compared to A-naïve ones. SAL-A produced anxiety-like behaviors and increased alcohol consumption/preference, especially/solely in the alcohol-withdrawal condition, while PF-04455242 augmented exploration with no effects on alcohol intake/preference. Our findings suggest a possible pharmacologic hyperreactivity of the KOR in PAG during alcohol withdrawal.

Defensive and Emotional Behavior Modulation by Serotonin in the Periaqueductal Gray.

Serotonin 5-hydroxytryptamine (5-HT) is a key neurotransmitter for the modulation and/or regulation of numerous physiological processes and psychiatric disorders (e.g., behaviors related to anxiety, pain, aggressiveness, etc.). The periaqueductal gray matter (PAG) is considered an integrating center for active and passive defensive behaviors, and electrical stimulation of this area has been shown to evoke behavioral responses of panic, fight-flight, freezing, among others. The serotonergic activity in PAG is influenced by the activation of other brain areas such as the medial hypothalamus, paraventricular nucleus of the hypothalamus, amygdala, dorsal raphe nucleus, and ventrolateral orbital cortex. In addition, activation of other receptors within PAG (i.e., CB1, Oxytocin, µ-opioid receptor (MOR), and γ-aminobutyric acid (GABAA)) promotes serotonin release. Therefore, this review aims to document evidence suggesting that the PAG-evoked behavioral responses of anxiety, panic, fear, analgesia, and aggression are influenced by the activation of 5-HT1A and 5-HT2A/C receptors and their participation in the treatment of various mental disorders.

Blockade of GPR55 in dorsal periaqueductal gray produces anxiety-like behaviors and evocates defensive aggressive responses in alcohol-pre-exposed rats.

GPR55 is a receptor expressed in several central nervous system areas, including the periaqueductal gray (PAG). Current knowledge of GPR55 physiology in PAG only covers pain integration, but it is involved in other actions such as anxiety, panic, motivated behaviors, and alcohol intake. In the present study, juvenile male Wistar rats were unexposed (alcohol-naïve group; A-naïve) or exposed to alcohol for 5 weeks (alcohol-pre-exposed group; A-pre-exposed). Posteriorly, animals received intra dorsal-PAG (D-PAG) injections of vehicle (10% DMSO), LPI (1 nmol/0.5 µl) and ML-193 (1 nmol/0.5 µl, a selective GPR55 antagonist). Finally, defensive burying behavior (DBB) paradigm and alcohol preference were evaluated. Compared to the A-naïve group, the A-pre-exposed vehicle group had higher (p < 0.05): (i) time of immobility; (ii) latency to and duration of burying; and (iii) alcohol consumption. In both groups (i.e., A-naïve and A-pre-exposed) treatment with LPI: (i) decreased duration of burying (p < 0.05); (ii) suppressed time of immobility; and (iii) increased alcohol intake (p < 0.05). On the other hand, treatment with ML-193: (i) decreased duration of immobility in A-pre-exposed (but not in A-naïve rats); (ii) promoted an aggressive response against the shock-probe in A-pre-exposed rats (p < 0.05); and (iii) increased alcohol intake (p < 0.05). Our results suggest that blockade of GPR55 in D-PAG is associated with anxiety-like behaviors, defensive aggressive behaviors, and higher alcohol intake, whereas LPI in D-PAG produced anxiolytic-like effects (probably GPR55-mediated), but not prevention of alcohol intake.

Experimental protocol for detecting higher alcohol consumers from a conventional rat line based on basal anxiety.

Predisposition for a high alcohol intake and the impact of alcohol-abstinence-relapse may be reliable experimentally performed in conventional adult rat lines if animals received juvenile exposure to alcohol (e.g., by forced consumption) and selecting those individuals with high basal anxiety levels during juvenile periods. Importantly, a forced alcohol consumption phase must be followed by an imposed withdrawal period to form an exposure-abstinence cycle (at least two cycles are required) which allow to obtain animals with notorious alcohol relapses. The easier way to test alcohol relapses is through voluntary ethanol intake models. On the other hand, the anxiety classification may be performance by classical paradigms such as an elevated plus maze test, defensive burying behavior test or any other. Here, we provide a step-by-step protocol description to detect higher alcohol consumers animals from male Wistar rats. This protocol should be especially useful for those interested in studying the participation of specific brain nucleus [e.g., periaqueductal gray (PAG)] and/or the neurotransmitters involved [e.g., neuropeptide Y (NPY)] in the alcohol intake phenomena if it is combined with stereotaxic surgery. However, every administration route of treatments or experimental design is appropriate; the limit is the own imagination, and the resources.

The Periaqueductal Gray and Its Extended Participation in Drug Addiction Phenomena.

The periaqueductal gray (PAG) is a complex mesencephalic structure involved in the integration and execution of active and passive self-protective behaviors against imminent threats, such as immobility or flight from a predator. PAG activity is also associated with the integration of responses against physical discomfort (e.g., anxiety, fear, pain, and disgust) which occurs prior an imminent attack, but also during withdrawal from drugs such as morphine and cocaine. The PAG sends and receives projections to and from other well-documented nuclei linked to the phenomenon of drug addiction including: (i) the ventral tegmental area; (ii) extended amygdala; (iii) medial prefrontal cortex; (iv) pontine nucleus; (v) bed nucleus of the stria terminalis; and (vi) hypothalamus. Preclinical models have suggested that the PAG contributes to the modulation of anxiety, fear, and nociception (all of which may produce physical discomfort) linked with chronic exposure to drugs of abuse. Withdrawal produced by the major pharmacological classes of drugs of abuse is mediated through actions that include participation of the PAG. In support of this, there is evidence of functional, pharmacological, molecular. And/or genetic alterations in the PAG during the impulsive/compulsive intake or withdrawal from a drug. Due to its small size, it is difficult to assess the anatomical participation of the PAG when using classical neuroimaging techniques, so its physiopathology in drug addiction has been underestimated and poorly documented. In this theoretical review, we discuss the involvement of the PAG in drug addiction mainly via its role as an integrator of responses to the physical discomfort associated with drug withdrawal.

Salvia divinorum increases alcohol intake and tonic immobility whilst decreasing food intake in Wistar rats.

The kappa-opioid system (KOP) is the key in drug abuse. Of all the compounds isolated from Salvia divinorum (S. divinorum), salvinorin-A (Sal-A) is predominant. Further, Sal-A is the only compound within S. divinorum which is reported to have psychoactive properties as a powerful kappa-opioid receptor (KOPr) agonist. Based on the key role of the KOP system in the consumption of drugs, S. divinorum extract (SDE) and Sal-A may modify the alcohol intake in Wistar rats. Assessing voluntary alcohol intake as a drug consummatory behavior, food intake as natural reward behavior and tonic immobility as indicative of anxiety-like behavior, the present study sought to identify the role of both SDE and Sal-A in the Wistar rat model. Forty-eight adult male rats were randomly divided into six groups: control, alcohol naive and vehicle, alcohol-naive and SDE, alcohol-naive and Sal-A, alcohol-consumption and vehicle, alcohol-consumption and SDE, and alcohol-consumption and Sal-A. Alcohol and food intake were assessed for two weeks. In the middle of these two weeks, vehicle, SDE (containing ~1 mg/kg of Sal-A) or Sal-A was injected intraperitoneally once a day for a week. Tonic immobility testing was performed once. The administration of SDE produced a significant increase in voluntary alcohol intake especially in rats with a history of forced alcohol consumption from a juvenile age, Sal-A elicited an increase in alcohol intake in animals with or without previous alcohol exposure, SDE and Sal-A prolonged the tonic immobility duration and decreased food intake. In conclusion, S. divinorum or Sal-A stimulated alcohol consumption in rats with a history of alcohol intake and independent of previous exposure respectively, also SDE or Sal-A elicited an anorexigenic effect, and increased tonic immobility as indicative of anxious-like behavior.The kappa-opioid system (KOP) is the key in drug abuse. Of all the compounds isolated from Salvia divinorum (S. divinorum), salvinorin-A (Sal-A) is predominant. Further, Sal-A is the only compound within S. divinorum which is reported to have psychoactive properties as a powerful kappa-opioid receptor (KOPr) agonist. Based on the key role of the KOP system in the consumption of drugs, S. divinorum extract (SDE) and Sal-A may modify the alcohol intake in Wistar rats. Assessing voluntary alcohol intake as a drug consummatory behavior, food intake as natural reward behavior and tonic immobility as indicative of anxiety-like behavior, the present study sought to identify the role of both SDE and Sal-A in the Wistar rat model. Forty-eight adult male rats were randomly divided into six groups: control, alcohol naive and vehicle, alcohol-naive and SDE, alcohol-naive and Sal-A, alcohol-consumption and vehicle, alcohol-consumption and SDE, and alcohol-consumption and Sal-A. Alcohol and food intake were assessed for two weeks. In the middle of these two weeks, vehicle, SDE (containing ~1 mg/kg of Sal-A) or Sal-A was injected intraperitoneally once a day for a week. Tonic immobility testing was performed once. The administration of SDE produced a significant increase in voluntary alcohol intake especially in rats with a history of forced alcohol consumption from a juvenile age, Sal-A elicited an increase in alcohol intake in animals with or without previous alcohol exposure, SDE and Sal-A prolonged the tonic immobility duration and decreased food intake. In conclusion, S. divinorum or Sal-A stimulated alcohol consumption in rats with a history of alcohol intake and independent of previous exposure respectively, also SDE or Sal-A elicited an anorexigenic effect, and increased tonic immobility as indicative of anxious-like behavior.

NPY-Y1 receptors in dorsal periaqueductal gray modulate anxiety, alcohol intake, and relapse in Wistar rats.

Neuropeptide Y (NPY) is likely the main endogenous anxiolytic neuromodulator involved in alcohol intake. NPY-Y1, a receptor for NPY, is highly expressed in the periaqueductal gray (PAG), a mesencephalic structure involved in integrating nervous activity to the performance of active and passive defensive behaviors related to fear and anxiety. Interestingly, anxiety and fear are some of the prevailing emotional negative states during alcohol abstinence. Moreover, an inverse relationship between NPY activity and alcohol consumption has been frequently reported, mainly in the extended amygdala. Nevertheless, both the roles of NPY and that of the receptor involved in these actions have been scarcely studied. Thus, the aim of this study was to analyze the pharmacological effect of NPY and NPY-Y1 receptor blockade into the dorsal periaqueductal gray (D-PAG) in an alcohol consumption and relapse paradigm in adult male Wistar rats. Ninety-six rats at postnatal day 42 (PND-42) were classified as having low and high anxiety (LA and HA), respectively, through the elevated plus maze test (EPM). Then, those animals were randomly divided into alcohol naïve (AN) and forced alcohol consumption (FAC) groups. A cannula was implanted in D-PAG to microinject vehicle (VEH), NPY, or BIBP-3226 (a selective NPY-Y1 receptor antagonist). A defensive burying behavior test (DBB) was performed to assess the anxiety-like state during withdrawal, followed by a 24-hour free choice voluntary alcohol intake test. Under our experimental conditions, NPY microinjection decreased alcohol consumption in HA rats, whereas NPY-Y1 receptor blockade in D-PAG produced a notably anxiogenic effect and higher alcohol intake and relapse. In conclusion, NPY in the D-PAG, most likely acting on NPY-Y1 receptors, induced a significant anxiolytic effect and prominently inhibited alcohol consumption and relapse in Wistar rats.

Differential effects of cholecystokinin (CCK-8) microinjection into the ventrolateral and dorsolateral periaqueductal gray on anxiety models in Wistar rats.

Cholecystokinin (CCK) is one of the main neurohormone peptide systems in the brain, and a major anxiogenic mediator. The periaqueductal gray (PAG) is a key midbrain structure for defensive behaviors, which could include anxiety, fear, or even panic. The CCK system has wide distribution in the PAG, where the dorsolateral region (DL) participates in active defensive behavior and the ventrolateral region (VL) in passive defensive behavior. The aim of this study was to assess the effect of CCK-8 microinjection into DL-PAG or VL-PAG on anxiety-like behavior through two tests: elevated plus maze (EPM) and defensive burying behavior (DBB). CCK-8 (0.5 and 1.0 µg/0.5 µL) presently microinjected into the DL-PAG produced an anxiogenic-like effect on the EPM evidenced by decreasing the time spent/number of entries in open arms compared to vehicle group. Additionally, the latency to burying decreased and burying time increased on the DBB test. Contrarily, CCK-8 microinjected into the VL-PAG resulted in greater open-arm time and more open-arm entries compared to the vehicle-microinjected group. The results on the DBB test confirmed an anxiolytic-like response of CCK-8 into the VL-PAG. In conclusion, CCK-8 microinjected into DL-PAG produced anxiety-like behavior on EPM, and for first time reported on DBB. Contrarily, CCK-8 microinjected into the VL-PAG reduced anxiety-like behavior also for first time reported using both behavioral models EPM and DBB.

Forced ethanol ingestion by Wistar rats from a juvenile age increased voluntary alcohol consumption in adulthood, with the involvement of orexin-A.

Human adolescents who drink alcohol are more likely to become alcoholics in adulthood. Alcohol administration (intraperitoneally) or drinking (in a 2-bottle free choice paradigm) during the juvenile/adolescent age of rats promotes voluntary alcohol consumption in adulthood. On the other hand, there is growing evidence that the orexinergic system plays a role in several rewarded behaviors, including alcohol ingestion. Since it is unknown what effect is exerted in adulthood by forced oral ethanol intake and/or administration of orexin-A (OX-A) in juvenile rats, the present study aimed to evaluate this question. A group of male Wistar rats was forced to drink ethanol (10% v/v) as the only liquid in the diet from weaning (postnatal day 21) to postnatal day 67 (46 days), followed by a forced withdrawal period. An age-matched group was raised drinking tap water (control). OX-A or its vehicle was microinjected intracerebroventricularly (i.c.v.) (1 nmol/0.6 µL) to explore its effect as well. Locomotor activity and voluntary ethanol consumption were later assessed in all groups. The rats forced to consume ethanol early in life showed an elevated level of ambulation and alcohol ingestion in adulthood. A single injection of OX-A increased locomotor activity and acute ethanol intake in rats with or without prior exposure to alcohol at the juvenile stage. In conclusion, forced ethanol consumption in juvenile rats led to increased voluntary alcohol drinking behavior during adulthood, an effect likely facilitated by OX-A.

Analgesic and anxiolytic effects of [Leu31,Pro34]-neuropeptide Y microinjected into the periaqueductal gray in rats.

Several reports have demonstrated that neuropeptide Y (NPY) is involved in food intake, epilepsy, circadian rhythms, drug seeking, pain and anxiety, and other physiological or pathological conditions. On the other hand, periaqueductal gray (PAG) is a key brain center for modulating pain, anxiety and fear. It is the main structure implicated in integrated defensive behaviors. One such behavior, tonic immobility (TI), resembles fear and is able to induce analgesia. After microinjection of [Leu31,Pro34]-Neuropeptide Y ([Leu31,Pro34]-NPY) into the PAG dorsal (D) or ventrolateral (VL) of adult male Wistar rats, the following parameters were assessed: i) the analgesic effect by means of the tail-flick test (TF), ii) the duration of TI as a passive defensive behavioral response and as an anxiety/fear model (considering both TF and TI as single behaviors), iii) TI-induced analgesia by the combination of TF/TI, and iv) the anxious-like state through the elevated plus maze (EPM), and defensive burying behavior (DBB). The results show that the microinjection of [Leu31,Pro34]-NPY into the PAG produced an analgesic effect (increasing the TF latency); overall decreased the TI duration, which might represent an important anti-fear effect. Moreover, [Leu31,Pro34]-NPY microinjected into the PAG allows for a TI-induced analgesic effect, as well as, a substantial anxiolytic effect (evidenced by the EPM and DBB models). Hence, [Leu31,Pro34]-NPY microinjected into the PAG, especially at 0.47nmol/0.5µL produces both analgesic and anxiolytic effects, in a higher magnitude within ventrolateral area.

Isolation stress and chronic mild stress induced immobility in the defensive burying behavior and a transient increased ethanol intake in Wistar rats.

Stress can be experienced with or without adverse effects, of which anxiety and depression are two of the most important due to the frequent comorbidity with alcohol abuse in humans. Historically, stress has been considered a cause of drug use, particularly alcohol abuse due to its anxiolytic effects. In the present work we exposed male Wistar rats to two different stress conditions: single housing (social isolation, SI), and chronic mild stress (CMS). We compared both stressed groups to group-housed rats and rats without CMS (GH) to allow the determination of a clear behavioral response profile related to their respective endocrine stress response and alcohol intake pattern. We found that SI and CMS, to a greater extent, induced short-lasting increased sucrose consumption, a transient increase in serum corticosterone level, high latency/immobility, and low burying behavior in the defensive burying behavior (DBB) test, and a transient increase in alcohol intake. Thus, the main conclusion was that stress caused by both SI and CMS induced immobility in the DBB test and, subsequently, induced a transient increased voluntary ethanol intake in Wistar rats with a free-choice home-cage drinking paradigm.

Effect of melatonin injection into the periaqueductal gray on antinociception and tonic immobility in male rats.

Melatonin (MLT) is a neurohormone with significant involvement in several biological functions, of which antinociception and tonic immobility (TI) may be the key neurobehavioral components to survive in adverse conditions such as a predator attack. TI-induced antinociception can be elicited, facilitated, or increased through opioid and γ-aminobutyric acid (GABA) among other chemical mediators at several levels of the central nervous system, mainly in the periaqueductal gray (PAG). The aim of this study was to assess the effect of the microinjection of MLT into the main PAG regions that are related to different integrated defensive responses, namely dorsal (D) and ventrolateral (VL), on both antinociception through the tail-flick (TF) test and TI duration as single behavioral response and on combined behavioral responses (TF/TI). We found that the microinjection of MLT into the main PAG areas produced antinociception but did not affect the TI duration. The microinjection of MLT into the D-PAG decreased TF latency during TI in the combined trial (TF/TI), which implies that TI-induced antinociception was blocked. The microinjection of MLT into the VL-PAG maintained the antinociceptive capability of the TI without addition or increase in the antinociceptive effects, implying a permissive effect by MLT on the TI-induced antinociception. MLT administration into the D-PAG decreased the TI duration on the TF/TI, whereas MLT administration into the VL-PAG had the opposite effect of significantly increasing TI duration with the TF/TI trial.

Involvement of opioid and GABA systems in the ventrolateral periaqueductal gray on analgesia associated with tonic immobility.

Ventrolateral periaqueductal gray (VL-PAG) contains key neuronal circuits related to the analgesic effect involved in integrated defensive behaviors such as immobility response (IR). The latter is characterized by a reversible state of motor inhibition that can be elicited in rats under several conditions including restriction of movements (tonic immobility: TI). It is known that IR-induced analgesia can be elicited by manipulations or drugs acting on the central nervous system (CNS) at different levels. The aim of this study was to assess the role of the opioid and the GABA systems in TI-elicited analgesia. After inducing TI in naïve rats by neck clamping, the analgesic effect was evaluated by the tail-flick (TF) test. Compared to the control group, rats with TI had increased TF latency evidencing an analgesic effect. An opioid receptor agonist and antagonist were injected systemically, as well as microinjected locally in VL-PAG, as well as GABAA receptor agonist and antagonist were microinjected into VL-PAG. Under both injection schemes, morphine increased TF latency and TI duration, while naloxone blocked TI-induced analgesia. Muscimol reduced TF latency and TI duration while bicuculline increased TF latency but not TI duration. This suggests that TI-elicited analgesia was mediated by opioids at different levels of the CNS especially in the VL-PAG by inhibition of intrinsic tonic GABAergic activity. There were no additive analgesic effects of morphine or bicuculline with tonic immobility, which probably means reach a certain upper limit under such conditions.

Corticosterone microinjected into nucleus pontis oralis increases tonic immobility in rats.

Tonic immobility (TI) is also known as "immobility response", "immobility reflex", "animal hypnosis", etc. It is an innate antipredatory behavior characterized by an absence of movement, varying degrees of muscular activity, and a relative unresponsiveness to external stimuli. Experimentally, TI is commonly produced by manually forcing an animal into an inverted position and restraining it in that position until the animal becomes immobile. Part of the neural mechanism(s) of TI involves the medullo-pontine reticular formation, with influence from other components of the brain, notably the limbic system. It has been observed that TI is more prolonged in stressed animals, and systemic injection of corticosterone (CORT) also potentiates this behavior. At present, the anatomical brain regions involved in the CORT modulation of TI are unknown. Thus, our study was made to determine if some pontine areas could be targets for the modulation of TI by CORT. A unilateral nucleus pontis oralis (PnO) microinjection of 1 µL of CORT (0.05 µg/1 µL) in rats resulted in clear behavioral responses. The animals had an increased duration of TI caused by clamping the neck (in this induction, besides of body inversion and restraint, there is also clamping the neck), with an enhancement in open-field motor activity, which were prevented by pretreatment injection into PnO with 1 µL of the mineralocorticoid-receptor antagonist spironolactone (0.5 µg/1 µL) or 1 µL of the glucocorticoid-receptor antagonist mifepristone (0.5 µg/1 µL). In contrast, these behavioral changes were not seen when CORT (0.05 µg/1 µL) was microinjected into medial lemniscus area or paramedian raphe. Our data support the idea that, in stressful situations, glucocorticoids released from adrenals of the prey reach the PnO to produce a hyper arousal state, which in turn can prolong the duration of TI.