Biochemical and immunohistochemical changes in delta-9-tetrahydrocannabinol-treated type 2 diabetic rats

The regulation of glucose, lipid metabolism and immunoreactivities of insulin and glucagon peptides by delta-9-tetrahydrocannabinol (Δ⁹-THC) in diabetes were examined in an experimental rat model. Male Sprague-Dawley rats were divided into four groups: (1) control, (2) Δ⁹-THC treated, (3) diabetic, and (4) diabetic + Δ⁹-THC. The type 2 diabetic rat model was established by intraperitoneal (i.p.) injection of nicotinamide (85 mg/kg body weight) followed after 15 min by i.p. injection of streptozotocin (STZ) at 65 mg/kg of body weight. Δ⁹-THC and Δ⁹-THC treated diabetic groups received 3 mg/kg/day of Δ⁹-THC for 7 days. The immunolocalization of insulin and glucagon peptides was investigated in the pancreas using a streptavidin–biotin–peroxidase technique. High density lipoprotein cholesterol (HDL), low density lipoprotein cholesterol (LDL), very low density lipoprotein cholesterol (VLDL), triglycerides (TG), total cholesterol (TC) and total protein (TP) levels were measured in serum. Total islet area percent of insulin immunoreactive cells slightly changed in diabetic + Δ⁹-THC rats compared to diabetic animals. However, the area percent of glucagon immunoreactive cells showed a decrease in diabetic + Δ⁹-THC rats compared to that of diabetic animals alone. Serum TC, HDL and LDL levels of diabetes + Δ⁹-THC group showed a decrease compared to the diabetic group. These results indicate that Δ⁹-THC may serve a protective role against hyperlipidemia and hyperglycemia in diabetic rats.     

Delta 9-tetrahydrocannabinol and the sleep-wakefulness cycle in rabbits

The effects of 0.5 and of 1.0 mg/kg of intravenously administered Δ⁹-tetrahydrocannabinol (Δ⁹-THC) on the sleep-wakefulness cylce were studied in six freely moving rabbits with chronically implanted electrodes. On three consecutive days 5-hr polygraphic recordings were made in each animal; the first record was begun immediately following administration of the drug, the second and third were made 24 and 48 hr later. At both dose levels, Δ⁹-THC evoked biphasic effects on the first day; an initial phase of extremely low amplitude fast activity of the alert state was followed by a period of predominantly slow wave sleep. During the initial alert phase spike discharges appeared in the motor cortex and hippocampus and behavioral alterations characterized chiefly by loss of motor coordination were observed. A decrease in the number of REM sleep episodes was mainly responsible for a significant reduction in the total time spent in REM sleep on the first day. On the second day the percent time of REM sleep was still below the control value although the difference was statistically insignificant, but by the third day it had returned to the control level.     

Effect of Δ9-Tetrahydrocannabinol on in Vitro Sensitization of Mouse Splenic Lymphocytes

The effects of Δ⁹-tetrahydrocannabinol (Δ⁹-THC) on the immune response of murine cells sensitized in vitro was determined using a plaque-forming cell (PFC) assay. Splenic lymphocytes from mice injected with Δ⁹-THC showed a depressed immunologic response when compared with cells from control animals which were identically semitized in vitro with sheep erythrocytes (SRBC). The direct addition of Δ⁹-THC to the culture media altered the im-munological response as demonstrated by a reduction in the number of PFC.     

Hyporesponsiveness to the Immunosuppressant Effects of Delta-8-Tetrahydrocannabinol

Abstract

Delta-9-tetrahydrocannabinol (Δ⁹-THC) and Δ⁸-THC have previously been shown to suppress humoral immunity in mice. The purpose of these investigations was to determine whether a hyporesponsiveness develops to the immunosuppressive effect of Δ⁸-THC. BALB/c mice were administered 60 mg/kg Δ⁸-THC i.p. 2 days after an i.p. immunizing dose of sheep erythrocytes (sRBC). Significant inhibition of direct hemolytic plaqueforming cells (PFC)/spleen was observed on days 3–6, with peak day of inhibition occurring on day 4. The effective inhibiting dose 50 (ED₅₀) measured on peak day of response was 40 mg/kg for PFC/10⁶ spleen cells and 38 mg/kg for PFC/spleen. When mice were pretreated daily for 5 days with different doses of Δ⁸-THC, similar ED₅₀'s were detected. Under this pretreatment regimen, 5 or 10 mg/kg produced no immunosuppression. Daily treatment with 5 or 10 mg/kg Δ⁸-THC for 5 days prior to sRBC and varying doses of Δ⁸-THC administered 2 days after sRBC resulted in significantly higher ED₅₀'s and increased values for the slopes of the dose response curves. These results suggest that a hyporesponsiveness develops to the immunosuppressive activity of Δ⁸-THC.     

Association between a cannabinoid receptor gene (CNR1) polymorphism and cannabinoid-induced alterations of the auditory event-related P300 potential

Numerous studies demonstrated a close relationship between cannabis abuse and schizophrenia with similar impairments in cognitive processing, particularly in P300 generation. Recently, an (AAT)n triplet repeat polymorphism within the cannabinoid receptor gene CNR1 has been found to be associated with both schizophrenia and substance dependence, and to modulate the P300 potential. As previously reported, both acute oral Δ⁹-tetrahydrocannabinol (Δ⁹-THC), the main psychoactive constituent of cannabis, and standardized cannabis extract containing Δ⁹-THC and cannabidiol (CBD) revealed a significant reduction of P300 amplitudes in healthy subjects but did not show any differences among each other. The aim of this study was to investigate whether the (AAT)n polymorphism differentially modulates the effects of Δ⁹-THC and cannabis extract on P300 generation in 20 healthy volunteers during an auditory choice reaction task. For the >10/>10 genotype, there was a significant decrease of P300 amplitude as well as a significant prolongation of P300 latency under pure Δ⁹-THC but not under cannabis extract. Moreover, we found a significant correlation between the number of AAT repeats and P300 variables for the Δ⁹-THC condition. Our data thus indicate that the CNR1 gene seems to be involved in the regulation of the P300 wave as a marker of selective attention and working memory. Moreover, it appears that variations within CNR1 may differentially alter the sensitivity to the acute effects of cannabinoids on P300 generation in healthy subjects.     

Social and solitary drinking: Effects on consumption and mood in male social drinkers

Rates of alcohol consumption and mood development were studied in four male social drinkers serving as their own controls: (a) in a real life social drinking situation of the subject's choice, and (b) under artificial solitary drinking conditions. Almost twice as much alcohol was consumed during party drinking, while solitary drinking was experienced as aversive and failed to induce the euphoric effects reported at the party. Implications for tension reduction theory and adjunctive behaviour theory were discussed.     

Alcohol and alkalosis enhance excystation of Opisthorchis viverrini metacercariae

The northeastern region of Thailand has long been known as an endemic area of the human liver fluke infection which is caused by Opisthorchis viverrini. Humans are infected by ingestion of uncooked cyprinoid fish in traditional dishes such as “koi-pla,” “pla-som,” “pla-jom,” and “pla-ra.” To date, the prevalence of this parasite infection remains high because of cultural behavior and local beliefs. The popular misunderstanding among people in this area is that alcohol, lemon juice, and fish sauce can kill the parasites. Thus, they believe that they can eat raw fish without the risk of infection. This study attempts to clarify the effects of ethyl alcohol and acidosis–alkalosis on O. viverrini metacercariae excystation. Metacercariae of O. viverrini were obtained from infected cyprinoid fish in a natural reservoir. Most metacercariae were obtained from small cyprinoid fish. Metacercariae were divided into three experimental groups and were treated with solutions containing four different concentrations of ethyl alcohol, four different concentrations of salt, and a range of acidic/basic pH. Metacercariae excystation was observed at the assigned times, and the data were then analyzed. Salt had no effect on excystation. Interestingly, the optimal conditions for O. viverrini excystation were pH 9 and 25 % ethyl alcohol. The present study suggests that raw fish should not be eaten while drinking alcohol or when consuming other ingredients with pH 9, because both alcohol and pH 9 could induce O. viverrini metacercariae excystation, leading to the early development of parasites in the hepatobiliary system.     

Ethanol-induced delta-opioid receptor modulation of glutamate synaptic transmission and conditioned place preference in central amygdala

Alcoholism involves compulsive behaviors of alcohol drinking, which is thought to be related at least initially to the rewarding effect of alcohol. It has been shown that mu-opioid receptors play an essential role in drug reward and dependence for many drugs of abuse including alcohol, but the function of delta-opioid receptors (DOR) in drug reward remains largely unknown at present. Previous animal studies using systemic approaches with DOR antagonists or DOR knockout animals have yielded inconsistent results, showing a decrease, an increase or no change in alcohol consumption and behaviors of alcohol reward after DOR inhibition or deletion. In the present study, we used ethanol-conditioned rats to investigate adaptive DOR function in neurons of the central nucleus of the amygdala (CeA), a key brain site for alcohol reward and addiction. We found that functional DOR was absent in glutamate synapses of CeA neurons from control rats, but it emerged and inhibited glutamate synaptic currents in CeA neurons from rats displaying ethanol-induced behavior of conditioned place preference (CPP). Analysis of paired-pulse ratios and miniature glutamate synaptic currents revealed that the recruited DOR was present on glutamatergic presynaptic terminals. Similar induction of functional DOR was also found on GABA synapses. Furthermore, microinjection of a DOR antagonist into the CeA reversed ethanol-induced CPP behavior in rats in vivo. These results suggest that repeated alcohol exposure recruits new functional DOR on CeA glutamate and GABA synapses, which may be involved in the expression or maintenance of ethanol-induced CPP behavior.     

Effects of stimulus displacement on adjunctive behaviour.

Food deprived animals were placed on an intermittent schedule (FI 60 sec) of food reinforcement and allowed to become polydipsic. When the drinking tube was empty animals showed a decrease but not complete cessation in licking. Animals whose drinking tube was empty or absent exhibited large increases in all other behaviours recorded (e.g. bar pressing, locomotor and exploratory behaviours, floor gnawing, grooming, attacking the drinking tube etc.) When a filled drinking tube was reinstated all behaviours returned to approximately their original levels. These changes were not observed in the normal wet tube group. The results are discussed in terms of current theories of adjunctive behaviours.     

Delta-9-Tetrahydrocannabinol Inhibits the Splenocyte Proliferative Response to Herpes Simplex Virus Type 2

Abstract

The present investigation was undertaken to determine the effect of in vivo Delta-9-tetrahydrocannabinol (Delta-9-THC) treatment on immune responsiveness to secondary exposure to herpes simplex virus type 2 (HSV2) antigens in vitro. A splenocyte proliferative assay, employing HSV2-infected mouse embyro fibroblasts as target cells, was used to measure immune responsiveness. Administration of 50mg/kg or 100mg/kg Delta-9-THC to B6C3F1 mice in concert with HSV2 infection resulted in suppression of the proliferative response to HSV2 cell-surface antigens expressed on virus-infected mouse embryo fibroblasts. Similarly, in vifero treatment of HSV2-infected cells with Delta-9-THC (10 ⁷M to ⁵M) resulted in a dose-dependent suppression of proliferative responsiveness of splenocytes of non-drug-treated HSV2-sensitized mice. These results suggest that Delta-9-THC inhibits immune responsiveness of B6C3F1 mice to homotypic challenge with HSV2. This inhibition may be resultant of drug action on both effector immunocytes and target HSV2 antigen-bearing cells.     

Effects of chronic d-amphetamine on the maintenance and acquisition of schedule-induced polydipsia in rats

The effects of chronic d-amphetamine on the acquisition of schedule-induced polydipsia and its maintenance by stimuli paired with food were evaluated in three interlocking experiments. In Experiment 1, polydipsia was induced in rats exposed to a response-independent fixed-time schedule in which a food pellet (US) was paired with a stimulus complex of lights and tone (CS) every 45 sec. When food was omitted and only the CS was presented rats drank very little water. Rats were then pretreated with 1 mg/kg d-amphetamine for 15 CS-US sessions and two or three subsequent CS-alone sessions. Animals remained polydipsic during CS-US sessions and drank little water during CS-alone sessions. However, d-amphetamine improved control exerted by the CS over drinking relative to no-drug sessions. In Experiment 2, acquisition of schedule-induced polydipsia during 10 sessions exposure to the periodic CS-US schedule was blocked in rats pretreated with 1 mg/kg d-amphetamine, compared with rats pretreated with buffer. During subsequent CS-alone sessions the temporal control of drinking by the CS was greater in the rats exposed to amphetamine. In Experiment 3, the rats that had not acquired polydipsia while under d-amphetamine in the previous experiment, all became polydipsic when pretreated with buffer. All rats remained polydipsic when re-exposed to amphetamine pretreatment. These results indicate that chronic d-amphetamine administration can facilitate control of licking and drinking by nonfood stimuli paired with food, and can block acquisition of schedule-induced polydipsia.     

Adjunctive behavior and smoking induced by a maze solving schedule in humans.

Adjunctive behavior has been reported as occurring in a variety of species and has been described primarily as a phenomenon of excessive drinking in the rat (schedule induced polydipsia). In an earlier study [9] it was shown that adjunctive behavior occurs in adult human subjects with an FI 60 schedule controlling game playing on a poker machine. In the present study 20 subjects, all University personnel or students, were paid to participate in an experiment where they were required to solve a maze under conditions of scheduled access (8 sec maze drawing). Four subjects were tested with 8, 60, 120 and 300 sec intervals, and 4 with FI 120 on all test sessions. An additional 12 subjects, who were all habitual smokers, were tested with FI 60 sec intervals on all test sessions. A comparison with baseline conditions, consisting of either listening to a tape recording or continuous problem solving, showed an increase in the amount of subjects' motor activity under schedule conditions. There was a significant increase in movement scores with an increase in schedule length, but no increase with repeated scheduled trials of equal duration. The amount of smoking during test sessions was significantly greater than during smoking baseline sessions. Allowing the subject to smoke or not to smoke on maze schedule sessions made no difference to the movement scores. Eight of the subjects were also given instructions to keep still. These instructions had no effect in reducing the amount of activity. These data show that the scheduling of cognitive tasks can also lead to adjunctive behavior.     

An assessment of adjunctive behavior in a safety-signal paradigm

Three groups of rats were exposed to 0.4 mA shock presentations on a VT 30-sec schedule. Behavior was monitored during 35-sec shock-free periods inserted randomly after 15 of the 60 shocks per session. In the no-signal and random-signal control groups the behavior patterns which developed were similar to defense or fear reactions. In the safety-signal group, in which a tone signalled the 35-sec no-shock periods, a pattern of adjunctive and facultative behaviors developed including marked increases in grooming and rearing behaviors. No evidence of adjunctive drinking was found in any of the three groups.     

Schedule-induced polydipsia in the guinea pig

Two of three food-deprived guinea pigs displayed schedule-induced polydipsia when 45 mg food pellets were delivered according to a fixed-time 1-min food schedule. Both animals displayed the typical postpellet pattern of adjunctive drinking. These data extend the generality of schedule-induced polydipsia to a new species.     

Adjunctive behavior induced by wheel running

Adjunctive behavior such as licking, nose poking, rearing, grooming and locomotion induced by intermittent wheel running were observed in four female hooded rats. All of these behaviors increased significantly in three of the four animals during the intermittent test session periods when the wheel was locked. When the wheel was kept locked continuously throughout the test sessions for two control animals little, if any, adjunctive behavior developed. The fact that adjunctive behavior can be produced in animals which are not deprived of food or water was emphasized and results were interpreted in terms of a nonspecific increase in motor excitability attributed to the intermittance of schedule associated stimulation.     

The course of aggressive behavior induced by a single injection of delta 9-tetrahydrocannabinol and its characteristics

Female, Wistar King A rats subjected to one day of isolated housing, during which all food was withheld for 22 hr and supplied for only 2 hr, and then given a single dose of Δ⁹-tetrahydrocannabinol (THC) markedly exhibited muricide and rod-attack behavior. This continued for 100 days after treatment as far as the animals remained in isolation. They displayed rat pup-killing behavior as well, although normal virgin female rats did not show such behavior. When the rats were transferred from isolated housing to group housing 60 days after THC treatment, rod-attack behavior disappeared completely in all rats and muricide disappeared in 13 of the 28 rats which exhibited muricide. However, when these rats were returned to isolation after a 15 day period of group housing, rod-attack behavior and muricide identical to that observed previously reappeared. It is concluded that housing condition plays a crucial role in the occurrence and maintenance of THC-induced aggression including muricide.     

Alcohol and aldehyde dehydrogenase polymorphisms and alcoholism

The alcohol-flush reaction occurs in Asians who inherit the mutantALDH2 ^*2 allele that produces an inactive aldehyde dehydrogenase enzyme. In these individuals, high blood acetaldehyde levels are believed to be the cause of the unpleasant symptoms that follow drinking. We measured the alcohol elimination rates and intensity of flushing in Chinese subjects in whom the alcohol dehydrogenaseADH2 andALDH2 genotypes were determined. We also correlatedADH2, ADH3, andALDH2 genotypes with drinking behavior in 100 Chinese men. We discovered thatADH2 ^*2 andADH3 ^*1, alleles that encode the high activity forms of alcohol dehydrogenase, as well as the mutantALDH2 ^*2 allele were less frequent in alcoholics than in controls. The presence ofALDH2 ^*2 was associated with slower alcohol metabolism and the most intense flushing. In those homozygous forALDH2 ^*1, the presence of twoADH2 ^*2 alleles correlated with slightly faster alcohol metabolism and more intense flushing, although a great deal of variability in the latter was noted.     

Effects of physostigmine and scopolamine on operant brightness discrimination in the rat

Performance in an operant brightness discrimination was facilitated through the mid-range of training by injections of 0.3 mg/kg physostigmine. Performance was facilitated, compared to saline injected controls, due to lower response rates during S^Δ while S^D response rates were not affected. Injection of 0.5 mg/kg scopolamine suppressed both S^D and S^Δ response rates proportionally and thus did not affect discrimination performance as reflected in the discrimination ratio. The facilitation of discrimination performance produced by physostigmine was interpreted as due to facilitation of inhibitory precesses and as consistent with the hypothesis of a central cholinergic inhibitory mechanism.     

Endogenous cannabinoids as an aversive or counter-rewarding system in the rat

Human use of marijuana (Cannabis sativa) is widely assumed to have rewarding properties, a notion supported by its widespread recreational use. However, no study has clearly demonstrated such effects in animal models. The purpose of this study was to test for the presumed rewarding effect of cannabinoids using a conditioned place preference paradigm. The results showed that animals failed to develop place conditioning at a low dose (1.5 mg/kg) and developed a place aversion at a high dose (15 mg/kg) of the active principle in marijuana, Δ⁹-tetrahydrocannabinol (Δ⁹-THC), a finding consistent with most previous studies. Moreover, the administration of the cannabinoid antagonist SR141716A induced a conditioned place preference at both a low (0.5 mg/kg) and a high (5 mg/kg) dose. In summary, cannabinoid antagonism produced place preference while cannabinoid agonism induced place aversion. These results suggest that endogenous cannabinoids serve normally to suppress reward or to induce aversion.     

Increased opioid release in specific brain areas in animals exposed to prenatal morphine and emotional stress later in life

Prenatal morphine treatment and emotional stress both have been shown to increase sensitivity to reward-related behaviors. It has been postulated that this increased sensitivity to rewarding stimuli may be the result of an enhanced release of endogenous opioids. In the present study, in vivo autoradiography was employed to investigate the endogenous opioid release in specific brain areas in rats. Pregnant animals were exposed to morphine or saline from day 8 of gestation till birth. Development of pups was monitored and play behavior was tested on postnatal day 21. Adult rats were exposed to repeated emotional stress or control treatment for five consecutive days and tested in a small open field 5 days later. [³H]-Diprenorphine was injected following this test to investigate endogenous opioid release.