Simultaneous comparison of cerebral dialysis and push-pull perfusion in the brain of rats: a critical review.

Over the last 30 years, studies of the in vivo activity of neurotransmitters and other endogenous factors in the brain have comprised a major effort in the neurosciences. Historically, the technology of push-pull perfusion was utilized as a major approach to investigations in this field. In the last 10 years, cerebral dialysis has been used as an alternative method essentially for the same scientific purpose, since the perfusion technique was viewed as difficult and excessively damaging to tissue. This review considers the representative literature in which both systems have been used to study local neurochemical responses to a drug or other chemical factor, a physiological condition or other situation. In addition, new experiments have been undertaken to compare, in the same animal and at the same time, the utility and properties inherent in the techniques of push-pull perfusion and cerebral dialysis in terms of the profile of a neurotransmitter activity and their local histopathological effects. A miniaturized 33/26 ga push-pull needle and a 24 ga dialysis probe were implanted simultaneously in the left and right caudate nuclei, respectively, in the anesthetized rat. An artificial cerebrospinal fluid (CSF) was perfused simultaneously through both devices at a rate of 10 microliters/min in the push-pull cannula and at 1.0 or 2.0 microliters/min in the dialysis probe. Within a series of 8-10 successive perfusions, excess K+ ions in a concentration of either 30 or 60 mM were incorporated in the CSF and delivered simultaneously to both the push-pull cannula and dialysis probe. Samples of perfusate and dialysate were assayed chromatographically by coulometric HPLC detector and quantitated in terms of the pg/min efflux of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA). The results showed that the resting level of DA was almost undetectable in dialysate samples from either structure; in push-pull perfusates the recovery of DA ranged between 7.0 to 10.0 pg/min, which was increased threefold by excess K+ ions. The recovery of DA and the three metabolites in samples of push-pull perfusate was two to four times that in samples of dialysate during the condition of excess K+ ions. Post-mortem histological analysis of the sites of perfusion and dialysis revealed little or no differences in the cytological damage induced by either the perfusion needle or dialysis probe. Finally, the advantages and limitations of each of these two experimental approaches to in vivo analysis of neurotransmitter efflux are reviewed in relation to the selection of an open or closed system for the on-line study of in vivo neurochemical events.

Drinking patterns in genetic low-alcohol-drinking (LAD) rats after systemic cyanamide and cerebral injections of THP or 6-OHDA.

A key question related to the role of acetaldehyde and aldehyde adducts in alcoholism concerns their relationship to the genetic mechanisms underlying drinking. Experimentally, the low-alcohol-drinking (LAD) rat represents a standard rodent model having a strong aversion to alcohol. In these experiments, preferences for water vs. alcohol, offered in concentrations from 3% to 30%, were determined over 10 days in adult LAD rats (N = 6 per group). Then a saline vehicle or either 10 or 20 mg/kg of the aldehyde dehydrogenase (AIDH) inhibitor, cyanamide, was injected s.c. twice daily for 3 days. Secondly, either 0.5 or 1.0 microg of tetrahydropapaveroline (THP) was infused i.c.v. twice daily for 3 days in LAD rats (N = 8) and, as a genetic control, THP also was infused identically in Sprague-Dawley (SD) rats (N = 8). The results showed that the lower and higher doses of cyanamide augmented alcohol intakes in 33% and 50% of the LAD rats, respectively, with the patterns of drinking resembling that of genetic high-alcohol-drinking HAD or P rats. Although i.c.v. infusions of THP had little effect on alcohol preference of LAD rats, alcohol drinking was enhanced significantly in the SD rats. In a supplementary study, 200 microg of 6-hydroxydopamine (6-OHDA) also was infused i.c.v. in LAD rats (N = 7) on two consecutive days; no change occurred in the characteristic aversion to alcohol. These findings suggest that in certain individuals, a perturbation in the synthesis of AIDH can modify the genetically based aversion to alcohol, thus precipitating the liability for alcoholism. In that neither THP nor 6-OHDA lesioning exerted any effect on the genetic nondrinking LAD animal suggests that an unknown endogenous factor in the brain must underlie the cyanamide-induced shift to alcohol preference. We conclude that the genetic elements that normally prevent the progression to addictive drinking in most individuals appear to be invariant and irreversible.

Differential efficacy of serotonergic drugs FG5974, FG5893, and amperozide in reducing alcohol drinking in P rats.

Amperozide (FG5606), a 5-HT2 receptor antagonist, is well known to suppress alcohol consumption in different rat models of drinking. The present study compared the efficacy of three drugs, FG5974, FG5893, and amperozide, which have differential affinities for 5-HT1A and 5-HT2A receptors, on alcohol drinking in the genetic alcohol-preferring (P) rat. After preference for alcohol vs. water was determined over 10 days when concentrations of alcohol were increased from 3% to 30%, the maximal concentration of alcohol preferred by each animal was selected for drug testing. A 4-day predrug preference test was followed by SC injection of the saline control vehicle or doses of 1.0 and 2.5 mg/kg FG5974, FG5893, or amperozide given at 1600 and 2200 h for 4 days. Alcohol preference testing concluded with a final 4-day interval. A total daily dose of 5.0 mg/kg FG5974 reduced absolute g/kg intake of alcohol and proportional intakes of the P rats significantly; the lower dose of FG5974 also reduced alcohol drinking significantly following treatment. The mixed 5-HT1A agonist/5-HT2A antagonist, FG5893, which suppresses drinking in cyanamide-treated rats, was without effect on alcohol ingested by the P rats. However, amperozide caused a dose-dependent decline in both absolute intakes and proportion of alcohol that was more intense than that of FG5974. The control vehicle failed to alter alcohol drinking and, like the FG compounds, did not affect food intake or body weight. Although the inhibition of alcohol drinking by amperozide corresponds precisely with previous findings, the effect of FG5974 contrasts to results obtained with a structurally analogous drug FG5893. Thus, the genetic strain of rat as well as the nature of the chemical characteristics of a 5-HT agonist/antagonist will determine the differential efficacy of a drug in influencing the volitional drinking of alcohol.

Neuropeptide Y perfused in the preoptic area of rats shifts extracellular efflux of dopamine, norepinephrine, and serotonin during hypothermia and feeding.

This study examined the localized action of neuropeptide Y (NPY) on monoamine transmitter activity in the hypothalamus of the unrestrained rat as this peptide induced hypothermia, spontaneous feeding or both responses simultaneously. A guide tube was implanted in the anterior hypothalamic pre-optic area (AH/POA) of Sprague-Dawley rats. Then either control CSF vehicle or NPY in a dose of either 100 ng/microliter or 250 ng/microliter was perfused by push-pull cannulae in this structure in the fully sated, normothermic rat. Successive perfusions were carried out at a rate of 20 microliters/min for 6.0 min with an interval of 6.0 min elapsing between each. Samples of perfusate were assayed by HPLC for their levels of dopamine (DA), norepinephrine (NE), serotonin (5-HT) and their respective metabolites. Whereas control CSF was without effect on body temperature (Tb) or feeding, repeated perfusions of NPY over 3.0 hr caused dose-dependent eating from 4 to 39 g of food, hypothermia of 0.9 to 2.3 degrees C or both responses concurrently. As the rats consumed 11-39 g of food, the efflux of NE, MHPG, DOPAC and 5-HT was enhanced significantly, whereas during the fall in Tb the efflux of NE, DOPAC and 5-HIAA from the AH/POA increased. When the Tb of the rat declined simultaneously with eating behavior, the levels in perfusate of DOPAC and HVA increased significantly while MHPG declined. During perfusion of the AH/POA with NPY the turnover of NE declined while DA and 5-HT turnover increased during hypothermia alone or when accompanied by feeding. These results demonstrate that the sustained elevation in NPY within the AH/POA causes a selective alteration in the activity of the neurotransmitters implicated in thermoregulation, satiety and hunger. These findings suggest that both DA and NE comprise intermediary factors facilitating the action of NPY on neurons involved in thermoregulatory and ingestive processes. The local activity of NPY on hypothalamic neurons apparently shifts the functional balance of serotonergic and catecholaminergic neurons now thought to play a primary role in the control of energy metabolism and caloric intake.

Suppression of alcohol preference in high alcohol drinking rats: efficacy of amperozide versus naltrexone.

The selectively bred high alcohol drinking (HAD) line of rat is considered as a potential model of one type of alcoholism. The purpose of the present experiments was to compare the efficacy of two drugs on the volitional drinking of the HAD rats: the 5-HT2A receptor antagonist, amperozide, and a nonselective antagonist of opiate receptors, naltrexone. To determine the pattern of alcohol drinking of the HAD rats, a standard preference test was used in which water was offered with alcohol increased in concentrations from 3% to 30% over 11 days. The maximally preferred concentration of alcohol of each rat was offered for 4 days and ranged from 7% to 20% with a mean intake of 6.9 g/kg per day. Initially, 1.0 mg/kg amperozide, 2.5 mg/kg naltrexone, or the saline vehicle were injected twice daily for 4 days at 1600 and 2200 hours. Secondly, 2.0 mg/kg amperozide, 5.0 mg/kg naltrexone, or the saline vehicle were administered also for 4 days. After the drug sequences, alcohol preference tests continued for another 4 days. Whereas the saline vehicle was without effect on drinking, the administration of either drug caused a significant dose-dependent reduction in the daily intake of alcohol by the HAD rats in terms of absolute g/kg and proportion of alcohol to water consumed. A comparison of the drinking response to the higher doses of the two drugs showed that amperozide was more efficacious in suppressing alcohol intake than naltrexone. Niether amperozide nor naltrexone exerted any significant effects on food and water intakes or on body weight. These results support the concept of a functional link in the brain between the serotonergic and opioidergic systems postulated to underlie, in part, the aberrant drinking of alcohol. A marked dissociation between the temporal patterns of drinking after naltrexone and amperozide treatment suggests that the opiate receptors mediate the immediate reinforcing effects of alcohol, whereas the more vegetative phenomena underlying addictive properties of alcohol are regulated by 5-HT2A receptors postsynaptic to serotonergic neurons. Finally, the inhibitory actions of both drugs imply that multiple receptor mechanisms within the mesolimbic and other systems in the brain underpin the addictive liability to alcohol.

Opiate and 5-HT2A receptors in alcohol drinking: preference in HAD rats is inhibited by combination treatment with naltrexone and amperozide.

Amperozide, a 5-HT2A receptor antagonist, and naltrexone, an opiate receptor antagonist, have been shown to suppress volitional drinking of alcohol in experimental animals. The present study examined the effects of the concurrent administration of both drugs on the volitional intake of alcohol in the selectively bred, high alcohol drinking (HAD) rat. Individual preferences for alcohol were determined by a standard 10-day test in which alcohol concentrations were increased from 3% to 30%. Following a 4-day predrug test during which water together with a maximally preferred concentration of 7% to 20% was offered to each HAD rat, amperozide and naltrexone were injected SC over a second 4-day period as follows: 1) amperozide at 1600 h and naltrexone at 2200 h; 2) the same drugs but in reversed temporal order; and 3) amperozide and naltrexone administered simultaneously at 1600 and 2200 h. Thereafter, alcohol preference testing continued for a third 4-day period. The alternate delivery of both drugs attenuated significantly the absolute g/kg and proportional intakes of alcohol in the HAD rats, whereas the saline vehicle was without effect. Although the simultaneous administration of naltrexone and amperozide produced an even greater decline in alcohol intake, without side effects on food and water intakes or on body weight, some residual drinking of alcohol persisted. Nevertheless, the results corroborate our previous findings on the suppression of alcohol drinking by antagonists of opiate and 5-HT2A receptors. Because amperozide and naltrexone together reduce the apparent reinforcing property of alcohol, the theory is supported that the addictive liability to alcohol is underpined by multiple receptor subtypes within the mesolimbic and other systems in the brain.

Genetics of alcoholism: simultaneous presentation of a chocolate drink diminishes alcohol preference in high drinking HAD rats.

Through selective crossbreeding of the N/Nih heterogeneous stock of rats, two genetic lines of rats have been developed that are categorized by their preference for ethyl alcohol as high alcohol drinking (HAD) and low alcohol drinking (LAD) animals. Corresponding to other strains of rat bred for alcohol selection or rejection, they were subdivided on the basis of their intake of a solution of 10% alcohol vs. water. The present experiments were designed to determine whether the HAD-1 and LAD-1 lines are similar to the P and NP rats in their profile of alcohol consumption. Five successive three-bottle preference tests for alcohol drinking in the presence of water were undertaken in both HAD (n = 9) and LAD (n = 10) rats as follows: 10% alcohol for 5 days; 3-30% concentrations of alcohol increased over 11 days; the maximally preferred concentration of alcohol for 5 days; this maximally preferred concentration of alcohol plus either chocolate Slender for 5 days, or an aspartame solution for 5 days. The intake of alcohol of the LAD rats during the 10% test was 0.4 g/kg/day, whereas during the 3-30% test, the maximum intake was 1.7 g/kg/day; their maximally preferred concentrations ranged between 7% and 9% alcohol. In contrast, the intake of 10% alcohol of the HAD rats was 6.5 g/kg/day, whereas during the 3-30% test the mean daily intake was 6.6 g/kg/day; the maximally preferred solutions of the HAD rats ranged between 13 to 20%, with the mean maximum intake of 10.57 g/kg/day reached at the 20% concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

5-HT2 receptor blockade by amperozide suppresses ethanol drinking in genetically preferring rats.

Previously, it was shown that the unique diphenylbutylpiperazinecarboxamide derivative, amperozide (FG 5606), inhibits the volitional drinking of ethanol induced in the rat by the inhibitor of aldehyde dehydrogenase, cyanamide. In this study, the efficacy of this long-acting psychotropic agent and potent 5-hydroxytryptamine2 (5-HT2) receptor antagonist was examined in the genetic line of ethanol-preferring (P) and -nonpreferring (NP) rats. In both lines, the pattern of drinking of ethyl alcohol was determined by a standard preference test for 3-30% ethanol vs. water. Then, the maximally preferred concentration of ethanol was determined for each individual, which ranged from 9-15% for P rats and 9-13% for NP animals. After a 4-day predrug test, either the saline control vehicle or amperozide was administered SC b.i.d. at 1600 and 2200 h. The drug was given over a 3-day period in one of three doses: 0.5, 1.0, or 2.5 mg/kg. The intake of ethanol of P rats was reduced significantly in a dose-dependent manner in terms of both absolute g/kg and proportion of ethanol to water during injections of amperozide. The same doses of amperozide had no effect on the low intake of ethanol in NP rats. The saline control vehicle also did not alter the consumption of ethanol of P or NP rats. Further, neither the consumption of food nor level of body weight was affected by amperozide either during or after its administration. These results demonstrate that in the individual predisposed genetically to drink ethanol amperozide exerts a palliative effect on the aberrant preference for ethanol consumed in a pharmacologically significant amount. Presently, dopaminergic and serotonergic synapses in the brain are implicated in the genetic differences in the patterns of ethanol consumption that distinguish the P from the NP line of rats. Because amperozide influences the functional activity of both dopaminergic and serotonergic neurons in the mesolimbic system, it is envisaged that the drug attenuates ethanol drinking by way of its direct action on these neurons.

Failure of the 5-HT2 receptor antagonist, ritanserin, to alter preference for alcohol in drinking rats.

The purpose of this study was to determine whether the 5-HT2 receptor antagonist, ritanserin, possesses the same sort of efficacy as another central 5-HT2 antagonist, amperozide, in reducing the pharmacologically induced preference for ethyl alcohol in the rat. Following the repeated administration of the inhibitor of aldehyde dehydrogenase, cyanamide, the preference for alcohol vs. water was determined in each of 20 Sprague-Dawley rats by a standard test using 3-30% concentrations. Then, each rat was offered water and its maximally preferred concentration of alcohol, which ranged from 9-15% and was consumed at a mean of 5.02 +/- 0.44 g/kg per day. After a 4-day predrug control test, either the saline control solution or 0.1, 0.3, or 1.0 mg/kg ritanserin was administered SC at 1600 h over 3 days. The daily intakes of alcohol of rats both during and after treatment with ritanserin were unchanged in terms of absolute g/kg and proportion of alcohol to total fluid consumed. Similarly, the control saline also was without any effect on alcohol consumption. Neither the consumption of food and total fluids nor the level of body weight was affected by these doses of ritanserin. Because our findings fail to coincide with previous reports on the effect of ritanserin on alcohol preference, it is envisaged that a methodological difference in earlier experimental procedures, such as the use of a weak 3% concentration of alcohol, could explain the discrepancy. Further, the present results contrast with the prolonged reduction in drinking produced by another 5-HT2 receptor antagonist, amperozide, which also acts centrally on dopaminergic neurons in the limbic system.(ABSTRACT TRUNCATED AT 250 WORDS)

Norepinephrine, dopamine, and 5-HT release from perfused hypothalamus of the rat during feeding induced by neuropeptide Y.

In the unrestrained rat, the hyperphagic-like ingestion of food evoked by the sustained elevation of neuropeptide-Y (NPY) in the hypothalamus was correlated with the release and turnover of monoaminergic transmitters in this structure. A single guide tube was implanted stereotaxically in the perifornical region of the hypothalamus for localized push-pull perfusion of an artificial CSF vehicle or NPY1-36 in a concentration of 10, 50, or 100 ng/1.0 microliters. After the rat was fully satiated, a site reactive to NPY was perfused repeatedly at a rate of 20 microliters/min for 6.0 min with an interval of 6.0-12 min elapsing between each perfusion. Samples of perfusate were analyzed by HPLC with coulometric detection for DA, HVA, DOPAC, NE, MHPG, 5-HT, and 5-HIAA. Although control perfusions were without effect on feeding or monoamine activity, NPY evoked mean cumulative intakes of food of 14 +/- 2.4, 25.6 +/- 3.0 and 26.5 +/- 3.2 g in response to 10, 50, or 100 ng/microliter concentrations of NPY, respectively, over the 4.0-5.0 hr test interval. HPLC analyses showed that during feeding the release of both NE and DA was enhanced significantly. The turnover of both catecholamines likewise increased significantly as reflected by the elevated levels of MHPG, DOPAC and HVA. However, neither the basal efflux of 5-HT nor its turnover, as reflected by the output of 5-HIAA, was affected during feeding induced by NPY perfused in the hypothalamus. These results suggest that a sustained elevation of NPY in the hypothalamus causes a perturbation in the basal activity of NE and DA which are both implicated in the neuronal mechanism regulating normal eating behavior. Thus, these catecholamine neurotransmitters are envisaged to comprise an intermediary step in the functional role played by NPY in the hypothalamus in integrating the control of energy metabolism and caloric intake.

Drinking of high concentrations of ethanol versus palatable fluids in alcohol-preferring (P) rats: valid animal model of alcoholism.

A genetically based animal model of alcoholism has been characterized in Wistar-derived rats in terms of their preference (P rats) or lack of preference (NP rats) for 10% ethanol over water. The present experiments were designed to determine: 1) whether a 10% solution of ethanol is the optimal concentration for differentiation of these lines; 2) what concentrations of ethanol are maximally preferred by P and NP rats; and 3) whether highly palatable fluids presented simultaneously with each rat's preferred solution of ethanol would alter the patterns of drinking by either the P or NP or both lines of rats. A three-bottle procedure was used to establish preference for ethanol in the presence of water as well as highly palatable solutions. The results showed that, when concentrations ranging from 3-30% were presented over a 12-day test interval, the mean absolute intake of ethanol of the P rats was 6.7 g/kg per day, with a maximum intake of 10.9 g/kg per day at the 25% concentration. These levels of intake were significantly higher than the 4.3 g/kg per day consumed during the presentation of the commonly used constant concentration of 10%. Similarly, the mean absolute intake of ethanol by the NP rats was also elevated significantly at concentrations of 15-30% (2.0 g/kg per day) above that consumed at the 10% concentration (0.4 g/kg).(ABSTRACT TRUNCATED AT 250 WORDS)