"Copulation-reward site" in the posterior hypothalamus.

Posterior hypothalamic selfstimulation of male rats, in which monopolar,platinum electrodes had been belaterally implanted, increased after systdmic injection of testosterone. constant stimulation to the same site elicited immediate copulation with estrous female rats. During constant stimulation males would press a bar to open a door for access to females. Even after ejaculation, males continued to open the door and to display sexual activity until stimulation terminated. Posterior hypothalamic stimulation is like normal sexual stimulation; it is rewarding, the reward varies with the amount of the sex hormone, and it elicits motivated copulation.

Overeating and obesity from damage to a noradrenergic system in the brain.

A discrete, ascending fiber system that supplies the hypothalamus with most of its noradrenergic terminals was destroyed at midbrain level, both electrolytically and with local injections of 6-hydroxydopamine, a destructive agent specific for catecholaminergic neurons. The result was hyperphagia leading to obesity. Fluorescence histochemical analysis showed that loss of noradrenergic terminals in ventral bundle termination areas such as the hypothalamus was necessary for hyperphagia. Damage to dorsal bundle or dopaminergic projections was not. Prior treatment with desmethylimipramine to selectively block uptake of 6-hydroxydopamine into noradrenergic neurons prevented both hyperphagia and loss of norepinephrine fluorescence. The lesions that produced hyperphagia also reduced the potency of d-amphetamine as an appetite suppressant. It is concluded that this noradrenergic bundle normally mediates suppression of feeding, thereby influences body weight, and serves as a substrate for d-amphetamine-induced loss of appetite.

Lateral hypothalamic control of killing: evidence for a cholinoceptive mechanism.

In rats that would not ordinarily kill mice, lateral hypothalamic injection of crystalline carbachol, a cholinomimetic, elicited killing. Norepinephrine, amphetamine, serotonin, and sodium salts were ineffective at the same site. Carbachol was ineffective when injected into the medial, dorsal, or ventral hypothalamus. As additional evidence for a cholinoceptive mechanism, neostigmine elicited killing, and, in spontaneous killers, methyl atropine blocked it. The results indicate that the lateral hypothalamus contains a cholinoceptive component of an innate system that activates killing, and anticholinergic treatment can be used as a means of suppressing killing.

Hyperphagia and obesity following serotonin depletion by intraventricular p-chlorophenylalanine.

Loss of brain serotonin was associated with overeating and increased body weight. Rats injected with p-chlorophenylalanine intraventricularly began overeating after 3 days and continued to display marked hyperphagia, primarily in the daytime, accompanied by increased body weight for 1 to 2 weeks. The effect was related to drug dose and to the degree and duration of serotonin depletion. Norepinephrine and dopamine levels were not significantly affected. It is concluded that p-chlorophenylalanine disinhibits feeding, as it does a number of other behaviors, by depleting serotonin. This suggests that hypothalamic lesions or dietary deficiencies which selectively and sufficiently deplete serotonin would lead to overeating.

Underweight rats have enhanced dopamine release and blunted acetylcholine response in the nucleus accumbens while bingeing on sucrose.

The present study tested whether rats release more accumbens dopamine (DA) during a sugar binge when they are underweight vs. normal weight. Since acetylcholine (ACh) in the nucleus accumbens (NAc) normally increases as a meal progresses and satiety ensues, we also tested whether ACh release is altered when an animal has lost weight. Rats were maintained on daily 8-h access to chow, with 10% sucrose solution available for the first 2 h. Microdialysis performed on day 21, at normal body weight, revealed an increase in extracellular DA to 122% of baseline in response to drinking sucrose. Extracellular ACh peaked at the end of the meal. Next, the rats were food and sucrose restricted so that by day 28 they were at 85% body weight. When retested, these animals released significantly more DA when drinking sucrose (179%), but ACh release failed to rise. A control group was tested in the same manner but given sugar only on days 1, 21 and 28. At normal body weight, control animals showed a non-significant rise in DA when drinking sucrose on day 21. On day 28, at 85% body weight, the controls showed a small increase (124%) in DA release; however, this was significantly lower than the 179% observed in the underweight rats with daily sugar access. These findings suggest that when an animal binges on sugar and then loses weight, the binge releases significantly more DA and less ACh than when animals are at a normal body weight.

Feeding and systemic D-amphetamine increase extracellular acetylcholine in the medial thalamus: a possible reward enabling function.

Acetylcholine neurons that project forward from the midbrain are known to enable dopaminergic reward functions in the ventral tegmental area. The question is whether acetylcholine might also be released in the mediodorsal thalamus for the same general purposes. Rats with a microdialysis probe lodged in the mediodorsal thalamus were allowed to eat chow for 20 min after 16-h food deprivation or were given varying doses of D-amphetamine when fed ad libitum. The result in both cases was a significant increase in extracellular acetylcholine. During feeding, acetylcholine increased to 177% of baseline. In response to d-amphetamine (2.5 mg/kg), acetylcholine increased to 184%, and with a higher dose (5 mg/kg) to 400% of baseline. It is concluded that midbrain projections to limbic portions of the thalamus provide acetylcholine for behavioral activation. This cholinergic function theoretically plays a role in enabling the limbic circuits that pass through the thalamus for reinforcement of feeding and psychostimulant abuse.

Sucrose sham feeding on a binge schedule releases accumbens dopamine repeatedly and eliminates the acetylcholine satiety response.

Drinking a sugar solution on an intermittent schedule can promote sugar bingeing and cause signs of dependence while releasing dopamine repeatedly like a drug of abuse. It is hypothesized that sweet taste alone is sufficient for this effect in sucrose bingeing rats. On the theory that acetylcholine in the nucleus accumbens plays a role in satiety, it is further hypothesized that purging the stomach contents will delay acetylcholine release. Rats with gastric fistulas and nucleus accumbens guide shafts for microdialysis were fed 12 h each day. During the first hour, fistulas were open for the sham-feeding group and closed for the real-feeding group, and 10% sucrose was the only food source. For the remaining 11 h, liquid rodent diet was available as well as the 10% sucrose to provide a balanced diet. In microdialysis tests during the first sugar meal on days 1, 2 and 21, extracellular dopamine increased at least 30% each day in both groups. Acetylcholine also increased during the sugar meals for the real-feeding animals, but not during sham feeding. In conclusion, the taste of sugar can increase extracellular dopamine in the nucleus accumbens without fail in animals on a dietary regimen that causes bingeing and sugar dependency. During sham feeding, the acetylcholine satiation signal is eliminated, and the animals drink more. These findings support the hypothesis that dopamine is released repeatedly in response to taste when bingeing on sweet food, and the acetylcholine satiety effect is greatly reduced by purging; this may be relevant to bulimia nervosa in humans.

Daily bingeing on sugar repeatedly releases dopamine in the accumbens shell.

Most drugs of abuse increase dopamine (DA) in the nucleus accumbens (NAc), and do so every time as a pharmacological response. Palatable food also releases accumbens-shell DA, but in naïve rats the effect can wane during a long meal and disappears with repetition. Under select dietary circumstances, sugar can have effects similar to a drug of abuse. Rats show signs of DA sensitization and opioid dependence when given intermittent access to sucrose, such as alterations in DA and mu-opioid receptors, cross-sensitization with amphetamine and alcohol, and behavioral and neurochemical signs of naloxone-precipitated withdrawal. The present experiment asks whether sucrose-dependent rats release DA each time they binge. We also predict that acetylcholine (ACh), which rises as the end of a meal, will be delayed in rats with intermittent access to sucrose. To create dependency, the experimental group (Daily Intermittent Sucrose) was maintained on a diet of 12-h food deprivation that extended 4 h into the dark, followed by 12-h access to a 10% sucrose solution and chow, daily, for 21 days. As the main result, these rats gradually increased their sucrose intake from 37 to 112 ml per day (from 13 to 20 ml in the first hour of access), and repeatedly increased extracellular DA to 130% of baseline as measured in the NAc shell by microdialysis during the first hour of sucrose access on day 1, day 2 and day 21. Three control groups failed to show a significant increase in extracellular DA on day 21: Sucrose only for 1 h on days 1 and 21 (Sucrose Twice), ad libitum access to sucrose and chow (Daily Ad libitum Sucrose), and intermittent chow instead of sucrose (Daily Intermittent Chow). Acetylcholine measured at the same time as DA, increased significantly toward the end and after each test meal in all groups. In the Daily Intermittent Sucrose group, the highest ACh levels (133%) occurred during the first sample after the sucrose meal ended. In summary, sucrose-dependent animals have a delayed ACh satiation response, drink more sucrose, and release more DA than sucrose- or binge-experienced, but non-dependent animals. These results suggest another neurochemical similarity between intermittent bingeing on sucrose and drugs of abuse: both can repeatedly increase extracellular DA in the NAc shell.

Galanin and alcohol dependence: neurobehavioral research.

It is known that microinjection of galanin (GAL) intraventricularly or in specific hypothalamic sites increases food consumption and, conversely, the intake of food increases the expression of GAL in hypothalamic sites. Ethanol (EtOH) is a calorie-rich food as well as a drug of abuse. The research reviewed here shows that GAL may play a similar role in alcohol intake. First, experiments in which GAL was microinjected into the third ventricle or the paraventricular nucleus (PVN) showed increases in EtOH consumption. The increase in EtOH consumption occurred during both the light and dark cycles after GAL injection in the third ventricle in rats with limited EtOH access. Injection of GAL did not increase food intake in rats that had been chronically drinking alcohol. GAL receptor blockade reversed these increases. Microinjection of GAL directly into the PVN also increased ad libitum EtOH intake and blockade of these receptors in the PVN inhibited ad libitum EtOH consumption. Secondly, rats administered EtOH showed increases in GAL in the PVN and related hypothalamic sites. EtOH injection and voluntary intake, both ad libitum and limited access, increased GAL gene and peptide expression in the PVN consistently across administration procedures. These experiments show that GAL injection increases alcohol intake and that the intake of alcohol increases GAL, suggesting a positive feedback relationship between alcohol intake and specific hypothalamic GAL systems. Such a relationship may contribute to the motivation to consume excessive alcoholic beverages and the development of alcohol dependence.

Brain neurotransmitters in food and drug reward.

The discovery of brain peptides as neurotransmitters opens a new chapter in the study of feeding from a neural point of view. This article reviews current studies suggesting a limbic system framework of chemical neuroanatomy in which peptides play an important role in the control of eating and behavior reinforcement. In this framework, food intake and a variety of anorectic drugs (amphetamine, phenylpropanolamine, and fenfluramine) act on dopamine, norepinephrine, and serotonin systems that ascend from the hindbrain and midbrain to modulate feeding and satiety systems in the hypothalamus. Opiate peptides (enkephalin), and brain-gut peptides (neurotensin and CCK) influence feeding and satiety when injected in the hypothalamus and reinforce behavior when injected in the midbrain. Locomotor stimulants (amphetamine and phencyclidine) reinforce behavior by an action in the nucleus accumbens. Thus the behavioral effects of foods and drugs can now be partially understood in the context of functional neuroanatomy.

Systemic morphine simultaneously decreases extracellular acetylcholine and increases dopamine in the nucleus accumbens of freely moving rats.

Microdialysis was used to measure extracellular levels of acetylcholine (ACh) and dopamine (DA) simultaneously in the nucleus accumbens (NAC) of freely moving rats. Systemic injection of morphine (20 mg/kg) significantly decreased ACh (30%, p less than .01) while it increased DA (55%, p less than .01). The effects of morphine were eliminated by naloxone. The results confirm that morphine increases DA and in addition, demonstrate an inhibitory influence of this opiate on extracellular levels of ACh in the NAC.

A diet promoting sugar dependency causes behavioral cross-sensitization to a low dose of amphetamine.

Previous research in this laboratory has shown that a diet of intermittent excessive sugar consumption produces a state with neurochemical and behavioral similarities to drug dependency. The present study examined whether female rats on various regimens of sugar access would show behavioral cross-sensitization to a low dose of amphetamine. After a 30-min baseline measure of locomotor activity (day 0), animals were maintained on a cyclic diet of 12-h deprivation followed by 12-h access to 10% sucrose solution and chow pellets (12 h access starting 4 h after onset of the dark period) for 21 days. Locomotor activity was measured again for 30 min at the beginning of days 1 and 21 of sugar access. Beginning on day 22, all rats were maintained on ad libitum chow. Nine days later locomotor activity was measured in response to a single low dose of amphetamine (0.5 mg/kg). The animals that had experienced cyclic sucrose and chow were hyperactive in response to amphetamine compared with four control groups (ad libitum 10% sucrose and chow followed by amphetamine injection, cyclic chow followed by amphetamine injection, ad libitum chow with amphetamine, or cyclic 10% sucrose and chow with a saline injection). These results suggest that a diet comprised of alternating deprivation and access to a sugar solution and chow produces bingeing on sugar that leads to a long lasting state of increased sensitivity to amphetamine, possibly due to a lasting alteration in the dopamine system.

Nucleus accumbens muscarinic receptors in the control of behavioral depression: antidepressant-like effects of local M1 antagonist in the Porsolt swim test.

Systemically administered cholinomimetics or cholinesterase inhibitors can depress behavior in humans and animals, whereas antimuscarinic agents reverse this effect or even produce euphoria. Although these effects have been well documented, the specific brain regions that mediate them remain largely unknown. In the present experiments, muscarinic agonists and antagonists were locally injected into the nucleus accumbens of female Sprague-Dawley rats to test for their effects on behavioral depression in the Porsolt swim test and locomotor activity. Local, microinjections of the drugs in the accumbens elicited behaviors that were similar to the systemic effects reported in other studies. Injection of the non-specific agonist arecoline (40 and 80 microg) dose-dependently inhibited swimming and escape behavior. This may be mediated in part by accumbens M1 receptors because blocking these receptors with the specific antagonist pirenzepine (17.5 and 35.0 microg) did the opposite by increasing swimming. Gallamine (0.13, 0.44, and 0.88 microg), an antagonist at M2 receptors, dose-dependently decreased swimming. Two-way microdialysis suggested that this was in part due to the release of ACh by blocking M2 autoreceptors. Scopolamine, a mixed M1/M2 receptor antagonist, also released ACh but did not decrease swimming, probably because the M1 receptors were blocked; the drug (1.0 microg) increased swimming time, much like pirenzepine. With the exception of arecoline, none of the drugs significantly affected locomotor activity in a photocell cage. Arecoline (40 microg), which had decreased swimming, reduced activity. The present study suggests that muscarinic receptors in the nucleus accumbens can control immobility in the Porsolt swim test. The onset of immobility may depend on the activation of post-synaptic M1 receptors.

Glutamate release in the nucleus accumbens is involved in behavioral depression during the PORSOLT swim test.

An abnormality in glutamate function has been implicated in the neural substrate of depressive disorders. To investigate this in rats, the Porsolt swim test was used to assess the role of glutamate in the nucleus accumbens. Glutamate injected into the nucleus accumbens dose-dependently decreased swimming time on the test day (day 2), whereas N-methyl-D-aspartate antagonists dizocilpine and 2-amino-5-phosphonovalerate increased swimming, like an antidepressant. Dizocilpine injected before the conditioning trial (day 1) did not modify the swimming times during the first day but abolished behavioral depression on day 2. Microdialysis coupled to capillary-zone electrophoresis was then used to determine in vivo changes in glutamate release in 1-min samples during the swim test. On day 1, glutamate increased significantly and reached a maximum of 222% after 3 min of swimming. On day 2, baseline glutamate levels were back to normal, but when the animal was placed in the water, glutamate increased to 419% during the first minute, and the animals swam significantly less. For comparison, tail pinch on consecutive days was used as a nonspecific, repeated stressor while accumbens glutamate levels were measured. Tail pinch on the first day increased glutamate similar to the effect obtained during the first day of swimming; however, a second day of tail pinch decreased glutamate levels, instead of the potentiated response observed during the second day of swimming. These results show that accumbens glutamate plays a role in causing the behavioral aspects of depressed behavior as modeled in the swim test. The accumbens may be a potential site of action for drugs that alter behavioral depression.

Activation of microsomal cytochrome P450 mono-oxygenase by Ca2+ store depletion and its contribution to Ca2+ entry in porcine aortic endothelial cells.

1. We investigated how microsomal cytochrome P450 mono-oxygenase (Cyp450 MO) is regulated in cultured porcine aortic endothelial cells. The hypothesis that a Cyp450 MO-derived metabolite links Ca2+ store depletion and Ca2+ entry was studied further. 2. Microsomal Cyp450 MO was monitored fluorometrically by dealkylation of 1-ethoxypyrene-3,6,8-tris-(dimethyl-sulphonamide; EPSA) in saponin permeabilized cells or in subcellular compartments. Endothelial Ca2+ signalling was measured by a standard fura-2 technique, membrane potential was determined with the potential-sensitive fluorescence dye, bis-(1,3-dibutylbarbituric acid) pentamethine oxonol (DiBAC4(5)) and tyrosine kinase was quantified by measuring the phosphorylation of a immobilized substrate with a horseradish peroxidase labelled phosphotyrosine specific antibody. 3. Depletion of cellular Ca2+ pools with inositol 1,4,5-trisphosphate (IP3), thapsigargin or cyclopiazonic acid activated microsomal Cyp450 MO. Similar to direct Ca2+ store depletion, chelating of intramicrosomal Ca2+ with oxalate stimulated Cyp450 MO activity, while changing cytosolic free Ca2+ failed to influence Cyp450 MO activity. These data indicate that microsomal Cyp450 MO is activated by depletion of IP3-sensitive stores. 4. Besides the common cytochrome P450 inhibitors, econazole, proadifen and miconazole, thiopentone sodium and methohexitone inhibited Cyp450 MO in a concentration-dependent manner. The physiological substrate of Cyp450 MO, arachidonic acid, inhibited EPSA dealkylation. In contrast to most other cytochrome P450 inhibitors used in this study, thiopentone sodium did not directly interfere with Ca2+ entry pathways, membrane hyperpolarization due to K+ channel activation or tyrosine kinase activity. 5. Inhibition of Cyp450 MO by thiopentone sodium diminished Ca2+/Mn2+ entry to Ca2+ store depletion by 43%, while it did not interfere with intracellular Ca2+ release by IP3 or thapsigargin. 6. Cyp450 MO inhibition with thiopentone sodium diminished autacoid-induced membrane hyperpolarization. 7. Induction of Cyp450 MO with dexamethasone/clofibrate for 72 h yielded increases in thapsigargin-induced Cyp450 MO activity (by 35%), Ca2+/Mn2+ entry (by 105%) and membrane hyperpolarization (by 40%). 8. The Cyp450 MO-derived compounds, 11,12 and 5,6-epoxyeicosatrienoic acids (EETs) yielded membrane hyperpolarization, insensitive to thiopentone sodium. 9. These data demonstrate that endothelial Cyp450 MO is activated by Ca2+ store depletion and Cyp450 MO produced compounds that hyperpolarize endothelial cells. 10. The data presented and our previous findings indicate that Cyp450 MO plays a crucial role in the regulation of store-operated Ca2+ influx. We propose that Cyp450 MO-derived EETs constitute a signal for Ca2+ entry activation and increase the driving force for Ca2+ entry by membrane hyperpolarization in porcine aortic endothelial cells.

Mechanisms of L-NG nitroarginine/indomethacin-resistant relaxation in bovine and porcine coronary arteries.

1. Coronary arteries from bovines (BCA) and pigs (PCA) were used for measuring endothelium-dependent relaxation in the presence of L-NG nitroarginine and indomethacin. As some compounds tested have been found to have an inhibitory effect on autacoid-activated endothelial Ca2+ signalling, endothelium-dependent relaxation was initiated with the Ca2+ ionophore A23187. 2. The common compounds for modulating arachidonic acid release/pathway, mepacrine and econazole only inhibited L-NG nitroarginine-resistant relaxation in BCA not in PCA. In contrast, proadifen (SKF 525A) diminished relaxation in BCA and PCA. Mepacrine and proadifen inhibited Hoe-234-initiated relaxation in BCA and PCA, while econazole only inhibited Hoe 234-induced relaxation in PCA. Due to the multiple effects of these compounds, caution is necessary in the interpretation of results obtained with these compounds. 3. The inhibitor of Ca(2+)-activated K+ channels, apamin, strongly attenuated A23187-induced L-NG nitroarginine-resistant relaxation in BCA while apamin did not affect L-NG nitroarginine-resistant relaxation in PCA. 4. Pertussis toxin blunted L-NG nitroarginine-resistant relaxation in BCA, while relaxation of PCA was not affected by pertussis toxin. 5. Thiopentone sodium inhibited endothelial cytochrome P450 epoxygenase (EPO) in PCA but not in BCA, while L-NG nitroarginine-resistant relaxation of BCA and PCA were unchanged. Protoporphyrine IX inhibited EPO in BCA and PCA and abolished L-NG nitroarginine-resistant relaxation of BCA not PCA. 6. An EPO-derived compound, 11,12-epoxy-eicosatrienoic acid (11,12-EET) yielded significant relaxation in BCA and PCA in three out of six experiments. 7. These findings suggest that L-NG nitroarginine-resistant relaxation in BCA and PCA constitutes two distinct pathways. In BCA, activation of Ca(2+)-activated K+ channels via a pertussis-toxin-sensitive G protein and EPO-derived compounds might be involved. In PCA, no selective inhibition of L-NG nitroarginine-resistant relaxation was found.

Hypothalamic serotonin in treatments for feeding disorders and depression as studied by brain microdialysis.

Microdialysis was used to measure changes in extracellular serotonin in the hypothalamus of rats while they engaged in feeding behavior or received drug treatments used to treat feeding disorders and affective disorders in humans. Hypothalamic serotonin increased significantly relative to controls in response to (1) intraperitoneal tryptophan after food deprivation, (2) the smell of food and eating a meal, (3) a conditioned taste aversion, (4) d-fenfluramine and fluoxetine, and (5) an amphetamine challenge test after chronic low doses of lithium. In some cases, increases correlated with nonspecific behavioral arousal were seen in the hippocampus. The results suggest that diet, drug, and behavioral therapies, alone or combined, can be used to preferentially modify hypothalamic serotonin in the control of behavioral, emotional, and endocrine problems.

Food intake and lateral hypothalamic self-stimulation covary after medial hypothalamic lesions or ventral midbrain 6-hydroxydopamine injections that cause obesity.

In rats with perifornical lateral hypothalamic (LH) electrodes that induced feeding, self-stimulation through the same electrodes increased immediately after ventromedial hypothalamic (VMH) lesions and did not return to normal until food intake normalized and the rats had become obese. In a second experiment a unilateral far-LH lesion decreased both feeding and contralateral perifornical LH self-stimulation. In Experiment 3, 6-hydroxydopamine (6-OHDA) injected into the midbrain to destroy the ventral noradrenergic bundle (VNAB) caused hyperphagia and increased LH self-stimulation. In summary, VMH or VNAB damage increased feeding and self-stimulation; contralateral far-LH damage decreased both. These results confirm the earlier suggestion that the VMH region is necessary for normal inhibition of feeding and feeding reward as reflected in self-stimulation rate. Although massive 6-OHDA-induced depletion of the dopamine system that passes through the LH can cause starvation and impair self-stimulation, the results suggest that selective catecholamine depletion of ventral midbrain neurons with sparing of the A9 and A10 dopaminergic cells can disinhibit feeding and self-stimulation. In all three experiments LH self-stimulation and food intake covaried, which suggests that they are functionally related.

Endogenous opiate reward induced by an enkephalinase inhibitor, thiorphan, injected into the ventral midbrain.

Opiates are known to be reinforcing when injected into the ventral tegmental area (VTA). The present study produced conditioned reinforcement with local injections of exogenous d-ala2-met5-enkephalinamide (DALA), a potent analogue of met-enkephalin, and with thiorphan , an enkephalinase inhibitor which protects endogenous opiates from enzymic degradation. In a conditioned place preference paradigm, rats received injections of DALA (1.0, 3.0, or 8.0 micrograms), thiorphan (60 micrograms), and/or naloxone (10 micrograms), or saline vehicle. Conditioned reinforcement was obtained with 8.0 micrograms of DALA and also with thiorphan but not with thiorphan plus naloxone. This suggests that reward can be generated by endogenous opiates in the VTA. Tests during the light phase and dark phase suggested that diurnal periodicity may play a role in opiate reward. It is concluded that the VTA can generate conditioned reward through transmitter-receptor interaction involving an endogenous opiate substrate which is probably enkephalinergic.

Bidirectional microdialysis in vivo shows differential dopaminergic potency of cocaine, procaine and lidocaine in the nucleus accumbens using capillary electrophoresis for calibration of drug outward diffusion.

Cocaine and two other local anesthetics were applied directly into the nucleus accumbens for 20 min by diffusion from a 4 mm microdialysis probe in freely moving rats. Cocaine (7.3 mM) increased the extracellular concentration of dopamine (DA). Equimolar procaine did also, but was not as potent as cocaine. Equimolar lidocaine had no effect. The concentration of these drugs outside the probe as measured by capillary electrophoresis in vitro was about 28% of that inside the probe, i.e. 72% remained inside. However, an in vivo test showed that about 53% cocaine and procaine, and 37% lidocaine remained in the perfusion fluid after passing through a probe inserted in the brain. This suggests that in vivo about 68 nmol cocaine diffused into the nucleus accumbens (NAC) during the 20 min. Five conclusions are drawn: (1) this confirms our earlier finding that local injection of cocaine increases extracellular DA, but in this case the cocaine was infused via the probe without disturbing the animal; (2) the action of cocaine on dopamine terminals in the accumbens is independent of local anesthesia; (3) procaine may enhance mood by a cocaine-like effect; (4) capillary electrophoresis has potential for measuring cocaine levels in small samples and (5) in vitro calibrations are of limited value to evaluate in vivo performance of microdialysis probes.