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Chronic opiate use is associated with increased impulsivity in both humans and animals, and previous studies suggest that acute morphine can increase impulsivity in non-dependent rats. However, the extent to which chronic opiate usage modulates the effect of acute morphine is unknown.Rats were trained to delay discount 20 % sucrose solution and then randomly assigned to either a dependent group that received a nightly 30 mg/kg subcutaneous dose of morphine or a non-dependent group that received a nightly saline injection. Once dependence was established, rats were then assigned to one of four acute morphine doses (0, 1.25, 2.5, 5 mg/kg). For 5 days, delay discounting curves were determined 22.5 h after maintenance doses and 1 h after their prescribed acute injections.In non-dependent rats, 2.5 and 5 mg/kg doses of morphine caused decreased preference for the large reward at all delays. Acute morphine had no effect on discounting curves in dependent rats.Morphine dependence can cause tolerance to the effects of acute morphine on delay discounting.

In old age there is evidence of waning motivation, and possibly lowering of mood. Physiologically there is a decline in the levels of brain neurotransmitters such as acetylcholine and dopamine, and loss of myelin. These changes might be expected to impair the functioning of brain circuitry for reinforcement, and to lead to impaired motivation. To evaluate the function of brain reinforcement mechanisms during aging we examined brain stimulation reward and its modulation by dopaminergic drugs in BN F344F1 rats aged from young adult (5 months) to old (37 months). Brain stimulation directly activates the neural circuitry for reinforcement, and the response rate-frequency tradeoff can be used to characterize the functioning of the system. Both young and old subjects readily learned to lever press for 0.6 s trains of 0.15 ms brain stimulation pulses, and there was no difference in the number of pulses per train required to maintain responding at 50% of the maximum rate (M50). Amphetamine (0.5 mg/kg) significantly reduced the M50, and the dopamine synthesis inhibitor alpha-methyl-p-tyrosine (100 mg/kg) increased the M50, but these effects were not influenced by the age of the subjects. The results suggest that in healthy animals dopaminergic modulation of reinforcement is functionally intact in old age.

Blocking the process of memory reconsolidation by means of amnestic agents may prove to have therapeutic applications. Here we used a morphine-induced conditioned place preference as an index of drug seeking. After inducing in rats a preference for a distinctive compartment paired with morphine, the memory for drug experience was reactivated by a 20-min test session and saline, the beta-antagonist propranolol, or the peripherally acting beta-antagonist nadolol were administered. Animals which received saline or nadolol upon reactivation, or propanolol without memory reactivation, maintained their preference for the drug-paired compartment 24h and seven days later. However, animals that received propranolol upon reactivation no longer displayed a morphine preference on either test, although these animals once again expressed a preference when given a morphine-primed retest at 10 days. Our results suggest that beta-blockers may have potential for attenuating the impact of cue-induced craving which is a major cause of relapse in detoxified addicts.

Protein synthesis inhibitors block consolidation of memory and may also block the reconsolidation of a reactivated memory in paradigms of aversive learning, but the evidence for reconsolidation effects is conflicting in appetitive paradigms. We now report that intra-cerebroventricular (ICV) anisomycin (400microg) prevents consolidation of morphine-induced place preference (CPP), but does not impair its reconsolidation unless the reactivation procedure associates anisomycin with the morphine context. Rats were injected alternately with morphine (5mg/kg, IP) or vehicle, and confined to one of two distinctive compartments in a three compartment apparatus. On a subsequent day rats were allowed to choose the compartment they preferred in a 20min test session. In the first experiment, rats that were injected with vehicle or with anisomycin before or 3h after training sessions, developed a CPP. However, rats that received anisomycin ICV immediately after training sessions did not develop a CPP. In experiment 2, rats received no ICV injections during initial training. Once a CPP was established, they received four additional training sessions on which they received vehicle or anisomycin ICV. All groups continued to prefer the morphine-paired compartment after reactivation sessions with vehicle or anisomycin ICV. In experiment 3, ICV anisomycin was administered selectively on morphine-paired reactivation trials or saline-paired reactivation trials and the CPP was weakened or strengthened, respectively. This suggests that associations between aversive effects of the amnestic treatment and the morphine context might mimic disruption of reconsolidation.

We investigated whether object recognition memory is modulated by estrogen in young (5 month) and aged (24 month) female C57Bl/6J mice, and if cholinergic muscarinic receptors might contribute to this response. Mice that were ovariectomized, or ovariectomized plus estradiol-treated three weeks before behavioral testing or quantitative autoradiography were compared to intact mice. Memory for a previously encountered object deteriorated significantly between 3 and 6h after initial exposure, regardless of animal age. In both young and aged mice, estradiol-treated mice showed significantly greater recall than did ovariectomized mice. In both age groups, the apparent number of [ 3 H]pirenzepine/M1-like and [ 3 H]AFDX384/M2-like muscarinic receptor binding sites was reduced in the basal forebrain as well as its projection areas following ovariectomy, but this decrease was not alleviated by estrogen. Aging poorly affected object memory, but reduced muscarinic binding in some cortical subregions and in the caudate nucleus. These findings suggest that estrogen effects on memory in C57Bl/6J mice are not due to changes in the number of muscarinic receptors. © 2002 Elsevier Science Inc. All rights reserved.

Opiate dependence is associated with increased impulsivity in both humans and animals. Although the state of withdrawal appears to contribute to this effect, a causal relationship has not been shown. Here, we test whether precipitating withdrawal in morphine-dependent rats through naloxone can cause increased impulsivity. Rats were trained on a delay discounting task and then randomly assigned to either a dependent group that received a nightly 30 mg/kg subcutaneous dose of morphine or a naive group that received nightly saline. Once dependence was established, 2-day test delay discounting curves were determined 1 h after three doses of naloxone (0, 0.25, 0.5 mg/kg). In dependent rats both doses of naloxone caused increased preference for the small reward at short delays. Naloxone had no effect on delay discounting in naive rats. We conclude that precipitating withdrawal in dependent rats can cause increased impulsivity.

The analgesic properties of the catecholamine uptake inhibitor nomifensine were investigated in the tail immersion, hot plate and formalin tests. Systemic administration of nomifensine produced analgesia only in the formalin test. The analgesia was dose-dependent (0.625-5 mg/kg), and the highest dose completely abolished nociceptive behaviors induced by 2% formalin. The analgesia was not affected by the opioid antagonist naltrexone (2.5-40 microg s.c.) but was dose-dependently reversed by the D2 antagonist eticlopride (181.3-270 microg/kg i.p.). Neither naltrexone nor eticlopride affected formalin pain scores. Nomifensine analgesia appears to be dopamine-mediated but independent of opioid mechanisms.

We have previously shown that T-maze learning impairments caused by lesions to the pedunculopontine tegmental nucleus (PPTg) can be reversed by the anxiolytic diazepam. We now report that diazepam also reverses the effect of PPTg lesions on conditioned place preference (CPP) to morphine but not to amphetamine. Rats with bilateral sham or N-methyl-D-aspartate lesions (0.1 or 0.05 M) to the PPTg were trained in a unbiased CPP paradigm with 2 mg/kg morphine or 2 mg/kg D-amphetamine associated with one compartment of the apparatus and vehicle injections in the alternative compartment. After three drug/saline-compartment pairings, the preference of the animals was assessed by allowing them to explore the entire apparatus for 20 min. In contrast to sham-lesioned subjects, the rats with PPTg lesions did not show a preference for the compartment paired with morphine or amphetamine. In two experiments the expression of a morphine CPP was restored by injecting the lesioned animals with 1 mg/kg of diazepam 30 min before the test session. Diazepam pre-treatment did not restore the expression of amphetamine CPP.

Rationale: The drug-abuse literature suggests that benzodiazepines may be preferentially abused in conjunction with opioids rather than stimulants. Objective: To investigate possible effects of diazepam on the reinforcing effects of morphine and amphetamine. Methods: The effects of diazepam (0.5, 1 or 2 mg/kg) on the formation and expression of conditioned place preferences (CPP) induced by morphine sulphate (0.3, 0.8, 2 and 8 mg/kg) or d-amphetamine (0.4, 0.8, 2 or 2.5 mg/kg) were studied in an unbiased CPP paradigm. The action of diazepam (1 mg/kg) on conditioned and unconditioned locomotion induced by morphine (2 mg/kg) or amphetamine (2 mg/kg) was assessed. Results: Rats that received conditioning injections of morphine in one environment displayed a preference for this environment. Pre-testing injections of diazepam did not alter the magnitude of this CPP. When diazepam was given with morphine during training, rats displayed a CPP for the environment paired with the two drugs. Injections of amphetamine in one environment also induced a preference for this environment. However, pre-testing injections of diazepam blocked the expression of amphetamine-induced CPP, and co-injections of diazepam blocked the formation of amphetamine CPP. Diazepam itself did not produce a CPP nor did it alter spontaneous place preferences. Diazepam equally blocked both morphine and amphetamine unconditioned and conditioned locomotor hyperactivity. This indicates that its effects on morphine and amphetamine CPP were not due to a differential effect on locomotion. Conclusions: Diazepam interferes with the reinforcing properties of amphetamines but not of morphine. The reinforcing effects of morphine and amphetamine are pharmacologically dissociable.

Electrolytic lesions of the pedunculopontine tegmental nucleus (PPTg) have been previously reported to increase anxiety-like behavior in rats. The aim of the present study was to compare these behavioral changes with those produced by an anxiogenic compound, the partial inverse agonist at benzodiazepine receptors, β-CCE. Three groups of rats, sham-lesioned treated with vehicle, sham-lesioned treated with 10 mg/kg of β-CCE, and PPTg-lesioned rats treated with vehicle, were tested in the elevated plus-maze, the social-interaction test, and for spontaneous locomotion. Histology showed that lesions were concentrated on the caudal half of the PPTg. Measures of both the PPTg-lesioned and β-CCE–treated rats indicated increased anxiety-like behavior in the elevated plus-maze and in the social-interaction test. Spontaneous locomotion, measured in the open- field arena, did not differ between sham controls and PPTg-lesioned rats, but was decreased in rats treated with β-CCE. Our results confirmed that electrolytic lesions of the caudal PPTg produce increased anxiety-like behavior. This behavior is quantitatively and qualitatively similar to that produced by 10 mg/kg of β-CCE.

A time-sampling method that allows up to eight rats to be tested simultaneously in the formalin test is described and compared to the continuous rating method. Time sampling the behavioural response to formalin every 1 or 2 min produces scores that are essentially identical to continuous rating for both the formalin concentration effect relationship and the morphine dose effect relationship, with no loss of statistical power. The most important advantage of the method is that it allows data on other aspects of the rats’ behaviour, such as behavioural state and the side effects of drugs to be scored during the formalin test. Formalin injection produces a dose-dependent decrease in locomotor and exploratory activity. The activity pattern of rats is normalized at morphine doses that produce about a 50% reduction in pain, while morphine doses high enough to completely suppress the pain response are accompanied by considerable sedation. The use of the jackknifing procedure to obtain unbiased estimates of the variability of parameters estimated from dose effect relationships is also described.

We report that a small population of neurons expresses both choline acetyltransferase and classical estrogen receptor immunoreactivity and they are found primarily in the bed nucleus of the stria terminalis. In short-term ovariectomized ageing mice (24 months, n = 5) there were 41.0 +/- 4.1% fewer of these double-labeled cells than in young (five months, n = 5) short-term ovariectomized C57BL/6J mice. To study cholinergic neuron estrogen responsiveness, young mice (n = 8) were ovariectomized at puberty (five weeks). After three months half of the mice (n = 4) were given physiological levels of 17beta estradiol for 10 days. Bed nucleus double-labeled neurons increased by 32.9% (P < or = 0.003) in the young mice given estrogen. In a gel shift assay, double-stranded oligonucleotides with putative estrogen response elements from the choline acetyltransferase gene were used as competitors against estrogen receptor binding to consensus estrogen response elements. A sequence with 60% homology to the vitellogenin estrogen response element was found to compete at 500- and 1000-fold excess. Young mice (five months) with ovaries demonstrated significantly (P < or = 0.04) better performance in the spontaneous alternation T-maze test than did old (19 month) mice with ovaries (young = 66.3 +/- 3.3% correct choices; vs old = 55.0 +/- 4.0% in old mice with ovaries). Young mice (five months old), ovariectomized for one month and treated with estrogen, showed significantly more spontaneous alternation than ovariectomized controls (69.1 +/- 2.8% vs 58.3 +/- 3.9%; P < or = 0.04). Estrogen also increased spontaneous alternation in old, short-term ovariectomized mice (61.5 +/- 2.7% vs 48 +/- 3.3%; P < or = 0.005). In either young or old ovariectomized mice, estrogen increased spontaneous alternation to levels seen in young animals with ovaries. Estrogen increases the number of choline acetyltransferase-immunoreactive and choline acetyltransferase/estrogen receptor-immunoreactive cells in old or young mice lacking estrogen, and enhances working memory in old or young mice lacking estrogen. Our data suggest that estrogen may act at the level of the choline acetyltransferase gene, but in view of the limited distribution of cholinergic cells expressing the classical estrogen receptor, it is unlikely that these cells can account for a memory enhancing effect of estrogen replacement.

Opioid-dependent humans are reported to show accelerated delay discounting of opioid rewards when compared to monetary rewards. It has been suggested that this may reflect a difference in discounting of consumable and non-consumable goods not specific to dependent individuals. Here, we evaluate the discounting of similar morphine and non-morphine oral rewards in dependent and non-dependent rats We first tested the analgesic and rewarding effects of our morphine solution. In a second experiment, we assigned rats randomly to either dependent or non-dependent groups that, 30 min after daily testing, received 30 mg/kg subcutaneous dose of morphine, or saline, respectively. Delay discounting of drug-free reward was examined prior to initiation of the dosing regimen. We tested discounting of the morphine reward in half the rats and retested the discounting of the drug-free reward in the other half. All tests were run 22.5 h after the daily maintenance dose. Rats preferred the morphine cocktail to the drug-free solution and consumed enough to induce significant analgesia. The control quinine solution did not produce these effects. Dependent rats discounted morphine rewards more rapidly than before dependence and when compared to discounting drug-free rewards. In non-dependent rats both reward types were discounted similarly. These results show that morphine dependence increases impulsiveness specifically towards a drug reward while morphine experience without dependence does not.

The present experiment examined the role of the ventral striatum in the rewarding effect of morphine and amphetamine by testing whether lesions of cell bodies within this region disrupt the development of a conditioned place preference to either drug. Bilateral, N-methyl-D-aspartate- or kainic acid-induced lesions of the ventral striatum block a conditioned place preference to amphetamine (1.5 mg/kg x 3 pairings) but not to morphine (2 mg/kg x 3 pairings). Because both lesions spared anterior portions of the ventral striatum, we examined the effect of larger or more selective ventral striatal lesions on a conditioned place preference induced by morphine. Destruction of the entire ventral striatum reduced, but did not eliminate, a conditioned place preference to morphine, whereas selective lesions of the anterior ventral striatum were ineffective. These results indicate that the ventral striatum is not critically involved in morphine's rewarding effect and support the suggestion that the rewarding effects of opiates and stimulants do not involve identical neural substrates.

The formalin test is increasingly used as a model of injury-produced pain but there is no generally accepted method of pain rating. To examine the properties of various pain rating methods we established dose-response relations for formalin injected in the plantar surface of one hind paw, and the analgesic effects of morphine and amphetamine using the most frequently reported behavioural measures of pain (favouring, lifting, licking and flinching/shaking of the injured paw) and combinations of these. Licking, elevation and favouring of the injected paw showed a biphasic response at all formalin doses. Flinching varied in form across the time course of formalin, and the biphasic nature of the behaviour was not as apparent. In untreated rats all these behaviours were infrequent. Flinching and favouring were increased after injection of local anaesthetic into the paw but remained negligible relative to the effect of formalin. Grooming other than that directed to the injected paw was elevated in a dose-dependent manner by formalin. Intercorrelations between the behaviours were different for the initial response and the second phase. Correlational analysis indicated that no single behavioural measure was a strong predictor of formalin, morphine and amphetamine dose. A simple sum of time spent licking plus elevating the paw, or the weighted pain score of Dubuisson and Dennis (1977), were superior to any single measure (r ranging from 0.75 to 0.86). Addition of flinching and favouring to the combined pain score using multiple regression did not increase variance explained. Depending on the measure used, a sedative dose of pentobarbital produced apparent analgesia, hyperalgesia or no effect. The interphase depression of pain, as well as the analgesic effects of morphine and amphetamine, were all associated with increased motor activation. Power analysis indicated that using a moderate dose of formalin and a combined pain score gave the greatest power to detect differences in pain. It was also found that pain scores increase with ambient temperature and that rat strains may differ in formalin pain sensitivity.

In the human opiate-dependent population, the most consistently reported deficit in executive functioning is impulsivity. Previous research has shown that acute and chronic exposure to drugs of abuse can increase impulsive choice; however, the extent to which opiate dependence contributes to increased impulsivity has not been examined. We report here the effects of morphine dependence on rats' delay discounting (DD) of a sucrose reward.We assigned rats randomly to either a dependent group that received a nightly 30 mg/kg subcutaneous dose of morphine or a morphine-naive group that received a nightly saline injection. DD of a sucrose reward was examined in rats prior to initiation of the dosing regimen, 22.5 h after the daily maintenance dose, and after a 14-day abstinence period.The groups did not differ at baseline, but rats showed accelerated DD while dependent on morphine. After withdrawal from morphine, DD in previously dependent rats was not significantly different from that of naive rats. Since dependent rats also showed reduced motivation to acquire the sucrose reinforcer, we performed a separate experiment to test whether such a decrease in motivation could cause an increase in impulsivity. We found that food-deprived rats switched to a free-feeding diet did not differ in DD from rats maintained at 85 % of free-feeding weight.An increase in impulsivity can result from physical dependence on morphine and cannot be attributed to changes in motivation to acquire sucrose-reinforced responses.

Publisher Summary Transgenic mouse models hold promise for elucidating the genetic basis of human pathophysiological conditions including addiction, schizophrenia, and dementia. These disorders typically involve changes in the regulation of emotion, behavioral flexibility, working memory, and decision making, behaviors that are critically dependent on different regions of the prefrontal cortex (PFC). The ability to use precise molecular genetic tools to study the cellular mechanisms underlying PFC function has resulted in a sudden increase in the use of mice in the study of brain and behavior. A very little is known about the structural organization of the mouse PFC including its morphology, and its connections with other brain regions. In addition, behavioral studies in mice depend critically on behavioral paradigms that are available to assess complex cognitive and emotional functions. The functional organization of the mouse PFC leans heavily on extrapolations from the rat PFC. This chapter reviews the anatomical and functional organization of the mouse prefrontal cortex. Consistent with the rat, anatomical staining techniques other than Nissl, such as SIM-32 and AChE clearly define boundaries between the dorsal, medial and orbital regions of the mouse PFC. Patterns of connectivity between selective PFC regions and cortical and subcortical structures reliably identifies specific cortical zones in the mouse PFC.

A mapping study was performed to determine where in the rat brain morphine acts to produce analgesia in the formalin test, which is an animal model of prolonged pain associated with tissue injury. A single dose (5 nmol) of morphine was bilaterally microinjected into a wide range of brain areas throughout the midbrain and forebrain. Strong analgesia was elicited from the posterior hypothalamic area, the periaqueductal gray and ventral tegmental area. Other sites from which analgesia was elicited were the nucleus accumbens and a few sites in the retrorubral field and caudate-putamen. Analgesia from the periaqueductal gray or nucleus accumbens was accompanied by decreased locomotor activity and catalepsy, whereas analgesia from the posterior hypothalamic area or ventral tegmentum was accompanied by a noticeable increase in locomotor activity and rearing. Morphine into various thalamic nuclei had no effect. These results indicate that the primary sites of action of morphine in the formalin test are probably the posterior hypothalamic area and periaqueductal gray, with an additional contribution from regions innervated by tegmental dopamine cells.

A stable memory can be disrupted if amnestic treatment is applied in conjunction with memory reactivation. Recent findings in the conditioned place preference (CPP) model suggest that blocking reconsolidation attenuates the ability of environmental cues to induce craving and relapse in drug addicts, but the impact of prior physical dependence has not been described. We examined the effect of post-reactivation amnestic treatment on reconsolidation of a CPP for morphine, in animals naïve to morphine, under chronic morphine experience or abstinent. Chronic morphine experience was induced by escalating doses of morphine from 10 mg/kg/day (s.c.), and maintained on 30 mg/kg/day during the course of conditioning and reactivation procedures, or conditioning alone. Naïve and morphine-experienced animals were trained in a three-compartment apparatus by four morphine (5 mg/kg, s.c.) and four saline experiences paired with either of two large conditioning compartments. The memory was then reactivated by a CPP test, and immediately afterward animals received an injection of the beta-adrenergic antagonist propranolol (10 mg/kg, s.c.), the GABAa agonist midazolam (1 mg/kg, i.p.), or saline. Morphine-naïve rats received only a single reconsolidation-blocking treatment (Experiment 1), while chronic morphine rats were given eight reactivation sessions each followed by amnestic treatment, either before (Experiment 2) or after 10 days of withdrawal (Experiment 3). Propranolol and midazolam disrupted reconsolidation in morphine-naïve rats, but failed to disrupt the CPP when rats were trained under chronic morphine treatment, even if they were recovered from chronic opiate exposure before reactivation. In fact, propranolol increased the preference for the drug-paired context in animals trained while maintained on chronic morphine. Midazolam had little effect. Morphine experience may produce neurochemical changes which alter memory storage processes and reduce the impact of amnestic treatments on reconsolidation.

The development of a conditioned place preference to morphine (2 mg/kg; three pairings) and the analgesic effect of morphine (0, 4 or 8 mg/kg) in the formalin test were studied in rats with sham or neurotoxin lesions of the pedunculopontine tegmental nucleus. Lesions were induced by bilateral infusions of N-methyl- d -aspartate (0.5 μ1 of 0.1 M solution) or vehicle over 10 min. No anti-seizure medication was administered in the first experiment, whereas animals in the second experiment were injected with diazepam (1 mg/kg) immediately after surgery. In Experiment 1, behaviour in the conditioned place preference and formalin tests was assessed in separate groups of lesioned and control rats. In Experiment 2, the same animals received both sets of tests. In both experiments lesions of the pedunculopontine tegmental nucleus blocked the development of a conditioned place preference to morphine, but had no effect on the behavioural response to formalin, or on its inhibition by morphine. Examination of cholinergic-stained cells found no correlation between the magnitude of behavioural effects and the number of acetylcholine cells destroyed by the lesions. These results confirm that the pedunculopontine tegmental nucleus mediates the development of a morphine-induced conditioned place preference, but not the analgesic effect of morphine.

Previously consolidated memories may become labile when they are reactivated and require reconsolidation. It has been suggested that when novel information is present at the time of memory reactivation reconsolidation is engaged but when no new information is present, reconsolidation may not occur, and extinction may be the dominant process instead. To test this idea we trained rats to associate a context with the rewarding properties of morphine (5 mg/kg, sc) over four conditioning pairings. Following training, animals were reactivated by a 30-min test session, once a day for 3 days. Rats were injected with the amnestic drug propranolol (10 or 40 mg/kg, sc) following reactivation either on the first or on the second day. They received saline on the alternate day. Propranolol disrupted reconsolidation for a conditioned place preference only when given on the first reactivation day, and this effect was more robust following the higher dose of propranolol. In contrast, animals given propranolol on the second reactivation day still displayed a preference for the morphine-paired context on the final test day. These results support the view that for memory to return to a labile state, the situation that evokes reactivation needs to be novel in some way. If the reactivation situation is familiar, reconsolidation may not occur.

Subcutaneous injection of dilute formalin induces pain in humans and behaviors indicative of pain in animals. The formalin test, which is based on these observations, is now widely used as a model of pain produced by tissue injury, but the neural mechanisms of pain and analgesia in this test have not been identified. Rats with transections of the brain rostral or caudal to the pons show behavioral reactions to formalin similar to those of normal rats, although the temporary abatement of pain 10-15 min after formalin is absent in transected animals. Doses of morphine that suppress the behavioral response to formalin in normal rats are not antinociceptive in the formalin test in decerebrate rats although sedation, catalepsy and inhibition of the tail-flick reflex still occur. These results suggest that the response to formalin is organized in the brain stem but the antinociceptive effect of morphine in this test is mediated by the diencephalon or forebrain.

The rewarding effects of brain stimulation and drugs are believed to depend on a common neural system. However, the pattern of responding produced by drug reinforcers is different from the pattern produced by conventional brain stimulation. Furthermore, pharmacological antagonists of reinforcement increase the rate of drug self-administration but depres self-stimulation. To test the hypothesis that the differences in the characteristics of brain stimulation and drugs as reinforcers are due to differences in the kinetics of drugs and brain stimulation, we modelled drug kinetics with frequency-modulated trains of brain stimulation. We report that animals will self-administer such brain stimulation in a manner that resembles drug self-administration and that, under these conditions, dopamine antagonists can increase the rate of self-stimulation.

Reactivation of memories may render them labile and subject to disruption by amnestic drugs thus reducing their impact on future behavior, but whether it is possible with well-established memories is not known. Here we examined the effect of two amnestic agents on reconsolidation of a conditioned place preference (CPP) for morphine when memory strength and memory age were varied. In a three-compartment apparatus animals received 4 or 8 experiences of morphine in one compartment and saline in the alternative compartment. The memory was then reactivated drug-free, and immediately afterwards animals received an injection of propranolol (10mg/kg, SC), midazolam (1mg/kg, IP), both amnestic agents combined, or saline. Animals conditioned with 4 pairings were re-tested 2 and 7 days after reactivation. After conditioning with 8 drug experiences memories were reactivated and treated 8 times, once every 48h, beginning 1 or 30 days after training. Propranolol, midazolam and their combination, disrupted reconsolidation for weak memories (4 pairings), but had little effect on stronger memories (8 pairings) reactivated 1 day after training. Extending the reactivation-amnestic treatments to 8 sessions did not disrupt the strong memory. Delaying reactivation sessions by 30 days enabled all three amnestic treatments to disrupt reconsolidation. Repeating amnestic treatment appeared to increase the effect of midazolam, but combining propranolol and midazolam did not enhance the amnestic effect. The amount of training and the age of the memory may be boundary conditions for reconsolidation.

The performance of an antiserum to progesterone and pregnane neurosteroids was assessed in two competitive assay setups: radioimmunoassay and enzyme-linked immunoassay with colorimetric detection, both with the same limit of detection of 2 pg. The enzyme-linked immunoassay was less labor-intensive and had better precision of measurement and was used to measure progesterone and six of its ring A-reduced metabolites in rat plasma. The measured levels of allopregnanolone and progesterone were in agreement with those reported previously when measured by gas chromatography/mass spectrometry and high-performance liquid chromatography coupled with radioimmunoassay and substantially lower than those previously measured by radioimmunoassay without chromatographic separation. Both isomers of dihydroprogesterone and all four isomers of pregnanolone were detected in rat plasma, indicating that progesterone is metabolized by reduction at the C5 and C3 position of the A ring, in both alpha and beta configurations. In addition to 5beta-dihydroprogesterone and isopregnanolone, which have not been previously detected in the rat, we found considerable amounts of pregnanolone, which is neuroactive, with similar potency to that of allopregnanolone but was previously thought not to be produced in rats.

The neurosphere assay can detect and expand neural stem cells (NSCs) and progenitor cells, but it cannot discriminate between these two populations. Given two assays have purported to overcome this shortfall, we performed a comparative analysis of the distribution and frequency of NSCs and progenitor cells detected in 400 μm coronal segments along the ventricular neuraxis of the adult mouse brain using the neurosphere assay, the neural colony forming cell assay (N-CFCA), and label-retaining cell (LRC) approach. We observed a large variation in the number of progenitor/stem cells detected in serial sections along the neuraxis, with the number of neurosphere-forming cells detected in individual 400 μm sections varying from a minimum of eight to a maximum of 891 depending upon the rostral-caudal coordinate assayed. Moreover, the greatest variability occurred in the rostral portion of the lateral ventricles, thereby explaining the large variation in neurosphere frequency previously reported. Whereas the overall number of neurospheres (3730 ± 276) or colonies (4275 ± 124) we detected along the neuraxis did not differ significantly, LRC numbers were significantly reduced (1186 ± 188, 7 month chase) in comparison to both total colonies and neurospheres. Moreover, approximately two orders of magnitude fewer NSC-derived colonies (50 ± 10) were detected using the N-CFCA as compared to LRCs. Given only 5% of the LRCs are cycling (BrdU+/Ki-67+) or competent to divide (BrdU+/Mcm-2+), and proliferate upon transfer to culture, it is unclear whether this technique selectively detects endogenous NSCs. Overall, caution should be taken with the interpretation and employment of all these techniques. Disclosure of potential conflicts of interest is found at the end of this article.

Rats received bilateral infusions of 6-hydroxydopamine into the olfactory tubercle, resulting in a substantial (89%) local depletion of dopamine. This treatment did not significantly alter conditioned place preference produced by (+)-amphetamine, suggesting that the mesolimbic dopaminergic projection to olfactory tubercle does not mediate the rewarding effects of (+)-amphetamine in rats.

The present study investigated the effect of acute systemic administration of six progesterone metabolites on formalin-induced pain in the rat. The 3alpha-hydroxylated metabolites allopregnanolone and pregnanolone are highly potent positive modulators at the GABA(A) receptor and produced a biphasic effect on pain in the formalin test. Dose-dependent antinociception was observed at lower doses (maximal antinociception at 0.16mg/kg) and was reversed at higher doses. Bicuculline abolished the antinociceptive effect. The 3beta-hydroxylated epipregnanolone and isopregnanolone are inactive or only weekly active at the GABA(A) receptor, and did not affect formalin-induced pain. 5alpha- and 5beta-dihydroprogesterone have also been shown to have low affinity for the GABA(A) receptor, but can be rapidly metabolized to their 3alpha-hydroxylated counterparts. In the formalin test, they produced a biphasic effect on pain similar to that of pregnanolone and allopregnanolone, but with lower potency. The effect was reversible by bicuculline, showing involvement of the GABA(A) receptor, and was blocked by indomethacin, implying that the antinociceptive effect is dependent on their conversion to allopregnanolone or pregnanolone. The results indicate that GABA-ergic progesterone metabolites modulate nociception. A change in levels of GABA-ergic progesterone metabolites, such as is observed in depression, chronic fatigue and premenstrual dysphoric disorder could, therefore, contribute to the pain complaints associated with these disorders.

Drugs of abuse, such as amphetamine and morphine, produce reward-related shifts on intracranial self-stimulation (ICSS) thresholds. The facilitatory effects on ICSS thresholds of drugs that act through the GABAergic system, however, are reported to be attributed to their antiseizure and anticonvulsant effects, rather than their reinforcing effects. Using a rate-frequency ICSS paradigm, we examined the effects of amphetamine (a reinforcing drug of abuse that acts via the catecholaminergic system), pentobarbital (a GABA(A) receptor agonist and reinforcing barbiturate with anticonvulsant properties), and gabapentin (a nonspecific GABAergic agonist and anticonvulsant with low abuse potential) on ICSS M(50) indices. All three doses of amphetamine (0.5, 1.0, and 2.0 mg/kg) and pentobarbital (2.5, 5.0, and 10.0 mg/kg) significantly lowered rate-frequency M(50) values. Gabapentin, on the other hand, significantly raised rate-frequency M(50) values, albeit only at the highest dose administered (30 mg/kg). Our results indicate that shifts in ICSS M(50) values produced by pentobarbital are associated with the reinforcing, not the anticonvulsant, effect of pentobarbital. These results are consistent with the view that there is a common system underlying the reinforcing effects of drugs and ICSS reinforcement, and suggest that the reinforcing and anticonvulsant effects of GABA agonists are dissociable.

There has been controversy as to whether the contribution of descending fibers from the rostral ventromedial medulla to opioid analgesia depends on the nature of the noxious stimulus eliciting pain. In the present study, inactivation of descending fibers by microinjection of muscimol (50 ng) in the rostral ventromedial medulla abolished morphine analgesia in the tail immersion and hot plate tests but decreased morphine analgesia by 60% in the formalin test. Analysis of the dose-response relation for morphine after inactivation of descending fibers revealed that, except for the tail immersion test, high doses of morphine could not overcome the block induced by muscimol. Also, morphine analgesia elicited supraspinally was not detectable when descending fibers were inactivated, suggesting that the analgesic effect of morphine in the brain requires a relay via the rostral ventromedial medulla. The analgesic effect of buprenorphine also depends on the integrity of descending fibers from the rostral ventromedial medulla. The results indicate that descending fibers from the rostral ventromedial medulla are critically important to the analgesic effect of opioids, regardless of the type of noxious stimulation eliciting pain. Residual analgesic effects of opioids after inactivation of descending fibers may be due to peripheral effects in the presence of inflammation.

Rationale: Drugs that are self-administered usually produce a conditioned place preference (CPP) but pentobarbital is self-administered by both animals and humans and is reported to be aversive in the CPP test. Objectives: We tested whether pentobarbital (5, 15, and 25 mg/kg; IP) could produce a place preference and examined the role of GABA, dopamine (DA) and opioid receptors in the pentobarbital CPP. Methods: Place conditioning was carried out in an apparatus consisting of two compartments connected by an alley at the rear. During the pre-exposure and test phase, the rats were free to wander in the apparatus for 20 min, and were drug-free. During the 6-day conditioning phase, rats were injected with drug (or vehicle), and confined to one compartment for 30 min and on alternate days were injected with vehicle (or drug) and confined to the other compartment. Upon obtaining a CPP, we examined whether pretreatment of the GABAA antagonists picrotoxin or bicuculline (0.5, 1.0, and 2.0 mg/kg; IP), the DA antagonist eticlopride (0.01, 0.05, and 0.25 mg/kg; SC), or the opioid antagonist naloxone (0.02, 0.20, and 2.0 mg/kg; IP) would block the CPP. Results: 15 mg/kg pentobarbital produced a CPP. The pentobarbital CPP was blocked by pretreatment of 1.0 and 2.0 mg/kg bicuculline, but not by 0.5, 1.0, or 2.0 mg/kg picrotoxin. The pentobarbital CPP was also blocked by 0.05 and 0.25 mg/kg eticlopride and by all of the doses of naloxone tested. Conclusions: Pretreatment with the antagonists bicuculline, eticlopride, and naloxone blocked a 15 mg/kg pentobarbital CPP. This indicates that GABAergic, dopaminergic, and opioid systems play a role in the reinforcing properties of pentobarbital.

We have examined the effects of muscimol and bicuculline microinjected in the rostral ventromedial medulla (RVM) on motor function and on nociception in three pain tests. In Exp. 1 microinjection of muscimol (6.25-400 ng in 1 microl) in the RVM dose-dependently decreased pain threshold of rats and the ED(50) for muscimol was the same in both the hot plate and tail immersion pain tests. In the hot plate test, but not in the tail immersion test, paw withdrawal latencies increased again with high doses of muscimol (75-400 ng). High doses also produced catalepsy. Exp. 2 examined the effects of muscimol (50 ng) and bicuculline (50 ng) over a range of formalin concentrations (0.25-4%) in the formalin test. Muscimol increased responsiveness to formalin and reduced the slope of the formalin dose-response relation. Bicuculline decreased responses to formalin and reduced the slope of the formalin dose-response relation. It is suggested that RVM cells with inhibitory projections to the dorsal horn are not subject to strong GABAergic influence under mild noxious stimulation. RVM cells are thus active, and spinal dorsal horn relay neurons are inhibited. On the other hand, intense noxious peripheral stimulation may stimulate the release of GABA onto RVM cells, which in turn shuts off descending inhibitory fibers to allow transmission of nociceptor input through the dorsal horn.

We have previously shown that diazepam blocks both the formation and the expression of amphetamine-induced conditioned place preference (CPP), but has no effect on the CPP induced by morphine. Because diazepam reduces dopamine activity in the nucleus accumbens, three experiments were conducted in order to investigate whether diazepam selectively blocks the expression of place conditioning dependent on mesolimbic dopamine. The first experiment found that systemic diazepam blocked the expression of conditioned place aversion induced by the kappa-receptor agonist U50-488. The second and third experiments demonstrated that intra-cranial injections of diazepam in the nucleus accumbens blocked the expression of amphetamine CPP but not of morphine CPP. It is concluded that diazepam interferes with mesolimbic dopamine-dependent motivational effects of drugs.

Bilateral N-methyl-d-aspartate lesions of the pedunculopontine tegmental nucleus (PPTg) blocked the acquisition of a delayed non-matching to position task (DNMP) performed in a T-maze. This acquisition impairment, however, was reversed by pre-testing injections of diazepam (1 mg/kg). These results, in addition to the finding that PPTg lesions elevated anxiety as measured by the elevated plus maze, suggest that PPTg is not involved in learning or memory, but in the regulation of anxiety.

To test the hypothesis that the pedunclopontine tegmental nucleus (PPTg) plays a role in anxiety, rats with electrolytic lesions of the PPTg were tested in two animal models of anxiety, the elevated plus maze and the social interaction tests. Histological analysis showed that lesions were concentrated on the caudal half of the PPTg. In the elevated plus maze, traditional as well as 'risk assessment' measures indicated significantly increased anxiety in the PPTg-lesioned rats vs sham-operated controls. In the social interaction test, behavioural scores were biased towards elevated anxiety in the PPTg-lesioned rats but only the number of social sniffs reached significance. Our results confirm that electrolytic lesions of the caudal PPTg produce increased anxiety.

The pedunculopontine tegmental nucleus (PPTg) is believed to play important roles in reward and learning. We examined the effect of PPTg lesions (0.5 μl of 0.1mNMDA injected bilaterally over 10 min) on the learning of an operant response for opiate reward. In 14 adult male Long–Evans rats, bilateral lesions of the PPTg disrupted the acquisition of responding for intravenous heroin (0.1 mg/kg infused at a rate of 0.25 ml/28 sec) on a fixed ratio-1 (FR-1) schedule of reinforcement. The 12 remaining lesioned animals increased their heroin intake over the acquisition sessions but did not reach the response levels of sham-lesioned animals on the 15th and final session. The sham- and PPTg-lesioned animals that learned the FR-1 task exhibited similar patterns of responding during extinction and reacquisition sessions. When tested on a progressive ratio (PR) schedule of reinforcement, however, PPTg-lesioned animals had lower break points than sham-lesioned animals. Asymmetric lesions, which destroyed the majority of the nucleus in one hemisphere only, did not produce any behavioral deficits. Rats that were lesioned after training also did not show deficits in responding under either FR or PR schedules. These findings suggest that PPTg lesions reduce the rewarding effect of opiates but do not disrupt the ability either to learn an operant response or the response requirements of a PR schedule.

The fact that centrally acting analgesics have abuse potential commensurate with their analgesic activity raises the question of whether these effects are related. The abuse potential of drugs depends on their ability to produce reinforcing effects, which are mediated by a neural system that includes the ventral tegmental dopamine cells and their connections with the ventral striatum. Morphine and amphetamine are both powerful analgesics and have high abuse potential. Their analgesic and reinforcing effects are mediated by similar receptors, similar sites of action, and overlapping neural substrates. These coincidences suggest that reinforcers may produce analgesia by transforming the aversive affective state evoked by pain into a more positive affective state. The implications of this hypothesis and its relation to other known mechanisms of analgesia are discussed. The hypothesis predicts that drugs with reinforcing effects should produce analgesia. A survey of drugs acting through 21 classes of receptors reveals that in 13 classes there is evidence for both analgesic and reinforcing effects that are approximately equipotent. The GABA(A) agonists were found to be the only drugs with confirmed abuse potential that lack analgesic activity. The interpretation of this and several other anomalous cases is discussed.

Bilateral microinjection of 5 nmol morphine into the posterior hypothalamic area (PHA), periaqueductal gray matter (PAG) or ventral tegmental area (VTA) elicits powerful suppression of nociceptive behaviors in the formalin test, an animal model of injury produced pain. The object of the present study was to determine whether analgesia in the formalin test (50 microl 2.5% formalin injected s.c. in one hindpaw) induced by systemically administered morphine requires opioid action at these sites, or other putative sites of opioid action. Morphine sulphate (6 mg/kg s.c.) produced almost complete analgesia in the second phase of the formalin test (30-50 min after formalin). Bilateral microinjection of the quaternary opioid antagonist naloxone methobromide (NxBr, 28 ng in 0.5 microl, 22 min after morphine) into the PHA completely abolished morphine analgesia, while NxBr into PAG partially reversed analgesia. Microinjection of NxBr into the VTA, central nucleus of the amygdala, habenula, striatum, nucleus accumbens or hypothalamic sites outside the PHA did not antagonize morphine analgesia, although microinjections into some of these sites appeared to reduce the cataleptogenic effects of morphine. The data indicate that the PHA and PAG are probably the primary sites of action of morphine in the formalin test.

There has been conflicting evidence concerning the role of descending bulbospinal influences on pain and opioid analgesia in the formalin test. We examined the effect of lesions of the dorsolateral funiculus (DLF) on dose-effect relations for formalin and morphine in the formalin test. Experiment 1 showed that DLF lesions reduced the effect of 5 mg/kg morphine on pain in the tail-flick test, and eliminated morphine's effect on pain produced by 2.5% formalin. When lower concentrations of formalin were used, DLF lesions produced hyperalgesia, indicated by a left shift (1.7-fold) of the formalin concentration-response curve. In experiment 2, DLF lesions increased the pain produced by 1.25% formalin and shifted the dose-response relation for the effect of morphine on the second phase of the pain response produced by 1.25% formalin to 2.5-fold higher doses. The data show that DLF lesions increase sensitivity to the pain-inducing effect of formalin, and this accounts for a substantial component of the effect of DLF lesions on morphine analgesia.

Excitotin lesions of the pedunculopontine tegmental nucleus have been found to block the acquisition of a conditioned place preference induced by morphine or amphetamine, and it has been suggested that such lesions may attenuate the primary reinforcing effects of these drugs and, possibly, other reinforcers. The present study examined the effects of pedunculopontine lesions on the reinforcing effects of brain stimulation. N -methyl- d -aspartate-induced lesions of the pedunculopontine nucleus prevented spontaneous acquisition of lever pressing for brain stimulation reinforcement during five daily 1 h sessions of training. The effective lesions damaged the retrorubral fields in addition to the pedunculopontine tegmental nucleus. N -methyl- d -aspartate (25 or 50 nmol) lesions of the retrorubral fields did not block acquisition of self-stimulation, however, controls reached their maximum rate of responding in the first session, responding of rats with retrorubral field lesions gradually increased over five days. When excitotoxin-induced lesions of the pedunculopontine nucleus were made after acquisition of self-stimulation, lesioned rats continued to respond to brain stimulation, but at a lower rate than controls. The results show that pedunculopontine lesions interfere with the learning and expression of a response reinforced by brain stimulation just as they block learning motivated by drugs and natural rewards. They also suggest that collateral damage to the retrorubral fields may contribute to the effects of pedunculopontine lesions on reinforced behaviour. These data support the view that the pedunculopontine tegmental nucleus is involved in the process by which reinforcers control purposive behaviour.

Partially decorticated rats were tested for their response to nociceptive stimulation in the formalin and tail flick tests, and for the effect of morphine on these responses and on motor activity. Undrugged rats showed vigorous responses to nociceptive stimulation in both tests, and exhibited the typical biphasic time course of pain in the formalin test. Morphine 4 and 8 mg/kg produced dose-dependent analgesia in both tests in sham operated rats, and in rats with lesions that removed all or part of the cortex from the midline to the rhinal fissure (excluding the occipital cortex). In rats with lesions that extended deep into the piriform cortex and damaged the amygdala morphine analgesia was eliminated or attenuated. These and other recent findings suggest that analgesia in the formalin test depends on ascending connections to the forebrain, probably the amygdala.

The cataleptic and locomotor depressant effects of morphine are mediated by a series of neural structures, all of which project to the pedunculopontine tegmental nucleus (PPTg). To test the idea that the PPTg is also involved in mediating morphine's effect on these behaviours, we examined catalepsy and spontaneous motor activity following bilateral lesions of the PPTg (0.5 μl of 0.1 M N- methyl- d -aspartate infused over 10 min). We also examined the effect of PPTg lesions on motor functioning by observing sensorimotor responses, limb use, muscle tone and locomotion. PPTg lesions completely abolished the catalepsy and decreased activity that normally follows morphine administration. In addition lesioned animals exhibited increased muscle tone, and impairments in limb use and righting reflexes. Although the deficits were subtle, these results confirm that damage to the PPTg is associated with motor abnormalities.

It has been proposed that the positive reinforcing and motor stimulating effects of drugs involve the activation of a common neural substrate. Reinforcing effects of food, drugs and brain stimulation are blocked by lesions of the pedunculopontine tegmental nucleus (PPTg), which is a component of the mesencephalic locomotor region. This has suggested that the PPTg may be involved in both positive reinforcement and forward locomotion. In four separate experiments, rats were prepared with NMDA (0.5 μ1 of 0.1 M solution) or sham lesions of the PPTg. Animals in the first two experiments were tested for the development of a conditioned place preference (CPP) to morphine (2 mg/kg × 3 pairings) or amphetamine (1.5 mg/kg × 3 pairings). Ten days later, spontaneous motor activity (SMA) was assessed in these animals following a subcutaneous injection of saline or amphetamine (1.5 mg/kg). In two further experiments, drug-naive lesioned and control animals were tested for SMA only (saline or 1.5 mg/kg amphetamine in Experiment 3, and saline, 0.5 mg/kg, or 3 mg/kg amphetamine in Experiment 4). Lesions of the PPTg blocked the development of a CPP to both morphine and amphetamine. In contrast, lesions had no effect on saline or amphetamine-stimulated SMA. The PPTg, therefore, appears to be involved in the reinforcing effects of amphetamine and morphine, but is not necessary for the expression of amphetamine-induced activity.

There are two phases to the behavioral response to injection of formalin. After an initial vigorous response, a period of reduced pain occurs 10 to 15 min after formalin, followed by reemergence of pain-related behaviors. These phases are believed to represent acute chemical stimulation of afferent neurons followed by injury-related inflammatory pain. Pentobarbital (10, 15, or 25 mg/kg), diazepam (0.5, 1.5, or 5.0 mg/kg), or ethanol (0.5, 1.0, or 1.5 g/kg) attenuated the diminution of pain between the two phases, so that pain was continuous throughout 60 min of testing, but had no effect on pain scores during the peaks of either phase. The effects of pentobarbital and diazepam were blocked by picrotoxin (2.5 mg/kg), which itself had no effect. Ro 15-1788 also blocked the effect of diazepam. Picrotoxin did not effectively antagonize the effect of ethanol. A high dose of picrotoxin (5.0 mg/kg) caused seizures in some rats and also eliminated the interphase depression of pain. The results suggest that the biphasic time course of formalin pain is produced by a central antinociceptive mechanism that is inhibited by GABAA receptors.