Chronic nicotine improves short-term memory selectively in a G72 mouse model of schizophrenia.

BACKGROUND AND PURPOSE: The prevalence of smoking in schizophrenia patients is exceptionally high; it is not known why but many researchers suggest that smoking constitutes a form of self-medication. Among the symptoms of schizophrenia that may be improved by nicotine are cognitive deficits. Hence, we studied the effects of long-term nicotine administration on cognition in a genetic animal model of schizophrenia susceptibility, G72-transgenic (G72Tg) mice. EXPERIMENTAL APPROACH: The effect of long-term nicotine or saline, administered by osmotic minipumps, on different cognitive domains was assessed in G72Tg mice and controls using a battery of behavioural tests. To investigate the mechanism underlying phenotypic differences, quantitative autoradiographic mapping of nACh receptor subtypes was performed in forebrain structures to explore effects of chronic nicotine exposure on nACh receptor density in wild-type (WT) and G72Tg mice. KEY RESULTS: Genotype significantly affected the cognitive effects of chronic nicotine administration. Whereas chronic nicotine disrupted cognitive performance in WT mice, it was effective at restoring impaired prepulse inhibition, working memory and social recognition in G72Tg mice. However, long-term spatial learning was further impaired by nicotine in transgenic animals. In contrast, associative learning was protected by G72-expression against the adverse nicotine effects seen in WT animals. G72-expression did not decisively influence nicotine-induced up-regulation of the α4ß2*subtype, whereas α7nACh receptor density was differentially altered by genotype or by a genotype·treatment interaction in specific brain areas, most notably hippocampal subregions. CONCLUSIONS AND IMPLICATIONS: Our data support the hypothesis that nicotine self-medication of schizophrenics improves cognitive symptoms, possibly by facilitating nicotine-induced α7nACh receptor activation in the hippocampus.

The stereotypy-inducing and OCD-like effects of chronic 'binge' cocaine are modulated by distinct subtypes of nicotinic acetylcholine receptors.

BACKGROUND AND PURPOSE: High rates of cigarette smoking occur in cocaine-dependent individuals, reflecting an involvement of nicotinic acetylcholine receptors (nAChRs) in cocaine-elicited behaviour. This study was designed to assess the contribution of different nAChR subtypes to the behavioural and neurochemical effects of chronic cocaine treatment. EXPERIMENTAL APPROACH: Cocaine (15 mg·kg(-1) , i.p.) was administered to male C57BL/6J mice in a chronic 'binge' paradigm, with and without the coadministration of the α7 preferring nAChR antagonist methyllycaconitine (MLA; 5 mg·kg(-1) , i.p.) or the ß2* nAChR antagonist dihydro-ß-erythroidine (DHßE; 2 mg·kg(-1) , i.p.). Quantitative autoradiography was used to examine the effect of cocaine exposure on α7 and α4ß2* nAChRs, and on the high-affinity choline transporter. KEY RESULTS: MLA+cocaine administration induced an intense self-grooming behaviour, indicating a likely role for α7 nAChRs in modulating this anxiogenic, compulsive-like effect of cocaine. In the major island of Calleja, a key area of action for neuroleptics, MLA+cocaine reduced choline transporter binding compared with cocaine (with or without DHßE) administration. DHßE treatment prevented the induction of stereotypy sensitisation to cocaine but prolonged locomotor sensitisation, implicating heteromeric ß2* nAChRs in the neuroadaptations mediating cocaine-induced behavioural sensitisation. 'Binge' cocaine treatment region-specifically increased α4ß2* nAChR binding in the midbrain dopaminergic regions: ventral tegmental area and substantia nigra pars compacta. CONCLUSIONS AND IMPLICATIONS: We have shown a differential, subtype-selective, contribution of nAChRs to the behavioural and neurochemical sequelae of chronic cocaine administration. These data support the clinical utility of targeting specific nAChR subtypes for the alleviation of cocaine-abuse symptomatology.

Deletion of the adenosine A(2A) receptor in mice enhances spinal cord neurochemical responses to an inflammatory nociceptive stimulus.

Knockout mice lacking the adenosine A(2A) receptor are less sensitive to nociceptive stimuli, and this may be due to the presence of pronociceptive A(2A) receptors on sensory nerves. In support of this hypothesis, we have recently shown that in A(2A) receptor knockout mice there are marked reductions in the changes of two markers of spinal cord neuronal activity, [(3)H]MK801 binding to NMDA receptors and uptake of [(14)C]-2-deoxyglucose, in response to formalin injection. We now report that following a more prolonged inflammatory stimulus, consisting of intraplantar injections of PGE(2) and paw pressure, there was in contrast an increase in [(3)H]MK801 binding and [(14)C]-2-deoxyglucose uptake in the spinal cords of the A(2A) receptor knockout mice which was much greater than in the wild-type mice. This increase suggests that when there is a pronounced inflammatory component to the stimulus, loss of inhibitory A(2A) receptors on inflammatory cells outweighs the loss of pronociceptive A(2A) receptors on peripheral nerves so that overall there is an increase in nociceptive signalling. This implies that although A(2A) antagonists have antinociceptive effects they may have only limited use as analgesics in chronic inflammatory pain.

Increased desensitization of dopamine D2 receptor-mediated response in the ventral tegmental area in the absence of adenosine A(2A) receptors.

G-protein coupled receptors interact to provide additional regulatory mechanisms for neurotransmitter signaling. Adenosine A(2A) receptors are expressed at a high density in striatal neurons, where they closely interact with dopamine D2 receptors and modulate effects of dopamine and responses to psychostimulants. A(2A) receptors are expressed at much lower densities in other forebrain neurons but play a more prominent yet opposing role to striatal receptors in response to psychostimulants in mice. It is, therefore, possible that A(2A) receptors expressed at low levels elsewhere in the brain may also regulate neurotransmitter systems and modulate neuronal functions. Dopamine D2 receptors play an important role in autoinhibition of neuronal firing in dopamine neurons of the ventral tegmental area (VTA) and dopamine release in other brain areas. Here, we examined the effect of A(2A) receptor deletion on D2 receptor-mediated inhibition of neuronal firing in dopamine neurons in the VTA. Spontaneous activity of dopamine neurons was recorded in midbrain slices, and concentration-dependent effects of the dopamine D2 receptor agonist, quinpirole, was compared between wild-type and A(2A) knockout mice. The potency of quinpirole applied in single concentrations and the expression of D2 receptors were not altered in the VTA of the knockout mice. However, quinpirole applied in stepwise escalating concentrations caused significantly reduced maximal inhibition in A(2A) knockout mice, indicating an enhanced agonist-induced desensitization of D2 receptors in the absence of A(2A) receptors. The A(2A) receptor agonist, CGS21680, did not exert any effect on dopamine neuron firing or response to quinpirole, revealing a novel non-pharmacological interaction between adenosine A(2A) receptors and dopaminergic neurotransmission in midbrain dopamine neurons. Altered D2 receptor desensitization may result in changes in dopamine neuron firing rate and pattern and dopamine release in other brain areas in response to persistent dopamine release and administration of psychostimulants.

Genetic deletion of the adenosine A(2A) receptor in mice reduces the changes in spinal cord NMDA receptor binding and glucose uptake caused by a nociceptive stimulus.

Mice lacking the adenosine A(2A) receptor are less sensitive to nociceptive stimuli, and A(2A) receptor antagonists have antinociceptive effects. We have previously shown a marked reduction in the behavioural responses to formalin injection in A(2A) receptor knockout mice. This may be due to the presence of pronociceptive A(2A) receptors on sensory nerves, and if so spinal cords from A(2A) receptor knockout mice may have altered neurochemical responses to a nociceptive stimulus. We tested this hypothesis by studying two parameters known to change with spinal cord activity, NMDA glutamate receptor binding and [(14)C]-2-deoxyglucose uptake, following intraplantar formalin injection in wild-type and A(2A) receptor knockout mice. In naïve untreated A(2A) knockout mice [(14)C]-2-deoxyglucose uptake in all regions of the spinal cord was significantly lower compared to the wild-type, similar to the reduced NMDA receptor binding that we have previously observed. Following formalin treatment, there was an decrease in [(3)H]-MK801 binding to NMDA receptors and an increase in [(14)C]-2-deoxyglucose uptake in the spinal cords of wild-type mice, and these changes were significantly reduced in the A(2A) knockout mice. In addition to altered behavioural responses, there are therefore corresponding reductions in spinal cord neurochemical changes induced by formalin in mice lacking adenosine A(2A) receptors. These observations support the hypothesis that activation of A(2A) receptors enhances nociceptive input into the spinal cord and suggests a possible role for A(2A) antagonists as analgesics.

Decrease of D2 receptor binding but increase in D2-stimulated G-protein activation, dopamine transporter binding and behavioural sensitization in brains of mice treated with a chronic escalating dose 'binge' cocaine administration paradigm.

Understanding the neurobiology of the transition from initial drug use to excessive drug use has been a challenge in drug addiction. We examined the effect of chronic 'binge' escalating dose cocaine administration, which mimics human compulsive drug use, on behavioural responses and the dopaminergic system of mice and compared it with a chronic steady dose (3 x 15 mg/kg/day) 'binge' cocaine administration paradigm. Male C57BL/6J mice were injected with saline or cocaine in an escalating dose paradigm for 14 days. Locomotor and stereotypy activity were measured and quantitative autoradiographic mapping of D(1) and D(2) receptors, dopamine transporters and D(2)-stimulated [(35)S]GTPgammaS binding was performed in the brains of mice treated with this escalating and steady dose paradigm. An initial sensitization to the locomotor effects of cocaine followed by a dose-dependent increase in the duration of the locomotor effect of cocaine was observed in the escalating but not the steady dose paradigm. Sensitization to the stereotypy effect of cocaine and an increase in cocaine-induced stereotypy score was observed from 3 x 20 to 3 x 25 mg/kg/day cocaine. There was a significant decrease in D(2) receptor density, but an increase in D(2)-stimulated G-protein activity and dopamine transporter density in the striatum of cocaine-treated mice, which was not observed in our steady dose paradigm. Our results document that chronic 'binge' escalating dose cocaine treatment triggers profound behavioural and neurochemical changes in the dopaminergic system, which might underlie the transition from drug use to compulsive drug use associated with addiction, which is a process of escalation.

Behavioural and biochemical responses to morphine associated with its motivational properties are altered in adenosine A(2A) receptor knockout mice.

BACKGROUND AND PURPOSE: The purinergic system through the A(2A) adenosine receptor regulates addiction induced by different drugs of abuse. The aim of the present study was to investigate the specific role of A(2A) adenosine receptors (A(2A)Rs) in the behavioural and neurochemical responses to morphine associated with its motivational properties. EXPERIMENTAL APPROACH: Mice lacking A(2A)Rs (A(2A) knockout (KO) mice) and wild-type littermates were used to evaluate behavioural responses induced by morphine. Antinociception was assessed using the tail-immersion and the hot-plate tests. Place-conditioning paradigms were used to evaluate the rewarding effects of morphine and the dysphoric responses of morphine withdrawal. Microdialysis studies were carried out to evaluate changes in the extracellular levels of dopamine in the nucleus accumbens of A(2A) KO mice after morphine administration. KEY RESULTS: The acute administration of morphine induced a similar enhancement of locomotor activity and antinociceptive responses in both genotypes. However, the rewarding effects induced by morphine were completely blocked in A(2A) KO mice. Also, naloxone did not induce place aversion in animals lacking the A(2A)Rs. CONCLUSIONS AND IMPLICATIONS: Our findings demonstrate that the rewarding and aversive effects associated with morphine abstinence were abolished in A(2A) KO mice, supporting a differential role of the A(2A) adenosine receptor in the somatic and motivational effects of morphine addiction. This study provides evidence for the role of A(2A)Rs as general modulators of the motivational properties of drugs of abuse. Pharmacological manipulation of these receptors may represent a new target in the management of drug addiction.

Adaptive changes in the expression of central opioid receptors in mice lacking the dopamine D2 receptor gene.

On the basis of numerous studies that have described interactions between the dopaminergic and opioidergic systems, we have investigated whether genetic deletion of dopamine D2 receptors (D2R) might influence the expression of central opioid receptors. The levels of mu, delta, kappa and nociceptin opioid peptide receptors were determined in the brains and spinal cords of D2R knockout mice using quantitative autoradiography. The significant changes in opioid receptor binding found in the brains of heterozygous and homozygous mice were mainly restricted to the basal ganglia. In homozygous mice, a down-regulation of mu and delta receptors was observed in the striatal and pallidal areas. This alteration may be an adaptive response to the increase in enkephalin levels previously described in the striatum of these mutant mice. On the contrary, an up-regulation of kappa receptors was found in the striatal and nigral regions and might be related to a change in dynorphin levels. Significant increases in nociceptin receptor binding were also observed in homozygous mice in brain areas involved in motor behavior. At the spinal level, only kappa and nociceptin receptor binding showed significant overall differences between genotypes. The functional consequences of these adaptive changes are discussed in relation to the findings of behavioral and neurochemical studies reported to date in D2R knockout mice.

Preprodynorphin mediates locomotion and D2 dopamine and mu-opioid receptor changes induced by chronic 'binge' cocaine administration.

Evidence suggests that the kappa-opioid receptor (KOP-r) system plays an important role in cocaine addiction. Indeed, cocaine induces endogenous KOP activity, which is a mechanism that opposes alterations in behaviour and brain function resulting from repeated cocaine use. In this study, we have examined the influence of deletion of preprodynorphin (ppDYN) on cocaine-induced behavioural effects and on hypothalamic-pituitary-adrenal axis activity. Furthermore, we have measured mu-opioid receptor (MOP-r) agonist-stimulated [(35)S]GTPgammaS, dopamine D(1), D(2) receptor and dopamine transporter (DAT) binding. Male wild-type (WT) and ppDYN knockout (KO) mice were injected with saline or cocaine (45 mg/kg/day) in a 'binge' administration paradigm for 14 days. Chronic cocaine produced an enhancement of locomotor sensitisation in KO. No genotype effect was found on stereotypy behaviour. Cocaine-enhanced MOP-r activation in WT but not in KO. There was an overall decrease in D(2) receptor binding in cocaine-treated KO but not in WT mice. No changes were observed in D(1) and DAT binding. Cocaine increased plasma corticosterone levels in WT but not in KO. The data confirms that the endogenous KOP system inhibits dopamine neurotransmission and that ppDYN may mediate the enhancement of MOP-r activity and the activation of the hypothalamic-pituitary-adrenal axis after chronic cocaine treatment.

Adenosine A2A receptors in ventral striatum, hypothalamus and nociceptive circuitry implications for drug addiction, sleep and pain.

Adenosine A2A receptors localized in the dorsal striatum are considered as a new target for the development of antiparkinsonian drugs. Co-administration of A2A receptor antagonists has shown a significant improvement of the effects of l-DOPA. The present review emphasizes the possible application of A2A receptor antagonists in pathological conditions other than parkinsonism, including drug addiction, sleep disorders and pain. In addition to the dorsal striatum, the ventral striatum (nucleus accumbens) contains a high density of A2A receptors, which presynaptically and postsynaptically regulate glutamatergic transmission in the cortical glutamatergic projections to the nucleus accumbens. It is currently believed that molecular adaptations of the cortico-accumbens glutamatergic synapses are involved in compulsive drug seeking and relapse. Here we review recent experimental evidence suggesting that A2A antagonists could become new therapeutic agents for drug addiction. Morphological and functional studies have identified lower levels of A2A receptors in brain areas other than the striatum, such as the ventrolateral preoptic area of the hypothalamus, where adenosine plays an important role in sleep regulation. Although initially believed to be mostly dependent on A1 receptors, here we review recent studies that demonstrate that the somnogenic effects of adenosine are largely mediated by hypothalamic A2A receptors. A2A)receptor antagonists could therefore be considered as a possible treatment for narcolepsy and other sleep-related disorders. Finally, nociception is another adenosine-regulated neural function previously thought to mostly involve A1 receptors. Although there is some conflicting literature on the effects of agonists and antagonists, which may partly be due to the lack of selectivity of available drugs, the studies in A2A receptor knockout mice suggest that A2A receptor antagonists might have some therapeutic potential in pain states, in particular where high intensity stimuli are prevalent.

Region selective up-regulation of micro-, delta- and kappa-opioid receptors but not opioid receptor-like 1 receptors in the brains of enkephalin and dynorphin knockout mice.

The role of endogenous opioid peptides and receptors has recently been investigated using knockout mice. Although the affinities of opioid peptides for opioid receptors has been known for many years there is still some uncertainty over which receptor is the endogenous target for each peptide. To address this issue we have studied using quantitative autoradiography the levels of all four opioid receptor subtypes (micro, delta, kappa and opioid receptor-like 1 [ORL1]) in brains sectioned from enkephalin and dynorphin knockouts, as well as from double knockouts. Because receptor up-regulation has been observed when its cognate ligand-peptide is genetically ablated, regional changes in receptor binding in knockout mice may reflect areas where the peptide ligand is tonically active at its receptor or played a role in receptor regulation. In addition, the study aimed to correlate previously observed behaviour in these animals with receptor modulation. Marked region-specific up-regulation of the micro, delta, and kappa opioid receptors but not ORL1 receptors was observed in proenkephalin and prodynorphin knockouts. In proenkephalin knockouts this was most pronounced for the micro- and delta-receptor and in prodynorphin knockouts for the kappa-receptor. Combinatorial double knockouts did not show any changes in addition to those observed in single knockouts. The largest changes were observed in limbic regions and our results suggest that proenkephalin peptides are tonically active at micro and delta-receptors predominantly in these areas. Prodynorphin peptides appear to regulate mostly the kappa-receptor but they are also modulators of micro- and delta-receptors.

Nociceptin/orphanin FQ knockout mice display up-regulation of the opioid receptor-like 1 receptor and alterations in opioid receptor expression in the brain.

The opioid receptor-like 1 receptor is a novel member of the opioid receptor family and its endogenous peptide ligand has been termed nociceptin and orphanin FQ. Activation of the opioid receptor-like 1 receptor by nociceptin/orphanin FQ in vivo produces hyperalgesia when this peptide is given supraspinally but analgesia at the spinal level. Nociceptin/orphanin FQ also reverses stress-induced analgesia, suggesting that the peptide has anti-opioid properties. Nociceptin/orphanin FQ knockout mice show alterations in pain sensitivity and stress responses and display increased morphine dependence, suggesting an interaction of the nociceptin/orphanin FQ system with classical opioid receptor function. To determine if the behavioural phenotype of nociceptin/orphanin FQ knockout mice reflects changes in either opioid receptor-like 1 or classical opioid receptor expression, we have carried out quantitative autoradiography of the opioid receptor-like 1, mu-, delta- and kappa-opioid receptors in the brains of these animals. Receptor density was measured on coronal sections from wild-type, heterozygous and homozygous mice using [(3)H]nociceptin, [(3)H][D-Ala(2)-N-methyl-Phe(4)-Gly(5) ol] enkephalin, [(3)H]deltorphin-I, or [(3)H](-)-N-methyl-N-[7-(1-pyrrodinyl)-1-oxospiro[4,5]dec-8-yl]-4-benzofuranacetamide to label opioid receptor-like 1, mu-, delta- and kappa-receptors, respectively. A region-specific up-regulation of the opioid receptor-like 1 receptor (up to 135%) was seen in brains from homozygous mice. Mu-Receptors also showed significant differences between genotypes whilst changes in delta- and kappa- receptors were minor. In conclusion the region-specific up-regulation of the opioid receptor-like 1 receptor indicates a tonic role for nociceptin/orphanin FQ in some brain structures and may suggest the peptide regulates the receptor expression in these regions. The changes in the opioid receptor-like 1 receptor may relate to the anxiogenic phenotype of these animals but the observed change in mu-receptors does not correlate with altered morphine responses.

Regional mapping of low-affinity kainate receptors in mouse brain using [(3)H](2S,4R)-4-methylglutamate autoradiography.

Recent data indicate that (2S,4R)-4-methylglutamate is a selective agonist for low affinity (GluR5 and GluR6) kainate receptor subunits. In the present study, we have employed [(3)H](2S,4R)-4-methylglutamate to examine low affinity kainate receptor distribution in mouse brain. [(3)H](2S,4R)-4-Methylglutamate labelled a single site in murine cerebrocortical membranes (K(d)=9.9+/-2.7 nM, B(max)=296.3+/-27.1 fmol mg protein(-1)). The binding of 8 nM [(3)H](2S,4R)-4-methylglutamate was displaced by several non-NMDA receptor ligands (K(i)+/-S.E.M.): domoate (1.1+/-0.2 nM)>kainate (7.1+/-1.1 nM) >> L-glutamate (187.6+/-31.9 nM) >> (S)-alpha-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) (>50 microM). [(3)H](2S,4R)-4-Methylglutamate autoradiography revealed a widespread regional distribution of low affinity kainate receptors. Highest binding densities occurred within deep layers of the cerebral cortex, olfactory bulb, basolateral amygdala and hippocampal CA3 subregion. Moderate labelling was also evident in the nucleus accumbens, dentate gyrus, caudate putamen, hypothalamus and cerebellar granule cell layer. These data show that [(3)H](2S,4R)-4-methylglutamate is a useful radioligand for selectively labelling low affinity kainate receptors.

Autoradiographic mapping of the opioid receptor-like 1 (ORL1) receptor in the brains of mu-, delta- or kappa-opioid receptor knockout mice.

The opioid receptor-like 1 (ORL1) receptor shares a high degree of sequence homology with the classical mu-, delta- and kappa-opioid receptors and a functional mutual opposition between these receptors has been suggested. To further address this possible interaction we have used mu-, delta- and kappa-opioid receptor knockout mice to determine autoradiographically if there are any changes in the number or distribution of the ORL1 receptor, labelled with [(3)H]nociceptin, in the brains of mice deficient in each of the opioid receptors. An up-regulation of ORL1 expression was observed across all brain regions in delta-knockouts with cortical regions typically showing a 15-30% increase in binding that was most marked in heterozygous mice. In contrast, ORL1 receptor expression was down-regulated in virtually all brain structures in heterozygous kappa-knockouts although the magnitude of this change was not as great as for the delta-knockouts. No significant alterations in ORL1 receptor expression were observed across brain regions in mu-receptor knockout mice and there were no qualitative differences in ORL1 receptor expression in any groups. These data suggest there are interactions between the ORL1 system and the classical opioid receptors and that the interactions are receptor-specific. The greater differences observed in heterozygous mice suggest that these interactions might be most relevant when there is only partial loss of receptor function.

Quantitative autoradiographic mapping of the ORL1, mu-, delta- and kappa-receptors in the brains of knockout mice lacking the ORL1 receptor gene.

Until recently the opioid receptor family was thought to consist of only the mu-, delta- and kappa-receptors. The cloning of opioid receptor like receptor (ORL1) and its endogenous ligand nociceptin/orphanin FQ, which displayed anti-opioid properties, has raised the issue of functional co-operativity of this system with the classical opioid system. ORL1 receptor knockout mice have been successfully developed by homologous recombination to allow the issue of potential heterogeneity of this receptor and also of compensatory changes in mu-, delta- or kappa-receptors in the absence of ORL1 to be addressed. We have carried out quantitative autoradiographic mapping of these receptors in the brains of mice that are wild-type, heterozygous and homozygous for the deletion of the ORL1 receptor. ORL1, mu-, delta- and kappa-receptors were labelled with [(3)H] leucyl-nociceptin (0.4 nM), [(3)H] DAMGO (4 nM), [(3)H] deltorphin-I (7 nM), and [(3)H] CI-977 (2.5 nM) respectively. An approximately 50% decrease in [(3)H] leucyl-nociceptin binding was seen in heterozygous ORL1 mutant mice and there was a complete absence of binding in homozygous brains indicating the single gene encodes for the ORL1 receptor and any putative subtypes. No significant gross changes in the binding to other opioid receptors were seen across genotypes in the ORL1 mutant mice demonstrating a lack of major compensation of classical opioid receptors in the absence of ORL1. There were a small number of region specific changes in the expression of classical opioid receptors that may relate to interdependent function with ORL1.

Analysis of [3H]bremazocine binding in single and combinatorial opioid receptor knockout mice.

Despite ample pharmacological evidence for the existence of multiple mu-, delta- and kappa-opioid receptor subtypes, only three genes encoding mu-(MOR), delta-(DOR) and kappa-(KOR) opioid receptor have been cloned. The KOR gene encodes kappa(1)-sites, which specifically bind arylacetamide compounds, and the possible existence of kappa-opioid receptor subtypes derived from another kappa-opioid-receptor gene, yet to be characterized, remains a very contentious issue. kappa(2)-Opioid receptors are described as binding sites typically labelled by the non-selective benzomorphan ligand [3H]bremazocine in the presence of mu-, delta- and kappa(1)-opioid receptor blocking ligands. To investigate the genetic origin of kappa(2)-opioid receptors, we have carried out homogenate binding experiments with [3H]bremazocine in brains of single MOR-, DOR-, KOR- and double MOR/DOR-deficient mice. Scatchard analysis showed that 68+/-12% of the binding sites arise from the MOR gene, 27+/-1% from the DOR gene and 14.5+/-0.2% from the KOR gene, indicating that the three known genes account for total [3H]bremazocine binding. Experiments in the presence of mu-, delta- and kappa(1)-opioid receptor suppressor ligands further showed that non-kappa(1)-opioid receptor labelling can be accounted for by binding to both the mu- and delta-opioid receptors. Finally, [3H]bremazocine binding experiments performed on brain membranes from the triple MOR/DOR/KOR-deficient mice revealed a complete absence of binding sites, confirming definitively that no additional gene is required to explain the total population of [3H]bremazocine binding sites. Altogether the data show that the putative kappa(2)-opioid receptors are in fact a mixed population of KOR, DOR and predominantly MOR gene products.

Influence of maternal milk on functional activation of delta-opioid receptors in postnatal rats.

Weaning rat pups at day 21 activates a delta-opioid receptor that mediates swim-stress-induced analgesia (swim SIA). We have addressed the possibility that removal of maternal milk is the stimulus for the weaning-induced delta-receptor activation by studying the effect of lactating and nonlactating surrogate mothers and two milk substitutes (casein-rich and casein-free) on opioid receptor control of swim SIA. The delta-receptor antagonist naltrindole (1 mg/kg) significantly antagonized swim SIA in 25-day-old weaned rats, in rats provided with a nonlactating surrogate, and those provided with casein-free milk substitute. Naltrindole had no effect in nonweaned pups, pups given a casein-rich substitute, or in pups from litters provided with a lactating surrogate from day 21 to day 25. Weaning-induced activation of delta-receptors involved in mediating swim SIA appears to be dependent on the loss of dietary casein, which is known to produce peptide fragments that can exert opioid activity. The data suggest that exposure to exogenous opioid peptides can influence the ontogenesis of mu- and delta-opioid receptors.

Postnatal naltrindole treatments induce behavioural modifications in preweanling rats.

To investigate the physiological role of the delta-opioid receptor during the preweanling period, we have studied the effects of chronic (daily injections from birth to postnatal day 19) and acute treatments with the selective delta-antagonist naltrindole (1 mg/kg), on behavioural and nociceptive responses in 20-day old male rats. Behavioural testing was performed using an open field paradigm. Acute naltrindole induced significant decreases in external and total ambulation (horizontal activity) and rearing behaviour (vertical activity), as well as a significant increase in grooming frequency. In animals chronically treated with naltrindole there was an increase in total ambulation one day after the discontinuation of the treatment. In a test of nociception (tail immersion) no significant effect of chronic naltrindole treatment on baseline latencies or of acute naltrindole on latency quotients (post-treatment latency/pre-treatment latency) were found. However, chronic naltrindole administration significantly decreased the latency quotients. The results show that the delta-opioid receptor participates in the tonic regulation of motor activity during the preweanling period and might be involved in certain aspects of stress responsiveness.

Neonatal naltrindole and handling differently affect morphine antinociception in male and female rats.

The effects of a daily injection of the delta selective opioid antagonist naltrindole (1 mg/kg), from birth to postnatal day 19, on antinociceptive responses to morphine (2 mg/kg) in 20-day-old rats of both sexes were investigated. The effects of postnatal handling were studied by including two control groups--one group receiving daily injections of saline, and a naive unhandled group. Antinociception was assessed using the tail-immersion test and time-response curves (5, 10, 15, and 30 min) were carried out for all experimental groups. In all treatment groups females showed greater sensitivity to the noxious stimuli compared to males. No significant effect of naltrindole treatment on baseline latencies was found. Postnatal handling increased sensitivity to thermal pain in both sexes, and reduced the effect of morphine in males. No significant effect of chronic naltrindole administration on morphine antinociception was found in this sex. Naltrindole-treated females showed an increased antinociception when compared to unhandled animals of the same gender. The results indicate that preweanling handling stress and chronic naltrindole treatment differentially affected morphine antinociception in male and female neonatal rats.

Effects of juvenile isolation and morphine treatment on social interactions and opioid receptors in adult rats: behavioural and autoradiographic studies.

The consequences of juvenile isolation and morphine treatment during the isolation period on (social) behaviour and mu-, delta- and kappa-opioid receptors in adulthood were investigated by using a social interaction test and in vitro autoradiography in rats. Juvenile isolation reduced social exploration in adults. Morphine treatment counteracted this reduction in isolated rats, but decreased social exploration in nonisolated rats. Self-grooming and nonsocial exploration were enhanced after juvenile isolation. Morphine treatment had no effect on self-grooming, but suppressed nonsocial exploration in isolated rats. With respect to the opioid receptors, juvenile isolation resulted in regiospecific increases in mu-binding sites with a 58% increase in the basolateral amygdala and a 33% increase in the bed nucleus of stria terminalis. Morphine treatment in isolated rats reversed this upregulation in both areas. The number of delta-binding sites did not differ between the experimental groups. A general upregulation of kappa-binding sites was observed after juvenile isolation, predominantly in the cortical regions, the hippocampus and the substantia nigra. Morphine treatment did not affect the upregulation of kappa-receptors. The results show that juvenile isolation during the play period causes long-term effects on social and nonsocial behaviours and on the number of mu- and kappa- but not delta-opioid receptors in distinct brain areas. The number of mu-receptors in the basolateral amygdala appears to be negatively correlated with the amount of social exploration in adult rats.