Glutamate receptor mGlu2 and mGlu3 knockout striata are dopamine supersensitive,with elevated D2High receptors and marked supersensitivity to the dopamine agonist (+)PHNO

The finding that the mGlu2/3 metabotropic glutamate receptor agonist, LY404039, improves clinical symptoms in schizophrenia warrants a search for a possible interaction between mGlu2/3 receptors and dopamine D2 receptors. Here, this topic is examined in striatal tissue of mice lacking either mGlu2 or mGlu3 receptor. Such mice are known to be behaviorally supersensitive to dopamine receptor agonists. Therefore, to determine the basis of this dopamine supersensitivity, the proportion of dopamine D2^High receptors was measured in the striata of mGlu2 and mGlu3 receptor knockout mice. The proportion of D2^High receptors was found to be elevated by 220% in the striata of both knockouts. To measure the functional dopamine supersensitivity, the D2 agonist (+)PHNO was used to stimulate the incorporation of GTP‐γ‐S in the striatal homogenates in the presence of drugs that blocked the dopamine D1, D3, and D5 receptors. Compared with control striata, the mGlu2 receptor knockout tissues were 67‐fold more sensitive to (+)PHNO, while the mGlu3 receptor knockout tissues were 17‐fold more sensitive. These data suggest that group II mGlu receptors—mGlu2 receptors in particular—may normally regulate D2 receptors by reducing the proportion of high‐affinity D2 receptors in membranes. Such regulation may contribute to the antipsychotic action of mGlu2/3 receptor agonists. Synapse 63:247–251, 2009. © 2008 Wiley‐Liss, Inc.     

Protective effect of LY379268, a selective group II metabotropic glutamate receptor agonist, on dizocilpine-induced neuropathological changes in rat retrosplenial cortex

In the present study, we examined the effects of LY379268, the group II metabotropic glutamate receptor (mGluR) agonist, on the neuropathological changes in the rat retrosplenial cortex induced by noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine ((+)-MK-801). Administration of LY379268 (1, 3, 10 mg/kg, i.p.) reduced dizocilpine (0.5 mg/kg, i.p.)-induced neuropathological changes in the retrosplenial cortex, in a dose-dependent manner. Co-administration of LY379268 (10 mg/kg, i.p.) with group II mGluR antagonist LY341495 (5 mg/kg, i.p.) blocked the effects of LY379268. Furthermore, LY379268 (10 mg/kg, i.p.) significantly reduced the expression of heat shock protein HSP-70, a marker of reversible neuronal injury, in the rat retrosplenial cortex after administration of dizocilpine (0.5 mg/kg, i.p.). Moreover, pretreatment with LY379268 (10 mg/kg, i.p.) significantly suppressed the increase in extracellular acetylcholine (ACh) levels in the retrosplenial cortex induced by administration of dizocilpine (0.5 mg/kg, i.p.). These results suggest that LY379268 has a protective effect on the neurotoxicity in the rat retrosplenial cortex after administration of NMDA receptor antagonists such as dizocilpine.     

Striatal dopamine metabolism in response to apomorphine: the effects of repeated amphetamine pretreatment

The dose-dependent decrease in striatal dopamine (DA) metabolites following apomorphine (APO) administration was utilized as an index of changes in DA receptor sensitivity following the repeated administration of amphetamine (AMPH). The results suggest that: (a) repeated AMPH pretreatment does not alter DA autoreceptor sensitivity; and (b) interpretations of the decline in striatal DA metabolites at high doses of APO (> 50 μg/kg), in terms of activation of postsynaptic DA receptors, may require re-evaluation.     

D(3) dopamine receptors are down-regulated in amphetamine sensitized rats and their putative antagonists modulate the locomotor sensitization to amphetamine

D(3) dopamine receptor agonists inhibit locomotor activity in rodents and modulate the reinforcing effect of psychostimulants; however, their functional role during behavioral sensitization remains unclear. In the present study, we intend to investigate if D(3) dopamine receptors alter during the amphetamine sensitization and test if manipulation of D(3) receptors would affect the development of locomotor sensitization to amphetamine. We have found that D(3) dopamine receptors are down-regulated in the limbic forebrain in chronic amphetamine-treated (5 mg/kg x 7 days) animals. The levels of both D(3) receptor protein (B(max) value) and mRNA decreased significantly in the behaviorally sensitized rats compared to the saline-treated controls. When animals were co-administered a putative D(3) receptor antagonist (U99194A or GR103691; 20 microg x 7 days; intracerebroventricle) and amphetamine (5 mg/kg x 7 days, i.p.), the locomotor sensitization to amphetamine was significantly inhibited. However, when the putative D(3) receptor antagonist U99194A was administered during the amphetamine withdrawal period at day 10, it did not affect the development of locomotor sensitization. Furthermore, pretreatment with the preferential D(3) agonist 7-hydroxydipropylaminotetralin partially blocked the inhibitory effect of U99194A on locomotor sensitization. These data prove the participation of D(3) dopamine receptors in the development of amphetamine sensitization and, in addition, suggest a potential application for D(3) antagonists in the prevention of amphetamine addiction.     

Repeated exposure to amphetamine disrupts dopaminergic modulation of excitatory synaptic plasticity and neurotransmission in nucleus accumbens

The mesolimbic dopamine system is essential for reward-seeking behavior, and drugs of abuse perturb the normal functioning of this pathway. The nucleus accumbens (NAc) is a major terminal field of the mesolimbic dopamine neurons and modifications in neuronal structure and function in NAc accompany repeated exposure to psychomotor stimulants and other addictive drugs. Glutamatergic afferents to the NAc are thought to be crucial to the development of several aspects of addictive behavior, including behavioral sensitization and relapse to cocaine self-administration. Here we examine glutamatergic neurotransmission and synaptic plasticity in NAc neurons in vitro before and after repeated amphetamine treatment in vivo. We find that dopamine attenuates the response of NAc neurons to repetitive activation of glutamatergic afferents and thereby blocks long-term potentiation (LTP) induced by high-frequency afferent stimulation. Dopamine's effects are mimicked by dopamine receptor agonists and by amphetamine. In a second set of experiments, animals were treated with amphetamine daily for 6 days and brain slices were prepared after 8-10 days of withdrawal. In these slices, LTP in the NAc appears normal. However, acute exposure of such slices to amphetamine no longer modulates synaptic transmission or LTP induction. Thus, repeated exposure to amphetamine produces long-lasting changes in the modulation of glutamatergic synaptic transmission by amphetamine in the NAc. Our results support the notion that after psychostimulant exposure, excitatory synapses on NAc neurons alter their response to further psychostimulant for long periods of time.     

Activation of group III metabotropic glutamate receptors by endogenous glutamate protects against glutamate-mediated excitotoxicity in the hippocampus in vivo

Perfusion of 4-aminopyridine (4-AP) by microdialysis in the hippocampus produces intense epileptiform behavioral and electrical activity and neurodegeneration, resulting from a stimulated release of glutamate from nerve endings. In contrast, accumulation of extracellular glutamate by blockade of its transport in vivo in anesthetized rats is innocuous, and studies in vitro in brain slices suggest that under these conditions glutamate may activate presynaptic group III metabotropic glutamate receptors (mGluRs) and inhibit its own release. Therefore, using microdialysis, EEG recording, and histological evaluation, we studied the effect of increased endogenous extracellular glutamate by blockade of its transport with pyrrolidine dicarboxylic acid (PDC) on the excitotoxic action of 4-AP in the hippocampus of awake rats. We found that up to a 20-fold increase in extracellular glutamate during >90 min with PDC does not induce any sign of excitotoxicity. On the contrary, this glutamate increase notably protected against the 4-AP-induced seizures and neurodegeneration, and, remarkably, this protection was dependent on the time of perfusion with PDC and thus on the duration of extracellular glutamate accumulation. To test whether this protective action was mediated by the activation of group III mGluRs, we used specific antagonists of these receptors and found that they clearly prevented the protective effect of PDC, without affecting the accumulation of extracellular glutamate. We conclude that the spillover of the excess extracellular glutamate activates presynaptic group III mGluRs and inhibits the stimulatory effect of 4-AP on its release, thus preventing the activation of postsynaptic N-methyl-D-aspartate receptors and its deleterious consequences.     

Effects of prenatal exposure to amphetamine in the medial prefrontal cortex of the rat

This study was designed to investigate the effects of prenatal exposure to amphetamine in the organization of the medial prefrontal cortex of the rat, by an evaluation of growth, morphometric and neurochemical parameters. Pregnant Wistar rats were given 10 mg/kg body weight/day of D-amphetamine sulfate, subcutaneously, from gestational days 8 to 22. Control groups of pregnant rats were injected with saline, pair-fed or non-manipulated; litters were culled to eight pups (four males and four females), weighed every other day until postnatal day 30 and every week until day 90. The Gompertz model was used to study body weight evolution and the estimated growth parameters were not significantly different in the experimental groups. At postnatal days 14 and 30, the volumes of the prefrontal cortex, the fraction of neuropile occupied by neurons and the number of neurons per unit surface area were determined. The number of neurons per unit volume of reference area was calculated using the stereological technique of the dissector. For neurochemical analysis, the medial prefrontal cortex was dissected to measure the concentration of dopamine, serotonin and their metabolites. The allometric relationship of forebrain/body growth pointed to a mechanism of sparing and compensatory growth in the amphetamine exposed group. The changes found in the number of neurons per unit volume at postnatal day 14 show a catch-up at postnatal day 30. A decrease in serotonin levels was found in the amphetamine group compared with the pair-fed control, which was reflected in the ratio of serotonin to its metabolite, 5-hydroxyindolacetic acid. These changes, whether permanent or transitory, raise the possibility that some of the effects of prenatal exposure to amphetamine may be due to modifications in the neurotransmitter levels of serotonin.     

Dopamine modulation of excitatory currents in the striatum is dictated by the expression of D1 or D2 receptors and modified by endocannabinoids

Striatal medium‐sized spiny neurons (MSSNs) receive glutamatergic inputs modulated presynaptically and postsynaptically by dopamine. Mice expressing the gene for enhanced green fluorescent protein as a reporter gene to identify MSSNs containing D1 or D2 receptor subtypes were used to examine dopamine modulation of spontaneous excitatory postsynaptic currents (sEPSCs) in slices and postsynaptic N‐methyl‐d ‐aspartate (NMDA) and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) currents in acutely isolated cells. The results demonstrated dopamine receptor‐specific modulation of sEPSCs. Dopamine and D1 agonists increased sEPSC frequency in D1 receptor‐expressing MSSNs (D1 cells), whereas dopamine and D2 agonists decreased sEPSC frequency in D2 receptor‐expressing MSSNs (D2 cells). These effects were fully (D1 cells) or partially (D2 cells) mediated through retrograde signaling via endocannabinoids. A cannabinoid 1 receptor (CB1R) agonist and a blocker of anandamide transporter prevented the D1 receptor‐mediated increase in sEPSC frequency in D1 cells, whereas a CB1R antagonist partially blocked the decrease in sEPSC frequency in D2 cells. At the postsynaptic level, low concentrations of a D1 receptor agonist consistently increased NMDA and AMPA currents in acutely isolated D1 cells, whereas a D2 receptor agonist decreased these currents in acutely isolated D2 cells. These results show that both glutamate release and postsynaptic excitatory currents are regulated in opposite directions by activation of D1 or D2 receptors. The direction of this regulation is also specific to D1 and D2 cells. We suggest that activation of postsynaptic dopamine receptors controls endocannabinoid mobilization, acting on presynaptic CB1Rs, thus modulating glutamate release differently in glutamate terminals projecting to D1 and D2 cells.     

Activation of group II metabotropic glutamate receptors reduces directional selectivity in retinal ganglion cells

In the mammalian retina, high levels of the group II metabotropic glutamate receptor (mGluR) subtype are expressed in starburst amacrine cells. A prominent role of starburst amacrine cells is the generation of directional selectivity in ON-OFF directionally selective retinal ganglion cells (DS RGCs). Extracellular microelectrodes were used to study the effects of activation of group II mGluRs on the responses of rabbit ON-OFF DS RGCs to a moving light stimulus. Directionally selective responses in these RGCs were substantially reduced by the selective group II mGluR agonist DCG-IV. DCG-IV brought out a response to movement in the null direction that was similar in magnitude and time course to the response to movement in the preferred direction. This effect of DCG-IV was reversed by the group II mGluR antagonist EGLU. Application of EGLU alone failed to alter directional selectivity in the RGCs but did reduce the response to movement in the preferred direction. To determine whether group II mGluRs modulate the release of the neurotransmitter acetylcholine from starburst amacrine cells, the effect of DCG-IV on ON-OFF DS RGCs was examined in the presence of ambenonium, an acetylcholinesterase inhibitor. When applied alone, ambenonium greatly prolonged the responses of ON-OFF DS RGCs to a light stimulus. This effect of ambenonium was completely abolished upon application of DCG-IV. Overall, the results suggest that postsynaptic group II mGluRs have the potential to influence directional selectivity in RGCs by inhibiting transmitter release from starburst amacrine cells.     

Increased locomotor response to amphetamine induced by the repeated administration of the selective kappa-opioid receptor agonist U-69593

Acute administration of kappa opioid receptor (KOR) agonists decreases both dopamine (DA) extracellular levels in the nucleus accumbens (NAc) and locomotor activity. Opposing to its acute effects, recent studies show that chronic administration of KOR agonists potentiates both stimulated DA release and induced locomotor activity. Since KOR agonists have been considered as potential treatment for stimulant dependence, the effects of their repeated administration on psychostimulant actions are of major concern. The present study was undertaken to investigate the in vivo effect of repeated administration of the KOR agonist U-69593 on DA extracellular levels in the NAc and on the locomotor activity challenged with amphetamine. Rats were injected once daily with the selective KOR agonist U-69593 or vehicle for four consecutive days. One-day after the last U-69593 injection, microdialysis studies assessing extracellular DA levels in the NAc and locomotor activity challenged with amphetamine were conducted. Microdialysis studies revealed that preexposure to U-69593 had no effect on basal DA levels but significantly augmented amphetamine-induced DA extracellular levels. Accordingly, amphetamine-induced locomotor activity was also significantly potentiated in U-69593 preexposed rats. These results suggest that long-term effect of KOR activation results in facilitation of amphetamine-induced DA extracellular levels in the NAc accompanied by sensitization of amphetamine-induced increase in locomotor activity.     

Enhanced sensitivity to group II mGlu receptor activation at corticostriatal synapses in mice lacking the familial parkinsonism-linked genes PINK1 or Parkin

An altered glutamatergic input at corticostriatal synapses has been shown in experimental models of Parkinson's disease (PD). In the present work, we analyzed the membrane and synaptic responses of striatal neurons to metabotropic glutamate (mGlu) receptor activation in two different mouse models of inherited PD, linked to mutations in PINK1 or Parkin genes.Both in PINK1 and Parkin knockout (^−/−) mice, activation of group I mGlu receptors by 3,5-DHPG caused a membrane depolarization coupled to an increase in firing frequency in striatal cholinergic interneurons that was comparable to the response observed in the respective wild-type (WT) interneurons. The sensitivity to group II and III mGlu receptors was tested on cortically-evoked excitatory postsynaptic potentials (EPSPs) recorded from medium spiny neurons (MSNs). Both LY379268 and L-AP4, agonists for group II and III, respectively, had no effect on intrinsic membrane properties, but dose-dependently reduced the amplitude of corticostriatal EPSPs. However, both in PINK1^−/− and Parkin^−/− mice, LY379268, but not L-AP4, exhibited a greater potency as compared to WT in depressing EPSP amplitude. Accordingly, the dose–response curve for the response to LY379268 in both knockout mice was shifted leftward. Moreover, consistent with a presynaptic site of action, both LY379268 and L-AP4 increased the paired-pulse ratio either in PINK1^−/− and Parkin^−/− or in WT mice. Acute pretreatment with L-dopa did not rescue the enhanced sensitivity to LY379268.Together, these results suggest that the selective increase in sensitivity of striatal group II mGlu receptors represents an adaptive change in mice in which an altered dopamine metabolism has been documented.     

Attenuation of seizures induced by homocysteic acid in immature rats by metabotropic glutamate group II and group III receptor agonists

Previous studies demonstrated that selected agonists for metabotropic glutamate group II and group III receptors can provide protection against seizures in adult animals. The present study has examined the potential effect of some of these compounds on seizures induced in immature rats by intracerebroventricular infusion of DL-homocysteic acid (DL-HCA, 600 nmol/side). Rat pups were sacrificed during generalised clonic-tonic seizures, 50--60 min after infusion. Comparable time intervals were used for sacrificing the pups which had received the protective drugs. The anticonvulsant effect was evaluated according to the suppression of behavioural manifestations of seizures and the protection of energy metabolite changes which normally accompany these seizures (large decreases of glucose and glycogen, and approximately 7- to 10-fold accumulation of lactate). Partial protection was exhibited by group II mGluR agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV, 0.6 nmol) and this effect was abolished after pretreatment with an antagonist for group II mGluRs (RS)-alpha-methyl-4-tetrazolylphenylglycine (MTPG, 100 nmol). In high doses (5--100 nmol), however, DCG IV evoked seizures which were prevented by AP7, suggesting that the convulsant effect was mediated by interaction with NMDA receptors. A pronounced anticonvulsant effect against DL-HCA-induced seizures was achieved with low doses of a highly selective group II mGluR agonist (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC, 0.6 nmol), group II agonist and group I mGluR antagonist (S)-4-carboxy-3-hydroxyphenylglycine ((S)-4-C3HPG, 0.6 nmol) and group III mGluR agonist (RS)-1-amino-3-(phosphonomethylene) cyclobutane-carboxylic acid (32 nmol). Generalised clonic--tonic seizures were completely suppressed and the metabolic changes were markedly ameliorated, there being only a 1.5-, 2- and 2.5-fold rise of lactate, respectively. Higher doses of (S)-4-C3HPG (1--100 nmol) were, however, less anticonvulsant than low doses. The present results have confirmed that mGluRs may be considered a potential target for treatment of epilepsy.     

Developmental exposure to the organochlorine insecticide endosulfan alters expression of proteins associated with neurotransmission in the frontal cortex

Exposure to environmental contaminants, such as organochlorine insecticides during critical periods of neurodevelopment has been shown to be a major contributor to several neuropsychological deficits seen in children, adolescence, and adults. Although the neurobehavioral outcomes resulting from exposure to these compounds are known the neurotransmitter circuitry and molecular targets that mediate these endpoints have not been identified. Given the importance of the frontal cortex in facilitating numerous neuropsychological processes, our current study sought to investigate the effects of developmental exposure to the organochlorine insecticide, endosulfan, on the expression of specific proteins associated with neurotransmission in the frontal cortex. Utilizing in vitro models we were able to show endosulfan reduces cell viability in IMR‐32 neuroblastoma cells in addition to reducing synaptic puncta and neurite outgrowth in primary cultured neurons isolated from the frontal cortex of mice. Elaborating these findings to an in vivo model we found that developmental exposure of female mice to endosulfan during gestation and lactation elicited significant alterations to the GABAergic (GAT1, vGAT, GABA_A receptor), glutamatergic (vGlut and GluN2B receptor), and dopaminergic (DAT, TH, VMAT2, and D2 receptor) neurotransmitter systems in the frontal cortex of male offspring. These findings identify damage to critical neurotransmitter circuits and proteins in the frontal cortex, which may underlie the neurobehavioral deficits observed following developmental exposure to endosulfan and other organochlorine insecticides. Synapse 68:485–497, 2014. © 2014 Wiley Periodicals, Inc.     

Difference in time course of modulation of synaptic transmission by group II versus group III metabotropic glutamate receptors in region CA1 of the hippocampus

We investigated the time course of modulation of synaptic transmission by group II and group III metabotropic glutamate receptors in region CA1 of the hippocampus. In the presence of 50 microM picrotoxin, pressure pulse application of 1 mM glutamate resulted in a fast onset of suppression of synaptic transmission in stratum lacunosum moleculare and a slower onset of suppression in stratum radiatum, with both effects returning to baseline over the course of several minutes. Application of 50 microM of the group II agonist (2R,4R)-APDC in stratum lacunosum moleculare resulted in the same fast onset of suppression while having no effect in stratum radiatum. Pressure pulse application of 100 microM DL-AP4 in stratum lacunosum moleculare and stratum radiatum resulted in a much slower onset of suppression of synaptic transmission than (2R,4R)-APDC. Suppression by (2R,4R)-APDC was accompanied by a rapid enhancement of paired pulse facilitation, indicative of a presynaptic mechanism. This demonstrates that activation of group II mGluRs in the hippocampus causes a fast onset of suppression in stratum lacunosum moleculare, while activation of group III mGluRs causes a slower onset of suppression. The difference in time course for group II vs. group III mGluRs suggests a different functional role, with group II playing a potential role in making synapses act as low pass filters.     

Long-term facilitation of amphetamine-induced rotational behavior and striatal dopamine release produced by a single exposure to amphetamine: Sex differences

Amphetamine (AMPH)-induced rotational behavior in non-lesioned rats and AMPH-stimulated dopamine (DA) release from striatal tissue fragments in vitro were used to study the long-term effects of a single injection of AMPH on activity in the mesostriatal DA system. A single injection of a low dose of AMPH (1.25 mg/kg) greatly enhanced the rotational behavior produced by a second injection of AMPH given 3–4 weeks later in intact female, ovariectomized female and castrated male rats. The effect of AMPH pretreatment in intact males differed from that in the other groups. When only 7–8 days separated the two test sessions both intact male and female rats showed sensitization of rotational behavior, but the magnitude of the change was greater in females. In addition, a single injection of 1.25 mg/kg of AMPH in vivo produced a long-lasting (3–5 weeks) enhancement of AMPH-stimulated DA release from striatal tissue in vitro. It is suggested that: (1) repeated injections of AMPH are not necessary to produce a long-lasting facilitation of behaviors mediated by the mesostriatal DA system; (2) gender and/or hormonal state influences the development of long-term changes in the mesostriatal DA system; and (3) changes in DA release from presynaptic terminals may contribute to the behavioral sensitization produced by stimulant drugs. The phenomena reported here may provide complementary in vitro and in vivo models for studying neuroplasticity in brain DA systems.     

The group I metabotropic glutamate receptor antagonist S-4-CPG modulates the locomotor response produced by the activation of D1-like, but not D2-like, dopamine receptors in the rat nucleus accumbens

Functional interactions between dopamine (DA) and glutamate neurotransmissions in both the dorsal and the ventral striatum have been described for long time. However, there is much controversy as to whether glutamate transmission stimulates or attenuates DA release and locomotor activity. We investigated the functional interactions on locomotor activity between group I metabotropic glutamatergic receptors (mGlu receptors) and both D1-like and D2-like DA receptors in the rat nucleus accumbens. Intra-accumbens administration of the selective group I mGlu receptor antagonist S-4-CPG (0.2 or 2 microg per side), which had no effect when injected alone, prevented the increase in locomotor activity produced by the selective D1-like receptor agonist SKF 38393 (1 microg per side). Co-administration with S-4-CPG of the group I mGlu receptor agonist DHPG, but not of the group II mGlu receptor agonist APDC or the group III mGlu receptor agonist AP4, reversed the antagonistic effect of S-4-CPG on the SKF 38393-induced increase in locomotor activity. This indicates that the antagonistic effect of S-4-CPG could result from an action at the group I mGlu receptors. In contrast, administration of S-4-CPG showed no effect on the locomotor responses produced by either the selective D2-like receptor agonist LY 171555 (1 microg per side) or a mixed solution of SKF 38393 + LY 171555 (1 microg per side each). Altogether, these results confirm that glutamate transmission may control locomotor function through mGlu receptors in a DA-dependent manner, and further indicate that group I mGlu receptors would interact with D1-like receptors, but not D2-like receptors, to modulate DA transmission and locomotor activity.     

Effects of blockade of glutamate NMDA receptors or of NO synthase on the development or the expression of associative or non-associative sensitization to locomotor activation by morphine

Summary. The sensitization to the pharmacological actions of morphine is probably a critical factor in the addictive properties of this drug. A discrimination between associative and non-associative type of sensitization might be relevant for possible differences in drug effects on sensitization phenomena. Furthermore, blockade of NMDA receptors might lead to an inhibition of NO-synthesis, and, accordingly, both of these effects might influence sensitization phenomena in a similar way. Male Wistar rats were sensitized to morphine by administrations of 3 mg/kg of morphine i.p. on day 1, 3, 5, and 7 and saline on days 2, 4, and 6. In part of the animals, the administration of morphine was performed in association with conditional stimuli, CS (test cage plus an auditory and an olfactory stimulus), in the other part not (pseudoconditioned, PCS). On day 17, the sensitization was more pronounced in the CS than the PCS group. The effects of dizocilpine (MK-801; 0.1 mg/kg i.p.), a blocker of NMDA glutamate receptors, or of L-NAME (N(G)-nitro-L-arginine methylester; 10 mg/kg i.p.), a non-specific inhibitor of NO synthase and the effect of N(ω)-propyl-L-arginine (20 mg/kg i.p.), a specific inhibitor of neuronal NO synthase, on expression or development of sensitization to morphine was studied. Neither MK-801 nor L-NAME influenced the development of associative and non-associative behavioural sensitization to morphine. The expression of sensitization to morphine was not influenced by MK-801. L-NAME inhibited the expression of associative, but not of non-associative sensitization. Surprisingly, inhibition of neuronal NO synthase, did not influence the expression of associative sensitization. We suggest that NMDA receptors were not involved in development or expression of both types of sensitization. Furthermore, the manifestation of the associative, but not the non-associative sensitization to morphine appeared to be dependent on a type of NO synthase, which is not the neuronal NO synthase, but probably the inducible NOS.