Antisense oligonucleotide-mediated correction of transcriptional dysregulation is correlated with behavioral benefits in the YAC128 mouse model of Huntington's disease.

BACKGROUND: Huntington's disease (HD) is a neurological disorder caused by mutations in the huntingtin (HTT) gene, the product of which leads to selective and progressive neuronal cell death in the striatum and cortex. Transcriptional dysregulation has emerged as a core pathologic feature in the CNS of human and animal models of HD. It is still unclear whether perturbations in gene expression are a consequence of the disease or importantly, contribute to the pathogenesis of HD. OBJECTIVE: To examine if transcriptional dysregulation can be ameliorated with antisense oligonucleotides that reduce levels of mutant Htt and provide therapeutic benefit in the YAC128 mouse model of HD. METHODS: Quantitative real-time PCR analysis was used to evaluate dysregulation of a subset of striatal genes in the YAC128 mouse model. Transcripts were then evaluated following ICV delivery of antisense oligonucleotides (ASO). Rota rod and Porsolt swim tests were used to evaluate phenotypic deficits in these mice following ASO treatment. RESULTS: Transcriptional dysregulation was detected in the YAC128 mouse model and appears to progress with age. ICV delivery of ASOs directed against mutant Htt resulted in reduction in mutant Htt levels and amelioration in behavioral deficits in the YAC128 mouse model. These improvements were correlated with improvements in the levels of several dysregulated striatal transcripts. CONCLUSIONS: The role of transcriptional dysregulation in the pathogenesis of Huntington's disease is not well understood, however, a wealth of evidence now strongly suggests that changes in transcriptional signatures are a prominent feature in the brains of both HD patients and animal models of the disease. Our study is the first to show that a therapeutic agent capable of improving an HD disease phenotype is concomitantly correlated with normalization of a subset of dysregulated striatal transcripts. Our data suggests that correction of these disease-altered transcripts may underlie, at least in part, the therapeutic efficacy shown associated with ASO-mediated correction of HD phenotypes and may provide a novel set of early biomarkers for evaluating future therapeutic concepts for HD.

Acute nicotine changes dynorphin and prodynorphin mRNA in the striatum.

RATIONALE: Nicotine displays rewarding and aversive effects, and while dopamine has been linked with nicotine's reward, the neurotransmitter(s) involved with aversion remains speculative. The kappa-dynorphinergic system has been associated with negative motivational and affective states, and whether dynorphin (Dyn) contributes to the behavioral pharmacology of nicotine is a pertinent question. OBJECTIVE: We determined whether administration of a single dose of nicotine alters the biosynthesis of Dyn in the striatum of mice. RESULTS: Nicotine free base, 1 mg/kg, sc, induced a biphasic, protracted increase of striatal Dyn, an initial rise by 1 h, which declined to control levels by 2 h, and a subsequent increase, between 6 and 12 h, lasting over 24 h. At 1 h, the nicotine effect was dose dependent, with doses>or=0.5 mg/kg inducing a response. Prodynorphin mRNA increased by 30 min for over 24 h, and in situ hybridization demonstrated elevated signal in caudate/putamen and nucleus accumbens. The nicotinic antagonist mecamylamine prevented the Dyn response, and a similar effect was observed with D1- and D2-like dopamine receptor antagonists, SCH 23390, sulpiride, and haloperidol. The glutamate NMDA receptor antagonist MK-801 reversed the nicotine-induced increase of Dyn, while the AMPA antagonist NBQX had a marginal effect. CONCLUSIONS: We interpret our findings to indicate that acute nicotine enhances the synthesis and release of striatal Dyn. We propose that nicotine influences Dyn primarily through dopamine release and that glutamate plays a modulatory role. A heightened dynorphinergic tone may contribute to the aversive effects of nicotine in naive animals and first-time tobacco smokers.

Antinociceptive and anti-allodynic effects of oral PL37, a complete inhibitor of enkephalin-catabolizing enzymes, in a rat model of peripheral neuropathic pain induced by vincristine.

Vincristine is a common anti-cancer therapy administered for the treatment of many types of tumors. Its dose-limiting side effect is the production of peripheral neuropathy, resulting in chronic neuropathic pain in many patients. An animal model of vincristine-induced sensory neuropathy was developed after repeated intraperitoneal injection in male rats and used in the present work to study the effects of PL37, an orally active complete dual inhibitor of enkephalin-catabolizing enzymes, on mechanical hypersensitivity and allodynia and on cold allodynia. We used the Electronic Von Frey filament (mechanical static allodynia), acetone test (cold allodynia), and a new behavioural test we first describe in this study, the "paint-brush test" which evaluates dynamic mechanical allodynia and dynamic mechanical hypersensitivity. We used a smooth paint brush leading to an innocuous stimulus, and a rough-one leading to an intense mechanical stimulus. Mechanical hypersensitivity and allodynia due to vincristine-induced neuropathy, but not cold allodynia, are strongly reduced by oral or i.p. injected PL37, the dose-dependent effects being reversed by naloxone-methiodide supporting the peripheral action of the dual inhibitor. These results show that enkephalins protected from degradation by PL37 could bind to peripheral opioid receptors expressed only on C- and Adelta-mechanonociceptors but not on cold thermonociceptors. The fact that PL37 is also active on small intensity mechanical stimulus could reveal an expression of opioid receptors on low threshold mechanoreceptors in the vincrisitine-evoked pathological conditions. Thus the increase in endogenous enkephalin levels induced by PL37 offers a new way to reduced neuropathic pain without the possible side effects of opiates.

Striatal output markers do not alter in response to circling behaviour in 6-OHDA lesioned rats produced by acute or chronic administration of the monoamine uptake inhibitor BTS 74 398.

The monoamine uptake inhibitor BTS 74 398 induces ipsilateral circling in 6-hydroxydopamine (6-OHDA) lesioned rats without induction of abnormal motor behaviours associated with L-dopa administration. We examined whether this was reflected in the expression of peptide mRNA in the direct and indirect striatal output pathways.6-OHDA lesioning of the nigrostriatal pathway increased striatal expression of PPE-A mRNA and decreased levels of PPT mRNA with PPE-B mRNA expression remaining unchanged. Acute L-dopa administration normalised PPE-A mRNA and elevated PPT mRNA while PPE-B mRNA expression remained unchanged. Acute administration of BTS 74 398 did not alter striatal peptide mRNA levels. Following chronic treatment with L-dopa, PPE-A mRNA expression in the lesioned striatum continued to be normalised and PPT mRNA was increased compared to the intact side. PPE-B mRNA expression was also markedly increased relative to the non-lesioned striatum. Chronic BTS 74 398 administration did not alter mRNA expression in the 6-OHDA lesioned striatum although small increases in PPT mRNA expression in the intact and sham lesioned striatum were observed. The failure of BTS 74 398 to induce changes in striatal neuropeptide mRNA correlated with its failure to induce abnormal motor behaviours or behavioural sensitisation but does not explain how it produces a reversal of motor deficits. An action in another area of the brain appears likely and may explain the subsequent failure of BTS 74 398 and related compounds to exert anti-parkinsonian actions in man.

Prenatal exposure to valproic acid disturbs the enkephalinergic system functioning, basal hedonic tone, and emotional responses in an animal model of autism.

RATIONALE: It has been suggested that behavioral aberrations observed in autism could be the result of dysfunction of the neuroregulatory role performed by the endogenous opioid peptides. Many of those aberrations have been recently modeled in rats exposed to valproic acid (VPA) on the 12th day of gestation (VPA rats). OBJECTIVES: The aim of the present study was to elucidate functioning of the enkephalinergic system, one of the endogenous opioid peptide systems strongly involved in emotional responses, in VPA rats using both biochemical and behavioral methods. MATERIALS AND METHODS: In situ hybridization was used to measure proenkephalin mRNA expression in adult VPA rats' central nucleus of the amygdala, the dorsal striatum, and the nucleus accumbens. Additional groups of animals were examined in a conditioned place aversion to naloxone, the elevated plus maze, and object recognition tests to assess their basal hedonic tone, anxiety, learning and memory, respectively. RESULTS: Prenatal exposure to VPA decreased proenkephalin mRNA expression in the dorsal striatum and the nucleus accumbens but not in the central nucleus of the amygdala. It also increased anxiety and attenuated conditioned place aversion to naloxone but had no impact on learning and memory. CONCLUSIONS: The present results suggest that prenatal exposure to VPA may lead to the decreased activity of the striatal enkephalinergic system and in consequence to increased anxiety and disregulated basal hedonic tone observed in VPA rats. Presented results are discussed in light of interactions between enkephalinergic, GABAergic, and dopaminergic systems in the striatum and mesolimbic areas of the brain.

Amphetamine triggers an increase in met-enkephalin simultaneously in brain areas and immune cells.

We analyzed effects of amphetamine on proenkephalin-derived peptides in brain areas and immune cells in rats. Acute, as well as a repeated amphetamine treatment, decreased the concanavalin-A-induced lymphocyte proliferation, concomitantly with an increase of free met-enkephalin in nucleus accumbens, prefrontal cortex, spleen, thymus and splenic macrophages. Proenkephalin protein increased in prefrontal cortex, thymus (32 kDa isoform), nucleus accumbens and spleen (44 kDa isoform), while proenkephalin mRNA levels decreased in brain stem. The influence of met-ENK in key brain areas for sensitization and in immune organs is consistent with the idea that changes on met-ENK could underlie amphetamine's effects on brain and IS.

Alterations in prodynorphin gene expression and dynorphin levels in different brain regions after chronic administration of 14-methoxymetopon and oxycodone-6-oxime.

Previous studies showed that opioid drugs-oxycodone-6-oxime and 14-methoxy-5-methyl-dihydromorphinone (14-methoxymetopon)-produced less respiratory depressive effect and slower rate of tolerance and dependence, respectively. It was also reported that morphine decreased the prodynorphin gene expression in the rat hippocampus, striatum and hypothalamus. In this study, we determined the prodynorphin gene expression and dynorphin levels in selected brain regions of opioid tolerant rats. We found that in the striatum morphine decreased, while oxycodone-6-oxime increased and 14-methoxymetopon did not alter the prodynorphin gene expression. In the nucleus accumbens, morphine and oxycodone-6-oxime did not change, while 14-methoxymetopon increased the prodynorphin gene expression. In the hippocampus both oxycodone-6-oxime and 14-methoxymetopon enhanced, whereas morphine did not alter the prodynorphin gene expression. In the rat striatum only oxycodone-6-oxime increased dynorphin levels significantly in accordance with the prodynorphin mRNA changes. In the hippocampus both opioid agonists increased the dynorphin levels significantly similarly to the augmented prodynorphin gene expression. In ventral tegmental area only 14-methoxymetopon increased dynorphin levels significantly. In nucleus accumbens and the temporal-parietal cortex the changes in the prodynorphin gene expression and the dynorphin levels did not correlate. Since the endogenous prodynorphin system may play a modulatory role in the development of opioid tolerance, the elevated supraspinal dynorphin levels appear to be partly responsible for the reduced degree of tolerance induced by the investigated opioids.

Variations in respiratory distress characterize the acute agonal period during heroin overdose death: relevance to postmortem mRNA studies.

AIMS: To determine whether there are factors during apparent rapid heroin overdose death that affect agonal state and thus brain pH (index of hypoxia) that can influence neurobiological systems linked to drug abuse. DESIGN AND METHODS: Brain specimens and autopsy/medical reports were investigated in subjects dying from heroin overdose (n=70) and compared to normal controls (n=45) as well as suicide victims (n=31) with a documented rapid cause of death. Detailed autopsy material was characterized as to positive and negative respiratory distress in relation to brain pH; drug toxicity and other demographic information was also evaluated. In situ hybridization histochemistry was used to study mRNA expression levels of dopamine (e.g., D2 receptor, dopamine transporter) and opioid (e.g., proenkephalin) related markers in various structures in relation to brain pH. FINDINGS: Brain pH was generally reduced in heroin overdose cases versus normal and suicide subjects. There was, however, significant variation in heroin overdose deaths related to differences in respiratory distress that differentially altered brain pH levels. Various factors such as vomit inhalation, resuscitation, pulmonary embolism and suffocation contributed to positive respiratory distress. Elevated brain pH was observed in heroin overdose with positive alcohol toxicity suggesting potentiated alcohol-induced rapidity of heroin deaths. mRNA expression levels of the dopamine-related genes and proenkephalin were positively correlated with brain pH. CONCLUSIONS: Respiratory distress contributes to variations in the acute agonal state during heroin overdose death that differentially alters brain pH levels and significantly impacts mRNA levels. Such findings should be considered for postmortem molecular/neurochemical neurobiological studies of opiate abusers.

Reversion of levodopa-induced motor fluctuations by the A2A antagonist CSC is associated with an increase in striatal preprodynorphin mRNA expression in 6-OHDA-lesioned rats.

The molecular mechanisms involved in the reversion of levodopa-induced motor fluctuations by the adenosine A2A antagonist 8-(3-chlorostryryl) caffeine (CSC) were investigated in rats with a 6-hydroxydopamine (6-OHDA)-induced lesion and compared with the ones achieved by the kappa-opioid agonist, U50,488. Animals were treated with levodopa (50 mg/kg/day) for 22 days and for one additional week with levodopa + CSC (5 mg/kg/day), levodopa + U50,488 (1 mg/kg/day), or levodopa + vehicle. The reversion of the decrease in the duration of levodopa-induced rotations by CSC, but not by U50,488, was maintained until the end of the treatment and was associated with a further increase in levodopa-induced preprodynorphin mRNA in the lesioned striatum, being higher in the ventromedial striatum. The increase in striatal preprodynorphin expression, particularly in the ventromedial striatum, may be related to the reversion of levodopa-induced motor fluctuations in the CSC-treated animals, suggesting a role of the direct striatal output pathway activity in the ventromedial striatum in the pathophysiology of motor fluctuations.

Chronic and acute effects of 3,4-methylenedioxy-N-methylamphetamine ('Ecstasy') administration on the dynorphinergic system in the rat brain.

The prodynorphin system is implicated in the neurochemical mechanism of psychostimulants. Exposure to different drugs of abuse can induce neuroadaptations in the brain and affect opioid gene expression. The present study aims to examine the possibility of a common neurobiological substrate in drug addiction processes. We studied the effects of single and repeated 3,4-methylenedioxy-N-methylamphetamine ('Ecstasy') on the gene expression of the opioid precursor prodynorphin, and on the levels of peptide dynorphin A in the rat brain. Acute (8 mg/kg, intraperitoneally) 3,4-methylenedioxy-N-methylamphetamine markedly raised, two hours later, prodynorphin mRNA levels in the prefrontal cortex, and in the caudate putamen, whereas it decreased gene expression in the ventral tegmental area. Chronic (8 mg/kg, intraperitoneally, twice a day for 7 days) 3,4-methylenedioxy-N-methylamphetamine increased prodynorphin mRNA in the nucleus accumbens, hypothalamus and caudate putamen and decreased it in the ventral tegmental area. Dynorphin A levels increased after chronic treatment in the ventral tegmental area and decreased after acute treatment in the nucleus accumbens, prefrontal cortex and hypothalamus. These findings confirm the role of the dynorphinergic system in mediating the effects of drugs of abuse, such as 3,4-methylenedioxy-N-methylamphetamine, in various regions of the rat brain, which may be important sites for the opioidergic mechanisms activated by addictive drugs.

CB1 receptor knockout mice show similar behavioral modifications to wild-type mice when enkephalin catabolism is inhibited.

Behavioral and biochemical studies have suggested a functional link between the endogenous cannabinoid and opioid systems. Different hypotheses have been proposed to explain the interactions between opioid and cannabinoid systems such as a common pathway stimulating the dopaminergic system, a facilitation of signal-transduction- and/or a cannabinoid-induced enhancement of opioid peptide release. However, at this time, all the studies have been performed with exogenous agonists (delta-9-tetrahydrocannabinol or morphine), leading to a generally excessive stimulation of receptors normally stimulated by endogenous effectors (anandamide or opioid peptides) in various brain structures. To overcome this problem, we have measured various behavioral responses induced by the stimulation of the endogenous opioid system using the dual inhibitor of enkephalin-degrading enzymes, RB101, in CB1 receptor knockout mice. Thus, analgesia, locomotor activity, anxiety and antidepressant-like effects were measured after RB101 administration (80 and 120 mg/kg i.p. or 10 mg/kg, i.v.) in CB1 receptor knockout mice and their wild-type littermates. In all the experiments, inhibition of enkephalin catabolism produced similar modifications in behavior observed in CB1 knockout and wild-type mice. These results suggest limited physiological interaction between cannabinoid and opioid systems.

Regulation of striatal preproenkephalin mRNA levels in MPTP-lesioned mice treated with estradiol.

We reported previously the protective effect of 17beta-estradiol (17beta-E(2)) on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopamine (DA) depletion. This protection was stereospecific, because 17beta-E(2) showed activity but 17alpha-estradiol (17alpha-E(2)) did not. The mechanisms by which estradiol exerts its beneficial effects, however, remain unknown. We investigated a possible implication of enkephalins (ENK) in neuroprotective activity of 17beta-E(2). Protection against MPTP-induced DA depletion was obtained with 17beta-E(2) but not 17alpha-E(2). MPTP lesion increased striatal preproenkephalin (PPE) mRNA levels and they remained elevated in 17alpha-E(2)-treated MPTP mice whereas 17beta-E(2) treatment decreased these levels to control values. This is the first report of estradiol modulation of striatal PPE mRNA in mice. Negative and significant correlations between DA levels, vesicular monoamine transporter (VMAT(2)) density, and PPE mRNA were observed in the striatum of lesioned animals. This effect of 17beta-E(2) on PPE mRNA after a lesion could be one of many mechanisms by which this steroid exerts its neuroprotective activity.

Candidate genes, pathways and mechanisms for bipolar (manic-depressive) and related disorders: an expanded convergent functional genomics approach.

Identifying genes for bipolar mood disorders through classic genetics has proven difficult. Here, we present a comprehensive convergent approach that translationally integrates brain gene expression data from a relevant pharmacogenomic mouse model (involving treatments with a stimulant--methamphetamine, and a mood stabilizer--valproate), with human data (linkage loci from human genetic studies, changes in postmortem brains from patients), as a bayesian strategy of crossvalidating findings. Topping the list of candidate genes, we have DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of 32 kDa) located at 17q12, PENK (preproenkephalin) located at 8q12.1, and TAC1 (tachykinin 1, substance P) located at 7q21.3. These data suggest that more primitive molecular mechanisms involved in pleasure and pain may have been recruited by evolution to play a role in higher mental functions such as mood. The analysis also revealed other high-probability candidates genes (neurogenesis, neurotrophic, neurotransmitter, signal transduction, circadian, synaptic, and myelin related), pathways and mechanisms of likely importance in pathophysiology.

The striopallidal pathway is involved in antiparkinsonian-like effects of the blockade of group I metabotropic glutamate receptors in rats.

The aim of the study was to examine the influence of the blockade of group I metabotropic glutamate receptors (mGluRs) on the haloperidol-induced catalepsy and proenkephalin mRNA expression in the rat striatum. Bilateral, intrastriatal injection of AIDA ((RS)-1-aminoindan-1,5-dicarboxylic acid, 3-15 microg/0.5 microl), a selective antagonist of group I mGluRs, inhibited catalepsy induced by haloperidol (0.5 mg/kg i.p.). Repeated intrastriatal AIDA administrations (3 x 15 microg/0.5 microl, 3 h apart) counteracted the haloperidol-induced (3 x 1.5 mg/kg s.c., 3 h apart) increase in the proenkephalin mRNA expression in that structure. The present study indicates that the blockade of the striatal group I mGluRs may inhibit parkinsonian akinesia by normalizing the function of the striopallidal pathway.

"Binge" cocaine differentially alters preproenkephalin mRNA levels in guinea pig brain.

Male Hartley guinea pigs were administered i.p. injections of cocaine or saline for 2 or 7 days in a "binge" paradigm. RNA was isolated from dissected brain regions and levels of preproenkephalin mRNA and total RNA were quantified by RNase protection assays. Following 2 days of "binge" cocaine administration, no significant alterations in preproenkephalin mRNA levels were detected in six brain regions. Following 7 days of cocaine administration, however, lower levels of preproenkephalin mRNA were observed in the nucleus accumbens and hypothalamus of cocaine-treated animals and higher levels in the frontal cortex and amygdala. These findings differed from previous studies in the rat, so an additional experiment was performed with animals treated at the 7 day time point. For increased statistical power, data from the two experiments were combined and examined by two-way ANOVAs; in this combined analysis, increases in preproenkephalin mRNA were observed in frontal cortex, amygdala, and hippocampus, decreases were found in the nucleus accumbens and hypothalamus, with no change in thalamus, caudate putamen, or cerebellum. These observed differences between guinea pigs and rats make this species an interesting model for neurobiological studies of cocaine-induced alterations in neuropeptide gene expression in the mammalian brain.

Ciproxifan, a histamine H3-receptor antagonist/inverse agonist, modulates the effects of methamphetamine on neuropeptide mRNA expression in rat striatum.

We have explored the effect of histamine H3-receptor ligands on the regulation of neuropeptide mRNA expression in the striatum by using in situ hybridization performed with proenkephalin, prodynorphin, substance P and proneurotensin riboprobes. Acute administration of ciproxifan, an H3-receptor antagonist/inverse agonist, or (R)-alpha-methylhistamine, an H3-receptor agonist, did not modify the striatal expression of the neuropeptides by itself. However, ciproxifan strongly and differentially modulated the effect of a single administration of 3 mg/kg methamphetamine on neuropeptide mRNA expression. This modulation was suppressed by the administration of (R)-alpha-methylhistamine and occurred in both the caudate-putamen and nucleus accumbens. Ciproxifan strongly potentiated the decrease of proenkephalin mRNA expression induced by methamphetamine. In contrast, it suppressed the increase in prodynorphin and substance P mRNA expression induced by methamphetamine. Methamphetamine alone or with ciproxifan did not modify proneurotensin mRNA expression. These neurochemical findings indicate that ciproxifan differentially regulates the effect of methamphetamine on the neuropeptides contained in striatonigral and striatopallidal neurons. They suggest that endogenous histamine and dopamine cooperate to modulate the activity of striatal projection neurons and strengthen the interest of H3-receptors as new targets for the treatment of psychotic disorders and drug abuse.

Unilateral striatal dopamine depletion: time-dependent effects on cortical function and behavioural correlates.

Previously, we showed that unilateral blockade of D1 dopamine receptors in the striatum inhibits immediate-early gene expression bilaterally throughout large parts of the cortex, including sensory-evoked expression in the barrel cortex. To further investigate this dopamine regulation of cortical function, we examined the effects of dopamine depletion on cortical gene regulation and behavioural correlates. Two days after unilateral infusion of 6-hydroxydopamine into the midbrain, rats displayed a (to some degree) bilateral reduction in cortical zif 268 expression that was more pronounced on the lesioned side. This decrease was found across motor, somatosensory, insular and piriform, but not cingulate, cortex, similar to the effects of blockade of striatal D1 receptors. Furthermore, whisker stimulation-evoked c-fos and zif 268 expression in the barrel cortex ipsilateral to the lesion was also attenuated by acute dopamine depletion. These cortical deficits were accompanied by a breakdown of spontaneous behaviours in an open-field test. In contrast, 21 days after dopamine depletion, both basal and sensory-evoked gene expression in the cortex were near-normal. This cortical recovery was paralleled by recovery in locomotion and in sensory-guided behaviour (scanning) related to the hemisphere contralateral to the lesion, but not in scanning by the dopamine-depleted hemisphere. Our results suggest that striatal dopamine exerts a widespread facilitatory influence on cortical function that is necessary, but not sufficient, for normal behaviour. Moreover, the mechanisms mediating this cortical facilitation appear to be subject to substantial neuroplasticity after dopamine perturbation.

Enkephalin release and opioid receptor activation does not mediate the antinociceptive or sedative/hypnotic effects of nitrous oxide.

In previous studies using Fos expression as a marker of neuronal activation, we showed that nitrous oxide (N(2)O) activates bulbospinal noradrenergic neurons in rats and that destruction of these neuronal pathways leads to loss of N(2)O antinociceptive action. Based on previous rat studies it has been proposed that these noradrenergic neurons are activated through opioid receptors through the release of endogenous opioid ligands in the periaqueductal gray. Using mice with a disrupted preproenkephalin gene (Penk2 -/-) and the opioid receptor antagonist naltrexone, we investigated the role of enkephalinergic mechanisms and opioid receptor activation in the behavioral and bulbospinal neuron responses to N(2)O in mice. The antinociceptive response to N(2)O was investigated using the tail-flick, hot-plate, and von Frey assays, the sedative/hypnotic response was measured using rotarod and loss of righting reflex, and bulbospinal neuronal activation was assessed with pontine Fos immunostaining. No differences were observed between wild-type and Penk2 -/- mice for the antinociceptive, sedative/hypnotic, and pontine neuronal activation effects of N(2)O. Similarly, naltrexone did not block N(2)O-induced antinociception, sedation, or hypnosis. We conclude that neither enkephalin nor opioid receptors participate in either the antinociceptive or the sedative/hypnotic actions of N(2)O in mice.

Effects of the potent analgesic enkephalin-catabolizing enzyme inhibitors RB101 and kelatorphan on respiration.

We investigated whether the enkephalin-catabolizing enzyme inhibitors RB101 and kelatorphan, which have been shown to be potent analgesics, depress respiration as do opioid analgesics. Ventilation was measured in cats and rodents by the barometric method, in the awake state and during anesthesia. Tissue distribution of the inhibitors was either generalized (RB101, 40-160 mg/kg i.p.), largely restricted by the blood-brain barrier to the periphery (kelatorphan, 0.7-20 mg/kg i.v.), or restricted to the brainstem (i.c.v. injection of RB101 in the fourth ventricle). RB101 did not affect ventilation in any condition tested, and large doses of kelatorphan produced a naloxone-reversible increase in ventilation and breathing frequency. Thus endogenous opioids released during conditions of normal ventilation do not exert any depressant neuromodulatory effect on this function, even when their extracellular concentrations are increased by peptidase inhibitors. The differential effect of these inhibitors on ventilation and nociception is discussed. We conclude that kelatorphan and RB101 are devoid of respiratory-depressant effects and might be interesting pharmacological alternatives to morphine and other opioid agonists.

Effects of spinorphin and tynorphin on synaptic transmission in rat hippocampal slices.

Spinorphin has been isolated from the bovine spinal cord as an endogenous inhibitor of enkephalin-degrading enzymes (aminopeptidase, dipeptidyl aminopeptidase III, angiotensin-converting enzyme and enkephalinase), and tynorphin has been synthesized as a more potent inhibitor of dipeptidyl aminopeptidase III. In this study, the effects of spinorphin and tynorphin on synaptic transmission were studied in rat isolated hippocampal slices. Field potentials were recorded from the CA1 region after stimulation of Schaffer collaterals. Spinorphin (1 microM), which alone had no effect, potentiated the facilitatory effects of enkephalin on the filed potentials at a stimulation interval of 15 s. At a stimulation interval of 10--4 s, spinorphin alone frequency dependently inhibited the field potential. On the other hand, tynorphin (1 microM), which alone had no effect at any stimulus interval, tended to potentiate the facilitatory effects of enkephalin. Spinorphin blocked long-term potentiation induced by tetanic stimulation (100 Hz, 1 s), whereas tynorphin had no effect on long-term potentiation. These results suggest that, at a low stimulation frequency, spinorphin potentiates the facilitatory effects of enkephalin by preventing degradation of enkephalin, whereas at a high stimulation frequency spinorphin use dependently inhibits synaptic transmission independently of enkephalin. On the other hand, tynorphin tends to potentiate the facilitatory effects of enkephalin without use-dependent inhibition.