Cannabinoid CB1 receptor-mediated inhibition of noradrenaline release in the human and guinea-pig hippocampus

We examined the question of whether cannabinoid receptors modulating noradrenaline release are detectable in the brain of humans and experimental animals. For this purpose, hippocampal slices from humans, guinea-pigs, rats and mice and cerebellar, cerebrocortical and hypothalamic slices from guinea-pigs were incubated with [³H]noradrenaline and then superfused. Tritium overflow was evoked either electrically (0.3 or 1Hz) or by introduction of Ca²⁺ ions (1.3μM) into Ca²⁺-free, K⁺-rich medium (25μM) containing tetrodotoxin 1μM. Furthermore, the cAMP accumulation stimulated by forskolin 10μM was determined in guinea-pig hippocampal membranes. We used the following drugs: the cannabinoid receptor agonists (–)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP-55,940) and R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone (WIN 55,212-2), the inactive S(–)-enantiomer of the latter (WIN 55,212-3) and the CB₁ receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (SR 141716). The electrically evoked tritium overflow from guinea-pig hippocampal slices was reduced by WIN 55,212-2 (pIC_30% 6.5) but not affected by WIN 55,212-3 up to 10μM. The concentration-response curve of WIN 55,212-2 was shifted to the right by SR 141716 (0.032μM) (apparent pA₂ 8.2), which by itself did not affect the evoked overflow. WIN 55,212-2 1μM also inhibited the Ca²⁺-evoked tritium overflow in guinea-pig hippocampal slices and the electrically evoked overflow in guinea-pig cerebellar, cerebrocortical and hypothalamic slices as well as in human hippocampal slices but not in rat and mouse hippocampal slices. SR 141716 (0.32μM) markedly attenuated the WIN 55,212-2-induced inhibition in guinea-pig and human brain slices. SR 141716 0.32μM by itself increased the electrically evoked tritium overflow in guinea-pig hippocampal slices but failed to do so in slices from the other brain regions of the guinea-pig and in human hippocampal slices. The cAMP accumulation stimulated by forskolin was reduced by CP-55,940 and WIN 55,212-2. The concentration-response curve of CP 55,940 was shifted to the right by SR 141716 (0.1μM; apparent pA₂ 8.3), which by itself did not affect cAMP accumulation. In conclusion, cannabinoid receptors of the CB₁ subtype occur in the human hippocampus, where they may contribute to the psychotropic effects of cannabis, and in the guinea-pig hippocampus, cerebellum, cerebral cortex and hypothalamus. The CB₁ receptor in the guinea-pig hippocampus is located presynaptically, is activated by endogenous cannabinoids and may be negatively coupled to adenylyl cyclase. Received: 5 June 1997 / Accepted: 6 August 1997     

Cannabinoid receptor-mediated inhibition of dopamine release in the retina

The possible occurrence of cannabinoid (CB) receptors was studied on superfused guinea-pig retinal discs preincubated with [3H]dopamine or [³H]noradrenaline. Tritium overflow was evoked either electrically (3 Hz) or by re-introduction of Ca²⁺, 1.3 mM after superfusion with Ca²⁺-free medium containing K⁺ 30 rnM. The accumulation of [³H]dopamine ([³H]DA) and [³H]noradrenaline ([³H]NA) was inhibited by the selective inhibitor of the neuronal dopamine transporter GBR-12909 (pIC_50% 7.29 and 7.41, respectively) but not by the selective inhibitor of the neuronal noradrenaline transporter desipramine (1 M). The electrically or Ca²⁺-evoked tritium overflow in retinal discs preincubated with [³H]DA or [³H]NA was reduced by the CB receptor agonists CP-55,940 and WIN 55,212-2 (pIC_50% in discs preincubated with [³H]NA, electrical stimulation: 7.03 and 6.70, respectively) but not affected by the inactive S(–)enantiomer of the latter, WIN 55,212-3 (up to 10 M). The concentration-response curve of WIN 55,212-2 was shifted to the right by the CB₁ receptor antagonist SR 141716 (apparent pA₂: 8.29) which, by itself, increased the evoked overflow. The facilitatory effect of SR 141716 was not affected by GBR-12909 and the dopamine receptor antagonist haloperidol. In conclusion, the dopaminergic neurones of the guinea-pig retina can be labelled by both [³H]DA and [³H]NA. Transmitter release from the dopaminergic neurones is inhibited by activation of cannabinoid receptors of the CB₁ type, which appear to be tonically activated by an endogenous CB receptor ligand.     

Cannabinoid CB<Subscript>1</Subscript> receptor-mediated inhibition of noradrenaline release in guinea-pig vessels,but not in rat and mouse aorta

Cannabinoids exert complex effects on blood pressure related to their interference with cardiovascular centres in the central nervous system and to their direct influence on vascular muscle, vascular endothelium and heart. In view of the relative lack of information on the occurrence of CB₁ receptors on the vascular postganglionic sympathetic nerve fibres, the aim of the present study was to examine whether cannabinoid receptor ligands affect the electrically evoked tritium overflow in superfused vessels (tissue pieces) from the guinea-pig, the rat and the mouse preincubated with ³H-noradrenaline. The cannabinoid receptor agonist WIN 55,212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]-pyrrolo[1,2,3-de]1,4-benzoxazinyl](1-naphthalenyl) methanone) inhibited the evoked tritium overflow in the guinea-pig aorta, but not in that of the rat or mouse. The concentration–response curve of WIN 55,212-2 was shifted to the right by the CB₁ receptor antagonist rimonabant, yielding an apparent pA₂ value of 7.9. The most pronounced (near-maximum) inhibition obtained at the highest WIN 55,212-2 concentration applied (3.2 μM) amounted to 40%. WIN 55,212-2 also inhibited the evoked overflow in guinea-pig pulmonary artery, basilar artery and portal vein, again in a manner sensitive to antagonism by rimonabant. The latter did not affect the evoked overflow by itself in the four vessels, but did increase the electrically evoked tritium overflow from superfused guinea-pig hippocampal slices preincubated with ³H-choline and from superfused guinea-pig retina discs preincubated with ³H-noradrenaline (labelling dopaminergic cells in this tissue). The inhibitory effect of 3.2 μM WIN 55,212-2 on the evoked overflow from the guineapig aorta was comparable in size to that obtained with agonists at the histamine H₃, κ opioid (KOP) and ORL₁ (NOP) receptor (1 or 10 μM, producing the respective near-maximum effects) whereas prostaglandin E₂ 1 μM caused a higher near-maximum inhibition of 70%. Prostaglandin E₂ also induced an inhibition by 65 and 80% in the rat and mouse aorta respectively, indicating that the present conditions are basically suitable for detecting presynaptic receptor-mediated inhibition of noradrenaline release. The results show that the postganglionic sympathetic nerve fibres in the guineapig aorta, but not in the rat or mouse aorta, are endowed with presynaptic inhibitory cannabinoid CB₁ receptors; such receptors also occur in guineapig pulmonary artery, basilar artery and portal vein. These CB₁ receptors are not subject to an endogenous tone and the extent of inhibition obtainable via these receptors is within the same range as that of several other presynaptic heteroreceptors, but markedly lower than that obtainable via receptors for prostaglandin E₂.     

Cannabinoid CB1 receptor-mediated inhibition of the neurogenic vasopressor response in the pithed rat

The effects of two cannabinoid receptor agonists, R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]-pyrrolo[1, 2, 3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone (WIN 55,212-2) and (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP-55,940), were studied on (i) the vasopressor response elicited in pithed rats by electrical stimulation of the sympathetic outflow and (ii) the release of ³H-noradrenaline and the vasoconstriction elicited in isolated rat tail arteries by transmural electrical stimulation. In pithed rats, the electrical (1Hz for 10s) stimulation of the preganglionic sympathetic nerve fibres increased diastolic blood pressure by about 30mmHg. This neurogenic vasopressor response (which under the conditions of our study was almost exclusively due to the release of catecholamines) was decreased by WIN 55-212,2 and CP-55,940 in a dose-dependent manner (inhibition by WIN 55,212-2 and CP-55,940, 0.1μmol/kg each, about 25–30%). The inhibition was identical in adrenalectomized rats and in animals with intact adrenals. The inhibitory action of WIN 55,212-2 and CP-55,940 was abolished by a dose of 0.03μmol/kg of the CB₁ receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlo- rophenyl)-4-methyl-3-pyrazole-carboxamide (SR 141716), which, by itself, had no effect. WIN 55,212-2, CP-55,940 and SR 141716 failed to affect the vasopressor response to exogenous noradrenaline (1nmol/kg), which also increased diastolic blood pressure by about 30mmHg. In isolated rat tail arteries, the electrically (0.4Hz) evoked tritium overflow and vasoconstriction were not modified by WIN 55,212-2 and CP-55,940 (1μmol/l each). In conclusion, the neurogenic vasopressor response in the pithed rat can be modulated via cannabinoid CB₁ receptors probably located presynaptically on the postganglionic sympathetic nerve fibres innervating resistance vessels. Received: 4 April 1997 / Accepted: 10 May 1997     

Inhibition of serotonin release in the mouse brain via presynaptic cannabinoid CB1 receptors

We studied whether serotonin release in the CNS is inhibited via cannabinoid receptors. In mouse brain cortex slices preincubated with [3H]serotonin and superfused with medium containing indalpine and metitepine, tritium overflow was evoked either electrically (3 Hz) or by introduction of Ca2+ (1.3 mM) into Ca2+-free K+-rich (25 mM) medium containing tetrodotoxin. The effects of cannabinoid receptor ligands on the electrically evoked tritium overflow from mouse brain cortex slices preincubated with [3H]choline and on the binding of [3H]WIN 55,212-2 and [35S]GTPgammaS to mouse brain cortex membranes were examined as well. In superfused mouse cortex membranes preincubated with [3H]serotonin, the electrically evoked tritium overflow was inhibited by the cannabinoid receptor agonist WIN 55,212-2 (maximum effect of 20%, obtained at 1 microM; pEC50=7.11) and this effect was counteracted by the CB1 receptor antagonist SR 141716 (apparent pA2=8.02), which did not affect the evoked tritium overflow by itself. The effect of WIN 55,212-2 was not shared by its enantiomer WIN 55,212-3 but was mimicked by another cannabinoid receptor agonist, CP-55,940. WIN 55,212-2 also inhibited the Ca2+-evoked tritium overflow and this effect was antagonized by SR 141716. Concentrations of histamine, prostaglandin E2 and neuropeptide Y, causing the maximum effect at their respective receptors, inhibited the electrically evoked tritium overflow by 33, 69 and 73%, respectively. WIN 55,212-2 (1 microM) inhibited the electrically evoked tritium overflow from mouse brain cortex slices preincubated with [3H]choline by 49%. [3H]WIN 55,212-2 binding to mouse cortex membranes was inhibited by CP-55,940, SR 141716 and WIN 55,212-2 (pKi=9.30, 8.70 and 8.19, respectively) but not by the auxiliary drugs indalpine, metitepine and tetrodotoxin (pKi<4.5). [35S]GTPgammaS binding was increased by WIN 55,212-2 (maximum effect of 80%, pEC50=6.94) but not affected by WIN 55,212-3. In conclusion, serotonin release in the mouse brain cortex is inhibited via CB1 receptors, which may be located presynaptically and are not activated by endogenous cannabinoids. The extent of inhibition is smaller than that obtained (1) via another three presynaptic receptors on serotoninergic neurones and (2) via CB1 receptors on cholinergic neurones in the same tissue.     

Defaecation, intestinal fluid accumulation and motility in rodents: implications of cannabinoid CB1 receptors

We have studied the effect of SR141716A (0.1–5 mg/kg, i.p.), a cannabinoid CB₁ receptor antagonist, and WIN (0.1–5 mg/kg, i.p.), a cannabinoid receptor agonist, on acute defaecation and gastrointestinal transit in mice and on intraluminal fluid accumulation in the rat small intestine. SR141716A increased while WIN 55,212-2 decreased defaecation, gastrointestinal transit and fluid accumulation. A per se non-effective dose of SR141716A (0.3 mg/kg) counteracted the inhibitory effect of WIN 55,212-2 (1 mg/kg) on gastrointestinal functions studied. The effect of SR141716 on both intestinal fluid accumulation in rats and gastrointestinal transit in mice was inhibited by atropine (1 mg/kg, i.p.), but not by hexamethonium (1 mg/kg, s.c.), SR140333 (20 μg/kg, i.p.) or SR48968 (20 μg/kg, i.p.), antagonists of NK₁ and NK₂ receptors, respectively. These results suggest that intestinal fluid accumulation and motility are inhibited by endogenous cannabinoid(s) acting at the cannabinoid CB₁ receptors. This effect may be mediated by mechanisms involving muscarinic cholinoceptors. Received: 13 July 1998 / Accepted: 25 September 1998     

Effects of the cannabinoid CB<Subscript>1</Subscript> receptor antagonist rimonabant on distinct measures of impulsive behavior in rats

Rationale Pathological impulsivity is a prominent feature in several psychiatric disorders, but detailed understanding of the specific neuronal processes underlying impulsive behavior is as yet lacking. Objectives As recent findings have suggested involvement of the brain cannabinoid system in impulsivity, the present study aimed at further elucidating the role of cannabinoid CB₁ receptor activation in distinct measures of impulsive behavior. Materials and methods The effects of the selective cannabinoid CB₁ receptor antagonist, rimonabant (SR141716A) and agonist WIN55,212-2 were tested in various measures of impulsive behavior, namely, inhibitory control in a five-choice serial reaction time task (5-CSRTT), impulsive choice in a delayed reward paradigm, and response inhibition in a stop-signal paradigm. Results In the 5-CSRTT, SR141716A dose-dependently improved inhibitory control by decreasing the number of premature responses. Furthermore, SR141716A slightly improved attentional function, increased correct response latency, but did not affect other parameters. The CB₁ receptor agonist WIN55,212-2 did not change inhibitory control in the 5-CSRTT and only increased response latencies and errors of omissions. Coadministration of WIN55,212-2 prevented the effects of SR141716A on inhibitory control in the 5-CSRTT. Impulsive choice and response inhibition were not affected by SR141716A at any dose, whereas WIN55,212-2 slightly impaired response inhibition but did not change impulsive choice. Conclusions The present data suggest that particularly the endocannabinoid system seems involved in some measures of impulsivity and provides further evidence for the existence of distinct forms of impulsivity that can be pharmacologically dissociated.     

Cannabinoid CB1 receptor-mediated inhibition of acetylcholine release in the brain of NMRI, CD-1 and C57BL/6J mice

Cannabinoid CB1 receptors occur as presynaptic receptors producing inhibition of neurotransmitter release. To elucidate their physiological role, experiments on tissues from CB1 receptor knockout mice would be helpful. We studied whether CB1 receptor-mediated inhibition of acetylcholine release is detectable in the brain of NMRI mice and of CD-1 and C57BL/6J mice (the latter two strains representing the wild-type strains of the two CB1 receptor knockout mouse models). Brain slices preincubated with [3H]choline were superfused and tritium overflow was evoked electrically (3 Hz) or by introduction of Ca2+ into Ca2+-free K+-rich medium (35 mM) containing tetrodotoxin. The eletrically evoked tritium overflow from NMRI mouse hippocampal slices was inhibited (maximally by 60%) by the cannabinoid receptor agonists CP-55,940 and WIN 55,212-2 but not affected by WIN 55,212-3 (the inactive enantiomer of WIN 55,212-2; pEC50=7.9, 7.4 and <5.5). The concentration-response curve of WIN 55,212-2 was shifted to the right by the CB1 receptor antagonist SR 141716 (apparent pA2=8.6). Compared to hippocampal slices from NMRI mice, WIN 55,212-2 1 microM inhibited the electrically evoked overflow (1) from cortical slices from NMRI mice to a lesser extent and from striatal slices not at all, (2) from hippocampal slices from CD-1 and C57BL/6J mice to an identical extent and (3) from hippocampal slices from Sprague-Dawley rats to at least the same extent. SR 141716 0.32 microM abolished the effect of WIN 55,212-2 1 microM in hippocampal slices from NMRI, CD-1 and C57BL/6J mice and in cortical slices from NMRI mice. The electrically evoked tritium overflow from NMRI mouse hippocampal slices was also inhibited by the muscarinic receptor agonist oxotremorine (maximum effect of 85%; pEC50=6.5) and this effect was antagonized by the muscarinic receptor antagonist AF-DX 384 (apparent pA2=8.3). The Ca2+-evoked tritium overflow from NMRI mouse hippocampal slices was inhibited by WIN 55,212-2 in a manner sensitive to SR 141716. In conclusion, the cholinergic axon terminals of the NMRI mouse hippocampus are endowed with presynaptic CB1 receptors. Such receptors are also detectable in the hippocampus of CD-1 and C57BL/6J mice. The maximum extent of the CB1 receptor-mediated inhibition of acetylcholine release is lower than the maximum effect mediated via the autoreceptor.     

Enhanced acetylcholine release in the hippocampus of cannabinoid CB(1) receptor-deficient mice

We examined whether acetylcholine release in the hippocampus and striatum and noradrenaline release in the hippocampus is altered in CB(1) receptor-deficient mice. The electrically evoked tritium overflow from hippocampal slices preincubated with [(3)H]-choline was increased by about 100% in CB(1)(-/-) compared to CB(1)(+/+) mice whereas the electrically evoked tritium overflow from striatal slices preincubated with [(3)H]-choline and from hippocampal slices preincubated with [(3)H]-noradrenaline did not differ. The cannabinoid receptor agonist, WIN 55,212-2, inhibited, and the CB(1) receptor antagonist, SR 141716, facilitated, the evoked tritium overflow from hippocampal slices (preincubated with [(3)H]-choline) from CB(1)(+/+) as opposed to CB(1)(-/-) mice. Both drugs did not affect the evoked tritium overflow from striatal slices (preincubated with [(3)H]-choline) and from hippocampal slices (preincubated with [(3)H]-noradrenaline) from CB(1)(+/+) and CB(1)(-/-) mice. The selective increase in acetylcholine release in CB(1)(-/-) mice may indicate that the presynaptic CB(1) receptors on the cholinergic neurones of the mouse hippocampus are tonically activated and/or constitutively active in vivo.     

Cannabinoid CB(1) receptor-mediated inhibition of hippocampal acetylcholine release is preserved in aged mice

1. The cannabinoid CB(1) receptor inverse agonist/antagonist SR 141716 increases acetylcholine release in rodent hippocampus and improves memory in some experimental paradigms. Since drugs like SR 141716 may represent a novel class of cognition-enhancing drugs, we wanted to check whether the function of the CB(1) receptor is preserved during ageing. 2. Hippocampal and striatal slices from 2- to 3- and 24- to 28-month-old C57BL/6J mice were preincubated with [(3)H]-choline or [(3)H]-noradrenaline ([(3)H]-NA) and superfused. 3. The cannabinoid receptor agonist WIN 55212-2 inhibited, and SR 141716 facilitated, the electrically (3 Hz) evoked tritium overflow in hippocampal slices (preincubated with [(3)H]-choline) from young and aged mice to the same extent. The evoked overflow per se was less by 33% in slices from aged animals. 4. WIN 55212-2 and SR 141716 did not affect, but the muscarinic receptor agonist oxotremorine inhibited, the evoked (3 Hz) overflow in striatal slices (preincubated with [(3)H]-choline) from young and aged mice to the same extent. The evoked overflow per se tended to be less in slices from aged animals. 5. The evoked (0.3 Hz) overflow in hippocampal slices (preincubated with [(3)H]-NA) was not affected by WIN 55212-2 and SR 141716, but was inhibited by histamine (via H(3) receptors) in slices from young mice and, to a somewhat less extent, in slices from aged mice. The evoked overflow per se did not differ between age groups. 6. In conclusion, the function of the CB(1) receptor involved in the tonic inhibition of hippocampal acetylcholine release is preserved in aged mice.     

Lack of CB1 receptors increases noradrenaline release in vas deferens without affecting atrial noradrenaline release or cortical acetylcholine release

1. We studied whether cannabinoid CB1 receptor gene disruption (to yield CB1-/- mice) affects the electrically evoked tritium overflow from vas deferens and atrial pieces preincubated with [3H]-noradrenaline (NA) ('noradrenaline release') and from cerebral cortex slices preincubated with [3H]-choline ('acetylcholine release'). 2. NA release was higher by 37% in vas deferens from CB1-/- mice than in vas deferens from CB1+/+ mice. The cannabinoid receptor agonist WIN 55,212-2 inhibited, and the CB1 receptor inverse agonist/antagonist SR 141716, increased NA release in vas deferens from CB1+/+ mice without affecting it in vas deferens from CB1-/- mice. 3. Atrial NA release did not differ between CB1+/+ and CB1-/- mice nor did WIN 55,212-2 affect NA release in either strain. 4. Cortical acetylcholine (Ach) release did not differ between CB1+/+ and CB1-/- mice. WIN 55,212-2 inhibited, but SR 141716 did not affect, Ach release in the cortex from CB1+/+ mice. Both drugs did not alter Ach release in the cortex from CB1-/- mice. 5. Tritium content did not differ between CB1+/+ and CB1-/- mice in any preparation. 6. In conclusion, the increase in NA release associated with CB1 receptor deficiency in the vas deferens, which cannot be ascribed to an alteration of tritium content of the preparations, suggests an endogenous tone at the CB1 receptors of CB1+/+ mice in this tissue. Furthermore, the effect of WIN 55,212-2 on NA release in the vas deferens and on cortical Ach release involves CB1 receptors, whereas the involvement of non-CB1-non-CB2 receptors can be excluded.     

CB<Subscript>1</Subscript>- and CB<Subscript>2</Subscript>-cannabinoid receptor-independent lipolysis induced by WIN 55,212-2 in male rat adipocytes

The expression of genes encoding the cannabinoid CB₁ and CB₂ receptors and fatty acid amide hydrolase (FAAH) and the lipolytic activity of cannabinoid agonists were investigated in rat adipose tissue.RT-PCR studies indicated that the genes encoding CB₁ and CB₂ receptors and FAAH are not expressed in epididymal adipocytes. In functional studies, the non-selective cannabinoid receptor agonist WIN 55,212-2 concentration-dependently (0.01–30 µM) induced glycerol release above baseline (E _max 96.1±6.2% of isoprenaline-induced lipolytic response). The selective CB₂ agonist JWH-015 (0.01–30 µM) had no lipolytic activity while the endocannabinoid 2-arachidonoylglycerol and the stable anandamide derivative, R(+)-methanandamide had, only a weak lipolytic effect at the highest concentrations employed (10 and 30 µM). The concentration/response relationship for WIN 55,212-2-mediated lipolytic activity, mimicked by the S(–)-enantiomer WIN 55,212-3, was shifted significantly to the right by the CB₁ antagonist AM 251 only at 10 µM, but was not modified by the -adrenoceptor antagonist propranolol (1 µM). The protein kinase inhibitor H-89, but not the two adenylyl cyclase inhibitors (±)N ⁶-R-phenylisopropyladenosine (R-PIA, 1 µM, a selective A₁ adenosine receptor agonist) or SQ 22,536 (50 µM) significantly reduced the glycerol efflux induced by WIN 55,212-2.Our data suggest that the cannabinoid drug WIN 55,212-2 may exert lipolytic activity in male rat adipocytes via an intracellular mechanism, not activated by CB₁ or CB₂ receptor stimulation, significantly reversed by H-89 but not clearly linked to stimulation of adenylyl cyclase.     

Facilitation and inhibition by capsaicin of cholinergic neurotransmission in the guinea-pig small intestine

The effects of capsaicin on [³H]acetylcholine release and muscle contraction were studied on the myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum preincubated with [³H]choline. Capsaicin concentration-dependently increased both basal [³H]acetylcholine release (pEC₅₀ 7.0) and muscle tone (pEC₅₀ 6.1). The facilitatory effects of capsaicin were antagonized by 1 μM capsazepine (pK _B 7.0 and 7.6), and by the combined blockade of NK₁ and NK₃ tachykinin receptors with the antagonists CP99994 plus SR142801 (each 0.1 μM). This suggests that stimulation by capsaicin of TRPV1 receptors on primary afferent fibres causes a release of tachykinins which, in turn, mediate via NK₁ and NK₃ receptors an increase in acetylcholine release. The capsaicin-induced acetylcholine release was significantly enhanced by the NO synthase inhibitor L-N^G-nitroarginine (100 μM). This indicates that tachykinins released from sensory neurons also stimulate nitrergic neurons and thus lead, via NO release, to inhibition of acetylcholine release. Capsaicin concentration-dependently reduced the electrically-evoked [³H]acetylcholine release (pEC₅₀ 6.4) and twitch contractions (pEC₅₀ 5.9). The inhibitory effects were not affected by either capsazepine, NK₁ and NK₃ receptor antagonists, the cannabinoid CB₁ antagonist SR141716A or by L-N^G-nitroarginine. Desensitization of TRPV1 receptors by a short exposure to 3 μM capsaicin abolished the facilitatory responses to a subsequent administration, but did not modify the inhibitory effects. In summary, capsaicin has a dual effect on cholinergic neurotransmission. The facilitatory effect is indirect and involves tachykinin release and excitation of NK₁ and NK₃ receptors on cholinergic neurons. The inhibition of acetylcholine release may be due to a decrease of Ca²⁺ influx into cholinergic neurons.     

Effects of selective h5-HT1B (SB-216641) and h5-HT1D (BRL-15572) receptor ligands on guinea-pig and human 5-HT auto- and heteroreceptors

Human cerebral cortical slices and synaptosomes, guinea-pig cerebral cortical slices and human right atrial appendages were used to study the effects of SB-216641, a preferential h5-HT_1B receptor ligand, and of BRL-15572, a preferential h5-HT_1D receptor ligand, on the presynaptic h5-HT_1B and h5-HT_1B-like autoreceptors in the human and guinea-pig brain preparations, respectively, and on the presynaptic h5-HT_1D heteroreceptors in the human atrium. The brain preparations, preincubated with [³H]serotonin ([³H]5-HT), and the segments of atrial appendages, preincubated with [³H]noradrenaline, were superfused with modified Krebs’ solution and tritium overflow was evoked electrically (human and guinea-pig cerebral cortex slices and human atrial appendages) or by high K⁺ (human cerebral cortex synaptosomes). The electrically evoked tritium overflow from guinea-pig cerebral cortex slices was reduced by the 5-HT receptor agonist 5-carboxamidotryptamine (5-CT). This effect was not modified by BRL-15572 (2μM; concentration 154 times higher than its K_i at h5-HT_1D receptors) but was antagonized by SB-216641 (0.1μM; concentration 100 times higher than its K_i at h5-HT_1B receptors; apparent pA₂ 8.45). SB-216641 (0.1μM) by itself facilitated, whereas BRL-15572 (2μM) did not affect, the evoked overflow. In human cerebral cortex slices SB-216641 (0.1μM) also facilitated, and BRL-15572 (2μM) again failed to affect, the electrically evoked tritium overflow. In human cerebral cortical synaptosomes, 5-CT reduced the K⁺-evoked tritium overflow. This response was unaffected by BRL-15572 (300nM) but antagonized by SB-216641 (15nM; drug concentrations 23 and 15 times higher than their K_i at h5-HT_1D and h5-HT_1B receptors, respectively). Both drugs, given alone, did not modify the K⁺-evoked tritium overflow. In human atrial appendages, the electrically evoked tritium overflow was inhibited by 5-HT in a manner susceptible to antagonism by BRL-15572 (300nM; 23 times K_i at h5-HT_1D receptors) but not by SB-216641 (30nM; 30 times K_i at h5-HT_1B receptors). Both drugs by themselves did not change the electrically evoked tritium overflow. In conclusion, SB-216641 behaves as a preferential antagonist at native human 5-HT_1B receptors and BRL-15572 as a preferential antagonist at native human 5-HT_1D receptors. These compounds are clearly useful tools for the differentiation between human 5-HT_1B and 5-HT_1D receptors in functional studies. Received: 14 March 1997 / Accepted: 18 May 1997     

Discriminative stimulus effects of the cannabinoid CB1 antagonist SR 141716A in rhesus monkeys pretreated with Δ9-tetrahydrocannabinol

Rationale Drug discrimination can be used to examine tolerance and dependence in agonist-treated animals by establishing an appropriate antagonist as a discriminative stimulus.Objective Establish intravenous SR 141716A as a discriminative stimulus in four rhesus monkeys pretreated with a relatively small dose of Δ⁹-tetrahydrocannabinol (Δ⁹-THC).Methods Rhesus monkeys received i.v. Δ⁹-THC (0.32 mg/kg) and discriminated i.v. SR 141716A (1 mg/kg) from vehicle while responding under a fixed ratio (FR) 5 schedule of stimulus-shock termination.Results The discriminative stimulus effects of SR 141716A were dose-dependent (ED₅₀=0.33 mg/kg) and were mimicked by the CB₁ antagonist AM 251 (ED₅₀=0.98 mg/kg), but not by a benzodiazepine (midazolam) or an N-methyl-D-aspartate antagonist (ketamine). An additional dose (0.32 mg/kg in addition to 0.32 mg/kg administered before the session) of Δ⁹-THC shifted the SR 141716A dose–effect curve 3-fold rightward. Omitting Δ⁹-THC before test sessions resulted in responding on the SR 141716A lever that was attenuated by subsequent administration of Δ⁹-THC (ED₅₀=0.13 mg/kg), CP 55940 (ED₅₀=0.013 mg/kg), and WIN 55212-2 (ED₅₀=0.35 mg/kg); midazolam and ketamine did not attenuate responding on the SR 141716A lever. SR 141716A (1 mg/kg) shifted the Δ⁹-THC and CP 55940 dose–effect curves 3.4-fold rightward; the WIN 55212-2 dose–effect curve was not significantly modified by a dose of 1 mg/kg of SR 141716A.Conclusions SR 141716A can be established as a discriminative stimulus in animals pretreated with Δ⁹-THC, and this assay is selective for cannabinoid activity. Differential antagonism of cannabinoids by SR 141716A might indicate that the mechanism of action of WIN 55212-2 is not identical to other cannabinoids. This study demonstrates that, under the appropriate conditions, drug discrimination has utility for examining cannabinoid dependence and withdrawal.     

Effects of cannabinoid receptor ligands on electrophysiological properties of myenteric neurones of the guinea-pig ileum

1. The effect of cannabinoid receptor agonists was studied in guinea-pig myenteric neurones in vitro by use of conventional intracellular recording techniques.
2. Exposure of myenteric neurones of the S-cell type to the cannabinoid receptor agonists WIN 55,212-2 (100 nM) and CP 55,940 (100 nM) reversibly and significantly depressed the amplitude of fast excitatory synaptic potentials (fast e.p.s.ps) by 46% and 37%, respectively.
3. The depressant effect of WIN 55,212-2 and CP 55,940 on fast e.p.s.p. amplitude (expressed as the area above the amplitude-time curve (mVs)) was significantly greater than that of the vehicle, Tween 80, which had no detectable effect.
4. The inhibitory effect of WIN 55,212-2 appeared to be concentration-dependent over the range 1–100 nM. WIN 55,212-3, its (−)-enantiomer (100 nM), was inactive.
5. The cannabinoid CB₁ receptor antagonist, SR141716A (1 μM), reversed the inhibitory effects of WIN 55,212-2 on fast e.p.s.ps in 38% of neurones tested (3/8) and acetylcholine (ACh)-induced depolarizations in 42% of neurones tested (5/12).
6. When tested on its own, SR141716A (1 μM) caused a 40–50% reduction in the amplitude of fast e.p.s.ps (n=9).
7. WIN 55,212-2 reversibly depressed the amplitude of the slow e.p.s.p. and, in 2 out of 7 neurones, this effect was reversed by SR141716A (1 μM).
8. It is concluded that cannabinoid-induced inhibition of fast cholinergic synaptic transmission occurred by reversible activation of both presynaptic and postsynaptic CB₁ receptors and that slow excitatory synaptic transmission can also be reversibly depressed by cannabinoids. Furthermore, it would seem that subpopulations of myenteric S-neurones and their synapsing cholinergic and non-cholinergic, non-adrenergic terminals are not endowed with cannabinoid receptors.

     

Involvement of central cannabinoid (CB1) receptors in the establishment of place conditioning in rats

The involvement of cannabinoid processes in positive reinforcement was studied using an unbiased, one-compartment, conditioned place preference (CPP) procedure in rats. This was achieved by examining the ability of the selective antagonist of the CB₁ cannabinoid receptor subtype, SR 141716, to counteract the CPP supported by classical reinforcers. The acquisition of CPP induced by cocaine (2 mg/kg), morphine (4 mg/kg) and food (standard chow and sucrose pellets) was dose-dependently blocked by pre-pairing administration of SR 141716 (0.03–3 mg/kg). However, SR 141716 (up to 10 mg/kg) did not significantly counteract the expression of cocaine-induced CPP. On the other hand, the synthetic CB receptor agonist, WIN 55212-2 (0.3–1 mg/kg), established a robust place aversion (CPA), as already described with other agonists, and CPP was never observed, even at 100-fold lower doses. The aversive effect of WIN 55212-2 was reversed by SR 141716 (0.3–1 mg/kg), suggesting that it was accounted for by the stimulation of CB₁ receptors. These findings indicate that, on their own, CB receptor agonists are unable to generate the processes necessary to induce a pleasurable state in animals, as assessed in place conditioning procedures. Nevertheless, a cannabinoid link may be involved in the neurobiological events, allowing the perception of the rewarding value of various kinds of reinforcers. However, a permanent endogenous cannabinoid tone seems unlikely to be necessary to ensure the organism a basal hedonic level since, given alone, SR 141716 supported neither CPP nor CPA. Received: 14 March 1997 / Final version: 28 July 1997     

Differential effects of acute cannabinoid drug treatment,mediated by CB<Subscript>1</Subscript> receptors,on the in vivo activity of tyrosine and tryptophan hydroxylase in the rat brain

The acute effects of cannabinoid drugs on the synthesis of noradrenaline, dopamine, and serotonin (5-HT) were assessed, simultaneously, using the accumulation of 3,4-dihydroxyphenylalanine (dopa) and 5-hydroxytryptophan (5-HTP) after decarboxylase inhibition as a measure of the rate of tyrosine and tryptophan hydroxylation in the rat brain in vivo. Treatment (1 h, i.p.) with ⁹-tetrahydrocannabinol (THC, 5, 10, and 20 mg/kg) and the cannabinoid receptor agonist WIN 55,212–2 (WIN, 2 and 4 mg/kg) increased dopa/noradrenaline synthesis (40–70%) in various brain regions enriched in this neurotransmitter (e.g., cerebral cortex, hippocampus, hypothalamus). In most brain regions, the content of noradrenaline was reduced by cannabinoid drugs (27–66%). For the effects of WIN (2 and 4 mg/kg), an inverse correlation (r=–0.61, P=0.036) was obtained between the accumulation of dopa and the content of noradrenaline in the hypothalamus. The stimulatory effect on dopa accumulation induced by THC was antagonized by the selective CB₁ receptor antagonists SR141716A and AM 281 (10 mg/kg). In contrast, THC and WIN decreased the synthesis of dopa/dopamine in the corpus striatum (16–37%) and that of 5-HTP/5-HT (20–35%) in brain regions enriched in 5-HT (e.g., cerebral cortex and hippocampus). These inhibitory effects of THC and WIN were also antagonized by AM 281 and/or SR141716A. THC did not alter the content of 5-HT or dopamine in the brain. The effects may be related to the activation of presynaptic inhibitory cannabinoid CB₁ receptors located on the neurones themselves (serotonin) and on facilitatory (dopamine) and inhibitory interneurones (noradrenaline).     

Inhibition of N-methyl-d-aspartate- and kainate-evoked noradrenaline release in human cerebral cortex slices by ethanol

Summary The effect of ethanol on the release of noradrenaline evoked by various stimuli was investigated in human cerebral cortical slices from patients undergoing neurosurgery. The slices were preincubated with [³H]noradrenaline and then superfused. Tritium overflow was stimulated by exposure to N-methyl-d-aspartate (NMDA; in slices superfused without Mg²⁺), kainic acid, veratridine or by increasing the K⁺ concentration.The NMDA evoked tritium overflow was concentration-dependently inhibited by ethanol; an inhibition by 37% occurred at 48 mmol/l ethanol. This ethanol concentration was not yet effective when kainic acid was used for stimulation, but ethanol 150 mmol/l strongly inhibited the tritium overflow evoked by kainic acid as well. The tritium overflow evoked by veratridine or high K⁺ was not affected by ethanol in the concentration range investigated.These findings are compatible with the suggestion that the NMDA receptor and, with less susceptibility, the kainate receptor are sites of action underlying the effect of ethanol in the human brain.Send offprint requests to M. Göthert at the above address     

WIN55,212-2 Inhibits Production of CX3CL1 by Human Astrocytes: Involvement of p38 MAP Kinase

CX3CL1 (fractalkine) has been shown not only to be neuroprotective but also may play a role in HIV-1-associated neuropathogenesis. In this study, we found that production of CX3CL1 by human astrocytes stimulated with interleukin (IL)-1β was inhibited in a concentration-dependent manner following pretreatment with the synthetic cannabinoid WIN55,212-2. The CB₂ receptor selective antagonist SR144528 significantly inhibited WIN55,212-2-mediated suppression of CX3CL1, suggesting a CB₂-receptor-related mechanism. IL-1β triggered the activation of p38 and ERK1/2 (p44/42) MAP kinase (MAPK) signaling pathways, but WIN55,212-2 mainly inhibited p38 MAPK phosphorylation. This finding was mirrored in experiments using known inhibitors of these MAPKs, suggesting that the suppression of CX3CL1 production by WIN55,212-2 involves inhibition of signaling via p38 MAPK. Our results support the concept that synthetic cannabinoids have anti-inflammatory properties and that these agents may have therapeutic potential for certain neuroinflammatory disorders.