Prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 and carbon monoxide reduces extracellular glutamate levels in primary rat cerebral cortex cell cultures

The effects of prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 (0.5 mg/kg s.c.), alone or in combination with carbon monoxide, on extracellular glutamate levels in primary rat cerebral cortical neuronal cultures, were investigated. Dam weight gain, pregnancy length and litter size at birth were not affected by prenatal treatment with WIN 55,212-2 and carbon monoxide alone or in combination. Basal and K(+)-evoked extracellular glutamate levels were reduced in cortical cultures from pups born to mothers exposed to WIN 55,212-2 and carbon monoxide alone or in combination compared to cultures from rats born to vehicle-treated mothers. In cultures obtained from rats exposed to vehicle or carbon monoxide alone during gestation, WIN 55,212-2 (0.01-100 nM) increased extracellular glutamate levels, displaying a bell-shaped concentration-response curve. In cultures from rats born to mothers exposed to WIN 55,212-2 alone or in combination with carbon monoxide the WIN 55,212-2 ( 1 nM)-induced increase in extracellular glutamate levels was lower than that observed in cultures from rats born to vehicle-treated mothers and similar at those observed at 10 and 100 nM concentrations. The selective CB1 receptor antagonist SR141716A (10 nM) counteracted the WIN 55,212-2-induced increase in extracellular glutamate levels in cultures exposed to vehicle or carbon monoxide during gestation, but failed to antagonise it in cultures from rats born to mothers exposed to WIN 55,212-2 alone or in combination with carbon monoxide. These findings provide evidence that prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 and carbon monoxide, alone or in combination, is associated with an impairment in cortical glutamatergic transmission. It could be speculated that such detrimental effects might be involved in the reported deficit in learning and memory associated with prenatal marijuana exposure.     

Ligand Binding and Modulation of Cyclic AMP Levels Depend on the Chemical Nature of Residue 192 of the Human Cannabinoid Receptor 1

Abstract: The human cannabinoid receptor associated with the CNS (CB1) binds Δ⁹-tetrahydrocannabinol, the psychoactive component of marijuana, and other cannabimimetic compounds. This receptor is a member of the seven transmembrane domain G protein-coupled receptor family and mediates its effects through inhibition of adenylyl cyclase. An understanding of the molecular mechanisms involved in ligand binding and receptor activation requires identification of the active site residues and their role. Lys¹⁹² of the third transmembrane domain of the receptor is noteworthy because it is the only nonconserved, charged residue in the transmembrane region. To investigate the properties of this residue, which are important for both ligand binding and receptor activation, we generated mutant receptors in which this amino acid was changed to either Arg (K192R), Gln (K192Q), or Glu (K192E). Wild-type and mutant receptors were stably expressed in Chinese hamster ovary cells and were evaluated in binding assays with the bicyclic cannabinoid CP-55,940 and the aminoalkylindole WIN 55,212-2. We found that only the most conservative change of Lys to Arg allowed retention of binding affinity to CP-55,940, whereas WIN 55,212-2 bound to all of the mutant receptors in the same range as it bound the wild type. Analysis of the ligand-induced inhibition of cyclic AMP production in cells expressing each of the receptors gave an EC₅₀ value for each agonist that was comparable to its binding affinity, with one exception. Although the mutant K192E receptor displayed similar binding affinity as the wild type with WIN 55,212-2, an order of magnitude difference was observed for the EC₅₀ for cyclic AMP inhibition with this compound. The results of this study indicate that binding of CP-55,940 is highly sensitive to the chemical nature of residue 192. In contrast, although this residue is not critical for WIN 55,212-2 binding, the data suggest a role for Lys¹⁹² in WIN 55,212-2-induced receptor activation.     

Cannabinoid-serotonin interactions in alcohol-preferring vs. alcohol-avoiding mice

Because cannabinoid and serotonin (5-HT) systems have been proposed to play an important role in drug craving, we investigated whether cannabinoid 1 (CB1) and 5-HT(1A) receptor ligands could affect voluntary alcohol intake in two mouse strains, C57BL/6 J and DBA/2 J, with marked differences in native alcohol preference. When offered progressively (3-10% ethanol) in drinking water, in a free-choice procedure, alcohol intake was markedly lower (approximately 70%) in DBA/2 J than in C57BL/6 J mice. In DBA/2 J mice, chronic treatment with the cannabinoid receptor agonist WIN 55,212-2 increased alcohol intake. WIN 55,212-2 effect was prevented by concomitant, chronic CB1 receptor blockade by rimonabant or chronic 5-HT(1A) receptor stimulation by 8-hydroxy-2-(di-n-propylamino)-tetralin, which, on their own, did not affect alcohol intake. In C57BL/6 J mice, chronic treatment with WIN 55,212-2 had no effect but chronic CB1 receptor blockade or chronic 5-HT(1A) receptor stimulation significantly decreased alcohol intake. Parallel autoradiographic investigations showed that chronic treatment with WIN 55,212-2 significantly decreased 5-HT(1A)-mediated [35S]guanosine triphosphate-gamma-S binding in the hippocampus of both mouse strains. Conversely, chronic rimonabant increased this binding in C57BL/6 J mice. These results show that cannabinoid neurotransmission can exert a permissive control on alcohol intake, possibly through CB1-5-HT(1A) interactions. However, the differences between C57BL/6 J and DBA/2 J mice indicate that such modulations of alcohol intake are under genetic control.     

Dopamine receptor antagonism disrupts social preference in zebrafish: a strain comparison study

Zebrafish form shoals in nature and in the laboratory. The sight of conspecifics has been found reinforcing in zebrafish learning tasks. However, the mechanisms of shoaling, and those of its reinforcing properties, are not known. The dopaminergic system has been implicated in reward among other functions and it is also engaged by drugs of abuse as shown in a variety of vertebrates including zebrafish. The ontogenetic changes in dopamine levels and, to a lesser degree, in serotonin levels, have been found to accompany the maturation of shoaling in zebrafish. Thus, we hypothesized that the dopaminergic system may contribute to shoaling in zebrafish. To test this we employed a D1-receptor antagonist and quantified behavioral responses of our subjects using a social preference (shoaling) paradigm. We found significant reduction of social preference induced by the D1-R antagonist, SCH23390, in the AB strain of zebrafish, an alteration that was not accompanied by changes in motor function or vision. We also detected D1-R antagonist-induced changes in the level of dopamine, DOPAC, serotonin and 5HIAA, respectively, in the brain of AB zebrafish as quantified by HPLC with electrochemical detection. We found the antagonist-induced behavioral changes to be absent and the levels of these neurochemicals to be lower in another zebrafish population, SF, demonstrating naturally occurring genetic variability in these traits. We conclude that this variability may be utilized to unravel the mechanisms of social behavior in zebrafish, a line of research that may be extended to other vertebrates including our own species.     

Fin-mutant female zebrafish (Danio rerio) exhibit differences in association preferences for male fin length

Females often choose to associate with males that have exaggerated traits. In fishes, this may reflect an overall preference for larger size in a potential mate. Female zebrafish (Danio rerio) prefer males with larger bodies but not longer fins. The availability of mutant and transgenic strains of zebrafish make this a unique model system in which to study the role of phenotypic variation in social and sexual behavior. We used mutant strains of zebrafish with truncated (short fin) and exaggerated (long fin) fins to further examine female preferences for fin length in dichotomous association tests. Wild type females showed no preferences between wild type males and short fin mutant males or between wild type males and long fin mutant males. short fin females also showed no preference for short fin males or wild type males while long fin females preferred to associate with long fin males over wild type males. These results suggest that the single gene long fin mutation that results in altered fin morphological may also be involved in a related female association preference.     

Acute and chronic alcohol dose: population differences in behavior and neurochemistry of zebrafish

The zebrafish has been in the forefront of developmental genetics for decades and has also been gaining attention in neurobehavioral genetics. It has been proposed to model alcohol-induced changes in human brain function and behavior. Here, adult zebrafish populations, AB and SF (short-fin wild type), were exposed to chronic treatment (several days in 0.00% or 0.50% alcohol v/v) and a subsequent acute treatment (1 h in 0.00%, 0.25%, 0.50% or 1.00% alcohol). Behavioral responses of zebrafish to computer-animated images, including a zebrafish shoal and a predator, were quantified using videotracking. Neurochemical changes in the dopaminergic and serotoninergic systems in the brain of the fish were measured using high-precision liquid chromatography with electrochemical detection. The results showed genetic differences in numerous aspects of alcohol-induced changes, including, for the first time, the behavioral effects of withdrawal from alcohol and neurochemical responses to alcohol. For example, withdrawal from alcohol abolished shoaling and increased dopamine and 3,4-dihydroxyphenylacetic acid in AB but not in SF fish. The findings show that, first, acute and chronic alcohol induced changes are quantifiable with automated behavioral paradigms; second, robust neurochemical changes are also detectable; and third, genetic factors influence both alcohol-induced behavioral and neurotransmitter level changes. Although the causal relationship underlying the alcohol-induced changes in behavior and neurochemistry is speculative at this point, the results suggest that zebrafish will be a useful tool for the analysis of the biological mechanisms of alcohol-induced functional changes in the adult brain.     

Culture of cells from zebrafish (Brachydanio rerio) embryo and adult tissues

The zebrafish is a popular model for studies of vertebrate development and toxicology. However, in vitro approaches with this organism have not been fully exploited because cell culture systems have been unavailable. We developed methods for the culture of cells from blastula-stage diploid and haploid zebrafish embryos, as well as cells from the caudal and pelvic fin, gill, liver, and viscera of adult fish. The haploid embryo-derived cells differentiated in culture to a pigmented phenotype and expressed, upon exposure to 2,3,7,8-tetrachlorodibenzo p-dioxin, a protein that was immunologically and functionally similar to rainbow trout cytochrome P450IA1 Zebrafish cultures were grown in a complex basal nutrient medium supplemented with insulin, trout embryo extract, and low concentrations of trout and fetal bovine serum; they could not be maintained in conventional culture medium containing a high concentration of mammalian serum. Using calcium phosphate-mediated transfection, a plasmid constructed for use in mammalian cells was introduced into zebrafish embryo cell cultures and expressed in a stable manner. These results indicated that the transfection procedures utilized in mammalian systems can also be applied to zebrafish cell cultures, providing a means for in vitro alteration of the genotype and phenotype of the cells.[/ p]Abbreviations TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin - EROD, 7-ethyoxyresorufin - HDPDS, 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - EDTA, ethylanediaminetetraacetic acid - FBS, fetal bovine serum - LDF, limit dilution factor - DMSO, dimethyl sulfoxide - ES, embryonal stem - PAH, polycylic aromatic hydrocarbons - ZG, zebrafish gill - ZBF, zebrafish pelvic fin - ZV, Zebrafish viscera - ZCF, zebrafish caudal fin - ZEM, diploid blastula-derived     

Agonist efficacy and receptor efficiency in heterozygous CB1 knockout mice: relationship of reduced CB1 receptor density to G-protein activation

Heterozygous CB1 receptor knockout mice were used to examine the effect of reduced CB1 receptor density on G-protein activation in membranes prepared from four brain regions: cerebellum, hippocampus, striatum/globus pallidus (striatum/GP) and cingulate cortex. Results showed that CB1 receptor levels were approximately 50% lower in heterozygous mice in all regions examined. However, maximal stimulation of [(35)S]guanosine-5'-(gamma-O-thio) triphosphate ([(35)S]GTPgammaS) binding by the high efficacy agonist WIN 55,212-2 was reduced by only 20-25% in most brain regions, with the exception of striatum/GP where the decrease in stimulation was as predicted (approximately 50%). Furthermore, although the efficacies of the cannabinoid partial agonists, methanandamide and (9)-tetrahydrocannabinol, were similarly lower in heterozygous mice, their relative efficacies compared with WIN 55,212-2 were generally unchanged. Saturation analysis of net WIN 55,212-2-stimulated [(35)S]GTPgammaS binding showed that decreased stimulation by WIN 55,212-2 in striatum/GP of heterozygous mice was caused by a decrease in the apparent affinity of net-stimulated [(35)S]GTPgammaS binding. The apparent maximal number of binding sites (B(max)) values of net WIN 55,212-2-stimulated [(35)S]GTPgammaS binding were unchanged in cerebellum and striatum/GP of heterozygous mice, but decreased in cingulate cortex, with a similar trend in hippocampus. Moreover, in every region except cingulate cortex, the maximal number of net-stimulated [(35)S]GTPgammaS binding sites per receptor was significantly increased in heterozygous mice. These results indicate region-dependent increases in the apparent efficiency of CB1 receptor-mediated G-protein activation in heterozygous CB1 knockout mice.     

Modulation of cannabinoid agonist binding by 5-HT in the rat cerebellum

Cross-talk between cannabinoid CB1 and serotonin 5-HT receptors in rat cerebellar membranes was investigated using radioligand binding. In competition against the CB1 antagonist, [3 H]SR141716A, the agonist, WIN 55,212-2 yielded a biphasic isotherm. The majority of binding was to a high-affinity state that was significantly reduced by the GTP analogue, Gpp(NH)p. Interestingly, 5-HT enhanced the high-affinity binding constant of WIN 55,212-2 while attenuating the proportion of high-affinity binding. 5-HT also significantly reduced the proportion of high-affinity binding of the cannabinoid agonist, HU 210, but had no effect on the agonist, CP 55,940. The effect of 5-HT on WIN 55,212-2 binding was inhibited by the 5-HT2 receptor antagonist ritanserin as well as Gpp(NH)p, suggesting a dependence on the 5-HT2 receptor and on G protein-receptor interactions, respectively. Subsequent [3 H]WIN 55,212-2 dissociation kinetic experiments revealed that 5-HT promoted a slower-dissociating species of radiolabelled agonist-receptor complex. Our findings support a membrane-delimited cross-talk between two G protein-coupled receptors that are co-localized in certain cells of the central nervous system. Intriguingly, the cannabinoid agonist dependence of the 5-HT modulatory effect suggests that agonist-specific conformations of the CB1 receptor may also be important in determining the extent of this cross-talk.     

Inhibition of striatal dopamine release by CB1 receptor activation requires nonsynaptic communication involving GABA, H2O2, and KATP channels

The main psychoactive component of marijuana, Delta9-tetrahydrocannabinol (THC), acts in the CNS via type 1 cannabinoid receptors (CB1Rs). The behavioral consequences of THC or synthetic CB1R agonists include suppression of motor activity. One explanation for movement suppression might be inhibition of striatal dopamine (DA) release by CB1Rs, which are densely localized in motor striatum; however, data from previous studies are inconclusive. Here we examined the effect of CB1R activation on locally evoked DA release monitored with carbon-fiber microelectrodes and fast-scan cyclic voltammetry in striatal slices. Consistent with previous reports, DA release evoked by a single stimulus pulse was unaffected by WIN55,212-2, a cannabinoid receptor agonist. However, when DA release was evoked by a train of stimuli, WIN55,212-2 caused a significant decrease in evoked extracellular DA concentration ([DA]o), implicating the involvement of local striatal circuitry, with similar suppression seen in guinea pig, rat, and mouse striatum. Pulse-train evoked [DA]o was not altered by either AM251, an inverse CB1R agonist, or VCHSR1, a neutral antagonist, indicating the absence of DA release regulation by endogenous cannabinoids with the stimulation protocol used. However, both CB1R antagonists prevented and reversed suppression of evoked [DA]o by WIN55,212-2. The effect of WIN55,212-2 was also prevented by picrotoxin, a GABAA receptor antagonist, and by catalase, a metabolizing enzyme for hydrogen peroxide (H2O2). Furthermore, blockade of ATP-sensitive K+ (KATP) channels by tolbutamide or glybenclamide prevented the effect of WIN55,212-2 on DA release. Together, these data indicate that suppression of DA release by CB1R activation within striatum occurs via a novel nonsynaptic mechanism that involves GABA release inhibition, increased generation of the diffusible messenger H2O2, and activation of KATP channels to inhibit DA release. In addition, the findings suggest a possible physiological substrate for the motor effects of cannabinoid agonist administration.     

Aggregation Behavior in Wildtype and Transgenic Zebrafish

Recent advances in the development and availability of genetically modified animals enable researchers to examine the effects of phenotypic characters on social behavior. In fish, shoaling behavior is known to be influenced by characteristics such as body coloration, striping pattern, body shape, and size. GloFish^TM are genetically engineered zebrafish (Danio rerio) that express red fluorescent protein (RFP), resulting in on overall red coloration under the dark longitudinal stripes. The GloFish pattern is distinct from the light body coloration underlying the dark longitudinal stripes seen in wildtype zebrafish. We presented wildtype and transgenic RFP zebrafish with same‐sex shoals of both strains of fish in dichotomous choice tests. No preference for either of the shoals was shown, however, both strains showed significant preferences for swimming near shoals vs. swimming near an empty tank compartment. When presented with opposite‐sex individuals of both strains, no preference was shown by either sex of either strain. Thus, the red body coloration of transgenic zebrafish does not appear to affect choice of social partner, in either a shoaling or a potentially reproductive context.     

Identification of the synthetic cannabinoid R(+)WIN55,212-2 as a novel regulator of IFN regulatory factor 3 activation and IFN-beta expression: relevance to therapeutic effects in models of multiple sclerosis

β-Interferons (IFN-βs) represent one of the first line treatments for relapsing-remitting multiple sclerosis, slowing disease progression while reducing the frequency of relapses. Despite this, more effective, well tolerated therapeutic strategies are needed. Cannabinoids palliate experimental autoimmune encephalomyelitis (EAE) symptoms and have therapeutic potential in MS patients although the precise molecular mechanism for these effects is not understood. Toll-like receptor (TLR) signaling controls innate immune responses and TLRs are implicated in MS. Here we demonstrate that the synthetic cannabinoid R(+)WIN55,212-2 is a novel regulator of TLR3 and TLR4 signaling by inhibiting the pro-inflammatory signaling axis triggered by TLR3 and TLR4, whereas selectively augmenting TLR3-induced activation of IFN regulatory factor 3 (IRF3) and expression of IFN-β. We present evidence that R(+)WIN55,212-2 strongly promotes the nuclear localization of IRF3. The potentiation of IFN-β expression by R(+)WIN55,212-2 is critical for manifesting its protective effects in the murine MS model EAE as evidenced by its reduced therapeutic efficacy in the presence of an anti-IFN-β antibody. R(+)WIN55,212-2 also induces IFN-β expression in MS patient peripheral blood mononuclear cells, whereas down-regulating inflammatory signaling in these cells. These findings identify R(+)WIN55,212-2 as a novel regulator of TLR3 signaling to IRF3 activation and IFN-β expression and highlights a new mechanism that may be open to exploitation in the development of new therapeutics for the treatment of MS.     

Role of receptor internalization in the agonist-induced desensitization of cannabinoid type 1 receptors

Agonist-induced internalization of G protein-coupled receptors (GPCRs) is an important mechanism for regulating signaling transduction of functional receptors at the plasma membrane. We demonstrate here that both caveolae/lipid-rafts- and clathrin-coated-pits-mediated pathways were involved in agonist-induced endocytosis of the cannabinoid type 1 receptor (CB1R) in stably transfected human embryonic kidney (HEK) 293 cells and that the internalized receptors were predominantly sorted into recycling pathway for reactivation. The treatment of CB1 receptors with the low endocytotic agonist Δ⁹-THC induced a faster receptor desensitization and slower resensitization than the high endocytotic agonist WIN 55,212-2. In addition, the blockade of receptor endocytosis or recycling pathway markedly enhanced agonist-induced CB1 receptor desensitization. Furthermore, co-expression of phospholipase D2, an enhancer of receptor endocytosis, reduced CB1 receptor desensitization, whereas co-expression of a phospholipase D2 negative mutant significantly increased the desensitization after WIN 55,212-2 treatment. These findings provide evidences for the importance of receptor endocytosis in counteracting CB1 receptor desensitization by facilitating receptor reactivation. Moreover, in primary cultured neurons, the low endocytotic agonist Δ⁹-THC or anandamide exhibited a greater desensitization of endogenous CB1 receptors than the high endocytotic agonist WIN 55,212-2, CP 55940 or 2-arachidonoyl glycerol, indicating that cannabinoids with high endocytotic efficacy might cause reduced development of cannabinoid tolerance to some kind cannabinoid-mediated effects.     

Cellular localization of cannabinoid receptors and activated G-proteins in rat anterior cingulate cortex

Cannabinoid receptors are found in moderate density throughout the cerebral cortex. The anterior cingulate cortex (ACC) is of particular interest due its high level of cannabinoid receptors and role in behaviors known to be modulated by cannabinoids. These studies were conducted to determine the cellular localization of cannabinoid receptors and to compare the level of cannabinoid receptor binding with receptor-mediated G-protein activity in the rat ACC. Either ibotenic acid or undercut lesions were made in ACC, and brains were processed for [3H]WIN 55,212-2 and WIN 55,212-2-stimulated [35S]GTPgammaS autoradiography. Both cannabinoid receptors and receptor-activated G-proteins were highest in laminae I and VI of ACC in control tissue. Although similar levels of receptor binding were found in these laminae, significantly higher levels of receptor-activated G-proteins were found in lamina VI. Ibotenic acid lesions that destroyed ACC neurons decreased [3H]WIN 55,212-2 binding by 60-70% and eliminated WIN 55,212-2-stimulated [35S]GTPgammaS binding. In contrast, deafferentation of the ACC with undercut lesions had no significant effect on cannabinoid receptor binding or G-protein activation. These results indicate that cannabinoid receptors in laminae I and VI of the ACC are located on somatodendritic elements or axons intrinsic to the ACC. In addition, differences in the relative levels of cannabinoid binding sites and activated G-proteins between cortical laminae indicate that the efficiency of cannabinoid receptors for G-protein activation may vary within a specific brain region.     

Adenosine A2A receptors enable the synaptic effects of cannabinoid CB1 receptors in the rodent striatum

Adenosine A_2A, cannabinoid CB₁ and metabotropic glutamate 5 (mGlu₅) receptors are all highly expressed in the striatum. The aim of the present work was to investigate whether, and by which mechanisms, the above receptors interact in the regulation of striatal synaptic transmission. By extracellular field potentials (FPs) recordings in corticostriatal slices, we demonstrated that the ability of the selective type 1 cannabinoid receptor (CB₁R) agonist WIN55,212-2 to depress synaptic transmission was prevented by the pharmacological blockade or the genetic inactivation of A_2ARs. Such a permissive effect of A_2ARs towards CB₁Rs does not seem to occur pre-synaptically as the ability of WIN55,212-2 to increase the R2/R1 ratio under a protocol of paired-pulse stimulation was not modified by ZM241385. Furthermore, the effects of WIN55,212-2 were reduced in slices from mice lacking post-synaptic striatal A_2ARs. The selective mGlu₅R agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) potentiated the synaptic effects of WIN55,212-2, and such a potentiation was abolished by A_2AR blockade. Unlike the synaptic effects, the ability of WIN55,212-2 to prevent NMDA-induced toxicity was not influenced by ZM241385. Altogether, these results show that the state of activation of A_2ARs regulates the synaptic effects of CB₁Rs and that A_2ARs may control CB₁ effects also indirectly, namely through mGlu₅Rs.     

Spectral differentiation of blue opsins between phylogenetically close but ecologically distant goldfish and zebrafish

Zebrafish and goldfish are both diurnal freshwater fish species belonging to the same family, Cyprinidae, but their visual ecological surroundings considerably differ. Zebrafish are surface swimmers in conditions of broad and shortwave-dominated background spectra and goldfish are generalized swimmers whose light environment extends to a depth of elevated short wavelength absorbance with turbidity. The peak absorption spectrum (lambdamax) of the zebrafish blue (SWS2) visual pigment is consistently shifted to short wavelength (416 nm) compared with that of the goldfish SWS2 (443 nm). Among the amino acid differences between the two pigments, only one (alanine in zebrafish and serine in goldfish at residue 94) was previously known to cause a difference in absorption spectrum (14-nm lambdamax shift in newt SWS2). In this study, we reconstructed the ancestral SWS2 pigment of the two species by applying likelihood-based Bayesian statistics and performing site-directed mutagenesis. The reconstituted ancestral photopigment had a lambdamax of 430 nm, indicating that zebrafish and goldfish achieved short wavelength (-14 nm) and long wavelength (+13 nm) spectral shifts, respectively, from the ancestor. Unexpectedly, the S94A mutation resulted in only a -3-nm spectral shift when introduced into the goldfish SWS2 pigment. Nearly half of the long wavelength shift toward the goldfish pigment was achieved instead by T116L (6 nm). The S295C mutation toward zebrafish SWS2 contributed to creating a ridge of absorbance around 400 nm and broadening its spectral sensitivity in the short wavelength direction. These results indicate that the evolutionary engineering approach is very effective in deciphering the process of functional divergence of visual pigments.     

The inhibitory action of exo- and endocannabinoids on [³H]GABA release are mediated by both CB₁and CB₂receptors in the mouse hippocampus

Exogenous and endogenous cannabinoids play an important role in modulating the release of neurotransmitters in hippocampal excitatory and inhibitory networks, thus having profound effect on higher cognitive and emotional functions such as learning and memory. In this study we have studied the effect of cannabinoid agonists on the potassium depolarization-evoked [(3)H]GABA release from hippocampal synaptosomes in the wild-type (WT) and cannabinoid 1 receptor (CB(1)R)-null mutant mice. All tested cannabinoid agonists (WIN55,212-2, CP55,940, HU-210, 2-arachidonoyl-glycerol, 2-AG; delta-9-tetra-hydrocannabinol, THC) inhibited [(3)H]GABA release in WT mice with the following rank order of agonist potency: HU-210>CP55,490>WIN55,212-2>2-AG>THC. By contrast, 2-AG and THC displayed the greatest efficacy eliciting almost complete inhibition of evoked [(3)H]GABA efflux, whereas the maximal inhibition obtained by HU-210, CP55,490, and WIN55,212-2 were less, eliciting not more than 40% inhibition. The inhibitory effect of WIN55,212-2, THC and 2-AG on evoked [(3)H]GABA efflux was antagonized by the CB(1) receptor inverse agonist AM251 (0.5 μM) in the WT mice. In the CB(1)R knockout mice the inhibitory effects of all three agonists were attenuated. In these mice, AM251 did not antagonize, but further reduced the [(3)H]GABA release in the presence of the synthetic agonist WIN55,212-2. By contrast, the concentration-dependent inhibitory effects of THC and 2-AG were partially antagonized by AM251 in the absence of CB(1) receptors. Finally, the inhibition of evoked [(3)H]GABA efflux by THC and 2-AG was also partially attenuated by AM630 (1 μM), the CB(2) receptor-selective antagonist, both in WT and CB(1) knockout mice. Our data prove the involvement of CB(1) receptors in the effect of exo- and endocannabinoids on GABA efflux from hippocampal nerve terminals. In addition, in the effect of the exocannabinoid THC and the endocannabinoid 2-AG, non-CB(1), probably CB(2)-like receptors are also involved.