An activation of parvocellular oxytocinergic neurons in the paraventricular nucleus in oxytocin-induced yawning and penile erection

Intracerebroventricular (ICV) or PVN local injections of oxytocin induce yawning and penile erection, for which a positive feedback mechanism for the PVN oxytocinergic activation is suggested, but this had not been directly substantiated in vivo. We have assessed the behavioral effects and activity of oxytocinergic neurons with double-staining for c-Fos and oxytocin in the PVN after ICV administration of oxytocin in adult male rats. ICV oxytocin injections (50 and 200 ng) dose-dependently induced yawning and penile erection and significantly increased the percentage of c-Fos positive oxytocin neurons in the medial, dorsal and lateral parvocellular subdivision of the PVN. However, increases in the magnocellular portion were not significant. We also found that lithium chloride (LiCl, 0.5 and l.0 mEq), a compound known to activate oxytocinergic neurons, also significantly increased the percentage of c-Fos positive oxytocin neurons in all PVN portions. However, LiCl did not induce yawning and penile erection, but counteracted the oxytocin-induced yawning and penile erection. These results suggest that if the activation of oxytocinergic neurons in the PVN is important for mediating oxytocin-induced yawning and penile erection, a selective activation of parvocellular oxytocinergic neurons in the PVN is likely to be involved.     

Central control of penile erection: role of the paraventricular nucleus of the hypothalamus

The paraventricular nucleus of the hypothalamus is an integration centre between the central and peripheral autonomic nervous systems. It is involved in numerous functions from feeding, metabolic balance, blood pressure and heart rate, to erectile function and sexual behaviour. In particular, a group of oxytocinergic neurons originating in this nucleus and projecting to extra-hypothalamic brain areas (e.g., hippocampus, medulla oblongata and spinal cord) control penile erection in male rats. Activation of these neurons by dopamine and its agonists, excitatory amino acids (N-methyl-D-aspartic acid) or oxytocin itself, or by electrical stimulation leads to penile erection, while their inhibition by gamma-amino-butyric acid (GABA) and its agonists or by opioid peptides and opiate-like drugs inhibits this sexual response. The activation of these neurons is secondary to the activation of nitric oxide synthase, which produces nitric oxide. Nitric oxide in turn causes, by a mechanism that is as yet unidentified, the release of oxytocin in extra-hypothalamic brain areas. Other compounds recently identified that facilitate penile erection by activating central oxytocinergic neurons are peptide analogues of hexarelin, a growth hormone releasing peptide, pro-VGF-derived peptides, endogenous peptides that may be released by neuronal nerve endings impinging on oxytocinergic cell bodies, SR 141716A, a cannabinoid CB1 receptor antagonist, and, less convincingly, adrenocorticotropin-melanocyte-stimulating hormone (ACTH-MSH)-related peptides. Paraventricular oxytocinergic neurons and similar mechanisms are also involved in penile erection occurring in physiological contexts, namely noncontact erections that occur in male rats in the presence of an inaccessible receptive female, and during copulation. These findings show that the paraventricular nucleus of the hypothalamus plays an important role in the control of erectile function and sexual activity. As the male rat is a model of sexual behaviour and penile physiology, which has largely increased in the last years our knowledge of peripheral and central mechanisms controlling erectile function (drugs that induce penile erection in male rats usually do so also in man), the above results may have great significance in terms of a human perspective for the treatment of erectile dysfunction.     

PD-168077, a selective dopamine D4 receptor agonist, induces penile erection when injected into the paraventricular nucleus of male rats

The effect of PD-168077 (N-methyl-4-(2-cyanophenyl)piperazynil-3-methylbenzamide maleate), a selective D4 dopamine receptor agonist, injected into the paraventricular nucleus of the hypothalamus on penile erection was studied in male rats. PD-168077 (1-200 ng) induced penile erection in a dose-dependent manner. The minimal effective dose was 50 ng, while the maximal response was found with 200 ng of the compound, which increased penile erection episodes from 0.3+/-0.03 to 1.7+/-0.21. The proerectile effect of PD-168077 was reduced almost completely by L-745,870 (3-(4-[chlorophenyl]piperazin-1-yl)-methyl-1H-pyrrolo[2,3-B]pyridine trihydrochloride), a selective D4 dopamine receptor antagonist, (1 microg) given into the paraventricular nucleus before the D4 dopamine agonist, and by other nonselective dopamine receptor antagonists, such as haloperidol (1 microg) and clozapine (1 microg), which block all dopamine receptor subtypes. The pro-erectile effect of PD-168077 was also reduced by the NO synthase inhibitor NG-nitro-L-arginine methylester (25 microg), but not by the oxytocin receptor antagonist d(CH2)5Tyr(Me)2-Orn8-vasotocin (1 microg), when given into the paraventricular nucleus. In spite of its inability to prevent the pro-erectile effect of PD-168077 when given in the paraventricular nucleus, d(CH2)5Tyr(Me)2-Orn8-vasotocin (1 microg) reduced almost completely PD-168077-induced penile erection when given into the lateral ventricles. The present results show that D4 dopamine receptors present in the paraventricular nucleus may influence penile erection by modulating the activity of paraventricular oxytocinergic neurons mediating erectile function.     

Functional expression of cell surface cannabinoid CB(1) receptors on presynaptic inhibitory terminals in cultured rat hippocampal neurons

At present, little is known about the mechanisms by which cannabinoids exert their effects on the central nervous system. In this study, fluorescence imaging and electrophysiological techniques were used to investigate the functional relationship between cell surface cannabinoid type 1 (CB(1)) receptors and GABAergic synaptic transmission in cultured hippocampal neurons. CB(1) receptors were labelled on living neurons using a polyclonal antibody directed against the N-terminal 77 amino acid residues of the rat cloned CB(1) receptor. Highly punctate CB(1) receptor labelling was observed on fine axons and at axonal growth cones, with little somatic labelling. The majority of these sites were associated with synaptic terminals, identified either with immunohistochemical markers or by using the styryl dye FM1-43 to label synaptic vesicles that had undergone active turnover. Dual labelling of neurons for CB(1) receptors with either the inhibitory neurotransmitter GABA or its synthesising enzyme glutamate decarboxylase, demonstrated a strong correspondence. The immunocytochemical data was supported by functional studies using whole-cell patch-clamp recordings of miniature inhibitory postsynaptic currents (mIPSCs). The cannabinoid agonist WIN55,212-2 (100nM) markedly inhibited (by 77+/-6.3%) the frequency of pharmacologically-isolated GABAergic mIPSCs. The effects of WIN55,212-2 were blocked in the presence of the selective CB(1) receptor antagonist SR141716A (100nM).In conclusion, the present data show that cell surface CB(1) receptors are expressed at presynaptic GABAergic terminals, where their activation inhibits GABA release. Their presence on growth cones could indicate a role in the targeting of inhibitory connections during development.     

Oxytocinergic innervation of autonomic nuclei controlling penile erection in the rat.

In the rat, spinal autonomic neurons controlling penile erection receive descending pathways that modulate their activity. The paraventricular nucleus of the hypothalamus contributes oxytocinergic fibers to the dorsal horn and preganglionic sympathetic and parasympathetic cell columns. We used retrograde tracing techniques with pseudorabies virus combined with immunohistochemistry against oxytocin and radioligand binding detection of oxytocinergic receptors to evidence the oxytocinergic innervation of thoracolumbar and lumbosacral spinal neurons controlling penile erection. Spinal neurons labelled with pseudo-rabies virus transsynaptically transported from the corpus cavernosum were present in the intermediolateral cell column and the dorsal gray commissure of the thoracolumbar and lumbosacral spinal cord. Confocal laser scanning microscopic observation of the same preparations revealed close appositions between oxytocinergic varicosities and pseudorabies virus-infected neurons, suggesting strongly the presence of synaptic contacts. Electron microscopy confirmed this hypothesis. Oxytocin binding sites were present in the superficial layers of the dorsal horn, the dorsal gray commissure and the intermediolateral cell column in both the thoracolumbar and lumbosacral segments. In rats, stimulation of the paraventricular nucleus induces penile erection, but the link between the nucleus and penile innervation remains unknown. Our findings support the hypothesis that oxytocin, released by descending paraventriculo-spinal pathways, activates proerectile spinal neurons.     

Cannabinoids inhibit striatonigral GABAergic neurotransmission in the mouse

The substantia nigra pars reticulata (SNR) belongs to the brain regions with the highest density of CB(1) cannabinoid receptors. Anatomical studies indicate that the great majority of CB(1) receptors in the SNR are localized on terminals of GABAergic axons arriving from the caudate-putamen (striatonigral axons). The aim of the present experiments was to clarify the role of CB(1) receptors on terminals of striatonigral axons.Oblique sagittal slices, including the caudate-putamen and the substantia nigra, were prepared from brains of young mice. Electrical stimulation in the caudate-putamen elicited GABAergic inhibitory postsynaptic currents (IPSCs) in the SNR, which were studied by patch-clamp techniques. The long latency of IPSCs (14+/-1 ms) suggests that striatonigral axons were indeed activated within the caudate-putamen. The synthetic CB(1)/CB(2) cannabinoid receptor agonist WIN55212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate; 10(-5) M) decreased the amplitude of IPSCs by 93+/-1%. CP55940 ((-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol; 10(-5) M), another CB(1)/CB(2) receptor agonist, also reduced IPSC amplitude, by 76+/-4%. The CB(1) cannabinoid receptor antagonist SR141716A (N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide; 10(-6) M) prevented the inhibition produced by WIN55212-2 (10(-5) M). Depolarization of SNR neurons led to suppression of IPSCs; this suppression was prevented by SR141716A (10(-6) M). Three observations indicate that the agonists inhibited neurotransmission presynaptically. (1) CP55940 (10(-5) M) enhanced the ratio of amplitudes of two IPSCs which were elicited by two electrical stimuli 100 ms apart (paired pulses). (2) WIN55212-2 (10(-5) M) did not change the amplitude of miniature IPSCs recorded in the presence of tetrodotoxin. (3) WIN55212-2 (10(-5) M) also had no effect on currents elicited in SNR neurons by ejection of the GABA(A) receptor agonist muscimol from a pipet.In summary, we have established a method which allows selective examination of GABAergic neurotransmission between striatonigral axons and SNR neurons. Using this method, the function of CB(1) cannabinoid receptors on terminals of striatonigral axons was unequivocally clarified. Activation of these receptors causes strong presynaptic inhibition of GABAergic neurotransmission between striatonigral axons and SNR neurons. This effect may be one explanation of the catalepsy observed in animals after cannabinoid administration. Endocannabinoids released from SNR neurons can modulate striatonigral neurotransmission by inhibiting GABA release from terminals of striatonigral axons.     

Cannabinoid CB1 receptors are localized primarily on cholecystokinin-containing GABAergic interneurons in the rat hippocampal formation.

Localization of cannabinoid CB 1 receptors on GABAergic interneurons in the rat hippocampal formation was studied by double-labeling immunohistochemistry with confocal microscopy. Virtually all CB1-immunoreactive neurons (95%) are GABAergic. CB 1 fluorescence showed a punctate pattern. In contrast, the GABA fluorescence was distributed homogeneously, suggesting that while CB 1 receptors and GABA exist in the same cells they are not localized in the same subcellular compartments. Although virtually all CB1 neurons were GABAergic, many GABAergic neurons did not contain CB1 receptors. GABAergic interneurons in the hippocampal formation can be further divided into subpopulations with distinct connections and functions, using cell markers such as neuropeptides and calcium binding proteins. CB1 receptors were highly co-localized with cholecystokinin and partially co-localized with calretinin and calbindin, but not with parvalbumin. This suggests that cannabinoids may modulate GABAergic neurotransmission at the synapses on the soma and at synapses on the proximal dendrites of the principal neurons, as well as at synapses on other GABAergic interneurons.     

Neural control of erection

Penile erection is a vascular event controlled by the autonomic nervous system. The spinal cord contains the autonomic preganglionic neurons that innervate the penile erectile tissue and the pudendal motoneurons that innervate the perineal striated muscles. Sympathetic pathways are anti-erectile, sacral parasympathetic pathways are pro-erectile, and contraction of the perineal striated muscles upon activity of the pudendal nerves improves penile rigidity. Spinal neurons controlling erection are activated by information from peripheral and supraspinal origin. Both peripheral and supraspinal information is capable of either eliciting erection or modulating or inhibiting an erection already present. Sensory information from the genitals is a potent activator of pro-erectile spinal neurons and elicits reflexive erections. Some pre-motor neurons of the medulla, pons and diencephalon project directly onto spinal sympathetic, parasympathetic and pudendal motoneurons. They receive in turn sensory information from the genitals. These spinal projecting pathways release a variety of neurotransmitters, including biogenic amines (serotonin, dopamine, noradrenaline, and adrenaline) and peptides that, through interactions with many receptor subtypes, exert complex effects on the spinal network that controls penile erection. Some supraspinal structures (e.g. the paraventricular nucleus and the medial preoptic area of the hypothalamus, the medial amygdala), whose roles in erection have been demonstrated in animal models, may not project directly onto spinal pro-erectile neurons. They are nevertheless prone to regulate penile erection in more integrated and coordinated responses of the body, as those occurring during sexual behavior. The application of basic and clinical research data to treatment options for erectile dysfunction has recently proved successful. Pro-erectile effects of phosphodiesterase type 5 inhibitors, acting in the penis, and of melanocortin agonists, acting in the brain, illustrate these recent developments.     

A beneficial aspect of a CB1 cannabinoid receptor antagonist: SR141716A is a potent inhibitor of macrophage infection by the intracellular pathogen Brucella suis

The psychoactive component of marijuana, delta9-tetrahydrocannabinol (THC) suppresses different functions of immunocytes, including the antimicrobicidal activity of macrophages. The triggering of cannabinoid receptors of CB1 and CB2 subtypes present on leukocytes may account for these effects. We investigated the influence of specific CB1 or CB2 receptor antagonists (SR141716A and SR144528, respectively) and nonselective CB1/CB2 cannabinoid receptor agonists (CP55,940 or WIN 55212-2) on macrophage infection by Brucella suis, an intracellular gram-negative bacteria. None of the compounds tested affected bacterial phagocytosis. By contrast, the intracellular multiplication of Brucella was dose-dependently inhibited in cells treated with 10-500 nM SR141716A and 1 microM SR141716A-induced cells exerted a potent microbicidal effect against the bacteria. SR144528, CP55,940, or WIN 55212-2 did not affect (or slightly potentiated) the growth of phagocytized bacteria. However, CP55,940 or WIN 55212-2 reversed the SR141716A-mediated effect, which strongly suggested an involvement of macrophage CB1 receptors in the phenomenon. SR141716A was able to pre-activate macrophages and to trigger an activation signal that inhibited Brucella development. The participation of endogenous cannabinoid ligand(s) in Brucella infection was discussed. Finally, our data show that SR141716A up-regulates the antimicrobial properties of macrophages in vitro and might be a pharmaceutical compound useful for counteracting the development of intramacrophagic gram-negative bacteria.     

Behavioral effects of cannabinoid agents in animals

Two subtypes of cannabinoid receptors have been identified to date, the CB1 receptor, essentially located in the CNS, but also in peripheral tissues, and the CB2 receptor, found only at the periphery. The identification of delta9-tetrahydrocannabinol (delta9-THC) as the major active component of marijuana (Cannabis sativa), the recent emergence of potent synthetic ligands and the identification of anandamide and sn-2 arachidonylglycerol as putative endogenous ligands for cannabinoid receptors in the brain, have contributed to advancing cannabinoid pharmacology and approaching the neurobiological mechanisms involved in physiological and behavioral effects of cannabinoids. Most of the agonists exhibit nonselective affinity for CB1/CB2 receptors, and delta9-THC and anandamide probably act as partial agonists. Some recently synthesized molecules are highly selective for CB2 receptors, whereas selective agonists for the CB1 receptors are not yet available. A small number of antagonists exist that display a high selectivity for either CB1 or CB2 receptors. Cannabinomimetics produce complex pharmacological and behavioral effects that probably involve numerous neuronal substrates. Interactions with dopamine, acetylcholine, opiate, and GABAergic systems have been demonstrated in several brain structures. In animals, cannabinoid agonists such as delta9-THC, WIN 55,212-2, and CP 55,940 produce a characteristic combination of four symptoms, hypothermia, analgesia, hypoactivity, and catalepsy. They are reversed by the selective CB1 receptor antagonist, SR 141716, providing good evidence for the involvement of CB1-related mechanisms. Anandamide exhibits several differences, compared with other agonists. In particular, hypothermia, analgesia, and catalepsy induced by this endogenous ligand are not reversed by SR 141716. Cannabinoid-related processes seem also involved in cognition, memory, anxiety, control of appetite, emesis, inflammatory, and immune responses. Agonists may induce biphasic effects, for example, hyperactivity at low doses and severe motor deficits at larger doses. Intriguingly, although cannabis is widely used as recreational drug in humans, only a few studies revealed an appetitive potential of cannabimimetics in animals, and evidence for aversive effects of delta9-THC, WIN 55,212-2, and CP 55,940 is more readily obtained in a variety of tests. The selective blockade of CB1 receptors by SR 141716 impaired the perception of the appetitive value of positive reinforcers (food, cocaine, morphine) and reduced the motivation for sucrose, beer and alcohol consumption, indicating that positive incentive and/or motivational processes could be under a permissive control of CB1-related mechanisms. There is little evidence that cannabinoid systems are activated under basal conditions. However, by using SR 141716 as a tool, a tonic involvement of a CB1-mediated cannabinoid link has been demonstrated, notably in animals suffering from chronic pain, faced with anxiogenic stimuli or highly motivational reinforcers. Some effects of SR 141716 also suggest that CB1-related mechanisms exert a tonic control on cognitive processes. Extensive basic research is still needed to elucidate the roles of cannabinoid systems, both in the brain and at the periphery, in normal physiology and in diseases. Additional compounds, such as selective CB1 receptor agonists, ligands that do not cross the blood brain barrier, drugs interfering with synthesis, degradation or uptake of endogenous ligand(s) of CB receptors, are especially needed to understand when and how cannabinoid systems are activated. In turn, new therapeutic strategies would likely to emerge.     

Ghrelin injected into the paraventricular nucleus of the hypothalamus of male rats induces feeding but not penile erection

The effect of ghrelin, a recently characterized endogenous receptor agonist for growth hormone (GH) secretagogue receptors, on feeding and penile erection was compared with that of EP 80661, a peptide analogue of the GH secretagogue hexarelin, previously identified for its pro-erectile activity when injected into the paraventricular nucleus of the hypothalamus of male rats. Ghrelin (0.01-1 microg), but not EP 80661 (0.02-1 microg), was found to be particularly effective in enhancing feeding. The minimal effective dose of ghrelin was 0.1 microg, which increased food intake by 88%, while the maximal response (355% above control values) was found with 1 microg of the peptide. The enhancing effect of ghrelin on feeding was prevented by the prior administration of the neuropeptide Y Y5 receptor antagonist (DTyr(2), DThr(32)) neuropeptide Y (NPY, 10 microg), but not by the GH-RH receptor antagonist MZ-4-71 (10 microg), or by EP 91073, a hexarelin analogue that antagonizes the pro-erectile effect of EP 80661 (10 microg), given into the lateral ventricles. In contrast, ghrelin failed to induce penile erection at all doses tested, while EP 80661 induced penile erection in a dose-dependent manner. The pro-erectile effect of EP 80661 was prevented by EP 91073 (10 microg), but not by (DTyr(2), DThr(32)) NPY (10 microg) or by the GH-RH receptor antagonist MZ 4-71 (10 microg), given into the lateral ventricles. The present results provide further support to the hypothesis that the GH secretagogue receptors mediating feeding are different from those mediating penile erection and activated by pro-erectile EP peptides.     

The role of oxytocin and the paraventricular nucleus in the sexual behaviour of male mammals

The paraventricular nucleus of the hypothalamus contains the cell bodies of a group of oxytocinergic neurons projecting to extrahypothalamic brain areas and to the spinal cord, which are involved in the control of erectile function and copulation. In male rats, these neurons can be activated by dopamine, excitatory amino acids, nitric oxide (NO), hexarelin analogue peptides and oxytocin itself to induce penile erection and facilitate copulation, while their inhibition by gamma-aminobutyric acid (GABA) and GABA agonists and by opioid peptides and opiate-like drugs inhibits sexual responses. The activation of paraventricular oxytocinergic neurons by dopamine, oxytocin, excitatory amino acids and hexarelin analogue peptides is apparently mediated by the activation of nitric oxide (NO) synthase. NO in turn activates, by a mechanism that is as yet unidentified, the release of oxytocin from oxytocinergic neurons in extrahypothalamic brain areas. Paraventricular oxytocinergic neurons and mechanisms similar to those reported above are also involved in the expression of penile erection in physiological contexts, namely, when penile erection is induced in the male by the presence of an inaccessible receptive female, which is considered a model for psychogenic impotence in man, as well as during copulation. These findings show that paraventricular oxytocinergic neurons projecting to extrahypothalamic brain areas and to the spinal cord and the paraventricular nucleus play an important role in the control of erectile function and male sexual behaviour in mammals.     

Electrophysiological actions of the dopamine agonist apomorphine in the paraventricular nucleus during penile erection

The ability to achieve and maintain penile erection is necessary for successful copulation. Studies have demonstrated that dopamine receptor stimulation in the paraventricular nucleus (PVN) of the hypothalamus induces penile erection in rodents, and the dopamine agonist apomorphine has been used to treat erectile dysfunction. The aim of this study was to determine the electrophysiological characteristics of PVN neuronal firing activity in anaesthetised rodents during apomorphine-induced erection. Our findings can be placed in two categories; those effects that occur immediately upon apomorphine administration and continue for up to several minutes prior to penile erection, deemed ‘pre-erectile’, and those effects that were only observed during penile erection and seminal emission. In the pre-erectile period, apomorphine acts on two different populations of PVN neurons to increase or decrease firing rates and increases alpha1 frequency band power in local field potentials. Decreased delta and increased theta frequency power in PVN local field potentials occur only during penile erection and seminal emission. These studies provide further understanding of the coordinated neuronal activity that occurs in the PVN during apomorphine-induced penile erection.     

GABAergic interneurons are the targets of cannabinoid actions in the human hippocampus

Cannabinoids have been shown to disrupt memory processes in mammals including humans. Although the CB1 neuronal cannabinoid receptor was identified several years ago, neuronal network mechanisms mediating cannabinoid effects are still controversial in animals, and even more obscure in humans. In the present study, the localization of CB1 receptors was investigated at the cellular and subcellular levels in the human hippocampus, using control post mortem and epileptic lobectomy tissue. The latter tissue was also used for [³H]GABA release experiments, testing the predictions of the anatomical data. Detectable expression of CB1 was confined to interneurons, most of which were found to be cholecystokinin-containing basket cells. CB1-positive cell bodies showed immunostaining in their perinuclear cytoplasm, but not in their somadendritic plasmamembrane. CB1-immunoreactive axon terminals densely covered the entire hippocampus, forming symmetrical synapses characteristic of GABAergic boutons. Human temporal lobectomy samples were used in the release experiments, as they were similar to the controls regarding cellular and subcellular distribution of CB1 receptors. We found that the CB1 receptor agonist, WIN 55,212-2, strongly reduced [³H]GABA release, and this effect was fully prevented by the specific CB1 receptor antagonist SR 141716A.

This unique expression pattern and the presynaptic modulation of GABA release suggests a conserved role for CB1 receptors in controlling inhibitory networks of the hippocampus that are responsible for the generation and maintenance of fast and slow oscillatory patterns. Therefore, a likely mechanism by which cannabinoids may impair memory and associational processes is an alteration of the fine-tuning of synchronized, rhythmic population events.     

Identification of the adrenoceptor subtypes expressed on GABAergic neurons in the anterior hypothalamic area and rostral zona incerta of GAD65-eGFP transgenic mice

GABA is a major neurotransmitter in the hypothalamus. In particular, neurons in the paraventricular nucleus (PVN) of the hypothalamus receive dense GABAergic inputs from peri-PVN regions. The noradrenergic system has been reported as a modulator of GABAergic transmission to the PVN. Previous electrophysiological and morphological studies support the presence of adrenoceptors on GABAergic neurons innervating the PVN. In this study, we identified three adrenoceptors on GABAergic neurons in the peri-PVN region, focusing on the anterior hypothalamic area (AHA) and rostral zona incerta (ZIr). GABAergic neurons were identified using enhanced green fluorescent protein (eGFP), followed by single cell RT-PCR analysis of the GABA synthetic enzymes, glutamic acid decarboxylase (GAD)65 and/or GAD67. Single cell RT-PCR data revealed the expression of alpha(1A)-, alpha(1B)- and alpha(2A)-adrenoceptor mRNA on GABAergic neurons in AHA and ZIr. Additionally, immunohistochemical studies showed that the immunoreactivities of alpha(1A)-, alpha(1B)- and alpha(2A)-adrenoceptor were colocalized with eGFP-expressing neurons in AHA and ZIr. The present findings suggest the contribution of adrenoceptors to the modulation of GABAergic neurons in AHA and ZIr.     

Glutamate synaptic inputs to ventral tegmental area neurons in the rat derive primarily from subcortical sources

Dopamine and GABA neurons in the ventral tegmental area project to the nucleus accumbens and prefrontal cortex and modulate locomotor and reward behaviors as well as cognitive and affective processes. Both midbrain cell types receive synapses from glutamate afferents that provide an essential control of behaviorally-linked activity patterns, although the sources of glutamate inputs have not yet been completely characterized. We used antibodies against the vesicular glutamate transporter subtypes 1 and 2 (VGlut1 and VGlut2) to investigate the morphology and synaptic organization of axons containing these proteins as putative markers of glutamate afferents from cortical versus subcortical sites, respectively, in rats. We also characterized the ventral tegmental area cell populations receiving VGlut1+ or VGlut2+ synapses according to their transmitter phenotype (dopamine or GABA) and major projection target (nucleus accumbens or prefrontal cortex). By light and electron microscopic examination, VGlut2+ as opposed to VGlut1+ axon terminals were more numerous, had a larger average size, synapsed more proximally, and were more likely to form convergent synapses onto the same target. Both axon types formed predominantly asymmetric synapses, although VGlut2+ terminals more often formed synapses with symmetric morphology. No absolute selectivity was observed for VGlut1+ or VGlut2+ axons to target any particular cell population. However, the synapses onto mesoaccumbens neurons more often involved VGlut2+ terminals, whereas mesoprefrontal neurons received relatively equal synaptic inputs from VGlut1+ and VGlut2+ profiles. The distinct morphological features of VGlut1 and VGlut2 positive axons suggest that glutamate inputs from presumed cortical and subcortical sources, respectively, differ in the nature and intensity of their physiological actions on midbrain neurons. More specifically, our findings imply that subcortical glutamate inputs to the ventral tegmental area expressing VGlut2 predominate over cortical sources of excitation expressing VGlut1 and are more likely to drive the behaviorally-linked bursts in dopamine cells that signal future expectancy or attentional shifting.     

Cannabinoid Receptor Antagonists SR141716 and SR144528 Exhibit Properties of Partial Agonists in Experiments on Isolated Perfused Rat Heart

We studied the effect of selective cannabinoid receptor ligands on contractility of isolated Langendorff-perfused rat heart. It was found that 10-min perfusion of rat heart with a solution containing selective agonist of CB1 and CB2 receptors HU-210 (10 nM) decreased left ventricular developed pressure and maximum rates of contraction and relaxation. However, HU-210 had no effect on heart rate and end-diastolic pressure. Treatment with selective CB1 receptor antagonist SR141716 (1 µM) and selective CB2 receptor antagonist SR144528 (1 µM) decreased left ventricular developed pressure and maximum rates of contraction and relaxation, but had no effect on heart rate and end-diastolic pressure. Ten-minute perfusion of rat heart with a solution containing selective agonist of CB1 and CB2 receptors HU-210 (10 nM) decreased cAMP concentration in the heart. CB receptor antagonists had little effect on cAMP concentration in the heart. The negative inotropic effect of HU-210 and CB receptor antagonists is probably mediated by activation of CB1 receptors. It can be hypothesized that the decrease in heart cAMP concentration is related to stimulation of CB2 receptors. Our results suggest that selective CB receptor antagonists SR141716 and SR144528 in a final concentration of 1 µM exhibit properties of partial CB receptor agonists.__________Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 5, pp. 512–516, May, 2005     

脂多糖对大鼠尾核神经元L型电压门控钙通道电流的影响和外源性2-AG的调制作用

目的:探讨内源性大麻素2-花生四烯酰甘油(2-AG)对脂多糖(LPS)损伤的大鼠尾核神经元L型电压门控钙通道(L-VGCC)电流的调制作用及其分子机制。方法:原代培养新生大鼠尾核神经元,分为对照组、LPS组、2-AG组、2-AG+LPS组、SR141716A(CB1受体反向激动剂)+2-AG+LPS组和AM630(CB2受体反向激动剂)+2-AG+LPS组,应用全细胞膜片钳记录2-AG对LPS损伤的大鼠尾核神经元L-VGCC电流的影响;采用Hoechst染色法观察2-AG对LPS诱导的尾核神经元损伤的影响,并用试剂盒测定尾核神经元caspase-3的活性。结果:(1)LPS能增强L-VGCC电流密度,且未影响L-VGCC激活及失活的电学特征;(2)2-AG能抑制LPS增强L-VGCC电流密度的作用;(3)LPS增强L-VGCC电流密度并非是通过CB1和CB2受体起作用的;(4)2-AG本身对尾核神经元L-VGCC电流密度、激活及失活等电流特性均不产生影响;(5)LPS诱导的尾核神经元caspase-3活性增强可被2-AG抑制,CB1受体反向激动剂SR141716A可取消2-AG的这种效应;(6)LPS可诱导尾核神经元表现出典型的凋亡特征,2-AG可使LPS诱导的核固缩细胞数目显著减少。结论:内源性大麻素2-AG可通过调节尾核神经元L-VGCC电流起抗炎作用和保护神经元的效应。     

Modulation of electrically evoked acetylcholine release through cannabinoid CB1 receptors: evidence for an endocannabinoid tone in the human neocortex

Cannabinoids are known to inhibit neurotransmitter release in the CNS through CB1 receptors. The present study compares the effects of synthetic cannabinoids on acetylcholine (ACh) release in human and mice neocortex. We further investigated a possible endocannabinoid tone on CB1 receptors in human neocortex caused by endogenous agonists like anandamide or 2-arachidonylglycerol. Brain slices, incubated with [3H]-choline, were superfused and stimulated electrically under autoinhibition-free conditions to evoke tritium overflow assumed to represent ACh release. The first series of experiments was performed with 26 pulses, 60 mA, at 0.1 Hz. In mice neocortical slices, the cannabinoid receptor agonist WIN55212-2 decreased ACh release (pIC50=6.68, I(max)=67%). In the human neocortex the concentration-response curve of WIN55212-2 was bell-shaped and flat (I(max observed) approximately 30%). The estimated maximum possible inhibition, however, was much larger: I(max derived)=79%. Lec, the negative logarithm (lg) of the biophase concentration of endocannabinoids in 'WIN55212-2 units,' was -6.52, the pKd of WIN55212-2 was 7.47. The CB1 receptor antagonist/inverse agonist SR141716 enhanced ACh release in the human neocortex (by 38%) and prevented the inhibitory effect of WIN55212-2. The concentration-response curve of WIN55212-2 was changed in its shape including a shift to the right due to the presence of SR141716. A pA2 of this antagonist between 11.60 and 11.18 was obtained. SR141716 alone had no effect in mice neocortical slices. A partial agonist without inverse agonistic activity, O-1184, enhanced ACh release in the human neocortex. The endocannabinoid uptake-inhibitor AM404 decreased ACh release in human, but not in mice, neocortical slices. Change of the stimulation parameters (eight trains of pseudo-one-pulse bursts (4 pulses, 76 mA, 100 Hz), spaced by 45 s intervals) led to a stronger inhibitory effect of WIN55212-2, and abolished the disinhibitory effect of SR141716 and O-1184. The results show that activation of CB1 cannabinoid receptors leads to inhibition of ACh release in the human and mouse neocortex. The endocannabinoid tone is high in the human, but not in the mouse neocortex and is dependent on neuronal activity. SR141716 acts as a competitive CB1 receptor antagonist.     

EEG alpha power changes reflect response inhibition deficits after traumatic brain injury (TBI) in humans

We studied the effect of cannabinoids on the activity of N-methyl-d-aspartate (NMDA) receptors in the locus coeruleus from rat brain slices by single-unit extracellular recordings. As expected, NMDA (100 microM) strongly excited (by nine fold) the cell firing activity of the locus coeruleus. Perfusion with the endocannabinoid anandamide (1 and 10 microM) or the anandamide transport inhibitor AM 404 (30 microM) enhanced the NMDA-induced excitation of locus coeruleus neurons. Similarly, the synthetic agonists R(+)-WIN 55212-2 (10 microM) and CP 55940 (30 microM) enhanced the effect of NMDA. In the presence of the CB(1) receptor antagonists SR 141716A (1 microM) or AM 251 (1 microM), the enhancement induced by anandamide (10 microM) was blocked. Our results suggest that cannabinoids modulate the activity of NMDA receptors in the locus coeruleus through CB(1) receptors.