Cannabidiol, a non-psychotropic component of cannabis, attenuates vomiting and nausea-like behaviour via indirect agonism of 5-HT(1A) somatodendritic autoreceptors in the dorsal raphe nucleus

BACKGROUND AND PURPOSE

To evaluate the hypothesis that activation of somatodendritic 5-HT_1A autoreceptors in the dorsal raphe nucleus (DRN) produces the anti-emetic/anti-nausea effects of cannabidiol (CBD), a primary non-psychoactive cannabinoid found in cannabis.

EXPERIMENTAL APPROACH

The potential of systemic and intra-DRN administration of 5-HT_1A receptor antagonists, WAY100135 or WAY100635, to prevent the anti-emetic effect of CBD in shrews (Suncus murinus) and the anti-nausea-like effects of CBD (conditioned gaping) in rats were evaluated. Also, the ability of intra-DRN administration of CBD to produce anti-nausea-like effects (and reversal by systemic WAY100635) was assessed. In vitro studies evaluated the potential of CBD to directly target 5-HT_1A receptors and to modify the ability of the 5-HT_1A agonist, 8-OH-DPAT, to stimulate [³⁵S]GTPγS binding in rat brainstem membranes.

KEY RESULTS

CBD suppressed nicotine-, lithium chloride (LiCl)- and cisplatin (20 mg·kg⁻¹, but not 40 mg·kg⁻¹)-induced vomiting in the S. murinus and LiCl-induced conditioned gaping in rats. Anti-emetic and anti-nausea-like effects of CBD were suppressed by WAY100135 and the latter by WAY100635. When administered to the DRN: (i) WAY100635 reversed anti-nausea-like effects of systemic CBD, and (ii) CBD suppressed nausea-like effects, an effect that was reversed by systemic WAY100635. CBD also displayed significant potency (in a bell-shaped dose–response curve) at enhancing the ability of 8-OH-DPAT to stimulate [³⁵S]GTPγS binding to rat brainstem membranes in vitro. Systemically administered CBD and 8-OH-DPAT synergistically suppressed LiCl-induced conditioned gaping.

CONCLUSIONS AND IMPLICATIONS

These results suggest that CBD produced its anti-emetic/anti-nausea effects by indirect activation of the somatodendritic 5-HT_1A autoreceptors in the DRN.

LINKED ARTICLES

This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7     

Antidepressant-like effects of cannabidiol in mice: possible involvement of 5-HT1A receptors

Background and purpose:

Cannabidiol (CBD) is a non-psychotomimetic compound from Cannabis sativa that induces anxiolytic- and antipsychotic-like effects in animal models. Effects of CBD may be mediated by the activation of 5-HT_1A receptors. As 5-HT_1A receptor activation may induce antidepressant-like effects, the aim of this work was to test the hypothesis that CBD would have antidepressant-like activity in mice as assessed by the forced swimming test. We also investigated if these responses depended on the activation of 5-HT_1A receptors and on hippocampal expression of brain-derived neurotrophic factor (BDNF).

Experimental approach:

Male Swiss mice were given (i.p.) CBD (3, 10, 30, 100 mg·kg⁻¹), imipramine (30 mg·kg⁻¹) or vehicle and were submitted to the forced swimming test or to an open field arena, 30 min later. An additional group received WAY100635 (0.1 mg·kg⁻¹, i.p.), a 5-HT_1A receptor antagonist, before CBD (30 mg·kg⁻¹) and assessment by the forced swimming test. BDNF protein levels were measured in the hippocampus of another group of mice treated with CBD (30 mg·kg⁻¹) and submitted to the forced swimming test.

Key results:

CBD (30 mg·kg⁻¹) treatment reduced immobility time in the forced swimming test, as did the prototype antidepressant imipramine, without changing exploratory behaviour in the open field arena. WAY100635 pretreatment blocked CBD-induced effect in the forced swimming test. CBD (30 mg·kg⁻¹) treatment did not change hippocampal BDNF levels.

Conclusion and implications:

CBD induces antidepressant-like effects comparable to those of imipramine. These effects of CBD were probably mediated by activation of 5-HT_1A receptors.     

Functional interactions between 5-HT2A and presynaptic 5-HT1A receptor-based responses in mice genetically deficient in the serotonin 5-HT transporter (SERT)

Background and purpose:

Despite decreased presynaptic 5-HT_1A and altered 5-HT_2A receptor function in genetically-deficient serotonin (5-HT) transporter (SERT) mice, the 5-HT_1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate salt (WAY 100635) still induced head twitches in these mice, a well-established 5-HT_2A receptor-mediated response.

Experimental approach:

Interactions between 5-HT_1A and 5-HT_2A receptors were assessed using the head-twitch response following 5-HT_1A and 5-HT_2A receptor agonists and antagonists in SERT wild-type (+/+), heterozygous (+/−), and knockout (−/−) mice. The role of brain 5-HT availability in WAY 100635 induced head twitches was also examined.

Key results:

WAY 100635 induced head twitches in a SERT gene-dose dependent manner, inducing 5-fold more head twitches in SERT −/− versus SERT +/+ mice. In SERT −/− mice, inhibition of 5-HT synthesis with p-chlorophenylalanine (PCPA) markedly depleted tissue 5-HT in all five brain areas examined and abolished WAY 100635 induced head twitches. Further, the selective 5-HT reuptake inhibitor fluvoxamine increased WAY 100635 induced head twitches in SERT +/+ and +/− mice. Head twitches following the 5-HT_2A receptor agonist (+/−)-2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI) were robust in SERT +/+ and +/− mice but much reduced in SERT −/− mice. DOI-induced head twitches were decreased by the 5-HT_1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) in SERT +/+ and +/− mice. All drug-induced head twitches were blocked by the 5-HT_2A receptor antagonist a-Phenyl-1-(2-phenylethyl)-4-piperidinemethanol (MDL 11,939).

Conclusions and implications:

These data show that indirect activation of 5-HT_2A receptors via blockade of presynaptic 5-HT_1A receptors potentiated head-twitch responses, suggesting functional interactions between these receptors, interactions affected by altered 5-HT availability. Our findings strongly support the correlation of WAY 100635 induced head twitches with increased 5-HT availability, induced genetically or pharmacologically.     

Cannabidiol inhibits paclitaxel-induced neuropathic pain through 5-HT1A receptors without diminishing nervous system function or chemotherapy efficacy

Background and Purpose

Paclitaxel (PAC) is associated with chemotherapy-induced neuropathic pain (CIPN) that can lead to the cessation of treatment in cancer patients even in the absence of alternate therapies. We previously reported that chronic administration of the non-psychoactive cannabinoid cannabidiol (CBD) prevents PAC-induced mechanical and thermal sensitivity in mice. Hence, we sought to determine receptor mechanisms by which CBD inhibits CIPN and whether CBD negatively effects nervous system function or chemotherapy efficacy.

Experimental Approach

The ability of acute CBD pretreatment to prevent PAC-induced mechanical sensitivity was assessed, as was the effect of CBD on place conditioning and on an operant-conditioned learning and memory task. The potential interaction of CBD and PAC on breast cancer cell viability was determined using the MTT assay.

Key Results

PAC-induced mechanical sensitivity was prevented by administration of CBD (2.5 – 10 mg·kg⁻¹) in female C57Bl/6 mice. This effect was reversed by co-administration of the 5-HT_1A antagonist WAY 100635, but not the CB₁ antagonist SR141716 or the CB₂ antagonist SR144528. CBD produced no conditioned rewarding effects and did not affect conditioned learning and memory. Also, CBD + PAC combinations produce additive to synergistic inhibition of breast cancer cell viability.

Conclusions and Implications

Our data suggest that CBD is protective against PAC-induced neurotoxicity mediated in part by the 5-HT_1A receptor system. Furthermore, CBD treatment was devoid of conditioned rewarding effects or cognitive impairment and did not attenuate PAC-induced inhibition of breast cancer cell viability. Hence, adjunct treatment with CBD during PAC chemotherapy may be safe and effective in the prevention or attenuation of CIPN.     

Further pharmacological characterization of 5-HT2C receptor agonist-induced inhibition of 5-HT neuronal activity in the dorsal raphe nucleus in vivo

Background and purpose:

Recent experiments using non-selective 5-hydroxytryptamine (5-HT)_2C receptor agonists including WAY 161503 suggested that midbrain 5-HT neurones are under the inhibitory control of 5-HT_2C receptors, acting via neighbouring gamma-aminobutyric acid (GABA) neurones. The present study extended this pharmacological characterization by comparing the actions of WAY 161503 with the 5-HT_2C receptor agonists, Ro 60-0275 and 1-(3-chlorophenyl) piperazine (mCPP), as well as the non-selective 5-HT agonist lysergic acid diethylamide (LSD) and the 5-HT releasing agent 3,4-methylenedioxymethamphetamine (MDMA).

Experimental approach:

5-HT neuronal activity was measured in the dorsal raphe nucleus (DRN) using extracellular recordings in anaesthetized rats. The activity of DRN GABA neurones was assessed using double-label immunohistochemical measurements of Fos and glutamate decarboxylase (GAD).

Key results:

Ro 60-0175, like WAY 161503, inhibited 5-HT neurone firing, and the 5-HT_2C antagonist SB 242084 reversed this effect. mCPP also inhibited 5-HT neurone firing (∼60% neurones) in a SB 242084-reversible manner. LSD inhibited 5-HT neurone firing; however, this effect was not altered by either SB 242084 or the 5-HT_2A/C receptor antagonist ritanserin but was reversed by the 5-HT_1A receptor antagonist WAY 100635. Similarly, MDMA inhibited 5-HT neurone firing in a manner reversible by WAY 100635, but not SB 242084 or ritanserin. Finally, both Ro 60-0275 and mCPP, like WAY 161503, increased Fos expression in GAD-positive DRN neurones.

Conclusions and implications:

These data strengthen the hypothesis that midbrain 5-HT neurones are under the inhibitory control of 5-HT_2C receptors, and suggest that the 5-HT_2C agonists Ro 60-0175, mCPP and WAY 161503, but not LSD or MDMA, are useful probes of the mechanism(s) involved.     

5-HT1A receptors are involved in the effects of xaliproden on G-protein activation,neurotransmitter release and nociception

Background and purpose:

Xaliproden (SR57746A) is a 5-HT_1A receptor agonist and neurotrophic agent that reduces oxaliplatin-mediated neuropathy in clinical trials. The present study investigated its profile on in vitro transduction, neurochemical responses and acute nociceptive pain tests in rats.

Experimental approach:

Xaliproden was tested on models associated with 5-HT_1A receptor activation including G-protein activation, extracellular dopamine and 5-HT levels measured by microdialysis and formalin-induced pain. Activation of 5-HT_1A receptors was confirmed by antagonism with WAY100635.

Key results:

Xaliproden exhibited high affinity for rat (r) and human (h) 5-HT_1A receptors (pK_i= 8.84 and 9.00). In [³⁵S]GTPγS (guanosine 5''-O-(3-[³⁵S]thio)triphosphate) assays it activated both hippocampal r5-HT_1A[pEC₅₀/E_MAX of 7.58/61% (%5-HT)] and recombinant h5-HT_1A receptors (glioma C6-h5-HT_1A: 7.39/62%; HeLa-h5-HT_1A: 7.24/93%). In functional [³⁵S]GTPγS autoradiography, xaliproden induced labelling in structures enriched with 5-HT_1A receptors (hippocampus, lateral septum, prefrontal and entorhinal cortices). Xaliproden inhibited in vivo binding of [³H]WAY100635 to 5-HT_1A receptors in mouse frontal cortex and hippocampus (ID₅₀: 3.5 and 3.3 mg·kg⁻¹, p.o. respectively). In rat, it increased extracellular dopamine levels in frontal cortex and reduced hippocampal 5-HT levels (ED₅₀: 1.2 and 0.7 mg·kg⁻¹, i.p. respectively). In a rat pain model, xaliproden inhibited paw licking and elevation (ED₅₀: 1 and 3 mg·kg⁻¹, i.p. respectively) following formalin injection in the paw. All effects were reversed by pretreatment with WAY100635.

Conclusions and implications:

These results indicate that activation of 5-HT_1A receptors is the principal mechanism of action of xaliproden and provide further support for the utility of 5-HT_1A receptor activation as an anti-nociceptive strategy.     

Cannabidiol inhibits synaptic transmission in rat hippocampal cultures and slices via multiple receptor pathways

BACKGROUND AND PURPOSE

Cannabidiol (CBD) has emerged as an interesting compound with therapeutic potential in several CNS disorders. However, whether it can modulate synaptic activity in the CNS remains unclear. Here, we have investigated whether CBD modulates synaptic transmission in rat hippocampal cultures and acute slices.

EXPERIMENTAL APPROACH

The effect of CBD on synaptic transmission was examined in rat hippocampal cultures and acute slices using whole cell patch clamp and standard extracellular recordings respectively.

KEY RESULTS

Cannabidiol decreased synaptic activity in hippocampal cultures in a concentration-dependent and Pertussis toxin-sensitive manner. The effects of CBD in culture were significantly reduced in the presence of the cannabinoid receptor (CB₁) inverse agonist, {"type":"entrez-nucleotide","attrs":{"text":"LY320135","term_id":"1257555575","term_text":"LY320135"}}LY320135 but were unaffected by the 5-HT_1A receptor antagonist, WAY100135. In hippocampal slices, CBD inhibited basal synaptic transmission, an effect that was abolished by the proposed CB₁ receptor antagonist, AM251, in addition to {"type":"entrez-nucleotide","attrs":{"text":"LY320135","term_id":"1257555575","term_text":"LY320135"}}LY320135 and WAY100135.

CONCLUSIONS AND IMPLICATIONS

Cannabidiol reduces synaptic transmission in hippocampal in vitro preparations and we propose a role for both 5-HT_1A and CB₁ receptors in these CBD-mediated effects. These data offer some mechanistic insights into the effects of CBD and emphasize that further investigations into the actions of CBD in the CNS are required in order to elucidate the full therapeutic potential of CBD.     

Evidence that 5-hydroxytryptamine7 receptors play a role in the mediation of afferent transmission within the nucleus tractus solitarius in anaesthetized rats

Background and purpose:

Central 5-hydroxytryptamine (5-HT)-containing pathways utilizing 5-HT₇ receptors are known to be critical for the mediation of cardiovascular reflexes. The nucleus tractus solitarius (NTS) is a site involved in the integration of cardiovascular afferent information. The present experiments examined the involvement of the 5-HT₇ receptor in the processing of cardiovascular reflexes in the NTS.

Experimental approach:

In anaesthetized rats extracellular recordings were made from 104 NTS neurones that were excited by electrical stimulation of the vagus nerve and/or activation of cardiopulmonary afferents. Drugs were applied ionophoretically in the vicinity of these neurones.

Key results:

The non-selective 5-HT₇ receptor agonist 5-carboxamidotryptamine maleate (5-CT) applied to 78 neurones increased the firing rate in 18 by 59% and decreased it in 38 neurones by 47%. Similarly, the 5-HT_1A agonist 8-OH-DPAT applied to 20 neurones had an excitatory (8), inhibitory (7) or no effect (5) on the 20 neurones tested. In the presence of the 5-HT₇ antagonist SB 258719 the 5-CT excitation was attenuated. Furthermore, the excitatory response of NTS neurones evoked by electrical stimulation of the vagus nerve or activation of cardiopulmonary afferents with intra atrial phenylbiguanide was attenuated by SB 258719. The inhibitory action of 5-CT was unaffected by SB 258719 and the 5-HT_1A antagonist WAY-100635. WAY-100635 failed to have any effect on 5-CT and vagal afferent-evoked excitations.

Conclusions and implications:

Vagal afferent-evoked excitation of NTS neurones can be blocked by SB 258719, a selective 5-HT₇ antagonist. This observation further supports the involvement of 5-HT neurotransmission in NTS afferent processing.     

Investigation of the role of 5-HT2 receptor subtypes in the control of the bladder and the urethra in the anaesthetized female rat

Background and purpose:

Micturition is controlled by central 5-HT-containing pathways. 5-HT₂ receptors have been implicated in this system especially in control of the urethra, which is a drug target for treating urinary incontinence. This study investigates the role of each of the three subtypes of this receptor with emphasis on sphincter regulation.

Experimental approach:

Recordings of urethral and bladder pressure, external urethral sphincter (EUS) EMG, as well as the micturition reflex induced by bladder distension along with blood pressure and heart rate were made in anaesthetized rats. The effects of agonists and antagonists for 5-HT₂ receptor subtypes were studied on these variables.

Key results:

The 5-HT_2C agonists Ro 60-0175, WAY 161503 and mCPP, i.v., activated the EUS, increased urethral pressure and inhibited the micturition reflex. The effects of Ro 60-0175 on the EUS were blocked by the 5-HT_2C antagonist SB 242084 and the 5-HT_2A antagonists, ketanserin and MDL 100907. SB 242084 also blocked the inhibitory action on the reflex, while the 5-HT_2B antagonist RS 127445 only blocked the increase in urethral pressure. The 5-HT_2A receptor agonist DOI given i.v. or i.t. but not i.c.v. activated the EUS.

Conclusions and implications:

5-HT_2A/2C receptors located in the sacral spinal cord activate the EUS, while central 5-HT_2C receptors inhibit the micturition reflex and 5-HT_2B receptors, probably at the level of the urethra, increase urethral smooth muscle tone. Furthermore, 5-HT_2B and 5-HT_2C receptors do not seem to play an important role in the physiological regulation of micturition.     

5-hydroxytryptamine induced relaxation in the pig urinary bladder neck

Background and purpose

5-Hydroxytryptamine (5-HT) is one of the inhibitory mediators in the urinary bladder outlet region. Here we investigated mechanisms involved in 5-HT-induced relaxations of the pig bladder neck.

Experimental approach

Urothelium-denuded strips of pig bladder were mounted in organ baths for isometric force recordings of responses to 5-HT and electrical field stimulation (EFS).

Key results

After phenylephrine-induced contraction, 5-HT and 5-HT receptor agonists concentration-dependently relaxed the preparations, with the potency order: 5-carboxamidotryptamine (5-CT) > 5-HT = RS67333 > (±)-8-hydroxy-2-dipropylaminotetralinhydrobromide > m-chlorophenylbiguanide > α-methyl-5-HT > ergotamine. 5-HT and 5-CT relaxations were reduced by the 5-HT₇ receptor antagonist (2R)-1-[(3-hydroxyphenyl)sulphonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrrolidine hydrochloride and potentiated by (S)-N-tert-butyl-3-(4-(2-methoxyphenyl)-piperazin-1-yl)-2-phenylpropanamide dihydrochloride (WAY 100135) and cyanopindolol, 5-HT_1A and 5-HT_1A/1B receptor antagonists respectively. Inhibitors of 5-HT_1B/1D, 5-HT₂, 5-HT_2B/2C, 5-HT₃, 5-HT₄, 5-HT_5A and 5-HT₆ receptors failed to modify 5-HT responses. Blockade of monoamine oxidase A/B, noradrenergic neurotransmission, α-adrenoceptors, muscarinic and purinergic receptors, nitric oxide synthase, guanylate cyclase and prostanoid synthesis did not alter relaxations to 5-HT. Inhibitors of Ca²⁺-activated K⁺ and ATP-dependent K⁺ channels failed to modify 5-HT responses but blockade of neuronal voltage-gated Na⁺-, Ca²⁺-and voltage-gated K⁺ (K_v)-channels potentiated these relaxations. Adenylyl cyclase activation and cAMP-dependent protein kinase (PKA) inhibition potentiated and reduced, respectively, 5-HT-induced responses. Under non-adrenergic, non-cholinergic, non-nitrergic conditions, EFS induced neurogenic, frequency-dependent, relaxations which were resistant to WAY 100135 and cyanopindolol.

Conclusions and implications

5-HT relaxed the pig urinary bladder neck through muscle 5-HT₇ receptors linked to the cAMP-PKA pathway. Prejunctional 5-HT_1A receptors and K_v channels modulated 5-HT-induced relaxations whereas postjunctional K⁺ channels were not involved in such responses. 5-HT₇ receptor antagonists could be useful in the therapy of urinary incontinence produced by intrinsic sphincter deficiency.     

Cannabidiolic acid prevents vomiting in Suncus murinus and nausea-induced behaviour in rats by enhancing 5-HT1A receptor activation

Background and Purpose

To evaluate the ability of cannabidiolic acid (CBDA) to reduce nausea and vomiting and enhance 5-HT_1A receptor activation in animal models.

Experimental Approach

We investigated the effect of CBDA on (i) lithium chloride (LiCl)-induced conditioned gaping to a flavour (nausea-induced behaviour) or a context (model of anticipatory nausea) in rats; (ii) saccharin palatability in rats; (iii) motion-, LiCl- or cisplatin-induced vomiting in house musk shrews (Suncus murinus); and (iv) rat brainstem 5-HT_1A receptor activation by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and mouse whole brain CB₁ receptor activation by CP55940, using [³⁵S]GTPγS-binding assays.

Key Results

In shrews, CBDA (0.1 and/or 0.5 mg·kg⁻¹ i.p.) reduced toxin- and motion-induced vomiting, and increased the onset latency of the first motion-induced emetic episode. In rats, CBDA (0.01 and 0.1 mg·kg⁻¹ i.p.) suppressed LiCl- and context-induced conditioned gaping, effects that were blocked by the 5-HT_1A receptor antagonist, WAY100635 (0.1 mg·kg⁻¹ i.p.), and, at 0.01 mg·kg⁻¹ i.p., enhanced saccharin palatability. CBDA-induced suppression of LiCl-induced conditioned gaping was unaffected by the CB₁ receptor antagonist, SR141716A (1 mg·kg⁻¹ i.p.). In vitro, CBDA (0.1–100 nM) increased the E_max of 8-OH-DPAT.

Conclusions and Implications

Compared with cannabidiol, CBDA displays significantly greater potency at inhibiting vomiting in shrews and nausea in rats, and at enhancing 5-HT_1A receptor activation, an action that accounts for its ability to attenuate conditioned gaping in rats. Consequently, CBDA shows promise as a treatment for nausea and vomiting, including anticipatory nausea for which no specific therapy is currently available.     

A spinal mechanism of action for duloxetine in a rat model of painful diabetic neuropathy

BACKGROUND AND PURPOSE

This study was designed to clarify mechanisms responsible for the anti-allodynic effects of duloxetine in diabetes.

EXPERIMENTAL APPROACH

The streptozotocin-induced diabetic rat model was used to compare the efficacy of duloxetine, 5-HT, the 5-HT_2A receptor agonist [1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI)] and two antagonists (ketanserin and pruvanserin) on tactile allodynia.

KEY RESULTS

Systemic or intrathecal injection of duloxetine alleviated tactile allodynia in diabetic rats. The effect of systemic duloxetine was reduced by intrathecal administration of ketanserin or pruvanserin, indicating participation of spinal 5-HT_2A receptors in the mechanism of action of duloxetine. In contrast to spinal delivery, systemic and local peripheral injections of ketanserin or pruvanserin alleviated tactile allodynia in diabetic rats. This effect was reversed immediately after systemic or local DOI injection.

CONCLUSIONS AND IMPLICATIONS

These results support the involvement of spinal 5-HT_2A receptors in the ability of duloxetine to ameliorate painful diabetic neuropathy. Our data also suggest that the role of 5-HT_2A receptors depends on the level of the neuraxis at which activation takes place, with peripheral activation contributing to tactile allodynia in diabetic rats, whereas spinal activation of this receptor alleviates tactile allodynia. The development of selective peripheral 5-HT_2A receptor antagonists may offer a novel approach for the treatment of diabetic neuropathic pain.     

Preferential in vivo action of F15599, a novel 5-HT1A receptor agonist, at postsynaptic 5-HT1A receptors

Background and purpose:

{"type":"entrez-nucleotide","attrs":{"text":"F15599","term_id":"1130739","term_text":"F15599"}}F15599, a novel 5-hydroxytryptamine (5-HT)_1A receptor agonist with 1000-fold selectivity for 5-HT compared with other monoamine receptors, shows antidepressant and procognitive activity at very low doses in animal models. We examined the in vivo activity of {"type":"entrez-nucleotide","attrs":{"text":"F15599","term_id":"1130739","term_text":"F15599"}}F15599 at somatodendritic autoreceptors and postsynaptic 5-HT_1A heteroreceptors.

Experimental approach:

In vivo single unit and local field potential recordings and microdialysis in the rat.

Key results:

{"type":"entrez-nucleotide","attrs":{"text":"F15599","term_id":"1130739","term_text":"F15599"}}F15599 increased the discharge rate of pyramidal neurones in medial prefrontal cortex (mPFC) from 0.2 µg·kg⁻¹ i.v and reduced that of dorsal raphe 5-hydroxytryptaminergic neurones at doses >10-fold higher (minimal effective dose 8.2 µg·kg⁻¹ i.v.). Both effects were reversed by the 5-HT_1A antagonist (±)WAY100635. {"type":"entrez-nucleotide","attrs":{"text":"F15599","term_id":"1130739","term_text":"F15599"}}F15599 did not alter low frequency oscillations (∼1 Hz) in mPFC. In microdialysis studies, {"type":"entrez-nucleotide","attrs":{"text":"F15599","term_id":"1130739","term_text":"F15599"}}F15599 increased dopamine output in mPFC (an effect dependent on the activation of postsynaptic 5-HT_1A receptors) with an ED₅₀ of 30 µg·kg⁻¹ i.p., whereas it reduced hippocampal 5-HT release (an effect dependent exclusively on 5-HT_1A autoreceptor activation) with an ED₅₀ of 240 µg·kg⁻¹ i.p. Likewise, application of {"type":"entrez-nucleotide","attrs":{"text":"F15599","term_id":"1130739","term_text":"F15599"}}F15599 by reverse dialysis in mPFC increased dopamine output in a concentration-dependent manner. All neurochemical responses to {"type":"entrez-nucleotide","attrs":{"text":"F15599","term_id":"1130739","term_text":"F15599"}}F15599 were prevented by administration of (±)WAY100635.

Conclusions and implications:

These results indicate that systemic administration of {"type":"entrez-nucleotide","attrs":{"text":"F15599","term_id":"1130739","term_text":"F15599"}}F15599 preferentially activates postsynaptic 5-HT_1A receptors in PFC rather than somatodendritic 5-HT_1A autoreceptors. This regional selectivity distinguishes {"type":"entrez-nucleotide","attrs":{"text":"F15599","term_id":"1130739","term_text":"F15599"}}F15599 from previously developed 5-HT_1A receptor agonists, which preferentially activate somatodendritic 5-HT_1A autoreceptors, suggesting that {"type":"entrez-nucleotide","attrs":{"text":"F15599","term_id":"1130739","term_text":"F15599"}}F15599 may be particularly useful in the treatment of depression and of cognitive deficits in schizophrenia.     

Characteristics of the actions by which 5-hydroxytryptamine affects electrical and mechanical activities in rabbit jugular vein graft

BACKGROUND AND PURPOSE

The vasomodulating actions of 5-HT in vein grafts, and the underlying mechanisms, remain to be fully clarified. Here, we characterized the actions by which 5-HT affects electrical and mechanical activities in rabbit autologous jugular vein grafts.

EXPERIMENTAL APPROACH

Smooth muscle cell (SMC) membrane potential and isometric tension were measured in vein grafts 4 weeks after implantation into carotid arteries. Changes in the expression of 5-HT receptor subtypes and in myosin heavy chain isoforms (SM1, SM2 and SMemb) were examined by immunohistochemistry and Western blot analysis.

KEY RESULTS

The walls of grafted veins displayed massive increases in the number of SM1- and SM2-positive SMCs. 5-HT induced a large depolarization and contraction that were each reduced by both 5-HT_2A- and 5-HT_1B/1D-receptor antagonists. The 5-HT-induced contraction was not modified by a 5-HT₇-receptor antagonist. The 5-HT₇-receptor-selective agonist AS 19 did not induce relaxation during the contraction to prostaglandin F_2α. Immunohistochemical and Western blot analyses revealed that immunoreactive responses against 5-HT_2A and 5-HT_1B/1D receptors were increased in the vein graft.

CONCLUSIONS AND IMPLICATIONS

5-HT is able to induce a large contraction in rabbit autologous jugular vein grafts through (i) an increased number of differentiated contractile SMCs; (ii) an increased number of SMCs expressing contractile 5-HT_2A- and 5-HT_1B/1D receptors; and (iii) a down-regulation of the function of the relaxant SMC 5-HT₇ receptors. These changes in the vein graft may help it to resist the higher pressure present on the arterial side of the circulation.     

Characteristics of the actions by which 5-HT affects electrical and mechanical activities in rabbit jugular vein

BACKGROUND AND PURPOSE

5-HT is known to be a potent vasospasmogenic agonist in various arteries. However, in veins the vasomodulating actions of 5-HT, and the underlying mechanisms, remain to be fully clarified. Here, we characterized the actions by which 5-HT affects electrical and mechanical activities in the rabbit jugular vein.

EXPERIMENTAL APPROACH

Membrane potential and isometric tension were measured in endothelium-intact and -denuded preparations. Localization of 5-HT receptor subtypes was examined immunohistochemically.

KEY RESULTS

5-HT induced a transient then a small, sustained smooth muscle cell hyperpolarization in endothelium-intact strips. In endothelium-denuded strips, 5-HT induced only a sustained hyperpolarization, and this was changed to a depolarization by the selective 5-HT₇ receptor inhibitor SB269970. This depolarization was inhibited by the 5-HT_2A receptor blocker sarpogrelate. 5-HT induced a relaxation of PGF_2α-induced contracted strips that was similar in endothelium-intact and -denuded preparations. The latter relaxation was changed to contraction by SB269970 and this contraction was inhibited by sarpogrelate. Immunoreactive responses against endothelial and smooth muscle 5-HT_2A receptors and smooth muscle 5-HT₇ receptors were identified in the vein. The 5-HT-induced relaxation of the PGF_2α contraction was inhibited by the cAMP-dependent protein kinase inhibitor Rp-cAMPS and by the AC inhibitor SQ22536.

CONCLUSIONS AND IMPLICATIONS

These results indicate that 5-HT activates both smooth muscle 5-HT₇ receptors (to produce relaxation) and smooth muscle 5-HT_2A receptors (to produce contraction) in rabbit jugular vein. We suggest that in this particular vein, the 5-HT_2A receptor-induced depolarization and contraction are masked by the 5-HT₇ receptor-induced responses, possibly via actions mediated by cAMP.     

Deletion of GIRK2 Subunit of GIRK Channels Alters the 5-HT1A Receptor-Mediated Signaling and Results in a Depression-Resistant Behavior

Background:

Targeting dorsal raphe 5-HT_1A receptors, which are coupled to G-protein inwardly rectifying potassium (GIRK) channels, has revealed their contribution not only to behavioral and functional aspects of depression but also to the clinical response to its treatment. Although GIRK channels containing GIRK2 subunits play an important role controlling excitability of several brain areas, their impact on the dorsal raphe activity is still unknown. Thus, the goal of the present study was to investigate the involvement of GIRK2 subunit-containing GIRK channels in depression-related behaviors and physiology of serotonergic neurotransmission.

Methods:

Behavioral, functional, including in vivo extracellular recordings of dorsal raphe neurons, and neurogenesis studies were carried out in wild-type and GIRK2 mutant mice.

Results:

Deletion of the GIRK2 subunit promoted a depression-resistant phenotype and determined the behavioral response to the antidepressant citalopram without altering hippocampal neurogenesis. In dorsal raphe neurons of GIRK2 knockout mice, and also using GIRK channel blocker tertiapin-Q, the basal firing rate was higher than that obtained in wild-type animals, although no differences were observed in other firing parameters. 5-HT_1A receptors were desensitized in GIRK2 knockout mice, as demonstrated by a lower sensitivity of dorsal raphe neurons to the inhibitory effect of the 5-HT_1A receptor agonist, 8-OH-DPAT, and the antidepressant citalopram.

Conclusions:

Our results indicate that GIRK channels formed by GIRK2 subunits determine depression-related behaviors as well as basal and 5-HT_1A receptor-mediated dorsal raphe neuronal activity, becoming alternative therapeutic targets for psychiatric diseases underlying dysfunctional serotonin transmission.     

High yield and efficient expression and purification of the human 5-HT3A receptor

Aim:

To establish a method for efficient expression and purification of the human serotonin type 3A receptor (5-HT_3A) that is suitable for structural studies.

Methods:

Codon-optimized cDNA of human 5-HT_3A was inserted into a modified BacMam vector, which contained an IgG leader sequence, an 8×His tag linked with two-Maltose Binding Proteins (MBP), and a TEV protease cleavage site. The BacMam construct was used to generate baculoviruses for expression of 5-HT_3A in HEK293F cells. The proteins were solubilized from the membrane with the detergent C₁₂E ₉, and purified using MBP affinity chromatography. The affinity tag was removed by TEV protease treatment and immobilized metal ion affinity chromatography. The receptors were further purified by size-exclusion chromatography (SEC). Western blot and SDS-PAGE were used to detect 5-HT_3A during purification. The purified receptor was used in crystallization and analyzed with negative stain electron microscopy (EM).

Results:

The BacMam system yielded 0.5 milligram of the human 5-HT_3A receptor per liter of cells. MBP affinity purification resulted in good yields with high purity and homogeneity. SEC profiles indicated that the purified receptors were pentameric. No protein crystals were obtained; however, a reconstructed 3D density map generated from the negative stain EM data fitted well with the mouse 5-HT_3A structure.

Conclusion:

With the BacMam system, robust expression of the human 5-HT_3A receptor is obtained, which is monodisperse, therefore enabling 3D reconstruction of an EM map. This method is suitable for high-throughput screening of different constructs, thus facilitating structural and biochemical studies of the 5-HT_3A receptor.     

Body temperature and cardiac changes induced by peripherally administered oxytocin,vasopressin and the non-peptide oxytocin receptor agonist WAY 267,464: a biotelemetry study in rats

Background and Purpose

There is current interest in oxytocin (OT) as a possible therapeutic in psychiatric disorders. However, the usefulness of OT may be constrained by peripheral autonomic effects, which may involve an action at both OT and vasopressin V_1A receptors. Here, we characterized the cardiovascular and thermoregulatory effects of OT, vasopressin (AVP) and the non-peptide OT receptor agonist WAY 267,464 in rats, and assessed the relative involvement of the OT and V_1A receptors in these effects.

Experimental Approach

Biotelemetry in freely moving male Wistar rats was used to examine body temperature and heart rate after OT (0.01 – 1 mg kg⁻¹; i.p.), AVP (0.001 – 0.1 mg kg⁻¹; i.p.) or WAY 267,464 (10 and 100 mg kg⁻¹; i.p.). The actions of the OT receptor antagonist Compound 25 (C25, 5 and 10 mg kg⁻¹) and V_1A receptor antagonist SR49059 (1 and 10 mg kg⁻¹) were studied, as well as possible V_1A receptor antagonist effects of WAY 267,464.

Key Results

OT and AVP dose-dependently reduced body temperature and heart rate. WAY 267,464 had similar, but more modest, effects. SR49059, but not C25, prevented the hypothermia and bradycardia induced by OT and AVP. WAY 267,464 (100 mg·kg⁻¹) prevented the effects of OT, and to some extent AVP.

Conclusions and Implications

Peripherally administered OT and AVP have profound cardiovascular and thermoregulatory effects that appear to principally involve the V_1A receptor rather than the OT receptor. Additionally, WAY 267,464 is not a simple OT receptor agonist, as it has functionally relevant V_1A antagonist actions.     

The phytocannabinoid, Δ9-tetrahydrocannabivarin,can act through 5-HT1A receptors to produce antipsychotic effects

Background and Purpose

This study aimed to address the questions of whether Δ⁹-tetrahydrocannabivarin (THCV) can (i) enhance activation of 5-HT_1A receptors in vitro and (ii) induce any apparent 5-HT_1A receptor-mediated antipsychotic effects in vivo.

Experimental Approach

In vitro studies investigated the effect of THCV on targeting by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) of 5-HT_1A receptors in membranes obtained from rat brainstem or human 5-HT_1A CHO cells, using [³⁵S]-GTPγS and 8-[³H]-OH-DPAT binding assays. In vivo studies investigated whether THCV induces signs of 5-HT_1A receptor-mediated antipsychotic effects in rats.

Key Results

THCV (i) potently, albeit partially, displaced 8-[³H]-OH-DPAT from specific binding sites in rat brainstem membranes; (ii) at 100 nM, significantly enhanced 8-OH-DPAT-induced activation of receptors in these membranes; (iii) produced concentration-related increases in 8-[³H]-OH-DPAT binding to specific sites in membranes of human 5-HT_1A receptor-transfected CHO cells; and (iv) at 100 nM, significantly enhanced 8-OH-DPAT-induced activation of these human 5-HT_1A receptors. In phencyclidine-treated rats, THCV, like clozapine (i) reduced stereotyped behaviour; (ii) decreased time spent immobile in the forced swim test; and (iii) normalized hyperlocomotor activity, social behaviour and cognitive performance. Some of these effects were counteracted by the 5-HT_1A receptor antagonist, WAY100635, or could be reproduced by the CB₁ antagonist, AM251.

Conclusions and Implications

Our findings suggest that THCV can enhance 5-HT_1A receptor activation, and that some of its apparent antipsychotic effects may depend on this enhancement. We conclude that THCV has therapeutic potential for ameliorating some of the negative, cognitive and positive symptoms of schizophrenia.