Substance P and its role in neural mechanisms governing learning, anxiety and functional recovery

The neurokinin Substance P (SP) is widely distributed in the central nervous system and has been extensively studied in various functional aspects. This review focuses on the behavioral relevance of SP. Here we show that SP can have memory-promoting, reinforcing and anxiolytic-like effects when administered systemically or into the nucleus basalis of the ventral pallidum. These effects seem to be mediated via the SP-preferring NK(1)receptor and differentially related to N- versus C-terminal fragments of the undecapeptide. Secondly, SP injection into the ventral pallidum can lead to increases of acetylcholine in frontal cortex and dopamine in nucleus accumbens, suggesting that the hypermnestic, positively reinforcing and anxiolytic effects observed upon basal forebrain injection of SP are mediated by activation of the nucleus accumbens-ventral pallidum circuitry. Furthermore, SP and certain SP-fragments may not only be considered to have beneficial behavioral effects in normal animals, but can also prevent lesion-induced functional deficits and improve the speed of recovery. This indicates that SP agonists might also have a neuroprotective capacity in parallel with recovery-promoting actions.     

Effect of neurokinin-I receptor antagonists on the function of 5-HT and noradrenaline neurons

Substance P antagonists have been proposed to be a new class of antidepressants. The present study aimed to determine the effect of the selective non-peptide rat neurokinin-1 (NK1) receptor antagonists WIN 51,708 and CP-96,345 on the firing activity of rat dorsal raphe serotonin (5-HT) and locus coeruleus noradrenaline (NA) neurons. While WIN51,708 (2mg/kg, i.v.) and CP-96,345 (0.15 mg/kg, i.v.) did not modify the firing activity of 5-HT and NA neurons, both antagonists attenuated the suppressant effect of the alpha2-adrenoceptor agonist clonidine on the firing activity of both types of neurons. In contrast, the responsiveness of 5-HT neurons to the i.v. administration of the 5-HT autoreceptor agonist LSD and the 5-HT1A receptor agonist 8-OH-DPAT remained unchanged. These findings suggest that NK1 receptor antagonists affect markedly the NA system via an attenuation of the function of alpha2-adrenoceptors on the cell body of NA neurons and, consequently, may also modulate 5-HT neurotransmission.     

Methamphetamine induces striatal neurokinin‐1 receptor endocytosis primarily in somatostatin/NPY/NOS interneurons and the role of dopamine receptors in mice

Methamphetamine (METH) is a psychostimulant that induces long‐term deficits of dopamine terminal markers and apoptotic cell death in the striatum. Our laboratory demonstrated that pharmacological blockade of the neurokinin‐1 receptor attenuated the METH‐induced damage to the striatal dopamine terminals and the apoptotic cell death of some striatal neurons. Here, we used histological methods to assess the effect of METH on neurokinin‐1 receptor trafficking in the striatum as an indirect index of signaling by the neuropeptide substance P (natural ligand for this receptor). Male mice received a single injection of METH (30 mg/kg, i.p.) and were sacrificed 30 min later. Immunohistofluorescence confocal microscopy confirmed that the neurokinin‐1 receptor is located on cholinergic and somatostatin interneurons of the striatum. METH induced the trafficking of the neurokinin‐1 receptor from the membrane into cytoplasmic endosomes primarily in the somatostatin/NPY/NOS interneurons, and this phenomenon was attenuated by antagonists of the dopamine D1 (SCH‐23390), D2 (raclopride), or neurokinin‐1 (WIN‐51,708) receptors. These data demonstrate that METH induces the trafficking of the striatal neurokinin‐1 receptors principally in the somatostatin/NPY/NOS interneurons and that this phenomenon is dependent on the activity of dopamine D1 and D2 receptors. Synapse, 2011. © 2010 Wiley‐Liss, Inc.     

Differential modulation of frontal cortex acetylcholine by injection of substance P into the nucleus basalis magnocellularis region in the freely-moving vs. the anesthetized preparation

In vivo microdialysis was used to assess the effects of unilateral substance P (SP) injection into the nucleus basalis magnocellularis on extracellular levels of acetylcholine (ACh) in the frontal cortex, either in freely moving or urethane-anesthetized rats. The results show that the neurochemical effects of SP are critically dependent on the choice of the experimental preparation: In the freely-moving rat, the injection procedure led to behavioral and concurrent bilateral cholinergic activation in the frontal cortex. This cholinergic activation was ipsilaterally reduced by intrabasalis injection of SP (1 ng), indicating that the peptide exerted an inhibitory influence on the neurochemical effect exerted by handling, intracranial needle insertion, and vehicle injection. In the anesthetized preparation, SP had a biphasic dose-dependent action on cortical ACh: a short-lasting ipsilateral increase immediately after injection (especially with 1 ng), and a delayed bilateral increase after more than 2 h (10, 100 ng). The procedure of inserting the injection needle moderately increased cortical ACh levels. Methodologically, these data are discussed with respect to the importance of using anesthetized vs. freely moving rats and the effects of intraparenchymal injections.     

Anxiolytic-like effects of ginseng in the elevated plus-maze model: comparison of red ginseng and sun ginseng

This study was performed to investigate the anxiolytic-like effects of red ginseng (RG, steamed raw ginseng at 98-100 degrees C) and sun ginseng (SG, heat-processed ginseng at higher temperature) in mice using the elevated plus-maze model. Furthermore, the anxiolytic-like effects of RG and SG were compared to a known active anxiolytic drug (diazepam). The RG butanol fraction (100 mg/kg) significantly increased the number of open arms entries and the time spent on the open arm (indicators of anxiolytic-like effects) compared with that of the saline group. However, lower doses of the SG total extract (50 mg/kg) and the SG butanol fraction (25 and 50 mg/kg) significantly increased the number of open arms entries and the time spent on the open arms. The RG total extract (100 mg/kg) and the SG total extract at a lower dose (25 mg/kg) did not increase the number of open arm entries or the time spent on the open arm. On the other hand, the RG butanol fraction (100 mg/kg), the SG total extract (50 mg/kg), and the SG butanol fraction (50 mg/kg) decreased locomotor activity in a manner similar to diazepam. These data indicate that ginseng has anxiolytic-like effects, and the anxiolytic potential of SG is stronger than that of RG in the elevated plus-maze model. Ginseng saponins have been suggested to play an important role in the anxiolytic effects of ginseng. We provide evidence that ginseng may be useful for the treatment of anxiety.     

Co-administration of fluoxetine and Sildenafil has benefits in anxiety behavior in mice

Fluoxetine, a selective serotonin reuptake inhibitor, has been widely prescribed drugs in psychiatry. However, it may produce controversial effects, including sexual dysfunction. Sildenafil, a phosphodiesterase inhibitor type 5, may reduce fluoxetine-induced behavior alterations, but its drug interaction profile has not been investigated. To evaluate the interaction between sildenafil and fluoxetine, we acutely or chronically administered adult male mice with intraperitoneal sildenafil (2 mg/kg); fluoxetine (10 mg/kg); a mixture of fluoxetine (10 mg/kg) and sildenafil (2 mg/kg); the anxiolytic benzodiazepine, diazepam (0.3 mg/kg); or saline, after which the mice were evaluated in an elevated plus-maze. The number of entries into the open arms, the time spent in the open arms, and the numbers of total entries into the arms were determined as measures of anxiety. Acute treatment with fluoxetine produced a behavioral profile consistent with anxiogenesis, while sildenafil produced an anxiolytic-like profile. Co-administration of fluoxetine with sildenafil reversed the anxiogenic effects of the former. In chronic treatment neither of drugs (alone) had any effects compared to controls. Their combination resulted in anxiolytic effect. These findings suggest that the anxiolytic sildenafil masked the effect of fluoxetine in the chronic experiment.     

Neurokinin receptor mRNA localization in human midbrain dopamine neurons

The structurally related neurokinin peptides, substance P and neurokinin A, are found in abundance within the substantia nigra of a variety of mammalian species. Although it has been established recently that the neurokinin-3 (NK3) receptor is the predominant neurokinin receptor found in rat substantia nigra and adjacent midbrain nuclei, the nature of the neurokinin receptor expressed in human midbrain has not been elucidated. In the present study, neurokinin receptor messenger RNA (mRNA) content within rat and human midbrain were directly compared by using quantitative in situ hybridization histochemistry. In contrast to the high abundance of NK3 receptor mRNA within dopamine (DA) cells of the rat midbrain, neurokinin-1 (NK1), but not NK3, receptor mRNA was localized to human midbrain DA cells. Within the human midbrain, the abundance of NK1 receptor mRNA differed significantly among the distinct DA cell-containing nuclei, with the highest level of expression seen in several subdivisions of the substantia nigra. Thus different neurokinin receptor subtypes apparently mediate the effects of substance P and neurokinin A on human versus rat DA neurons. J. Comp. Neurol. 382:394-400, 1997. © 1997 Wiley-Liss, Inc.     

Substance P excites globus pallidus neurons in vivo

Substance P is a member of the neurokinin family. Previous studies have reported the existence of substance P and its high-affinity receptor, neurokinin-1 receptor, in globus pallidus. Employing in vivo extracellular recording combined with behavioural tests, the effects of substance P in globus pallidus of rats were studied. Micropressure ejection of the selective neurokinin-1 receptor agonist [Sar9,Met(O2)11] substance P increased the spontaneous firing rate of pallidal neurons in a concentration-dependent manner, with increases of 27.3% at 0.01, 33.4% at 0.03, 45.5% at 0.1, 38.4% at 0.3 and 36.4% at 1.0 mm. The selective neurokinin-1 receptor antagonist SR140333B prevented the excitatory effects induced by [Sar9,Met(O2)11] substance P. In behaving rats, we observed the postural effects of neurokinin-1 receptor activation in the globus pallidus. Consistent with electrophysiological results, unilateral microinjection of [Sar9,Met(O2)11] substance P (0.1 mm) led to a SR140333B-sensitive contralateral deflection in the presence of systemic haloperidol administration. Combining electrophysiological and behavioural findings, we concluded that substance P produces excitatory effects on globus pallidus neurons via neurokinin-1 receptors.     

Diphenyl diselenide exerts anxiolytic-like effect in Wistar rats: putative roles of GABAA and 5HT receptors

Diphenyl diselenide [(PhSe)2] is an organoselenium compound which presents pharmacological antioxidant, anti-inflammatory, antinociceptive and antidepressant properties. The present study was designed to investigate the anxiolytic effect of (PhSe)2 in rats, employing the elevated plus maze task. The involvement of 5HT and GABA receptors in the anxiolytic-like effect was also evaluated. (PhSe)2 (5, 25 and 50 micromol/kg, i.p.) did not affect locomotor activity as evaluated in the open open-field test, and learning and memory when assessed in the inhibitory foot-shock avoidance task. However, (PhSe)2 at the 50 micromol/kg dose produced signs of an anxiolytic action, namely a decreased number of fecal boli in the open-field arena and an increased time spent in as well as an increased number of entries to the open arms of the elevated plus maze test. To evaluate the role of GABA and 5HT receptors in the anxiolytic-like effect of (PhSe)2, a selective GABAA receptor antagonist bicuculline, (0.75 mg/kg, i.p.), a non-selective 5HT2A/2C receptor antagonist, ritanserin (2 mg/kg, i.p.), a selective 5HT2A receptor antagonist, ketanserin (1 mg/kg, i.p.), and a selective 5HT1A receptor antagonist, WAY100635 (0.1 mg/kg, i.p.) were used. All the antagonists used were able to abolish the anxiolytic effect of (PhSe)2 suggesting that GABAA and 5HT receptors may play a role in the pharmacological property of this selenocompound in the central nervous system.     

The Role of Spinal Neurokinin-2 Receptors in the Processing of Nociceptive Information from the Joint and in the Generation and Maintenance of Inflammation-evoked Hyperexcitability of Dorsal Horn Neurons in the Rat

In spinal cord neurons in anesthetized rats, the role of neurokinin A and neurokinin-2 receptors in the processing of nociceptive information from the knee joint was studied. The specific non-peptide antagonist at the neurokinin-2 receptor, SR48968, its inactive R -enantiomer, SR48965, neurokinin A, substance P and ( R, S )-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), were administered ionophoretically close to neurons with input from the knee joint. SR48968 reduced the effects of exogenous neurokinin A, but not those of exogenous substance P and AMPA, indicating selective blockade of neurokinin-2 receptors. In most neurons with input from the normal knee joint, SR48968 reduced dose-dependently the responses to noxious pressure applied to the knee, and in ˜50% of the neurons the responses to innocuous pressure. The administration of SR48968 during the induction of an experimental joint inflammation markedly attenuated the development of inflammation-evoked hyperexcitability. In hyperexcitable neurons with input from the inflamed joint, SR48968 reduced the responses to noxious and innocuous pressure. The relative reduction of the responses was more pronounced than in neurons with input from the normal joint. None of the effects of SR48968 was mimicked by SR48965. These data show that neurokinin-2 receptors are involved in the spinal processing of nociceptive information from the normal joint. Furthermore, neurokinin-2 receptors must be coactivated at an early stage of inflammation, to allow the generation of hyperexcitability. Finally, neurokinin-2 receptors are involved in the maintenance of hyperexcitability during inflammation. In summary, spinal neurokinin-2 receptors are important in the generation of pain in the normal and inflamed joint.     

Involvement of substance P and central opioid receptors in morphine modulation of the CHS response

Morphine administration prior to challenge with the antigen 2,4-dinitro-fluorobenzene increases the contact hypersensitivity (CHS) response in rats. The present study extended these findings by showing that central, but not systemic, administration of N-methylnaltrexone antagonized the morphine-induced enhancement of the CHS response. The importance of the neuroimmune mediator substance P was shown via the attenuation of the morphine-induced enhancement following both systemic and topical administration of the NK-1 antagonist WIN51,708. Taken together, the findings of the present study provide new data showing that central opioid receptors and peripheral substance P are involved in the morphine-induced enhancement of the CHS response.     

Tachykinin NK(3)receptor involvement in anxiety

This study investigates the effects of intracerebroventricular injection of selective agonists and antagonists of tachykinin NK(3)receptor on performance of mice in the elevated plus-maze test. Mice were treated with either vehicle or 1, 10, 100 or 500 pmol of neurokinin B or senktide ([succinil-Asp(6), MePhe(8)]substance P(6-11), a natural and synthetic selective NK(3)receptor agonists, respectively. Other mice received similar doses of [Trp(7)beta-Ala(8)]NKA(4-10)or SR 142801 ((S)-N-(1-(3-(1-benzoyl-3-(3, 4-dichlorophenyl)-piperidin-3-yl)propyl)-4-phenyl-piperidin- 4-yl)-N-m ethylacetamide) tachykinin NK(3)receptor selective peptide and non-peptide antagonists, respectively. Senktide significantly increased the frequency of entries and the time spent in the open arms, which is compatible with an anxiolytic action. Neurokinin B treatment did not alter the plus-maze parameters in a significant way. Conversely, the NK(3)peptide antagonist [Trp(7)beta-Ala(8)]NKA(4-10), but not SR142801 non-peptide antagonist, showed a reverse effect, i.e. an anxiogenic profile of action, reducing the frequency and the time spent in the open arms. Co-injection of either senktide plus [Trp(7)beta-Ala(8)]NKA((4-10)), or senktide plus SR 142801, blocked the effects promoted by senktide, indicating that centrally-administered NK(3)receptor agonists and antagonists can modulate experimental anxiety.     

The NK1 receptor antagonist NKP608 lacks anxiolytic-like activity in Swiss-Webster mice exposed to the elevated plus-maze

The selective non-peptide NK(1) receptor antagonist NKP608 has been shown to exert potent anxiolytic-like effects in the rat and gerbil social interaction tests. In vitro binding of NKP608 in cortical, striatal and rest-of-brain tissue samples from mice, rats and gerbils indicated comparable pIC(50) values for rats and mice (in all three tissues) and only slightly higher values for gerbils. It would therefore be expected that doses previously found to produce anxiolytic-like effects in rats and gerbils would also be active in mice. The present study evaluated NKP608 in one of the most widely-used animal models of anxiety, the mouse elevated plus-maze. Two consecutive experiments were conducted in which the effects of NKP608 (0.0003-10.0 mg/kg, p.o.) were compared to those produced by the prototypical benzodiazepine anxiolytic, chlordiazepoxide (CDP, 15 mg/kg, p.o.). Ethological scoring methods were used to provide comprehensive behavioural profiles for each compound. In both experiments, acute CDP treatment resulted in significant anxioselective effects, i.e., reductions in measures of open arm avoidance without any alteration in general activity levels (closed arm entries and rearing). Although the results of Experiment 1 (0.001-10.0 mg/kg NKP608) suggested a weak anxiolytic-like action of NKP608 at 0.001 mg/kg (significant increase in percent open arm entries), Experiment 2 failed both to replicate this effect or to find any behavioural activity at lower (0.0003 mg/kg) or higher (0.03 mg/kg) doses. Present findings suggest that the anxiolytic efficacy of this NK(1) receptor antagonist may be test-specific and thus limited to particular subtypes of anxiety. These new data are also discussed in relation to the general difficulty of relating the behavioural profiles of NK(1) receptor antagonists to their potency at NK(1) receptors.     

The effects of CRA 1000, a non-peptide antagonist of corticotropin-releasing factor receptor type 1, on adaptive behaviour in the rat

Intracerebrally administered CRF has been demonstrated to elicit several behavioural deficits in novel and potentially stressful experimental paradigms, and to promote activity in familiar situations. This study examined the effect of CRA 1000, a novel non-peptide antagonist of CRF(1)receptors, on rat behaviour in tests of anxiolytic and antidepressant activity and novelty-oriented behaviour. CRA 1000 (1.25-10 mg/kg) had no major effect in elevated plus-maze and social interaction tests. However, CRA 1000 (5 mg/kg) significantly reduced immobility in the forced swimming test, suggesting an antidepressant-like effect. In the exploration box test, CRA 1000 (1.25 mg/kg) had an anxiolytic effect on rat exploratory behaviour both in intact rats and after lesioning of the projections of locus coeruleus by DSP-4 (50 mg/kg) treatment. A higher dose of CRA 1000 (5 mg/kg) tended to have anxiolytic-like effects in DSP-4 pretreated rats, but in intact animals this dose prevented the increase in exploration which develops with repeated exposure to initially anxiety-provoking situations. Taken together, these experiments demonstrate that CRF1 receptor blockade by CRA 1000 has antidepressant-like effects, does not have a robust anti-anxiety effect in non-stressed animals, but does have anxiolytic-like effects in more complex tasks, which can be observed also after denervation of the locus coeruleus projections. However, large doses of CRF1 receptor antagonists may reduce motivation of exploratory behaviour in familiar environments.     

Anxiolytic-like behavior in rats is induced by the neonatal intracranial injection of apotransferrin

To determine whether neonatal intracranial injection of apotransferrin (aTf), which increases myelin deposition, has behavioral effects in rats, 3-day-old rats were intracranially injected with 350 ng of aTf and tested at 25 and 60 days of age. An anxiolytic-like behavior was observed in aTf-treated rats, evidenced by an increase in the exploration of open arms in the plus maze test without changes in the locomotor activity. This behavioral profile persists until adulthood. Intraperitoneal injection of 0.75 mg/kg of picrotoxin, a GABA(A) receptor channel antagonist, abolished this anxiolytic-like behavior, indicating that neonatal aTf induces a long-lasting increase in GABA(A) receptor functionality.     

Substance P fragments and striatal endogenous dopamine outflow: interaction with substance P

Accumulating evidence shows that N- and C-terminal substance P fragments have significant biological activity. Substance P(1–9) and substance P(6–11) have been reported to be major substance P metabolites in rat striatum. We investigated the effects of these fragments on endogenous dopamine outflow in rat striatal slices. Substance P-(1–9) and substance P-(6–11) induced a significant increase in dopamine outflow at 0.1 and 1 nM. The effects of substance P-(6–11) (1 nM) were reversed by the tachykinin NK₁ antagonist WIN 51,708 (17β-hydroxy-17α-ethynyl-5α-androstano[3,2-b]pyrimido[1,2-a]benzimidazole) (2.5 nM), whereas the effects of substance P-(1–9) were not modified by the antagonist. Substance P-(1–9) and substance P-(6–11) (1 nM) did not increase the dopamine overflow induced by 25 mM KCl. The effects of the two fragments were reversed by the muscarinic antagonist atropine (1 μM) but not by nicotinic antagonists dihydro-β-erythroidine (0.5 μM) and pempidine (10 μM). The co-incubation of tissue with substance P and each fragment in a 1/1 or 10/1 ratio of substance P to metabolite revealed a negative interaction between parent and fragments. A similar pattern was observed when substance P was co-administered with the active fragments substance P(1–4), substance P(1–7), substance P(5–11) and substance P(8–11). The data show that substance P-(1–9) and substance P-(6–11) have modulatory effects similar to substance P. However, the presence of active substance P metabolites does not appear to amplify the signal mediated by the parent peptide.     

Participation of GABAA receptors in the modulation of experimental anxiety by tachykinin agonists and antagonists in mice

Mice were acutely intraperitoneally treated with diazepam (DZP), pentylenetetrazol (PTZ) or NaCl 0.9% (control group), and 15 min later, the DZP-treated group received substance P (SP), neurokinin A (NKA; NK1 and NK2 natural preferential agonists), [Trp7 beta-Ala8] NKA(4-10) (Trp-7; NK3 antagonist) or vehicle intracerebroventricularly, whereas the PTZ-treated group was intracerebroventricularly administered with FK 888, SR 48968 (NK1 and NK2 antagonists, respectively) or senktide (SENK--[succinil-Asp6, MePhe8] substance P(6-11); NK3 agonist), or vehicle immediately before they were submitted to the elevated plus-maze (EPM) test. Another group of animals was repeatedly treated with increasing doses of DZP or NaCl 0.9% intraperitoneally for 28 days, and 3 days after the last injection (test day), animals received DZP, FK 888, SR 48968, SENK or vehicle intracerebroventricularly, or DZP (NaCl 0.9%) intraperitoneally before the EPM evaluation. The anxiolytic action of the acute treatment with DZP was inhibited by the central administration of NKA and Trp-7 but not by SP. NK1 and NK2 antagonists, but not NK3 agonist, blocked the anxiogenic action of PTZ, as evaluated in the plus-maze test. Flumazenil (FLM), a benzodiazepine antagonist, was not able to inhibit the anxiolytic profile of action induced by the NK2 antagonist. Central administration of FK 888 and SR 48968 promoted anxiolytic effects in both control and DZP-withdrawn animals, suggesting a clear relationship between the GABAergic and the tachykinergic systems, mostly involving NK1 and NK2 receptors, in the modulation of experimental anxiety in mice.     

The paraventricular nucleus of the hypothalamus is a neuroanatomical substrate for the inhibition of palatable food intake by neuropeptide S

Neuropeptide S (NPS) is a recently discovered neurotransmitter that binds to its cognate G-protein coupled receptor, NPSR. Previous studies have shown that central administration of this peptide induces anxiolytic-like effects, promotes arousal and inhibits feeding in the same dose range. In the present study, we sought to investigate further the unique physiopharmacological profile of the NPS system by characterizing its effects on palatable food consumption in rats and comparing it with the effect of the classical anxiolytic benzodiazepine midazolam. The results demonstrated that midazolam (5.0 or 10.0 mg/kg) increases palatable food consumption, while intracerebroventricular (ICV) administration of NPS markedly reduces it. The anorectic effect of NPS (0.1–1.0 nmol per rat, ICV) was prevented by ICV pretreatment with the NPSR antagonist [ d -Cys(tBU)⁵]NPS (20.0–60.0 nmol per rat). Pretreatment with the nonselective corticotrophin-releasing factor receptor (CRF) antagonist alpha-helical CRF 9–41 (6.25 and 12.5 nmol per rat) completely reversed the hypophagic action of CRF (0.4 nmol per rat, ICV) but did not prevent the anorectic effect of ICV NPS (1.0 nmol per rat). Brain site-specific microinjection experiments revealed that NPS markedly inhibits palatable food intake if administered into the paraventricular nucleus of the hypothalamus (PVN). A similar but smaller and shorter lasting reduction of feeding was observed following intra-lateral hypothalamus administration, whereas no effect was observed following injection into the central amygdala. The present study demonstrates that NPS evokes a potent inhibition of palatable food consumption and that the PVN is an important site of action for its effect.     

Evidence for a potential role for TRPV1 receptors in the dorsolateral periaqueductal gray in the attenuation of the anxiolytic effects of cannabinoids

Several studies have shown anxiolytic effects of cannabinoids after systemic or central injections. The periaqueductal gray matter is a midbrain structure involved in the control of anxiety states. Intra-cerebral administration of cannabidiol, a phytocannabinoid, or anandamide, an endocannabinoid, into the dorsolateral portion of periaqueductal gray (dlPAG) promotes anxiolytic-like effects in several animal models of anxiety with bell-shaped dose–response curves. The reasons for these curves are still unclear, but since these drugs can also activate TRPV1 receptors and increase glutamate release, we hypothesized that, at high doses, cannabidiol and WIN 55,212-2, a CB1 receptor agonist, could activate TRPV1 receptors, facilitating glutamate neurotransmission and anxiety responses. To test this hypothesis male Wistar rats with cannulae aimed toward the dlPAG were submitted to the following intra-dlPAG treatments: Experiment 1. Vehicle (0.2 µL) or WIN 55,212-2 (3–30 pmol); Experiment 2. Capsazepine (CPZ, 10 nmol, a TRPV1 receptor antagonist) or vehicle followed, 5 min later, by vehicle or WIN 55, 212-2 (10 or 30 pmol); Experiment 3. CPZ (10 nmol) or vehicle followed, 5 min later, by cannabidiol (30 or 60 nmol). Ten minutes after the last injection the animals were tested in the elevated plus maze (EPM). WIN 55,212-2 and cannabidiol induced anxiolytic effects at lower doses that disappeared at the higher dose. Although CPZ + WIN 10 or CPZ + WIN 30 pmol groups were not different from control (CPZ + V), capsazepine prevented the decrease in open arm exploration caused by the higher of dose of WIN 55,212-2 (30 nmol) relative to the lower dose of WIN 55,212-2 (10 nmol) and, in the case of cannabidiol (60 nmol), increased open arm exploration (V + CBD 60 group versus CPZ + CBD 60 group). These results suggest that TRPV1 receptors in the dlPAG modulate anxiety and that activation of these receptors by high doses of cannabinoids could be involved in the bell-shaped dose–response curves observed with these compounds.     

Involvement of NMDA receptor complex in the anxiolytic-like effects of chlordiazepoxide in mice

In the present study, we demonstrated that low, ineffective doses of N-methyl-d-aspartic acid (NMDA) receptor antagonists [competitive NMDA antagonist, CGP 37849, at 0.312 mg/kg intraperitoneally (i.p.), antagonist of the glycine_B sites, L-701,324, at 2 mg/kg i.p., partial agonist of glycine_B sites, d-cycloserine, at 2.5 mg/kg i.p.] administered jointly with an ineffective dose of the benzodiazepine, chlordiazepoxide (CDP, 2.5 mg/kg i.p.), significantly increased the percentage of time spent in the open arms of the elevated plus-maze (index of anxiolytic effect). Furthermore, CDP-induced anxiolytic-like activity (5 mg/kg i.p.) was antagonized by NMDA (75 mg/kg i.p.) and by an agonist of glycine_B sites of the NMDA receptor complex, d-serine [100 nmol/mouse intracerebroventricularly (i.c.v.)]. The present study showed a positive interaction between γ-aminobutyric acid (GABA) and glutamate neurotransmission in the anxiolytic-like activity in the elevated plus-maze test in mice and this activity seems to particularly involve the NMDA receptors.