Hypothalamic Paraventricular Stimulation Inhibits Nociceptive Wide Dynamic Range Trigeminocervical Complex Cells via Oxytocinergic Transmission.

Oxytocinergic transmission blocks nociception at the peripheral, spinal, and supraspinal levels through the oxytocin receptor (OTR). Indeed, a neuronal pathway from the hypothalamic paraventricular nucleus (PVN) to the spinal cord and trigeminal nucleus caudalis (Sp5c) has been described. Hence, although the trigeminocervical complex (TCC), an anatomical area spanning the Sp5c, C1, and C2 regions, plays a role in some pain disorders associated with craniofacial structures (e.g., migraine), the role of oxytocinergic transmission in modulating nociception at this level has been poorly explored. Hence, in vivo electrophysiological recordings of TCC wide dynamic range (WDR) cells sensitive to stimulation of the periorbital or meningeal region were performed in male Wistar rats. PVN electrical stimulation diminished the neuronal firing evoked by periorbital or meningeal electrical stimulation; this inhibition was reversed by OTR antagonists administered locally. Accordingly, neuronal projections (using Fluoro-Ruby) from the PVN to the WDR cells filled with Neurobiotin were observed. Moreover, colocalization between OTR and calcitonin gene-related peptide (CGRP) or OTR and GABA was found near Neurobiotin-filled WDR cells. Retrograde neuronal tracers deposited at the meningeal (True-Blue, TB) and infraorbital nerves (Fluoro-Gold, FG) showed that at the trigeminal ganglion (TG), some cells were immunopositive to both fluorophores, suggesting that some TG cells send projections via the V1 and V2 trigeminal branches. Together, these data may imply that endogenous oxytocinergic transmission inhibits the nociceptive activity of second-order neurons via OTR activation in CGRPergic (primary afferent fibers) and GABAergic cells.

Clavulanic Acid and its Potential Therapeutic Effects on the Central Nervous System.

Clavulanic acid (CLAV) is a non-antibiotic ß-lactam that has been used since the late 1970s as a ß-lactamase inhibitor in combination with amoxicillin, another ß-lactam with antibiotic activity. Its long-observed adverse reaction profile allows it to say that CLAV is a well-tolerated drug with mainly mild adverse reactions. Interestingly, in 2005, it was discovered that ß-lactams enhance the astrocytic expression of GLT-1, a glutamate transporter essential for maintaining synaptic glutamate homeostasis involved in several pathologies of the central nervous system (CNS). This finding, along with a favorable pharmacokinetic profile, prompted the appearance of several studies that intended to evaluate the effect of CLAV in preclinical disease models. Studies have revealed that CLAV can increase GLT-1 expression in the nucleus accumbens (NAcc), medial prefrontal cortex (PFC), and spinal cord of rodents, to affect glutamate and dopaminergic neurotransmission, and exert an anti-inflammatory effect by modulating the levels of the cytokines TNF-α and interleukin 10 (IL-10). CLAV has been tested with positive results in preclinical models of epilepsy, addiction, stroke, neuropathic and inflammatory pain, dementia, Parkinson's disease, and sexual and anxiety behavior. These properties make CLAV a potential therapeutic drug if repurposed. Therefore, this review aims to gather information on CLAV's effect on preclinical neurological disease models and to give some perspectives on its potential therapeutic use in some diseases of the CNS.

Serotonergic Modulation of Neurovascular Transmission: A Focus on Prejunctional 5-HT Receptors/Mechanisms.

5-Hydroxytryptamine (5-HT), or serotonin, plays a crucial role as a neuromodulator and/or neurotransmitter of several nervous system functions. Its actions are complex, and depend on multiple factors, including the type of effector or receptor activated. Briefly, 5-HT can activate: (i) metabotropic (G-protein-coupled) receptors to promote inhibition (5-HT1, 5-HT5) or activation (5-HT4, 5-HT6, 5-HT7) of adenylate cyclase, as well as activation (5-HT2) of phospholipase C; and (ii) ionotropic receptor (5-HT3), a ligand-gated Na+/K+ channel. Regarding blood pressure regulation (and beyond the intricacy of central 5-HT effects), this monoamine also exerts direct postjunctional (on vascular smooth muscle and endothelium) or indirect prejunctional (on autonomic and sensory perivascular nerves) effects. At the prejunctional level, 5-HT can facilitate or preclude the release of autonomic (e.g., noradrenaline and acetylcholine) or sensory (e.g., calcitonin gene-related peptide) neurotransmitters facilitating hypertensive or hypotensive effects. Hence, we cannot formulate a specific impact of 5-HT on blood pressure level, since an increase or decrease in neurotransmitter release would be favoured, depending on the type of prejunctional receptor involved. This review summarizes and discusses the current knowledge on the prejunctional mechanisms involved in blood pressure regulation by 5-HT and its impact on some vascular-related diseases.

The role of the endocannabinoid 2-arachidonoylglycerol in the in vivo spinal oxytocin-induced antinociception in male rats.

Oxytocin receptor (OTR) activation at the spinal level produces antinociception. Some data suggest that central OTR activation enhances social interaction via an increase of endocannabinoids (eCB), but we do not know if this could occur at the spinal level, modulating pain transmission. Considering that oxytocin via OTR stimulates diacylglycerol formation, a key intermediate in synthesizing 2-arachidonylglycerol (2-AG), an eCB molecule, we sought to test the role of the eCB system on the spinal oxytocin-induced antinociception. Behavioral and electrophysiological experiments were conducted in naïve and formalin-treated (to induce long-term mechanical hypersensitivity) male Wistar rats. Intrathecal RHC 80267 injections, an inhibitor of the enzyme diacylglycerol lipase (thus, decreasing 2-AG formation), produces transient mechanical hypersensitivity, an effect unaltered by oxytocin but reversed by gabapentin. Similarly, in in vivo extracellular recordings of naïve spinal wide dynamic range cells, juxtacellular picoinjection of RHC 80267 increases the firing of nociceptive Aδ-, C-fibers, and post-discharge, an effect unaltered by oxytocin. Interestingly, in sensitized rats, oxytocin picoinjection reverses the RHC 80627-induced hyperactivity of Aδ-fibers (but not C- or post-discharge activity). In contrast, a sub-effective dose of JZL184 (a monoacylglycerol lipase inhibitor, thus favoring 2-AG levels), which does not have per se an antinociceptive effect in the formalin-induced hypernociception, the oxytocin-induced antinociception is boosted. Similarly, electrophysiological experiments suggest that juxtacellular JZL184 diminishes the neuronal firing of nociceptive fibers, and co-injection with oxytocin prolongs and enhances the antinociceptive effect. These data may imply that 2-AG formation may play a role in the spinal antinociception induced by oxytocin.

The differential in vivo contribution of spinal α2A- and α2C-adrenoceptors in tonic and acute evoked nociception in the rat.

Spinal α2-adrenoceptor induces analgesia by neuronal inhibition of primary afferent fibers. This family receptor coupled to G i/o proteins can be subdivided into three functional subtypes: α2A, α2B, and α2C-adrenoceptors, and current evidence on spinal analgesia supports the relevance of α2A and seems to exclude the role of α2B, but the functional contribution of α2C-adrenoceptors remains elusive. The present study was designed to pharmacologically dissect the contribution of spinal α2-adrenoceptor subtypes modulating tonic or acute peripheral nociception. Using male Wistar rats, we analyzed the effect of spinal clonidine (a non-selective α2A/α2B/α2C-adrenoceptor agonist) and/or selective subtype α2-adrenoceptor antagonists on: 1) tonic nociception induced by subcutaneous formalin (flinching behavior) or 2) acute nociception induced by peripheral electrical stimulus in in vivo extracellular recordings of spinal dorsal horn second-order wide dynamic range (WDR) neurons. Clonidine inhibited the nocifensive behavior induced by formalin, an effect blocked by BRL 44408 (α2A-adrenoceptor antagonist) but not by imiloxan (α2B-adrenoceptor antagonist) or JP 1302 (α2C-adrenoceptor antagonist). Similarly, spinal BRL 44408 reversed the clonidine-induced inhibition of nociceptive WDR activity. Interestingly, spinal JP 1302 per se produced behavioral antinociception (an effect blocked by bicuculline, a preferent GABAA channel blocker), but no correlation was found with the electrophysiological experiments. These data imply that, at the spinal level, 1) presynaptic α2A-adrenoceptor activation produces antinociception during acute or tonic nociceptive stimuli; and 2) under tonic nociceptive (inflammatory) input, spinal α2C-adrenoceptors are pronociceptive, probably by the inactivation of GABAergic transmission. This result supports a differential role of α2A and α2C-adrenoceptors modulating nociception.

The glial cell's role in antinociceptive differential effects of oxytocin upon female and male rats.

BACKGROUND: Sex plays a crucial role in pain processing and response to analgesic drugs. Indeed, spinal glia seems to be significant in the sexual dimorphism observed in the above effects. Recently, studies have associated oxytocin with antinociceptive effects, but these have been mainly performed in male animals; consequently, the influence of sex has been poorly explored. METHODS: Using a model of spinal nociception that produces pain through activation of the spinal glia, that is, intrathecal (i.t.) lipopolysaccharide (LPS) injection, we analysed the changes in the analgesic response to i.t. oxytocin in female and male rats by behavioural (punctate mechanical hypersensitivity), electrophysiological (unitary extracellular recordings of wide dynamic range [WDR] cells) and molecular biology (real-time PCR of proinflammatory genes) experiments. RESULTS: We found that LPS-induced hypersensitivity was longer in female (>96 h) than in male (≈4 h) rats. Besides, spinal oxytocin preferentially prevents the LPS-induced hypersensitivity in male rather than female rats. Indeed, LPS increases the spinal neuronal-evoked activity associated with the activation of peripheral Aδ- and C-fibres and post-discharge in males, whereas only C-fibre discharge was enhanced in females. The electrophysiological data correlate with the fact that spinal oxytocin only prevented TNF-α and IL-1ß synthesis in male rats. CONCLUSIONS: Therefore, these data suggest that oxytocin-mediated analgesia depends on a sexual dimorphism involving activation of the spinal glia. These results reinforced the idea that different strategies are required to treat pain in men and women, and that oxytocin could be used preferentially to treat pain with a significant inflammatory component in men. SIGNIFICANCE STATEMENT: Oxytocin is a molecule that emerges as a potent analgesic in preclinical and clinical studies. We investigated the contribution of glia to the response of oxytocin-induced analgesia and how sex influences in this response show that different strategies are required to treat pain in men and women, and that oxytocin could be used preferentially to treat pain with a significant inflammatory component in men.

Ultrastructural Evidence for Oxytocin and Oxytocin Receptor at the Spinal Dorsal Horn: Mechanism of Nociception Modulation.

Oxytocin is a hypothalamic neuropeptide involved in the inhibition of nociception transmission at spinal dorsal horn (SDH) level (the first station where the incoming peripheral signals is modulated). Electrophysiological, behavioral, and pharmacological data strongly support the role of this neuropeptide and its receptor (the oxytocin receptor, OTR) as a key endogenous molecule with analgesic properties. Briefly, current data showed that oxytocin release from the hypothalamus induces OTR activation at the SDH, inducing selective inhibition of the nociceptive Aδ- and C-fibers (probably peptidergic) activity, but not the activity of proprioceptive fibers (i.e. Aß-fibers). The above inhibition could be a direct presynaptic mechanism, or a mechanism mediated by GABAergic interneurons. However, the exact anatomical localization of oxytocin and OTR remains unclear. In this context, the present study set out to analyze the role of OTRs, GABAergic cells and CGRP fibers in the SDH in rats by using electron microscopy. Ultrastructural analyses of the SDH tissue show that: (i) oxytocin and OTR are found in asymmetrical synapsis; (ii) OTR is found in GABAergic interneurons (near unmyelinated fibers), CGRPergic fibers and glial cells; (iii) whereas oxytocin is present in supraspinal descending projection fibers. These anatomical data strongly support the notion that oxytocin released at the SDH could presynaptically inhibit the nociceptive input from the peripheral primary afferent fibers. This inhibitory action could be direct or use a GABA interneuron. Furthermore, our findings that OTR is exhibited in glial tissue at the SDH requires further exploration in nociception assays.

The impact of CGRPergic monoclonal antibodies on prophylactic antimigraine therapy and potential adverse events.

Migraine is a prevalent medical condition and the second most disabling neurological disorder. Regarding its pathophysiology, calcitonin gene-related peptide (CGRP) plays a key role, and, consequently, specific antimigraine pharmacotherapy has been designed to target this system. Hence, apart from the gepants, the recently developed monoclonal antibodies (mAbs) are a novel approach to treat this disorder. In this review we consider the current knowledge on the mechanisms of action, specificity, safety, and efficacy of the above mAbs as prophylactic antimigraine agents, and examine the possible adverse events that these agents may trigger. Antimigraine mAbs act as direct scavengers of CGRP (galcanezumab, fremanezumab, and eptinezumab) or against the CGRP receptor (erenumab). Due to their long half-lives, these molecules have revolutionized the prophylactic treatment of this neurovascular disorder. Moreover, because of their physicochemical properties, these agents are hepato-friendly and do not cross the blood-brain barrier (highlighting the relevance of peripheral mechanisms in migraine). Nevertheless, apart from potential cardiovascular side effects, the interaction with AMY1 receptors and immunogenicity induced by autoantibodies against mAbs could be a concern for the safety of long-term treatment with these molecules.

In Vivo Dissection of Two Intracellular Pathways Involved in the Spinal Oxytocin-Induced Antinociception in the Rat.

Behavioral and electrophysiological data show that at the spinal level, oxytocin inhibits pain transmission by activation of oxytocin receptors (OTRs). Canonically, OTRs are coupled to Gq proteins, which induce a rise of intracellular Ca2+ by activating the phospholipase C (PLC). However, in vitro data showed that OTRs cause a plethora of intracellular events, some related to the activation of Gi proteins. Using a behavioral approach, we analyzed the main in vivo intracellular pathway elicited by spinal oxytocin during a peripheral inflammatory/persistent nociceptive stimulus. Intrathecal oxytocin reduces early (number of flinches) and late (mechanical allodynia) formalin-induced nociception, an effect abolished by the OTR antagonist (L-368,899). Furthermore, the antinociception observed during the early phase (acute inflammatory) was also reverted by U-73122 (PLC inhibitor) but not by pertussis toxin (Gαi/o protein inhibitor) or gallein (Gßγ subunit inhibitor). In contrast, the late oxytocin-induced behavioral analgesia was blocked by pertussis and gallein but not by U-73122. Since oxytocin's effects during the early phase were also antagonized by Nω-nitro-l-arginine methyl ester, ODQ, or glibenclamide (inhibitors of nitric oxide synthase [NOS], soluble guanylyl cyclase [GC], and K+ATP channels, respectively), the role of two differential pathways elicited by oxytocin is supported. Hence, we showed in in vivo experiments that oxytocin recruits two differential spinal intracellular pathways mediated by Gq (PLC/NOS/GC/K+ATP) or Gi proteins during a peripheral nociceptive stimulus.

A critical review of the neurovascular nature of migraine and the main mechanisms of action of prophylactic antimigraine medications.

INTRODUCTION: Migraine involves neurovascular, functional, and anatomical alterations. Migraineurs experience an intense unilateral and pulsatile headache frequently accompanied with vomiting, nausea, photophobia, etc. Although there is no ideal preventive medication, frequency in migraine days may be partially decreased by some prophylactics, including antihypertensives, antidepressants, antiepileptics, and CGRPergic inhibitors. However, the mechanisms of action involved in antimigraine prophylaxis remain elusive. AREAS COVERED: This review recaps some of the main neurovascular phenomena related to migraine and currently available preventive medications. Moreover, it discusses the major mechanisms of action of the recommended prophylactic medications. EXPERT OPINION: In the last three years, migraine prophylaxis has evolved from nonspecific to specific antimigraine treatments. Overall, nonspecific treatments  mainly involve neural actions, whereas specific pharmacotherapy (represented by CGRP receptor antagonists and CGRPergic monoclonal antibodies) is predominantly mediated by neurovascular mechanisms that may include, among others: (i) reduction in the cortical spreading depression (CSD)-associated events; (ii) inhibition of pain sensitization; (iii) blockade of neurogenic inflammation; and/or (iv) increase in cranial vascular tone. Accordingly, the novel antimigraine prophylaxis promises to be more effective, devoid of significant adverse effects (unlike nonspecific treatments), and more beneficial for the quality of life of migraineurs.

An outlook on the trigeminovascular mechanisms of action and side effects concerns of some potential neuropeptidergic antimigraine therapies.

Introduction: In addition to serotonin (5-hydroxytryptamine; 5-HT) and other (neuro)mediators, the role of neuropeptides in migraine pathophysiology is relevant. Indeed, while some molecules interfering with calcitonin gene-related peptide (CGRP) transmission have recently been approved for clinical antimigraine use, other neuropeptides with translational use are in the pipeline. Among others, hypothalamic neuropeptides such as pituitary adenylate cyclase-activating peptide (PACAP), oxytocin (OT), and orexins stand out as potential novel targets to treat this neurovascular disorder. Areas covered: Based on the aforementioned findings, the present review: (i) summarizes the current knowledge on the role of the above neuropeptides in the trigeminovascular system, and migraine pathophysiology; and (ii) discusses some issues related with the mechanisms of action and side effects concerns that could be elicited when targeting the CGRPergic, PACAPergic, oxytocinergic and orexinergic systems. Expert opinion: Specific antimigraine pharmacotherapies have evolved from the enhancement of serotonergic 5-HT1B/1D/1F transmission to the use of compounds interacting with neuropeptidergic systems. Canonically, neuropeptides cause an array of complex intracellular mechanisms that, after modifying neuronal and/or vascular transmission, result in antimigraine action and also potential side effects. Furthermore, due to the chemical nature of some molecules targeting the above neuropeptidergic transmission (e.g., monoclonal antibodies, peptides), there are some limiting pharmacokinetics issues.

Cortical Modulation of Nociception.

Nociception is the neuronal process of encoding noxious stimuli and could be modulated at peripheral, spinal, brainstem, and cortical levels. At cortical levels, several areas including the anterior cingulate cortex (ACC), prefrontal cortex (PFC), ventrolateral orbital cortex (VLO), insular cortex (IC), motor cortex (MC), and somatosensory cortices are involved in nociception modulation through two main mechanisms: (i) a descending modulatory effect at spinal level by direct corticospinal projections or mostly by activation of brainstem structures (i.e. periaqueductal grey matter (PAG), locus coeruleus (LC), the nucleus of raphe (RM) and rostroventral medulla (RVM)); and by (ii) cortico-cortical or cortico-subcortical interactions. This review summarizes evidence related to the participation of the aforementioned cortical areas in nociception modulation and different neurotransmitters or neuromodulators that have been studied in each area. Besides, we point out the importance of considering intracortical neuronal populations and receptors expression, as well as, nociception-induced cortical changes, both functional and connectional, to better understand this modulatory effect. Finally, we discuss the possible mechanisms that could potentiate the use of cortical stimulation as a promising procedure in pain alleviation.

CLARITY with neuronal tracing and immunofluorescence to study the somatosensory system in rats.

BACKGROUND: The CLARITY technique enables researchers to visualize different neuronal connections along the nervous system including the somatosensory system. NEW METHOD: The present work describes the antero-lateral and dorsal column pathways until the thalamic and cortical stations, as well as descending oxytocinergic and vasopressinergic innervations by means of combined CLARITY, neuronal tracing, and immunofluorescence techniques. We used male Sprague-Dawley rats of 13, 30, and 60 days. RESULTS: The main results are as follows: A) CLARITY is a reliable technique that can be combined with fluorescent neuronal tracers and immunofluorescence techniques without major procedure modifications; B) at spinal level, some primary afferent fibers were labeled by CGRP, as well as the presence of neuronal populations that simultaneously project to the gracile and ventral posterolateral thalamic nuclei; C) corticothalamic connections were visible when retrograde tracers were injected at thalamic level; D) oxytocin receptors were expressed in the spinal dorsal horn by GABAergic-positive neurons, reinforcing previous outcomes about the possible mechanism for oxytocin blocking the primary afferent sensory input. COMPARISON WITH EXISTING METHODS AND CONCLUSIONS: The CLARITY technique lets us observe in a transparent way the entire processed tissue compared with classical histological methods. CLARITY is a potentially useful tool to describe neuroanatomical structures and their neurochemical stratus.

Intrathecal Oxytocin Improves Spontaneous Behavior and Reduces Mechanical Hypersensitivity in a Rat Model of Postoperative Pain.

The first few days post-surgery, patients experience intense pain, hypersensitivity and consequently tend to have minor locomotor activity to avoid pain. Certainly, injury to peripheral tissues produces pain and increases sensitivity to painful (hyperalgesia) and non-painful (allodynia) stimuli. In this regard, preemptive pharmacological treatments to avoid or diminish pain after surgery are relevant. Recent data suggest that the neuropeptide oxytocin when given at spinal cord level could be a molecule with potential preemptive analgesic effects, but this hypothesis has not been properly tested. Using a validated postoperative pain model (i.e. plantar incision), we evaluated in male Wistar rats the potential preemptive antinociceptive effects of intrathecal oxytocin administration measuring tactile hypersensitivity (across 8 days) and spontaneous motor activity (across 3 days). Hypersensitivity was evaluated using von Frey filaments, whereas spontaneous activity (total distance, vertical activity episodes, and time spent in the center of the box) was assessed in real time using a semiautomated open-field system. Under these conditions, we found that animals pretreated with spinal oxytocin before plantar incision showed a diminution of hypersensitivity and an improvement of spontaneous behavior (particularly total distance and vertical activity episodes). This report provides a basis for addressing the therapeutic relevance of oxytocin as a potential preemptive analgesic molecule.

The locus of Action of CGRPergic Monoclonal Antibodies Against Migraine: Peripheral Over Central Mechanisms.

Migraine is a complex neurovascular disorder characterized by attacks of moderate to severe unilateral headache, accompanied by photophobia among other neurological signs. Although an arsenal of antimigraine agents is currently available in the market, not all patients respond to them. As Calcitonin Gene-Related Peptide (CGRP) plays a key role in the pathophysiology of migraine, CGRP receptor antagonists (gepants) have been developed. Unfortunately, further pharmaceutical development (for olcegepant and telcagepant) was interrupted due to pharmacokinetic issues observed during the Randomized Clinical Trials (RCT). On this basis, the use of monoclonal antibodies (mAbs; immunoglobulins) against CGRP or its receptor has recently emerged as a novel pharmacotherapy to treat migraines. RCT showed that these mAbs are effective against migraines producing fewer adverse events. Presently, the U.S. Food and Drug Administration approved four mAbs, namely: (i) erenumab; (ii) fremanezumab; (iii) galcanezumab; and (iv) eptinezumab. In general, specific antimigraine compounds exert their action in the trigeminovascular system, but the locus of action (peripheral vs. central) of the mAbs remains elusive. Since these mAbs have a molecular weight of ∼150 kDa, some studies rule out the relevance of their central actions as they seem unlikely to cross the Blood-Brain Barrier (BBB). Considering the therapeutic relevance of this new class of antimigraine compounds, the present review has attempted to summarize and discuss the current evidence on the probable sites of action of these mAbs.

Oxytocin prevents neuronal network pain-related changes on spinal cord dorsal horn in vitro.

Recently, oxytocin (OT) has been studied as a potential modulator of endogenous analgesia by acting upon pain circuits at the spinal cord and supraspinal levels. Yet the detailed action mechanisms of OT are still undetermined. The present study aimed to evaluate the action of OT in the spinal cord dorsal horn network under nociceptive-like conditions induced by the activation of the N-methyl-d-aspartate (NMDA) receptor and formalin injection, using calcium imaging techniques. Results demonstrate that the spontaneous Ca2+-dependent activity of the dorsal horn cells was scarce, and the coactivity of cells was mainly absent. When NMDA was applied, high rates of activity and coactivity occurred in the dorsal horn cells; these rates of high activity mimicked the activity dynamics evoked by a neuropathic pain condition. In addition, although OT treatment increased activity rates, it was also capable of disrupting the conformation of coordinated activity previously consolidated by NMDA treatment, without showing any effect by itself. Altogether, our results suggest that OT globally prevents the formation of coordinated patterns previously generated by nociceptive-like conditions on dorsal horn cells by NMDA application, which supports previous evidence showing that OT represents a potential therapeutic alternative for the treatment of chronic neuropathic pain.

The Rostral Agranular Insular Cortex, a New Site of Oxytocin to Induce Antinociception.

The rostral agranular insular cortex (RAIC) is a relevant structure in nociception. Indeed, recruitment of GABAergic activity in RAIC promotes the disinhibition of the locus ceruleus, which in turn inhibits (by noradrenergic action) the peripheral nociceptive input at the spinal cord level. In this regard, at the cortical level, oxytocin can modulate the GABAergic transmission; consequently, an interaction modulating nociception could exist between oxytocin and GABA at RAIC. Here, we tested in male Wistar rats the effect of oxytocin microinjection into RAIC during an inflammatory (by subcutaneous peripheral injection of formalin) nociceptive input. Oxytocin microinjection produces a diminution of (1) flinches induced by formalin and (2) spontaneous firing of spinal wide dynamic range cells. The above antinociceptive effect was abolished by microinjection (at RAIC) of the following: (1) L-368899 (an oxytocin receptor [OTR] antagonist) or by (2) bicuculline (a preferent GABAA receptor blocker), suggesting a GABAergic activation induced by OTR. Since intrathecal injection of an α2A-adrenoceptor antagonist (BRL 44408) partially reversed the oxytocin effect, a descending noradrenergic antinociception is suggested. Further, injection of L-368899 per se induces a pronociceptive behavioral effect, suggesting a tonic endogenous oxytocin release during inflammatory nociceptive input. Accordingly, we found bilateral projections from the paraventricular nucleus of the hypothalamus (PVN) to RAIC. Some of the PVN-projecting cells are oxytocinergic and destinate GABAergic and OTR-expressing cells inside RAIC. Aside from the direct anatomic link between PVN and RAIC, our findings provide evidence about the role of oxytocinergic mechanisms modulating the pain process at the RAIC level.SIGNIFICANCE STATEMENT Oxytocin is a neuropeptide involved in several functions ranging from lactation to social attachment. Over the years, the role of this molecule in pain processing has emerged, showing that, at the spinal level, oxytocin blocks pain transmission. The present work suggests that oxytocin also modulates pain at the cortical insular level by favoring cortical GABAergic transmission and activating descending spinal noradrenergic mechanisms. Indeed, we show that the paraventricular hypothalamicnucleus sends direct oxytocinergic projections to the rostral agranular insular cortex on GABAergic and oxytocin receptor-expressing neurons. Together, our data support the notion that the oxytocinergic system could act as an orchestrator of pain modulation.

Monoaminergic Receptors as Modulators of the Perivascular Sympathetic and Sensory CGRPergic Outflows.

Blood pressure is a highly controlled cardiovascular parameter that normally guarantees an adequate blood supply to all body tissues. This parameter is mainly regulated by peripheral vascular resistance and is maintained by local mediators (i.e., autacoids), and by the nervous and endocrine systems. Regarding the nervous system, blood pressure can be modulated at the central level by regulating the autonomic output. However, at peripheral level, there exists a modulation by activation of prejunctional monoaminergic receptors in autonomic- or sensory-perivascular fibers. These modulatory mechanisms on resistance blood vessels exert an effect on the release of neuroactive substances from the autonomic or sensory fibers that modify blood pressure. Certainly, resistance blood vessels are innervated by perivascular: (i) autonomic sympathetic fibers (producing vasoconstriction mainly by noradrenaline release); and (ii) peptidergic sensory fibers [producing vasodilatation mainly by calcitonin gene-related peptide (CGRP) release]. In the last years, by using pithed rats, several monoaminergic mechanisms for controlling both the sympathetic and sensory perivascular outflows have been elucidated. Additionally, several studies have shown the functions of many monoaminergic auto-receptors and hetero-receptors expressed on perivascular fibers that modulate neurotransmitter release. On this basis, the present review: (i) summarizes the modulation of the peripheral vascular tone by adrenergic, serotoninergic, dopaminergic, and histaminergic receptors on perivascular autonomic (sympathetic) and sensory fibers, and (ii) highlights that these monoaminergic receptors are potential therapeutic targets for the development of novel medications to treat cardiovascular diseases (with some of them explored in clinical trials or already in clinical use).

Potential Mechanisms Involved in Palmitoylethanolamide-Induced Vasodepressor Effects in Rats.

Palmitoylethanolamide is an endogenous lipid that exerts complex vascular effects, enhances the effects of endocannabinoids and induces a direct hypotension, but the mechanisms involved have been poorly explored. Hence, this study investigated in Wistar pithed rats the role of CB1, CB2, TRPV1 and GPR55 receptors in the inhibition by palmitoylethanolamide of the vasopressor responses produced by sympathetic stimulation or exogenous noradrenaline. Frequency- and dose-dependent vasopressor responses were analysed before and during intravenous (i.v.) continuous infusions of palmitoylethanolamide in animals receiving i.v. bolus of the antagonists NIDA41020 (CB1), AM630 (CB2), capsazepine (TRPV1), and/or cannabidiol (GPR55). Palmitoyletha-nolamide (0.1-3.1 µg/kg/min) dose-dependently inhibited the sympathetically induced and noradrenaline-induced vasopressor responses. Both inhibitions were: (i) partially blocked by 100 µg/kg NIDA41020, 100 µg/kg capsazepine, or 31 µg/kg cannabidiol; (ii) unaffected by 310 µg/kg AM630; and (iii) abolished by the combination NIDA41020 + capsazepine + cannabidiol (100, 100, and 31 µg/kg, respectively). The resting blood pressure was decreased by palmitoylethanolamide (effect prevented by NIDA41020, capsazepine or cannabidiol, but not by AM630). These results suggest that: (i) palmitoylethanolamide inhibits the vasopressor responses to sympathetic stimulation and exogenous noradrenaline and that it induces hypotension; and (ii) all these effects are mediated by prejunctional and vascular CB1, TRPV1 and probably GPR55, but not by CB2, receptors.