Alexandre Charlet

Evoked Axonal Oxytocin Release in the Central Amygdala Attenuates Fear Response

The hypothalamic neuropeptide oxytocin (OT), which controls childbirth and lactation, receives increasing attention for its effects on social behaviors, but how it reaches central brain regions is still unclear. Here we gained by recombinant viruses selective genetic access to hypothalamic OT neurons to study their connectivity and control their activity by optogenetic means. We found axons of hypothalamic OT neurons in the majority of forebrain regions, including the central amygdala (CeA), a structure critically involved in OT-mediated fear suppression. In vitro, exposure to blue light of channelrhodopsin-2-expressing OT axons activated a local GABAergic circuit that inhibited neurons in the output region of the CeA. Remarkably, in vivo, local blue-light-induced endogenous OT release robustly decreased freezing responses in fear-conditioned rats. Our results thus show widespread central projections of hypothalamic OT neurons and demonstrate that OT release from local axonal endings can specifically control region-associated behaviors.

A New Population of Parvocellular Oxytocin Neurons Controlling Magnocellular Neuron Activity and Inflammatory Pain Processing

Oxytocin (OT) is a neuropeptide elaborated by the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. Magnocellular OT neurons of these nuclei innervate numerous forebrain regions and release OT into the blood from the posterior pituitary. The PVN also harbors parvocellular OT cells that project to the brainstem and spinal cord, but their function has not been directly assessed. Here, we identified a subset of approximately 30 parvocellular OT neurons, with collateral projections onto magnocellular OT neurons and neurons of deep layers of the spinal cord. Evoked OT release from these OT neurons suppresses nociception and promotes analgesia in an animal model of inflammatory pain. Our findings identify a new population of OT neurons that modulates nociception in a two tier process: (1) directly by release of OT from axons onto sensory spinal cord neurons and inhibiting their activity and (2) indirectly by stimulating OT release from SON neurons into the periphery.

Fast non-genomic effects of progesterone-derived neurosteroids on nociceptive thresholds and pain symptoms.

Abstract Fast Inhibitory controls mediated by glycine (GlyRs) and GABAA receptors (GABAARs) play an important role to prevent the apparition of pathological pain symptoms of allodynia and hyperalgesia. The use of positive allosteric modulators of these receptors, specifically expressed in the spinal cord, may represent an interesting strategy to limit or block pain expression. In this study, we have used stereoisomers of progesterone metabolites, acting only via non‐genomic effects, in order to evaluate the contribution of GlyRs and GABAARs for the reduction of mechanical and thermal heat hypernociception. We show that 3α neurosteroids were particularly efficient to elevate nociceptive thresholds in naive animal. It also reduced mechanical allodynia and thermal heat hyperalgesia in the carrageenan model of inflammatory pain. This effect is likely to be mediated by GABAA receptors since 3β isomer was inefficient. More interestingly, 3α5β neurosteroid was only efficient on mechanical allodynia while having no effect on thermal heat hyperalgesia. We characterized these paradoxical effects of 3α5β neurosteroid using the strychnine and bicuculline models of allodynia. We clearly show that 3α5β neurosteroid exerts an antinociceptive effect via a positive allosteric modulation of GABAARs but, at the same time, is pronociceptive by reducing GlyR function. This illustrates the importance of the inhibitory amino acid receptor channels and their allosteric modulators in spinal pain processing. Moreover, our results indicate that neurosteroids, which are synthesized in the dorsal horn of the spinal cord and have limited side effects, may be of significant interest in order to treat pathological pain symptoms.

Differentiating Thermal Allodynia and Hyperalgesia Using Dynamic Hot and Cold Plate in Rodents

UNLABELLED In animal studies, thermal sensitivity is mostly evaluated on the basis of nociceptive reaction latencies in response to a given thermal aversive stimulus. However, these techniques may be inappropriate to differentiate allodynia from hyperalgesia or to provide information differentiating the activation of nociceptor subtypes. The recent development of dynamic hot and cold plates, allowing computer-controlled ramps of temperature, may be useful for such measures. In this study, we characterized their interest for studying thermal nociception in freely moving mice and rats. We showed that escape behavior (jumps) was the most appropriate parameter in C57Bl/6J mice, whereas nociceptive response was estimated by using the sum of paw lickings and withdrawals in Sprague-Dawley rats. We then demonstrated that this procedure allows the detection of both thermal allodynia and hyperalgesia after peripheral pain sensitization with capsaicin in mice and in rats. In a condition of carrageenan-induced paw inflammation, we observed the previously described thermal hyperalgesia, but we also revealed that rats exhibit a clear thermal allodynia to a cold or a hot stimulus. These results demonstrate the interest of the dynamic hot and cold plate to study thermal nociception, and more particularly to study both thermal allodynia and hyperalgesia within a single paradigm in awake and freely moving rodents. PERSPECTIVE Despite its clinical relevance, thermal allodynia is rarely studied by researchers working on animal models. As shown after stimulation of capsaicin-sensitive fibers or during inflammatory pain, the dynamic hot and cold plate validated in the present study provides a useful tool to distinguish between thermal allodynia and thermal hyperalgesia in rodents.

Reduction and prevention of vincristine-induced neuropathic pain symptoms by the non-benzodiazepine anxiolytic etifoxine are mediated by 3α-reduced neurosteroids

ABSTRACT The central processing of peripheral nociceptive messages is highly controlled by the activity of local inhibitory networks in the spinal cord and supraspinal centers. Recently, it has been shown that endogenous 3α‐reduced neurosteroids (3αNS) exert a significant spinal antinociception by potentiating GABAA receptor function. Because endogenous 3αNS can be produced in many relay structures of the nociceptive system, we tested the potential analgesic efficacy of promoting the production of neurosteroids by using etifoxine (ETX, 50 mg/kg i.p.). This prescribed non‐benzodiazepine anxiolytic was shown previously to stimulate neurosteroidogenesis in its early step after binding to the mitochondrial translocator protein complex (TSPO). Using an animal model of generalized neuropathic pain resulting from a 2‐ week treatment with the antitumoral agent vincristine sulfate (VCR, 0.1 mg/kg i.p.), we show that injections of ETX (50 mg/kg i.p.) given every day reduced the VCR‐induced mechanical and thermal pain symptoms but also prevented their appearance, if used in prophylaxia 1 week before VCR. Both the curative and preventive effects of ETX on pain symptoms were mediated by the production of 3αNS as demonstrated in animals treated with the enzymatic inhibitor provera (6‐medroxyprogesterone acetate; 20 mg/kg s.c.). Altogether, this study shows for the first time that promoting 3αNS could be a possible therapeutic strategy to treat neuropathic pain symptoms. Since ETX is already available as an anxiolytic, its use in humans, provided that its analgesic properties are confirmed, could be rapidly considered.

Nociceptive thresholds are controlled through spinal β2-subunit-containing nicotinic acetylcholine receptors.

Summary β2∗‐nAChRs are receptors essential for setting nociceptive thresholds by controlling GABAergic inhibition in the spinal cord. The participation of β2∗‐nAChRs in the modulation of nociceptive transmission suggest that these receptors might be targets of interest for developing selective pharmacological compounds in the context of pain treatment. ABSTRACT Although cholinergic drugs are known to modulate nociception, the role of endogenous acetylcholine in nociceptive processing remains unclear. In the current study, we evaluated the role of cholinergic transmission through spinal β2‐subunit‐containing nicotinic acetylcholine receptors in the control of nociceptive thresholds. We show that mechanical and thermal nociceptive thresholds are significantly lowered in β2∗‐knockout (KO) mice. Using nicotinic antagonists in these mice, we demonstrate that β2∗‐nAChRs are responsible for tonic inhibitory control of mechanical thresholds at the spinal level. We further hypothesized that tonic β2∗‐nAChR control of mechanical nociceptive thresholds might implicate GABAergic transmission since spinal nAChR stimulation can enhance inhibitory transmission. Indeed, the GABAA receptor antagonist bicuculline decreased the mechanical threshold in wild‐type but not β2∗‐KO mice, and the agonist muscimol restored basal mechanical threshold in β2∗‐KO mice. Thus, β2∗‐nAChRs appeared to be necessary for GABAergic control of nociceptive information. As a consequence of this defective inhibitory control, β2∗‐KO mice were also hyperresponsive to capsaicin‐induced C‐fiber stimulation. Our results indicate that β2∗‐nAChRs are implicated in the recruitment of inhibitory control of nociception, as shown by delayed recovery from capsaicin‐induced allodynia, potentiated nociceptive response to inflammation and neuropathy, and by the loss of high‐frequency transcutaneous electrical nerve stimulation (TENS)–induced analgesia in β2∗‐KO mice. As high‐frequency TENS induces analgesia through Aβ‐fiber recruitment, these data suggest that β2∗‐nAChRs may be critical for the gate control of nociceptive information by non‐nociceptive sensory inputs. In conclusion, acetylcholine signaling through β2∗‐nAChRs seems to be essential for setting nociceptive thresholds by controlling GABAergic inhibition in the spinal cord.

Long-Lasting Spinal Oxytocin Analgesia Is Ensured by the Stimulation of Allopregnanolone Synthesis Which Potentiates GABAA Receptor-Mediated Synaptic Inhibition

Hypothalamospinal control of spinal pain processing by oxytocin (OT) has received a lot of attention in recent years because of its potency to reduce pain symptoms in inflammatory and neuropathic conditions. However, cellular and molecular mechanisms underlying OT spinal antinociception are still poorly understood. In this study, we used biochemical, electrophysiological, and behavioral approaches to demonstrate that OT levels are elevated in the spinal cord of rats exhibiting pain symptoms, 24 h after the induction of inflammation with an intraplantar injection of λ-carrageenan. Using a selective OT receptor antagonist, we demonstrate that this elevated OT content is responsible for a tonic analgesia exerted on both mechanical and thermal modalities. This phenomenon appeared to be mediated by an OT receptor-mediated stimulation of neurosteroidogenesis, which leads to an increase in GABAA receptor-mediated synaptic inhibition in lamina II spinal cord neurons. We also provide evidence that this novel mechanism of OT-mediated spinal antinociception may be controlled by extracellular signal-related protein kinases, ERK1/2, after OT receptor activation. The oxytocinergic inhibitory control of spinal pain processing is emerging as an interesting target for future therapies since it recruits several molecular mechanisms, which are likely to exert a long-lasting analgesia through nongenomic and possibly genomic effects.

Neuropeptide S Activates Paraventricular Oxytocin Neurons to Induce Anxiolysis

Neuropeptides, such as neuropeptide S (NPS) and oxytocin (OXT), represent potential options for the treatment of anxiety disorders due to their potent anxiolytic profile. In this study, we aimed to reveal the mechanisms underlying the behavioral action of NPS, and present a chain of evidence that the effects of NPS within the hypothalamic paraventricular nucleus (PVN) are mediated via actions on local OXT neurons in male Wistar rats. First, retrograde studies identified NPS fibers originating in the brainstem locus coeruleus, and projecting to the PVN. FACS identified prominent NPS receptor expression in PVN-OXT neurons. Using genetically encoded calcium indicators, we further demonstrated that NPS reliably induces a transient increase in intracellular Ca2+ concentration in a subpopulation of OXT neurons, an effect mediated by NPS receptor. In addition, intracerebroventricular (i.c.v.) NPS evoked a significant somatodendritic release of OXT within the PVN as assessed by microdialysis in combination with a highly sensitive radioimmunoassay. Finally, we could show that the anxiolytic effect of NPS seen after i.c.v. or intra-PVN infusion requires responsive OXT neurons of the PVN and locally released OXT. Thus, pharmacological blockade of OXT receptors as well as chemogenetic silencing of OXT neurons within the PVN prevented the effect of synthetic NPS. In conclusion, our results indicate a significant role of the OXT system in mediating the effects of NPS on anxiety, and fill an important gap in our understanding of brain neuropeptide interactions in the context of regulation of emotional behavior within the hypothalamus. SIGNIFICANCE STATEMENT Given the rising scientific interest in neuropeptide research in the context of emotional and stress-related behaviors, our findings demonstrate a novel intrahypothalamic mechanism involving paraventricular oxytocin neurons that express the neuropeptide S receptor. These neurons respond with transient Ca2+ increase and somatodendritic oxytocin release following neuropeptide S stimulation. Thereby, oxytocin neurons seem essential for neuropeptide S-induced anxiolysis, as this effect was blocked by pharmacological and chemogenetic inhibition of the oxytocin system.

Radiotelemetric and Symptomatic Evaluation of Pain in the Rat After Laparotomy: Long-Term Benefits of Perioperative Ropivacaine Care

UNLABELLED Effective relief of acute and long-term postoperative pain is of utmost importance to patients undergoing surgery. Here, we worked on a controlled procedure of abdominal surgery in the rat inducing persistent postoperative pain symptoms for up to 10 days and tested the efficacy of perioperative care with the local anesthetic ropivacaine. Laparotomy was likewise used to implant radiotelemetric probes by which electrocardiogram, body temperature, and locomotor activity were recorded in freely moving animals. We showed that postoperative pain symptoms (mechanical allodynia) measured in periphery of the scar were associated over time with persistent tachycardia, elevated heart rate variability, and loss of mobility. Furthermore, a single subcutaneous infiltration of the local anesthetic ropivacaine in the periphery of the abdominal incision was sufficient to prevent the appearance of allodynia and the associated cardiac and motor signs of pain, monitored by radiotelemetry. These beneficial effects were observed when the infiltration was performed in the perioperative period, but not later. This study on freely moving animals exhibiting long-lasting postoperative pain symptoms and altered autonomic/motor function illustrates well the importance of the timing of preemptive analgesia care with long-acting local anesthetics. Moreover, it emphasizes the utility of monitoring heart rate variability to quantify spontaneous expression of long-lasting postoperative pain. PERSPECTIVE Speeding the recovery time after surgery using perioperative ropivacaine care is of significant clinical relevance because it might limit the risk of chronic pain and postoperative complications. In humans, chronobiological analysis of heart rate variability could also help quantify spontaneous pain expression with minimal emotional bias.

Oxytocin Signaling in Pain: Cellular, Circuit, System, and Behavioral Levels

Originally confined to the initiation of parturition and milk ejection after birth, the hypothalamic nonapeptide oxytocin (OT) is now recognized as a critical determinant of social behavior and emotional processing. It accounts for the modulation of sensory processing and pain perception as OT displays a potent analgesic effect mediated by OT receptors (OTRs) expressed in the peripheral and central nervous systems. In our chapter, we will first systemically analyze known efferent and afferent OT neuron projections, which form the anatomical basis for OT modulation of somatosensory and pain processing. Next, we will focus on the synergy of distinct types of OT neurons (e.g., magno- and parvocellular OT neurons) which efficiently control acute inflammatory pain perception. Finally, we will describe how OT signaling mechanisms in the spinal cord control nociception, as well as how OT is able to modulate emotional pain processing within the central amygdala. In the conclusions at the end of the chapter, we will formulate perspectives in the study of OT effects on pain anticipation and pain memory, as well as propose some reasons for the application of exogenous OT for the treatment of certain types of pain in human patients.