Rostroventral Medulla Research Articles

Pain behavior and the expression of Fos in the related brain regions of rats with chronic construction injury

Objective To observe pain behavior and the expression of Fos in the related brain regions, including Anterior Cingulate Cortex (ACC), Periaqueductal Gray (PAG), Rostral ventromedial nucleus (RVM) in chronic constrictive injury (CCI) rats and to explore whether ACC modulate spinal nociceptive transmission through endogenous descending facilitatory system. Methods A total of 48 male SD rats were randomly divided into six groups: Naive group, Sham group (just separated the left sciatic nerves without ligation), CCI group (the left sciatic nerve was ligated), CCI+ P group (on the 14 th day after surgery, intraperitoneal injection of 10 mg/kg paroxetine 45 min before behavior test), ACC-Sham group (bilateral microinjection of 0.9% NaCl in ACC, 1μl/each side) and ACC-AP5 group (bilateral microinjection of AP5 25 mM in ACC, 1 μl/each side on the 13th day after surgery). On the 14th day after light-dark transition test, forced swimming test, paw-withdrawal mechanical threshold(PWMT) and paw-withdrawal thermal patency(PWTL) were performed, the rats were terminally anesthetized and ACC, PAG, RVM and the spinal cord was rapidly removed, and then the expression of c-fos was measured by immunohistochemistry. Results (1)Rats in CCI group demonstrated nociceptive hypersensitivity and depressive-like behaviors compared with Naive rats, while rats in CCI+ P group and in ACC-AP5 group showed less nociceptive hypersensitivity and less depressive-like behaviors compared with CCI group. (2)Compared with Naive group, the number of c-fos positive neurons in the bilateral ACC (left, surgery side, 4920.6±1053.7; right, 2059.3±409.1), VIPAG (9074.8±2320.3), RVM (6195.4±895.0) and bilateral spinal cord (left, 15148.8±3080.2; right, 6400.2±1558.4) was significantly enhanced in CCI group, especially in the left side. In contrast, the amount of fos labeled neurons declined in the bilateral ACC (left, 2776.4±820.1; right, 1120.5±141.4), VIPAG (4002.2±1171.8), RVM (2938.9±910.3) and bilateral spinal cord (left, 8742.0±1131.0; right, 3933.1±858.9) in CCI+ P group and also declined in the bilateral ACC (left, 3623.1±667.4; right, 696.5±164.8), VIPAG (5668.8±1403.3), RVM (3972.3±851.7) and bilateral spinal cord (left, 10675.4±1725.3; right, 3818.3±1085.1) in ACC-AP5 group. Conclusion Endogenous descending facilitatory system may contribute to ACC modulating spinal nociceptive transmission. Key words: Anterior cingulate cortex; Spinal; Periaqueductal grey; Rostroventral medulla; Fos protein

Inhibition of CaMKIIα in the Central Nucleus of Amygdala Attenuates Fentanyl-Induced Hyperalgesia in Rats.

Opioid-induced hyperalgesia (OIH) is a less-studied phenomenon that has been reported in both preclinical and clinical studies. Although the underlying cause is not entirely understood, OIH is a real-life problem that affects millions of patients on a daily basis. Research has implicated the important contribution of Ca(2+)/calmodulin-dependent protein kinase IIα (CaMKIIα) to OIH at the level of spinal nociceptors. To expand our understanding of the entire brain circuitry driving OIH, in this study we investigated the role of CaMKIIα in the laterocapcular division of the central amygdala (CeLC), the conjunctive point between the spinal cord and rostro-ventral medulla. OIH was produced by repeated fentanyl administration in the rat. Correlating with the development of mechanical allodynia and thermal hyperalgesia, CaMKIIα activity was significantly elevated in the CeLC in OIH. In addition, the frequency and amplitude of spontaneous miniature excitatory postsynaptic currents (mEPSCs) in CeLC neurons were significantly increased in OIH. 2-[N-(2-hidroxyethyl)-N-(4-methoxy-benzenesulfonyl)]-amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, a CaMKIIα inhibitor, dose dependently reversed sensory hypersensitivity, activation of CeLC CaMKIIα, and mEPSCs in OIH. Taken together, our data for the first time implicate a critical role of CeLC CaMKIIα in OIH.

Dysfunctional Relationship Between the Prefrontal Cortex and Amygdala for Explaining Posttraumatic CRPS Syndrome.

Dear Editor, After limb trauma, the first short lasting response corresponds to a facilitatory supraspinal influence exerted at the spinal cord with sympathoexcitation. A descending sympathoexcitatory response with analgesia, peripheral vasoconstriction, and facilitated motor reflexes could be an active adaptive response for helping to maintain the capacity to use an injured body part for flight or fight in case of emergency. After that, a descending sympathoinhibitory response with hyperalgesia, vasodilatation, and muscle weakness could be a passive adaptive response, where facilitation of pain might promote protection from new injury and vasodilatation and immobilization might help the healing process of the injured region. Persistent sympathoinhibition has been implicated for developing posttraumatic complex regional pain syndrome (CRPS). However, there is little known about the underlying neuroanatomical pathways involved in persistent sympathoinhibition in CRPS patients. Periaqueductal gray (PAG) has been seen as a center for defensive reactions that is capable of coordinating the sensory, motor, and autonomic outputs of the stress response. According to Vianna [1], four main longitudinal cell-rich subdivisions exist in the PAG, namely the dorsomedial (dmPAG), dorsolateral (dlPAG), lateral, and ventrolateral (vlPAG) subdivisions. Activation of neurons in the lateral/dorsolateral column of the PAG (l/dlPAG) results in sympathoexcitation that accompany the fight-or-flight response [2,3]. Activation of neurons in the vlPAG results in the sympathoinhibition that can accompany deep pain [2,3]. These PAG columns have extensive, viscerotopically organized, descending projections to sympathetic premotor neurons in the rostroventral medulla (RVM) [4]. Differential activation of l/dlPAG columns and vlPAG columns could provide a substrate for the parallel activation or inhibition of premotor neurons in the RVM and Locus Coeruleus thus explaining different patterns of autonomic activity supporting active and passive coping behaviors. Afferent signals from the autonomic nervous system terminating in lamina …

Identifying local and descending inputs for primary sensory neurons.

Primary pain and touch sensory neurons not only detect internal and external sensory stimuli, but also receive inputs from other neurons. However, the neuronal derived inputs for primary neurons have not been systematically identified. Using a monosynaptic rabies viruses-based transneuronal tracing method combined with sensory-specific Cre-drivers, we found that sensory neurons receive intraganglion, intraspinal, and supraspinal inputs, the latter of which are mainly derived from the rostroventral medulla (RVM). The viral-traced central neurons were largely inhibitory but also consisted of some glutamatergic neurons in the spinal cord and serotonergic neurons in the RVM. The majority of RVM-derived descending inputs were dual GABAergic and enkephalinergic (opioidergic). These inputs projected through the dorsolateral funiculus and primarily innervated layers I, II, and V of the dorsal horn, where pain-sensory afferents terminate. Silencing or activation of the dual GABA/enkephalinergic RVM neurons in adult animals substantially increased or decreased behavioral sensitivity, respectively, to heat and mechanical stimuli. These results are consistent with the fact that both GABA and enkephalin can exert presynaptic inhibition of the sensory afferents. Taken together, this work provides a systematic view of and a set of tools for examining peri- and extrasynaptic regulations of pain-afferent transmission.

Surgical injury in the neonatal rat alters the adult pattern of descending modulation from the rostroventral medulla.

Neonatal pain and injury can alter long-term sensory thresholds. Descending rostroventral medulla (RVM) pathways can inhibit or facilitate spinal nociceptive processing in adulthood. As these pathways undergo significant postnatal maturation, the authors evaluated long-term effects of neonatal surgical injury on RVM descending modulation. Plantar hind paw or forepaw incisions were performed in anesthetized postnatal day (P)3 Sprague-Dawley rats. Controls received anesthesia only. Hind limb mechanical and thermal withdrawal thresholds were measured to 6 weeks of age (adult). Additional groups received pre- and post-incision sciatic nerve levobupivacaine or saline. Hind paw nociceptive reflex sensitivity was quantified in anesthetized adult rats using biceps femoris electromyography, and the effect of RVM electrical stimulation (5-200 μA) measured as percentage change from baseline. In adult rats with previous neonatal incision (n = 9), all intensities of RVM stimulation decreased hind limb reflex sensitivity, in contrast to the typical bimodal pattern of facilitation and inhibition with increasing RVM stimulus intensity in controls (n = 5) (uninjured vs. neonatally incised, P < 0.001). Neonatal incision of the contralateral hind paw or forepaw also resulted in RVM inhibition of hind paw nociceptive reflexes at all stimulation intensities. Behavioral mechanical threshold (mean ± SEM, 28.1 ± 8 vs. 21.3 ± 1.2 g, P < 0.001) and thermal latency (7.1 ± 0.4 vs. 5.3 ± 0.3 s, P < 0.05) were increased in both hind paws after unilateral neonatal incision. Neonatal perioperative sciatic nerve blockade prevented injury-induced alterations in RVM descending control. Neonatal surgical injury alters the postnatal development of RVM descending control, resulting in a predominance of descending inhibition and generalized reduction in baseline reflex sensitivity. Prevention by local anesthetic blockade highlights the importance of neonatal perioperative analgesia.

Slow breathing and hypoxic challenge: cardiorespiratory consequences and their central neural substrates.

Controlled slow breathing (at 6/min, a rate frequently adopted during yoga practice) can benefit cardiovascular function, including responses to hypoxia. We tested the neural substrates of cardiorespiratory control in humans during volitional controlled breathing and hypoxic challenge using functional magnetic resonance imaging (fMRI). Twenty healthy volunteers were scanned during paced (slow and normal rate) breathing and during spontaneous breathing of normoxic and hypoxic (13% inspired O2) air. Cardiovascular and respiratory measures were acquired concurrently, including beat-to-beat blood pressure from a subset of participants (N = 7). Slow breathing was associated with increased tidal ventilatory volume. Induced hypoxia raised heart rate and suppressed heart rate variability. Within the brain, slow breathing activated dorsal pons, periaqueductal grey matter, cerebellum, hypothalamus, thalamus and lateral and anterior insular cortices. Blocks of hypoxia activated mid pons, bilateral amygdalae, anterior insular and occipitotemporal cortices. Interaction between slow breathing and hypoxia was expressed in ventral striatal and frontal polar activity. Across conditions, within brainstem, dorsal medullary and pontine activity correlated with tidal volume and inversely with heart rate. Activity in rostroventral medulla correlated with beat-to-beat blood pressure and heart rate variability. Widespread insula and striatal activity tracked decreases in heart rate, while subregions of insular cortex correlated with momentary increases in tidal volume. Our findings define slow breathing effects on central and cardiovascular responses to hypoxic challenge. They highlight the recruitment of discrete brainstem nuclei to cardiorespiratory control, and the engagement of corticostriatal circuitry in support of physiological responses that accompany breathing regulation during hypoxic challenge.

The roles of neurotensin and its analogues in pain.

Neurotensin (NT) is an endogenous 13 amino acid neuropeptide with profound opioid-independent analgesic effects. This role of NT is thought to be mediated by both neurotensin receptor subtype 1 (NTS1) and neurotensin receptor subtype 2 (NTS2). NT and its receptors are widely distributed in the pain circuits in central nervous system. Thus NT might modulate pain in many structures of pain pathway, such as spinal cord, rostroventral medulla (RVM) and periaqueductal gray (PAG). Actually either intrathecal application of NT or direct injection of NT into RVM or PAG or intracerebroventricular injection of NT showed analgesic effects. NT exerted its antinociceptive effects in both acute pain and chronic pain models. The analgesic effects of NT were originally found in acute pain experiments. In the case of pathological pain, for example, formalin injection induced inflammatory pain and sciatic nerve constriction induced neuropathic pain, NT also shows antinociceptive effects. The effects exist in somatic pain as well as visceral pain induced by noxious colorectal distension (CRD) or writhing test. It should be noted that NT plays an important role in stress-induced antinociception (SIAN), especially in higher intensity stress experiments. However as a neuropeptide, NT is susceptible to degradation by peptidases and cannot cross the blood-brain barrier (BBB). Great efforts have been made to find NT analogues that are more biologically stable and could inhibit pain by systematic administration. The present review focuses on the analgesic role and the underlying mechanisms of NT and its analogues in pain, especially in chronic pain models.

The selectivity of rostroventral medulla descending control of spinal sensory inputs shifts postnatally from A fibre to C fibre evoked activity

Brainstem descending control is crucial in maintaining the balance of excitation and inhibition in spinal sensory networks. In the adult, descending inhibition of spinal dorsal horn circuits arising from the brainstem rostroventral medial medulla (RVM) is targeted to neurons with a strong nociceptive C fibre input. Before the fourth postnatal week, the RVM exerts a net facilitation of spinal networks but it is not known if this is targeted to specific dorsal horn neuronal inputs. As the maturation from descending facilitation to inhibition occurs only after C fibre central synaptic maturation is complete, we hypothesized that RVM facilitation in young animals is targeted to A fibre afferent inputs. To test this, the RVM was stimulated while recording dorsal horn neuronal activity in vivo under isoflurane anaesthesia at postnatal day (P) 21 and P40 (adult). Electrical thresholds for A and C fibre evoked activity, spike counts and wind-up characteristics at baseline and during RVM stimulation (10–100 µA, 10 Hz) were compared. In adults, RVM stimulation selectively increased the threshold for C fibre evoked activity while at P21, it selectively decreased the threshold for A fibre evoked activity and these effects were correlated to the wind-up characteristics of the neuron. Thus, the postnatal shift in RVM control of dorsal horn circuits is not only directional but also modality specific, from facilitation of A fibre input in the young animal to inhibition of nociceptive C input in the adult, with additional contextual factors. The descending control of spinal sensory networks serves very different functions in young and adult animals.

Visceral analgesic effect of 5-HT4 receptor agonist in rats involves the rostroventral medulla (RVM)

The 5-HT4 receptor agonist tegaserod (TEG) has been reported to modulate visceral pain. However, the underlying mechanism remains unknown. The objective of the present study was to examine the analgesic mechanism and site of action of TEG. In male rats, visceral pain was assessed by measuring visceromotor response (VMR) to colorectal distension (CRD). Inflammation was induced by intracolonic injection of tri-nitrobenzene sulfonic acid (TNBS). The effect of TEG on the VMR was tested by injecting intraperitoneal (i.p.), intrathecal (i.t.), intracerebroventricular (i.c.v) or in the rostroventral medulla (RVM). The effect of the drug was also tested on responses of CRD-sensitive pelvic nerve afferents (PNA) and lumbo-sacral (LS) spinal neurons. Systemic injection of TEG attenuated VMR in naive and TNBS-treated rats. Similarly, supraspinal, but not spinal, injection of TEG attenuated the VMR. While GR113808, (selective 5-HT4 antagonist) blocked the effect, naloxone (NLX) an opioid receptor antagonist reversed the effect of TEG. Although i.t. NLX did not block the inhibitory effect of TEG in VMR study, i.t. injection of α2-adrenergic receptor antagonist yohimbine blocked the effect of TEG when given systemically. While TEG had no effect on the responses of CRD-sensitive PNA, it inhibited the responses of CRD-sensitive LS neurons in spinal intact condition. This inhibition was blocked by GR113808, NLX and β-funaltrexamine (β-FNA) when injected into the RVM. Results indicate that TEG produces analgesia via activation of supraspinal 5-HT4 receptors which triggers the release of opioids at supraspinal site, which activates descending noradrenergic pathways to the spinal cord to produce analgesia.

Modulation of Peripheralμ-Opioid Analgesia byσ1Receptors

We evaluated the effects of σ1-receptor inhibition on μ-opioid-induced mechanical antinociception and constipation. σ1-Knockout mice exhibited marked mechanical antinociception in response to several μ-opioid analgesics (fentanyl, oxycodone, morphine, buprenorphine, and tramadol) at systemic (subcutaneous) doses that were inactive in wild-type mice and even unmasked the antinociceptive effects of the peripheral μ-opioid agonist loperamide. Likewise, systemic (subcutaneous) or local (intraplantar) treatment of wild-type mice with the selective σ1 antagonists BD-1063 [1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine dihydrochloride] or S1RA [4-[2-[[5-methyl-1-(2-naphthalenyl)1H-pyrazol-3-yl]oxy]ethyl] morpholine hydrochloride] potentiated μ-opioid antinociception; these effects were fully reversed by the σ1 agonist PRE-084 [2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate) hydrochloride], showing the selectivity of the pharmacological approach. The μ-opioid antinociception potentiated by σ1 inhibition (by σ1-receptor knockout or σ1-pharmacological antagonism) was more sensitive to the peripherally restricted opioid antagonist naloxone methiodide than opioid antinociception under normal conditions, indicating a key role for peripheral opioid receptors in the enhanced antinociception. Direct interaction between the opioid drugs and σ1 receptor cannot account for our results, since the former lacked affinity for σ1 receptors (labeled with [(3)H](+)-pentazocine). A peripheral role for σ1 receptors was also supported by their higher density (Western blot results) in peripheral nervous tissue (dorsal root ganglia) than in several central areas involved in opioid antinociception (dorsal spinal cord, basolateral amygdala, periaqueductal gray, and rostroventral medulla). In contrast to its effects on nociception, σ1-receptor inhibition did not alter fentanyl- or loperamide-induced constipation, a peripherally mediated nonanalgesic opioid effect. Therefore, σ1-receptor inhibition may be used as a systemic or local adjuvant to enhance peripheral μ-opioid analgesia without affecting opioid-induced constipation.

Postnatal maturation of endogenous opioid systems within the periaqueductal grey and spinal dorsal horn of the rat

Significant opioid-dependent changes occur during the fourth postnatal week in supraspinal sites (rostroventral medulla [RVM], periaqueductal grey [PAG]) that are involved in the descending control of spinal excitability via the dorsal horn (DH). Here we report developmentally regulated changes in the opioidergic signalling within the PAG and DH, which further increase our understanding of pain processing during early life. Microinjection of the μ-opioid receptor (MOR) agonist DAMGO (30ng) into the PAG of Sprague-Dawley rats increased spinal excitability and lowered mechanical threshold to noxious stimuli in postnatal day (P)21 rats, but had inhibitory effects in adults and lacked efficacy in P10 pups. A tonic opioidergic tone within the PAG was revealed in adult rats by intra-PAG microinjection of CTOP (120ng, MOR antagonist), which lowered mechanical thresholds and increased spinal reflex excitability. Spinal adminstration of DAMGO inhibited spinal excitability in all ages, yet the magnitude of this was greater in younger animals than in adults. The expression of MOR and related peptides were also investigated using TaqMan real-time polymerase chain reaction and immunohistochemistry. We found that pro-opiomelanocortin peaked at P21 in the ventral PAG, and MOR increased significantly in the DH as the animals aged. Enkephalin mRNA transcripts preceded the increase in enkephalin immunoreactive fibres in the superficial dorsal horn from P21 onwards. These results illustrate that profound differences in the endogenous opioidergic signalling system occur throughout postnatal development.

The Dilemma of Opioid-Induced Hyperalgesia and Tolerance in Chronic Opioid Therapy = معضلة فرط التألم و حالة التحمل الناتج عن العلاج طويل الأمد بالأدوية ( المسكنات ) الأفيونية

The Dilemma of Opioid-Induced Hyperalgesia and Tolerance in Chronic Opioid Therapy = معضلة فرط التألم و حالة التحمل الناتج عن العلاج طويل الأمد بالأدوية ( المسكنات ) الأفيونية

A critical period in the supraspinal control of pain: opioid-dependent changes in brainstem rostroventral medulla function in preadolescence

We have previously shown that the balance of electrically evoked descending brainstem control of spinal nociceptive reflexes undergoes a switch from excitation to inhibition in preadolescent rats. Here we show that the same developmental switch occurs when μ-opioid receptor agonists are microinjected into the rostroventral medulla (RVM). Microinjections of the μ-opioid receptor agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) into the RVM of lightly anaesthetised adult rats produced a dose-dependent decrease in mechanical nociceptive hindlimb reflex electromyographic activity. However, in preadolescent (postnatal day 21 [P21]) rats, the same doses of DAMGO produced reflex facilitation. RVM microinjection of δ-opioid receptor or GABAA receptor agonists, on the other hand, caused reflex depression at both ages. The μ-opioid receptor-mediated descending facilitation is tonically active in naive preadolescent rats, as microinjection of the μ-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) into the RVM at this age decreases spinal nociceptive reflexes while having no effect in adults. To test whether tonic opioid central activity is required for the preadolescent switch in RVM descending control, naloxone hydrochloride was delivered continuously from subcutaneous osmotic mini-pumps for 7-day periods, at various postnatal stages. Blockade of tonic opioidergic activity from P21 to P28, but not at earlier or later ages, prevented the normal development of descending RVM inhibitory control of spinal nociceptive reflexes. Enhancing opioidergic activity with chronic morphine over P7 to P14 accelerated this development. These results show that descending facilitation of spinal nociception in young animals is mediated by μ-opioid receptor pathways in the RVM. Furthermore, the developmental transition from RVM descending facilitation to inhibition of pain is determined by activity in central opioid networks at a critical period of periadolescence.

Association of Mu-opioid and NMDA Receptors in the Periaqueductal Gray: What Does it Mean for Pain Control?

The brain has an endogenous descending control system to modulate pain. This system is thought to arise from the activation of output neurons in the periaqueductal gray area (PAG) that project to the rostroventral medulla, with a relay to the spinal cord to modulate incoming pain signals (Basbaum and Fields, 1984). Electrical stimulation or microinjection of opioids into the ventral portion of the PAG results in analgesia. Opioids activate the pathway by inhibiting tonic GABA release (or disinhibition). The disinhibition hypothesis predicts that morphine and glutamate stimulate the descending pathway via activation of different cell populations, opioids by inhibiting GABA interneurons or inhibitory projection neurons into the PAG, and glutamate by directly activating PAG output neurons. In this issue of Neuropsychopharmacology, Rodriguez-Munoz et al provide evidence for a close association of NMDA and mu-opioid (MOR) receptors in the PAG, suggesting that these two receptors are not only colocalized within the same cell population, but that there is bidirectional regulation of the NMDA/MOR interaction during acute morphine tolerance.

Endogenous descending modulation: spatiotemporal effect of dynamic imbalance between descending facilitation and inhibition of nociception

In conscious rats, we investigated the change of nociceptive paw withdrawal reflexes elicited by mechanical and heat stimuli during intramuscular (i.m.) 5.8% hypertonic (HT) saline elicited muscle nociception. i.m. injection of HT saline caused rapid onset, long lasting (around 7 days), bilateral mechanical hyperalgesia, while it induced bilateral, slower onset (1 day after the HT saline injection), long-term (about 1-2 weeks) heat hypoalgesia. Ipsilateral topical pre-treatment of the sciatic nerve with 1% capsaicin significantly prevented the occurrence of both the bilateral mechanical hyperalgesia and the contralateral heat hypoalgesia. Intrathecal administration of either 6-hydroxydopamine hydrobromide (6-OHDA) or 5,7-dihydroxytryptamine (5,7-DHT), and intraperitoneal injection of naloxone all markedly attenuated the HT saline induced bilateral heat hypoalgesia, but not the mechanical hyperalgesia. Combined with experiments with lesioning of the rostroventral medulla with kainic acid, the present data indicate that unilateral i.m. injection of HT saline elicits time-dependent bilateral long-term mechanical hyperalgesia and heat hypoalgesia, which were modulated by descending facilitatory and inhibitory controls, respectively. We hypothesize that supraspinal structures may function to discriminate between afferent noxious inputs mediated by Aδ- and C-fibres, either facilitating Aδ-fibre mediated responses or inhibiting C-fibre mediated activities. However, this discriminative function is physiologically silent or inactive, and can be triggered by stimulation of peripheral C-fibre afferents. Importantly, in contrast to the rapid onset of descending facilitation, the late occurrence of descending inhibition suggests a requirement of continuous C-fibre input and temporal summation. Thus, a reduction of C-fibre input using exogenous analgesic agents, i.e. opioids, may counteract the endogenous descending inhibition.

The changing balance of brainstem–spinal cord modulation of pain processing over the first weeks of rat postnatal life

Brainstem-spinal cord connections play an essential role in adult pain processing, and the modulation of spinal pain network excitability by brainstem nuclei is known to contribute to hyperalgesia and chronic pain. Less well understood is the role of descending brainstem pathways in young animals when pain networks are more excitable and exposure to injury and stress can lead to permanent modulation of pain processing. Here we show that up to postnatal day 21 (P21) in the rat, the rostroventral medulla of the brainstem (RVM) exclusively facilitates spinal pain transmission but that after this age (P28 to adult), the influence of the RVM shifts to biphasic facilitation and inhibition. Graded electrical microstimulation of the RVM at different postnatal ages revealed a robust shift in the balance of descending control of both spinal nociceptive flexion reflex EMG activity and individual dorsal horn neuron firing properties, from excitation to inhibition, beginning after P21. The shift in polarity of descending control was also observed following excitotoxic lesions of the RVM in adult and P21 rats. In adults, RVM lesions decreased behavioural mechanical sensory reflex thresholds, whereas the same lesion in P21 rats increased thresholds. These data demonstrate, for the first time, the changing postnatal influence of the RVM in spinal nociception and highlight the central role of descending brainstem control in the maturation of pain processing.

Inputs to serotonergic neurons revealed by conditional viral transneuronal tracing

Descending projections arising from brainstem serotonergic (5HT) neurons contribute to both facilitatory and inhibitory controls of spinal cord "pain" transmission neurons. Unclear, however, are the brainstem networks that influence the output of these 5HT neurons. To address this question, here we used a novel neuroanatomical tracing method in a transgenic line of mice in which Cre recombinase is selectively expressed in 5HT neurons (ePet-Cre mice). Specifically, we injected the conditional pseudorabies virus recombinant (BA2001) that can replicate only in Cre-expressing neurons. Because BA2001 transports exclusively in a retrograde manner, we were able to reveal a subset of the neurons and circuits that are located upstream of the Cre-expressing 5HT neurons. We show that diverse brainstem regions differentially target the 5HT neurons of the dorsal raphe (DR) and the nucleus raphe magnus of the rostroventral medulla (RVM). Among these are several catecholaminergic and cholinergic cell groups, the periaqueductal gray, several brainstem reticular nuclei, and the nucleus of the solitary tract. We conclude that a brainstem 5HT network integrates somatic and visceral inputs arising from various areas of the body. We also identified a circuit that arises from projection neurons of deep spinal cord laminae V-VIII and targets the 5HT neurons of the NRM, but not of the DR. This spinoreticular pathway constitutes an anatomical substrate through which a noxious stimulus can activate 5HT neurons of the NRM and in turn could trigger descending serotonergic antinociceptive controls.

Genetically expressed transneuronal tracer reveals direct and indirect serotonergic descending control circuits

Despite the evidence for a significant contribution of brainstem serotonergic (5HT) systems to the control of spinal cord "pain" transmission neurons, attention has turned recently to the influence of nonserotonergic neurons, including the facilitatory and inhibitory controls that originate from so-called "on" and "off" cells of the rostroventral medulla (RVM). Unclear, however, is the extent to which these latter circuits interact with or are influenced by the serotonergic cell groups. To address this question we selectively targeted expression of a transneuronal tracer, wheat germ agglutinin (WGA), in the 5HT neurons so as to study the interplay between the 5HT and non-5HT systems. In addition to confirming the direct medullary 5HT projection to the spinal cord we also observed large numbers of non-5HT neurons, in the medullary nucleus reticularis gigantocellularis and magnocellularis, that were WGA-immunoreactive, i.e., were transneuronally labeled from 5HT neurons. FluoroGold injections into the spinal cord established that these reticular neurons are not only postsynaptic to the 5HT neurons of the medulla, but that most are also at the origin of descending, bulbospinal pathways. By contrast, we found no evidence that neurons of the midbrain periaqueductal gray that project to the RVM are postsynaptic to midbrain or medullary 5HT neurons. Finally, we found very few examples of WGA-immunoreactive noradrenergic neurons, which suggests that there is considerable independence of the monoaminergic bulbospinal pathways. Our results indicate that 5HT neurons influence "pain" processing at the spinal cord level both directly and indirectly via feedforward connections with multiple non-5HT descending control pathways.

Modulation of Brainstem Opiate Analgesia in the Rat by σ1Receptors: A Microinjection Study

sigma(1) Receptors have been implicated in the modulation of opioid analgesia. In the current study, we examined the role of sigma(1) systems in the periaqueductal gray (PAG), the rostroventral medulla (RVM), and the locus coeruleus (LC) of the rat, regions previously shown to be sensitive to morphine. Morphine was a potent analgesic in all three regions. Coadministration of the sigma(1) agonist (+)-pentazocine diminished the analgesic actions of morphine in all three regions, although the PAG was far less sensitive than the other two regions. Blockade of the sigma(1) receptors with haloperidol in the RVM markedly enhanced the analgesic actions of coadministered morphine, implying a tonic activity of the sigma(1) system in this region. This effect was mimicked by down-regulation of RVM sigma(1) receptors using an antisense approach. However, no tonic sigma(1) activity was observed in either the LC or the PAG. The RVM also was important in modulating analgesia elicited from morphine microinjected into the PAG. The analgesic actions of morphine given into the PAG could be attenuated by (+)-pentazocine placed into the RVM, whereas haloperidol in the RVM enhanced PAG morphine analgesia. These studies illustrate the pharmacological importance of sigma(1) receptors in the brainstem modulation of opioid analgesia.

Baroreceptor‐mediated inhibition of respiration after peripheral and central administration of a 5‐HT1A receptor agonist in neonatal piglets

Inhibition of neurones in the ventral medulla accentuates the respiratory inhibition associated with acute blood pressure elevation in piglets. Activation of presynaptic 5-HT(1A) receptors inhibits serotonergic neurones in the ventral medulla and caudal raphé, and we tested the hypothesis that administration of 8-hydroxydipropylaminotetralin (8-OH-DPAT), a 5-HT(1A) agonist, within the rostroventral medulla and caudal raphé would enhance baroreceptor-mediated inhibition of respiratory activity in decerebrate, neonatal piglets. Baroreceptor stimulation was achieved by inflating a balloon in the distal aorta to elevate carotid blood pressure. After two to four control trials of baroreceptor stimulation, each piglet was given either a single intravenous (i.v.) dose of 10 microg kg(-1) 8-OH-DPAT or treated by adding 10 or 30 mm 8-OH-DPAT to the dialysate for approximately 10 min to inhibit serotonergic neurones, after which the baroreceptor stimulation trials were repeated. Baroreceptor stimulation reduced respiratory activity, particularly the respiratory frequency, which diminished from 35.7 +/- 3.3 to 33.8 +/- 3.1 breaths min(-1) (P < 0.02) and, following i.v. 8-OH-DPAT, baroreceptor-mediated inhibition of respiratory output was significantly accentuated (P < 0.05); the respiratory frequency declined from 34.5 +/- 3.6 to 26.5 +/- 2.9 breaths min(-1). Increasing aortic blood pressure reduced the respiratory frequency (P < 0.01), but focal dialysis of 10 or 30 mm 8-OH-DPAT had, on average, no effect on the ventilatory inhibition associated with an acute elevation of blood pressure. We conclude that activation of 5-HT(1A) receptors after systemic administration of 8-OH-DPAT enhanced baroreflex-mediated inhibition of ventilation, but this effect cannot be attributed to 5-HT(1A) receptor activation within the rostroventral medulla and caudal raphé.