Probiotic and high-fat diet: effects on pain assessment, body composition, and cytokines in male and female adolescent and adult rats.

Obesity in adolescence is increasing in frequency and is associated with elevated proinflammatory cytokines and chronic pain in a sex-dependent manner. Dietary probiotics may mitigate these detrimental effects of obesity. Using a Long-Evans adolescent and adult rat model of overweight (high fat diet - 45% kcal from fat from weaning; HFD), we determined the effect of a single strain dietary probiotic (L. plantarum 299v [Lp299v] from weaning) on the theoretically increased neuropathic injury-induced pain phenotype and inflammatory cytokines. We found that although HFD increased fat mass, it did not markedly affect pain phenotype, particularly in adolescence, but there were subtle differences in pain in adult male vs. female rats. The combination of HFD and Lp299v augmented the increase in leptin in adolescent females. There were many non-interacting, main effects of age, diet and probiotic on an array of cytokines and adipokines with adults being higher than adolescents, HFD higher than the control diet, and a decrease with probiotic compared to placebo. Of particular interest were the probiotic-induced increases in IL12p70 in female adolescents on a HFD. We conclude that a more striking pain phenotype could require a higher and longer duration caloric diet or different etiology of pain. A major strength of our study was that a single strain probiotic had a wide range of inhibiting effects on most pro-inflammatory cytokines. The positive effect of probiotic on leptin in female adolescent rats is intriguing and worthy of exploration.

Fatty acid amide hydrolase activity in the dorsal periaqueductal gray attenuates neuropathic pain and associated dysautonomia.

The complexity of neuropathic pain and its associated comorbidities, including dysautonomia, make it difficult to treat. Overlap of anatomical regions and pharmacology of sympathosensory systems in the central nervous system (CNS) provide targets for novel treatment strategies. The dorsal periaqueductal gray (dPAG) is an integral component of both the descending pain modulation system and the acute stress response and is critically involved in both analgesia and the regulation of sympathetic activity. Local manipulation of the endocannabinoid signaling system holds great promise to provide analgesia without excessive adverse effects and also influence autonomic output. Inhibition of fatty acid amide hydrolase (FAAH) increases brain concentrations of the endocannabinoid N-arachidonoylethanolamine (AEA) and reduces pain-related behaviors in neuropathic pain models. Neuropathic hyperalgesia and reduced sympathetic tone are associated with increased FAAH activity in the dPAG, which suggests the hypothesis that inhibition of FAAH in the dPAG will normalize pain sensation and autonomic function in neuropathic pain. To test this hypothesis, the effects of systemic or intra-dPAG FAAH inhibition on hyperalgesia and dysautonomia developed after spared nerve injury (SNI) were assessed in male and female rats. Administration of the FAAH inhibitor PF-3845 into the dPAG reduces hyperalgesia behavior and the decrease in sympathetic tone induced by SNI. Prior administration of the CB1 receptor antagonist AM281, attenuated the antihyperalgesic and sympathetic effects of FAAH inhibition. No sex differences were identified. These data support an integrative role for AEA/CB1 receptor signaling in the dPAG contributing to the regulation of both hyperalgesia behavior and altered sympathetic tone in neuropathic pain.

Cardiovascular regulation in response to multiple hemorrhages: analysis and parameter estimation.

Mathematical models can provide useful insights explaining behavior observed in experimental data; however, rigorous analysis is needed to select a subset of model parameters that can be informed by available data. Here we present a method to estimate an identifiable set of parameters based on baseline left ventricular pressure and volume time series data. From this identifiable subset, we then select, based on current understanding of cardiovascular control, parameters that vary in time in response to blood withdrawal, and estimate these parameters over a series of blood withdrawals. These time-varying parameters are first estimated using piecewise linear splines minimizing the mean squared error between measured and computed left ventricular pressure and volume data over four consecutive blood withdrawals. As a final step, the trends in these splines are fit with empirical functional expressions selected to describe cardiovascular regulation during blood withdrawal. Our analysis at baseline found parameters representing timing of cardiac contraction, systemic vascular resistance, and cardiac contractility to be identifiable. Of these parameters, vascular resistance and cardiac contractility were varied in time. Data used for this study were measured in a control Sprague-Dawley rat. To our knowledge, this is the first study to analyze the response to multiple blood withdrawals both experimentally and theoretically, as most previous studies focus on analyzing the response to one large blood withdrawal. Results show that during each blood withdrawal both systemic vascular resistance and contractility decrease acutely and partially recover, and they decrease chronically across the series of blood withdrawals.

Upregulation of fatty acid amide hydrolase in the dorsal periaqueductal gray is associated with neuropathic pain and reduced heart rate in rats.

Nerve damage can induce a heightened pain response to noxious stimulation, which is termed hyperalgesia. Pain itself acts as a stressor, initiating autonomic and sensory effects through the dorsal periaqueductal gray (dPAG) to induce both sympathoexcitation and analgesia, which prior studies have shown to be affected by endocannabinoid signaling. The present study addressed the hypothesis that neuropathic pain disrupts autonomic and analgesic regulation by endocannabinoid signaling in the dPAG. Endocannabinoid contents, transcript levels of endocannabinoid signaling components, and catabolic enzyme activity were analyzed in the dPAG of rats at 21 days after painful nerve injury. The responses to two nerve injury models were similar, with two-thirds of animals developing hyperalgesia that was maintained throughout the postinjury period, whereas no sustained change in sensory function was observed in the remaining rats. Anandamide content was lower in the dPAG of rats that developed sustained hyperalgesia, and activity of the catabolic enzyme fatty acid amide hydrolase (FAAH) was higher. Intensity of hyperalgesia was correlated to transcript levels of FAAH and negatively correlated to heart rate and sympathovagal balance. These data suggest that maladaptive endocannabinoid signaling in the dPAG after nerve injury could contribute to chronic neuropathic pain and associated autonomic dysregulation. This study demonstrates that reduced anandamide content and upregulation of FAAH in the dPAG are associated with hyperalgesia and reduced heart rate sustained weeks after nerve injury. These data provide support for the evaluation of FAAH inhibitors for the treatment of chronic neuropathic pain.

Components of the cannabinoid system in the dorsal periaqueductal gray are related to resting heart rate.

The present study was undertaken to examine whether variations in endocannabinoid signaling in the dorsal periaqueductal gray (dPAG) are associated with baseline autonomic nerve activity, heart rate, and blood pressure. Blood pressure was recorded telemetrically in rats, and heart rate and power spectral analysis of heart rate variability were determined. Natural variations from animal to animal provided a range of baseline values for analysis. Transcript levels of endocannabinoid signaling components in the dPAG were analyzed, and endocannabinoid content and catabolic enzyme activity were measured. Higher baseline heart rate was associated with increased anandamide content and with decreased activity of the anandamide-hydrolyzing enzyme, fatty acid amide hydrolase (FAAH), and it was negatively correlated with transcript levels of both FAAH and monoacylglycerol lipase (MAGL), a catabolic enzyme for 2-arachidonoylglycerol (2-AG). Autonomic tone and heart rate, but not blood pressure, were correlated to levels of FAAH mRNA. In accordance with these data, exogenous anandamide in the dPAG of anesthetized rats increased heart rate. These data indicate that in the dPAG, anandamide, a FAAH-regulated lipid, contributes to regulation of baseline heart rate through influences on autonomic outflow.

Pontine µ-opioid receptors mediate bradypnea caused by intravenous remifentanil infusions at clinically relevant concentrations in dogs.

Life-threatening side effects such as profound bradypnea or apnea and variable upper airway obstruction limit the use of opioids for analgesia. It is yet unclear which sites containing µ-opioid receptors (µORs) within the intact in vivo mammalian respiratory control network are responsible. The purpose of this study was 1) to define the pontine region in which µOR agonists produce bradypnea and 2) to determine whether antagonism of those µORs reverses bradypnea produced by intravenous remifentanil (remi; 0.1-1.0 µg·kg(-1)·min(-1)). The effects of microinjections of agonist [D-Ala(2),N-Me-Phe(4),Gly-ol(5)]-enkephalin (DAMGO; 100 µM) and antagonist naloxone (NAL; 100 µM) into the dorsal rostral pons on the phrenic neurogram were studied in a decerebrate, vagotomized, ventilated, paralyzed canine preparation during hyperoxia. A 1-mm grid pattern of microinjections was used. The DAMGO-sensitive region extended from 5 to 7 mm lateral of midline and from 0 to 2 mm caudal of the inferior colliculus at a depth of 3-4 mm. During remi-induced bradypnea (~72% reduction in fictive breathing rate) NAL microinjections (~500 nl each) within the region defined by the DAMGO protocol were able to reverse bradypnea by 47% (SD 48.0%) per microinjection, with 13 of 84 microinjections producing complete reversal. Histological examination of fluorescent microsphere injections shows that the sensitive region corresponds to the parabrachial/Kölliker-Fuse complex.

Anandamide in the dorsal periaqueductal gray inhibits sensory input without a correlation to sympathoexcitation.

There is growing literature supporting cannabinoids as a potential therapeutic for pain conditions. The development of chronic pain has been associated with reduced concentrations of the endogenous cannabinoid anandamide (AEA) in the midbrain dorsal periaqueductal gray (dPAG), and microinjections of synthetic cannabinoids into the dPAG are antinociceptive. Therefore, the goal of this study was to examine the role of the dPAG in cannabinoid-mediated sensory inhibition. Given that cannabinoids in the dPAG also elicit sympathoexcitation, a secondary goal was to assess coordination between sympathetic and antinociceptive responses. AEA was microinjected into the dPAG while recording single unit activity of wide dynamic range (WDR) dorsal horn neurons (DHNs) evoked by high intensity mechanical stimulation of the hindpaw, concurrently with renal sympathetic nerve activity (RSNA), in anesthetized male rats. AEA microinjected into the dPAG decreased evoked DHN activity (n = 24 units), for half of which AEA also elicited sympathoexcitation. AEA actions were mediated by cannabinoid 1 receptors as confirmed by local pretreatment with the cannabinoid receptor antagonist AM281. dPAG microinjection of the synaptic excitant DL-homocysteic acid (DLH) also decreased evoked DHN activity (n = 27 units), but in all cases this was accompanied by sympathoexcitation. Thus, sensory inhibition elicited from the dPAG is not exclusively linked with sympathoexcitation, suggesting discrete neuronal circuits. The rostrocaudal location of sites may affect evoked responses as AEA produced sensory inhibition without sympathetic effects at 86 % of caudal compared to 25 % of rostral sites, supporting anatomically distinct neurocircuits. These data indicate that spatially selective manipulation of cannabinoid signaling could provide analgesia without potentially harmful autonomic activation.

Association between spatial working memory and Re-experiencing symptoms in PTSD.

BACKGROUND AND OBJECTIVES: Few studies have evaluated the link between working memory (WM) and post-traumatic stress disorder (PTSD). Further, it is unknown whether this relationship is accounted for by other relevant variables including negative affect, emotional dysregulation, or general non-WM-related cognitive control deficits, which are associated with PTSD. The purpose of this study was to determine the extent to which a computerized WM task could predict PTSD symptomology incrementally beyond the contribution of other relevant variables associated with PTSD. METHODS: Thirty veterans were eligible to complete emotional symptom questionnaires, a heart-rate variability measure, and computerized tasks (i.e., emotional Stroop and automated complex span tasks). A three-stage hierarchical regression was conducted with the PCL-5 total score and symptom clusters (i.e., re-experiencing, avoidance, hyperarousal, and negative cognition/mood) as the dependent variable. RESULTS: Results revealed that only the re-experiencing symptom cluster was significantly predicted by executive, verbal, and visuospatial WM tasks, which explained an additional 29.7% of the variance over and above other relevant variables. Most notably, the visuospatial task was the only WM task that significantly explained PCL-5 re-experiencing symptoms. LIMITATIONS: This study was based on a small sample of veterans with PTSD and causality cannot be determined with this cross-sectional study. CONCLUSIONS: Overall, the results suggest that deficits in visuospatial WM are significantly associated with PTSD re-experiencing symptoms after controlling for other relevant variables. Further research should evaluate whether an intervention to improve visuospatial WM capacity can be implemented to reduce re-experiencing symptoms.

Heterogeneity in patterns of pain development after nerve injury in rats and the influence of sex.

The genesis of neuropathic pain is complex, as sensory abnormalities may differ between patients with different or similar etiologies, suggesting mechanistic heterogeneity, a concept that is largely unexplored. Yet, data are usually grouped for analysis based on the assumption that they share the same underlying pathogenesis. Sex is a factor that may contribute to differences in pain responses. Neuropathic pain is more prevalent in female patients, but pre-clinical studies that can examine pain development in a controlled environment have typically failed to include female subjects. This study explored patterns of development of hyperalgesia-like behavior (HLB) induced by noxious mechanical stimulation in a neuropathic pain model (spared nerve injury, SNI) in both male and female rats, and autonomic dysfunction that is associated with chronic pain. HLB was analyzed across time, using both discrete mixture modeling and rules-based longitudinal clustering. Both methods identified similar groupings of hyperalgesia trajectories after SNI that were not evident when data were combined into groups by sex only. Within the same hyperalgesia development group, mixed models showed that development of HLB in females was delayed relative to males and reached a magnitude similar to or higher than males. The data also indicate that sympathetic tone (as indicated by heart rate variability) drops below pre-SNI level before or at the onset of development of HLB. This study classifies heterogeneity in individual development of HLB and identifies sexual dimorphism in the time course of development of neuropathic pain after nerve injury. Future studies addressing mechanisms underlying these differences could facilitate appropriate pain treatments.

Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats.

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are limited resources directing scientists in the techniques for measuring and analyzing these variables. This protocol describes the methods for measuring RSNA and mean arterial pressure in anesthetized rats. The protocol also includes the approaches for accessing the brain during RSNA recordings for central nervous system (CNS) manipulations. The RSNA recording technique is compatible with pharmacologic, optogenetic, or electrical stimulation of the CNS. The approach is useful when an investigator will measure short-term (min to h) autonomic responses in non-survival experiments to correlate anatomically with CNS nuclei. The approach is not intended to be used to obtain chronic (survival) recordings of RSNA in rats. Discharges in RSNA, averaged rectified RSNA, and mean arterial pressure can be quantified and analyzed further using parametric statistical tests. Methods for obtaining venous access, recording mean arterial pressure telemetrically, and brain fixation for future histological analysis are also described in the article.

Retrograde labeling reveals extensive distribution of genioglossal motoneurons possessing 5-HT2A receptors throughout the hypoglossal nucleus of adult dogs.

Inspiratory hypoglossal motoneurons (IHMNs) innervate the muscles of the tongue and play an important role in maintaining upper airway patency. However, this may be reduced during sleep and by sedatives, potent analgesics, and volatile anesthetics. The genioglossal (GG) muscle is the main protruder and depressor muscle of the tongue and contributes to upper airway patency during inspiration. In vitro data suggest that serotonin (5-hydroxytryptamine, 5-HT), via the 5-HT(2A) receptor (5-HT(2A)R) subtype, plays a key role in controlling the excitability of IHMNs. The distribution of GG motoneurons (GGMNs) within the hypoglossal (XII) nucleus has not been studied in the adult dog. Further, it is uncertain whether the 5-HT(2A)R is located on GGMNs in the adult dog. We therefore used the cholera toxin B (CTB) subunit as a retrograde tracer to map the location of GGMNs in combination with immunofluorescent labeling to determine the presence and colocalization of 5-HT(2A)R within the XII nucleus in adult mongrel dogs. Injection of CTB into the GG muscle resulted in retrogradely labeled cells in a compact column throughout the XII nucleus, extending from 0.75 mm caudal to 3.45 mm rostral to the obex. Fluorescence immunohistochemistry revealed extensive 5-HT(2A)R labeling on CTB-labeled GGMNs. Identification of the 5-HT(2A)R on GGMNs in the XII nucleus of the adult dog supports in vitro data and suggests a physiological role for this receptor subtype in controlling the excitability of GGMNs, which contribute to the maintenance of upper airway patency.

The pedunculopontine tegmentum controls renal sympathetic nerve activity and cardiorespiratory activities in nembutal-anesthetized rats.

Elevated renal sympathetic nerve activity (RSNA) accompanies a variety of complex disorders, including obstructive sleep apnea, heart failure, and chronic kidney disease. Understanding pathophysiologic renal mechanisms is important for determining why hypertension is both a common sequelae and a predisposing factor of these disorders. The role of the brainstem in regulating RSNA remains incompletely understood. The pedunculopontine tegmentum (PPT) is known for regulating behaviors including alertness, locomotion, and rapid eye movement sleep. Activation of PPT neurons in anesthetized rats was previously found to increase splanchnic sympathetic nerve activity and blood pressure, in addition to altering breathing. The present study is the first investigation of the PPT and its potential role in regulating RSNA. Microinjections of DL-homocysteic acid (DLH) were used to probe the PPT in 100-µm increments in Nembutal-anesthetized rats to identify effective sites, defined as locations where changes in RSNA could be evoked. A total of 239 DLH microinjections were made in 18 rats, which identified 20 effective sites (each confirmed by the ability to evoke a repeatable sympathoexcitatory response). Peak increases in RSNA occurred within 10-20 seconds of PPT activation, with RSNA increasing by 104.5 ± 68.4% (mean ± standard deviation) from baseline. Mean arterial pressure remained significantly elevated for 30 seconds, increasing from 101.6 ± 18.6 mmHg to 135.9 ± 36.4 mmHg. DLH microinjections also increased respiratory rate and minute ventilation. The effective sites were found throughout the rostal-caudal extent of the PPT with most located in the dorsal regions of the nucleus. The majority of PPT locations tested with DLH microinjections did not alter RSNA (179 sites), suggesting that the neurons that confer renal sympathoexcitatory functions comprise a small component of the PPT. The study also underscores the importance of further investigation to determine whether sympathoexcitatory PPT neurons contribute to adverse renal and cardiovascular consequences of diseases such as obstructive sleep apnea and heart failure.

Uptake blockade of endocannabinoids in the NTS modulates baroreflex-evoked sympathoinhibition.

Previous studies supporting a possible physiological role for an endogenous cannabinoid, arachidonylethanolamide (AEA, anandamide), showed a significant increase in AEA content in the nucleus tractus solitarius (NTS) after an increase in blood pressure (BP) and prolonged baroreflex inhibition of renal sympathetic nerve activity (RSNA) after exogenous AEA microinjections into the NTS. These results, along with other studies, support the hypothesis that endogenous AEA can modulate the baroreflex through cannabinoid CB(1) receptor activation within the NTS. This study was performed to characterize the physiological role of endogenously released cannabinoids (endocannabinoids) in regulating baroreflex control of RSNA through actions in the NTS. Endocannabinoid effects were assessed by measuring the RSNA baroreflex response to increased pressure after bilateral microinjections of AM404, an endocannabinoid transport inhibitor, into the NTS of adult male Sprague Dawley rats. AM404 blocks uptake of endocannabinoids and enhances the effects of any endocannabinoids released [M. Beltramo, et al., Functional role of high-affinity anandamide transport, as revealed by selective inhibition, Science 277 (5329) (1997) 1094-1097.] into the NTS. Therefore, it was hypothesized that microinjections of AM404 should exhibit effects similar to microinjections of exogenous AEA. In this study, AM404 microinjections into the NTS were found to significantly prolong baroreflex inhibition of RSNA compared to control, similar to effects of exogenous AEA. This effect is thought to result from an increased endocannabinoid presence in the NTS, leading to prolonged CB(1) receptor activation. These results indicate that endocannabinoids released in the NTS have the potential to modulate baroreflex control at this site in the central baroreflex pathway.

Effects of endocannabinoids on discharge of baroreceptive NTS neurons.

Previously, we have shown that microinjection of endocannabinoids (ECBs) into the nucleus tractus solitarius (NTS) can modulate baroreflex control of blood pressure (BP), prolonging pressor-induced inhibition of renal sympathetic nerve activity. This suggests that ECBs can modulate excitability of baroreceptive neurons in the NTS. Studies by others have shown that neural cannabinoid (CB1) receptors are present on fibers in the NTS, suggesting that some presynaptic modulation of transmitter release could occur in this region which receives direct afferent projections from arterial baroreceptors and cardiac mechanoreceptors. This study, therefore, was performed to determine the effects of ECBs on NTS baroreceptive neuronal discharge. Picoinjection of the ECB anandamide (AEA) was found to significantly increase discharge of baroreceptive neurons (20 of 23). Picoinjection of the ECB uptake inhibitor, AM404, which enhances endogenous ECB activity, also significantly increased discharge of baroreceptive neurons (8 of 10 neurons). To determine if effects of ECBs involved a GABAA mechanism, the neuronal responses to AEA and AM404 were tested after prior blockade of postsynaptic GABAA receptors by bicuculline (BIC) or SR 95531 hydrobromide (gabazine--SR 95531), which would eliminate any effects due to modulation of GABA input. The increase in neuronal discharge to both AEA and AM404 was significantly attenuated following BIC or SR 95531, which alone significantly increased discharge of baroreceptive neurons tested. These results support the hypothesis that ECBs enhance baroreflex function through increases in NTS baroreceptive neuronal activity, due in part to modulation of GABAergic inhibitory effects at the neuronal level.

Differential activation of medullary vagal nuclei during different phases of swallowing in the cat.

The aim of this study was to identify the medullary vagal nuclei involved in the different phases of swallowing activated physiologically in a species with an esophagus similar to human. In decerebrate cats, the pharyngeal (0.5-1.0 ml water in pharynx (N=6)) or esophageal (1-3 ml air in esophagus (N=5)) phases of swallowing were stimulated separately once per minute for 3 h, and we compared the resulting c-fos immunoreactivity within neuronal cell nuclei of the dorsal motor nucleus (DMN), nucleus tractus solitarius (NTS) and nucleus ambiguus (NA) with a sham control group (N=5). We found that the pharyngeal phase was associated with an elevated number of c-fos positive neurons in the intermediate (NTSim), interstitial (NTSis), ventromedial (NTSvm) subnuclei of the NTS, caudal DMN, and dorsal NA; and the esophageal phase was associated with an elevated number of c-fos positive neurons in the central (NTSce), ventral, dorsolateral, ventrolateral subnuclei of the NTS, rostral DMN, and ventral NA. We concluded that the pharyngeal and esophageal phases of swallowing are associated with different sets of NTS subnuculei; and the DMN and NA may contain functionally different populations of motor neurons situated rostrocaudally and dorsoventrally associated with the different phases of swallowing. The central pattern generator (CPG) for swallowing probably receives significant peripheral feedback, and the NTSvm may participate in the transition of the pharyngeal to the esophageal phase of swallowing.

Hemorrhagic sympathoinhibition mediated through the periaqueductal gray in the rat.

In response to hemorrhage in the anesthetized rat, an initial renal sympathoexcitation is followed by profound sympathoinhibition and hypotension with increasing blood loss. Microinjection of the gamma-aminobutyric acid(A) agonist muscimol to block neurotransmission through the sympathoinhibitory region of the ventrolateral periaqueductal gray matter (vlPAG) did not alter resting sympathetic nerve activity or blood pressure. However, the response to hemorrhage was converted to a maintained renal sympathoexcitation with a delayed and attenuated accompanying hypotension. These data indicate that neurons in the vlPAG mediate the sympathetic and cardiovascular responses to severe hemorrhage.

Pain tests provoke modality-specific cardiovascular responses in awake, unrestrained rats.

Nociception modulates heart rate (HR) and mean arterial pressure (MAP), suggesting their use of HR and MAP as indicators of pain in animals. We explored this with telemetric recording in unrestrained control and neuropathic (spinal nerve ligation) rats. Plantar stimulation was performed emulating techniques commonly used to measure pain, specifically brush stroke, von Frey fiber application, noxious pin stimulation, acetone for cooling, and radiant heating, while recording MAP, HR, and specific evoked somatomotor behaviors (none; simple withdrawal; or sustained lifting, shaking, and grooming representing hyperalgesia). Pin produced elevations in both HR and MAP, and greater responses accompanied hyperalgesia behavior compared to simple withdrawal. Von Frey stimulation depressed MAP, and increased HR only when stimulation produced hyperalgesia behavior, suggesting that minimal nociception occurs without this behavior. Brush increased MAP even when no movement was evoked. Cold elevated both HR and MAP whether or not there was withdrawal, but MAP increased more when withdrawal was triggered. Heating, consistently depressed HR and MAP, independent of behavior. Other than a greater HR response to pin in animals made hyperalgesic by injury, cardiovascular events evoked by stimulation did not differ between control and neuropathic animals. We conclude that (a) thermoregulation rather than pain may dominate responses to heat and cooling stimuli; (b) brush and cooling stimuli may be perceived and produce cardiovascular activation without nocifensive withdrawal; (c) sensations that produce hyperalgesia behavior are accompanied by greater cardiovascular activation than those producing simple withdrawal; and (d) von Frey stimulation lacks cardiovascular evidence of nociception except when hyperalgesia behavior is evoked.

Retrograde release of endocannabinoids inhibits presynaptic GABA release to second-order baroreceptive neurons in NTS.

In prior studies, we found that activation of cannabinoid-1 receptors in the nucleus tractus solitarii (NTS) prolonged baroreflex-induced sympathoinhibition in rats. In many regions of the central nervous system, activation of cannabinoid-1 receptors presynaptically inhibits γ-aminobutyric acid (GABA) release, disinhibiting postsynaptic neurons. To determine if cannabinoid-1 receptor-mediated presynaptic inhibition of GABA release occurs in the NTS, we recorded miniature inhibitory postsynaptic currents in anatomically identified second-order baroreceptive NTS neurons in the presence of ionotropic glutamate receptor antagonists and tetrodotoxin. The cannabinoid-1 receptor agonists, WIN 55212-2 (0.3-30 µM) and methanandamide (3 µM) decreased the frequency of miniature inhibitory postsynaptic currents in a concentration-dependent manner, an effect that was blocked by the cannabinoid-1 receptor antagonist, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM 251, 5 µM). Importantly, depolarization of second-order baroreceptive neurons decreased the frequency of miniature inhibitory postsynaptic currents; an effect which was blocked by the cannabinoid-1 receptor antagonist. The data indicate that depolarization of second-order baroreceptive NTS neurons induces endocannabinoid release from the neurons, leading to activation of presynaptic cannabinoid-1 receptors, inhibition of GABA release and subsequent enhanced baroreflex signaling in the NTS. The data suggest that endocannabinoid signaling in the NTS regulates short-term synaptic plasticity and provide a mechanism for endocannabinoid modulation of central baroreflex control.

Baroreceptor reflex is suppressed in rats that develop hyperalgesia behavior after nerve injury.

The baroreceptor reflex buffers autonomic changes by decreasing sympathetic activity and increasing vagal activity in response to blood pressure elevations, and by the reverse actions when the blood pressure falls. Because of the many bidirectional interactions of pain and autonomic function, we investigated the effect of painful nerve injury by spinal nerve ligation (SNL) on heart rate (HR), blood pressure (BP) and their regulation by the baroreceptor reflex. Rats receiving SNL were separated into either a hyperalgesic group that developed sustained lifting, shaking and grooming of the foot after plantar punctate nociceptive stimulation by pin touch or a group of animals that failed to show this hyperalgesic behavior after SNL. SNL produced no effect on resting BP recorded telemetrically in unrestrained rats compared to control rats receiving either skin incision or sham SNL. However, two tests of baroreceptor gain showed depression only in animals that developed sustained hyperalgesia after SNL. The animals that failed to develop hyperalgesia after SNL were found to have elevations in HR both before and for the first 4 days after SNL, and HR variability analysis gave indications of decreased vagal control of resting HR and elevated sympatho-vagal balance at these same time intervals. In human patients, other research has shown that blunted baroreceptor reflex sensitivity predicts poor outcome during conditions such as hypertension, congestive heart failure, myocardial infarction, and stroke. If baroreceptor reflex suppression is also found in human subjects during chronic neuropathic pain, this may adversely affect survival.

Microinjection of a cannabinoid receptor antagonist into the NTS increases baroreflex duration in dogs.

Baroreceptor afferent fibers synapse in the nucleus tractus solitarius (NTS) of the medulla. Neuronal cannabinoid (CB)(1) receptors are expressed in the NTS and central administration of CB(1) receptor agonists affect blood pressure (BP) and heart rate. In addition, there is evidence that endocannabinoids are produced in the brain stem. This study examined whether changes in CB(1) receptor activity in the NTS modulated the baroreceptor reflex, contributing to changes seen in BP and heart rate. Baroreflexes were evoked in anesthetized dogs by pressure ramp stimulations of the isolated carotid sinus before and after microinjection of CB(1) receptor agonist WIN-55212-2 (1.25-1.50 pmol) or antagonist SR-141716 (2.5-3.0 pmol) into cardiovascular regions of the NTS. Microinjection of the SR-141716 did not affect baseline BP or baroreflex sensitivity. However, SR-141716 significantly prolonged the time needed to return to the baseline level of BP after the pressure ramp. Microinjection of WIN-55212-2 had no effect on the baroreflex. These data suggest that endocannabinoids can modulate the excitability of NTS neurons involved in the baroreceptor reflex, leading to modulation of baroreflex regulation.