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.

Endothelin-1 mediates attenuated carotid baroreceptor activity by intermittent hypoxia.

The objectives of the present study were to examine the effects of intermittent hypoxia (IH) on arterial baroreflex function and assess the underlying mechanism(s). Experiments were performed on adult male rats treated with 14 days of IH (15 s of hypoxia, 5 min of normoxia; 8 h/day) or normoxia (control). Arterial blood pressures were elevated in IH-treated rats, and this effect was associated with attenuated heart rate and splanchnic sympathetic nerve responses to arterial baroreflex activation. In IH-treated rats, carotid baroreceptor responses to elevated sinus pressures were attenuated. Endothelin-1 (ET-1) levels were elevated in the carotid sinus region of IH-treated rats, and this effect was associated with increased endothelin converting enzyme (ECE) activity, which generates biologically active ET-1. ET(A) receptor antagonist prevented the effects of IH on carotid baroreceptor activity. In IH-treated rats, reactive oxygen species (ROS) levels were elevated in the carotid sinus region, and antioxidant treatment prevented the effects of IH on ET-1 levels, ECE activity, carotid baroreceptor activity, and baroreflex function. These results demonstrate that 1) IH attenuates arterial baroreflex function, which is in part due to reduced carotid baroreceptor responses to elevated carotid sinus pressure, and 2) IH-induced carotid baroreceptor dysfunction involves reactive oxygen species-dependent upregulation of ET-1 signaling in the carotid sinus region.

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.

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.

Substance P in the baroreceptor reflex: 25 years.

Twenty-five years ago, very little was known about chemical communication in the afferent limb of the baroreceptor reflex arc. Subsequently, considerable anatomic and functional data exist to support a role for the tachykinin, substance P (SP), as a neuromodulator or neurotransmitter in baroreceptor afferent neurons. Substance P is synthesized and released from baroreceptor afferent neurons, and excitatory SP (NK1) receptors are activated by baroreceptive input to second-order neurons. SP appears to play a role in modulating the gain of the baroreceptor reflex. However, questions remain about the specific role and significance of SP in mediating baroreceptor information to the central nervous system (CNS), the nature of its interaction with glutaminergic transmission, the relevance of colocalized agents, and complex effects that may result from mediation of non-baroreceptive signals to the CNS.

Anandamide content and interaction of endocannabinoid/GABA modulatory effects in the NTS on baroreflex-evoked sympathoinhibition.

Cannabinoids have been shown to modulate central autonomic regulation and baroreflex control of blood pressure (BP). The presence of cannabinoid CB(1) receptors on fibers in the nucleus tractus solitarius (NTS) suggests 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 mechanism(s) of effects of microinjection of an endocannabinoid, arachidonylethanolamide (anandamide, AEA), into the NTS on baroreflex sympathetic nerve responses produced by phenylephrine-induced pressure changes in anesthetized rats. AEA prolonged reflex inhibition of renal sympathetic nerve activity (RSNA), suggesting an increase in baroreflex sensitivity. This effect of AEA was blocked by prior microinjection of SR-141716 to block cannabinoid CB(1) receptors. To determine whether this baroreflex enhancement by AEA involved a GABA(A) mechanism, the baroreflex response to AEA was tested after prior blockade of postsynaptic GABA(A) receptors by bicuculline, which would eliminate any effects due to modulation of GABA activity. After bicuculline, which alone prolonged the baroreflex inhibition of RSNA, AEA shortened the duration of RSNA inhibition, suggesting a possible presynaptic inhibition of glutamate release previously obscured by a more dominant GABA(A) effect. To support a possible physiological role for AEA, AEA concentration in the NTS was measured after a phenylephrine-induced increase in BP. AEA content in the NTS was increased significantly over that in normotensive animals. These results support the hypothesis that AEA content is increased by brief periods of hypertension and suggest that AEA can modulate the baroreflex through activation of CB(1) receptors within the NTS, possibly modulating effectiveness of GABA and/or glutamate neurotransmission.

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.