Ultrastructural analysis of rat ventrolateral periaqueductal gray projections to the A5 cell group.

Stimulation of neurons in the ventrolateral periaqueductal gray (PAG) produces antinociception as well as cardiovascular depressor responses that are mediated in part by pontine noradrenergic neurons. A previous report using light microscopy has described a pathway from neurons in the ventrolateral PAG to noradrenergic neurons in the A5 cell group that may mediate these effects. The present study used anterograde tracing and electron microscopic analysis to provide more definitive evidence that neurons in the ventrolateral PAG form synapses with noradrenergic and non-catecholaminergic A5 neurons in Sasco Sprague-Dawley rats. Deposits of anterograde tracer, biotinylated dextran amine, into the rat ventrolateral PAG labeled a significant number of axons in the region of the rostral subdivision of the A5 cell group, and a relatively lower number in the caudal A5 cell group. Electron microscopic analysis of anterogradely-labeled terminals in both rostral (n=127) and caudal (n=70) regions of the A5 cell group indicated that approximately 10% of these form synapses with noradrenergic dendrites. In rostral sections, about 31% of these were symmetric synapses, 19% were asymmetric synapses, and 50% were membrane appositions without clear synaptic specializations. In caudal sections, about 22% were symmetric synapses, and the remaining 78% were appositions. In both rostral and caudal subdivisions of the A5, nearly 40% of the anterogradely-labeled terminals formed synapses with non-catecholaminergic dendrites, and about 45% formed axoaxonic synapses. These results provide direct evidence for a monosynaptic pathway from neurons in the ventrolateral PAG to noradrenergic and non-catecholaminergic neurons in the A5 cell group. Further studies should evaluate if this established monosynaptic pathway may contribute to the cardiovascular depressor effects or the analgesia produced by the activation of neurons in the ventrolateral PAG.

Neurotensin-produced antinociception in the rostral ventromedial medulla is partially mediated by spinal cord norepinephrine.

Microinjection of neurotensin (NT) into the rostral ventromedial medulla (RVM) produces dose-dependent antinociception. Here we show that antinociception produced by intra-RVM microinjection of neurotensin (NT) or the selective NT receptor subtype 1 (NTR1) agonist PD149163 can be partially blocked by intrathecal (i.t.) yohimbine, an alpha2-adrenoceptor antagonist and by methysergide, a serotonin receptor antagonist. Antinociception produced by the NTR2 agonist beta-lactotensin (beta-LT) is blocked by intrathecal (i.t.) yohimbine, but not by methysergide i.t. It is not known which noradrenergic cell group is involved in this newly identified noradrenergic component of NTR-mediated antinociception. These experiments provide the first evidence that selective activation of NTR2 in the RVM produces antinociception. These results also provide evidence that activation of NTR1 in the RVM produces antinociception through spinal release of norepinephrine (NE) and serotonin, and that activation of NTR2 in the RVM produces antinociception mediated by spinal release of NE.

Neurotensin activation of the NTR1 on spinally-projecting serotonergic neurons in the rostral ventromedial medulla is antinociceptive.

Microinjection of neurotensin (NT) in the rostral ventromedial medulla (RVM) produces dose-dependent antinociception. The NTR1 (Neurotensin Receptor Subtype 1) may mediate part of this response, however definitive evidence is lacking, and the spinal mediators of NTR1-induced antinociception are unknown. In the present study, we used immunohistochemical techniques to show that the NTR1, but not the NTR2 is expressed by spinally projecting serotonergic neurons of the RVM. We also show that microinjection of NT or the NTR1-selective agonist PD149163 in the RVM both produce dose-dependent antinociception in the tail-flick test that is blocked by the NTR1-selective antagonist SR48692. The antinociception produced by NT or PD149163 is also blocked by intrathecal administration of the non-selective serotonergic receptor antagonist methysergide. The results of these experiments provide anatomical and behavioral evidence that activation of NTR1-expressing spinally projecting neurons in the RVM produces antinociception through release of serotonin in the spinal dorsal horn. These results support the conclusion that the NTR1 plays an important role in the central modulation of nociception.

Separate populations of neurons in the rostral ventromedial medulla project to the spinal cord and to the dorsolateral pons in the rat.

Activation of neurons in the rostral ventromedial medulla (RVM) directly modulates spinal nociceptive transmission by projections to the spinal cord dorsal horn and indirectly by projections to neurons in the dorsolateral pons (DLP) that project to the spinal cord dorsal horn. However, it is not known whether the same neurons in the RVM produce both direct and indirect modulation of nociception. Deposits of the retrograde tracers Fluoro-Gold (FG) in the spinal cord dorsal horn and DiI in the DLP were used to determine whether the same RVM neurons project to both of these regions. Only 0.9+/-0.1% of RVM neurons retrogradely labeled with Fluoro-Gold from the spinal cord were also labeled with DiI placed in the DLP. In addition, spinally projecting RVM neurons were significantly larger than RVM neurons that project to the DLP. Finally, spinally projecting neurons were found predominantly on the midline and within the RVM; neurons that project to the DLP had a wider distribution and were present both within and outside of the RVM. Thus, separate and morphologically distinct populations of RVM neurons appear to modulate nociception by direct and indirect descending pathways.

Ultrastructural analysis of ventrolateral periaqueductal gray projections to the A7 catecholamine cell group.

Stimulation of neurons in the ventrolateral periaqueductal gray produces antinociception that is mediated in part by pontine noradrenergic neurons. Previous light microscopic analysis provided suggestive evidence for a direct projection from neurons in the ventrolateral periaqueductal gray to noradrenergic neurons in the A7 cell group that innervate the spinal cord dorsal horn. Therefore, the present ultrastructural study used anterograde tracing combined with tyrosine hydroxylase immunoreactivity to provide definitive evidence that neurons in the ventrolateral periaqueductal gray form synapses with the somata and dendrites of noradrenergic neurons of the A7 cell group. Injections of the anterograde tracers biotinylated dextran amine or Phaseolus vulgaris leucoagglutinin into the ventrolateral periaqueductal gray of Sasco Sprague-Dawley rats yielded a dense innervation in the region of the lateral pons containing the A7 cell group. Electron microscopic analysis of anterogradely labeled terminals (n=401) in the region of the A7 cell group indicated that approximately 10% of these formed plasmalemmal appositions to tyrosine hydroxylase-immunoreactive dendrites with no intervening astrocytic processes. About 23% of these were asymmetric synapses, 10% were symmetric synapses, and 67% did not exhibit clearly differentiated synaptic specializations. The majority of anterogradely labeled terminals (60%) formed plasmalemmal appositions with dendrites and somata that lacked detectable tyrosine hydroxylase immunoreactivity. About 35% of these were symmetric synapses, 9% were asymmetric synapses and 56% did not form synaptic specializations. Approximately 30% of all anterogradely labeled terminals displayed features characteristic of axo-axonic synapses.The present results provide direct ultrastructural evidence to support the hypothesis that the analgesia produced by stimulation of neurons in the ventrolateral periaqueductal gray is mediated, in part, by activation of spinally projecting noradrenergic neurons in the A7 catecholamine cell group.

Periaqueductal gray neurons monosynaptically innervate extranuclear noradrenergic dendrites in the rat pericoerulear region.

Previous reports using light microscopy have provided anatomical evidence that neurons in the ventrolateral periaqueductal gray (PAG) innervate the medial pericoerulear dendrites of noradrenergic neurons in the nucleus locus coeruleus (LC). The present study used anterograde tracing and electron microscopic analysis to provide more definitive evidence that neurons in the ventrolateral PAG form synapses with the somata or dendrites of noradrenergic LC neurons. Deposits of either biotinylated dextran amine or Phaseolus vulgaris leucoagglutinin into the rat ventrolateral PAG labeled a moderate to high number of axons in the region of the medial pericoerulear region and Barrington's nucleus, but a relatively low number were labeled in the nuclear core of the LC. Ultrastructural analysis of anterogradely labeled terminals at the levels of the rostral (n = 233) and caudal (n = 272) subdivisions of the LC indicated that approximately 20% of these form synapses with tyrosine hydroxylase-immunoreactive dendrites; most of these were located in the medial pericoerulear region. In rostral sections, about 12% of these were symmetric synapses, 9% were asymmetric synapses, and 79% were membrane appositions without clear synaptic specializations. In caudal sections, about 30% were symmetric synapses, 11% were asymmetric synapses, and 59% were appositions. In both rostral and caudal sections, 60% of the anterogradely labeled terminals formed synapses with noncatecholamine dendrites, and 20% formed axoaxonic synapses. These results provide direct evidence for monosynaptic projections from neurons in the ventrolateral PAG to the extranuclear dendrites of noradrenergic LC neurons. This monosynaptic pathway may mediate in part the analgesia, reduced responsiveness to external stimuli, and decreased excitability of somatic motoneurons produced by stimulation of neurons in the ventrolateral PAG.

Bidirectional modulation of nociception by GABA neurons in the dorsolateral pontine tegmentum that tonically inhibit spinally projecting noradrenergic A7 neurons.

The A7 catecholamine cell group in the dorsolateral pontine tegmentum constitutes an important part of the descending pathways that modulate nociception. Evidence from immunocytochemical studies demonstrate that noradrenergic A7 neurons are densely innervated by GABA terminals arising from GABA neurons that are located in the dorsolateral pontine tegmentum medial to the A7 cell group. GABA(A) receptors are also located on the somata and dendrites of noradrenergic A7 neurons. These findings suggest that noradrenergic neurons in the A7 cell group may be under tonic inhibitory control by GABA neurons. To test this hypothesis, the GABA(A) antagonist bicuculline methiodide in doses of 0.2 or 1.0nmol was microinjected into sites located dorsal to the A7 cell group and the resulting effects on tail flick and nociceptive foot withdrawal responses were measured. Both doses of bicuculline produced significant increases in tail flick latencies and small, but significant, increases in foot withdrawal latencies. Intrathecal injection of the alpha(2)-adrenoceptor antagonist yohimbine, in a dose of 76.7nmol (30microg), attenuated the antinociceptive effect of bicuculline on both the tail and the feet. In contrast, the alpha(1)-adrenoceptor antagonist WB4101, in a nearly equimolar dose of 78.6nmol (30microg), increased the antinociceptive effect of bicuculline on both the tail and the feet. Intrathecal injection of the antagonists alone did not consistently alter nociceptive responses of either the feet or the tail. These findings suggest that noradrenergic neurons in the A7 cell group are tonically inhibited by local GABA neurons. Furthermore, these findings suggest that inhibition of GABA(A) receptors located on spinally-projecting A7 noradrenergic neurons disinhibits, or activates, two populations of A7 neurons that have opposing effects on nociception. One of these populations facilitates nociception by an action mediated by alpha(1)-adrenoceptors in the spinal cord dorsal horn and the other population inhibits nociception by an action mediated by alpha(2)-adrenoceptors.

Synergistic interactions between two alpha(2)-adrenoceptor agonists, dexmedetomidine and ST-91, in two substrains of Sprague-Dawley rats.

Several lines of evidence indicate that the antinociception produced by intrathecal administration of the alpha(2)-adrenoceptor agonists dexmedetomidine or ST-91 is mediated by different subtypes of the alpha(2)-adrenoceptor. We recently provided additional pharmacologic evidence for this idea, as well as for differences in the function of these receptors between Harlan and Sasco rats, two widely-used outbred substrains of Sprague-Dawley rat. The present study used isobolographic analysis to further characterize the receptors at which intrathecally administered ST-91 and dexmedetomidine act in these two substrains. The rationale for these studies derives from the assumption that if dexmedetomidine and ST-91 act as agonists at the same receptor then they should interact in an additive manner. However, if they interact in a supra-additive manner, then they must act at different subtypes of the alpha(2)-adrenoceptor. In the tail-flick test, the dose-effect relationship for a 1:3 mixture of dexmedetomidine and ST-91 was shifted significantly to the left of the theoretical dose-additive line in both Harlan and Sasco Sprague-Dawley rats. A similar finding was made in the hot-plate test despite the fact that the dose-response characteristics of the agonists were different in this test. Thus, in Harlan rats, in which ST-91 is a full agonist and dexmedetomidine is essentially inactive, the dose-effect relationship for the mixture of dexmedetomidine and ST-91 was shifted far to the left of the dose-additive line. Similarly, in Sasco rats, in which ST-91 is a partial agonist and dexmedetomidine is inactive, co-administration of the two agonists also shifted the dose-response relationship to the left of the dose-additive line. The consistent finding that these two alpha(2)-adrenoceptor agonists interact in a supra-additive manner provides strong evidence that dexmedetomidine and ST-91 produce antinociception by acting at different alpha(2)-adrenoceptor subtypes in the spinal cord. This conclusion is consistent with the earlier proposal that dexmedetomidine acts predominantly at alpha(2A)-adrenoceptors whereas ST-91 acts predominantly at non-alpha(2A)-adrenoceptors. Recent anatomical evidence indicates that these non-alpha(2A) adrenoceptors may be of the alpha(2C) type. The synergistic combination of an alpha(2A)- and an alpha(2C)-adrenoceptor agonist may provide a unique and highly effective drug combination for the treatment of pain without the sedation produced by an equianalgesic dose of a single alpha(2)-adrenoceptor agonist.

Ultrastructural evidence that substance P neurons form synapses with noradrenergic neurons in the A7 catecholamine cell group that modulate nociception.

Potent antinociception can be produced by electrical stimulation of spinally projecting noradrenergic neurons in the A7 catecholamine cell group and this effect is blocked by intrathecal injection of alpha2-adrenoceptor antagonists. Microinjection of substance P near A7 neurons also produces antinociception that is blocked by intrathecal injection of alpha2-adrenoceptor antagonists. These observations suggest that substance P produces antinociception by activating noradrenergic A7 neurons. However, it is not known whether this effect of substance P is produced by a direct or an indirect action on A7 neurons. Although light microscopic studies have demonstrated the existence of both substance P-containing axon terminals and neurokinin-1 receptors in the region of the A7 cell group, it is not known whether substance P terminals form synapses with noradrenergic A7 neurons. These experiments used double-labeling immunocytochemical methods and electron microscopic analysis to determine whether substance P-containing axons form synapses with noradrenergic neurons in the A7 cell group. Pre-embedding immunocytochemistry, combined with light and electron microscopic analysis, was used to provide ultrastructural evidence for synaptic connections between substance P-immunoreactive terminals labeled with immunoperoxidase and tyrosine hydroxylase-immunoreactive A7 neurons labeled with silver-enhanced immunogold. Tyrosine hydroxylase labeling was found in perikarya and dendrites in the A7 region, and substance P labeling was found in axons and synaptic terminals. Substance P-labeled terminals formed asymmetric synapses with tyrosine hydroxylase-labeled dendrites, but only a few of these were present on tyrosine hydroxylase-labeled somata. Substance P-labeled terminals also formed asymmetric synapses with unlabeled dendrites, and many unlabeled terminals formed both symmetric and asymmetric synapses with tyrosine hydroxylase-labeled dendrites. These results demonstrate that substance P neurons form a significant number of synapses with the dendrites of noradrenergic A7 neurons and support the conclusion that microinjection of substance P in the A7 cell group produces antinociception by direct activation of spinally projecting noradrenergic neurons.

Microinjection of morphine in the A7 catecholamine cell group produces opposing effects on nociception that are mediated by alpha1- and alpha2-adrenoceptors.

Stimulation of neurons in the ventromedial medulla produces antinociception in part by inhibiting nociceptive dorsal horn neurons. This antinociceptive effect is mediated in part by spinally projecting noradrenergic neurons located in the A7 catecholamine cell group. Methionine-enkephalin-immunoreactive neurons in the ventromedial medulla project to an area that includes the A7 cell group, and these enkephalin neurons may mediate part of the antinociception produced by stimulation of sites in the ventromedial medulla. This possibility was tested by determining the effects of microinjecting morphine near the A7 cell group on nociceptive foot and tail responses. Microinjection of a 3.75 nmol dose of morphine in the A7 region did not alter nociceptive responses, but a higher dose of 7.5 nmol facilitated these responses. In contrast, a higher dose of 15 nmol of morphine did not alter nociceptive responses. Selective alpha-adrenoceptor antagonists were injected intrathecally to determine whether the hyperalgesia produced by morphine is mediated by spinally projecting noradrenergic A7 neurons. Intrathecal injection of the alpha2-adrenoceptor antagonist yohimbine did not alter the hyperalgesic effect produced by the 7.5 nmol dose of morphine, but the alpha1 antagonist WB4101 reversed the hyperalgesia and produced antinociception that lasted for nearly 30 min. Although the 15 nmol dose of morphine did not alter nociceptive responses, intrathecal injection of yohimbine after the microinjection of morphine produced a significant facilitation of nociception, and intrathecal injection of WB401 produced a significant antinociceptive effect. Intrathecal injection of the antagonists alone did not consistently alter nociception. These findings, and those of published reports, suggest that morphine indirectly activates two populations of spinally projecting A7 noradrenergic neurons that have opposing effects on nociception. One of these populations facilitates nociception by an action mediated by alpha1-adrenoceptors in the spinal cord dorsal horn and the other population inhibits nociception by an action mediated by alpha2-adrenoceptors. These results suggest that some of the methionine-enkephalin neurons located in the ventromedial medulla that project to the A7 cell group can exert bidirectional control of nociceptive responses.

Projections of neurons in the periaqueductal gray to pontine and medullary catecholamine cell groups involved in the modulation of nociception.

Stimulation of neurons in the periaqueductal gray (PAG) produces antinociception that is mediated in part by noradrenergic neurons that innervate the spinal cord dorsal horn. Because norepinephrine-containing neurons are not found in the PAG, noncatecholamine neurons in the PAG must project to, and activate, spinally projecting catecholamine neurons located in the pons or medulla. The present studies determined the projections of neurons in the ventrolateral PAG to the A5, A6 (locus coeruleus), and A7 catecholamine cell groups that are known to contain spinally projecting noradrenergic neurons. The anterograde tracer biotinylated dextran amine (BDA) was injected into the ventrolateral PAG, and labeled axon terminal profiles were identified near noradrenergic neurons that were visualized by processing tissue sections for tyrosine hydroxylase immunoreactivity. Highly varicose, anterogradely labeled terminal profiles were found apposed to the dendrites and somata of tyrosine-hydroxylase-immunoreactive neurons and non-tyrosine-hydroxylase-immunoreactive neurons in the dorsolateral and ventrolateral pontine tegmentum. These axon terminal profiles were more dense on the side ipsilateral to the BDA deposit, and both A7 and locus coeruleus neurons received a more dense innervation than did the A5 neurons. Although definitive evidence for a direct pathway from PAG neurons to spinally projecting A7 neurons requires ultrastructural studies, the results of the present studies provide presumptive evidence for direct projections from neurons in the PAG to noradrenergic A7 neurons that innervate the spinal cord dorsal horn and modulate pain perception. If neurons in the ventrolateral PAG do form synapses with noradrenergic A7 neurons, these spinally projecting catecholamine neurons may mediate part of the analgesic effect produced by systemic administration of morphine. In contrast, the projections of PAG neurons to the A5 cell group and the locus coeruleus may mediate the cardiovascular and motor effects produced by stimulation of sites in the ventrolateral PAG.

The antinociception produced by microinjection of a cholinergic agonist in the ventromedial medulla is mediated by noradrenergic neurons in the A7 catecholamine cell group.

Activation of neurons in the ventromedial medulla by electrical stimulation or by microinjection of opioid or cholinergic agonists produces antinociception that is mediated in part by spinally-projecting noradrenergic neurons. Several lines of evidence indicate that these noradrenergic neurons are located in the pontine A7 catecholamine cell group. For example, anatomical studies have demonstrated that neurons in the ventromedial medulla project to the noradrenergic neurons in the A7 catecholamine cell group that provide the major noradrenergic innervation of the spinal cord dorsal horn. In addition, electrical and chemical stimulation of A7 neurons produces antinociception that can be reduced by intrathecal injection of alpha2-adrenoceptor antagonists. The present studies provide more direct evidence that activation of neurons in the ventromedial medulla produces antinociception by activating noradrenergic neurons in the A7 cell group. Neurons in the ventromedial medulla were stimulated by microinjecting the cholinergic agonist carbachol (5 microg) into sites in the nucleus raphe magnus or the nucleus gigantocellularis pars alpha of pentobarbital anesthetized Sprague-Dawley rats. In some experiments, the local anesthetic tetracaine (10 microg) was then microinjected near the A7 cell group to inactivate the spinally-projecting noradrenergic neurons. In other experiments, cobalt chloride (100 mM) was microinjected near the A7 cell group to block synaptic activation of spinally-projecting noradrenergic neurons. Microinjection of carbachol into sites in the ventromedial medulla produced antinociception, assessed using the tail flick test, that lasted more than 60 min. However, the effects of carbachol were attenuated by microinjection of either tetracaine or cobalt into sites near the A7 cell group neurons identified by tyrosine hydroxylase-immunoreactivity. Similar injections of tetracaine or cobalt more than 500 microm from the A7 neurons did not alter the antinociceptive effect of carbachol. These results support the conclusion that the antinociception produced by activating neurons in the ventromedial medulla is mediated in part by the subsequent activation of spinally-projecting noradrenergic neurons in the A7 cell group.

Antinociception produced by microinjection of morphine in the rat periaqueductal gray is enhanced in the foot, but not the tail, by intrathecal injection of alpha1-adrenoceptor antagonists.

Antinociception produced by microinjection of morphine in the ventrolateral periaqueductal gray is mediated in part by alpha2-adrenoceptors in the spinal cord dorsal horn. However, several recent reports demonstrate that microinjection of morphine in the ventrolateral periaqueductal gray inhibits nociceptive responses to noxious heating of the tail by activating descending neuronal systems that are different from those that inhibit the nociceptive responses to noxious heating of the feet. More specifically, alpha2-adrenoceptors appear to mediate the antinociception produced by morphine using the tail-flick test, but not that using the foot-withdrawal or hot-plate tests. The present study extended these findings and determined the role of alpha1-adrenoceptors in mediating the antinociceptive effects of morphine microinjected into the ventrolateral periaqueductal gray using both the foot-withdrawal and the tail-flick responses to noxious radiant heating in lightly anesthetized rats. Intrathecal injection of selective antagonists was used to determine whether the antinociceptive effects of morphine were modulated by alpha1-adrenoceptors. Injection of the selective alpha1-adrenoceptor antagonists prazosin or WB4101 potentiated the increase in the foot-withdrawal response latency produced by microinjection of morphine in the ventrolateral periaqueductal gray. In contrast, either prazosin or WB4101 partially reversed the increase in the tail-flick response latency produced by morphine. These results indicate that microinjection of morphine in the ventrolateral periaqueductal gray modulates nociceptive responses to noxious heating of the feet by activating descending neuronal systems that are different from those that inhibit the nociceptive responses to noxious heating of the tail. More specifically, alpha1-adrenoceptors mediate a pro-nociceptive action of morphine using the foot-withdrawal response, but in contrast, alpha1-adrenoceptors appear to mediate part of the antinociceptive effect of morphine determined using the tail-flick test.

Enkephalin neurons that project to the A7 catecholamine cell group are located in nuclei that modulate nociception: ventromedial medulla.

The location of methionine enkephalin neurons in the medulla oblongata that project to the dorsolateral pontine tegmentum was investigated using anterograde and retrograde tract tracing combined with immunocytochemical neurotransmitter identification. The results of these experiments demonstrate that enkephalinergic neurons from areas known to modulate nociception project to the region of the A7 catecholamine cell group in the dorsolateral pontine tegmentum. The medullary nuclei that contain these enkephalinergic neurons include the nucleus raphe magnus and the nucleus reticularis gigantocellularis pars alpha in the ventromedial medulla. While some of these enkephalinergic axons appose the somata and dendrites of A7 neurons, the majority of these axons appear to contact non-catecholamine neurons in the dorsolateral pontine tegmentum. Unidentified neurons located in the nucleus raphe magnus, the nucleus reticularis gigantocellularis pars alpha, and the nucleus reticularis gigantocellularis also project to the A7 area. Many of the neurons in the nucleus reticularis gigantocellularis pars alpha appear to contact both noradrenergic A7 neurons and non-catecholamine neurons in the dorsolateral pontine tegmentum, whereas most of those in the nucleus raphe magnus appear to contact non-catecholamine neurons. The anatomical findings described in this report and the results of preliminary behavioral studies provide evidence to support a model in which activation of the enkephalin-containing neurons in the ventromedial medulla facilitates nociception, while the non-enkephalin neurons mediate part of the antinociception produced by stimulating sites in the ventromedial medulla.

Differences in the antinociceptive effects of alpha-2 adrenoceptor agonists in two substrains of Sprague-Dawley rats.

In this study, we examined whether Sprague-Dawley rats obtained from two different vendors, Harlan and Sasco, differ with respect to the types of alpha-2 adrenoceptors in the spinal cord that mediate antinociception. This hypothesis was tested using two alpha-2 adrenoceptor agonists, dexmedetomidine and ST-91, which are relatively selective for alpha-2A and alpha-2B adrenoceptors, respectively, and two different measures of nociception, the tail-flick and the 55 degrees C hot-plate test. Dexmedetomidine and ST-91 each increased tail-flick latency to a similar extent in both Harlan and Sasco rats, although dexmedetomidine was more efficacious than ST-91 in each substrain. However, the efficacy of these agonists was markedly different in Harlan and Sasco rats when the hot-plate test was used. For example, ST-91 was a full agonist in the hot-plate test in Harlan rats but a weak partial agonist in Sasco rats. Dexmedetomidine was a very weak partial agonist in Harlan rats and ineffective in the hot-plate test in Sasco rats. These findings suggest that (1) both spinal alpha-2A and alpha-2B receptors modulate nociceptive responses in the tail-flick test in both Harlan and Sasco rats; (2) hot-plate responses are mediated predominantly by alpha-2B adrenoceptors, with a minimal contribution by alpha-2A adrenoceptors in the Harlan rat and (3) hot-plate responses are not appreciably affected by either alpha-2A or alpha-2B adrenoceptors in the Sasco rat. These findings confirm previous reports that intrathecal administration of alpha-2 adrenoceptor agonists produces thermal antinociception in the rat. However, the magnitude of the antinociceptive effect is dependent on the receptor selectivity of the agonist used, cutaneous tissue stimulated to elicit nociceptive responses and substrain of rat.

Spinal cholinergic and monoamine receptors mediate the antinociceptive effect of morphine microinjected in the periaqueductal gray on the rat tail, but not the feet.

The antinociceptive effects of morphine (5 micrograms) microinjected into the ventrolateral periaqueductal gray were determined using both the tail flick and the foot withdrawal responses to noxious radiant heating in lightly anesthetized rats. Intrathecal injection of appropriate antagonists was used to determine whether the antinociceptive effects of morphine were mediated by alpha 2-noradrenergic, serotonergic, opioid, or cholinergic muscarinic receptors. The increase in the foot withdrawal response latency produced by microinjection of morphine in the ventrolateral periaqueductal gray was reversed by intrathecal injection of the cholinergic muscarinic receptor antagonist atropine, but was not affected by the alpha 2-adrenoceptor antagonist yohimbine, the serotonergic receptor antagonist methysergide, or the opioid receptor antagonist naloxone. In contrast, the increase in the tail flick response latency produced by morphine was reduced by either yohimbine, methysergide or atropine. These results indicate that microinjection of morphine in the ventrolateral periaqueductal gray inhibits nociceptive responses to noxious heating of the tail by activating descending neuronal systems that are different from those that inhibits the nociceptive responses to noxious heating of the feet. More specifically, serotonergic, muscarinic cholinergic and alpha 2-noradrenergic receptors appear to mediate the antinociception produced by morphine using the tail flick test. In contrast, muscarinic cholinergic, but not monoamine receptors appear to mediate the antinociceptive effects of morphine using the foot withdrawal response.

The function of noradrenergic neurons in mediating antinociception induced by electrical stimulation of the locus coeruleus in two different sources of Sprague-Dawley rats.

Although noradrenergic neurons in the nucleus locus coeruleus are known to project to the spinal cord, these neurons appear to innervate different regions of the spinal cord in Sprague-Dawley rats obtained from two different vendors. Recent anatomical studies demonstrated that the noradrenergic neurons in the locus coeruleus in Sasco Sprague-Dawley rats primarily innervate the ventral horn, whereas Harlan Sprague-Dawley rats have coeruleospinal projections that terminate in the dorsal horn of the spinal cord. This report describes the results of behavioral experiments that were designed to determine the functional significance of these anatomical differences. Electrical stimulation of neurons in the locus coeruleus produced antinociception in both Harlan and Sasco rats. The antinociception in Harlan rats was readily reversed by intrathecal injection of yohimbine, a selective alpha 2-adrenoceptor antagonist, or by phentolamine, a non-selective alpha 2-adrenoceptor antagonist. In contrast, these antagonists did not alter the antinociception produced by locus coeruleus stimulation in Sasco rats. Finally, the alpha 2-antagonist, idazoxan, did not alter the antinociceptive effect of locus coeruleus stimulation in either group of rats. These observations indicate that coeruleospinal noradrenergic neurons in Harlan and Sasco Sprague-Dawley rats have different physiological functions. Thus, electrical stimulation of noradrenergic neurons in the locus coeruleus that innervate the spinal cord dorsal horn (Harlan rats) produces antinociception, but stimulation of coeruleospinal noradrenergic neurons that project to the ventral horn (Sasco rats) does not produce antinociception. It is likely that genetic differences between these outbred stocks of rats account for the fundamental differences in the projections of coeruleospinal neurons and their function in controlling nociception.

The projections of noradrenergic neurons in the A5 catecholamine cell group to the spinal cord in the rat: anatomical evidence that A5 neurons modulate nociception.

Brainstem noradrenergic neurons located in the A5, A6, and A7 catecholamine cell groups provide the entire noradrenergic innervation of the spinal cord. We have previously demonstrated that noradrenergic neurons in the A6 and A7 cell groups innervate the ventral and dorsal horns, respectively. Since the specific spinal cord terminations of the A5 cell group have not been clearly delineated, the present experiments were designed to trace the projections from this noradrenergic cell group to the spinal cord, using the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) in combination with dopamine-beta-hydroxylase immunocytochemistry. The results of these experiments indicate that A5 noradrenergic neurons project ipsilaterally through the dorsolateral funiculus in cervical, thoracic, and lumbar segments. In cervical segments, these axons terminate primarily in the ipsilateral deep dorsal horn (laminae IV-VI) and the intermediate zone (lamina VII). In thoracic segments, the intermediolateral cell column is heavily innervated by A5 axons. In lumbar segments, the concentration of A5 axons is more diffuse and more widely distributed than that in cervical and thoracic segments. Although there is a higher density of axons in the deep dorsal horn and the intermediate zone, there are also scattered axons in the dorsal and ventral horns. The innervation of these regions of the spinal cord by A5 neurons provides anatomical support for the conclusion that these noradrenergic neurons are involved in modulating cardiovascular reflexes and nociceptive transmission in the spinal cord.

Anatomical evidence for genetic differences in the innervation of the rat spinal cord by noradrenergic locus coeruleus neurons.

Pontospinal noradrenergic neurons located in the A5, A6 (locus coeruleus, LC), and A7 cell groups are the major source of the noradrenergic innervation of the spinal cord. We have recently examined the specific terminations of these three cell groups in the spinal cord and found that the LC provides the major noradrenergic innervation of the ventral horn, while the A7 and A5 cell groups innervate the dorsal horn and intermediate zone, respectively. However, the results of similar experiments from another laboratory have shown that noradrenergic neurons in the locus coeruleus primarily innervate the dorsal horn, while the A5 and A7 innervate the intermediate zone and the ventral horn. These conflicting results may be due to fundamental genetic differences between the rats used in our experiments (Sasco Sprague-Dawley) and those used by the other laboratory (Harlan Sprague-Dawley). This possibility was examined by determining the projections of coeruleospinal neurons in these two rat substrains using the anterograde tracer Phaseolus vulgaris leucoagglutinin. The results indicate that in Sasco rats the LC neurons project through the ipsilateral ventromedial funiculus and terminate almost exclusively in the medial part of laminae VII and VIII, the motoneuron pool of lamina IX, and lamina X. In contrast, LC neurons in Harlan rats project bilaterally through the superficial dorsal horn and the dorsolateral funiculus and terminate most heavily in dorsal horn laminae I-IV. In addition, the LC neurons of Sasco rats innervate cervical spinal cord segments more densely than lumbar spinal cord segments, while in Harlan rats the lumbar spinal cord is more densely innervated than the cervical spinal cord. These results indicate that the projections of coeruleospinal neurons in Sasco rats are fundamentally different from those in Harlan rats and suggest that noradrenergic LC neurons may have different physiological functions in these two rat substrains.

Antinociception induced by microinjection of substance P into the A7 catecholamine cell group in the rat.

Stimulation of neurons in the ventromedial medulla produces antinociception that is mediated in part by indirect activation of pontospinal noradrenergic neurons. Substance P-containing neurons located in the ventromedial medulla project to the A7 catecholamine cell group and may serve as an excitatory link between these two cell groups. Thus, the antinociception induced by stimulation of the neurons in ventromedial medulla may be mediated by substance P released from these projections which activates spinally projecting noradrenergic neurons in the A7 cell group. This hypothesis was tested by determining whether microinjection of various doses of substance P into the A7 cell group of the rat could induce antinociception. The results indicated that substance P induced dose-dependent antinociception that was more pronounced in the hindlimb ipsilateral to the microinjections. This observation is consistent with anatomical observations that noradrenergic A7 neurons project predominantly to the ipsilateral spinal cord dorsal horn. Moreover, the antinociceptive effects of substance P microinjection appear to be mediated at least in part by activation of spinally projecting noradrenergic neurons in the A7 cell group, because intrathecal injections of the alpha-2 noradrenergic antagonists yohimbine and idazoxan blocked these antinociceptive effects. The results of these experiments support the hypothesis that the antinociception induced by stimulation of neurons in the ventromedial medulla is mediated in part by activation of substance P-containing neurons that project to, and activate, spinally projecting noradrenergic neurons located in the A7 catecholamine cell group.