Baicalin Activates Glycine and γ-Aminobutyric Acid Receptors on Substantia Gelatinosa Neurons of the Trigeminal Subsnucleus Caudalis in Juvenile Mice.

The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) receives nociceptive afferent inputs from thin-myelinated A[Formula: see text] fibers and unmyelinated C fibers and has been shown to be involved in the processing of orofacial nociceptive information. Scutellaria baicalensis Georgi (Huang-Qin, SbG), one of the 50 fundamental herbs of Chinese herbology, has been used historically as anti-inflammatory and antineoplastic medicine. Baicalin, one of the major compounds of SbG, has been reported to have neuroprotective, anti-inflammatory and analgesic effects. However, the receptor type activated by baicalin and its precise action mechanism on the SG neurons of Vc have not yet been studied. The whole-cell patch clamp technique was performed to examine the ion channels activated by baicalin on the SG neurons of Vc. In high Cl[Formula: see text] pipette solution, the baicalin (300[Formula: see text][Formula: see text]M) induced repeatable inward currents ([Formula: see text][Formula: see text]pA, [Formula: see text]) without desensitization on all the SG neurons tested. Further, the inward currents showed a concentration (0.1-3[Formula: see text]mM) dependent pattern. The inward current was sustained in the presence of tetrodotoxin (0.5[Formula: see text][Formula: see text]M), a voltage sensitive Na[Formula: see text] channel blocker. In addition, baicalin-induced inward currents were reduced in the presence of picrotoxin (50[Formula: see text][Formula: see text]M), a GABAA receptor antagonist, flumazenil (100[Formula: see text][Formula: see text]M), a benzodiazepine-sensitive GABAA receptor antagonist, and strychnine (2[Formula: see text][Formula: see text]M), a glycine receptor antagonist, respectively. These results indicate that baicalin has inhibitory effects on the SG neurons of the Vc, which are due to the activation of GABAA and/or the glycine receptor. Our results suggest that baicalin may be a potential target for orofacial pain modulation.

GABAergic influence on temporomandibular joint-responsive spinomedullary neurons depends on estrogen status.

Sensory input from the temporomandibular joint (TMJ) to neurons in superficial laminae at the spinomedullary (Vc/C1-2) region is strongly influenced by estrogen status. This study determined if GABAergic mechanisms play a role in estrogen modulation of TMJ nociceptive processing in ovariectomized female rats treated with high- (HE) or low-dose (LE) estradiol (E2) for 2days. Superficial laminae neurons were activated by ATP (1mM) injections into the joint space. The selective GABAA receptor antagonist, bicuculline methiodide (BMI, 5 or 50µM, 30µl), applied at the site of recording greatly enhanced the magnitude and duration of ATP-evoked responses in LE rats, but not in units from HE rats. The convergent cutaneous receptive field (RF) area of TMJ neurons was enlarged after BMI in LE but not HE rats, while resting discharge rates were increased after BMI independent of estrogen status. By contrast, the selective GABAA receptor agonist, muscimol (50µM, 30µl), significantly reduced the magnitude and duration of ATP-evoked activity, resting discharge rate, and cutaneous RF area of TMJ neurons in LE and HE rats, whereas lower doses (5µM) affected only units from LE rats. Protein levels of GABAA receptor ß3 isoform at the Vc/C1-2 region were similar for HE and LE rats. These results suggest that GABAergic mechanisms contribute significantly to background discharge rates and TMJ-evoked input to superficial laminae neurons at the Vc/C1-2 region. Estrogen status may gate the magnitude of GABAergic influence on TMJ neurons at the earliest stages of nociceptive processing at the spinomedullary region.

Bilateral descending hypothalamic projections to the spinal trigeminal nucleus caudalis in rats.

Several lines of evidence suggest that the hypothalamus is involved in trigeminal pain processing. However, the organization of descending hypothalamic projections to the spinal trigeminal nucleus caudalis (Sp5C) remains poorly understood. Microinjections of the retrograde tracer, fluorogold (FG), into the Sp5C, in rats, reveal that five hypothalamic nuclei project to the Sp5C: the paraventricular nucleus, the lateral hypothalamic area, the perifornical hypothalamic area, the A11 nucleus and the retrochiasmatic area. Descending hypothalamic projections to the Sp5C are bilateral, except those from the paraventricular nucleus which exhibit a clear ipsilateral predominance. Moreover, the density of retrogradely FG-labeled neurons in the hypothalamus varies according to the dorso-ventral localization of the Sp5C injection site. There are much more labeled neurons after injections into the ventrolateral part of the Sp5C (where ophthalmic afferents project) than after injections into its dorsomedial or intermediate parts (where mandibular and maxillary afferents, respectively, project). These results demonstrate that the organization of descending hypothalamic projections to the spinal dorsal horn and Sp5C are different. Whereas the former are ipsilateral, the latter are bilateral. Moreover, hypothalamic projections to the Sp5C display somatotopy, suggesting that these projections are preferentially involved in the processing of meningeal and cutaneous inputs from the ophthalmic branch of the trigeminal nerve in rats. Therefore, our results suggest that the control of trigeminal and spinal dorsal horn processing of nociceptive information by hypothalamic neurons is different and raise the question of the role of bilateral, rather than unilateral, hypothalamic control.

Ocular surface-evoked Fos-like immunoreactivity is enhanced in trigeminal subnucleus caudalis by prior exposure to endotoxin.

Endotoxin-induced uveitis (EIU) is a common animal model for anterior uveitis in humans that causes long-term changes in trigeminal brain stem neurons. This study used c-fos immunohistochemistry to assess the effects of different routes of administration of endotoxin on activation of trigeminal brain stem neurons produced by ocular surface stimulation. A single dose of endotoxin (lipopolysaccharide (LPS)) given to male rats by systemic (i.p., 1 mg/kg) or intraocular (ivt, 20 microg) routes increased the number of Fos-positive neurons in rostral (trigeminal subnucleus interpolaris/subnucleus transition (Vi/Vc)) and caudal portions of trigeminal subnucleus caudalis (trigeminal subnucleus caudalis/upper cervical spinal cord transition (Vc/C(1-2))) by 20% mustard oil (MO) applied to the ocular surface 7 days, but not at 2 days, after LPS compared with naïve rats. I.c.v. (20 microg) LPS did not affect MO-evoked Fos. To determine if the pattern of enhanced Fos expression after systemic LPS also depended on the nature of the ocular surface stimulus, additional groups received ocular stimulation by 10% histamine or dry eye conditions. Seven days, but not 2 days, after i.p. LPS both histamine- and dry eye-evoked Fos was increased at the Vi/Vc transition, while smaller effects were seen at other regions. These results suggested that EIU modulation of trigeminal brain stem neuron activity was mediated mainly by peripheral actions of LPS. Enhancement of Fos at the Vi/Vc region after MO, histamine and dry eye conditions supports the hypothesis that this region integrates innocuous as well as noxious sensory information, while more caudal portions of Vc process mainly nociceptive signals from the eye.

Glutamine uptake contributes to central sensitization in the medullary dorsal horn.

Mustard oil application to tooth pulp produces central sensitization in rat medullary dorsal horn (MDH) nociceptive neurons, which has been implicated in persistent pain mechanisms. We found that superfusion onto MDH of methylaminoisobutyric acid, a competitive inhibitor of the neuronal system A transporter for presynaptic uptake of glutamine (a glutamate precursor released from astroglia), significantly depressed development of mustard oil-induced central sensitization in rat MDH nociceptive neurons. This finding indicates that the system A transporter is required for the expression of central sensitization and confirms the important roles of astroglia, glutamine and presynaptic modulation of glutamate release in the development of central sensitization.

Effect of persistent monoarthritis of the temporomandibular joint region on acute mustard oil-induced excitation of trigeminal subnucleus caudalis neurons in male and female rats.

The effect of persistent inflammation of the temporomandibular (TMJ) region on Fos-like immunoreactivity (Fos-LI) evoked by acute noxious stimulation of the same or opposite TMJ was assessed in male and cycling female rats. Two weeks after inflammation of the TMJ by complete Freund's adjuvant (CFA, 25 microg) the selective small fiber excitant, mustard oil (MO, 20%), was injected into the arthritic or opposite TMJ under barbiturate anesthesia. MO stimulation of the arthritic TMJ increased Fos-LI ipsilateral, but not contralateral, to MO compared to naïve subjects in superficial laminae at the trigeminal subnucleus caudalis/upper cervical cord (Vc/C2) junction independent of sex hormone status. Unexpectedly, MO stimulation of the opposite TMJ in arthritic rats also produced a greater Fos-LI response ipsilateral to MO than naïve animals. Fos-LI produced in the dorsal paratrigeminal region (dPa5) and Vc/C2 junction after MO stimulation of the normal TMJ was significantly greater in proestrous than diestrous females or male monoarthritic rats. In contrast to naïve animals, Fos-LI was produced in deep laminae at the Vc/C2 junction ipsilateral to MO in CFA-treated animals independent of the site of prior CFA inflammation or sex hormone status. These results indicated that persistent monoarthritis of the TMJ region enhanced the excitability of trigeminal brainstem neurons to subsequent TMJ injury that occurred bilaterally in multiple regions of the lower trigeminal brainstem complex and depended on sex hormone status.

Endogenous ATP involvement in mustard-oil-induced central sensitization in trigeminal subnucleus caudalis (medullary dorsal horn).

Central sensitization represents a sustained hypersensitive state of dorsal horn nociceptive neurons that can be evoked by peripheral inflammation or injury to nerves and tissues. It reflects neuroplastic changes such as increases in neuronal spontaneous activity, receptive field size, and responses to suprathreshold stimuli and a decrease in activation threshold. We recently demonstrated that purinergic receptor mechanisms in trigeminal subnucleus caudalis (Vc; medullary dorsal horn) are also involved in the initiation and maintenance of central sensitization in brain stem nociceptive neurons of trigeminal subnucleus oralis. The aim of the present study was to investigate whether endogenous ATP is involved in the development of central sensitization in Vc itself. The experiments were carried out on urethan/alpha-chloralose anesthetized and immobilized rats. Single neurons were recorded and identified as nociceptive-specific (NS) in the deep laminae of Vc. During continuous saline superfusion (0.6 ml/h it) over the caudal medulla, Vc neuronal central sensitization was readily induced by mustard oil application to the tooth pulp. However, this mustard-oil-induced central sensitization could be completely blocked by continuous intrathecal superfusion of the wide-spectrum P2X receptor antagonist pyridoxal-phosphate-6-azophenyl-2, 4-disulphonic acid tetra-sodium (33-100 microM) and by apyrase (an ectonucleotidase enzyme, 30 units/ml). Superfusion of the selective P2X1, P2X3 and P2X(2/3) receptor antagonist 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (6-638 microM) partially blocked the Vc central sensitization. The two P2X receptor antagonists did not significantly affect the baseline nociceptive properties of the Vc neurons. These findings implicate endogenous ATP as an important mediator contributing to the development of central sensitization in nociceptive neurons of the deep laminae of the dorsal horn.

Distribution of trigeminothalamic and spinothalamic lamina I terminations in the macaque monkey.

Thalamic terminations from trigeminal, cervical, and lumbosacral lamina I neurons were investigated with Phaseolus vulgaris leucoagglutinin (PHA-L) and labeled dextrans. Iontophoretic injections guided by physiological recordings were restricted to lamina I or laminae I-II. PHA-L-labeled trigemino- and spinothalamic (TSTT) terminations were identified immunohistochemically. TRITC- and FITC-labeled dextrans were injected at different levels to confirm topography. Terminations consistently occurred in two main locations: a distinguishable portion of posterolateral thalamus identified cytoarchitectonically as the posterior part of the ventral medial nucleus (VMpo) and a portion of posteromedial thalamus designated as the ventral caudal part of the medial dorsal nucleus (MDvc). In addition, isolated fibers bearing boutons of passage were observed in the ventral posterior medial and lateral (VPM and VPL) nuclei, and spinal terminations occurred in the ventral posterior inferior nucleus (VPI). Isolated terminations occasionally occurred in other sites (e.g., suprageniculate, zona incerta, hypothalamic paraventricular n.). Terminations in MDvc occurred in concise foci that were weakly organized topographically (posteroanterior = rostrocaudal). Terminations in VMpo consisted of dense clusters of ramified terminal arbors bearing multiple large boutons that were well organized topographically (anteroposterior = rostrocaudal). Terminations in VMpo colocalized with a field of calbindin-immunoreactive terminal fibers; double-labeled terminals were documented at high magnification. This propitious marker was especially useful at anterior levels, where VMpo can easily be misidentified as VPM. These findings demonstrate phylogenetically novel primate lamina I TSTT projections important for sensory and motivational aspects of pain, temperature, itch, muscle ache, sensual touch, and other interoceptive feelings from the body.

Mustard oil has differential effects on the response of trigeminal caudalis neurons to heat and acidity.

Topical application of mustard oil (allyl isothiocyanate) to the skin or injection into joints induces hyperalgesia, allodynia, and neuroinflammation. However, when applied to the oral or nasal mucosa, mustard oil evokes a desensitizing pattern of irritation. Presently we investigated the responses of neurons in superficial laminae of trigeminal subnucleus caudalis (Vc) to noxious thermal (53 degrees C) and chemical (pentanoic acid; 200 mM) stimuli prior to and following lingual mustard oil application. A low concentration of mustard oil (0.125%) applied by constant flow (0.5 ml/min; 15 min), initially excited Vc neurons followed by partial desensitization. Responses to noxious heat were unchanged following mustard oil. A high concentration of mustard oil (1.25%) initially excited Vc neurons followed quickly (within 20 s) by nearly complete desensitization. The desensitization was transient since reapplication of mustard oil approximately 20 min later elicited a comparable response that also rapidly desensitized. Mustard oil also transiently cross-desensitized Vc responses to pentanoic acid (to 52%), in striking contrast to noxious heat-evoked responses which were significantly sensitized to approximately 160% of pre-mustard oil levels. The data suggest that the effect of mustard oil on subsequent lingual nociceptive responses is concentration dependent, transient, and modality specific.

Anandamide is able to inhibit trigeminal neurons using an in vivo model of trigeminovascular-mediated nociception.

Arachidonylethanolamide (anandamide, AEA) is believed to be the endogenous ligand of the cannabinoid CB(1) and CB(2) receptors. CB(1) receptors have been found localized on fibers in the spinal trigeminal tract and spinal trigeminal nucleus caudalis. Known behavioral effects of anandamide are antinociception, catalepsy, hypothermia, and depression of motor activity, similar to Delta(9)-tetrahydocannanbinol, the psychoactive constituent of cannabis. It may be a possible therapeutic target for migraine. In this study, we looked at the possible role of the CB(1) receptor in the trigeminovascular system, using intravital microscopy to study the effects of anandamide against various vasodilator agents. Anandamide was able to inhibit dural blood vessel dilation brought about by electrical stimulation by 50%, calcitonin gene-related peptide (CGRP) by 30%, capsaicin by 45%, and nitric oxide by 40%. CGRP(8-37) was also able to attenuate nitric oxide (NO)-induced dilation by 50%. The anandamide inhibition was reversed by the CB(1) receptor antagonist AM251. Anandamide also reduced the blood pressure changes caused by CGRP injection, this effect was not reversed by AM251. It would seem that anandamide acts both presynaptically, to prevent CGRP release from trigeminal sensory fibers, and postsynaptically to inhibit the CGRP-induced NO release in the smooth muscle of dural arteries. CB(1) receptors seem to be involved in the NO/CGRP relationship that exists in causing headache and dural blood vessel dilation. It also seems that some of the blood pressure changes caused by anandamide are mediated by a noncannabinoid receptor, as AM251 was unable to reverse these effects. It can be suggested that anandamide is tonically released to play some form of modulatory role in the trigeminovascular system.

Response properties of TMJ units in superficial laminae at the spinomedullary junction of female rats vary over the estrous cycle.

Neurons responsive to stimulation of the temporomandibular joint (TMJ) region were recorded from superficial laminae at the trigeminal subnucleus caudalis/upper cervical cord (Vc/C(2)) junction region of cycling female rats under barbiturate anesthesia. To determine if receptive field (RF) properties or sensitivity to algesic chemicals of TMJ units vary over the estrous cycle, animals were selected from proestrous (high estrogen) or early diestrous (low estrogen) stages. More than 90% of TMJ units from each group received convergent nociceptive input [wide dynamic range (WDR) or nociceptive specific (NS)-like] from facial skin. The cutaneous high-threshold RF areas of WDR units from proestrous rats were 30% larger than diestrous units, while RF areas of NS units were similar. Bradykinin (BK, 0.1-10 microM) injection into the TMJ region excited a high percentage of units (>80% of total) from both groups in a dose-related manner. However, BK-evoked response magnitude (R(mag), +140%) and duration (+64%) were greater for proestrous than diestrous units. Both WDR and NS-like TMJ units of proestrous females displayed enhanced BK-evoked R(mag) values and response duration. Glutamate or mustard oil excitation of TMJ units was not affected by stage of the estrous cycle. Several TMJ units from proestrous and diestrous females were activated antidromically from the contralateral posterior thalamus, indicating that projection and nonprojection units were included in the sample population. These results were consistent with the hypothesis that factors related to stage of the estrous cycle modify the processing of deep craniofacial inputs by superficial dorsal horn neurons at the spinomedullary junction, a key region for the initial integration of sensory signals from the TMJ.

Vagus nerve stimulation in awake rats reduces formalin-induced nociceptive behaviour and fos-immunoreactivity in trigeminal nucleus caudalis.

Besides its well-established efficacy in epilepsy, vagus nerve stimulation (VNS) may be of potential interest in pain treatment. It has, however, not yet been assessed in animal pain models with the devices and stimulation protocols used in humans. We have therefore studied in awake rats the effects of left cervical VNS on trigeminal nociception using an implantable electrode and stimulator (NCP-Cyberonics). VNS was applied for 24h at 2 mA intensity, 20 Hz frequency, 0.5 ms pulse width and a duty cycle of 20s ON/18s OFF. As a nociceptive stimulus, we injected formalin into the left mystacial vibrissae, assessed behaviour for 45 min and sacrificed the animals 45 min later. Fos-immunoreactive (Fos-Ir) neurons were counted in laminae I-II of trigeminal nucleus caudalis (TNC) on both sides. We used three groups of control animals: VNS without formalin, formalin without VNS and sham VNS (implanted without stimulation or formalin). Whereas sham VNS had no significant effect, VNS alone increased Fos expression in ipsilateral TNC in addition to the expected increase in nucleus tractus solitarius. It also significantly attenuated the increase of Fos-Ir neurons observed in ipsilateral TNC laminae I-II after formalin injection. If the proper VNS effect on Fos-expression was subtracted, the reduction of formalin-induced nociceptor activation was 55%. VNS also reduced nociceptive behaviour on average by 96.1% during the early phase (0-6 min) and by 60.7% during the late phase (6-45 min) after the formalin injection. These results suggest that VNS applied with a device used in human therapy may have in awake rats a significant antinociceptive effect in a model of trigeminal pain.

P2X receptors in trigeminal subnucleus caudalis modulate central sensitization in trigeminal subnucleus oralis.

This study investigated the role of trigeminal subnucleus caudalis (Vc) P2X receptors in the mediation of central sensitization induced in nociceptive neurons in subnucleus oralis (Vo) by mustard oil (MO) application to the tooth pulp in anesthetized rats. MO application produced a long-lasting central sensitization reflected in neuroplastic changes (i.e., increases in neuronal mechanoreceptive field size and responses to innocuous and noxious mechanical stimuli) in Vo nociceptive neurons. Twenty minutes after MO application, the intrathecal (i.t.) administration to the rostral Vc of the selective P2X(1), P2X(3), and P2X(2/3) receptor antagonist, 2'-(or 3'-)O-trinitrophenyl-ATP (TNP-ATP), significantly and reversibly attenuated the MO-induced central sensitization for more than 15 min; saline administration had no effect. Administration to the rostral Vc of the selective P2X(1), P2X(3), and P2X(2/3) receptor agonist, alpha,beta-methylene ATP (alpha,beta-meATP, i.t.) produced abrupt and significant neuroplastic changes in Vo nociceptive neurons, followed by neuronal desensitization as evidenced by the ineffectiveness of a second i.t. application of alpha,beta-meATP and subsequent MO application to the pulp. Administration to the rostral Vc of the selective P2X(1) receptor agonist beta,gamma-methylene ATP (beta,gamma-meATP, i.t.) produced no significant neuroplastic changes per se and did not affect the subsequent MO-induced neuroplastic changes in Vo nociceptive neurons. These results suggest that P2X(3) and possibly also the P2X(2/3) receptor subtypes in Vc may play a role in the initiation and maintenance of central sensitization in Vo nociceptive neurons induced by MO application to the pulp.

Separate, parallel sensory and hedonic pathways in the mammalian somatosensory system.

We propose that separate sensory and hedonic representations exist in each of the primary structures of the somatosensory system, including brainstem, thalamic and cortical components. In the dorsal horn of the spinal cord, the hedonic representation, which consists primarily of nociceptive-specific, wide dynamic range, and thermoreceptive neurons, is located in laminae I and II, while the sensory representation, composed primarily by low-threshold and wide dynamic range neurons, is found in laminae III through V. A similar arrangement is found in the caudal spinal trigeminal nucleus. Based on the available anatomical and electrophysiological data, we then determine the corresponding hedonic and sensory representations in the area of the dorsal column nuclei, ventrobasal and posterior thalamic complex, and cortex. In rodent primary somatosensory cortex, a hedonic representation can be found in laminae Vb and VI. In carnivore and primate primary and secondary somatosensory cortical areas no hedonic representation exists, and the activities of neurons in both areas represent the sensory aspect exclusively. However, there is a hedonic representation in the posterior part of insular cortex, bordering on retroinsular cortex, that receives projections from two thalamic areas in which hedonics are represented. The functions of the segregated components of the system are discussed, especially in relation to the subjective awareness of pain.

Intrathecal administration of 5-HT(3) receptor agonist modulates jaw muscle activity evoked by injection of mustard oil into the temporomandibular joint in the rat.

The effect of intrathecal administration of the 5-HT(3) receptor agonist 2-methyl-5-hydroxytryptamine (2m-5HT) on jaw muscle activity evoked by mustard oil (MO) injection into the temporomandibular joint of anesthetized rats was examined. One microgram or 100 microg of 2m-5HT significantly enhanced or suppressed jaw muscle responses, respectively. Pre-administration of tropisetron, a 5-HT(3) receptor antagonist, attenuated the effect of 2m-5HT. These results indicate that activation of 5-HT(3) receptors can modulate trigeminal nociceptive responses.

Protein kinase C gamma-like immunoreactivity of trigeminothalamic neurons in the medullary dorsal horn of the rat.

We examined protein kinase C gamma-like immunoreactivity (PKCgamma-LI) of trigeminothalamic neurons in the rat medullary dorsal horn (MDH) after injecting a retrograde tracer, Fluoro-Gold (FG), into the thalamus. Over 90% of FG-labeled neurons in the marginal layer (lamina I) and a few FG-labeled neurons in the superficial part of the magnocellular layer (lamina III) showed PKCgamma-LI. No PKCgamma-neurons in the substantia gelatinosa (lamina II) were labeled with FG. PKCgamma-mediated regulation of trigeminothalamic neurons may contribute to the changes in MDH activity during persistent pain.

Central mu opioid receptor mechanisms modulate mustard oil-evoked jaw muscle activity.

The injection of the small-fibre excitant and inflammatory irritant mustard oil (MO) into the temporomandibular joint (TMJ) region of rats evokes a sustained and reversible increase in electromyographic (EMG) activity of jaw muscles. The 'rekindling' of this nociceptive reflex by intrathecal (i.t.) administration of the opiate antagonist naloxone and mu but not delta and kappa selective opioid antagonist, suggests that it may be modulated by endogenous opioid inhibitory mechanisms.

Influence of serotonin receptor antagonists on substance P and serotonin release evoked by tooth pulp stimulation with electro-acupuncture in the trigeminal nucleus cudalis of the rabbit.

We studied the effect of NAN-190 (5-HT(1A) antagonist), ketanserin (5-HT(2) antagonist) and ICS 205-930 (5-HT(3) antagonist) on tooth pulp stimulation (TPS)-induced 5-HT release and substance P (SP) release in the superficial layers of the trigeminal nucleus caudalis (SpVc-I,II) in the presence or absence of electro-acupuncture (EAP). TPS slightly increased 5-HT release and significantly increased SP release. In combination with EAP, TPS-induced 5-HT release was remarkably enhanced, whereas SP release was significantly suppressed. Pretreatment with NAN-190 (3.5 mg/kg, i.v.) significantly enhanced the increase in TPS-induced 5-HT release in the presence of EAP. On the other hand, the increase of 5-HT release induced following TPS in the presence of EAP was inhibited by pretreatment with ketanserin (2.5 mg/kg, i.v.) and ICS 205-930 (1 mg/kg, i.v.). When NAN-190 was pre-treated in the animals combined TPS and EAP, the amount of SP release was significantly reduced compared with the absence of this drug. On the other hand, pretreatment with ketanserin and ICS 205-930 reversed the inhibitory effect of EAP on the TPS-generated SP release, especially ICS 205-930, which remarkably enhanced TPS-induced SP release compared with the absence of this drug. On the basis of the obtained results, we concluded that NAN-190 and ICS 205-930 act on EAP-induced analgesia positively and suppressively, respectively, by regulation of TPS-generated SP release through activation of their subtype receptors. On the other hand, ketanserin does not affect TPS-induced 5-HT release and SP release in the presence of EAP.

Cornea-responsive medullary dorsal horn neurons: modulation by local opioids and projections to thalamus and brain stem.

Previously, it was determined that microinjection of morphine into the caudal portion of subnucleus caudalis mimicked the facilitatory effects of intravenous morphine on cornea-responsive neurons recorded at the subnucleus interpolaris/caudalis (Vi/Vc) transition region. The aim of the present study was to determine the opioid receptor subtype(s) that mediate modulation of corneal units and to determine whether opioid drugs affected unique classes of units. Pulses of CO(2) gas applied to the cornea were used to excite neurons at the Vi/Vc ("rostral" neurons) and the caudalis/upper cervical spinal cord transition region (Vc/C1, "caudal" neurons) in barbiturate-anesthetized male rats. Microinjection of morphine sulfate (2.9-4.8 nmol) or the selective mu receptor agonist D-Ala, N-Me-Phe, Gly-ol-enkephalin (DAMGO; 1.8-15.0 pmol) into the caudal transition region enhanced the response in 7 of 27 (26%) rostral units to CO(2) pulses and depressed that of 10 units (37%). Microinjection of a selective delta ([D-Pen(2,5)] (DPDPE); 24-30 pmol) or kappa receptor agonist (U50488; 1.8-30.0 pmol) into the caudal transition region did not affect the CO(2)-evoked responses of rostral units. Caudal units were inhibited by local DAMGO or DPDPE but were not affected by U50,488H. The effects of DAMGO and DPDPE were reversed by naloxone (0.2 mg/kg iv). Intravenous morphine altered the CO(2)-evoked activity in a direction opposite to that of local DAMGO in 3 of 15 units, in the same direction as local DAMGO but with greater magnitude in 4 units, and in the same direction with equal magnitude as local DAMGO in 8 units. CO(2)-responsive rostral and caudal units projected to either the thalamic posterior nucleus/zona incerta region (PO/ZI) or the superior salivatory/facial nucleus region (SSN/VII). However, rostral units not responsive to CO(2) pulses projected only to SSN/VII and caudal units not responsive to CO(2) projected only to PO/ZI. It was concluded that the circuitry for opioid analgesia in corneal pain involves multiple sites of action: inhibition of neurons at the caudal transition region, by intersubnuclear connections to modulate rostral units, and by supraspinal sites. Local administration of opioid agonists modulated all classes of corneal units. Corneal stimulus modality was predictive of efferent projection status for rostral and caudal units to sensory thalamus and reflex areas of the brain stem.