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Differentiation of oligodendroglial progenitor cells (OPCs) into myelinating oligodendrocytes is known to be regulated by the microenvironment where they differentiate. However, current research has not verified whether or not oligodendroglial lineage cells (OLCs) derived from different anatomical regions of the central nervous system (CNS) respond to microenvironmental cues in the same manner. Here, we isolated pure OPCs from rat neonatal forebrain (FB) and spinal cord (SC) and compared their phenotypes in the same in vitro conditions. We found that although FB and SC OLCs responded differently to the same external factors; they were distinct in proliferation response to mitogens, oligodendrocyte phenotype after differentiation, and cytotoxic responses to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-type glutamate receptor-mediated excitotoxicity at immature stages of differentiation in a cell-intrinsic manner. Moreover, transcriptome analysis identified genes differentially expressed between these OPC populations, including those encoding transcription factors (TFs), cell surface molecules, and signaling molecules. Particularly, FB and SC OPCs retained the expression of FB- or SC-specific TFs, such as Foxg1 and Hoxc8, respectively, even after serial passaging in vitro. Given the essential role of these TFs in the regional identities of CNS cells along the rostrocaudal axis, our results suggest that CNS region-specific gene regulation by these TFs may cause cell-intrinsic differences in cellular responses between FB and SC OLCs to extracellular molecules. Further understanding of the regional differences among OPC populations will help to improve treatments for demyelination in different CNS regions and to facilitate the development of stem cell-derived OPCs for cell transplantation therapies for demyelination. Cover Image for this issue: doi. 10.1111/jnc.13809., (© 2017 International Society for Neurochemistry.)

Itch is relayed to higher centers by projection neurons in the spinal and medullary dorsal horn. We employed a double-label method to map the ascending projections of pruriceptive and nociceptive trigeminal and spinal neurons. The retrograde tracer fluorogold (FG) was stereotaxically injected into the right thalamus or lateral parabrachial area (LPb) in mice. Seven days later, mice received intradermal (id) microinjection of histamine, chloroquine, capsaicin, or vehicle into the left cheek. Histamine, chloroquine, and capsaicin intradermally elicited similar distributions of Fos-positive neurons in the medial aspect of the superficial medullary and spinal dorsal horn from the trigeminal subnucleus caudalis to C2. Among neurons retrogradely labeled from the thalamus, 43%, 8%, and 22% were Fos-positive following id histamine, chloroquine, or capsaicin. Among the Fos-positive neurons following pruritic or capsaicin stimuli, ∼1-2% were retrogradely labeled with FG. Trigeminoparabrachial projection neurons exhibited a higher incidence of double labeling in the superficial dorsal horn. Among the neurons retrogradely labeled from LPb, 36%, 29%, and 33% were Fos positive following id injection of histamine, chloroquine, and capsaicin, respectively. Among Fos-positive neurons elicited by id histamine, chloroquine, and capsaicin, respectively, 3.7%, 4.3%, and 4.1% were retrogradely labeled from LPb. The present results indicate that, overall, relatively small subpopulations of pruriceptive and/or nociceptive neurons innervating the cheek project to thalamus or LPb. These results imply that the vast majority of pruritogen- and algogen-responsive spinal neurons are likely to function as interneurons relaying information to projection neurons and/or participating in segmental nocifensive circuits., (© 2015 Wiley Periodicals, Inc.)

This chapter summarizes recent findings regarding the central transmission of acute and chronic itch. Itch is transduced by cutaneous pruriceptors that transmit signals to neurons in the superficial spinal cord. Spinal itch-signaling circuits utilize several neuropeptides whose receptors represent novel targets to block itch transmission. Itch is relieved by scratching, which activates spinal interneurons to inhibit itch-transmitting neurons. Spinal itch transmission is also thought to be modulated by descending pathways. Itch is transmitted rostrally via ascending pathways to activate a variety of brain regions involved in sensory discrimination of affective and motor responses to itch. The pathophysiological mechanisms of chronic itch are poorly understood but likely involve sensitization of itch-signaling pathways and/or dysfunction of itch-inhibitory circuits. Improved understanding of central itch mechanisms has identified a number of novel targets for the development of antipruritic treatment strategies., (© 2016 S. Karger AG, Basel.)

Eugenol and carvacrol from clove and oregano, respectively, are agonists of the warmth-sensitive transient receptor potential channel TRPV3 and the irritant-sensitive transient receptor potential ankyrin (TRPA)-1. Eugenol and carvacrol induce oral irritation that rapidly desensitizes, accompanied by brief enhancement of innocuous warmth and heat pain in humans. We presently investigated if eugenol and carvacrol activate nociceptive primary afferent and higher order trigeminal neurons and enhance their heat-evoked responses, using calcium imaging of cultured trigeminal ganglion (TG) and dorsal root ganglion (DRG) neurons, and in vivo single-unit recordings in trigeminal subnucleus caudalis (Vc) of rats. Eugenol and carvacrol activated 20-30% of TG and 7-20% of DRG cells, the majority of which additionally responded to menthol, mustard oil and/or capsaicin. TG cell responses to innocuous (39°) and noxious (42 °C) heating were enhanced by eugenol and carvacrol. We identified dorsomedial Vc neurons responsive to noxious heating of the tongue in pentobarbital-anesthetized rats. Eugenol and carvacrol dose-dependently elicited desensitizing responses in 55% and 73% of heat-sensitive units, respectively. Responses to noxious heat were briefly enhanced by eugenol and carvacrol. Many eugenol- and carvacrol-responsive units also responded to menthol, cinnamaldehyde and capsaicin. These data support a peripheral site for eugenol and carvacrol to enhance warmth- and noxious heat-evoked responses of trigeminal neurons, and are consistent with the observation that these agonists briefly enhance warmth and heat pain on the human tongue., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

We investigated roles for substance P (SP), gastrin-releasing peptide (GRP), and glutamate in the spinal neurotransmission of histamine-dependent and -independent itch. In anesthetized mice, responses of single superficial dorsal horn neurons to intradermal (i.d.) injection of chloroquine were partially reduced by spinal application of the α-amino-3-hydroxy-5-methyl-4-isoxazole proprionate acid (AMPA)/kainate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Co-application of CNQX plus a neurokinin-1 (NK-1) antagonist produced stronger inhibition, while co-application of CNQX, NK-1, and GRP receptor (GRPR) antagonists completely inhibited firing. Nociceptive-specific and wide dynamic range-type neurons exhibited differential suppression by CNQX plus either the GRPR or NK-1 antagonist, respectively. Neuronal responses elicited by i.d. histamine were abolished by CNQX alone. In behavioral studies, individual intrathecal administration of a GRPR, NK-1, or AMPA antagonist each significantly attenuated chloroquine-evoked scratching behavior. Co-administration of the NK-1 and AMPA antagonists was more effective, and administration of all 3 antagonists abolished scratching. Intrathecal CNQX alone prevented histamine-evoked scratching behavior. We additionally employed a double-label strategy to investigate molecular markers of pruritogen-sensitive dorsal root ganglion (DRG) cells. DRG cells responsive to histamine and/or chloroquine, identified by calcium imaging, were then processed for co-expression of SP, GRP, or vesicular glutamate transporter type 2 (VGLUT2) immunofluorescence. Subpopulations of chloroquine- and/or histamine-sensitive DRG cells were immunopositive for SP and/or GRP, with >80% immunopositive for VGLUT2. These results indicate that SP, GRP, and glutamate each partially contribute to histamine-independent itch. Histamine-evoked itch is mediated primarily by glutamate, with GRP playing a lesser role. Co-application of NK-1, GRP, and AMPA receptor antagonists may prove beneficial in treating chronic itch., (Copyright © 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

Scratching relieves itch, but the underlying neural mechanisms are poorly understood. We presently investigated a role for the inhibitory neurotransmitters GABA and glycine in scratch-evoked inhibition of spinal itch-signaling neurons in a mouse model of chronic dry skin itch. Superficial dorsal horn neurons ipsilateral to hindpaw dry skin treatment exhibited a high level of spontaneous firing that was significantly attenuated by cutaneous scratching, pinch and noxious heat. Scratch-evoked inhibition was nearly abolished by spinal delivery of the glycine antagonist, strychnine, and was markedly attenuated by respective GABA(A) and GABA(B) antagonists bicuculline and saclofen. Scratch-evoked inhibition was also significantly attenuated (but not abolished) by interruption of the upper cervical spinal cord, indicating the involvement of both segmental and suprasegmental circuits that engage glycine- and GABA-mediated inhibition of spinal itch-signaling neurons by noxious counterstimuli.

Intradermal cheek injection of pruitogens or algogens differentially elicits hindlimb scratching or forelimb wiping, suggesting that these behaviors distinguish between itch and pain. We studied whether pruritogens and algogens excite separate or overlapping populations of primary afferent and second-order trigeminal neurons in mice. Calcium imaging of primary sensory trigeminal ganglion (TG) cells showed that 15.4% responded to histamine, 5.8% to the protease-activated receptor (PAR)-2 agonist, 13.4% to allyl isothiocyanate (AITC), and 36.7% to capsaicin. AITC and/or capsaicin activated the vast majority of histamine- and PAR-2 agonist-sensitive TG cells. A chemical search strategy identified second-order neurons in trigeminal subnucleus caudalis (Vc) responsive to histamine, the PAR-2 agonist, or AITC. A minority of histamine or PAR-2 agonist-responsive Vc neurons responded to the other pruritogen, whereas a large majority of puritogen-responsive Vc neurons responded to capsaicin and/or AITC. A minority of AITC-responsive Vc neurons responded to pruritogens, whereas most responded to capsaicin. These data indicate that most primary and higher-order trigeminal sensory neurons are activated by both pruritic and algesic stimuli, although a minority exhibit selectivity. The results are discussed in terms of population codes for itch and pain that result in distinct behavioral responses of hindlimb scratching and forelimb wiping that are mediated at lumbar and cervical segmental levels, respectively.

Recent studies have suggested the existence of separate transduction mechanisms and sensory pathways for histamine and nonhistaminergic types of itch. We studied whether histamine and an agonist of the protease-activated receptor (PAR)-2, associated with nonhistaminergic itch, excite murine dorsal horn neurons. Single units were recorded in superficial lumbar dorsal horn of adult ICR mice anesthetized with pentobarbital. Unit activity was searched using a small intradermal hindpaw injection of histamine or the PAR-2 agonist SLIGRL-NH2. Isolated units were subsequently challenged with intradermal histamine followed by SLIGRL-NH2 (each 50 microg/1 microl) or reverse order, followed by mechanical, thermal, and algogenic stimuli. Forty-three units were classified as wide dynamic range (62%), nociceptive specific (22%), or mechano insensitive (16%). Twenty units gave prolonged (mean, 10 min) discharges to intradermal injection of histamine; 76% responded to subsequent SLIGRL-NH2, often more briefly. Units additionally responded to noxious heat (63%), cooling (43%), topical mustard oil (53%), and intradermal capsaicin (67%). Twenty-two other units gave prolonged (mean, 5 min) responses to initial intradermal injection of SLIGRL-NH2; 85% responded to subsequent intradermal histamine. They also responded to noxious heat (75%), mustard oil (93%), capsaicin (63%), and one to cooling. Most superficial dorsal horn neurons were excited by both histamine and the PAR-2 agonist, suggesting overlapping pathways for histamine- and non-histamine-mediated itch. Because the large majority of pruritogen-responsive neurons also responded to noxious stimuli, itch may be signaled at least partly by a population code.

The TRPA1 agonist mustard oil (allyl isothiocyanate=AITC) induces heat hyperalgesia and mechanical allodynia in human skin and sensitizes rat spinal wide dynamic range (WDR) neuronal responses to noxious skin heating. We presently used electrophysiological methods to investigate if AITC affects the responsiveness of individual spinal WDR neurons to intense skin cooling. Recordings were made from cold-sensitive WDR neurons in lamina I and deeper dorsal horn; 21/23 also responded to noxious skin heating. Topical application of AITC excited 8/18 units and significantly enhanced their responses to noxious heat while not significantly affecting responses to the cold stimulus. Vehicle (mineral oil) had no effect on thermal responses. The data confirm a role for the TRPA1 agonist AITC in enhancing heat nociception without significantly affecting cold sensitivity.

Mustard oil [allyl isothiocyanate (AITC)] and cinnamaldehyde (CA), agonists of the ion channel TRPA1 expressed in sensory neurons, elicit a burning sensation and heat hyperalgesia. We tested whether these phenomena are reflected in the responses of lumbar spinal wide-dynamic range (WDR) neurons recorded in pentobarbital-anesthetized rats. Responses to electrical and graded mechanical and noxious thermal stimulation were tested before and after cutaneous application of AITC or CA. Repetitive application of AITC initially increased the firing rate of 52% of units followed by rapid desensitization that persisted when AITC was reapplied 30 min later. Responses to noxious thermal, but not mechanical, stimuli were significantly enhanced irrespective of whether the neuron was directly activated by AITC. Windup elicited by percutaneous or sciatic nerve electrical stimulation was significantly reduced post-AITC. These results indicate that AITC produced central inhibition and peripheral sensitization of heat nociceptors. CA did not directly excite WDR neurons, and significantly enhanced responses to noxious heat while not affecting windup or responses to skin cooling or mechanical stimulation, indicating a peripheral sensitization of heat nociceptors.

Most cold-sensitive subnucleus caudalis (Vc) neurons are also excited by the TRPM8 agonist menthol and the TRPA1 agonist cinnamaldehyde (CA). We investigated how interactions among menthol, CA and noxious cooling and heating of the tongue affected responses of superficial Vc units recorded in thiopental-anesthetized rats. Units responded to 1% CA which enhanced cold- and heat-evoked responses 5 min later. They responded more strongly to 10% CA which initially depressed cold responses, followed by enhancement at 5 min without affecting responses to heat. Following 10% CA, the mean response to 1% menthol was significantly lower than when menthol was tested first. After menthol, the subsequent response to CA was significantly weaker compared to the mean CA-evoked response when it was tested first. These results demonstrate mutual cross-desensitization between CA and menthol. The response to CA was enhanced following prior application of 10% ethanol (menthol vehicle). Prior application of menthol did not prevent the biphasic effect of 10% CA on cold-evoked responses, nor did prior application of CA prevent menthol enhancement of cold-evoked responses. Responses to noxious heat were unaffected by 10% CA and menthol regardless of the order of chemical presentation. These data indicate that superficial Vc neurons receive convergent input from primary afferents expressing TRPM8 and TRPA1. The mutual cross-desensitization between CA and menthol, and differential modulation of cold- vs. heat-evoked responses, suggests a direct inhibition of TRPM8 and TRPA1 expressed in peripheral nerve endings by CA and menthol, respectively, rather than a central site of interaction.

Background: Because of the logistical obstacles to measurement under hyperbaric conditions, the effect of nitrous oxide (N2O) alone on spinal neuronal responses has not been tested. We hypothesized that, like other inhaled anesthetics, N2O would depress spinal neuronal responses to noxious stimulation., Methods: The lumbar spinal cord was exposed in rats anesthetized with isoflurane. Mechanically ventilated rats were placed into a hyperbaric chamber and needle electrodes were inserted into the hindpaws. Isoflurane administration was discontinued and anesthesia converted to N2O by pressurizing the chamber with N2O. A microelectrode was inserted into the lumbar cord using computer-controlled motors and a hydraulic microdrive. Neuronal responses to electrical stimulation of the hindpaw were sought at 1.5, 2, and 2.5 atm N2O (0.8-1.3 minimum alveolar concentration)., Results: Increasing N2O partial pressures variably affected neuronal responses to a 2 s 100-Hz electrical stimulus. Neuronal depth and neuronal response were correlated, with superficial neurons tending to be facilitated, while deeper neurons were depressed; (overall responses were 1331 +/- 408, 1594 +/- 383, and 1578 +/- 500 impulses/min at 1.5, 2, and 2.5 atm N2O, respectively; mean, standard error). N2O did not affect neuronal responses to a repetitive "windup" stimulus. Infusion of the N-methyl-d-aspartate blocker MK-801 into separate rats increased the neuronal response to the 100-Hz stimulus (from 781 +/- 216 to 1352 +/- 269 impulses/min, P < 0.05)., Conclusions: N2O facilitated superficial spinal neuronal responses to noxious stimulation while depressing deeper neurons. These results suggest that anesthetic partial pressures of N2O have divergent effects on spinal neuronal responses to noxious stimulation, the specific responses depending on the depth of the spinal neurons.

The recent discoveries of cold-sensitive transient receptor potential (TRP) channels prompted us to investigate the responses of neurons in trigeminal subnucleus caudalis (Vc) to intraoral cooling and agonists of TRPM8 and TRPA1. Single units responsive to lingual cooling were recorded in superficial laminae of Vc in thiopental-anesthetized rats. All units responded to noxious heat and 88% responded to menthol. Responses increased with menthol concentration from 0.1 to 1% (6.4-64 mM) and plateaued at 10% (640 mM). Noxious cold-evoked responses were significantly enhanced after menthol in a concentration-dependent manner. Constant-flow application of 1% menthol elicited a phasic discharge that adapted over 2-8 min and significantly enhanced subsequent cold-evoked but not heat-evoked responses; vehicle (10% ethanol) was ineffective. Reapplication of menthol 15 min later elicited a significantly reduced response (self-desensitization). Vc units were similarly excited phasically by 1% menthol dissolved in 40% ethanol. The 40% ethanol briefly excited Vc units during the first minute and reduced subsequent responses to noxious heat and cold while exhibiting neither self-desensitization nor cross-desensitization to menthol. Menthol cross-desensitized Vc responses to 40% ethanol. Most menthol-responsive units also responded to the TRPA1 agonists cinnamaldehyde and mustard oil, and the TRPV1 agonist capsaicin. Units in superficial Vc receive convergent input from primary afferents that express TRPM8, TRPA1, and/or TRPV1 channels, either directly or indirectly via intersubnuclear pathways. The convergent nature of these units suggests a general role in signaling noxious stimuli.

Windup is a progressive increase in responses of nociceptive spinal cord neurons to repeated electrical C fiber stimulation. We hypothesized that isoflurane would depress windup at approximately the minimum alveolar anesthetic concentration (MAC) required to suppress purposeful movement in response to noxious stimulation. We recorded windup responses in single lumbar spinal neurons (n = 17) to a series of 15 repetitive electrical stimuli delivered at 1 Hz to the hindpaw at C fiber strength; hindpaw withdrawal force was simultaneously recorded. The total number of action potentials per 15 stimuli (mean +/- sem as a percentage of each neuron's maximal response) was 83% +/- 5%, 84% +/- 5%, 67% +/- 7%, and 57% +/- 8% at 0.7, 0.9, 1.1, and 1.4 MAC, respectively. The 0.9 and 1.1 MAC values differed significantly from each other, whereas the 0.7 and 0.9 MAC values differed from the 1.4 MAC value (P < 0.05). The reduced firing was attributed to a depression of the initial C fiber-evoked responses in most units, and a reduction in windup slope over the initial 5 stimuli in 6 units. Muscle force was 67%, 11%, and 4% of the 0.7 MAC value at 0.9, 1.1, and 1.4 MAC, respectively. Isoflurane depressed excitability and variably affected windup of lumbar spinal cord neurons, while uniformly depressing windup of limb withdrawals in a concentration-dependent manner.

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.

We describe a method to record single unit neuronal activity from mouse spinal cord using volatile anesthesia. The small size of the mouse can complicate usual methods that are used for single-unit recording in rats, but simple modifications can significantly increase the number of successful recordings. Stabilization of the vertebral column is particularly important, as are adequate ventilation of the animal, control of body temperature and accurate determination of anesthetic concentrations in respiratory gas samples., (Copyright 2004 Elsevier B.V.)

Background: Isoflurane acts primarily in the spinal cord to block movement; however, it is unclear how supraspinal sites might contribute to anesthetic effects on quantified parameters of movement such as force., Methods: The authors investigated the effects of isoflurane on spontaneous and noxious heat-evoked activity of nociceptive reflex-modulating ON and OFF cells in the rostral ventromedial medulla of rats. Single ON or OFF neurons were recorded simultaneously with hind-limb withdrawal force elicited by graded noxious thermal hind paw stimulation. Isoflurane concentrations were administered in reference to each animal's minimum alveolar concentration (MAC) of isoflurane., Results: From 0.65 to 1.15 MAC, isoflurane dose-dependently reduced spontaneous activity of ON cells by 70% (P < 0.001). OFF-cell spontaneous activity was dose-dependently increased 138% (P < 0.001). ON-cell heat-evoked activity was depressed 95% by isoflurane from 0.65 to 1.15 MAC (P < 0.001). Isoflurane-induced changes in ON- and OFF-cell activity paralleled similar reductions in withdrawal force, with the largest change in both neuronal activity and withdrawal force occurring between 0.85 and 1.15 MAC. For the lowest stimulus temperature, excitatory responses of ON cells and inhibitory responses of OFF cells were significantly greater for trials in which withdrawals occurred than for trials in which no withdrawal occurred, suggesting that responses in both classes of neurons were related to movement rather than the stimulus alone., Conclusions: The results show that isoflurane modulation of ON- and OFF-cell activity corresponds to anesthetic-induced reductions in hind-limb withdrawal force, and therefore, the effects of isoflurane on these classes of neurons in rostral ventromedial medulla might contribute to motor depression.

We investigated the role of neuronal nicotinic acetylcholine receptors (nAChRs) in nicotine cross-desensitization of chemonociceptive responses of trigeminal subnucleus caudalis (Vc) neurons in rats. Vc responses to lingually applied pentanoic acid were significantly reduced following nicotine, and this was prevented when the nAChR antagonist mecamylamine was applied before or after nicotine. A peripheral site of nicotine cross-desensitization is suggested via a nAChR-mediated reduction in acidic excitation of lingual nociceptors that project to Vc.

We investigated the possible role of amiloride-sensitive ion channels of the ENaC/DEGenerin superfamily in the activation of trigeminal nociceptive neurons elicited by noxious chemical stimulation of the oral mucosa using two methodologies, single-unit recording and c-fos immunohistochemistry. In pentobarbital-anesthetized rats, single-unit recordings were made from neurons in superficial laminae of dorsomedial trigeminal subnucleus caudalis (Vc) that responded to noxious thermal and chemical stimuli applied to the dorsal tongue. Successive application of each of three chemicals (250 mM pentanoic acid, n=6 units; 250 mM citric acid, n=8; 5 M NaCl, n=6) evoked responses that were not affected following topical application of amiloride (1 mM). In separate experiments, pentobarbital-anesthetized rats received one of the following stimuli delivered to the dorsal tongue: 250 mM pentanoic acid (n=6); 1 mM amiloride followed by 250 mM pentanoic (N=6); 5 M NaCl (n=5); or 1 mM amiloride followed by 5 M NaCl (n=5). Two hours later they were perfused with 4% paraformaldehyde and the brain stems processed for c-fos immunoreactivity. Both pentanoic acid and 5 M NaCl evoked similar numbers and patterns of fos-like immunoreactivity (FLI) in dorsomedial Vc and other brain stem regions, with no significant difference in counts of FLI in animals pretreated with amiloride. These results suggest that amiloride-sensitive Na(+) channels are not essential in mediating the activation of intraoral trigeminal nociceptors.

Conflicting reports exist regarding the ability of quinine to activate neurons in the trigeminal system. We used the complementary approaches of single-unit electrophysiology and c-fos immunohistochemistry to investigate whether quinine (100 mM) activates chemonociceptive cells in the brainstem trigeminal subnucleus caudalis (Vc). In electrophysiological experiments, 38 units responded to noxious mechanical, thermal and chemical (200 mM pentanoic acid) stimuli applied to the tongue with an increase in firing rate; none responded to lingual quinine whether the quinine was presented before or after application of pentanoic acid. In the c-fos immunohistochemical experiment, both quinine and water elicited equivalent levels of fos-like immunoreactivity (FLI) in dorsomedial Vc that were significantly lower than the level of FLI evoked by pentanoic acid. These data collectively indicate that quinine does not elicit activity in chemonociceptive Vc neurons.

The method of c-fos immunohistochemistry was used to identify the brain stem distribution of neurons activated following irritant chemical stimulation of the laryngopharyngeal mucosa. In pentobarbital-anesthetized rats, either water (control), nicotine (600 mM, 1 ml) or capsaicin (330 microM, 1 ml) was applied to the pharynx via a cannula placed posterior to the soft palate. Following nicotine and capsaicin, there was a significant increase in fos-like immunoreactivity (FLI) compared with controls in the following areas: nucleus of the solitary tract from the level of the pyramidal decussation caudally to the level of the area postrema rostrally; dorsomedial aspect of trigeminal subnucleus caudalis (Vc); and paratrigeminal islands interspersed in the spinal trigeminal tract. There was significantly more FLI in Vc and paratrigeminal nuclei following capsaicin than following nicotine, while the reverse was true for NTS. In addition, there was a significant increase in FLI in area postrema and the ventrolateral medullary region dorsal to the lateral reticular nucleus following nicotine but not capsaicin. The distributions of FLI in NTS, area postrema, Vc, and paratrigeminal nuclei are consistent with prior anatomical tract-tracing studies and suggest roles for these brain stem regions in mediating sensory and reflex responses to irritant chemical stimulation of the upper respiratory mucosa.

In human studies, repeated intraoral application of strong acidic or salt stimuli induces irritation that progressively increases across trials (sensitization), whereas irritation elicited by nicotine progressively decreases (desensitization). We investigated whether nociceptive neurons in trigeminal subnucleus caudalis (Vc) exhibit increasing or decreasing patterns of firing to the intraoral application of these irritants. In rats anesthetized with halothane and thiopental, single-unit recordings were made from nociceptive neurons in superficial layers of dorsomedial Vc that responded to mechanical and noxious thermal and chemical stimulation of the tongue. NaCl (5M), citric acid (300 mM), pentanoic acid (300 mM) or nicotine (600 mM) were separately delivered to the tongue by constant flow (0.32 ml/min) for 15 or 25 min. NaCl, citric acid and pentanoic acid each elicited a progressive, significant increase in Vc neuronal firing over the initial 10 min to a plateau level that was maintained for the stimulus duration. Nicotine induced a significant increase in firing rate of Vc neurons within 6 min, followed by a decline back to the baseline level over the ensuing 10 min. Following a rest period, reapplication of nicotine no longer activated Vc neurons, indicative of self-desensitization. We additionally tested for nicotine cross-desensitization to acid. After recording the responses of Vc neurons to pentanoic acid and noxious heat, nicotine was then applied for 15 min. Post-nicotine responses to pentanoic acid were markedly reduced (to 13% of control), indicative of cross-desensitization; responses to noxious heat were also reduced to a lesser degree (to 71% of control). The progressive increase in Vc neuronal firing elicited by NaCl and acid, and the decline in firing after initial nicotinic excitation, resemble psychophysical patterns of sensitization and desensitization, respectively, and support the involvement of Vc neurons in the signaling of oral irritant sensations.

Aversion to capsaicin (0.1-10 ppm) was assessed using a two-bottle paired preference paradigm, before and after intracisternal injection of substance P conjugated to saporin (SP-SAP) to ablate neurons in superficial medullary and cervical dorsal horn that express NK-1 receptors. Before SP-SAP, there was a concentration-dependent decrease in consumption of capsaicin with a threshold of 0.1-0.3 ppm. Following SP-SAP, significantly more capsaicin solution was consumed at 1- and 10-ppm concentrations. These results support a role for substance P in the mediation of high, but not low, levels of capsaicin-induced oral irritation., (Copyright 2002 Elsevier Science B.V.)

The direct and indirect effects of nitrous oxide (N2O) on the nociceptive responses of lumbar dorsal horn neurons, and the indirect effects on midbrain reticular formation (MRF) neurons and thalamic neurons were determined in goats anaesthetized with isoflurane. The technique used enabled the differential delivery of N2O to either the torso or the cerebral circulation, thus allowing assessment of the direct spinal and indirect brain effects of N2O. Systemic delivery of N2O appeared to have divergent effects, facilitating (4/11) or depressing (7/11) the responses of dorsal horn neurons. Such divergent effects were also observed when N2O was differentially delivered to the circulation in the torso (i.e. the spinal cord). Likewise, MRF and thalamic responses to noxious stimulation were variably affected by administration of N2O to the torso, with some cells facilitated (7/13 MRF neurons, 3/8 thalamic neurons) and others depressed (6/13 MRF neurons, 5/8 thalamic neurons). It appears that N2O has variable effects on the caprine CNS. The facilitatory action of N2O might partially explain why it is a relatively weak anaesthetic.

Repeated application of capsaicin at a 1-min interstimulus interval (ISI) to the tongue induces a progressively increasing irritant sensation (sensitization), followed after a rest period by reduced sensitivity to further capsaicin (desensitization). Sequential reapplication of capsaicin induces irritation that eventually increases to initial levels: stimulus-induced recovery (SIR). In contrast, repeated application of nicotine elicits a declining irritant sensation across trials. To investigate possible neural correlates of these phenomena, we recorded from single units in superficial laminae of the dorsomedial trigeminal subnucleus caudalis (Vc) that responded to noxious thermal (54 degrees C) and chemical (1 M pentanoic acid) stimulation of the tongue of anesthetized rats. We then recorded responses to either capsaicin (330 microM) or nicotine (0.6 M), delivered either once, repeatedly at 1-min ISI, or continually by constant flow. After the initial capsaicin application and a rest period, the capsaicin was reapplied in the identical manner to test for SIR. The mean response of 14 Vc units to sequential application of pentanoic acid did not vary significantly across trials, indicating lack of tachyphylaxis or sensitization. The averaged response of 11 Vc units to repeated capsaicin increased significantly across the first eight trials and then plateaued. Following the rest period, spontaneous firing had returned to the precapsaicin level. With capsaicin reapplication, the averaged response increased again after a significant delay (due to desensitization), but did not reattain the peak firing rate achieved in the initial series (partial SIR). Constant-flow application of capsaicin induced an identical sensitization followed by nearly complete SIR. A single application of capsaicin induced a significant rise in firing in eight other units, but the rate of rise and maximal firing rate were both much lower compared with repetitive or constant-flow capsaicin. When capsaicin was reapplied once after the rest period, there was no change in firing rate indicating absence of SIR. These results indicate that maintenance of the capsaicin concentration induces a progressive increase in neuronal response that parallels sensitization. With recurrent capsaicin application, desensitization can be overcome to result in a delayed recovery of Vc responses similar to SIR. In contrast, the averaged response of 17 Vc units to repeated or constant-flow application of nicotine increased only over the first 3 min, and then decreased to spontaneous levels even as nicotine was still being applied. These results are consistent with the decrease in the perceived irritation elicited by sequential application of nicotine in humans.

To characterize the role of neuronal nicotinic acetylcholine receptors (nAChRs) in oral irritation and pain, we employed the method of c-fos immunohistochemistry to map the locations and numbers of brainstem neurons that express the immediate-early gene, c-fos, after application of nicotine to the tongue, either alone or after pretreatment with cholinergic antagonists. Groups of anesthetized rats received the following chemicals delivered bilaterally to the dorsal tongue: (1) 0.9% NaCl followed by nicotine (1%, 61 mM), (2) the nAChR antagonist, mecamylamine 0.1% (= 4.9 mM) followed by nicotine, (3) the muscarinic antagonist atropine (0.1% 1.46 mM) followed by nicotine, (4) atropine (1%, 14.6 mM) followed by nicotine, (5) 0.9% NaCl as a control, and (6) unstimulated controls. Two hours later, animals were perfused with phosphate-buffered saline followed by 4% paraformaldehyde through the aorta. Post-fixed brainstems were cut in 50-micron frozen sections and immunohistochemically processed for fos-like immunoreactivity (FLI). Following application of nicotine, there were significant increases in FLI compared with saline-treated controls in dorsomedial and ventrolateral aspects of the trigeminal caudalis. Pretreatment with either mecamylamine or the high (1%) concentration of atropine significantly reduced nicotine-evoked FLI in these areas, while pretreatment with the low (0.1%) atropine concentration did not significantly affect FLI. These results are consistent with the idea that nicotine activates nAChRs residing on lingual nociceptive fibers, which, in turn, excite neurons in trigeminal caudalis.

Nicotine evokes pain in the skin and oral mucosa and excites a subpopulation of cutaneous nociceptors, but little is known about the central transmission of chemogenic pain. We have investigated the responses of lumbar spinal wide dynamic range (WDR)-type dorsal horn neurons to intracutaneous (ic) microinjection of nicotine in pentobarbital-anesthetized rats. Nearly all (97%) units responded to nicotine microinjected ic (1 microl) into the low-threshold region of the hind-paw mechanosensitive receptive field in a concentration-related manner (0.01-10%). Responses to repeated injections of 10% nicotine exhibited tachyphylaxis at 5-, 10-, and 15-min interstimulus intervals. Significant tachyphylaxis was not seen with 1% nicotine. All nicotine-responsive units tested (n = 30) also responded to ic histamine (1 microl, 3%) and did not exhibit tachyphylaxis to repeated histamine. However, there was significant cross-tachyphylaxis of nicotine to histamine. Thus 5 min after ic nicotine, histamine-evoked responses were attenuated significantly compared with the initial histamine-evoked response prior to nicotine, with partial recovery over the ensuing 15 min. Neuronal excitation by ic nicotine was not mediated by histamine H1 receptors because ic injection of the H1 receptor antagonist, cetirizine, had no effect on ic nicotine-evoked responses, whereas it significantly attenuated ic histamine-evoked responses in the same neurons. The lowest-threshold portion of cutaneous receptive fields showed a significant expansion in area at 20 min after ic nicotine 10%, indicative of sensitization. Responses to 1% nicotine were significantly reduced after ic injection of the nicotinic antagonist, mecamylamine (0.1% ic), with no recovery over the ensuing 40-60 min. These data indicate that nicotine ic excites spinal WDR neurons, partly via neuronal nicotinic acetylcholine receptors that are presumably expressed in cutaneous nociceptor terminals. Repeated injections of high concentrations of nicotine led to tachyphylaxis and cross-tachyphylaxis with histamine, possibly relevant to peripheral analgesic effects of nicotine.

Carbonated drinks elicit a sensation that is highly sought after, yet the underlying neural mechanisms are ill-defined. We hypothesize that CO(2) is converted via carbonic anhydrase into carbonic acid, which excites lingual nociceptors that project to the trigeminal nuclei. We investigated this hypothesis using three methodological approaches. Electrophysiological methods were used to record responses of single units located in superficial laminae of the dorsomedial aspect of trigeminal subnucleus caudalis (Vc) evoked by lingual application of carbonated water in anesthetized rats. After pretreatment of the tongue with the carbonic anhydrase inhibitor dorzolamide, neuronal responses to carbonated water were significantly attenuated, followed by recovery. Using c-Fos immunohistochemistry, we investigated the distribution of brainstem neurons activated by intraoral carbonated water. Fos-like immunoreactivity (FLI) was significantly higher in the superficial laminae of dorsomedial and ventrolateral Vc in animals treated with carbonated water versus controls. Dorzolamide pretreatment significantly reduced FLI in dorsomedial Vc. We also examined the sensation elicited by carbonated water in human psychophysical studies. When one side of the tongue was pretreated with dorzolamide, followed by bilateral application of carbonated water, a significant majority of subjects chose the untreated side as having a stronger sensation and assigned significantly higher intensity ratings to that side. Dorzolamide did not reduce irritation elicited by pentanoic acid. The present data support the hypothesis that carbonated water excites lingual nociceptors via a carbonic anhydrase-dependent process, in turn exciting neurons in Vc that are presumably involved in signaling oral irritant sensations.

Background: The spinal cord appears to be the site at which isoflurane suppresses movement that occurs in response to a noxious stimulus. In an attempt to localize its site of suppressant action, the authors determined the effect of isoflurane on dorsal horn neuronal responses to supramaximal noxious stimulation at end-tidal concentrations that just permitted and just prevented movement., Methods: Rats (n = 14) were anesthetized with isoflurane, and after lumbar laminectomy, the minimum alveolar concentration (MAC) for each rat was determined using a supramaximal mechanical stimulus. In these same rats, after extracellular microelectrode placement in the lumbar spinal cord, dorsal horn neuronal responses to the supramaximal stimulus were determined at the concentrations of isoflurane that bracketed each rat's MAC (0.1% higher and lower than MAC). The MAC of isoflurane was then re-determined., Results: Dorsal horn neuronal response was 1,757+/-892 impulses/min at 0.9 MAC and 1,508+/-988 impulses/min at 1.1 MAC, a 14% decrease (P < 0.05). Cell responses varied, with some cells increasing their response at the higher concentration of isoflurane. The MAC of isoflurane was 1.38+/-0.2% before and 1.34+/-0.2% after determination of dorsal horn neuronal responses., Conclusions: Isoflurane, at concentrations that bracket MAC, has a variable and minimal depressant effect on dorsal horn cell responses to noxious mechanical stimulation. These data suggest that the major action of isoflurane to suppress movement evoked by a noxious stimulus might occur primarily at a site other than the dorsal horn.

Itch sensation is reduced by cooling the skin. We tested whether lowering skin temperature attenuates responses of spinal dorsal horn neurons elicited by intracutaneous (i.c.) microinjection of histamine in anesthetized rats. Cooling the skin to 3 degrees C significantly and reproducibly reduced (to a mean of 48%) i.c. histamine-evoked responses in 20 of 24 wide dynamic range-type dorsal horn neurons. Histamine-evoked responses recovered to control levels after rewarming the skin. Assuming that such neurons play a role in signaling itch, depression of their responses during skin cooling may account for the psychophysical observation that skin cooling relieves itch in humans.

To investigate the role of trigeminal subnucleus caudalis in neural mechanisms of irritation, we recorded single-unit responses to application of a variety of irritant chemicals to the tongue or ocular mucosa in thiopental-anesthetized rats. Recordings were made from wide dynamic range (WDR) and nociceptive-specific units in superficial layers of the dorsomedial caudalis (0-3 mm caudal to obex) responsive to mechanical stimulation and noxious heating of the ipsilateral tongue ("tongue" units) and from WDR units in ventrolateral caudalis (0-2 caudal to obex) responsive to mechanical and noxious thermal stimulation of cornea-conjunctiva and frequently also surrounding skin ("cornea-conjunctival" units). The following chemicals were delivered topically (0.1 ml) onto the dorsal anterior tongue or instilled into the ipsilateral eye: capsaicin (0.001-1% = 3.3 x 10(-2) to 3.3 x 10(-5) M), ethanol (15-80%), histamine (0.01-10% = 9 x 10(-1) to 9 x 10(-4) M), mustard oil (allyl-isothiocyanate, 4-100% = 4 x 10(-1) to 10 M), NaCl (0.5-5 M), nicotine (0.01-10% = 6 x 10(-1) to 6 x 10(-4) M), acidified phosphate buffer (pH 1-6), piperine (0.01-1% = 3.5 x 10(-2) to 3.5 x 10(-4) M), serotonin (5-HT; 0.3-3% = 1.4 x 10(-1) to 1.4 x 10(-2) M), and carbonated water. The dose-response relationship and possible tachyphylaxis were tested for each chemical. Of 32 tongue units, 31 responded to one or more, and frequently all, chemicals tested. The population responded to 75.3% of the various chemicals tested (

Histamine elicits the sensation of itch at the site of skin application as well as alloknesis (itch elicited by innocuous mechanical stimuli) in a surrounding area in humans and expansion of the low-threshold mechanosensitive receptive field area of spinal wide dynamic range (WDR)-type dorsal horn neurons in rats. We presently tested if the histamine-evoked expansion of neuronal receptive field area depends on a spinal N-methyl-D-aspartate (NMDA) receptor-mediated process. In pentobarbital sodium-anesthetized rats, mechanical receptive field areas of single WDR-type dorsal horn neurons were mapped with graded von Frey filaments before and 10 min after intracutaneous (ic) microinjection of histamine (1 microl; 1, 3, or 10%) at a low-threshold site within the receptive field. Intracutaneous microinjection of histamine evoked dose-related increases in firing rate, as well as a dose-dependent expansion in mean receptive field area 10 min after 3 and 10%, but not 1%, histamine doses. When a noncompetitive or competitive NMDA receptor antagonist dizocilpine [MK-801; D(-)-2-amino-5-phosphonovalerate (APV), respectively; 1 microM] was first applied topically to the surface of the spinal cord, there was no significant change in mean receptive field area after ic microinjection of 10% histamine. The mean neuronal response to histamine in the presence of spinal MK-801 or APV was not significantly different from the mean response to histamine in the absence of these drugs. These results suggest that spinal NMDA receptors are involved in histamine-induced expansion of mechanical receptive field area, a neural event possibly involved in the development of alloknesis.

In this study we investigated the receptive field properties, responses to mechanical and thermal stimuli, and sensitivity to systemic administration of pentobarbital sodium and morphine of single neurons recorded in the sacral spinal cords of pentobarbital-anesthetized rats. Fifty-three neurons responded to innocuous mechanical stimulation of the tail. Of 45 neurons that were additionally tested with noxious thermal stimulation, 62% responded and were classified as wide-dynamic-range or multireceptive neurons. Recording sites were located mainly in the middle layers of the S2-S4 dorsal horn. Mechanosensitive receptive fields on the tail varied widely in size (range 0.14-35 cm2, mean 10.33 cm2) and form, and were in nearly all cases bilateral. Most neurons responded with a high-frequency discharge followed by a more slowly adapting response to pressure stimuli delivered with von Frey hairs. Responses (maximal frequency and total number of impulses) increased in a graded manner to pressure stimuli ranging from 1.2 to 447 g. For neurons responsive to noxious heating of the tail, responses increased in a linear manner over the range of 38-54 degrees C and often leveled off at higher temperatures. Of nine neurons tested with both graded von Frey and noxious heat stimuli, mean responses (maximal frequency and total number of impulses) evoked by the strongest pressure stimuli were larger than those evoked by the most intense heat stimuli, but the difference was not statistically significant. Responses to repeated 48 degrees C stimuli were significantly attenuated within 8 min after systemic administration of morphine (1 or 2 mg/kg ip), reaching maximal suppression (to 37.3%; N = 13) after 18 min, with recovery following systemic naloxone. After morphine (1 and 2 mg/kg ip), the slope of the population stimulus-response function for noxious heat was reduced (51.8%), and the threshold was increased (by 4 degrees C). Responses to noxious heat were significantly depressed (to a mean of 54%; N = 10) by supplemental administration of pentobarbital (mean 17 mg/kg over 5 min). On the basis of similarities between the present data and previous behavioral measures of tail flick stimulus-response functions and their modulation, it is suggested that some of the present neurons might function as interneurons in the tail flick reflex arc.

Many chemicals including nicotine, capsaicin and piperine (pungent chemicals in red and black peppers, respectively) evoke oral pain and irritation via largely unknown neural mechanisms. As a first step in defining the central pathway for oral chemical irritation, we have used an immunohistochemical method to map locations of brainstem neurons expressing the nuclear protein, c-Fos (a putative nociceptive marker), following application of various irritants to the tongue. In barbiturate-anesthetized rats, one of the following was applied to the dorsal surface of the tongue: nicotine (0.5%), capsaicin (0.1%), histamine (2 or 20%), piperine (0.2%), acetylcholine (10%) or vehicle control (0.9% saline, dH2O, 70% ethanol). After 2 h the rat was perfused with fixative and the brainstem removed, sectioned, and processed immunohistochemically. Following application of each irritant, fos-immunoreactive nuclei were consistently observed in the superficial dorsal horn of dorsomedial trigeminal nucleus caudalis (-3 to +0.5 mm relative to obex), interstitial (paratrigeminal) nucleus, and area postrema. Approximately equal numbers were observed bilaterally even with unilateral application to the tongue. Fos-immunoreactive nuclei were observed in dorsomedial trigeminal caudalis bilaterally when a restricted area on the tip of the tongue was stimulated with capsaicin, but were located predominantly ipsilaterally following stimulation of the lateral tongue. Few or no Fos-immunoreactive nuclei were seen in these areas in control rats. Numbers of Fos-immunoreactive nuclei were significantly increased following nicotine and capsaicin in ventrolateral trigeminal nucleus caudalis and nucleus of the solitary tract. Fos-immunoreactivity was also seen consistently in the ventrolateral medulla dorsal to the lateral reticular nucleus, and vestibular and cochlear nuclei, and less consistently in nucleus raphe pallidus and inferior olive, in both irritant and in control groups, indicating that it was not stimulus-evoked. These results have identified a population of neurons in the dorsomedial trigeminal nucleus caudalis likely to be involved in signaling chemical irritation of the tongue. Increases in Fos-immunoreactivity observed in the nucleus of the solitary tract, area postrema, and ventrolateral trigeminal caudalis also suggest roles for these areas in autonomic responses consequent to oral irritation.

In rats anesthetized with sodium pentobarbital, we tested the effects of systemic or intrathecal administration of the opiate antagonist naloxone, the serotonergic antagonist methysergide, or the adrenergic antagonist phentolamine, on inhibition produced by electrical stimulation in midbrain periaqueductal gray (PAG) or lateral reticular formation (LRF) of the responses of single lumbar dorsal horn neurons to noxious heating (50-54 degrees C, 10 s) of hindpaw skin. Systemically administered naloxone (1-10 mg/kg i.v.) reduced (greater than 20% below predrug inhibition) inhibition from PAG and/or LRF in 5/12 units and had no effect in the remainder. Systemic methysergide (2-6 mg/kg i.v.) selectively reduced PAG-evoked inhibition in 6 units, while inhibitions from both PAG and LRF stimulation were reduced in one unit and unaffected in 8 units. Systemic phentolamine (2-4 mg/kg) reduced LRF-evoked inhibition in 4 units, while inhibitions from PAG and LRF were reduced in one unit and unaffected in 5. Intrathecally applied methysergide reduced or abolished PAG-evoked inhibition in 8/16 units and reduced or abolished LRF-evoked inhibition in 6/14 units. Reductions in the level of inhibition were reversible in one-half of the cases, whereas they persisted for over 2 h in the remainder. The mixed effects of both systemically and intrathecally administered drugs suggest that monoamines and opiates may be partly involved in spinal inhibitory mechanisms activated from the midbrain.

In the present study, an attempt was made to correlate the neuronal responsiveness of individual preoptic-septal (POA/S) units to iontophoretically applied GnRH with the onset of sexual receptivity. In both behavioral and electrophysiological studies, ovariectomized, estrogen-primed rats were used. In behaviorally tested rats, lordosis quotients (LQ) were determined at varying times following progesterone (P) injection. For electrophysiological studies, P was given 1 hr after the start of recording. GnRH was iontophoretically applied for 30 sec at 16 nA on spontaneously discharging cells. A unit was deemed excited or inhibited if a repeatable 30% change in discharge rate was observed. From 2-10 hours as the LQ increased from 17 to 90 the total number of GnRH sensitive cells did also. The majority of responsive cells were excited by the peptide. As receptivity displayed a sharp increase from 2 to 6 hours the mean responsiveness of cells excited by GnRH was significantly elevated over inhibitory responses. These findings confirm the E/P biasing effect on POA/S unit responses to GnRH. Moreover, they suggest that a dynamic relationship exists between GnRH responses at the cellular level and sexual behavior throughout the course of steroid-induced receptivity.

Single units responsive to noxious heating of glabrous hindfoot skin were recorded in the lumbar spinal cord of rats anesthetized with sodium pentobarbital. Unit responses to heat stimuli (e.g. 50 degrees C, 10 s) delivered at 2-min intervals were stable and were markedly suppressed during stimulation (100-ms pulse trains at 100 Hz, 3/s, 25-400 microA) in the midbrain periaqueductal gray (PAG) or lateral reticular formation (LRF). Inhibition did not appear to outlast the midbrain stimulation period. By systematically varying the position of an array of 3 or 5 stimulating electrodes, we observed that stimulation in PAG and subjacent tegmental areas, as well as in widespread regions of the LRF bilaterally, suppressed unit responses to noxious skin heating. The degree of suppression of unit responses increased with graded increases in PAG or LRF stimulation intensity. LRF appeared to be more efficacious than PAG stimulation, based on lower currents at threshold, as well as on significantly greater slopes in current-inhibition plots for LRF compared to PAG stimulation. Unit responses increased linearly with graded increases in stimulus temperature from 42 to 54 degrees C. Slopes of temperature-response lines were reduced during PAG stimulation with no change in response threshold, while temperature-response lines were shifted in a parallel manner toward higher temperatures during LRF stimulation with an increase in response threshold. The results suggest that differential inhibitory systems are activated by PAG or LRF stimulation and are discussed in relation to previous findings in the cat and as a possible mechanism of stimulation-produced analgesia.

Eugenol and carvacrol from clove and oregano, respectively, are agonists of the warmth-sensitive transient receptor potential channel TRPV3 and the irritant-sensitive transient receptor potential ankyrin (TRPA)-1. Eugenol and carvacrol induce oral irritation that rapidly desensitizes, accompanied by brief enhancement of innocuous warmth and heat pain in humans. We presently investigated if eugenol and carvacrol activate nociceptive primary afferent and higher order trigeminal neurons and enhance their heat-evoked responses, using calcium imaging of cultured trigeminal ganglion (TG) and dorsal root ganglion (DRG) neurons, and in vivo single-unit recordings in trigeminal subnucleus caudalis (Vc) of rats. Eugenol and carvacrol activated 20-30% of TG and 7-20% of DRG cells, the majority of which additionally responded to menthol, mustard oil and/or capsaicin. TG cell responses to innocuous (39°) and noxious (42 °C) heating were enhanced by eugenol and carvacrol. We identified dorsomedial Vc neurons responsive to noxious heating of the tongue in pentobarbital-anesthetized rats. Eugenol and carvacrol dose-dependently elicited desensitizing responses in 55% and 73% of heat-sensitive units, respectively. Responses to noxious heat were briefly enhanced by eugenol and carvacrol. Many eugenol- and carvacrol-responsive units also responded to menthol, cinnamaldehyde and capsaicin. These data support a peripheral site for eugenol and carvacrol to enhance warmth- and noxious heat-evoked responses of trigeminal neurons, and are consistent with the observation that these agonists briefly enhance warmth and heat pain on the human tongue.

While considerable effort has been made to investigate the neural mechanisms of pain, much less effort has been devoted to itch, at least until recently. However, itch is now gaining increasing recognition as a widespread and costly medical and socioeconomic issue. This is accompanied by increasing interest in the underlying neural mechanisms of itch, which has become a vibrant and rapidly-advancing field of research. The goal of the present forefront review is to describe the recent progress that has been made in our understanding of itch mechanisms.

Itch, an unpleasant sensation associated with the desire to scratch, is symptomatic of dermatologic and systemic disorders that often resist antihistamine treatment. Histamine-independent itch mediators include serotonin (5-HT) and agonists of the protease-activated receptor-2 (PAR-2). We used behavior, Fos immunohistochemistry, and electrophysiology to investigate if these mediators activate spinal dorsal horn neurons in a manner consistent with itch. Intradermal (id) injection of the PAR-2 agonist SLIGRL-NH2 in the rostral back evoked bouts of directed hindlimb scratches over 20 -30 min. Hindpaw injection of SLIGRL-NH2 produced Fos staining in superficial dorsal horn which was then targeted for single-unit recording. Small id microinjections of SLIGRL-NH2 or 5-HT identified responsive single units in the superficial dorsal horn of mice anesthetized with pentobarbital. Thirty-eight units characterized as wide dynamic range, nociceptive specific, or mechanically insensitive exhibited significantly increased firing after id SLIGRL-NH2 for 9 min, to partial (25%) tachyphylaxis with repeated injection. A majority additionally responded to 5-HT (70%), mustard oil (79%), and capsaicin (71%). Seven units isolated with the 5-HT search stimulus exhibited significant and prolonged responses to 5-HT with tachyphylaxis to repeated injections. The majority also responded to SLIGRL-NH2 , mustard oil, and capsaicin. The prolonged responses of superficial dorsal horn neurons to SLIGRL-NH2 and 5-HT suggest a role in signaling itch. However, their responsiveness to algogens is inconsistent with itch specificity. Alternatively, such neurons may signal itch, whereas noxious stimulus levels recruit these and a larger population of pruritogen-insensitive cells to signal pain which masks or occludes the itch signal. [ABSTRACT FROM AUTHOR]

The method of c-fos immunodetection was used to map the distributions of neurons in the lumbar spinal dorsal horn that were activated following intracutaneous (i.c.) microinjection, or iontophoretic application, of different irritant chemicals to the lateral hindpaw of rats. Microinjections (1 µl) of histamine, serotonin (5-HT), nicotine, capsaicin, or formalin each elicited similar distributions of Fos-like immunoreactivity (FLI) in laminae I-II of the ipsilateral superficial dorsal horn, with little or no FLI in deeper laminae or contralaterally. In laminae I and II, FLI cell counts were significantly higher following i.c. histamine, 5-HT, capsaicin, formalin, and noxious pinch, compared to i.c. saline controls. Capsaicin-evoked FLI was dose-dependent. Multivariate analysis of variance revealed no significant difference in spatial distributions of FLI induced by any of the chemical or pinch stimuli. Iontophoretic application of histamine, 5-HT, or nicotine also elicited similar distributions of FLI in the superficial dorsal horn, and cell counts of FLI were significantly higher compared to controls receiving iontophoretic vehicle (methyl cellulose). These results indicate either that individual laminae I-II neurons are activated by each of the irritant chemicals, or that neurons selectively responsive to a given irritant are comingled without any apparent laminar segregation. [ABSTRACT FROM AUTHOR]

Highlights: [•] Intradermal injection of pruritogens in the calf elicits biting behavior. [•] After histamine, touch elicited biting behavior (alloknesis). [•] Capsaicin elicited licking; postcapsaicin touch elicited flinching (allodynia). [•] Histamine-responsive dorsal horn neurons exhibit enhanced response to touch. [•] Model is useful to study spinal mechanisms of alloknesis and allodynia. [Copyright &y& Elsevier]

Abstract: Menthol is used in pharmaceutical applications because of its desired cooling and analgesic properties. The neural mechanism by which topical application of menthol decreases heat pain is not fully understood. We investigated the effects of topical menthol application on lumbar dorsal horn wide dynamic range and nociceptive-specific neuronal responses to noxious heat and cooling of glabrous hindpaw cutaneous receptive fields. Menthol increased thresholds for responses to noxious heat in a concentration-dependent manner. Menthol had a biphasic effect on cold-evoked responses, reducing the threshold (to warmer temperatures) at a low (1%) concentration and increasing threshold and reducing response magnitude at high (10%, 40%) concentrations. Menthol had little effect on responses to innocuous or noxious mechanical stimuli, ruling out a local anesthetic action. Application of 40% menthol to the contralateral hindpaw tended to reduce responses to cooling and noxious heat, suggesting a weak heterosegmental inhibitory effect. These results indicate that menthol has an analgesic effect on heat sensitivity of nociceptive dorsal horn neurons, as well as biphasic effects on cold sensitivity, consistent with previous behavioral observations. [Copyright &y& Elsevier]

Abstract: TRPA1 agonists cinnamaldehyde (CA) and mustard oil (allyl isothiocyanate=AITC) induce heat hyperalgesia and mechanical allodynia in human skin, and sensitize responses of spinal and trigeminal dorsal horn neurons to noxious skin heating in rats. TRPA1 is also implicated in cold nociception. We presently used behavioral methods to investigate if CA affects sensitivity to thermal and mechanical stimuli in rats. Unilateral intraplantar injection of CA (5–20%) induced a significant, concentration-dependent reduction in latency for ipsilateral paw withdrawal from a noxious heat stimulus, peaking (61.7% of pre-injection baseline) by 30min with partial recovery at 120min. The highest dose of CA also significantly reduced the contralateral paw withdrawal latency. CA significantly reduced mechanical withdrawal thresholds of the injected paw that peaked sooner (3min) and was more profound (44.4% of baseline), with no effect contralaterally. Bilateral intraplantar injections of CA resulted in a significant cold hyperalgesia (cold plate test) and a weak enhancement of innocuous cold avoidance (thermal preference test). The data are consistent with roles for TRPA1 in thermal (hot and cold) hyperalgesia and mechanical allodynia. [Copyright &y& Elsevier]

Abstract: The retrograde tracer cholera toxin beta-subunit (CTB) was used to trace long ascending propriospinal projections from neurons in the lumbosacral spinal cord to the upper cervical (C3) gray matter in adult male Sprague–Dawley rats. Following large 0.5μl CTB injections restricted mainly to the upper cervical ventral horn (n =5), there were many lumbosacral CTB-positive neurons (14–17/section) in the intermediate gray and ventral horn (dorsal lamina VIII, medial VII extending into X) contralaterally, with fewer at corresponding ipsilateral locations. Labeled cells (4–8/section) were also observed in contralateral laminae IV–VI and the lateral spinal nucleus, with fewer ipsilaterally. Few labeled cells (<2/section) were observed in superficial laminae I–II. Smaller (0.15μl) microinjections of CTB restricted to the upper cervical ventral gray matter labeled cells in contralateral laminae VII–VIII (~6–9/section) with fewer ipsilaterally. There were relatively fewer (<2/section) in the intermediate dorsal horn and very few (<1/section) in lamina I. Larger (0.5μl) CTB injections encompassing the C3 dorsal and ventral gray matter on one side labeled significantly more CTB-positive neurons (>6/section) in contralateral lamina I compared to ventral horn injections. These results suggest direct projections from ventromedially located neurons of lumbar and sacral segments to the contralateral ventral gray matter of upper cervical segments, as well as from neurons in the intermediate but not superficial dorsal horn. They further suggest that some lumbosacral superficial dorsal horn neurons project to the upper cervical dorsal horn. These propriospinal projections may be involved in coordinating head and neck movements during locomotion or stimulus-evoked motor responses. [Copyright &y& Elsevier]