Anatomical evidence of pruriceptive trigeminothalamic and trigeminoparabrachial projection neurons in mice.

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.

Eugenol and carvacrol excite first- and second-order trigeminal neurons and enhance their heat-evoked responses.

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.

A tingling sanshool derivative excites primary sensory neurons and elicits nocifensive behavior in rats.

Szechuan peppers contain hydroxy-α-sanshool that imparts desirable tingling, cooling, and numbing sensations. Hydroxy-α-sanshool activates a subset of sensory dorsal root ganglion (DRG) neurons by inhibiting two-pore potassium channels. We presently investigated if a tingle-evoking sanshool analog, isobutylalkenyl amide (IBA), excites rat DRG neurons and, if so, if these neurons are also activated by agonists of TRPM8, TRPA1, and/or TRPV1. Thirty-four percent of DRG neurons tested responded to IBA, with 29% of them also responding to menthol, 29% to cinnamic aldehyde, 66% to capsaicin, and subsets responding to two or more transient receptor potential (TRP) agonists. IBA-responsive cells had similar size distributions regardless of whether they responded to capsaicin or not; cells only responsive to IBA were larger. Responses to repeated application of IBA at a 5-min interstimulus interval exhibited self-desensitization (tachyphylaxis). Capsaicin did not cross-desensitize responses to IBA to any greater extent than the tachyphylaxis observed with repeated IBA applications. These findings are consistent with psychophysical observations that IBA elicits tingle sensation accompanied by pungency and cooling, with self-desensitization but little cross-desensitization by capsaicin. Intraplantar injection of IBA elicited nocifensive responses (paw licking, shaking-flinching, and guarding) in a dose-related manner similar to the effects of intraplantar capsaicin and serotonin. IBA had no effect on thermal sensitivity but enhanced mechanical sensitivity at the highest dose tested. These observations suggest that IBA elicits an unfamiliar aversive sensation that is expressed behaviorally by the limited response repertoire available to the animal.

Excitation of mouse superficial dorsal horn neurons by histamine and/or PAR-2 agonist: potential role in itch.

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.

Mustard oil enhances spinal neuronal responses to noxious heat but not cooling.

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.

Modulation of oral heat and cold pain by irritant chemicals.

Common food irritants elicit oral heat or cool sensations via actions at thermosensitive transient receptor potential (TRP) channels. We used a half-tongue, 2-alternative forced-choice procedure coupled with bilateral pain intensity ratings to investigate irritant effects on heat and cold pain. The method was validated in a bilateral thermal difference detection task. Capsaicin, mustard oil, and cinnamaldehyde enhanced lingual heat pain elicited by a 49 degrees C stimulus. Mustard oil and cinnamaldehyde weakly enhanced lingual cold pain (9.5 degrees C), whereas capsaicin had no effect. Menthol significantly enhanced cold pain and weakly reduced heat pain. To address if capsaicin's effect was due to summation of perceptually similar thermal and chemical sensations, one-half of the tongue was desensitized by application of capsaicin. Upon reapplication, capsaicin elicited little or no irritant sensation yet still significantly enhanced heat pain on the capsaicin-treated side, ruling out summation. In a third experiment, capsaicin significantly enhanced pain ratings to graded heat stimuli (47 degrees C to 50 degrees C) resulting in an upward shift of the stimulus-response function. Menthol may induce cold hyperalgesia via enhanced thermal gating of TRPM8 in peripheral fibers. Capsaicin, mustard oil, and cinnamaldehyde may induce heat hyperalgesia via enhanced thermal gating of TRPV1 that is coexpressed with TRPA1 in peripheral nociceptors.

Effects of TRPA1 agonists mustard oil and cinnamaldehyde on lumbar spinal wide-dynamic range neuronal responses to innocuous and noxious cutaneous stimuli in rats.

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.

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.

Oral irritation by mustard oil: self-desensitization and cross-desensitization with capsaicin.

We investigated the temporal pattern of oral irritation elicited by sequential application of mustard oil (allyl-isothiocyanate), and whether it exhibits self-desensitization and cross-desensitization with capsaicin. Mustard oil (0.125%, 40 micro l) was sequentially applied to one side of the tongue at 1 min intervals, and subjects rated the intensity of the irritant sensation elicited by each stimulus. Ratings successively declined across trials, indicating desensitization. In contrast, sequential application of capsaicin (10 ppm) elicited irritation that increased in intensity across trials (sensitization). To test for self-desensitization by mustard oil, a 10 min hiatus was imposed following the series of unilateral mustard oil stimuli, after which mustard oil was applied to both sides of the tongue. In a two-alternative forced-choice paradigm, subjects chose which side had stronger irritation and also independently rated the irritant intensity on each side. A significant majority of subjects chose the side not previously receiving mustard oil as more intense, and assigned significantly higher intensity ratings to that side, indicating self-desensitization. In two additional sessions, the same paradigm was used to show mustard oil cross-desensitization of irritation elicited by capsaicin, and capsaicin cross-desensitization of irritation from mustard oil. In a final session, sequential application of mustard oil at faster (20 s) intervals initially evoked a sensitizing pattern followed by desensitization. The temporal patterns of oral irritation exhibited by mustard oil, and its reciprocal cross-desensitization with capsaicin, are similar to those of menthol and nicotine.

Taste suppression following lingual capsaicin pre-treatment in humans.

The effect of oral capsaicin on taste sensations in humans was reinvestigated with attention to methodological issues raised in previous studies, including the mode of presentation and temperature of the tastant stimulus, as well as the sensitizing and desensitizing properties of capsaicin. One-half of the dorsal anterior tongue was pre-treated with capsaicin, followed by bilateral tastant application (sucrose, NaCl, quinine, monosodium glutamate and citric acid). Subjects indicated on which side the taste intensity was greater in a two-alternative, forced-choice procedure and also rated taste intensity independently on each side of the tongue. Each of the five tastants was tested sequentially, with reapplication of capsaicin between trials in order to maintain a constant level of burn. Four experiments were conducted: (i) a high concentration (33 p.p.m.) (109 microM) capsaicin effect on taste intensity elicited by high tastant concentrations; (ii) a high concentration capsaicin effect on taste intensity elicited by low tastant concentrations; (iii) a low concentration (1.5 p.p.m.) (4.9 microM) capsaicin effect on taste intensity elicited by low tastant concentrations; and (iv) validation of the method for localizing taste by pre-treating one side of the tongue with Gymnema sylvestre, followed by bilateral application of sucrose. In the first experiment, a significant proportion of the subjects chose the non-treated side in the two-alternative, forced-choice procedure and assigned significantly higher ratings to that side for sucrose-induced sweetness, quinine-induced bitterness and glutamate-induced umami sensations. Salty and sour sensations were not different between sides. A 15 min break was imposed in order to allow the capsaicin burn to disappear and desensitization to set in, followed by reapplication of the tastant test solutions. There were no bilateral differences in the intensity of the sensations elicited by any of the five tastants. Similar results were obtained in experiments 2 and 3. In the fourth experiment, all 15 subjects tested chose the side not treated with Gymnema sylvestre as having a stronger sweet taste and assigned significantly higher ratings to that side, thereby validating the method for taste localization. These results indicate that oral capsaicin reduces certain but not all taste sensations and are discussed in terms of possible physiological and cognitive interactions.

Variable effects of nitrous oxide at multiple levels of the central nervous system in goats.

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.

Isoflurane depresses electroencephalographic and medial thalamic responses to noxious stimulation via an indirect spinal action.

UNLABELLED: Anesthetics such as isoflurane act in the spinal cord to suppress movement in response to noxious stimulation. Spinal anesthesia decreases hypnotic/sedative requirements, possibly by decreasing afferent transmission of stimuli. We hypothesized that isoflurane action in the spinal cord would similarly depress the ascending transmission of noxious input to the thalamus and cerebral cortex. In six isoflurane-anesthetized goats, we measured electroencephalographic (EEG) and thalamic single-unit responses to a clamp applied to the forelimb. Cranial bypass permitted differential isoflurane delivery to the torso and cranial circulations. When the cranial-torso isoflurane combination was 1.3% +/- 0.2%-1.0% +/- 0.4% the noxious stimulus did not evoke significant changes in the EEG or thalamic activity: 389 (153-544) to 581 (172-726) impulses/min, (median, 25th-75th percentile range, P: > 0.05). When the cranial-torso isoflurane combination was 1.3% +/- 0.2%-0.3% +/- 0.2%, noxious stimulation increased thalamic activity: 804 (366-1162) to 1124 (766-1865) impulses/min (P: < 0.05), and the EEG "desynchronized": total EEG power decreased from 25 +/- 20 microV(2) to 12 +/- 8 microV(2) (P: < 0.05). When the cranial-torso isoflurane was 1.7% +/- 0.1%-0.3% +/- 0.2%, the noxious stimulus did not significantly affect thalamic: 576 (187-738) to 1031 (340-1442) impulses/min (P: > 0.05), or EEG activity. The indirect torso effect of isoflurane on evoked EEG total power (12.6 +/- 2.7 microV(2)/vol%, mean +/- SE) was quantitatively similar to the direct cranial effect (17.7 +/- 3.0 microV(2)/vol%; P: > 0.05). These data suggest that isoflurane acts in the spinal cord to blunt the transmission of noxious inputs to the thalamus and cerebral cortex, and thus might indirectly contribute to anesthetic endpoints such as amnesia and unconsciousness. IMPLICATIONS: Isoflurane action in the spinal cord diminished the transmission of noxious input to the brain. Because memory and consciousness are likely dependent on the "arousal" state of the brain, this indirect action of isoflurane could contribute to anesthetic-induced amnesia and unconsciousness.

Isoflurane action in the spinal cord blunts electroencephalographic and thalamic-reticular formation responses to noxious stimulation in goats.

BACKGROUND: Isoflurane depresses the electroencephalographic (EEG) activity and exerts part of its anesthetic effect in the spinal cord. The authors hypothesized that isoflurane would indirectly depress the EEG and subcortical response to noxious stimulation in part by a spinal cord action. METHODS: Depth electrodes were inserted into the midbrain reticular formation (MRF) and thalamus of six of seven isoflurane-anesthetized goats, and needle-electrodes were placed into the skull periosteum. In five of seven goats, an MRF microelectrode recorded single-unit activity. The jugular veins and carotid arteries were isolated to permit cranial bypass and differential isoflurane delivery. A noxious mechanical stimulus (1 min) was applied to a forelimb dewclaw at each of two cranial-torso isoflurane combinations: 1.1+/-0.3%-1.2+/-0.3% and 1.1+/-0.3-0.3+/-0.1% (mean +/- SD). RESULTS: When cranial-torso isoflurane was 1.1-1.2%, the noxious stimulus did not alter the EEG. When torso isoflurane was decreased to 0.3%, the noxious stimulus activated the MRF, thalamic, and bifrontal-hemispheric regions (decreased high-amplitude, low-frequency power). For all channels combined, total (-33+/-15%), delta(-51+/-22%), theta (-33+/-19%), and alpha (-26+/-16%) power decreased after the noxious stimulus (P<0.05); beta power was unchanged. The MRF unit responses to the noxious stimulus were significantly higher when the spinal cord isoflurane concentration was 0.3% (1,286+/-1,317 impulses/min) as compared with 1.2% (489+/-437 impulses/min, P<0.05). CONCLUSIONS: Isoflurane blunted the EEG and MRF-thalamic response to noxious stimulation in part via an action in the spinal cord.

Isoflurane blunts electroencephalographic and thalamic-reticular formation responses to noxious stimulation in goats.

BACKGROUND: Anesthetics, including isoflurane, depress the electroencephalogram (EEG). Little is known about the quantitative effects of isoflurane on EEG and subcortical electrical activity responses to noxious stimulation. The authors hypothesized that isoflurane would depress the results of EEG and subcortical response to noxious stimulation at concentrations less than those needed to suppress movement. Furthermore, determination of regional differences might aid in elucidation of sites of anesthetic action. METHODS: Ten goats were anesthetized with isoflurane, and minimum alveolar concentration (MAC) was determined using a noxious mechanical stimulus. Depth electrodes were inserted into the midbrain reticular formation and thalamus. Needle electrodes placed in the skull periosteum measured bifrontal and bihemispheric EEG. The noxious stimulus was applied at each of four anesthetic concentrations: 0.6, 0.9, 1.1, and 1.4 MAC. RESULTS: At an isoflurane concentration of 0.6 MAC, the noxious stimulus activated the midbrain reticular formation, thalamic, and bifrontal-hemispheric regions, as shown by decreased high-amplitude, low-frequency power. For all channels combined (mean +/- SD), total (-33+/-7%), delta (-47+/-12%), theta (-23+/-12%), and alpha (-21+/-6%) power decreased after the noxious stimulus (P < 0.001); beta power was unchanged. At 0.9 MAC, total (-35+/-5%), delta (-42+/-7%), theta (-35+/-8%), and alpha (-23+/-11%) power decreased after the noxious stimulus (P < 0.001); beta power was unchanged. At 1.1 MAC only one site, and at 1.4 MAC, no site, had decreased power after the noxious stimulus. CONCLUSIONS: Isoflurane blunted EEG and midbrain reticular formation-thalamus activation response to noxious stimulation at concentrations (1.1 MAC or greater) necessary to prevent movement that occurred after noxious stimulation. It is unknown whether this is a direct effect or an indirect effect via action in the spinal cord.

Activation of neurons in rat trigeminal subnucleus caudalis by different irritant chemicals applied to oral or ocular mucosa.

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 (

Interneurons presynaptic to rat tail-flick motoneurons as mapped by transneuronal transport of pseudorabies virus: few have long ascending collaterals.

The method of transneuronal retrograde transport of the Bartha strain of the swine alpha-herpes virus, pseudorabies virus, was used to identify putative interneurons presynaptic to motoneurons that supply a tail-flick muscle in the rat. We also investigated whether these interneurons also contribute to ascending somatosensory pathways. Two to five days after injection of pseudorabies virus into the left abductor caudae dorsalis muscle, and cholera toxin B into the right somatosensory thalamus and midbrain, rats were perfused and spinal cord sections processed immunohistochemically in a two-step procedure to stain cholera toxin B-immunoreactive cells black and pseudorabies virus-immunoreactive cells brown. At short (two-day) survivals, the first spinal neurons to be pseudorabies virus-immunoreactive were in the ipsilateral abductor caudae dorsalis motoneuron pool (S3-S4) and intermediolateral cell column (T12-L2), with a few (0 to five/section) bilaterally in the intermediate zone and around the central canal (all lumbosacral levels). With longer (three- to four-day) survival, more cells were noted (20-50/section) bilaterally (ipsilateral preponderance) in the dorsal and ventral horns of the lumbosacral cord. Many were in lamina I (marginal layer), while few were in lamina II (substantia gelatinosa). At four- and five-day survivals, the numbers of cells increased (20 to 100/section) bilaterally and now included lamina II. The fact that unilateral rhizotomy at L4-Co1 failed to change the distribution of spinal pseudorabies virus labeling suggests that the labeling was due to retrograde transport via the ventral root. In support, bilateral removal of the lumbar sympathetic ganglia, which receive their preganglionic innervation through the ventral root, reduced pseudorabies virus immunoreactivity throughout the thoracic and rostral lumbar spinal cord. These data indicate that there are (i) direct projections from intermediate and dorsal horn cells to abductor caudae dorsalis motoneurons, and (ii) disynaptic connections from dorsal horn (possibly including lamina II) cells to more ventral last-order interneurons. We also suggest that some lamina II cells are presynaptic to lamina I cells that project directly to abductor caudae dorsalis motoneurons. We observed cholera toxin B-immunoreactive cells (five to 20/section) in the expected locations (contralateral lamina I, deep dorsal horn and intermediate zone; lateral spinal nucleus bilaterally). Double-labeled (i.e. pseudorabies virus- and cholera toxin B-immunoreactive) neurons were only occasionally seen in the lateral spinal nucleus and were absent in the spinal gray matter, indicating that segmental interneurons do not collateralize in long ascending sensory pathways to the midbrain and somatosensory thalamus.

Demonstration of extensive brainstem projections to medial and lateral thalamus and hypothalamus in the rat.

We use the method of retrograde transport of colloidal gold with silver intensification to map locations of brainstem neurons projecting to various nuclei of the medial and lateral thalamus (Menetrey, Histochemistry 83, 391-395, 1985; Seeley and Field, Brain Res. 449, 177-191, 1988). In rats injections of less than 1.0 microliters of a gold-wheatgerm agglutinin-horseradish peroxidase complex were restricted to the centrum medianum, centralis lateralis, medialis dorsalis, zona incerta, ventrobasal thalamic complex, or medial or lateral hypothalamus. Injections in the centralis lateralis, with some involvement of centrum medianum and medial medialis dorsalis, labeled numerous neurons throughout the rostrocaudal extent of the midbrain periaqueductal gray, and adjacent reticular formation, dorsal raphe nucleus and substantia nigra. These labeled neurons were distributed bilaterally, but with a preponderance ipsilaterally. Numerous neurons in the deep layers of the ipsilateral superior colliculus and in the anterior pretectal nucleus were also labeled. Many neurons in the pontomedullary raphe nuclei (including nucleus raphe magnus), locus coeruleus and dorsolateral parabrachial nuclei, and in the pontomedullary reticular formation, were labeled bilaterally. Fewer were seen bilaterally in the lateral reticular nucleus and nucleus of the solitary tract, with relatively few in the dorsal aspect of the trigeminal nucleus caudalis. Injections restricted largely to the centrum medianum labeled fewer cells in the brainstem, but with similar distributions. An injection restricted to the centralis lateralis also resulted in a similar distribution of labeled neurons in midbrain and nucleus raphe magnus but did not label neurons at more caudal medullary levels. An injection restricted to zona incerta labeled numerous neurons in a distribution similar to that following medial thalamic injections, except that many more were observed in the contralateral sensory trigeminal nucleus and in the dorsal column nuclei. Injections of the ventrobasal thalamic nucleus labeled many neurons in the trigeminal and dorsal column nuclei, but many fewer neurons in the midbrain, periaqueductal gray and reticular formation compared with medial thalamic injections. Labeled neurons were also seen in the superior and inferior colliculi, due presumably to involvement by the injection of the lateral posterior and magnocellular medial geniculate nuclei, respectively. Injections in the lateral hypothalamus labeled numerous neurons in a distribution similar to that seen following medial thalamic injections.(ABSTRACT TRUNCATED AT 400 WORDS)

Hypothalamic inhibition of rat dorsal horn neuronal responses to noxious skin heating.

The responses of single lumbar dorsal horn neurons to noxious radiant heat stimuli (42-54 degrees C, 10 sec, 1/2 min) applied to glabrous hind paw skin were recorded in rats anesthetized with sodium pentobarbital. Unit responses to 50 or 52 degrees C stimuli were constant over time and were consistently and powerfully inhibited during bipolar stimulation (three 100 msec trains/sec at 100 Hz, 200 microA) in the medial hypothalamus. Inhibition was also evoked by stimulation in medial and ventrobasal thalamic nuclei, lateral hypothalamus and adjacent cerebral peduncle, and amygdala. Inhibition increased with graded increases in intensity of hypothalamic stimulation, with a mean inhibitory threshold of 71 +/- 43 (S.D.) microA for 13 units. The responses of dorsal horn units to graded increases in the temperature of noxious heat stimuli were inhibited during hypothalamic stimulation, such that slopes of the linear temperature-response functions were reduced with no change in response threshold (mean: approximately 44 degrees C). Inhibition was blocked or reduced in 4/7 units following systemic administration of the 5-hydroxytryptamine (5-HT) antagonist methysergide. The results confirm and extend previous work in the cat and are discussed in relation to analgesic mechanisms.