Roles of glutamate, substance P, and gastrin-releasing peptide as spinal neurotransmitters of histaminergic and nonhistaminergic itch.

Electrophysiology

Experiments were performed using 118 adult male C57BL/6 mice (18–33 g) under a protocol approved by the UC Davis Animal Care and Use Committee. The single-unit recording from the lumbar spinal cord was conducted as previously detailed [ 3 ; 8 ]. Anesthesia was induced by sodium pentobarbital (60 mg/kg ip) and maintained by supplemental injections (10–20 mg/kg/hr). A gravity-driven perfusion system allowed artificial cerebrospinal fluid (Krebs: 117 mM NaCl, 3.6 mM KCl, 2.5 mM CaCl 2 , 1.2 mM MgCl 2 , 1.2 mM NaH 2 PO 4 , 25 mM NaHCO 3 and 11 mM glucose which was equilibrated with 95% O 2 and 5% CO 2 at 37°C) to be superfused continually over the exposed lumbosacral spinal cord [ 7 ]. A tungsten microelectrode recorded single-unit activity in the lumbar spinal cord. A chemical search strategy [ 3 ; 8 ] was used to identify and isolate chloroquine-responsive units. Our search strategy was intended to maximize the chance of isolating a chloroquine-responsive neuron; we assume that such neurons either gave rise to ascending projections, or served as interneurons in segmental scratch-reflex circuitry, and no attempt was presently made to distinguish between these possibilities. Briefly, a small (~0.25 μl) intradermal (id) microinjection of chloroquine (100 μg/μl) was made in the ventral hindpaw and a spontaneously active unit in the superficial lumbar dorsal horn (depth < 300 um) was isolated. After the spontaneous activity had waned, chloroquine (1 μl, 100 μg/μl) was injected again at the same site through the same needle. Only units exhibiting an increase of >30% in firing to this second test microinjection of chloroquine were selected for further study. Responses were usually recorded for at least 30 min, although in many cases unit firing declined over a shorter period. During the time that the unit exhibited a relatively stable level of chloroquine-evoked firing (usually 1 min post-injection), one of the following antagonists were successively delivered directly at spinal cord for 1 min; the GRP receptor antagonist RC-3095 (20 μM), the NK-1 receptor antagonist L-733060 (200 μM), the AMPA/kainate receptor antagonist CNQX (100 μM), a combination of CNQX (100 μM) and L-733060 (200 μM), or a combination of RC-3095 (20 μM), CNQX (100 μM) and L-733060 (200 μM). When unit firing to the second chloroquine injection declined and reached a steady level, the unit’s mechanosensitive receptive field was determined. The perimeter of the mechanical receptive field was mapped using a von Frey filament (55 mN bending force) by determining sites at which the unit either did (within receptive field) or did not (outside receptive field) respond to at least 3 of 5 repeated applications. The rationale for choosing this bending force is that it was sufficient to map the maximum extent of the mechanical receptive field as assessed by comparison of receptive field sizes mapped using a range of von Frey stimuli (0.7 mN: 1.2±0.6 mm 2 , 6.9 mN: 3.7±0.9 mm 2 , 55mN: 9.9±1.4 mm 2 , 758 mN: 10.3±1.4 mm 2 ). Units were classified as wide dynamic range (WDR) –type if they responded in a graded manner to innocuous mechanical stimulation (brushing, cotton wisp) and noxious pinch, or nociceptive-specific (NS) if they responded to noxious pinch (and to the 55 mN von Frey stimulus) but not to the cotton wisp or brush stimuli. The properties are similar to those of WDR and NS units shown in previous studies to respond to histamine, serotonin, the PAR-2/ MrgprC11 agonist SLIGRL, or chloroquine [ 3 ; 8 ]. In some units, at least 5 min after the noxious pinch stimulus, either the NK-1 receptor antagonist, the AMPA/kainate receptor antagonist, or a mixture of both, was superfused directly over the spinal cord for 1 min. At the end of the antagonist superfusion, the noxious pinch stimulus was delivered again at the same site on the receptive field. Thirty min later, the noxious pinch stimulus was delivered in the same manner. Following mechanical stimulation, histamine (50 μg) was injected id within the same receptive field at a different location via a separate injection cannula. Following the id histamine injection, we tested the effects of antagonists for the receptors of NK1, GRP, or AMPA/kainate in the same manner as described above for chloroquine. Units were then tested with topical hindpaw application of allyl isothiocyanate (AITC; mustard oil, Sigma; 75% in mineral oil, 2 μl). Following the AITC application, we tested the effects of the NK-1 receptor antagonist, the AMPA/kainate receptor antagonist, or a combination of both antagonists in the same manner as described above for chloroquine. Action potentials were recorded to a computer and counted using Chart software (AD Instruments, Colorado Springs CO) and Spike2 software (CED Instruments). Ongoing responses elicited by chloroquine, histamine, or AITC were averaged at 20-sec intervals before, during and after the antagonist application, and compared by one way repeated-measures analysis of variance (ANOVA) followed by post-hoc Bonferroni test, with p < 0.05 set as significant. The mean firing rate was calculated over a 20-sec period 40 sec after the antagonist application, and compared by one way ANOVA followed by post-hoc Bonferroni test, with p < 0.05 set as significant. The criterion for decrease in ongoing firing was >70% decrease below the ongoing activity elicited by pruritogen over a 20-sec period 40 sec after the antagonist application. Mean peak responses elicited by noxious pinch were compared by one way repeated-measures ANOVA followed by post-hoc Bonferroni test, with p < 0.05 set as significant. At the end of each experiment, an electrolytic lesion was made at the spinal cord recording site. The spinal cord was postfixed in 10% buffered formalin, cut in 50 μm frozen sections, and examined under the light microscope to identify lesions.

Behavior

Experiments were conducted using adult male C57BL/6 mice (Simonsen, Gilroy, CA; 19–25 g) under a protocol approved by the UC Davis Animal Care and Use Committee. The fur on the rostral back was shaved and mice were habituated to the Plexiglas recording arena one week prior to testing. For intrathecal injections, either vehicle (saline), the GRP receptor antagonist RC-3095 (0.3 nmol; Sigma-Aldrich, St. Louis MO), the NK-1 antagonist L-733060 (22.7 nmol; Tocris Bioscience, Minneapolis, MN), the AMPA/kainate antagonist CNQX (20 nmol; Tocris Bioscience), a combination of CNQX (20 nmol) and L-733060 (22.7 nmol), or a combination of RC-3095 (0.3 nmol), CNQX (20 nmol) and L-733060 (22.7 nmol), was administered by lumbar puncture, followed 5 min later by id injection (10 μl) of either chloroquine (193 nmol; Sigma-Aldrich) or histamine (271 nmol; Sigma-Aldrich). Microinjections were made id in the nape of the neck using a 30 G needle attached to a Hamilton microsyringe by PE-50 tubing. Immediately after the id injection the mouse was placed into the arena and videotaped from above for 30 min. Generally 3–4 mice were injected and videotaped simultaneously. Immediately after commencing videotaping all investigators left the room. Videotapes were reviewed by investigators blinded to the treatment, and the number of scratch bouts was counted at 5-min intervals. A scratch bout was defined as one or more rapid back-and-forth hind paw motions directed toward and contacting the injection site, and ending with licking or biting of the toes and/or placement of the hind paw on the floor. Hind paw movements directed away from the injection site (e.g., ear-scratching) and grooming movements were not counted. One-way ANOVA followed by the Bonferroni post-test was used to compare the total number of scratch bouts across pretreatment groups. In all cases p