Article Figures & Data
Figures
- Fig. 1.
Effect of N2O on Fos induction in the neurons of the A5, LC, and A7 noradrenergic nuclei. Rats were exposed for 90 min to 70% ATA N2O (n = 7) or room air (n = 7), then transcardially perfused, and the brain was removed. Pontine sections were stained for Fos and TH. The double-stained neurons in each region were counted section by section, and the four sections with the highest counts were summed for each rat. Nitrous oxide exposure dramatically increased Fos expression in the A5 (14 ± 3 vs 5 ± 1 in air), LC (124 ± 17 vs 35 ± 7 in air), and A7 (5 ± 1 vs 0.1 ± 0.1 in air) noradrenergic neurons. **p < 0.01; ***p < 0.001 versus air.
- Fig. 2.
TH (light brown) and Fos (blue) staining in A5 neurons. A,Immunohistochemistry after exposure to air. B,Arrow points to double-stained neuron in A5 cell group of rat exposed to N2O. Note the increase in double-labeled neurons with N2O exposure.
- Fig. 3.
TH (light brown) and Fos (blue) staining in LC noradrenergic neurons.A, Representative section in rat exposed to air.B, Marked increase in double-stained neurons after N2O exposure. Also note the numerous Fos-positive nuclei in the LC region in TH-negative cells.
- Fig. 4.
TH (light brown) and Fos (blue) staining in LC noradrenergic neurons. A Fos-positive nucleus is clearly seen in a TH-positive LC neuron (arrow).
- Fig. 5.
TH (light brown) and Fos (blue) staining in A7 sections. A,Representative section in rat exposed to air. B, Marked increase in double-stained neurons after N2O exposure. Also note the numerous Fos-positive nuclei in TH-negative cells.
- Fig. 6.
Immunolesioning of brainstem noradrenergic neurons with intracerebroventricular DβH–saporin. Rats were injected with saline (3 μl) or DβH–saporin (3 μg/3 μl; n= 14 for each group). Two weeks after injection the rats underwent behavioral testing and then were transcardially perfused, and the brain was removed. The brainstem sections were stained for TH, and the number of TH-positive neurons in A5 and A7 were totaled for all sections. Data were standardized by the mean of the saline group. Approximately 30% of noradrenergic neurons survived in A5 (31 ± 6%) and A7 (28 ± 6%) after lesioning. Counting was not performed for the LC region because TH-positive neurons are densely packed and are difficult to count in the LC. Furthermore, not a single neuron survived in LC region after DβH–saporin treatment. ***p < 0.001 versus saline-treated.
- Fig. 7.
Immunohistochemistry of noradrenergic neurons in the A5 cell group. A, TH of a saline-treated rat.B, Profound reduction in TH-stained neurons in the immunolesioned rat.
- Fig. 8.
Immunohistochemistry of noradrenergic neurons in the LC. A, Very dense TH staining is observed in the LC region of the saline-treated rat. B, No immunoreactivity is observed after DβH–saporin treatment.
- Fig. 9.
Immunohistochemistry of noradrenergic neurons in the A7 cell group. A, TH immunohistochemistry in the A7 cell group of a saline-treated rat. B, Note the absence of TH-stained neurons in the immunolesioned rat.
- Fig. 10.
Effect of noradrenergic lesioning on the antinociceptive and sedative effects of nitrous oxide.A, Tail-flick latency was measured before (air) and after N2O exposure. There was no significant difference in baseline latencies of the saline (n = 11)- and DβH–saporin (n = 12)-treated rats, using the same intensity setting for the radiant heat source. The N2O antinociceptive effect was observed in the saline treatment group (27.7 ± 6.1%MPE), but not in the DβH–saporin-treated rats (1.0 ± 6.7%MPE). Three rats in the control group and two rats in the LC lesion group were excluded from the tail-flick data because of an excessive change in tail temperature after N2O exposure.B, There was no difference between saline (n = 10; 112 ± 3%)- and DβH–saporin (n = 10; 116 ± 3%)-treated rats in the sedative effect of N2O, as measured by the concentration (ATA), which prevented a righting reflex in half the rats (ED50 LORR). *p < 0.05 versus air; ###p < 0.001 versus saline.
- Fig. 11.
A, N2O antinociceptive effect on hot-plate assay was reduced in α2BKO. This figure illustrates the antinociceptive effects of N2O on the hot-plate assay in the α2A−/−, α2C−/− knock-out mice (α2AKO and α2CKO), the D79N mutant mice with nonfunctional α2A adrenoceptors, and in their genetically matched WT controls (on a C57BL/6J congenic background). The α2B−/− (α2BKO) mice were on a different genetic background (C57BL/6J × 129SvJ hybrid), so they required their own genetically matched WT controls (WT for α2B). There were no differences in baseline hot-plate latencies between the various mouse strains (data not shown). Only the α2BKO mice had reduced N2O antinociception on the hot-plate assay (reduced 65%, 24 ± 9 vs 69 ± 9%MPE in WT for α2B), indicating that the α2B adrenoceptor subtype mediates N2O antinociception for this supraspinal response. The N2O antinociceptive responses (%MPE) in the other knock-out and mutant mouse strains were: WT, 62 ± 10; α2AKO, 62 ± 11; α2CKO, 49 ± 18; and D79N, 72 ± 9 (n = 16 for each group). B,N2O antinociceptive effect on tail-flick assay was lost in α2BKO. There were no differences in baseline tail-flick latencies of the α2BKO mice and their WT controls. The WT for α2B mice had a significant N2O antinociceptive effect on the tail-flick assay (14.7 ± 6.2%MPE), but the α2BKO mice had no N2O antinociceptive response on the tail-flick assay (1.3 ± 2.1%MPE), indicating that the α2B adrenoceptor subtype mediates N2O antinociception (n = 10 for each group). N2O had no effect on tail-flick latencies in the C57BL/6J WT controls for the D79N, α2AKO and α2CKO mice, so these strains were not tested for N2O tail-flick effects. *p < 0.05; **p < 0.01 versus air; #p < 0.05; ###p < 0.001 versus genetically matched WT controls.
- Fig. 12.
N2O sedative effect was intact in the α2B−/− knock-out mice. The sedative effects of N2O (35 and 70% ATA) in the α2A−/−, α2B−/−, and α2C−/− knock-out mice (A KO, B KO, and C KO, respectively), the D79N mice, and in their respective WT controls (n = 8 for each group). There were no differences in baseline rotarod latencies among the various mouse strains. The sedative effect was observed on the rotarod assay in all the mouse strains including the B KO mice. An enhanced sedative effect was observed in the C KO mice with the lower concentration of N2O. *p < 0.05 versus air.
- Fig. 13.
Detection of mRNA for α2Badrenoceptor in various tissues from wild-type mice (n = 2). RT-PCR analysis of total RNA prepared from kidney, spinal cord, and heart tissue revealed detectable levels of message for this receptor were found in the kidney and spinal cord but not in heart tissue. The bottom panel demonstrates that total RNA from each of these tissues was of similar quality and yielded the expected ribosomal bands after ethidium staining.
In this issue
