Reduction in ␣ 2A -AR-IR and ␣ 2C -AR-IR after dorsal rhizotomy. Dorsal rhizotomies were performed to determine whether the ␣ 2 -ARs were synthesized in dorsal root ganglia neurons and trafficked centrally into the spinal cord. After rhizotomy, a dramatic reduction in ␣ 2A -AR-IR was observed ipsilateral to the lesion ( A ). A reduction in ␣ 2C -AR-IR was also observed ( C ), although to a lesser extent than for the ␣ 2A -AR. SP-IR, however, was reduced to a similar extent in both tissue sections, indicating that the efficacy of the treatment was similar in each case ( B , D ).

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Differential distribution of alpha2A and alpha2C adrenergic receptor immunoreactivity in the rat spinal cord

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Sep 1998

alpha2-Adrenergic receptors (alpha2-ARs) mediate a number of physiological phenomena, including spinal analgesia. We have developed subtype-selective antisera against the C termini of the alpha2A-AR and alpha2C-AR to investigate the relative distribution and cellular source or sources of these receptor subtypes in the rat spinal cord. Immunoreactiv...

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... open- ing inwardly rectif ying potassium channels (Surprenant and North, 1988; Shen et al., 1992), by decreasing presynaptic calcium influx (Ewald et al., 1989; Surprenant et al., 1990), and by inhib- iting adenylyl cyclase (Andrade and Aghajanian, 1985; Uhl ́n and Wikberg, 1988; Uhl ́n and Wikberg, 1989). Three subtypes of ␣ 2 -ARs have been cloned in human and rat, corresponding to the pharmacological subtypes ␣ 2A , ␣ 2B , and ␣ 2C , respectively (for review, see Bylund et al., 1994). Because of the lack of subtype- selective pharmacological agents, it is unclear which subtype or subtypes contribute to spinal adrenergic analgesia, although a major role has been suggested for the ␣ 2A -AR (Millan, 1992; Millan et al., 1994; Stone et al., 1997b). In situ hybridization studies with probes directed against the ␣ 2A -AR have detected mRNA in a subset of dorsal root ganglion (DRG) and spinal cord neurons (Nicholas et al., 1993). In addition, ␣ 2A -AR mRNA and immunoreactivity have been detected in the cell bodies of neurons in almost all supraspinal noradrenergic nuclei (Nicholas et al., 1993; Rosin et al., 1993; Scheinin et al., 1994). The ␣ 2A -AR may therefore act as an autoreceptor on some noradrenergic terminals. In addition, ␣ 2A -AR immunoreactivity ( ␣ 2A -AR-IR) has been reported in the superficial dorsal horn of the rat spinal cord (Rosin et al., 1993) and in DRG (Gold et al., 1997). Interestingly, although studies have detected ␣ 2C -AR mRNA in a large number of DRG neurons (Nicholas et al., 1993; Gold et al., 1997), spinal ␣ 2C -AR-IR has only been detected in cell bodies in the ventral horn (Rosin et al., 1996). Based on this information, spinal ␣ 2 -ARs may originate from three possible sources. First, the receptors may be synthesized in the cell bodies of DRG neurons and trafficked centrally into the spinal cord, where they would serve a presynaptic function to modulate release of transmitter from these terminals. Second, these receptors may be synthesized by second-order spinal neurons in which they may modulate primary afferent activity either postsynaptically or presynaptically within circuits intrinsic to the spinal cord. Third, these receptors may be synthesized by supraspinal neurons and trafficked to axons and nerve terminals of descending noradrenergic fibers in which they would act as inhibitory autoreceptors controlling noradrenaline release in the spinal cord. The goal of this study was to determine the source or sources of spinal ␣ 2A -AR-IR and ␣ 2C -AR-IR. We have thus generated anti-peptide antisera directed against the C termini of the pre- dicted sequences of the ␣ -AR and ␣ -AR. Dorsal rhizotomy, neonatal capsaicin treatment, and double-labeling experiments were used to determine the origin and cytochemical profile of ␣ 2 -AR immunoreactive fibers and terminals observed in the dorsal horn. Part of these results have been presented in abstract form (Stone et al., 1996, 1997a,c). MDCK cells, transfected with the ␣ 2A -AR, ␣ 2B -AR, or ␣ 2C -AR or untransfected, were stained with rabbit-derived antisera gen- erated against the ␣ 2A -AR or ␣ 2C -AR. ␣ 2A -AR-IR was observed only on cells transfected with the ␣ 2A -AR (Fig. 1 A – D ), whereas ␣ 2C -AR-IR was observed only on cells transfected with the ␣ 2C -AR (Fig. 1 E – H ). The ␣ 2A -AR-IR was blocked by preabsorp- tion of the antiserum with the peptide corresponding to ␣ 2A -AR but not by that corresponding to the ␣ 2C -AR (Fig. 1 I , J ). Simi- larly, ␣ 2C -AR-IR was blocked by preabsorption of the antiserum with the peptide corresponding to the ␣ 2C -AR but not the ␣ 2A -AR (Fig. 1 K , L ). Guinea pig-derived anti- ␣ 2C -AR antisera generated similar results (data not shown). These results demon- strate that both the ␣ 2A -AR and ␣ 2C -AR antisera recognize the receptors against which they were generated and do not cross- react with other known ␣ -AR subtypes. In rat spinal cord, both ␣ 2A -AR-IR and ␣ 2C -AR-IR were ob- served in nerve terminals and varicosities. No definitive evidence for cell body labeling was obtained. These immunoreactive ter- minals were most highly concentrated in laminae I and II of the dorsal horn at all levels of the spinal cord (Fig. 2 A , C ). In addition, ␣ 2A -AR-IR was observed in the area surrounding the central canal and in the intermediolateral cell column of the thoracic cord (data not shown). Strong ␣ 2C -AR-IR was observed in the lateral spinal nucleus and was more prevalent in deeper layers of the dorsal horn than ␣ 2A -AR-IR. Staining was blocked in both cases by preincubation of each antisera with its cognate peptide (Fig. 2 B , D ). To determine whether the spinal ␣ 2A -AR-IR and ␣ 2C -AR-IR were of primary afferent origin, dorsal rhizotomies were per- formed to deplete the spinal cord of primary afferent input. We observed a dramatic reduction in ␣ 2A -AR-IR ipsilateral to dorsal rhizotomy (Fig. 3 A ). In contrast, dorsal rhizotomy yielded only a slight reduction in ␣ 2C -AR-IR (Fig. 3 C ). Double-labeling each section with antiserum directed against SP confirmed that SP-IR was also reduced by dorsal rhizotomy to approximately the same extent (Fig. 3 B , D ) in both sections, indicating that the efficacy of the treatment was similar in each case. To determine whether one or both subtypes is expressed in capsaicin-sensitive neurons, we treated neonatal rats with capsa- icin and examined their spinal cords at 5 weeks of age. Capsaicin, the active ingredient in hot peppers, acts as a neurotoxin on small diameter primary afferents, many of which are SP-containing (Jancso et al., 1977; Nagy et al., 1981). Sensitivity of sensory neurons to capsaicin has been associated with a role in nocicep- tion (Nagy et al., 1980; Jancso et al., 1987; Hammond and Ruda, 1991). We observed a dramatic reduction in ␣ 2A -AR-IR but not ␣ 2C -AR-IR (Fig. 4), indicating that the subset of DRG cells that express the ␣ 2A -AR is likely to consist of small diameter, capsaicin-sensitive nociceptors. In addition, these results suggest that the two subtypes may exist on different populations of neurons. Double-labeling experiments were conducted to determine the relationship between ␣ 2A -AR-IR and ␣ 2C -AR-IR in the superfi- cial dorsal horn. Sections of rat spinal cord double-labeled with anti- ␣ 2A -AR and anti- ␣ 2C -AR antisera produced robust immu- nostaining for each receptor in the superficial dorsal horn (Fig. 5 A , B ). The resultant digital images were then digitally merged (Fig. 5 C ). In merged images, the appearance of yellow may indicate colocalization, although higher resolution is required to distinguish colocalization from close apposition or superimposi- tion. When examined at high resolution, instances of receptor colocalization were rare (Fig. 5 D ). This observation further sug- gests that the two subtypes are located primarily on different neuronal populations. Double-labeling with anti- ␣ 2A -AR and anti-SP antisera revealed extensive colocalization in the spinal cord (Fig. 5 E – H ). Thus, ␣ 2A -ARs appear to be present on the spinal terminals of SP- containing primary afferent fibers. Examination of sections double-labeled for ␣ 2A -AR-IR and CGRP-IR also revealed a significant degree of colocalization (Fig. 5 I – L ). In contrast, stain- ing for the neuropeptide leuenkephalin failed to colocalize with ␣ 2A -AR-IR (Fig. 5 M – P ). To determine whether ␣ 2A -ARs exist as autoreceptors on de- scending noradrenergic terminals, we used antibodies directed against dopamine- ␤ -hydroxylase (D ␤ H) and tyrosine hydroxylase (TH), enzymes involved in the biosynthesis of noradrenaline. Spinal cord sections double-labeled for either ␣ 2A -AR-IR and D ␤ H-IR or ␣ 2A -AR-IR and TH-IR showed no instances of colocalization (Fig. 5 Q – X ). This trend was observed throughout the spinal cord. However, examples of close apposition between D ␤ H-IR or TH-IR and ␣ 2A -AR-IR fibers and terminals were occasionally observed. Whether such close appositions represent axoaxonic synaptic relationships remains to be established. Double-labeling experiments indicated that ␣ 2C -AR-IR and SP-IR are rarely colocalized in the spinal cord (Fig. 6 A – D ). However, ␣ 2C -AR-IR was present on some CGRP-IR (Fig. 6 E – H ) and somatostatin-IR (Fig. 6 I – L ) fibers. Interestingly, some overlap was detected between enkephalin-IR and ␣ 2C - AR-IR (Fig. 6 M – P ). Because enkephalin is not believed to be present in primary afferent fibers (Hokfelt et al., 1977; Johansson et al., 1978; Seybold and Elde, 1980), this result is consistent with a spinal source of ␣ 2C -AR. No colocalization was observed be- tween ␣ 2C -AR-IR and the endogenous opioid peptide preprodynorphin-IR (data not shown). In an effort to further characterize the phenotype of spinal cells that express the ␣ 2C -AR, double-labeling was performed with antisera directed against the NK-1 receptor (Fig. 6 Q – T ). We observed that the ␣ 2C -AR is not detected on either NK-1- expressing neurons or astrocytes, indicated by the rare colocal- ization of the glial marker GFAP with ␣ 2C -AR-IR (Fig. 6 U – X ). ␣ 2C -AR-IR failed to colocalize with either D ␤ H-IR (Fig. 6 Y – BB ) or TH-IR (data not shown), suggesting that neither the ␣ 2A -AR nor the ␣ 2C -AR is likely to be present on the terminals of descending supraspinal noradrenergic neurons. We have developed subtype-specific anti-peptide antisera in rab- bits and guinea pigs directed against the C-terminal portions of the ␣ 2A -AR and ␣ 2C -AR. The resultant antisera recognized their corresponding receptors and did not cross-react with other known ␣ 2 -AR subtypes. In addition, labeling was blocked by preabsorption with the cognate peptide in both transfected cells and tissue sections. Immunoreactivity for both ␣ 2A -AR-IR and ␣ 2C -AR-IR was observed predominantly in fibers in the superfi- cial layers of the dorsal horn of the spinal cord. Both dorsal rhizotomy and neonatal capsaicin treatment re- sulted in ...

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... et al., 1990), and by inhib- iting adenylyl cyclase (Andrade and Aghajanian, 1985; Uhl ́n and Wikberg, 1988; Uhl ́n and Wikberg, 1989). Three subtypes of ␣ 2 -ARs have been cloned in human and rat, corresponding to the pharmacological subtypes ␣ 2A , ␣ 2B , and ␣ 2C , respectively (for review, see Bylund et al., 1994). Because of the lack of subtype- selective pharmacological agents, it is unclear which subtype or subtypes contribute to spinal adrenergic analgesia, although a major role has been suggested for the ␣ 2A -AR (Millan, 1992; Millan et al., 1994; Stone et al., 1997b). In situ hybridization studies with probes directed against the ␣ 2A -AR have detected mRNA in a subset of dorsal root ganglion (DRG) and spinal cord neurons (Nicholas et al., 1993). In addition, ␣ 2A -AR mRNA and immunoreactivity have been detected in the cell bodies of neurons in almost all supraspinal noradrenergic nuclei (Nicholas et al., 1993; Rosin et al., 1993; Scheinin et al., 1994). The ␣ 2A -AR may therefore act as an autoreceptor on some noradrenergic terminals. In addition, ␣ 2A -AR immunoreactivity ( ␣ 2A -AR-IR) has been reported in the superficial dorsal horn of the rat spinal cord (Rosin et al., 1993) and in DRG (Gold et al., 1997). Interestingly, although studies have detected ␣ 2C -AR mRNA in a large number of DRG neurons (Nicholas et al., 1993; Gold et al., 1997), spinal ␣ 2C -AR-IR has only been detected in cell bodies in the ventral horn (Rosin et al., 1996). Based on this information, spinal ␣ 2 -ARs may originate from three possible sources. First, the receptors may be synthesized in the cell bodies of DRG neurons and trafficked centrally into the spinal cord, where they would serve a presynaptic function to modulate release of transmitter from these terminals. Second, these receptors may be synthesized by second-order spinal neurons in which they may modulate primary afferent activity either postsynaptically or presynaptically within circuits intrinsic to the spinal cord. Third, these receptors may be synthesized by supraspinal neurons and trafficked to axons and nerve terminals of descending noradrenergic fibers in which they would act as inhibitory autoreceptors controlling noradrenaline release in the spinal cord. The goal of this study was to determine the source or sources of spinal ␣ 2A -AR-IR and ␣ 2C -AR-IR. We have thus generated anti-peptide antisera directed against the C termini of the pre- dicted sequences of the ␣ -AR and ␣ -AR. Dorsal rhizotomy, neonatal capsaicin treatment, and double-labeling experiments were used to determine the origin and cytochemical profile of ␣ 2 -AR immunoreactive fibers and terminals observed in the dorsal horn. Part of these results have been presented in abstract form (Stone et al., 1996, 1997a,c). MDCK cells, transfected with the ␣ 2A -AR, ␣ 2B -AR, or ␣ 2C -AR or untransfected, were stained with rabbit-derived antisera gen- erated against the ␣ 2A -AR or ␣ 2C -AR. ␣ 2A -AR-IR was observed only on cells transfected with the ␣ 2A -AR (Fig. 1 A – D ), whereas ␣ 2C -AR-IR was observed only on cells transfected with the ␣ 2C -AR (Fig. 1 E – H ). The ␣ 2A -AR-IR was blocked by preabsorp- tion of the antiserum with the peptide corresponding to ␣ 2A -AR but not by that corresponding to the ␣ 2C -AR (Fig. 1 I , J ). Simi- larly, ␣ 2C -AR-IR was blocked by preabsorption of the antiserum with the peptide corresponding to the ␣ 2C -AR but not the ␣ 2A -AR (Fig. 1 K , L ). Guinea pig-derived anti- ␣ 2C -AR antisera generated similar results (data not shown). These results demon- strate that both the ␣ 2A -AR and ␣ 2C -AR antisera recognize the receptors against which they were generated and do not cross- react with other known ␣ -AR subtypes. In rat spinal cord, both ␣ 2A -AR-IR and ␣ 2C -AR-IR were ob- served in nerve terminals and varicosities. No definitive evidence for cell body labeling was obtained. These immunoreactive ter- minals were most highly concentrated in laminae I and II of the dorsal horn at all levels of the spinal cord (Fig. 2 A , C ). In addition, ␣ 2A -AR-IR was observed in the area surrounding the central canal and in the intermediolateral cell column of the thoracic cord (data not shown). Strong ␣ 2C -AR-IR was observed in the lateral spinal nucleus and was more prevalent in deeper layers of the dorsal horn than ␣ 2A -AR-IR. Staining was blocked in both cases by preincubation of each antisera with its cognate peptide (Fig. 2 B , D ). To determine whether the spinal ␣ 2A -AR-IR and ␣ 2C -AR-IR were of primary afferent origin, dorsal rhizotomies were per- formed to deplete the spinal cord of primary afferent input. We observed a dramatic reduction in ␣ 2A -AR-IR ipsilateral to dorsal rhizotomy (Fig. 3 A ). In contrast, dorsal rhizotomy yielded only a slight reduction in ␣ 2C -AR-IR (Fig. 3 C ). Double-labeling each section with antiserum directed against SP confirmed that SP-IR was also reduced by dorsal rhizotomy to approximately the same extent (Fig. 3 B , D ) in both sections, indicating that the efficacy of the treatment was similar in each case. To determine whether one or both subtypes is expressed in capsaicin-sensitive neurons, we treated neonatal rats with capsa- icin and examined their spinal cords at 5 weeks of age. Capsaicin, the active ingredient in hot peppers, acts as a neurotoxin on small diameter primary afferents, many of which are SP-containing (Jancso et al., 1977; Nagy et al., 1981). Sensitivity of sensory neurons to capsaicin has been associated with a role in nocicep- tion (Nagy et al., 1980; Jancso et al., 1987; Hammond and Ruda, 1991). We observed a dramatic reduction in ␣ 2A -AR-IR but not ␣ 2C -AR-IR (Fig. 4), indicating that the subset of DRG cells that express the ␣ 2A -AR is likely to consist of small diameter, capsaicin-sensitive nociceptors. In addition, these results suggest that the two subtypes may exist on different populations of neurons. Double-labeling experiments were conducted to determine the relationship between ␣ 2A -AR-IR and ␣ 2C -AR-IR in the superfi- cial dorsal horn. Sections of rat spinal cord double-labeled with anti- ␣ 2A -AR and anti- ␣ 2C -AR antisera produced robust immu- nostaining for each receptor in the superficial dorsal horn (Fig. 5 A , B ). The resultant digital images were then digitally merged (Fig. 5 C ). In merged images, the appearance of yellow may indicate colocalization, although higher resolution is required to distinguish colocalization from close apposition or superimposi- tion. When examined at high resolution, instances of receptor colocalization were rare (Fig. 5 D ). This observation further sug- gests that the two subtypes are located primarily on different neuronal populations. Double-labeling with anti- ␣ 2A -AR and anti-SP antisera revealed extensive colocalization in the spinal cord (Fig. 5 E – H ). Thus, ␣ 2A -ARs appear to be present on the spinal terminals of SP- containing primary afferent fibers. Examination of sections double-labeled for ␣ 2A -AR-IR and CGRP-IR also revealed a significant degree of colocalization (Fig. 5 I – L ). In contrast, stain- ing for the neuropeptide leuenkephalin failed to colocalize with ␣ 2A -AR-IR (Fig. 5 M – P ). To determine whether ␣ 2A -ARs exist as autoreceptors on de- scending noradrenergic terminals, we used antibodies directed against dopamine- ␤ -hydroxylase (D ␤ H) and tyrosine hydroxylase (TH), enzymes involved in the biosynthesis of noradrenaline. Spinal cord sections double-labeled for either ␣ 2A -AR-IR and D ␤ H-IR or ␣ 2A -AR-IR and TH-IR showed no instances of colocalization (Fig. 5 Q – X ). This trend was observed throughout the spinal cord. However, examples of close apposition between D ␤ H-IR or TH-IR and ␣ 2A -AR-IR fibers and terminals were occasionally observed. Whether such close appositions represent axoaxonic synaptic relationships remains to be established. Double-labeling experiments indicated that ␣ 2C -AR-IR and SP-IR are rarely colocalized in the spinal cord (Fig. 6 A – D ). However, ␣ 2C -AR-IR was present on some CGRP-IR (Fig. 6 E – H ) and somatostatin-IR (Fig. 6 I – L ) fibers. Interestingly, some overlap was detected between enkephalin-IR and ␣ 2C - AR-IR (Fig. 6 M – P ). Because enkephalin is not believed to be present in primary afferent fibers (Hokfelt et al., 1977; Johansson et al., 1978; Seybold and Elde, 1980), this result is consistent with a spinal source of ␣ 2C -AR. No colocalization was observed be- tween ␣ 2C -AR-IR and the endogenous opioid peptide preprodynorphin-IR (data not shown). In an effort to further characterize the phenotype of spinal cells that express the ␣ 2C -AR, double-labeling was performed with antisera directed against the NK-1 receptor (Fig. 6 Q – T ). We observed that the ␣ 2C -AR is not detected on either NK-1- expressing neurons or astrocytes, indicated by the rare colocal- ization of the glial marker GFAP with ␣ 2C -AR-IR (Fig. 6 U – X ). ␣ 2C -AR-IR failed to colocalize with either D ␤ H-IR (Fig. 6 Y – BB ) or TH-IR (data not shown), suggesting that neither the ␣ 2A -AR nor the ␣ 2C -AR is likely to be present on the terminals of descending supraspinal noradrenergic neurons. We have developed subtype-specific anti-peptide antisera in rab- bits and guinea pigs directed against the C-terminal portions of the ␣ 2A -AR and ␣ 2C -AR. The resultant antisera recognized their corresponding receptors and did not cross-react with other known ␣ 2 -AR subtypes. In addition, labeling was blocked by preabsorption with the cognate peptide in both transfected cells and tissue sections. Immunoreactivity for both ␣ 2A -AR-IR and ␣ 2C -AR-IR was observed predominantly in fibers in the superfi- cial layers of the dorsal horn of the spinal cord. Both dorsal rhizotomy and neonatal capsaicin treatment re- sulted in a dramatic decrease in ␣ 2A -AR-IR. The present results suggest that the majority of spinal ␣ 2A -ARs are synthesized by DRG neurons and trafficked centrally. These ...

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... al., 1987; Hoehn et al., 1988). Activation of ␣ 2 -ARs can decrease neuronal excitation by open- ing inwardly rectif ying potassium channels (Surprenant and North, 1988; Shen et al., 1992), by decreasing presynaptic calcium influx (Ewald et al., 1989; Surprenant et al., 1990), and by inhib- iting adenylyl cyclase (Andrade and Aghajanian, 1985; Uhl ́n and Wikberg, 1988; Uhl ́n and Wikberg, 1989). Three subtypes of ␣ 2 -ARs have been cloned in human and rat, corresponding to the pharmacological subtypes ␣ 2A , ␣ 2B , and ␣ 2C , respectively (for review, see Bylund et al., 1994). Because of the lack of subtype- selective pharmacological agents, it is unclear which subtype or subtypes contribute to spinal adrenergic analgesia, although a major role has been suggested for the ␣ 2A -AR (Millan, 1992; Millan et al., 1994; Stone et al., 1997b). In situ hybridization studies with probes directed against the ␣ 2A -AR have detected mRNA in a subset of dorsal root ganglion (DRG) and spinal cord neurons (Nicholas et al., 1993). In addition, ␣ 2A -AR mRNA and immunoreactivity have been detected in the cell bodies of neurons in almost all supraspinal noradrenergic nuclei (Nicholas et al., 1993; Rosin et al., 1993; Scheinin et al., 1994). The ␣ 2A -AR may therefore act as an autoreceptor on some noradrenergic terminals. In addition, ␣ 2A -AR immunoreactivity ( ␣ 2A -AR-IR) has been reported in the superficial dorsal horn of the rat spinal cord (Rosin et al., 1993) and in DRG (Gold et al., 1997). Interestingly, although studies have detected ␣ 2C -AR mRNA in a large number of DRG neurons (Nicholas et al., 1993; Gold et al., 1997), spinal ␣ 2C -AR-IR has only been detected in cell bodies in the ventral horn (Rosin et al., 1996). Based on this information, spinal ␣ 2 -ARs may originate from three possible sources. First, the receptors may be synthesized in the cell bodies of DRG neurons and trafficked centrally into the spinal cord, where they would serve a presynaptic function to modulate release of transmitter from these terminals. Second, these receptors may be synthesized by second-order spinal neurons in which they may modulate primary afferent activity either postsynaptically or presynaptically within circuits intrinsic to the spinal cord. Third, these receptors may be synthesized by supraspinal neurons and trafficked to axons and nerve terminals of descending noradrenergic fibers in which they would act as inhibitory autoreceptors controlling noradrenaline release in the spinal cord. The goal of this study was to determine the source or sources of spinal ␣ 2A -AR-IR and ␣ 2C -AR-IR. We have thus generated anti-peptide antisera directed against the C termini of the pre- dicted sequences of the ␣ -AR and ␣ -AR. Dorsal rhizotomy, neonatal capsaicin treatment, and double-labeling experiments were used to determine the origin and cytochemical profile of ␣ 2 -AR immunoreactive fibers and terminals observed in the dorsal horn. Part of these results have been presented in abstract form (Stone et al., 1996, 1997a,c). MDCK cells, transfected with the ␣ 2A -AR, ␣ 2B -AR, or ␣ 2C -AR or untransfected, were stained with rabbit-derived antisera gen- erated against the ␣ 2A -AR or ␣ 2C -AR. ␣ 2A -AR-IR was observed only on cells transfected with the ␣ 2A -AR (Fig. 1 A – D ), whereas ␣ 2C -AR-IR was observed only on cells transfected with the ␣ 2C -AR (Fig. 1 E – H ). The ␣ 2A -AR-IR was blocked by preabsorp- tion of the antiserum with the peptide corresponding to ␣ 2A -AR but not by that corresponding to the ␣ 2C -AR (Fig. 1 I , J ). Simi- larly, ␣ 2C -AR-IR was blocked by preabsorption of the antiserum with the peptide corresponding to the ␣ 2C -AR but not the ␣ 2A -AR (Fig. 1 K , L ). Guinea pig-derived anti- ␣ 2C -AR antisera generated similar results (data not shown). These results demon- strate that both the ␣ 2A -AR and ␣ 2C -AR antisera recognize the receptors against which they were generated and do not cross- react with other known ␣ -AR subtypes. In rat spinal cord, both ␣ 2A -AR-IR and ␣ 2C -AR-IR were ob- served in nerve terminals and varicosities. No definitive evidence for cell body labeling was obtained. These immunoreactive ter- minals were most highly concentrated in laminae I and II of the dorsal horn at all levels of the spinal cord (Fig. 2 A , C ). In addition, ␣ 2A -AR-IR was observed in the area surrounding the central canal and in the intermediolateral cell column of the thoracic cord (data not shown). Strong ␣ 2C -AR-IR was observed in the lateral spinal nucleus and was more prevalent in deeper layers of the dorsal horn than ␣ 2A -AR-IR. Staining was blocked in both cases by preincubation of each antisera with its cognate peptide (Fig. 2 B , D ). To determine whether the spinal ␣ 2A -AR-IR and ␣ 2C -AR-IR were of primary afferent origin, dorsal rhizotomies were per- formed to deplete the spinal cord of primary afferent input. We observed a dramatic reduction in ␣ 2A -AR-IR ipsilateral to dorsal rhizotomy (Fig. 3 A ). In contrast, dorsal rhizotomy yielded only a slight reduction in ␣ 2C -AR-IR (Fig. 3 C ). Double-labeling each section with antiserum directed against SP confirmed that SP-IR was also reduced by dorsal rhizotomy to approximately the same extent (Fig. 3 B , D ) in both sections, indicating that the efficacy of the treatment was similar in each case. To determine whether one or both subtypes is expressed in capsaicin-sensitive neurons, we treated neonatal rats with capsa- icin and examined their spinal cords at 5 weeks of age. Capsaicin, the active ingredient in hot peppers, acts as a neurotoxin on small diameter primary afferents, many of which are SP-containing (Jancso et al., 1977; Nagy et al., 1981). Sensitivity of sensory neurons to capsaicin has been associated with a role in nocicep- tion (Nagy et al., 1980; Jancso et al., 1987; Hammond and Ruda, 1991). We observed a dramatic reduction in ␣ 2A -AR-IR but not ␣ 2C -AR-IR (Fig. 4), indicating that the subset of DRG cells that express the ␣ 2A -AR is likely to consist of small diameter, capsaicin-sensitive nociceptors. In addition, these results suggest that the two subtypes may exist on different populations of neurons. Double-labeling experiments were conducted to determine the relationship between ␣ 2A -AR-IR and ␣ 2C -AR-IR in the superfi- cial dorsal horn. Sections of rat spinal cord double-labeled with anti- ␣ 2A -AR and anti- ␣ 2C -AR antisera produced robust immu- nostaining for each receptor in the superficial dorsal horn (Fig. 5 A , B ). The resultant digital images were then digitally merged (Fig. 5 C ). In merged images, the appearance of yellow may indicate colocalization, although higher resolution is required to distinguish colocalization from close apposition or superimposi- tion. When examined at high resolution, instances of receptor colocalization were rare (Fig. 5 D ). This observation further sug- gests that the two subtypes are located primarily on different neuronal populations. Double-labeling with anti- ␣ 2A -AR and anti-SP antisera revealed extensive colocalization in the spinal cord (Fig. 5 E – H ). Thus, ␣ 2A -ARs appear to be present on the spinal terminals of SP- containing primary afferent fibers. Examination of sections double-labeled for ␣ 2A -AR-IR and CGRP-IR also revealed a significant degree of colocalization (Fig. 5 I – L ). In contrast, stain- ing for the neuropeptide leuenkephalin failed to colocalize with ␣ 2A -AR-IR (Fig. 5 M – P ). To determine whether ␣ 2A -ARs exist as autoreceptors on de- scending noradrenergic terminals, we used antibodies directed against dopamine- ␤ -hydroxylase (D ␤ H) and tyrosine hydroxylase (TH), enzymes involved in the biosynthesis of noradrenaline. Spinal cord sections double-labeled for either ␣ 2A -AR-IR and D ␤ H-IR or ␣ 2A -AR-IR and TH-IR showed no instances of colocalization (Fig. 5 Q – X ). This trend was observed throughout the spinal cord. However, examples of close apposition between D ␤ H-IR or TH-IR and ␣ 2A -AR-IR fibers and terminals were occasionally observed. Whether such close appositions represent axoaxonic synaptic relationships remains to be established. Double-labeling experiments indicated that ␣ 2C -AR-IR and SP-IR are rarely colocalized in the spinal cord (Fig. 6 A – D ). However, ␣ 2C -AR-IR was present on some CGRP-IR (Fig. 6 E – H ) and somatostatin-IR (Fig. 6 I – L ) fibers. Interestingly, some overlap was detected between enkephalin-IR and ␣ 2C - AR-IR (Fig. 6 M – P ). Because enkephalin is not believed to be present in primary afferent fibers (Hokfelt et al., 1977; Johansson et al., 1978; Seybold and Elde, 1980), this result is consistent with a spinal source of ␣ 2C -AR. No colocalization was observed be- tween ␣ 2C -AR-IR and the endogenous opioid peptide preprodynorphin-IR (data not shown). In an effort to further characterize the phenotype of spinal cells that express the ␣ 2C -AR, double-labeling was performed with antisera directed against the NK-1 receptor (Fig. 6 Q – T ). We observed that the ␣ 2C -AR is not detected on either NK-1- expressing neurons or astrocytes, indicated by the rare colocal- ization of the glial marker GFAP with ␣ 2C -AR-IR (Fig. 6 U – X ). ␣ 2C -AR-IR failed to colocalize with either D ␤ H-IR (Fig. 6 Y – BB ) or TH-IR (data not shown), suggesting that neither the ␣ 2A -AR nor the ␣ 2C -AR is likely to be present on the terminals of descending supraspinal noradrenergic neurons. We have developed subtype-specific anti-peptide antisera in rab- bits and guinea pigs directed against the C-terminal portions of the ␣ 2A -AR and ␣ 2C -AR. The resultant antisera recognized their corresponding receptors and did not cross-react with other known ␣ 2 -AR subtypes. In addition, labeling was blocked by preabsorption with the cognate peptide in both transfected cells and tissue sections. Immunoreactivity for both ␣ 2A -AR-IR and ␣ 2C -AR-IR was observed predominantly in fibers in the superfi- cial layers of the dorsal horn ...

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The differential in vivo contribution of spinal α2A- and α2C-adrenoceptors in tonic and acute evoked nociception in the rat

Article

Full-text available

Nov 2022

Spinal α2-adrenoceptor induces analgesia by neuronal inhibition of primary afferent fibers. This family receptor coupled to G i/o proteins can be subdivided into three functional subtypes: α2A, α2B, and α2C-adrenoceptors, and current evidence on spinal analgesia supports the relevance of α2A and seems to exclude the role of α2B, but the functional contribution of α2C-adrenoceptors remains elusive. The present study was designed to pharmacologically dissect the contribution of spinal α2-adrenoceptor subtypes modulating tonic or acute peripheral nociception. Using male Wistar rats, we analyzed the effect of spinal clonidine (a non-selective α2A/α2B/α2C-adrenoceptor agonist) and/or selective subtype α2-adrenoceptor antagonists on: 1) tonic nociception induced by subcutaneous formalin (flinching behavior) or 2) acute nociception induced by peripheral electrical stimulus in in vivo extracellular recordings of spinal dorsal horn second-order wide dynamic range (WDR) neurons. Clonidine inhibited the nocifensive behavior induced by formalin, an effect blocked by BRL 44408 (α2A-adrenoceptor antagonist) but not by imiloxan (α2B-adrenoceptor antagonist) or JP 1302 (α2C-adrenoceptor antagonist). Similarly, spinal BRL 44408 reversed the clonidine-induced inhibition of nociceptive WDR activity. Interestingly, spinal JP 1302 per se produced behavioral antinociception (an effect blocked by bicuculline, a preferent GABAA channel blocker), but no correlation was found with the electrophysiological experiments. These data imply that, at the spinal level, 1) presynaptic α2A-adrenoceptor activation produces antinociception during acute or tonic nociceptive stimuli; and 2) under tonic nociceptive (inflammatory) input, spinal α2C-adrenoceptors are pronociceptive, probably by the inactivation of GABAergic transmission. This result supports a differential role of α2A and α2C-adrenoceptors modulating nociception.

Ascending noradrenergic excitation from the locus coeruleus to the anterior cingulate cortex

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Full-text available

May 2022

Anterior cingulate cortex (ACC) plays important roles in sensory perception including pain and itch. Neurons in the ACC receive various neuromodulatory inputs from subcortical structures, including locus coeruleus noradrenaline (LC-NA) neurons. Few studies have been reported about synaptic and behavioral functions of LC-NA projections to the ACC. Using viral-genetic method (AAV-DIO-eYFP) on DBH-cre mice, we found that LC-NA formed synaptic connections to ACC pyramidal cells but not interneurons. This is further supported by the electron microscopic study showing NAergic fibers contact the presynaptic inputs and post-synaptic areas of the pyramidal cells. NA application produced both pre-and post-synaptic potentiation effects in ACC excitatory transmission in vivo and in vitro. Activation of LC-NA projection to the ACC by optogenetic method produced enhancement of excitatory transmission in vitro and induced scratching and behavioral sensitization for mechanical stimulation. Our results demonstrate that LC-NA projections enhance or facilitate brain responses to pain and itch by potentiating glutamatergic synaptic transmissions in the ACC.

The Mesencephalic Locomotor Region: Beyond Locomotor Control

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Full-text available

May 2022

The mesencephalic locomotor region (MLR) was discovered several decades ago in the cat. It was functionally defined based on the ability of low threshold electrical stimuli within a region comprising the cuneiform and pedunculopontine nucleus to evoke locomotion. Since then, similar regions have been found in diverse vertebrate species, including the lamprey, skate, rodent, pig, monkey, and human. The MLR, while often viewed under the lens of locomotion, is involved in diverse processes involving the autonomic nervous system, respiratory system, and the state-dependent activation of motor systems. This review will discuss the pedunculopontine nucleus and cuneiform nucleus that comprises the MLR and examine their respective connectomes from both an anatomical and functional angle. From a functional perspective, the MLR primes the cardiovascular and respiratory systems before the locomotor activity occurs. Inputs from a variety of higher structures, and direct outputs to the monoaminergic nuclei, allow the MLR to be able to respond appropriately to state-dependent locomotion. These state-dependent effects are roughly divided into escape and exploratory behavior, and the MLR also can reinforce the selection of these locomotor behaviors through projections to adjacent structures such as the periaqueductal gray or to limbic and cortical regions. Findings from the rat, mouse, pig, and cat will be discussed to highlight similarities and differences among diverse species.

Effect of Pregabalin Combined with Duloxetine and Tramadol on Allodynia in Chronic Postischemic Pain and Spinal Nerve Ligation Mouse Models

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Mar 2022

Although there are various drugs for Neuropathic pain (NP), the effects of single drugs are often not very satisfactory. The analgesic effects of different combinations of pregabalin, duloxetine, and tramadol or the combination of all three are still unclear. Mixtures of two or three drugs at low and high concentrations (7.5, 10, 15, and 20 mg/kg pregabalin; 7.5, 10, 15, and 30 mg/kg duloxetine; 5 and 10 mg/kg tramadol) were administered to chronic postischemic pain (CPIP) and spinal nerve ligation (SNL) model mice. The effects of these combinations of drugs on mechanical allodynia were investigated. The expression of the glial fibrillary acidic protein (GFAP) in the spinal cord and dorsal root ganglia (DRGs) was measured. The combination of pregabalin, duloxetine, and tramadol significantly alleviated mechanical hyperalgesia in mice with CPIP and SNL. After the administration of this drug combination, the expression of GFAP in the spinal cord and DRGs was lower in the CPIP and SNL model mice than in control mice. This result suggests that the combination of these three drugs may be advantageous for the treatment of NP because it can reduce side effects by preventing the overuse of a single drug class and exert increased analgesic effects via synergism.

Suppression of P2X3 receptor‐mediated currents by the activation of α2A‐adrenergic receptors in rat dorsal root ganglion neurons

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Full-text available

Dec 2021
CNS NEUROSCI THER

Aims The α2‐adrenergic receptor (α2‐AR) agonists have been shown to be effective in the treatment of various pain. For example, dexmedetomidine (DEX), a selective α2A‐AR agonist, can be used for peripheral analgesia. However, it is not yet fully elucidated for the precise molecular mechanisms. P2X3 receptor is a major receptor processing nociceptive information in primary sensory neurons. Herein, we show that a functional interaction of α2A‐ARs and P2X3 receptors in dorsal root ganglia (DRG) neurons could contribute to peripheral analgesia of DEX. Methods Electrophysiological recordings were carried out on rat DRG neurons, and nociceptive behavior was quantified in rats. Results The activation of α2A‐ARs by DEX suppressed P2X3 receptor‐mediated and α,β‐methylene‐ATP (α,β‐meATP)‐evoked inward currents in a concentration‐dependent and voltage‐independent manner. Pre‐application of DEX shifted the α,β‐meATP concentration‐response curve downwards, with a decrease of 50.43 ± 4.75% in the maximal current response of P2X3 receptors to α,β‐meATP in the presence of DEX. Suppression of α,β‐meATP‐evoked currents by DEX was blocked by the α2A‐AR antagonist BRL44408 and prevented by intracellular application of the Gi/o protein inhibitor pertussis toxin, the adenylate cyclase activator forskolin, and the cAMP analog 8‐Br‐cAMP. DEX also suppressed α,β‐meATP‐evoked action potentials through α2A‐ARs in rat DRG neurons. Finally, the activation of peripheral α2A‐ARs by DEX had an analgesic effect on the α,β‐meATP‐induced nociception. Conclusions These results suggested that activation of α2A‐ARs by DEX suppressed P2X3 receptor‐mediated electrophysiological and behavioral activity via a Gi/o proteins and cAMP signaling pathway, which was a novel potential mechanism underlying analgesia of peripheral α2A‐AR agonists.

Dexmedetomidine Inhibits ASIC Activity via Activation of α2A Adrenergic Receptors in Rat Dorsal Root Ganglion Neurons

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May 2021

Dexmedetomidine (DEX), a selective α2 adrenergic receptor (α2-AR) agonist, has been shown to have peripheral analgesic effects in a variety of pain conditions. However, the precise molecular mechanisms have not yet been fully elucidated. Acid sensing ion channels (ASICs) are the major player in pain associated with tissue acidosis. Given that both α2-ARs and ASICs exist in dorsal root ganglia (DRG) neurons, we therefore investigated the effects of DEX on the functional activity of ASICs. Herein, whole-cell patch-clamp recordings demonstrated that DEX suppressed ASIC-mediated and acid-evoked currents and action potentials in dissociated rat DRG neurons. DEX shifted downwards concentration-response curve to protons, with a decrease of 35.83 ± 3.91% in the maximal current response to pH 4.5. DEX-induced inhibition of ASIC currents was blocked by the α2A-AR antagonist BRL44408 in DRG neurons. DEX also inhibited ASIC3 currents in CHO cells co-expressing ASIC3 and α2A-ARs, but not in ASIC3 transfected CHO cells without α2A-ARs expression. DEX-induced inhibition of ASIC currents was mimicked by the protein kinase A inhibitor H-89, and blocked by intracellular application of the Gi/o protein inhibitor pertussis toxin and the cAMP analog 8-Br-cAMP. In addition, peripherally administration of DEX dose-dependently relieved nociceptive responses to intraplantar injection of acetic acid in rats through local α2A-ARs. Our results indicated that DEX inhibited the functional activity of ASICs via α2A-ARs and intracellular Gi/o proteins and cAMP/protein kinase A signaling pathway in rat DRG neurons, which was a novel potential mechanism that probably mediated peripheral analgesia of DEX.

Ascending noradrenergic excitation from the locus coeruleus to the anterior cingulate cortex

Article

Full-text available

Dec 2020

Anterior cingulate cortex (ACC) plays important roles in sensory perception including pain and itch. Neurons in the ACC receive various neuromodulatory inputs from subcortical structures, including locus coeruleus noradrenaline (LC-NA) neurons. Few studies have been reported about synaptic and behavioral functions of LC-NA projections to the ACC. Using viral-genetic method (AAV-DIO-eYFP) on DBH-cre mice, we found that LC-NA formed synaptic connections to ACC pyramidal cells but not interneurons. This is further supported by the electron microscopic study showing NAergic fibers contact the presynaptic inputs and post-synaptic areas of the pyramidal cells. NA application produced both pre- and post-synaptic potentiation effects in ACC excitatory transmission in vivo and in vitro. Activation of LC-NA projection to the ACC by optogenetic method produced enhancement of excitatory transmission in vitro and induced scratching and behavioral sensitization for mechanical stimulation. Our results demonstrate that LC-NA projections enhance or facilitate brain responses to pain and itch by potentiating glutamatergic synaptic transmissions in the ACC.

Is Spinal Dexmedetomidine Aggravating Hypotension after Tourniquet Deflation?

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Full-text available

Jul 2020

Background and aims: The addition of dexmedetomidine to spinal anesthesia decreases the incidence of tourniquet pain but may aggravate hypotension after tourniquet deflation. Methods: Fifty patients were included in this prospective, double-blinded, randomized study, randomly divided into two equal groups of 25 patients each. Spinal anesthesia was performed using 2.5 mL of 0.5% hyperbaric bupivacaine plus 0.5 mL of normal saline in control group (Group C) or 2.5 mL of 0.5% hyperbaric bupivacaine plus 0.5 mL (5 μg) of dexmedetomidine in (Group D). Tourniquet pain was treated by 50 mg of meperidine and repeated in a dose of 20 mg, and the total meperidine consumption was calculated. After tourniquet deflation, heart rate and mean blood pressure were measured for 15 min in the operating room and at these times: before induction of anesthesia (baseline), after inflating tourniquet (inflation), 1 min before deflating tourniquet (predeflation), after tourniquet deflation (10 min postdeflation), and maximum blood pressure and heart rate changes. Duration of time that started before the minimum blood pressure and maximum heart rate was changed until recovery was recorded. Results: Pain after torniquet inflation was significantly higher in the Group C compared to the Group D. The maximal change of blood pressure was lower in the dexmedetomidine than in the control group. The mean time between the maximal change in blood pressure reached and started to recover was 135 ± 14 s in the dexmedetomidine group and 80 ± 31 s in the control group (P < 0.01) and maximal heart rate change was lower in dexmedetomidine group than the control group. The time between the maximal heart rate changes until recovery was 113.2 ± 19 s in the dexmedetomidine group and 53.2 ± 11 s in the control group P < 0.01. Conclusion: Adding dexmedetomidine to spinal anesthesia decreases the incidence of tourniquet pain but aggravates the hemodynamic effect of tourniquet deflation.

Activation of α-adrenoceptors depresses synaptic transmission of myelinated afferents and inhibits pathways mediating primary afferent depolarization (PAD) in the in vitro mouse spinal cord

Article

Full-text available

May 2020
EXP BRAIN RES

Somatosensory afferent transmission strength is controlled by several presynaptic mechanisms that reduce transmitter release at the spinal cord level. We focused this investigation on the role of α-adrenoceptors in modulating sensory transmission in low-threshold myelinated afferents and in pathways mediating primary afferent depolarization (PAD) of neonatal mouse spinal cord. We hypothesized that the activation of α-adrenoceptors depresses low threshold-evoked synaptic transmission and inhibits pathways mediating PAD. Extracellular field potentials (EFPs) recorded in the deep dorsal horn assessed adrenergic modulation of population monosynaptic transmission, while dorsal root potentials (DRPs) recorded at root entry zone assessed adrenergic modulation of PAD. We found that noradrenaline (NA) and the α1-adrenoceptor agonists phenylephrine and cirazoline depressed synaptic transmission (by 15, 14 and 22%, respectively). DRPs were also depressed by NA, phenylephrine and cirazoline (by 62, 30, and 64%, respectively), and by the α2-adrenoceptor agonist clonidine, although to a lower extent (20%). We conclude that NA depresses monosynaptic transmission of myelinated afferents onto deep dorsal horn neurons via α1-adrenoceptors and inhibits interneuronal pathways mediating PAD through the activation of α1- and α2-adrenoceptors. The functional significance of these modulatory actions in shaping cutaneous and muscle sensory information during motor behaviors requires further study.

Cellular Mechanisms for Antinociception Produced by Oxytocin and Orexins in the Rat Spinal Lamina II—Comparison with Those of Other Endogenous Pain Modulators

Article

Full-text available

Sep 2019

Eiichi Kumamoto

Much evidence indicates that hypothalamus-derived neuropeptides, oxytocin, orexins A and B, inhibit nociceptive transmission in the rat spinal dorsal horn. In order to unveil cellular mechanisms for this antinociception, the effects of the neuropeptides on synaptic transmission were examined in spinal lamina II neurons that play a crucial role in antinociception produced by various analgesics by using the whole-cell patch-clamp technique and adult rat spinal cord slices. Oxytocin had no effect on glutamatergic excitatory transmission while producing a membrane depolarization, γ-aminobutyric acid (GABA)-ergic and glycinergic spontaneous inhibitory transmission enhancement. On the other hand, orexins A and B produced a membrane depolarization and/or a presynaptic spontaneous excitatory transmission enhancement. Like oxytocin, orexin A enhanced both GABAergic and glycinergic transmission, whereas orexin B facilitated glycinergic but not GABAergic transmission. These inhibitory transmission enhancements were due to action potential production. Oxytocin, orexins A and B activities were mediated by oxytocin, orexin-1 and orexin-2 receptors, respectively. This review article will mention cellular mechanisms for antinociception produced by oxytocin, orexins A and B, and discuss similarity and difference in antinociceptive mechanisms among the hypothalamic neuropeptides and other endogenous pain modulators (opioids, nociceptin, adenosine, adenosine 5’-triphosphate (ATP), noradrenaline, serotonin, dopamine, somatostatin, cannabinoids, galanin, substance P, bradykinin, neuropeptide Y and acetylcholine) exhibiting a change in membrane potential, excitatory or inhibitory transmission in the spinal lamina II neurons.

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