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The effect of rhizotomy on NADPH diaphorase staining in the lumbar spinal cord of the rat
349
Brain Research, 607 (1993) 349-353 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 25600
The effect of rhizotomy on NADPH diaphorase staining in the lumbar spinal cord of the rat M a r g a r e t A. V i z z a r d , S u s a n L. E r d m a n a n d W i l l i a m C. de G r o a t University of Pittsburgh, School of Medicine, Department of Pharmacology, Pittsburgh, PA 15261 (USA) (Accepted 29 December 1992)
Key words: NADPH diaphorase; Rhizotomy; Lumbar spinal cord; Nitric oxide; Primary afferent; Sacral parasympathetic nucleus
In the L6-S1 spinal segments of the rat NADPH diaphorase fiber-like staining extending along the lateral edge of the dorsal horn to the sacral parasympathetic nucleus was eliminated 1-3 weeks following dorsal-ventral L6-S1 rhizotomy. However, other NADPH diaphorase staining in the dorsal horn was not affected by rhizotomy. Rhizotomy also doubled the number (5 versus 10 cells/section) of NADPH diaphorase positive neurons in the region of the sacral parasympathetic nucleus (SPN). These data indicate that NADPH diaphorase is present in primary afferent projections to the SPN and that NADPH diaphorase activity in SPN neurons can be enhanced by neuronal injury. The functional significance of the NADPH diaphorase histochemical stain remains to be determined; however, if NADPH diaphorase is nitric oxide synthase then this would suggest that NO has an important role in the neural pathways to the pelvic viscera.
Nitric oxide (NO) has been implicated as a neurotransmitter or neuromodulator at various sites in the mammalian nervous system8'1°'18. Neurons containing NO have been identified histochemically 17 by the presence of NADPH diaphorase activity or immunocytochemically7-9 with an antibody for NO synthase, the enzyme responsible for NO synthesis. A one-to-one correlation between NADPH diaphorase positive neurons and NO synthase immunoreactive neurons has been described in various sites in the central nervous system7'9. Thus, it appears that NADPH diaphorase histochemistry provides a specific histochemical marker for neurons containing NO. In previous studies 23 we used the NADPH diaphorase histochemical reaction in combination with retrograde axonal transport of a fluorescent dye injected into the major pelvic ganglion of rats to examine the distribution of NO synthase in afferent and preganglionic efferent pathways to pelvic organs. We found that a large percentage of visceral afferent neurons in the L6-S1 dorsal root ganglia and preganglionic neurons in the sacral parasympathetic nucleus (SPN) of the L6-S1 spinal cord exhibit NADPH diaphorase
activity. In addition, in the L6-S1 spinal segments a prominent band of NADPH diaphorase staining was detected on the lateral edge of the dorsal horn extending from Lissauer's tract to the region of the SPN. This band was not present in adjacent spinal segments (L4L5 and $2). Since this pattern of staining resembles the central projections of afferent pathways from the pelvic viscera 15'19 and since it has similar segmental distribution as pelvic visceral afferents ~3, this raised the possibility that the staining represented primary afferent projections to the SPN. This was confirmed in the present experiments by examining the effect of unilateral transection of the L6-S1 dorsal and ventral roots on NADPH diaphorase staining in the spinal cord. In the course of these experiments we also discovered that transection of the dorsal and ventral roots increased the number of neurons in the region of the SPN which exhibited NADPH diaphorase staining. Unilateral L6-S1 dorsal and ventral rhizotomies were performed on adult, male Wistar rats (200-300 g) (n = 6). Ventral roots as well as dorsal roots were cut to eliminate the necessity of identifying the two roots and ensured that all afferent pathways were inter-
Correspondence: M.A. Vizzard, University of Pittsburgh School of Medicine, Department of Pharmacology, Biomedical Science Tower, Pittsburgh, PA 15261, USA. Fax: (1) (412) 648-1945.
350 rupted. In some animals the roots were cut on the left side and in others on the right side. Three control animals with intact spinal roots were also included in the study. After 1-3 weeks, the animals were deeply anesthetized with pentobarbital (50 mg/kg) and then sacrificed via intracardiac perfusion first with Krebs buffer followed by 4% paraformaldehyde fixative. Tissues were removed and postfixed for 6-10 h in the same fixative solution. After rinsing with phosphate buffered saline (0.1 M NaCI in phosphate buffer, pH 7.2), tissue was placed in ascending concentrations of sucrose (10-30%) in phosphate buffer for cryoprotection. Sections of the spinal cord (L5 to $2) (42 Izm) were then processed on slide for N A D P H diaphorase activity according to the procedure of Vincent et al. 22. Sections were incubated in 0.1 M phosphate buffer (pH 7.4), containing 0.3% Triton X-100, 0.1 m g / m l nitroblue tetrazolium and 1.0 m g / m l b-NADPH at 37°C for 30 min to 2.5 h. Following the reaction, the sections were rinsed in phosphate buffer (pH 7.4), air-dried overnight and coverslipped with Entellan. All N A D P H diaphorase positive cells in the region of the sacral parasympathetic nucleus (lateral lamina V-VII) and around the central canal in L6 and S1 were counted in 30-50 sections/segment on the control and rhizotomized sides. The central canal region was visually divided along the dorso-ventral axis to count cells on the right and left sides. Cell counts are presented as average number of cells per section (mean _+ S.E.M.). The results are not corrected for double counting. The number of N A D P H diaphorase positive cells on the control and rhizotomized sides were compared using paired Student's t-test. A probability level of P < 0.05 was accepted as significant. In control animals with intact spinal roots, N A D P H diaphorase staining was similar on the left and right sides of the L6-S1 spinal segments. Neurons exhibiting N A D P H diaphorase activity were present in the region of the SPN, around the central canal, in the dorsal commissure and in laminae I-II of the dorsal horn 1'6'16'21 (Fig. IA). Fiber-like staining was present in the superficial layers of the dorsal horn and dorsal commissure as well as in Lissauer's tract and in a bundle extending from Lissauer's tract to the region of the SPN. This bundle was not present in every section suggesting that
it may occur intermittently along the rostral-caudal axis as noted for visceral afferent projections labeled with wheat germ agglutinin horseradish peroxidase (WGAHRP) or horseradish peroxidase (HRPY 5'1~. The lateral bundle was not present in L5 or $2 spinal segments and its appearance in L6 coincided with the appearance of N A D P H diaphorase positive cells in the region of the SPN. In animals with unilateral spinal rhizotomy, the lateral bundle was eliminated ipsilateral to the rhizotomy (Fig. 1C) but other fiber-like staining in the dorsal horn and dorsal commissure was not detectably changed. The numbers of N A D P H diaphorase positive cells were also differentially affected by rhizotomy. In the region of the central canal in the L6 segment the number of N A D P H diaphorase positive cells were similar on the control and rhizotomy sides. When the central canal region was visually divided along the dorso-ventral axis, an average of 5 cells/sections were N A D P H diaphorase positive on the rhizotomy side and 5.2 cells/section were N A D P H diaphorase positive on the intact side. However, in the region of the SPN, the control side of the L6 spinal segments had an average of 4.5 _+ 0.2 cells/section (n = 5 animals) that were N A D P H diaphorase positive (Fig. 1D); whereas the rhizotomized side had an average of 9.5 + 0.3 cells/section that were N A D P H diaphorase positive (Fig. 1E). Similarly, in the region of the SPN of the SI spinal segments, an average of 5.8 + 0.5 cells/section (n = 5 animals) on the control side and 9.1_+0.5 cells/section on the rhizotomized side were N A D P H diaphorase positive. Differences in cell counts between control and rhizotomized sides were statistically significant for both L6 and S1 ( P < 0.05). Different intensities of N A D P H diaphorase cell staining were observed in the region of the SPN on both the control and rhizotomized sides. N A D P H diaphorase stained cells were either very darkly stained, moderately stained or lightly stained. The distribution of N A D P H diaphorase positive cells among these three groups was unaltered after rhizotomy. When N A D P H diaphorase cells in the region of the SPN on the intact side of spinal segment L6 (n = 3 animals) were subdivided among these three groups based upon staining intensity, 36.5% of cells were very darkly stained, 37.5%
Fig. 1. Distribution of NADPH diaphorase staining in the L6 spinal cord of rat following right, dorsal and ventral rhizotomy. A: low-power photomicrograph showing NADPH diaphorase positive neurons in the region of the sacral parasympathetic nucleus (SPN), the central canal and the dorsal horn. NADPH diaphorase fiber-like staining is also present in the dorsal horn and in a thin bundle extending from Lissauer's tract to the region of the SPN (B, arrows) Note the absence of this bundle on the right side of the spinal cord after rhizotomy (C). Distribution of NADPH diaphorase positive cells in the region of the SPN on the intact (D) and axotomized sides (E) of the L6 spinal cord. In addition to the increase in the number of NADPH diaphorase positive cells (E) following axotomy, NADPH diaphorase positive cells also occupied a greater area in the region of the SPN. Calibration bar in (E) represents 320 mm in A, 170 mm in B-C and 70 mm in D-E.
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352 were moderately stained and 25.9% were lightly stained. Within the region of the SPN on the rhizotomized side, 38.5% of cells were very darkly stained, 32.6% were moderately stained and 28.9% were lightly stained. In addition to increased numbers of N A D P H diaphorase positive cells within the region of the SPN on the rhizotomized side, the area occupied by these cells in the L6 spinal segment (n = 3 animals) was also increased. On the rhizotomized side, cells were distributed over a broader dorso-ventral distance (123.9 + 13.7 mm in control versus 183.3 + 27.8 mm in rhizotomized side). In the S1 segment, no change was noted in the distribution of cells after rhizotomy (145.3 + 38.7 mm versus 144.6 +_ 34 mm). This study revealed that transection of the dorsal and ventral roots in the L6-S1 spinal segments of the rat alters fiber-like and neuronal N A D P H diaphorase staining in the region of the sacral parasympathetic nucleus. For example, the thin band of N A D P H diaphorase staining extending from Lissauer's tract to the SPN was eliminated by section of the spinal roots indicating that this band consists of primary afferent projections. However, fiber-like N A D P H diaphorase staining in the regions of the dorsal horn and dorsal commissure was not changed. Thus this staining must originate in the central nervous system or enter the spinal cord via other dorsal roots. The bundle of N A D P H diaphorase positive afferents projecting to the SPN is very similar to the central projections of pelvic visceral afferents that have been identified by transganglionic transport of H R P or W G A - H R P 15'~9. Since a large percentage of pelvic visceral afferent neurons in the L6-S1 dorsal root ganglia also exhibit N A D P H diaphorase staining it seems reasonable to conclude that part of the N A D P H diaphorase afferent bundle consists of visceral afferent projections. This view is also supported by studies in which c-fos gene expression was used to examine the central projections of pelvic visceral afferents z-5. Electrical stimulation of the pelvic nerve 5 or stimulation of afferent pathways in the lower urinary tract 2'3 or colon 2° of the rat induced c-los expression in a large population of neurons in the region of the SPN and lateral dorsal horn. Gene expression was induced by nociceptive as well as non-nociceptive stimuli and occurred in spinal tract neurons (spinopontine and spinohypothalamic), interneurons and parasympathetic preganglionic neurons 4. Somatic afferents from the distal urethra which travel in the pudendal nerve 2 also project to and induce c-los expression in neurons in this region of the SPN. Thus, the intermediolateral area of L6-S1 spinal gray matter receiving N A D P H diaphorase containing afferent input is likely to play an important role in
viscerosomatic integration which is essential for urogenital and distal bowel function. The present histochemical observations raise the possibility that nitric oxide may be a neurotransmitter or neuromodulator in the afferent projections to this spinal integrating center. Spinal rhizotomy also influenced the N A D P H diaphorase staining in neurons in the SPN. As noted in previous studies from this laboratory 23, a large percentage of preganglionic efferent neurons involved in pelvic visceral function exhibit N A D P H diaphorase activity; however, under normal circumstances, not all of these neurons stained. The doubling of the number of N A D P H diaphorase positive neurons from 5 to 10 cells per section on the side of the rhizotomy may reflect the induction or upregulation of N A D P H diaphorase enzymatic activity in preganglionic neurons following axotomy. This change in staining appeared to be selective for SPN neurons since there was no change in the numbers of N A D P H diaphorase positive neurons surrounding the central canal and no N A D P H diaphorase activity in motoneurons in the ventral horn, which also were axotomized. A similar increase in N A D P H diaphorase staining has been noted in vagal efferent neurons in the nucleus ambiguus and dorsal motor nucleus of the vagus 2 to 10 days following cervical vagotomylL Ventral root avulsion also induces the novel expression of N A D P H diaphorase activity in motoneurons in the lumbar spinal cord of the rat 24. Although in the present experiments we did not detect an increase in enzymatic activity in axotomized motoneurons, it is clear that N A D P H diaphorase activity in spinal cord and brainstem efferent neurons is labile and can be upregulated by neural damage. However, N A D P H diaphorase staining could reflect the activity of other enzymes in addition to nitric oxide synthase, therefore in future experiments it will be important to demonstrate that upregulation of N A D P H diaphorase activity in the sacral parasympathetic nucleus following rhizotomy does indeed reflect a change in nitric oxide synthase. Further studies will also be necessary to determine whether all L6-S1 preganglionic neurons exhibit N A D P H diaphorase activity following rhizotomy and whether this change occurs following selective dorsal or ventral rhizotomies. It is noteworthy that on both the normal and rhizotomized sides of the spinal cord that neurons in the SPN region exhibited considerable variability in N A D P H diaphorase staining. Neurons were grouped into three categories: light, moderate and dark staining. The relative proportions of these three groups were not altered by rhizotomy. Variations in the intensity of the staining (light and dark) of vagal pregan-
353 glionic neurons have also been reported 11. Similar variations were also noted after vagotomy. The functional significance of NADPH diaphorase staining and variations in the staining in parasympathetic preganglionic neurons is uncertain. However, it is tempting to speculate that nitric oxide may be released from the preganglionic perikarya in the spinal cord and/or from preganglionic nerve terminals in the peripheral ganglia and act as a cotransmitter or neuromodulator at these sites. Variations in NADPH diaphorase staining may reflect different roles of nitric oxide in different subpopulations of preganglionic neurons (e.g. colon, bladder and sex organs). It has been speculated that light and dark NADPH diaphorase staining in dorsal root ganglion cells reflects local modulation and transmitter functions, respectively, of nitric oxide in these cells TM. In summary, this study has demonstrated that NADPH diaphorase staining on the lateral edge of the dorsal horn was contained in afferent fibers. Furthermore, it was demonstrated that NADPH diaphorase staining can be induced or upregulated in neurons in the region of the SPN following axotomy. The functional significance of the NADPH diaphorase histochemical stain remains to be determined; however, if NADPH diaphorase is nitric oxide synthase 12 then this would suggest that NO has an important role in the neural pathways to the pelvic viscera. This study was supported by NIH Grants DK 37241 and DK 42369, NSF Grant BNS-890-8934 and NIMH Training Grant MH 18273. 1 Anderson, C.R., NADPH diaphorase-positive neurons in the rat spinal cord include a subpopulation o f autonomic preganglionic neurons, Neurosci. Lett., 139 (1992) 280-284. 2 Birder, L.A. and de Groat, W.C., Increased c-fos expression in spinal neurons after irritation of the lower urinary tract in the rat, J. Neurosci., in press. 3 Birder, L.A. and de Groat, W.C., The effect of glutamate antagonists on c-los expression induced in spinal neurons by irritation of the lower urinary tract, Brain Res., 580 (1992) 115-120. 4 Birder, L.A., Roppolo, J.R. and de Groat, W.C., c-los as a marker for subsets of visceral second order neurons in the rat lumbosacral spinal cord, Soc. Neurosci. Abstr., 16 (1990) 703. 5 Birder, L.A., Roppolo, J.R., Iadarola, M.J. and de Groat, W.C., Electrical stimulation of visceral afferent pathways in the pelvic nerve increases c-los in the rat lumbosacral spinal cord, Neurosci. Lett., 129 (1992) 193-196. 6 Blottner, D. and Baumgarten, H., Nitric oxide synthetase (NOS) containing sympathoadrenal cholinergic neurons of the rat IMLcell column: evidence from histochemistry, immunohistochemistry and retrograde labeling, J. Comp. Neurol., 316 (1992) 45-55. 7 Bredt, D.S., Glatt, C.E., Hwang, P.M., Fotuhi, M., Dawson, T.M.
and Snyder, S.H., Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase, Neuron, 7 (1991) 615624. 8 Bredt, D.S., Hwang, P.M. and Snyder, S.H., Localization of nitric oxide synthase indicating a neural role for nitric oxide, Nature, 347 (1990) 768-770. 9 Dawson. T.M., Bredt, D.S., Fotuhi, M., Hwang, P.M. and Snyder, S.H., Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues, Proc. Natl. Acad. Sci. USA, 88 (1991) 7797-7801. 10 Gaily, J.A., Montague, P.R., Reeke, G.N. and Edelman, G.M., The NO hypothesis: possible effects of a short-lived, rapidly diffusible signal in the development and function of the nervous system, Proc. NatL Acad. Sci. USA, 87 (1990) 3547-3551. 11 Gonzalez, M.F., Sharp, F.R. and Sagar, S.M., Axotomy increases NADPH diaphorase staining in rat vagal motor neurons, Brain Res. Bull., 18 (1987) 417-427. 12 Hope, B.T., Michael, G.J., Knigge, K.M. and Vincent, S.R., Neuronal NADPH diaphorase is a nitric oxide synthase, Proc. Natl. Acad. Sci. USA, 88 (1991) 2811-2814. 13 Keast, J.R. and de Groat, W.C., Segmental distribution and peptide content of primary afferent neurons innervating the urogenital organs and colon of male rats, J. Comp. Neurol., 319 (1992) 615-623. 14 Morris, R., Southam, E., Braid, D.M. and Garthwaite, J., Nitric oxide may act as a messenger between dorsal root ganglion neurones and their satellite cells, Neurosci. Lett., 137 (1992) 29-32. 15 Nadelhaft, I, and Booth, A.M., The location and morphology of preganglionic neurons and the distribution of visceral afferents from the rat pelvic nerve: a horseradish peroxidase study, J. Comp. Neurol., 226 (1984) 238-245. 16 Saito, A., Kidd, G.J., Hanley, D.F., Bredt, D.S., Dawson, T.M., Wilson, D., Traystman, R.J. and Snyder, S.H., Rat spinal cord neurons contain nitric oxide synthase, Soc. Neurosci. Abstr., 18 (1) (1992) 858. 17 Scherer-Singler, U., Vincent, S.R., Kimura, H. and McGeer, E.G., Demonstration of a unique population of neurons with NADPH diaphorase histochemistry, J. Neurosci. Methods, 8 (1983) 229-234. 18 Southam,. E., Morris, R. and Garthwaite, J., Sources and targets of nitric oxide in rat cerebellum, Neurosci. Lett., 137 (1992) 241-244. 19 Steers, W.D., Ciambotti, J., Etzel, B., Erdman, S. and de Groat, W.C., Alterations in afferent pathways from the urinary bladder of the rat in response to partial urethral obstruction, J. Comp. Neurol. , 310 (1991) 1-10. 20 Traub, R.J. and Gebhart, G.F., Noxious and non-noxious colorectal distension results in c-fos induction in the rat spinal cord, Soc. Neurosci. Abstr., 16 (1990) 704. 21 Valtschanoff, J.G., Weinberg, R.J. and Rustioni, A., NADPH diaphorase in the spinal cord of rats, J. Comp. l~eurol., 321 (1992) 209-222. 22 Vincent, S.R. and Kimura, H., Histochemical mapping of nitric oxide synthase in the rat brain, Neuroscience, 46 (4) (1992) 755-784. 23 Vizzard, M.A., Erdman,.S.L. and de Groat, W.C., Localization of nitric oxide (NO) in pelvic afferent and efferent pathways of the rat, Neurosci. Lett., (submitted). 24 Wu, W., Neuronal NADPH diaphorase are related to survival and regeneration after severe neuronal damage, Soc. Neurosci. Abstr., 18 (1) (1992) 860.
Brain Research, 607 (1993) 349-353 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 25600
The effect of rhizotomy on NADPH diaphorase staining in the lumbar spinal cord of the rat M a r g a r e t A. V i z z a r d , S u s a n L. E r d m a n a n d W i l l i a m C. de G r o a t University of Pittsburgh, School of Medicine, Department of Pharmacology, Pittsburgh, PA 15261 (USA) (Accepted 29 December 1992)
Key words: NADPH diaphorase; Rhizotomy; Lumbar spinal cord; Nitric oxide; Primary afferent; Sacral parasympathetic nucleus
In the L6-S1 spinal segments of the rat NADPH diaphorase fiber-like staining extending along the lateral edge of the dorsal horn to the sacral parasympathetic nucleus was eliminated 1-3 weeks following dorsal-ventral L6-S1 rhizotomy. However, other NADPH diaphorase staining in the dorsal horn was not affected by rhizotomy. Rhizotomy also doubled the number (5 versus 10 cells/section) of NADPH diaphorase positive neurons in the region of the sacral parasympathetic nucleus (SPN). These data indicate that NADPH diaphorase is present in primary afferent projections to the SPN and that NADPH diaphorase activity in SPN neurons can be enhanced by neuronal injury. The functional significance of the NADPH diaphorase histochemical stain remains to be determined; however, if NADPH diaphorase is nitric oxide synthase then this would suggest that NO has an important role in the neural pathways to the pelvic viscera.
Nitric oxide (NO) has been implicated as a neurotransmitter or neuromodulator at various sites in the mammalian nervous system8'1°'18. Neurons containing NO have been identified histochemically 17 by the presence of NADPH diaphorase activity or immunocytochemically7-9 with an antibody for NO synthase, the enzyme responsible for NO synthesis. A one-to-one correlation between NADPH diaphorase positive neurons and NO synthase immunoreactive neurons has been described in various sites in the central nervous system7'9. Thus, it appears that NADPH diaphorase histochemistry provides a specific histochemical marker for neurons containing NO. In previous studies 23 we used the NADPH diaphorase histochemical reaction in combination with retrograde axonal transport of a fluorescent dye injected into the major pelvic ganglion of rats to examine the distribution of NO synthase in afferent and preganglionic efferent pathways to pelvic organs. We found that a large percentage of visceral afferent neurons in the L6-S1 dorsal root ganglia and preganglionic neurons in the sacral parasympathetic nucleus (SPN) of the L6-S1 spinal cord exhibit NADPH diaphorase
activity. In addition, in the L6-S1 spinal segments a prominent band of NADPH diaphorase staining was detected on the lateral edge of the dorsal horn extending from Lissauer's tract to the region of the SPN. This band was not present in adjacent spinal segments (L4L5 and $2). Since this pattern of staining resembles the central projections of afferent pathways from the pelvic viscera 15'19 and since it has similar segmental distribution as pelvic visceral afferents ~3, this raised the possibility that the staining represented primary afferent projections to the SPN. This was confirmed in the present experiments by examining the effect of unilateral transection of the L6-S1 dorsal and ventral roots on NADPH diaphorase staining in the spinal cord. In the course of these experiments we also discovered that transection of the dorsal and ventral roots increased the number of neurons in the region of the SPN which exhibited NADPH diaphorase staining. Unilateral L6-S1 dorsal and ventral rhizotomies were performed on adult, male Wistar rats (200-300 g) (n = 6). Ventral roots as well as dorsal roots were cut to eliminate the necessity of identifying the two roots and ensured that all afferent pathways were inter-
Correspondence: M.A. Vizzard, University of Pittsburgh School of Medicine, Department of Pharmacology, Biomedical Science Tower, Pittsburgh, PA 15261, USA. Fax: (1) (412) 648-1945.
350 rupted. In some animals the roots were cut on the left side and in others on the right side. Three control animals with intact spinal roots were also included in the study. After 1-3 weeks, the animals were deeply anesthetized with pentobarbital (50 mg/kg) and then sacrificed via intracardiac perfusion first with Krebs buffer followed by 4% paraformaldehyde fixative. Tissues were removed and postfixed for 6-10 h in the same fixative solution. After rinsing with phosphate buffered saline (0.1 M NaCI in phosphate buffer, pH 7.2), tissue was placed in ascending concentrations of sucrose (10-30%) in phosphate buffer for cryoprotection. Sections of the spinal cord (L5 to $2) (42 Izm) were then processed on slide for N A D P H diaphorase activity according to the procedure of Vincent et al. 22. Sections were incubated in 0.1 M phosphate buffer (pH 7.4), containing 0.3% Triton X-100, 0.1 m g / m l nitroblue tetrazolium and 1.0 m g / m l b-NADPH at 37°C for 30 min to 2.5 h. Following the reaction, the sections were rinsed in phosphate buffer (pH 7.4), air-dried overnight and coverslipped with Entellan. All N A D P H diaphorase positive cells in the region of the sacral parasympathetic nucleus (lateral lamina V-VII) and around the central canal in L6 and S1 were counted in 30-50 sections/segment on the control and rhizotomized sides. The central canal region was visually divided along the dorso-ventral axis to count cells on the right and left sides. Cell counts are presented as average number of cells per section (mean _+ S.E.M.). The results are not corrected for double counting. The number of N A D P H diaphorase positive cells on the control and rhizotomized sides were compared using paired Student's t-test. A probability level of P < 0.05 was accepted as significant. In control animals with intact spinal roots, N A D P H diaphorase staining was similar on the left and right sides of the L6-S1 spinal segments. Neurons exhibiting N A D P H diaphorase activity were present in the region of the SPN, around the central canal, in the dorsal commissure and in laminae I-II of the dorsal horn 1'6'16'21 (Fig. IA). Fiber-like staining was present in the superficial layers of the dorsal horn and dorsal commissure as well as in Lissauer's tract and in a bundle extending from Lissauer's tract to the region of the SPN. This bundle was not present in every section suggesting that
it may occur intermittently along the rostral-caudal axis as noted for visceral afferent projections labeled with wheat germ agglutinin horseradish peroxidase (WGAHRP) or horseradish peroxidase (HRPY 5'1~. The lateral bundle was not present in L5 or $2 spinal segments and its appearance in L6 coincided with the appearance of N A D P H diaphorase positive cells in the region of the SPN. In animals with unilateral spinal rhizotomy, the lateral bundle was eliminated ipsilateral to the rhizotomy (Fig. 1C) but other fiber-like staining in the dorsal horn and dorsal commissure was not detectably changed. The numbers of N A D P H diaphorase positive cells were also differentially affected by rhizotomy. In the region of the central canal in the L6 segment the number of N A D P H diaphorase positive cells were similar on the control and rhizotomy sides. When the central canal region was visually divided along the dorso-ventral axis, an average of 5 cells/sections were N A D P H diaphorase positive on the rhizotomy side and 5.2 cells/section were N A D P H diaphorase positive on the intact side. However, in the region of the SPN, the control side of the L6 spinal segments had an average of 4.5 _+ 0.2 cells/section (n = 5 animals) that were N A D P H diaphorase positive (Fig. 1D); whereas the rhizotomized side had an average of 9.5 + 0.3 cells/section that were N A D P H diaphorase positive (Fig. 1E). Similarly, in the region of the SPN of the SI spinal segments, an average of 5.8 + 0.5 cells/section (n = 5 animals) on the control side and 9.1_+0.5 cells/section on the rhizotomized side were N A D P H diaphorase positive. Differences in cell counts between control and rhizotomized sides were statistically significant for both L6 and S1 ( P < 0.05). Different intensities of N A D P H diaphorase cell staining were observed in the region of the SPN on both the control and rhizotomized sides. N A D P H diaphorase stained cells were either very darkly stained, moderately stained or lightly stained. The distribution of N A D P H diaphorase positive cells among these three groups was unaltered after rhizotomy. When N A D P H diaphorase cells in the region of the SPN on the intact side of spinal segment L6 (n = 3 animals) were subdivided among these three groups based upon staining intensity, 36.5% of cells were very darkly stained, 37.5%
Fig. 1. Distribution of NADPH diaphorase staining in the L6 spinal cord of rat following right, dorsal and ventral rhizotomy. A: low-power photomicrograph showing NADPH diaphorase positive neurons in the region of the sacral parasympathetic nucleus (SPN), the central canal and the dorsal horn. NADPH diaphorase fiber-like staining is also present in the dorsal horn and in a thin bundle extending from Lissauer's tract to the region of the SPN (B, arrows) Note the absence of this bundle on the right side of the spinal cord after rhizotomy (C). Distribution of NADPH diaphorase positive cells in the region of the SPN on the intact (D) and axotomized sides (E) of the L6 spinal cord. In addition to the increase in the number of NADPH diaphorase positive cells (E) following axotomy, NADPH diaphorase positive cells also occupied a greater area in the region of the SPN. Calibration bar in (E) represents 320 mm in A, 170 mm in B-C and 70 mm in D-E.
m 0
352 were moderately stained and 25.9% were lightly stained. Within the region of the SPN on the rhizotomized side, 38.5% of cells were very darkly stained, 32.6% were moderately stained and 28.9% were lightly stained. In addition to increased numbers of N A D P H diaphorase positive cells within the region of the SPN on the rhizotomized side, the area occupied by these cells in the L6 spinal segment (n = 3 animals) was also increased. On the rhizotomized side, cells were distributed over a broader dorso-ventral distance (123.9 + 13.7 mm in control versus 183.3 + 27.8 mm in rhizotomized side). In the S1 segment, no change was noted in the distribution of cells after rhizotomy (145.3 + 38.7 mm versus 144.6 +_ 34 mm). This study revealed that transection of the dorsal and ventral roots in the L6-S1 spinal segments of the rat alters fiber-like and neuronal N A D P H diaphorase staining in the region of the sacral parasympathetic nucleus. For example, the thin band of N A D P H diaphorase staining extending from Lissauer's tract to the SPN was eliminated by section of the spinal roots indicating that this band consists of primary afferent projections. However, fiber-like N A D P H diaphorase staining in the regions of the dorsal horn and dorsal commissure was not changed. Thus this staining must originate in the central nervous system or enter the spinal cord via other dorsal roots. The bundle of N A D P H diaphorase positive afferents projecting to the SPN is very similar to the central projections of pelvic visceral afferents that have been identified by transganglionic transport of H R P or W G A - H R P 15'~9. Since a large percentage of pelvic visceral afferent neurons in the L6-S1 dorsal root ganglia also exhibit N A D P H diaphorase staining it seems reasonable to conclude that part of the N A D P H diaphorase afferent bundle consists of visceral afferent projections. This view is also supported by studies in which c-fos gene expression was used to examine the central projections of pelvic visceral afferents z-5. Electrical stimulation of the pelvic nerve 5 or stimulation of afferent pathways in the lower urinary tract 2'3 or colon 2° of the rat induced c-los expression in a large population of neurons in the region of the SPN and lateral dorsal horn. Gene expression was induced by nociceptive as well as non-nociceptive stimuli and occurred in spinal tract neurons (spinopontine and spinohypothalamic), interneurons and parasympathetic preganglionic neurons 4. Somatic afferents from the distal urethra which travel in the pudendal nerve 2 also project to and induce c-los expression in neurons in this region of the SPN. Thus, the intermediolateral area of L6-S1 spinal gray matter receiving N A D P H diaphorase containing afferent input is likely to play an important role in
viscerosomatic integration which is essential for urogenital and distal bowel function. The present histochemical observations raise the possibility that nitric oxide may be a neurotransmitter or neuromodulator in the afferent projections to this spinal integrating center. Spinal rhizotomy also influenced the N A D P H diaphorase staining in neurons in the SPN. As noted in previous studies from this laboratory 23, a large percentage of preganglionic efferent neurons involved in pelvic visceral function exhibit N A D P H diaphorase activity; however, under normal circumstances, not all of these neurons stained. The doubling of the number of N A D P H diaphorase positive neurons from 5 to 10 cells per section on the side of the rhizotomy may reflect the induction or upregulation of N A D P H diaphorase enzymatic activity in preganglionic neurons following axotomy. This change in staining appeared to be selective for SPN neurons since there was no change in the numbers of N A D P H diaphorase positive neurons surrounding the central canal and no N A D P H diaphorase activity in motoneurons in the ventral horn, which also were axotomized. A similar increase in N A D P H diaphorase staining has been noted in vagal efferent neurons in the nucleus ambiguus and dorsal motor nucleus of the vagus 2 to 10 days following cervical vagotomylL Ventral root avulsion also induces the novel expression of N A D P H diaphorase activity in motoneurons in the lumbar spinal cord of the rat 24. Although in the present experiments we did not detect an increase in enzymatic activity in axotomized motoneurons, it is clear that N A D P H diaphorase activity in spinal cord and brainstem efferent neurons is labile and can be upregulated by neural damage. However, N A D P H diaphorase staining could reflect the activity of other enzymes in addition to nitric oxide synthase, therefore in future experiments it will be important to demonstrate that upregulation of N A D P H diaphorase activity in the sacral parasympathetic nucleus following rhizotomy does indeed reflect a change in nitric oxide synthase. Further studies will also be necessary to determine whether all L6-S1 preganglionic neurons exhibit N A D P H diaphorase activity following rhizotomy and whether this change occurs following selective dorsal or ventral rhizotomies. It is noteworthy that on both the normal and rhizotomized sides of the spinal cord that neurons in the SPN region exhibited considerable variability in N A D P H diaphorase staining. Neurons were grouped into three categories: light, moderate and dark staining. The relative proportions of these three groups were not altered by rhizotomy. Variations in the intensity of the staining (light and dark) of vagal pregan-
353 glionic neurons have also been reported 11. Similar variations were also noted after vagotomy. The functional significance of NADPH diaphorase staining and variations in the staining in parasympathetic preganglionic neurons is uncertain. However, it is tempting to speculate that nitric oxide may be released from the preganglionic perikarya in the spinal cord and/or from preganglionic nerve terminals in the peripheral ganglia and act as a cotransmitter or neuromodulator at these sites. Variations in NADPH diaphorase staining may reflect different roles of nitric oxide in different subpopulations of preganglionic neurons (e.g. colon, bladder and sex organs). It has been speculated that light and dark NADPH diaphorase staining in dorsal root ganglion cells reflects local modulation and transmitter functions, respectively, of nitric oxide in these cells TM. In summary, this study has demonstrated that NADPH diaphorase staining on the lateral edge of the dorsal horn was contained in afferent fibers. Furthermore, it was demonstrated that NADPH diaphorase staining can be induced or upregulated in neurons in the region of the SPN following axotomy. The functional significance of the NADPH diaphorase histochemical stain remains to be determined; however, if NADPH diaphorase is nitric oxide synthase 12 then this would suggest that NO has an important role in the neural pathways to the pelvic viscera. This study was supported by NIH Grants DK 37241 and DK 42369, NSF Grant BNS-890-8934 and NIMH Training Grant MH 18273. 1 Anderson, C.R., NADPH diaphorase-positive neurons in the rat spinal cord include a subpopulation o f autonomic preganglionic neurons, Neurosci. Lett., 139 (1992) 280-284. 2 Birder, L.A. and de Groat, W.C., Increased c-fos expression in spinal neurons after irritation of the lower urinary tract in the rat, J. Neurosci., in press. 3 Birder, L.A. and de Groat, W.C., The effect of glutamate antagonists on c-los expression induced in spinal neurons by irritation of the lower urinary tract, Brain Res., 580 (1992) 115-120. 4 Birder, L.A., Roppolo, J.R. and de Groat, W.C., c-los as a marker for subsets of visceral second order neurons in the rat lumbosacral spinal cord, Soc. Neurosci. Abstr., 16 (1990) 703. 5 Birder, L.A., Roppolo, J.R., Iadarola, M.J. and de Groat, W.C., Electrical stimulation of visceral afferent pathways in the pelvic nerve increases c-los in the rat lumbosacral spinal cord, Neurosci. Lett., 129 (1992) 193-196. 6 Blottner, D. and Baumgarten, H., Nitric oxide synthetase (NOS) containing sympathoadrenal cholinergic neurons of the rat IMLcell column: evidence from histochemistry, immunohistochemistry and retrograde labeling, J. Comp. Neurol., 316 (1992) 45-55. 7 Bredt, D.S., Glatt, C.E., Hwang, P.M., Fotuhi, M., Dawson, T.M.
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