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A biochemically distinct sub-population of neurons in the human substantia gelatinosa
Journal of the Neurological Sciences, 1982, 55 : 175-183
175
Elsevier Biomedical Press
A B I O C H E M I C A L L Y D I S T I N C T S U B - P O P U L A T I O N OF N E U R O N S THE HUMAN SUBSTANTIA GELATINOSA
IN
Study with G-6-PD Histochemistry
BRUCE A. BEUTLER*, KARl STEFANSSON and BARRY G. W. ARNASON** Department of Neurology, The University of Chicago, The Division of the Biological Sciences and the Pritzker School of Medicine, 950 East 59th Street, Chicago, IL 60637 (U.S.A.)
(Received 9 November, 1981) (Accepted 20 January, 1982)
SUMMARY A method for localization of glucose-6-phosphate dehydrogenase (G-6-PD; D-glucose-6-phosphate: N A D P + oxidoreductase; E.C. 1.1.1.49) activity has been applied to human nervous tissue. Intensely staining cells, not definable by conventional histologic techniques, have been identified in the human spinal cord, with highest numbers present in the substantia gelatinosa of the sacral region. The cells have a neuron-like morphology and express neuronal-specific antigen but are heterogeneous in size and shape. They are not detectable in infant spinal cord, but stain heavily in adults. We propose that these cells are homologous to the G-6-PDactive dorsal medullary cells first noted by Sakharova et al. (1979) and together with the latter group, may comprise a hitherto unrecognized system of neurons in the h u m a n central nervous system.
Key words: C e n t r a l nervous s y s t e m - -
Development
--
Maturation
--
Neuro-
anatomy -- Neurohistology
This work was supported by a grant from the Amyotrophic Lateral Sclerosis Society of America. *Present address: Department of Neurology, University of Texas, Southwestern Medical School, 5324 Harry Hines Boulevard, Dallas, TX 75235, U.S.A. **To whom correspondence should be addressed. 002-510X/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
176 INTRODUCTION
In a recent publication, Sakharova et al. (1979) reported the presence of a group of "peculiar cells" in the mammalian medulla oblongata. Located predominantly in the region of the gracile nucleus, the cells stained intensely with reagents specific for the enzyme glucose-6-phosphate dehydrogenase (G-6-PD; D-glucose-6-phosphate : NADP + oxidoreductase; E.C. 1.1.1.49), but had escaped comment by earlier investigators of G-6-PD in nervous tissue (Friede et al. 1962). Sakharova et al. (1979) speculated that the cells were an unusual form of neuron, reminiscent of neurons described some years earlier by Sakharov in gastropod ganglia (1974) and suggested that they might subserve a secretory function, requiring a high activity of enzymes of the hexose-monophosphate shunt as a source of NADPH to be used in reductive synthesis. Sakharova et al. (1979) did not report on the expression of other pyridine nucleotide-linked dehydrogenases by these cells, did not mention the presence of comparable cells outside the mammalian medulla oblongata, and did not present their data with consideration given to the age of their subjects. Here, we report our experience with a procedure for localizing pyridine nucleotide-linked dehydrogenases, incorporating eosin counterstaining to enhance morphologic detail. We have noted a group of cells in the spinal cord of man which appears identical to the cells of the mammalian medulla oblongata described by Sakharova et al. (1979), and which may represent the caudal portion of a craniosacral system of cells. MATERIALS A N D METHODS
Human autopsy specimens were obtained as soon as possible after death, Age, sex, race, cause of death, post-mortem interval, and G-6-PD genotype for each of the 19 patients studied are presented in Table 1. G-6-PD assay was performed on cadaveric blood from females, according to the WHO method (Betke et al. 1967) at 37 °C. In males, the G-6-PD fluorescent spot test was used (Beutler 1966). For both males and females, electrophoresis on Titan III-H cellulose acetate plates (Helena Laboratories, Beaumont, TX) was performed according to the method of Sparkes et al. (1969). All blood samples for assay and for electrophoresis were collected in ACD solution, and stored at 4 °C until used. Enzyme histochemistry was adapted from the method of Hess et al. 0958). Tissue specimens were quick-frozen in liquid nitrogen after mounting on cork slabs, 20-/~m sections were cut at -20 °C and affixed to room temperature slides. The sections were then incubated for periods ranging from 15 to 90 min at 37 °C with one of the reaction mixtures prepared as follows:
177 TABLE 1 CHARACTERISTICS OF THE AUTOPSY SPECIMENS STUDIED The 19 subjects studied are shown arranged in order of increasing age. Asterisks imply that both cord a n d brain were available; in other cases, only cord was available. In males, G-6-PD positivity or negativity ( + o r - ) was assessed by the fluorescent spot test (Betke et al. 1967) while in females, a quantitative assay (IU/g hemoglobin) was employed to evaluate genotype. An activity below 5.5 IU/g H b was considered indicative of heterozygosity for the A- allele. In 3 cases, no blood was available for assay or electrophoresis; the patients were considered to be B + homozygotes on the basis o f race alone. Age (yr)
Sex
Post-mortem interval (h)
Race
G-6-PD activity (IU/g Hb)
1/52
F
25
W
1/52" 7/52
F M
20 6
W B
+
B+
B+/B+ B+/Y
1/52 1"
M F
44 10
B W
+ 6.1
A+ B+
A+/Y B+/B+
38 39* 49 51"
F F F F
23 28 28 32
W B B B
12.9 5.4 5.4
A+/B+ B+ B+
B+/B+ A+/B+ A -/B+ A -/B+
52 58
F F
74 18
B B
6.3 8.1
B+ B+
B+/B+ B+/B+
60 60 62* 62 71" 76 77 83*
F M F F M M M M
24 25 22 18 23 33 24 27
B W B B W B W W
9.6 + 7.4 7.1 + + + +
A+/B+ B+ B+ B+ B+ A+ B+ B+
A+/B+ B+/Y B+/B+ B+/B+ B+/Y A+/Y B+/Y B+/Y
A. Glucose-6-phosphate dehydrogenase: 1. Disodium glucose-6-phosphate (G-6-P), 1.0 M 2. N A D P ÷, 1.0 M 3. MgCI2, 0.05 M 4. Sodium azide, 0.1 M 5. Tris-HCl, 0.2 M, p H 6.8 6. Sodium fluoride, 0.01 M 7. Polyvinylpyrrolidone, 75 m g / m l 8. Nitro-blue tetrazolium (NBT), 2 mg/ml
Electrophoretic mobility
Presumed genotype
Cause of death
B+/B+
31 wk gestation ; tracheoesophageal fistula; intracranial bleeding Hypoplastic left heart Hepatic arteriovenous malformation Respiratory failure Down's syndrome; atrioventricular canal Alcoholic cirrhosis Renal failure Alcoholic cirrhosis Chronic myelogenous leukemia; blastic crisis Acute myelogenous leukemia Adenocarcinoma; primary unknown Stroke Multiple myeloma Myocardial infarction Renal failure Myocardial infarction Septic shock Respiratory and renal failure Cardiac arrest; post hip surgery
0.1 ml 0.025 ml 0.1 ml 0.1 ml 0.25 ml 0.05 ml 1.0 ml 0.375 ml
B. Hexose-6-phosphate dehydrogenase (H-6-PD; glucose d e h y d r o g e n a s e ; fl-D-glucose : NAD(P) + oxidoreductase; E.C. 1.1.47) : 1. Disodium galactose-6-phosphate (Gal-6-P), 1.0 M 0.1 ml 2. N A D +, 1.0 M 0.025 ml 3. through 8. as above C. 6-Phosphogluconate dehydrogenase (6-PGD; 6-phospho-D-gluconate: N A D P + oxidoreductase (decarboxylating); E.C. 1.1.1.44) 1. Disodium 6-phosphogluconate (6-PG), 1.0 M 0.1 ml 2. N A D P +, 1.0 M 0.025 ml 3. through 8. as above
178 D. NADH dehydrogenase (NADH-D; NADH: (acceptor) oxidoreductasc; E.C. 1.6.99.3): E. Lactate dehydrogenase (LDH; L-lactate: NAD + oxidoreductase: E.C, 1.1,1.27) F. Glutamate dehydrogenase (GD; L-glutamate: NAD + oxidoreductase (deaminating); E.C. 1.4.1.2)
as per Hess, et al, (1958)
G. Glycerol-3-phosphate dehydrogenase (e-GPD; sn-glycerol-3-phosphate: NAD + 2-oxidoreductase; E.C. 1.1.1.8) H, Alcohol dehydrogenase (ADH ; alcohol: NAD + oxidoreductase; E.C. 1.I.1.1)
NBT, G-6-P, Gal-6-P, 6-PG, e-glycerolphosphate, monosodium glutamate, NAD +, NADP + , and NADH were all obtained from Sigma Chemical Co., St. Louis, MO. Lactic acid was obtained from J.T. Baker Chemical Co., Phillipsburg, NJ. All staining solutions were frozen at -20 °C in small aliquots until use. Control staining solutions were prepared as above, with the substrate omitted. Following incubation, slides were fixed for at least 10 min in 10~ phosphatebuffered formalin, pH 7.0 (Fisher Scientific Co., Fair Lawn, N J). They were then dehydrated for 90 s with 80~o ethanol, and counterstained for 90 s in eosin. After further dehydration, through 95 and 100~ ethanol, the slides were stirred in xylen~ and mounted with Permount. Since excessive exposure to xylene appears to dissolve the formazan precipitate, immersion in xylene was limited to 10 s. Sections adjacent to those examined for activity of the above mentioned enzymes were stained with antiserum to the neuronal specific protein 14-3-2 (Moore and Perez 1968) using a specific antiserum and the peroxidase antiperoxidase method of Sternberger (1979). The antiserum to 14-3-2 was a generous gift from Dr. Blake W. Moore. RESULTS
The method of G-6-PD histochemistry described above yields sharply defined staining of both neuronal elements and the neuropil. No difference is found between A, B, and A- isozymes in terms of staining intensity in any portion of the CNS. We will confine ourselves to a description of two groups of cells in the central nervous system (CNS) of adults that stain more intensely than any other cellular structure in the CNS. One is in the medulla oblongata and the other in the spinal cord. Neither of these is found in infants. The medullary cells correspond to the group described by Sakharova et al. (1979) and lie, for the most part, within the gracile nucleus. In the spinal cord, virtually all the intensely staining ceils occupy Rexed laminae II and III, and therefore lie within the substantia gelatinosa. These intensely staining cells in both groups are also stained with antiserum to 14-3-2. In the spinal cord, the cell number is greatest at mid-sacral level (Fig. 1). At low lumbar level, the number of cells is smaller, and they are only situated medially (Fig. 2). By mid-lumbar level, the cells are scarce. Darkly staining cells are scarce or absent in 10 of 11 specimens of adult thoracic cord, and in 11 of 12 specimens of adult cervical cord examined (Table 2),
179
,I ~
J
°
~
~"
Fig. 1. Mid-sacral cord, 30 min incubation. Darkly stainmg cells of the spinal group are seen in large numbers at this level, and stand out sharply from the majority of the neurons of the substantia gelatinosa, which are G-6-PD-negative. × 100.
Fig. 2. Low lumbar cord, 30 min incubation. The group is now principally distributed within the mcdial substantia gelatinosa, and contains fewer cells than in Fig. 1. × 100.
180 TABLE 2 DISTRIBUTION
OF DARKLY
STAINING
CELLS AT VARIOUS
LEVELS OF THE NEURAXIS
19 s u b j e c t s studied are shown arranged in order of increasing age. The stated levels o f the neuraxis were examined for the presence of darkly staining ceils after treatment with G - 6 - P D r e a g e n t , a n d s c o r e d as f o l l o w s : 0 = n o cells visible in the s e c t i o n ; + = 1 - 1 0 cells visible; + + = 1 1 - 5 0 cells visible; + + + = o v e r 50 cells visible. N A : data were not available.
The
Age
Medulla
C-cord
T-cord
L/S-cord
NA 0
0 0
0 0
0 0
4 mo
NA NA
NA NA
0 0
NA 0
1 yr
0
NA
0
0
NA +++ NA +++ NA NA NA NA +++ NA +++ NA NA +++
+ + + + NA 0 + 0 NA + + +++ + +
+ + + + 0 + + NA + + + ++ NA NA
+ + +++ + + +++ + + + + + + + +++ NA +++ +++ + +++
Infants 1 wk 1 wk 7 wk
Adults 38 yr 39yr 49 y r 51yr 52 yr 58 y r 60 y r 60 y r 62yr 62 yr 71yr 76yr 77 yr 83yr
+ +
+ + +
as well as in the spinal trigeminal nucleus. However, in the gracile nucleus of the dorsal medulla similar cells are apparent in all specimens from adults, as de~'ibed by Sakharova et al. (1979). Within planes of section, the cells of both medullary and sacral groups appear heterogeneous in size and shape. Occasional cells of both groups display stained processes (Fig. 3), some of which can be followed for several cell diameters. The youngest adult studied, a 38-year-old white female, shows darkly staining cells in the spinal cord in numbers comparable to those found in older individ~aals. Likewise, in the youngest adult (age 39) from whom a brain was obtained, both the medulla oblongata and spinal cord contain darkly staining cells in numbers comparable to those found in older patients. No darkly staining cells are found at any level in infant cord. Two infant medulla oblongata specimens are also negative for dartdy staining celts. In addition to G-6-PD, seven other oxidoreductase,s were stu~ed. These are NADH-dehydrogenase (NADH-D), lactate dehydrogenase (LDH), glutamate dehydrogenase (GD), 6-phosphogluconate dehydrogenase (6-PGD), glycerol-3-phosphate dehydrogenase (~-GPD), glucose dehydrogenase (H-6-PD), and alcohol de-
181
Fig. 3. Darkly staining cells of the sacral group at higher magnification. Note the different size and shape of the two cells pictured here, and the formazan-stained process extending from the larger cell. 90 min incubation; x 2000.
TABLE 3 S T A I N I N G I N T E N S I T Y FOR SEVERAL O X I D A T I V E E N Z Y M E S Intensity of staining of medullary and lumbosacral cells is assessed for a panel of 8 different oxidoreductases (abbreviations in text). + + + + = very intense staining; + + + = intense staining; + + = moderate staining; + = light staining; 0 = negative staining. Staining time = 90 min. Control contained no substrate.
Enzyme
Lumbosacral cells
Medullary cells
NADH-D
++++
++++
G-O-PD LDH GD
+++ + + + +++
++ + + + + +++
6-PGD
+ +
+ +
c~-GPD
+
+
H-6-PD ADH
0 to + 0 to +
0 to + 0 to +
Control
0
0
182 hydrogenase (ADH). The results of these studies are outlined in Table 3. Medullary and lumbosacral cells of the type under discussion show concordant staining lor each enzyme. Staining varies in intensity depending upon the enzyme studied. For each enzyme tested, other cells stain less intensely than the clusters of densely G-6-PD-positive cells. Hematoxylin and eosin staining of serial sections of medulla oblongata and sacral cord demonstrate that the G-6-PD positive cells comprise approximately 1/5 to 1/10 of the total population of neurons present in the gracile nucleus and in the substantia gelatinosa of the sacral cord. In thoracic and cervical cord sections, G-6-PD-positive cells constitute a negligible fraction of the neurons of the substantia gelatinosa. DISCUSSION We have described the distribution, enzymological characteristics, and an agerelated acquisition of G-6-PD activity of a group of cells in the human substantia gelatinosa, not mentioned previously by investigators studying the oxidative enzymes of the CNS (Thomas et al. 1961 ; Friede and Fleming 1962). On morphologic grounds, the cells appear indistinguishable from the dorsal medullary cells described in humans, rats, and rabbits by Sakharova et al. (1979), and thought by these workers to be neurons. We are inclined to believe that both the medullary and sacral groups of cells are neurons, based on the ultrastructural description of the medullary cells (1979), and the possession, by both groups of cells, of processes that appear to be axons. The immunohistochemical staining for 14-3-2 protein which in the human CNS is confined to neurons (Stefansson and Wollmann 1981) also indicates thai the cells are neurons. The medullary and spinal cells exhibit concordant staining properties when tested for the expression of 8 different enzymes. They appear developmentally similar in view of the late acquisition of enzyme activity common to both groups. The enzymologic and morphologic similarity of the cells could argue for a similarity of function, and perhaps implies that the two groups of cells constitute cranial and caudal divisions of a single system of neurons. Based on Golgi impregnation studies (Gobel 1975), the intrinsic cells of the substantia gelatinosa have been divided into 3 morphologic types, distinguishable by the appearance and orientation of their dendrites. With conventional neuroanatomic methods, the architecture and morphotogic appearance of neurons of the substantia gelatinosa is similar at all levels of the spinal cord. We show here, however, that the sacral spinal cord (and, to a minor degree, the lumbar cord) contains a biochemically distinct population of cells in the substantia gelatinosa. Thus, it appears that the lumbosacral substantia gelatinosa differs from the substantia gelatinosa at other levels. Perhaps these cells serve a function distinct from that of the non-staining population. From their dorsal location, we may infer that the cells are somehow involved in afferent transmission. The confinement of the cells to the lumbosacral cord, as well as their activity in adults but not in infants may hint at a role in sexual function.
183 ACKNOWLEDGEMENTS We thank Dr. Robert Wollmann
a n d D r . S t e p h a n i e H o l t f o r t h e i r h e l p in
obtaining autopsy specimens, and Miss Michele Poon for her technical assistance. REFERENCES Betke, K., E. Beutler, G.J. Brewer, H.N. Kirkman, L. Luzzatto, A.G. Motulsky, B. Ramot and M. Siniscalco (1967) WHO Tech. Rep. Set. No. 366, pp. 30-32. Beutler, E. (1966) A series of new screening procedures for pyruvate kinase deficiency, glucose-6phosphate dehydrogenase deficiency and glutathione reductase deficiency, Blood, 28 : 553-562. Friede, R.L. and L.M. Fleming (1962) A mapping of oxidative enzymes in the human brain, J. Neurochem., 9: 179-198. Gobel, S. (1975) Golgi studies of the substantia gelatinosa neurons in the spinal trigeminal nucleus, J. comp. Neurol., 162: 397416. Hess, R., D. Scarpelli and A. G. E. Pearse (1958) The cytochemical localization of oxidative enzymes, J. Biophys. Biochem. Cytol., 4:747 760. Moore, B.W. and V.J. Perez (1968) Specific acidic proteins of the nervous system. In: F.D. Carlson (Ed.), Physiological and Biochemical Aspects of Nervous Integration, Prentice-Hall, Englewood Cliffs, N J, p. 343. Sakharov, D.A. (1974) The Genealogy of Neurons, Nauka, Moscow. Sakharova, A.V., N.B. Salimova and D.A. Sakharov (1979) Peculiar cells notable for very high activity of glucose-6-phosphate dehydrogenase in the mammalian medulla oblongata - - A histochemical and electron microscopic study, Neuroscience, 4:1173-1177. Sparkes, R.S., M.C. Baluda and D.E. Townsend (1969) Cellusose acetate electrophoresis of human glucose-6-phosphate dehydrogenase, J. Lab. clin. Med., 73: 531-534. Stefansson, K. and R. Wollmann (1981) Distribution of the neuronal specific protein, 14-3-2, in central nervous system lesions of tuberous sclerosis, Acta neuropath. (Berl.), 53:113-117. Sternberger, L.A. (1979)Immunocytochemistry, 2nd edition, J. Wiley and Sons, New York,'NY, pp. 104-130. Thomas, E. and A. G. E. Pearse (1961) The fine localization of dehydrogenases in the nervous system, Histochemie, 2: 266282.
175
Elsevier Biomedical Press
A B I O C H E M I C A L L Y D I S T I N C T S U B - P O P U L A T I O N OF N E U R O N S THE HUMAN SUBSTANTIA GELATINOSA
IN
Study with G-6-PD Histochemistry
BRUCE A. BEUTLER*, KARl STEFANSSON and BARRY G. W. ARNASON** Department of Neurology, The University of Chicago, The Division of the Biological Sciences and the Pritzker School of Medicine, 950 East 59th Street, Chicago, IL 60637 (U.S.A.)
(Received 9 November, 1981) (Accepted 20 January, 1982)
SUMMARY A method for localization of glucose-6-phosphate dehydrogenase (G-6-PD; D-glucose-6-phosphate: N A D P + oxidoreductase; E.C. 1.1.1.49) activity has been applied to human nervous tissue. Intensely staining cells, not definable by conventional histologic techniques, have been identified in the human spinal cord, with highest numbers present in the substantia gelatinosa of the sacral region. The cells have a neuron-like morphology and express neuronal-specific antigen but are heterogeneous in size and shape. They are not detectable in infant spinal cord, but stain heavily in adults. We propose that these cells are homologous to the G-6-PDactive dorsal medullary cells first noted by Sakharova et al. (1979) and together with the latter group, may comprise a hitherto unrecognized system of neurons in the h u m a n central nervous system.
Key words: C e n t r a l nervous s y s t e m - -
Development
--
Maturation
--
Neuro-
anatomy -- Neurohistology
This work was supported by a grant from the Amyotrophic Lateral Sclerosis Society of America. *Present address: Department of Neurology, University of Texas, Southwestern Medical School, 5324 Harry Hines Boulevard, Dallas, TX 75235, U.S.A. **To whom correspondence should be addressed. 002-510X/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
176 INTRODUCTION
In a recent publication, Sakharova et al. (1979) reported the presence of a group of "peculiar cells" in the mammalian medulla oblongata. Located predominantly in the region of the gracile nucleus, the cells stained intensely with reagents specific for the enzyme glucose-6-phosphate dehydrogenase (G-6-PD; D-glucose-6-phosphate : NADP + oxidoreductase; E.C. 1.1.1.49), but had escaped comment by earlier investigators of G-6-PD in nervous tissue (Friede et al. 1962). Sakharova et al. (1979) speculated that the cells were an unusual form of neuron, reminiscent of neurons described some years earlier by Sakharov in gastropod ganglia (1974) and suggested that they might subserve a secretory function, requiring a high activity of enzymes of the hexose-monophosphate shunt as a source of NADPH to be used in reductive synthesis. Sakharova et al. (1979) did not report on the expression of other pyridine nucleotide-linked dehydrogenases by these cells, did not mention the presence of comparable cells outside the mammalian medulla oblongata, and did not present their data with consideration given to the age of their subjects. Here, we report our experience with a procedure for localizing pyridine nucleotide-linked dehydrogenases, incorporating eosin counterstaining to enhance morphologic detail. We have noted a group of cells in the spinal cord of man which appears identical to the cells of the mammalian medulla oblongata described by Sakharova et al. (1979), and which may represent the caudal portion of a craniosacral system of cells. MATERIALS A N D METHODS
Human autopsy specimens were obtained as soon as possible after death, Age, sex, race, cause of death, post-mortem interval, and G-6-PD genotype for each of the 19 patients studied are presented in Table 1. G-6-PD assay was performed on cadaveric blood from females, according to the WHO method (Betke et al. 1967) at 37 °C. In males, the G-6-PD fluorescent spot test was used (Beutler 1966). For both males and females, electrophoresis on Titan III-H cellulose acetate plates (Helena Laboratories, Beaumont, TX) was performed according to the method of Sparkes et al. (1969). All blood samples for assay and for electrophoresis were collected in ACD solution, and stored at 4 °C until used. Enzyme histochemistry was adapted from the method of Hess et al. 0958). Tissue specimens were quick-frozen in liquid nitrogen after mounting on cork slabs, 20-/~m sections were cut at -20 °C and affixed to room temperature slides. The sections were then incubated for periods ranging from 15 to 90 min at 37 °C with one of the reaction mixtures prepared as follows:
177 TABLE 1 CHARACTERISTICS OF THE AUTOPSY SPECIMENS STUDIED The 19 subjects studied are shown arranged in order of increasing age. Asterisks imply that both cord a n d brain were available; in other cases, only cord was available. In males, G-6-PD positivity or negativity ( + o r - ) was assessed by the fluorescent spot test (Betke et al. 1967) while in females, a quantitative assay (IU/g hemoglobin) was employed to evaluate genotype. An activity below 5.5 IU/g H b was considered indicative of heterozygosity for the A- allele. In 3 cases, no blood was available for assay or electrophoresis; the patients were considered to be B + homozygotes on the basis o f race alone. Age (yr)
Sex
Post-mortem interval (h)
Race
G-6-PD activity (IU/g Hb)
1/52
F
25
W
1/52" 7/52
F M
20 6
W B
+
B+
B+/B+ B+/Y
1/52 1"
M F
44 10
B W
+ 6.1
A+ B+
A+/Y B+/B+
38 39* 49 51"
F F F F
23 28 28 32
W B B B
12.9 5.4 5.4
A+/B+ B+ B+
B+/B+ A+/B+ A -/B+ A -/B+
52 58
F F
74 18
B B
6.3 8.1
B+ B+
B+/B+ B+/B+
60 60 62* 62 71" 76 77 83*
F M F F M M M M
24 25 22 18 23 33 24 27
B W B B W B W W
9.6 + 7.4 7.1 + + + +
A+/B+ B+ B+ B+ B+ A+ B+ B+
A+/B+ B+/Y B+/B+ B+/B+ B+/Y A+/Y B+/Y B+/Y
A. Glucose-6-phosphate dehydrogenase: 1. Disodium glucose-6-phosphate (G-6-P), 1.0 M 2. N A D P ÷, 1.0 M 3. MgCI2, 0.05 M 4. Sodium azide, 0.1 M 5. Tris-HCl, 0.2 M, p H 6.8 6. Sodium fluoride, 0.01 M 7. Polyvinylpyrrolidone, 75 m g / m l 8. Nitro-blue tetrazolium (NBT), 2 mg/ml
Electrophoretic mobility
Presumed genotype
Cause of death
B+/B+
31 wk gestation ; tracheoesophageal fistula; intracranial bleeding Hypoplastic left heart Hepatic arteriovenous malformation Respiratory failure Down's syndrome; atrioventricular canal Alcoholic cirrhosis Renal failure Alcoholic cirrhosis Chronic myelogenous leukemia; blastic crisis Acute myelogenous leukemia Adenocarcinoma; primary unknown Stroke Multiple myeloma Myocardial infarction Renal failure Myocardial infarction Septic shock Respiratory and renal failure Cardiac arrest; post hip surgery
0.1 ml 0.025 ml 0.1 ml 0.1 ml 0.25 ml 0.05 ml 1.0 ml 0.375 ml
B. Hexose-6-phosphate dehydrogenase (H-6-PD; glucose d e h y d r o g e n a s e ; fl-D-glucose : NAD(P) + oxidoreductase; E.C. 1.1.47) : 1. Disodium galactose-6-phosphate (Gal-6-P), 1.0 M 0.1 ml 2. N A D +, 1.0 M 0.025 ml 3. through 8. as above C. 6-Phosphogluconate dehydrogenase (6-PGD; 6-phospho-D-gluconate: N A D P + oxidoreductase (decarboxylating); E.C. 1.1.1.44) 1. Disodium 6-phosphogluconate (6-PG), 1.0 M 0.1 ml 2. N A D P +, 1.0 M 0.025 ml 3. through 8. as above
178 D. NADH dehydrogenase (NADH-D; NADH: (acceptor) oxidoreductasc; E.C. 1.6.99.3): E. Lactate dehydrogenase (LDH; L-lactate: NAD + oxidoreductase: E.C, 1.1,1.27) F. Glutamate dehydrogenase (GD; L-glutamate: NAD + oxidoreductase (deaminating); E.C. 1.4.1.2)
as per Hess, et al, (1958)
G. Glycerol-3-phosphate dehydrogenase (e-GPD; sn-glycerol-3-phosphate: NAD + 2-oxidoreductase; E.C. 1.1.1.8) H, Alcohol dehydrogenase (ADH ; alcohol: NAD + oxidoreductase; E.C. 1.I.1.1)
NBT, G-6-P, Gal-6-P, 6-PG, e-glycerolphosphate, monosodium glutamate, NAD +, NADP + , and NADH were all obtained from Sigma Chemical Co., St. Louis, MO. Lactic acid was obtained from J.T. Baker Chemical Co., Phillipsburg, NJ. All staining solutions were frozen at -20 °C in small aliquots until use. Control staining solutions were prepared as above, with the substrate omitted. Following incubation, slides were fixed for at least 10 min in 10~ phosphatebuffered formalin, pH 7.0 (Fisher Scientific Co., Fair Lawn, N J). They were then dehydrated for 90 s with 80~o ethanol, and counterstained for 90 s in eosin. After further dehydration, through 95 and 100~ ethanol, the slides were stirred in xylen~ and mounted with Permount. Since excessive exposure to xylene appears to dissolve the formazan precipitate, immersion in xylene was limited to 10 s. Sections adjacent to those examined for activity of the above mentioned enzymes were stained with antiserum to the neuronal specific protein 14-3-2 (Moore and Perez 1968) using a specific antiserum and the peroxidase antiperoxidase method of Sternberger (1979). The antiserum to 14-3-2 was a generous gift from Dr. Blake W. Moore. RESULTS
The method of G-6-PD histochemistry described above yields sharply defined staining of both neuronal elements and the neuropil. No difference is found between A, B, and A- isozymes in terms of staining intensity in any portion of the CNS. We will confine ourselves to a description of two groups of cells in the central nervous system (CNS) of adults that stain more intensely than any other cellular structure in the CNS. One is in the medulla oblongata and the other in the spinal cord. Neither of these is found in infants. The medullary cells correspond to the group described by Sakharova et al. (1979) and lie, for the most part, within the gracile nucleus. In the spinal cord, virtually all the intensely staining ceils occupy Rexed laminae II and III, and therefore lie within the substantia gelatinosa. These intensely staining cells in both groups are also stained with antiserum to 14-3-2. In the spinal cord, the cell number is greatest at mid-sacral level (Fig. 1). At low lumbar level, the number of cells is smaller, and they are only situated medially (Fig. 2). By mid-lumbar level, the cells are scarce. Darkly staining cells are scarce or absent in 10 of 11 specimens of adult thoracic cord, and in 11 of 12 specimens of adult cervical cord examined (Table 2),
179
,I ~
J
°
~
~"
Fig. 1. Mid-sacral cord, 30 min incubation. Darkly stainmg cells of the spinal group are seen in large numbers at this level, and stand out sharply from the majority of the neurons of the substantia gelatinosa, which are G-6-PD-negative. × 100.
Fig. 2. Low lumbar cord, 30 min incubation. The group is now principally distributed within the mcdial substantia gelatinosa, and contains fewer cells than in Fig. 1. × 100.
180 TABLE 2 DISTRIBUTION
OF DARKLY
STAINING
CELLS AT VARIOUS
LEVELS OF THE NEURAXIS
19 s u b j e c t s studied are shown arranged in order of increasing age. The stated levels o f the neuraxis were examined for the presence of darkly staining ceils after treatment with G - 6 - P D r e a g e n t , a n d s c o r e d as f o l l o w s : 0 = n o cells visible in the s e c t i o n ; + = 1 - 1 0 cells visible; + + = 1 1 - 5 0 cells visible; + + + = o v e r 50 cells visible. N A : data were not available.
The
Age
Medulla
C-cord
T-cord
L/S-cord
NA 0
0 0
0 0
0 0
4 mo
NA NA
NA NA
0 0
NA 0
1 yr
0
NA
0
0
NA +++ NA +++ NA NA NA NA +++ NA +++ NA NA +++
+ + + + NA 0 + 0 NA + + +++ + +
+ + + + 0 + + NA + + + ++ NA NA
+ + +++ + + +++ + + + + + + + +++ NA +++ +++ + +++
Infants 1 wk 1 wk 7 wk
Adults 38 yr 39yr 49 y r 51yr 52 yr 58 y r 60 y r 60 y r 62yr 62 yr 71yr 76yr 77 yr 83yr
+ +
+ + +
as well as in the spinal trigeminal nucleus. However, in the gracile nucleus of the dorsal medulla similar cells are apparent in all specimens from adults, as de~'ibed by Sakharova et al. (1979). Within planes of section, the cells of both medullary and sacral groups appear heterogeneous in size and shape. Occasional cells of both groups display stained processes (Fig. 3), some of which can be followed for several cell diameters. The youngest adult studied, a 38-year-old white female, shows darkly staining cells in the spinal cord in numbers comparable to those found in older individ~aals. Likewise, in the youngest adult (age 39) from whom a brain was obtained, both the medulla oblongata and spinal cord contain darkly staining cells in numbers comparable to those found in older patients. No darkly staining cells are found at any level in infant cord. Two infant medulla oblongata specimens are also negative for dartdy staining celts. In addition to G-6-PD, seven other oxidoreductase,s were stu~ed. These are NADH-dehydrogenase (NADH-D), lactate dehydrogenase (LDH), glutamate dehydrogenase (GD), 6-phosphogluconate dehydrogenase (6-PGD), glycerol-3-phosphate dehydrogenase (~-GPD), glucose dehydrogenase (H-6-PD), and alcohol de-
181
Fig. 3. Darkly staining cells of the sacral group at higher magnification. Note the different size and shape of the two cells pictured here, and the formazan-stained process extending from the larger cell. 90 min incubation; x 2000.
TABLE 3 S T A I N I N G I N T E N S I T Y FOR SEVERAL O X I D A T I V E E N Z Y M E S Intensity of staining of medullary and lumbosacral cells is assessed for a panel of 8 different oxidoreductases (abbreviations in text). + + + + = very intense staining; + + + = intense staining; + + = moderate staining; + = light staining; 0 = negative staining. Staining time = 90 min. Control contained no substrate.
Enzyme
Lumbosacral cells
Medullary cells
NADH-D
++++
++++
G-O-PD LDH GD
+++ + + + +++
++ + + + + +++
6-PGD
+ +
+ +
c~-GPD
+
+
H-6-PD ADH
0 to + 0 to +
0 to + 0 to +
Control
0
0
182 hydrogenase (ADH). The results of these studies are outlined in Table 3. Medullary and lumbosacral cells of the type under discussion show concordant staining lor each enzyme. Staining varies in intensity depending upon the enzyme studied. For each enzyme tested, other cells stain less intensely than the clusters of densely G-6-PD-positive cells. Hematoxylin and eosin staining of serial sections of medulla oblongata and sacral cord demonstrate that the G-6-PD positive cells comprise approximately 1/5 to 1/10 of the total population of neurons present in the gracile nucleus and in the substantia gelatinosa of the sacral cord. In thoracic and cervical cord sections, G-6-PD-positive cells constitute a negligible fraction of the neurons of the substantia gelatinosa. DISCUSSION We have described the distribution, enzymological characteristics, and an agerelated acquisition of G-6-PD activity of a group of cells in the human substantia gelatinosa, not mentioned previously by investigators studying the oxidative enzymes of the CNS (Thomas et al. 1961 ; Friede and Fleming 1962). On morphologic grounds, the cells appear indistinguishable from the dorsal medullary cells described in humans, rats, and rabbits by Sakharova et al. (1979), and thought by these workers to be neurons. We are inclined to believe that both the medullary and sacral groups of cells are neurons, based on the ultrastructural description of the medullary cells (1979), and the possession, by both groups of cells, of processes that appear to be axons. The immunohistochemical staining for 14-3-2 protein which in the human CNS is confined to neurons (Stefansson and Wollmann 1981) also indicates thai the cells are neurons. The medullary and spinal cells exhibit concordant staining properties when tested for the expression of 8 different enzymes. They appear developmentally similar in view of the late acquisition of enzyme activity common to both groups. The enzymologic and morphologic similarity of the cells could argue for a similarity of function, and perhaps implies that the two groups of cells constitute cranial and caudal divisions of a single system of neurons. Based on Golgi impregnation studies (Gobel 1975), the intrinsic cells of the substantia gelatinosa have been divided into 3 morphologic types, distinguishable by the appearance and orientation of their dendrites. With conventional neuroanatomic methods, the architecture and morphotogic appearance of neurons of the substantia gelatinosa is similar at all levels of the spinal cord. We show here, however, that the sacral spinal cord (and, to a minor degree, the lumbar cord) contains a biochemically distinct population of cells in the substantia gelatinosa. Thus, it appears that the lumbosacral substantia gelatinosa differs from the substantia gelatinosa at other levels. Perhaps these cells serve a function distinct from that of the non-staining population. From their dorsal location, we may infer that the cells are somehow involved in afferent transmission. The confinement of the cells to the lumbosacral cord, as well as their activity in adults but not in infants may hint at a role in sexual function.
183 ACKNOWLEDGEMENTS We thank Dr. Robert Wollmann
a n d D r . S t e p h a n i e H o l t f o r t h e i r h e l p in
obtaining autopsy specimens, and Miss Michele Poon for her technical assistance. REFERENCES Betke, K., E. Beutler, G.J. Brewer, H.N. Kirkman, L. Luzzatto, A.G. Motulsky, B. Ramot and M. Siniscalco (1967) WHO Tech. Rep. Set. No. 366, pp. 30-32. Beutler, E. (1966) A series of new screening procedures for pyruvate kinase deficiency, glucose-6phosphate dehydrogenase deficiency and glutathione reductase deficiency, Blood, 28 : 553-562. Friede, R.L. and L.M. Fleming (1962) A mapping of oxidative enzymes in the human brain, J. Neurochem., 9: 179-198. Gobel, S. (1975) Golgi studies of the substantia gelatinosa neurons in the spinal trigeminal nucleus, J. comp. Neurol., 162: 397416. Hess, R., D. Scarpelli and A. G. E. Pearse (1958) The cytochemical localization of oxidative enzymes, J. Biophys. Biochem. Cytol., 4:747 760. Moore, B.W. and V.J. Perez (1968) Specific acidic proteins of the nervous system. In: F.D. Carlson (Ed.), Physiological and Biochemical Aspects of Nervous Integration, Prentice-Hall, Englewood Cliffs, N J, p. 343. Sakharov, D.A. (1974) The Genealogy of Neurons, Nauka, Moscow. Sakharova, A.V., N.B. Salimova and D.A. Sakharov (1979) Peculiar cells notable for very high activity of glucose-6-phosphate dehydrogenase in the mammalian medulla oblongata - - A histochemical and electron microscopic study, Neuroscience, 4:1173-1177. Sparkes, R.S., M.C. Baluda and D.E. Townsend (1969) Cellusose acetate electrophoresis of human glucose-6-phosphate dehydrogenase, J. Lab. clin. Med., 73: 531-534. Stefansson, K. and R. Wollmann (1981) Distribution of the neuronal specific protein, 14-3-2, in central nervous system lesions of tuberous sclerosis, Acta neuropath. (Berl.), 53:113-117. Sternberger, L.A. (1979)Immunocytochemistry, 2nd edition, J. Wiley and Sons, New York,'NY, pp. 104-130. Thomas, E. and A. G. E. Pearse (1961) The fine localization of dehydrogenases in the nervous system, Histochemie, 2: 266282.