16
Neuro~'cience Letters, 1 I0 (1990) 16 21 Elsevier Scientitic Publishers Ireland Ltd.
NSL 06701
Substance P-like immunoreactivity in the human trigeminal ganglion M a r i n a Del Fiacco, M a r i n a Q u a r t u , Alessandra Floris and G i a c o m o Diaz Dipartimento di Citomorlblo~ia, University o! Cagliari, Ca~liari (Italy) (Received 26 July 1989: Revised version received 26 October 1989; Accepted 8 November 1989) Key word.w Substance P: lmmunoreactivity; Trigeminal ganglion; H u m a n Presence of substance P-like immunoreactive neurons and nerve fibers is demonstrated in the trigeminal ganglion of newborn and adult h u m a n subjects by the indirect immunofluorescence technique. Two populations of neurons containing high and low densities of immunoreactive material, respectively, are identilied. Morphometric analyses indicate that (i) most of positive neurons are medium and small sized; (ii) immunoreactive perikarya grow in size from newborns to adults, with up to a 50% increase in diameter. Percent frequency of positive perikarya, on the other hand, is higher in newborns (23.6%) and decreases in adults ( 16.7 %).
Neurons immunoreactive to the neuropeptide substance P (SP) have been shown in the trigeminal (gasserian) ganglion of several animal species [3, 6, 7, 8, 10, 17]. These cells are considered as primary sensory neurons mainly involved in neurotransmission of painful peripheral stimuli [9]. Although generally described as a fairly homogeneous population of small and medium sized neurons, species specific differences appear to exist in their dimensions as well as in their percent frequency (cf. refs. 7, 8, 17). The existence of SP-containing neurons in the human gasserian ganglion is suggested by data on the SP-positive innervation of the central and peripheral trigeminal territories, such as the sensory nucleus of the 5th cranial nerve [4], eye [16, 18], nasal mucosa [12] and cerebral blood vessels [5]. In this paper we give evidence for the presence of SP-like immunoreactive neurons in the human gasserian ganglion and provide morphometric and percent frequency estimates on such cell population. Because of the enhanced possibility of detecting neuronal somata by immunohistochemistry at early stages of ontogenesis [4], we examined tissues from both newborn and adult subjects. Specimens of gasserian ganglion were obtained at autopsy from premature and full-term newborns, who died within 5 days from birth, and from adult subjects, aged Corre~ondence: M. Del Fiacco, Dipartimento di Citomorfologia, University of Cagliari, via G.T. Porcell 2, 09124 Cagliari, Italy. 0304-3940/90/$ 03.50
1990 Elsevier Scientific Publishers Ireland Ltd.
17
Fig. 1. Trigeminal ganglion from premature newborn (25 weeks of gestation) (A), full-term newborn (B, C, E, F) and adult (D) subjects. A D: immunoreactive cell bodies of different size and fluorescence intensity. Short arrows indicate positive proximal processes; long arrows indicate beaded fibres; thick arrow indicates autofluorescent lipofuscins. E: thicker (long arrows) and thinner (short arrows) immunoreactive beaded fibers. F: bundle of beaded and non-beaded fibres of various caliber. A, B, D, E: x 260; C: x 265; F: x 175.
67-70 years. Postmortem delay before fixation ranged from 24 to 40 h. Fixation was performed by immersion in 4% buffered paraformaldehyde, pH 7.3, for 4 h at room temperature. Specimens were rinsed overnight in phosphate buffer 0.1 M, pH 7.3, containing 5-20% sucrose. Ten-~tm-thick cryostat sections collected on coated slides were processed by the indirect immunofluorescence technique of Coons et al. [1]. Incubation with sP monoclonal rat antibody, raised and characterized by Cuello et al. [2], diluted 1:300, was followed by incubation with fluorescein isothiocyanate (FITC)-conjugated anti-rat serum (Dakopatts), diluted 1:40. Specificity of the reac-
18 tion was checked by substituting the primary antibody with non-immune rat serum or with the diluted antibody pre-absorbed with 200 tag/ml of synthetic SP. Slides coverslipped with glycerol/phosphate buffer saline 3:1 (V/V) were observed in a Leitz Dialux 20 microscope, equipped with epifluorescence optics. Micrographs were taken on Ilford HP5 film (400 ASA) by means of a Vario Orthomat photographic apparatus. After observation and photography, slides were counterstained with Cresyl violet. Morphometric analysis was performed on line drawings of neuronal somata profiles traced from photographic prints. Taking as reference the occurrence of the nucleus, only sections of neurons cut through the central portion of the cell body were considered. The surface area of each neuron, calculated by a digitizing tablet connected to a microcomputer, was transformed into the corresponding diameter of a circle of equivalent area. Statistical parameters (mean, median, S.E.M., skewness, kurtosis) were estimated for diameters of different classes of neurons, recognized on the basis of the immunoreactivity pattern and subject age. The percent frequency of positive perikarya was calculated by the ratio of the total number of labelled cells found in different sections to the total number of cells found in the same sections counterstained with Cresyl violet. Both in specimens from newborn and adult subjects the specific immunoreaction revealed the presence of neuronal perikarya and varicose and non-varicose nerve fibres. Such immunoreactive structures were absent in control preparations. In specimens from adult subjects non-specifically autofluorescent lipofuscins were present in the tissue, often in large accumulations within neuronal somata. SP-like immunoreactive perikarya of various size were detected over the ganglion (Fig. 1A-D). The immunoreactive product appeared in form of granular fluorescent material distributed in the cytoplasmic compartment. Density of such granular product varied so that two classes of positive cell bodies were identified: low-density immunoreactive (LDI) neurons, in which distinct fluorescent granules were homogeneously sparse, and high-density immunoreactive (HDI) neurons, in which granules were so abundant and closely packed together as to give most of the cytoplasm a compact strong brilliancy. Thin filamentous or thicker tubular processes filled with immunoreactive material were frequently seen at their emergence from the perikaryon (Fig. I A,C,D). Strongly immunofluorescent beaded and non-beaded nerve fibers of different calibre were sparse among the cell bodies (Fig. 1A) and ran among the cell clusters either in little number (Fig. 1E), or grouped to tbrm thick bundles (Fig. 1F). Morphometric data are statistically summarized in Table I and graphically visualized in Fig. 2. The perikaryon size was found to be smaller in newborns than in adults. Such difference was statistically significant in both groups of neurons and particularly relevant in LDI cells, which showed a 50 % increase in diameter, corresponding to a volume growth of about 3.4 times, in adults compared to newborn subjects. The percent frequency of immunoreactive cells in adult subjects amounted to 16.7_+ 2 % (mean frequency + 95 % confidence limits), that is, a sixth of the neuronal population (253 positive of 1516 total cells found in 6 sections). In newborns, the
19
'°f 35
el,
uJ
t
30
IuJ
:E 0 ~r
o
25
i
m
u
w
u
15 Fig. 2. Diameters of perikarya classified according to the immunoreaction pattern. Paired points indicate the increase from newborn (11) to adult ( 0 ) for each cell type. Vertical bars represent standard errors. Distribution parameters are numerically summarized in Table I. LDI, low-density immunoreactive cells; HDI, high-density immunoreactive cells.
frequency was 23.6 + 3 %, corresponding to a 4th of the neuronal population (427 positive of 1806 total cells found in 10 sections). The difference between the frequency of positive cells in newborn and adult was statistically significant (corrected Chisquare 24.07 with 1 degree of freedom, P < < 0.0001). Similarly to what was observed in other animal species, the human trigeminal ganglion contains a population of SP-like immunoreactive neurons. Morphometric data indicate that most of them are small and medium size neurons. However, perikarya measuring up to 55 lam in diameter are also detectable in the adult tissue. Correspondingly, immunoreactive fibers of different caliber are present. In agreement with previous data obtained in rabbit [17], immunohistochemical results indicate that it is possible to discriminate the positive neurons on the basis of density of immunoreactive material in their perikaryon. Morphometric characterization confirms the qualitative distinction between LDI and HDI neurons. However, contrary to what was observed in rabbit, the diameter of LDI cells is significantly smaller than that of HDI cells, both in newborn and in adult. According to the positive correlation between cell body size and size of the peripheral territory innervated [13], both classes of immunoreactive neurons (LDI, HDI) show an increase from newborn to adult. Percent frequency of immunoreactive cells,
20 TABLE 1 DESCRIPTIVE STATISTICAL PARAMETERS OF THE DIAMETER OF HUMAN TRIGEMINAL GANGLION PERIKARYA CLASSIFIED ACCORDING TO DENSITY OF SP-LIKE IMMUNOREACTIVE MATERIAL AND SUBJECT AGE Values are expressed in gm. LDI, low-density immunoreactive cells: HDI, high-density immunoreactive cells. Parameters
LDI
HDI
LDI + HDI
Newborn Mean Median S.D. Standardized skewness Standardized kurtosis Number of cells
20.3 20.3 2.4 0.5 0.0 23
28.2 28.6 5.3 -0.3 (1.7 32
24.9 23.1 0.7 1.5 (].4 55
31.2 32.(I 5.4 - 0.2 0.3 21
34.0 34.9 7,6 - 0.0 1.3 24
32.7 32.9 6.8 0.4 1.5 45
Adult Mean Median S.D. Standardized skewness Standardized kurtosis Number of cells
o n the o t h e r h a n d , is h i g h e r in n e w b o r n t h a n in a d u l t subjects. T h e e n h a n c e d possibility o f d e t e c t i n g n e u r o n a l s o m a t a by i m m u n o h i s t o c h e m i s t r y
in j u v e n i l e life [4]
m i g h t a c c o u n t for such difference w i t h age; a l t e r n a t i v e l y , it m i g h t be d u e to g a n g l i o n cell d e a t h in o l d e r p e o p l e (see ref. 1 1). It has b e e n s u g g e s t e d t h a t s m a l l cells o f s e n s o r y g a n g l i a are specifically n o c i c e p t i v e (see ref. I !) a n d m o r p h o l o g i c a l a n d b e h a v i o r a l e v i d e n c e i n d i c a t e s t h a t SP acts in the n e u r o t r a n s m i s s i o n o f n o x i o u s stimuli in t h e t r i g e m i n a l s y s t e m [14]. H o w e v e r , c e n t r a l a n d p e r i p h e r a l d i s t r i b u t i o n o f S P - p o s i t i v e i n n e r v a t i o n suggests t h a t the p e p t i d e is i n v o l v e d in a v a r i e t y o f s e n s o r y m o d a l i t i e s [4, 9, 15]. T h e h e t e r o g e n e i t y o b s e r v e d in the S P - p o s i t i v e cell p o p u l a t i o n o f the t r i g e m i n a l g a n g l i o n m i g h t p e r h a p s reflect differences in f u n c t i o n a l i m p l i c a t i o n s o f the p e p t i d e in the h u m a n t r i g e m i n a l system.
1 Coons, A.H. and Kaplan, M.H., Localization of antigens in tissue cells. II. Improvements in a method f\~r the detection of antigen by means of fluorescent antibody, J. Exp. Med., 91 (1950) 1 9. 2 Cuello, A.C., Galfre, G. and Milstein, C., Detection of substance P in the central nervous system by a monoclonal antibody, Proc. Natl. Acad. Sci. U.S.A., 76 (19791 3532 3536. 3 Del Fiacco, M. and Cuello, A.C., Substance P- and enkephalin-containing neurones in the rat trigeminal system, Neuroscience, 5 (19801 803 815.
21 4 Del Fiacco, M., Dessi, M.L. and Levanti, M.C., Topographical localization of substance P in the human post-mortem brainstem. An immunohistochemical study in the newborn and adult tissue, Neuroscience, 12 (1984) 591~11. 5 Edvinsson L. and Uddman, R., Immunohistochemical localization and dilatatory effect of substance P on human cerebral vessels, Brain Res., 232 (1982) 466471. 6 H6kfelt, T., Kellerth, J.-O., Nilsson, G. and Pernow, B., Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons, Brain Res., 100 (1975a) 235-252. 7 H6kfelt, T., Kellerth, J.-O., Nilsson, G. and Pernow, B., Substance P: localization in the central nervous system and in some primary sensory neurons, Science, 190 (1975b) 889-890. 8 Kuwayama, Y., Terenghi, G., Polak, J.M., Trojanowski, J.Q. and Stone, R.A., A quantitative correlation of substance P-, calcitonin gene-related peptide- and cholecystokinin-like immunoreactivity with retrogradely labeled trigeminal ganglion cells innervating the eye, Brain Res., 405 (1987) 220-226. 9 Leeman, S.E. and Gamse, R., Substance P in sensory neurons. Trends Pharmac. Sci.,2(1981) 119 121. 10 Lehtosalo, J.I., Uusitalo H., Stjernschantz J. and Palkama A., Substance Pqike immunoreactivity in the trigeminal ganglion. A fluorescence, light and electron microscope study, Histochemistry, 80 (1984) 421-427. 11 Lieberman, A.R., Sensory ganglia. In D.N. Landon (Ed.), The Peripheral Nerve, Chapman and Hall, London, 1976, pp. 188 278. 12 Lundblad, L., Lundberg, J.M., Brodin, E. and Anggard, A., Capsaicin-sensitive substance P-immunoreactive afferent nerves in the nasal mucosa of various species. In P. Skrabanek and D. Powell (Eds.), Substance P, Boole, Dublin 1983, pp. 98-99. 13 Ohta, M., Offord, K. and Dyck, P.J., Morphometric evaluation of first sacral ganglia of man, J. Neurol. Sci, 22 (1974) 73 82. 14 Paxinos, G., O' Brien, M., Cuello, A.C. and Del Fiacco, M., Substance P in primary afferents and pain. In C. Peck and M. Wallace (Eds.), Problems in Pain, Pergamon, Sydney, 1980, pp. 64-72. 15 Pioro, E.P., Hughes, J.T. and Cuello, A.C., Demonstration of substance P immunoreactivity in the nucleus dorsalis of human spinal cord, Neurosci. Lett., 51 (1984) 61~55. 16 Stone, R.A. and Kuwayama, Y., Substance P-like immunoreactive nerves in the human eye, Arch, Ophthalmol., 103/8 (1985) 1207 1211. 17 Tervo, K., Tervo, T., Er/ink6, L., Erfink6, O. and Cuello, A.C., lmmunoreactivity for substance P in the Gasserian ganglion, ophthalmic nerve and anterior segment of the rabbit eye, Histochem. J., 13 (1981) 435-443. 18 Tervo, T., Tervo, K., Erfink6, L., Vannas, A., Erfink6, O. and Cuello, A.C., Substance P immunoreaction and acetylcholinesterase activity in the cornea and Gasserian ganglion, Ophtalm. Res., 15 (1983) 280 288.
Neuro~'cience Letters, 1 I0 (1990) 16 21 Elsevier Scientitic Publishers Ireland Ltd.
NSL 06701
Substance P-like immunoreactivity in the human trigeminal ganglion M a r i n a Del Fiacco, M a r i n a Q u a r t u , Alessandra Floris and G i a c o m o Diaz Dipartimento di Citomorlblo~ia, University o! Cagliari, Ca~liari (Italy) (Received 26 July 1989: Revised version received 26 October 1989; Accepted 8 November 1989) Key word.w Substance P: lmmunoreactivity; Trigeminal ganglion; H u m a n Presence of substance P-like immunoreactive neurons and nerve fibers is demonstrated in the trigeminal ganglion of newborn and adult h u m a n subjects by the indirect immunofluorescence technique. Two populations of neurons containing high and low densities of immunoreactive material, respectively, are identilied. Morphometric analyses indicate that (i) most of positive neurons are medium and small sized; (ii) immunoreactive perikarya grow in size from newborns to adults, with up to a 50% increase in diameter. Percent frequency of positive perikarya, on the other hand, is higher in newborns (23.6%) and decreases in adults ( 16.7 %).
Neurons immunoreactive to the neuropeptide substance P (SP) have been shown in the trigeminal (gasserian) ganglion of several animal species [3, 6, 7, 8, 10, 17]. These cells are considered as primary sensory neurons mainly involved in neurotransmission of painful peripheral stimuli [9]. Although generally described as a fairly homogeneous population of small and medium sized neurons, species specific differences appear to exist in their dimensions as well as in their percent frequency (cf. refs. 7, 8, 17). The existence of SP-containing neurons in the human gasserian ganglion is suggested by data on the SP-positive innervation of the central and peripheral trigeminal territories, such as the sensory nucleus of the 5th cranial nerve [4], eye [16, 18], nasal mucosa [12] and cerebral blood vessels [5]. In this paper we give evidence for the presence of SP-like immunoreactive neurons in the human gasserian ganglion and provide morphometric and percent frequency estimates on such cell population. Because of the enhanced possibility of detecting neuronal somata by immunohistochemistry at early stages of ontogenesis [4], we examined tissues from both newborn and adult subjects. Specimens of gasserian ganglion were obtained at autopsy from premature and full-term newborns, who died within 5 days from birth, and from adult subjects, aged Corre~ondence: M. Del Fiacco, Dipartimento di Citomorfologia, University of Cagliari, via G.T. Porcell 2, 09124 Cagliari, Italy. 0304-3940/90/$ 03.50
1990 Elsevier Scientific Publishers Ireland Ltd.
17
Fig. 1. Trigeminal ganglion from premature newborn (25 weeks of gestation) (A), full-term newborn (B, C, E, F) and adult (D) subjects. A D: immunoreactive cell bodies of different size and fluorescence intensity. Short arrows indicate positive proximal processes; long arrows indicate beaded fibres; thick arrow indicates autofluorescent lipofuscins. E: thicker (long arrows) and thinner (short arrows) immunoreactive beaded fibers. F: bundle of beaded and non-beaded fibres of various caliber. A, B, D, E: x 260; C: x 265; F: x 175.
67-70 years. Postmortem delay before fixation ranged from 24 to 40 h. Fixation was performed by immersion in 4% buffered paraformaldehyde, pH 7.3, for 4 h at room temperature. Specimens were rinsed overnight in phosphate buffer 0.1 M, pH 7.3, containing 5-20% sucrose. Ten-~tm-thick cryostat sections collected on coated slides were processed by the indirect immunofluorescence technique of Coons et al. [1]. Incubation with sP monoclonal rat antibody, raised and characterized by Cuello et al. [2], diluted 1:300, was followed by incubation with fluorescein isothiocyanate (FITC)-conjugated anti-rat serum (Dakopatts), diluted 1:40. Specificity of the reac-
18 tion was checked by substituting the primary antibody with non-immune rat serum or with the diluted antibody pre-absorbed with 200 tag/ml of synthetic SP. Slides coverslipped with glycerol/phosphate buffer saline 3:1 (V/V) were observed in a Leitz Dialux 20 microscope, equipped with epifluorescence optics. Micrographs were taken on Ilford HP5 film (400 ASA) by means of a Vario Orthomat photographic apparatus. After observation and photography, slides were counterstained with Cresyl violet. Morphometric analysis was performed on line drawings of neuronal somata profiles traced from photographic prints. Taking as reference the occurrence of the nucleus, only sections of neurons cut through the central portion of the cell body were considered. The surface area of each neuron, calculated by a digitizing tablet connected to a microcomputer, was transformed into the corresponding diameter of a circle of equivalent area. Statistical parameters (mean, median, S.E.M., skewness, kurtosis) were estimated for diameters of different classes of neurons, recognized on the basis of the immunoreactivity pattern and subject age. The percent frequency of positive perikarya was calculated by the ratio of the total number of labelled cells found in different sections to the total number of cells found in the same sections counterstained with Cresyl violet. Both in specimens from newborn and adult subjects the specific immunoreaction revealed the presence of neuronal perikarya and varicose and non-varicose nerve fibres. Such immunoreactive structures were absent in control preparations. In specimens from adult subjects non-specifically autofluorescent lipofuscins were present in the tissue, often in large accumulations within neuronal somata. SP-like immunoreactive perikarya of various size were detected over the ganglion (Fig. 1A-D). The immunoreactive product appeared in form of granular fluorescent material distributed in the cytoplasmic compartment. Density of such granular product varied so that two classes of positive cell bodies were identified: low-density immunoreactive (LDI) neurons, in which distinct fluorescent granules were homogeneously sparse, and high-density immunoreactive (HDI) neurons, in which granules were so abundant and closely packed together as to give most of the cytoplasm a compact strong brilliancy. Thin filamentous or thicker tubular processes filled with immunoreactive material were frequently seen at their emergence from the perikaryon (Fig. I A,C,D). Strongly immunofluorescent beaded and non-beaded nerve fibers of different calibre were sparse among the cell bodies (Fig. 1A) and ran among the cell clusters either in little number (Fig. 1E), or grouped to tbrm thick bundles (Fig. 1F). Morphometric data are statistically summarized in Table I and graphically visualized in Fig. 2. The perikaryon size was found to be smaller in newborns than in adults. Such difference was statistically significant in both groups of neurons and particularly relevant in LDI cells, which showed a 50 % increase in diameter, corresponding to a volume growth of about 3.4 times, in adults compared to newborn subjects. The percent frequency of immunoreactive cells in adult subjects amounted to 16.7_+ 2 % (mean frequency + 95 % confidence limits), that is, a sixth of the neuronal population (253 positive of 1516 total cells found in 6 sections). In newborns, the
19
'°f 35
el,
uJ
t
30
IuJ
:E 0 ~r
o
25
i
m
u
w
u
15 Fig. 2. Diameters of perikarya classified according to the immunoreaction pattern. Paired points indicate the increase from newborn (11) to adult ( 0 ) for each cell type. Vertical bars represent standard errors. Distribution parameters are numerically summarized in Table I. LDI, low-density immunoreactive cells; HDI, high-density immunoreactive cells.
frequency was 23.6 + 3 %, corresponding to a 4th of the neuronal population (427 positive of 1806 total cells found in 10 sections). The difference between the frequency of positive cells in newborn and adult was statistically significant (corrected Chisquare 24.07 with 1 degree of freedom, P < < 0.0001). Similarly to what was observed in other animal species, the human trigeminal ganglion contains a population of SP-like immunoreactive neurons. Morphometric data indicate that most of them are small and medium size neurons. However, perikarya measuring up to 55 lam in diameter are also detectable in the adult tissue. Correspondingly, immunoreactive fibers of different caliber are present. In agreement with previous data obtained in rabbit [17], immunohistochemical results indicate that it is possible to discriminate the positive neurons on the basis of density of immunoreactive material in their perikaryon. Morphometric characterization confirms the qualitative distinction between LDI and HDI neurons. However, contrary to what was observed in rabbit, the diameter of LDI cells is significantly smaller than that of HDI cells, both in newborn and in adult. According to the positive correlation between cell body size and size of the peripheral territory innervated [13], both classes of immunoreactive neurons (LDI, HDI) show an increase from newborn to adult. Percent frequency of immunoreactive cells,
20 TABLE 1 DESCRIPTIVE STATISTICAL PARAMETERS OF THE DIAMETER OF HUMAN TRIGEMINAL GANGLION PERIKARYA CLASSIFIED ACCORDING TO DENSITY OF SP-LIKE IMMUNOREACTIVE MATERIAL AND SUBJECT AGE Values are expressed in gm. LDI, low-density immunoreactive cells: HDI, high-density immunoreactive cells. Parameters
LDI
HDI
LDI + HDI
Newborn Mean Median S.D. Standardized skewness Standardized kurtosis Number of cells
20.3 20.3 2.4 0.5 0.0 23
28.2 28.6 5.3 -0.3 (1.7 32
24.9 23.1 0.7 1.5 (].4 55
31.2 32.(I 5.4 - 0.2 0.3 21
34.0 34.9 7,6 - 0.0 1.3 24
32.7 32.9 6.8 0.4 1.5 45
Adult Mean Median S.D. Standardized skewness Standardized kurtosis Number of cells
o n the o t h e r h a n d , is h i g h e r in n e w b o r n t h a n in a d u l t subjects. T h e e n h a n c e d possibility o f d e t e c t i n g n e u r o n a l s o m a t a by i m m u n o h i s t o c h e m i s t r y
in j u v e n i l e life [4]
m i g h t a c c o u n t for such difference w i t h age; a l t e r n a t i v e l y , it m i g h t be d u e to g a n g l i o n cell d e a t h in o l d e r p e o p l e (see ref. 1 1). It has b e e n s u g g e s t e d t h a t s m a l l cells o f s e n s o r y g a n g l i a are specifically n o c i c e p t i v e (see ref. I !) a n d m o r p h o l o g i c a l a n d b e h a v i o r a l e v i d e n c e i n d i c a t e s t h a t SP acts in the n e u r o t r a n s m i s s i o n o f n o x i o u s stimuli in t h e t r i g e m i n a l s y s t e m [14]. H o w e v e r , c e n t r a l a n d p e r i p h e r a l d i s t r i b u t i o n o f S P - p o s i t i v e i n n e r v a t i o n suggests t h a t the p e p t i d e is i n v o l v e d in a v a r i e t y o f s e n s o r y m o d a l i t i e s [4, 9, 15]. T h e h e t e r o g e n e i t y o b s e r v e d in the S P - p o s i t i v e cell p o p u l a t i o n o f the t r i g e m i n a l g a n g l i o n m i g h t p e r h a p s reflect differences in f u n c t i o n a l i m p l i c a t i o n s o f the p e p t i d e in the h u m a n t r i g e m i n a l system.
1 Coons, A.H. and Kaplan, M.H., Localization of antigens in tissue cells. II. Improvements in a method f\~r the detection of antigen by means of fluorescent antibody, J. Exp. Med., 91 (1950) 1 9. 2 Cuello, A.C., Galfre, G. and Milstein, C., Detection of substance P in the central nervous system by a monoclonal antibody, Proc. Natl. Acad. Sci. U.S.A., 76 (19791 3532 3536. 3 Del Fiacco, M. and Cuello, A.C., Substance P- and enkephalin-containing neurones in the rat trigeminal system, Neuroscience, 5 (19801 803 815.
21 4 Del Fiacco, M., Dessi, M.L. and Levanti, M.C., Topographical localization of substance P in the human post-mortem brainstem. An immunohistochemical study in the newborn and adult tissue, Neuroscience, 12 (1984) 591~11. 5 Edvinsson L. and Uddman, R., Immunohistochemical localization and dilatatory effect of substance P on human cerebral vessels, Brain Res., 232 (1982) 466471. 6 H6kfelt, T., Kellerth, J.-O., Nilsson, G. and Pernow, B., Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons, Brain Res., 100 (1975a) 235-252. 7 H6kfelt, T., Kellerth, J.-O., Nilsson, G. and Pernow, B., Substance P: localization in the central nervous system and in some primary sensory neurons, Science, 190 (1975b) 889-890. 8 Kuwayama, Y., Terenghi, G., Polak, J.M., Trojanowski, J.Q. and Stone, R.A., A quantitative correlation of substance P-, calcitonin gene-related peptide- and cholecystokinin-like immunoreactivity with retrogradely labeled trigeminal ganglion cells innervating the eye, Brain Res., 405 (1987) 220-226. 9 Leeman, S.E. and Gamse, R., Substance P in sensory neurons. Trends Pharmac. Sci.,2(1981) 119 121. 10 Lehtosalo, J.I., Uusitalo H., Stjernschantz J. and Palkama A., Substance Pqike immunoreactivity in the trigeminal ganglion. A fluorescence, light and electron microscope study, Histochemistry, 80 (1984) 421-427. 11 Lieberman, A.R., Sensory ganglia. In D.N. Landon (Ed.), The Peripheral Nerve, Chapman and Hall, London, 1976, pp. 188 278. 12 Lundblad, L., Lundberg, J.M., Brodin, E. and Anggard, A., Capsaicin-sensitive substance P-immunoreactive afferent nerves in the nasal mucosa of various species. In P. Skrabanek and D. Powell (Eds.), Substance P, Boole, Dublin 1983, pp. 98-99. 13 Ohta, M., Offord, K. and Dyck, P.J., Morphometric evaluation of first sacral ganglia of man, J. Neurol. Sci, 22 (1974) 73 82. 14 Paxinos, G., O' Brien, M., Cuello, A.C. and Del Fiacco, M., Substance P in primary afferents and pain. In C. Peck and M. Wallace (Eds.), Problems in Pain, Pergamon, Sydney, 1980, pp. 64-72. 15 Pioro, E.P., Hughes, J.T. and Cuello, A.C., Demonstration of substance P immunoreactivity in the nucleus dorsalis of human spinal cord, Neurosci. Lett., 51 (1984) 61~55. 16 Stone, R.A. and Kuwayama, Y., Substance P-like immunoreactive nerves in the human eye, Arch, Ophthalmol., 103/8 (1985) 1207 1211. 17 Tervo, K., Tervo, T., Er/ink6, L., Erfink6, O. and Cuello, A.C., lmmunoreactivity for substance P in the Gasserian ganglion, ophthalmic nerve and anterior segment of the rabbit eye, Histochem. J., 13 (1981) 435-443. 18 Tervo, T., Tervo, K., Erfink6, L., Vannas, A., Erfink6, O. and Cuello, A.C., Substance P immunoreaction and acetylcholinesterase activity in the cornea and Gasserian ganglion, Ophtalm. Res., 15 (1983) 280 288.
