Neuroscienee Letters, 142 (1992) 1 l 1 114 ~"~ 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved I)304-3940/92/$ 05.00
111
NSL 08802
Distribution of 5-HT1 binding sites in cat spinal cord Lillian M. Pubols a~b'd, Nicola A. Bernau a, Lynn A. K a n e a, Susanne D. Dawsona, Anne L. Burleigh a'a and A r t h u r S. Polans a.c "R. S. Dow Neurological Sciences Institute, Portland, OR (USA), bDepartment of Neurosurgery and 'Department of Ophthalmology. Good Samaritan Hospital and Medical Center, Portland, OR (USA) and aDepartment of Physiology, Oregon Health Sciences University, Portland, OR ( USA ) (Received 8 April 1992; Revised version received 30 April 1992: Accepted 1 May 1992)
Key words." Serotonin; Spinal cord; Cat; Receptor binding autoradiography Quantitative analysis of high affinity [3H]5-HT binding to 5-HT~ receptors in the cervical, thoracic, lumbar, and sacral spinal cord of the cat revealed specific binding throughout the grey matter, with the highest levels of binding in laminae II and IlI, and the lowest levels in laminae I and VII. Relatively high levels were also observed in the thoracic intermediolateral cell column. There were no significant differences in the degree of binding between various segmental levels. Comparison of these data with published maps of 5-HT immunoreactivity reveals with the exception of lamina I - a close correspondence between the degree of immunoreactivity and the degree of 5-HT binding. These results suggest that 5-HT plays an important role in a variety of spinal cord sensory, motor and autonomic functions.
The serotonin (5-hydroxytryptamine: 5-HT) innervation of the spinal cord grey matter is a subject of considerable interest, particularly with regard to the role of 5HT in modulating nociception and spinal cord reflexes. Immunoreactivity to 5-HT is found throughout the spinal cord grey matter, with high concentrations in the superficial dorsal horn and area around the central canal (e.g. refs. 8, 9, 18, 22, 25). Much of this immunoreactivity is contained in the axons and terminals of pathways descending from the raphe nuclei [2]. The distribution of 5-HT receptors in the rat's spinal cord has been characterized in some detail (e.g. refs. 10, 13, 20). However, while the cat has been widely used for studies of the physiological effects of 5-HT in the spinal cord (e.g. refs. 1, 3, 4, 7, 11, 21, 23), much less is known about the distribution of 5-HT receptors in the cat's spinal cord. One previous autoradiographic study, using [3H]LSD to label both 5-HTI and 5-HT 2 receptors in the cat's lumbar spinal cord, found high levels of specific binding in the superficial dorsal horn and in the area around the central canal, with a lack of binding in the ventral horn [l 9]. The purpose of the present study was to provide an anatomically detailed and quantitative description of the distribution of 5-HT~ receptors in the dorsal and ventral Correspondence." L.M. Pubols, R.S. Dow Neurological Sciences Institute, 1120 N.W. 20th Avenue, Portland, OR 97209-1595, USA. Fax: (1) (503) 229-7229.
horns of the cat's spinal cord at a variety of segmental levels. In addition, we compared the observed pattern of binding with published data on the pattern of 5-HT innervation in the spinal cord. Subjects for these experiments were three adult female cats. The binding protocol was a modification of that used in a previous study of 5-HT receptors [19]. Animals were deeply anesthetized with sodium pentobarbital and transcardially perfused with 0.9% phosphate-buffered saline at 4°C, followed by 0.1% paraformaldehyde and 5% sucrose in 0.I M phosphate buffer, pH 7.4, at 4 ° C. For each cat, blocks of tissue were taken from five different levels of the spinal cord: C7 ~, T~ 9, L~, L~ and S~ > All tissue was immediately frozen, stored at -70°C, sectioned serially at 20 y m on a cryostat, and mounted on chrome-alum-gelatin coated slides. Three successive sections were mounted on each slide. Alternate slides were used for total and non-specific binding conditions. Binding sites were labeled using 2 nM [~H]5-HT creatinine sulphate (20 26 Ci/mmol, New England Nuclear). There is general agreement that 2 nM [3H]5-HT labels predominantly the 5-HT~ binding sites, with little labelling of 5-HT2, 5-HT3, or 5H-T uptake sites [13, 15, 19, 24]. Slides containing tissue sections were preincubated at 37°C for 10 min in 0.17 M Tris buffer, pH 7.7, containing 0.4 mM CaC12, 0.1% ascorbate, and 10/3M pargyline. Slides were further incubated in buffer at 22°C for 15 min. For slides used in evaluating nonspecific binding the second buffer also contained an excess of
tl2
Fig. I. A: dark-field autoradiogram of grains over one side o [ a lhoracic spinal cord section J¥om one cat. Incubation with 2 nM [;tf]5-1 [T i I,,mi binding), B: dark-field autoradiogram of an adjacent section incubated aith 2 nM [~H]5-HT and 10/JM 5-HI (non-specific binding). ( : brighl-ticid photomicrograph of a Kluver Barrera-stained section from (he same tis~,ue block. Arrows indicate the intermediolateral cell ~.ohnnn I hc cc))!)~! calla[ in A and ( contains India ink. Bar 200,am.
unlabelled 5-HT (10/zM). Slides were gently agitated for 1 hr at 22°C in buffer containing 2 nM [3H]5-HT, with or without unlabelled 5-HT (10/~M). The concentration of [3H]5-HT used is close to the Kd for the displacement of [3H]5-HT by 10/tM 5-HT (3.8 nM) (see ref. 14). Sections were rinsed in two changes (7 s and 2 min) of 4°C Tris buffer, pH 7.1, followed by a 10 sec rinse in 4°C deionized water. To determine that specific binding had occurred, some sections from each incubation were wiped from the slides into vials and counted in a liquid scintillation counter. Slides for autoradiography were allowed to dry at room temperature. Then the sections were tightly covered with an NTB3 emulsion- coated coverslip glued to the end of the slide, and placed in the dark at 4°C for 10 weeks. After development of the coverslips in D-19, the sections were postfixed in Carnoy's fixative and lightly stained with Safranin O. The coverslips were then mounted with Permount. Quantification of grain density was performed using the video counting program (VCTME) of a Bioquant image analysis system. A part (3400 #m 2) of each lamina, defined cytoarchitectonically [17], was viewed at a magnification of x640. The microscope was then focused on the overlying silver grains, and a grey levels threshold was set such that only the image of the grains was above the threshold. Grain density within the selected area was analyzed by counting the total number ofpixels occupied by grains. One sample per laminar locus was taken from the right side of each section. A minimum of 5 (mean of 11.9) sections per segmental level for both total and non-
specific conditions were analyzed tbr each animal. Plastic tritium standards (Amersham low range), sectioned at 20 /2m, were exposed and developed with the material from each cat. Grain density was linearly correlated (r > 0.99) with radioactivity of the standards. Densities for the experimental material fell within the range of the standards. Together with data provided by the manufacturer. the standards were used to convert pixel counts to fmol/ mg of tissue. Specific binding was defined as total minus non-specific binding. Binding to 5-HT receptors was found throughout the spinal cord grey matter, but the amount of binding varied significantly among the laminae. Fig. I shows an autoradiogram for total and non specific binding at the thoracic level, and a bright field photomicrograph of a stained section from the same tissue block. The mean specific binding for laminae ! X of all segments was 26.7 fmol/mg. In the dorsal horn, lamina I had a consistently low level of binding, while binding in laminae II and II1 was consistently above the mean (Fig. 2). In general, binding decreased dorsoventrally from lamina II to lamina VII. Binding in laminae Vl and VII was consistently below the mean. In the ventral horn higher levels of binding were found in laminae VIII-IX than in lamina VII, The intermediolateral cell column and Clarke's nucleus showed above average levels of binding. A repeated measures analysis of variance of the data shown in Fig. 2 for laminae common to all segments (l V and VII-X) revealed a significant effect of Lamina (/'=5.172. df~8/16, P0.05), or SegmentxLamina (F= 1.203, df=32/64, P>0.05), To probe the nature of the significant effect of Lamina, t-tests comparing the mean for lamina II with each of the other laminae were performed. These showed that binding in lamina II was significantly (P
111
NSL 08802
Distribution of 5-HT1 binding sites in cat spinal cord Lillian M. Pubols a~b'd, Nicola A. Bernau a, Lynn A. K a n e a, Susanne D. Dawsona, Anne L. Burleigh a'a and A r t h u r S. Polans a.c "R. S. Dow Neurological Sciences Institute, Portland, OR (USA), bDepartment of Neurosurgery and 'Department of Ophthalmology. Good Samaritan Hospital and Medical Center, Portland, OR (USA) and aDepartment of Physiology, Oregon Health Sciences University, Portland, OR ( USA ) (Received 8 April 1992; Revised version received 30 April 1992: Accepted 1 May 1992)
Key words." Serotonin; Spinal cord; Cat; Receptor binding autoradiography Quantitative analysis of high affinity [3H]5-HT binding to 5-HT~ receptors in the cervical, thoracic, lumbar, and sacral spinal cord of the cat revealed specific binding throughout the grey matter, with the highest levels of binding in laminae II and IlI, and the lowest levels in laminae I and VII. Relatively high levels were also observed in the thoracic intermediolateral cell column. There were no significant differences in the degree of binding between various segmental levels. Comparison of these data with published maps of 5-HT immunoreactivity reveals with the exception of lamina I - a close correspondence between the degree of immunoreactivity and the degree of 5-HT binding. These results suggest that 5-HT plays an important role in a variety of spinal cord sensory, motor and autonomic functions.
The serotonin (5-hydroxytryptamine: 5-HT) innervation of the spinal cord grey matter is a subject of considerable interest, particularly with regard to the role of 5HT in modulating nociception and spinal cord reflexes. Immunoreactivity to 5-HT is found throughout the spinal cord grey matter, with high concentrations in the superficial dorsal horn and area around the central canal (e.g. refs. 8, 9, 18, 22, 25). Much of this immunoreactivity is contained in the axons and terminals of pathways descending from the raphe nuclei [2]. The distribution of 5-HT receptors in the rat's spinal cord has been characterized in some detail (e.g. refs. 10, 13, 20). However, while the cat has been widely used for studies of the physiological effects of 5-HT in the spinal cord (e.g. refs. 1, 3, 4, 7, 11, 21, 23), much less is known about the distribution of 5-HT receptors in the cat's spinal cord. One previous autoradiographic study, using [3H]LSD to label both 5-HTI and 5-HT 2 receptors in the cat's lumbar spinal cord, found high levels of specific binding in the superficial dorsal horn and in the area around the central canal, with a lack of binding in the ventral horn [l 9]. The purpose of the present study was to provide an anatomically detailed and quantitative description of the distribution of 5-HT~ receptors in the dorsal and ventral Correspondence." L.M. Pubols, R.S. Dow Neurological Sciences Institute, 1120 N.W. 20th Avenue, Portland, OR 97209-1595, USA. Fax: (1) (503) 229-7229.
horns of the cat's spinal cord at a variety of segmental levels. In addition, we compared the observed pattern of binding with published data on the pattern of 5-HT innervation in the spinal cord. Subjects for these experiments were three adult female cats. The binding protocol was a modification of that used in a previous study of 5-HT receptors [19]. Animals were deeply anesthetized with sodium pentobarbital and transcardially perfused with 0.9% phosphate-buffered saline at 4°C, followed by 0.1% paraformaldehyde and 5% sucrose in 0.I M phosphate buffer, pH 7.4, at 4 ° C. For each cat, blocks of tissue were taken from five different levels of the spinal cord: C7 ~, T~ 9, L~, L~ and S~ > All tissue was immediately frozen, stored at -70°C, sectioned serially at 20 y m on a cryostat, and mounted on chrome-alum-gelatin coated slides. Three successive sections were mounted on each slide. Alternate slides were used for total and non-specific binding conditions. Binding sites were labeled using 2 nM [~H]5-HT creatinine sulphate (20 26 Ci/mmol, New England Nuclear). There is general agreement that 2 nM [3H]5-HT labels predominantly the 5-HT~ binding sites, with little labelling of 5-HT2, 5-HT3, or 5H-T uptake sites [13, 15, 19, 24]. Slides containing tissue sections were preincubated at 37°C for 10 min in 0.17 M Tris buffer, pH 7.7, containing 0.4 mM CaC12, 0.1% ascorbate, and 10/3M pargyline. Slides were further incubated in buffer at 22°C for 15 min. For slides used in evaluating nonspecific binding the second buffer also contained an excess of
tl2
Fig. I. A: dark-field autoradiogram of grains over one side o [ a lhoracic spinal cord section J¥om one cat. Incubation with 2 nM [;tf]5-1 [T i I,,mi binding), B: dark-field autoradiogram of an adjacent section incubated aith 2 nM [~H]5-HT and 10/JM 5-HI (non-specific binding). ( : brighl-ticid photomicrograph of a Kluver Barrera-stained section from (he same tis~,ue block. Arrows indicate the intermediolateral cell ~.ohnnn I hc cc))!)~! calla[ in A and ( contains India ink. Bar 200,am.
unlabelled 5-HT (10/zM). Slides were gently agitated for 1 hr at 22°C in buffer containing 2 nM [3H]5-HT, with or without unlabelled 5-HT (10/~M). The concentration of [3H]5-HT used is close to the Kd for the displacement of [3H]5-HT by 10/tM 5-HT (3.8 nM) (see ref. 14). Sections were rinsed in two changes (7 s and 2 min) of 4°C Tris buffer, pH 7.1, followed by a 10 sec rinse in 4°C deionized water. To determine that specific binding had occurred, some sections from each incubation were wiped from the slides into vials and counted in a liquid scintillation counter. Slides for autoradiography were allowed to dry at room temperature. Then the sections were tightly covered with an NTB3 emulsion- coated coverslip glued to the end of the slide, and placed in the dark at 4°C for 10 weeks. After development of the coverslips in D-19, the sections were postfixed in Carnoy's fixative and lightly stained with Safranin O. The coverslips were then mounted with Permount. Quantification of grain density was performed using the video counting program (VCTME) of a Bioquant image analysis system. A part (3400 #m 2) of each lamina, defined cytoarchitectonically [17], was viewed at a magnification of x640. The microscope was then focused on the overlying silver grains, and a grey levels threshold was set such that only the image of the grains was above the threshold. Grain density within the selected area was analyzed by counting the total number ofpixels occupied by grains. One sample per laminar locus was taken from the right side of each section. A minimum of 5 (mean of 11.9) sections per segmental level for both total and non-
specific conditions were analyzed tbr each animal. Plastic tritium standards (Amersham low range), sectioned at 20 /2m, were exposed and developed with the material from each cat. Grain density was linearly correlated (r > 0.99) with radioactivity of the standards. Densities for the experimental material fell within the range of the standards. Together with data provided by the manufacturer. the standards were used to convert pixel counts to fmol/ mg of tissue. Specific binding was defined as total minus non-specific binding. Binding to 5-HT receptors was found throughout the spinal cord grey matter, but the amount of binding varied significantly among the laminae. Fig. I shows an autoradiogram for total and non specific binding at the thoracic level, and a bright field photomicrograph of a stained section from the same tissue block. The mean specific binding for laminae ! X of all segments was 26.7 fmol/mg. In the dorsal horn, lamina I had a consistently low level of binding, while binding in laminae II and II1 was consistently above the mean (Fig. 2). In general, binding decreased dorsoventrally from lamina II to lamina VII. Binding in laminae Vl and VII was consistently below the mean. In the ventral horn higher levels of binding were found in laminae VIII-IX than in lamina VII, The intermediolateral cell column and Clarke's nucleus showed above average levels of binding. A repeated measures analysis of variance of the data shown in Fig. 2 for laminae common to all segments (l V and VII-X) revealed a significant effect of Lamina (/'=5.172. df~8/16, P0.05), or SegmentxLamina (F= 1.203, df=32/64, P>0.05), To probe the nature of the significant effect of Lamina, t-tests comparing the mean for lamina II with each of the other laminae were performed. These showed that binding in lamina II was significantly (P
