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Postnatal development of calcitonin gene-related peptide (CGRP)-, substance P (SP)-, galanin (GAL)- and thyrotrophin releasing hormone (TRH)-like immunoreactivity in the rat lumbar spinal cord
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POSTNATAL DEVELOPMENTOF CALCITONIN GENE-RELATED PEPTIDE (CGRP)-, SUBSTANCE P (SP)-, GALANIN (GAL)- AND THYROTROPHIN RELEASING HORMONE(TRH)-LIKE IMMUNOREACTIVITY IN THE RAT LUMBARSPINAL CORD
*M. Koltzenburg, S.J. Gibson, *M. Fitzgerald, °S.R. Bloom and J.M. Polak. Departments of Histochemistry and °Medicine, Royal Postgraduate Medical School, London; *Department of Anatomy, University College London, London. In the rat major p h y s i o l o g i c a l , behavioural and neurochemical changes occur during the f i r s t three postnatal weeks. In p a r t i c u l a r , primary afferents mature, descending i n h i b i t o r y systems are established and there are changes in reflexogenic responses. We have investigated the d i s t r i b u t i o n of CGRP, SP, GAL and TRH in the lumbar spinal cord during the postnatal period. Rat pups ( I , 5, i 0 , 15, 18 and 24 days) and a group treated with capsaicin (or vehicle) at day 2 of l i f e and k i l l e d at day 18, were fixed by perfusion with benzoquinone (0.4%) or paraformaldehyde (4%) s o l u t i o n . Spinal cord sections were immunostained with antisera to CGRP, SP, GAL or TRH using the PAP method. The d i s t r i b u t i o n of SP and CGRP was s i m i l a r , except for the presence of CGRP-positive motoneurons. In the dorsal horn the number of fibres increased u n t i l day 15-20. At day I a few GAL-IR fibres were seen in the dorsal horn and dorsal columns. Numbers increased in the dorsal horn un t i l day 15. GAL-IR motoneurons and efferent axons were notably v i s i b l e at day 5 - i0 and became less apparent at l a t e r times. TRH was most abundant in fibres in the ventral horn at day 5; at day i0 a transient immunoreact i v i t y was localised to lamina I I . By day 18 the d i s t r i b u t i o n of a l l peptides was s i m i l a r to the adult. Capsaicin pretreatment induced a loss of CGRP-, SP- and GAL-IR fibres from laminae I and I I . The results demonstrate early organisation of peptidergic pathways, both i n t r i n s i c and e x t r i n s i c , in the spinal cord. RECEPTORS TO GOMBESIN-LIKE PEPTIDES DEHONSTRATED USING A DIVALENT LIGAND AND COLLOIDAL GOLD PROBE ELECTRON IHHUNOCYTOCHEMISTRY
P.~i.Lackie I , F . C u t t i t t a 3, D.R.Springall 1, J.D.Minna 3, S.R.Bloom 2 and J.M.Polak I Departments of iHistochemistry and 2Medicine, Royal Postgraduate Medical School, London WI20HS, U.K. and 3NCI-Navy Medical Oncology Branch, NCI-National Navy Medical Center, Bethesda, MD 20814, U.S.A. Growth factors have recently been implicated in tumour growth. Moreover, some regulatory peptides have been shown to exert growth-promoting actions, e.g. bombesin and i t s mammalian counterpart g a s t r i n - r e l e a s i n g peptide (GRP) influence the growth of small cell carcinoma cultures when applied exogenously. Bombesin/GRP is known to be produced by small cell carcinoma c e l l cultures and the peptide(s) can therefore be considered to ~ autotrophic. Indeed, bombesin/GRP receptors have been detected in cultures of small cell carcinoma ~ receptor-ligand assay. Divalent bombesin was synthesised and incubated with c e l l s derived from small cell carcinoma of the lung (SCCL; NCI-345 grown in RPMI 1640 + 10% foetal c a l f serum) f o r 30 min. The l a b e l l e d c e l l s were then f i x e d , pelleted and cryosectioned for electron microscopy. Sites of ligand binding were visualised on grid-mounted cryosections using a mouse monoclonal anti-bombesin immunoglobulin ( 2 A l l ; i : I 0 0 d i l u t i o n ) which was subsequently l a b e l l e d with goat anti-mouse IgG adsorbed to 5nm c o l l o i d a l gold. Appropriate controls including omission of divalent bombesin, preadsorption with monovalent bombesin etc. were carrfed out. Bombesin/GRP receptors were l o c a l i s e d on the surface of the SCCL c e l l s without capping. This novel method of receptor l o c a l i s a t i o n has considerable potential in both cell biological and pathological studies of receptor l o c a l i s a t i o n and turnover. I t is quite possible that receptor sites could be q u a n t i f i e d with s l i g h t modification to the basic procedure.
POSTNATAL DEVELOPMENTOF CALCITONIN GENE-RELATED PEPTIDE (CGRP)-, SUBSTANCE P (SP)-, GALANIN (GAL)- AND THYROTROPHIN RELEASING HORMONE(TRH)-LIKE IMMUNOREACTIVITY IN THE RAT LUMBARSPINAL CORD
*M. Koltzenburg, S.J. Gibson, *M. Fitzgerald, °S.R. Bloom and J.M. Polak. Departments of Histochemistry and °Medicine, Royal Postgraduate Medical School, London; *Department of Anatomy, University College London, London. In the rat major p h y s i o l o g i c a l , behavioural and neurochemical changes occur during the f i r s t three postnatal weeks. In p a r t i c u l a r , primary afferents mature, descending i n h i b i t o r y systems are established and there are changes in reflexogenic responses. We have investigated the d i s t r i b u t i o n of CGRP, SP, GAL and TRH in the lumbar spinal cord during the postnatal period. Rat pups ( I , 5, i 0 , 15, 18 and 24 days) and a group treated with capsaicin (or vehicle) at day 2 of l i f e and k i l l e d at day 18, were fixed by perfusion with benzoquinone (0.4%) or paraformaldehyde (4%) s o l u t i o n . Spinal cord sections were immunostained with antisera to CGRP, SP, GAL or TRH using the PAP method. The d i s t r i b u t i o n of SP and CGRP was s i m i l a r , except for the presence of CGRP-positive motoneurons. In the dorsal horn the number of fibres increased u n t i l day 15-20. At day I a few GAL-IR fibres were seen in the dorsal horn and dorsal columns. Numbers increased in the dorsal horn un t i l day 15. GAL-IR motoneurons and efferent axons were notably v i s i b l e at day 5 - i0 and became less apparent at l a t e r times. TRH was most abundant in fibres in the ventral horn at day 5; at day i0 a transient immunoreact i v i t y was localised to lamina I I . By day 18 the d i s t r i b u t i o n of a l l peptides was s i m i l a r to the adult. Capsaicin pretreatment induced a loss of CGRP-, SP- and GAL-IR fibres from laminae I and I I . The results demonstrate early organisation of peptidergic pathways, both i n t r i n s i c and e x t r i n s i c , in the spinal cord. RECEPTORS TO GOMBESIN-LIKE PEPTIDES DEHONSTRATED USING A DIVALENT LIGAND AND COLLOIDAL GOLD PROBE ELECTRON IHHUNOCYTOCHEMISTRY
P.~i.Lackie I , F . C u t t i t t a 3, D.R.Springall 1, J.D.Minna 3, S.R.Bloom 2 and J.M.Polak I Departments of iHistochemistry and 2Medicine, Royal Postgraduate Medical School, London WI20HS, U.K. and 3NCI-Navy Medical Oncology Branch, NCI-National Navy Medical Center, Bethesda, MD 20814, U.S.A. Growth factors have recently been implicated in tumour growth. Moreover, some regulatory peptides have been shown to exert growth-promoting actions, e.g. bombesin and i t s mammalian counterpart g a s t r i n - r e l e a s i n g peptide (GRP) influence the growth of small cell carcinoma cultures when applied exogenously. Bombesin/GRP is known to be produced by small cell carcinoma c e l l cultures and the peptide(s) can therefore be considered to ~ autotrophic. Indeed, bombesin/GRP receptors have been detected in cultures of small cell carcinoma ~ receptor-ligand assay. Divalent bombesin was synthesised and incubated with c e l l s derived from small cell carcinoma of the lung (SCCL; NCI-345 grown in RPMI 1640 + 10% foetal c a l f serum) f o r 30 min. The l a b e l l e d c e l l s were then f i x e d , pelleted and cryosectioned for electron microscopy. Sites of ligand binding were visualised on grid-mounted cryosections using a mouse monoclonal anti-bombesin immunoglobulin ( 2 A l l ; i : I 0 0 d i l u t i o n ) which was subsequently l a b e l l e d with goat anti-mouse IgG adsorbed to 5nm c o l l o i d a l gold. Appropriate controls including omission of divalent bombesin, preadsorption with monovalent bombesin etc. were carrfed out. Bombesin/GRP receptors were l o c a l i s e d on the surface of the SCCL c e l l s without capping. This novel method of receptor l o c a l i s a t i o n has considerable potential in both cell biological and pathological studies of receptor l o c a l i s a t i o n and turnover. I t is quite possible that receptor sites could be q u a n t i f i e d with s l i g h t modification to the basic procedure.