Neuroscience Vol. 6. No. 4, pp. 713 to 723, 1981

Printed in Great Britain

03064522/81/040713-1 ISO2.00/0

Pergamon Press Ltd

0 1981 IBRO

HUMAN SPINAL NEURONS: INNERVATION BY BOTH SUBSTANCE P AND ENKEPHALIN

CAROLE C. LAMOTTE and NIHAL C. DE LANEROLLE* Section of Neuroanatomy and Section of Neurosurgery* Yale University School

of Medicine, New Haven, Connecticut, U.S.A.

Abstract-The distribution of the peptides substance P and methionine-enkephalin in relation to neurons in the human thoracic and lumbar spinal cord has been studied with the unlabeled peroxidase anti-peroxidase method employing antibodies directed against substance P and methionine-enkephalin. The peptides have been localized in varicosities and terminal-like processes in close apposition to the soma and proximal dendrites of neurons in the marginal zone, the intermediolateral nucleus, intermedio- medial nucleus, the reticular nucleus of lamina V and the ventral horn; some cells in all these regions are innervated by both peptides.

The findings provide morphological evidence of these peptides in the human spinal cord. The demon- stration of innervation by both peptides of single neurons in laminae I and V provides some morpho- logical basis to models that postulate the interaction of substance P and enkephalin in pain modulation. The innervation of other neuron types indicates that these neuropeptides may be involved in other spinal functions as well.

SEVERAL studies have reported the immunohistoche-

mica1 localization of substance P (NIL~s~N, H&FELT & PERNOW, 1974; H~KFELT, KELLERTH, NIL%G~N & PERNOW, 1975; PICKEL, REIS & LEEMAN, 1977; CHAN-

PALAY & PALAY, 1977; CUELLO & KANAZAWA, 1978; and BARBER, VAUGHN, SLEMMON, SALVATRRA, ROBERTS & LEEMAN, 1979) and met-enkephalin (ELDE, H~KFELT, JOHAN~Z~~N & TERENIUS 1976; SIMANTOV, KUHAR, UHL & SNYDER, 1977; WATSON, AKIL, SULLI- VAN & BARCHAS, 1977; H&FELT, ELDE, JOHANSSON, TERENIUS & STEIN, 1977; and SAR, STUMPF, MILLER, CHANG & CUATRECASAS, 1978) in axons and terminals of the rat spinal cord. Both of these neuropeptides

were found in high concentration in the dorsal horn, especially the marginal zone and the substantia gelati- nosa. Lighter staining occurred in the intermediate

and ventral grey matter. Attention has been mainly focused on the possible

roles of these peptides in spinal mechanisms of pain transmission. There is strong evidence that at least some of the substance P in the spinal grey matter is associated with primary afferents, particularly noci- ceptive afferents (TAKAHASHI & OTSUKA, 1974; H~K- FELT, ELDE, JOHANSSON, LUFT, NIL.%S~N & ARIMURA,

1976). Further, substance P has been shown to poten- tiate the excitation of nociceptive neurons evoked by cutaneous afferent stimulation (HENRY, 1976; RANDIC 8~ MILETIC, 1977; SASTRY, 1979a). In contrast, enke- phalin has not been identified in primary afferents; however, enkephalin-positive cell bodies have been found in the rat spinal cord (H~KFELT er al., 1977), and the distribution of enkephahn correlates closely with that of opiate receptors (LAMOTTE, PERT & SNYDER, 1976; ATWEH & KUHAR, 1977). Met-enke-

Abbreviations: DAB, diaminobenzidine; PBS, phosphate- buffered saline

phahn and morphine have been shown to potentiate presynaptic inhibition of primary afferents (SASTRY, 19796), inhibit the release of substance P from primary afferents (JESSEL & IVERSEN, 1977), and reduce

the responses of dorsal horn neurons to painful stimu- lation of their receptive fields (DUGGAN, HALL & HEADLEY, 1976; MILETIC, KOVACS & RANDIC, 1977).

As part of an investigation of the location of these substances in the human spinal cord and their interre-

lationships, we report the apparent dual innervation

of certain types of spinal neurons by both substance P and enkephahn terminals. Some of these results have been described in a preliminary communication (DELANEROLLE & LAMOTTE, 1980).

EXPERIMENTAL PROCEDURES

Human thoracic and lumbar spinal cord from 3 male subjects was obtained at autopsy (4-12 h post-mortem), and fixed by immersion in picric acid-paraformaldehyde in phosphate buffer solution (STEFANINI, MARTINO & ZAM- BONI, 1967) for 24 h. This fixative was chosen as it pen- etrated the immersed tissue more rapidly, and conse- quently fixed autopsy tissue better than the fixatives more commonly used for immunohistochemistry, i.e., 4% para- formaldehyde or 4% paraformaldehyde-glutaraldehyde mixtures. As a result of the better preservation of morphol- ogy, the distribution of the peptides could be localized better. After fixation, the tissue was washed in several changes of a phosphate buffer (KARLWN & SCHULTZ, 1965),

and finally in 70% ethyl alcohol to remove any remaining traces of picric acid. It was then dehydrated and embedded in paraffin wax. Ten micron serial sections were made in transverse, sagittal and horizontal planes. Consecutive sections were alternately stained for substance P or met- enkephahn. These methods of fixation, embedding and staining were selected following a comparison of trials using several other methods, including cryostat sectioning, wax embedding and immunofluorescence. Although tissue

713

714 CAKOLE C. LAMOTT and NIHAL C. DE LANEROLLE

preservation in human autopsy material is not comparable

to that of perfused experimental animals. our methods

yielded considerable tissue and peptide preservation.

The sections were treated with xylene to remove paraffin,

and then incubated in the following solutions: phosphate-

butTered saline (PBS), pH 7.2 (15-20 min); 3”: normal goat

serum in PBS containing 0.3:<, Triton X-100 (30 min): rab-

bit antimet-enkephalin or antisubstance P (Immunonuc-

lear, Stillwater. Minnesota) in PBS containing I’?; normal

goat serum and 0.3”,; Triton X-100. for 48 h at 4-C; Goat

anti-rabbit serum (Cappell Laboratories, I :20 in PBS for

30 min at room temperature); peroxidase-anti-peroxidase

complex (Accurate Chemical & Scientific Corporation,

New York) I: 100 for 30 min at room temperature;

diaminobenzidine hydrochloride (DAB. Sigma) in 0.05 M

Tris buffer, pH 7.6 (0.05”‘,) for l5-30min: DAB in am-

monium acetate buffer pH 5.5 (VAC(.A. HEWETT & Woon-

sots, 1978) and O.Ol”,, hydrogen peroxide for 4 min. The final reaction was stopped by rinsing with distilled water. Sections were counter-stained with cresyl violet and mounted with permount under a coverslip.

The sections were rinsed in two changes of PBS for 5 min each between each incubation except after the 3”; nor- mal goat serum; the latter was merely removed by suction. Rabbit anti met-enkephalin and anti substance P were used in dilutions varying from I ; 500 to I : 2ooO. A dilution

of 1:800 gave optimal results for both antibodies. Pre-incu- bation with Tris-DAB followed by reaction with am- monium acetate-DAB containing the hydrogen peroxide was found to give the best immunospecific staining with the least background stain.

As controls for antibody specificity. adjacent sections were stained as above but instead of the primary antibody we used (a) primary antibody pre-absorbed with the specific antigen (substance P or met-enkephalin, 50 pg to I mg peg tide per I ml antibody solution I :800), or (b) antibody pre- treated with pcptides other than the one under study (somatostatin. neurotensin. and met-enkephalin or sub- stance P for substance P and met-enkephalin respectively, I mg peptide per I ml of antibody I:800 dilution). As a tissue control. the primary antibody was replaced by PBS with I”; normal goat serum containing 0.3”,, Triton X-100 (the vehicle for antibody dilution).

RESULTS

Serial sections, alternately stained for substance P and met-enkephaiin, revealed single cells whose soma and proximal dendrites were surrounded by both sub stance P and met-enkephalin-like immunoreactivity localized in varicosities and terminal-like structures. (Fig. 1). Definite identification of dual labelled cells could be made in the marginal zone, the intermediola- teral nucleus, the intermediomedial nucleus, the re- ticular nucleus of lamina V and the ventral horn. Since these cell types are among the largest of the spinal cord, sequential identification of one cell over several serial sections was possible. Smaller cell classes, such as those in the substantia gelatinosa, were difficult to identify positively from section to section, although terminal labelling in the neuropil was heavy in that region. Thus, no conclusion about possible dual innervation of other spinal cord cell classes can presently be drawn.

We conclude that the immunoreaction observed 01, the above cells was specific for substance P and tnet-

enkephalin based on the results of control studies (Figs 2A--C). Anti-serum to substance P pre-absorbed with enkephalin, neurotensin and somatostatin yielded a pattern of staining exactly similar to thar with only anti-substance P. Comparable controls for antiserum to met-enkephalin also yielded the same staining as for anti met-enkephalin alone. Secondly. the application of anti-substance P pre-absorbed with

substance P, and anti met-enkephalin pre-absorbed with metenkephalin did not produce staining for sub-

stance P or met-enkaphalin. respectively. Finally. when either primary antibody was omitted no stain- ing was observed.

Large marginal neurons, particularly those located in the surrounding white matter of the dorsal columns or the lateral funiculus received both types of termin- als (Figs 2&G and 3). Usually, two to three cells per 10 pm section were found to be doubly labelled: man) other cells in the marginal zone received only one or the other type of innervation. In the intcrmediolateral nucleus, both soma and dendrites of large and small preganglionic sympathetic neurons were nested in ter- minals of both substance P and enkephalin; most cells received a dense innervation by both types of terminals (Figs 4A, B). In each section. at least one small cell of the intermediomedial nucleus was seen to receive a moderate to high density of both substance

P and enkephalin along their soma and dendrites

(Figs 4C, D). Terminals and fibers were moderately dense in the reticular nucleus of lamina V and it was common to find one to three large cells clearly out- lined with both substance P and met-enkephalin ter- minals in any 2 adjacent sections (Figs 5A -D). Many large and medium sized cells of the ventral horn received substance P terminals; fewer received enke- phalin terminals (Figs SE-H). Doubly innervated motoneurons were less common than the other cell classes described above. (Differentiation of the cells as motoneurons or intemeurons could not be definitive without an additional labelling technique to specifi- cally identify motoneurons, such as retrograde filling of the cells with horseradish peroxidase.)

DISCUSSION

This study demonstrates the presence of substance P and met-enkephalin-like immunoreatiivity in ter- minals and axons of the human spinal cord; the close apposition of these types of terminals to marginal, reticular, sympathetic preganglionic, intermediome- dial and ventral horn neurons; and the apparent dual innervation of some of the cells in each class by both types of terminals.

CUELLQ POUK & F’EARSE. (1976) have reported a similar overall distribution of substance P-positive axons in human spinal cord; varicosities were seen around motoneurons, although other cell types were not described. Previous studies in rat spinal cord have

Neuro~ptides in human spinal cord

FIG. 1. Photomicrographs of adjacent transverse sections through the dorsal horn of the lumbar region of the human spinal cord. Substance P-like immunoreactivity (A) and met-enkephalin-like immunoreao tivity (B) identified by the PAP reaction product appears as the darker structures, and are particularly dense.in Laminae I and II. Parts of the same marginal neuron (solid arrow-head) and Lamina V neuron (open arrow-head) are seen in both sections outlined by terminal-like structures immunopositive for

both substance P and met-enkephalin. Magnification x 70

FIG. 2. A-C: Photomicrographs of transverse serial sections through a marginal neuron !ocated along lateral border of Lamina I. A: Section incubated according to described methods in antisera to sub- stance P. Reaction product occurs around neuron soma and dendrites and fibres and terminals also seen in laminae I and iI. 8: Control section in which substance P antisera is pre-absorbed with met- enkephalin, neurotensin and somatostatin. Specificity of the immunoreaction is indicated by staining equal to that in figure A. C: Section incubated in substance P antisera pre-absorbed with substance P. Specificity of antisera is indicated by total absence of staining. The small round structures appearing as black dots are nuclei of glia. I&G: Photomicrographs of four transverse serial sections of another marginal neuron in lateral lamina I. Sections D and F were stained with met-enkephalin antisera. Sections E and G were stained with substance P antisera, The soma and proximal dendrites are outlined

by immunor~ctive structures. Ma~ifications x 427.5

FIG. 3. Photomicrographs of sagittal serial sections through two marginal neurons (cell 1 :A and B: cell 2: C and D). outlined with substance P (A&C) and met-enkephalin (B&D) terminal-like staining

Magnification x 427.5

FIG. 4. A and B: Photomicrograph of adjacent sagittal sections through the same cluster of intermdio- lateral neurons reacted for substance P (A) and met-enkephalin (B). C and D: Adjacent sagittai sections through an inte~ediom~ial neuron, showing substance P-staining around soma and dendrites (C) and

met-enkephalin-staining around dendrite (D). Magnification x 665

FIG. 5. A-D: Transverse serial sections through neuron found in reticular nucleus of lamina V. Soma and long dendrites are outlined in A and C by met-enkephalin terminals and B and D by substance P terminals. E-H: Transverse serial sections through large neuron (motoneuron?) found in ventral horn, heavily outlined by substance P terminals in E and G, and lightly outlined by met-enkephalin terminals

in F and H. Magnification x 427.5

716

718

Neuro~ptides in human spinal cord 721

observed the proximity of substance P terminals to marginal cells (BARBER et al, 1979; H~KFELT et al, 1975), to preganglionic sympathetic neurons (H&- FELT et al., 1975) and to motoneurons (BARBER et al., 1979). BARBER et al., 1979, have also shown electron micrographs of substance P-labelled terminals in con- tact with rat marginal neuron proximal dendrites. Another autonomic preganglionic nucleus, the dorsal motor nucleus of the vagus, is heavily innervated by substance P in the rat (KATZ & KARTEN, 1979). The origins or physiolo~cal significance of the innervation by substance P and enkephalin of cells with widely different functional roles is unknown.

The source of the substance P axons in the spinal cord is not exclusively from primary afferents. LUNG DAHL, H&FELT & NILS~ON (1978) have identified sub- stance P-positive cells in laminae III, IV and V and the sympathetic preganglionic nucleus (inte~~iola- teral neurons) in the rat cord, and TESSLER, ARTY- MYSHYN, MURRAY & GOLDBERGER (1979) reported positive cells in laminae VII, VIII, and X in the cat lumbar cord. Further, enkephalin-positive cells have been found in the substantia gelatinosa of the trigem- inal nucleus of the cat (Urn, GOODMAN, KUHAR, CHILDERS & SNYDER, 1979) and in the marginal zone, laminae IV and V, the sacral parasympathetic nu- cleus, and medial to the central canal in the cat sacral cord (GLAZER & BASBAUM, 1979). In all the studies on animals referred to above, peptide-containing cell- bodies were visualized only in tissue from animals pretreated with colchicine prior to being killed for immunohistochemistry. Such manipulations are unac- ceptable in humans, and thus the existence or absence of substance P or met-enkephalin-like immunoreac- tive cell-bodies in the human spinal cord cannot be verified. However, the existence of such cell-bodies in a number of mammalian species makes it probable that they occur in the human too. It is, then, particu- larly interesting that some marginal neurons in the cat stain positive for enkephalin (GLASER & BASBAUM, 1979) and preganglionic sympathetic neurons in the rat stain positive for substance P (LJUNGDAHL et al., 1978) and that our results indicate these cell classes commonly receive both substance P and enkephalin terminals in man. Thus, it appears possible that cells producing a given peptide may also be innervated by other cells using the same or other peptides.

Electron microscope identification of substance P and met-enkepha~n-la~lled profiles is in progress to determine if they may establish direct synaptic con- tact onto these cell classes or if some of the contacts are indirect, e.g., a terminal or varicosity may form axoaxonic contacts with other terminals impinging on the cell. Similar materiaf from monkey and cat spinal

cords have been examined with the electron micro- scope in our laboratory and the positive-staining material located in unmyelinated axons and terminal- like profiles. BARBER et al. (1979) have reported axo- dendritic and axosomatic substance P contacts with cells in the marginal zone and the substantia gelati- nosa in the rat spinal cord, and also substance P ter- minals that were presynaptic or postsynaptic to other axon terminals. Thus, several forms of contact may exist for both substance P and enkephalin terminals, althou~ no enkephalin terminals in the spinal cord have been demonstrated with electron microscopy. Future observations can be tested against models of synaptic interrelationships in the spinal cord. For example, ZIEGLGANSBERGER & TULLOCH (1979) and JE~%L & IVERSEN (1977), have proposed circuits in the dorsal horn in which substance P terminals directly contact the dendrites of spinothalamic cells located in the marginal zone and lamina V. They also suggest local enkephalin neurons could impinge directly on these same cells or form axoaxonic contacts on the substance P terminals. This model has been suggested to account for the facilitatory action of substance P and the ‘presynaptic’ inhibitory action of enkepha~ on the excitability of dorsal horn neurons that occurs with iontophoresis of these peptides. However the existence of presynaptic enkephalinergic axoaxonic contacts has yet to have strong anatomical support (see also, HUNT, KELLY & EMISON, 1980; Ed.) Recently PICKEL, SUMAL, BECKLEY, MILLER & REIS (1980) have found the majority of enkephalin-positive terminals in the rat neostriatum to make axodendritic contacts, rather than axoaxonic. Further, as discussed by the author in an earlier paper (LAMOTTE et al, 1976) the loss of dorsal horn opiate receptors following rhizo- tomy could occur from either degeneration of recep- tor sites on the primary afferents or trans-synaptic degeneration of cell receptor sites postsynaptic to the primary afferents. Thus, either axoaxonic or axoden- dritic positions would be possible for enkephalinergic terminals in the dorsal horn. Only definitive electron microscopic studies can settle this point. Further, the dual innervation observed on several cell types in human cord indicates a variety of interactions of these terminal types may also be in effect in other spinal nuclei.

~c~ow~e~geme~f~-We are very grateful to Mr M. WADE (Chief Technician, Neuropathological Services, Yale Uni- versity) for providing us with the human spinal cords, to Mrs DOLORES MONTOYA for expert technical assistance, and to Dr R. P. ELDE for critical reading of this manu- script. The work was supported by NIH grant NS13335.

722 CAR~LE C. LAMOTTE and NIHAL C. DE LANEROL.I.F

REFERENCES

ATWEH S. F. & KUHAR M. J. (1977) Autoradiographic localization of opiate receptors in rat brain. 1. Spinal cord and medulla. Brain Res. 124, 53-67.

BARBER R. P., VAUGHN J. E., SLEMMON J. R., SALVATRRA P. M., ROBERTS E. & LEEMAN S. E. (1979) The origin, distribution and synaptic relationships of substance P axons in rat spinal cord. J. camp. Neural. 184, 331-352.

CHAN-PALAY V. & PALAY S. L. (1977) Uitrastructural identification of substance P cells and their processes in rat sensory ganglia and their terminals in the spinal cord by immunocytochemistry. Proc. natn. Acad. Sci. U.S.A. 74, 405@5054.

CUELLO A. C. & KANAZAWA I. (1978) The distribution of substance P immunoreactive fibers in the rat central neurons system J. camp. Neural. 178, 129-156.

CUELLO A. C., POLAK J. & PEARSE A. G. E. (1976) Substance P: a naturally occurring transmitter in human spinal cord. Lancet 2, 1054-1056.

DE LANEROLLE N. & LAMO~E C. (1980) Distribution of Substance P and met-enkephalin in human and monkey spinal cord Anat. Rec. l%, 44A.

DUGGAN A. W., HALL J. G. & HEADLEY P. M. (1976) Morphine, enkephalin and the substantia gelatinosa. Nature, Land. 264, 456458.

ELDE R., HGKFELT T., JOHJ\NSSON 0. & TERENIUS L. (1976) Immunohistochemical studies using antibodies to leucine- enkephalin: initial observations on the nervous system of the rat. Neuroscience 1, 349-355.

GLAZER E. J. & BASBAUM A. I. (1979) Enkephahn perikarya in the marginal layer and sacral autonomic nucleus of the cat spinal cord. Sot. for Neurosci. Abst. 5, 2434.

HENRY J. L. (1976) Effects of substance P on functionally identified units in cat spinal cord. Brain Res. 114, 439451. H~~KFELT T., ELDE R., JOHANSWN O., LUFT R., N~LSWN G. & ARIMURA A. (1976) Immunohistochemical evidence for

separate populations of somatostatin-containing and substance P-containing primary afferent neurons in the rat. Neuroscience 1, 131-136.

H~~KFELT T., ELDE R., JOHANWN O., TERENIUS L. & STEIN L. (1977) The distribution of enkephalin immunoreactive cell bodies in the rat central nervous system. Neuroscience Letters 5, 25-31.

H~~KFELT T., KELLERTH J. 0.. NILSSON G. & PERNOW B. (1975) Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons. Brain Res. 100, 235-252.

HUNT S. P., KELLY J. S. & EMSON P. C. (1980) The electron microscopic localization of methionine-enkephalin within the superficial layers (I & II) of the spinal cord. Neuroscience 5, 1871-1890.

JESSEL T. M. & IVERSEN L. L. (1977) Opiate analgesics inhibit substance P release from rat trigeminal nucleus. Nature Land. 268, 549-551.

KARLSSON V. & SCHULTZ R. L. (1965) Fixation of the central nervous system for electron microscopy by aldehyde perfusion. I. Preservation with aldehyde perfusates versus direct perfusion with osmium tetroxide with special reference to membranes and extracellular space. J. Ultrastruct. Res. 12, 16&186.

KATZ D. M. & KARTEN H. J. (1979) Substance P-like immunoreactivity (SPLI) and target-organ representation within the nucleus solitarius (nS) and vagal motor nucleus (DMNX). Socfor Neurosci. Abst. 5, 531.

LAMO~TE C., PERT C. B. & SNYDER S. H. (1976) Opiate receptor binding in primate spinal cord: distribution and changes after dorsal root section. Brain Res. 112, 407-412.

LJUNGDAHL A., HGKFEL? T. & NILSSON G. (1978) Distribution of substance P-like immunoreactivity in the central nervous system of the rat. I. Cell bodies and nerve terminals. Neuroscience 3, 861-943.

MILETIC V., KOVACS M. S. & RANDIC M. (1977) Actions of somatostatin and methionine-enkephalin on cat dorsal horn neurons activated by noxious stimuli. Sot. for Neurosci. Abst. 3, 488.

NILSSON G., H~~KFELT T. & PERNOW B. (1974) Distribution of substance P-like immunoreactivity in the rat central nervous system as revealed by immunohistochemistry. Med. Biol. 52, 424-427.

PICKEL V. M., REIS D. J. & LEEMAN S. E. (1977) Ultrastructural localization of substance P in neurons of rat spinal cord. Brain Res. 122. 534-540.

P~CKEL V. M., SUMAL K. K., BECKLEY S. C., MILLER R. J. & Rus D. J. (1980) Immunocytochemical localization of enkephalin in the neostriatum of rat brain: a light and electron microscopic study. J. Camp. Neural: 189, 721-740.

RANDIC M. & MILE~C V. (1977) Effect of substance P in cat dorsal horn neurones activated by noxious stimuli. Brain Res.

128, 164-169. SAR M., STUMPF W. E., MILLER R. J., CHANG K. J. & CUATRECASAS P. (1978) Immunohistochemical IocalizatioI: of

enkephalin in rat brain and spinal cord. J. cotnp. Neural. 182, 17-38. SASTRY B. R. (1979a) Substance P effects on spinal nociceptive neurons. Life Sci. 24, 2169-2177. SASTRY B. R. (1979b) Presynaptic effects of morphine and methionine-enkephalin in feline spinal cord Neuropharmacoloyy

18, 367-375. SIMANTOV R., KUHAR M., UHL G. & SNVDER S. (1977) O@oid peptide enkephalin: immunohistochemicat mapping in the

rat central nervous system. Proc. Natn. Acad. Sci. (Wash.) 74; 2167-2171. S~FANINI M., DEMARTINO C. & ZAMBONI L. (1967) Fixation of ejaculated spermatozoa for electron microscopy. Nature

Land. 216, 173-I 74. TAKAHASHI T. & OTSUKA M. (1974) Distribution of substance P in cat spinal cord and the alteration following unilateral

dorsal root section. Jap. J. Pharmacol. 24, Suppl. 105. TESSLER A., ARTYMYSHYN R., MURRAY M. & GOLDBERGER M. (1979) Interneurons as possible source for the return of

substance P (SP) after lumbosacral detierentation. Soc.for Neurosci. Abst. 5, 636. UHL G. R., GCXJDMAN R. R., KUHAR M. J., CHILDERS S. R. & SNYDER S. H. (1979) Immunohistochemical mapping of

enkephalin containing cell bodies,

Neuropeptides in human spinal cord 723

VACCA L. L., HEWETT D. & WOODSON G. (1978) A comparison of methods using diaminobenzidine (DAB) to localize peroxidases in erythrocytes, neutrophils, and peroxidase-antiperoxidase complex. Stain Technol. 53, 331-336.

WATSON S., AKIL H., SULLIVAN S. & BARCHAS J. (1977) Immunocytochemical localization of methionine enkephalin: preliminary observations. L@ Sci. 21, 733-738.

ZIEGLGANSBERGER W. & TULLOCH I. (1979) The effects of methionine-and leucine-enkephalin on spinal neurons of the cat. Brain Res. 167, 53-64.

(Accepted 21 September 1980)