0306-4522/91 $3.00 + 0.00 PergamonPress plc 0 1991 IBRO

NeuroscienceVol. 45, No. 1, PP. 185-193, 1991 Printedin Great Britain

SUBSTANCE P- AND CALCITONIN GENE-RELATED PEPTIDE IMMUNOREACTIVITY IN PRIMARY AFFERENT NEURONS OF THE CAT’S KNEE JOINT U.

HANESCH,*

B.

HEPPELMANN

and R. F. SCHMIDT

Physiologisches Institut der Universitat Wiirzburg, Riintgenring 9, D-8700 Wtirxburg, F.R.G. distribution of substance P and calcitonin gene-related peptide was determined in primary aBerent neurons of the medial and posterior articular nerve of the cat’s knee joint. Perikarya of articular afferents were visualized by retrograde lahelling with the fluorescent dye Fast Blue which was applied at the transected end of the peripheral nerves. Substance P was found in about 17% of labelled medial articular tierents and in about 16% of labelled posterior articular afferents, respectively, whereas calcitonin gene-related peptide was present in about 35 and 32% of the medial and posterior articular nerve cells, respectively. Taking into account that these neuropeptides are known to be co-localized, probably not more than one-third of the joint afferents contain substance P and/or calcitonin gene-related peptide. Quantification of cell diameters revealed that substance P was found only in small- or Abstract-The

intermediate-sized perikarya (< 50 pm) indicating that this peptide is predominantly found in unmyelinated neurons. Calcitonin gene-related peptide was present mainly in small- and intermediate- but also in some large-sized neurons (> 50 pm) providing evidence that this peptide is found in unmyelinated and to a lesser extent in myelinated neurons. This is consistent with previous studies that show that substance P and calcitonin gene-related peptide are present primarily in unmyelinated and thinly myelinated primary afferents. When the portion of substance P-positive neurons of the medial articular nerve is compared to the number of articular afferents displaying a nociceptive function as determined in earlier electrophysiological studies, it can be calculated that at most 30% of the nociceptive-specific articular afferents contain this neuropeptide. Since this finding parallels observations in other combined electrophysiological and immunocytochemical investigations, these data support the idea that substance P may be a functionally important neuropeptide in a small particular subgroup of nociceptive articular afferents but is not an essential neuropeptide in all articular nociceptive nerve fibres.

During recent years a variety of neuroactive peptides lease of CGRP has been demonstrated in the cat have been identified in the peripheral nervous system spinal cord in viva,” and iontophoretic application of (for review see Refs 23 and 28). Some evidence exists CGRP produced a slow but prolonged excitation of that they play a role in the transmission of sensory neurons in the dorsal horn of cats.49 Taken together, information, mainly acting as neuromodulatory this evidence suggests that CGRP is associated with substances.49*65p67 the processing of nociceptive information. Substance P (SP) was first described by von Euler The co-localization of CGRP with SP in cell and Gaddum” and has been demonstrated by bodies of sensory ganglia and primary afferent nerve immunohistochemistry to be localized in small- to fibres has been confirmed by many group~.“.*.“,~~*~ intermediate-sized perikarya of the dorsal root Therefore, it is conceivable that CGRP and SP ganglia22342347 and in afferent C- and A6 -fibres,‘8*“*39*47are released simultaneously,‘* influencing the release some of them responding specifically to noxious and the effects of other neuropeptides or neurostimuli. 39 Together with the fmding that noxious modulators.55*63@ stimulation leads to a release of SP in the spinal The findings that both neuropeptides occur in cord 10~11~31~37~55 these results indicate that this neuroperipheral afferent nerve fibres and that there exist peptide is involved in the transmission of noxious neuropeptide binding sites in the innervated tissue& information. suggest that the function of primary afferents is not Calcitonin gene-related peptide (CGRP), a recently only restricted to the conveyance of (nociceptive) discovered neuropeptide, 57 has also been shown to information from the periphery to central targets in occur in dorsal root ganglion cell bodiesi4,‘6,30and in the spinal cord, but that they display an efferent primary sensory axons. ‘,56,61Stimulus-dependent reregulating role in the innervated peripheral tissue.35@ In fact, antidromic stimulation of primary afferents evokes an enhanced release of SP into the extracellu*To whom correspondence should be addressed. Abbreviations: CGRP, calcitonin gene-related peptide; lar space of the eye,) as well as into the intra-articular DAB, 3,3’-diaminobenxidine; HRP, horseradish peroxifluid of the knee joint,66 mediating vasodilation and dase; -LI, -like immunoreactivity; MAN, medial articuextravasation of plasma proteins and erythrolar nerve; NGS, normal goat serum; PAN, posterior cytes,13s” which are the main characteristics of articular nerve; PAP, peroxidaseantiperoxidase; PBS, phosphate-buffered saline; SP, substance P. an inflammatory process. Therefore, the release 185

of neuropeptides such as SP and CGRP may play important role in mediating the neurogenic component of inflammation. In recent years, the knee joint of the cat has been

an

used as a model to study the mechanisms of nociception in normal and inflamed tissue. In contrast to the wealth of knowledge concerning the innervation pattern and the responses of afferent nerve fibres to different stimuli, sparse information exists on the peptidergic innervation of the knee joint and its role in the development of inflammatory diseases. Therefore, in this study we examined the distribution of SP and CGRP in knee joint afferents. The approach we used was the identification of afferents by retrograde labelling of dorsal root ganglion cells with the fluorescent dye Fast Blue followed by an immunocytochemical identification of the neuropeptide content of these cells. The percentage of SP- and CGRP-like immunoreactivity (-LI) was determined by counting the fluorescent-labelled and immunopositive perikarya. Additionally, a morphometrical analysis was performed to determine the soma size distribution of the peptidergic articular neurons. EXPERIMENTAL

PROCEDURES

Nine adult cats of either sex with a body weight of 1.5-2.2 kg were anesthetized with sodium pentobarbital (Nembutal; 44mg/kg body weight, i.p.) prior to surgery. During the operation, additional sodium pentobarbital was administered intravenously in amounts of about 10 mg/h to maintain a deep anaesthesia. Retrograde tracing

In each cat, one articular nerve per side was labelled. The preparation of the medial articular nerve (MAN) and the posterior articular nerve (PAN) was performed as described previously.8 The nerves, lying in a small plastic trough closed at both ends with wax, were cut and exposed to the fluorescent dye Fast Blue (5%, w/v) dissolved in distilled water or in 2% dimethylsulphoxide. After an incubation time of about 2 h the suspension was removed and the trough washed out with a 10% serum albumin solution. The trough was coated with a small sheath of plastic and a drop of agarose, which ensured a proteinaceous environment for the axon stumps. The incision was closed and the animals were allowed to recover from narcosis. After the surgery, the cats were treated with 0.1 g ampicilline (Binotal, i.m.) to avoid infection. Fixation and tissue preparation

Four days (seven animals) to five days (two animals) after the operation, the animals were deeply anaesthetized with sodium pentobarbital(60 mg/kg, i.p.) and perfused intracardially with I1 of 0.1 M phosphate-buff&d saline (PBS; pH 7.4) followed by 1.5 1 of a neutral fixative solution consisting of 4% paraformaldehyde and 0.5% picric acid in PBS. A survival time of four days was chosen in most of the experiments, as this led to an optimal labelling of the perikarya without any leakage of the fluorescent dye from the labelled cells into surrounding neurons. The relevant dorsal root ganglia [L5/L6 (MAN) and L6/L7 (PAN)8 resp.] and the lumbosacral spinal cord were removed and postfixed for a further 4 h. After extensive washing in a solution of 10% sucrose in PBS, the tissue was stored either for one day at 20°C or for two days at 4°C. Dorsal root ganglia were cut longitudinally in 50-ym sections with a freezing microtome, collected alternately in two series

on gelatuuzed slides (for immunostaining with SP- ;md CGRP-antibodies), and air-dried. To examine if there was a leakage of the fluorescent tracer during the incubation out of the trough into the surrounding muscle tissue, lumbosacral spinal cord segments were cut horizontally into 75-pm serial sections and mounted for fluorescence microscopy. No fluorescent-labelled motoneurons, however, were found in all experiments indicating a specific labelling of articular afferents. Analysis

ofjuorescence

labeiling

Sections were examined on a Leitz fluorescence microscope equipped with a filter system having an excitation wave length of 340-380 nm (Leitz A filter). Cells which had taken up the fluorescent tracer were photographed using an image intensifier camera and contrast enhancement with an image processing system (IPS, Kontron). Immunocytochemical procedures

Sections were processed for the immunocytochemical localization of SP and CGRP using the peroxidase-antiperoxidase (PAP) technique. Both section series were hydrated in PBS (PH 7.4) and preincubated subsequently in 5% normal goat serum (NGS) in PBS with 0.5% Triton X-100 for 30min at room temperature. They were then placed in primary antisera raised in rabbits, either anti-SP (1:4000; Calbiochem) or anti-CGRP (1: 5000; Amersham) and stored for two days in a humid atmosphere at 4°C. All antisera applied were diluted in PBS containing 1% NGS and 0.5% Triton X-100. Following five washes of 10 min in PBS, the sections were incubated for 2 h with goat anti-rabbit IgG (1:40; Peninsula) at room temperature. intensely washed for another hour and finally placed in rabbit PAP complex (1:400; Sternberger) for 2 h. After further rinsing the peroxidase label was developed in 0.025% 3,3’-diaminobenzidine tetrahydrochloride (DAB; Sigma), activated with 0.017% hydrogen peroxide. Reaction product in excess was washed out with PBS and the sections were dehydrated in ethanol, cleared in xylene, and mounted for light microscopy. Specljicity of immunostaining

The specificity of immunostaining was tested either by omitting the primary antiserum or by preabsorbing the antibodies with 100 pg/ml of SP (Sigma) or with 50 pg/ml of synthetic rat CGRP (Peninsula) overnight at 4°C. In both cases no staining was obtained. Since cross-reactivity of the antisera with other peptides cannot be fully excluded, positive staining is termed SP-LI and CGRP-LI. Quantitative analysis

The percentage of articular dorsal root ganglion cells showing SP-LI or CGRP-LI was determined by comparing the photographs of the fluorescent-labelled perikarya with the immunopositive cells in the tissue sections. This method appeared to be necessary, as we found a marked reduction of the fluorescent labelling during the immunoreaction process. Cell body size of articular afferents was examined in the immunostained SO-pm-thick ganglion sections of seven animals The contours of identified and immunopositive or immunonegative perikarya were drawn using a camera lucida. The area was measured with a computer-aided analysis (HISTOL, List-electronic) and the diameter was calculated by converting the area in an equivalent circle. Only cells with a clearly visible nucleus were included. RESULTS

Ident$cation

of joint aflerents by retrograde labelling with the @orescent dye Fast Blue

The application of Fast Blue to the transsected ends of the MAN and the PAN resulted in its

SP and CGRP in articular afferent neurons

retrograde transport to the neuronal perikarya in the dorsal root ganglia. Fluorescence was detected in the cytoplasm of cell bodies of all sixes, distributed throughout the ganglia with no somatotopic pattern. In the individual experiments about 150 to 540 cells were stained (not corrected for double counting). Although the cell body size of MAN and PAN afferents had been determined in the previous horseradish peroxidase (HRP) study,* we undertook a morphometrical analysis for two reasons. Firstly, we used the fluorescent dye Fast Blue, which may have a different uptake and rate of transport than HRP. Secondly, the fixation procedure and tissue processing used with Fast Blue may have produced slight variations in somal size compared to the HRP study.

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Medial articular nerve afferent neurons Labelling of four MANS in four different cats resulted in a total of 746 cell bodies in ganglia LS/L6 containing the fluorescent dye. The unimodal distribution of the sampled soma sizes of the articular afferent neurons is shown in Fig. 1. They were in a range of 24-86 pm with a peak at about 44 pm.

0

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somo size (pm)

Fig. 1. Cell-diameter distributions @-pm-wide classes) of articular afferent neurons in lumbar dorsal root ganglia retrogradely labelled with Fast Blue from the -MAN (top; n = 746) and the PAN (bottom; n = 1239). In both cases the data of four experiments were pooled.

Posterior articular nerve ajierent neurons After labelling of six PANS in five cats, a total of 2037 fluorescent afferent neurons were identified in ganglia L6/L7. The diameter of labelled perikarya ranged from 24 up to 100 pm (Fig. 1). Compared to the MAN, the soma size distribution of the PAN showed a shift to larger perikarya.

Distribution of substance P-like immunoreactivity in the articular afferents Immunocytochemistry for detection of SP was performed on the lumbar dorsal root ganglia sections of six animals which contained the fluorescent cell bodies of labelled articular afferents. SP-LI appeared to be homogeneously distributed (Fig. 2) within the cytoplasm of approximately 15% of all dorsal root ganglion cell bodies in segments L5, L6 and L7. In addition to the perikarya, immunoreactive axons could be observed within the ganglia. Some of them encircled presumable SP-negative cell bodies in a basket-like manner. The percentage of SP-positive neurons of four MANS and four PANS was determined by comparing photographs of sections from spinal ganglia containing Fast Blue-labelled neurons with the same sections following processing for SP-LI (Fig. 2). It .was found that the immunostained cells represented 17.3 + 2.2% of the total MAN afferents and 15.7 rf: 0.3% of the PAN neurons (Table 1). The SP-LI-containing cells of both articular nerves were generally of small and intermediate size with diameters ranging from 24 to 54gm (Fig. 3). The distribution of the soma size was unimodal with a maximum at about 38 pm in both cases.

Distribution of calcitonin gene-related peptide-like immunoreactivity in the articular afferents Lumbar dorsal root ganglia sections of seven animals showing Fast Blue-labelled cell bodies of the MAN or PAN were processed for CGRP-LI (Fig. 2). CGRP-positive perikarya were found to be more abundant than SP-positive ones. About 35% of all cell bodies in dorsal root ganglia L5, L6 and L7 contained CGRP-immunoreactive material. CGRPLI occurred in a variety of patterns: as a bright and even staining across the cytoplasm, as a patchy staining in granule-like structures forming a perinuclear ring, as a pun&ate, granule-like staining evenly distributed within the cytoplasm, or as any combination of these forms. In some of the cell bodies it could be clearly seen that in addition to the perinuclear ring, there was also an immunoreactivity more diffusely distributed across the perikaryon. Nevertheless, the soma size of neurons exhibiting different staining patterns was in a similar range. By comparing the photographs of the Fast Blue distribution in the ganglia after labelling of four MANS and five PANS with the immunoreacted sections, the percentage of CGRP-positive articular afferents was calculated. The variation between the Table 1. Proportion (%) of neurons in dorsal root ganglia cells projecting to the knee joint in the cat containing substance P- or calcitonin gene-related peptide-like immunoreactivity (mean f SD.) MAN PAN

SP (%)

CGRP (%)

17.3 f 2.2 (?I = 4) 15.7 * 0.3 (n = 4)

34.8 f 1.1 (n = 4) 32.2+1.9(11=5)

1X8

II. HANESCH rt ul

Fig. 2. Left: fluorescence photomicrographs of labelled articular afferent cell bodies in the sixth lumbar dorsal root ganglion after exposure of the MAN to Fast Blue (FB). Right: photomicrographs of the same sections showing SP- and CGRP-like immunoreactive neurons (taken from two different experiments) after immunostaining with the PAP procedure. Arrows indicate perikarya of articular afferents retrogradely tabelled with Fast Blue and displaying SP- or CGRP-LI. Scale bars = 50 pm.

individual experiments was very low. Taken together, 34.8 f 1.1% of the Fast Blue-labelled MAN cells and 31.8 f 2.0% of the fluorescent PAN neurons contained CGRP-LI (Table 1).

CCRP-LI was mainly found in small- and intermediate-sized as well as in some large articular afferent perikarya, in contrast to SP-LI, which was restricted to small- and intermediate-sized cells.

189

SP and CGRP in articular afferent neurons

MAN

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Fig. 3. Soma size distribution of Fast Blue-labelled articular afferent cell bodies of the MAN (top; n = 397 and 309) and PAN (bottom; n = 601 and 537) and proportion of cells (black bars) additionally showing SP-LI (left) or CGRP-LI (right). Data were pooled from articular nerves of four animals each.

CGRP-positive somata of the MAN ranged in size from 28 to 68 pm, whereas those of the PAN were in a range from 26 up to 78 pm (Fig. 3). A unimodal, skewed distribution of the diameter was apparent with a maximum at about 38 pm in both nerves. DISCUSSION

In recent years, the neuropeptides SP and CGRP have been detected and quantified in atTerents of different tissues. In the knee joint, they are thought to play an important role in the development of inflammation by mediating vasodilatation and extravasation of plasma proteins, as well as in the transmission of nociceptive information to the spinal cord. So far, a quantitative investigation of the distribution of SP and CGRP in articular alferents has been performed only in the rat.” In this study, retrograde labelling of articular dorsal root ganglion cells was performed by injecting the fluorescent dye into the cavity of the knee joint. Injecting the dye into the knee joint cavity, however, does not result in a complete staining of all knee joint alferents and nonspecific labelling of afferents in the adjacent muscles, tendon and connective tissue may occur. Therefore, only a small number of cells could be examined in this study. In the present study, the MAN was cut and incubated with the fluorescent dye Fast Blue resulting in a highly specific and efficient (up to about 540 cells in one experiment) labelling of the articular dorsal root ganglia cells. The disadvantage of this technique, however, is that one has to accept a possible reduction of the neuropeptide-containing cells of about 30% in the case of SP, as after sectioning the sciatic nerve in rats the content of mRNA coding for SP and

of the neuropeptide itself was found to be reduced. The expression of preprotachykinin mRNA in lumbar dorsal root ganglia cells is decreased to about 40% of the control level after three daysS2 The number of cells containing preprotachykinin mRNA is reduced to about 70%.53 Finally, the amount of SP in dorsal root ganglia also shows a reduction of about 30% after the same period.29@ The content of CGRP, however, is not influenced by axotomy.62 The size distribution of the articular cell bodies is shifted to higher diameters compared to the results of the HRP tracing experiments previously described by Craig et al.* This discrepancy is probably caused by changes of the soma size during different histochemical treatments of the tissue. Combining the axonal tracing technique with the immunocytochemical procedure enabled us to examine the distribution of the neuropeptides SP and CGRP in the articular neurons of the dorsal root ganglia. As not all afferent neurons supplying the knee joint were labelled, these data provide information about the relative proportions of SP- and CGRP-LI afferents rather than giving absolute values. In several immunocytochemical investigations, additional treatment of the ganglia with colchicine was performed to block the axoplasmatic transport and to increase the amount of peptides in the perikarya.e.g.41 In our study we did not use colchicine to avoid possible complications from further surgery and colchicine toxicity. In every experiment the intensity of the immunoreaction seemed to be sufficient to identify the articular somata containing SP and CGRP. Nevertheless, some cells with a very low content of the investigated neuropeptides may have escaped detection by our staining method.

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U. HANESCH et ul.

Distrihtion of‘ substance P- and calcitonin generelated peptide-like immunoreacti~~ity in dorsal root ganglion cells of different neroes

The proportion of SP-positive neurons in nerves innervating different tissues varies from approximately 10 to 60%. Our results indicate that the percentage of SP-containing perikarya contributing axons to the MAN is about 17% and to the PAN about 16%. These values are similar to the percentages of pelvic (24%),34 pudendal (21%)‘4 and hypogastric nerves (23%)” as well as of afferents of the kidney (24%),36 the bladder (18%)32 and the diaphragm (11 %)26 in the cat. Comparable results are found in perikarya of the saphenous nerve (lO%).sO the gastrocnemius nerve (15%)% and in afferents of the bladder (16%)59 in the rat. On the other hand, thoracic visceral afferent nerves contain a higher proportion of SP-containing perikarya: in the cat 61%33 and in the rat 50%% of the neurons of the splanchnic nerve exhibit a SP-LI. Afferents of the oesophagus (39%) and stomach (56%) in the rat show SP-LI in a similar range.” The proportion of CGRP-LI fibres of nerves innervating different tissues, however, varies in a wider range. In this study of articular afferents about 35% of the MAN neurons and 32% of the PAN aRerents contain this peptide. Nerves innervating the skin and the muscle exhibit a similar percentage of CGRP-LI: 20% of the saphenous and 30% of the gastrocnemius nerve afferents in the rat contain the peptide.s0 In contrast. visceral nerves differ markedly in their CGRP content from the somatic ones. In the rat, 90% of sensory neurons supplying the kidney and ureter,@’ 60% of bladder afferents,” 70% of oesophagus afferents,” 82% of stomach afferents17 and 95% of splanchnic nerve afferents’* are CGRP immunoreactive. These results led to the idea that CGRP is a marker for neurons in visceral rather than in somatic tissues.” Even on the assumption that the two neuro~ptides are present in distinct subpopuiations, SF and CGRP-positive neurons constitute not more than half of the articular nerve afferents. In fact, neuropeptides were found to be co-localized in primary afferent neurons and their peripheral nerve fibres. Moreover, most of the cells which contain SP-LI are also immunoreactive for CGRP.6JS,4’*42,64 This means that the number of knee joint afferents containing SPand/or CGRP-LI may be diminished to about onethird, if one assumes that these neuropeptides coexist in the same neuron. Distribution of substance P- and calcitonin generelated peptide-like immunoreactioity in fine articular afferen ts Our results show that perikarya of articular afferents immunoreactive for SP are almost entirely of small and intermediate size, which is consistent with earlier studies.24*36 Combined electronhvsiolonical I , -r-~

and morphological examinations of dorsal root ganglion cells in the cat revealed that perikarya giving rise to unmyelinated group IV fibres (or C-fibres) range to about 50~rn.~~ For intermediate-sized cells (35550pm) the size of the cell body bears no predictable relation to the conduction velocity of the peripheral fibre. 4o For large cells with a diameter above 50,um a correlation to impulse conduction velocities in the A range (groups I, II and III) exists.4” Comparable correlations between soma size and conduction velocity of the peripheral fibre were also shown in further studies.s,19 In peripheral nerve fibres, SP-LI was found in unmyelinated fibres of group IV.‘.g.27.45 This led to the assumptjon that, either SP does not exist in myelinated nerve fibres, or that in peripheral nerve fibres the concentration of this peptide is too low for a positive immunoreaction. in the superficial laminae of the monkey dorsal horn, however, ultrastructural studies have demonstrated SP-LI in thinly myelinated axons9 and, moreover, light microscopic investigations combined with el~trophysiolog~~l recordings showing SP-LI in the lumbar dorsal root ganglia cells giving rise to peripheral nerve fibres belonging to group III.47 The observation of SP-LI in cell bodies of the articular nerves whose diameter is that of small- and intermediate-sized cells (< 50 pm) indicates that this peptide is predominantly localized in neurons giving rise to unmyelinated group IV flbres and, furthermore, may be found in some thinly myelinated group III fibres. In the articular afferent neurons CGRP-LI was found in small- and inte~ediate-size as well as in large perikarya. Therefore, this neuropeptide presumably exists in unmyelinated and in myelinated peripheral nerve fibres. The existence of CGRP-LI in alTerent neurons giving rise to myelinated nerve fibres has been described by several authors.‘,‘6*4EIn the present study about 25% of the CGRP-positive articular nerve cell bodies were large (>.50pm) and are thus likely groups III, II and perhaps group I type cells. However, about 75% of neurons containing CGRP-LI are in the range of small- and intermediatesized neurons which, as discussed for SP-positive cells, give rise to small unmyelinated and, to a lesser extent, to thinly myelinated axons. The slight differences in the distribution of SP- and CGRP-LI in the MAN and PAN can be explained by the diversity in the fibre composition of the articular nerves. Both neuropeptides were found in a higher percentage in MAN afferents. This corresponds to the greater proportion of groups III and IV fibres in this nerve.” Comparison of substance P- and calcitonin geerelated peptide-Zike immunoreactivit~~ distribution with the sensory characteristics qf jne articular afferents Quantitative morphological examinations of the MAN revealed a total number of 630 afferent nerve fibres.38 Based on the diameter distribution, about

SP and CGRP in articular afferent neurons

21% or 130 fibres belong to group III and about 70% or 440 fibres belong to group IV.21 Electrophysiological examination of MAN units revealed that these fibres differ in their response behaviour to passive non-noxious and noxious joint movements5* About 45% of group III fibres and about 29% of group IV fibres were activated by non-noxious movements.” About 55% (group III) and about 71% (group IV) of the fibres exhibited a high mechanical threshold, as they could be activated only by noxious movements or they did not respond to any joint movement.20 Combining the morphological and the functional data, one can calculate that about 60% of all afferents in the MAN have a high mechanical threshold probably reflecting nociceptive afferents. In the present immunohistochemical examination, SP-LI was found in no more than 17% of all perikarya of the MAN. This means that only up to about 30% of the nociceptive knee joint afferents may contain SP. This percentage, however, must bc diminished in view of the fact that SP may exist in mechanosensitive neurons as we11.39 In fact, combined electrophysiological and immunocytochemical investigations revealed no correlation between the existence of a given neuropeptide and the sensory function of primary afferent neurons. Only a part of the nociceptive afferents contained SP-LI, whereas this neuropeptide was present in some neurons responding to innocuous stimuli.39 The role of CGRP in the synaptic transmission of sensory information in the spinal cord, however, is still unclear. Some interactions with SP, e.g. potentiating and prolonging the duration of the SP action, could be revealed.43,55*6” The coherence of the CGRP distribution with definite sensory characteristics of fine articular afferents, however, remains to be determined. Neuropeptides such as SP and CGRP are trans-

191

ported not only to the central terminals in the spinal cord, but also to the peripheral branches of the primary atferent neurons. In fact, it is reported that the majority of the individual bioactive substance takes the latter route..’ Stimulation of sensory afferents of the cat sciatic nerve at intensities exciting groups III and IV fibres or by local application of capsaicin resulted in a release of SP into the synovial cavity,& suggesting that neuropeptides like SP mediate the local effector function of sensory alferents. Moreover, it is reasonable to speculate that neuropeptides are mediators of neurogenic inflammation, as antidromic stimulation of cat articular afferents of the knee joint resulted in vasodilation and substantial plasma and erythrocyte extravasation (which are components of inflammation) into the synovial fluid,13 and the application of a SP antagonist resulted in a potent inhibition of plasma extravasation.7*13 Based on the discovery that several neuropeptides are synthesized in sensory neuron?’ it is likely that there is a broad spectrum of neuroactive substances in the articular nerves and it is tempting to speculate that most of them do not only act simply as neurotransmitters, but may display peripheral local effector activities. The principle of multiple peptide comessenger functions, claimed by Hiikfelt et al.,25 may therefore be extended to a principle of multiple peptide co-effector and co-messenger function. To understand the mechanisms of neurogenic control of the inflammatory response, more work remains to be done to identify the elements that bring their modulatory influences to bear.

Acknowledgements-We

wish to thank Dr W. D. Hutchison for critical reading of the manuscript. Thanks also to Mrs Ch. Jansen and Mrs B. Trost for skilful technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft.

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14 Muy 1991)