Neurokinin A-like immunoreactivity in articular afferents of the cat

Brain Research, 586 (1992) 332-335 ~-~ 1992 Elsevier Science Publishers B.V, All rights reserved 0006-8993/92/$05.00

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Short Communications

Neurokinin A-like immunoreactivity in articular afferents of the cat U. Hanesch, B. H e p p e l m a n n and R.F. S c h m i d t Physiologisches hlstitut der Unirersitiit Wiirzburg. Wiirzburg (FRG) (Accepted 28 April 1992)

Key words: Knee joint: Dorsal root ganglion. Colchicine; Neuropeptide; Tachykinin; Nociception; Neurokinin A; Substance P

Dorsal root ganglion cells with axons innervating the cat's knee joint via the medial articular nerve were retrogradely labelled with Fast blue. Neurokinin A-like immunorcactivity was found in 4.5:i: 1.1% (mean+S,D. of 5 nerves and 695 cells) of the articular afferents. Colchicine treatment of the ganglia increased the percentage of immunopositive cells to 8.5 ::1:0,7% (mean :t: S,D. of 6 nerves and 554 cells) after 3-22 h. The diameter distribution of the immunopositive somata ranged from 20 to 50/,tin with a maximum at 26-30 #m. Comparing the proportions of neurokinin A-immunopostvve cells with those of substance P. it can be calculated on the basis of mRNA encoding that neurokinin A is synthetized in about half of the substance P-containing primary articular afferents,

Tachykinins. a structurally related family of neuropcptides, are widely distributed in the peripheral and central nervous system, They are encoded by two genes which give rise to different messenger RNAs encoding four distinct protein precursors, the pre-protachykinins. The three major tachykinins substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) are generated by proteolytic cleavage of the precursor molecules (cf. ref. 13), In rat spinal cord, dorsal root~; and dorsal root ganglia SP and NKA are distributed in a very similar manner with fairly constant molar ratios, whereas NKB seems to be absent from primary afferent neurons t t. Using immunohistochcmicai methods it has been shown that SP and NKA are colocalized: primary afferents which are immunopositive for NKA are also immunopositive for SP, but not all SP-positive cells contain NKA 3. Both tachykinins are released in the dorsal horn of the cat spinal cord after noxious mechanical or thermal stimulation of the skin suggesting that these neuropeptides are involved in the transmission of noxious information 4.s As increased levels of immunoreactive NKA persist beyond the duration of the noxious stimuli and ~re detected over the whole depth of the dorsal horn even extending into the adjacent white matter, it was

suggested that NKA has a modulator role in long-term alterations in spinal cord excitability following even brief noxious stimulation 5. To examine the mechani,,;ms of nocieeption in norm:d and inflamed tissue, the knee joint of the cat has been used in numerous studies, An acute inflammation also leads to a marked release of both tachykinins in the lumbar spinal cord with a similar pattern obtained after a noxious stimulation of the skin v'=". As demonstrated in a recent study some 17% of the primary afferents of the medial articular nerve (MAN) of the cat's knee joint are immunopositive for SP ¢'. The proportion of joint afferents containing NKA is unknown, Therefore, in the present study we determined the proportion of cat knee joint afferents with a positive immunoreactivity for NKA after a retrograde labelling of the respective perikarya in the dorsal root ganglia, To enhance the concentration of this neuropeptide and to get more precise information about the proportion of afferents containing NKA, some of the ganglia were additionally incubated with colchicine which is known to inhibit axonal transport 2,,~ In total, seven adult cats of either sex were used in this study, They were anesthetized with sodium pentobarbital (Nembutal; 44 mg/kg body weight, i.p.) prior

Corrt'sl~mdence: B, Heppelmann, Physiologisches Institut der Universit~it Wiirzburg, RBntgenring 9, D-8700 Wiirzburg, FRG. Fax: (49) (931) 54553.

333 and untreated ganglia were removed, postfixed for a further 4 h in the same fixative and stored for one to two days at 4°C in a solution of 30% sucrose in PBS. All ganglia were cut longitudinally in 30/~m sections using a freezing microtome, collected alternately in 4 series on gelatinized slides, and air-dried. For this study, all cells in one of the section series which were labelled by FB were photographed with a Zeiss fluorescence microscope (Fig. 1). Rehydrated sections were incubated with primary antiserum raised in rabbit (Peninsula, code no.: RAS 7359-N, lot no.: 010527-1) for 2 days at 4°C. Previous test series with dilutions ranging from 1 : 100 to 1 : 1000 revealed an optimal antibody dilution of 1 : 120 in cat's dorsal root ganglia. Thereafter, sections were incubated with goat anti-rabbit IgG (1:40, DAKO) for 2 h

to surgery. During the operation, additional sodium pentobarbital was administered intravenously in amounts of about 10 m g / h to maintain deep anesthesia. In each cat, one MAN was cut and exposed to Fast blue (FB) (5%, w / v ) dissolved in 2% dimethyisulphoxide for 1.5 h 6. The incision was closed and the animals were allowed to recover from narcosis. After 4 days, one of the two dorsal root ganglia containing the perikarya of the MAN (L5 and L6) n was exposed by a hemilaminectomy. A small ball of cotton soaked with 10% colchicine in tyrode solution was placed on the ganglia for 30 min. After 3-22 h the animals were perfused intracardially with one liter of 0.1 M phosphate-buffered saline (PBS; pH 7.4) followed by 1.5 liters of a neutral fixative consisting of 4% paraformaldehyde and 0.5% picric acid in PBS. Treated

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Fig. I. Top: fluorescence photomicrograph of labelled articular afferent cell bodies in the sixth lumbar dorsal root ganglion after exposure of the MAN to Fast blue (FB). Bottom: photomicrograph of the same section showing NKA-like immunoreactive neurons after immunostaining with the PAP-procedure. Arrows indicate a perikaryon of an articular afferent labelled with FB and displaying NKA-Ii. Bar = 50 ~m.

334 and finally with rabbit peroxidase antiperoxidase (PAP) complex (1 : 4(X); Sternberger) for 2 h, both incubations at room temperature. The peroxidase label was developed in 0.O~'~c~c 3,3-diaminobenzidine tetrahydrochloride (DAB; Sigma), activated with 0.017% hydrogen peroxide. The specificity of immunostaining was tested by omitting the primary antiserum and by preabsorbing the antibodies with increasing concentrations (0.1-100 nmol/ml diluted antiserum) of synthetic NKA (Calbiochem). A preabsorption with 0.1 nmol/ml of NKA reduced the number of immunopositive cells. No staining was obtained at concentrations of 1, 10, and 100 mmol/ml of NKA and after omitting the primary antiserum. As polyclonal antibodies to NKA may cross-react with other peptides, mainly with SP (3¢/c cross-reactivity tested by radioimmunoassay; Peninsula), all positive staining is termed "NKA-like immunoreactivity' ( N K A - l i ) . "ro examine if cross-reactivity between NKA and SP may influence our immunohistochemical results, we tested the specificity of the immunoreaction by preabsorbing the antiserum with 1, 10 and Ill0 mmol synthetic substance P (Peninsula) per ml diluted antiserum. Using a control ganglion, 865-1295 perikarya were examined in 4 series of 5 sections each. in sections without :t SP-preabsorption 4.4 ± (),5c~, of tit,: cell bodies were immunopositive. No significant

r~duction of this proportion was I'ound in sections with a Sl).preabsorption of the NKA antiserum (1 nmol/nd SP: 4.5 ± ().5C;', i() nmol/ml SP: 4,(I ± O.gq;', I()() nmol/ml SP: 4.3 ± (i.I~rA: tested with the Student's t-test). This demonstrates that cross-reactivity of the antihodies used was not a problem and did not influence the results of the present study in any significant way. The proportion of joint affcrcnts which show a positive NKA-li were determined by comparing the

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Fig. 2. Influence of a ¢olchicinc Ireatment tm the pmp,)rtion of NKA-like immuu,)pt)silive perikarya of retrogradely lal~clled knee joint affcrents, A: percentage of NKA-immunol~)sitive cell bodies revealed from mntrol ganglia and from ganglia incuhatcd fi~r 3-22 h, B: mean proportion of NKA-like immunolmsitivc afferents ( +_S,D,) in untreated (5 nerves and 6~5 cells) und coichicine treated (6 nerves and 554 ceils) ganglia, The difference is signilicant (P < ILfX|I, Student's/-I~:st).

photographs of the fluorescence labelling with the distribution of immunopositive perikarya within the same sections after the immunoreaction (Fig, 1). This procedure was used, as the peroxidase reaction influences the fluorescence labelling. It has the advantage that the immunoreactivity can be analyzed without a fading of a fluorescent dye. Cell body sizes were determined after the immunoreaction. To avoid errors by analyzing offcuts, only contours of cells with a clearly visible nucleus were included in this examination. The contours were drawn using a camera lucida, the area was measured with a computer-aided analysis system (HISTOL; List Electronic), and the diameter was calculated by convcrting the area to an equivalent circle. incubation of the MAN with FB resulted in an intense fluorescent labelling of the corresponding perikarya (Fig. i). In the individual experiments, 55-18{) labelled cells were found in the examined sections. The size of the fluorescent labelled perikarya showed a unimodal, skewed distribution with a range from 16 to 94 #m and a peak at about 30/.tin (Fig. 3A). NKA-li appeared homogeneously distributed within tile cytoplasm of the ganglion cells (Fig. !). Without colchicine treatment, 4.5 =1: 1.1% (mean :!: S.D. of 5 nerves :rod 695 cells) of FB-labelled neurons exhibited NKA-li (Fig. 2). They generally were of small and intermediate size with diameters ranging from 20 to 50 /~m with a n~aximum at 26=30/~m (Fig, 3A). inhibitiotl of axonal transport with ¢olchicine enhanced the proportion of immunopositive articular al't'er~nts (Fig, 2), Three hours after the incubation, %8% o1' the al'f~rcnts showed a positive immunoreactivity,

This value was further increased to 9,4% alter a 22 h period. As there was only a slight difference in the proportion of positive cell bodies between shorter and longer times after the colchicine treatment, we pooled the data from all incubated ganglia, resulting in an average increase of the proportion of NKA-li containing cells to 8,5 ± 0,7~;~ (mean + S,D. of 6 nerves and 554 cells, P_< 0,0Ill, Studcnrs Hest), The soma size distribution was not influenced by the colchicine treatment (compare black bars in Fig, 3B with 3A), These results reveal that only a very low proportion of joint affcrcnts show a NKA-li either with or without colchicine treatment of the dorsal root ganglia. The marked increase of immunopositive neurons after colchicine treatment indicates that inhibition of axonal transport is necessary for the determination of the number of NKA containing primary affcrents. As the pre-protachykinins contain the sequences of either SP and NKA, or SP alone ~, one would expect that only a portion of SP-immunopositive primary af-

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small proportion of nociceptive-specific afferents may be involved in long term alterations in spinal cord excitibility following brief noxious stimulations or during the first hours of an acute peripheral inflammation by the release of NKA.

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Fig. 3. Soma size distribution of Fast blue labelled perikarya of the MAN (white bars) and proportion of cells (black bars) additionally showing a NIG~-Ii. A: soma sizes of articular afferents after labelling of 5 MANs without colchicine treatment (n = 695 cells). B: pooled data of labelled perikarya (n = 554) in ganglia which were fixed 3-22 h after incubation with colchicine (n = 6 nerves).

ferents should exhibit a colocalization of SP and NKA. With the present data it is possible to calculate the extent of this eolocalization in knee joint afferents. As shown in a previous study ~' about 17% of the cat's knee joint afferents show a SP-li. The present data reveal that about 9% of these joint afferents are NKAlike immunopositive, Assuming that NKA is always synthetized together with SP, it can be calculated that about half of the SP.containing joint affercnts contain NKA as a colocalized peptide - derived from the pre-protachykinin A (/3 and ?). Based on morphological and electrophysiological data, at most some 30% of cat's knee joint afferents, which have a high mechanical threshold probably reflecting nociceptive afferents, contain SP ¢'. According to this calculation, NKA may therefore be present only in up to 15% of nociceptive-specific articular afferents. This proportion, however, has to be reduced further, as it w~s shown that there exists no clear correlation between the function and the neuropeptide content of a primary afferent neuron me. Therefore, only a very

We wish to thank Dr. P.C. Emson for critical reading of the manuscript. This study has been supported by the Deutsche Forschungsgemeinschaft. ! Craig, A.D., Heppelmann, B. and Schaible, H.-G., The projection of the medial and posterior articular nerves of the cat's knee to the spinal cord, J. Comp. NeuroL, 276 (1988) 279-288. 2 Dahlstr6m, A., Effect o! colchicine on transport of amine storage granules in sympathetic nerves of the cat, Era: J. Pharmacol., 5 (1968) 111-112. 3 Di~isgaard, C.-J., ltaegerstrand, A., Theodorsson-Norheim, E., Brodin, E. and H6kfelt, T., Neurokinin A like immunoreactivity in rat primary sensory neurons: coexistance with substance P, Histochenlistry, 83 (1985) 37-40. 4 Duggan, A.W., Hendi~j, I.A., Morton, C.R., Hutchison, W.D. and Zhao, Z.Q., Cutaneous stimuli releasing immunoreactive substance P in the dorsal horn of the cat, Brah~ Res., 451 (1988) 261-273. 5 Duggan, A.W., Hope, PJ., Jarrott, B., Schaible, H.-G. and Fleetwood-Walker, S,M., Release, spread and persistence of immunoreactive neurokinin A in the dorsal horn of the cat following noxious cutaneous stimulation. Studies with antibody microprobes, Neuroscience, 35 (1990) 195-202. 6 Hanesch, U., Iteppelmann, B. and Sc.hmidt. R.F., Substance Pand calcitonin gene-related peptide-in~unoreactivity in primary afferent neurons of the cat's knee join~, Neurosciem'e, 45 (1991) 185-193. 7 l iope, P.J., Jarmtt, B., Schaible, H.-G., Clarke, R.W. and Duggan, A.W., R01ease and spread of immuno~'~ctive neurokinin A in the cat spinal c()rd in a model of acute arthritis, Brain Rt,s., 533 (1990) 292-299. 8 Krause, J,E,, Chirgwin, J.M., Carter, M.S., Xu, Z.S. and ltershey, A., Three rut preprotachykinin mRNAs enc(Jde the neumpeptides substance P and neurokinin A, Pmc'. Natl. Acad. St'i. USA, 84 (1987) 881-885. 9 Kreutzber8, G,, Neuronal dynamics and flow. IV. Blockage of intraaxonai enzyme transport by colehicine, Proc. NatL A¢,ad. SoL USA, 62 (1959) 722-728, Leah, J,D., Cameron, A.A. and Snow, P.J,, Neuropcptides in physiologically identified mammali:m sensory neurons, Nt,urosci. Lett., 56 (1985) 257-263. Ogawa, T., Kanazawa, I. and Kimura, S., Regional di:~trjbution of substance P, neurokinin A and neurokinin B in rat spil,al cord, nerve roots and dorsal root gan~,lia and the effects of di:fsal root section or spinal tnmssection, Brain Res., 359 (1985) 152-157. Schaible, H.-G., Jarrott, B., Hope, P.J. and Duggan, A,W., Release of immunoreactive substance P in the spinal cord during development of acute artllritis in the knee joint of the cat: a study with antibody microprobes, Brain Res., 529 (1990) 214-223. Spampinato, S. and Ferri, S., Pharmacology of spinal peptides affecting and motor functions: dynorphins, :.omatostatins and tachykinins, Phannacol. Res., 23 (1991) 113-127.