Autoradiographic evidence of serotonin1 binding sites on primary afferent fibres in the dorsal horn of the rat spinal cord

Neuroscience Letters, 83 (1987) 71 76 Elsevier Scientific Publishers Ireland Ltd.

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NSL 05000

Aiatoradiographic evidence of serotoninl binding sites on primary afferent fibres in the dorsal horn of the rat spinal cord Genevi6ve Daval 1, Daniel Verg6 l, Allan I. Basbaum 2, Sylvie Bourgoin 3 and Michel H a m o n 3 IDbpartement de Cytologie, Institut des Neurosciences, C.N.R.S. U.A. 4199, Universitk Pierre et Marie Curie, Paris (France), 2Department of Anatomy, University of California, San Francisco, CA 94143 (U.S.A.) and 3INSERM U288, Neurobiologie Cellulaire et Fonctionnelle, Facultk de Mbdecine Pitib-Salp~tri~re, Paris (France) (Received 15 June 1987; Revised version received 20 August 1987; Accepted 27 August 1987) Key words:

Serotonin receptor; Opiate receptor; Rat spinal cord; Quantitative autoradiography; Dorsal rhizotomy; Capsaicin

Spinal serotonin~ (5-HT0 (labelled by [3H]5-HT), 5-HTtA (labelled by [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT)), /~- (labelled by [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol ([3H]DAGO) and [3H]naloxone) and g-opiate (labelled by [3H]Tyr-o-Ser-Gly-Phe-Leu-Thr ([3H]DSTLE)) receptor binding sites were studied in adult rats using quantitative autoradiography after either neonatal treatment with capsaicin or unilateral cervical dorsal rhizotomy. Both treatments produced a significant loss of 5-HT ( - 2 0 to -30%) and opiate ( - 3 0 to -45%) binding sites within the superficial layers of the dorsal horn, suggesting they are partly located presynaptically on primary afferent fibres. Thus, 5-HT, as well as opiates, might generate analgesia by acting - at least partly - on primary afferent nociceptive fibres at the spinal level.

A serotoninergic pathway, which originates mainly from the nucleus raphe magnus (NMR) and terminates in the superficial layers of the dorsal horn of the spinal cord [20], is involved in the descending control of nociception [1]. Electrical stimulation of N M R evokes the release of serotonin (5-HT) in the spinal grey [19], inhibits nociceptive neurons in the dorsal horn [18], reduces the excitability of primary afferent C fibres [8], and produces a profound analgesia [15]. Similarly the antinociceptive effect of an intrathecal administration of 5-HT (see ref. 24) is associated with a postsynaptic inhibition of nociceptive neurons [7] and/or a negative influence of the indoleamine on the excitability of primary afferent fibres [2]. Such pre- and postsynaptic mechanisms have also been proposed to explain the analgesic effect of opiates at the spinal level [23], and binding studies with radioactive opiate agonists and antagonists confirmed that some opiate receptors are located presynaptically on primary afferent fibres [4, 5]. In this study, we used a similar approach to examine whether 5-HT Correspondence: G. Daval, Departement de Cytologie, lnstitut des Neurosciences, C.N.R.S.U.A. 4199, Universit6 Pierre et Marie Curie, 7 Quai Saint-Bernard, F-75005 Paris, France. 0304-3940/87/$ 03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd.

72 receptors are also located on primary afferent fibres, as expected from a presynaptic action of the indoleamine [2]. 5-HT receptor binding sites were labelled by [3H]5-HT or [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) [22] and studied by quantitative autoradiography. Unilateral dorsal rhizotomy [10] or neonatal treatment with capsaicin [13] was used to destroy primary afferent fibres and examine the associated changes in 5-HT receptor binding sites at the spinal level. For comparison, the fate of/~- and 6-opiate receptors selectively labelled by [3H]naloxone, [3H]Tyr-DAla-Gly-(Me)Phe-Gly-ol ([3H]DAGO) [6] and [3H]Tyr-D-Ser-Gly-Phe-Leu-Thr ([3H]DSTLE) [3] respectively was studied in the same animals. Capsaicin (50 mg/kg, s.c.) was administered to 3 male Sprague Dawley rats on the 2nd day after birth. This treatment causes a selective destruction of unmyelinated primary afferent fibres [13]. At 3 months of age, these animals and corresponding controls (treated with the vehicle) were anaesthetized with chloral hydrate (350 mg/ kg, i.p.), and sacrificed by an intracardial perfusion with 500 ml of S6rensen buffer (0.15 M sodium phosphate, pH 7.4). One cm of cervical spinal cord was dissected and immediately frozen at - 4 0 ° C in isopentane cooled with liquid nitrogen. Unilateral rhizotomy of dorsal roots C4 to C6 was performed in 3 adult male Sprague Dawley rats (3 months old; 25~300 g body weight) as previously described [10]. To prevent the autotomy behaviour that occurs in rhizotomized rats, operated animals were housed with unoperated females (see ref. 10). Seven days after surgery, the rats were sacrificed and the tissue was treated as above. Sections of 16-/~m of the spinal cord were cut at - 2 0 ° C in a cryostat and thawmounted onto gelatin-coated slides. Sections were preincubated for 30 min in 0.17 M Tris-HCl buffer, pH 7.6, then incubated for 1 h at 20°C in the same buffer, in the presence of 1 nM [3H]naloxone (55 Ci/mmol, New England Nuclear) of 1 nM [3H]DAGO (60 Ci/mmol, Amersham) to label /l-opiate binding sites, 1 nM [3H]DSTLE (30.5 Ci/mmol, New England Nuclear) for 6-opiate binding sites, 2 nM [3H]5-HT (11.7 Ci/mmol, New England Nuclear) for 5-HTI binding sites and 2 nM [3H]8-OH-DPAT (85 Ci/mmol, Service des Mol6cules Marqu6es, CEN Saclay, France) for 5-HTIA binding sites. After two 5-min washes in ice-cold buffer, the sections were dipped into ice-cooled distilled water and dried with cold air. Sections were exposed in the dark at 4°C to tritium-sensitive film (LKB) for 2 months ([3H]8OH-DPAT), 3 months ([3H]5-HT, [3H]naloxone) or 5 months ([3H]DAGO, [3H]DSTLE). Autoradiograms were developed in Microdol (Kodak). Non-specific binding was estimated from adjacent sections incubated with the same labelled ligand in the presence of 50/tM naloxone (opiate binding sites) or 10/tM 5-HT (5-HT binding sites). Specific binding in the dorsal horn was measured with a Quantimet 720 densitometer (Cambridge, U.K.), and expressed in optical density (O.D.) units (see ref. 22). Measurements made on different films were calibrated with tritium standards (Amersham). Fig. 1 illustrates that 5-HTI binding sites like those for opiates are most concentrated in the superficial layers of the dorsal horn (lamina I and ll). One week after dorsal rhizotomy,, there was a decreased labelling of both opiate and 5-HT binding sites in the dorsal horn ipsilateral to the lesion (Fig. 1), but no change was detected

73

cont rol~ i d erhizotomized side

B

A

o

C

D

Fig. 1. Autoradiographic images of cervical spinal cord sections labelled with tritiated ligands after unilateral (right) dorsal rhizotomy. Coronal sections were incubated with: A: 2 n M [3H]8-OH-DPAT; B: 2 n M [3H]5-HT; C: 1 n M [3H]DAGO; D: 1 n M [3H]naloxone. Increasing density of labelling goes from white to black•

in other areas of the spinal grey matter. Densitometric measurements showed that all types of binding sites were affected (Table I). Compared to the unoperated side, there was a 43% reduction of [3H]DAGO and [3H]naioxone binding in the superficial layers of the dorsal horn ipsilateral to the rhizotomy. The loss was less pronounced but still significant for 5-HT binding sites ( - 2 3 % with [3H]5-HT; -22% with [3H]8OH-DPAT) (Table I). Since rhizotomy causes a non-selective degeneration of primary afferent fibres, we used neonatal treament with capsaicin to selectively destroy unmyelinated fibres [13]. This treatment also produced a significant loss of 5-HT binding in the superficial layers of the dorsal horn (Table I). As indicated in Table I, the capsaicin effect was simi-

5-HT~ 5-HT~A /~ I~

¢5

[3H]5-HT [3H]8-OH-DPAT [3H]DAGO [3H]nal°x°ne

[3H]DSTLE

~Contralateral side; bipsilateral side.

Binding sites

[3H]Ligand

0.081 _+0.009

0.088 _+0.007 0.115_+0.006 0.162+0.015 -

Control

0.052+0.004**

0.064 +0.007* 0.081+0.003"* 0.112+0.011"*

Treated

Capsaicin

(36)

(27) (30) (31)

(%)

0.068 + 0.005* 0.095_+0.005* 0.100_+0.008"* 0.069 ± 0.004**

0.122 _+0.012

Lesioned b

0.088 __+0.004 0.122+0.010 0.176+0.007

Control .

Rhizotomy

(43)

(23) (22) (43)

(%)

O.D. measurements were made at the cervical level of the spinal cord. Each value is the m e a n i S . E . M , of 6-10 individual determinations in 3 rats for each assay condition. The percent reduction due to each treatment is indicated in parentheses. * P < 0 . 0 5 , **P<0.01 (Student's t-test) when compared to respective control values.

EFFECT OF N E O N A T A L T R E A T M E N T W I T H C A P S A I C I N O R U N I L A T E R A L D O R S A L R H I Z O T O M Y ON 5-HT A N D OPIATE B I N D I N G SITES IN THE S U P E R F I C I A L LAYERS OF T H E D O R S A L H O R N

TABLE I

75 larly pronounced when 5-HT binding sites were labelled with either [3H]8-OH-DPAT ( - 3 0 % ) or [3H]5-HT ( - 2 7 % ) . Consistent with previous studies [5], opiate binding was also significantly reduced in capsaicin-treated rats; the reduction of 3H-ligand binding was approximately the same ( ~ - 3 0 to - 3 5 % ) whether [3H]DAGO or [3H]DSTLE was used to label/t- or 0-opiate receptors respectively (Table I). Previous autoradiographic studies have demonstrated that both 5-HT1A and 5HTIB receptor binding sites are most highly concentrated in lamina I and II of the dorsal horn [17]. The present data confirm this observation and further show that the degeneration of primary afferent fibres due to dorsal rhizotomy or neonatal capsaicin treatment is associated with a significant reduction in the density of 5-HT~ binding sites within the superficial dorsal horn. Although it cannot be totally excluded that this change resulted from some transynaptic events consecutive to the lesions, two series of observations rather support that it reflected the disappearance of 5-HTI receptor binding sites located on unmyelinated primary afferent fibres. First, a loss of opiate binding sites also occurs at the same level after both treatments (see also refs. 4, 5), and evidence has been provided by several groups (see refs. in ref. 23) that it is the direct consequence of the disappearance of opiate receptors located on primary afferent fibres. Second, previous demonstration of the presynaptic location of 5-HTI binding sites on sensory (visual) afferent fibres has been achieved in another region where 5-HT exerts a presynaptic control, i.e. the superior colliculus in the rat [21]. Although convergent observations support that the functional primary afferent opiate effect is a decrease of substance P (SP) release [9, 11],the evidence for 5-HT is less clear. Thus Mudge et al. [12] found an inhibitory influence of 5-HT upon SP release from cultured dorsal root ganglion neurons, but Pang and Vasko [16] failed to detect a comparable action in spinal cord slices. Since SP is but one of many putative neurotransmitters of primary afferent neurons, further studies will be necessary to identify which are regulated through 5-HTj receptors. Even after complete destruction of primary afferent fibres by dorsal rhizotomy, there was only a 20 25% reduction of [3H]5-HT and [3H]8-OH-DPAT binding sites. This suggests that most 5-HT1 (labelled by [3H]5-HT) and particularly 5-HTIA (labelled by [3H]8-OH-DPAT) receptors are located postsynaptically, as expected from anatomical data which revealed that bulbospinal serotoninergic fibres contact postsynaptic neurons but not primary afferent fibres within the dorsal horn [14, 20]. In conclusion our data are consistent with studies that demonstrated that 5-HTI (but not 5-HT2, see ref. 24) receptors are involved in both pre- [2, 8] and postsynaptic [7, 20] controls on the transmission of nociceptive messages at the spinal level. This research has been supported by grants from C.N.R.S. (ATP), I N S E R M and DRET. We are very grateful to A. Patey and P. Mailly for excellent assistance in densitometry, and D. Touret for preparing photographs. 1 Basbaum, A.I. and Fields, H.C., Endogenous pain control systems: brain stem spinal pathways and endorphin circuitry,Annu. Rev. Neurosci.,7 (1984) 309-338. 2 Carstens, E., Klumpp, D., Randic, M. and Zimmermann, M., Effect of iontophoreticallyapplied 5hydroxytryptamineon the excitabilityof single primary afferentC- and A-fibresin the cat spinal cord,

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