THE JOURNAL OF COMPARATIVE NEUROLOGY 261~98-104 (1987)

Prenatal Growth of Fine-Diameter Primary Afferents Into the Rat Spinal Cord:

A Transganglionic Tracer Study

MARIA FITZGERALD Cerebral Functions Research Group, Department of Anatomy,

University College London, London WClE 6BT, England

ABSTRACT The aim of this study was to examine the growth of fine-diameter, A6

and C, primary afferents into the dorsal horn of the spinal cord. To do this, A6 and C primary afferents were transganglionically labeled with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) in fetal rats at var- ious ages and their growth was traced into the lumbar spinal cord. Sciatic or common peroneal nerves were injected with WGA-HRP at E18-E21.5 (just before birth) and the fetuses were returned to the uterus for 24 h before histochemical analysis. The results show fine-diameter afferent fibers grow- ing into the L4L5 spinal cord a t El9 when they reach the white matter overlying the dorsal horn and begin to penetrate lamina I. Twelve hours later at E19.5 terminals can be seen in lamina 110 and by E20 they are increasing in density and have reached IG. By birth they have achieved the density in laminae I and I1 that is found in the neonate and young rat. Labeling of only the peroneal branch of the sciatic nerve, leaving the tibia1 unlabeled, demonstrated that the somatotopic specificity of central terminal fields is apparent from the time fine afferent fibers first grow into the spinal cord.

Key words: development, dorsal roots, substantia gelatinosa, unmyelinated fibers, dorsal horn

Dorsal roots grow towards the thoracic and lumbar cord at embryonic day (E) 13.5-14.5 but they do not penetrate the grey matter until E15.5 (Windle and Baxter, '36; Smith, '83; Altman and Bayer, '84). The first afferents, presumably from muscle, grow down into the motoneuron pool in the ventral horn. These are followed by fibers terminating in the deep dorsal horn on E16.5-17.5, which are likely to be low-threshold cutaneous afferents. These data agree well with studies on reflex development. Hindlimb reflexes can first be evoked from cutaneous stimulation "in vivo" on E17.5 (Angulo y Gonzalez, '32; Narayanan et al., '71) but dorsal root stimulation evokes a ventral root reflex at E15.5 "in vitro" (Saito, '79). At birth (E21.5) cells in the dorsal horn respond to electrical stimulation of the fastest-con- ducting afferents, and cells with low- as well as high-thresh- old cutaneous receptive fields can be recorded (Fitzgerald, '85a).

The arrival and growth into the dorsal horn of A6- and C- fiber afferents and their termination in lamina I and I1 or substantia gelatinosa are less well established. Nociceptive

reflexes are evident at birth (Fitzgerald and Gibson, '84) but there is some postnatal delay in the maturation of C- fiber evoked responses in the spinal cord (Fitzgerald, '85a). Previous studies using Golgi impregnation (Scheibel and Scheibel, '681, HRP loading of thoracic nerves (Smith, '83) and electron microscope (EM) studies of C terminals (Pig- natelli et al., '85) have suggested that C fibers do not grow into the dorsal horn until birth or immediately thereafter. Peptides such as substance P and somatostatin, which are located in fine-diameter afferents, can be seen in laminae I and I1 just before birth (Semba et al., '82), whereas FRAP, which is present in dorsal root ganglion cells from El5 (Schoenen, '78), does not appear in the cord until 12 h after birth (Fitzgerald and Gibson, '84). A6 and C fibers make up the majority of primary afferents and the time of their arrival and termination in the dorsal horn is of obvious importance for understanding the developmental organiza-

Accepted January 21,1987.

0 1987 ALAN R. LISS, INC.

PRENATAL GROWTH OF FINE-DIAMETER PRIMARY AFFERENTS 99

tion of the spinal cord. Furthermore, while it has been clearly demonstrated that larger primary afferents, termi- nating deep in the dorsal horn, grow in a somatotopically ordered fashion (Smith, '83) it is not known whether fine afferent fibers also demonstrate this specificity.

To study the timing and pattern of A6- and C-fiber growth and early terminations in the dorsal horn, we have used transganglionic transport of wheat germ agglutinin-horse- radish peroxidase (WGA-HRP) from sciatic nerve branches in utero. WGA-HRP has been demonstrated to be prefer- entially taken up by small dorsal root ganglion cells in the adult rat (Robertson and Arvidsson, '85) and to exclusively label terminals in laminae I and I1 of the dorsal horn in the spinal cord (Swett and Woolf, '85). Here it has been used in fetuses to label growing fine afferents.

METHODS Wistar rats were mated in the departmental animal house

and conception was confirmed by the presence of a vaginal plug. Embryonic day (E) 0 was the day on which the plug was first detected. At E18-E21.5 (just before birth) rats were anesthetized with 40 mgkg pentobarbitone i.p. and placed on a heated pad. Under sterile conditions, the uter- ine horn on one side was exposed. Before an incision was made in the uterine wall, the position of the hindlimb of one rat fetus was located and a purse-string suture was sewn in a circle of about 0.5-cm diameter through the uter- ine muscle and amnion directly overlying the limb. An incision was then made in the center of the sutured area and the fetal limb was gently pulled through the hole. All the time, the purse strings were held with sufficient tension to hold the limb in place and prevent any more of the fetus from pushing through the incision and yet not so tightly as to constrict blood flow. The sciatic nerve was exposed at midthigh level in the fetal thigh and 0.2 p1 of 7.5% HRP- WGA (horseradish peroxidase conjugated with wheat germ agglutinin) was slowly injected into it over 5 min with a micropipette and microsyringe. Only if a brown swelling resulting from HRP under the neural sheath could be clearly observed under the microscope was the procedure continued.

In most cases the whole sciatic nerve was injected at a very proximal point at the sciatic notch so that its thigh branches as well as most distal divisions were labeled. In some cases, however, injections were made more distally and restricted to the common peroneal branch of the sciatic, leaving the tibial branch unlabeled. This was possible be- cause the peroneal lies lateral to the tibial and the dissec- tion through the lateral thigh of the fetus exposes the peroneal upmost. Indeed it was easier to inject this nerve alone than to ensure filling of the whole sciatic nerve with- out leakage of HRP-WGA into the underlying muscle. Pre- cision of injection site was not critical for assessing the time of fine afferent fiber growth into the dorsal horn, but it was essential for examining the somatotopic location of ingrow- ing afferents, and the peroneal injections were used for this part of the study.

Following injection, the thigh muscles and skin were su- tured and the fetal limb was gently pushed back into the uterus while the purse strings were drawn tight. When the limb was back in position and clearly free from the incision, the purse string suture was tied off and the uterine horn pushed back into the abdominal cavity. Two such injections

total time of fetal hindlimb surgery did not exceed 15 min. Longer than this often resulted in a deterioration of placen- tal blood flow and subsequent fetal death. It was essential to keep temperature and blood loss to a minimum.

The pregnant rat and her fetuses recovered uneventfully and abortion never occurred. Twenty-four hours after sur- gery they were reanesthetized with 40 mgkg pentobarbi- tone and the fetal cords were removed. In the case of animals operated on at E21, successful birth of the treated rat pups and their littermates had taken place in this interim period. Attempts at transplacental perfusion were not successful, and the operated fetuses were removed from the uterus, given further i.p. pentobarbitone, and perfused transcardially with 1.25% glutaraldehyde and 1.0% form- aldehyde in 0.1 M phosphate buffer at 0°C. The lumbar cord was removed and the positions of the L4 and L5 seg- ments were marked with insect pins after being traced back to the sciatic nerve entry. The cords were postfixed for 2 hr in a solution of 1:l fixative in 0.1 M phosphate buffer and 10% sucrose in 0.1 M phosphate buffer and then stored at 0°C in the sucrose solution alone. Transverse frozen sec- tions were cut at 30 pm and every section was stained for HRP labeling by the TMB method and counterstained with neutral red. Crystal artifact was a greater problem in fetal tissue than in neonatal or adult tissue, but it did not inter- fere with the results.

RESULTS

Growth of fine afferent fibers and their terminals in the dorsal horn

Fetal sciatic nerves or branches of them were injected with WGA-HRP on El8 (n = 21, E18.5 (n = 3), El9 (n = 3), E19.5 (n = 2), E20.5 (n = 3), and E21.5 (i.e., just before parturition) (n = 2).

The first age at which label was observed in primary afferents by this method was at E19, when the sciatic nerve had been injected at E18. Figure 1 shows an example of this. Fibers containing WGA-HRP are clearly observed in the dorsal roots and a small number reach the white matter overlying the dorsal horn. A very few fibers begin to pene- trate lamina I of the grey matter at this age and in one animal one or two terminal-like processes are observed in lamina I in a few sections (Fig. 2A). Twelve hours laters at E19.5 (the sciatic nerve having been injected at E18.5) the fine-diameter fiber afferents are seen to have grown consid- erably. Figure 2B shows that the afferent labeling is now located in lamina I1 of the dorsal horn. The terminals are still sparse at this age, however, and are largely restricted to the outer part of lamina I1 (110). Twelve hours later at E20 (injection at E19), the density of fine afferent terminals has increased and has now penetrated into the inner part of lamina I1 (111) (Fig. 2C). Just before birth at E21.5 (injec- tion at E20.5) the terminal labeling in laminae I and I1 is dense and prominent throughout the laminae (Fig. 3). It is now comparable with the early neonatal WGA-HRP label- ing of C fibers reported previously (Fitzgerald and Swett, '83) and that illustrated in Figure 4 at P1.

Somatotopy of growing fine-diameter afferents Another important feature of the growth of fine-diameter

afferents into lamina I1 is that it was found to be somatotop- icallv specific. In Figure 2, only the peroneal branch of the

were attempted per pregnant rat, one in each horn. The sciatic nerve was injected with WGA-HRP at E18.5 and

100 M. FITZGERALD

Fig. 1. A 30-pm section of L4 spinal cord at E19, following WGA-HRP application to the whole sciatic nerve 24 h earlier at E18. Label can be seen in the dorsal root (dr) and in the white matter (wm) overlying the dorsal horn. Some fibers have penetrated lamina I. Scale: 100 pm.

E19, whereas the tibial branch was left unlabeled. As can be seen in Figure 2, the afferent terminal label was in this case restricted, in the mediolateral plane, to the central part of the dorsal horn, which is the area normally occupied by peroneal afferents in the adult (Swett and Woolf, '85). The medial part of the dorsal horn, which is normally occupied by tibial nerve afferents in the adult, and the most lateral part, normally occupied by proximal thigh nerves, are not invaded by ingrowing peroneal nerve afferents. In Figure 3 the label was applied at E20.5 and also excluded the tibial nerve. Here the section is through L5, where the peroneal nerve terminals spread more laterally. In Figure 3 a very clear medial border can be seen abutting the unlabeled terminal region of tibial nerve afferents. In Fig- ure 4, the whole sciatic nerve is labeled at the sciatic notch, therefore including proximal thigh nerves, and the whole mediolateral extent of lamina I1 is full of labeled afferent terminals.

DISCUSSION By injection of peripheral nerves in the hindlimb with

WGA-HRP at various times during fetal life, the growth of fine-diameter, A6 and C afferents into the lumbar dorsal horn and the development of terminals in lamina I1 or the substantia gelatinosa has been demonstrated. C fibers are known to terminate in both lamina I and lamina 11 of the dorsal horn in the adult (Willis and Coggeshall, '78; Dodd et al., '83; Sugiura et al., '86), whereas A6 terminals are

found in lamina I and deeper in lamina V (Willis and Coggeshall, '78). There is evidence that fine-diameter affer- ents, particularly C fibers, are labeled preferentially by WGA-HRP when it is applied to adult peripheral nerve or injected into the skin. The tracer is transported to small dorsal root ganglion cells (Robertson and Arvidsson, '85) and then transganglionically t o the terminals in lamina I and I1 (Swett and Woolf, '85). The same preferential label- ing is achieved in the newborn rat (Fitzgerald and Swett, '83). It seems reasonable, therefore, to assume that the majority of the primary afferents labeled in the present study are also C fibers. Some A6 fibers may also be labeled, but only those in lamina I, since no labeling was found in deeper laminae. In light microscope studies such as these, the structures stained may well be axons as well as termi- nals. EM studies will be needed t o clarify this.

The results demonstrate that fine afferent fibers grow into the lumbar dorsal roots and dorsal root entry zone at El9 but do not yet penetrate the dorsal horn at the time. They may reach the entry zone earlier than this, since earlier ages were not tested here. It is possible that before El9 A6 and C terminals are present in lamina I1 but cannot transport WGA-HRP, but this seems unlikely in view of the clear ability of growing axons to transport tracers to their growing tips as demonstrated in other systems (Scott, '82; Sretevan and Schatz, '84). Transport rate is also appar- ently unaffected by age (Ochs, '73). Our results show that 12 h later at E19.5 they have penetrated into the dorsal

Fig. 2. Sections 30 pm thick of L4 spinal cord at E l 9 (A) E19.5 (B) and growth of afferent fibers through the dorsal roots (dr) and the proliferation E20 (C) following WGA-HRP application to the common peroneal branch of of terminals (t) in lamina I, lamina 110, and Hi can be followed over this 24- the sciatic nerve 24 h earlier at E18, E18.5, and E19, respectively. The h period. Scale: 100 pm.

Figure 2

102

Fig. 3. A 30-pm section of L5 spinal cord just before birth at E21.5 following WGA-HRP application to the common peroneal 24 h earlier at E20.5. The terminal labeling in lamina I1 is dense and the sharp medial border with unlabeled tibia1 nerve afferent territory can be seen. Scale: 100 pm.

Fig. 4. A 30-pm section of L4 spinal cord at P1 following WGA-HRP application to the whole sciatic nerve at the sciatic notch (therefore including proximal thigh branches as well as distal nerves) 24 h earlier a t E21.5 cust before birth). The whole of laminae I and I1 is full of afferent fiber terminals. Scale: 100 wm.

M. FITZGERALD

PRENATAL GROWTH OF FINE-DIAMETER PRIMARY AFFERENTS 103

horn and formed sparse terminals mainly in lamina 110. By E20 they have increased in density and reached IIi. By birth at E21.5, the terminal labeling in laminae I and I1 is comparable to that seen in the first neonatal week (Fitzger- ald and Swett, '83).

The question whether C-fiber derents , as defined in adult sensory nerves, actually exist as a separate subgroup at these prenatal stages is an important one. In a sense, one could argue that in early development all primary afferents are C fibers in that they are all unmyelinated (Sima, '74; Fitzgerald, '85a). However, it is clear that particular fea- tures of future A-fiber and C-fiber afferents are distinguish- able long before myelination. Their cell body size differences in the dorsal root ganglia are apparent from the time of the cells' birth (Lawson et al., '74; Altman and Bayer, '84). The membrane properties and action potentials of A and C cells are also clearly distinguishable before myelination (Fulton, '87). Histochemical markers of fine afferent fibers such as fluoride-resistant phosphatase and substance P are ob- served in a subset of dorsal root ganglion cells from El5 and E19, respectively (Semba et al., '82; Schoenen, '78). Recordings of the physiological properties of primary affer- ents in the newborn rat show that C-fiber-type polymodal nociceptors, AG-type high-threshold mechanoreceptors, and AP-fiber-type low-threshold afferents are already distin- guishable groups at this time despite the uniformly low conduction velocities (Fitzgerald, '87).

The results here add to the existing evidence that fibers grow sequentially into the dorsal horn in order of decreas- ing size and terminate progressively more dorsally. Smith ('83) demonstrated that in thoracic cord the first fibers to grow into the grey matter at E15.5 appeared to be muscle afferents and penetrated deep into the ventral horn moto- neuron pool. These were followed by small fibers that ter- minated more dorsally in the deep dorsal horn, presumably cutaneous A afferents a t E16.5-17.5. Flame-shaped hair fol- licle afferents are first observed in lamina I11 at El9 (Beal, '82). Here we have shown that A6 and C fibers begin to penetrate the dorsal horn last of all, on E19, and produce terminals in superficial dorsal horn on E19.5-20. The dor- soventral sequential positioning of A and C afferents may be partly a straightforward question of space. If at E19, low- threshold A-fiber-type cutaneous afferents are occupying lamina 111, when C fibers arrive at El9 only lamina I1 is left unoccupied. However, clearly more sophisticated sig- naling systems must also play a part, perhaps in the form of specific cell surface antigens (Dodd and Jessell, '85). The results here show that fine afferent fibers grow into their exact somatotopic location just as has been reported for A fibers in deeper laminae (Smith, '83). None of the exuberant projections demonstrated in the developing neuromuscular junction or visual system (see Hopkins and Brown, '84) could be observed here. Nevertheless, this precision of early terminal growth can be easily disrupted if a nearby nerve is sectioned at birth. In this case nearby C fibers will grow into the area normally occupied by the cut nerve terminals (Fitzgerald, '85b).

The growth of fine afferent fibers into the dorsal horn beginning a t El9 and the elaboration of their terminals in the late fetal period agree with EM reports of the appear- ance of glomeruli and terminal structures attributable to C fibers in lamina I1 in the early neonatal period (Pignatelli et al., '85). C-polymodal nociceptors may be a clearly iden- tifiable functional group at birth (Fitzgerald, '87), but the

present results indicate that their central actions are likely to mature later in the postnatal period. This is interesting in view of the changes in physiological and anatomical organization of the dorsal horn that occur at this time. Projection neurons develop prenatally but the interneurons that make up the majority of lamina I1 neurons develop only postnatally (Bicknell and Beal, '84). C-fiber evoked responses cannot be generated in deep dorsal horn until P7-8, and dorsal horn cells have large receptive fields and long afterdischarges, and show little convergence in the early neonatal period (Fitzgerald, '85a). It is possible that the maturation of functional C-fiber terminals in the neo- natal period triggers the postnatal organization of dorsal horn cells.

ACKNOWLEDGMENTS The author gratefully acknowledges the assistance of P.

Ainsworth in the histological preparation of the tissue and of J. Middleton and M. Gardner in the care of the animals. The work was supported by the MRC.

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