Properties of Individual Embryonic Primary Afferents and Their Spinal Projections in the Rat

Károly Mirnics and H. Richard Koerber

Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Mirnics, Károly and H. Richard Koerber. Properties of individual embryonic primary afferents and their spinal projections in the rat. J. Neurophysiol. 78: 1590-1600, 1997. Embryonic (E19-E20) and early postnatal (P2) spinal cords with intact saphenous and sciatic nerves were isolated and placed in aerated artificial cerebral spinal fluid (CSF). Intracellular recordings were made from cells in the L2-L6 dorsal root ganglia using microelectrodes filled with 3 M potassium acetate or 5% neurobiotin (NB) in 1 M potassium acetate. Several physiological properties of adequately impaled cells were measured, including peripheral conduction velocity, action potential (AP) amplitude and duration, duration of afterhyperpolarization (AHP), input impedance, rheobase, presence of inward rectifying current, and maximum somal firing frequency. The extent to which these properties are correlated also was determined. One cell per ganglion was injected with NB. Stained somata and their central projections in the spinal cord were visualized in serial 50 µm sections. Cell size was determined and the central morphology of the central projections examined. Although some fibers were in the process of growing into the spinal cord, others had established projections over several millimeters in the dorsal columns. Although most of these fibers supported projections in the gray matter, 22% only maintained fibers in the dorsal columns. Fibers with projections in the dorsal horn exhibited three types of morphology: projections confined to the superficial dorsal horn (laminae I, II); terminals confined to laminae III-V; and projections spanning laminae II-V. In addition, some embryonic fibers maintained projections to the dorsal horn that extended over five lumbar segments. Somal APs could be divided into two groups: broad spikes with inflections on their falling phase and narrow spikes without inflections. On average, cells with broad spikes (BS) had the following characteristics: slower peripheral conduction velocity, larger amplitude, higher rheobase and input impedance, longer AHP duration, and lower maximum firing frequency. There were significant correlations between conduction velocity and several of the physiological properties. Conduction velocity was negatively correlated with AP duration, rheobase, and input impedance and positively correlated with maximum firing frequency. Comparisons between spike shape and central morphology revealed that cells lacking collaterals in the gray matter and those with projections in the superficial dorsal horn always had broad somal spikes with inflections. Those with projections confined to laminae III-V always had narrow somal spikes (NS).

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Primary sensory neurons in the dorsal root ganglia have diverse but characteristic physiological properties. These properties have been shown to be associated closely with the type of peripheral receptor innervated in the periphery. For example, afferent fibers innervating nociceptors in the periphery always have broad somal spikes with inflections on the falling phase of the spike, whereas nonnociceptors have narrow somal spikes without inflections (Koerber et al. 1988; Lawson et al. 1988; Rose et al. 1986; Snow and Myers 1985). Although the central projections of primary afferent fibers also exhibit a great deal of diversity, the terminations of individual fiber collaterals often form morphologically distinct patterns in the spinal gray (Brown 1981; Koerber et al. 1991; Woolf 1986, 1987). These morphological phenotypes are associated uniquely with specific peripheral response properties (Koerber et al. 1991; Woolf 1986, 1987). In addition to this distinct central morphology, primary afferent projections are organized somatotopically, forming a precisely mapped presynaptic neuropil in the dorsal horn (Brown et al. 1992; Koerber and Brown 1980, 1982; Rivero-Melián and Grant 1990, 1991; Shortland et al. 1989).

Development of the distinct primary afferent fiber physiological and morphological phenotypes and the somatotopic organization of their projections to the dorsal horn has been the subject of numerous studies. Classic Golgi studies described the general morphology of these projections in the embryonic and neonatal rat and cat (Ramón y Cajal 1909; Scheibel and Scheibel 1968; Szentágothai and Réthelyi 1973). Subsequent studies employed horseradish peroxide (HRP) transport in bulk labeling techniques to examine more closely the timing of the growth of fibers in the spinal cord and the establishment of somatotopic organization in the dorsal horn (Fitzgerald 1987; Kudo and Yamada 1987; Smith 1983). The discovery of new lipophilic tracers, such as DiI, have enabled investigators to use fixed tissue, allowing a more comprehensive description of this process (Honig and Hume 1986; Mendelson et al. 1992; Mirnics and Koerber 1995a,b; Ruit and Snider 1991).

Results from the more recent studies using bulk labeling techniques have suggested that primary afferent fiber projections display much of the adult specificity early in the developmental process. Fibers apparently grow directly to somatotopically appropriate locations in the dorsal horn (Mirnics and Koerber 1995b; Smith 1983) and establish connections within specific laminae (Mendelson et al. 1992; Mirnics and Koerber 1995b; Ruit and Snider 1991). One notable exception to this specificity is the extension of flame-shaped arbors into the superficial dorsal horn during embryonic and neonatal development (Fitzgerald et al. 1994; Mirnics and Koerber 1995b; Ramón y Cajal 1909; Scheibel and Scheibel 1968; Szentágothai and Réthelyi 1973). In the adult, these fibers primarily innervate guard and tylotrich hair follicles and rarely extend into laminae IIi (Brown 1981). Fitzgerald et al. (1994) recently have examined the timing of this retraction and found that, in the rat, flame-shaped arbors assume their mature form ~3 wk of age.

Other studies have examined the development of physiological properties of primary sensory neurons. Many of these studies have used dissociated cell culture techniques to examine the development of ionic currents underlying the action potential (Nowycky 1992; Ransom and Holtz 1977; Spitzer 1983; Spitzer and Lamborghini 1976). In vitro studies examining the physiological properties of neonatal primary afferents also have suggested that the diversity of physiological phenotypes is evident at birth (Fitzgerald andFulton 1992; Fulton 1987).

Although these studies suggest that primary sensory neurons exhibit adult phenotypes from very early stages of development, technical limitations preclude verification of the appropriateness of single fiber projections. For example,previous studies could not determine if an individual fiber supported collaterals that projected to both somatotopically appropriate and inappropriate locations in the dorsal horn. Similarly, it was not possible to determine if all the projections of a single fiber were associated with specific laminae as in the adult. To address these questions and others, we have taken advantage of the outstanding diffusion properties of the new intracellular dye neurobiotin (NB) in an isolated spinal cord/dorsal root ganglion (DRG) in vitro preparation (Mirnics and Koerber 1995c; Mirnics et al. 1993). In this preparation, we penetrate individual DRG cell somas, physiologically characterize the cell, and iontophoretically inject the neuron with NB. Neurobiotin rapidly diffuses into the spinal cord, and the central projections of the single fiber can be visualized.

The experiments described here were designed to determine if the correlations between morphology and physiology of primary afferents present in adults are also present in the late prenatal and postnatal rat. A specific question addressed is, are the relationships between spike shape, afterhyperpolarization, conduction velocity, somatotopic organization, and laminar targets of the developing fibers the same as in the adult? A preliminary report has been published previously (Mirnics et al. 1993).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

The electrophysiological experiments were carried out using rats aged from embryonic day 19 (E19) to postnatal day 2 (P2). The dam was anesthetized with an intramuscular injection of a mixture of ketamine (90 mg/kg) and xylazine (2.5 mg/kg). The abdomen was opened by a transverse 5 cm cut, and a single embryo was dissected for the electrophysiology experiment. The dam was maintained under deep anesthesia using supplemental doses of ketamine (50 mg/kg) when necessary. Additional embryos were removed (if needed) by the same method until a viable preparation was obtained. Once the recording experiments were underway, the rest of the embryos were removed and transcardially perfused with 0.9% NaCl followed by chilled (+4°C) 4% paraformaldehyde in 0.2 M phosphate buffered saline. These embryos were stored for later use in fluorescent dye tracing studies. The dam was killed by an anesthetic overdose.

The dissected embryos and postnatal pups used in the in vitro electrophysiology experiments were anesthetized by cooling and staged according to morphological criteria (Hebel and Stromberg 1986). The embryos and pups were put into a dissecting chamber and submerged in aerated (95% O2-5% CO2) and chilled (+4°C) artificial cerebrospinal fluid (ACSF) containing (in mM) 127.0 NaCl, 1.9 KCl, 1.2 KH2PO4, 1.3 MgSO4, 2.4 CaCl2, 26.0 NaHCO3, and 10.0 D-glucose with a high-speed flow rate (>10 ml/min). During the dissection, the CSF gradually warmed to room temperature. The chest was opened, the sternum removed, and the embryos and pups were perfused transcardially with 5-15 ml of the same chilled and aerated CSF. After evisceration, the spinal cord and DRGs were exposed by a ventral approach. Individual peripheral nerves were identified and dissected with an intact lumbosacral plexus and DRGs attached. The nerves and plexus were freed from the surrounding tissue, while the L2-L6 DRGs were left embedded in the bony structures. The ventral roots were cut. To achieve the desired stability, the preparation was pinned down with 0.1-mm diam 2- to 4-mm-long insect pins to the bottom of the recording chamber [the bottom was covered with 2 mm silicone elastomer (Sylguard)] with the medial face of the DRGs and the ventral side of the spinal cord facing upward. A schematic representation of the preparation is shown in Fig. 1.


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FIG. 1. Schematic drawing of the experimental preparation.

The recording chamber with the preparation was supplied with a continuous inflow (>5 ml/min) of the oxygenated ACSF which was gradually warmed to 30-31°C. Individual nerves (saphenous, femoral, obturator, sciatic, or sural, depending on the experiment) were sucked into stimulation electrodes (Fig. 1). After a stabilization time of 30 min, individual DRGs were impaled intracellularly with glass microelectrodes containing 3 M potassium acetate or 5% NB in 1 M potassium acetate. Neurons with a resting potential of <= 50 mV and with an action potential overshoot were accepted for further study. After recording the somal action potential and conduction delay, both depolarizing and hyperpolarizing 50-ms current pulses were injected into the soma to determine rheobase, input impedance, and the presence of any inward rectification. In addition, the maximum firing rate was determined by passing brief depolarizing current pulses (200 µs) from 1 to 200 Hz. The DC recorded voltage responses were digitized (22 kHz) and stored on tape for off-line analysis. After the experiments, the somal spikes and the response to the various current pulses were averaged and plotted. Each somal action potential was classified as either a broad (BS) or narrow spike (NS) based on the presence or absence of an inflection on the falling phase of the spike. The determination of the presence of an inflection was made by calculating and plotting the first derivative (dV/dt) of the averaged trace (e.g., Koerber et al. 1988). It should be noted that in this study baseline duration of the action potential (AP) was not a criterion in distinguishing between broad and narrow spikes.

After the electrophysiological characterization, one cell per DRG was injected with NB (+1-2 nA, 75% duty cycle, 10-20 nA·min total current). When satisfactory transport to the spinal cord was reached (4-6 h depending on the injected DRGs and age of embryos), the DRGs were dissected from the bony structures and submerged in 4% paraformaldehyde in PBS. The dorsal and ventral roots were removed from the cord, and the appropriate segments (+2 segments from the most rostral and caudal injections) were cut and submerged in the same 4% paraformaldehyde. After 1 h postfixation, the DRGs and spinal cord were placed in 4% paraformaldehyde in 25% sucrose.

The next day serial 50 µm cross-sections were obtained by cutting both the cord and DRGs on the freezing microtome. The sections were mounted on double subbed slides and allowed to dry for several hours or overnight. Dried sections were submerged in cold 4% paraformaldehyde for 30 min to help prevent the loss of sections. After pretreatment with 1% bovine serum albumin (20 min) and 0.3% hydrogen peroxide (30 min), the sections were reacted for 2.5 h with avidin-HRP conjugate (VECTOR Laboratories, Elite kit). The sections were incubated in NiSO4-3,3'-diaminobenzidine chromogen and visualized by H2O2. After the washing period of 15 min, the sections were counterstained lightly by Neutral red, dehydrated, and mounted. Laminar boundaries were identified by morphological criteria as described in adult and developmental studies (Mirnics and Koerber 1995b; Rivero-Melian and Grant 1991).

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Anatomy

DRG. A total of 60 primary afferent somata was injected in 25 experimental preparations. Of these, 32 were judged to be stained adequately and were used for further analysis. Fibers were judged to be stained adequately if they were stained uniformly over the extent of the projection, i.e., the stain did not slowly fade out with distance in the dorsal columns or in the gray matter, and the fibers either extended beyond the sampled tissue or ended abruptly sometimes in growth cones. Fibers were recovered from three different age groups (E19, 13 fibers; E20, 14 fibers; P2, 5 fibers) that had projections in the spinal cord. In the embryonic preparations, the somata ranged in size from 10 to 32 µm. Although most stained cells were similar in appearance to surrounding cells (Fig. 2A), on some occasions, the cells were shaped very irregularly (Fig. 2B). It is not clear whether this is due to direct intracellular effects of NB, histological processing, or neuronal deterioration under the in vitro conditions. Regardless of the somal morphology, each cell recovered gave rise to a single fiber that bifurcated into a peripheral and central process with the peripheral branch usually having the appearance of being slightly larger in diameter (Fig. 2). In addition to the two main branches, some fibers also exhibited small collateral fibers that remained in the DRG. These finer collaterals arose from either main fiber (i.e., central or peripheral branch) and often branched extensively giving rise to several en passant and terminal axon swellings (Fig. 3). Although it is impossible to determine whether these swellings are synaptic boutons at the light microscopic level, they are similar in size (0.8-4.0 µm) and appearance to axonal swellings found in the dorsal horn.


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FIG. 2. Photomicrographs of 2 neurobiotin (NB)-stained dorsal root ganglion (DRG) cells in an E20 preparation. right-arrow, central processes; black-triangle, point to peripheral processes; *, collaterals within the DRG. A: this afferent had a broad somal spike and supported central collaterals only within the superficial laminae. Calibration bar = 20 µm. B: this afferent had a narrow somal spike and supported central collaterals only within laminae III, IV, and V. Calibrartion bar = 30 µm.


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FIG. 3. High-power photomicrograph of a NB-labeled afferent within a L4 DRG in a P2 rat. *, central process; right-arrow, terminal and en passant swellings on a short collateral within the DRG. This putative low-threshold mechanoreceptor (LTMR) supported boutons within dorsal horn laminae II, III, and IV (Fig. 7) and had a broad somal action potential (AP). Calibration bar = 20 µm.

Spinal cord

Central fibers that entered the dorsal columns ranged from 0.5 to 1.2 µm in diameter. Fibers usually bifurcated into a main rostral and caudal branch shortly after entering the cord. Although the general morphology of the embryonic and early postnatal fibers varied, the fibers examined in the present study fell into two basic groups. The first group of fibers entered the spinal cord, bifurcated, and extended rostrally and caudally but did not give rise to collaterals descending to the gray matter (n = 14; E19 = 6, E20 = 6, P2 = 2). The rostrocaudal extent of the main collaterals of these fibers ranged from 0.1 to 2.2 mm. In some cases, growth cones were evident in the dorsal columns near the dorsal root entry zone, indicating that the fibers just recently had reached the spinal cord and were actively growing. In other cases, the fibers already had traversed three to four spinal segments without generating additional collaterals. Several fibers (3 of 14) supported very short sprouts that only extended 10-30 µm from the main collateral and remained in the white matter.

Fibers in the second group (n = 18; E19 = 7, E20 = 8, P2 = 3) did give rise to descending collaterals that entered the gray matter (Fig. 4). Embryonic fibers (E19-20) gave rise to an average of eight collaterals (1-21) with a spacing of 25-950 µm. The main difference between projections of these fibers was laminar location of the terminal arborizations. Within this second group of fibers, there were three basic morphological types observed. One type of fiber exhibited projections confined to the superficial laminae (n = 3; E19 = 1, E20 = 2; Fig. 5) whereas a second group supported projections confined to laminae III-V (n = 13; E19 = 6, E20 = 6, P2 = 1; Figs. 4 and 6). The third set of fibers (P2 = 2) exhibited collaterals that terminated in both the superficial (laminae II) and deep dorsal horn (laminae III, IV, and V; Fig. 7). In addition to collaterals extending into the gray matter, afferents often would extend small collateral sprouts. These sprouts were usually 20-30 µm in length and remained confined to the dorsal columns.


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FIG. 4. Low-power photomicrograph of a cross-section from an E19 rat spinal cord. The cell soma had a narrow noninflected spike and was injected in the L4 DRG. The section was taken midway through the L4 segment. Note that this putative LTMR fiber arborized only within the laminae III and IV (*). right-arrow points to the main collateral in the dorsal columns. DH, dorsal horn; DC, dorsal column; CC, central canal; VH, ventral horn. Calibration bar = 100 µm.


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FIG. 5. High-power photomicrograph of a cross-section from an E20 dorsal horn. This afferent had a broad somal spike with an inflection. This putative nociceptive fiber had a central projection that was confined to the superficial dorsal horn. right-arrow, main collateral; d, dorsal; v, ventral; m, medial; l, lateral. Calibration bar = 50 µm.


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FIG. 6. Photomicrographs of cross-sections of dorsal horn from 2 different E20 preparations. Both afferents had noninflected narrow somal spikes. These putative LTMR fibers had central projections arborizing in laminae III-V. A: calibration bar = 50 µm; B: calibration bar = 20 µm.


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FIG. 7. Camera lucida reconstruction of flame-shaped arbor from a P2 preparation. This putative hair follicle afferent had an inflected broad somal spike. Note that the projection arborizes in laminae II-IV.

At these early stages of development, fibers with projections confined to the superficial dorsal horn had relatively simple branching patterns. These fibers were small [axon diameter, 0.5-1.0 µm; peripheral conduction velocity (CV), 0.2-0.8 m/s] and supported collateral branches that expanded mediolaterally and longitudinally within the superficial laminae (Fig. 5). These branches usually contain multiple axonal swellings and ended in terminal bouton-like structures or growth cones. At the same embryonic stages(E19-20), larger fibers (axon diameter, 0.8-1.2 µm; CV, 0.5-1.4 m/s) projecting to the deeper dorsal horn laminae supported more collaterals and exhibited a more complex branching pattern. Although descending collaterals of these fibers were observed to branch within the superficial laminae, these side branches always pointed toward the laminae III and IV and ended in growth cones without en passant swellings (Fig. 8). Fibers supporting collaterals with these two distinct central morphologies are most likely representative of developing cutaneous nociceptors and low-threshold mechanoreceptors (LTMRs). In older preparations, the central projections of these morphological types become more extensive and the branching more complex, but they remain within the same laminar boundaries.


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FIG. 8. Photomicrograph of a cross-section of dorsal horn from an E20 preparation. Afferent had a noninflected narrow somal spike. This putative LTMR fiber had a central projection that was confined to the deeper dorsal horn laminae. Note that the short branch coming off the penetrating collateral in lamina II (black-triangle) does not support putative synaptic swellings and is growing toward laminae III and IV. right-arrow, main collateral. Calibration bar = 50 µm.

The third set of fibers with a distinct central morphology had central projections that terminated in laminae II-V (Fig. 7). These fibers were relatively large in diameter and supported characteristic flame-shaped arbors in the dorsal horn. These arbors were very similar to those reported in adults for fibers innervating guard hair follicles (Brown 1981; Woolf 1986, 1987). In the adult, flame-shaped arbors occasionally extend into the ventral parts of laminae IIi, but for the most part they are confined to laminae III and IV with some diffuse projections to deeper laminae (e.g., Brown 1981). However, during development, these flame-shaped arbors have been shown to extend well into the superficial dorsal horn traversing all of laminae II (e.g., Ramón y Cajal 1909). In this series of experiments, these fibers were only observed in postnatal preparations.

In summary, with the exception of the distinct morphology of the developing flame-shaped arbors, individual primary afferent fibers exhibited characteristic and uniform morphological phenotypes during development. These results suggest that cutaneous fibers sending collaterals into the gray matter already have acquired a functional phenotype (e.g., nociceptor or LMTR). However, another population of unbranched fibers remained apparently uncommitted in the dorsal columns. Furthermore, it should be noted that during these late embryonic stages, fibers still are entering the spinal cord (Fig. 9). Apparently some inappropriate collaterals and fibers do exist (Fig. 10), suggesting a postnatal pruning of afferent collaterals to their adult somatotopic form.


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FIG. 9. High-power photomicrograph of the growth cone of an afferent located in the L5 dorsal root. This afferent fiber was activated by electrical stimulation of the saphenous nerve of an E20 preparation and had a broad inflected somal spike. DR, dorsal root. Calibration bar = 10 µm.


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FIG. 10. Photomicrograph of a cross-section E20 dorsal horn. Afferent had a noninflected narrow somal spike. This putative LTMR fiber had a central projection that was confined to the deeper dorsal horn laminae. Note the apparently inappropriate laterally growing branch coming off the main collateral in the dorsal columns (black-triangle). This collateral ended in a growth cone. right-arrow, main collateral. Calibration bar = 50 µm.

Long-ranging projections

As mentioned above, fibers supported as many as 21 collaterals descending into the dorsal horn. This is a significantly higher number than reported in adults (Shortland et al. 1989; Woolf 1986, 1987). In addition, these projections extend rostrocaudally over as many as 4.5 mm in the embryonic dorsal horn. Thus a single fiber can maintain projections over five to six lumbar segments. As depicted in Fig. 11, individual fibers project over roughly the same rostrocaudal extent in the dorsal horn as whole dorsal roots. The average projection length of cutaneous afferents in the adult rat dorsal horn is ~3-3.5 mm (Shortland et al. 1989), which is equivalent to about one lumbar segment. It should be noted that the average adult distance may be artifactually low as NB has been shown to be far superior to HRP in revealing long-ranging spinal projections of cutaneous fibers in the cat (Koerber and Mirnics 1995; Wilson et al. 1996). Regardless of the possible underestimation of the extent of single fiber projections in the adult, the findings presented here suggest the potential for postnatal reshaping of spinal projections by pruning of inappropriate collateral branches and growth of appropriate ones.


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FIG. 11. Drawings on the left of A and B represent the location and density of afferent fiber projections (dorsal view maps of lamina II/IV boundary) from the L3 and L5 DRGs to the E20 rat dorsal horn (taken from Mirnics and Koerber 1995b). Drawings on the right of A and B represent the rostrocaudal origin of collateral branches of putative LTMR fibers from 2 different E19 preparations injected in the corresponding DRGs. right-arrow, zone of entry; thick vertical lines, main collaterals in the dorsal columns; horizontal lines, origin of collaterals in the gray matter. Note that these afferents gave rise to terminals in the gray matter over 3.5 (A) and 4.5 (B) spinal segments. This distance is roughly equivalent to 90% of the length of the entire DRG projection.

Physiological properties

Intracellular recordings were made from 80 cells in embryonic and postnatal preparations (E19 = 26; E20 = 44; P2 = 10). In each case, the somal spike was recorded, and, when possible, the conduction delay after peripheral nerve stimulation was recorded along with the voltage response to hyperpolarizing and depolarizing current pulses (Fig. 12). At both embryonic stages studied, somal spikes exhibited the same diversity seen in the adult (Fitzgerald and Fulton 1992; Koerber et al. 1988; Ritter and Mendell 1992; Rose et al. 1986). Somal action potentials could be very broad (>11 ms baseline duration) with pronounced inflections on the falling phase of the spike, or they could be very brief (<2 ms) without an inflection (Fig. 12).


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FIG. 12. Examples of electrophysiological recordings and measurements. A: averaged traces of intracellular recordings (top) made from 4 afferent somas in response to peripheral nerve stimulation. Bottom: first derivatives of the APs. All spikes were recorded in the same E20 preparation. Note the different shapes and duration of the somal spikes. B: voltage response of 2 (E20) afferents to 50-ms hyperpolarizing current pulses of different intensities injected into the cell soma. Note the absence of inward rectification. C: voltage response of an E19 afferent to repetitive intracellular depolarizing current injections (200 µs pulse, 50 Hz). Eight traces of consecutive stimulation are superimposed. Note the failure of the afferent to respond to each stimulus with an action potential. right-arrow, time of intracellular current injection. D: voltage response of an E19 afferent to the saphenous nerve stimulation at 0.5 Hz. Five traces of consecutive stimulation are superimposed. Note the arrival of the dorsal root reflex spike in the cell soma at variable latencies following the spike evoked by stimulation of the saphenous nerve. right-arrow, onset of the nerve stimulation current pulses.

Comparisons were made between the different physiological parameters measured in E19 and E20 preparations, and no statistically significant differences were found. Therefore, the results from these two stages have been combined for further analysis.

In adults, it has been shown that there is a negative correlation between spike duration and conduction velocity. Although sensory neurons with the slowest conducting fibers always had BS and those with fastest conducting fibers NS, there was significant overlap in the CV ranges of these groups of cells (Harper and Lawson 1985; Koerber et al. 1988; Rose et al. 1986). In the embryonic preparation, this was also the case (Fig. 13A). This overlap during development suggests the parallel maturation of different fiber types. Although there was an overlap in spike duration, significant differences were observed between the average spike and afterhyperpolarization (AHP) duration of BS and NS cells (Table 1). BS and NS had the same average resting membrane potential, but BS cells had significantly larger spike amplitude, higher rheobase, and input impedance. NS cells also were able to respond with APs to repetitive intracellular stimulation at much higher rates than BS cells. These results are summarized in Table 1.


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FIG. 13. Plots of relationships between the electrical properties of the embryonic cells that showed significant correlation. Each symbol represents a single cell.

 
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TABLE 1. Summary of somal membrane properties

Comparisons between peripheral conduction velocity and a number of other parameters measured revealed strong relationships. As noted above, CV was correlated negatively with AP duration (r = 0.68; P < 0.001; Fig. 13A). In addition, there was a negative correlation with rheobase (r = 0.31; P < 0.05; Fig. 13B) and input impedance (r = 0.56; P < 0.001; Fig. 13C). There was a strong positive correlation between AP duration and AHP duration (r = 0.82; P < 0.001; Fig. 13D). Maximum firing frequency was correlated negatively with AHP duration (r = 0.41; P < 0.05; Fig. 13E) and positively correlated with conduction velocity (r = 0.38; P < 0.05; Fig. 13F). All correlations shown here are similar to those seen in adult preparations.

Another interesting finding in these preparations was the generation of dorsal root reflexes (DRR) in these afferent fibers (Fig. 12D). DRR could be activated by either stimulation of the peripheral nerve containing the afferent fiber or in nerves innervating adjacent regions of the hindlimb. Previous studies have shown that functional connections between afferent fibers and dorsal horn cells are established around E16-17 (Fitzgerald 1991; Kudo and Yamada 1987; Saito 1979). The results presented here suggest that functional axo-axonal connections between dorsal horn cells and primary afferents are present by E19, but could be established earlier.

Although many of these findings were similar to those observed in the adult, other findings differed. For example, there was no inward rectification in response to hyperpolarizing current pulses, and no cells were observed to fire multiple spikes in response to sustained depolarizing currents. This suggests that although many of the physiological properties of adult sensory neurons are present at these early stages of development, others develop later.

Correlation of between spike shape and central projections

The correlation between spike shape and central morphology was quite clear. Cells with NS (no inflections on their falling phase) always had central projections confined to dorsal horn laminae III-V. Cells with BS (with inflections) had central projections confined to laminae I and II. In addition, all cells that had central projections confined to the dorsal columns or were in the process of growing into the spinal cord always had BS. The two cells from postnatal preparations (P2) that supported flame-shaped arbors in laminae II-IV also had BS. Therefore, another consistent finding was that only fibers with BS maintained terminals within laminae I-II. Likewise, those with projections confined to dorsal horn laminae III-V always had NS.

Interestingly, one type of adult sensory fiber morphology that was not observed in these preparations and in ongoing studies in the mouse (Mirnics and Koerber 1995c; Mirnics et al. 1996) is that of the myelinated high-threshold mechanoreceptors, i.e., projections into both laminae I and laminae V (Light and Perl 1979).

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

The results from these studies have provided detailed information on the anatomic and physiological properties of individual developing primary afferents and their projections to the spinal cord. These findings both confirm and expand on earlier findings, which either examined primary afferent projections using bulk labeling techniques or in vitro preparations to record somal membrane properties. In agreement with earlier studies, fibers that entered the dorsal horn grew to very specific mediolateral and laminar locations in the dorsal horn, suggesting an early establishment of modality and somatotopic order (Fitzgerald 1987; Mendelson et al. 1992; Mirnics and Koerber 1995b; Smith 1983). However, some single fibers supported projections to the dorsal horn over the entire lumbar enlargement and thus have extensive projections that may be considered somatotopically inappropriate. Staining of individual fibers also has revealed additional details of the afferent fiber morphology in both the DRG and the spinal cord. Finally, correlation of somal membrane properties and the central projections of the individual developing fibers has allowed us to determine that many adult phenotypes are present during early development.

Collateral fibers in the DRG

One of the more interesting findings of this study was axon collaterals and bouton-like swellings within the DRG. Afferent fibers supporting intraganglionic arborizations originally were described by Ramón y Cajal (1909). However, these projections have not been investigated in any detail. Although there is no good evidence that DRG cells communicate by means of chemical synapses, sensory neurons do contain receptors for many neuroactive substances (reviewed in Willis and Coggeshall 1991) as well as neurotrophic molecules (for review, see Snider and Wright 1996). Therefore, it is quite possible that these projections could be functioning in a neuromodulatory role.

The results of the present study do not reveal any correlation between the appearance of these intraganglionic collaterals and any specific physiological or anatomic properties of the individual sensory neurons. Furthermore, we have not observed any evidence of synaptic transmission between afferent fibers during the intracellular recordings. However, it is possible that any potentials produced by these inputs are either very small or have a very slow rise time. The results of the present study do not suggest what function might be mediated by these projections during development. However, it is possible that direct communication between sensory neurons could have a trophic effect on fibers innervating the same peripheral targets or they could aid in the refinement of the somatotopic organization of central projections. For example, fibers innervating the same peripheral location or target tissue, could provide positive reinforcement (e.g., depolarization of membrane potential) allowing for more synchronized activity and strengthening of appropriate central connections.

Laminar distribution of terminals

At these early stages of development, individual afferent fibers exhibited a high degree of specificity in their central termination patterns. In many respects, these patterns are similar to adult morphological phenotypes, suggesting that individual embryonic fibers had acquired a specific identity. For example, fibers with slow conduction velocities and projections confined to the superficial dorsal horn most likely will mature as C-fiber nociceptors. Those fibers with faster embryonic conduction velocities and projections confined to deeper dorsal horn laminae most likely will mature as myelinated LTMRs.

One exception to this early appearance of adult morphological phenotypes is the existence of relatively fast conducting fibers that support flame-shaped arbors in laminae II-V. While it is known that these fibers retract from the superficial dorsal horn to occupy their adult locations around the third postnatal week (Fitzgerald et al. 1994), they may have a critical function during this period of development. For example, because these large fibers enter and establish projections before many of the C-fiber projections (Fitzgerald et al. 1991; Mirnics and Koerber 1995b), they may provide the afferent limb of nociceptive reflexes, such as the withdrawal reflex, during the early postnatal period (see also Fitzgerald et al. 1994). Another possible function of these fibers could be to provide somatotopic cues to the later arriving C fibers, as projections from both fiber types are in a somatotopic register in laminae II and III of adult animals (e.g., Mirnics and Koerber 1995b; Rivero-Melián and Grant 1990, 1991). This type of guidance mechanism could be either contact mediated (Sperry 1963) or activity dependent (Goodman and Shatz 1993; Hebb 1949).

Somatotopic organization

Another striking finding of this study is the extremely long rostrocaudal extent of some single fiber projections to the dorsal horn, i.e., the same extent as seen from labeling the entire dorsal root. This means that the area of skin innervated by a single primary afferent is represented across the same rostrocaudal extent as the skin included in the entire dermatome. This suggests a lack of somatotopic order in the rostrocaudal axis. However, projections in the dorsal horn occupied restricted mediolateral locations in the dorsal horn that were comparable with those seen in adult animals (Brown et al. 1991; Shortland et al. 1989), suggesting that even at the single fiber level, somatotopic order in the mediolateral axis of the dorsal horn exists very early in development.

One explanation for these long-ranging fibers would be that during early development fibers initially grow extensive projections that are pruned back later in development. However, recent anatomic studies using intracellular injection of NB in cat cutaneous afferents have shown that similar long-ranging functional projections exist in the adult. These collaterals are spaced up to a segment (4 mm) apart and are morphologically distinct from collaterals found in the center of the projection zone (Koerber and Mirnics 1995). Additional physiological evidence from P. D. Wall and colleagues (e.g., Wall and Shortland 1991; Wall and Werman 1976) suggests that afferent fiber projections may extend even further than has been anatomically documented. It has been suggested that these collaterals may be ineffective under normal conditions but can become functional after injury (e.g., Wall and MacMahon 1994). Thus instead of representing extensive diffuse projections that are pruned back later during development, these long-ranging collaterals may be maintained and have a specific function in the mature system (Koerber and Mirnics 1995).

The long-ranging cutaneous afferent projections to the spinal dorsal horn help clarify the results of previous studies examining plasticity in the projections of developing fibers. Denervation studies of neonatal spinal cord have shown a significant rearrangement in the projections of mature single fibers (Wilson and Snow 1991; Woolf et al. 1992). The anatomic substrate of this extensive plasticity very well could be the long-ranging collaterals seen during development. Expansions of primary afferent terminals in adult plasticity (LaMotte et al. 1989; Molander et al. 1988) also could employ denervation-induced growth of the intact long-ranging projections. Regardless of the degree of somatotopic appropriateness of these collaterals, their presence suggests the ability of the developing afferent fiber projections to exhibit abundant plasticity.

Fibers lacking dorsal horn projections

Another novel finding in this study is the group of fibers that appear to remain suspended in the dorsal columns. An obvious concern over this finding is whether these fibers were stained adequately. We have been very careful to include in this group only those fibers that were very darkly stained over their entire length. Although a few of these fibers ended in growth cones in the dorsal columns a short distance (100-200 µm) from the entry zone, the majority of these fibers traversed a segment or more without producing collaterals. The existence of a population of uncommitted fibers during perinatal development could help explain some recent findings suggesting that alterations in neurotrophin levels could alter the fate of a subpopulation of sensory neurons (Ritter et al. 1991). Specifically, after neonatal anti-nerve growth factor (NGF) treatment a subset of small myelinated fibers, which normally would become mechanical nociceptors, apparently switched phenotypes to mature as LTMRs. Taken together these results suggests that there may be a population of uncommitted fibers present in the spinal cord during late embryonic and early postnatal development that are responsive to variations in NGF levels. Further support for this possibility comes from the fact that one morphological phenotype not seen in the present study and in ongoing studies in mice (Mirnics and Koerber 1995c; Mirnics et al. 1996) is that of myelinated nociceptors, suggesting that they may mature later in development.

Physiological properties

The functional properties of the individual primary afferent fibers were much as expected from previous studies in both adult and developing preparations (Fitzgerald and Fulton 1992; Fulton 1987; Harper and Lawson 1985; Koerber et al. 1988; Ritter and Mendell 1992). Somal spikes were both broad with inflections on their falling phase and narrow lacking inflections. In addition, differences in the properties of the cells with different spike shapes were as expected. For example, on average, cells with broad spikes had slower conduction velocities, were larger in amplitude, and had higher input impedance and higher rheobase measurements. The relationships between conduction velocity and other physiological measurements were as expected from previous studies in the adult (e.g., Koerber et al. 1988; Ritter and Mendell 1992). Therefore, the physiological results also suggest that many afferent fibers have acquired mature functional phenotypes early in development.

It has been suggested that the narrowing of the somal spike and loss of the inflection in the A-fiber population were coincident with myelination of the peripheral fiber (Fitzgerald and Fulton 1992; Fulton 1987). In the present study, it is shown that narrow spikes without inflections can be seen as early as E19. Because peripheral myelination does not begin until after birth (Mirsky et al. 1980; Winter et al. 1982), it is probably not the determining factor in the change in somal spike shape.

Other functional properties of mature sensory neurons were not present in the developing animal. First, unlike mature cells, these cells were not capable of firing multiple action potentials in response to sustained depolarizing current pulses. Second, developing cells did not show any signs of the time and voltage-dependent inward rectification (Mayer and Westbrook 1983) in response to hyperpolarizing current pulses. These findings very well may be related as the inward rectification helps return the cell to a more depolarized level, which would be beneficial for repetitive generation of action potentials. Support for this possibility can be seen in adult preparations where it has been shown that sensory neurons that exhibit strong inward rectification, fire repetitively to a sustained depolarization (Belmonte and Gallego 1983; Harper 1986; Koerber et al. 1988).

Another interesting result of the present study was the early appearance of DRRs in the primary afferent fibers. Although the function of primary afferent depolarization and resulting DRRs during development is not clear, there are some possible roles for this gamma -aminobutyric acid-mediated current. First, during these early stages of development, the descending brain stem inhibitory systems have not been established (Fitzgerald and Koltzenburg 1986). Thus primary afferent depolarization may be the only effective means of inhibiting afferent input. On the other hand, it may have a very different function in the developing system. For example, it could serve as a form of positive feedback for sculpting dorsal horn cell receptive fields or pruning back inappropriate connections.

In summary, these physiological results show that developing afferent fibers have many of the functional attributes of adult sensory neurons. However, some other properties, e.g., some of the voltage-gated currents, apparently lag behind.

Structure/function relationship

The relationship between somal spike shape and the pattern of single fiber projections in the dorsal horn was quite consistent and resembled that seen in the adult. Cells with broad-inflected spikes maintained projections in the superficial dorsal horn, and those with narrow spikes had projections outside the superficial dorsal horn. The only novel fiber type seen was that which had inflected somal spikes but maintained flame-shaped arbors in laminae II-IV. Presumably, as these fibers retract their projections from the superficial dorsal horn they simultaneously loose the inflection on the falling phase of their somal action potential. Interestingly, it also has been suggested that the adult LMTRs that sprout into the dorsal horn in response to peripheral injury also develop inflections on the falling phase of the somal AP (Koerber et al. 1995).

Those fibers that remained confined to the dorsal columns, which are apparently still in the process of growing or waiting for additional cues to develop further, also have broad-inflected somal spikes. These results show a strong relationship between somal action potential shape and the location of central projections, suggesting that even at early stages of development somal properties can be a good indicator of the developmental state of the fiber.

Conclusions

The results of this study provide a detailed description of the anatomic and functional properties of individual developing sensory fibers. Anatomic results show that most fibers project to laminar and somatotopic locations in the dorsal horn that are appropriate for a single submodality and somatotopic order. Physiological results are also in agreement with these findings. Correlations between morphology and function of the developing fibers reveal that adult phenotypes are present during late fetal development. Finally, although there is a great deal of order and precision in the development of primary sensory neurons, the existence of long-ranging afferent projections and the population of apparently uncommitted cells in the dorsal columns, suggests that they retain the ability to respond to alterations in the normal developmental process.

    FOOTNOTES

  Address reprint requests to H. R. Koerber.

  Received 22 July 1996; accepted in final form 23 May 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society