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Previous Article | Next Article
Volume 17, Number 14, Issue of July 15, 1997 pp. 5536-5548
Copyright ©1997 Society for NeuroscienceCells in Laminae III and IV of the Rat Spinal Cord that Possess the Neurokinin-1 Receptor and Have Dorsally Directed Dendrites Receive a Major Synaptic Input from Tachykinin-Containing Primary Afferents
Magda Naim, Rosemary C. Spike, Christine Watt, Safa A. S. Shehab, and Andrew J. Todd Laboratory of Human Anatomy, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom
ABSTRACT
Many neurons with cell bodies in laminae III or IV of the spinal dorsal horn possess the neurokinin 1 receptor and have dorsal dendrites that arborize in the superficial dorsal horn. We have performed a confocal microscopic study to determine whether these cells receive inputs from substance P-containing primary afferents. All neurons of this type received contacts from substance P-immunoreactive axons, and in most cases the contacts onto dorsal dendrites were very numerous. A great majority (90-100%) of substance P-immunoreactive varicosities in contact with these cells were also immunoreactive with antibody to calcitonin gene-related peptide, indicating that they were of primary afferent origin. The density of contacts from substance P-immunoreactive varicosities onto these cells was significantly higher than that seen on cholinergic neurons in lamina III (which do not possess the receptor). Electron microscopy revealed that synapses were present at points of contact between substance P-immunoreactive boutons and dorsal dendrites of cells with the neurokinin 1 receptor. Some cells of this type belong to the spinothalamic tract, and we therefore examined neurons with cell bodies in laminae III or IV that possessed the neurokinin 1 receptor and were labeled retrogradely after thalamic injection of cholera toxin B subunit. These cells also received contacts from substance P-immunoreactive axons on their dorsal dendrites. The results of this study indicate that neurons of this type are a major target for substance P-containing primary afferents.
Key words: substance P; substance P receptor; tachykinins; confocal microscopy; electron microscopy; spinothalamic tract
INTRODUCTION
The primary afferent input to the spinal dorsal horn is arranged in a highly ordered manner, with fine-diameter fibers terminating mainly in the superficial part (laminae I-II), whereas larger afferents reach the deeper laminae (Light and Perl, 1979
; Sugiura et al., 1987
) axons, contain the neuropeptide substance P (McCarthy and Lawson, 1989
Although C fibers terminate mainly in laminae I and II, neurons with cell bodies in deeper laminae (III-V) frequently respond to C fiber input, and two mechanisms are thought to underlie this phenomenon. Activity may be relayed by neurons in superficial laminae with axons that pass ventrally (Light and Kavookjian, 1988
Antibodies recently have been raised against the neurokinin 1 (NK1) receptor on which substance P acts (Vigna et al., 1994
MATERIALS AND METHODS
Immunofluorescence. Seven adult albino Swiss rats (either sex, 220-310 gm) were anesthetized deeply and perfused with a fixative containing 4% freshly depolymerized formaldehyde in 0.1 M phosphate buffer (PB). Lumbar spinal cord segments were removed, stored in fixative for at least 4 hr, rinsed in PB, and cut into transverse or parasagittal sections (60 or 70 µm thick) with a vibratome. The sections were treated with 50% ethanol for 30 min to enhance antibody penetration (Llewellyn-Smith and Minson, 19926 M; Sigma, Poole, UK) or omission of the NK1 receptor antibody selectively abolished the corresponding type of fluorescent staining.
Sections were examined with a Bio-Rad (Hemel Hempstead, UK) MRC 1024 confocal laser scanning microscope equipped with a krypton-argon laser. They were viewed initially with epifluorescence, and NK1 receptor-immunoreactive neurons with cell bodies in laminae III or IV and dendrites that entered the superficial dorsal horn were identified. Because laminar boundaries cannot be identified accurately in material prepared in this way, only cells that were at least 150 µm below the dorsal white matter were studied. Each cell initially was scanned with a 20× objective lens and the 488 nm line of the krypton-argon laser to excite fluorescein (NK1 receptor-like immunoreactivity). Then the immunoreactive neurons were scanned with all three lines of the laser with a 40× or 60× oil immersion objective lens, and an extensive search was made for contacts formed onto the dendrites and cell bodies by varicosities that were substance P-immunoreactive (see Fig. 1). In sections reacted with CGRP antiserum, the dendrites and cell bodies of the NK1 receptor-immunoreactive neurons were scanned sequentially with the three lines of the laser to distinguish varicosities that contained only substance P immunoreactivity from those that were also CGRP-immunoreactive. At least 90 cells were examined in this part of the study, and over one-half of these were from sections that had been reacted with the CGRP antiserum. 
Fig. 1. An NK1 receptor-immunoreactive neuron with the cell body 290 µm below the dorsal white matter, which received numerous contacts from substance P-immunoreactive varicosities. This cell is number 10 in Figure 3 and Table 1. NK1 receptor immunoreactivity is shown in green (a-d), substance P immunoreactivity in blue (b-d), and CGRP immunoreactivity in red (b-d). a, A low-magnification view of the cell body and dendritic tree revealed with antibody to the NK1 receptor. The boxes show the regions represented in b-d. b-d, Three regions of the dendritic tree shown at higher magnification. In each region axons with only substance P (blue) or CGRP (red) immunoreactivity are visible, but many show both types of peptide immunoreactivity and appear purple. Many varicosities with both substance P and CGRP immunoreactivity are in contact with the dendrites of the NK1 receptor-immunoreactive neuron; where these overlap with the NK1 receptor immunoreactivity, this appears white. a was obtained from 35 optical sections 1 µm apart, b and d from four and five sections, respectively, at 0.7 µm, and c from nine sections at 0.5 µm apart. Scale bars: a, 65 µm; b-d, 10 µm.
[View Larger Version of this Image (143K GIF file)]
Quantitative analysis. A detailed analysis of the density of contacts from substance P-immunoreactive varicosities was performed on 12 NK1 receptor-immunoreactive neurons (four each from three rats) taken from sections reacted with substance P and CGRP antibodies and, for comparison, on six ChAT-immunoreactive neurons in lamina III (two each from three rats). These cells were selected at random from sagittal sections in which penetration of the peptide immunostaining apparently was complete. Complete series of scans at different focal planes (z series) initially were made through the cell with the 20× objective lens to reveal the dendritic arborization (immunostained with NK1 receptor or ChAT antibodies). Projections of these z series were used to reconstruct the cells with the aid of a computer drawing program. The boundary between lamina I and the overlying white matter and the ventral limit of the plexus of substance P-/CGRP-immunoreactive axons were indicated on the drawings. Then the cells were reexamined with the confocal microscope as described above, and the positions of all peptide-immunoreactive varicosities that contacted them were plotted onto these drawings (see Figs. 2, 3, 4). 
Fig. 2. Drawings of 6 of the 12 NK1 receptor-immunoreactive neurons for which a detailed analysis of the contacts from substance P-immunoreactive varicosities was performed. The cells are all from parasagittal sections. In each case the upper dashed line represents the border between lamina I and the dorsal white matter, whereas the lower line indicates the ventral limit of the dense plexus of peptide-immunoreactive axons. Filled circles show contacts from varicosities with both substance P and CGRP immunoreactivities; open circles are those with only substance P immunoreactivity. Scale bar, 100 µm.
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Fig. 3. Drawings of the other six cells for which a detailed analysis of contacts was performed. The lines and symbols are the same as those used in Figure 2. Scale bar, 100 µm.
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Fig. 4. Drawings of six ChAT-immunoreactive neurons seen in parasagittal sections. In each case the dashed line represents the ventral limit of the plexus of substance P-containing axons, and filled circles indicate contacts from substance P-immunoreactive varicosities onto the cells. Scale bar, 100 µm.
[View Larger Version of this Image (16K GIF file)]
To estimate the density of contacts onto different parts of the dendritic trees of the NK1 receptor-immunoreactive neurons, we divided segments of the dendrites of these cells into three groups: superficial dorsal dendrites (which lay within the plexus of peptide-immunoreactive axons in lamina I and II); deep dorsal dendrites (which lay deep to this plexus), and ventrally directed dendrites (which were defined as those terminating ventral to the cell body). The ChAT-immunoreactive neurons did not have significant ventrally directed dendrites, although in five of six cases, dendrites did extend into the plexus of substance P-immunoreactive axons. For these cells portions of the dendrites within the plexus and those parts that lay below it were analyzed separately.
Because two-dimensional views of the neurons underestimate the length of dendrites that are passing obliquely through the section, measurements of the depth at different points on the dendritic tree within the vibratome section were determined from the z series. For each cell these measurements were made at points at which dendrites originated from the soma, terminated, left the section, or branched and also where the gradient changed significantly. It was assumed that in between these points dendrites traveled in a straight line and the "real" length of the intervening dendritic segment was, therefore, determined as the square root of the sum of the squares of measured length and difference in depths. Lengths were measured with an image analysis program (KS400, Kontron Electronik, Munich, Germany).
Electron microscopy. To combine confocal and electron microscopy, we made some modifications to the immunofluorescence procedure described above. Three male albino Swiss rats (270-350 gm) were anesthetized and perfused with a fixative consisting of 0.2% glutaraldehyde and 4% formaldehyde in PB. Parasagittal sections (70 µm) were cut with a vibratome, treated with 50% ethanol and then with 1% sodium borohydride, each for 30 min, and rinsed extensively. Then they were incubated for 3 d in the antibodies to NK1 receptor, substance P, and CGRP at the same concentrations as described above, rinsed, and incubated overnight in the following secondary antibodies: anti-rabbit IgG conjugated to fluorescein, anti-goat IgG conjugated to lissamine rhodamine, and anti-rat IgG conjugated to Cy5 (all raised in donkey, diluted 1:100; Jackson ImmunoResearch). The diluent for all antibodies consisted of PBS containing 5% donkey serum, but without Triton X-100. After rinsing, the sections were mounted in Vectashield antifade mounting medium. Sections were viewed with the confocal microscope as described above, and from each of the three rats a single NK1 receptor-immunoreactive neuron with a cell body in lamina III or IV and dorsal dendrites that received numerous contacts from substance P-immunoreactive varicosities was selected. For each of these cells z series were obtained with the 488 nm line and 20× objective to reveal the dendritic tree, and then a selected region of the dendritic tree was scanned sequentially with each of the three laser lines and the 60× objective to record the positions of contacts from substance P- and CGRP-immunoreactive varicosities.
Then the vibratome sections that contained these neurons were processed to reveal NK1 receptor and substance P immunoreactivities with diaminobenzidine (DAB). The sections were removed from the coverslips, rinsed in PBS, and incubated for 3 d in a mixture of species-specific biotinylated secondary antibodies (donkey anti-rabbit IgG and donkey anti-rat IgG, diluted 1:200 in PBS with 5% donkey serum; Jackson ImmunoResearch). After further rinsing, they were incubated for 3 d in extravidin-peroxidase (diluted 1:1000 in PBS; Sigma), and peroxidase activity was revealed with DAB in the presence of hydrogen peroxide. The sections were treated with 1% osmium tetroxide for 30 min, dehydrated in acetone, and flat-embedded in Durcupan between glass coverslips. They were examined with the light microscope, and the selected parts of the NK1 receptor-immunoreactive neurons were identified and, in two cases, photographed. Then one of the coverslips was removed, and the sections were mounted onto blocks of cured resin. Ultrathin sections through the region of interest were cut with a diamond knife, collected on single-slot grids coated with Formvar, stained with lead citrate, and viewed on a Philips CM100 transmission electron microscope.
To identify the immunoreactive structures seen with the electron microscope, we printed the electron micrographs so that the final magnification was exactly equal to that of the corresponding confocal image. Photographs of the immunoreactive dendrite taken with the light microscope were printed at this magnification also. By a comparison of these images it was possible to determine the corresponding fluorescent profile for each DAB-labeled structure.
Labeling of spinothalamic neurons. Because we wanted to identify spinothalamic tract neurons, three adult male albino Swiss rats (280-320 gm) received stereotaxic injections of 4 µl of 1% cholera toxin B subunit (CTb) into the left thalamus. The procedure was performed exactly as described previously (Marshall et al., 1996
Antibody specificity. The NK1 receptor antibody was raised against a synthetic peptide corresponding to the 15 amino acid residues at the C terminus of the rat NK1 receptor, which was coupled to bovine thyroglobulin (Vigna et al., 1994
The monoclonal antibody raised against substance P (Cuello et al., 1979 -CGRP conjugated to bovine serum albumin with glutaraldehyde.
RESULTS
Confocal microscopy
Many large NK1 receptor-immunoreactive neurons were seen at depths between 150 and 300 µm below the dorsal white matter, and in most cases dorsal dendrites of these cells could be followed into the superficial laminae, where they often branched extensively. With confocal microscopy these branches could be followed even when they entered the dense plexus of immunoreactive dendrites, which is present in lamina I and the dorsal part of lamina II. The dendrites of these cells often extended for long distances in rostral and caudal directions but appeared to be more compact in the mediolateral axis, and the neurons therefore were seen best in parasagittal sections (Figs. 1-3) although, even in these, most cells had some dendrites that left the section. The dendritic morphology of the cells was variable, but, as reported by Brown et al. (1995)
Immunostaining with the substance P and CGRP antibodies was similar to that reported previously in the dorsal horn. An extensive plexus of immunoreactive fibers and varicosities was present in the superficial part (laminae I and II), and these structures were also present at a lower density throughout the remainder of the dorsal horn. Many axons possessed both types of peptide immunoreactivity; however, axons showing only substance P or only CGRP immunoreactivity also were present (Fig. 1). Although penetration of immunostaining with the substance P and CGRP antibodies appeared to be complete in transverse sections (because approximately equal numbers of immunoreactive structures were seen throughout the depth of the vibratome section), the penetration of peptide immunostaining in parasagittal sections was more variable, and in some cases there was a clear reduction in the density of immunoreactive profiles away from the cut surfaces of the section.
All of the dorsal dendrites belonging to these neurons that could be followed up into the superficial dorsal horn received contacts from substance P-immunoreactive varicosities (Figs. 1, 2, 3). These contacts were often very numerous, particularly on the large primary and secondary dendrites as they passed through lamina III and lamina II, where often it was possible to follow individual substance P-immunoreactive axons that ran along the length of the dorsal dendrites and on which the varicosities formed many contacts. Substance P-immunoreactive varicosities also formed contacts onto the cell bodies and ventrally directed dendrites of most of these neurons, although these contacts were much less numerous than those on the dorsal dendrites. In sections reacted with antibodies to both peptides, a great majority of substance P-immunoreactive varicosities that contacted all parts of these neurons were also CGRP-immunoreactive (Figs. 1, 2, 3), although varicosities that possessed only substance P immunoreactivity also were seen in contact with most of the cells. In addition, the cells received some contacts from varicosities with CGRP, but not substance P, immunoreactivity. Quantitative analysis
The 12 NK1 receptor-immunoreactive neurons analyzed quantitatively are illustrated in Figures 2 and 3. The parts of the cells that were included in the vibratome sections received contacts from between 100 and 434 substance P-immunoreactive varicosities. The proportion of these varicosities that were also CGRP-immunoreactive varied from 90.2 to 100% (Table 1). The superficial parts of dorsal dendrites of these neurons received between 13.5 and 26.9 contacts from substance P-containing varicosities per 100 µm (mean = 18.8 ± 4.0 SD), whereas the density of contacts per 100 µm on deeper parts of the dorsal dendrites was between 7.7 and 20.1 (mean 13.2 ± 3.9 SD) and on ventral dendrites was between 1.3 and 15.7 (mean 5.5 ± 4.6 SD).
Table 1. The numbers of contacts formed by varicosities with substance P immunoreactivity onto the 12 NK1 receptor-immunoreactive neurons illustrated in Figures 2 and 3
Cell number Total SP contacts Substance P + CGRP Substance P only Percentage of substance P also CGRP 1 434 414 20 95.4 2 252 247 5 98 3 135 123 12 91.1 4 188 181 7 96.3 5 160 160 0 100 6 164 148 16 90.2 7 137 127 10 92.7 8 201 194 7 96.5 9 232 222 10 95.7 10 285 269 16 94.4 11 100 94 6 94 12 280 272 8 97.1
The six ChAT-immunoreactive neurons analyzed are shown in Figure 4. None of these neurons showed NK1 receptor immunoreactivity. Although all of the cells were contacted by substance P-immunoreactive varicosities, the density of contacts was much lower than that seen on the NK1 receptor-immunoreactive neurons (Fig. 4). The portions of dendrites of these cells that lay within the plexus of substance P-containing axons received between 0.9 and 6.9 contacts per 100 µm from substance P-immunoreactive varicosities (mean 3.8 ± 2.3 SD), whereas parts of the dendrites that lay below the plexus received between 0.3 and 2.5 contacts per 100 µm (mean 1.3 ± 0.9 SD).
The differences between the densities of contacts from substance P-immunoreactive varicosities onto the dorsal dendrites of NK1 receptor-immunoreactive or ChAT-immunoreactive neurons were highly significant, both for dendrites within the plexus of substance P-immunoreactive axons (p < 0.0001, unpaired t test) and for those that lay below the plexus (p < 0.0001, unpaired t test). Electron microscopy
In sections reacted with biotinylated anti-rabbit and anti-rat IgG followed by avidin-peroxidase, profiles that had been labeled with fluorescein (NK1 receptor-immunoreactive) or Cy5 (substance P-immunoreactive) were stained with DAB and could be identified with both light and electron microscopy (Figs. 5-7). For each confocal image an approximately corresponding ultrathin section could be identified, although because the ultrathin sections were thinner than the "optical sections" obtained with the confocal microscope and the two may not have been perfectly parallel, not all of the profiles seen on a particular confocal image were present in a single ultrathin section and vice versa. However, by comparing the positions of DAB-labeled profiles in a series of ultrathin sections with profiles that were labeled with Cy5 in the confocal images, it was possible to identify the substance P-immunoreactive profiles that were in contact with the NK1 receptor-immunoreactive dendrites (Fig. 6b,d). Twenty-four substance P-immunoreactive varicosities that contacted these dendrites were identified with the electron microscope (six to ten from each of the three rats), and 22 of these were also CGRP-immunoreactive. At all of these contacts the substance P-immunoreactive varicosity was presynaptic to the immunoreactive dendrite at an asymmetrical synapse (Fig. 7).
Fig. 5. One of the NK1 receptor-immunoreactive neurons that was used for the electron microscopic analysis, seen in a parasagittal section. The cell body is 150 µm below the dorsal white matter, and one of the primary dendrites passes obliquely toward the superficial dorsal horn. This image was constructed from three optical sections at 1.5 µm intervals and shows the NK1 receptor immunoreactivity. The boxed area shows the region represented in Figure 6. Scale bar, 50 µm.
[View Larger Version of this Image (113K GIF file)]
Fig. 6. Combined confocal and electron microscopy of part of the dorsal dendrite of the cell shown in Figure 5. a, NK1 receptor immunoreactivity in a series of seven optical sections taken at 0.5 µm intervals. The arrow shows a small branch given off from the main dendritic shaft (D). b, A confocal image obtained from a single optical section, which was taken from the series used to generate a. In this case all three types of immunoreactivity are shown. Green represents NK1 receptor immunoreactivity, whereas blue and red represent substance P and CGRP immunoreactivity, respectively. Many structures show both types of peptide immunoreactivity and appear purple. Several substance P-immunoreactive varicosities are in contact with the NK1 receptor-immunoreactive dendrite (D) or its small branch (large arrow), and four of these are indicated with the numbered small arrows. The varicosity numbered 2 shows only substance P immunoreactivity, whereas those numbered 1, 3, and 4 have both substance P and CGRP immunoreactivity. c, The corresponding region seen with light microscopy after the immunoperoxidase reaction and at a focal depth approximately equivalent to the confocal image in b. The main dendritic shaft (D) and part of its small branch (large arrow) are seen clearly, whereas most of the right-hand branch is out of focus. Many of the substance P-immunoreactive varicosities are also visible, including the ones indicated in b (numbered small arrows). d, A low-magnification electron micrograph of the corresponding region. This section is also at a depth nearly equivalent to the confocal image in b. The main dendritic shaft (D) and its small branch (large arrow) can be seen. The substance P-immunoreactive varicosities indicated in b are visible again (numbered small arrows). Scale bar, 10 µm.
[View Larger Version of this Image (155K GIF file)]
Fig. 7. High-magnification electron micrographs to show synapses between the substance P-immunoreactive varicosities indicated with numbered arrows in Figure 6b-d and the NK1 receptor-immunoreactive dendrite belonging to the cell illustrated in Figure 5. The micrographs are taken either from the ultrathin section illustrated in Figure 6d (b, d) or else from nearby sections in the series (a, c, e). a, In a nearby section the substance P-immunoreactive axon (A; arrow 1 in Fig. 6b-d) forms a synapse onto the small branch (B), which was given off from the main dendritic shaft. b-d, The substance P-immunoreactive axonal boutons (A) (numbered 2-4 in Fig. 6b-d, respectively) form synapses onto the dendrite (D) of the NK1 receptor-immunoreactive neuron. e, The synapse shown in d is seen more clearly in a nearby ultrathin section. In each case an asymmetrical synaptic specialization is visible (between arrows). Scale bar, 0.5 µm.
[View Larger Version of this Image (189K GIF file)]
Spinothalamic tract neurons
The spread of tracer resulting from injection of CTb into the thalamus was similar in all three experiments, and one example is illustrated in Figure 8. In each case CTb immunoreactivity virtually filled the left thalamus, but there was no spread of tracer to the hypothalamus or midbrain. The appearance and distribution of labeled spinothalamic neurons in the lumbar spinal cord were the same as those reported previously (Marshall et al., 1996
Fig. 8. Drawings of the thalamic injection sites in one animal. The area of damage caused by the injection is shown in black, and the spread of tracer is indicated by shading. The numbers associated with each section refer to the distance in millimeters anterior to the ear bar. AM, Anteromedial thalamic nucleus; MG, medial geniculate nucleus; PF, parafascicular thalamic nucleus; PO, posterior thalamic nuclear group; VM, ventromedial thalamic nucleus; VL, ventrolateral thalamic nucleus; VPM, ventroposterior thalamic nucleus (medial); VPL, ventroposterior thalamic nucleus (lateral).
[View Larger Version of this Image (25K GIF file)]
Fig. 9. Substance P-immunoreactive contacts onto a spinothalamic neuron with the NK1 receptor. NK1 receptor immunoreactivity is shown in green (a-f), CTb immunoreactivity in blue (b), and substance P immunoreactivity in red (c-f). a, A low-magnification confocal image showing only the NK1 receptor immunoreactivity. The cell body is located 235 µm below the dorsal white matter, and dendrites extend up into the superficial dorsal horn. Boxes indicate the regions illustrated in b-f. b, NK1 receptor and CTb immunoreactivity in a single optical section through the cell body. c-f, Confocal images showing NK1 receptor and substance P immunoreactivity. The dendrites of this neuron receive numerous contacts from substance P-immunoreactive varicosities. a was obtained from nine optical sections 1.5 µm apart, c and e-f from four sections, and d from seven optical sections, each 0.5 µm apart. Scale bars: a, 50 µm; b, 20 µm; c-f, 10 µm.
[View Larger Version of this Image (143K GIF file)]
On the remaining five spinothalamic neurons, fewer contacts were observed; however, these cells were present in sections in which the penetration of substance P immunostaining clearly was incomplete.
DISCUSSION
The main finding of this study was that neurons with cell bodies in lamina III or IV that possess the NK1 receptor and have dendrites that penetrate the superficial dorsal horn receive numerous contacts from substance P-immunoreactive boutons. Although most of these contacts are located in laminae I and II (where substance P-containing axons are common), many of the cells also received contacts on the proximal parts of these dendrites and also on dendrites that arborize in the deeper laminae. A great majority of these boutons are apparently of primary afferent origin because they also contain CGRP, and, from the sample examined with electron microscopy, it appears that most or all of them form asymmetrical synapses onto the dendrites.
To confirm that this relationship was specific, we also examined the distribution of contacts formed by substance P-immunoreactive axons onto another population of lamina III neurons: those that contained ChAT. These were chosen because the ChAT immunoreactivity gives a Golgi-like filling of the neurons and therefore allows contacts on all parts of the dendritic tree to be identified. Although these cells did receive some contacts from substance P-immunoreactive boutons, the density of contacts was significantly lower than that on the dendrites of the NK1 receptor-immunoreactive neurons in the equivalent part of the dorsal horn. Technical considerations
Immunofluorescence and confocal microscopy offered advantages over other techniques for the present study, in particular the possibility of distinguishing three fluorochromes (Brelje et al., 1993Cells in laminae III and IV with the NK1 receptor
Neurons with cell bodies in laminae III and IV of the dorsal horn and dendrites that penetrate the superficial laminae initially were observed after Golgi impregnation. Although cells of this type were illustrated by Ramón y Cajal (1909)
De Koninck et al. (1992)
NK1 receptor-immunoreactive neurons with cell bodies in lamina V and dendrites that reach the superficial laminae are seen occasionally in rat dorsal horn (Brown et al., 1995
Because many substance P-containing primary afferents are thought to be nociceptors (Kantner et al., 1985
We have reported previously that some cells of this type belong to the spinothalamic tract (Marshall et al., 1996
FOOTNOTES
Received Feb. 26, 1997; revised April 30, 1997; accepted May 6, 1997.
This work was supported by grants from the Wellcome Trust and the Robertson Trust, which are gratefully acknowledged. We thank Dr. S. Vigna for donating antiserum to the neurokinin-1 receptor and Mr. M. Neilson for technical assistance.
Correspondence should be addressed to Dr A. J. Todd, Laboratory of Human Anatomy, University of Glasgow, Glasgow G12 8QQ, U.K.
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