Cobalt accumulation in neurons expressing ionotropic excitatory amino acid receptors in young rat spinal cord: Morphology and distribution

THE JOURNAL OF COMPARATIVE NEUROLOGY 344:321-335 (1994)

Cobalt Accumulation in Neurons Expressing Ionotropic Excitatory Amino

Acid Receptors in Young Rat Spinal Cord: Morphology and Distribution

I S T V ~ NAGY, CLIFFORD J. WOOLF, ANDY DRAY, AND LASZLO URBAN Department of Anatomy and Developmental Biology, University College London (I.N., C.J.W.)

and Department of Pharmacology, Sandoz Institute for Medical Research (A.D., L.U.), London WClE 6BN, England; Department of Anatomy,

University Medical School of Debrecen, 4012 Debrecen, Hungary (I.N.)

ABSTRACT Excitatory amino acids (EAA) acting on N-methyl-D-aspartate (NMDA), a-amino-3-hydroxy-

5-methyl-4-isoxazole propionic acid (AMPA) and kainate receptors play an important role in synaptic transmission in the spinal cord. Quantitative autoradiography and physiological experiments suggest that NMDA receptors are localized mainly in lamina I1 while kainate and AMPA receptors are found on both dorsal and ventral horn neurons. However the cell types expressing EAA receptors and their laminar distribution is not known. We have used a cobalt uptake method to study the morphology and distribution of spinal cord neurons expressing AMPA, kainate, or NMDA excitatory amino acid receptors in the lumbar enlargement of the rat spinal cord. The technique involved superfusion of hemisected spinal cords of 14 day-old rat pups in vitro with excitatory amino acid receptor ligands in the presence of CoC12. Cobalt has been shown to enter cells through ligand-gated ion channels in place of Ca2+. Cells which accumulated cobalt ions following activation by ionotropic excitatory amino acid receptors were visualised histochemically. The cobalt uptake generated receptor-specific labeling of cells, as the NMDA receptor antagonist D-(-)-2-amino-(5)-phosphonovaleric acid (D-AP-5) (20 yM) blocked the NMDA, but not kainate-induced cobalt uptake. The kainate-induced cobalt labeling was reduced by the non-selective excitatory amino acid receptor antagonist kynurenic acid (4 mM). Passive opening of the voltage-gated Ca2+-channels by KC1 (50 mM) did not result in cobalt uptake, indicating that cobalt enters the cells through ligand-gated Ca2+-channels.

AMPA (500 FM), kainate (500 yM), or NMDA (500 yM) each induced cobalt uptake with characteristic patterns and distributions of neuronal staining. Overall, kainate induced cobalt uptake in the greatest number of neuronal perikarya while NMDA-induced uptake was the lowest. AMPA and kainate, but not NMDA superfusion, resulted in cobalt labeling of glial cells.

Our results show that the cobalt uptake technique is a useful way to study the morphology and distribution of cells expressing receptors with ligand-gated Ca2+ channels. o 1994 Wiley-Liss, Inc.

Key words: cobalt uptake, AMPA receptor, kainate receptor, NMDA receptor

Excitatory amino acids ( E M ) play a major role in the synaptic transmission of motor and sensory systems in the spinal cord (for review, see Headley and Grillner, 1991). Excitatory amino acids act on two major types of receptors: ionotropic and metabotropic (Mayer and Westbrook, 1987; Collingridge and Lester, 1989; Monaghan et al., 1989; Miller, 1991a,b). Activation of the ionotropic receptors results in a fast excitatory postsynaptic depolarization (EPSP) by opening ligand-gated ion channels incorporated in the receptors (Barnard and Henley, 1990; Betz, 1990;

Nakanishi et al., 1990). Three types of ionotropic EAA receptors have been recognised on the basis of their pharma- cological characteristics: the a-amino-3-hydroxy-5-methyl- 4-isoxazole propionic acid (AMPA), the kainate, and N- methyl-n-aspartic acid (NMDA) receptors (Watkins et Evans, 1981; Mayer and Westbrook, 1987; Monaghan et al.,

Accepted December 13, 1993. Address reprint requests to Dr. Lasz16 Urban, Sandoz Institute for

Medical Research, 5 Gower Place, London WClE 6BN, England.

o 1994 WILEY-LISS, INC.

Figu

re 1

EM-EVOKED COBALT UPTAKE IN SPINAL CORD CELLS

LAMINA I LAMINA II

1989; Barnard and Henley, 1990). Each receptor type consists of subunits, which have been cloned (Wada et al., 1989; Bettler et al., 1990, 1992; Boulter et al., 1990; Keinanen et al., 1990; Moriyoshi et al., 1991; Werner et al., 1991). It has been shown that during activation of EAA receptors by any of the agonists, a substantial amount of Ca2+ enters the cells through the ligand-gated ion channels (Murphy et al., 1987; Murphy and Miller, 1989; Iino et al., 1990; Gilbertson et al., 1991).

Excitatory amino acid receptor ligands excite different groups of spinal cord neurons (Jahr and Jessell, 1985; Evans et al., 1987; Davies et al., 1988; King et al., 1988; Schneider and Perl, 1988; Jeftinija, 1989; Yoshimura and Jessell, 1990; Gerber and Randic, 1991). One view has been that AMPA and kainate receptors play a role in the generation of fast EPSPs at the first synapse between primary afferents and secondary sensory neurons (Schneider and Perl, 1988; Yoshimura and Jessell, 1990) or motoneurons (Buhrle and Sonnhof, 1983; Jahr and Yoshioka, 1986), while NMDA receptors are involved in the excitatory transmission through polysynaptic pathway in the dorsal horn (Watkins and Evans, 1981; Jessell et al., 1986; Headley and Grillner, 1991). Another has been that activation of NMDA receptors is responsible for the prolonged phase of EPSPs (Yoshimura and Jessell, 1990; Yoshimura and Nishi, 1993) and consequently for the long-lasting excitation of dorsal horn cells in response to C-fiber input (Woolf and Thomp- son, 1991). NMDA receptor activation has also been shown to be important for prolonged changes of spinal excitability such as occurs during the windup of action potentials in response to repeated C-fiber inputs (Davies and Lodge, 1987; Dickenson and Sullivan, 1987; Thompson et al., 19901, central sensitisation (Schaible et al., 1991; Woolf and Thompson, 1991; Ren et al., 1992) and hypoxic excitation (Gill et al., 1987; Urban et al., 1989). Several studies have shown differences in the sensitivity of spinal cord neurons located in different laminae to NMDA and non-NMDA receptor agonists (King et al., 1988; Schneider and Perl, 1988; Yoshimura and Jessell, 1990).

In spite of a relatively large body of physiological data, the distribution and morphology of spinal cord neurons express-

KAINATE

Fig. 1. A Low-power magnification of transverse sections of hemisected spinal cords after superfusing the cords with 1 mM CoC12 and (a) AMPA (500 pM), or (c) NMDA (500 pM), or (d) NMDA (500 pM) + D-AP5 (20 pM). The figure also shows the result of the control experiments (b), where no receptor agonist was added to the superfusate containing the cobalt. The pia mater and vessels entering the spinal cord were labeled in every case. Cells near to the cut surface were labeled more strongly than those elsewhere in the cord. In the AMPA-stimulated cord, cells were labeled in the white and grey matter, while in the NMDA-treated cords, only cells in the grey matter were labeled. When the selective NMDA receptor antagonist D-AP5 (20 pM) was superfused prior to, during, and after the NMDA application the staining of cells was almost completely blocked (d). In control experiments (b) only a few very faintly stained neurons were scattered throughout the grey matter. B: Low-power magnification of transverse sections of hemisected spinal cords after superfusing the cords with 1 mM CoCl, in the presence of (a) kainate (500 pM), or (b) kainate (500 pM) + kynurenic acid (4 mM), or (c) kainate (500 pM) + D-AP5 (20 pM), or (d) KC1 (50 mM). A great number of cells were labeled both in the white and grey matter after kainate application to the superfusate. This labeling was substantially reduced by the application of kynurenic acid. On the other hand the selective NMDA receptor antagonist AF'5 did not effect staining produced by kainate. Depolarization of spinal cord cells with KCl in the presence of CoClz did not produce apattern of labeling of cells which differed from the control experiment. Bar = 500 pm.

LAMINA I I I P 323

AM PA

LAMINA 111 LAMINA 1V LAMINA V

VENTRAL HORN vI k 0 20 40 60

PERCENTAGE OF LABELLED CELL BODIES

LAMINA IV LAMINA V LAMINA VI

VENTRAL HORN 0 20 40 60

PERCENTAGE OF LABELLED CELL BODIES

LAMINA II ' I 3 LAMINA 1 1 1 LAMINA IV LAMINA V LAMINA VI

VENTRAL HORN

NMDA

0 20 40 60 PERCENTAGE OF LABELLED CELL BODIES

Fig. 2. Laminar distribution of the relative number of cobalt- labeled perikarya after superfusing the preparation with 500 pM of AMPA (top), kainate (middle), and NMDA (bottom). Values were calculated from the results of cell counting in five randomly chosen sections and expressed as a percentage of Nissl-stained cells (see Materials and Methods).

ing different EAA receptors have not been studied in detail. Several methods can be used to visualize EAA receptors in the central nervous system. Autoradiography shows the regional location of binding sites but lacks the capacity to reveal the type of cell expressing the receptors (Monaghan and Cotman, 1985; Monaghan et al., 1989; Jansen et al., 1990; Mitchell and Anderson, 1991; Kalb et al., 1992). Studies using in situ hybridization (Furuyama et al., 1993) and immunocytochemistry (Martin et al., 1993) in spinal cord have revealed cells expressing different subunit mRNAs and subunit proteins of EAA receptors. However, the exact subunit composition of the functioning EAA receptors is not yet known. The accumulation of intracellular Ca2+ during the specific activation of ionotropic EAA receptors can be used to visualize neurons in vitro by means of either the calcium-sensitive dyes such as fura 2 or by substituting Co2+ for Ca2+ in the medium. While fura-2 can be used successfully on tissue culture, cobalt uptake can also be used on intact tissue (Hogan, 1983; Winter, 1987; Wood et al., 1988; Pruss et al., 1991; Fulton et al., 1992; Hu-Tsai et al., 1992; Williams et al., 1992; Nagy et al., 1993b). Williams et al. (1992) have shown that this technique produces a specific labeling of neurons as the cobalt entry induced by NMDA or non-NMDA receptor agonists was reduced in a

324 I. NAGY ET AL.

DENSITY OF LABELLED NEURONES

Fig. 3. Relative density of labeled perikarya after stimulating the cells with AMPA, kainate, and NMDA in the presence of cobalt (see Materials and Methods). The red colour represents the highest density while black represents the lowest density. The medial part of the dorsal horn shows higher density than the lateral part in each case. In lamina IV there is a high-density area in kainate and NMDA-treated spinal

TABLE 1. Distribution of Cobalt-Labeled Cells Sensitive to Excitatory Amino Acid Lipands in the Spinal Cord

Laminaitme of neuron AMPA Kainate NMDA

Lamina I Fusiform Flattened Pyramidal Multipolar

Lamina I1 Islet Stalked Transverseux of Cajal Multipolar

Lamina I11 Central Antenna Pyramidal

Lamina IV Central Antenna Pyramidal

+ 0 +

++ + -

++ + +

++ + + +

++ 0 +

+ -

+ + + -

t + + t

+ + 0

+ + ++ + 0 + -

t + 0

t + + +

dose-dependent manner by the co-application of selective NMDA or non-NMDA receptor antagonists, respectively.

Here we have studied the morphology and distribution of spinal cord cells expressing EAA receptors in young rats by using a modification of the cobalt uptake technique. Prelimi- nary results have been communicated in brief form (Nagy et al., 1993~).

cords. In the intermediate zone labeled cells are distributed homoge- neously after non-NMDA receptor agonist application. In the motoneuron pool a large proportion of cells were labeled after AMPA treatment, whereas kainate and NMDA produced staining in fewer motoneurons. The density of labeling was the highest in lateral spinal nucleus after kainate stimulation.

MATERIALS AND METHODS Preparation of hemisected spinal cord and

stimulation of cobalt uptake Under enflourane (Abbot) anesthesia the spinal cords of

14 day-old rats (Sprague-Dawley) were removed and imme- diately transferred into ice-cold buffer solution (solution A) containing in mM NaCl 118; KC1 4.6; MgS04 1.2; glucose

Fig. 4. High-power magnification of cobalt-labeled superficial dorsal horn neurons from transverse sections of spinal cords stimulated by AMPA (500 pM). A A typical triangular shaped perikaryon of a pyramidal lamina I cell (arrow). B: Cobalt-stained lamina I cell of the fusiform B type with a round cell body (arrow) and a proximal dendrite running toward the deeper laminae. C: Labeled cells in the middle part of lamina I (small arrow) and I1 (large arrow). Note the bulging appearance of some cell bodies in lamina I characteristic of multipolar cells and in particular the transversely oriented flattened cell in the outer part of lamina 11. D Round cobalt-labeled perikarya in lamina I and 110 (arrows). E: Round cobalt-labeled cells in lamina I (open arrows) and a fusiform neuron in lamina 110 (solid arrow). Its dendrites are running toward both the more superficial and deeper layers. F: Round (open arrow) and multipolar (solid arrow) cobalt-labeled perikarya in lamina 110. G A labeled cell (large arrow) with a ventrally running proximal dendrite (small arrows) in lamina IIi. Bar = 20 pm.

Figure 5

EAA-EVOKED COBALT UPTAKE IN SPINAL CORD CELLS

10; Na-HEPES 25, pH 7.4. After the dura mater was removed, the spinal cords were hemisected and placed in a dish where they were continuously superfused (2.5-3 ml/ min) with a buffer solution (solution B) containing NaCl 57.5 mM; KCl5 mM; CaClz 1 mM; MgClz 2 mM; HEPES 10 mM; glucose 12 mM; sucrose 139 mM; tetrodotoxin (TTX) 300 nM (pH 7.6). After a 5 minute saturation, 1 mM CoClz and 500 pM of a selective excitatory amino acid (EAA) receptor agonists, AMPA, kainate, or NMDA were added to the solution for 20 minutes, followed by a 20 minute wash in solution B (Pruss et al., 1991; Williams et al., 1992). After the spinal cords were rinsed in 2 mM ethylenediamine- tetra-acetic acid (EDTA, Pruss et al., 19911, they were transferred into solution B saturated with HzS, for 5 minutes, to transform the water-soluble CoClz taken up by the stimulated cells into insoluble CoS. Tissues were fixed in 4% paraformaldehyde for 24-48 hours and washed in 0.1 M phosphate buffer. The concentration of the agonists were chosen according to their activities described by Murphy et al. (1987) and Williams et al. (1992).

Histology Forty micron-thick transverse sections from the L3-L5

segments of the hemisected spinal cords were cut on a cryostat, mounted on gelatin-coated slides, and dried. Sec- tions were washed in distilled water and the CoS precipitate intensified by a silver-containing physical developer (Szbkely and Gallyas, 1975) for 17-18 minutes. Neutral red was used as a counterstain. The sections were dehydrated in alcohol, cleared in histoclear, and coverslipped with DPX.

Forty micron-thick transverse sections of a non-treated spinal cord were Nissl stained.

Control studies In control experiments no EAA receptor agonists were

added to solution B containing CoC12. The specificity of the cobalt labeling induced by the EAA receptor agonists was challenged by the selective NMDA receptor antagonist D-(-)-2-amino-5-phosphonovaleric acid (D-AP-5; 20 pM) and the non-selective EAA antagonist kynurenic acid (4 mM). Antagonists were superfused 5 minutes prior to the application of the agonists and were present continuously during the challenge with agonists and during the period of washing. Cobalt entry into cells was also studied by a depolarizing challenge with 50 mM KC1 in the presence of 1 mM CoClz for 20 minutes.

Fig. 5. High-power magnification of cobalt-labeled dorsal horn neurons in transverse sections of spinal cords stimulated by kainate (500 *MI. In lamina I a great diversity of labeled cells were observed. A Triangular-shaped cells (open arrows) of the pyramidal type with transversely oriented proximal dendrites are shown in the middle part of lamina I and round cells (solid arrows) in the outer part of lamina 11. B: A flattened cell in lamina I (black arrow) and round perikarya in lamina 110. Note the labeled fibres running transversely in lamina I in (A) and (B; white arrows). C: Round heavily labeled neurons in the lateral part of lamina I (white arrowheads) and outer lamina I1 (black arrowheads). The majority of these cells had no labeled proximal dendrite in the plane of the sections indicating that in lamina I they belong to the fusiform A type. D: In the medial part of the first lamina multipolar cells (arrows) were strongly stained. E: Round (arrowheads) and elongated (arrows) perikarya were stained in the third lamina. Their proximal dendrites were running toward the more superficial layers. F: Multipolar perikarya (arrow) in lamina 111. Bar = 40 wm.

327

Quantification of data The laminar distribution, percentage of labeled cells, and

density of the cobalt-stained cells were calculated as fol- lows. A grid was generated on microphotographs of five non-contiguous sections chosen randomly from treated spinal cords and from Nissl-stained non-treated sections. The grid consisted of “horizontal” lines at 30 pm intervals, following the border of laminae. The medio-lateral exten- sion of the grey matter was divided by 12 vertical lines. The cobalt-labeled and Nissl-stained perikarya found in each square were counted, the counts were averaged for the five sections, and the percentages of labeled neurons in squares and laminae were calculated. Profiles showing characteristic nuclear structure in both Nissl-stained and cobalt- stained cells were considered perikarya. If the perikaryon fell on a gridline, then it was counted in that square where its largest proportion was located.

Characterization of different neuron types was under- taken by careful visual observation of at least ten sections from each spinal cord with high-power magnification (loox, oil immersion objectives) by use of a Nikon microscope. Cell types were identified on the basis of the appearance of the proximal dendrites and their major orientation and of the shape of the soma and compared with those in the literature. Unfortunately ventral to lamina IV there is no systematic description of cell types. Photomicrographs were taken at 40 x and 100 x magnification.

RESULTS Application of excitatory amino acid receptor agonists to

the superfusate resulted in a black staining of many spinal cord cells. Labeled neurons were found in all parts of the grey matter, indicating good penetration of the agonists into the spinal cord (Fig. 1). In sections from control experiments only a few motoneurons and interneurons near the cut medial surface were lightly labeled (Fig. 1). Reticular fibres of the pia mater and some of the blood vessels gave a non-specific background staining in the spinal cords (Fig. 1).

The selective NMDA receptor antagonist D-AP-5 (20 pM) blocked the cobalt labeling induced by 500 (LM NMDA (Fig. 1). On the other hand the same concentration of D-AP-5 did not have any effect on the labeling produced by kainate superfusion (Fig. 1). Co-application of kynurenic acid (4 mM) with kainate (500 (LM) blocked staining of glial cells in the white matter and substantially reduced labeling of neuronal profiles in the grey matter (Fig. 1). Potassium chloride (50 mM) superfusion to the spinal cord did not result in staining of neurons or glial cells (Fig. 1).

Laminar distribution of labeled neurons The total number of labeled neurons were different in

spinal cords stimulated by the three different EAA receptor agonists. Kainate superfusion produced the highest, while NMDA superfusion the lowest number of labeled cell bodies per section. Mean values of stained neurons in 40 pM thick transverse sections were (mean * S.E.M.) kainate: 748.6 t 49.5; AMPA: 427.2 * 8.1; NMDA 411.6 ? 24.0; which were 33.5%; 19.1%, and 18.4% of the number of neurons found in Nissl-stained sections, respectively.

Figure 2 shows the laminar distribution of cobalt labeled neurons after AMPA, kainate, and NMDA treatment. The highest proportion of labeled cells per lamina was induced

Figure 6

EAA-EVOKED COBALT UPTAKE IN SPINAL CORD CELLS 329

by kainate application. The relative laminar patterns were, however, similar, with the highest number of labeled cells in lamina I, lamina VI, and ventral horn. Induction of cobalt uptake was the lowest in lamina I11 for all three agonists. NMDA produced relatively fewer labeled cells in lamina I1 than kainate and AMPA with maximal labeling in laminae I and IV.

Density of labeled neurons The relative density of labeled neurons is shown in

Figure 3, which reveals that labeling within laminae were non-uniform. In the superficial and deep dorsal horn, for example, a higher percentage of neurons was labeled in the medial part than in the lateral part after stimulation of the spinal cords by either of the agonists. The ventral horn of the spinal cords showed high density of stained perikarya in response to all agonists. However, more neurons in the ventro-lateral motoneuron pool were stained after AMPA than kainate or NMDA superfusion.

In the lateral spinal nucleus virtually all cells were labeled in kainate-stimulated spinal cords, while only 70% and 40% of neurons were stained in the AMPA and NMDA-treated cords, respectively (Fig. 3).

Cobalt-labeled cells in different part of the spinal cord

Dorsal horn. Stimulation of neurons with the different EAA receptor agonists resulted in staining of different cell types in the dorsal horn laminae. Table 1 summarises the results obtained from laminae I-IV for AMPA, kainate, or NMDA-treated spinal cords.

Four major types of lamina I neurons, similar to those described by Lima and Coimbra (1986), were found. AMPA resulted in staining of pyramidal cells with triangular cell bodies (Fig. 4A), fusiform cells with round perikarya (Fig. 4B), and multipolar cells with polygonal perikarya often showing a bulging contour (Fig. 4C). The latter cells were the most abundant in the AMPA-treated cords. Fusiform cells usually had short, ventrally running dendrites entering lamina I1 (Fig. 4B); therefore they could belong to the B type group. Kainate induced cobalt labeling of all types of lamina I neurons (Fig. 5A-D); however, fusiform neurons were the most numerous (Fig. 5C). In contrast to the fusiform cells found in AMPA-treated spinal cords, only a few of these had ventrally running dendrites (A type fusiform neurons). A great number of labeled dendrites running parallel with the surface were encoun- tered in the kainate-treated cords (Fig. 5A-D). NMDA treatment resulted in labeling predominantly in multipolar cells (Fig. 6A. pyramidal and flattened cells). In addition a few flattened and fusiform labeled neurons were observed.

Lamina 1.

Fig. 6. High-power magnification of cobalt-labeled dorsal horn neurons in transverse sections of spinal cords stimulated by NMDA (500 pM). A In the medial part of lamina I and outer lamina I1 multipolar cells (arrows) were labeled after NMDA superfusion. B: A few weakly labeled round cell bodies (arrowheads) were stained in the middle part of lamina 110. C: A stained perikarya (arrowhead) with labeled proximal dendrite (small arrows) directed dorsally in lamina IIi. D Another lamina IIi cobalt-stained cell (arrowhead) with a proximal dendrite (small arrows) entering the third lamina. E: Central (arrowhead) and antenna cells (asterisk) in lamina IV. Note the sharp border of the “dotted” background at the border of the third and fourth lamina (dotted line). F: Cobalt-labeled motoneurons (asterisks). Bar = 20 pm.

AMPA and NMDA stimulation also produced a low- and high-density dotted background staining, respectively.

Four types of neurons, similar to those described by several authors (Ramon y Cajal, 1895; Gobel, 1978; Schoenen, 1982; Todd and Lewis, 1986; Beal et al., 1989) were labeled by cobalt in lamina 11. Islet cells with round perikarya, stalked cells, “transverseux” cells of Cajal having medio-laterally running dendrites, and multipolar neurons were found. AMPA produced staining in all these types of neurons (Fig. 4C-G). However, stalked cells were the most abundant type of stained neurons in AMPA- treated spinal cords. Kainate-induced cobalt entry predominantly in islet cells (Fig. 5A-C). Beside them a few multipolar (Fig. 5D) vertical and stalked neurons were also stained. In the NMDA-stimulated spinal cords cobalt labeled islet, multipolar, and stalked cells (Fig. 6A-D), with islet cells the most abundant. AMPA and NMDA produced a low- and high-density dotted background, respectively (Figs. 4C-F,

All together three types of neurons, similar to those characterized in previous studies (Rethelyi and Szentagothai, 1969, 1973; Brown, 1981; Maxwell et al., 1983; Maxwell, 1985), were found in lamina 111. In the AMPA and kainate-treated spinal cords all these cell types were stained (Figs. 5E-F, 7A-C, 8B); however, antenna cells with elongated perikarya and central cells with round or polygonal cell bodies were the most common in each case. Dendrites of antenna and pyramidal cells entered the more superficial laminae. NMDA stimulation resulted in cobalt staining of antenna and central cells similar to those found in the non-NMDA receptor agonist-treated spinal cords. Similar to lamina I and I1 a high-density dotted background was observed in this lamina after NMDA stimulation (Fig. 6E).

Lamina IV contained three kinds of labeled neurons similar to those described by Rethelyi and Szen- tagothai (1973). AMPA induced cobalt uptake predominantly in antenna cells with elongated perikarya and dendrites penetrating the superficial layers (Fig. 7D). A few central neurons with polygonal perikarya and short proximal dendrites were also observed (Fig. 7E). In sections of kainate-treated spinal cords central cells similar to those found after AMPA stimulation were the most abundant. Some pyramidal (Fig. 8C) and antenna cells having triangular or elongated cell bodies, respectively, and dendrites entering lamina I11 were also labeled. NMDA application resulted in cobalt labeling of antenna cells and central neurons with round perikarya (Fig. 6E).

In the lateral part of lamina V, each of the agonists produced cobalt labeling in a number of large multipolar neurons. Dendrites of these cells were directed laterally and entered the lateral spinal nucleus. Figure 8D shows such a neuron after kainate stimulation. In the other parts of lamina V and in lamina VI different-sized multipolar neurons were labeled following treatment with each EAA receptor agonist.

In the intermediate part of the grey matter and in the ventral horn different-sized multipolar perikarya were labeled after the stimulation of the spinal cords with any of the EAA receptor agonists. Figure 6F shows a cobalt-labeled motoneuron after NMDA stimulation.

Multipolar neurons in the lateral spinal nucleus (Gwyn and Waldron, 1968) were heavily labeled by cobalt after stimulating the spinal cords with

Lamina IZ.

6A-D) . Lamina III.

Lamina IV.

Base of the dorsal horn (laminae V-VZ).

Intermediate zone and ventral horn.

Lateral spinal nucleus.

Figure 7


Fig. 8. High-power magnification of cobalt-labeled deep dorsal horn neurons in transverse sections of spinal cords stimulated by kainate (500 pM). A A labeled lamina IV neuron with a proximal dendrite penetrating the lamina 111. B: Triangular-shaped cell in lamina 111 with

an apical dendrite running toward the lamina 11. C: A triangular- shaped perikaryon in lamina IV. D: At the lateral edge of lamina V large multipolar neurons with dendrites entering the lateral spinal nucleus were labeled. Bar = 40 pm.

either of the EAA receptor agonists. Figure 7F shows a typical neuron in this nucleus after AMPA stimulation.

Staining of glial cells. AMPA and kainate also produced staining of glial cells. In the kainate-treated spinal cord labeled glial cells were found only in the white matter, while after AMPA treatment they were scattered both in the white and grey matter.

DISCUSSION In this study a cobalt uptake technique has been used to

visualise cells expressing different kinds of EAA receptors

Fig. 7. High-power magnification of deep dorsal horn cobalt-labeled neurons in transverse sections of spinal cord stimulated by AMPA. A: A triangular-shaped cobalt-labeled cell body from the medial part of lamina 111. Its apical dendrite penetrates deeply the more superficial lamina. B: A spindle-shaped perikaryon in the lateral part of lamina 111. C: A cobalt-labeled cell in lamina I11 with a proximal dendrite running toward lamina 11. D: A lamina IV neuron with dendrite running toward the most superficial laminae. E: Multipolar cobalt-stained perikaryon in lamina IV. F A multipolar perikaryon in the lateral spinal nucleus. Bar = 20 pm.

in the spinal cords of young rats. A large number of different neurons were labeled throughout the spinal grey matter after the activation of cells by different EAA receptor agonists. Glial cells in the white and grey matter were also stained after AMPA or kainate stimulation.

The stimulated cobalt uptake method introduced by Hogan (1983) has recently been used to label neurons and glial cells expressing capsaicin, bradykinin, and EAA receptors in tissue culture, dorsal root ganglia, hippocampal and cerebellar slices, and glial cells in optic nerve (Winter, 1987; Wood et al., 1988; Pruss et al., 1991; Fulton et al., 1992; Hu-Tsai et al., 1992; Williams et al., 1992; Nagy et al., 1993b). It has been suggested that cobalt enters the cells through ligand-gated Ca2+-channels coupled to the activated EAA receptors (Pruss et al., 1991; Fulton et al., 1992). Our findings are in agreement with this as passive opening of voltage-gated Ca2+-channels with 50 mM potassium did not result in cobalt staining of spinal cord cells. Results of our control experiments show that the cobalt labeling induced by the stimulation of spinal cord cells with EAA receptor agonists was specific. Williams et al. (1992) have also found that this technique provides specific label-

332

ing of neurons expressing EAA receptors in the hippocam- pus.

Electrophysiological experiments have found that the in vitro hemisected spinal cord preparation retains full activity only when obtained from young animals (up to 14 days postnatal (Thompson et al., 1990; Nagy et al., 1993a) and consequently our experiments were done on young rat tissue. However, in the newborn rat spinal cord, the expression of NMDA receptor is higher in the deep dorsal horn, intermediate zone, and ventral horn than in the adult, and the adult pattern is only attained 21 days postnatal (Kalb et al., 1992). Thus any comparison of our results to previous data obtained in adult animals by autoradiography, in situ hybridization, or immunocytochemistry must take into account possible developmentally regulated differences in receptor expression.

In the dorsal horn autoradiographic studies have shown that superficial laminae contain the highest density of EAA binding sites (Monaghan and Cotman, 1985; Monaghan et al., 1989; Jansen et al., 1990; Mitchell and Anderson, 1991). However, Furuyama et al. (1993) have recently reported that the expression of the kainate receptor subunit mRNAs is low with a homogeneous distribution of positive cells throughout the rest of the dorsal horn. They also found that NMDA receptor subunit mRNA expression is higher in laminae IV-V than in the more superficial laminae and that the expression of the AMPA receptor subunits GluR1-3 mRNA and protein is the highest in lamina I1 and the outer part of lamina 111. Martin et al. (1993) have also found a high density of cells expressing the AMPA receptor subunit GluRl and GluR2/3/4c in the superficial laminae. There is, therefore, an apparent discrepancy between the autoradiographic binding studies and the distribution of mRNA for the EAA receptors and EAA-sensitive cobalt-labeled neurons, which is particularly prominent for NMDA. NMDA binding is highest in lamina 11, the very lamina that shows the lowest level of NMDA receptor mRNA or NMDA- stimulated cobalt-stained cells. The low number of lamina I1 neurons with NMDA receptor in the latter two cases is in agreement, though, with electrophysiological results showing that synaptic transmission in lamina I1 is overwhelm- ingly non-NMDA receptor dependent (Schneider and Perl, 1988; Yoshimura and Jessell, 1991; Yoshimura and Nishi, 1993). The present finding of a high density of background cobalt labeling in lamina 1-111 following NMDA application may resolve this discrepancy as these cobalt-labeled struc- tures may represent dendrites originating from neurons with cell bodies located in lamina I and/or the deep dorsal horn. Dendrites of these cells have been shown to penetrate lamina I1 (Rambn y Cajal, 1895; RBthelyi and Szentagothai, 1973; Gobel, 1978; Brown, 1981; Lima and Coimbra, 1986), and lamina 111-IV neurons have been shown receiving synaptic input from primary afTerents in lamina I1 (Woolf and King, 1987; Todd, 1989). Cobalt uptake might also occur without any staining of perikarya, as 70% of dorsal horn cells have been shown to be more sensitive to NMDA on the dendritic tree than on the soma (Arancio et al., 1993). Intrinsic lamina I1 cells might not be, therefore, NMDA responsive, but lamina I1 might, as a result of dendrites of cells with perikarya in the superficial or deep laminae, contain many NMDA-responsive elements.

There is also a discrepancy between the distribution of binding sites for AMPA and kainate in laminae 1-111 (Monaghan and Cotman, 1985; Monaghan et al., 1989; Jansen et al., 1990; Mitchell and Anderson, 1991), receptor

I. NAGY ET AL.

subunits (Furuyama et al., 1993; Martin et al., 19931, subunit mRNAs (Furuyama et al., 1993) and the distribution of cobalt-labeled cells after AMPA and kainate application. The GluR1-4 subunits are highly sensitive to AMPA (Boulter et al., 1990; Keinanen et al., 1990) and respond to kainate but not to NMDA (Boulter et al., 1990; Keinanen et al., 1990). I t has been also shown that glutamate receptor subunits form heteromultimeric receptor complexes containing at least two distinct subunits (Nakanishi et al., 1990; Wenthold et al., 19921, but the exact composition of these complexes is not known. However, Hollmann et al. (1991) have reported that AMPA receptor complexes containing the GluR2 subunit are not permeable to Ca2+ (Hollmann et al., 1991). Thus a possible explanation for the discrepancy between cobalt label and mRNA distribution and binding sites might be that kainate and AMPA stimulate cobalt uptake in cells with kainate and AMPA receptor complexes that do not contain the GluR2 subunit.

The density of cobalt-labeled perikarya in the lateral part of the dorsal horn was lower than in the medial part. This labeling pattern cannot be the result of an insufficient penetration of the chemicals because neurons in the lateral spinal nucleus next to the dorsal horn were heavily stained in every case. Interestingly, substance P immunoreactivity is also higher in the medial than in the lateral part of the superficial dorsal horn (Knyihar-Csillik et al., 1990) while the trans- l-aminocyclopentane-1,3-dicarboxylate (t-ACPD) induced inositol 1,4,5-triphosphate (IP3) formation shown by Fotuhi et al. (1993) was higher in the lateral than in the medial part of the spinal cord. These differences may refer to the somatotopic organization of the spinal cord in the lumbar enlargement with medial areas innervating the distal limbs, particularly the glabrous skin and lateral parts, more proximal and hairy skin (Swett and Woolf, 1985).

No data are available on the distribution of intermediate zone neurons expressing EAA receptors, receptor subunits, or receptor subunit mRNA. Cells in this area receive synaptic input mainly from muscle primary afferents (A- stermark and Kummel, 1986) and project to motor nuclei, cerebellum, and other parts of the brain (Edgley and Jankowska, 1988; Bras et al., 19891, and a substantial proportion of them are inhibitory interneurons (Jankowska and Lindstrom, 1972). The higher proportion of cobalt- labeled neurons after non-NMDA receptor activation sug- gests that these receptors may be more important than NMDA receptors in the integration of motor information.

In the ventral horn, Martin et al. (1993) have reported that the AMPA receptor subunits GluR1, GluR2/3/4c, and GluR4 were all expressed in motoneurons particularly in the lateral nuclei. Furuyama et al. (1993) have found that the density of motoneurons expressing AMPA receptor subunit GluR3 and GluR4 mRNA and NMDA receptor subunit NR1 mRNA was high while that of cells expressing the kainate receptor subunit GluRS mRNA was low. Auto- radiographic experiments have shown low activity of [H3]- EAA receptor agonists in the whole ventral horn (Mon- aghan and Cotman, 1985; Monaghan et al., 1989; Jansen et al., 1990; Mitchell and Anderson, 1991). The high percentage of motoneurons labeled by cobalt after AMPA and NMDA perfusion is in agreement with the findings of the in situ hybridization and immunocytochemical studies (Fu- ruyama et al., 1993; Martin et al., 1993). However, we also found a substantial number of motoneurons labeled by cobalt after kainate stimulation. Electrophysiological stud-


ies have confirmed the expression of AMPA, kainate, and NMDA receptors by motoneurons (Holehean et al., 1992).

None of the in situ hybridization, the immunocytochemical, or the autoradiographic studies have reported on the expression of EAA receptor in the lateral spinal nucleus, but we found very high levels of EAA-sensitive neurons in this nucleus. Previously, neurons in this nucleus have been shown containing substance P, vasoactive intestinal polypeptide, bombesin, dynorphin, glutamate decarboxylase, and cholinesterase (Navaratnam and Lewis, 1970; Fuji et al., 1985; Giesler and Elde, 1985; Nahin, 1987; Leah et al., 1988; Sasek et al., 1991) and receive inputs from neuropep- tide containing fibres (Menetrey et al., 1950; Bresnahan et al., 1984; Giesler and Elde, 1985; Robertson and Grant, 1985; Cliffer et al., 1988; Knyihh-Csillik et al., 1990).

The non-NMDA receptor agonists induced cobalt labeling in glial cells. This result is in agreement with previous findings in hippocampal, cerebellar, and optic nerve cell cultures and slices, where kainate and quisqualate induced cobalt labeling in 0-2A progenitor and type-2 astrocytes (Pruss et al., 1991; Fulton et al., 1992), and confirm that some glial cells in the central nervous system express ionotropic, non-NMDA excitatory amino acid receptors.

Our findings suggest that a loose correlation may exist in the dorsal horn between the morphological characteristics of neurons and the EAA receptors they express. Thus NMDA receptors are expressed relatively more in lamina I-IV cells with rostro-caudally distributed dendritic trees and in lamina 111-IV cells with dendrites penetrating the superficial laminae. No clear correlation has been found between the morphology of dorsal horn neurons and characteristics of the peripheral stimuli they respond to (Bennett et al., 1980, 1981; Light et al., 1979; Maxwell et al., 1983; Woolf and Fitzgerald, 1983; Woolf and King, 1987; Rethelyi et al., 1989). However, a correlation has been found between the morphology and the immunocytochemical characteristics of lamina I-IV cells (Todd and McKenzie, 1989; Antal et al., 1990, 1991; Todd and Sullivan, 1990; Mather and Ho, 1992; Lima et al., 1993). Comparing the immunocytochemical distribution of the inhibitory neurotransmitter y-aminobutyric acid (GABA) and glycine (Todd and McKen- zie, 1989; Todd and Sullivan, 1990; Antal et al., 1991; Mather and Ho, 1992; Powell and Todd, 1992) with the cobalt-stained cells, it seems that lamina I-IV inhibitory interneurons express potentially all types of EAA receptors.

ACKNOWLEDGMENTS We thank Dr. D. Lima for her valuable comments and

Miss J. Head for the help in the statistical analyses. The authors also thank P. Ainsworth for the excellent technical assistance. I. Nagy was supported by a Welcome Trust European Research Fellowship. C.J. Woolf is the recipient of a Bristol-Myers Squibb unrestricted pain research grant.

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Cobalt accumulation in neurons expressing ionotropic excitatory amino acid receptors in young rat spinal cord: Morphology and distribution