Brain Research, 581 (1992) 67-80 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00

67

BRES 17748

Glutamate-immunoreactive synapses on retrogradely-labelled sympathetic preganglionic neurons in rat thoracic spinal cord I.J. Llewellyn-Smith a'b, K.D. Phendb~ J.B. Minson a, P.M.

Pilowsky a and

J.P.

Chalmers a

~Department of Medicine and Centre for Neuroscience, Flinders University, Bedford Park, S.A. (Australia~ and ~'Department of Cell Biology and Anatomy, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill. NC 27599-7090 (USA) (Accepted 31 December 1991)

Key words: Colloidal gold; Cholera toxin B subunit; Glutamate; lmmunocytochemistry; Spinal cord: Sympathetic preganglionic neuron: Rat; Retrograde tracing; Ultrastructure

Retrograde tracing with cholera toxin B subunit (CTB) combined with post-embedding immunogold labelling was used to demonstrate the presence of glutamate-immunoreactive synapses on sympathetic preganglionic neurons that project to the adrenal medulla or to the superior cervical ganglion in rat thoracic spinal cord. At the electron microscope level, glutamate-immunoreactive synapses were found on retrogradely labelled nerve cell bodies and on dendrites of all sizes. Two-thirds of the vesicle-containing axon profiles that were directly apposed to, or synapsed on, CTB-immunoreactive sympathoadrenal neurons were glutamate positive. The proportion of glutamate-immunoreactive contacts and synapses on sympathoadrenal neurons decreased to zero when the anti-glutamate antiserum was absorbed with increasing concentrations of glutamate from 0.1 mM to 10 mM. Double immunogold labelling for glutamate and 7-aminobutyric acid (GABA) showed that glutamate-immunoreactive profiles did not contain GABA and that GABA-immunoreactive profiles did not contain glutamate. These results suggest that glutamate is the major excitatory neurotransminer to sympathoadrenal neurons and possibly to other sympathetic preganglionic neurons in the intermediolateral cell column of the spinal cord. INTRODUCTION Sympathetic preganglionic n e u r o n s are responsible for mediating many autonomic reflexes. They supply a source of tonic excitation to the adrenal medulla and to postganglionic sympathetic n e u r o n s that innervate a variety of target organs, including the heart and blood vessels. Sympathetic preganglionic n e u r o n s are in turn subject to both excitatory and inhibitory influences from neurons located in the spinal cord, brainstem and higher centers. Since a knowledge of the chemicals that mediate these effects is necessary for u n d e r s t a n d i n g the regulation of sympathetic outflow, many recent studies have examined the n e u r o t r a n s m i t t e r content of n e u r o n s that provide synaptic inputs to sympathetic preganglionic neurons. At the ultrastructural level, nerve fibers containing neuropeptides, m o n o a m i n e s and v-aminobutyric acid ( G A B A ) have been shown to form synapses on retrogradely labelled sympathetic preganglionic n e u r o n s 6'8' 9,13,14,25,30,43. F u r t h e r m o r e , application of m a n y of these chemicals in the vicinity of sympathetic preganglionic n e u r o n s elicits changes in their activity, firing rate or m e m b r a n e potential 2-5't5-19"23"26'32'33"46. O n e chemical that is an important candidate for reg-

ulating the activity of sympathetic preganglionic neurons is the excitatory amino acid, L-glutamate. Iontophoretic application of glutamate has been shown to increase the firing rate of sympathetic preganglionic n e u r o n s 2. Intracellular studies have demonstrated that stimulation of N-methyl-D-aspartate receptors induces excitatory postsynaptic potentials in these neurons in neonatal rats 33. Excitatory amino acid antagonists administered intrathecally lower basal blood pressure and partially prevent the rise in blood pressure and sympathoexcitation seen after stimulation of the rostral ventrolateral medulla ~9~ 27 29,45 F u r t h e r m o r e , microdialysis results from our laboratory indicate that this stimulation also increases the release of glutamate in the lateral horn of the spinal cord 22. Immunohistochemical studies have shown that glutamate-immunoreactive neurons are present in the rostral ventrolateral medulla 36'4°, an important center for the tonic and reflex control of blood pressure (for review see ref. 12) and that phosphate-activated glutaminase ( P A G ) , an enzyme involved in the biosynthesis of glutamate, is present in adrenaline- or serotonin-containing n e u r o n s in this area 2~. More recently, we have found that in the rostral ventrolateral medulla P A G occurs in all of the phenylethanolamine-N-methyltrans-

Correspondence." l.J. Llewellyn-Smith, Department of Medicine, Flinders Medical Centre, Bedford Park, S.A. 5042, Australia. Fax: (61) (8) 204-5268.

68 ferase ( P N M T ) - c o n t a i n i n g n e u r o n s and all of the serotonin-containing

neurons

retrogradely

labelled

with

c h o l e r a toxin B ( C T B ) - g o l d f r o m the i n t e r m e d i o l a t e r a l cell c o l u m n 31. W e also identified a large p o p u l a t i o n of b u l b o s p i n a l P A G n e u r o n s in the rostral v e n t r a l m e d u l l a that c o n t a i n e d n e i t h e r P N M T

n o r s e r o t o n i n 31. G l u t a -

m a t e - i m m u n o r e a c t i v e t e r m i n a l s , h a v e b e e n f o u n d in the i n t e r m e d i o l a t e r a l cell c o l u m n and f o r m synapses t h e r e w' 34. M o s t of t h e s e t e r m i n a l s are of supraspinal origin 34 and s o m e arise f r o m n e r v e cell b o d i e s in the rostral v e n t r o l a t e r a l m e d u l l a 35. H o w e v e r , it has not yet b e e n established precisely which n e u r o n s in the i n t e r m e d i o l a t e r a l

cubated with continuous agitation in a 1:25.00t~ dilution of antiCTB antiserum (List Biological Laboratories) containing 10% NHS for 2-4 days, then in a 1:200 dilution of biotinylated anti-sheep immunoglobulin (Sigma B-7390) containing l% NHS for 24 h and finally in a 1:1500 dilution of ExtrAvidin-horseradish peroxidase (Sigma E-2886) overnight. All incubations were done at room temperature. All immunoreagents were diluted in phosphate-buffered saline containing I0 mM Tris and 0.05% thimerosal (TPBS). After each incubation, sections were washed 3 x 3(I rain in TPBS. As controls, some sections were stained with anti-CTB antiserum that had been pre-absorbed with l ~tg/ml or 5 ~g/ml CTB. Other sections were incubated in TPBS containing 10% NHS without primary antiserum. CTB-immunoreactive neurons were revealed by a nickel-intensified diaminobenzidine reaction with peroxide ions being generated by glucose oxidase 25. The reaction was stopped after 7-12 min by the addition of a large volume of phosphate buffer.

cell c o l u m n r e c e i v e g l u t a m a t e synapses. In the p r e s e n t study, we h a v e used r e t r o g r a d e tracing with u n c o n j u g a t e d C T B and p o s t - e m b e d d i n g i m m u n o c y t o c h e m i s t r y for g l u t a m a t e

to d e m o n s t r a t e

that gluta-

m a t e - i m m u n o r e a c t i v e synapses o c c u r o n s y m p a t h e t i c preg a n g l i o n i c n e u r o n s p r o j e c t i n g to t h e a d r e n a l m e d u l l a o r to the s u p e r i o r cervical g a n g l i o n in the rat. W e also det e r m i n e d the p r o p o r t i o n o f g l u t a m a t e - i m m u n o r e a c t i v e synapses o n s y m p a t h o a d r e n a l n e u r o n s . In a d d i t i o n , we s h o w e d that g l u t a m a t e and G A B A

are p r e s e n t in sepa-

r a t e p o p u l a t i o n s o f n e r v e fibers that f o r m synapses on sympathetic preganglionic neurons.

MATERIALS AND METHODS

Tracer injections and tissue processing Male Wistar-Kyoto rats weighing 250-350 g were anesthetized with sodium pentobarbital (Nembutal; 60 mg/kg) and chloral hydrate (100 mg/kg). Three rats received injections of approximately 10/~1 of a 1% solution of CTB (List Biological Laboratories; Campbell, CA) into the adrenal medulla. Approximately 5/~1 of 1% CTB was injected into the superior cervical ganglion of a fourth rat. Three days after the injection of tracer, the animals were anesthetized with sodium pentobarbital (100 mg/kg). Heparin (1,000 IU) was injected through a cannula placed in the ascending aorta and blood was cleared from the vascular system by flushing with 150200 ml of oxygenated DMEM/Ham's F12 tissue culture medium (Sigma D-8900). The rats were then fixed by perfusion with 1 liter of fixative containing either 2.5% glutaraldehyde, 0.5% formaldehyde, 0.1% picric acid (one rat with an adrenal medulla injection) or 2.5% glutaraldehyde (the remaining rats) in 0.1 M phosphate buffer, pH 7.4. Segments of thoracic spinal cord were removed (TT-T9 for the rats with CTB in the adrenal medulla, T1-T 3 for the rat with CTB in the superior cervical ganglion), divided either dorsoventrally along the mid-line or into spinal segments and postfixed for 45 rain at room temperature with continuous agitation on a shaker. The tissue was washed briefly in several changes of phosphate buffer and cut at 70/~m on a vibratome, either parasagittally or transversely.

Post-embedding immunocytochemistry After several washes in phosphate buffer, sections were postfixed in 0.5% OsO4 in phosphate buffer for 60 min, dehydrated through alcohol and propylene oxide and embedded flat in Durcupan (Fluka). Silver to pale gold ultrathin sections of retrogradely labelled sympathetic preganglionic neurons were cut with a diamond knife and mounted on either nickel mesh grids or formvarcoated nickel single slot grids. Glutamate immunoreactivity was detected by post-embedding immunocytochemistry using a modification 39 of the method of de Zeeuw et al. 47. The grids were first washed in 50 mM Tris, pH 7.6, containing 0.9% NaC1 and 0.1% Triton X-100 (TBS-Triton) and then incubated overnight at room temperature in drops of a 1:i0,000 (single slot grids) or 1:20,000 dilution (mesh grids) of rabbit anti-glutamate antiserum (Code 645a2, prepared by Dr. Peter Petrusz) in TBS-Triton, pH 7.6. After several washes in TBS-Triton, pH 7.6, and one wash in TBSTriton, pH 8.2, the grids were incubated for 1 h at room temperature in a 1:25 dilution of rabbit anti-immunoglobulin adsorbed to 10 nm gold particles (AuroProbe EM GAR G10; Amersham, UK). The grids were washed in TBS-Triton, pH 7.6, and then in water, dried, stained with 5% aqueous uranyl acetate followed by Reynold's lead citrate and examined with an electron microscope. To test the specificity of the anti-glutamate antiserum, grids were incubated in anti-glutamate antiserum that had been absorbed with either 0.1 mM, 1 mM or 10 mM L-glutamate or in anti-glutamate antiserum that had been absorbed with either 0.1 mM, 1.0 mM or 10 mM L-aspartate. To confirm that absorption with glutamate abolished glutamate immunoreactivity in axon terminals, adjacent series of 2-3 ultrathin sections mounted on formvar-coated single slot grids were stained either with anti-glutamate or with anti-glutamate that had been absorbed with 10 mM glutamate. Two methods were used to determine whether glutamate-positive terminals were also positive for GABA. First, adjacent series of 2-3 serial sections on single slot grids were stained either with anti-glutamate antiserum or with anti-GABA antiserum (code 483a2; ref. 20) diluted 1:2500 in TBS-Triton, pH 7.6. Second, double-staining was done to reveal glutamate and GABA on the same section 39. After glutamate immunoreactivity had been localized with anti-rabbit IgG conjugated to 10 nm gold particles, the grids were exposed for 1 h to paraformaldehyde vapors at 80°C. They were then incubated in anti-GABA antiserum (1:5000 dilution in TBS-Triton, pH 7.6), which was localized with anti-rabbit IgG linked to 15 nm gold particles (AuroProbe EM GAR G15; Amersham). As further controls, some grids were incubated in normal rabbit serum or in TBS-Triton instead of primary antiserum.

Pre-embedding immunocytochemistry for CTB Retrogradely transported CTB was detected by pre-embedding immunocytochemistry as previously described 25. Sections of spinal cord were pretreated for 30 min with 50% ethanol in distilled water immediately after they were cut to improve antibody penetration 24 and washed several times in phosphate buffer. Non-specific antibody binding sites were blocked by incubating the sections for 30 min in 10% normal horse serum (NHS). Sections were then in-

Quantitation and analysis Vesicle-containing axon profiles that were directly apposed to, or synapsed on, CTB-immunoreactive nerve cell bodies and dendrites were assessed for glutamate immunoreactivity by counting gold particles either on electron micrographs or on the screen of the electron microscope. Ultrathin sections through sympathoadrenal neurons were quantified for two rats, one fixed with 2.5% glutaral-

69

Fig. 1. Electron micrographs of a glutamate-immunoreactive synapse on the cell body of a sympathetic preganglionic neuron that was retrogradely labelled with CTB from the adrenal medulla. A: low power micrograph showing a portion of the cell body of the neuron (NCB). The neuron contains deposits of electron-dense peroxidase reaction product, indicating the presence of CTB immunoreactivity. The vesiclecontaining axon profiles indicated by 1 and 2 are shown at higher power in B. BV, blood vessel. Bar = 1/~m. B: micrograph showing axon profiles 1 and 2 from A at higher magnification. There are many 10 nm gold particles over synaptic vesicles in axon profile 1. The density of gold particles is 129.81pm 2 (excluding mitochondria). Profile 1 forms a synapse (arrowheads) on the CTB-immunoreactive nerve cell body. Profile 2 shows few 10 nm gold particles. The gold particle density over profile 2 is 17.1/#m 2 (excluding mitochondria). Profile 2 also forms a synapse on the retrogradely labelled nerve cell body. Bar = 500 nm.

70

Fig. 2. Electron micrograph showing a glutamate-immunoreactive axon profile, which is covered with many 10 nm gold particles, forming a synapse (arrowheads) on a dendrite (D), which is positive for CTB that was retrogradely transported from the adrenal medulla. The gold particle density over this profile is 75.6//tm 2 (excluding mitochondria). An adjacent vesicle-containing axon profile (uAP) shows only a few gold particles, indicating that it is non-immunoreactive for glutamate. Bar = 250 nm.

dehyde and the other with 2.5% glutaraldehyde, 0.5% formaldehyde, 0.1% picric acid. Synapses were identified by clustering of vesicles presynaptically and by the presence of postsynaptic membrane specializations. Quantitation on electron micrographs. For assessing glutamate immunoreactivity from electron micrographs, photographs of vesiculated axon profiles that directly contacted or synapsed on CTBimmunoreactive neurons were printed to a final magnification of 60,000-80,000×. The areas of the axon profiles and the mitochondria within them were measured with a digitizer pad. Gold particles were counted manually. Particles over mitochondria were excluded from the counts since the anti-glutamate antiserum used here is known to cross-react with intermediates in the Krebs cycle (L. Abdullah and P. Petrusz, personal communication). (This was confirmed here ultrastructurally by the concentration of gold particles over mitochondria in cell bodies, in dendrites and in boutons containing either gold-labelled or unlabelled vesicle populations.) The value for the area of each profile less its mitochondrial area was used to calculate the number of gold particlesd/~m z. Gold particle densities were also determined over the following structures: (1) CTB-negative somata and dendrites that were intermixed with CTB-positive neurons in the intermediolateral cell column, (2) C'I~-negative cell bodies in the ventral horn and (3) the lumina of blood vessels, which were examined to assess background labelling over empty resin. In the rat fixed with 2.5% glutaraldehyde, gold particle densities over CTB-positive neurons were also measured. A vesiculated axon profile was judged to be glutamate-immunoreactive if the concentration of gold particles over the profile exceeded the 99% confidence limit (mean + 2.576 standard devia-

tions) of the concentration of gold particles over CTB-negative neurons in the intermediolateral cell column. The total numbers of glutamate-immunoreactive and non-immunoreactive axon profiles contacting or synapsing on all CTB-immunoreactive structures in a single section from each rat were used to determine the proportion of the synaptic input to sympathoadrenal neurons that was glutamatergic. Quantitation on the electron microscope screen. For counting on the screen of the electron microscope, profiles were classified as glutamate immunoreactive if there were at least 20 gold particles over axon profiles (usually many more). Gold particles over mitochondria were excluded from the counts (see above). Profiles with fewer than 20 gold particles in non-mitochondrial areas were considered to be non-immunoreactive. To determine proportions of glutamate-positive axon profiles, the number of glutamate-positive and glutamate-negative terminals were counted in a single ultrathin section for each animal or treatment. A total of at least 35 vesiclecontaining axon profiles were categorized as either glutamate-positive or glutamate-negative for each sample.

RESULTS

Retrograde labelling o f sympathetic preganglionic neurons with C T B C T B i m m u n o r e a c t i v i t y c o u l d b e d e t e c t e d b y light mic r o s c o p y in s y m p a t h e t i c p r e g a n g l i o n i c n e u r o n s

in vi-

71 100

8O

60

.A,

• '''"

"''"

- . Aspar?;e

40

N o electron dense-reaction product, indicating the presence of CTB immunoreactivity, was found in any of the h u n d r e d s of vesicle-containing axon profiles that were examined during the course of this study. A b s o r p t i o n of the anti-CTB antiserum with 1/~g/ml or 5 ktg/ml CTB abolished labelling in sympathetic preganglionic neurons. L a b e l l e d neurons were also absent from sections that had b e e n incubated without primary antiserum. G l u t a m a t e - i m m u n o r e a c t i v e synaptic inputs to sympathetic preganglionic neurons

~

20

e

i

i

,uw

0.1

1.0

10

Concentration of absorbing amino acid (mM) Fig. 3. Effect of absorption of the anti-glutamate antiserum with L-glutamate or L-aspartate on the percentage of the total number of profiles contacting or synapsing on retrogradely labelled sympathetic preganglionic neurons that are immunoreactive for glutamate.

b r a t o m e sections b o t h before and after processing for electron microscopy (see ref. 25) and in semithin (1/~m) sections that were cut from r e s i n - e m b e d d e d v i b r a t o m e sections. The great majority of the CTB-labelled neurons were located in the i n t e r m e d i o l a t e r a l cell column pars principalis; some C T B - i m m u n o r e a c t i v e cell bodies occurred in the dorsolateral funiculus and occasional labelled neurons were found in the intercalated nucleus or the central autonomic area. A t the ultrastructural level, C T B - i m m u n o r e a c t i v e neurons were identified by the presence of electron-dense peroxidase reaction product in their cytoplasm (Figs. 1, 2 and 4 - 7 ) . Most C T B - i m m u n o r e a c t i v e dendrites and nerve cell bodies were m o d e r a t e l y to lightly labelled. The CTB-positive cell bodies were m a r k e d by variably sized deposits of intense peroxidase reaction product, particularly over the Golgi apparatus (Fig. 1); the rest of the cytoplasm either had no label or contained homogeneously distributed m o d e r a t e to faint electron-dense reaction product (Fig. 7). R a n d o m l y distributed deposits of intense reaction product m a r k e d most of the labelled dendrites and there was often m o d e r a t e to faint electron density associated with microtubules and the web-like c o m p o n e n t s of the cytoplasmic matrix (Figs. 2, 5 and 6). A few dendrites were so heavily labelled that their cytoplasm was completely filled with electron-dense material.

G l u t a m a t e immunoreactivity in axon terminals was indicated by the presence of many 10 nm gold particles over synaptic vesicles (Figs. 1B, 2, 4A, 5B, 6A, 7). A x o n profiles that were positive for glutamate contained large numbers of small clear vesicles and, in some cases, a few large dense-cored vesicles (Fig. 7A). C T B - i m m u n o r e a c tive neurons that projected either to the superior cervical ganglion or to the adrenal medulla received synapses from g l u t a m a t e - i m m u n o r e a c t i v e axon profiles. These synapses occurred on cell bodies (Figs. 1, 4 A and 7A) and on large, medium-sized and small dendrites (Figs. 2, 5 and 6). The glutamate-positive synapses often had thick postsynaptic densities (Figs. 1, 2 and 4 - 7 ) and filamentous material in the synaptic cleft (Figs. 5B and 7A). Terminals containing vesicles that were labelled with many gold particles sometimes lay next to or near terminals containing vesicles that were labelled with very few gold particles (Figs. 2 and 5).

TABLE I Gold particle density over neurons and blood vessels (particles/ ~m 2 +-- S.D.)

Rat 1 was perfused with 2.5% glutaraldehyde; rat 2 was perfused with 2.5% glutaraldehyde, 0.5% formaldehyde, 0.2% picric acid. Rat 1

Rat 2

IML neurons without CTB-IR Number counted Total area measured

33.3 + 10.3 10 67.0/~m 2

25.2 + 10.0 10 62.9/~m z

VH neurons without CTB-IR Number counted Total area measured

42.7 + 12.9 5 36.2 ~m 2

29.5 + 7.1 5 35.2 ~m 2

IML neurons with CTB-IR Number counted Total are measured

38.1 + 5.2 10 76.2/~m 2

Blood vessel lumina Number counted Total area measured

1.2 + 2.3 6 53.7 ~m 2

0.9 + 0.8 6 52.4 #m 2

IML, intermediolateral cell column; VH, ventral horn.

72

Quantitation of glutamate synapses Spinal cords from two rats in which CTB had been in-

retrogradely labelled neurons. Glutamate-positive axon profiles that were not in contact with retrogradely la-

jected into the adrenal medulla, one fixed with 2.5% glutaraldehyde, 0.5% formaldehyde, 0.1% picric acid, and

belled n e u r o n s were excluded from the counts. Quantitation on electron micrographs. In the rat per-

the other with 2.5% glutaraldehyde, were sectioned

fused with 2.5% glutaraldehyde, 0.5% formaldehyde, 0.1% picric acid, the density of gold particles over the

parasagittally so that a single ultrathin section contained many CTB-immunoreactive neurons. These sections were used to quantify glutamate-immunoreactive axon profiles that were directly apposed to or synapsed on the

cytoplasm of n o n - i m m u n o r e a c t i v e cell bodies and dendrites in the intermediolateral cell column was 25.2 + 10.0 gold particles//~m2 (mean + S.D.. n = 10; Table I).

qlF

'~

~!!~ '~~i

%

~

~?i

4

Fig. 4. Electron micrographs showing semi-serial sections through an axon profile that formed a synapse (arrowheads) on the cell body of a sympathoadrenal neuron (NCB) that was lightly immunoreactive for CTB. A: appearance of the profile after staining with unabsorbed antiglutamate antiserum (GLU). The synaptic vesicles in the axon profile are labelled with many 10 nm gold particles. B: profile after staining with anti-glutamate antiserum that had been absorbed with 10 mM glutamate (GLU abs). There are very few gold panicles over synaptic vesicles. Bar in B, which applies to A and B, = 250 nm.

73

Fig. 5. Electron micrographs showing post-embedding staining for glutamate after absorption of the anti-glutamate antiserum with 1 mM aspartate. A: low power micrograph showing a dendrite (D) that was retrogradely labelled from the adrenal medulla. The dendrite contains heavy deposits of electron-dense peroxidase reaction product, indicating the presence of CTB immunoreactivity. Two vesicle-containing axon profiles (1 and 2) are shown at higher power in B and C, respectively. Bar = 1 #m. B: micrograph showing axon profile 1 from A at higher power. The profile forms a synapse (arrowheads) on the retrogradely labelled dendrite (D) and there are only a few 10 nm gold particles over synaptic vesicles, indicating that the profile is not immunoreactive for glutamate. The gold particle density over profile 1 is 13.3//zm 2 (excluding mitochondria). Bar = 250 nm. C: micrograph showing axon profile 2 from A at higher power. The profile forms a synapse (arrowheads) on the retrogradely labelled dendrite (D) and the profile is covered by many 10 nm gold particles, indicating that the profile is glutamate immunoreactive in spite of absorption of the anti-glutamate antiserum with aspartate. The the mitochondria in this profile are heavily labelled with gold particles because the anti-glutamate antiserum cross-reacts with intermediates in the Krebs cycle. The gold particle density over profile 2 (excluding mitochondria) is 107.7/#m 2. Bar = 500 nm.

74 Fig. 6. Electron micrographs showing double post-embedding staining for both glutamate and G A B A on semi-serial uhrathin ~cclions. (il,~ tamate was localized with 10 nm gold particles. G A B A was localized with 15 n m gold particles. A glutamate-positive axon protile( I ), wt~ich is covered with m a n y 10 n m gold particle but few i5 nm gold particles, forms a synapse (arrowheads) on the CTB-immunoreactive (asterisks) dendrite (D) of a neuron that projects to the adrenal medulla. A neighboring profile (2) contacts the retrogradely labelled dendritc bul ~s tlt)~ associated with a clear postsynaptic m e m b r a n e specialization. Profile 2 is covered with m a n y 15 n m gold particles and few lit nm gold particles, indicating that it is G A B A immunoreactive. Bar in B. which applies to A and B, = 250 n m o_..~

With this gold particle density as the level of non-specific background labelling over neurons, any axon profile with a gold particle density equal to or greater than 51.0 gold particles//~m 2 (mean + 2.576 x S.D.; 99% confidence limit) was assumed to be glutamate immunoreactive. Of 56 axon profiles that contacted or synapsed on CTB-positive neurons in a single section, 39 (70%) were labelled with 51 or more gold particles. The highest level of labelling seen in an axon profile contacting or synapsing on a CTB-positive neuron in this rat was 136.6 gold particles/~m 2 and the mean level of labelling in the positive profiles was 78.4 + 21.3 (S.D.) gold particles//~m 2. In the rat perfused with 2.5% glutaraldehyde, the density of gold particles over the cytoplasm of non-immunoreactive cell bodies and dendrites in the intermediolateral cell column was 33.3 + 10,3 gold particles/#m 2 (mean + S.D., n = 10; Table I). The gold particle density for 99% confidence that an axon profile was positive for glutamate was 59.9 gold particles/ktm 2. Of 63 axon profiles that contacted or synapsed on CTB-positive neurons in a single section, 42 (67%) were labelled with 60 or more gold particles. The highest level of labelling seen in an axon profile contacting or synapsing on a CTB-positive neuron in this rat was 183 gold particles/ #m 2 and and the mean level of labelling in the positive profiles was 99.1 _+ 28.6 (S.D.) gold particles/#m 2. Quantitation on the electron microscope screen. In the rat perfused with 2.5% glutaraldehyde, 0.5% formaldehyde, 0.1% picric acid, a total of 187 of 295 profiles (63%) on sympathetic preganglionic neurons were glutamate positive on the basis of counts on the electron microscope screen. These data came from 3 runs of postembedding staining on ultrathin sections that were separated by at least 8 ~m to prevent double-counting of varicosities. In the 3 runs, the percentage of profiles that were glutamate immunoreactive ranged from 63% to 65%. In the rat perfused with 2.5% glutaraldehyde, 191 of 338 vesicle-containing axon profiles (57%) that contacted or synapsed on retrogradely labelled sympathetic preganglionic neurons were immunoreactive for glutamate on the basis of on-screen counts.

Immunogold labelling of cell bodies and blood vessel lumina Light microscopic immunohistochemical evidence has

suggested that the neurons in the intermediolateral cell column are glutamatergic 34. This hypothesis was tested by comparing the gold particle density over CTB-negative neurons in the intermediolateral cell column with neurons in the ventral horn (Table I). For both rats, analysis of variance showed that the gold particle densities over the two types of neurons were not significantly different. The gold particle density over sympathetic preganglionic neurons retrogradely labelled with CTB was also measured for the rat perfused with 2.5°A glutaraldehyde (Table I), which had the highest gold particle density over terminals. By analysis of variance, the gold particle density over CTB-positive neurons in this rat was not signficantly different from the gold particle density over CTB-negative neurons in either the intermediolateral cell column or the ventral horn. For both rats the level of background labelling over the lumina of blood vessels (i.e. empty resin) was about 1 gold particle/pro 2 (Table I), a level equivalent to that seen in experiments considered to be the most successful by Ottesen 37.

Specificity controls Since the on-screen counting method was much quicker than the on-micrograph counting method, gave reproducible results and consistently underestimated the proportion of glutamate-positive profiles in comparison to the on-micrograph counting method (see above), the resuits of specificity test were assessed by gold particle counts on the microscope screen. Absorption with glutamate. In an experiment in which ultrathin sections from the rat perfused with 2.5% glutaraldehyde, 0.5% formaldehyde, 0.1% picric acid were used, absorption of the anti-glutamate antiserum with 0.1 mM, 1 mM or 10 mM glutamate decreased from 65% to zero the proportion of profiles that contacted or synapsed on retrogradely labelled sympathetic preganglionic neurons and contained glutamate immunoreactivity (Fig. 3). After staining with unabsorbed antiserum, 43 of 66 profiles on CTB-immunoreactive neurons were positive for glutamate. Absorption with 0.1 mM glutamate resuited in 33 of 73 profiles with glutamate immunoreactivity. Twenty-three of 110 profiles were glutamate positive using antiserum absorbed with 1 mM glutamate. (In two other experiments, one on the same rat, and one on the rat fixed with 2.5% glutaraldehyde, absorption with

o •

a

:~ •

%

O ¸~

o~

76 1 m M glutamate also decreased the p r o p o r t i o n of glutamate-positive profiles by about two-thirds.) A b s o r p t i o n with 10 m M glutamate resulted in no glutamate-immunoreactive profiles (0/89) contacting or synapsing on retrogradely labelled neurons. The abolition of staining after absorption with 10 m M glutamate was confirmed with adjacent series of 2 - 3 ultrathin sections that were stained either with anti-glutamate or with anti-glutamate absorbed with 10 m M glutamate (Fig. 4). In 10 out of 10 cases, profiles that were labelled with many gold parti-

cles after staining with the unabsorbed antiserum were not labelled in adjacent sections after staining with the a b s o r b e d antiserum. Absorption with aspartate. A b s o r p t i o n of the anti-glutamate antiserum with aspartate had no significant effect on the p r o p o r t i o n of axon profiles that contacted or synapsed on CTB-positive neurons and were glutamate immunoreactive (Fig. 3). Treatment with 0.1 m M aspartate increased the p r o p o r t i o n of glutamate-positive profiles (86/109 glutamate positive) c o m p a r e d to the u n a b s o r b e d

Fig. 7. Electron micrographs showing double staining for glutamate and GABA on the same ultrathin section. Glutamate was localized with 10 nm gold particles. GABA was localized with 15 nm gold particles. Bar in B, which applies to both A and B, = 250 nm. A: the cell body (NCB) of a retrogradely labelled sympathoadrenal neuron receives a synapse (arrowheads) from a gtutamate-immunoreactive axon profile. The profile is covered with many 10 nm gold particles but only a few 15 nm gold particles. A deposit of electron-dense peroxidase reaction product (asterisk) indicates the presence of CTB immunoreactivity in the cell body. A large dense core vesicle is indicated by a small arrow. B: the cell body (NCB) of a retrogradely labelled sympathoadrenal neuron receives a synapse (arrowheads) from a GABA-positive axon profile. The profile is covered with many 15 nm gold particle but only a very few 10 nm gold particles. Deposits of electron-dense reaction product (asterisks) indicate the presence of CTB immunoreactivity in the cell body.

77 antiserum (55/87 glutamate positive). After absorption with 1 mM aspartate, the proportion of labelled profiles (40/64 glutamate positive) was the same as with the unabsorbed antiserum (and see Fig. 5). Absorption with 10 mM aspartate slightly decreased the proportion of glutamate-positive profiles (38/68 glutamate positive) on retrogradely labelled neurons. Staining with normal serum or buffer. Only unlabelled boutons were found in sections that had been labelled with normal rabbit serum or with buffer instead of primary antiserum.

Staining for glutamate and G A B A in the same profiles Axon profiles that were immunoreactive for glutamate were not immunoreactive for G A B A . When one series of 2-3 ultrathin sections was stained with anti-glutamate antiserum and an adjacent series of 2-3 sections with anti-GABA antiserum, profiles that were glutamate positive were never G A B A positive and vice versa. Similarly, when the same ultrathin section was stained to reveal glutamate immunoreactivity with 10 nm gold particles and G A B A immunoreactivity with 15 nm gold particles (Figs. 6 and 7), glutamate-positive axon profiles were labelled with many 10 nm gold particles but had few 15 nm gold particles. Conversely, GABA-positive profiles had many 15 nm particles but few 10 nm particles. DISCUSSION This study has shown that glutamate-immunoreactive synapses occur on the cell bodies and dendrites of sympathetic preganglionic neurons retrogradely labelled from CTB injections into the adrenal medulla or into the superior cervical ganglion. Quantitation of axon profiles that were directly apposed to or synapsed on neurons projecting to the adrenal medulla revealed that twothirds were immunoreactive for glutamate. Furthermore, glutamate immunoreactivity and G A B A immunoreactivity were shown to be in separate populations of axon terminals that synapsed on sympathoadrenal neurons by staining consecutive series of sections with glutamate or G A B A , or by using double immunogold labelling. In a recent study of the synaptic input to sympathoadrenal preganglionic neurons, Bacon and Smith 6 found that about one-third of all axon terminals were G A B A immunoreactive. In combination, our results and those of Bacon and Smith suggest that the vast majority, if not all, of the nerve fibers that synapse on sympathetic preganglionic neurons contain either an excitatory or inhibitory amino acid neurotransmitter. If this is the case, then these amino acid-immunoreactive nerve fibers probably also contain other neurotransmitter candidates, such

as adrenaline, substance P or enkephalin, since nerve fibers containing these substances or their synthetic enzymes have previously been shown to form synapses with sympathetic preganglionic n e u r o n s 6'8'9't3'14'25'30'43. Glutamate is known to excite sympathetic preganglionic neurons 2. Administration of excitatory amino acid antagonists into the spinal subarachnoid space attenuates the increase in blood pressure caused by stimulation of the vasopressor areas of the rostral ventrolateral medulla 19'27-29'45 and glutamate release in the spinal cord is raised by this stimulation 22. These results, in combination with the anatomical findings made here, suggest that glutamate not only provides the major excitatory input to sympathoadrenal neurons but is also likely to be a major excitatory transmitter to other types of sympathetic preganglionic neurons. The cell bodies of origin of the glutamate-containing synapses on sympathetic preganglionic neurons have yet to be conclusively identified. The vast majority of the glutamate-immunoreactive terminals in the lateral horn disappear after spinal transection at T 3 (ref. 34), suggesting that supraspinal neurons are the major source of the glutamate input to sympathetic preganglionic neurons. At least some of these terminals are likely to come from neurons in the vasopressor areas of the rostral ventral medulla, since our recent work has shown that many PAG-immunoreactive neurons in this area project to the intermediolateral cell column 3~. Furthermore, we have demonstrated that a proportion of the bulbospinal PAGimmunoreactive neurons also contains the adrenalinesynthesizing enzyme, PNMT, while another group contains serotonin 3~. Some or all of these groups of bulbospinal neurons could form glutamate-immunoreactive synapses on retrogradely labelled neurons. In fact, Morrison et al. 35 have recently shown that a small number of terminals that were anterogradely labelled from injections of Phaseolus vulgaris leucoagglutinin into the rostral ventrolateral medulla and synapsed onto unidentified dendrites in the intermediolateral cell column were immunoreactive for glutamate. A monosynaptic pathway from the area of the midline serotonin-containing neurons to the intermediolateral cell column has also been demonstrated 7. As well as synapses of bulbospinal origin, it is possible that a few glutamate-positive terminals on sympathetic preganglionic neurons could come from the neurons in the dorsal horn that a c c u m u l a t e [3H]Daspartate after lateral horn injections 1. There could also be an input to the intermediolateral cell column from primary afferent neurons, which are well-known to be glutamate-positive (for review see ref. 41), although degeneration studies in monkey thoracic spinal cord 11'42 suggest that, if such a projection exists, it is not a significant one.

78 Essential requirements for any immunocytochemical studies on glutamate are to show that the antibody is detecting n e u r o t r a n s m i t t e r glutamate rather than glutamate in metabolic pools and that the antibody is specific for glutamate. We found here that the cytoplasm of C T B - i m m u n o r e a c t i v e and n o n - i m m u n o r e a c t i v e cell bodies and dendrites (except for mitochondria, see Materials and Methods; Table I) had about 3 times fewer gold particles//~m2 than glutamate-positive axon profiles. This difference in labelling intensity corresponds to that seen by van den Po144 between postsynaptic dendrites and glut a m a t e - i m m u n o r e a c t i v e presynaptic axon terminals in rat hypothalamus. F u r t h e r m o r e , convincingly negative axon profiles were found next to or near convincingly positive profiles. These observations argue in favor of our detecting n e u r o t r a n s m i t t e r glutamate rather than metabolic glutamate in this study. O u r m e t h o d of counting glutamate-positive profiles (defined as showing 20 or m o r e gold particles over vesicles) on r e t r o g r a d e l y labelled sympathetic preganglionic neurons on the screen of the electron microscope p r o v e d to be useful for assessing the specificity of the anti-glutamate antiserum. Using this quick but reliable m e t h o d (which consistently underestim a t e d the p r o p o r t i o n of g l u t a m a t e - i m m u n o r e a c t i v e profiles on r e t r o g r a d e l y labelled neurons), we were able to show that, as the concentration of glutamate used to absorb the anti-glutamate antiserum increased from 0 to 10 m M , the p r o p o r t i o n of labelled profiles contacting or synapsing on C T B - i m m u n o r e a c t i v e neurons decreased from about 2/3 of all profiles to none. On the o t h e r hand, absorption with aspartate had no significant effect. These ultrastructural results c o m p l e m e n t the light microscopic findings of Petrusz et al. 38, who showed densitometrically that dorsal horn staining for glutamate was slightly increased by absorption with low concentrations of aspartate. We also showed, using either staining of alternate series of sections for glutamate and G A B A or double-staining the same section for these two substances, that glutamate-positive terminals were negative for G A B A and that G A B A - p o s i t i v e terminals were negative for glutamate. Taken together, all of these results

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indicate that the chemical which we were localizing in nerve terminals in this study was probably glutamate. The possibility that glutamate may be a neurotransmitter in sympathetic preganglionic neurons has been raised by light microscopic immunohistochemical observations34; high levels of glutamate immunoreactivity were found in nerve cell bodies in the intermediolateral cell column and the dorsal horn but not in any o t h e r areas of the spinal gray matter. In the present study, gold particle densities over neurons in the intermediolateral cell column (both retrogradely labelled with CTB and unlabelled) and over neurons in the ventral horn were comp a r e d statistically. G l u t a m a t e labelling over intermediolateral neurons was not significantly different from that over ventral horn neurons. Since our ultrastructural results contradict the light microscopic observations 34, it seems inadvisable to presume at this stage that sympathetic preganglionic neurons use glutamate as a neurotransmitter. In conclusion, this study has shown that sympathetic preganglionic neurons that send axons to the adrenal medulla or to the superior cervical ganglion receive synapses that are immunoreactive for glutamate. Two-thirds of the axon profiles that directly contacted or synapsed on s y m p a t h o a d r e n a l neurons were positive for glutamate. These results suggest that glutamate m a y be a major excitatory neurotransmitter regulating autonomic function at the spinal cord level.

Acknowledgements. The post-embedding staining was performed while I.L-S. was a visitor in the laboratory of Dr. Aldo Rustioni, Department of Anatomy and Cell Biology, University of North Carolina at Chapel Hill. Dr. Rustioni also donated the anti-glutamate antiserum and critically reviewed the manuscript. Without his assistance and generosity, this work would not have been possible. We are very grateful to him. We would also like to thank Dr, Peter Petrusz for helpful discussions and Drs. Petrusz and Richard Weinberg for comments on the manuscript. This work was supported by the National Health and Medical Research Council of Australia, the National Heart Foundation of Australia, the National Sudden Infant Death Syndrome Council of Australia and the Flinders Medical Centre Research Foundation. Adrian Wright, Margaret McLaren and Rachael Coffey provided expert technical assistance.

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