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Glutamate and GABA content of calbindin-immunoreactive nerve terminals in the rat intermediolateral cell column
Autonomic Neuroscience: Basic and Clinical 98 (2002) 7 – 11 www.elsevier.com/locate/autneu
Glutamate and GABA content of calbindin-immunoreactive nerve terminals in the rat intermediolateral cell column I.J. Llewellyn-Smith*, C.L. Martin, J.B. Minson Cardiovascular Neuroscience Group, Cardiovascular Medicine and Centre for Neuroscience, Flinders University, Bedford Park, South Australia 5042, Australia
Abstract Immunoreactivity for calbindin-D28K (calbindin) occurs in some bulbospinal vasopressor neurons in the rostral ventrolateral medulla and calbindin-immunoreactive terminals form synapses in the intermediolateral cell column (IML), where the cell bodies of sympathetic preganglionic neurons are located. In this study, we used post-embedding immunogold labelling to determine whether calbindin terminals in the IML contained the excitatory amino acid neurotransmitter glutamate. We also assessed GABA immunoreactivity in semi-serial sections through the same terminals since this inhibitory amino acid transmitter is present in the inputs to sympathetic preganglionic neurons that lack glutamate. Analysis of 42 calbindin-positive terminals whose postsynaptic targets were not identified revealed two major groups on the basis of amino acid content. One group was immunoreactive for glutamate; and the other, for GABA. In addition, about 20% of the calbindin terminals were positive for both glutamate and GABA. Our anatomical methods cannot differentiate whether this third group is a subset of the GABAergic terminals or a separate population capable of co-releasing the two amino acids. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Amino acids; Blood pressure; Post-embedding immunogold labelling; Spinal cord; Ultrastructure
1. Introduction Calbindin-D28K (calbindin) is one of several related calcium-binding proteins that are found in neurons (Celio, 1990; Miller, 1991). Although calbindin is likely to buffer intracellular calcium and thereby help to protect neurons from damage caused by release of excess calcium from internal stores (Miller, 1991, 1995; Baimbridge et al., 1992; Monje et al., 2001), calbindin’s function in neurons has not yet been precisely identified. Nevertheless, calbindin and the other neuronal calcium-binding proteins have proved to be useful markers for defining functional subsets of central and peripheral autonomic neurons (e.g., Grkovic and Anderson, 1995, 1997; Anderson, 1998). In this context, we have previously shown that calbindin-immunoreactivity delineates a subpopulation of bulbospinal adrenaline-synthesizing neurons in the rostral ventrolateral medulla (Goodchild et al., 2000). We also showed that, in the intermediolateral cell column (IML), calbindin-immunoreactive nerve termi*
Corresponding author. Department of Medicine, Flinders Medical Centre, Bedford Park, South Australia 5042, Australia. Tel.: +61-8-82044456; fax: +61-8-8204-5268. E-mail address: [email protected] (I.J. Llewellyn-Smith).
nals apposed retrogradely labelled sympathetic preganglionic neurons and formed synapses at the electron microscope (EM) level. Furthermore, many of the synapses made by calbindin terminals in the IML were asymmetric, suggesting that the terminals might be excitatory (Gray, 1959). Since glutamate is a major excitatory transmitter released onto sympathetic preganglionic neurons and critical for the control of blood pressure by these neurons (Guyenet et al., 1987; Mills et al., 1988, 1989; Verberne et al., 1990; Bazil and Gordon, 1991; Kapoor et al., 1992; Inokuchi et al., 1992; Deuchars et al., 1995), we examined the calbindin-immunoreactive terminals in the IML for the presence of glutamate immunoreactivity using post-embedding immunogold labelling. Because GABA is present in the glutamate-negative terminals that synapse on sympathetic preganglionic neurons (Llewellyn-Smith et al., 1992, 1998), we also assessed the calbindin terminals for the presence of GABA immunoreactivity.
2. Materials and methods Three male Wistar-Kyoto rats (250 –350 g) were anaesthetised (Nembutal; 100 mg/kg, i.p.), injected with heparin (1000 IU) and perfused with oxygenated tissue culture
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medium (Sigma) followed by 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Thoracic spinal cords were removed, divided into pieces and postfixed for 2 h at room temperature on a shaker in the same fixative. The tissue was washed in phosphate buffer and cut parasagittally at 50 Am on a Vibratome. Sections were exposed to 50% ethanol in distilled water for 60 min (Llewellyn-Smith and Minson, 1992) and then to 10% normal horse serum (NHS) in Tris – phosphate buffered saline (TPBS) for 30 min. Sections were incubated on a shaker at room temperature in a rabbit polyclonal antibody against calbindin-D28K (1:150,000 in 10% NHS –TPBS; Swant, Bellinzona, Switzerland) for 3 – 5 days, in donkey anti-rabbit immunoglobulin (1:500 in 1% NHS – TPBS; Jackson ImmunoResearch, West Grove, PA) for 24 h and then in ExtrAvidin-peroxidase (1:1500 in
TPBS; Sigma) for 24 h. Sections were washed 3 30 min in TPBS after each incubation. A nickel-intensified diaminobenzidine (DAB) reaction was used to visualize calbindin-immunoreactivity (Llewellyn-Smith et al., 1993). Stained sections were osmicated, dehydrated in acetone and embedded flat in Durcupan. We detected glutamate- and GABA-immunoreactivity in semi-serial sections through calbindin-immunoreactive terminals in the IML by post-embedding immunocytochemistry (Llewellyn-Smith and Weaver, 2001). Ultrathin sections were cut with a diamond knife and mounted in pairs on formvar-coated single slot grids. The grids were washed and then incubated overnight at room temperature in rabbit antiglutamate (1:25,000; Biogenesis, Poole, UK) or rabbit antiGABA (1:5000; Sigma) antiserum diluted in Tris-buffered
Fig. 1. Electron micrographs of semi-serial ultrathin sections through a terminal that synapses (arrowheads) on a dendrite in the IML. The terminal contains deposits of DAB reaction product (stars), indicating that it is immunoreactive for calbindin. (A) Immunogold labelling for glutamate indicates that the terminal is positive for glutamate (GLU+). The gold particle density over the terminal is 77.3 particles/Am2. The minimum density for positivity on this section was 59.8 particles/Am2. (B) The terminal in (A) in an adjacent section, which was stained to show GABA immunoreactivity. The terminal is negative for this amino acid (GABA ), the gold particle density over the terminal being 14.2 particles/Am2 and the minimum density for positivity on this section being 22. 6 particles/Am2. Scale bar in (B), which also applies to (A), 500 nm.
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Fig. 2. Electron micrographs of semi-serial ultrathin sections through two terminals (1 and 2) that contact dendrites in the IML. The two terminals contain DAB reaction product (stars), indicating that they are immunoreactive for calbindin. Terminal 1 is only lightly labelled whereas DAB reaction product fills most of terminal 2. (A) Immunogold labelling for GABA indicates that terminal 1 is positive for this amino acid (GABA+). The gold particle density over terminal 1 is 135.3 particles/Am2. The minimum density for positivity on this section was 23.2 particles/Am2. (B) The terminals in (A) in an adjacent section, which was stained to show glutamate immunoreactivity. Terminal 1 is negative for glutamate (GLU ), the gold particle density over the terminal being 45.1 particles/Am2 and the minimum density for positivity on this section being 82.8 particles/Am2. Scale bar in (B), which also applies to (A), 500 nm.
saline containing Triton X-100 (TBS-Triton). The anti-glutamate and anti-GABA antisera have previously been shown to recognize only the respective antigens against which they were raised (Saha et al., 1995; Murphy et al., 1996). After washing, the grids were incubated in anti-rabbit immunoglobulin conjugated to 10-nm gold particles (1:25 in TBSTriton; AuroProbe EM GAR, Amersham, UK). Finally, the grids were washed, dried and stained with uranyl acetate and lead citrate. We characterized calbindin-immunoreactive terminals as glutamate- or GABA-immunoreactive as described previously (Llewellyn-Smith and Weaver, 2001). We counted gold particles over synaptic vesicles in the portions of terminals that lacked or had minimal deposition of nickel-DAB reaction product and measured the areas over which particles were counted. To establish whether a terminal was positive for glutamate or GABA, its labelling density (gold particles/Am2) was ascertained and compared to a statistically defined ‘‘background’’ labelling density. This density was calculated from 25 elliptical samples of cytoplasm that were randomly selected from somata in the IML and that contained predominantly rough endoplasmic reticulum and/ or Golgi apparatus for each ultrathin section that was analyzed. Gold particles over mitochondrial profiles were excluded from both terminal and background counts.
and some formed synapses on dendrites and cell bodies, confirming our earlier observations (Goodchild et al., 2000). Small, clear and occasional large granular vesicles were present in the calbindin terminals. Variable amounts of electron-dense nickel-DAB reaction product occurred in the calbindin-positive terminals in the IML (Figs. 1 and 2). Some of the positive terminals were so heavily labelled that it was not possible to analyze either their vesicle content or their gold particle labelling (not shown). Other terminals contained identifiable synaptic vesicles, but much of their volume was filled with reaction product (for example, terminal 2 in Fig. 2). Yet in other terminals, only a small amount of nickel-DAB reaction product was present (Fig. 1 and terminal 1 in Fig. 2). Only those terminals in which gold particles and vesicles could be resolved were analyzed for their content of glutamate and GABA. Immunoreactivity for glutamate and/or GABA could be detected in a total of 42 calbindin-immunoreactive terminals from the IML of three rats. Of these terminals, 17 (41%; Fig. 1) were immunoreactive for glutamate alone; and 16 (38%; Fig. 2, terminal 1), for GABA alone. The remaining nine terminals (21%) showed immunoreactivity for both amino acids.
4. Discussion 3. Results At the EM level, calbindin-immunoreactive vesicle-containing axon terminals were scattered throughout the IML
These results suggest that at least two neurochemically identifiable populations of calbindin-immunoreactive terminals innervate the IML. One major calbindin-positive pop-
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ulation shows immunoreactivity for glutamate; and the other, immunoreactivity for GABA. It is possible that a third group of calbindin terminals, supplying the IML, contains both amino acids. The calbindin/glutamate terminals demonstrated in this study could come from supraspinal or intraspinal sources. At least some of the terminals with this phenotype are likely to arise from the bulbospinal calbindin/adrenaline neurons in the ventral medulla (Granata and Chang, 1994; Goodchild et al., 2000). The occurrence of glutamate in these medullary inputs is consistent with the fact that all of the bulbospinal adrenaline-synthesizing neurons in this region contain phosphate-activated glutaminase (Minson et al., 1991), an enzyme considered to be a marker for neurons that use glutamate as a neurotransmitter, and that these neurons are sympathoexcitatory (Lipski et al., 1995; Schreihofer and Guyenet, 1997; Verberne et al., 1999). Glutamate/ calbindin terminals may also come from the calbindin neurons in the dorsal horn that accumulate H3-D-aspartate after injection of this radioactive excitatory amino acid into the intermediate gray (Antal et al., 1991). These aspartateaccumulating calbindin neurons may be the same population as the calbindin neurons that show glutamate immunoreactivity after light microscopic immunohistochemistry and project to the nucleus tractus solitarius (Gamboa-Esteves et al., 2001); but this is not yet known. The origins of the GABA/calbindin terminals in the IML are unknown. Calbindin-positive somata occur throughout the gray matter of the thoracic spinal cord (Antal et al., 1990). Some of these neurons may contain GABA since these two neurochemicals co-localize in many different kinds of central neurons (e.g., Hendry et al., 1989; Webster et al., 1990; Miettinen et al., 1992). Supraspinal neurons might also supply calbindin/GABA terminals to the IML, which receives a long GABAergic projection that either comes from or passes through the ventral medulla (Llewellyn-Smith et al., 1995). All of the bulbospinal calbindin neurons in the medulla are catecholaminergic (Goodchild et al., 2000) and are therefore, likely to be excitatory (i.e., glutamatergic). Furthermore, the calbindin neurons in the A5 region are not bulbospinal (Goodchild et al., 2000). Hence, any supraspinal calbindin/GABA neurons that might supply the IML should lie in other areas of the medulla or pons or in higher centres. It is not possible to determine whether the calbindin/ glutamate/GABA terminals in the IML are a separate population of inputs from the calbindin/glutamate and the calbindin/GABA terminals on the basis of immunogold labelling data alone. The co-localization of glutamate and GABA in calbindin terminals may relate to the fact that glutamate is necessary for GABA synthesis. An interesting alternative explanation is that both amino acids might be released from the calbindin/glutamate/GABA terminals to act post- and/or presynaptically. There is increasing evidence that single synaptic vesicles can take up, store and co-release both GABA and glutamate (Takamori et al., 2000a,b). Further-
more, co-release of glutamate and GABA is supported anatomically by the occurrence of group I metabotropic glutamate receptors postsynaptic to GABA-positive boutons in the globus pallidus (Hanson and Smith, 1999), and physiologically by the reduction of GABA-mediated thalamic inhibition, through activation of presynaptic metabotropic glutamate receptors (Salt et al., 1996). Although glutamate and GABA are probably localized in discrete subsets of nerve fibers that synapse on sympathetic preganglionic neurons in the IML of intact spinal cord (LlewellynSmith et al., 1992, 1998), studies on a completely transected cord indicate that, after injury, about 5% of all of the inputs to these neurons contain both amino acids (Llewellyn-Smith et al., 1997; Llewellyn-Smith and Weaver, 2001). Furthermore, caudal to a transection, the proportion of the total input to SPN that is GABAergic increases significantly by 2 weeks after injury (Llewellyn-Smith and Weaver, 2001). These findings indicate plasticity in the amino acid innervation of the IML and it will be interesting to see if the populations of calbindin-immunoreactive fibers change either qualitatively or quantitatively after spinal cord injury.
Acknowledgements This work was supported by the National Health and Medical Research Council of Australia, the National Heart Foundation of Australia and the Australian Research Council. We thank Lee Travis for expert technical assistance.
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Llewellyn-Smith, I.J., Arnolda, L.F., Pilowsky, P.M., Chalmers, J.P., Minson, J.B., 1998. GABA- and glutamate-immunoreactive synapses on sympathetic preganglionic neurons projecting to the superior cervical ganglion. J. Auton. Nerv. Syst. 71, 96 – 110. Miettinen, R., Gulyas, A.I., Baimbridge, K.G., Jacobowitz, D.M., Freund, T.F., 1992. Calretinin is present in non-pyramidal cells of the rat hippocampus — II. Co-existence with other calcium binding proteins and GABA. Neuroscience 48, 29 – 43. Miller, R.J., 1991. The control of neuronal Ca2 + homeostasis. Prog. Neurobiol. 37, 255 – 285. Miller, R.J., 1995. Regulation of calcium homoeostasis in neurons: the role of calcium-binding proteins. Biochem. Soc. Trans. 23, 629 – 632. Mills, E.H., Minson, J.B., Pilowsky, P.M., Chalmers, J.P., 1988. N-MethylD-aspartate receptors in the spinal cord mediate pressor responses to stimulation of the rostral ventrolateral medulla in the rat. Clin. Exp. Pharmacol. Physiol. 15, 147 – 155. Mills, E., Minson, J., Drolet, G., Chalmers, J.P., 1989. Effect of intrathecal amino acid receptor antagonists on basal blood pressure and pressor responses to brainstem stimulation in normotensive and hypertensive rats. J. Cardiovasc. Pharmacol. 15, 877 – 883. Minson, J., Pilowsky, P., Llewellyn-Smith, I., Kaneko, T., Kapoor, V., Chalmers, J., 1991. Glutamate in spinally projecting neurons of the rostral ventral medulla. Brain Res. 555, 326 – 331. Monje, M.L., Phillips, R., Sapolsky, R., 2001. Calbindin overexpression buffers hippocampal cultures from the energetic impairments caused by glutamate. Brain Res. 911, 37 – 42. Murphy, S.M., Pilowsky, P.M., Llewellyn-Smith, I.J., 1996. Vesicle shape and amino acids in synaptic inputs to phrenic motoneurons: do all inputs contain either glutamate or GABA? J. Comp. Neurol. 373, 200 – 219. Saha, S., Batten, T.F.C., McWilliam, P.N., 1995. Glutamate, g-aminobutyric acid and tachykinin-immunoreactive synapses in the cat nucleus tractus solitarii. J. Neurocytol. 24, 55 – 74. Salt, T.E., Eaton, S.A., Turner, J.P., 1996. Characterization of the metabotropic glutamate receptors (mGluRs) which modulate GABA-mediated inhibition in the ventrobasal thalamus. Neurochem. Int. 29, 317 – 322. Schreihofer, A.M., Guyenet, P.G., 1997. Identification of C1 presympathetic neurons in rat rostral ventrolateral medulla by juxtacellular labeling in vivo. J. Comp. Neurol. 387, 524 – 536. Takamori, S., Riedel, D., Jahn, R., 2000a. Immunoisolation of GABAspecific synaptic vesicles defines a functionally distinct subset of synaptic vesicles. J. Neurosci. 20, 4904 – 4911. Takamori, S., Rhee, J.S., Rosenmund, C., Jahn, R., 2000b. Identification of a vesicular glutamate transporter that defines a glutamatergic phenotype in neurons. Nature 407, 189 – 194. Verberne, A.J.M., Widdop, R.E., Maccarrone, C., Jarrott, B., Beart, P.M., Louis, W.J., 1990. Intrathecal kynurenate reduces arterial pressure, heart rate and baroreceptor-heart rate reflex in conscious rats. Neurosci. Lett. 114, 309 – 315. Verberne, A.J.M., Stornetta, R.L., Guyenet, P.G., 1999. Properties of C1 and other ventrolateral medullary neurones with hypothalamic projections in the rat. J. Physiol. 517, 477 – 494. Webster, W.R., Batini, C., Buisseret-Delmas, C., Compoint, C., Guegan, M., Thomasset, M., 1990. Colocalization of calbindin and GABA in medial nucleus of the trapezoid body of the rat. Neurosci. Lett. 111, 252 – 257.
Glutamate and GABA content of calbindin-immunoreactive nerve terminals in the rat intermediolateral cell column I.J. Llewellyn-Smith*, C.L. Martin, J.B. Minson Cardiovascular Neuroscience Group, Cardiovascular Medicine and Centre for Neuroscience, Flinders University, Bedford Park, South Australia 5042, Australia
Abstract Immunoreactivity for calbindin-D28K (calbindin) occurs in some bulbospinal vasopressor neurons in the rostral ventrolateral medulla and calbindin-immunoreactive terminals form synapses in the intermediolateral cell column (IML), where the cell bodies of sympathetic preganglionic neurons are located. In this study, we used post-embedding immunogold labelling to determine whether calbindin terminals in the IML contained the excitatory amino acid neurotransmitter glutamate. We also assessed GABA immunoreactivity in semi-serial sections through the same terminals since this inhibitory amino acid transmitter is present in the inputs to sympathetic preganglionic neurons that lack glutamate. Analysis of 42 calbindin-positive terminals whose postsynaptic targets were not identified revealed two major groups on the basis of amino acid content. One group was immunoreactive for glutamate; and the other, for GABA. In addition, about 20% of the calbindin terminals were positive for both glutamate and GABA. Our anatomical methods cannot differentiate whether this third group is a subset of the GABAergic terminals or a separate population capable of co-releasing the two amino acids. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Amino acids; Blood pressure; Post-embedding immunogold labelling; Spinal cord; Ultrastructure
1. Introduction Calbindin-D28K (calbindin) is one of several related calcium-binding proteins that are found in neurons (Celio, 1990; Miller, 1991). Although calbindin is likely to buffer intracellular calcium and thereby help to protect neurons from damage caused by release of excess calcium from internal stores (Miller, 1991, 1995; Baimbridge et al., 1992; Monje et al., 2001), calbindin’s function in neurons has not yet been precisely identified. Nevertheless, calbindin and the other neuronal calcium-binding proteins have proved to be useful markers for defining functional subsets of central and peripheral autonomic neurons (e.g., Grkovic and Anderson, 1995, 1997; Anderson, 1998). In this context, we have previously shown that calbindin-immunoreactivity delineates a subpopulation of bulbospinal adrenaline-synthesizing neurons in the rostral ventrolateral medulla (Goodchild et al., 2000). We also showed that, in the intermediolateral cell column (IML), calbindin-immunoreactive nerve termi*
Corresponding author. Department of Medicine, Flinders Medical Centre, Bedford Park, South Australia 5042, Australia. Tel.: +61-8-82044456; fax: +61-8-8204-5268. E-mail address: [email protected] (I.J. Llewellyn-Smith).
nals apposed retrogradely labelled sympathetic preganglionic neurons and formed synapses at the electron microscope (EM) level. Furthermore, many of the synapses made by calbindin terminals in the IML were asymmetric, suggesting that the terminals might be excitatory (Gray, 1959). Since glutamate is a major excitatory transmitter released onto sympathetic preganglionic neurons and critical for the control of blood pressure by these neurons (Guyenet et al., 1987; Mills et al., 1988, 1989; Verberne et al., 1990; Bazil and Gordon, 1991; Kapoor et al., 1992; Inokuchi et al., 1992; Deuchars et al., 1995), we examined the calbindin-immunoreactive terminals in the IML for the presence of glutamate immunoreactivity using post-embedding immunogold labelling. Because GABA is present in the glutamate-negative terminals that synapse on sympathetic preganglionic neurons (Llewellyn-Smith et al., 1992, 1998), we also assessed the calbindin terminals for the presence of GABA immunoreactivity.
2. Materials and methods Three male Wistar-Kyoto rats (250 –350 g) were anaesthetised (Nembutal; 100 mg/kg, i.p.), injected with heparin (1000 IU) and perfused with oxygenated tissue culture
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medium (Sigma) followed by 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Thoracic spinal cords were removed, divided into pieces and postfixed for 2 h at room temperature on a shaker in the same fixative. The tissue was washed in phosphate buffer and cut parasagittally at 50 Am on a Vibratome. Sections were exposed to 50% ethanol in distilled water for 60 min (Llewellyn-Smith and Minson, 1992) and then to 10% normal horse serum (NHS) in Tris – phosphate buffered saline (TPBS) for 30 min. Sections were incubated on a shaker at room temperature in a rabbit polyclonal antibody against calbindin-D28K (1:150,000 in 10% NHS –TPBS; Swant, Bellinzona, Switzerland) for 3 – 5 days, in donkey anti-rabbit immunoglobulin (1:500 in 1% NHS – TPBS; Jackson ImmunoResearch, West Grove, PA) for 24 h and then in ExtrAvidin-peroxidase (1:1500 in
TPBS; Sigma) for 24 h. Sections were washed 3 30 min in TPBS after each incubation. A nickel-intensified diaminobenzidine (DAB) reaction was used to visualize calbindin-immunoreactivity (Llewellyn-Smith et al., 1993). Stained sections were osmicated, dehydrated in acetone and embedded flat in Durcupan. We detected glutamate- and GABA-immunoreactivity in semi-serial sections through calbindin-immunoreactive terminals in the IML by post-embedding immunocytochemistry (Llewellyn-Smith and Weaver, 2001). Ultrathin sections were cut with a diamond knife and mounted in pairs on formvar-coated single slot grids. The grids were washed and then incubated overnight at room temperature in rabbit antiglutamate (1:25,000; Biogenesis, Poole, UK) or rabbit antiGABA (1:5000; Sigma) antiserum diluted in Tris-buffered
Fig. 1. Electron micrographs of semi-serial ultrathin sections through a terminal that synapses (arrowheads) on a dendrite in the IML. The terminal contains deposits of DAB reaction product (stars), indicating that it is immunoreactive for calbindin. (A) Immunogold labelling for glutamate indicates that the terminal is positive for glutamate (GLU+). The gold particle density over the terminal is 77.3 particles/Am2. The minimum density for positivity on this section was 59.8 particles/Am2. (B) The terminal in (A) in an adjacent section, which was stained to show GABA immunoreactivity. The terminal is negative for this amino acid (GABA ), the gold particle density over the terminal being 14.2 particles/Am2 and the minimum density for positivity on this section being 22. 6 particles/Am2. Scale bar in (B), which also applies to (A), 500 nm.
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Fig. 2. Electron micrographs of semi-serial ultrathin sections through two terminals (1 and 2) that contact dendrites in the IML. The two terminals contain DAB reaction product (stars), indicating that they are immunoreactive for calbindin. Terminal 1 is only lightly labelled whereas DAB reaction product fills most of terminal 2. (A) Immunogold labelling for GABA indicates that terminal 1 is positive for this amino acid (GABA+). The gold particle density over terminal 1 is 135.3 particles/Am2. The minimum density for positivity on this section was 23.2 particles/Am2. (B) The terminals in (A) in an adjacent section, which was stained to show glutamate immunoreactivity. Terminal 1 is negative for glutamate (GLU ), the gold particle density over the terminal being 45.1 particles/Am2 and the minimum density for positivity on this section being 82.8 particles/Am2. Scale bar in (B), which also applies to (A), 500 nm.
saline containing Triton X-100 (TBS-Triton). The anti-glutamate and anti-GABA antisera have previously been shown to recognize only the respective antigens against which they were raised (Saha et al., 1995; Murphy et al., 1996). After washing, the grids were incubated in anti-rabbit immunoglobulin conjugated to 10-nm gold particles (1:25 in TBSTriton; AuroProbe EM GAR, Amersham, UK). Finally, the grids were washed, dried and stained with uranyl acetate and lead citrate. We characterized calbindin-immunoreactive terminals as glutamate- or GABA-immunoreactive as described previously (Llewellyn-Smith and Weaver, 2001). We counted gold particles over synaptic vesicles in the portions of terminals that lacked or had minimal deposition of nickel-DAB reaction product and measured the areas over which particles were counted. To establish whether a terminal was positive for glutamate or GABA, its labelling density (gold particles/Am2) was ascertained and compared to a statistically defined ‘‘background’’ labelling density. This density was calculated from 25 elliptical samples of cytoplasm that were randomly selected from somata in the IML and that contained predominantly rough endoplasmic reticulum and/ or Golgi apparatus for each ultrathin section that was analyzed. Gold particles over mitochondrial profiles were excluded from both terminal and background counts.
and some formed synapses on dendrites and cell bodies, confirming our earlier observations (Goodchild et al., 2000). Small, clear and occasional large granular vesicles were present in the calbindin terminals. Variable amounts of electron-dense nickel-DAB reaction product occurred in the calbindin-positive terminals in the IML (Figs. 1 and 2). Some of the positive terminals were so heavily labelled that it was not possible to analyze either their vesicle content or their gold particle labelling (not shown). Other terminals contained identifiable synaptic vesicles, but much of their volume was filled with reaction product (for example, terminal 2 in Fig. 2). Yet in other terminals, only a small amount of nickel-DAB reaction product was present (Fig. 1 and terminal 1 in Fig. 2). Only those terminals in which gold particles and vesicles could be resolved were analyzed for their content of glutamate and GABA. Immunoreactivity for glutamate and/or GABA could be detected in a total of 42 calbindin-immunoreactive terminals from the IML of three rats. Of these terminals, 17 (41%; Fig. 1) were immunoreactive for glutamate alone; and 16 (38%; Fig. 2, terminal 1), for GABA alone. The remaining nine terminals (21%) showed immunoreactivity for both amino acids.
4. Discussion 3. Results At the EM level, calbindin-immunoreactive vesicle-containing axon terminals were scattered throughout the IML
These results suggest that at least two neurochemically identifiable populations of calbindin-immunoreactive terminals innervate the IML. One major calbindin-positive pop-
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ulation shows immunoreactivity for glutamate; and the other, immunoreactivity for GABA. It is possible that a third group of calbindin terminals, supplying the IML, contains both amino acids. The calbindin/glutamate terminals demonstrated in this study could come from supraspinal or intraspinal sources. At least some of the terminals with this phenotype are likely to arise from the bulbospinal calbindin/adrenaline neurons in the ventral medulla (Granata and Chang, 1994; Goodchild et al., 2000). The occurrence of glutamate in these medullary inputs is consistent with the fact that all of the bulbospinal adrenaline-synthesizing neurons in this region contain phosphate-activated glutaminase (Minson et al., 1991), an enzyme considered to be a marker for neurons that use glutamate as a neurotransmitter, and that these neurons are sympathoexcitatory (Lipski et al., 1995; Schreihofer and Guyenet, 1997; Verberne et al., 1999). Glutamate/ calbindin terminals may also come from the calbindin neurons in the dorsal horn that accumulate H3-D-aspartate after injection of this radioactive excitatory amino acid into the intermediate gray (Antal et al., 1991). These aspartateaccumulating calbindin neurons may be the same population as the calbindin neurons that show glutamate immunoreactivity after light microscopic immunohistochemistry and project to the nucleus tractus solitarius (Gamboa-Esteves et al., 2001); but this is not yet known. The origins of the GABA/calbindin terminals in the IML are unknown. Calbindin-positive somata occur throughout the gray matter of the thoracic spinal cord (Antal et al., 1990). Some of these neurons may contain GABA since these two neurochemicals co-localize in many different kinds of central neurons (e.g., Hendry et al., 1989; Webster et al., 1990; Miettinen et al., 1992). Supraspinal neurons might also supply calbindin/GABA terminals to the IML, which receives a long GABAergic projection that either comes from or passes through the ventral medulla (Llewellyn-Smith et al., 1995). All of the bulbospinal calbindin neurons in the medulla are catecholaminergic (Goodchild et al., 2000) and are therefore, likely to be excitatory (i.e., glutamatergic). Furthermore, the calbindin neurons in the A5 region are not bulbospinal (Goodchild et al., 2000). Hence, any supraspinal calbindin/GABA neurons that might supply the IML should lie in other areas of the medulla or pons or in higher centres. It is not possible to determine whether the calbindin/ glutamate/GABA terminals in the IML are a separate population of inputs from the calbindin/glutamate and the calbindin/GABA terminals on the basis of immunogold labelling data alone. The co-localization of glutamate and GABA in calbindin terminals may relate to the fact that glutamate is necessary for GABA synthesis. An interesting alternative explanation is that both amino acids might be released from the calbindin/glutamate/GABA terminals to act post- and/or presynaptically. There is increasing evidence that single synaptic vesicles can take up, store and co-release both GABA and glutamate (Takamori et al., 2000a,b). Further-
more, co-release of glutamate and GABA is supported anatomically by the occurrence of group I metabotropic glutamate receptors postsynaptic to GABA-positive boutons in the globus pallidus (Hanson and Smith, 1999), and physiologically by the reduction of GABA-mediated thalamic inhibition, through activation of presynaptic metabotropic glutamate receptors (Salt et al., 1996). Although glutamate and GABA are probably localized in discrete subsets of nerve fibers that synapse on sympathetic preganglionic neurons in the IML of intact spinal cord (LlewellynSmith et al., 1992, 1998), studies on a completely transected cord indicate that, after injury, about 5% of all of the inputs to these neurons contain both amino acids (Llewellyn-Smith et al., 1997; Llewellyn-Smith and Weaver, 2001). Furthermore, caudal to a transection, the proportion of the total input to SPN that is GABAergic increases significantly by 2 weeks after injury (Llewellyn-Smith and Weaver, 2001). These findings indicate plasticity in the amino acid innervation of the IML and it will be interesting to see if the populations of calbindin-immunoreactive fibers change either qualitatively or quantitatively after spinal cord injury.
Acknowledgements This work was supported by the National Health and Medical Research Council of Australia, the National Heart Foundation of Australia and the Australian Research Council. We thank Lee Travis for expert technical assistance.
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