Co-localization of AMPA receptor subunits in the nucleus of the solitary tract in the rat

Brain Research 958 (2002) 454–458 www.elsevier.com / locate / brainres

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Co-localization of AMPA receptor subunits in the nucleus of the solitary tract in the rat Sue A. Aicher*, Sarita Sharma, Jennifer L. Mitchell Neurological Sciences Institute, Oregon Health & Science University, 505 NW 185 th Ave., Beaverton, OR 97006, USA Accepted 18 September 2002

Abstract AMPA-type glutamate receptors in the caudal portions of nucleus of the solitary tract (NTS) are critical for responses to excitatory afferents from the viscera, including baroreceptors. Using immunocytochemistry combined with electron microscopy, the cellular distributions of different AMPA receptor subunits in the caudal NTS were found to be distinct. GluR2 / 3 was found at pre- and postsynaptic sites, and in astrocytic glia; while GluR1 was found primarily in small dendrites and spines. In dual-labeling studies, GluR1 and GluR2 were co-localized in large dendrites, but GluR1 was more often found alone in dendritic spines. Therefore, single neurons in the NTS contain both subunits, but there is differential trafficking of GluR1 to potential sites for synaptic plasticity.  2002 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters and receptors Topic: Excitatory amino acid receptors: structure; function and expression Keywords: Glutamate receptors; Nucleus tractus solitarius; Baroreflex; Immunocytochemistry

The caudal portions of the nucleus of the solitary tract (NTS) receive glutamatergic primary afferents from a variety of visceral tissues [14], including the baroreceptors that are sensitive to distention of the aortic arch and large vessels surrounding the heart [6]. Stretch of the baroreceptors can activate a reflex within the brain that is mediated through several brain stem and spinal nuclei. The net effect of baroreflex activation is a reduction in sympathetic output and heart rate [3]. Dysfunction of the baroreflex can lead to hypertension [11]. Extensive evidence has implicated the a-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)-type glutamate receptors [12] in mediating the effects of baroreceptor stimulation within the NTS [5,10,23]. Recent studies have shown that three of the four known AMPA receptor subunits are present within the NTS [7,15]. The present results confirm and extend this observation by providing quantitative analyses of the subunit distribution within neurons in the caudal NTS. In addition, dual*Corresponding author. Tel.: 11-503-418-2550; fax: 11-503-4182501. E-mail address: [email protected] (S.A. Aicher).

labeling studies with subunit-selective antibodies examined the co-localization of subunits GluR1 and GluR2 in some neurons. Our results suggest that while some caudal NTS neurons make multiple AMPA receptor subunits, these subunits are differentially trafficked to various portions of the dendritic tree. Male Sprague–Dawley rats (Taconic Labs., 250–350 g, n58) were used for these experiments and all methods were approved by the IACUCs at Weill Medical College of Cornell University and at Oregon Health & Science University. Rats were overdosed with pentobarbital sodium (150 mg / kg) and perfused transcardially with the following sequence of solutions: (1) heparinized saline; (2) 3.8% acrolein in 2% paraformaldehyde; and (3) 2% paraformaldehyde in 0.1 M phosphate buffer (PB). The brainstem was sectioned (40 mm) on a vibrating microtome and collected into 0.1 M PB. Sections through the NTS were processed for immunoperoxidase localization of each antigen. Polyclonal antibodies were raised in rabbit and directed against the R1 (GluR1), R2 / 3 (GluR2 / 3) and the R4 (GluR4) subunits of the AMPA receptor were obtained from Chemicon (Temecula, CA, USA). A dilution series (1–8 mg / ml) was tested for each antibody and an optimal

0006-8993 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03693-4

S. A. Aicher et al. / Brain Research 958 (2002) 454–458

dilution was chosen empirically for further analysis. All incubations were for 40 h at 4 8C, and the bound antibody was detected with a biotinylated goat anti-rabbit IgG (1:400; Vector Labs., Burlingame, CA, USA). Colocalization of GluR1 and GluR2 was examined using combined immunoperoxidase and immunogold methods [2,8] with subunit-selective primary antibodies obtained from Chemicon (rabbit anti-GluR1 IgG; mouse anti-GluR2 IgG) that have been extensively characterized [21]. The dilution for GluR1 was 1:25 for both methods; while the dilutions for GluR2 were 1:150 for immunoperoxidase and 1:15 for immunogold. Appropriate secondary antibodies were used for each set of experiments: biotinylated secondary antibodies (1:400) (goat anti-rabbit IgG (Vector Labs.), goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA)) and colloidal gold labeled secondary antibodies (1:50) (goat anti-rabbit IgG and goat anti-mouse IgG; Amersham Pharmacia Biotech, Piscataway, NJ, USA) and were visualized sequentially. Colloidal gold particles were visualized by silver intensification using the IntenSEM kit (Amersham Pharmacia Biotech). Control procedures where the primary antibody or secondary antibody was excluded from the procedure failed to yield labeling at the light microscopic level, similar to the labeling seen with GluR4 (see below).

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Tissue sections were osmicated and embedded in plastic following immunocytochemical procedures. Vibratome sections through NTS at the level of the area postrema (Fig. 1a) were selected and ultrathin sections (75 nm) through the surface of each section were collected onto copper grids, counterstained, and examined using a Phillips CM10 electron microscope. The region sampled was confined to the area between the area postrema and the medial edge of the solitary tract and contains primarily the medial subnucleus [14] (Fig. 1a). For single labeling studies, two to three vibratome sections from three different animals were included in the analysis for each antibody. For the dual-labeling studies, two vibratome sections from two different animals were included in the analysis, and two different labeling directions were examined (immunoperoxidase versus immunogold) for each antigen. In addition, only sections from the tissue–plastic interface were examined to avoid under detection of the immunogold antigen. For the analysis in the dual-labeling study, cross-sectional diameters of dendrites containing either GluR1 alone, GluR2 alone, or both antigens were compared using a Kruskal–Wallis one-way ANOVA, followed by Dunn’s posthoc comparisons, a 5 0.05. We examined the distribution of antibodies directed against AMPA receptor subunits in the caudal NTS at both

Fig. 1. AMPA receptor subunits GluR1 (a, b), GluR2 / 3 (c), and GluR4 (d) had distinct distributions in the caudal NTS. (a) GluR1 was densely localized within the area postrema (AP) and was seen primarily in punctate profiles (arrowheads). The boxed area indicates the region of NTS sampled in quantitative studies and is shown at higher magnification in (b). (b) GluR1 labeling was dense in punctate profiles (arrowheads) and was also seen in neurons (open arrow). (c) GluR2 / 3 was found in many neurons (open arrows) and in some punctate profiles. (d) GluR4 was not detected within the NTS in this study. ts5solitary tract Scale bars5100 mm (a, c, d), 50 mm (b).

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the light and electron microscopic levels and found that the cellular distribution of each AMPA receptor subunit was distinct within this region of NTS (Fig. 1). At the light microscopic level, GluR1 was found primarily in small punctate structures and also in cell bodies (Fig. 1a and b). GluR2 / 3 was found in both cells and processes (Fig. 1c), while GluR4 was not detected in this nucleus at the dilutions tested (1–8 mg / ml) and was not studied further (Fig. 1d). At the electron microscopic level, GluR2 / 3 had a mixed presynaptic (Fig. 2a) and postsynaptic distribution (Fig. 2b) and was also found in glial processes (Fig. 2b). In contrast, GluR1 was found primarily at postsynaptic sites, particularly in small dendrites (,1.5 mm) and dendritic spines (,0.5 mm and receiving an asymmetric synapse)

(Fig. 2c and d). The contrasting cellular distributions of GluR1 and GluR2 / 3 (Table 1) suggest that different NTS neurons and afferents to NTS may contain distinct complements of AMPA receptor subunits [15]. However, even within the caudal NTS, the distinct postsynaptic distributions of GluR1 and GluR2 / 3 (Table 2) led us to speculate that these receptor subunits may be found in the same neurons, but trafficked to different portions of the dendritic tree of these cells [18]. To test this hypothesis, we dually labeled sections using subunit-selective antibodies to GluR1 and GluR2. As with the GluR2 / 3 antibody, the distribution of GluR2 was mixed at presynaptic and postsynaptic sites (Fig. 3), while GluR1 was primarily found in small dendrites and spines (Fig. 3b and c). The two subunits were occasionally co-

Fig. 2. Electron microscopy shows that AMPA receptor subunits were found at pre- and postsynaptic sites in the caudal NTS, as well as in glial processes. GluR2 / 3 was found within presynaptic axons (R2 / 3-a) (a) and in dendrites (R2 / 3-d) (b) that received asymmetric synapses (filled curved arrow) from unlabeled terminals (ut). Within glial processes (R2 / 3-g), the peroxidase labeling was often seen along tight appositions that resemble gap junctions. GluR1 was seen in dendritic spines (R1-s) (c) and in small dendrites (R1-d) (d) that received asymmetric synapses (filled curved arrows) from unlabeled terminals. Scale bars50.25 mm (a–c), 0.50 mm (d).

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Table 1 Ultrastructural distribution of AMPA receptor subunits in caudal NTS Antigen

Somata

Dendrites

Axons

Terminals

Glia

Total

GluR1 GluR2 / 3

1 (1%) 0 (0%)

164 (90%) 123 (47%)

8 (4%) 75 (29%)

2 (1%) 9 (3%)

7 (4%) 55 (21%)

182 262

Table 2 Dendritic distribution of AMPA receptor subunits in caudal NTS Antigen

Large dendrite

Small dendrite

Spine

Subtotal

GluR1 GluR2 / 3

2 (1%) 12 (10%)

116 (71%) 98 (80%)

46 (28%) 13 (10%)

164 123

localized within large dendritic processes (Fig. 3a) that were significantly larger than dendrites containing only GluR1 (see statistics in legend of Fig. 3). These results suggest that some neurons synthesize both subunits, but GluR1 is preferentially trafficked to dendritic spines. Our results show that each of the subunits of the AMPA

Fig. 3. AMPA receptor subunits GluR1 and GluR2 were occasionally co-localized in large dendrites. (a) Large dendrite (R11R2-d) contained immunoperoxidase labeling for GluR2 (filled arrow), as well as immunogold labeling for GluR1 (arrowheads). Some of the immunogold labeling and the peroxidase labeling were closely associated with the plasma membrane. An unmyelinated axon contained GluR2 (R2-a). (b) Small dendrite (R1-d) contained immunogold labeling for GluR1 that was associated with nonsynaptic portions of the plasma membrane, away from an apposition (open arrow) with an unlabeled terminal (ut). An unmyelinated axon (R2-a) contained immunoperoxidase labeling for GluR2. (c) Small dendrite (R1-d) contained immunogold labeling for GluR1 and received an asymmetric synapse (filled curved arrow) from an unlabeled terminal (ut1), and was also apposed to other unlabeled axon terminals (ut2, ut3). Dendrites containing only GluR1 (n541) were significantly smaller than dendrites containing either GluR2 alone (n513) or dendrites containing both GluR1 and GluR2 (n514) (Kruskal–Wallis one-way ANOVA on Ranks P50.0003; Dunn’s posthoc pairwise comparisons, GluR1 alone different from dual-labeled and from GluR2 alone). Only dendrites were included in the quantitative analysis, but GluR2-labeled axons, somata, and glial processes were also seen. Scale bars50.25 mm.

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receptor has a distinct cellular distribution in the caudal NTS, consistent with observations from other laboratories [4]; although the presence of GluR4 has been detected by other investigators in cat and rat NTS [4,17]. While GluR1 was found primarily at postsynaptic sites, GluR2 was located at both pre- and postsynaptic sites in the NTS, similar to the distribution of the NMDAR1 receptor subunit in the NTS [1]. Together these results suggest that glutamate may act a multiple neuronal and glial sites in NTS. The finding that many of the AMPA receptive neurons in the subpostremal region of NTS are barosensitive [7] supports the notion that the receptors examined in the present study may be involved in baroreceptor transmission, although a role in other glutamatergic functions cannot be excluded. In dual-labeling studies, we found that GluR1 and GluR2 are co-localized in some neurons, but only in large, presumably proximal dendrites. These results indicate that cells containing multiple AMPA receptor subunits can traffick these subunits to different portions of the dendritic tree [16]. This suggests [20] that the signal(s) controlling receptor trafficking are probably distinct not only for different classes of receptors, but even for subunits of a single type of receptor. Further, GluR1 was often found in dendritic spines that do not contain GluR2. This intriguing result suggests that while fairly sparse within the nucleus, these spines probably contain AMPA receptors that are permeable to calcium [9,13,22] and may therefore be important in synaptic plasticity [19].

Acknowledgements The authors thank Alla Goldberg and Kristin Swanson for technical assistance. Some of the experiments were conducted at the Weill Medical College of Cornell University. Grant support from the American Heart Association and NIH HL56301.

[5]

[6] [7]

[8]

[9]

[10]

[11] [12] [13]

[14]

[15]

[16]

[17]

[18] [19] [20]

References [21] [1] S.A. Aicher, S. Sharma, V.M. Pickel, N-Methyl-D-aspartate receptors are present in vagal afferents and their dendritic targets in the nucleus tractus solitarius, Neuroscience 91 (1999) 119–132. [2] S.A. Aicher, B.-I. Hahn, T.A. Milner, N-Methyl-D-aspartate-type glutamate receptors are found in postsynaptic targets of adrenergic terminals in the thoracic spinal cord, Brain Res. 856 (2000) 1–11. [3] S.A. Aicher, T.A. Milner, V.M. Pickel, D.J. Reis, Anatomical substrates for baroreflex sympathoinhibition, Brain Res. Bull. 51 (2000) 107–110. [4] R. Ambalavanar, C.L. Ludlow, R.J. Wenthold, Y. Tanaka, M. Damirjian, R.S. Petralia, Glutamate receptor subunits in the nucleus

[22]

[23]

of the tractus solitarius and other regions of the medulla oblongata in the cat, J. Comp. Neurol. 402 (1998) 75–92. M.C. Andresen, M.Y. Yang, Non-NMDA receptors mediate sensory afferent synaptic transmission in medial nucleus tractus solitarius, Am. J. Physiol. Heart Circ. Physiol. 90 (1990) H1307–H1311. M.C. Andresen, D.L. Kunze, Nucleus tractus solitarius—gateway to neural circulatory control, Annu. Rev. Physiol. 56 (1994) 93–116. S.A. Botsford, C. Dean, F.A. Hopp, J.L. Seagard, Presence of glutamate receptor subtypes on barosensitive neurons in the nucleus tractus solitarius of the dog, Neurosci. Lett. 261 (1999) 113–117. J. Chan, C. Aoki, V.M. Pickel, Optimization of differential immunogold–silver and peroxidase labeling with maintenance of ultrastructure in brain sections before plastic embedding, J. Neurosci. Meth. 33 (1990) 113–127. H.S. Engelman, T.B. Allen, A.B. MacDermott, The distribution of neurons expressing calcium-permeable AMPA receptors in the superficial laminae of the spinal cord dorsal horn, J. Neurosci. 19 (1999) 2081–2089. F.J. Gordon, C. Leone, Non-NMDA receptors in the nucleus of the tractus solitarius play the predominant role in mediating aortic baroreceptor reflexes, Brain Res. 568 (1991) 319–322. G.A. Head, Baroreflexes and cardiovascular regulation in hypertension, J. Cardiovasc. Pharmacol. 26 (Suppl. 2) (1995) S7–S16. M. Hollmann, S. Heinemann, Cloned glutamate receptors, Annu. Rev. Neurosci. 17 (1994) 31–108. R.I. Hume, R. Dingledine, S.F. Heinemann, Identification of a site in glutamate receptor subunits that controls calcium permeability, Science 253 (1991) 1028–1031. M. Kalia, J.M. Sullivan, Brainstem projections of sensory and motor components of the vagus nerve in the rat, J. Comp. Neurol. 211 (1982) 248–264. J.P. Kessler, A. Baude, Distribution of AMPA receptor subunits GluR1-4 in the dorsal vagal complex of the rat: a light and electron microscope immunocytochemical study, Synapse 34 (1999) 55–67. D.V. Lissin, R.C. Carroll, R.A. Nicoll, R.C. Malenka, M. Von Zastrow, Rapid, activation-induced redistribution of ionotropic glutamate receptors in cultured hippocampal neurons, J. Neurosci. 19 (1999) 1263–1272. R.S. Petralia, R.J. Wenthold, Light and electron immunocytochemical localization of AMPA-selective glutamate receptors in the rat brain, J. Comp. Neurol. 318 (1992) 329–354. M.E. Rubio, R.J. Wenthold, Differential distribution of intracellular glutamate receptors in dendrites, J. Neurosci. 19 (1999) 5549–5562. B.L. Sabatini, M. Maravall, K. Svoboda, Ca 21 signaling in dendritic spines, Curr. Opin. Neurobiol. 11 (2001) 349–356. S.H. Shi, Y. Hayashi, R.S. Petralia, S.H. Zaman, R.J. Wenthold, K. Svoboda, R. Malinow, Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation, Science 284 (1999) 1811–1816. S.J. Siegel, W.G. Janssen, J.W. Tullai, S.W. Rogers, T. Moran, S.F. Heinemann, J.H. Morrison, Distribution of the excitatory amino acid receptor subunits GluR2(4) in monkey hippocampus and colocalization with subunits GluR5-7 and NMDAR1, J. Neurosci. 15 (1995) 2707–2719. M.S. Washburn, M. Numberger, S. Zhang, R. Dingledine, Differential dependence on GluR2 expression of three characteristic features of AMPA receptors, J. Neurosci. 17 (1997) 9393–9406. J.C. Yen, J.Y.H. Chan, S.H.H. Chan, Differential roles of NMDA and non-NMDA receptors in synaptic responses of neurons in nucleus tractus solitarii of the rat, J. Neurophysiol. 81 (1999) 3034–3043.