Somatostatin immunoreactivity in axon terminals in rat nucleus tractus solitarii arising from central nucleus of amygdala: coexistence with GABA and postsynaptic expression of sst2A receptor
Introduction
The central nucleus of the amygdala (CeA) is a forebrain nucleus that participates in autonomic regulation during emotional behaviour through its reciprocal connections with the nucleus tractus solitarii (NTS) in the medulla oblongata (Price et al., 1987, Dampney, 1994, Spyer, 1994). Axons originating in the various divisions within the CeA were shown to specifically target different areas within the dorsal vagal complex (i.e. NTS subnuclei and dorsal vagal nucleus) by studies using different anterograde tracers (Veening et al., 1984, Danielsen et al., 1989, Liubashina et al., 2000), and terminals of CeA axons synapsing on NTS neurones have been shown to be γ-aminobutyric acid (GABA)-immunoreactive (-IR, Jia et al., 1997, Saha et al., 2000). It has previously been noted that a substantial proportion of the GABAergic terminals in the NTS contain large dense-cored vesicles (Maqbool et al., 1991). This suggests that they could release additional peptide or amine co-transmitters and originate from specific projections distinct from terminals that probably arise from local sources and co-release GABA and glycine (Saha et al., 1999).
One candidate for a co-transmitter in GABAergic terminals of the NTS is somatostatin (SOM). SOM-IR fibres are found throughout the NTS (Finley et al., 1981, Kalia et al., 1984, Maley, 1996), and evidence for the co-existence of SOM and GABA has been reported in many brain areas (Oertel et al., 1983, Hendry et al., 1984, Somogyi et al., 1984, Van den Pol, 1986, Kubota et al., 1994). SOM-IR cells are abundant in the CeA (Bennett-Clarke et al., 1980, Finley et al., 1981, Roberts, 1992, Wray and Hoffman, 1983, Moga and Gray, 1985, Cassell et al., 1986, Shimada et al., 1989), and furthermore, SOM-immunoreactivity was observed in some CeA neurones that were retrogradely labelled by tracer injected into the dorsal vagal complex (Gray and Magnuson, 1987, Veening et al., 1984, Higgins and Schwaber, 1983). Thus, it is possible that many of the GABA-IR CeA terminals in the NTS could also contain SOM immunoreactivity.
SOM occurs in two biologically active forms, with the shorter peptide, SOM-14 predominating over the N-terminal extended form, SOM-28 throughout the brain (Johansson et al., 1984, Epelbaum, 1986, Patel, 1992). These peptides can have excitatory, as well as inhibitory actions on neurones (Dodd and Kelly, 1978, Pittman and Siggins, 1981, Mueller et al., 1986) mediated by G-protein coupled receptors, which may be linked to delayed rectifier K+ channels (Wang et al., 1989). Five receptor subtypes (sst1–sst5) have been cloned, with all except sst5 having higher affinity for SOM-14 than SOM-28 (Meyerhof et al., 1992, Yamada et al., 1992, Hoyer et al., 1995, Reisine and Bell, 1995, Viollet et al., 1995, Schindler et al., 1996). SOM has modulatory actions on cardiovascular reflexes, producing hypotension and bradycardia when microinjected in the NTS of anesthetised rats (Koda et al., 1985). Intracerebroventricular administration of SOM-14 facilitates phenylephrine-induced baroreceptor responses (Lin et al., 1991), but, in conscious rats, SOM-28 promotes hypertension and tachycardia (Brown, 1988). Both forms of SOM depress the excitability of NTS neurones through hyperpolarisation resulting from augmentation of outward K+ conductance (Jacquin et al., 1988).
The purpose of this study was to investigate whether SOM immunoreactivity is present in anterogradely labelled CeA terminals in the caudal, cardioregulatory zone of NTS, using light and electron microscopic immunolabelling methods. Since we have previously shown that the majority of CeA terminals in the NTS are GABAergic (Saha et al., 2000), we have also combined pre- and post-embedding labelling methods to examine the extent of colocalisation of SOM and GABA immunoreactivities. Furthermore, we have sought evidence for a functional role of SOM release from CeA terminals by examining the relationship of these terminals to NTS neurones expressing the SOM receptor subtype sst2A.
Section snippets
Injection of anterograde tracer
Eleven adult male rats (300–315 g, bred at the University of Leeds) were used in this study. Experimental procedures were performed in accordance with the regulations of the UK Animals (Scientific Procedures) Act, 1986 and European Council Directive 86/609/EEC. Surgery was performed under sodium pentobarbitone (40 mg/kg, i.p.) anaesthesia and buprenorphine (0.1 mg/kg) analgesia, and the animals received daily postoperative care after the surgery. Biotinylated dextran amine (BDA, mol. wt. 10 000
Results
In four of the seven animals that received injections of BDA into the CeA, the tracer involved the rostrocaudal extent of the medial CeA, with limited spread into the adjacent lateral and capsular divisions of the nucleus (Fig. 1). In three cases the injections were centred in the lateral part of the CeA, but with many neurones also labelled in the medial CeA. As reported in our previous study (Saha et al., 2000), peroxidase labelling revealed that the BDA injections centred in both the medial
Discussion
The present results provide the first evidence that GABAergic terminals within the NTS that originate from CeA neurones contain SOM. When immunolocalisation of SOM was combined with post-embedding GABA immunohistochemistry, the great majority (>80%) of SOM-IR terminals in the caudal NTS were found to be GABA-immunopositive, and in an earlier study (Saha et al., 2000), we have shown that almost all the CeA terminals in the NTS contain GABA immunoreactivity (Table 1). Furthermore, by triple
Acknowledgements
This study was supported by a British Heart Foundation Project Grant (PG98095) and by Wellcome Trust and Medical Research Council JREI equipment awards. We thank Jean C. Kaye for her expert technical assistance, and Dr D.V. Pow (Brisbane, Australia) for provision of GABA antiserum.
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