Somatostatin immunoreactivity in axon terminals in rat nucleus tractus solitarii arising from central nucleus of amygdala: coexistence with GABA and postsynaptic expression of sst_2A receptor

Abstract

Axon terminals synapsing on neurones in the nucleus tractus solitarii (NTS) that originate from the central nucleus of the amygdala (CeA) have been shown to contain γ-aminobutyric acid (GABA) immunoreactivity. Here we investigated whether such terminals also contain somatostatin (SOM), a neuropeptide found in axons distributed throughout the NTS and in somata in the CeA, and known to modulate cardiovascular reflexes when microinjected into the NTS. With fluorescence microscopy, SOM immunoreactivity was seen in the varicosities of some axons throughout the NTS that were anterogradely labelled with biotin dextran amine injected into the CeA. Such varicosities were frequently observed in close proximity to dendrites of NTS neurones that were immunoreactive for the SOM receptor sst_2A subtype, and in many cases also for catecholamine synthesising enzymes. In the caudal, cardioregulatory zone of NTS, SOM immunoreactivity was localised by electron microscopic pre-embedding gold labelling to boutons containing dense-cored and clear pleomorphic vesicles and forming symmetrical synapses, mostly onto dendrites. Additional post-embedding gold labelling for GABA suggested that a subpopulation (29%) of GABAergic terminals sampled in this area of NTS contained SOM. Almost all boutons anterogradely labelled from the amygdala were GABA-immunoreactive (-IR) and 21% of these were SOM-IR. A similar proportion of these boutons (22%) formed synapses onto dendrites containing immunoreactivity for the SOM receptor sst_2A subtype. These observations provide evidence that some of the GABAergic projection neurones in the CeA that inhibit baroreceptor reflex responses in the NTS in response to fear or emotional stimuli could release SOM, which might modulate the activity of NTS neurones via an action on sst_2A receptors.

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 sst_2A.