GABA ρ-receptor subunits assemble as homo- or heteromeric ligand-gated channels with other ρ-subunits to form GABAC receptors. GABA ρ-subunits were initially discovered in the retina but have been reported in CNS regions such as the cerebellum, hippocampus, cortex and superior colliculus (Zhang et al. 2001). In the present study we have investigated the presence of the ρ1-subunit in the adult rat medulla oblongata.
Male rats (100-150 g, n = 3) were anaesthetised with Sagatal (60 mg kg-1 I.P.), perfused transcardially with artificial cerebrospinal fluid in which NaCl was replaced with sucrose (217 mM) and the brain removed. Total RNA was isolated from medulla and reverse transcribed followed by 35 cycles of PCR using ρ1-subunit-specific primers. Sections of medulla (10 µm) were cryostat cut, and mounted on slides that had been pretreated with 3-aminopropyltriethoxysilane (Sigma). A 202 bp cDNA fragment from the amino-terminal of the ρ1-subunit was cloned into pGEM T-easy (Promega) and used to generate DIG-labelled sense and antisense RNA probes. In situ hybridisation was carried out using a modified version of the manufacturer protocol (www.biochem.roche.com). For immunohistochemistry rats (n = 3) were anaesthetised as above and perfused transcardially with 4 % para-formaldehyde and 0-0.5 % glutaraldehyde in 0.1 M phosphate buffer. Free-floating 50 µm sections of medulla oblongata were incubated in an affinity-purified antibody raised in rabbit against the amino-terminal of the ρ1-subunit (1:200; a gift from Dr Maurice Garret, Bordeaux, France). Sections were then washed and placed in either donkey anti-rabbit conjugated Cy3 (1:1000, Jackson) or a biotinylated anti-rabbit (1:200, Vector), followed by extravidin peroxidase conjugate (1:1500, Sigma) visualised with diaminobenzidine (Sigma). Sections were then prepared for either light or electron microscopic analysis.
RT-PCR shows the presence of the ρ1-subunit transcript in medulla. In situ hybridisation detects ρ1 mRNA in neurones throughout the medulla, including the inferior olive, lateral reticular nucleus and dorsal vagal nucleus, nucleus of the solitary tract (NTS) and the hypoglossal nucleus. Similar populations of neurones were also labelled using immunohistochemistry. Preliminary ultrastructural analysis of the ρ1-subunit in the NTS revealed immunoreactivity in astrocyte processes surrounding presynaptic terminals, the endoplasmic reticulum of neurones and at the postsynaptic specialisation of some synaptic junctions. These results clearly demonstrate that the ρ1-subunit is expressed in the adult rat medulla oblongata and may be present in both glia and neurones.This work was supported by The Wellcome Trust.
- Zhang, D., Pan, Z.-H., Awobuluyi, M. & Lipton, S.A. (2001). TiPS 22, 121-132.