Experimental approaches for the study of oxytocin and vasopressin gene expression in the central nervous system

Abstract

Intron-specific probes measure heteronuclear RNA (hnRNA) levels and thus approximate the transcription rates of genes, in part because of the rapid turnover of this intermediate form of RNA in the cell nucleus. Previously, we used oxytocin (Oxt)- and vasopressin (Avp)- intron-specific riboprobes to measure changes in Oxt and Avp hnRNA levels in the supraoptic nucleus (SON) by quantitative in situ hybridization (ISH) after various classical physiological perturbations, including acute and chronic salt loading, and lactation. In the present experiments, we used a novel experimental model to study the neurotransmitter regulation of Oxt and Avp gene expression in the rat SON in vivo. Bilateral cannulae connected via tubing to Alzet osmotic mini-pumps were positioned over the SON. In every experiment, one SON was infused with PBS and served as the control SON in each animal, and the contralateral SON received infusions of various neurotransmitter agonists and antagonists. Using this approach, we found that Avp but not Oxt gene expression increased after acute (2-5h) combined excitatory amino acid agonist and GABA antagonist treatment, similar to what we found after an acute hyperosmotic stimulus. Since both OXT and AVP are known to be comparably and robustly secreted in response to acute osmotic stimuli in vivo and glutamate agonists in vitro, our results indicate a dissociation between OXT secretion and Oxt gene transcription in vivo.

Fig. 1

Vasopressin gene expression and excitatory amino acid stimulation in the SON. (A) Schematic diagram of in vivo experimental system is illustrated. Stereotaxic surgery was performed on adult male rats and bilateral cannulae were directed over the SON. Cannulae were connected via PE50 tubing to Alzet mini-pumps containing one of three different solutions: an excitatory cocktail of NMDA, AMPA and bicuculline (NAB); TTX or PBS. In all experiments, the left SON of each male served as an internal control and received vehicle infusion (PBS), while the right SON was infused with either PBS (control solution) or an experimental solution NAB or TTX. Representative photomicrographs of Avp hnRNA ISH results are shown in (B–C). (B) PBS was infused over both SONs for 3 h. (C) A cocktail of NAB was infused over the right SON for 2.5 h. (D) TTX was infused over the right SON for 2 days, and 1.5 M NaCl was injected i.p. to increase Avp hnRNA expression acutely for 2 h. Note that the left uninhibited SON showed enhanced Avp hnRNA comparable to the NAB stimulated SON in (C), whereas the TTX inhibited SON (right side) did not (see text). Abbreviations: PBS, phosphate buffered saline (vehicle); EXP, experimental drug; PVN, paraventricular nucleus; SCN, suprachiasmatic nucleus; SON, supraoptic nucleus; OX, optic chiasm; LV, lateral ventrical; 3V, third ventrical; f, fornix; ic, internal capsule; cc, corpus callosum.

Fig. 2

Quantitative determinations of vasopressin (A) and oxytocin (B) hnRNA levels in control (unstimulated) SONs and SONs stimulated by infusions of the excitatory amino acid cocktail (NAB). The data are expressed as percentage changes in the NAB-treated (right side) SON as compared to the PBS-control (left) SON (see Fig. 1C). The control bars in the graph represent the mean control data from five animals in which both sides of the SON were equally treated (e.g., by PBS infusion only, Fig. 1B). The bars labelled NAB are from animals in which the right SON infused with PBS (n = 5). *Equals significant difference (p<0.05) in hnRNA level between PBS and NAB stimulated SONs.