Materials and methods Animals

Adult male Sprague-Dawley rats (250-350 g body weight) were obtained from Taconic Farms (Germantown, NY, USA) and group housed until the time of surgery and then subsequently separated into individual cages. The animals were kept under temperature- (22+ 1°C) and light-controlled conditions (12-h light-dark cycle, with lights on at 06:00 am). Regular rat chow and water were provided ad libitum. All experiments were conducted in accordance with the guidelines set forth by the NIH Animal Care and Use Committee.

Stereotaxic surgery

The surgical procedures were similar to those described elsewhere (Shahar et al., 2004). Briefly, male rats were weighed and anaesthetized with isoflurane, administered via a Stoelting gas anaesthesia adaptor for the stereotaxic instrument. Once anaesthetized, the rat was placed in the stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). Using the stereotaxic coordinates from the Paxinos and Watson (1986) atlas, bilateral intra-cerebral cannulae connected to osmotic mini pumps were directed over the SON (see Fig. 1A). A small (1.5 mm diameter), parasagittal hole was made on each side of the skull (1.8 mm lateral to the sagittal suture and 1.30 mm caudal to the bregma) using dental burr. Two sterile stainless steel cannulae (28 gauge) 8.80 mm long, were inserted down to a level of 0.3 mm up to the dorsal border of the SON, using the following stereotaxic coordinates: 1.30mm posterior to bregma; 1.80mm medial lateral on each side; 8.80mm dorsal ventral. Each cannula (Plastics One, Inc. 6591 Merriman Road, S.W. Roanoke, VA 24018) was attached via sterile PE50 tubing (Plastics One, Inc.) to a sterile model 2002, ALZET osmotic mini-pump (DURECT Corporation, Cupertino, CA, USA), and fixed to the skull with sterile acrylic dental cement (Plastics One, Inc.) bonded to sterile stainless steel screws (Plastics One, Inc.) inserted in the skull. The two ALZET osmotic mini-pumps, connected to the PE50 tubes, were placed subcutaneously on the dorsal aspect of the rat, in between the two scapulae through subcutaneous tunnels which were formed using haemostatic forceps, starting from the mid-sagittal skin incision. One pump (for the left SON) always contained the vehicle solution (sterile PBS), while the other pump (for the right SON) contained either PBS or the experimental drug. All solutions were filtered through a 0.22 mM Millipore filter. The pumps were filled at least 12 h prior to the surgery in a sterile environment and placed in the incubator (37°C) while immersed in 0.9% saline for the purpose of osmotic activation.

Following the above neurosurgical interventions, the sagittal skin incision was closed with surgical stainless steel clips and Ketoprofen was administered (5mg/kg, diluted in 0.9% NaCl). Animals were killed within 2-5 h or 2 days of surgery. Prior to being killed, animals were anesthetized with isoflurane and immediately perfused transcardially with ice-cold 50 ml PBS followed by 250 ml of fixative solution (4% paraformaldehyde, 0.19% picric acid in 0.1 M phosphate buffer, pH 7.4). Brains were removed and post-fixed overnight in the same fixative solution (diluted 1:4 with PBS). Next, each brain was slowly agitated in a 30% sucrose solution (30% sucrose in 0.9% normal saline) for four nights changing the solution at least twice.

Experimental procedures

Control animals consisted of males that had either undergone stereotaxic surgery and infused bilaterally with PBS (n — 2) or males that had simply been killed and perfused without any surgical manipulation (n — 3). Of the males that had bilateral cannulae, one was killed 3h after surgery, and the other was killed 2 days after surgery. The experimental male rats (NAB) received varying doses of NMDA (Sigma, St. Louis, MO, M-3262), AMPA (Sigma A1455) and bicuculline (Sigma B6889) infused over the right SON and PBS over the left SON for 2-5 h. Three animals had 100 mM NMDA and AMPA and either 50 mM, 500 mM or 2.5 mM bicuculline. Two males received a cocktail of 500 mM each of NMDA, AMPA and bicuculline (NAB). There are no significant differences in hnRNA responses between these NAB cocktails containing different doses, and these data are averaged. Three male rats received 3 mM TTX (Sigma Corp) over the right SON and PBS over the left SON for 2 days. These rats also received an osmotic stress (1.5 M NaCl i.p., 1 ml/100 kg) 2h prior to the cardiac perfusions.

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 2h. 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.

Was this article helpful?

0 0

Post a comment