Effects of the hippocampus on the motor expression of augmented breaths

Anaesthesia and surgeryUrethane at a dose of 1.5 g/kg of body weight was administered intraperitoneally. The depth of anaesthesia was frequently monitored by checking corneal and hind paw-pinch stretch reflexes. Prophylactic doses of atropine methyl nitrate (0.05 mg/100 g) were also administered intramuscularly before surgery to reduce respiratory secretions. Femoral artery and venous cannulation were routinely performed to connect the rat to blood pressure transducers and replacement fluid line, respectively. Supplementary doses of urethane (0.􏰅15–0.􏰅3 g/kg) were administered intravenously through the femoral vein during experi- ments, as required.Chest wall displacement. A force transducer (WPI, Inc) was used to monitor chest wall displacement. A string of stainless steel wire (0.003" bare, 0.0055" coated; A-M Systems) was anchored to the loose skin on the dorsum of the thoracic cavity and then attached to the transducer, which was mounted on the stereotaxic frame. The accuracy of the force transducer was further validated by electromyogram (EMG) from the crural diaphragmatic activity, where bursts of muscle activity represent inspiration.Diaphragm EMG. In each animal, a transverse incision was made through the rectus abdominis muscle at the level of the xiphoid cartilage. The liver was then reflected to expose the crural diaphragm. A pair of fine platinum wire electrodes was implanted in the crural dia- phragm (0.003@ bare, 0.0055@ coated; A-M Systems). Electrode lengths of about 20 cm were used for each recording. A portion of the Teflon coating was stripped at the end and middle of the electrodes (approximately 5 mm each). The electrodes were then threaded through the cru- ral diaphragm by secure attachment to a 17 mm 1⁄2 circle reverse cutting suture needle. The electrodes were pulled through until the bare middle portion was in contact with the dia- phragm. The loop was knotted to maintain contact and so that it was not easily pulled out of the muscle. Caution was applied to prevent pneumothorax. The stripped end was then con- nected to a bio-amplifier through a wire relay. A reference electrode was attached to the body. The abdominal incision was closed with a continuous suture pattern, holding both the muscles and skin together to maintain contractile pressure. Skin adhesives were also applied to secure the stitch. At the end of each experiment, the diaphragm was carefully removed to assess elec- trode placement. Diaphragmatic EMG during an augmented breath shows prolonged muscu- lar activity during inspiration with peak amplitudes higher than normal breaths. Also, the EMG tracing clearly shows a period of respiratory pause after each augmented breath. Coordination of both methods, forced transducer and DEMG, was used to enhance precision in calculating the duration of inspiration and expiration. A heating pad was used to maintain the body temperature between 37–38 oC.<br>Inspiratory duration (Ti): Each burst of diaphragmatic electromyogram activity was consid- ered an inspiration. The diaphragm is mostly active during inspiration and passive during expiration. Thus, the start of diaphragmatic muscle firing, as captured by the EMG tracing, was indicative of the start of inspiration and the entire period of firing was defined as the inspiratory duration (Ti). The precise duration was calculated offline by identifying such periods of activity in the LabChart software and obtaining its numerical value in seconds from the ‘Data Pad’ function of the same software.Expiratory duration (Te): The duration from the end of an inspiratory period to the start of the next was defined as expiratory duration. This period was extracted following the same pro- cedure used to extract Ti.Craniotomy and microinjectionsAnimals were placed in a prone position and carefully fitted in a stereotaxic apparatus (Nar- ishige Scientific Instrument Lab.). An incisor and ear bars was used to firmly anchor the head. Protective ophthalmic ointment (GenTeal 1) was applied, and the scalp was shaved and disin- fected with an antiseptic solution (Betadine1) or 70% ethanol solution. A midline incision of about 2 cm was then made over the scalp using a scalpel blade or cauterizer (Erbe ICC 200) to minimise bleeding while exposing the skull. Bregma and lambda were identified and used to ensure that the skull was in a flat position by adjusting them to the same stereotaxic position. Small boreholes (􏰝 2 mm in diameter) were drilled using a craniotomy drill (Microtorque II, SDR Scientific) to expose and allow access to the brain. A nick was made in the dura mater to enhance precision and prevent destroying the fragile tip of glass micropipettes. Glass micropi- pettes with tip diameters of 20–30 μm were pulled using a Sutter pipette puller (P-97, Novato, CA). The micropipettes were calibrated and coupled to Hamilton syringes to enable delivery of precise volumes of drugs into the brain. Coordinates of the anterior-posterior position, medial- lateral position as well as the depth of Bregma were recorded, and calculations were made for various target regions according to Paxinos and Watson, 2007. The ventral hippocampus was chemically mapped with microinjections of the excitatory amino acid, D, L-Homocysteic acid (DLH, 50 mM, 200–400 nL) until areas that have influence over the expression of aug- mented breaths were identified. The dorsal hippocampus was also examined for control and comparison. Quantitative analyses of the typical characteristics of augmented breaths were ana- lysed using 30 representative augmented breaths selected randomly from six animals, selected mostly during baseline breathing periods. Tracings of augmented breaths that were distorted by external factors such as adjusting the animals’ position during experimental/surgical manipula- tions of were excluded. The breaths surrounding each augmented breath were also assessed. For this, the five breaths each immediately preceding and succeeding an augmented were analysed for both inspiratory and expiratory duration. The rats were spontaneously breathing room air throughout the period of the experiment, and in a prone position.<br>