Thirteen rabbits (NZW) weighing 1.85-2.4kg were anaesthetised with a 4mg/kg intravenous bolus injection of alphaxalone-alphadolone ("Saffan") through the marginal ear vein and supplemented when required with bolus injections of 0.6-
1.2mg/kg iv. The left femoral artery and vein were then cannulated for blood pressure monitoring and to provide a maintenance infusion of Saffan (3.75-15mg/kg/hr), respectively. Saffan was the anaesthetic of choice for these experiments since it has been shown not to disturb forebrain induced cardiovascular responses in the cat
(Timms, 1981). Drugs were given via the marginal ear vein. The trachea was cannulated for subsequent artificial ventilation and the bladder was drained and cannulated (see method 2(a)). Animals' rectal temperature was maintained at 38±0.5®C by using a thermostatically controlled heating blanket. All rabbits were paralysed using 0.6mg/kg iv bolus injections of atracurium ("Tracrium"), after testing blink, pupil and pedal withdrawal reflexes, and ventilated artificially (see method 2(a)). Supplementary bolus injections of iracrium were given every 30-45minutes after testing the depth of anaesthesia. Tracrium was chosen since it has a large safety margin between the doses required for neuromuscular blockade and those likely to lead to cardiovascular side effects (Sutherland, Squire, Gibb & Marshall, 1983). End tidal CO2 was maintained between 4 and 4.5% by altering the minute volume and/or by iv
bolus injections of molar sodium bicarbonate solution. In addition, blood gases were measured and pH was maintained in the range of 7.33-7.41, PCO2 between 28.4 and
42. ImmHg and P0 2 was in excess of lOOmmHg in all animals. Craniotomies were
performed over the posterior cerebellar vermis and left ACe or left HDA. Anaesthetic has been shown to abolish the cardiovascular effects to amygdaloid stimulation in the cat (Stock, Schlor, Heidt & Buss, 1978), therefore in cases where typical ACe responses could not be evoked, the left HDA was then stimulated. Regions from which typical ACe responses were elicited were located at the level of bregma, 5-5.5mm lateral to the midline and 10.2-11.9mm ventral to the surface of the brain. Stimulations o f ACe, at around threshold for obtaining a cardiovascular response, consisted o f 4s trains of 210-250pA at lOOHz with a pulse duration of 0.5ms. Activation of lobule IXb, at around threshold, also consisted of 4s trains but of 110-300pA at lOOHz with a pulse duration of 0.2ms. The cerebellar electrode was placed 0.3-0.5mm ventral to the surface o f the cortex.
Regions of the brain where typical HDA responses were evoked were located 3.5- 5mm caudal to bregma, 0.5-lmm lateral from the midline and 12.4-13.5mm ventral to the surface of the brain. Stimulation of the HDA and lobule IXb in this group, at around threshold, consisted of 6s trains of 120-180pA at 70Hz with a pulse duration of
0.5ms and 6s trains of 80-400p.A at lOOHz with a pulse duration o f 0.2ms, respectively.
In eight of the hypothalamic/amygdaloid experiments the electrode used consisted o f a stainless steel hypodermic needle with an outside diameter of 300pM and internal diameter o f 125pM. The electrode was varnished to 0.3mm from the tip, and a custom built plastic luer head, with calibrated capillary glass embedded into it and glued secure, was fixed into the steel luer fitting at the top of the electrode. A pressure line was attached to the capillary glass for microinjection of drugs and the outer barrel of the needle was used for electrical stimulation. In the other five experiments, stainless steel tubing (300pM ext. diam. and lOOpM int. diam.) was varnished to 0.3mm from the tip and the steel barrel was used for electrical stimulation. pplO tubing was used to connect the electrode to a Ipl Hamilton syringe and the lumen was employed for microinjection over 15s. The cerebellar electrode was made from a single pulled capillary glass filled with a 50:50 mixture of "Wood's" metal and indium (ext. diam 0.1-0.2mm).
PNA (n=7), RSNA (n=5), femoral blood fiow (n=7) and renal blood fiow (n=l) were measured in some animals (for details see method 3(b) and 3(c)).
5 (b) Experimental protocol
At the beginning of each experiment the cardioinhibitory component of the baroreceptor reflex was tested by a bolus intravenous injection o f phenylephrine (30 p g per animal; See method 3(d) for details).
Upon locating a region of the left hypothalamus or amygdala from where typical cardiovascular responses were evoked, the stimulus intensity was decreased to around threshold for obtaining a response. Lobule IXb was then stimulated followed 1 minute later by activation of either the HDA or ACe. After a subsequent minute had passed both structures were activated simultaneously and the procedure was repeated another four times. The largest changes in both blood pressure and heart rate to be induced by the end of the stimulus were used in the quantification of results.
The stimulus intensity was then increased to produce a much larger cardiovascular response for the second part of the study. 100-200nl of filtered (0.22pm) acidified saline (pH 6.6) was then injected into the ACe or HDA as a vehicle control for microinjections of L-glutamate. Acidified saline was used as a control to determine if an increase in extracellular volume or decrease in pH would cause some form of non specific mechanical or chemical activation of the neurons in the ACe or HDA. In addition, the effect of saline on the electrically evoked responses was observed. The electrode was then taken out of the brain, flushed through and filled with filtered L- glutamate (50-100mM, pH6.6-6.8), made up in saline (0.9%), and replaced to the same site. The magnitude of the evoked response to electrical stimulation was checked, to indicate replacement of the electrode to the same position. 75-200nl o f L-glutamate was then microinjected to elucidate whether cell bodies in the region of the electrode tip were capable of evoking responses similar to those produced by electrical stimulation. The effect of the L-glutamate on the magnitude and profile of cardiovascular response evoked by electrical stimulation were then monitored for up to 50 minutes after the injection. At the end o f the experiment in two animals, 200nl of filtered acidified saline was microinjected into lobule IXb. After 20 minutes the same volume of filtered L-glutamate (lOOmM) was injected into lobule IXb and the cardiovascular effects observed.
For later identification of the stimulation sites, 100-200nl of a solution o f filtered pontamine sky blue dye (PSB) was injected through the lumen of the electrode (n=3) or an electrolytic lesion was made by passing 0.2mA DC current through the outer barrel of the electrode for 20s (n=10). Although the diffusion coefficient of the dye will be different from that of L-glutamate, it provides an approximate indication of spread. In the experiments where dye was injected, the electrode was removed after injecting the dye and replaced to the same site. A small electrolytic lesion was made (0.1mA DC for 5s) to confirm that the electrode returned to the same site each time it was removed.
5 r d Analysis o f results
For histological processing to identify stimulation sites see methods 2(e) and 3(e). The % change in femoral and renal vascular conductances were calculated as in method 3(e). Where heart intervals are mentioned, they were calculated as in method 2(c). For statistical analysis, mean ACe or HD A and lobule IXb elicited responses were compared against the respective simultaneously induced response, as well as the arithmetically added responses of IXb alone plus either the HDA or ACe using the two-way ANOVA with Tukey-Kramer post tests. Differences are considered as significant where p<0.05. The data are expressed as mean ± s.e.m. (n=no. of animals and t=no of stimulations).
5. RESULTS