After completion of the surgery, animals were allowed to stabilise for at least 30 minutes. Experiments were only performed in animals in which the baseline variables were stable. Only those experiments in which stable control reflexes were obtained have been included in these studies.
In all experiments, the sympathetic activity to the heart was blocked by pre- treatment with the selective adrenoceptor antagonist atenolol (1 mg kg-'' i.v.). Atenolol was used since it does not bind to 5-HT receptors (Middlemiss et al.,
1977) and poorly penetrates the central nervous system (Street et al., 1979). In the presence of such a sympathetic blockade, changes in R-R interval are indicative of changes in cardiac vagal activity. To confirm that this dose of atenolol was adequate to block the sympathetic drive to the heart throughout the duration of the experiments, atropine methylnitrate was given at the end of the longest protocol (107.5 minutes, protocol 1b) and the reflex stimulations were repeated. After treatment with atropine, the reflex increases in R-R interval evoked by stimulating baroreceptor or cardiopulmonary afferents were abolished.
All i.c injections of test drugs and vehicle were given in a volume of 20 |il over a period of 20 seconds. Test drugs injected i.v. were given in a volume of 1 ml also over a period of 2 0 seconds.
Schematic diagrams of the different protocols used are shown in figures 2.1-2.4.
Protocol 1
In protocol 1 (figure 2.1 a), the effects of the 5-HT-ia receptor ligand buspirone were examined on the reflex responses evoked by stimulating cardiopulmonary afferents with PBG and baroreceptor afferents by electrical stimulation of the aortic nerve. Since the cardiopulmonary reflex has been previously shown to be modulated by buspirone (Dando et al., 1994), stimulation of these afferents in this protocol served as a positive control.
5 minutes after administration of atenolol, alternate PBG and aortic nerve stimulations were carried out to elicit the cardiopulmonary and baroreceptor reflexes respectively. 2.5 minutes after a PBG stimulation, the aortic nerve was stimulated at six frequencies (5, 10, 20, 40, 80 and 160 Hz at 2 minute
intervals). 2.5 minutes after the last aortic nerve stimulation, another PBG stimulus was given and the cycle repeated.
After a control period consisting of three PBG stimulations and two sets of aortic nerve stimulations, the test drug or vehicle was administered and 5
minutes later, the alternate PBG and aortic nerve stimulations were resumed for a period of 45 minutes. In these experiments, the test drug was the 5-HTia receptor ligand buspirone administered either i.v. or i.c. and the vehicle control was saline administered i.e.
To determine whether the effects of buspirone were due to selective agonist actions at 5-HTia receptors, the above protocol was altered slightly in some experiments. In these experiments, i.c. injection of buspirone was performed 2 0 minutes after i.v. injection of the selective S-HT^a receptor antagonist WAY-100635 (figure 2.1 b).
Figure 2.1 Protocol 1
Schematic diagram illustrating the experimental protocol used to investigate:-
a) the effect of vehicle (saline) or buspirone applied i.c. or i.v. on the reflex responses evoked by stimulating cardiopulmonary and baroreceptor afferents.
b) the effect of buspirone applied i.e. on the reflex responses evoked by stimulating cardiopulmonary and baroreceptor afferents following i.v. pre-treatment with
WAY-100635.
Cardiopulmonary afferents were stimulated by right atrial injection of phenylbiguanide (PBG).
Baroreceptor afferents were activated by electrical stimulation of the left aortic nerve at 5, 10, 20, 40, 80 and 160 Hz. a) PBG PBG PBG PBG PBG PBG PBG I Aortic stim(Hz)| I I I I I I 5 10 20 40 80 160 j | | | | |
liilillliiiliii liilililiiiiiilliiliil
^ ^ lOminAtenolol Saline Buspirone
1 mg kg ' i.v. 20 |il 200 ^g kg ' i.c. or i.v.
b)
PBG PBG PBG PBG PBG PBG PBG PBG
Hz) 160
Î
Î
Î
Atenolol WAY-100635 Buspirone
1 mg kg ' i.v. 100 pg kg ' i.v. 200 |ig kg ' i.e.
Protocol 2
In protocol 2, the effects of the 5-HT-iA receptor ligand buspirone were examined on the reflex responses evoked by stimulating carotid chemoreceptor afferents using NaCN. Again the cardiopulmonary afferents were also
stimulated in this protocol so as to provide a positive control.
5 minutes after the administration of atenolol, alternate PBG and NaCN stimulations were performed at 5 minutes intervals to elicit the cardiopulmonary and chemoreceptor reflexes respectively (figure 2 . 2 a).
After a control period consisting of three PBG and NaCN stimulations, the test drug or vehicle was administered and 5 minutes later, the alternate PBG and NaCN stimulations were resumed for a period of 35 minutes. As with protocol 1 a, the test drug was the 5-HT-ia receptor ligand buspirone
administered either i.v. or i.c. and the vehicle control was saline administered i.c.
The above protocol was also modified in some experiments to determine whether the effects of buspirone were due to selective agonist actions at 5- HT-ia receptors. In these experiments, i.c. injection of buspirone was performed 20 minutes after i.v. injection of the 5-HTiA receptor antagonist WAY-100635 (figure 2 .2 b).
Figure 2.2 Protocol 2
Schematic diagram illustrating the experimental protocol used to investigate:-
a) the effect of vehicle (saline) or buspirone applied i.c. or i.v. on the reflex responses evoked by stimulating cardiopulmonary and chemoreceptor afferents.
b) the effect of buspirone applied i.c. on the reflex responses evoked by stimulating cardiopulmonary and chemoreceptor afferents following i.v. pre-treatment with WAY-100635.
Cardiopulmonary afferents were stimulated by right atrial injection of phenylbiguanide (PBG).
Chemoreceptor afferents were stimulated by injection of sodium cyanide (NaCN) into the left lingual artery.
a)
PBG PBG PBG PBG PBG PBG PBG
I NaCN I NaCN I NaCN I NaCN I NaCN I NaCN I NaCN
Î
Î
Atenolol Saline Buspirone