S .J. McDougall et al. Brain Research 883 2000 148 –156
149
initiation of the stress response [15] and can regulate in diameter, Plastic Labs, Lansing, MI for 60 min between
sympathetic vasomotor outflow [24,41]. A number of 900 and 1200 h. This procedure of restraint stress was
neurotransmitters have been implicated in central car- performed once for the acute study and for 10 consecutive
diovascular control including arginine vasopressin AVP days in the chronic study, as previously described [37,22].
and angiotensin II Ang II [18]. Elevations in plasma AVP reset arterial baroreflex control of sympathetic nerve
2.2. Receptor autoradiography activity and heart rate HR to lower pressures, an effect
mediated by V receptors in rabbits [20]. Similarly in rats,
Frozen brainstems and kidneys were equilibrated from
1A
AVP is known to facilitate the baroreceptor heart-rate 2808C to 2178C and then sectioned in a cryostat Cryocut
reflex and sensitize low and high pressure baroreceptors 1800, Leica. Coronal sections 14 mm were collected
[13]. Conversely, Ang II inhibits baroreflex function at the from specific brainstem regions. In the kidney, sagittal
level of the NTS [5]. AVP and Ang II are also involved in sections incorporating both cortex and medulla were cut on
the control of the stress response. Thus AVP is known to a cryostat and sections were thaw-mounted onto gelatin-
become a major ACTH stimulator with repeated restraint chrome alum coated slides. All tissue slices were stored at
immobilisation stress [32], while it has been suggested that 2808C until autoradiography experiments. For the three
Ang II plays a role in the control of SAS activity due to autoradiography assays, tissue slices from all treatment
immobilisation-induced stress response [16]. groups day 0, 1 and 10 days of stress and from both
Since we have recently documented the differential strains WKY and SHR were processed simultaneously
effects of restraint on cardiovascular regulation between for any particular receptor assay, to ensure a valid com-
WKY and SHR [22], the present study was designed to parison between groups.
investigate whether this differential coping between WKY The vasopressin V
receptor autoradiography technique
1A 125
and SHR with chronic restraint was associated with using
[ I]HO-LVA
0.03 nM
sequence: 4-HO-
differential alterations in neuropeptide receptor binding. To Phenylacetyl-
D
-TyrMe-Phe-Gln-Asn-Arg-Pro-Arg-NH ;
2
achieve this aim, quantitative in vitro autoradiography was specific activity: 2000 Ci mmol; Auspep, Parkville, Aus-
employed to assess the effect of restraint stress upon the tralia was adapted from a published protocol [2] by
density of V , AT and AT
receptors in selected increasing bovine serum albumin concentration in the
1A 1
2
brainstem regions and the kidneys of acutely and chroni- incubation medium from 0.1 to 1; the addition of a 15
cally stressed WKY and SHR. min pre-incubation of tissue in incubation medium at room
temperature; 1 mM AVP Sigma, St. Louis, USA was used to define non-specific binding. Following the assay incuba-
2. Methods tion, brain sections were washed for 735 min in ice-cold
Tris–HCl 50 mM, pH7.4 while kidney sections had 635 Experimental procedures were approved by the Monash
min washes.
125
University Animal Ethics Committee and performed ac- For angiotensin receptor autoradiography, [
I]Sar-Ile cording to the National Health and Medical Research
Ang II 0.5 nM specific activity: 2000 Ci mmol; Auspep, Council of Australia guidelines for animal experimenta-
Parkville, Australia binding was performed according to a tion. All rats were obtained from the Austin Hospital,
published protocol [9], except that the assay incubation Heidelberg, Victoria and maintained on a 12 h light dark
was for 60 min and the post incubation wash consisted of cycle with standard laboratory rat chow and water avail-
433 min washes. AT and AT
receptor binding was
1 2
able ad libitum. determined in the presence of PD123319 10 mM gift
from Dr J. Keiser, Parke-Davis, USA and losartan 10 2.1. Restraint stress
mM gift from Dr. R. Smith, DuPont, USA, respectively. Non-specific binding was defined by 20 mM Ang II
Age matched 15–16 weeks male WKY n515 and Auspep, Parkville, Australia.
SHR n515 rats were divided into three groups n55 per After washing and rinsing, all tissue sections were dried
group: i control no-stress, ii acute 60 min and iii under a gentle stream of cool air and desiccated overnight.
chronic 60 min daily for 10 days stress groups. Those Subsequently, all slides were apposed to X-ray film
rats designated for the ‘chronic’ paradigm had telemetry Kodak XAR-5 alongside standard microscales American
probes implanted 11 days prior to the onset of restraint; Radiolabelled Chemicals, Inc.. Film apposition time dif-
telemetry data are published elsewhere [22]. Rats subjected fered for various regions as determined by preliminary
to restraint were housed singularly while control rats assays, and autoradiograms were subsequently developed
maintained social contact. Previous studies have demon- in a Kodak automatic developer 100 Plus. Binding of
125 125
strated that isolation housing of adult rats per se is not [
I]HO-LVA and [ I]Sar-Ile Ang II to brainstem and
stressful, indeed rats need to be isolated from weaning to kidney structures was quantified from autoradiograms
show signs of stress anxiety [31]. The stress imposed using a micro-computer imaging densitometry MCID M4
involved confining rats inside aerated perspex tubes 6 cm image analysis system Imaging Research Incorporated,
150 S
Canada, under constant illumination, as previously de- strains Fig. 2 and Table 1. Binding densities varied
scribed [19,37]. Briefly, images were captured and regions significantly, with SHR showing greater V
density in the
1A 2
of interest delineated such that unit activity dpm per mm hypoglossal nucleus 17 and kidney cortex 129,
could be determined with reference to the standard mi- but lower V
density in the LC 221 and Sp5 215
1A
croscales. Brain nuclei were defined using counterstained as compared to WKY. Differences in binding to Ang II
0.1 thionin slices and reference to a stereotaxic atlas receptors were also found with SHR showing a greater
[26]. All slices were of a constant thickness, cut on the density of AT
receptors in the medial nucleus tractus
1
same cryostat, thereby negating any potential confounding solitarius SolM 112, commissural nucleus tractus
factor of tissue thickness on signal density. solitarius SolC 126, AP 137 and RVLM
19, but decreased AT density in the kidney medulla
1
2.3. Statistical analysis 219 and cortex 212 as compared to WKY Fig. 2
and Table 1. Binding to AT receptors was significantly
2
All data are represented as mean6standard error of the higher in the LC 111, SolM 121, and IO 122
mean. For analysis of quantitative autoradiography data, an of SHR as compared to WKY Fig. 2.
unpaired t-test was employed for a strain comparison controls only. A one-way ANOVA, followed by post-hoc
3.2. Effect of restraint Dunnett’s test, was used to compare different stress
paradigms within strains, while a 2-way ANOVA was used Changes in V , AT and AT receptor binding within
1A 1
2
to assess the effect of strain on the temporal nature of the the brainstem and kidney of WKY and SHR associated
response to the stressor. In this instance data were normal- with ongoing restraint are shown in Fig. 2 and Table 1,
ised to preclude any potentially confounding effects of respectively. Significant decreases in V
binding after one
1A
different basal values between strains. P,0.05 was consid- restraint session acute group occurred in the LC and
ered significant. hypoglossal nucleus of the WKY, while in the SHR
significant increases were observed in the SolM, hypo- glossal nucleus and Sp5. Within the WKY group subjected
3. Results to chronic restraint stress, V
