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Olfactory thresholds in normal and adrenalectomized rats
Physiology and Behavior, Vol. 9, pp. 495-500, Brain Research Publications Inc., 19"/2. Printed in U.S.A.
Olfactory Thresholds in Normal and Adrenalectomized Rats ' PETER C. SAKELLARIS 2
A m e s Research Center-NASA, Code-239-17, M o f f e t t Field, California, 94035
(Received 21 March 1972)
SAKELLARIS, P. C. Olfactory thresholds in normal and adrenalectomized rats. PHYSIOL. BEHAV. 9 (4) 495-500, 1972.- Olfactory thresholds for pyridine were examined in normal and adrenalectomized rats. The animals were trained to suppress bar-pressing on a VR 10:1 schedule in the presence of the odorant. Adrenalectomized animals were found to have a lower threshold than normais. When one normal animal was subsequently adrenalectomized and another sham operated the threshold decreased for the first but did not change in the latter. The administration of corticosterone to both animals resulted in a shift in limen toward normal levels for the adrenalectomized rat but no change for the intact rat. Olfactory thresholds Corticosterone Adrenalectomy Olfactometer Neural-hormonal interaction Pyridin~
THE INFLUENCE of adrenal steroids on sensory processes has received minimal investigative attention. In the case of olfactory sensitivity only two reports have been published to date, both utilizing adrenal insufficient patients. In each case, however, dramatic alterations in threshold were reported. Henkin and Bartter [6] observed as much as a 100,000-fold drop in olfactory threshold for odorants such as NaCI, HCI, pyridine and nitrobenzene in adrenal insufficient patients who w e r e not on glucocorticoid maintenance. The limens returned to normal levels following the administration of glucocorticoids but not m i n e r a l o c o r t i c o i d s . Pruszewicz and Kosowicz [10] reported a greater sensitivity to citric oil and to ground coffee in patients with adrenal failure than in normal, healthy humans. Unfortunately, in both a rigorous quantification of the olfactory shifts had not been achieved. Henkin and Bartter evalutated smell sensitivity using a sniff technique while blast olfactometry was employed by Pruszewicz and Kosowicz. I n the sniff method, the detection thresholds were specified in terms of the concentration of the odorant solution in a test bottle, which is a coarse and unreliable estimate of the concentration of odorant molecules in the gas phase entering the animal's nares. For the blast technique there is a problem with interference due to proprioceptive stimulation of nasal tissues by the air stream. The present report explores the generality of the threshold shift phenomenon in the olfactory modality. More importantly, an attempt was made to control the
Water deprivation Sensory Processes
Response suppression Psychophysics
relevant parameters of the olfactory situation in order to establish the magnitude and limits of these changes. METHOD
Animals Four naive hooded male rats of the Long-Evans strain were used.
Apparatus The training and testing chamber (Fig. 1) was made of stainless steel plating, 12-in. long and 4-in. square in cross section, open at each end. At the forward end, on what would constitute the right side of the chamber, a dipper mechanism was installed such that a water dipper could be presented to the animal without interfering with air flow or contaminating air purity. A negative pressure outside the chamber assured the flow of air out of the dipper receiving hole. A horizontal bar projected from the middle of the floor, at the front of the enclosure, and six l/8-in, vertical stainless steel rods set 1/2-in. apart and installed foward of the bar confined" the animal within the chamber. At the rear of the enclosure a rectangular cap, also containing vertical stainless steel rods, was put into position after the animal was introduced into the response chamber. This cap was attached to a 4-in. dia. flexible, accordian-type hose. The opposite end of the hose was attached to a venting hood and, thus, provided efficient active evacuation of air
Submitted in partial fulfillment of the requirements for the Doctor of Philosophy Degree, Department of Medical Psychology, University of Oregon Medical School. ZThe author is indebted to Dr. F. Robert Brush and Dr. David S. Phillips for their valuable counsel. 495R
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\ FIG. 1. Response chamber and accessory apparatus. A. response chamber; B. diffusion chamber; C. horizonal bar; D. dipper receiving hole; E. speaker; F. air inlet tubes: G. enclosure vent; H. exhaust hose.
introduced into the c h a m b e r from the front. Periodic tests were c o n d u c t e d to assure that air flows were in the appropriate directions, that is, that the c h a m b e r was being properly exhausted by the action of the h o o d and that air flowed out of the test chamber at the receiving hole. A speaker was connected to the external surface at the front end of the chamber to provide c o n t i n u o u s background noise. A glass sleeve which could be easily removed for cleaning following each animal's daily session was designed to fit inside the stainless steel chamber. The entire b a r-m e c h a nism-dipper-mechanism-chamber arrangement was housed in a refrigerator shell and was m o u n t e d on a waterproofed w o o d e n frame so that it could be removed and appropriately cleaned and dried. The achievement and control of air purity was realized through the i n c o r p o r a t i o n of a flow-dilution o l f a c t o m e t e r (Fig. 2). T w o air systems were utilized, one to provide the steady stream of deodorized background air and a second to facilitate the presentation of odorous molecules. A T arrangement at an outlet valve on the laboratory's compressed air line provided the two air supplies. Each of these was led through a National Cylinder Gas regulating valve to a 20-1 glass carboy reservoir which served to d a m p e n any significant oscillations in air pressure within the lines. F r o m these tanks air passed to a series of three gas diffusion bottles containing Purafil [ 4 ] , calcium chloride, and a layered c o m b i n a t i o n of charcoal and silica gel, respectively. Each system then passed through a coil to a flowmeter. F r o m this point the deodorized background air, moving at a p p r o x i m a t e l y 33.3 l/min entered a manifold where it was fractionated into six equal streams. Each of the six lines then led to a glass T. The air stream to be odorized, moving al 175 cc/min. also entered a 6-outlet manifold. Each of the output streams from the manifold led to a stainless steel microregulating valve, then to a gas diffusion bottle. The air was odorized by sparging through the odorant solution. The
odorous air was then directed to a three-way stopcock. The normally open (flowing) pathway of each stopcock was directed to a ventilating hood by way of a six-outlet reducing manifold. Thus, a continuous air flow through each solution bottle was assured, and variability of odor concentrations within the gas was avoided. A f l o w m e t e r was used to m o n i t o r the rate of flow out of tile individual stopcocks. On tile downstream [normally closed} side of each stopcock a glass T was connected (the same T to which deodorized background air was flowing). The third arm of the T i o u t p u t ) was directed, by 1A-in. O.D. teflon tubing, through a wall of the refrigerator shell to a pyramidal-shaped, stainless steel diffusion chamber. Finally, the latter was latched to the animal chamber when an animal was to be run. The sparging bottles and the coils were immersed in a constant temperature bath (glass) which contained distilled water recirculated through a charcoal-glasswool filter. Each bottle, stopcock, and related connectors and tubing were removed at the end of each day's session, washed and dried. O d o r a n t solutions were freshly prepared just prior to the start of each animal's session and put into particular predetermined bottles.
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FIG. 2. Schematic of the olfactometer. A. air supply; B. national cylinder gas regulators; C. 20-liter carboys; D. purafil filter; E. calcium chloride dryers; F. charcoal-silica gel filters; G. 8-ft. glass coils; H. flowmeters; 1. manifolds; J. microregulating valves; K. gas diffusion, odorant bottles; L. 3-way glass-teflon stopcocks; M. glass T's.
Procedure At a p p r o x i m a t e l y 40 days of age two animals were bilaterally adrenalectomized (ADX) using the dorsal approach under ether anesthesia. All animals were at least 100 days old when tile experiment was formally begun. The animals were motivated by depriving them of water for a p p r o x i m a t e l y 23.5 hr before each experimental session. Following each session, at the beginning of the experiment, each animal was given an a m o u n t of water which was sufficient to maintain his body weight at 80% of ad lib weight (as determined at 100 days of age). However, as the experiment progressed and the animals became more efficient at the discrimination, their weights were progres-
OLFACTORY THRESHOLDS sively increased so t h a t p r i o r to t h e d e t e r m i n a t i o n of t h r e s h o l d s for p y r i d i n e t h e y weighed b e t w e e n 100% a n d 115% of t h e i r ad lib weight. T h e A D X animals received a s o l u t i o n of 1% saline a n d 5% d e x t r o s e b o t h as a reinforcem e n t a n d for m a i n t e n a n c e , and t h e i n t a c t a n i m a l s received tap water. T h e animals h a d P u r i n a rat c h o w c o n s t a n t l y available in the individual h o m e cages. All animals were first t r a i n e d t o b a r press for w a t e r reinforcement on a continuous reinforcement (CRF) schedule. T h e n t h e y were i n t r o d u c e d to a 3 : 1 variable ratio s c h e d u l e a n d progressively s h i f t e d to a final variable ratio of 10:1. As r e s p o n d i n g o n t h e final s c h e d u l e i m p r o v e d , d e o d o r i z e d air was passed t h r o u g h t h e r e s p o n s e c h a m b e r . O n c e a stable r e s p o n s e rate at 10:1 was observed discrimin a t i o n t r a i n i n g was begun. F o u r c h a n n e l s o f t h e olfactom e t e r were used, t w o c o n t a i n i n g o d o r o u s s o l u t i o n s o f similar c o n c e n t r a t i o n a n d t w o c o n t a i n i n g d e o d o r i z e d ( b l a n k ) solutions. A n o d o r was p r e s e n t e d at a n i n t e n s e c o n c e n t r a t i o n d u r i n g w h i c h n o r e i n f o r c e m e n t was available. If t h e a n i m a l s t o p p e d r e s p o n d i n g for f r o m 8 - 1 0 sec, t h e o d o r was t e r m i n a t e d a n d t h e r e i n f o r c e m e n t s c h e d u l e was r e i n s t a t e d . H o w e v e r , if n o s u p p r e s s i o n of b a r pressing occurred, the odor and the withholding of water continued for 2 min. T h e r e a f t e r , o d o r was d i s c o n t i n u e d a n d reinforcem e n t r e i n s t a t e d . O n occasions, a b l a n k r a t h e r t h a n a n o d o r was p r e s e n t e d a n d r e i n f o r c e m e n t was c o n t i n u e d . A trial ( t h e p r e s e n t a t i o n of a n o d o r or a b l a n k ) o c c u r r e d after. every 7 - 1 5 r e i n f o r c e m e n t s for a t o t a l o f 24 ( 1 2 o d o r a n d 12 b l a n k ) trials per session. D i s c r i m i n a t i o n and, t h e r e f o r e , s u p p r e s s i o n was said t o have o c c u r r e d w h e n n o t m o r e t h a n o n e b a r press was m a d e d u r i n g a n 8-sec interval b e g i n n i n g 2 sec a f t e r o d o r onset. T h e c o n c e n t r a t i o n was decreased b y a f a c t o r o f 10 w h e n t h e a n i m a l d i s c r i m i n a t e d o n 10 o f 12 o d o r trials for t h r e e c o n s e c u t i v e sessions. W h e n c r i t e r i o n was r e a c h e d at a s o l u t i o n c o n c e n t r a t i o n o f 1 X 1 0 - 4 . 5 M t h e p r o c e d u r e was altered. Previous research had e s t a b l i s h e d t h a t this c o n c e n t r a t i o n was a p p r o x i m a t e l y o n e a n d a h a l f log steps a b o v e t h r e s h o l d for n o r m a l , i n t a c t rats. E a c h of f o u r o d o r a n t b o t t l e s was filled w i t h a pyridined e o d o r i z e d w a t e r s o l u t i o n of d i f f e r e d c o n c e n t r a t i o n (generally o n e - h a l f log step d i f f e r e n c e b e t w e e n c o n c e n t r a t i o n s ) a n d t w o b o t t l e s were filled w i t h d e o d o r i z e d w a t e r as blanks. S t i m u l u s p r e s e n t a t i o n , e i t h e r o d o r or b l a n k , was accomplished by changing the state of the stopcock on a selected c h a n n e l so t h a t air f r o m t h e sparging b o t t l e was d i r e c t e d i n t o t h e d e o d o r i z e d , b a c k g r o u n d airstream. E a c h s t i m u l u s was p r e s e n t e d for 15 sec a n d t h e n u m b e r o f responses o c c u r r i n g d u r i n g a n interval b e g i n n i n g 2 sec a f t e r s t i m u l u s o n s e t d e f i n e d as t u r n i n g t h e s t o p c o c k , a n d t e r m i n a t i n g 12 sec a f t e r s t i m u l u s o n s e t was r e c o r d e d . A p r o g r a m was d e v e l o p e d such t h a t o d o r a n d b l a n k stimuli were r a n d o m l y p r e s e n t e d w i t h i n t e r t r i a l intervals averaging 30 sec. T h e o r d e r o f s t i m u l u s p r e s e n t a t i o n is i n d i c a t e d in T a b l e 1. Using P r o g r a m A, t h e first s t i m u l u s (trial) t h e a n i m a l received was a b l a n k f r o m o n e of t h e t w o c h a n n e l s c o n t a i n i n g d e o d o r i z e d solution. T h e s e c o n d stimulus, following an intertrial interval o f p r e d e t e r m i n e d d u r a t i o n was also a b l a n k b u t f r o m a d i f f e r e n t c h a n n e l t h a n t h e first, etc. T h u s , t h e 2 0 t h trial c o n s i s t e d o f a n o d o r p r e s e n t a t i o n f r o m o d o r C h a n n e l # 4 and the 21st trial an o d o r p r e s e n t a t i o n f r o m o d o r C h a n n e l #3. A f t e r t w o experim e n t a l sessions a d i f f e r e n t p r o g r a m was selected at r a n d o m , w h i c h c h a n g e d t h e p o i n t of e n t e r i n g t h e sequence. F o r e x a m p l e , a f t e r t w o sessions in w h i c h P r o g r a m A was used, P r o g r a m D m i g h t be e m p l o y e d . Here the first s t i m u l u s o f
497R TABLE 1 PROGRAMS FOR STIMULUS PRESENTATION ORDER OF PRESENTATION PROGRAM
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21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
9 l0 It 12 13 14 15 16 17 18 19 20 21 22
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t h e session given t h e subject w o u l d b e an o d o r f r o m o d o r Channel #1, the second stimulus an odor from odor C h a n n e l # 3 , etc. T h e p r o g r a m was c o m p l e t e d at least twice in each e x p e r i m e n t a l session f o r each animal. T h e s t i m u l u s associated w i t h each c h a n n e l was also r a n d o m l y c h a n g e d every t w o sessions b u t n o t c o i n c i d e n t w i t h c h a n g e s in t h e p r o g r a m . T h u s , B l a n k # 2 m i g h t have b e e n in C h a n n e l 3 d u r i n g o n e session b u t in C h a n n e l 5 in a n o t h e r session. Similarly, O d o r # 4 m i g h t have b e e n in C h a n n e l 6 d u r i n g o n e session b u t in C h a n n e l 1 in a n o t h e r session. Since a n y p a r t i c u l a r c h a n n e l m i g h t have b e e n used for a b l a n k o n o n e d a y b u t for a n o d o r o n t h e n e x t , rigorous cleaning o f the a p p a r a t u s b e t w e e n sessions was a b s o l u t e l y essential. Initially, all o d o r c h a n n e l s c o n t a i n e d s o l u t i o n s at suprat h r e s h o l d c o n c e n t r a t i o n s differing b y ½ log steps. As t h e s t u d y progressed, the f o u r c o n c e n t r a t i o n s were systematically lowered b y ½ log steps until a p o i n t was r e a c h e d at w h i c h t w o of the c h a n n e l s were above t h e a n i m a l ' s t h r e s h o l d and t w o were below. P r e l i m i n a r y o b s e r v a t i o n s had i n d i c a t e d t h a t d e t e c t i o n c o u l d be said to have o c c u r r e d w h e n the ratio of t h e n u m b e r of responses d u r i n g an o d o r to t h e m e a n n u m b e r of responses during t h e b l a n k s for the session was 0 . 6 2 5 or less. T h u s , a d i c h o t o m o u s classific a t i o n was a d o p t e d . T h e c o n c e n t r a t i o n having a 0.50 p r o b a b i l i t y of d e t e c t i o n , so d e f i n e d , was t a k e n to be t h e t h r e s h o l d point. T h i s p o i n t was c o n s i d e r e d to have b e e n reliably identified w h e n the a n i m a l ' s p e r f o r m a n c e was stable over five c o n s e c u t i v e sessions. T h e t w o n o r m a l , i n t a c t a n i m a l s were selected for f u r t h e r research. O n e a n i m a l was bilaterally a d r e n a l e c t o m i z e d and t h e o t h e r received a sham o p e r a t i o n . B o t h animals were p u t o n saline m a i n t e n a n c e in c o n j u n c t i o n w i t h the d e p r i v a t i o n schedule. A p p r o x i m a t e l y 18 hr p o s t - o p e r a t i v e l y and for 4
498R
SAKELLARIS -5 .I .2
days thereafter the olfactory threshold of each animal was determined. On Day 3 both animals were given two subcutaneous injections, 8 hr apart, of 0.5 ml corticosterone (Steraloids, In.) at 6 mg/ml in propylene glycol (therefore 7 mg/kg b-wt). Thresholds were again assessed twelve hours after the last injection.
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The detection thresholds for pyridine for intact animals were higher than those of the ADX group (Fig. 3). The data were transformed to X'= x / X in order to effect homogeneity of within-cell variance. A two-way analysis of variance for repeated measures [ 15] showed only the Groups effect to be significant (p<0.01; F=46.5, dr=l,2). The effects of adrenalectomy or a sham operation on the previously normal animals are seen in Fig. 4. Thresholds for the intact animal did not change whereas for the ADX rat an obvious drop occurred. Steroid administration had no effect on the limen for #1856 (normal) but appeared to shift that for animal #1857 (ADX) toward the preoperative level. Normalization of performance of the bar pressing task was observed for the ADX animals during training and testing (Fig. 5). There were no differences found between the ADX and Normal groups for mean bar presses per min in intertrial periods (ADX: M=87.7, SD=12.0; Normal: M=80.3, SD=7.5). Similarly, no differences in response rate were seen for 4 post-operative sessions between # 1856 and #1857 (ADX (1857): M=76.4, SD=3.5; Sham (1856): M=85.6; SD=6.9).
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The difference in odor sensitivity to pyridine between normal and ADX rats was significant. This 5-fold divergence, although notably less than the shift observed in human patients with adrenal cortical insufficiency, leads to the speculation of some sort of interaction between neural tissues and hormones besides that known for the control of elaboration and secretion of the substances themselves. In the studies using human patients the experimental groups included individuals with hypophyseal pathology and, therefore, depressed ACTH levels, in order to determine whether the sensory alterations were mediated by ACTH or by glucocorticoids alone. The possibility existed that lowered thresholds in adrenal insufficiency were the result of increased ACTH secretion. Since detection thresholds for patients with panhypopituitarism or adrenal cortical insufficiency did not differ, either during maintenance periods or when steroid therapy was terminated, an
O L F A C T O R Y THRESHOLDS
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ACTH effect was not likely. In the present study it cannot be unequivocally concluded that ACTH played no role in the differences seen. However, the data of Hodges and Vernikos [7] would suggest that 24 hr after adrenalectomy the plasma ACTH concentration of subject #1857 was minimal yet his olfactory sensitivity increased. Since olfactory thresholds were not determined for the ADX group in infancy, that is before bilateral adrenalectomy, it was not immediately clear whether or not these animals had inherent odor detection capabilities for pyridine greater than those of the normal animals and unrelated to adrenal state. It was assumed that, if that were the case, normal males adrenalectomized in adulthood following threshold assessment would have no change in sensitivity. The data did not support this assumption. The threshold of the adrenalectomized animal dropped to the range of those of the ADX group and remained there, while the sham animal showed no shift. Further, steroid maintenance raised the ADX's threshold but had no effect on that of the intact animal. These results are consistent with the hypothesis of an adrenal mediation of sensory sensitivity. The loss of adrenal hormones, even in the rat, can lead eventually to a general debilitation manifested by a precipitous decrease in locomotor and grooming activity plus a rapid drop in body weight. However, a saline and dextrose maintenance program results in a return to normal of body weight and general activity. Normalization of performance of the bar pressing task was observed for the ADX animals during the entire period of their training. As is seen in Fig. 5, these animals actually had a mean response rate during intertrial periods slightly greater than the normals. The data support the conclusion that ADX animals had no muscular-capacitative deficit, at least for the task involved, which led to differences in response rate between these animals and the normals, recorded during the test trials. Ordinarily, a water deprivation regimen would be expected to lead to some degree of body dehydration and could result in an alteration of epithelial nasal tissues. The effect would be magnified in adrenalectomized animals due to their increased water loss and could in an unexplained way lead to differences in odor sensitivity. However, in the
present study the watering schedule was gradually adjusted in order to increase body weight to 100% or greater of ad lib values and consequently restore total water intake. Even though the animals were given water only during a short period every 24 hr, the regimen resulted in a daily water intake for all animals which did not differ from t h a t observed in intact and ADX rats on ad lib food and water maintenance. It is unlikely that the observed threshold shift was mediated by an alteration in olfactory mucus secretions because of the changes in glucocorticoid concentrations. Adrenalectomy will most likely lead to a small decrease in mucus secretions in many body areas and is probably related to the effects of the adrenal extirpation on systemic and cellular fluid and electrolyte balances [ 1 ], as well as to losses in available carbohydrates, the latter being an important fraction of mucus macromolecules [9]. Henkin and Bartter [6] have shown that restoring electrolyte balance will not return thresholds to normal in patients with adrenal cortical insufficiency. Even if a dramatic mucosal change were to occur as a result of adrenalectomy or adrenal pathology it would lead to an effect opposite to the one observed. The moist mucus surface is believed to be the medium in which olfactory molecules are dissolved [2] and maximal olfactory sensitivity is observed when the olfactory epithelium is red, swollen, and wet, provided that the airways are not significantly obstructed [ 12 ]. The identification of a similar threshold shift phenomenon in humans and rats is consistent with observations on the interspecies generality of many characteristics of the h y p o t h a l a m o - a d e n o h y p o p h y s e a l - a d r e n a l axis (Sandor, [ 11 ] ; Vernikos-Danellis and Marks, [ 13 ] ) while the salient disparity in the magnitude of the threshold changes between the two species is consonant with data showing differences in the dynamics of the human and rat systems (yon Euler and Heller, [ 141 ; Gorbman and Bern,J3] ). Although it has been proposed that increased sensory sensitivity following adrenalectomy is related to an enhanced neural excitability associated with decreased plasma glucocorticoid concentrations [5], the mechanism(s) mediating the threshold-shift phenomenon is not known [8].
REFERENCES 1. Bang, B. G. and F. B. Bang. Effect of water deprivation on nasal mucous flow. Proc. Soc. exp. Biol. Med. 106: 516-521, 1961. 2. Bojsen-MoUer, F. Topography of the nasal glands in rats and some other mammals. Anat. Rec. 150:11-24, 1964. 3. Gorbman, A. and H. A. Bern. A Textbook of Comparative Endocrinology. New York: John Wiley and Sons, 1962. 4. Hanna, G. F., R. L. Kuehner, J. D. Karnes and R. Garbowicz. A chemical method for odor control. Ann. N. Y. Acad. Sci. 116: 663-675, 1964. 5. Henkin, R. 1. The effects of corticosteroids and ACTH on sensory systems. In: Progress in Brain Research: Pituitary, Adrenal and the Brain, edited by D. DeWied and J. A. W. M. Weijnen. Amsterdam: Elsevier, 1970, pp. 270-293. 6. Henkin, R. I. and F. C. Bartter. Studies on olfactory thresholds in normal man and in patients with adrenal insufficiency: The role of adrenal cortical steroids and of serum sodium concentration. J. clin. Invest. 45: 1631-1639, 1966. 7. Hodges, J. R. and J. Vernikos. Circulating corticotrophin in normal and adrenalectomized rats after stress. Acta Endocr. 30: 188-196, 1959.
8. Koranyi, L., C. Beyer and C. Guzman-Flores. Effect of ACTH and hydrocortisone on multiple unit activity in the forebrain and hypothalamus in response to reticular stimulation. Physiol. Behav. 7:331-335, 1971. 9. Platt, H. A. The application of biophysical chemistry to the study of human cervical mucus. Ann. N. Y. Acad. Sci. 130: 925-939, 1966. 10. Pruszewicz, A. and J. Kosowicz. Quantatative and qualitative studies of taste and smell in adrenal failure. Pol. Endocr. 17: 188-193, 1966. 11. Sandor, T. A comparative survey of steroids and steroidgenic pathways throughout the vertebrates. In: General and Comparative Endocrinology: Supplement 2., edited by M. R. N. Prasad. New York: Academic Press, 1969, pp. 284-298. 12. Schneider, R. A. and S. Wolf. Relation of olfactory acuity to nasal membrane function. J. appl. Physiol. 15: 914-920, 1960. 13. Vernikos-Danellis, J. and B. H. Marks. Epinephrine-induced release of ACTH in normal human subjects: a test of pituitary function. Endocrinology 70:525 -531, 1962,
500R
Euler, U. S. and J. Heller (Eds.). Comparative Endocrinology, Vol. 1. New York: Academic Press, 1963.
14. yon
SAKELLARIS
15. Winer, B. J. Statistical Principles in ExperimentaI Design. New York: McGraw-Hill, 1962.
Olfactory Thresholds in Normal and Adrenalectomized Rats ' PETER C. SAKELLARIS 2
A m e s Research Center-NASA, Code-239-17, M o f f e t t Field, California, 94035
(Received 21 March 1972)
SAKELLARIS, P. C. Olfactory thresholds in normal and adrenalectomized rats. PHYSIOL. BEHAV. 9 (4) 495-500, 1972.- Olfactory thresholds for pyridine were examined in normal and adrenalectomized rats. The animals were trained to suppress bar-pressing on a VR 10:1 schedule in the presence of the odorant. Adrenalectomized animals were found to have a lower threshold than normais. When one normal animal was subsequently adrenalectomized and another sham operated the threshold decreased for the first but did not change in the latter. The administration of corticosterone to both animals resulted in a shift in limen toward normal levels for the adrenalectomized rat but no change for the intact rat. Olfactory thresholds Corticosterone Adrenalectomy Olfactometer Neural-hormonal interaction Pyridin~
THE INFLUENCE of adrenal steroids on sensory processes has received minimal investigative attention. In the case of olfactory sensitivity only two reports have been published to date, both utilizing adrenal insufficient patients. In each case, however, dramatic alterations in threshold were reported. Henkin and Bartter [6] observed as much as a 100,000-fold drop in olfactory threshold for odorants such as NaCI, HCI, pyridine and nitrobenzene in adrenal insufficient patients who w e r e not on glucocorticoid maintenance. The limens returned to normal levels following the administration of glucocorticoids but not m i n e r a l o c o r t i c o i d s . Pruszewicz and Kosowicz [10] reported a greater sensitivity to citric oil and to ground coffee in patients with adrenal failure than in normal, healthy humans. Unfortunately, in both a rigorous quantification of the olfactory shifts had not been achieved. Henkin and Bartter evalutated smell sensitivity using a sniff technique while blast olfactometry was employed by Pruszewicz and Kosowicz. I n the sniff method, the detection thresholds were specified in terms of the concentration of the odorant solution in a test bottle, which is a coarse and unreliable estimate of the concentration of odorant molecules in the gas phase entering the animal's nares. For the blast technique there is a problem with interference due to proprioceptive stimulation of nasal tissues by the air stream. The present report explores the generality of the threshold shift phenomenon in the olfactory modality. More importantly, an attempt was made to control the
Water deprivation Sensory Processes
Response suppression Psychophysics
relevant parameters of the olfactory situation in order to establish the magnitude and limits of these changes. METHOD
Animals Four naive hooded male rats of the Long-Evans strain were used.
Apparatus The training and testing chamber (Fig. 1) was made of stainless steel plating, 12-in. long and 4-in. square in cross section, open at each end. At the forward end, on what would constitute the right side of the chamber, a dipper mechanism was installed such that a water dipper could be presented to the animal without interfering with air flow or contaminating air purity. A negative pressure outside the chamber assured the flow of air out of the dipper receiving hole. A horizontal bar projected from the middle of the floor, at the front of the enclosure, and six l/8-in, vertical stainless steel rods set 1/2-in. apart and installed foward of the bar confined" the animal within the chamber. At the rear of the enclosure a rectangular cap, also containing vertical stainless steel rods, was put into position after the animal was introduced into the response chamber. This cap was attached to a 4-in. dia. flexible, accordian-type hose. The opposite end of the hose was attached to a venting hood and, thus, provided efficient active evacuation of air
Submitted in partial fulfillment of the requirements for the Doctor of Philosophy Degree, Department of Medical Psychology, University of Oregon Medical School. ZThe author is indebted to Dr. F. Robert Brush and Dr. David S. Phillips for their valuable counsel. 495R
496R
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introduced into the c h a m b e r from the front. Periodic tests were c o n d u c t e d to assure that air flows were in the appropriate directions, that is, that the c h a m b e r was being properly exhausted by the action of the h o o d and that air flowed out of the test chamber at the receiving hole. A speaker was connected to the external surface at the front end of the chamber to provide c o n t i n u o u s background noise. A glass sleeve which could be easily removed for cleaning following each animal's daily session was designed to fit inside the stainless steel chamber. The entire b a r-m e c h a nism-dipper-mechanism-chamber arrangement was housed in a refrigerator shell and was m o u n t e d on a waterproofed w o o d e n frame so that it could be removed and appropriately cleaned and dried. The achievement and control of air purity was realized through the i n c o r p o r a t i o n of a flow-dilution o l f a c t o m e t e r (Fig. 2). T w o air systems were utilized, one to provide the steady stream of deodorized background air and a second to facilitate the presentation of odorous molecules. A T arrangement at an outlet valve on the laboratory's compressed air line provided the two air supplies. Each of these was led through a National Cylinder Gas regulating valve to a 20-1 glass carboy reservoir which served to d a m p e n any significant oscillations in air pressure within the lines. F r o m these tanks air passed to a series of three gas diffusion bottles containing Purafil [ 4 ] , calcium chloride, and a layered c o m b i n a t i o n of charcoal and silica gel, respectively. Each system then passed through a coil to a flowmeter. F r o m this point the deodorized background air, moving at a p p r o x i m a t e l y 33.3 l/min entered a manifold where it was fractionated into six equal streams. Each of the six lines then led to a glass T. The air stream to be odorized, moving al 175 cc/min. also entered a 6-outlet manifold. Each of the output streams from the manifold led to a stainless steel microregulating valve, then to a gas diffusion bottle. The air was odorized by sparging through the odorant solution. The
odorous air was then directed to a three-way stopcock. The normally open (flowing) pathway of each stopcock was directed to a ventilating hood by way of a six-outlet reducing manifold. Thus, a continuous air flow through each solution bottle was assured, and variability of odor concentrations within the gas was avoided. A f l o w m e t e r was used to m o n i t o r the rate of flow out of tile individual stopcocks. On tile downstream [normally closed} side of each stopcock a glass T was connected (the same T to which deodorized background air was flowing). The third arm of the T i o u t p u t ) was directed, by 1A-in. O.D. teflon tubing, through a wall of the refrigerator shell to a pyramidal-shaped, stainless steel diffusion chamber. Finally, the latter was latched to the animal chamber when an animal was to be run. The sparging bottles and the coils were immersed in a constant temperature bath (glass) which contained distilled water recirculated through a charcoal-glasswool filter. Each bottle, stopcock, and related connectors and tubing were removed at the end of each day's session, washed and dried. O d o r a n t solutions were freshly prepared just prior to the start of each animal's session and put into particular predetermined bottles.
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FIG. 2. Schematic of the olfactometer. A. air supply; B. national cylinder gas regulators; C. 20-liter carboys; D. purafil filter; E. calcium chloride dryers; F. charcoal-silica gel filters; G. 8-ft. glass coils; H. flowmeters; 1. manifolds; J. microregulating valves; K. gas diffusion, odorant bottles; L. 3-way glass-teflon stopcocks; M. glass T's.
Procedure At a p p r o x i m a t e l y 40 days of age two animals were bilaterally adrenalectomized (ADX) using the dorsal approach under ether anesthesia. All animals were at least 100 days old when tile experiment was formally begun. The animals were motivated by depriving them of water for a p p r o x i m a t e l y 23.5 hr before each experimental session. Following each session, at the beginning of the experiment, each animal was given an a m o u n t of water which was sufficient to maintain his body weight at 80% of ad lib weight (as determined at 100 days of age). However, as the experiment progressed and the animals became more efficient at the discrimination, their weights were progres-
OLFACTORY THRESHOLDS sively increased so t h a t p r i o r to t h e d e t e r m i n a t i o n of t h r e s h o l d s for p y r i d i n e t h e y weighed b e t w e e n 100% a n d 115% of t h e i r ad lib weight. T h e A D X animals received a s o l u t i o n of 1% saline a n d 5% d e x t r o s e b o t h as a reinforcem e n t a n d for m a i n t e n a n c e , and t h e i n t a c t a n i m a l s received tap water. T h e animals h a d P u r i n a rat c h o w c o n s t a n t l y available in the individual h o m e cages. All animals were first t r a i n e d t o b a r press for w a t e r reinforcement on a continuous reinforcement (CRF) schedule. T h e n t h e y were i n t r o d u c e d to a 3 : 1 variable ratio s c h e d u l e a n d progressively s h i f t e d to a final variable ratio of 10:1. As r e s p o n d i n g o n t h e final s c h e d u l e i m p r o v e d , d e o d o r i z e d air was passed t h r o u g h t h e r e s p o n s e c h a m b e r . O n c e a stable r e s p o n s e rate at 10:1 was observed discrimin a t i o n t r a i n i n g was begun. F o u r c h a n n e l s o f t h e olfactom e t e r were used, t w o c o n t a i n i n g o d o r o u s s o l u t i o n s o f similar c o n c e n t r a t i o n a n d t w o c o n t a i n i n g d e o d o r i z e d ( b l a n k ) solutions. A n o d o r was p r e s e n t e d at a n i n t e n s e c o n c e n t r a t i o n d u r i n g w h i c h n o r e i n f o r c e m e n t was available. If t h e a n i m a l s t o p p e d r e s p o n d i n g for f r o m 8 - 1 0 sec, t h e o d o r was t e r m i n a t e d a n d t h e r e i n f o r c e m e n t s c h e d u l e was r e i n s t a t e d . H o w e v e r , if n o s u p p r e s s i o n of b a r pressing occurred, the odor and the withholding of water continued for 2 min. T h e r e a f t e r , o d o r was d i s c o n t i n u e d a n d reinforcem e n t r e i n s t a t e d . O n occasions, a b l a n k r a t h e r t h a n a n o d o r was p r e s e n t e d a n d r e i n f o r c e m e n t was c o n t i n u e d . A trial ( t h e p r e s e n t a t i o n of a n o d o r or a b l a n k ) o c c u r r e d after. every 7 - 1 5 r e i n f o r c e m e n t s for a t o t a l o f 24 ( 1 2 o d o r a n d 12 b l a n k ) trials per session. D i s c r i m i n a t i o n and, t h e r e f o r e , s u p p r e s s i o n was said t o have o c c u r r e d w h e n n o t m o r e t h a n o n e b a r press was m a d e d u r i n g a n 8-sec interval b e g i n n i n g 2 sec a f t e r o d o r onset. T h e c o n c e n t r a t i o n was decreased b y a f a c t o r o f 10 w h e n t h e a n i m a l d i s c r i m i n a t e d o n 10 o f 12 o d o r trials for t h r e e c o n s e c u t i v e sessions. W h e n c r i t e r i o n was r e a c h e d at a s o l u t i o n c o n c e n t r a t i o n o f 1 X 1 0 - 4 . 5 M t h e p r o c e d u r e was altered. Previous research had e s t a b l i s h e d t h a t this c o n c e n t r a t i o n was a p p r o x i m a t e l y o n e a n d a h a l f log steps a b o v e t h r e s h o l d for n o r m a l , i n t a c t rats. E a c h of f o u r o d o r a n t b o t t l e s was filled w i t h a pyridined e o d o r i z e d w a t e r s o l u t i o n of d i f f e r e d c o n c e n t r a t i o n (generally o n e - h a l f log step d i f f e r e n c e b e t w e e n c o n c e n t r a t i o n s ) a n d t w o b o t t l e s were filled w i t h d e o d o r i z e d w a t e r as blanks. S t i m u l u s p r e s e n t a t i o n , e i t h e r o d o r or b l a n k , was accomplished by changing the state of the stopcock on a selected c h a n n e l so t h a t air f r o m t h e sparging b o t t l e was d i r e c t e d i n t o t h e d e o d o r i z e d , b a c k g r o u n d airstream. E a c h s t i m u l u s was p r e s e n t e d for 15 sec a n d t h e n u m b e r o f responses o c c u r r i n g d u r i n g a n interval b e g i n n i n g 2 sec a f t e r s t i m u l u s o n s e t d e f i n e d as t u r n i n g t h e s t o p c o c k , a n d t e r m i n a t i n g 12 sec a f t e r s t i m u l u s o n s e t was r e c o r d e d . A p r o g r a m was d e v e l o p e d such t h a t o d o r a n d b l a n k stimuli were r a n d o m l y p r e s e n t e d w i t h i n t e r t r i a l intervals averaging 30 sec. T h e o r d e r o f s t i m u l u s p r e s e n t a t i o n is i n d i c a t e d in T a b l e 1. Using P r o g r a m A, t h e first s t i m u l u s (trial) t h e a n i m a l received was a b l a n k f r o m o n e of t h e t w o c h a n n e l s c o n t a i n i n g d e o d o r i z e d solution. T h e s e c o n d stimulus, following an intertrial interval o f p r e d e t e r m i n e d d u r a t i o n was also a b l a n k b u t f r o m a d i f f e r e n t c h a n n e l t h a n t h e first, etc. T h u s , t h e 2 0 t h trial c o n s i s t e d o f a n o d o r p r e s e n t a t i o n f r o m o d o r C h a n n e l # 4 and the 21st trial an o d o r p r e s e n t a t i o n f r o m o d o r C h a n n e l #3. A f t e r t w o experim e n t a l sessions a d i f f e r e n t p r o g r a m was selected at r a n d o m , w h i c h c h a n g e d t h e p o i n t of e n t e r i n g t h e sequence. F o r e x a m p l e , a f t e r t w o sessions in w h i c h P r o g r a m A was used, P r o g r a m D m i g h t be e m p l o y e d . Here the first s t i m u l u s o f
497R TABLE 1 PROGRAMS FOR STIMULUS PRESENTATION ORDER OF PRESENTATION PROGRAM
STIMULUS
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B
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D
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[ 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
23 24 1 2 3 4 5 6 7
22 23 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
9 l0 It 12 13 14 15 16 17 18 19 20 21 22
BLANK BLANK BLANK ODOR ODOR BLANK BLANK ODOR BLANK BLANK ODOR BLANK BLANK BLANK ODOR BLANK ODOR BLANK BLANK ODOR ODOR BLANK BLANK BLANK
2 1 1 1 3 2 1 2 I 2 4 2 2 1 2 2 1 1 1 4 3 2 1 2
t h e session given t h e subject w o u l d b e an o d o r f r o m o d o r Channel #1, the second stimulus an odor from odor C h a n n e l # 3 , etc. T h e p r o g r a m was c o m p l e t e d at least twice in each e x p e r i m e n t a l session f o r each animal. T h e s t i m u l u s associated w i t h each c h a n n e l was also r a n d o m l y c h a n g e d every t w o sessions b u t n o t c o i n c i d e n t w i t h c h a n g e s in t h e p r o g r a m . T h u s , B l a n k # 2 m i g h t have b e e n in C h a n n e l 3 d u r i n g o n e session b u t in C h a n n e l 5 in a n o t h e r session. Similarly, O d o r # 4 m i g h t have b e e n in C h a n n e l 6 d u r i n g o n e session b u t in C h a n n e l 1 in a n o t h e r session. Since a n y p a r t i c u l a r c h a n n e l m i g h t have b e e n used for a b l a n k o n o n e d a y b u t for a n o d o r o n t h e n e x t , rigorous cleaning o f the a p p a r a t u s b e t w e e n sessions was a b s o l u t e l y essential. Initially, all o d o r c h a n n e l s c o n t a i n e d s o l u t i o n s at suprat h r e s h o l d c o n c e n t r a t i o n s differing b y ½ log steps. As t h e s t u d y progressed, the f o u r c o n c e n t r a t i o n s were systematically lowered b y ½ log steps until a p o i n t was r e a c h e d at w h i c h t w o of the c h a n n e l s were above t h e a n i m a l ' s t h r e s h o l d and t w o were below. P r e l i m i n a r y o b s e r v a t i o n s had i n d i c a t e d t h a t d e t e c t i o n c o u l d be said to have o c c u r r e d w h e n the ratio of t h e n u m b e r of responses d u r i n g an o d o r to t h e m e a n n u m b e r of responses during t h e b l a n k s for the session was 0 . 6 2 5 or less. T h u s , a d i c h o t o m o u s classific a t i o n was a d o p t e d . T h e c o n c e n t r a t i o n having a 0.50 p r o b a b i l i t y of d e t e c t i o n , so d e f i n e d , was t a k e n to be t h e t h r e s h o l d point. T h i s p o i n t was c o n s i d e r e d to have b e e n reliably identified w h e n the a n i m a l ' s p e r f o r m a n c e was stable over five c o n s e c u t i v e sessions. T h e t w o n o r m a l , i n t a c t a n i m a l s were selected for f u r t h e r research. O n e a n i m a l was bilaterally a d r e n a l e c t o m i z e d and t h e o t h e r received a sham o p e r a t i o n . B o t h animals were p u t o n saline m a i n t e n a n c e in c o n j u n c t i o n w i t h the d e p r i v a t i o n schedule. A p p r o x i m a t e l y 18 hr p o s t - o p e r a t i v e l y and for 4
498R
SAKELLARIS -5 .I .2
days thereafter the olfactory threshold of each animal was determined. On Day 3 both animals were given two subcutaneous injections, 8 hr apart, of 0.5 ml corticosterone (Steraloids, In.) at 6 mg/ml in propylene glycol (therefore 7 mg/kg b-wt). Thresholds were again assessed twelve hours after the last injection.
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The detection thresholds for pyridine for intact animals were higher than those of the ADX group (Fig. 3). The data were transformed to X'= x / X in order to effect homogeneity of within-cell variance. A two-way analysis of variance for repeated measures [ 15] showed only the Groups effect to be significant (p<0.01; F=46.5, dr=l,2). The effects of adrenalectomy or a sham operation on the previously normal animals are seen in Fig. 4. Thresholds for the intact animal did not change whereas for the ADX rat an obvious drop occurred. Steroid administration had no effect on the limen for #1856 (normal) but appeared to shift that for animal #1857 (ADX) toward the preoperative level. Normalization of performance of the bar pressing task was observed for the ADX animals during training and testing (Fig. 5). There were no differences found between the ADX and Normal groups for mean bar presses per min in intertrial periods (ADX: M=87.7, SD=12.0; Normal: M=80.3, SD=7.5). Similarly, no differences in response rate were seen for 4 post-operative sessions between # 1856 and #1857 (ADX (1857): M=76.4, SD=3.5; Sham (1856): M=85.6; SD=6.9).
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FIG. 4. Threshold concentrations for animals #1856 (Sham-op) and #1857 (ADX) preoperative, postoperative, and postinjection.
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FIG. 3. Median threshold concentrations for Normal and ADX groups.
The difference in odor sensitivity to pyridine between normal and ADX rats was significant. This 5-fold divergence, although notably less than the shift observed in human patients with adrenal cortical insufficiency, leads to the speculation of some sort of interaction between neural tissues and hormones besides that known for the control of elaboration and secretion of the substances themselves. In the studies using human patients the experimental groups included individuals with hypophyseal pathology and, therefore, depressed ACTH levels, in order to determine whether the sensory alterations were mediated by ACTH or by glucocorticoids alone. The possibility existed that lowered thresholds in adrenal insufficiency were the result of increased ACTH secretion. Since detection thresholds for patients with panhypopituitarism or adrenal cortical insufficiency did not differ, either during maintenance periods or when steroid therapy was terminated, an
O L F A C T O R Y THRESHOLDS
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ACTH effect was not likely. In the present study it cannot be unequivocally concluded that ACTH played no role in the differences seen. However, the data of Hodges and Vernikos [7] would suggest that 24 hr after adrenalectomy the plasma ACTH concentration of subject #1857 was minimal yet his olfactory sensitivity increased. Since olfactory thresholds were not determined for the ADX group in infancy, that is before bilateral adrenalectomy, it was not immediately clear whether or not these animals had inherent odor detection capabilities for pyridine greater than those of the normal animals and unrelated to adrenal state. It was assumed that, if that were the case, normal males adrenalectomized in adulthood following threshold assessment would have no change in sensitivity. The data did not support this assumption. The threshold of the adrenalectomized animal dropped to the range of those of the ADX group and remained there, while the sham animal showed no shift. Further, steroid maintenance raised the ADX's threshold but had no effect on that of the intact animal. These results are consistent with the hypothesis of an adrenal mediation of sensory sensitivity. The loss of adrenal hormones, even in the rat, can lead eventually to a general debilitation manifested by a precipitous decrease in locomotor and grooming activity plus a rapid drop in body weight. However, a saline and dextrose maintenance program results in a return to normal of body weight and general activity. Normalization of performance of the bar pressing task was observed for the ADX animals during the entire period of their training. As is seen in Fig. 5, these animals actually had a mean response rate during intertrial periods slightly greater than the normals. The data support the conclusion that ADX animals had no muscular-capacitative deficit, at least for the task involved, which led to differences in response rate between these animals and the normals, recorded during the test trials. Ordinarily, a water deprivation regimen would be expected to lead to some degree of body dehydration and could result in an alteration of epithelial nasal tissues. The effect would be magnified in adrenalectomized animals due to their increased water loss and could in an unexplained way lead to differences in odor sensitivity. However, in the
present study the watering schedule was gradually adjusted in order to increase body weight to 100% or greater of ad lib values and consequently restore total water intake. Even though the animals were given water only during a short period every 24 hr, the regimen resulted in a daily water intake for all animals which did not differ from t h a t observed in intact and ADX rats on ad lib food and water maintenance. It is unlikely that the observed threshold shift was mediated by an alteration in olfactory mucus secretions because of the changes in glucocorticoid concentrations. Adrenalectomy will most likely lead to a small decrease in mucus secretions in many body areas and is probably related to the effects of the adrenal extirpation on systemic and cellular fluid and electrolyte balances [ 1 ], as well as to losses in available carbohydrates, the latter being an important fraction of mucus macromolecules [9]. Henkin and Bartter [6] have shown that restoring electrolyte balance will not return thresholds to normal in patients with adrenal cortical insufficiency. Even if a dramatic mucosal change were to occur as a result of adrenalectomy or adrenal pathology it would lead to an effect opposite to the one observed. The moist mucus surface is believed to be the medium in which olfactory molecules are dissolved [2] and maximal olfactory sensitivity is observed when the olfactory epithelium is red, swollen, and wet, provided that the airways are not significantly obstructed [ 12 ]. The identification of a similar threshold shift phenomenon in humans and rats is consistent with observations on the interspecies generality of many characteristics of the h y p o t h a l a m o - a d e n o h y p o p h y s e a l - a d r e n a l axis (Sandor, [ 11 ] ; Vernikos-Danellis and Marks, [ 13 ] ) while the salient disparity in the magnitude of the threshold changes between the two species is consonant with data showing differences in the dynamics of the human and rat systems (yon Euler and Heller, [ 141 ; Gorbman and Bern,J3] ). Although it has been proposed that increased sensory sensitivity following adrenalectomy is related to an enhanced neural excitability associated with decreased plasma glucocorticoid concentrations [5], the mechanism(s) mediating the threshold-shift phenomenon is not known [8].
REFERENCES 1. Bang, B. G. and F. B. Bang. Effect of water deprivation on nasal mucous flow. Proc. Soc. exp. Biol. Med. 106: 516-521, 1961. 2. Bojsen-MoUer, F. Topography of the nasal glands in rats and some other mammals. Anat. Rec. 150:11-24, 1964. 3. Gorbman, A. and H. A. Bern. A Textbook of Comparative Endocrinology. New York: John Wiley and Sons, 1962. 4. Hanna, G. F., R. L. Kuehner, J. D. Karnes and R. Garbowicz. A chemical method for odor control. Ann. N. Y. Acad. Sci. 116: 663-675, 1964. 5. Henkin, R. 1. The effects of corticosteroids and ACTH on sensory systems. In: Progress in Brain Research: Pituitary, Adrenal and the Brain, edited by D. DeWied and J. A. W. M. Weijnen. Amsterdam: Elsevier, 1970, pp. 270-293. 6. Henkin, R. I. and F. C. Bartter. Studies on olfactory thresholds in normal man and in patients with adrenal insufficiency: The role of adrenal cortical steroids and of serum sodium concentration. J. clin. Invest. 45: 1631-1639, 1966. 7. Hodges, J. R. and J. Vernikos. Circulating corticotrophin in normal and adrenalectomized rats after stress. Acta Endocr. 30: 188-196, 1959.
8. Koranyi, L., C. Beyer and C. Guzman-Flores. Effect of ACTH and hydrocortisone on multiple unit activity in the forebrain and hypothalamus in response to reticular stimulation. Physiol. Behav. 7:331-335, 1971. 9. Platt, H. A. The application of biophysical chemistry to the study of human cervical mucus. Ann. N. Y. Acad. Sci. 130: 925-939, 1966. 10. Pruszewicz, A. and J. Kosowicz. Quantatative and qualitative studies of taste and smell in adrenal failure. Pol. Endocr. 17: 188-193, 1966. 11. Sandor, T. A comparative survey of steroids and steroidgenic pathways throughout the vertebrates. In: General and Comparative Endocrinology: Supplement 2., edited by M. R. N. Prasad. New York: Academic Press, 1969, pp. 284-298. 12. Schneider, R. A. and S. Wolf. Relation of olfactory acuity to nasal membrane function. J. appl. Physiol. 15: 914-920, 1960. 13. Vernikos-Danellis, J. and B. H. Marks. Epinephrine-induced release of ACTH in normal human subjects: a test of pituitary function. Endocrinology 70:525 -531, 1962,
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Euler, U. S. and J. Heller (Eds.). Comparative Endocrinology, Vol. 1. New York: Academic Press, 1963.
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SAKELLARIS
15. Winer, B. J. Statistical Principles in ExperimentaI Design. New York: McGraw-Hill, 1962.