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The olfactory control of meal pattern in rats
Physiology and Behavior, Vol. 9, pp. 817-821. Brain Research Publications Inc., 1972. Printed in U. S. A.
The Olfactory Control of Meal Pattern in Rats CHRISTIANE G. LARUE AND JACQUES LE MAGNEN
Laboratoire de Neurophysiologie sensorielle et comportementale du C.N.R.S. Collbge de France, Paris 5e, France
(Received 28 December 1971)
LARUE, C. G. AND J. LE MAGNEN. The olfactory control of meal pattern in rats. PHYSIOL. BEHAV. 9(5) 817-821, 1972.- The role of the olfactory input in the control system regulating the free food intake of rats has been investigated. In a first experiment it was shown that the mean daily intake of 20 adult male and female rats, made anosrnic by the bilateral ablation of olfactory bulbs, did not change significantly after surgery. But, as a result of the bulbar removal, the day to day fluctuation of this daily intake was significantly increased. In a second experiment the pre and postoperative recording of the daily feeding pattern (meal size and intervals) revealed that the olfactory bulbs ablation induced a more or less persistant and typical nibbling pattern. The stereotaxie electrolytic lesion of the cortico-medial amygdaioid olfactory projection, interrupting one of the olfacto-hypothalamic pathways, induced the same disrupted feeding pattern. The results are discussed in relation to control systems regulating meal sizes and meal to meal intervals in normal rats. It is suggested that the orosensory and particularly the olfactory control is responsible for determining the normal feeding pattern made of large and consistant meals followed by long intervals of nonfeeding. Olfactory bulbs removal
Food intake
Meal pattern
THE RESPECTIVE roles of orosensory and of metabolic postingestive factors in the control of meal size is still open to discussion. It is known that, for a given state of food deprivation, the satiating amount eaten in a meal of each particular food is sensory specific. This fact, among others, argues that the meal size is mainly or exclusively the behavioral response to the orosensory stimulating properties of each food. The innate and learned level of this response is defined as the palatability of the food. Various sensory stimulating activities of food in the m o u t h contribute to this palatability of the food and therefore to the control of meal size. Hitherto for reasons of experimental simplicity, the role of gustatory stimulations has been mainly investigated. By contrast, the study of the role of the mechanical and, most of all, of the olfactory control of food intake has been widely neglected. However, it has been shown that in learned specific [4] or caloric [12] appetite the amount of food eaten may be entirely dependent on an arbitrary added odor. In order to study further the role of olfactory stimuli and olfactory neural pathways in the oral metering of food, experiments have been undertaken to control the effects of the interruption of these oropharyngeal sensory afferents upon feeding responses in rats. In a previous work it has been shown that the olfactory bulb removal in a rat fed ad lib with a familiar food, did not change strikingly the 24 hr intake [9]. This daily intake of food and its relative constancy from day to day are dependent on the relationship between the two basic parameters: namely meal sizes and meal to meal intervals. Meal to meal intervals are adjusted to varying meal size in the so-called frequency regulation of food intake [10, 11, 14]. The fact that the 24 hr intake of anosmic rats was not significantly modified suggested that
Amygdaloid lesions
the olfactory afferent was not involved in these processes of adjustment. However results of new experiments, here reported, have shown: (1) that the mechanism which regulates the day to day constancy of intake is, nevertheless, impaired in anosmic rats; and, (2) that under the influence of the interruption of olfactory pathways to critical hypothalamic centers, a striking alteration of the ad lib daily feeding pattern is introduced mainly due to a disruption of the normal control of meal size. EXPERIMENT 1 Ten adult female (190 g) and l 0 adult male (265 g) Wistar rats were used. The animals were individually caged and had free access to food (standard powder dietSorolabo) and water at all times. Bilateral ablation of bulbs was performed under light nembutal anesthesia and their removal was by suction through a perforation of the skull, care being taken to scrape the cribriform plate in order to ensure the complete suction of olfactory fibres. The daffy food intake of these operated animals was then recorded for 37 days and compared with their preoperative intake. At the conclusion of the experiment rats were sacrificed by an overdose of nembutal. Brains, perfused with normal saline and fixed in situ by 10% formalin, were removed for histological examination. Eight female and 10 male rats were sham operated (anesthesia, opening of the skull, no olfactory bulb removal) and submitted to the same postoperative procedure.
Results As shown in Table 1, the mean daily intakes in sham operated and bulbectomized males are not significantly 817
818
LARUE AND LE MAGNEN TABLE 1
Mean daily food intake (g) ± SD
INDIVIDUAL SUBJECTS
Group mean 2 SD
Female sham operated sham operation
Female bulbectomized bulbectomy
Before 10 days
A~er 37 days
Before 10 days
A~er 37 days
15.00±2.54 16.90±3.37 15.9022.73 15.80±3.20 18.38±2.79 17.1822.90 18.95±2.82 t7.94±2.25
17.47±2.09 18.6722.72 17.4522.06 16.9121.62 18.81±2.20 19.0022.90 19.8023.52 19.6022.59
19.18±1.50 16.5021.78 18.00±2.33 18.04±1.90 16.7223.06 17.5422.81 19.55±1.57 19.0422.14 17.40±1.04 17.45±1.15
18.96±3.64 19.0723.41 20.56±4.65 21.2124.93 19.8525.43 21.5524.83 20.56±3.79 20.4823.84 19.66±4.44 18.62±3.01
17.3822.82
18.47±2.46
17.9421.93
20.0424.20
Male sham operated sham operation Before l0 days
After 37 days
Male bulbectomized bulbectomy Before 10 days
After 37 days
29.87±4.12 26.2123.88 23.5522.23 ' 23.1722.03 27.35±2.49 24.81±2.61 22.8122.64 22.7522.25 25.4520.89 24.29±2.40 24.7521.73 28.4723.21 23.9522.58 23.2022.62 24.5922.57 24.4223.38 25.4121.77 27.1822.26 29.5023.40 29.2522.82
22.5021.48 23.27±1.29 25.8021.77 24.4021.79 26.7522.08 23.6421.92 26.18±1.01 28.6821.80 25.9022.40 31.0022.98
24.9723.53 28.5023.83 23.84±2.50 22.3823.10 26.98±2.79 26.3622.37 26.09±5.78 28.0923.64 28.58±2.80
25.7222.44
25.47±1.74
26.6823.33
25.3722.74
Statistical difference of group means
t = 2.389 p<0.05
t = 4.64 p<0.001
t = 0.042 p>0.2
t = 1.16 p>0.05
Statistical difference of SD's
t = 1.486 p>0.2
t = 7.037 p<0.001
t = 0.899 p>0.2
t = 4.74 p<0.001
different (t = 1.207). The difference is slightly significant in female group (t = 3.30 0.01>p>0.001). However, the examination of individual SD of daily intakes before and after surgery revealed that a striking increase of the fluctuation of the successive 24 hr intake was induced by removal of the olfactory bulb. The SD is without exception higher after the bulbectomy than before. This difference is absent in sham operated controls. Taking the mean of the male and female groups the difference between the SD before and after the operation is highly significant.
EXPERIMENT 2 By using the food recording technique routinely used in the laboratory it was possible to look f o r an effect of the olfactory bulb removal on the food intake by analysing the detailed feeding pattern: meal size and meal to meal interval, within the 24-hr cycle.
Method Six male and 7 female adult Wistar rats were used. Their normal feeding patterns were recorded using the continuous and automatic food cup weighing device [18]. This preoperative feeding pattern was recorded for 5 days at least. The removal of olfactory bulbs was performed as before. Postoperatively, the food intake was again recorded for 20 days.
Results After surgery, in almost every animal, a greatly modified feeding pattern appeared, particularly during the night period (Fig. 1). This abnormal feeding pattern consisted of very long, slow and large meals. Meals are conventionally defined as a feeding period preceded and followed by an interval of nonfeeding longer than 40 rain [10] and [unpublished data]. Such large meals reaching 1 2 - 1 5 g each are made up of successive and small bouts of feeding separated by many short pauses. The frequency distribution of the time of interruptions between each bout gives a good picture of the new meal pattern induced by the bulbectomy (Fig. 2). Such a pattern is commonly identified and named a nibbling pattern. The number of pauses shorter than 40 min was highly increased, t = 10.22 p<0.001. The number of intervals longer than 40 min was unchanged in day time t = 0.86. The nibbling pattern leads to a modification of the frequency distribution of meal size, the meal being defined as above (Fig. 3). Histological examination showed that the bulbectomy was well localized (Fig. 5). In one rat (male No. 7) the removal of olfactory bulb was associated with a lesion extended to neighbouring structures. This particular rat, excluded from the above results, was slightly hyperphagic (150%). However, in contrast with the feeding behavior of VMH lesioned rats, the typical nibbling pattern of bulbectomized rats was also present.
OLFACTORY CONTROL OF MEAL PATTERN IN RATS
819
g 10 ] 0
.
,"
/
,° t
I
12h
NIGHT
DAY
FIG. 1. Typical recorded feeding pattern of a typical rat before surgery (top) and after removal of olfactory bulb (bottom) on a typical day. FIG. 4. Histological section of a bulbectomized rat. I00
EXPERIMENT 3
ml N
5Lo '~
.~ ~
The preceding results suggested that the suppression of the olfactory input to the critical hypothalamic centers was responsible for this typical impairment of the meal size control. One of the known olfacto-hypothalamic pathways is the cortico-medial amygdala projections of the lateral olfactory tract, and from this region by fibres reaching the stria terminalis and the supra and postcommissural pathways. In order to test this suggestion it was attempted to place lesions in various points of this pathway to see if such lesions were associated with the appearance of the same nibbling pattern. Method
~
FIG. 2. Frequency distribution over 12 days of the duration of feeding interruptions of a typical animal. Left: shorter than 40 min (pauses). Right: longer than 40 rain (intervals). A. Before bulbectomy. B. After Bulbectomy.
O•no 1 NIGHT
0
2
4
6
8
0
2
4
6
8g
DAY
10
12
14
16 g
0
Results
2g
8
0
Twenty-two female rats were used in this experiment. The bilateral lesion of the cortico-medial region of the amygdala was made stereotaxically under nembutal anesthesia using the following coordinates: A P = 6 . 8 mm; H = + 1.2 mm from the zero of the ear bars and 3.8 mm lateral to the center of the sagittal sinus. Lesions were made by passing either an anodal current (8 rats) or cathodal one (14 rats) of 1 mA for 30 sec through a stainless steel electrode insulated except for 0.30 mm at the tip. The feeding pattern of rats was recorded for 5 days before and 10 days after surgery.
2g
FIG. 3. Frequency distribution of meals size for 4 days. A. Before bulbectomy. B. 16 days after bulbectomy.
The typical nibbling pattern was clearly observed in 7 out of 22 rats. However, in the remaining 15 rats the feeding pattern was to a greater or lesser extent intermediate between the nibbling and normal feeding patterns. In order to evaluate the degree of efficiency of the lesion upon the feeding response, it was thus necessary to use a quantitative estimate of this pattern. The t-test between the mean number of pauses and intervals before and after surgery in the night period appeared as a good estimate of an effective nibbling. By using this criteria the above 7 rats were tested as significantly nibblers p< 0.01; out of the 15 others, 7 are also significantly nibblers 0.05 >p>0.01. The feeding pattern of the remaining 8 rats was not significantly modified p> 0.05. Histological examination revealed that in the 7 very significant nibblers the lesions were bilaterally and accurately located in the cortico-medial amygdala region, and
820
LARUE AND LE MAGNEN cases far from the cortico-medial amygdala (usually too high, Fig. 7). Lesions produced by the cathodal technique were less extensive than those using the anode. DISCUSSION
FIG. 5. Histological section showing well located electrolytic lesions in the cortico-medial area of the amygdala.
FIG. 6. Histological section showing less correct lesions.
FIG. 7. Histological section showing lesions placed out of the cortico-medial area of the amygdala.
were superimposable for all these animals (Fig. 5). In the second group of less significant nibblers, lesions were less perfect with, in some cases, a clear bilateral dissymmetry of the lesion or unilateral lesion (Fig. 6). In the last nonsignificant group, lesions were obviously misplaced and in some
The total amount of food eaten ad lib by rats over 24 hr is the sum of amounts eaten in a given number of daily meals. This period of 24 hr is the shortest over which normal rats demonstrate a relative constancy of cumulative intake. Such a cumulative daily intake (the body weight being stable) corresponds to the steady state of body energy balance. It has been shown that this metabolically adjusted and constant daily food intake is dependent on the regulation of post meal intervals according to variable meal sizes, and on a relation between the night and day time feeding [ 1, 10, 11, 21, 22]. The same process of adjustment of meal to meal intervals to meal sizes is operative to rapidly modify the intake level after shifts of the caloric intake or expenditure. The reduced accuracy of such a day to day regulation of food intake resulting from bulbectomy suggests that the process involved in this accuracy is somewhat impaired after the loss of olfactory cues. Such a slight deficit in the day to day regulation, demonstrated in Experiment 1, is presumably a direct consequence upon the adjusting process of the highly modified meal pattern induced by the removal of olfactory bulbs shown in Experiment 2. Within the long lasting nibbled meals, in which short feeding bouts separated by short pauses are substituted for large meals separated by long intervals, a new control system of the meal sizes seems to be effective as a consequence of suppression of the olfactory input. It is of interest to note that the mean rate of eating in meals defined as before, made of short feeding bouts with frequent and short interruptions, is low. For example in male No. 8, 92-+6 m/min, versus 216-+5 m/min in the more continuous meals before bulbectomy. The speed of eating or feeding rate during the meal has been shown to be dependent on the palatability of the food and was proposed as a measure of the relative palatability of food [ 15]. The observed lowering of the mean feeding rate of a food as a result of bulbectomy could therefore be interpreted as a reduction of the palatability level of that food due to the suppression of its odorous stimulatory activity. The olfactory projections to VMH nuclei and to the lateral hypothalamic region, the first through the corticomedial area of amygdala and the stria terminalis, the second via the medial forebrain bundle, have been anatomically well identified [3, 6, 7, 19]. A modification of the single unit or multiunit electrical activity under the influence of a stimulation by food odors has been demonstrated in VMH [2] and in LH [5, 16, 20]. It has been suggested that these two olfacto-hypothalamic pathways account partly for the oral positive and negative feed back mechanisms controlling the meal size. The interruption of olfactory signals to hypothalamic centers would have resulted in the loss of the main rewarding effect normally indispensable to sustain large and continuous meals, and to induce at the end of such meals a persistent satiety. The remaining oral cues would be able to reinforce only short bouts of eating interrupted by brief pauses. However the fact that the nibbling pattern may be obtained by the cortico-medial amygdaloid lesion cannot be
O L F AC T OR Y CONTROL OF MEAL PATTERN IN RATS interpreted in favor of the hypothesis that the disruption of the meal size control is caused by the interruption of the olfacto VMH pathway. Normally the nibbling pattern is not exhibited by the hyperphagic VMH lesioned rat. However it has been shown that the removal of olfactory bulbs prior to or after VMH lesion produces hyperphagic nibblers (unpublished data). In contrast to the VMH lesioned rat, the LH recovered rat has been shown to exhibit the same nibbling pattern [ 8 ] ; this pattern is not due to prandial drinking since desalivate, but neurologically normal, rats which are prandial drinkers do not show a disruption of meal pattern. It has been recently shown [ 15] that the unilateral LH damage introduces a sensory neglect toward olfactory, visual and tactile stimuli presented on the contralateral side. The aphagia in the bilaterally lesioned rat could be the result of an impairment of the integration of the sensory and particularly the olfactory input in the control o f the motor feeding performance. Although such a sensory neglect after unilateral LH lesion disappeared after a time period identical to that needed for the first stage of
821 recovery from aphagia, it may be presumed that the nibbling pattern in the fully recovered LH rat could be the result of a residual impairment of the olfactory input in controlling the meal size. Either the suppression by bulbectomy or the loss of the efficiency of the olfactory input by the LH lesion could result in the same disruption of the meal pattern by the disappearance of its normal olfactory control through the olfactory LH pathway. The induction of an identical nibbling pattern by the cortico-medial amygdaloid lesion is more difficult to explain since the lesion of the VMH at the terminus of the bulbo-amygdalo hypothalamic pathway does not induce this pattern. This region is known to be a projection of fibers coming not from the main olfactory bulb but rather from the accessory olfactory bulb and from the Jacobsen or vomeronasal organ [23 ]. However, the hypothesis that this accessory olfactory organ might be involved in the orosensory control of feeding has been excluded by showing that the section of vomeronasal nerves does not induce an altered feeding pattern [unpublished data].
REFERENCES 1. Balagura, S. and D. V. Coscina. Periodicity of food intake in the rat as measured by an operant response. Physiol. Behav. 3: 641-643, 1968. 2. Campbell, J. F., D. Bindra, H. Krebs and R. P. Ferenchak. Response of single units of the hypothalamic ventromedial nucleus to environmental stimuli. Physiol. Behav. 4: 183-187, 1969. 3. Dreifuss, J. P., J. T. Murphy and P. Gloor. Contrasting effects of two identified amygdaloid efferent pathways on single hypothalamic neurons. J. Neurophysiol. 23: 237-248, 1968. 4. Harris, L. J., J. Clay, F. J. Hargreaves and A. Ward. Appetite and choice of diet: the ability of the vitamin B deficient rat to discriminate between diets containing and lacking the vitamin. Prec. r. Soc. 113: 161-190, 1963. 5. Hamburg M. D. Hypothalamic unit activity and eating behavior. Am. J. Physiol. 220: 980-985, 1971. 6. Heimer, L. Synaptic distribution of centripetal and centrifugal nerve fibers in the olfactory system in the rat. J. Anat. 103: 413-432, 1968. 7. Heimer, L. and J. H. Nauta. The hypothalamic distribution of the stria terminalis in the rat. Brain Res. 13: 284-297, 1969. 8. Kissileff, H. R. Free feeding in normal and recovered lateral rats monitored by a pellet detecting eatometer. Physiol. Behav. 5: 163-173, 1970. 9. LaRue, C. and J. Le Magnen. R@onses alimentaires du rat anosmique. C.R. Soc. Biol. 00: 161-572, 1967. 10. Le Magnen, J. and S. TaUon. Enregistrement et analyse pr61iminaire de la "p~riodicit6 alimentaire spontan6e" chez le rat blanc. Z Physiol., Paris 55: 286-297, 1963. 11. Le Magnen, J. and S. TaUon. La p6riodicit6 spontan6e de la prise d'aliments ad libitum du rat blanc. J. Physiol., Paris 958: 323-349, 1966. 12. Le Magnen, J. Peripheral and systemic actions of food in the caloric regulation of intake. Ann. N. Y. Acad. ScL 157: 1126-1157, 1969.
13. Le Magnen, J. et M. Devos. Le substrat m6tabolique de la faim. Etude des facteurs de d6clenchement de la prise alimentaire du rat nourri ad libitum. C. R. Acad. Sci. Paris 268: 3107-3110, 1969. 14. Le Magnen, J. and M. Devos. Metabolic correlates of the meal onset in the free food intake of rats. Physiol. Behav. 5: 805-814, 1970. 15. Le Magnen, J. Advances in studies on the physiological control and regulation of food intake. Progr. Physiol. Psychol. 4: 203-261, 1971. 16. Marshall. J. F., B. H. Turner and P. Teitelbaum. Sensory neglect produced by lateral hypothalamic damage. Science 174: 523-525, 1971. 17. Oomura, Y., H. Ooyama, T. Yamamoto and F. Naka. Reciprocal relationship of the lateral and ventromedial hypothalamus in the regulation of food intake. Physiol. Behav. 2: 97-115, 1967. 18. Pokrovsky, V. and J. Le Magnen. R6alisation d'un dispositif d'enregistrement graphique continu et automatique de la consommation alimentaire du rat blanc. J. Physiol. 455: 318-000, 1963. 19. Price, J. L. and T. P. S. PoweU. An experimental study of the origin and the course of the centrifugal fibers to the olfactory bulb in the rat. J. Anat. 107: 2, 215-237, 1970. 20. Scott, J. W. and C. Pfaffmann. Olfactory input to the hypothalamus: Electrophysiological evidence. Science 158: 1592-1594, 1967. 21. Snowdon, C. T. Motivation, regulation and the control of meal parameters with oral and intragastric feeding. J. comp. physiol. Psychol. 69: 91-100, 1969. 22. Thomas, D. W. and J. Mayer. Meal taking and regulation of food intake by normal and hypothalamic hyperphagia rats. J. comp. physiol. Psychol. 66: 642-653, 1968. 23. Winans, S. and F. Scalia. Amygdaloid nucleus: new afferent input from the vomeronasal organ. Science 170: 330-332, 1970.
The Olfactory Control of Meal Pattern in Rats CHRISTIANE G. LARUE AND JACQUES LE MAGNEN
Laboratoire de Neurophysiologie sensorielle et comportementale du C.N.R.S. Collbge de France, Paris 5e, France
(Received 28 December 1971)
LARUE, C. G. AND J. LE MAGNEN. The olfactory control of meal pattern in rats. PHYSIOL. BEHAV. 9(5) 817-821, 1972.- The role of the olfactory input in the control system regulating the free food intake of rats has been investigated. In a first experiment it was shown that the mean daily intake of 20 adult male and female rats, made anosrnic by the bilateral ablation of olfactory bulbs, did not change significantly after surgery. But, as a result of the bulbar removal, the day to day fluctuation of this daily intake was significantly increased. In a second experiment the pre and postoperative recording of the daily feeding pattern (meal size and intervals) revealed that the olfactory bulbs ablation induced a more or less persistant and typical nibbling pattern. The stereotaxie electrolytic lesion of the cortico-medial amygdaioid olfactory projection, interrupting one of the olfacto-hypothalamic pathways, induced the same disrupted feeding pattern. The results are discussed in relation to control systems regulating meal sizes and meal to meal intervals in normal rats. It is suggested that the orosensory and particularly the olfactory control is responsible for determining the normal feeding pattern made of large and consistant meals followed by long intervals of nonfeeding. Olfactory bulbs removal
Food intake
Meal pattern
THE RESPECTIVE roles of orosensory and of metabolic postingestive factors in the control of meal size is still open to discussion. It is known that, for a given state of food deprivation, the satiating amount eaten in a meal of each particular food is sensory specific. This fact, among others, argues that the meal size is mainly or exclusively the behavioral response to the orosensory stimulating properties of each food. The innate and learned level of this response is defined as the palatability of the food. Various sensory stimulating activities of food in the m o u t h contribute to this palatability of the food and therefore to the control of meal size. Hitherto for reasons of experimental simplicity, the role of gustatory stimulations has been mainly investigated. By contrast, the study of the role of the mechanical and, most of all, of the olfactory control of food intake has been widely neglected. However, it has been shown that in learned specific [4] or caloric [12] appetite the amount of food eaten may be entirely dependent on an arbitrary added odor. In order to study further the role of olfactory stimuli and olfactory neural pathways in the oral metering of food, experiments have been undertaken to control the effects of the interruption of these oropharyngeal sensory afferents upon feeding responses in rats. In a previous work it has been shown that the olfactory bulb removal in a rat fed ad lib with a familiar food, did not change strikingly the 24 hr intake [9]. This daily intake of food and its relative constancy from day to day are dependent on the relationship between the two basic parameters: namely meal sizes and meal to meal intervals. Meal to meal intervals are adjusted to varying meal size in the so-called frequency regulation of food intake [10, 11, 14]. The fact that the 24 hr intake of anosmic rats was not significantly modified suggested that
Amygdaloid lesions
the olfactory afferent was not involved in these processes of adjustment. However results of new experiments, here reported, have shown: (1) that the mechanism which regulates the day to day constancy of intake is, nevertheless, impaired in anosmic rats; and, (2) that under the influence of the interruption of olfactory pathways to critical hypothalamic centers, a striking alteration of the ad lib daily feeding pattern is introduced mainly due to a disruption of the normal control of meal size. EXPERIMENT 1 Ten adult female (190 g) and l 0 adult male (265 g) Wistar rats were used. The animals were individually caged and had free access to food (standard powder dietSorolabo) and water at all times. Bilateral ablation of bulbs was performed under light nembutal anesthesia and their removal was by suction through a perforation of the skull, care being taken to scrape the cribriform plate in order to ensure the complete suction of olfactory fibres. The daffy food intake of these operated animals was then recorded for 37 days and compared with their preoperative intake. At the conclusion of the experiment rats were sacrificed by an overdose of nembutal. Brains, perfused with normal saline and fixed in situ by 10% formalin, were removed for histological examination. Eight female and 10 male rats were sham operated (anesthesia, opening of the skull, no olfactory bulb removal) and submitted to the same postoperative procedure.
Results As shown in Table 1, the mean daily intakes in sham operated and bulbectomized males are not significantly 817
818
LARUE AND LE MAGNEN TABLE 1
Mean daily food intake (g) ± SD
INDIVIDUAL SUBJECTS
Group mean 2 SD
Female sham operated sham operation
Female bulbectomized bulbectomy
Before 10 days
A~er 37 days
Before 10 days
A~er 37 days
15.00±2.54 16.90±3.37 15.9022.73 15.80±3.20 18.38±2.79 17.1822.90 18.95±2.82 t7.94±2.25
17.47±2.09 18.6722.72 17.4522.06 16.9121.62 18.81±2.20 19.0022.90 19.8023.52 19.6022.59
19.18±1.50 16.5021.78 18.00±2.33 18.04±1.90 16.7223.06 17.5422.81 19.55±1.57 19.0422.14 17.40±1.04 17.45±1.15
18.96±3.64 19.0723.41 20.56±4.65 21.2124.93 19.8525.43 21.5524.83 20.56±3.79 20.4823.84 19.66±4.44 18.62±3.01
17.3822.82
18.47±2.46
17.9421.93
20.0424.20
Male sham operated sham operation Before l0 days
After 37 days
Male bulbectomized bulbectomy Before 10 days
After 37 days
29.87±4.12 26.2123.88 23.5522.23 ' 23.1722.03 27.35±2.49 24.81±2.61 22.8122.64 22.7522.25 25.4520.89 24.29±2.40 24.7521.73 28.4723.21 23.9522.58 23.2022.62 24.5922.57 24.4223.38 25.4121.77 27.1822.26 29.5023.40 29.2522.82
22.5021.48 23.27±1.29 25.8021.77 24.4021.79 26.7522.08 23.6421.92 26.18±1.01 28.6821.80 25.9022.40 31.0022.98
24.9723.53 28.5023.83 23.84±2.50 22.3823.10 26.98±2.79 26.3622.37 26.09±5.78 28.0923.64 28.58±2.80
25.7222.44
25.47±1.74
26.6823.33
25.3722.74
Statistical difference of group means
t = 2.389 p<0.05
t = 4.64 p<0.001
t = 0.042 p>0.2
t = 1.16 p>0.05
Statistical difference of SD's
t = 1.486 p>0.2
t = 7.037 p<0.001
t = 0.899 p>0.2
t = 4.74 p<0.001
different (t = 1.207). The difference is slightly significant in female group (t = 3.30 0.01>p>0.001). However, the examination of individual SD of daily intakes before and after surgery revealed that a striking increase of the fluctuation of the successive 24 hr intake was induced by removal of the olfactory bulb. The SD is without exception higher after the bulbectomy than before. This difference is absent in sham operated controls. Taking the mean of the male and female groups the difference between the SD before and after the operation is highly significant.
EXPERIMENT 2 By using the food recording technique routinely used in the laboratory it was possible to look f o r an effect of the olfactory bulb removal on the food intake by analysing the detailed feeding pattern: meal size and meal to meal interval, within the 24-hr cycle.
Method Six male and 7 female adult Wistar rats were used. Their normal feeding patterns were recorded using the continuous and automatic food cup weighing device [18]. This preoperative feeding pattern was recorded for 5 days at least. The removal of olfactory bulbs was performed as before. Postoperatively, the food intake was again recorded for 20 days.
Results After surgery, in almost every animal, a greatly modified feeding pattern appeared, particularly during the night period (Fig. 1). This abnormal feeding pattern consisted of very long, slow and large meals. Meals are conventionally defined as a feeding period preceded and followed by an interval of nonfeeding longer than 40 rain [10] and [unpublished data]. Such large meals reaching 1 2 - 1 5 g each are made up of successive and small bouts of feeding separated by many short pauses. The frequency distribution of the time of interruptions between each bout gives a good picture of the new meal pattern induced by the bulbectomy (Fig. 2). Such a pattern is commonly identified and named a nibbling pattern. The number of pauses shorter than 40 min was highly increased, t = 10.22 p<0.001. The number of intervals longer than 40 min was unchanged in day time t = 0.86. The nibbling pattern leads to a modification of the frequency distribution of meal size, the meal being defined as above (Fig. 3). Histological examination showed that the bulbectomy was well localized (Fig. 5). In one rat (male No. 7) the removal of olfactory bulb was associated with a lesion extended to neighbouring structures. This particular rat, excluded from the above results, was slightly hyperphagic (150%). However, in contrast with the feeding behavior of VMH lesioned rats, the typical nibbling pattern of bulbectomized rats was also present.
OLFACTORY CONTROL OF MEAL PATTERN IN RATS
819
g 10 ] 0
.
,"
/
,° t
I
12h
NIGHT
DAY
FIG. 1. Typical recorded feeding pattern of a typical rat before surgery (top) and after removal of olfactory bulb (bottom) on a typical day. FIG. 4. Histological section of a bulbectomized rat. I00
EXPERIMENT 3
ml N
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The preceding results suggested that the suppression of the olfactory input to the critical hypothalamic centers was responsible for this typical impairment of the meal size control. One of the known olfacto-hypothalamic pathways is the cortico-medial amygdala projections of the lateral olfactory tract, and from this region by fibres reaching the stria terminalis and the supra and postcommissural pathways. In order to test this suggestion it was attempted to place lesions in various points of this pathway to see if such lesions were associated with the appearance of the same nibbling pattern. Method
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FIG. 2. Frequency distribution over 12 days of the duration of feeding interruptions of a typical animal. Left: shorter than 40 min (pauses). Right: longer than 40 rain (intervals). A. Before bulbectomy. B. After Bulbectomy.
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Twenty-two female rats were used in this experiment. The bilateral lesion of the cortico-medial region of the amygdala was made stereotaxically under nembutal anesthesia using the following coordinates: A P = 6 . 8 mm; H = + 1.2 mm from the zero of the ear bars and 3.8 mm lateral to the center of the sagittal sinus. Lesions were made by passing either an anodal current (8 rats) or cathodal one (14 rats) of 1 mA for 30 sec through a stainless steel electrode insulated except for 0.30 mm at the tip. The feeding pattern of rats was recorded for 5 days before and 10 days after surgery.
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FIG. 3. Frequency distribution of meals size for 4 days. A. Before bulbectomy. B. 16 days after bulbectomy.
The typical nibbling pattern was clearly observed in 7 out of 22 rats. However, in the remaining 15 rats the feeding pattern was to a greater or lesser extent intermediate between the nibbling and normal feeding patterns. In order to evaluate the degree of efficiency of the lesion upon the feeding response, it was thus necessary to use a quantitative estimate of this pattern. The t-test between the mean number of pauses and intervals before and after surgery in the night period appeared as a good estimate of an effective nibbling. By using this criteria the above 7 rats were tested as significantly nibblers p< 0.01; out of the 15 others, 7 are also significantly nibblers 0.05 >p>0.01. The feeding pattern of the remaining 8 rats was not significantly modified p> 0.05. Histological examination revealed that in the 7 very significant nibblers the lesions were bilaterally and accurately located in the cortico-medial amygdala region, and
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LARUE AND LE MAGNEN cases far from the cortico-medial amygdala (usually too high, Fig. 7). Lesions produced by the cathodal technique were less extensive than those using the anode. DISCUSSION
FIG. 5. Histological section showing well located electrolytic lesions in the cortico-medial area of the amygdala.
FIG. 6. Histological section showing less correct lesions.
FIG. 7. Histological section showing lesions placed out of the cortico-medial area of the amygdala.
were superimposable for all these animals (Fig. 5). In the second group of less significant nibblers, lesions were less perfect with, in some cases, a clear bilateral dissymmetry of the lesion or unilateral lesion (Fig. 6). In the last nonsignificant group, lesions were obviously misplaced and in some
The total amount of food eaten ad lib by rats over 24 hr is the sum of amounts eaten in a given number of daily meals. This period of 24 hr is the shortest over which normal rats demonstrate a relative constancy of cumulative intake. Such a cumulative daily intake (the body weight being stable) corresponds to the steady state of body energy balance. It has been shown that this metabolically adjusted and constant daily food intake is dependent on the regulation of post meal intervals according to variable meal sizes, and on a relation between the night and day time feeding [ 1, 10, 11, 21, 22]. The same process of adjustment of meal to meal intervals to meal sizes is operative to rapidly modify the intake level after shifts of the caloric intake or expenditure. The reduced accuracy of such a day to day regulation of food intake resulting from bulbectomy suggests that the process involved in this accuracy is somewhat impaired after the loss of olfactory cues. Such a slight deficit in the day to day regulation, demonstrated in Experiment 1, is presumably a direct consequence upon the adjusting process of the highly modified meal pattern induced by the removal of olfactory bulbs shown in Experiment 2. Within the long lasting nibbled meals, in which short feeding bouts separated by short pauses are substituted for large meals separated by long intervals, a new control system of the meal sizes seems to be effective as a consequence of suppression of the olfactory input. It is of interest to note that the mean rate of eating in meals defined as before, made of short feeding bouts with frequent and short interruptions, is low. For example in male No. 8, 92-+6 m/min, versus 216-+5 m/min in the more continuous meals before bulbectomy. The speed of eating or feeding rate during the meal has been shown to be dependent on the palatability of the food and was proposed as a measure of the relative palatability of food [ 15]. The observed lowering of the mean feeding rate of a food as a result of bulbectomy could therefore be interpreted as a reduction of the palatability level of that food due to the suppression of its odorous stimulatory activity. The olfactory projections to VMH nuclei and to the lateral hypothalamic region, the first through the corticomedial area of amygdala and the stria terminalis, the second via the medial forebrain bundle, have been anatomically well identified [3, 6, 7, 19]. A modification of the single unit or multiunit electrical activity under the influence of a stimulation by food odors has been demonstrated in VMH [2] and in LH [5, 16, 20]. It has been suggested that these two olfacto-hypothalamic pathways account partly for the oral positive and negative feed back mechanisms controlling the meal size. The interruption of olfactory signals to hypothalamic centers would have resulted in the loss of the main rewarding effect normally indispensable to sustain large and continuous meals, and to induce at the end of such meals a persistent satiety. The remaining oral cues would be able to reinforce only short bouts of eating interrupted by brief pauses. However the fact that the nibbling pattern may be obtained by the cortico-medial amygdaloid lesion cannot be
O L F AC T OR Y CONTROL OF MEAL PATTERN IN RATS interpreted in favor of the hypothesis that the disruption of the meal size control is caused by the interruption of the olfacto VMH pathway. Normally the nibbling pattern is not exhibited by the hyperphagic VMH lesioned rat. However it has been shown that the removal of olfactory bulbs prior to or after VMH lesion produces hyperphagic nibblers (unpublished data). In contrast to the VMH lesioned rat, the LH recovered rat has been shown to exhibit the same nibbling pattern [ 8 ] ; this pattern is not due to prandial drinking since desalivate, but neurologically normal, rats which are prandial drinkers do not show a disruption of meal pattern. It has been recently shown [ 15] that the unilateral LH damage introduces a sensory neglect toward olfactory, visual and tactile stimuli presented on the contralateral side. The aphagia in the bilaterally lesioned rat could be the result of an impairment of the integration of the sensory and particularly the olfactory input in the control o f the motor feeding performance. Although such a sensory neglect after unilateral LH lesion disappeared after a time period identical to that needed for the first stage of
821 recovery from aphagia, it may be presumed that the nibbling pattern in the fully recovered LH rat could be the result of a residual impairment of the olfactory input in controlling the meal size. Either the suppression by bulbectomy or the loss of the efficiency of the olfactory input by the LH lesion could result in the same disruption of the meal pattern by the disappearance of its normal olfactory control through the olfactory LH pathway. The induction of an identical nibbling pattern by the cortico-medial amygdaloid lesion is more difficult to explain since the lesion of the VMH at the terminus of the bulbo-amygdalo hypothalamic pathway does not induce this pattern. This region is known to be a projection of fibers coming not from the main olfactory bulb but rather from the accessory olfactory bulb and from the Jacobsen or vomeronasal organ [23 ]. However, the hypothesis that this accessory olfactory organ might be involved in the orosensory control of feeding has been excluded by showing that the section of vomeronasal nerves does not induce an altered feeding pattern [unpublished data].
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13. Le Magnen, J. et M. Devos. Le substrat m6tabolique de la faim. Etude des facteurs de d6clenchement de la prise alimentaire du rat nourri ad libitum. C. R. Acad. Sci. Paris 268: 3107-3110, 1969. 14. Le Magnen, J. and M. Devos. Metabolic correlates of the meal onset in the free food intake of rats. Physiol. Behav. 5: 805-814, 1970. 15. Le Magnen, J. Advances in studies on the physiological control and regulation of food intake. Progr. Physiol. Psychol. 4: 203-261, 1971. 16. Marshall. J. F., B. H. Turner and P. Teitelbaum. Sensory neglect produced by lateral hypothalamic damage. Science 174: 523-525, 1971. 17. Oomura, Y., H. Ooyama, T. Yamamoto and F. Naka. Reciprocal relationship of the lateral and ventromedial hypothalamus in the regulation of food intake. Physiol. Behav. 2: 97-115, 1967. 18. Pokrovsky, V. and J. Le Magnen. R6alisation d'un dispositif d'enregistrement graphique continu et automatique de la consommation alimentaire du rat blanc. J. Physiol. 455: 318-000, 1963. 19. Price, J. L. and T. P. S. PoweU. An experimental study of the origin and the course of the centrifugal fibers to the olfactory bulb in the rat. J. Anat. 107: 2, 215-237, 1970. 20. Scott, J. W. and C. Pfaffmann. Olfactory input to the hypothalamus: Electrophysiological evidence. Science 158: 1592-1594, 1967. 21. Snowdon, C. T. Motivation, regulation and the control of meal parameters with oral and intragastric feeding. J. comp. physiol. Psychol. 69: 91-100, 1969. 22. Thomas, D. W. and J. Mayer. Meal taking and regulation of food intake by normal and hypothalamic hyperphagia rats. J. comp. physiol. Psychol. 66: 642-653, 1968. 23. Winans, S. and F. Scalia. Amygdaloid nucleus: new afferent input from the vomeronasal organ. Science 170: 330-332, 1970.