Alterations in feeding behaviour of female rats at varying phases of their reproductive sequence as a result of lesions in various parts of the amygdala Rachel K MacDonald City University The topic of this proposed research is the role of the amygdala in hunger and satiety following damage in female infant and adult rats as a result of sexual and maternal activity. Data will be collected from 200 female albino rats, 50 infants and 150 adults of the Sprague-Dawley strain. Hunger and satiety will be analysed following feeding behaviour analysis of body weight regulation, spontaneous food and water intake, and the responses to glucose gavage and long-term food deprivation. The results should provide evidence that the aforementioned feeding mechanisms are affected by lesions in some parts of the amygdala and should lead to the safe conclusion that the amygdala does play an equal role in hunger and satiety as it does in appetite. Specific aims: (abstract / summary ) The broad, long-term objectives of this research include; assessing the role of the amygdala (See Figure. 1 for location) on feeding behaviour in the female rat.

In order to realise the role of neural mechanisms and signals underlying hunger and satiation. This specific research is intended to accomplish a greater understanding of the effect of lesions in varying areas of the amygdala on body weight regulation, unprompted food and water intake, responses to glucose gavage and long-term food deprivation. These feeding factors will be analysed at various points throughout the rats' reproductive and maternal activities and the sample will therefore include infant female rats and adult female rats. As the amygdala is comprised of a collection of nuclei in the temporal lobe the basolateral complex is in red, and the lateral nucleus is in yellow The hippocampus is also displayed in blue, with its cell bodies in black.

The green is a collection of various nerve fibres contributing to the internal and external capsule and corpus callosum. The main assumptions underlying this research are that lesions in altered parts of the amygdala will affect feeding behaviour of the rats in diverse ways as different parts of the amygdala are responsible for divergent functions and these differences will alter depending on whether the female rat in 'in heat', pregnant, or nursing as the amygdala plays roles in reproductive behaviour as well is ingestion. The general goal is therefore to observe both similarities and differences in feeding behaviour observed in Schoenfeld & Hamilton's (1981) report by therefore extending their prior research and applying the findings to a larger rat population. A further goal is to verify or refute Schoenfeld & Hamilton's vague statement that "Body weight regulation, spontaneous food and water intake, and the responses to glucose gavage and long-term food deprivation were not altered by lesions in the amygdala. Hence the wide-ranging research question the proposed study attempts to answer is 'what exact role does the amygdala play in feeding in the female rat as a function of age and sexual eminence.' This question shall be answered by firstly asking which of the following behavioural tests; body weight regulation, spontaneous food and water intake, the responses to glucose gavage and feeding response to long-term food deprivation, are altered by lesions in the amygdala of female rats. This latter question enables us to assess the role the amygdala plays in hunger and satiety in the female rat.

It is consequently important to make the distinction between appetite, hunger, and satiety, as the latter ones are the only ones that require thorough testing. This is because although various theories have been put forward generally the whole topic in question is poorly understood. To understand the complexities it is essential to recognize what the terms hunger, appetite and satiety mean. Hunger is the craving for food that is associated with objective sensations. It is a general sensation localised to the stomach region; it appears when the stomach is empty and coincides with contractions detected by mechanoreceptors in the stomach wall.

Appetite is the desire for specific types and quantity of food. Satiety is the opposite of hunger and results from a filling meal. Schoenfeld & Hamilton (1984) felt that some of the tests they conducted did not show disruption of satiety or hunger at all, just appetite. This proposed research will put that assumption to test again, with another variable of gender and reproductive phase. These questions are important because it is imperative to learn if Schoenfeld & Hamilton's (1981) conclusions extend to female rats, and also whether lesions of the amygdala affect the feeding behaviour of the rat differently in different reproductive stages. The first experiment, on food deprivation is fundamental as although the topic has been a critical factor in research on amygdaloid mechanisms in feeding behaviour.

Food deprivation in rats is still largely misunderstood, especially as concerning female rats. The relevant theoretical framework to the proposed research involves producing lesions bilaterally in the various sites in and around the amygdala and then assessing brain damage using standardized gridded plates from a rat brain atlas, to see if lesions in particular areas affect feeding behaviour in differing ways. The hypotheses will be tested thoroughly by using similar methods as stated in Schoenfeld & Hamilton's method section but using female rats instead of male, and at different points in their life cycles. The factor of age will be carefully controlled as will the factor of weight. Background and Significance: The theoretical and empirical background for this proposed research chiefly originates from Schoenfeld & Hamilton's (1981) investigation into disruption of appetite following small lesions in the amygdala of male rats. Klver and Bucy (1939) are most often cited as having initially suggested that the amygdala plays an important role in feeding behaviour.

They came to this conclusion through observing the functions of the temporal lobes in monkeys. Others researchers that have worked on similar problems include; Nachman and Ashe (1974), however they extended the original idea to the study of rats. Unfortunately, the methods used in these previous studies led to a lack of specificity. The consequences of the research led to more unanswered questions, as no conclusions had been uncovered about the fixed limits or boundary of the amygdala's involvement in feeding behaviour. Even though Nachman and Ashe did manage to incorporate behavioural specificity with anatomical specificity in a reasonable way it is still unclear whether differences in results between experiments are due to differing procedures, anatomical destruction, or some other distinction. For this reason the proposed experimental procedure shall closely follow Schoenfeld & Hamilton's, as displayed in their 1981 publication.

The results and conclusions of previous research are quite contradictory. For instance Pujols (1966) study, and Kemble & Schwartzbaum's (1969) study on the effect of lesions on the response to brief exposure to 8% sucrose solutions yielded completely inconsistent findings even though they were extremely similar experiments. Another important contradiction relates to the theory behind experiment 1. Dacey and Grossman (1977) compared the effects of lesions restricted to only the amygdala and only to the striatum. They found that lesions of the central amygdaloid nucleus do not alter spontaneous feeding and drinking.

Conversely, observations by Box and Mogenson (1975) were conflicting. Clearly, further tests need to be carried out in order to shed more light on the subject. The proposed study will therefore be limited to the investigation of infant and adult female rats with lesions in various part of their amygdala (see Table 1), on subsequent testing in four measures of their feeding behaviour. Experiment Lesions sites: Experiment 1: Ventromedial area Transition zone Caudoventral area Midlateral area Central nucleus Experiment 2: Lateral amygdala Medial amygdala Amygdalaostriatal transition zone Striatum Table 1. Area of brain damage in each experiment The work that I intend to execute will take this previous research and not only extend it but also challenge some of its core conclusions. It will also explore a previously ignored area of research by investigating the interaction between reproductive behaviour and appetite following brain damage of the amygdala.

Following thorough analysis and evaluation of the current knowledge available, it becomes apparent that the research proposed would fill in an imperative gap in the literature, i. e. the absent data from females of the Sprague-Dawley strain, at differing stages of reproductive age and activity. Pfaff and Keiner (1972) found that the amygdala contains some cell groups, which are targets for estrogens. It is therefore sensible to assume that lesions in particular areas will cause interference of feedings behaviour in most female rats (except for the ones that are not sexually mature). The future study will also challenge prior knowledge by retesting the claim that "the amygdala may play a greater role in appetite than in hunger or satiety." This extended assessment will only analyse the previously dismissed feeding behaviour, which damage to different parts of the amygdala was concluded as having not been changed.

In summary, this proposal comprises a replication of the Schoenfeld & Hamilton (1981) study plus an essential gender extension. Research Design and Methods: The experimental design is completely between. The main factor that will be manipulated is the region of damage within the amygdala (See Table. 1).

However the main independent variable is of reproductive status. This has 4 levels: (1) 6 weeks of age or younger (therefore not sexually mature) (2) 4 months and above (undergoing usual reproductive cycles) (3) Pregnant (4) Nursing The dependant variable differs within each experiment with consideration of the behaviour under inspection. The variable concerning the reproductive aspect of the rats is based on the following specifics: Rats reach sexual maturity at 5 weeks of age Females of breeding age come into heat every 4-5 days. Female rats don't come into heat if they are pregnant or nursing.

The best time to breed a female is after 4 months of age. The gestation period is normally 22 days. Babies can be weaned at 4-5 weeks. The proposed experiment will contain two independent studies: Experiment 1 - brief food consumption following food deprivation or glucose gavage.

Experiment 2 - body weight regulation and spontaneous consumption of food pellets and water. The latter study will be a single focus of behavioural observation, unlike in Schoenfeld & Hamilton's study when in was analysed within other experiments. The procedures apparent within the proposed research will closely follow Schoenfeld and Hamilton's (1981) techniques. (See main method below for divergences) The data will be analysed like such: Figure 2. A flow diagram of the proposed data analysis Outcomes that are inconsistent with the results that are expected could arise due to differing procedural details or differing anatomical destruction and should be consequently interpreted as ambiguous. Alternatively, inconsistent findings could arise due to the gender of the rats; this aspect is therefore not contradictory as some dissimilarity between the two sexes should be expected due to the differing role of the amygdala in male and female reproductive behaviour as this brain structure is sexually dimorphic.

Any refutations would suggest that Schoenfeld & Hamilton were correct in postulating that lesions in any areas of the amygdala do not alter certain feeding behaviours, i. e. food deprivation and body weight regulation. Results that portray no difference between male and female rats, regardless of reproductive state would simply imply that the amygdala is more similar in males and females that we might otherwise have thought. Even these findings would for that reason would still add relevant data to the general problem of the role of amygdala in rats. Outcomes that are consistent with the hypotheses include relevant evidence that lesions in particular areas of the amygdala do affect body weight regulation, spontaneous food and water intake, response to glucose gavage and long term food deprivation.

This substantiating data would imply that the amygdala (or certain areas within it) do play a role in hunger and / or satiety and not just appetite. In experiment 1 rats with Caudoventral area damage will have increased intake following deprivation but to a decreased level if an infant (under 6 weeks). In accordance with Cole's (1974) findings it is likely that the amygdaloid lesions in a dorsomedial region will result in less absolute food consumption following overnight deprivation than that shown by controls. Increased food consumption will take place in pregnant rats due to increased activation of the medial amygdala. Lesions of the ventromedial area will also produce this result.

Schoenfeld & Hamilton stated that male rats "overeat to a brief period of food restriction." This shall be demonstrated in female rats, except the ones that are pregnant or nursing. This experiment will display hunger (rather than just appetite) if rats respond to long-term deprivation with a normal increase in food intake. In experiment 2 a difference in nursing and pregnant rats from the other groups would be expected if their lesions were in the medial amygdala. This is because cells In the medial amygdala contain estrogen receptors and it is reasonable to assume that this will interact with the feeding mechanisms controlled by the medial amygdala. However as the medial nucleus of the amygdala plays an important role in reproductive behaviour the results from the rats in usual reproductive cycles may differ significantly. The medial nucleus of the amygdala receives olfactory information from the olfactory bulb and accessory olfactory bulb.

As it is involved in the effects of odors and pheromones on reproductive behaviour lesions in this area could cause different results in rats with a normal reproductive cycle. This is especially true if they are 'in heat' and / or a male rat is brought close to them. The results that are expected from experiment 1 would illustrate that body weight loss is more closely associated with damage to the amygdala itself. Especially in pregnant or nursing rats, as the medial amygdala plays a central role in reproduction. Also a high level of body weight and intake should be associated with the control group only, regardless of age, but more so in pregnant rats. The body weight levels of animals with amygdaloid lesions should differ significantly from the controls.

Also in experiment 2: unless the rat is six weeks or under, the body weight of the striatal damaged rats will drop significantly. General Method Subjects - Two hundred female albino rats of the Sprague-Dawley strain will be used. Fifty of these will be approximately 6 weeks of age (infant, not reached sexual maturity) and 150 will be approximately 4 months (adult). The animals will be housed individually with Purina Lab Chow and tap water freely available except as noted. Surgery and Histology These initial lesion-making procedures will be taken directly from Schoenfeld & Hamilton's (1981) procedure, p.

566. However for Experiment 1, reproduced grid plates of the damage will be composed of 1313 mm squares, each of which will correspond to an area of 1 mm represented on the atlas plates. Yet, for experiment 2, the grid will be composed of 1212 mm squares, each of which will correspond to an area of 8 mm on the atlas plates. Lesions scores from corresponding sites or squares on each side will be combined to make one bilateral score for each site in each rat, by using the two hypotheses followed in Schoenfeld & Hamilton's article. The exact same procedures for the statistical analyses (p. 567) will also be employed.

Experiment 1- Response to food deprivation and glucose gavage This experiment shall examine the role of the amygdala in the response to hunger and satiety. Food deprivation and glucose gavage shall be used to approximate these states. Subjects - one hundred rats (25 at 6 weeks, 25 at 4 months, 25 pregnant, and 25 nursing), weighing approx 370 g will be assigned to one of 5 surgical groups, or a control group. The procedure is as stated on p. 579 of Schoenfeld & Hamilton's publication.

A large number of lesions sites will be analysed because Schoenfeld & Hamilton found that "changes in behaviour was correlated with damage that encompassed several sites rather then just one." Six experimental groups will be formed: 1. Ventromedial area 2. Transition zone 3. Caudoventral area 4.

Midlateral area 5. Central nucleus 6. Operated controls Four of each category of rat will be in each group. Experiment 2 - Body weight and spontaneous intake This experiment will examine the extent to which actual damage to amygdaloid tissues may account for symptoms of reduced intake and lowered body weight following lesions in the amygdala itself to lesions invading striatal tissues dorsal to the amygdala. Subjects - one hundred rats, weighing approximately 270 g at the beginning of the experiment will be used.

The pregnant rats will undergo testing at the beginning of their pregnancy so they will not weigh significantly more than subjects in other groups. Twenty-five will be roughly 6 weeks old (not sexually mature), 25 will be 4 months old and in usual reproductive cycle, 25 will be pregnant, and 25 will be nursing at the time of testing. Subjects will be randomly assigned to 4 surgical groups or a control group (anesthetized only). Lesions in surgical groups: 1. Lateral amygdala 2. Medial amygdala 3.

Amygdala-striatal transition zone 4. Striatum There will consequently be 5 rats (of different sort) randomly assigned to one of the surgical groups or the control group. The procedure for this experiment will also be carried out as dictated in Schoenfeld & Hamilton's article (p. 568). References: Box, B. M.

, & Mogenson, G. J. Alterations in infestive behaviours after bilateral lesions of the amygdala in the rat. Physiology and Behaviour, 1975, 15, 679-688 Dacey, D. M. , & Grossman, S.

P. Aphasia, adi psia, and sensory-motor deficits produced by amygdala lesions: a function of extra-amygdaloid damage. Physiology and Behaviour, 1977, 19, 389-395. Fon berg, E. The relation between alimentary and emotional amygdala regulation.

In D. Nov in, W. Wyrwicka, & G. Bray (Eds. ) Hunger: Basic mechanisms and clinical implications. New York: Raven Press, 1976.

Klver, H. , & Bucy, P. C. Preliminary analysis of functions of the temporal lobes in monkeys. Archives of Neurology and Psychiatry, Chicago, 1939, 42, 979-1000 Nachman, M.

, & Ashe, J. H. Effects of basolateral amygdala lesions on neo phobia, learned taste aversion, and sodium appetite in rats. Journal of Comparative and Physiological Psychology, 1974, 87, 622-643. Pfaff, D.

W. , & Keiner, M. Estradiol-concentrating cells in the rat amygdala as part of a limbic-hypothalamic hormone-sensitive system. In B.

E. Eleftheriou (Ed. ), The neurobiology of the amygdala. New York: Plenum Press, 1972. Schoenfeld, T. A.

, & Hamilton, L. W. Disruption of Appetite but not Hunger or Satiety following small lesions in the Amygdala of Rats. Journal of Comparative and Physiological Psychology, vol.

95, No. 4, 565-587.