Behavioral analyses of sugar processing in choice, feeding, and learning in larval Drosophila

Gustatory stimuli have at least 2 kinds of function: They can support immediate, reflexive responses (such as substrate choice and feeding) and they can drive internal reinforcement. We provide behavioral analyses of these functions with respect to sweet taste in larval Drosophila. The idea is to use the dose-effect characteristics as behavioral "fingerprints" to dissociate reflexive and reinforcing functions. For glucose and trehalose, we uncover relatively weak preference. In contrast, for fructose and sucrose, preference responses are strong and the effects on feeding pronounced. Specifically, larvae are attracted to, and feeding is stimulated most strongly for, intermediate concentrations of either sugar: Using very high concentrations (4 M) results in weakened preference and suppression of feeding. In contrast to such an optimum function regarding choice and feeding, an asymptotic dose-effect function is found for reinforcement learning: Learning scores reach asymptote at 2 M and remain stable for a 4-M concentration. A similar parametric discrepancy between the reflexive (choice and feeding) and reinforcing function is also seen for sodium chloride (Niewalda T, Singhal S, Fiala A, Saumweber T, Wegener S, Gerber B, in preparation). We discuss whether these discrepancies are based either on inhibition from high-osmolarity sensors upon specifically the reflexive pathways or whether different sensory pathways, with different effective dose-response characteristics, may have preferential access to drive either reflex responses or modulatory neurons mediating internal reinforcement, respectively.     

Behavioural analyses of quinine processing in choice, feeding and learning of larval Drosophila

Gustatory stimuli can support both immediate reflexive behaviour, such as choice and feeding, and can drive internal reinforcement in associative learning. For larval Drosophila, we here provide a first systematic behavioural analysis of these functions with respect to quinine as a study case of a substance which humans report as "tasting bitter". We describe the dose-effect functions for these different kinds of behaviour and find that a half-maximal effect of quinine to suppress feeding needs substantially higher quinine concentrations (2.0 mM) than is the case for internal reinforcement (0.6 mM). Interestingly, in previous studies (Niewalda et al. 2008, Schipanski et al 2008) we had found the reverse for sodium chloride and fructose/sucrose, such that dose-effect functions for those tastants were shifted towards lower concentrations for feeding as compared to reinforcement, arguing that the differences in dose-effect function between these behaviours do not reflect artefacts of the types of assay used. The current results regarding quinine thus provide a starting point to investigate how the gustatory system is organized on the cellular and/or molecular level to result in different behavioural tuning curves towards a bitter tastant.     

The carrot,not the stick: appetitive rather than aversive gustatory stimuli support associative olfactory learning in individually assayed <Emphasis Type="Italic">Drosophila</Emphasis> larvae

The ability to learn is universal among animals; we investigate associative learning between odors and tastants in larval Drosophila melanogaster. As biologically important gustatory stimuli, like sugars, salts, or bitter substances have many behavioral functions, we investigate not only their reinforcing function, but also their response-modulating and response-releasing function. Concerning the response-releasing function, larvae are attracted by fructose and repelled by sodium chloride and quinine; also, fructose increases, but salt and quinine suppress feeding. However, none of these stimuli has a nonassociative, modulatory effect on olfactory choice behavior. Finally, only fructose but neither salt nor quinine has a reinforcing effect in associative olfactory learning. This implies that the response-releasing, response-modulating and reinforcing functions of these tastants are dissociated on the behavioral level. These results open the door to analyze how this dissociation is brought about on the cellular and molecular level; this should be facilitated by the cellular simplicity and genetic accessibility of the Drosophila larva.     

Dietary salt levels affect salt preference and learning in larval Drosophila

Drosophila larvae change from exhibiting attraction to aversion as the concentration of salt in a substrate is increased. However, some aversive concentrations appear to act as positive reinforcers, increasing attraction to an odour with which they have been paired. We test whether this surprising dissociation between the unconditioned and conditioned response depends on the larvae's experience of salt concentration in their food. We find that although the point at which a NaCl concentration becomes aversive shifts with different rearing experience, the dissociation remains evident. Testing larvae using a substrate 0.025 M above the NaCl concentration on which the larvae were reared consistently results in aversive choice behaviour but appetitive reinforcement effects.     

Frustrated appetitive foraging behavior, stereotypic pacing, and fecal glucocorticoid levels in snow leopards (Uncia uncia) in the Zurich Zoo

This study hypothesized that permanently frustrated, appetitive-foraging behavior caused the stereotypic pacing regularly observed in captive carnivores. Using 2 adult female snow leopards (Uncia uncia), solitarily housed in the Zurich Zoo, the study tested this hypothesis experimentally with a novel feeding method: electronically controlled, time-regulated feeding boxes. The expected result of employing this active foraging device as a successful coping strategy was reduced behavioral and physiological measures of stress, compared with a control-feeding regime without feeding boxes. The study assessed this through behavioral observations and by evaluating glucocorticoid levels noninvasively from feces. Results indicated that the 2 snow leopards did not perform successful coping behavior through exercising active foraging behavior or through displaying the stereotypic pacing. The data support a possible explanation: The box-feeding method did not provide the 2 snow leopards with the external stimuli to satisfy their appetitive behavioral needs. Moreover, numerous other factors not necessarily or exclusively related to appetitive behavior could have caused and influenced the stereotypic pacing.     

Get in My Belly: Food Preferences Trigger Approach and Avoidant Postural Asymmetries

Appetitive motivational states are fundamental neural and behavioral mechanisms underlying healthy and abnormal eating behavior, though their dynamic influence on food-related behavior is unknown. The present study examined whether personal food-related preferences would activate approach and avoidance systems, modulating spontaneous postural sway toward and away from food items. Participants stood on a balance board that collected real-time data regarding postural sway along two axes (x, y) while they viewed a series of images depicting food items varying in nutritional value and individual preferences. Overall, participants showed reliable postural sway toward highly preferred and away from highly non-preferred items. This effect became more pronounced over time; sway along the mediolateral axis showed no reliable variation by preference. Results carry implications for two-factor (homeostatic versus hedonic) neurobehavioral theories of hunger and appetitive motivation, and carry applied clinical implications for the measurement and management of abnormal eating behavior.     

“Sex, drugs and the brain”: The interaction between drugs of abuse and sexual behavior in the female rat

Preclinical and clinical research investigating female sexual motivation has lagged behind research on male sexual function. The present review summarizes recent advances in our understanding of the specific roles of various brain areas, as well as our understanding of the role of dopaminergic neurotransmission in sexual motivation of the female rat. A number of behavioral paradigms that can be used to thoroughly evaluate sexual behavior in the female rat are first discussed. Although traditional assessment of the reflexive, lordosis posture has been useful in understanding the neuroanatomical and neurochemical systems that contribute to copulatory behavior, the additional behavioral paradigms described in this review have helped us expand our understanding of appetitive and consumatory behavioral patterns that better assess sexual motivation - the equivalent of “desire” in humans. A summary of numerous lesion studies indicates that different areas of the brain, including forebrain and midbrain structures, work together to produce the complex repertoire of female sexual behavior. In addition, by investigating the effects of commonly addictive drugs, we are beginning to elucidate the role of dopaminergic neurotransmission in female sexual motivation. Consequently, research in this area may contribute to meaningful advances in the treatment of human female sexual dysfunction.     

Maximizing the effectiveness of environmental enrichment: Suggestions from the experimental analysis of behavior

Environmental enrichment programs provide benefits to both captive animals and the facilities that house them, but cost time and resources to design, implement, and maintain. As yet, there have been few theoretically based guidelines to assist animal care staff in establishing cost-efficient enrichment methods that both elicit the desired behavioral changes and maintain their success over time. We describe several well-studied principles from the field of experimental analysis of behavior, including intrinsic reinforcement, extrinsic reinforcement, habituation, extinction, and schedules of reinforcement that could be very useful for evaluating the short- and long-term effectiveness of enrichment. We use this theoretical framework to generate testable hypotheses and provide examples of enrichment studies relevant to our predictions. In particular, we suggest that enrichment devices that offer extrinsic reinforcement (food, social access, etc. as a result of performing behaviors) should produce greater and more prolonged changes in behavior than devices that rely on the behavior itself being reinforcing to the animal. For techniques that provide no extrinsic reinforcement, using stimuli that are novel, are more different from the environment, have been withheld or altered in some way, or are presented less frequently may help reduce habituation. For techniques that provide extrinsic reinforcement, making reinforcement more difficult to obtain and providing more or higher quality reinforcers may increase the long-term success of the enrichment program. In addition, enrichment may be more effective if animal care staff avoid continuously reinforcing behaviors after they are established, enriching immediately after feeding, and exposing animals to enrichment when reinforcement is no longer available. While the current enrichment literature supports the application of behavior analytic theory, empirical evaluation of many of our predictions is still needed.     

Activity of neurons in the pedunculopontine nucleus in conditioned instrumental appetitive reflex

Activity of 91 neurons in the compact and diffuse parts of the pedunculopontine tegmental nucleus (PPTg) was recorded in freely moving rabbits during execution of instrumental appetitive conditioning. Of the recorded neurons, 37.4% changed their activity in response to a conditioned stimulus, which is the evidence for the PPTg involvement in instrumental conditioning. Excitatory conditioned neuronal responses to the conditioned stimulus and food reinforcement significantly prevailed over inhibitory reactions. Neuronal responses to the conditioned stimulus were classified in several basic patterns reflecting stimulus effects, structure of the behavioral act, and reinforcement properties. These reactions indicate the involvement of the PPTg in attention, motor learning, and reinforcement. The revealed differences in associative reactive properties of the compact and diffuse parts of the PPTg to the conditioned stimulus and reinforcement point to the functional heterogeneity of this structure and suggest the leading role of the cholinergic part of the PPTg in instrumental appetitive conditioning and reinforcement as well as the essential contribution of the diffuse part into classical appetitive conditioning.     

Assessment of Potential Sublethal Effects of Various Insecticides on Key Biological Traits of The Tobacco Whitefly,Bemisia tabaci

The tobacco whitefly Bemisia tabaci is one of the most devastating pests worldwide. Current management of B. tabaci relies upon the frequent applications of insecticides. In addition to direct mortality by typical acute toxicity (lethal effect), insecticides may also impair various key biological traits of the exposed insects through physiological and behavioral sublethal effects. Identifying and characterizing such effects could be crucial for understanding the global effects of insecticides on the pest and therefore for optimizing its management in the crops. We assessed the effects of sublethal and low-lethal concentrations of four widely used insecticides on the fecundity, honeydew excretion and feeding behavior of B. tabaci adults. The probing activity of the whiteflies feeding on treated cotton seedlings was recorded by an Electrical Penetration Graph (EPG). The results showed that imidacloprid and bifenthrin caused a reduction in phloem feeding even at sublethal concentrations. In addition, the honeydew excretions and fecundity levels of adults feeding on leaf discs treated with these concentrations were significantly lower than the untreated ones. While, sublethal concentrations of chlorpyrifos and carbosulfan did not affect feeding behavior, honeydew excretion and fecundity of the whitefly. We demonstrated an antifeedant effect of the imidacloprid and bifenthrin on B. tabaci, whereas behavioral changes in adults feeding on leaves treated with chlorpyrifos and carbosulfan were more likely caused by the direct effects of the insecticides on the insects'' nervous system itself. Our results show that aside from the lethal effect, the sublethal concentration of imidacloprid and bifenthrin impairs the phloem feeding, i.e. the most important feeding trait in a plant protection perspective. Indeed, this antifeedant property would give these insecticides potential to control insect pests indirectly. Therefore, the behavioral effects of sublethal concentrations of imidacloprid and bifenthrin may play an important role in the control of whitefly pests by increasing the toxicity persistence in treated crops.     

Pheromones linked to sexual behaviors excite the appetitive phase of feeding behavior of Aplysia fasciata II. Excitation of C-PR, a neuron involved in the generation of appetitive behaviors

Pheromones presumably released by conspecifics amplify both the appetitive and the consummatory components of feeding in Aplysia. These effects can be mimicked by administering homogenate of the large hermaphroditic duct containing atrial gland tissue, as well as peptides from the bag cells. Identified cerebro-pedal regulator (C-PR) neuron is thought to command various behaviors that comprise the appetitive phase of feeding. In a reduced preparation, we investigated the effects on the C-PR of applying these substances to the rhinophores, the sensory organs which detect pheromones. Stimuli that excite feeding in the animal were also found to affect the C-PR. Large hermaphroditic duct homogenate caused a doubling in the firing rate of the C-PR, and amplified the response of the C-PR to other excitatory stimuli, such as touch of food to the rhinophores. Bag cell peptides (α, β and γ bag cell peptide, and egg-laying hormone) caused smaller increases in the firing rate of the C-PR. These data are consistent with the hypothesis that pheromones facilitate appetitive feeding behavior in part via their excitation of C-PR. Accepted: 28 November 1997     

Contrasting Role of Octopamine in Appetitive and Aversive Learning in the Crab Chasmagnathus

Background

Biogenic amines are implicated in reinforcing associative learning. Octopamine (OA) is considered the invertebrate counterpart of noradrenaline and several studies in insects converge on the idea that OA mediates the reward in appetitive conditioning. However, it is possible to assume that OA could have a different role in an aversive conditioning.

Methodology/Principal Findings

Here we pharmacologically studied the participation of OA in two learning processes in the crab Chasmagnathus granulatus, one appetitive and one aversive. It is shown that the aversive memory is impaired by an OA injection applied immediately or 30 minutes after the last training trial. By contrast, the appetitive memory is blocked by OA antagonists epinastine and mianserine, but enhanced by OA when injected together with the supply of a minimum amount of reinforcement. Finally, double-learning experiments in which crabs are given the aversive and the appetitive learning either successively or simultaneously allow us to study the interaction between both types of learning and analyze the presumed action of OA. We found that the appetitive training offered immediately, but not one hour, after an aversive training has an amnesic effect on the aversive memory, mimicking the effect and the kinetic of an OA injection.

Conclusions/Significance

Our results demonstrate that the role of OA is divergent in two memory processes of opposite signs: on the one hand it would mediate the reinforcement in appetitive learning, and on the other hand it has a deleterious effect over aversive memory consolidation.     

Properties of a halophil nicotinamide–adenine dinucleotide phosphate-specific isocitrate dehydrogenase. Preliminary studies of the salt relations and kinetics of the crude enzyme

The effects of chlorides on NADP-specific isocitrate dehydrogenase from Halobacterium salinarium were investigated. The enzyme is stabilized by potassium chloride and sodium chloride and this effect is discussed in relation to the Hill (1913) equation. Kinetics of the enzyme were studied within a range of concentrations of potassium chloride and sodium chloride. Apparent Michaelis constants for both substrates were affected by salt concentration, the effect being greater in sodium chloride than in potassium chloride. Minimal apparent Michaelis constants for both substrates were similar to the corresponding constants reported for yeast isocitrate dehydrogenase. V(max.) was maximal in each salt at a concentration of about 1m. The maximum was higher in sodium chloride than in potassium chloride. At salt concentrations above about 2.3m, the apparent V(max.) was lower in sodium chloride than in potassium chloride, and at salt concentrations below 0.75-1.0m, each salt behaved as a linear activator of the enzyme. Within this concentration range salt and NADP(+) acted competitively; the activation by salt was overcome at finite concentrations of NADP(+). At concentrations above about 1m, potassium chloride was a linear non-competitive inhibitor of the enzyme. Within the range 1.0-2.5m, sodium chloride was also a linear non-competitive inhibitor, but above 2.5m it caused more pronounced inhibition.     

Rapid effects of aromatase inhibition on male reproductive behaviors in Japanese quail

Non-genomic effects of steroid hormones on cell physiology have been reported in the brain. However, relatively little is known about the behavioral significance of these actions. Male sexual behavior is activated by testosterone partly through its conversion to estradiol via the enzyme aromatase in the preoptic area (POA). Brain aromatase activity (AA) changes rapidly which might in turn be important for the rapid regulation of behavior. Here, acute effects of Vorozole, an aromatase inhibitor, injected IP at different doses and times before testing (between 15 and 60 min), were assessed on male sexual behavior in quail. To limit the risk of committing both types of statistical errors (I and II), data of all experiments were entered into a meta-analysis. Vorozole significantly inhibited mount attempts (P < 0.05, size effect [g] = 0.527) and increased the latency to first copulation (P < 0.05, g = 0.251). The treatment had no effect on the other measures of copulatory behavior. Vorozole also inhibited appetitive sexual behavior measured by the social proximity response (P < 0.05, g = 0.534) or rhythmic cloacal sphincter movements (P < 0.001, g = 0.408). Behavioral inhibitions always reached a maximum at 30 min. Another aromatase inhibitor, androstatrienedione, induced a similar rapid inhibition of sphincter movements. Radioenzyme assays demonstrated that within 30 min Vorozole had reached the POA and completely blocked AA measured in homogenates. When added to the extracellular milieu, Vorozole also blocked within 5 min the AA in POA explants maintained in vitro. Together, these data demonstrate that aromatase inhibition rapidly decreases both consummatory and appetitive aspects of male sexual behavior.     

Similar effect of caerulein on intracranial self-stimulation in vagotomized and non-vagotomized rats

Electrical stimulation eliciting self-stimulation behavior from postero-lateral hypothalamic implanted electrode was controlled by factors that control normal feeding. In this idea, lateral hypothalamic stimulation possessed an appetite whetting property and this is experienced as rewarding. The octapeptide cholecystokinin, a gut hormone, has been experimented upon to produce the complete behavioral sequence of satiety in rats. We observed that an i.p. injection of caerulein (an analog of cholecystokinin) did decrease, in a dose-related manner, the rate for brain self-stimulation. However, a similar effect on the rate of ICSS was measured after a bilateral cut of the vagus nerve at a subdiaphragmatic level. This result suggests that the decreasing effect on ICSS after an i.p. injection of caerulein is not strictly related to feeding. We interpret the decrease of reinforcement induced by caerulein as the action of a general satiety for any object presenting a rewarding value for behavior.     

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces^1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates^5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)^7-10.Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar^11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement ⁹. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups⁹.     

Cooling as Reinforcing Stimulus in Aplysia

An experiment was carried out to investigate the role of temperaturein the previously reported reinforcing effect of an increasein sea water level in Aplysia. In the present experiment, itwas found that the reinforcing effect of water level changeon rod-pressing behavior in Aplysia depends on a decrease intemperature associated with water level change. In order studymodification of rod pressing behavior produced by contingentincrease in water level and decrease in temperature, the rateand latency of rod-press responses in experimental animals wherecompared with those of yoked control animals exposed to non-contingentwater level and temperature change. Higher response rates andshorter response latencies were obtained from experimental overyoked control animals but only the shorter latencies of experimentalanimals were attributed to a behavioral change resulting fromcontingent water level and temperature reinforcement.     

Intravascular food reward

Consumption of calorie-containing sugars elicits appetitive behavioral responses and dopamine release in the ventral striatum, even in the absence of sweet-taste transduction machinery. However, it is unclear if such reward-related postingestive effects reflect preabsorptive or postabsorptive events. In support of the importance of postabsorptive glucose detection, we found that, in rat behavioral tests, high concentration glucose solutions administered in the jugular vein were sufficient to condition a side-bias. Additionally, a lower concentration glucose solution conditioned robust behavioral responses when administered in the hepatic-portal, but not the jugular vein. Furthermore, enteric administration of glucose at a concentration that is sufficient to elicit behavioral conditioning resulted in a glycemic profile similar to that observed after administration of the low concentration glucose solution in the hepatic-portal, but not jugular vein. Finally using fast-scan cyclic voltammetry we found that, in accordance with behavioral findings, a low concentration glucose solution caused an increase in spontaneous dopamine release events in the nucleus accumbens shell when administered in the hepatic-portal, but not the jugular vein. These findings demonstrate that the postabsorptive effects of glucose are sufficient for the postingestive behavioral and dopaminergic reward-related responses that result from sugar consumption. Furthermore, glycemia levels in the hepatic-portal venous system contribute more significantly for this effect than systemic glycemia, arguing for the participation of an intra-abdominal visceral sensor for glucose.     

Current research in amphibians: studies integrating endocrinology, behavior, and neurobiology

Amphibian behavioral endocrinology has focused on reproductive social behavior and communication in frogs and newts. Androgens and estrogens are critical for the expression of male and female behavior, respectively, and their effects are relatively clear. Corticosteroids have significant modulatory effects on the behavior of both sexes, as does the peptide neuromodulator arginine vasotocin in males, but their effects and interactions with gonadal steroids are often complex and difficult to understand. Recent work has shown that the gonadal hormones and social behavior are mutually reinforcing: engaging in social interactions increases hormone levels just as increasing hormone levels change behavior. The reciprocal interactions of hormones and behavior, as well as the complex interactions among gonadal steroids, adrenal steroids, and peptide hormones have implications for the maintenance and evolution of natural social behavior, and suggest that a deeper understanding of both endocrine mechanisms and social behavior would arise from field studies or other approaches that combine behavioral endocrinology with behavioral ecology.     

The evolution of magnanimity

Conspicuous consumption associated with status reinforcement behavior can be explained in terms of costly signaling, or strategic handicap theory, first articulated by Zahavi and later formalized by Grafen. A theory is introduced which suggests that the evolutionary raison d’être of status reinforcement behavior lies not only in its effects on lifetime reproductive success, but in its positive effects on the probability of survival through infrequent, unpredictable demographic bottlenecks. Under some circumstances, such “wasteful” displays may take the form of displays of altruistic behavior and generosity on the part of high status individuals, in that is signals the ability to bear the short-term costs of being generous or “cooperative,” while at the same time reinforcing the long-term benefits of higher status. James L. Boone is an associate professor in the Department of Anthropology at the University of New Mexico, where he carries out research in behavioral ecology and the archaeology of complex societies. His current interests are in the evolution of social status reinforcement behavior and variation in patterns of conspicuous consumption in human history.