This tome is meant to collect my research over the last six years and hand it in as the cumulative “Habilitationsleistung” at the Freie Universität Berlin. For this purpose I will now briefly list the selected publications and ex-plain their connection to each other as well as to my current research. The neurobiology of spontaneous behavior and the operant learning it allows have been my research topic from the very beginning. With neurobiology ideally being studied at the genetic, physiological and behavioral level, two comple-menting model systems were chosen which both exhibited spontaneous be-havior and operant learning, and where one was more accessible genetically and the other was more accessible physiologically.
Because of the superior physiological access to the individual neurons which generate behavior in the marine snail Aplysia, we extended previous work in this model system to also incorporate in vivo operant learning (Brembs et al., 2002) and an in vitro preparation in which both operant and classical processes can be studied simultaneously (Brembs et al., 2004).
These experiments on Aplysia feeding behavior revealed how an identified neuron (B51) which is involved in determining what behavior is generated is modified by dopamine-mediated contingent reward such that future behavior will be biased towards the rewarded behavior. In a single-cell analogue of op-erant learning, we demonstrated how activity-dependent plasticity changed input resistance and burst threshold in B51 only in neurons which had received iontophoretic pulses of dopamine contingent with bursting activity and not in unpaired neurons (Brembs et al., 2002). Because B51 is active only late dur-ing the behavior, it cannot be critically involved in the generation of the be-havior, only in determining what behavior is to be produced. Therefore, part of my research effort is currently focused on the optophysiology of spontaneously active isolated Aplysia buccal ganglia to investigate the circuitry involved.
Because of the superior genetic accessibility of the fruit fly Drosophila, we used transgenic and wildtype flies to study the neurobiology of spontaneous behavior and operant learning in both freely behaving and tethered Droso-phila. Stricken by the spontaneous outbursts of aggression and the subse-quent development of strict territoriality in freely behaving flies, we initiated the research on the neurobiological determinants of aggressive behavior (Baier et al., 2002). Interestingly, two of these determinants were the bio-genic amines octopamine and dopamine, which later turned out to be involved in processing appetitive and aversive stimuli, respectively, during learning.
Receptors for both amines are preferentially expressed in the mushroom-bodies and blocking output from this neuropil reduces the level of aggression.
Another important factor was β-alanine, the concentration of which is regu-lated by the actions of the black and ebony genes, respectively. Further char-acterizing the black gene locus, we found that black
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mutant flies lack a pyri-doxal-5-phosphate, PLPdependent decarboxylase, Dgad2. This mutant, be-sides showing reduced levels of aggression, also behaves abnormally in Buri-dan’s paradigm, which cannot be explained by a lack of first order visual func-tion as no electroretinogram or target recognifunc-tion defects were detected (Phillips et al., 2005). The Dgad2 gene is an excellent example for the plei-otropy of genes involved in behavior which warrants more sophisticated
inter-Björn Brembs operant learning 4
ventions than constitutive gene knock-outs. Further demonstrating this fact is a study which involved stationary flying Drosophila (Brembs et al., 2007).
Combining lines of evidence from several topics, this study investigated the influence of octopamine and its precursor, tyramine, on flight performance.
With octopamine being critically involved in flight performance in several in-sect species as well as in initiating aggressive behaviors and in mediating ap-petitive stimuli during learning, it became necessary to find out if mutants lacking octopamine (Tyramine β -Hydroxylase mutants) are suited for learning experiments in tethered flight at the flight simulator. Our transgenic and pharmacological treatments revealed a complex, degenerate orchestration of flight performance in which lack of either octopamine or tyramine could be compensated for and only an ablation of all tyraminergic/octopaminergic neu-rons completely abolished sustained flight. These results are best explained with a wide range of subpopulations of tyraminergic and octopaminergic neu-rons which each contribute to any of the observed phenotypes in aggression, motor control and learning.
Wildtype flies, tethered in stationary flight as in the previous experi-ments, can fly continuously for several hours. Attached to a torque meter, they reveal a striking variability in their turning behavior. Analyzing the tem-poral structure of the yaw torque of wildtype flies in various situations with and without re-afferent feedback revealed that the variability in the behavior of the flies is best explained by a non-linear mechanism (Maye et al., 2007).
This result rules out simple stochastic processes and instead suggests that even seemingly random variability in the fly’s behavior is generated spontane-ously and endogenspontane-ously by the fly’s brain. These data dovetail nicely with a number of neurobiological, evolutionary and ecological findings which indicate that spontaneous behavioral variability is an evolved trait with a neurobiologi-cal basis (Brembs, 2008, subm.). Because spontaneous behavior is also a prerequisite for operant learning, we studied various forms of operant learning with tethered Drosophila at the torque meter.
To study learning in tethered Drosophila, a rigorous breeding regime is required, as well as sophisticated mechanical setup which allows the exquisite control of the fly’s environment. These experimental procedures have recently been described for the first time in a peer-reviewed video publication (Brembs, 2008). This setup allowed us to observe a peculiar effect in higher-order learning which had already been observed in simple pattern learning be-fore: operant control of external stimuli facilitates learning about these stimuli (Brembs and Wiener, 2006). In this case, operant control of the colors which determined which one of two visual patterns was being punished, al-lowed the animals to solve this occasion setting situation, whereas classical presentation of the colors did not lead to significant learning. The mushroom-bodies were not required for the operant facilitation of occasion setting and just as wildtype flies, flies with blocked mushroom-body output also failed the classical version. Occasion setting leads to a form of context-dependent mem-ory: in one occasion (e.g. green coloration), one of two patterns is punished, in the other occasion (e.g., blue coloration), the other pattern is punished.
Flies which have learned this relationship have developed a pattern-memory which is dependent on the color context. Further exploiting this new occasion setting paradigm as well as a previously developed paradigm to study context-independent memory (i.e., context generalization), we found that
generaliza-Björn Brembs operant learning 5
tion and discrimination rely on two different parameters of the colors used (Brembs and Hempel de Ibarra, 2006). Specifically, generalization occurs only if the chromaticity is sufficiently similar, whereas discrimination learning relies on brightness differences.
Generalization and discrimination are also at the heart of the set of ex-periments which aimed at understanding the genetic basis for operant learning and how operant learning interacts with other forms of learning, such as clas-sical learning. Our genetic study showed a double dissociation of the molecular processes involved in operant and classical learning (Brembs and Plendl, 2008, subm.). Specifically, the rutabaga (rut-)adenylyl cyclase was re-quired for classical learning, but not for operant learning, whereas protein kinase C (PKC) was required for operant but not for classical learning. Impor-tantly, this double dissociation could only be observed if the operant learning paradigm did not include any predictive stimuli at all (‘pure’ operant learning).
As soon as a predictive stimulus was present, learning about this stimulus dominated the experiment. This result corroborated and extended a previous experiment from my diploma and PhD thesis where wildtype animals general-ized such an operantly controlled stimulus across behavioral contexts. In other words, predictive stimuli contained in operant learning situations become equivalent to classical stimuli not only because they are acquired independ-ently of the behavior with which they were controlled during training, but also because of the genes required for the learning task. Because the mushroom-bodies are involved in some forms of generalization, I trained flies with blocked mushroom-body output in a situation with both operant and classical predictors and then tested them for any operant component and generaliza-tion of any classical component (Brembs, 2008, in prep.). The results indi-cate that the dominance of the classical stimuli in such composite learning situations is mediated by the mushroom-bodies inhibiting operant learning.
Corroborating the results from higher-order learning, the mushroom-bodies seem not to be involved in the facilitation of classical learning in these experi-ments either. Thus, these data are consistent with the hypothesis that there are reciprocal interactions between a rut-dependent classical system and a PKC-dependent operant system. The classical system dominates in learning situations where predictive stimuli are present and inhibits operant learning via the mushroom-bodies. A component of the operant system (operant be-havior) facilitates the classical system via unknown, non-mushroom-body pathways. The proposed function of this reciprocal arrangement is to prevent the operant system from interfering with generalization of classical memory.
In this view, the interfering action of the operant system consists of storing behavioral memories as habits.
