The beauty of the network in the brain and the origin of the mind in the control of behavior.

Behavior is not adequately described as a stimulus-response process. It is initiated by the animal and is generated because of its expected outcome in the future. The outcome can be good or bad for the animal. The brain is in charge of the selection process. This is the basic function of the brain. Taking Drosophila as a study case, this paper discusses initiating activity, several examples of outcome expectations, trying out (the internal search for a suitable behavior), chaining of actions, and the functional roles of chance in action selection. It takes mental processes and states such as goals, intentions, feelings, memories, cognition, and attention as higher levels of behavioral control that have their origin in biological evolution.

'Humans and other animals'-on the scope of brain science.

Abstract: This essay is dedicated to Obaid on the occasion of his 80th birthday. We both worked on the behavior of Drosophila and on what underlies behavior in the fly brain. Is that the fly's mind? The essay is about some limitations of brain science. It is just a little piece of writing. It is meant to honor Obaid for his contributions to Drosophila neurogenetics in 40 years and to science in India. I hope he takes it instead of a bowl of flowers-adding to the praise.

Are the structural changes in adult Drosophila mushroom bodies memory traces? Studies on biochemical learning mutants.

The pre-imaginal development of Drosophila mushroom bodies is under the influence of an unknown variable which causes populations of wild-type flies at eclosion to differ in the average number of Kenyon cell fibers. During the first week of adult life the number adjusts to an intermediate level which depends upon the experience of the flies. Under olfactory deprivation or social isolation it reaches a lower level than under favorable rearing conditions (J. Neurogenet., 1 (1984) 113-126). The biochemical learning mutants dance and rutabaga show no experience-dependent modulation of fiber number (Fig. 2). In both strains the mushroom bodies of young adults seem to develop abnormally; in dance a loss of about 600 fibers is observed, in rutabaga fiber number is low at eclosion and does not increase (Fig. 1a). The following model for long-term memory is proposed: in mushroom bodies outgrowth and decay of Kenyon cell fibers occur simultaneously. The fibers randomly form transient synapses onto extrinsic output neurons of the mushroom bodies and receive synapses from modulating neurons. Experience consolidates certain synapses, thus prolonging survival of the respective Kenyon cell fibers and increasing the steady state level of fiber number (Fig. 3).

The central complex of Drosophila melanogaster is involved in flight control: studies on mutants and mosaics of the gene ellipsoid body open.

Visual flight control is studied in three mutant alleles of the gene ellipsoid body open (ebo) of Drosophila melanogaster. In mutant ebo flies the central complex is disturbed to varying degrees. Defects range from a small opening in the ellipsoid body to the dissociation of the ring into two parts, a cleft in the fan-shaped body and hypoplasia in the protocerebral bridge. Other parts of the brain are not visibly affected. Flight behavior is normal with respect to the amplitude of the optomotor response and to the object response (single rotating stripe). A reduced amplitude in the small random oscillations of the torque trace (yaw torque activity), however, is found in all three alleles. In two of them the frequency of torque spikes is reduced. In the allele ebo(678) the dynamics of the optomotor response is altered. Upon reversal of the direction of rotation mutant flies take longer than wild type to shift their yaw torque to the new response level (optomotor reversal time). Finally, these flies also behave abnormally in the flight simulator in which their yaw torque controls the angular velocity of the panorama. Many ebo(678) flies fixate a single stripe less persistently than normal flies, some even trying to fly away from it (antifixation). In ebo(678) gynandromorphs the four behavioral phenotypes ("yaw torque activity", "torque spike frequency", "on-target-fixation" and "optomotor reversal time") are all highly correlated with the phenotype of the ellipsoid body. Yaw torque activity and torque spike frequency in addition are correlated with the phenotype of the thorax suggesting that these behavioral defects are in part caused by mutant influences on the ventral ganglion. The results support the hypothesis that the central complex is involved in the control of flight behavior.

[Architecture of the X chromosome, expression of LIM kinase 1, and recombination in the agnostic mutants of Drosophila: a model of human Williams syndrome]. / Osobennosti arkhitektury X-khromosomy, ékspressii LIM kinazy 1 i rekombinatsii u mutantov drozofily lokusa agnostic: model' sindroma uil'iamsa cheloveka.

As the Human Genome and Drosophila Genome Projects were completed, it became clear that functions of human disease-associated genes may be elucidated by studying the phenotypic expression of mutations affecting their structural or functional homologs in Drosophila. Genomic diseases were identified as a new class of human disorders. Their cause is recombination, which takes place at gene-flanking duplicons to generate chromosome aberrations such as deletions, duplications, inversions, and translocations. The resulting imbalance of the dosage of developmentally important genes arises at a frequency of 10(-3) (higher than the mutation rate of individual genes) and leads to syndromes with multiple manifestations, including cognitive defects. Genomic DNA fragments were cloned from the Drosophila melanogaster agnostic locus, whose mutations impair learning ability and memory. As a result, the locus was exactly localized in X-chromosome region 11A containing the LIM kinase 1 (LIMK1) gene (CG1848), which is conserved among many species. Hemizygosity for the LIMK1 gene, which is caused by recombination at neighboring extended repeats, underlies cognitive disorders in human Williams syndrome. LIMK1 is a component of the integrin signaling cascade, which regulates the functions of the actin cytoskeleton, synaptogenesis, and morphogenesis in the developing brain. Immunofluorescence analysis revealed LIMK1 in all subdomains of the central complex and the visual system of Drosophila melanogaster. Like in the human genome, the D. melanogaster region is flanked by numerous repeats, which were detected by molecular genetic methods and analysis of ectopic chromosome pairing. The repeats determined a higher rate of spontaneous and induced recombination. including unequal crossing over, in the agnostic gene region. Hence, the agnostic locus was considered as the first D. melanogaster model suitable for studying the genetic defect associated with Williams syndrome in human.

Visual learning in individually assayed Drosophila larvae.

An understanding of associative learning is facilitated if it can be analyzed in a simple animal like the fruit fly Drosophila. Here, we introduce the first visual associative learning paradigm for larval Drosophila; this is remarkable as larvae have an order of magnitude fewer neurons than adult flies. Larvae were subjected to either of two reciprocal training regimes: Light+/Dark- or Light-/Dark+. Subsequently, all larvae were individually tested for their preference between Light versus Dark. The difference between training regimes was therefore exclusively which visual situation was associated with which reinforcer; differences observed during the test thus reflected exclusively associative learning. For positive reinforcement (+) we used fructose (FRU), and for negative reinforcement (-) either quinine or sodium chloride (QUI, NaCl). Under these conditions, associative learning could be reproducibly observed in both wild-type strains tested. We then compared the effectiveness of training using differential conditioning, with both positive and negative reinforcement, to that using only positive or only negative reinforcement. We found that FRU only, but neither QUI nor NaCl, was in itself effective as a reinforcer. This is the first demonstration of appetitive learning in larval Drosophila. It is now possible to investigate the behavioral and neuronal organization of appetitive visual learning in this simple and genetically easy-to-manipulate experimental system.

The role of central parts of the brain in the control of sound production during courtship in Drosophila melanogaster.

The question of the roles of the two main parts of the insect brain, the mushroom bodies and the central complex, in controlling motor coordination and triggering a variety of behavioral programs, including sound production, remains controversial. With the aim of improving our understanding of this question, we studied the parameters of songs used by five-day-old males during courtship for fertilized wild-type females (Canton-S, C-S) over 5-min periods at 25 degrees C; males were of two wild-type Drosophila melanogaster lines (Berlin and C-S). Berlin males lacking mushroom bodies because of treatment with hydroxyurea during development (chemical removal of the mushroom bodies) were used, along with two mutants with defects in the mushroom bodies (mbm1 and mud1), two mutants with defects in the central complex (ccbKS127 and cexKS181), and mutant cxbN71 with defects in both the mushroom bodies and the central complex. The experiments reported here showed that courtship songs in males lacking mushroom bodies were virtually identical to those of wild-type males. The main parameters of pulsatile song in mutants mbm1 and mud1 (interpulse interval and train duration) were insignificantly different from those of the songs of wild-type flies, though the stability of the pulse oscillator was the same. Flies of these lines were no different from wild-type flies in terms of courtship success (percentage of copulating pairs in 10-min tests). Conversely, the songs of mutants with defects in the central complex differed from those of wild-type males. Firstly, there was degradation of the stability of the pulse oscillator and interpulse intervals were very variable. In addition, pulses were often significantly longer and appeared multicyclic, as in the well-known cacophony mutant, while the mean train duration was significantly shorter. Males of the line cexKS181 usually courted very intensely, though abnormal sounds were generally emitted. Mutants cexKS181 and ccbKS127 were significantly less successful in courtship than wild-type flies. These data show that the central complex appears to play a very important role in controlling song, while the mushroom bodies are not related to this function.

Conditioning with compound stimuli in Drosophila melanogaster in the flight simulator.

Short-term memory in Drosophila melanogaster operant visual learning in the flight simulator is explored using patterns and colours as a compound stimulus. Presented together during training, the two stimuli accrue the same associative strength whether or not a prior training phase rendered one of the two stimuli a stronger predictor for the reinforcer than the other (no blocking). This result adds Drosophila to the list of other invertebrates that do not exhibit the robust vertebrate blocking phenomenon. Other forms of higher-order learning, however, were detected: a solid sensory preconditioning and a small second-order conditioning effect imply that associations between the two stimuli can be formed, even if the compound is not reinforced.

[Brain central regions in courtship sound production in Drosophila melanogaster]. / Rol' tsentral'nykh otdelov mozga v kontrole zvukoizlucheniia pri ukhazhivanii in Drosophila melanogaster.

There are debates about the function of the two main central brain structures of insects--mushroom bodies and the central complex--in the control of motor co-ordination and triggering of different behaviour programs including sound production. To throw additional light onto this problem we analysed the parameters of the love song produced by 5-day old males courting for 5 minutes a fertilised CS female at 25 degrees C, in two wild-type strains of Drosophila melanogaster (Berlin and CS), hydroxyurea (HU)-treated flies (chemical ablation of the mushroom bodies) two mushroom body mutants (mbm1 and mud1), two central complex mutants (ccbKS127 and cexKS181) and a mutant cxbN71 with defects both in the mushroom bodies and in the central complex. It was found that the love song of HU-treated flies devoid of the mushroom bodies is very similar to that of wild-type flies. In mbm1 and mud1 the main parameters of the song (interpulse interval, IPI, and train duration) are slightly shifted from those of wild type but the sharpness of tuning of the pulse oscillator is the same. The flies of all these strains are equal to wild-type strains in mating success (% of copulations with virgins in 10-min test). On the contrary, the songs of the central complex mutants differ from those of wild-type flies. First of all, the sharpness of tuning of the pulse oscillator is destroyed,--the IPIs become highly variable. The pulses often are much longer and polycyclic as in well known cacophony mutant. The mean duration of pulse trains is much shorter. The males of the mutant cexKS181 usually court violently, but in most cases abnormal sounds are produced. Both cexKS181 and ccbKS127 males are much less successful in matings in comparison to wild-type flies. One can conclude that the central complex plays probably a very important role in the control of singing, whereas the mushroom bodies are practically not involved in this function.

Flexibility in a single behavioral variable of Drosophila.

The flexibility of behavior is so rich, and its components are so exquisitely interwoven, that one may be well advised to turn to an isolated behavioral module for study. Gill withdrawal in Aplysia, the proboscis extension reflex in the honeybee, and lid closure in mammals are such examples. We have chosen yawing, a single component of flight orientation in Drosophila melanogaster, for this approach. A specialty of this preparation is that the behavioral output can be reduced beyond the single module by one further step. It can be studied in tethered animals in which all turns are blocked while the differentially beating wings still provide the momentum. These intended yaw turns are measured by a torque meter to which the fly is hooked. The fly is held horizontally as if cruising at high speed. The head is glued to the thorax. It can bend its abdomen, extend its proboscis, and move its legs but cannot shift its direction of gaze or its orientation in space. Evidently, a fly hardly ever encounters this bizarre situation in the wild. We describe here the flexibility in this single behavioral variable. It provides insights into the relation between classical and operant conditioning, the processing of and interactions between the conditioned visual stimuli, early visual memory, visual pattern recognition, selective attention, and several other experience-dependent properties of visual orientation behavior. We start with a brief summary of visual flight control at the torque meter.

Age-dependent memory loss, synaptic pathology and altered brain plasticity in the Drosophila mutant cardinal accumulating 3-hydroxykynurenine.

A search for Drosophila mutants with phenotypes similar to human diseases might help to unravel evolutionary conserved genes implicated in polygenic human disorders. Among these are neurodegenerative diseases, characterized by a late onset disturbance of memory, synaptic and glial pathology, structural brain impairments and altered content of the intermediates of the kynurenine pathway, the modulators of glutamate excito- and oxidative toxicity. This pathway is conserved in insects, in rodents, and in humans. We tested the Drosophila mutants cardinal (3-hydroxykynurenine excess) and cinnabar (kynurenic acid excess) for age-dependent changes in memory, synaptic pathology, structural brain plasticity and glial immunoreactivity. The mutant cardinal demonstrated a decline in learning and memory from the 12th to the 29th day of life in a paradigm of conditioned courtship suppression. Memory decline was accompanied by a sharp decrease in immunoreactivity to the synaptic cysteine string protein, and alterations in volumetric parameters of the mushroom bodies, the brain structures implicated in memory.

Mushroom body defect, a gene involved in the control of neuroblast proliferation in Drosophila, encodes a coiled-coil protein.

Neurogenesis relies on the establishment of the proper number and precisely controlled proliferation of neuroblasts, the neuronal precursor cells. A role for the mushroom body defect (mud) gene in both of these aspects of neuroblast behavior, as well as possible roles in other aspects of fruit fly biology, is implied by phenotypes associated with mud mutations. We have localized mud by determining the sequence change in one point mutant, identifying a predicted ORF affected by the mutation, and showing that an appropriate segment of the genome rescues mud mutant phenotypes. An analysis of mud cDNAs and a survey of mud transcripts by Northern blotting indicate that the gene is subject to differential splicing and is expressed primarily during embryogenesis but also, at lower levels, during subsequent developmental stages in a sexually dimorphic manner. The gene is predicted to encode a polypeptide without obvious homologs but with two prominent structural features, a long coiled coil that constitutes the central core of the protein and a carboxyl-terminal transmembrane domain.

Localization of a short-term memory in Drosophila.

Memories are thought to be due to lasting synaptic modifications in the brain. The search for memory traces has relied predominantly on determining regions that are necessary for the process. However, a more informative approach is to define the smallest sufficient set of brain structures. The rutabaga adenylyl cyclase, an enzyme that is ubiquitously expressed in the Drosophila brain and that mediates synaptic plasticity, is needed exclusively in the Kenyon cells of the mushroom bodies for a component of olfactory short-term memory. This demonstrates that synaptic plasticity in a small brain region can be sufficient for memory formation.

Tissue-specific expression of a type I adenylyl cyclase rescues the rutabaga mutant memory defect: in search of the engram.

Most attempts to localize physical correlates of memory in the central nervous system (CNS) rely on ablation techniques. This approach has the limitation of defining just one of an unknown number of structures necessary for memory formation. We have used the Drosophila rutabaga type I Ca(2+)/CaM-dependent adenylyl cyclase (AC) gene to determine in which CNS region AC expression is sufficient for memory formation. Using pan-neural and restricted CNS expression with the GAL4 binary transcription activation system, we have rescued the memory defect of the rutabaga mutant in a fast robust spatial learning paradigm. The ventral ganglion, antennal lobes, and median bundle are likely the CNS structures sufficient for rutabaga AC- dependent spatial learning.

The operant and the classical in conditioned orientation of Drosophila melanogaster at the flight simulator.

Ever since learning and memory have been studied experimentally, the relationship between operant and classical conditioning has been controversial. Operant conditioning is any form of conditioning that essentially depends on the animal's behavior. It relies on operant behavior. A motor output is called operant if it controls a sensory variable. The Drosophila flight simulator, in which the relevant behavior is a single motor variable (yaw torque), fully separates the operant and classical components of a complex conditioning task. In this paradigm a tethered fly learns, operantly or classically, to prefer and avoid certain flight orientations in relation to the surrounding panorama. Yaw torque is recorded and, in the operant mode, controls the panorama. Using a yoked control, we show that classical pattern learning necessitates more extensive training than operant pattern learning. We compare in detail the microstructure of yaw torque after classical and operant training but find no evidence for acquired behavioral traits after operant conditioning that might explain this difference. We therefore conclude that the operant behavior has a facilitating effect on the classical training. In addition, we show that an operantly learned stimulus is successfully transferred from the behavior of the training to a different behavior. This result unequivocally demonstrates that during operant conditioning classical associations can be formed.

Central complex substructures are required for the maintenance of locomotor activity in Drosophila melanogaster.

In Drosophila melanogaster, former studies based on structural brain mutants have suggested that the central complex is a higher control center of locomotor behavior. Continuing this investigation we studied the effect of the central complex on the temporal structure of spontaneous locomotor activity in the time domain of a few hours. In an attempt to dissect the internal circuitry of the central complex we perturbed a putative local neuronal network connecting the four neuropil regions of the central complex, the protocerebral bridge, the fan-shape body, the noduli and the ellipsoid body. Two independent and non-invasive methods were applied: mutations affecting the neuroarchitecture of the protocerebral bridge, and the targeted expression of tetanus toxin in small subsets of central complex neurons using the binary enhancer trap P[GAL4] system. All groups of flies with a disturbed component of this network exhibited a common phenotype: a drastic decrease in locomotor activity. While locomotor activity was still clustered in bouts and these were initiated at the normal rate, their duration was reduced. This finding suggests that the bridge and some of its neural connections to the other neuropil regions of the central complex are required for the maintenance but not the initiation of walking.

Context generalization in Drosophila visual learning requires the mushroom bodies.

The world is permanently changing. Laboratory experiments on learning and memory normally minimize this feature of reality, keeping all conditions except the conditioned and unconditioned stimuli as constant as possible. In the real world, however, animals need to extract from the universe of sensory signals the actual predictors of salient events by separating them from non-predictive stimuli (context). In principle, this can be achieved if only those sensory inputs that resemble the reinforcer in their temporal structure are taken as predictors. Here we study visual learning in the fly Drosophila melanogaster, using a flight simulator, and show that memory retrieval is, indeed, partially context-independent. Moreover, we show that the mushroom bodies, which are required for olfactory but not visual or tactile learning, effectively support context generalization. In visual learning in Drosophila, it appears that a facilitating effect of context cues for memory retrieval is the default state, whereas making recall context-independent requires additional processing.

Age-dependent changes in memory and mushroom bodies in the Drosophila mutant vermilion deficient in the kynurenine pathway of tryptophan metabolism.

Evolutionary conservation of homologous genes that cause related phenotypes in humans and Drosophila help to unravel genes implicated in polygenic human diseases. Among them are neurodegenerative disorders, such as Huntington, Parkinson, Alzheimer and HIV-induced diseases. They are characterized by a late onset disturbances of memory, changes in volumetric indices of the brain structures involved in memory formation, synaptic and glial pathology, and altered content of the intermediates of the kynurenine pathway, the endogenous modulators of the NMDA receptors. This pathway in conserved in insects, rodents and humans. We, therefore, studied the effects of aberrant tryptophan metabolism on memory, brain plasticity, synaptic and glial immunoreactivity in the Drosophila mutants vermilion (no kynurenines) and cinnabar (excess of neuroprotective kynurenic acid) over the life time. The mutant vermilion demonstrated gradual decline of 3-th memory performance and complete memory failure on the 28th day of life in a paradigm of conditioned courtship suppression. A drastic increase in the volume of the calyces of the mushroom bodies, and a decay in immunochemical staining of this brain structure with antibodies to synaptic protein csp and glia, precede the age-dependent memory defect and develop from the 12th day of adult life.