A genetic screen for mutations that disrupt an auditory response in Drosophila melanogaster

Hearing is one of the last sensory modalities to be subjected to genetic analysis in Drosophila melanogaster. We describe a behavioral assay for auditory function involving courtship among groups of males triggered by the pulse component of the courtship song. In a mutagenesis screen for mutations that disrupt the auditory response, we have recovered 15 mutations that either reduce or abolish this response. Mutant audiograms indicate that seven mutants reduced the amplitude of the response at all intensities. Another seven abolished the response altogether. The other mutant, 5L3, responded only at high sound intensities, indicating that the threshold was shifted in this mutant. Six mutants were characterized in greater detail. 5L3 had a general courtship defect; courtship of females by 5L3 males also was affected strongly. 5P1 males courted females normally but had reduced success at copulation. 5P1 and 5N18 showed a significant decrement in olfactory response, indicating that the defects in these mutations are not specific to the auditory pathway. Two other mutants, 5M8 and 5N30, produced amotile sperm although in 5N30 this phenotype was genetically separable from the auditory phenotype. Finally, a new adult circling behavior phenotype, the pirouette phenotype, associated with massive neurodegeneration in the brain, was discovered in two mutants, 5G10 and 5N18. This study provides the basis for a genetic and molecular dissection of auditory mechanosensation and auditory behavior.

Acoustic and neural bases for innate recognition of song

In behavior reminiscent of the responsiveness of human infants to speech, young songbirds innately recognize and prefer to learn the songs of their own species. The acoustic and physiological bases for innate recognition were investigated in fledgling white-crowned sparrows lacking song experience. A behavioral test revealed that the complete conspecific song was not essential for innate recognition: songs composed of single white-crowned sparrow phrases and songs played in reverse elicited vocal responses as strongly as did normal song. In all cases, these responses surpassed those to other species’ songs. Although auditory neurons in the song nucleus HVc and the underlying neostriatum of fledglings did not prefer conspecific song over foreign song, some neurons responded strongly to particular phrase types characteristic of white-crowned sparrows and, thus, could contribute to innate song recognition.

A preference for own-subspecies' song guides vocal learning in a song bird

In many song birds, males develop their songs as adults by imitating the songs of one or more tutors, memorized previously during a sensitive phase early in life. Previous work using two assays, the production of imitations by adult males and playback-induced calling by young birds during the sensitive phase for memorization, has shown that song birds can discriminate between their own and other species' songs. Herein I use both assays to show that male mountain white-crowned sparrows, Zonotrichia leucophrys oriantha, must learn to sing but have a genetic predisposition to memorize and learn the songs of their own subspecies. Playback tests to young naive birds before they even begin to sing reveal that birds give begging calls more in response to oriantha song than to songs of another species. After 10 days of tutoring with songs of either their own or another subspecies, birds continue to give stronger call responses to songs of their own subspecies, irrespective of whether they were tutored with them, and are more discriminating in distinguishing between different dialects of their own subspecies. The memory processes that facilitate recognition and discrimination of own-subspecies' song may also mediate the preferential imitation of song of a bird's own subspecies. Such perceptual biases could constrain the direction and rate of cultural evolution of learned songs.

Song selectivity and sensorimotor signals in vocal learning and production

Bird song, like human speech, is a learned vocal behavior that requires auditory feedback. Both as juveniles, while they learn to sing, and as adults, songbirds use auditory feedback to compare their own vocalizations with an internal model of a target song. Here we describe experiments that explore a role for the songbird anterior forebrain pathway (AFP), a basal ganglia-forebrain circuit, in evaluating song feedback and modifying vocal output. First, neural recordings in anesthetized, juvenile birds show that single AFP neurons are specialized to process the song stimuli that are compared during sensorimotor learning. AFP neurons are tuned to both the bird's own song and the tutor song, even when these stimuli are manipulated to be very different from each other. Second, behavioral experiments in adult birds demonstrate that lesions to the AFP block the deterioration of song that normally follows deafening. This observation suggests that deafening results in an instructive signal, indicating a mismatch between feedback and the internal song model, and that the AFP is involved in generating or transmitting this instructive signal. Finally, neural recordings from behaving birds reveal robust singing-related activity in the AFP. This activity is likely to originate from premotor areas and could be modulated by auditory feedback of the bird's own voice. One possibility is that this activity represents an efference copy, predicting the sensory consequences of motor commands. Overall, these studies illustrate that sensory and motor processes are highly interrelated in this circuit devoted to vocal learning, as is true for brain areas involved in speech.

A locus for female discrimination behavior causing sexual isolation in Drosophila

The genetic basis of sexual isolation that contributes to speciation is one of the unsolved questions in evolutionary biology. Drosophila ananassae and Drosophila pallidosa are closely related, and postmating isolation has not developed between them. However, females of both species discriminate their mating partners, and this discrimination contributes to strong sexual isolation between them. By using surgical treatments, we demonstrate that male courtship songs play a dominant role in female mate discrimination. The absence of the song of D. pallidosa dramatically increased interspecies mating with D. ananassae females but reduced intraspecies mating with D. pallidosa females. Furthermore, genetic analysis and chromosomal introgression by repeated backcrosses to D. pallidosa males identified possible loci that control female discrimination in each species. These loci were mapped on distinct positions near the Delta locus on the middle of the left arm of the second chromosome. Because the mate discrimination we studied is well developed and is the only known mechanism that prevents gene flow between them, these loci may have played crucial roles in the evolution of reproductive isolation, and therefore, in the speciation process between these two species.

Chemical basis of courtship in a beetle (Neopyrochroa flabellata): cantharidin as precopulatory "enticing" agent.

Male Neopyrochroa flabellata have a natural affinity for cantharidin (Spanish fly). They are attracted to cantharidin baits in the field and feed on the compound if it is offered to them in the laboratory. Males that ingest cantharidin secrete cantharidin from a cephalic gland. Females sample secretion from this gland during courtship and mate preferentially with males that had fed on cantharidin. Cantharidin-unfed males can be rendered acceptable to females if cantharidin is added to their cephalic gland.

Chemical basis of courtship in a beetle (Neopyrochroa flabellata): Cantharidin as "nuptial gift".

The amount of cantharidin (Spanish fly) that the Neopyrochroa flabellata male presents to the female as a glandular offering during courtship represents only a small fraction of the total cantharidin the male accumulates systemically following ingestion of the compound. A major fraction of the acquired cantharidin is stored by the male in the large accessory glands of the reproductive system. At mating, the male transfers this supply, presumably as part of the sperm package, to the spermatheca of the female. The female in turn allocates the gift to the eggs. Eggs endowed with cantharidin proved relatively invulnerable to attack by a predaceous beetle larva (Coleomegilla maculata).

Functional testicular tissue does not masculinize development of the zebra finch song system.

Current theories of sexual differentiation maintain that ovarian estrogen prevents masculine development of the copulatory system in birds, whereas estrogen derived from testicular androgens promotes masculine sexual differentiation of neuroanatomy and sexual behavior in mammals. Paradoxically, some data suggest that the neural song system in zebra finches follows the mammalian pattern with estrogenic metabolites of testicular secretions causing masculine development. To test whether the removal of estrogen from males during early development would prevent the development of masculine song systems, zebra finches were treated embryonically with an inhibitor of estrogen synthesis. In addition, this treatment in genetic female zebra finches induced both functional ovarian and testicular tissue to develop, thus allowing the assessment of the direct effects of testicular secretions on song system development. In males, the inhibition of estrogen synthesis before hatching had a small but significant effect in demasculinizing one aspect of the neural song system. In treated females, the song systems remained morphologically feminine. These results suggest that masculinization of the song system is not determined solely by testicular androgens or their estrogenic metabolites.

Misexpression of the white (w) gene triggers male-male courtship in Drosophila.

We report here that the general ectopic expression of a tryptophan/guanine transmembrane transporter gene, white (w), induces male-male courtship in Drosophila. Activation of a hsp-70/miniwhite (mini-w) transgene in mature males results in a marked change in their sexual behavior such that they begin to vigorously court other mature males. In transformant populations containing equal numbers of both sexes, most males participate, thus forming male-male courtship chains, circles, and lariats. Mutations that ablate the w transgene function also abolish this inducible behavior. Female sexual behavior does not appear to be altered by ectopic w expression. By contrast, when exposed to an active homosexual courtship environment, non-transformant males alter their behavior and actively participate in the male-male chaining. These findings demonstrate that, in Drosophila, both genetic and environmental factors play a role in male sexual behavior.

Feedback circuitry within a song-learning pathway.

The song system of birds consists of several neural pathways. One of these, the anterior forebrain pathway, is necessary for the acquisition but not for the production of learned song in zebra finches. It has been shown that the anterior forebrain pathway sequentially connects the following nuclei: the high vocal center, area X of lobus parolfactorius, the medial portion of the dorsolateral thalamic nucleus, the lateral magnocellular nucleus of anterior neostriatum (IMAN), and the robust nucleus of the archistriatum (RA). We now show in zebra finches (Taeniopygia guttata) that IMAN cells that project to RA also project to area X, forming a feedback loop within the anterior forebrain pathway. The axonal endings of the IMAN projection into area X form cohesive and distinct domains. Small injections of tracer in subregions of area X backfill a spatially restricted subset of cells in IMAN, that, in turn, send projections to RA that are arranged in horizontal layers, which may correspond to the functional representation of vocal tract muscles demonstrated by others. We infer from our data that there is a myotopic representation throughout the anterior forebrain pathway. In addition, we suggest that the parcellation of area X into smaller domains by the projection from IMAN highlights a functional architecture within X, which might correspond to units of motor control, to the representation of acoustic features of song, or both.

Decrements in auditory responses to a repeated conspecific song are long-lasting and require two periods of protein synthesis in the songbird forebrain.

Earlier work showed that playbacks of conspecific song induce expression of the immediate early gene ZENK in the caudo-medial neostriatum (NCM) of awake male zebra finches and that this response disappears with repeated presentations of the same stimulus. In the present study, we investigated whether repetitions of a song stimulus also elicited a decrement in the electrophysiological responses in the NCM neurons of these birds. Multiunit auditory responses in NCM were initially vigorous, but their amplitude decreased (habituated) rapidly to repeated stimulation, declining to about 40% of the initial response during the first 50 iterations. A similar time course of change was seen at the single unit level. This habituation occurred specifically for each song presented but did not occur when pure tones were used as a stimulus. Habituation to conspecific, but not heterospecific, song was retained for 20 h or longer. Injections of inhibitors of protein or RNA synthesis at the recording site did not affect the initial habituation to a novel stimulus, but these drugs blocked the long-term habituation when injected at 0.5-3 h and at 5.5-7 h after the first exposure to the stimulus. Thus, at least two waves of gene induction appear to be necessary for long-lasting habituation to a particular song.