Functional mapping of the auditory midbrain during mate call reception

Abstract

We examined patterns of neural activity as assayed by changes in gene expression to localize representation of acoustic mating signals in the auditory midbrain of frogs. We exposed wild-caught male Physalaemus pustulosus to conspecific mating calls that vary in their behavioral salience, nonsalient mating calls, or no sound. We measured expression of the immediate early gene egr-1 (also called ZENK, zif268, NGFI-A, and krox-24) throughout the torus semicircularis, the auditory midbrain homolog of the inferior colliculus. Differential egr-1 induction in response to the acoustic stimuli occurred in the laminar, midline, and principal nuclei of the torus semicircularis, whereas the ventral region did not show significant effects of stimulus. The laminar nucleus differentially responded to conspecific mating calls compared with nonsalient mating calls, whereas the midline and principal nuclei responded preferentially to one of two conspecific calls. These responses were not explained by simple acoustic properties of the stimuli, and they demonstrate a functional heterogeneity of auditory processing of complex biological signals within the frog midbrain. Moreover, using analyses that assess the ability of the torus semicircularis as a whole to discriminate among acoustic stimuli, we found that activity patterns in the four regions together provide more information about biologically relevant acoustic stimuli than activity in any single region.

Figure 1.

Acoustic stimuli presented to frogs. Sonograms (top graphs) and waveforms (bottom graphs) of treatment stimuli (top to bottom): conspecific P. pustulosus whine (whine), conspecific whine plus three chucks (whine-chuck), three chucks (chuck), and heterospecific whine (P. enesefae). Peak-to-peak amplitudes of stimuli were matched. Total call energy relative to P. pustulosus whine: whine chuck 1.36 dB, P. enesefae 7.82 dB, and chuck, -15.4 dB.

Figure 2.

Photomicrographs showing typical cytoarchitecture and egr-1 expression within the torus semicircularis of animals hearing acoustic stimuli. A, Sections hybridized with the antisense probe showed high egr-1 expression in the torus semicircularis (TS) but little expression within the optic tectum (OT). Scale bar, 0.1 mm. B, An example of a photograph used for quantitative analysis. Black spots were counted to determine number of silver grains within the field of view, and cell bodies were measured to determine area of frame covered by cells. All images for quantification were photographed at this magnification. Scale bar, 0.01 mm. C, Section stained with cresyl violet to show toral subdivisions. Left half of panel is mirror image of photograph on right half for clarity. Laminar nucleus (L) cells formed layers oriented mediolaterally extending from the ventrolateral edge of the optic tectum (OT). Principal nucleus (P) cells were densely packed below the laminar nucleus layers. The midline nucleus included the cells clustered along the dorsoventral extent of the midline. The ventral region (V) included the sparse cells between the principal nucleus and anterodorsal tegmentum (AD). Scale bar, 0.1 mm.

Figure 3.

Mean egr-1 expression differs in subdivisions of the toral semicircularis. Mean egr-1 levels in each toral subdivision combining animals in all treatment groups, with error bars indicating SE. Midline and principal regions had the highest egr-1 expression, whereas the ventral nucleus had the lowest expression.

Figure 4.

Effects of biologically relevant acoustic stimulation on egr-1 expression within the four subdivisions of the torus semicircularis. Each panel shows mean egr-1 levels of all frogs in each of the five stimulus conditions with error bars indicating SE. Note y-axis scales differ on each graph. A, Egr-1 expression in the laminar nucleus increases in response to sound. Natural conspecific mating calls (whine and whine-chuck) show greater induction than other mating call stimuli (heterospecific whine and chuck only). B, Egr-1 levels in the midline region of animals that heard whines are significantly higher than those in animals exposed to whine-chucks. C, In the principal nucleus, egr-1 levels in animals that heard whines are higher than levels in animals presented with whine-chucks. D, The ventral region did not show significant egr-1 elevation based on acoustic stimulation. S, Silence; C, chuck; E, P. enesefae whine; W, P. pustulosus whine; WC, P. pustulosus whine-chuck.

Figure 5.

Discriminant function analysis classifies individuals based on acoustic stimulus using mean egr-1 expression values for all four toral subdivisions. Classification was based on three functions, the primary two shown here on the horizontal (function 1) and vertical (function 2) axes. Egr-1 expression of each individual is represented by a symbol positioned at the calculated values for function 1 and function 2. Symbol shape indicates which acoustic stimulus the individual heard (key on the right of the figure; abbreviations as in Fig. 4). The degree of clustering of each symbol shows how well animals presented with that stimulus were classified based on egr-1 expression patterns.