Optical Imaging

Procedures were performed as described previously [26,27]. Briefly, the birds were anesthetized by injection of 0.1 ml of 20% urethane intramuscularly. The birds’ heads were fixed using a stereotaxic head-holder for small birds [31], modified to hold the head in a position where the beak was oriented horizontally. A craniotomy was performed on the left hemisphere to expose the visual wulst, leaving the dura mater intact. The exposed brain area was then covered with warm agarose (2.5% in saline) and a glass cover slip. The contralateral eye was opened by retracting and fixating the lower eyelid in an open position by Histoacryl (Braun, Melsungen), keeping the nictitating membrane intact. If moistening by the nictitating membrane appeared to be not sufficient, silicon oil was applied to keep the sclera in good condition. At the end of the experiment, the birds were sacrificed by an overdose of sodium pentobarbital.

Neuronal activity in the left visual wulst of zebra finches was captured using autofluorescent flavoprotein imaging [27, 32,33]. To image sensory-driven activity, a temporally periodic stimulus was continuously presented to the bird and the brain’s response at the stimulus frequency was extracted by Fourier analysis [34]. Optical images of visual wulst activation were obtained using a CCD camera (Dalsa 1M30) and a 130 x 55 mm lens with an aperture of 1.2 (Nikon, Tokyo, Japan), controlled by custom software. The acquired image covered 4.6x4.6 mm of the brain surface. The camera was focused below the wulst surface at a depth of 500 μm, and neuronal activity was captured using blue light (455 ± 10 nm). Frames were acquired at a rate of 30 Hz, binned to 7.5 Hz and stored as 512x512 pixel images after spatial binning of the camera image.

A high refresh rate monitor (Flatron LCD 295LM, 100 Hz, 46.5×30 cm), covering 75°×50° of the visual field, was placed at an angle of 60° (position where the fovea is directed) at a distance of 30 cm from the eyes. Vertically, the monitor was positioned such that the horizontal line from the eye to the monitor was 7 cm above the lower rim of the monitor. Visual stimuli consisted of vertical bars moving horizontally back and forth (azimuth) or horizontal bars moving vertically (elevation), 4° wide and moving at a speed of 10°/s.

Activity maps were calculated from the acquired frames by Fourier analysis using custom software [34]. The phase component of the signal was used for the calculation of retinotopy and visualized in so-called phase maps, which are colour-coded with respect to the position of the stimulus eliciting this activation, with blue coding the center of the monitor. The amplitude component represents the intensity of neuronal activation, i.e. response magnitude expressed as fractional change in reflectance x 10⁻⁴. The activity distribution of each experiment is visualized in activity maps. The combined information of the magnitude of neuronal activation and retinotopy is displayed in so-called polar maps. Topographically ordered areas are characterized by the sequence of colours—blue which codes the center of the screen (the direction into which the fovea is "looking") flanked by red/violet coding the right and green/yellow coding the left side of the screen in the trials with azimuth stimuli. In the vertical, the horizontal plane (7 cm above the lower rim of the monitor) is coded by light/greenish blue and is flanked by blue-violet-red for areas dorsal and green-yellow for areas ventral from the horizontal. The colour codes used to display the retinotopic polar maps are shown along with each polar map.

As described in our previous paper [13], we also constructed iso-azimuth and iso-elevation line maps. Iso-azimuth and iso-elevation lines describe, similar to the colour codes used in the polar maps, the line of activation within the brain which is triggered by a vertical bar moving horizontally back and forth over the stimulus screen (azimuth stimulus), or the line of activation induced by a horizontal bar moving up and down (elevation stimulus). The numbers on the iso-lines indicate the corresponding viewing angles. Foveal direction (60°) is denoted as 0° for azimuth lines. For elevation lines, 0° denotes the direction of the horizon. Positive values are lateral from the foveal direction for the azimuth and dorsal for the elevation changes, negative values correspond to frontal direction for azimuth and caudal for elevation changes.

Electrophysiology

Seventeen adult male zebra finches (Taeniopygia guttata) from the stock of the Bielefeld Behavioral Biology department were examined. Birds were anesthetized by an injection of urethane (SIGMA Diagnostics, 0.01 ml, 20% PBS) into the flight muscle. When reflexes on pinging the toes were not observed any longer, the bird’s head was attached to a stereotaxic head holder [31]. Xylocain Gel (2%, Astra Zeneca GmbH, Wedel, Germany) was applied to the skin of the ear holes for additional local anaesthesia.

Feathers were removed and the skin was incised and retracted to expose the skull at the desired positions for electrode placement. Then, the skull was opened by removing the two bone layers. The dura was incised using a sharp injection needle at the coordinate determined for electrode insertion. The eyelid of the right eye was fixed by surgical adhesive in an open position shortly before the experiment started. The nictitating membrane was kept intact to protect the eye from desiccation during the experiment. During anaesthesia the nictitating membrane is open and blinks in response to slight touch stimulation.

The reference electrode was clamped to the skin of the head and moistened with saline (0.9%). The recording electrode (tungsten in glass micro-electrode TM31A10, World Precision Instruments, Inc., Sarasota, USA, 0.9–1.0 MΩ, tip diameter 1–2 μm, 1 μm insulation) was positioned according to the coordinates of the topographic maps which were determined in previous imaging studies [13,26] using the coordinate system of the stereotaxic atlas of the zebra finch [35]. After penetration of the dura, a grounded hood of fine wire mesh was attached to the stand to shield bird and electrode from electromagnetic noise caused by the stimulus screen. The hood did not essentially obscure the bird’s view.

Visual stimuli were provided by a computer screen with 100 Hz refresh rate located at the same position relative to the experimental animal as the stimulus screen in the optical imaging experiments (see above). Stimuli identically to those used in the optical imaging experiments were produced with help of the Vision Egg library [36]. White stripes of 5 cm width were moving into four directions (up, down, left, right) over the screen with a speed of 4.1 degrees/ second (see optical imaging methods). In addition, we were using short flashing illumination of the whole screen during the searching phase when advancing the electrode, and also for determination of the latency of isolated neurons. 40 sweeps were performed for each direction and latency determination.

The electrode was advanced slowly in steps of 2 μm by a motorized micro drive. The amplified neuronal responses were visualized by an oscilloscope and also monitored acoustically by a loudspeaker. When a neuron was responding to the moving flashlight, its responses were tested as described below. After reaching a depth of 2000 μm, the electrode was retracted completely and re-inserted at a slightly different coordinate for a new approach. The distance between recording sites was at least 50 μm to avoid recording the same neuron twice.

The received signal was amplified (x1000) and band pass filtered (300 Hz lower, 20 kHz upper cut-off frequency; A-M Systems Model 1800) before it was digitized (CED 1401mkII, Cambridge Electronic Design) and stored ("Spike 2" recording software, Cambridge Electronic Design).

The activity of single neurons within a recording was separated offline using the spike sorting function provided by Spike 2 (a template matching procedure). The resulting files were further analysed with custom made Matlab (Mathworks) scripts including a separately published toolbox ‘CircStat’ [37].

At the end of each experiment, the birds were sacrificed by an overdose of sodium pentobarbital and the position of the last electrode track was marked by an electrical lesion. The brain was removed and stored for at least two days in fixative (4% paraformaldehyde in phosphate buffered saline (PFA) followed by 30% saccharose in PFA). Coronal 40 μm sections were cut, mounted on glass slides and stained with Giemsa dye (SIGMA Diagnostics, St. Louis, USA).

Neuroanatomy

13 male zebra finches from the institute’s stock were used for this study. The birds were anaesthetized by 0.04 ml of equithesin [38] and placed in a stereotaxic device especially constructed for small birds [31], which secures the head in the same position as depicted by the stereotaxic atlas of the zebra finch [35]. After removing the feathers, making a cross like incision to the skin and pulling the skin flaps aside, a section of bone and dura was removed from the skull to expose the visual wulst of the bird at the left side of the brain or on both hemispheres depending on the intended experiment. Injections of fluorescent tracers were made using glass micropipettes, with tip diameters of 20–30 μm, which were advanced into the brain using a Narishige microdrive. Injections were then performed with a custom made pressure system comparable to the commercially avilable "picospritzer". Two different fluorescent dextrans were used, either micro-ruby (red) or micro-emerald (green) (both 3,000K molecular weight; Molecular Probes, Eugene, OR). We attempted to inject one of the tracers in the posterior retinotopic map which was visible at the same place quite reliably in any of our experiments; the second injection was aimed towards a more anterior map which was also visible in many, but not in all experiments, and the location of which was somewhat more variable. As already described for the electrophysiological recordings, the micropipettes were positioned according to the coordinates of the topographic maps which were determined in previous imaging studies [13,26] using the coordinate system of the stereotaxic atlas of the zebra finch [35]. We also made experiments with coordinates outside of the anterior or the posterior map, also one experiment where we injected into the right instead of the left hemisphere, two bilateral injections at coordinates between the anterior and the posterior map, and two retinal injections. However, the results of these injections will not be reported here in detail because there were too few cases to draw reliable conclusions.

In four cases, optical imaging was done as already described above, prior to the tracer injections. The only exception was that we used halothane for anesthesia because the birds had to wake up at the end of the experiment for a survival period of 24 hours. We then determined the location of the maps by overlaying the brain surface vascular pattern (taken by the CCD camera device of the optical imaging setup, using a green filter which intensifies the contrast) and the retinotopic phase map. Using the overlaid vascular pattern and the phase map image, we then propagated the glass pipette with a stereotaxic microdrive to the desired positions, advanced the tip into the brain by 500 μm (the depth of the input layer of the visual wulst) and injected the tracer with help of a nanoliter injection device (General Valve, Fairfield, NJ).

After the injections were made, the craniotomy was covered with the skin flaps which were then agglutinated using medical cyanoacrylic glue (Histoacryl), and the wound was anaesthetized by local anaesthesia (2% xylocaine gel). 24 hours later the birds were deeply anesthetized with sodium pentobarbital (100 mg/kg) and immediately perfused transcardially with phosphate-buffered saline (PBS; 0.9% NaCl, 0.1 M phosphate buffer) followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4). The brain was removed from the skull and immersed in paraformaldehyde for another 24 hours at 4°C. For cryoprotection, the brain was then placed in 30% sucrose in 0.1M PBS until it sank to the bottom of the jar.

Using a freezing stage microtome, 40 μm coronal sections were cut and stored in individual wells containing PBS. The sections were mounted on gelatinized glass slides, dried, coverslipped using SlowFade Gold antifade reagent with DAPI (Invitrogen, Eugene, OR).

Sections were viewed with a compound light microscope (Zeiss Axioscope 2plus) equipped with the appropriate fluorescence filters (rhodamine and FITC). Images (5184*3456 pixels) were acquired using a Nikon digital camera under remote control by an IMac computer. Brightness and contrast of the photos were adjusted in some cases by Adobe Photoshop. Overlays of the fluorescein and the rhodamine pictures were made by using the "merge channels" function of the NIH "ImageJ" program.

Visualizing wulst retinotopic maps by intrinsic signal optical imaging

Basic features of our optical imaging experiments were already described in previous papers [13,26,27]. Visual stimulation with moving bars resulted in activation of distinct areas of the visual wulst, which turned out to be topographically ordered neuronal maps, visible best in a depth of 500 μm, and outspread parallel to the brain surface. It was not possible to relate the maps to a distinct layer; but because the map quality became worse when we focussed the camera deeper (1000 μm) or more superficial (100 μm) we presume that the input layer IHA may be the source of the mapped activity, but that the topographical order extends to HA and HD also (see also [13]). In all successful experiments, we obtained at least one retinotopic map, here referred to as the posterolateral map, because additional maps not always obtained were located more anterior and medial to this map. In 17 out of 32 experiments, imaging revealed a single map, 13 experiments visualized multiple maps, and 2 just a faint second map in addition to the posterolateral one. In addition to these experiments with wild type birds, we also recorded topographic wulst maps in 6 individuals of the white morph of the zebra finch which has been shown to express a slightly deviating visual system [39,40]. In 5 of the 6 birds, there were multiple wulst retinotopic maps, while the 6^th bird had only one map. Because the other results concerning the white birds were not different from those of the wild type (coloured) animals, we do not present them separately.

Besides this variation in numbers, there was quite a big variation in the exact appearance of the single maps and in the relation of the maps to each other. However, comparing all experiments, we were able to define an arrangement of three maps, which was found in 9 of the 13 experiments emanating multiple maps. Fig 1 provides examples of multiple maps from three different zebra finches. The upper panel of Fig 1 shows colour-coded polar maps of retinotopy (colour-code shown on top of the figure; for further description, see methods), displaying retinotopic organization as a sequence of colour for both azimuth and elevation; the corresponding activity maps are illustrated in the lower panel. Both azimuth and elevation maps clearly show at least three distinct representations of the visual field of the contralateral eye. It is obvious that the distribution of the maps within the imaged area was not identical from brain to brain; however, in all cases, a topographical mapping was visible in the lower left corner corresponding to the posterolateral part of the visual wulst. This posterolateral map was the most stable one, it was detected in every successful imaging experiment. Another more variable map was detected anterior to the posterolateral map. Because it was in most cases slightly displaced medially, we call it the anteromedial map. A third, even more variable map, was identified medial to the posterolateral map (posteromedial map). The lower panel of Fig 1 depicts the activity distribution within the recorded wulst area. It shows that the posterolateral map area was always strongly activated by the visual stimuli, while the activation of the other maps was quite variable, as was the position of the activation maxima.

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Fig 1. Optically imaged retinotopic maps in the zebra finch visual wulst are variable.

Activation in the left visual wulst was induced by visual stimulation of the right eye with moving vertical and horizontal bars (top). Colour coded polar maps of azimuth and elevation (upper row) and grey-scale coded activity maps (lower row) from three zebra finches (ZF 2, ZF 77 and ZF 587), showing at least three separate representations of the visual field. The magnitude of stimulus-driven neuronal activation is given as a number in the upper right corner of the activity maps. A/B, E/ F, I/ J: azimuth and activity maps from three different birds, showing interindividual differences in both retinotopic details and activation strength. C/D, G/ H, K/ L: elevation and activity maps from the same birds as above. Note that e.g. the anterior retinotopic maps in C are mirror images of the posterior retinotopic map. Scale bar 500 μm.

https://doi.org/10.1371/journal.pone.0154927.g001

Fig 2 shows the iso-azimuth and iso-elevations lines of three examples of multiple retinotopic maps, marked by green circles. Each iso-line on the maps indicates the longitude (azimuth maps) and latitude (elevation maps) of the visual field area, which evoked activity in the visual wulst. The maps centers are characterized by iso-azimuth lines that are quite parallel to each other. The iso-elevation lines, which are also parallel to each other and roughly perpendicular to the iso-azimuth lines, are more widely spaced in all three maps, a feature which we have described already for the posterolateral map in a previous paper [13].

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Fig 2. Iso-azimuth and iso-elevation line maps from three examples of multiple maps in the visual wulst.

Iso-azimuth (blue) and iso-elevation lines (red) from the same birds shown in Fig 1 are illustrated. The roughly parallel arrangement of iso-azimuth and iso-elevation lines indicates an orderly retinotopic representation of the multiple maps, indicated by green circles. Note that iso-azimuth and iso-elevation lines tend to be perpendicular to each other. Scale bar 500 μm.

https://doi.org/10.1371/journal.pone.0154927.g002

Another common feature was the different orientation of the three maps (Figs 1 and 2). In the posterolateral map, the gradient along the fronto-lateral axis of the visual field was oriented rostro-caudally, while the dorso-ventral axis was oriented latero-medially. The posteromedial map was rotated anticlockwise against the posterolateral map by ~ 45°, and the topographic relation between the frontolateral and the dorsoventral axes was preserved. Compared to the posterolateral map, the azimuth representation within the anterior map was rotated clockwise by ~ 90°, and so were the iso-elevation lines, which were also mirrored compared with those of the posterolateral map. Although we did not make measurements, it might be useful to point out that the spacing of the elevation lines of the posteromedial map appeared to be similar to that of the posterolateral map, in which the elevation spacing was more than double of the azimuth line spacing [13]. Likewise, the spacing of the iso-elevation lines appeared to be even wider in the anterior map.

Fig 3 illustrates the interindividual variability of the map positions obtained in the different imaging experiments. We marked the approximate positions of the retinotopic maps by coloured circles in the azimuth polar maps (these were chosen because they best show the spatial limits of the maps) obtained in the experiments and then superimposed the coloured circles onto one exemplary multiple wulst map. It is obvious that the position of the posterolateral map (red circles) is the most stable one, those of the anteromedial (green circles) and the posteromedial (yellow circles) are more variable. Blue circles depict the position of a fourth map, which we saw in only a few experiments. The hatched circles in Fig 3 depict the areas that we subsequently investigated by extracellular electrophysiology (see below). The properties of the other two areas were not further investigated in this series of experiments.

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Fig 3. Visualization of the interindividual differences in multiple map locations.

Left panel: Superimposition of the retinotopic map positions from different experiments. Coloured circles circumvent the centers of all imaged wulst retinotopic maps in 9 zebra finches: the posterolateral map (P-l, red), the antero-medial (A-m, green) and the postero-medial map (P-m, yellow). In some cases, a fourth map was discernible (blue ovals). White dotted circles indicate the location of the electrophysiological recordings and the tracer injections. Scale bar 500 μm. Right panel: Top view of the zebra finch brain illustrating the location of the maps. Arrow: origin of the coordinate system used. The grid has a width of 1mm. Scale bar 1mm [35].

https://doi.org/10.1371/journal.pone.0154927.g003

Electrophysiology

Fig 4 shows an overview of the electrophysiological recordings. Overall, 74 of 88 neurons responded reliably to flashes as well as to moving bars (see methods). The response to the four stimulus directions is shown for each recorded neuron, averaged over 40 trials and normalized to the maximum firing rate. Rows correspond to neurons; time is on the x-axis. Dark colors correspond to low firing rate, hot colors to high firing rates. Each block shows cells from the four spatial regions in which we sampled, indicated to the left. Black bars above each block indicate the stimulus duration (the transition of the moving bar across the monitor, 0.48 s for vertical motion, 0.64 s for horizontal motion), and arrows show the direction of the bar movement. It is obvious that the responses of neurons are not uniform: while all neurons exhibited a response starting at the onset of the stimulus, only 27 also exhibited an off-response. Onset and offset is not identical with the leading or trailing edge of the stimulus. Instead, the neurons were activated when the moving bar entered the receptive field of the recorded neuron, and the off response was, with some delay, elicited when the bar was leaving the receptive field.

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Fig 4. Visual response properties of neurons in different visual wulst regions: Overview of the electrophysiological recordings.

The neuronal responses of each recorded cell to each visual stimulus is displayed after averaging over 40 trials and normalizing to the maximum firing rate of the cell. Rows correspond to cells, time is on the x-axis. Dark colors correspond to low firing rate, hot colors to high firing rates. Each block (A-D) comprises cells from the four different wulst regions in which we sampled (C and D are not further treated in the paper because the samples were too small). Black bars above each block indicate the stimulus duration (the transition of the moving bar across the monitor, 0.48 sec for vertical motion, 0.64 sec for horizontal motion), and arrows show the direction of the bar movement.

https://doi.org/10.1371/journal.pone.0154927.g004

Examples of both types of neurons are depicted in Figs 5 and 6. In both figures, the left panels show raster plots and peristimulus-time histograms (PSTH) of four examples each with on-responses (Fig 5) and with on-off responses (Fig 6). The shaded area depicts the time of visibility of the stimulus on the stimulation monitor. Red bars in the PSTH indicate an activation two standard deviations above background. The highest amplitudes within these activation bouts were used to construct the circular diagrams in the medial panels of Figs 5 and 6, illustrating the differences in direction preferences between the different neurons. Red lines connect the maximal "on" responses to each of the four stimulus directions, the blue lines in Fig 6 those of the "off" responses. It is obvious that most neurons were, to different degrees, direction selective. The line emanating from the center is the mean vector calculated from the four directional responses. The right panels in Figs 5 and 6 show a rough reconstruction of the receptive field of the given neuron, which we made by combining the "on" responses to all movement directions. The size of the receptive fields differed from quite small ones (eg. Fig 5D) to very big ones that were extending over the limits of the stimulus monitor.

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Fig 5. Four examples of neurons from the posterolateral wulst area with on–responses (A-D).

Left two panels: Raster plots (40 sweeps) and PSTH´s. Grey shaded areas: Time of stimulus transition across the monitor. Arrows indicate the direction of stimulus movement. Medial panel: Directional responses of the same neurons as circular diagrams. The line originating in the center is the main vector. Its length is a measure for the directionality of the neuron. Right panel: Estimation of the receptive fields by combining the responses to all four directions. Light colors indicate strong activation.

https://doi.org/10.1371/journal.pone.0154927.g005

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Fig 6. Neurons from the posterolateral and anteromedial wulst areas with on (red) and off (blue) responses.

Note that the off responses are often maximal with a stimulus direction opposite to that of the on–response. Other features as in Fig 5.

https://doi.org/10.1371/journal.pone.0154927.g006

Fig 7A shows the spatial distribution of our recording sites on the visual wulst as seen from above. Most recordings were located within the posterolateral and the anteromedial area (p-l and a-m, see also Fig 3). The colours of the recording points code the direction of the mean vector (coloured circle, the direction of movement of the stimulus bar on the computer screen is indicated by the arrows). The distribution of the coloured recording points already indicates that the direction preferences differed between the two areas. This is quantified in the two circular diagrams on the right (Fig 7B and 7C), which show the distribution of the directionality vectors calculated from the direction preferences of the single neurons. While the antero-medial map (Fig 7B) shows maxima with upward directions, the maximum in the postero-lateral map (Fig 7C) is directed downwards. This also indicates that the preferred orientation of the bars in both cases may be horizontal; however, it is not possible to really disentangle the orientational and directional preferences on the basis of our experiments because we did not measure both parameters independently.

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Fig 7. Distribution of directional responses within the anteromedial (a-m) and the posterolateral (p-l) wulst field.

A: Positions of the recording sites on the surface of the visual wulst of the left hemisphere. x-axis depicts the distance from the midline of the brain, y-axis the distance from the Y-point (see methods). The compass rose at the top left indicates the colour coding of the different directions used to mark the directions at the recording sites. B: directional response distribution of the anteromedial map, 0 degrees: downwards, 180 degrees: upwards, 90 degrees: forwards (rostral), 270 degrees: backwards (caudal). C: posteromedial map, explanations see B. Red colour indicates on responses, blue colour indicates off responses.

https://doi.org/10.1371/journal.pone.0154927.g007

In addition to directionality, the two areas also differ in the latency of neuronal responses as measured by flashing illumination of the entire stimulus screen (Fig 8). The neurons of the anteromedial area comprise significantly (Mann Whitney U-test, 2-sided, z = 2.07, p = 0.0385) longer latencies (median 49,5 ms) compared with those of the posterolateral ones (median 29 ms).

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Fig 8. Latencies of neurons within the anteromedial (a-m) and the posterolateral (p-m) map.

Blue dots: individual latencies, horizontal orange line: median.

https://doi.org/10.1371/journal.pone.0154927.g008

Tracing

Our tracing attempts were not aimed to provide a complete analysis of the projection patterns of the thalamic visual nuclei to the visual wulst. Instead, we focused on investigating whether the posterolateral and anteromedial maps received distinct afferents from thalamic regions. We restricted our analysis to those maps (in the left hemisphere) because they were also investigated with electrophysiological methods (see above).

Principally, our results are congruent with those previously described by other authors (e.g. [22,41,42,43]) and reviewed by Güntürkün et al. [21]. We here found projections to the visual wulst from the following thalamic areas: SPC (n. superficialis parvocellularis), DLAmc (n. dorsolateralis anterior, pars magnocellularis), DLLd and DLLv (n. dorsolateralis pars dorsalis/ pars ventralis), LdOPT (n. lateralis dorsalis nuclei optici principalis thalami), n. SpRt (n. suprarotundus).

Most of these nuclei projected to the ipsi- and the contralateral visual wulst, as has already been shown in the above mentioned papers. In general, the strength of the projections to the anteromedial map was smaller than of those to the posterolateral one, but there was no case where we could find a projection exclusively to the one or the other area. On the other hand, there were no single neurons projecting to both wulst areas, as we did not find any colocalization of the two tracers within individual neurons of the thalamic nuclei.

Within the thalamic nuclei, there were clusters of neurons projecting exclusively to one of the two areas (Fig 9A–9D), indicating that these nuclei comprise separate subdivisions serving only one of the two wulst maps. There was no obvious pattern of systematic arrangement like medial to lateral or anterior to posterior. The clusters were found in both hemispheres, confirming earlier studies that the thalamo-wulst projection is bilateral. Fig 9A shows an example (Zf45) where the dorsal part of DLL contains red fluorescent neurons retrogradely labeled from the anterior map (rhodamine injection) while the ventral part shows green fluorescent neurons labeled from the posterior map. Fig 9B shows the same area in another case (Zf 56) where we injected the green tracer into the anterior and the red tracer into the posterior map, and got a very similar picture just with interchanged colours. Fig 9C depicts a clustered label within SPC where the neurons projecting to the anterior map are labeled in red, and those projecting to the posterior map in green (Zf 320). In areas SRt and SPC, we also observed non-clustered, overlapping projections: Fig 9D shows a case (Zf 45) where neurons retrogradely labeled from the anterior or the posterior map are intermingled. As mentioned before, there was no case of double labeled neurons, which would have been expressed here in yellow.

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Fig 9. Tracing of thalamo-wulst afferents into the posterior and anterior map.

A-D: Retrogradely labeled neurons within various thalamic relay nuclei of the thalamofugal system. Scale bars, 50 μm. E, F: Wulst injection sites with efferents from the second injection site in the other retinotopic wulst map. Scale bars 100 μm. For details see text. G: Percentage of ipsi- (blue), contra- (green) and bilateral (red) projections to the anterior and the posterior wulst map.

https://doi.org/10.1371/journal.pone.0154927.g009

Our tracing experiments also revealed that there are interhyperpallial connections between the two visual representations. At both injection sites, anterogradely labeled fibers from the other injection site were visible (Fig 9E and 9F), indicating that connections between these two areas are reciprocal.

To get some quantitative information on possible differences between the afferents of the two maps from the ipsi- and contralateral side, we selected all cases with successful injections into the two areas, and counted how often we observed projections exclusively to the ipsilateral or to the contralateral hemisphere, or bilateral projections. Fig 9G illustrates the percentages of ipsi-, bi- and contralateral thalamic efferents to the anterior and the posterior map, respectively. Obviously, while almost half of the map afferents were bilateral (around 50%), the anterior map received more contra- than ipsilateral projections, and the posterior map more ipsi- than contralateral ones. This difference was significant (Chi squared test on the original numbers, df = 2, Chi squared = 8.77, p<0.05).