Alignment of EphA4 and ephrin-B2 expression patterns with developing modularity in the lateral cortex of the inferior colliculus

Abstract

In the multimodal lateral cortex of the inferior colliculus (LCIC), there are two neurochemically and connectionally distinct compartments, termed modular and extramodular zones. Modular fields span LCIC layer 2 and are recipients of somatosensory afferents, while encompassing extramodular domains receive auditory inputs. Recently, in developing mice, we identified several markers (among them glutamic acid decarboxylase, GAD) that consistently label the same modular set, and a reliable extramodular marker, calretinin, (CR). Previous reports from our lab show similar modular-extramodular patterns for certain Eph-ephrin guidance members, although their precise alignment with the developing LCIC neurochemical framework has yet to be addressed. Here we confirm in the nascent LCIC complementary GAD/CR-positive compartments, and characterize the registry of EphA4 and ephrin-B2 expression patterns with respect to its emerging modular-extramodular organization. Immunocytochemical approaches in GAD67-GFP knock-in mice reveal patchy EphA4 and ephrin-B2 domains that precisely align with GAD-positive LCIC modules, and are complementary to CR-defined extramodular zones. Such patterning was detectable neonatally, yielding discrete compartments prior to hearing onset. A dense plexus of EphA4-positive fibers filled modules, surrounding labeled ephrin-B2 and GAD cell populations. The majority of observed GABAergic neurons within modular boundaries were also positive for ephrin-B2. These results suggest an early compartmentalization of the LCIC that is likely instructed in part through Eph-ephrin guidance mechanisms. The overlap of developing LCIC neurochemical and guidance patterns is discussed in the context of its seemingly segregated multimodal input-output streams.

Keywords: Eph; RRID: AB_2619710; RRID:AB_2095679; RRID:AB_2095700; RRID:AB_2099371; RRID:AB_2278725; ephrin; guidance; immunocytochemistry; inferior colliculus; modularity; multimodal; patch-matrix.

Figure 1.

Colocalization of GAD67-GFP (green) and GAD67 immunostaining (red) in IC neurons in a P14 knock-in mouse. Separate channels (a, b) and corresponding digital merge (c) of a mid-rostrocaudal IC section at low magnification reveal overlapping patterns. CNIC patterns appear homogeneous, unlike observed modular clustering in the LCIC (c, dashed contours). High magnification confocal images show representative double-labeled IC neurons (a-c insets, i, ii, iii). Arrowheads in (c) highlight the position of depicted neurons: (i) in a dorsal LCIC module, (ii) in a ventral LCIC module, and (iii) in the CNIC. Scale bars in (a-c) = 200µm, (i-iii) = 10µm.

Figure 2.

Complementary GAD and CR LCIC patterns in pre- and post-hearing mice. GAD-positive LCIC modules (green) with complementary CR-labeling (blue) are evident at birth (a-c) and even more prominent by P14 (d-f). At P0, GAD modules appear less apparent, although primitive clustering is evident within layer 2 (a). CR labeling at this age is concentrated in layers 1 and 3 and already exhibits an extra modular pattern (b, dashed contours), surrounding primitive GAD clusters (c). At P14, the complementary organization is further sharpened, with distinct GAD layer 2 modules (d) encompassed by CR expression (e, f, dashed contours). At this later stage, CR-positive intermodular bridges linking layers 1 and 3 (e, IMB) are present, further delineating modular boundaries. Scale bars in (a-c) = 50µm, (d-f) = 100µm.

Figure 3.

Developmental progression of GAD/EphA4 modular overlap. GAD (green) and EphA4 (red) expression at P0 (a-c), P4 (d-f), P8 (g-i) and P12 (j-l). GAD layer 2 modules are present at all ages (a, d, g, j), becoming more obvious by hearing onset (j). EphA4 modules (b, e, h, k, dashed contours) are easily discernible at all ages and restricted to layer 2. Digital merges (c, f, i, l) show precision of EphA4 and GAD modular overlap (dashed contours). EphA4 labeling was consistent with a dense plexus of fibers and presumptive terminals, surrounding many of the GAD-positive modular neurons. Scale bars = 50µm.

Figure 4.

Early periodic and aligned GAD/EphA4 LCIC patterns. Developmental images (a, c, e, and corresponding brightness plot profiles (b, d, f, h) for GAD (green) and EphA4 (red) expression in the mid-rostrocaudal LCIC. Signal fluctuations are periodic and in-phase (arrows), highlighting overlap of GAD and EphA4 signals. Autocorrelation analyses revealed strong periodic signals for both channels (periodicity > 0.5) at each of the ages with no statistically significant difference between ages (p > 0.05). Period length was also not significantly different between GAD and EphA4 at each age (p > 0.05), but was shown to significantly increase from P0/P4 to P4/P8 (p < 0.5). Cross-correlation functions assessing relative overlap for the two channels with zero spatial shift was strong at each of the ages ( > 0.5), with no significant change (p > 0.5) across ages. White curves (a, c, e, g) indicate trajectory of LCIC layer 2 sampling. Dashed contours highlight layer 2 modules corresponding to peaks in adjacent brightness plot profiles (arrows). Scale bars in (a, c, e, g) = 100µm.

Figure 5.

Alignment of EphA4 with GAD and complementary CR expression at P4. LCIC magnification series (a-d, e-h, i-l) of separate channels (GAD = green, EphA4 = red, CR = blue) with digital merges. Inset boxes in (a) and (e) shown at higher magnification in subsequent rows. LCIC patterning is readily apparent at low magnification (a-d), with clear discontinous EphA4 modules that occupy CR voids. GAD clustering into layer 2 modules is more easily observed at higher magnification (e-h, i-l), as is the fibrous-like appearance of EphA4 labeling, in contrast to cellular GAD and CR staining. At depicted mid-rostrocaudal LCIC level, upwards of six developing GAD/EphA4-positive modules (h, arrowheads) are observed situated amongst surrounding extra modular CR labeling. Scale bar in (a-d) = 200 µm, (e-h) = 100 µm, (i-l) = 50 µm.

Figure 6.

EphA4 registry with developing LCIC neurochemical framework at P8. LCIC digital merges (GAD = green, EphA4 = red, CR = blue) at low (a) and high magnification (b, inset box in a). Similar to P4, multiple GAD/EphA4-positive modules are readily apparent spanning LCIC layer 2 (b, arrowheads), with surrounding complementary CR extra modular labeling. Scale bar in (a) = 200 µm and (b) = 100 µm.

Figure 7.

Development of GAD/ephrin-B2 modular overlap. GAD (green) and ephrin-B2 (red) expression at P0 (a-c), P4 (d-f), P8 (g-i) and P12 (j-l). GAD modules again become increasingly more apparent with age (a, d, g, j). Ephrin-B2 modules (b, e, h, k, dashed contours) are easily discernible at all ages and highly localized to layer 2, similar to EphA4. Digital merges (c, f, i, l) show spatial registry of GAD and ephrin-B2 patterns (dashed contours). Scale bars = 50µm.

Figure 8.

Ontogeny of alignment of GAD/ephrin-B2 LCIC patterns. Developmental images (a, c, e, g) and corresponding brightness plot profiles (b, d, f, h) for GAD (green) and ephrin-B2 (red) expression in the mid-rostrocaudal LCIC. Signals are periodic and in-phase especially at the three older stages (c, e, g, dashed contours; d, f, h, arrows), while statistically weaker at birth (a, b). Period length was not significantly different between GAD and ephrin-B2 at each age (p > 0.05), but was shown to significantly increase from P0/P4 to P4/P8 (p < 0.5). Cross-correlations were strong at each of the ages (> 0.5), with a trend of more precise overlap with age. White curves (a, c, e, g) indicate trajectory of LCIC layer 2 sampling. Dashed contours highlight layer 2 modules corresponding to peaks in adjacent brightness plot profiles (arrows). Scale bars in (a, c, e, g) = 100µm.

Figure 9.

Ephrin-B2 alignment with LCIC modularity at P4 and P8. LCIC magnification series of digital merges (GAD = green, ephrin-B2 = red, CR =blue) in a P4 (a-c) and P8 (d-f) mouse. Inset boxes in (a, b; d, e) are shown at higher magnification in (b; e) and (c; f) respectively. At P4, multiple GAD/ephrin-B2-positive modules span LCIC layer 2 (b, arrowheads), with complementary CR labeling. A similar arrangement is seen at P8, with increasing clarity of the emerging guidance and neurochemical framework (e, arrowheads). A single ephrin-B2 module is highlighted for each of the ages (c, f, dashed contours). Scale bars in (a, d) = 200 µm, (b, e) = 100 µm, and (c, f) = 50 µm.

Figure 10.

Cellular examination of GAD/EphA4/ephrin-B2 modular spatial registry. Triple-labeling (GAD = green, EphA4 = red, ephrin-B2 = blue) showing low magnification overview along with higher magnification (a, inset box) confocal images (b, c, d, e) at P4. In contrast to GAD (b) and ephrin-B2 (d) somata labeling, EphA4 expression consists of a dense, punctate fibrous network (c). Most modular GAD neurons are also ephrin B2 positive, and surrounded by EphA4 labeling (compare b-e, arrowheads). However, there are several single-labeled ephrin-B2 modular neurons present that are also situated in EphA4 lacunae. Scale bars (a) = 100µm, (b-e) = 15µm.

Figure 11.

Caudal to rostral overlap of GAD/EphA4/ephrin-B2 patterning. LCIC triple-labeling (GAD = green, EphA4 = red, ephrin-B2 = blue) throughout the caudorostral extent of the LCIC (a-d). Caudally, modules are interconnected (a), appearing as a strip along layer 2. In the mid-rostrocaudal zones the discontinuous modular morphology is most readily apparent (b, c), prior to converging and taking up a deeper position in the rostral extreme (d). Matching of GAD, EphA4, and ephrin B2 LCIC patterns is consistent throughout the caudorostral dimension. The observed shape changes are consistent with that previously described for other modular markers (Dillingham et al., 2017). Scale bars (a-d) = 100µm.

Figure 12.

Schematic diagram depicting the modular-extra modular multimodal LCIC framework. Modules are positive for GAD, EphA4, and ephrin-B2 and receive somatosensory inputs converging from multiple levels. Surrounding extra modular zones are CR positive, receiving inputs of auditory origin arising largely from auditory cortex and adjacent IC subdivisions.