Inhibitory Neuropeptide Y Neurons Send Widespread Cross-Connections in the Auditory Midbrain

To quantify and determine the distribution of CoIC projections of NPY neurons we performed retrograde tracing using red fluorescent Retrobeads (RB, “red beads, ” Luma-Fluor, Inc., Naples, FL, USA; 1:2–1:4 dilution). Injections were made into the right IC of 10 NPY-hrGFP mice aged P22-P62 of both sexes. Throughout the procedure, mice were anesthetized with isoflurane (1.5%–3%), and their body temperature maintained with a homeothermic heating pad. Mice were injected subcutaneously with the analgesic carprofen (5 mg/kg, CarproJect, Henry Schein Animal Health, Portland, ME, USA). Following this, the scalp was shaved, and a rostro-caudal incision was made to expose the skull. The injection sites were mapped using stereotaxic coordinates relative to the lambda suture, and injection depths were relative to the surface of the skull. A single craniotomy was made above the injection site using a micromotor drill (K 1050, Foredom Electric Co., Bethel, CT, USA) with a 0.5 mm burr (Fine Science Tools, Foster City, CA, USA). For the first set of experiments, we made multi-subdivision RB injections into the right IC through two penetrations: penetration 1–900 μm caudal, 1,000 μm lateral, and 2,000 μm deep; penetration 2–900 μm caudal, 1,250 μm lateral, and 2,000 μm deep. One injection of 40–60 nl of RB was performed in each penetration, resulting in two RB deposits with a total volume of 80–120 nl. For the second set of experiments, we made focal injections of 10 nl of RB in one penetration: 900 μm caudal, 1,000–1,150 μm lateral, and 1,600–1,750 μm deep for the ICc injections and 890 μm caudal, 800 μm lateral, and 1,675 μm deep for the ICd injection.

RBs were injected with a Nanoject III nanoliter injector (Drummond Scientific Company, Broomall, PA, USA) connected to an MP-285 micromanipulator (Sutter Instruments, Novato, CA, USA). Glass injection pipettes were pulled from 1.14 mm outer diameter, 0.53 mm inner diameter capillary glass (cat# 3-000-203-G/X, Drummond Scientific Company) with a P-1000 microelectrode puller (Sutter Instrument). The injector tip was cut, and front filled with RB. After the injection was complete, the scalp was sutured using Ethicon 6–0 (0.7 metric) nylon sutures (Ethicon USA LLC, Somerville, NJ, USA) or glued using 3 M Vetbond tissue adhesive (3 M, St. Paul, MN, USA), and the incision was treated with 0.5 ml 2% lidocaine hydrochloride jelly (Akorn Inc., Lake Forest, IL, USA). After recovery from anesthesia, mice were returned to the vivarium and were monitored daily.

Five to 10 days after RB injection, mice were anesthetized with isoflurane and perfused transcardially with phosphate-buffered saline (PBS), pH = 7.4, for 1 min and then with a 10% buffered formalin solution (Millipore Sigma, cat# HT501128, Burlington, MA, USA) for 10 min. Brains were collected and stored in the same formalin solution for 2 h then cryoprotected overnight at 4°C in PBS containing 20% sucrose. Brains were cut into 40–50 μm coronal sections on a vibratome or freezing microtome. Sections were washed in PBS (three washes of 10 min each) and then treated with 10% normal donkey serum (Jackson ImmunoResearch Laboratories, catalog #017-000-121, West Grove, PA, USA) and 0.3% Triton X-100 in PBS for 2 h. Sections were then incubated for 24–40 h at 4°C in mouse anti-GAD67 (1:1,000; Sigma Millipore, catalog #MAB5406), rabbit anti-NeuN (1:500; Sigma Millipore, catalog #ABN78), and/or guinea pig anti-GlyT2 (1:2,000; Synaptic Systems, catalog #272-004, Goettingen, Germany). After the incubation period, sections were rinsed in PBS and incubated in AlexaFluor-647-tagged goat anti-mouse IgG (1:100; Thermo Fisher Scientific, catalog #A21039), AlexaFluor-647-tagged donkey anti-mouse IgG, AlexaFluor-647-tagged donkey anti-rabbit IgG, and/or AlexaFluor-594-tagged goat anti-guinea pig IgG (1:500; Thermo Fisher Scientific, catalog #A-21202, #A-21206, and #A-11076) for 1.5-2 h at room temperature. Sections were then mounted on Superfrost Plus microscope slides (Thermo Fisher Scientific, catalog #12-550-15, Waltham, MA, USA) and coverslipped using Fluoromount-G (SouthernBiotech, catalog #0100-01, Birmingham, AL, USA) or DPX (Sigma Millipore, catalog #06522). For the multi-subdivision RB injections, images were collected using a Zeiss AxioImager.Z2 microscope. Low magnification images were collected using a 5× objective. High-magnification images are maximum intensity projections of image stacks obtained with a 63× oil-immersion objective (NA = 1.4) and structured illumination (Apotome 2, Zeiss) to provide optical sectioning at 0.5 μm z-steps. For the focal RB injections, images of the IC were collected using a 10x objective, 20× objective, 1.30 NA 40× oil-immersion objective (1,024 × 1,024 resolution, 1 μm z-step, 0.75 zoom), or a 1.40 NA 63× oil-immersion objective (1,024 × 1,024 resolution, 1 μm z-step, 0.75 zoom) on a Leica TCS SP8 laser scanning confocal microscope.

GABAergic neurons were identified using the mouse monoclonal anti-GAD67 antibody (Sigma Millipore, catalog #MAB5406). The 67 kDA isoform of glutamic acid-decarboxylase (GAD) is required for the synthesis of the neurotransmitter GABA. Anti-GAD67 antibodies were raised against this isoform. The vendor reported no cross-reactivity for GAD65—the 65 kDA isoform of GAD—using a Western blot analysis. The same anti-GAD67 antibody has been used in several IC studies to identify GABAergic neurons (Ito et al., 2009; Beebe et al., 2016; Goyer et al., 2019; Silveira et al., 2020). To label glycinergic terminals, we used a guinea pig polyclonal anti-GlyT2 antibody (Synaptic Systems, catalog #272004). Glycine transporter 2 (GlyT2) is a transmembrane protein involved in the removal of extracellular glycine and primarily labels axons and terminals. The manufacturer reported complete specificity for the 100 kDA isoform of GlyT2 using Western blot analysis in mouse brain tissue. To visualize neuronal cell bodies, we performed anti-NeuN staining with a rabbit polyclonal antibody (Sigma Millipore, catalog #ABN78). Using a Western blot analysis on mouse brain tissue, this antibody selectively bound a set of closely associated protein isoforms of NeuN, a neuron-specific protein which binds to DNA in neurons. Several studies have used this antibody as a general neuronal marker in the IC (Mellott et al., 2014; Beebe et al., 2016; Goyer et al., 2019; Silveira et al., 2020; Beebe and Schofield, 2021).

Due to fluorescence overlap, immunofluorescence to delineate IC subdivisions was not performed in sections from mice injected with RB. Instead, we prepared a reference series of sections from a control C57BL/6J mouse, aged P49. These sections were immunolabeled for GAD67 and GlyT2 and imaged using the 20× objective of a Leica TCS SP8 confocal microscope. The pattern of GAD67 and GlyT2 immunolabeling has previously been used to identify IC subdivisions (Buentello et al., 2015; Silveira et al., 2020; Beebe and Schofield, 2021). For each RB injection case, IC subdivision boundaries were drawn based on the distribution of GABA and glycinergic terminals in the most similar reference sections. The outer edges of the IC were identified based on fluorescence from the NeuN immunolabeling. The outline and subdivision borders of the IC were drawn using Neurolucida 11 software (MBF Bioscience, Williston, VT, USA).

Following multi-subdivision RB injections, a series of every third section through the rostro-caudal extent of the contralateral IC in each case was examined for hrGFP+ (NPY) cells, and for RB-labeled cells (which were presumed to project across the CoIC). Cells of either type or those where both markers were present, were plotted using a Zeiss AxioImager. Z2 microscope, and Neurolucida 11 software (MBF Bioscience). Marker counts and plots were exported from Neurolucida Explorer and prepared using Microsoft Excel and Adobe Illustrator, respectively.

Following focal RB injections, confocal images were taken from a series of 5–8 coronal sections evenly spaced along the rostral-caudal extent of the IC and imported into Neurolucida 11 software (MBF Bioscience) for analysis. NPY neurons were identified based on hrGFP fluorescence and marked when RB clearly filled and co-labeled an hrGFP+ cell body. Similarly, non-NPY neurons were identified and marked when RB clearly filled and co-labeled a NeuN+/hrGFP- cell body. Contours and plots of the left IC (contralateral to the RB injection site) were exported from Neurolucida 11 and assembled into figures in Adobe Illustrator 2022.

For cases where the RB injection was centered in the ICc, the analyzed sections were imported into the serial section manager of Neurolucida 11. The sections were oriented and stacked in 3D from caudal (bottom) to rostral (top). To assess if RB-labeled NPY neurons had a different distribution than the RB-labeled non-NPY neurons, we compared the distances of each RB-labeled neuron to a point, which we refer to as the centroid, located approximately homotopic to the center of mass of the RB injection site in the contralateral IC. To define the centroid point, we first collected a 10× image of the IC slice containing the strongest RB labeling on the injected side (the right side) of the IC. We then imported this image to Neurolucida and used contours to outline the perimeter of the IC and the main body of the RB labeling corresponding to the injection site. These contours were then reflected across the midline of the IC to yield a “mirror-image” projection of the injection site, which was aligned to the previously prepared reconstruction of the left IC. To prevent bias in this alignment procedure, cell markers were hidden so that only contours were visible. The coordinates of the centroid of the mirror-image injection site were calculated using the Contour Analysis function in Neurolucida Explorer (MBF Bioscience). A locus marker was placed at the centroid coordinates, and the distances, in μm, between each neuron marker and the centroid were calculated using the Marker Analysis functions in Neurolucida Explorer. Distances for neurons located in the ICc were imported to Igor Pro 9 (WaveMetrics), and for each injection case, distances were sorted into 150 μm bins and the proportions of NPY and non-NPY neurons in each bin were calculated as NPY/(NPY + non-NPY) and non-NPY/(NPY + non-NPY), respectively. These proportions were then compared to the overall proportions of NPY and non-NPY neurons found throughout the ICc for each injection case. A summary analysis was conducted by calculating the mean ± standard error for each of the 150 μm bins across the five ICc injection cases. This analysis was then repeated for the ICc injection cases, but with RB-labeled neurons from all IC subdivisions included in the analysis. Similarly, for the ICd injection case, proportion analysis was conducted with only RB-labeled ICd neurons included and also with all RB-labeled IC neurons included.

Serial sections were aligned and stacked in Neurolucida as described above for the NPY vs. Non-NPY Proportion Analysis. X and Y coordinates from each neuron were then extracted in Neurolucida Explorer and imported into MATLAB (MathWorks, Natick, MA, USA). In MATLAB, the numbers of neurons in 100 μm × 100 μm bins covering the left ICc or all subdivisions of the left IC were counted using the neuron coordinates and the “hist3” function. The resulting bivariate histograms were plotted as heatmaps where the intensity of the color corresponds to the densities of NPY and non-NPY neurons. Overlays of the NPY and non-NPY neuron heatmaps were prepared using the “imfuse” function in MATLAB.

Multi-subdivision injections included two or more IC subdivisions and extended across more of the IC rostro-caudal axis as compared to focal injections. Figure 1A shows an injection that included lateral parts of the ICd and dorsal parts of the ICc and IClc. Other cases included more ventral parts of the ICc and IClc. These multi-subdivision injections typically resulted in many RB-labeled neurons in auditory centers, including the contralateral IC. RB-labeled cells were present widely across the subdivisions of the contralateral IC and could be NPY+ or NPY-. Figure 1B shows examples of RB-labeled cells in each subdivision of the contralateral IC, some of which are NPY+ (magenta arrows), and some of which are NPY- (“non-NPY”; cyan arrowheads). NPY+ cells that lacked retrograde tracer were also observed in the contralateral IC (green arrowheads). Figure 1C shows the distribution of RB-labeled commissural cells, including non-NPY cells (cyan squares) and NPY+ cells (magenta triangles) in the IC contralateral to an RB injection in one case. Across four cases with multi-subdivision injections, an average of 11.1% ± 2.1% of CoIC-projecting cells were NPY+, with similar proportions (10%–12%) in individual IC subdivisions (Figure 1D). This suggests that NPY cells make up a substantial portion, potentially a majority, of inhibitory commissural cells, which have been estimated to be 10%–40% of the overall CoIC pathway in other studies (Malmierca et al., 1995; González-Hernández et al., 1996; Nakamoto et al., 2013; Chen et al., 2018).

Previous studies showed that the commissural projections of ICc neurons are predominately homotopic and tonotopic, connecting similarly tuned isofrequency lamina between the two sides of the IC (Saldaña and Merchán, 1992; Malmierca et al., 1995, 2009). However, these studies did not distinguish among neuron types, and since GABAergic neurons comprise only ~25% of IC neurons and ~10%–40% of commissural neurons, it is likely that past results mainly reflect the projection patterns of excitatory IC neurons. To test whether NPY neurons follow the projection pattern previously described, we made small, focal injections of RB in the right ICc of five NPY-hrGFP mice. Following each injection, the left IC contained NPY neurons and non-NPY neurons labeled with RB, indicating that they formed commissural projections to the injection site in the right IC (Figure 2).

For each ICc injection case, we analyzed the location of the RB injection site in the right IC and the distribution of RB-labeled neurons in the left IC. In Case 25, nearly all the RB deposit was in a narrow tract in the dorsal half of the right ICc (Figure 3A). In the left IC, non-NPY neurons labeled with RB formed a densely packed cluster that extended throughout the ICc along a ventrolateral to dorsomedial axis, with a relatively narrow spread perpendicular to this main axis. Fewer RB-labeled non-NPY neurons were present in the ICd and very few were observed in the IClc (Figure 3B, cyan squares). This pattern remained similar across much of the rostrocaudal extent of the ICc but tapered off in more rostral sections. RB-labeling was particularly dense at the location mirroring the injection site in the right IC (Figure 3B, gold outline in section 3). These results are consistent with the commissural projection patterns detailed in previous studies, suggesting that the commissural projections of non-NPY neurons mainly connect the corresponding isofrequency lamina on the two sides of the ICc. In contrast, RB-labeled NPY neurons within the ICc were more common on the outer edges of the region defined by the RB-labeled, non-NPY neurons.

In Case 40, the RB injection site was in the dorsal half of the right ICc and was more caudal than the injection site for Case 25 (Figure 3C). RB-labeled NPY and non-NPY neurons were present in the left ICc and ICd, with less labeling in the IClc (Figure 3D). As with Case 25, RB-labeling of non-NPY neurons in the left ICc was dense at the site homotopic to the injection site (Figure 3D, gold outline in section 2) and mainly extended in a band matching the presumed orientation of the isofrequency laminae (Figure 3D, cyan squares). The distribution of RB-labeled NPY neurons partly overlapped the distribution of non-NPY neurons but also appeared more dispersed (Figure 3D, magenta triangles).

In Case 39, the RB deposit was similar in location but larger than that in Case 25, located in the dorsal half of the right ICc, slightly rostral to the midpoint of the rostral-caudal axis of the IC (Figure 3E). RB-labeling of non-NPY neurons in the left ICc was strongest around the site homotopic to the injection site and extended through the ICc in a pattern consistent with the labeling of a small number of isofrequency laminae (Figure 3F). In contrast, RB-labeled NPY neurons again appeared more common in ICc regions surrounding the region where RB-labeled non-NPY neurons were densest (Figure 3F). RB-labeled NPY and non-NPY neurons were also common in the ICd and rarer in the IClc, although there were more RB-labeled neurons in the IClc in this case than in the previous cases. Despite the variation in the size of the tracer deposit and its rostro-caudal location, the results from the three cases with injections in the dorsal ICc showed similar results.

In Case 22, the RB injection site included some dorsal ICc but extended will into the ventral ICc, producing a deposit much more spread out than in the previous cases (Figure 3G). In the left IC, RB-labeled NPY and non-NPY neurons were again common in the ICc and ICd, but rare in the IClc. Within the ICc, the labeling of non-NPY neurons did not produce a distribution as narrow as in previous cases. Nonetheless, the distribution of RB-labeled NPY neurons in the left IC appeared more dispersed and less prevalent at the site homotopic to the RB injection site compared to non-NPY neurons (Figure 3H).

In Case 08, the final ICc injection case, the RB injection site was in a caudal and ventral portion of the ICc, closer to the midline than in the previous cases (Figure 3I). RB-labeled non-NPY neurons were dense at the site homotopic to the injection site and again extended out from the homotopic site following a laminar pattern that extended through the ICc (Figure 3J, sections 1 and 2). Once again, RB-labeled NPY neurons were more common in the regions surrounding the homotopic site (Figure 3J).

To summarize the distribution of RB-labeled neurons in the ICc across coronal sections, we aligned IC sections according to the three-dimensional shape of the IC, compressed neuron distributions into two dimensions, removed markers for neurons not located in the ICc, and generated heatmaps to separately show the density distributions of NPY and non-NPY neurons in the ICc (Figure 4). For Case 25, the heatmaps show that the densest commissural projection of non-NPY neurons was from a site homotopic to the RB injection site (Figure 4A, left). The remainder of the non-NPY neuron projection came from an angled tract consistent with the presumed orientation of the isofrequency laminae, with the density of labeled cells decreasing with increasing distance from the homotopic site. In contrast, the distribution of RB-labeled NPY neurons was densest at sites surrounding the homotopic site, with a comparatively low density at the homotopic site (Figure 4A, middle). An overlay of the non-NPY and NPY heatmaps shows that the NPY neuron distribution was broader than the non-NPY distribution along the dorsolateral-ventromedial axis (i.e., perpendicular to the presumed isofrequency axis; Figure 4A, right). The two cell populations spanned a similar distance along the isofrequency axis. A similar pattern was apparent in the heatmaps for Case 40, which again showed that RB-labeled non-NPY neurons tended to follow a laminar distribution (Figure 4B), while RB-labeled NPY neurons were more dispersed, with the highest densities in regions away from the peak densities of non-NPY neurons.

In Cases 39, 22, and 08, heatmaps again showed that non-NPY neurons were densest in regions that were mainly homotopic to the RB injection site, while NPY neurons were more widely distributed and generally denser in regions surrounding the area where non-NPY neurons were densest (Figures 4C–E). This trend was apparent even though the distribution of non-NPY neurons was less obviously following the arrangement of the isofrequency laminae in these cases. Together, heatmap analysis of the ICc injection cases suggests that the commissural projections of NPY neurons are less homotopic and more divergent than those of non-NPY neurons. Furthermore, given the tonotopic organization of the ICc, the larger spread of NPY neurons in the dorsolateral-ventromedial axis, corresponding to spread across more isofrequency laminae, suggests that commissural NPY neurons can provide cross-frequency inhibition to neurons in the contralateral IC.

Since the ICc injection cases also yielded RB-labeled neurons in the ICd and IClc, we performed a second heatmap analysis, this time including RB-labeled neurons from all IC subdivisions (Figure 5). Near the site homotopic to the injection site, the all-subdivision heatmaps recapitulate the results shown above for the ICc-only data set. However, the expanded heatmaps also show that RB-labeled NPY and non-NPY neurons tended to have only partially overlapping distributions in the ICd and IClc, as evident by the many heatmap bins that are cyan or magenta. This is particularly apparent in Cases 25, 40, and 22 (Figures 5A–D). In contrast, Cases 39 and 08 exhibited more overlap of RB-labeled NPY and non-NPY neurons in the ICd, as can be seen in the gray and black bins, although there were also regions closer to the borders of the IC in these cases where NPY or non-NPY neurons predominated (Figures 5C,E). Overall, these results suggest that the NPY and non-NPY neurons that project to a particular subregion in the contralateral ICc exhibit only partially overlapping distributions, even when the originating neurons are in the ICd or IClc.

To quantitatively assess the tendencies of NPY and non-NPY neurons in the ICc to make homotopic commissural projections, we mapped for each ICc case a point in the left IC homotopic to the centroid (center of mass) of the RB injection site in the right IC and measured the three-dimensional distance of each RB-labeled ICc neuron to this centroid. We then sorted these distances into 150 μm bins and within each distance bin, calculated the relative proportion of RB-labeled neurons that were NPY neurons vs. non-NPY neurons (Figures 6A–E). The within-bin proportions were compared to the overall proportions of RB-labeled NPY and non-NPY neurons found in the ICc (horizontal black lines, Figures 6A–E). This revealed distance bins where NPY neurons were under- or over-represented. We used 150 μm bins for this analysis because previous work showed that isofrequency laminae in the rat are 90–150 μm apart (Malmierca et al., 1993), and we, therefore, predict that neurons located >150 μm apart in the mouse ICc have either different frequency tuning or participate in different circuits compared to neurons located within 150 μm of each other. In all five ICc cases, the proportion of RB-labeled NPY neurons found within 150 μm of the injection site centroid was lower than the overall proportion of RB-labeled NPY neurons in the ICc. This pattern was not evident at distances farther from the centroid.

To summarize these results, we calculated the mean proportions of RB-labeled NPY and non-NPY neurons across the ICc cases for the first four distance bins (Figure 6F). We then compared these results to the means of the overall proportions of NPY and non-NPY neurons across the five ICc cases (horizontal black lines, Figure 6F). This summary analysis showed that the proportion of RB-labeled NPY neurons in the 150 μm-radius region surrounding the injection site centroid was approximately half that found on average throughout the ICc (0–150 μm bin NPY mean = 0.057 vs. overall mean = 0.118). A two-tailed t-test comparing the proportions of RB-labeled NPY neurons in the 0–150 μm bin to the overall mean proportion of RB-labeled NPY neurons in the ICc gave a p value of 0.04 (n = 5, t₄ = −2.983). We also computed the median distances of RB-labeled neurons from the injection site centroid, and in each case, NPY neurons had a larger median distance than non-NPY neurons (Figure 6G). A two-tailed paired t-test comparing these median distances gave a p-value of 0.04 (n = 5, t₄ = −2.908). Together, these results support the hypothesis that commissural NPY neurons in the ICc form a more distributed, less homotopic projection than non-NPY neurons.

We wondered whether different patterns would emerge if RB-labeled neurons from all IC subdivisions were included in the quantitative analysis, and we, therefore, repeated the above quantitative analyses for the ICc injection cases, this time including all RB-labeled neurons found in the IC. As with the previous analysis, in all five ICc cases the proportion of RB-labeled NPY neurons located within 150 μm of the injection site centroid was lower than the overall proportion of RB-labeled NPY neurons (Figures 7A–E). Summary analysis again revealed that the proportion of NPY neurons in the 0–150 μm bin was less than half the overall proportion of RB-labeled NPY neurons in the IC (0–150 μm bin NPY mean = 0.057 vs. overall mean = 0.145; Figure 7F). A two-tailed t-test comparing the proportions of RB-labeled NPY neurons in the 0–150 μm bin to the overall mean proportion of RB-labeled NPY neurons in the ICc gave a p-value of 0.01 (n = 5, t₄ = −4.325). In addition, a comparison of the median distances of RB-labeled neurons from the injection site centroid again showed that RB-labeled NPY neurons tended to be farther from the centroid than non-NPY neurons (Figure 7G). A two-tailed paired t-test comparing these median distances gave a p value of 0.002 (n = 5, t₄ = −7.289).

Combined, results from the five ICc injection cases indicate that the commissural projections of non-NPY neurons in the ICc are predominately homotopic and often appear to follow the laminar topology of the ICc. In contrast, the commissural projections of NPY neurons in the ICc were more heterotopic, commonly originating from contralateral sites that likely had different frequency tuning or participated in non-mirror image circuits compared to the injection site. Thus, non-NPY commissural neurons in the ICc may predominately provide excitation or inhibition to neurons with similar receptive fields in the contralateral ICc, while NPY commissural neurons in the ICc are more likely to provide lateral inhibition and possibly sideband inhibition to the contralateral ICc. The ICc injection cases also showed that both NPY and non-NPY neurons in the ICd send a large projection to the contralateral ICc. The commissural projection from the ICd to the ICc did not reveal a consistent pattern of differences between non-NPY and NPY neurons, although the heatmap analysis showed that RB-labeled non-NPY and NPY neurons often did not overlap in the ICd (Figure 5). Commissural projections from the IClc were less common and, when present, also lacked a clear organization.

In one mouse, Case 36, the RB injection was located entirely within the right ICd (Figure 8A). Previous studies report that commissural projections to the ICd largely arise from locations distributed throughout the contralateral ICd (i.e., a heterotopic, divergent projection) and to a lesser extent from regions of the contralateral ICc that have similar frequency tuning to the ICd region under study (i.e., a tonotopic projection; Saldaña and Merchán, 1992; Malmierca et al., 2009). Consistent with the former observation, most of the RB-labeled NPY and non-NPY neurons in the left IC of Case 36 were in the ICd, with no clear organization within the ICd other than a tendency to be denser near the site homotopic to the injection site (Figure 8B). A smaller number of RB-labeled NPY and non-NPY neurons were observed in the left ICc, along with an even smaller number in the left IClc, but there was no clear organization to these distributions. Heatmaps of the density of RB-labeled neurons located in the ICd (ICc and IClc neurons removed) revealed a different pattern than observed with the ICc injection sites: RB-labeling for both non-NPY neurons and NPY neurons largely overlapped and was densest at the site homotopic to the ICd injection site (Figure 8C). Analysis of the proportions of RB-labeled ICd neurons located in 150 μm distance bins from the injection site centroid showed that NPY neurons were slightly overrepresented in the 0–150 μm bin compared to the overall proportion of RB-labeled NPY neurons in the ICd (Figure 8D). The median distance of RB-labeled NPY neurons to the centroid was 329 μm compared to 339 μm for non-NPY neurons. These patterns differ from those observed for the ICc injection cases (compare to Figure 6). We also ran the heatmap analysis and proportion analysis for the ICd injection case with RB-labeled neurons from all IC subdivisions included in the analysis (Figures 8E,F). The results again revealed a large amount of spatial overlap of RB-labeled NPY and non-NPY neurons, with NPY neurons slightly overrepresented in the 0–150 μm distance bin. With RB-labeled neurons from all IC subdivisions included, the median distance of NPY neurons to the centroid was 423 μm compared to 452 μm for non-NPY neurons.

As these data come from one injection case, they are not sufficient to support strong conclusions about ICd commissural connections, but we include the results here because they are consistent with past studies and therefore bolster support for the hypothesis that commissural projections targeting the ICd primarily originate in the contralateral ICd. Furthermore, the lack of a clear difference in the distribution of NPY and non-NPY commissural neurons targeting the contralateral ICd contrasts with the differences observed for NPY and non-NPY commissural neurons targeting the contralateral ICc. Together, these results suggest that the organizing principles and corresponding functional roles of commissural projections differ depending on the IC subdivision that is targeted and the neuron type forming the projection.