A micro-CT-based standard brain atlas of the bumblebee

Abstract

In recent years, bumblebees have become a prominent insect model organism for a variety of biological disciplines, particularly to investigate learning behaviors as well as visual performance. Understanding these behaviors and their underlying neurobiological principles requires a clear understanding of brain anatomy. Furthermore, to be able to compare neuronal branching patterns across individuals, a common framework is required, which has led to the development of 3D standard brain atlases in most of the neurobiological insect model species. Yet, no bumblebee 3D standard brain atlas has been generated. Here we present a brain atlas for the buff-tailed bumblebee Bombus terrestris using micro-computed tomography (micro-CT) scans as a source for the raw data sets, rather than traditional confocal microscopy, to produce the first ever micro-CT-based insect brain atlas. We illustrate the advantages of the micro-CT technique, namely, identical native resolution in the three cardinal planes and 3D structure being better preserved. Our Bombus terrestris brain atlas consists of 30 neuropils reconstructed from ten individual worker bees, with micro-CT allowing us to segment neuropils completely intact, including the lamina, which is a tissue structure often damaged when dissecting for immunolabeling. Our brain atlas can serve as a platform to facilitate future neuroscience studies in bumblebees and illustrates the advantages of micro-CT for specific applications in insect neuroanatomy.

Keywords: Bombus terrestris; Insect standard brain atlas; Iterative shape averaging; Neuropils; Reconstruction.

Fig. 1

Workflow for reconstruction of neuropils from micro-CT data of Bombus terrestris. (a) Virtual frontal 2D slice from the micro-CT data showing the position of the brain in the head capsule. Red arrows: muscles; green arrows: retina. (b) Same slice as in (a) but cropped to exclude all non-neuronal tissues. (c) The cropped slice with all neuropil regions of interest manually labeled. The color code of the neuropils is consistent with Brandt et al. (2005) and Kurylas et al. (2008). (d) Surface model of an individual brain reconstruction with all neuropils labeled. The remaining neuropils (RN) were labeled semi-transparent to make the central complex (CX) better visible. Neuropils: antennal lobes (AL), anterior optic tubercle (AOTU), basal ring (BR) central complex (CX), collar (CO), lamina (LA), lip (LIP), lobula (LO), medulla (ME), ocellar synaptic plexi (OC), peduncle (PED), and remaining neuropils (RN). Scale bars = 1000 µm

Fig. 2

Standard brain atlas of Bombus terrestris. Left column: Shape-based average of surface reconstruction from a frontal, lateral, posterior, dorsal, and ventral perspective. The color code at the bottom represents the colors of the reconstructed neuropils. Right column: Direct volume rendering of averaged raw data from a frontal, lateral, posterior, dorsal, and ventral perspective. For better visibility of the neuropils in the central brain, the remaining neuropils (RN) were excluded in this figure. The whole standard including the RN is provided in the supplementary information (Supplementary Fig. S2). Scale bar = 1000 µm

Fig. 3

Optic Lobes. (a) Frontal view of a shape-based average of surface reconstruction showing the primary visual neuropils including lamina (LA), medulla (ME), and lobula (LO). (a′) Frontal view of one slice of the micro-CT scans showing an optic lobe. (a′′) Frontal optical section (confocal image) of optic lobe, stained against synapsin. Red arrows show damage of the LA that occurred during dissection. Scale bars = 200 µm

Fig. 4

Ocellar synaptic plexi. Shape based average of surface reconstruction of the ocellar synaptic plexi (OC) of B. terrestris standard brain atlas in frontal view (a) and dorsal view (b). Micro-CT scans of the OC in frontal view (a′) and dorsal view (b′). Scale bars = 200 µm

Fig. 5

Mushroom bodies. (a) Shape-based average of surface reconstruction of the mushroom body (MB) with pedunculus (PED), vertical lobe (VL), medial lobe (ML), and the calyx (basal ring: BR, collar: CO, lip: LIP). (b) Virtual section through 3D reconstruction shown in (a). (a′ and b′) Anterior view of micro-CT scans at two different levels showing the compartments of the MB. (a′) Anterior slice shows the BR, CO, LIP, and VL. (b′) Posterior slice shows the BR, CO, LIP, and PED. (a″ and b″) Confocal image (frontal optical slice) of the MB, stained with an antiserum against synapsin. Optical section levels correspond to those in (a′) and (b′). (c, c′, and c″) The optic tracts (OT) consisting of the anterior superior optic tract (ASOT), the anterior inferior optic tract (AIOT), and the lobula optic tract (LOT). The tracts are shown here in different depths of the standard brain micro-CT data. Since they run in parallel, they cannot be distinguished here. Scale bars = 200 µm (a-b″), 500 µm (c-c″)

Fig. 6

Central Complex. (a) Shape-based average of central complex surface reconstruction showing the upper division of the central body (CBU), the lower division of the central body (CBL), the protocerebral bridge (PB), and the paired noduli (NO). (a′) Posterior view of (a) shows the complete PB and NO. (b, c, and d) Frontal view of CX in different depths of micro-CT scan shows all neuropils comprising the CX. (b′, c′, and d′) Anti-synapsin immunolabeling showing the CX neuropils at similar depths. Scale bars = 200 µm

Fig. 7

The anterior optic tubercle. (a) Shape-based average of surface reconstruction of the right anterior optic tubercle (AOTU). (a′) One slice of the micro-CT scan of the left AOTU in frontal view. (a″) Optical horizontal plane of a synapsin staining of the left AOTU. (b, b′, and b″) Frontal slices of the standardized grey data of the micro-CT scans in different planes. The figures present the course of the anterior optic tract (AOT) from the AOTU to the optic lobes (OL). AL antennal lobe, LUC lower unit complex of the AOTU, UU upper unit of the AOTU. Scale bars = 50 µm (a–a″), 200 µm (b–b″)

Fig. 8

Antennal Lobes. (a) Anterior view of shape-based average of surface reconstruction showing the left antennal lobe (AL). (a′) Anterior view of one slice of the micro-CT scans showing the AL. (a″) Frontal confocal slice of a synapsin stained B. terrestris brain showing the AL including single glomeruli. Scale bars = 200 µm

Fig. 9

Volumetric analysis of the bumblebee brain. (a) Mean volume and standard deviation of the different neuropils in the ten individual bumblebee brains. For better visibility of the smaller volumes the remaining neuropils were left out here. (b) Upper illustration shows the shape-based average of reconstructed neuropils. The lower figure highlights the proportion of the reconstructed neuropils. Neuropils: antennal lobes (AL), anterior optic tubercle (AOTU), basal ring (BR), central complex (CX), collar (CO), lamina (LA), lip (LIP), lobula (LO), medulla (ME), ocellar synaptic plexi (OC), peduncle (PED), and remaining neuropils (RN)

Fig. 10

Registration of neurons into the standard brain. (a) Projection view of a confocal scan of neurobiotin/Streptavidin-Alexa568 labeled tangential neurons in the central complex. (b) Direct volume rendering of tangential neurons registered into the standard brain. To show the individual branches of the neurons in more detail, only the CBU and CBL of the standard brain have been illustrated here. (c) Anterior and (c′) posterior view of the registered tangential neurons in the entire standard brain to show the localization of the neurons. Neuropils: antennal lobes (AL), anterior optic tubercle (AOTU), lower division of the central body (CBL), upper division of the central body (CBU), central complex (CX), lamina (LA), lobula (LO), mushroom body (MB), medulla (ME), ocellar synaptic plexi (OC), remaining neuropils (RN). Scale bars = 100 µm (a and b), 1000 µm (c and c′)