Synaptic organization in the adult honey bee brain is influenced by brood-temperature control during pupal development

Abstract

Recent studies have shown that the behavioral performance of adult honey bees is influenced by the temperature experienced during pupal development. Here we explore whether there are temperature-mediated effects on the brain. We raised pupae at different constant temperatures between 29 and 37 degrees C and performed neuroanatomical analyses of the adult brains. Analyses focused on sensory-input regions in the mushroom bodies, brain areas associated with higher-order processing such as learning and memory. Distinct synaptic complexes [microglomeruli (MG)] within the mushroom body calyces were visualized by using fluorophore-conjugated phalloidin and an antibody to synapsin. The numbers of MG were different in bees that had been raised at different temperatures, and these differences persisted after the first week of adult life. In the olfactory-input region (lip), MG numbers were highest in bees raised at the temperature normally maintained in brood cells (34.5 degrees C) and significantly decreased in bees raised at 1 degrees C below and above this norm. Interestingly, in the neighboring visual-input region (collar), MG numbers were less affected by temperature. We conclude that thermoregulatory control of brood rearing can generate area- and modality-specific effects on synaptic neuropils in the adult brain. We propose that resulting differences in the synaptic circuitry may affect neuronal plasticity and may underlie temperature-mediated effects on multimodal communication and learning.

Fig. 1.

Immunofluorescence labeling of MG in the MB calyx (CA) of the honey bee brain. (A) Overview of the central brain labeled with phalloidin (green) and propidium iodide (red). (B) Higher magnification of MB calyces (BR, basal ring; CO, collar; LP, lip) and peduncle (PED). (C–E) Double labeling of the calyx lip (box shown in B: synapsin-IR, red; phalloidin, green). Merged images in E with Inset show an individual MG. (F–J) Antennal lobe PNs (G, red) and phalloidin (H, green); the image is merged in F and J. The arrow in F indicates PN axons. (K and L) KC dendrites (K, red; 16.2-μm stack of 27 sections) and phalloidin labeling (L, green). Merged images of single optical sections are shown in L; the arrows indicate MG and overlap. (M) 3D reconstruction of MG (synapsin-IR, red; phalloidin, green). AL, antennal lobe; CC, central complex. [Scale bars: A, 200 μm; B, 100 μm; C–E, 20 μm (Inset, 3 μm); F, 50 μm; G–J, 20 μm; K, 20 μm; L, 10 μm; and M, 3μm.]

Fig. 2.

Changes in MG with different rearing temperatures (1-day-old bees). (Upper) Branch of the MB calyx labeled with phalloidin (measured areas are indicated). (Scale bar, 50 μm.) (Lower) The top histogram shows changes in cross-sectional areas of the lip (Left) and collar [Right; white bars indicate the loose region (L), and gray bars indicate the dense region (D)]. The middle histograms show the number of MG profiles in the lip (Left) and dense portion of the collar (Right) (a and b indicate significance). The bottom histograms show estimated density of MG in the lip (Left) and dense region of the collar (Right) (normalized to 1,000 μm²).

Fig. 3.

Changes in MG with different rearing temperatures (7-day-old bees). (Top) Cross-sectional areas in the lip (LP, Left) and collar (CO, Right; white bars indicate the loose region, and gray bars indicate the dense region). (Middle) Number of MG profiles in the lip (Left) and the dense region of the collar (Right) (a and b indicate significance). (Bottom) Estimated density of MG in the lip (Left) and dense region of the collar (Right) (normalized to 1,000 μm²).

Fig. 4.

Thermoregulation matches optimal developmental time, emergence rate, and synaptic-neuropil development (shown for the MB-calyx lip). Shortest development (rectangles), highest emergence rate (triangles), and highest number of MG in the lip (circles) overlap in the narrow range normally maintained by thermoregulation in central brood cells (gray area).