Experience- and age-related outgrowth of intrinsic neurons in the mushroom bodies of the adult worker honeybee

Abstract

A worker honeybee performs tasks within the hive for approximately the first 3 weeks of adult life. After this time, it becomes a forager, flying repeatedly to collect food outside of the hive for the remainder of its 5-6 week life. Previous studies have shown that foragers have an increased volume of neuropil associated with the mushroom bodies, a brain region involved in learning, memory, and sensory integration. We report here that growth of the mushroom body neuropil in adult bees occurs throughout adult life and continues after bees begin to forage. Studies using Golgi impregnation asked whether the growth of the collar region of the mushroom body neuropil was a result of growth of the dendritic processes of the mushroom body intrinsic neurons, the Kenyon cells. Branching and length of dendrites in the collar region of the calyces were strongly correlated with worker age, but when age-matched bees were directly compared, those with foraging experience had longer, more branched dendrites than bees that had foraged less or not at all. The density of Kenyon cell dendritic spines remained constant regardless of age or behavioral state. Older and more experienced foragers therefore have a greater total number of dendritic spines in the mushroom body neuropil. Our findings indicate that, under natural conditions, the cytoarchitectural complexity of neurons in the mushroom bodies of adult honeybees increases as a function of increasing age, but that foraging experience promotes additional dendritic branching and growth.

Fig. 1.

The mushroom bodies of the adult bee. Kenyon cell somata (K) occupy a region entirely distinct from the neuropil. Kenyon cell axons make up the pedunculus (P) and the lobes [only the β lobe (β) is seen in this view]. Kenyon cell dendrites form the calyx, which is subdivided into three regions, the lip (L), collar (C), and basal ring (BR). Dendrites of the collar region only (outlined) were analyzed in this study. CC, Central complex. Scale bar, 100 μm.

Fig. 2.

Ontogeny of mushroom body neuropil volume increase in bees taken from a typical colony. An increase in neuropil volume is positively correlated with increasing age and experience (Exp). Sample sizes for each group are as follows: day 1 adult, n = 11; first orientation flight,n = 16; fifth orientation flight,n = 12; first foraging flight,n = 9; 1 week forager, n = 6; and 2 week forager, n = 6. Lettersindicate significant differences between groups as indicated bypost hoc comparison (Student–Newman–Keuls test). Groups assigned the same letters are statistically similar to each other.

Fig. 3.

Golgi-stained dendritic arbors of collar Kenyon cells in bees of different ages. A, B, Kenyon cell dendrites in P7 bees. C, D, Kenyon cell dendrites in 4-week-old adults. Dendrites of older bees were typically longer and more spread out than those of pupae. The main branch point of each dendritic tree is clearly visible as a sudden termination of the main neurite (arrows). Scale bar, 10 μm.

Fig. 4.

Differences in dendritic spine morphology between P7 bees and D1 adults. A, Dendritic spines on pupal Kenyon cell dendrites are thin and filamentous (arrows).B, Kenyon cell dendritic spines of D1 adults. The spines appear shorter and thicker, and many have a knobbed shape (arrows). Scale bar, 10 μm.

Fig. 5.

Mushroom body total neuropil and collar neuropil volume estimates for honeybees of differing ages and experience. The first five groups are arranged in each graph in order of increasing age, with the bottom two bars representing groups of the same age and differing amounts of foraging experience.A, Total volume of the mushroom body neuropil, including the collar region of the calyx. B, Volume of the collar region of the mushroom bodies only. Sample sizes for each behavioral group are listed in Table 1. Letters indicate significant differences in neuropil volume between groups as determined by post hoc pairwise comparisons (Fisher's protected LSD). Groups assigned the same letters are statistically similar to each other.

Fig. 6.

Total number of dendritic segments in the collar Kenyon cells of bees of differing ages and experience. Sample sizes for each behavioral group are listed in Table 1. Groups and statistical analysis are as in Figure 5.

Fig. 7.

Age- and experience-related changes in the distribution of intersections with the Sholl ring diagram in bees.A, Comparison of three groups of same-age bees differing only in the amount of flight experience. B, Comparison of very young (P7) bees with very old (Four Week Old) bees. Sample sizes for each behavioral group are listed in Table 1. Letters indicate significant differences in segment number observed over all groups for each 10 μm interval, as determined by post hocpairwise comparisons (least square means comparisons). Groups assigned the same letters are statistically similar to each other.

Fig. 8.

Distribution of dendritic branch order for bees of differing ages and experience. A, Comparison of three groups of same-age bees differing only in flight experience.B, Comparison of young, inexperienced bees (Nurse) with older, experienced bees (Forager). Sample sizes for each behavioral group are listed in Table 1. Letters indicate significant differences in segment number for each branch order as determined bypost hoc pairwise comparisons (least square means comparison). Groups assigned the same letters are statistically similar to each other.

Fig. 9.

Representative dendritic trees of nurses, new precocious foragers, and experienced precocious foragers. Large arrows indicate the main branch point; small arrows show dendritic branches arising directly from the neurite, before the main branch point. Scale bar, 10 μm.

Fig. 10.

Schematic diagram representing how branch order distribution patterns observed in nurses, new precocious foragers, and experienced precocious foragers may be achieved by experience-dependent dendritic sprouting. New precocious foragers appear to have more dendrites projecting from the main neurite than do nurses and experienced precocious foragers (dotted lines). Experienced precocious foragers appear to lose these main neurite dendritic projections, with a subsequent increase in higher branch orders. This progression could be explained if some of the new branches seen in precocious foragers continued branching toward the calyx base instead of perpendicular to the neurite (dotted lines), causing the main branch point to shift upward. Branching at distal tips also contributes to the increase in the number of higher-order branches seen in experienced precocious foragers. Numbersindicate branch orders of each dendritic segment.