Valence of social information is encoded in different subpopulations of mushroom body Kenyon cells in the honeybee brain

Abstract

Over 600 Myr of evolutionary divergence between vertebrates and invertebrates is associated with considerable neuroanatomical variation both across and within these lineages. By contrast, valence encoding is an important behavioural trait that is evolutionarily conserved across vertebrates and invertebrates, and enables individuals to distinguish between positive (potentially beneficial) and negative (potentially harmful) situations. We tested the hypothesis that social interactions of positive and negative valence are modularly encoded in the honeybee brain (i.e. encoded in different cellular subpopulations) as in vertebrate brains. In vertebrates, neural activation patterns are distributed across distinct parts of the brain, suggesting that discrete circuits encode positive or negative stimuli. Evidence for this hypothesis would suggest a deep homology of neural organization between insects and vertebrates for valence encoding, despite vastly different brain sizes. Alternatively, overlapping localization of valenced social information in the brain would imply a 're-use' of circuitry in response to positive and negative social contexts, potentially to overcome the energetic constraints of a tiny brain. We used immediate early gene expression to map positively and negatively valenced social interactions in the brain of the western honeybee Apis mellifera. We found that the valence of a social signal is represented by distinct anatomical subregions of the mushroom bodies, an invertebrate sensory neuropil associated with social behaviour, multimodal sensory integration, learning and memory. Our results suggest that the modularization of valenced social information in the brain is a fundamental property of neuroanatomical organization.

Keywords: behavioural neurobiology; brain evolution; immediate early genes; neurogenomics; social behaviour; valence encoding.

Figure 1.

Neural activity map of immediate early gene (IEG) localization following agonistic and affiliative contexts. (a) Generalized anatomical organization of the honeybee olfactory pathway, which transfers environmental information from the antennal lobe (AL) to the mushroom bodies (MBs), regions of higher-order sensory processing, learning and memory. Olfactory receptor neurons are housed in antennal sensilla and project axons to glomeruli in the AL. After local processing, the AL projects axons via antennocerebral tracts to the MBs and lateral horn (LH, not shown). The MBs are composed of neurons called Kenyon cells (KCs), which are divided into three subpopulations: the inner compact cells, outer compact cells and non-compact KCs. Left side of brain diagram shows generalized anatomical structures in the olfactory pathway; right side shows detailed anatomy and representative labelling. Red dots on right side of brain diagram show regions of high hr38 and egr1 transcriptional activity at 15 min following guarding or nursing behaviour, relative to controls; IEG activity in these regions was bilaterally distributed. We analysed both sides of the bee brain and only detected consistent IEG transcriptional activity in the AL and MBs. LCA: lateral calyx; MCA: medial calyx. Class I KCs include inner (pink) and non-compact (yellow) KCs; Class II KCs contain outer compact KCs (purple). (b) Representative magnified portion of AL showing clear outline of neurons and distinction between IEG⁺ (solid arrowheads) and IEG⁻ (hollow arrowheads) cell. Scale bar, 10 µm. Coronal sections showing representative patterns of hr38 (C) and egr1 (D) induction in the AL and MBs at 15 min following exposure to an inanimate object (control), intruder bee (guard) or queen larva (nurse). Coronal sections are rendered in greyscale. Scale bar, 50 µm. (Online version in colour.)

Figure 2.

Immediate early gene (IEG) transcription is transiently induced following an aggressive interaction in an agonistic context. Time-course analysis shows peak activity of hr38 and egr1 transcription in the (a,b) antennal lobe and (c,d) mushroom bodies at 15 min following a display of guarding behaviour that included biting and stinging an unfamiliar intruder bee. Median values divide boxes, ends of box show lower and upper quartiles (25% and 75%, respectively), extreme lines show the minimum and maximum values excluding outliers, and extreme values (points 1.5 times the interquartile range above or below the upper or lower quartile, respectively) are represented by hollow circles. ^n.s.p > 0.05, *p < 0.05, **p < 0.005, ***p < 0.001, Welch's t-test, n = 5–7 bees per group per time point. Brain regions analysed here are highlighted in figure 1. (Online version in colour.)

Figure 3.

Anatomical localization of immediate early gene (IEG) transcriptional activity following a social stimulus is dependent on the valence of the social interaction. Both hr38 (a,c,e,g) and egr1 (b,d,f,h) showed similar patterns of activation at 15 min following an interaction with an inanimate object (control), an unfamiliar, aggressive bee (guard) or a caregiving interaction with a queen larva in a wax cup (nurse). Boxplots show IEG transcriptional activity in the antennal lobe (a,b), total Kenyon cells (KCs) in the mushroom bodies (c,d), Class I KCs (e,f) and Class II KCs (g,h) using the same designation as in figure 2. Significantly different groups are denoted by different letters (p < 0.05, Tukey–Kramer's honestly significant difference test after ANOVA, n = 9–15 bees per group). Brain regions analysed here are highlighted in figure 1. (Online version in colour.)

Figure 4.

Principal component analysis (PCA) separates controls, guards and nurses based on normalized immediate early gene expression in the four regions of the honeybee olfactory pathway analysed in this study (figure 1 and Methods). We included normalized expression of (a) hr38 and (b) egr1 in the antennal lobe, Class I Kenyon cells (inner compact + non-compact) and Class II Kenyon cells (outer compact). Analyses were conducted at 15 min post-assay, where we found peak hr38 and egr1 expression. Largest shape denotes centroid for each group. n = 9–15 bees per group. (Online version in colour.)