Alarm cues and alarmed conspecifics: neural activity during social learning from different cues in Trinidadian guppies

Abstract

Learning to respond appropriately to novel dangers is often essential to survival and success, but carries risks. Learning about novel threats from others (social learning) can reduce these risks. Many species, including the Trinidadian guppy (Poecilia reticulata), respond defensively to both conspecific chemical alarm cues and conspecific anti-predator behaviours, and in other fish such social information can lead to a learned aversion to novel threats. However, relatively little is known about the neural substrates underlying social learning and the degree to which different forms of learning share similar neural mechanisms. Here, we explored the neural substrates mediating social learning of novel threats from two different conspecific cues (i.e. social cue-based threat learning). We first demonstrated that guppies rapidly learn about threats paired with either alarm cues or with conspecific threat responses (demonstration). Then, focusing on acquisition rather than recall, we discovered that phospho-S6 expression, a marker of neural activity, was elevated in guppies during learning from alarm cues in the putative homologue of the mammalian lateral septum and the preoptic area. Surprisingly, these changes in neural activity were not observed in fish learning from conspecific demonstration. Together, these results implicate forebrain areas in social learning about threat but raise the possibility that circuits contribute to such learning in a stimulus-specific manner.

Keywords: anti-predator behaviour; conditioned threat learning; fear conditioning; pS6; social information; social learning.

Figure 1.

Overview of behavioural paradigms. (a) Training trial for Experiment 1. Fish were trained with one of three stimulus combinations: light + alarm cue (pictured), no light + alarm cue or light + water. Following a 2 min pre-stimulus period, fish (excluding the no light + alarm cue group) were presented with the light stimulus. Alarm cue (or water control) was presented 45 s after the onset of the light stimulus, which stayed on for a total of 2 min. (b) Training trial for Experiment 2. Fish were exposed to one of two demonstrator shoal types: ‘trained' demonstrators or ‘sham' demonstrators (4 demonstrators per tank). Following a two-min pre-stimulus period, fish were presented with the light stimulus for a total of two min. (c) Testing trial for both experiments. Following a two-min pre-stimulus period, isolated fish were presented with the light stimulus for two min. The proportion of time spent in the bottom third of the tank (substrate use) was recorded as a measure of defensive behaviour. (Online version in colour.)

Figure 2.

Fish exposed to paired light and alarm cue stimuli show increased defensive behaviour at test compared to controls (a), and differential neural activity during learning acquisition (b,c). (a) Experiment 1a: fish trained with a paired light + alarm cue stimulus increased substrate use when presented with the light stimulus alone during testing. Fish trained with a light + water stimulus decreased substrate use under testing conditions, and post-stimulus substrate use was similar between the two control conditions. Box plots show group medians with whiskers indicating upper and lower quartiles, points represent individuals’ data. (b) Experiment 1b: photomicrograph of DAPI staining (blue; top) and pS6 immunoreactivity (red) in area Vv. Top panel depicts 10× coronal image, with dashed box representing the approximate area examined for pS6 quantification. Bottom panels are representative images of pS6 expression in fish exposed to no cue versus light + alarm cue. Scale bars are 20 µm. (c) Experiment 1b: relative to light + water, alarm cue and no-cue controls, fish exposed to light + alarm cue pairings showed a significantly greater density of pS6-expressing neurons in areas Vv and POA. In the POA, no cue control fish showed significantly greater pS6 expression than no light + alarm cue control fish. ‘No cue' treatment applies only to Experiment 1b (c; see 'Material and methods'). Bar plot values represent square root transformed least square means (±s.e.m.). ***p < 0.001; **p < 0.01; *p < 0.05. (Online version in colour.)

Figure 3.

Fish exposed to trained demonstrators show increased defensive behaviour during testing compared to controls (a), but no significant differences in pS6 expression compared to both control groups during learning acquisition (b). (a) Experiment 2a: fish from both demonstration groups showed similar pre-stimulus substrate use during testing. Only fish previously exposed to trained demonstrators were observed to increase substrate use following light cue presentation during testing. Box plots show group medians with whiskers indicating upper and lower quartiles, points represent individuals’ data. (b) Experiment 2b: relative to fish exposed to sham demonstrators, fish exposed to uncued shoals or trained demonstrators showed significantly greater density of pS6-expressing neurons in area Vs. ‘Uncued Shoal' treatment applies only to Experiment 2b (b; see 'Material and methods'). Bar plot values represent square-root-transformed least-square means (±s.e.m.). **p< 0.01; *p < 0.05.