Pigs as a new behavioral model for studying Pavlovian eyeblink conditioning

Intro Pigs have been an increasingly popular preclinical model in nutritional neuroscience, as their anatomy, physiology, and nutrition requirements are highly comparable to those of humans. Eyeblink conditioning is one of the most well-validated behavioral paradigms in neuroscience to study underlying mechanisms of learning and memory formation in the cerebellum. Eyeblink conditioning has been performed in many species but has never been done on young pigs. Therefore, our aim here was to develop and validate an eyeblink conditioning paradigm in young pigs. Method Eighteen intact male pigs were artificially reared from postnatal day 2 to 30. The eyeblink conditioning setup consisted of a sound-damping box with a hammock that pigs were placed in, which allowed the pig to remain comfortable yet maintain a typical range of head motion. In a delay conditioning paradigm, the conditional stimulus (CS) was a 550 ms blue light-emitting diode (LED), the unconditional stimulus (US) was a 50 ms eye air-puff, the CS-US interval was 500 ms. Starting at postnatal day 14, pigs were habituated for five days to the eyeblink conditioning setup, followed by 5 daily sessions of acquisition training (40 paired CS-US trials each day). Results The group-averaged amplitude of eyelid responses gradually increased over the course of the five days of training, indicating that pigs learned to make the association between the LED light CS and the air-puff US. A similar increase was found for the conditioned response (CR) probability: the group-averaged CR probability on session 1 was about 12% and reached a CR probability of 55% on day 5. The latency to CR peak time lacked a temporal preference in the first session, but clearly showed preference from the moment that animals started to show more CRs in session 2 and onwards whereby the eyelid was maximally closed exactly at the moment that the US would be delivered. Conclusion We concluded that 4-week-old pigs have the capability of performing in a cerebellar classical association learning task, demonstrating for the first time that eyeblink conditioning in young pigs has the potential to be a valuable behavioral tool to measure neurodevelopment.


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The use of pigs as an experimental animal model has been increasing in various fields, 56 including neuroscience (Gieling et (Odle et al., 2014). For these reasons, the use of young pigs as a translational model in 66 nutritional and developmental neuroscience is increasing (Mudd & Dilger, 2017).

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In the field of nutritional and developmental neuroscience, pigs have been tested in a variety 69 of behavioral tasks, including T-maze, radial arm maze, and novel object recognition focus on the function of the hippocampus and/or the neocortex. However, pigs have not been 72 extensively investigated in a task that focuses specifically on the cerebellar motor response 73 in eyeblink conditioning. Therefore, our aim was to establish procedures to conduct 74 Pavlovian eyeblink conditioning in pigs, which is a cerebellar-dependent learning task. 75 During eyeblink conditioning, subjects typically hear a short beep or see a light flash 76 (conditional stimulus, CS), followed several hundred milliseconds later by an air-puff on the 77 eye (unconditional stimulus, US). In a cerebellar-dependent 'delay paradigm', the CS and US 78 have different onset delays but co-terminate ( Figure 1A, B). As a result of repeated CS-US 79 pairings, subjects eventually associate the CS with US, and in anticipation of the US learn to 80 close their eyes in response to the CS. This anticipatory behavior to close the eye after the CS 81 but before the US is called the conditioned response (CR) (Ten Brinke, 2013; Heck et al., 2013;82 Freeman & Steinmetz, 2011). Eyeblink conditioning became a popular learning model 83 because it is simple in its form but is discrete in that it specifically measures associative and 84 sensory-motor learning (Heiney et al., 2014). As a result, the neural circuits and plasticity 85 mechanisms in cerebellum involved in eyeblink conditioning have been studied in very high 86 detail ( Figure 1C). Eyeblink  with a custom-designed hammock securely attached, in which the pig rested during trials.

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A blue light-emitting diode (LED) was attached inside the testing unit, approximately 10 cm 120 from the anticipated location of the pig's head. This blue LED light was used as a 121 conditional stimulus (CS). Additionally, an airline connected to a regulator was attached to 122 the pig's head, just below the eye, and it was used as an unconditional stimulus (US). These 123 pieces were attached using medical-grade surgical tape and glue ( Figure 1A). Individual   There were a total of 5 blocks of trials per day, and each block contained 1 US-only trial, 6 143 paired trials, and 1 CS-only trial. The first 500 ms of each trial was a baseline period, followed by the onset of CS ( Figure 1B). The onset of US was 1000 ms after the beginning of the trial 145 with 500 ms inter-stimulus interval (ISI), and US and CS co-terminated at 1050 ms after 50 146 ms of temporal overlap of CS and US. Inter-trial interval was determined by the following 147 criteria: (1) a random duration between 8-12 sec at the minimum must pass; (2) the eye  CR was present; (4) the latency to CR onset in trials wherein a CR was present; (5) the latency 180 to CR peak in trials wherein a CR was present.

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Because pigs responded with a partial eye opening to the CS at the start of training, we     Table 1). 228 The averaged FEC₁₅₀₋₅₀₀ showed a statistically significant effect of session (F(4,68) = 6.05, p 229 = 0.0003, ANOVA on LME). On average, pigs started with a FEC₁₅₀₋₅₀₀ of 0.12 (±0.06 95% CI) 230 on session 1 and reached a value of 0.39 (±0.12 95% CI) on day 5 ( Figure 3C, Table 1). 231 Similarly, for FEC₅₀₀ there was a main effect of day (F(4,48)=11.79, p<0.0001, ANOVA on 232 LME). On average, pigs started with a FEC500ms of -0.19 (±0.10 95% CI) on day 1 and 233 reached a value of 0.33 (±0.12 95% CI) at the end of training ( Figure 3D, Table 1). Note the 234 negative value on day 1 for FEC₅₀₀, reflecting the partial eyelid opening, that is not masked 235 when only looking at the FEC₁₅₀₋₅₀₀. Finally, we also found a small but significant main effect (2) latency in milliseconds to the maximum peak of the CR in the 246 interval between 150 and 500 ms after CS onset. Note that the onset of the air-puff US is at 247 500 ms after CS onset. In addition, we looked at the variability of the latencies to CR peak, 248 since we observed in the raw traces that over the course of training the timing of these CR 249 peaks became more precise, i.e., became more centered around the onset of the air-puff US.

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The latency to CR peak time lacked a temporal preference in the first session, but clearly 252 showed preference from the moment that animals started to show CRs more reliably in 253 session 2 and onwards. The averaged latency to CR peak showed a statistically significant  Figure 4D, Table 1). Based on these timing parameters of the eyeblink CR, we conclude 268 that pigs were able to adaptively time their eyeblink CR.

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The main purpose of the present study was to develop and validate the eyeblink conditioning 272 paradigm in young pigs. We found that pigs were indeed able to learn the eyeblink 273 conditioning task: both CR probability and the CR amplitude showed a gradual increase over the course of five days of training. Moreover, the eyeblink CR were properly timed, in the 275 sense that the eyelid was maximally closed exactly around the onset of the air-puff US, 276 herewith providing the optimal protection against the aversive air-puff while perturbing the   As mentioned above, we observed that pigs often responded with a further opening of the 303 eye in response to the CS during the first two training sessions (Figure 2A, B, Figure 3A). 304 Examination of the MDMT signal and videos during the eyeblink conditioning test taught us 305 that pigs often had the eyelid partly closed, whereby the upper eyelid was dropped down a 306 bit. This partial eye closure was the neutral position of the pig's eye and was not due to any human's life in terms of their total brain volume growth (Thibault & Margulies, 1998). Thus, 323 our 4-to-5-wk-old pigs can be considered as 4-to-5-month-old infants. Interestingly, infants 324 aged four to five months, or even younger, have shown their capability of performing delay 325 eyeblink conditioning ( Lintz et al., 1967;Ivkovich et al., 1999;Herbert et al., 2003). During 326 this critical period, delay eyeblink conditioning can serve as a sensitive behavioral paradigm 327 to study cerebellar development.

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The authors declare that the research was conducted in the absence of any commercial or 396 financial relationships that could be construed as a potential conflict of interest.  confidence interval. A statistically significant effect found of session was found for the SD of 656 the latency to CR peak, indicating that the variability in the CR peak times get smaller over 657