Optogenetic stimulation of mPFC pyramidal neurons as a conditioned stimulus supports associative learning in rats

Abstract

It is generally accepted that the associative learning occurs when a behaviorally neutral conditioned stimulus (CS) is paired with an aversive unconditioned stimulus (US) in close temporal proximity. Eyeblink conditioning (EBC) is a simple form of associative learning for motor responses. Specific activation of a population of cells may be an effective and sufficient CS for establishing EBC. However, there has been no direct evidence to support this hypothesis. Here, we show in rats that optogenetic activation of the right caudal mPFC pyramidal neurons as a CS is sufficient to support the acquisition of delay eyeblink conditioning (DEC). Interestingly, the associative memory was not stably expressed during the initial period of daily conditioning session even after the CR acquisition reached the asymptotic level. Finally, the intensity and consistency of the CS were found to be crucial factors in regulating the retrieval of the associative memory. These results may be of importance in understanding the neural cellular mechanisms underlying associative learning and the mechanisms underlying retrieval process of memory.

Figure 1. Selective labelling and optogenetic activation of the right caudal mPFC neurons.

(a) The rats were stereotactically injected with pAAV 2/8-CaMKIIα-ChR2-mCherry targeting the right caudal mPFC. (b) Example of ChR2-mCherry expression in the right caudal mPFC. (c) Representative images showing cell-specific ChR2-mCherry expression (red) in pyramidal neurons (green) of the right caudal mPFC. (d) Statistics of expression in the right caudal mPFC pyramidal neurons (502 cells, from five mice). (e) Percentage of c-Fos-positive cells among ChR2-mCherry-expressing cells (324/334 cells) or mCherry-expressing cells (21/320 cells) after light stimulation (n = 3 rats each; ^***P < 0.001; two-tailed unpaired Student’s t-test). (f) In vivo right caudal mPFC “optrode” recording setup. (g) Multi-unit activity in the right caudal mPFC from a rat injected with pAAV 2/8-CaMKIIα-ChR2-mCherry in response to trains of 7 light pulses (470 nm, 10 mW/mm², 20 Hz, 10 ms pulse duration). Blue bars represent light on. Data are represented as mean ± s.e.m.

Figure 2. Optogenetic stimulation CS supports the acquisition of associative eyeblink conditioning.

(a) Rats were implanted with stimulating electrodes in the subdermally caudal to the left eye for delivery of unconditioned stimuli (US) and with electrodes for recording the electromyographic (EMG) activity of the ipsilateral orbicularis oculi (O.O.) muscle. An optrode and a guide cannula were targeted to the right caudal mPFC for optical stimulation, for recording local field potentials, and for drug injection. (b) Rats were trained in a sound- and light-attenuating chamber. (c) Upper panel: the conditioning paradigm illustrating the timing of the CS and the US. Middle panel: representative O.O. EMG before learning. Lower panel: representative O.O. EMG after learning. (d, e) the CR% (d) and EMG response topographies (e) across two habituation and ten acquisition training sessions in ChR2/paired, ChR2/unpaired, and mCherry/paired groups ( = 8 rats each; ^* and ^# indicate significant differences between the ChR2/paired group and the ChR2/unpaired and mCherry/paired groups; ^*or ^#P < 0.05, ^** or ^##P < 0.01, ^*** or ^###P < 0.001; two-way ANOVA with repeated measures followed by Tukey post-hoc test). Data are represented as mean ± s.e.m. The rat drawing was drawn by Guang-yan Wu according to the present experiment.

Figure 3. Muscimol injection prevents the expression of CR.

(a) Injection of GABA_A receptor agonist muscimol (red) or ACSF (blue) into the right caudal mPFC. (b) Top: experimental scheme, Bottom: muscimol markedly impaired the CR expression compared with ACSF (left). However, this impairment was reversible when the same rats were retested 24 h later in the absence of muscimol (right; n = 9 rats for ACSF group and n = 10 rats for muscimol group; N.S., not significant, ^***P < 0.001; two-tailed unpaired Student’s t-test). Data are represented as mean ± s.e.m.

Figure 4. A period of time is required for stable performance of CR.

(a–c) Top: training and testing scheme. Bottom: modification of intertrial interval (ITI) had significant effect on trials to criterion (b), but had no effect on CR% (a) or latency to criterion (c; n = 9 rats each; N.S., not significant, ^*P < 0.05, ^***P < 0.001; one-way ANOVA followed by Tukey post-hoc test). (d–f) Top: training and testing scheme. Bottom: modification of the proportion of habituation and conditioning training had no effect on CR% (d), trials to criterion (e), or latency to criterion (f; n = 9 rats for 0-trial Hab., control and 40-trial Hab. groups, n = 8 rats for 20-trial Hab. group, and n = 9 rats for 30-trial Hab. group; N.S., not significant, one-way ANOVA followed by Tukey post-hoc test). Data are represented as mean ± s.e.m.

Figure 5. The CS intensity and consistency affect the memory retrieval.

(a–c) Top: training and testing scheme. Bottom: decreasing the intensity of cue produced significant deficits in DEC retrieval, but increasing the intensity of cue produced significant improvements in CR performance (n = 8 rats for both reduced 10 + 40 and enhanced 10 + 40 groups and n = 9 rats for Medium 10 + 40, control group; N.S., not significant, ^*P < 0.05; one-way ANOVA followed by Tukey post-hoc test). (d–f) Top: training and testing scheme. Bottom: changing the consistency of CS produced significant deficits in DEC retrieval (n = 8 rats for 2 + 8 group and n = 9 rats for Medium 10 + 40, control group; N.S., not significant, ^*P < 0.05, ^**P < 0.01; two-tailed unpaired Student’s t-test). (g–i) Top: training and testing scheme. Bottom: decreasing the intensity and consistency of cue produced significant deficits in DEC retrieval, whereas decreasing the consistency and increasing the intensity of cue produced significant improvements in CR performance (n = 8 rats for 5 + 45 group, n = 9 rats for Medium 10 + 40, control group, and n = 10 rats for 30 + 20 group; N.S., not significant, ^*P < 0.05; one-way ANOVA followed by Tukey post-hoc test). Data are represented as mean ± s.e.m.