Limits...
Trace Eyeblink Conditioning in Mice Is Dependent upon the Dorsal Medial Prefrontal Cortex, Cerebellum, and Amygdala: Behavioral Characterization and Functional Circuitry(1,2,3).

Siegel JJ, Taylor W, Gray R, Kalmbach B, Zemelman BV, Desai NS, Johnston D, Chitwood RA - eNeuro (2015)

Bottom Line: To identify the circuitry involved, we made restricted lesions of the medial prefrontal cortex (mPFC) and found that learning was prevented.Anatomical data from these critical regions showed that mPFC and amygdala both project to the rostral basilar pons and overlap with eyelid-associated pontocerebellar neurons.The data further reveal a specific role for the amygdala as providing a conditioned stimulus-associated input to the cerebellum.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Learning and Memory, University of Texas at Austin , Austin, Texas 78712.

ABSTRACT
Trace eyeblink conditioning is useful for studying the interaction of multiple brain areas in learning and memory. The goal of the current work was to determine whether trace eyeblink conditioning could be established in a mouse model in the absence of elicited startle responses and the brain circuitry that supports this learning. We show here that mice can acquire trace conditioned responses (tCRs) devoid of startle while head-restrained and permitted to freely run on a wheel. Most mice (75%) could learn with a trace interval of 250 ms. Because tCRs were not contaminated with startle-associated components, we were able to document the development and timing of tCRs in mice, as well as their long-term retention (at 7 and 14 d) and flexible expression (extinction and reacquisition). To identify the circuitry involved, we made restricted lesions of the medial prefrontal cortex (mPFC) and found that learning was prevented. Furthermore, inactivation of the cerebellum with muscimol completely abolished tCRs, demonstrating that learned responses were driven by the cerebellum. Finally, inactivation of the mPFC and amygdala in trained animals nearly abolished tCRs. Anatomical data from these critical regions showed that mPFC and amygdala both project to the rostral basilar pons and overlap with eyelid-associated pontocerebellar neurons. The data provide the first report of trace eyeblink conditioning in mice in which tCRs were driven by the cerebellum and required a localized region of mPFC for acquisition. The data further reveal a specific role for the amygdala as providing a conditioned stimulus-associated input to the cerebellum.

No MeSH data available.


Related in: MedlinePlus

Acquisition rates, example behavior, and CR amplitudes for mice trained using a trace 50–250 protocol. A, CR rates for 16 mice over 12 training sessions. Note that mice showed very low spontaneous blink rates during acclimation sessions, during which no conditioning stimuli were presented (left graph). Most mice (12/16, black lines) learned to a criterion of >50% CRs within 12 sessions (median CR rate each session for learners is shown in red; light gray lines show mice that failed to meet criterion, and light red shows median CR rates for all 16 mice). When aligned to criterion sessions the data show that mice increased expression at similar rates (right graph, red markers denote median CR rates), even though the onset of learning varied across mice. B, Example acclimation and acquisition sessions for one mouse. Like all mice studied here, this mouse showed low spontaneous blink rates during acclimation and initial training sessions (left waterfall plots). Note the expression of CRs reached asymptotic levels by Acq 9 (right waterfall plots), whereas the amplitude of CRs continued to increase over several additional sessions (most clearly observed in overlaid probe trials, bottom graphs). C, Left graph shows the median (black line) and interquartile range (gray shaded area) for the amplitude of CRs measured from probe trials for the 12 learners. Most mice showed significant increases in the amplitude of CRs between initial expression and the last training session (middle graph, paired t test, t = 2.73, df = 11, p = 0.02; examples for 3 mice given in right graphs, including one of the two mice that showed the opposite trend, numbers indicate median amplitude for that session).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4596016&req=5

Figure 2: Acquisition rates, example behavior, and CR amplitudes for mice trained using a trace 50–250 protocol. A, CR rates for 16 mice over 12 training sessions. Note that mice showed very low spontaneous blink rates during acclimation sessions, during which no conditioning stimuli were presented (left graph). Most mice (12/16, black lines) learned to a criterion of >50% CRs within 12 sessions (median CR rate each session for learners is shown in red; light gray lines show mice that failed to meet criterion, and light red shows median CR rates for all 16 mice). When aligned to criterion sessions the data show that mice increased expression at similar rates (right graph, red markers denote median CR rates), even though the onset of learning varied across mice. B, Example acclimation and acquisition sessions for one mouse. Like all mice studied here, this mouse showed low spontaneous blink rates during acclimation and initial training sessions (left waterfall plots). Note the expression of CRs reached asymptotic levels by Acq 9 (right waterfall plots), whereas the amplitude of CRs continued to increase over several additional sessions (most clearly observed in overlaid probe trials, bottom graphs). C, Left graph shows the median (black line) and interquartile range (gray shaded area) for the amplitude of CRs measured from probe trials for the 12 learners. Most mice showed significant increases in the amplitude of CRs between initial expression and the last training session (middle graph, paired t test, t = 2.73, df = 11, p = 0.02; examples for 3 mice given in right graphs, including one of the two mice that showed the opposite trend, numbers indicate median amplitude for that session).

Mentions: Before training began, mice were first given experience on the running wheel (15–20 min for 1–2 d) followed by three baseline acclimation sessions (1 per day) to determine spontaneous blink rates while the mice became familiar with experimental procedures. During this phase, sessions were identical to those used during training except no conditioning stimuli were presented. Mice showed very low spontaneous blink rates during acclimation sessions (0–5%; Fig. 2A,B, left). With training, 75% of mice learned to express conditioned eyeblink responses (>50% CR rate) within 5–12 sessions, with asymptotic performance rates ranging between 65–95% (Fig. 2A, left graph). Conditioned responses were expressed unilaterally (verified in 2 mice, 75% and 85% CRs rates in trained eyes, 0% and 12% CR rates in untrained eyes, data not shown). Critically, startle responses (eyelid responses <50 ms after CS onset) were not observed in any of the mice (Figs. 1C, 2B). To ensure that the camera sampling rate and analysis was sensitive enough to have detected startle-associated eyelid responses, we presented four mice with the blue light CS (20 trials) followed by trials in which an intentionally startling tone was presented instead (Fig. 1E). Startle-associated eyelid responses to the light CS were never observed, even when sampling at 1500 fps, whereas startle responses to the tone were readily observed at 200 fps (Fig. 1E). Furthermore, downsampling from 1500 to 187 fps suggested that little if any information was lost at the lower sampling rate. Three of the four mice showed tone-evoked startle that was readily detected by our eyelid analysis at 200 fps (Fig. 1E, Mice A–C), whereas the fourth mouse did not show detectable eyelid startle responses even at the higher sampling rate (Mouse D). As a second measure, potential freezing in response to presentation of the light CS was analyzed in six mice in which wheel rotation was measured throughout the first training session. Trials preceded by wheel movement (for the 250 ms prior to CS onset, mean pre-CS speed = 4.4 ± 1.5 cm/s) did not show systematic decreases in speed during the 250 ms following CS onset (mean post-CS speed = 4.3 ± 1.4 cm/s, paired t = −1.31, df = 5, p = 0.25a; Fig. 1D), indicating that the mice were not freezing to the light CS.


Trace Eyeblink Conditioning in Mice Is Dependent upon the Dorsal Medial Prefrontal Cortex, Cerebellum, and Amygdala: Behavioral Characterization and Functional Circuitry(1,2,3).

Siegel JJ, Taylor W, Gray R, Kalmbach B, Zemelman BV, Desai NS, Johnston D, Chitwood RA - eNeuro (2015)

Acquisition rates, example behavior, and CR amplitudes for mice trained using a trace 50–250 protocol. A, CR rates for 16 mice over 12 training sessions. Note that mice showed very low spontaneous blink rates during acclimation sessions, during which no conditioning stimuli were presented (left graph). Most mice (12/16, black lines) learned to a criterion of >50% CRs within 12 sessions (median CR rate each session for learners is shown in red; light gray lines show mice that failed to meet criterion, and light red shows median CR rates for all 16 mice). When aligned to criterion sessions the data show that mice increased expression at similar rates (right graph, red markers denote median CR rates), even though the onset of learning varied across mice. B, Example acclimation and acquisition sessions for one mouse. Like all mice studied here, this mouse showed low spontaneous blink rates during acclimation and initial training sessions (left waterfall plots). Note the expression of CRs reached asymptotic levels by Acq 9 (right waterfall plots), whereas the amplitude of CRs continued to increase over several additional sessions (most clearly observed in overlaid probe trials, bottom graphs). C, Left graph shows the median (black line) and interquartile range (gray shaded area) for the amplitude of CRs measured from probe trials for the 12 learners. Most mice showed significant increases in the amplitude of CRs between initial expression and the last training session (middle graph, paired t test, t = 2.73, df = 11, p = 0.02; examples for 3 mice given in right graphs, including one of the two mice that showed the opposite trend, numbers indicate median amplitude for that session).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4596016&req=5

Figure 2: Acquisition rates, example behavior, and CR amplitudes for mice trained using a trace 50–250 protocol. A, CR rates for 16 mice over 12 training sessions. Note that mice showed very low spontaneous blink rates during acclimation sessions, during which no conditioning stimuli were presented (left graph). Most mice (12/16, black lines) learned to a criterion of >50% CRs within 12 sessions (median CR rate each session for learners is shown in red; light gray lines show mice that failed to meet criterion, and light red shows median CR rates for all 16 mice). When aligned to criterion sessions the data show that mice increased expression at similar rates (right graph, red markers denote median CR rates), even though the onset of learning varied across mice. B, Example acclimation and acquisition sessions for one mouse. Like all mice studied here, this mouse showed low spontaneous blink rates during acclimation and initial training sessions (left waterfall plots). Note the expression of CRs reached asymptotic levels by Acq 9 (right waterfall plots), whereas the amplitude of CRs continued to increase over several additional sessions (most clearly observed in overlaid probe trials, bottom graphs). C, Left graph shows the median (black line) and interquartile range (gray shaded area) for the amplitude of CRs measured from probe trials for the 12 learners. Most mice showed significant increases in the amplitude of CRs between initial expression and the last training session (middle graph, paired t test, t = 2.73, df = 11, p = 0.02; examples for 3 mice given in right graphs, including one of the two mice that showed the opposite trend, numbers indicate median amplitude for that session).
Mentions: Before training began, mice were first given experience on the running wheel (15–20 min for 1–2 d) followed by three baseline acclimation sessions (1 per day) to determine spontaneous blink rates while the mice became familiar with experimental procedures. During this phase, sessions were identical to those used during training except no conditioning stimuli were presented. Mice showed very low spontaneous blink rates during acclimation sessions (0–5%; Fig. 2A,B, left). With training, 75% of mice learned to express conditioned eyeblink responses (>50% CR rate) within 5–12 sessions, with asymptotic performance rates ranging between 65–95% (Fig. 2A, left graph). Conditioned responses were expressed unilaterally (verified in 2 mice, 75% and 85% CRs rates in trained eyes, 0% and 12% CR rates in untrained eyes, data not shown). Critically, startle responses (eyelid responses <50 ms after CS onset) were not observed in any of the mice (Figs. 1C, 2B). To ensure that the camera sampling rate and analysis was sensitive enough to have detected startle-associated eyelid responses, we presented four mice with the blue light CS (20 trials) followed by trials in which an intentionally startling tone was presented instead (Fig. 1E). Startle-associated eyelid responses to the light CS were never observed, even when sampling at 1500 fps, whereas startle responses to the tone were readily observed at 200 fps (Fig. 1E). Furthermore, downsampling from 1500 to 187 fps suggested that little if any information was lost at the lower sampling rate. Three of the four mice showed tone-evoked startle that was readily detected by our eyelid analysis at 200 fps (Fig. 1E, Mice A–C), whereas the fourth mouse did not show detectable eyelid startle responses even at the higher sampling rate (Mouse D). As a second measure, potential freezing in response to presentation of the light CS was analyzed in six mice in which wheel rotation was measured throughout the first training session. Trials preceded by wheel movement (for the 250 ms prior to CS onset, mean pre-CS speed = 4.4 ± 1.5 cm/s) did not show systematic decreases in speed during the 250 ms following CS onset (mean post-CS speed = 4.3 ± 1.4 cm/s, paired t = −1.31, df = 5, p = 0.25a; Fig. 1D), indicating that the mice were not freezing to the light CS.

Bottom Line: To identify the circuitry involved, we made restricted lesions of the medial prefrontal cortex (mPFC) and found that learning was prevented.Anatomical data from these critical regions showed that mPFC and amygdala both project to the rostral basilar pons and overlap with eyelid-associated pontocerebellar neurons.The data further reveal a specific role for the amygdala as providing a conditioned stimulus-associated input to the cerebellum.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Learning and Memory, University of Texas at Austin , Austin, Texas 78712.

ABSTRACT
Trace eyeblink conditioning is useful for studying the interaction of multiple brain areas in learning and memory. The goal of the current work was to determine whether trace eyeblink conditioning could be established in a mouse model in the absence of elicited startle responses and the brain circuitry that supports this learning. We show here that mice can acquire trace conditioned responses (tCRs) devoid of startle while head-restrained and permitted to freely run on a wheel. Most mice (75%) could learn with a trace interval of 250 ms. Because tCRs were not contaminated with startle-associated components, we were able to document the development and timing of tCRs in mice, as well as their long-term retention (at 7 and 14 d) and flexible expression (extinction and reacquisition). To identify the circuitry involved, we made restricted lesions of the medial prefrontal cortex (mPFC) and found that learning was prevented. Furthermore, inactivation of the cerebellum with muscimol completely abolished tCRs, demonstrating that learned responses were driven by the cerebellum. Finally, inactivation of the mPFC and amygdala in trained animals nearly abolished tCRs. Anatomical data from these critical regions showed that mPFC and amygdala both project to the rostral basilar pons and overlap with eyelid-associated pontocerebellar neurons. The data provide the first report of trace eyeblink conditioning in mice in which tCRs were driven by the cerebellum and required a localized region of mPFC for acquisition. The data further reveal a specific role for the amygdala as providing a conditioned stimulus-associated input to the cerebellum.

No MeSH data available.


Related in: MedlinePlus