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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

Apparatus and equipment configuration used for trace eyeblink conditioning in head-fixed mice (A), analysis of high-speed video (B), and example behavioral sessions from two mice (C). Mice did not show freezing (D) or startle responses (E) to presentation of a blue light CS. A, Mice were surgically implanted with a headplate to allow for fixation on a running wheel during training. CS included a blue LED CS and an air puff US. Eyelid behavior was monitored during trials with an infrared sensitive high-speed camera (200–250 fps). The black fur around the eye was illuminated with infrared light to contrast with a mouse’s eye and allow for analysis (see below). B, Standard trace conditioning trials consisted of a 200 ms baseline, 50 ms blue light CS, and a 250 ms stimulus-free trace interval followed by a 20 ms air puff US (top). Each frame of a trial was analyzed by calculating the ratio of white–black pixels within a specified region of interest (green rectangle in example sample frames; dashed lines show borders of white and black thresholded pixels), yielding the FEC at each sampled time point during a trial (right graphs; upward deflection indicates closure, red dashed line shows the minimum FEC to qualify as a CR, 0.1). Initially the mouse closed his eye only in response to the air puff (top), but with continued training learned to close his eye in response to the light CS and in anticipation of the air puff (bottom, CR). C, Waterfall plots show each trial of a session from two well trained mice (blue indicates presentation of light CS, gray indicates presentation of air puff US). Training sessions consisted of 60 trials, with CS-only probe trials presented every fifth trial (arrows) to allow for analysis of CRs in the absence of reflexive US responses. Note the upward deflection of the black lines prior to the air puff presentation for many of the trials, indicative of a CR. Bottom graphs show probe trials from the example sessions, which were used to calculate the latencies and amplitude of CRs for each mouse/session (see text). Note the absence of apparent startle responses just after CS presentation and the ramping topography of eyelid closures such that the peaks of CRs were well timed to US presentation (which were not presented during probe trials). D, Example wheel-running behavior from one mouse during its first trace eyeblink conditioning session (pseudocolor plot, each row is one trial, “+” indicates trials in which mouse was running before CS onset and included in analysis), and the averaged momentary speed before and after CS presentation (middle graph, median ± IQR). Bottom graph shows the averaged momentary speed for six mice (black lines), indicating that mice did not decrease locomotion (freeze) in response to the light CS (paired t = −1.31, df = 5, p = 0.25, n.s.). E. Example eyelid responses from four mice subjected to a startle-eliciting tone (top and bottom rows) or blue light CS (middle row), sampling at either 200 or 1500 fps as indicated. Startle responses to the tone were clearly detected when sampling at 200 fps, and were never observed in response to the light CS, even when sampling at 1500 fps.
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Figure 1: Apparatus and equipment configuration used for trace eyeblink conditioning in head-fixed mice (A), analysis of high-speed video (B), and example behavioral sessions from two mice (C). Mice did not show freezing (D) or startle responses (E) to presentation of a blue light CS. A, Mice were surgically implanted with a headplate to allow for fixation on a running wheel during training. CS included a blue LED CS and an air puff US. Eyelid behavior was monitored during trials with an infrared sensitive high-speed camera (200–250 fps). The black fur around the eye was illuminated with infrared light to contrast with a mouse’s eye and allow for analysis (see below). B, Standard trace conditioning trials consisted of a 200 ms baseline, 50 ms blue light CS, and a 250 ms stimulus-free trace interval followed by a 20 ms air puff US (top). Each frame of a trial was analyzed by calculating the ratio of white–black pixels within a specified region of interest (green rectangle in example sample frames; dashed lines show borders of white and black thresholded pixels), yielding the FEC at each sampled time point during a trial (right graphs; upward deflection indicates closure, red dashed line shows the minimum FEC to qualify as a CR, 0.1). Initially the mouse closed his eye only in response to the air puff (top), but with continued training learned to close his eye in response to the light CS and in anticipation of the air puff (bottom, CR). C, Waterfall plots show each trial of a session from two well trained mice (blue indicates presentation of light CS, gray indicates presentation of air puff US). Training sessions consisted of 60 trials, with CS-only probe trials presented every fifth trial (arrows) to allow for analysis of CRs in the absence of reflexive US responses. Note the upward deflection of the black lines prior to the air puff presentation for many of the trials, indicative of a CR. Bottom graphs show probe trials from the example sessions, which were used to calculate the latencies and amplitude of CRs for each mouse/session (see text). Note the absence of apparent startle responses just after CS presentation and the ramping topography of eyelid closures such that the peaks of CRs were well timed to US presentation (which were not presented during probe trials). D, Example wheel-running behavior from one mouse during its first trace eyeblink conditioning session (pseudocolor plot, each row is one trial, “+” indicates trials in which mouse was running before CS onset and included in analysis), and the averaged momentary speed before and after CS presentation (middle graph, median ± IQR). Bottom graph shows the averaged momentary speed for six mice (black lines), indicating that mice did not decrease locomotion (freeze) in response to the light CS (paired t = −1.31, df = 5, p = 0.25, n.s.). E. Example eyelid responses from four mice subjected to a startle-eliciting tone (top and bottom rows) or blue light CS (middle row), sampling at either 200 or 1500 fps as indicated. Startle responses to the tone were clearly detected when sampling at 200 fps, and were never observed in response to the light CS, even when sampling at 1500 fps.

Mentions: Head-fixed mice were trained in custom-built sound-attenuating chambers. Animals were positioned on a cylindrical wheel that allowed them to run freely, according to the methods of Medina and colleagues (Fig. 1A; Chettih et al., 2011; Heiney et al., 2014). Training chambers contained an infrared light source for video illumination and a high-speed infrared camera (Prosilica GC 650 or GE 680, Allied Vision Technologies) to capture eyelid position at 200 or 250 fps, respectively, which was counterbalanced within training groups. Higher sampling rates than the maximum full-frame rate possible for each camera were achieved by specifying a subframe that surrounded the eye from the full frame and only sampling from that subframe during trials (Fig. 1B). Care was taken to ensure that the infrared light used to illuminate the fur surrounding the eye could not induce photothermal retinal damage by using a dispersed infrared LED array (FY-4748, Shenzhen Feyond Technology). The CS was delivered with a blue LED (HLMP-AB74-WXBDD, Avago Technologies; current = 6.2 mA at 2.78 V) mounted in view of both eyes 3–5 cm in front of and above the head. The air puff US was delivered using a 24 gauge stainless steel cannula positioned 2–4 mm lateral to the right eye (Fig. 1A). Each training chamber also included a loudspeaker to provide white noise during training (65 dB at the site of head fixation).


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)

Apparatus and equipment configuration used for trace eyeblink conditioning in head-fixed mice (A), analysis of high-speed video (B), and example behavioral sessions from two mice (C). Mice did not show freezing (D) or startle responses (E) to presentation of a blue light CS. A, Mice were surgically implanted with a headplate to allow for fixation on a running wheel during training. CS included a blue LED CS and an air puff US. Eyelid behavior was monitored during trials with an infrared sensitive high-speed camera (200–250 fps). The black fur around the eye was illuminated with infrared light to contrast with a mouse’s eye and allow for analysis (see below). B, Standard trace conditioning trials consisted of a 200 ms baseline, 50 ms blue light CS, and a 250 ms stimulus-free trace interval followed by a 20 ms air puff US (top). Each frame of a trial was analyzed by calculating the ratio of white–black pixels within a specified region of interest (green rectangle in example sample frames; dashed lines show borders of white and black thresholded pixels), yielding the FEC at each sampled time point during a trial (right graphs; upward deflection indicates closure, red dashed line shows the minimum FEC to qualify as a CR, 0.1). Initially the mouse closed his eye only in response to the air puff (top), but with continued training learned to close his eye in response to the light CS and in anticipation of the air puff (bottom, CR). C, Waterfall plots show each trial of a session from two well trained mice (blue indicates presentation of light CS, gray indicates presentation of air puff US). Training sessions consisted of 60 trials, with CS-only probe trials presented every fifth trial (arrows) to allow for analysis of CRs in the absence of reflexive US responses. Note the upward deflection of the black lines prior to the air puff presentation for many of the trials, indicative of a CR. Bottom graphs show probe trials from the example sessions, which were used to calculate the latencies and amplitude of CRs for each mouse/session (see text). Note the absence of apparent startle responses just after CS presentation and the ramping topography of eyelid closures such that the peaks of CRs were well timed to US presentation (which were not presented during probe trials). D, Example wheel-running behavior from one mouse during its first trace eyeblink conditioning session (pseudocolor plot, each row is one trial, “+” indicates trials in which mouse was running before CS onset and included in analysis), and the averaged momentary speed before and after CS presentation (middle graph, median ± IQR). Bottom graph shows the averaged momentary speed for six mice (black lines), indicating that mice did not decrease locomotion (freeze) in response to the light CS (paired t = −1.31, df = 5, p = 0.25, n.s.). E. Example eyelid responses from four mice subjected to a startle-eliciting tone (top and bottom rows) or blue light CS (middle row), sampling at either 200 or 1500 fps as indicated. Startle responses to the tone were clearly detected when sampling at 200 fps, and were never observed in response to the light CS, even when sampling at 1500 fps.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Apparatus and equipment configuration used for trace eyeblink conditioning in head-fixed mice (A), analysis of high-speed video (B), and example behavioral sessions from two mice (C). Mice did not show freezing (D) or startle responses (E) to presentation of a blue light CS. A, Mice were surgically implanted with a headplate to allow for fixation on a running wheel during training. CS included a blue LED CS and an air puff US. Eyelid behavior was monitored during trials with an infrared sensitive high-speed camera (200–250 fps). The black fur around the eye was illuminated with infrared light to contrast with a mouse’s eye and allow for analysis (see below). B, Standard trace conditioning trials consisted of a 200 ms baseline, 50 ms blue light CS, and a 250 ms stimulus-free trace interval followed by a 20 ms air puff US (top). Each frame of a trial was analyzed by calculating the ratio of white–black pixels within a specified region of interest (green rectangle in example sample frames; dashed lines show borders of white and black thresholded pixels), yielding the FEC at each sampled time point during a trial (right graphs; upward deflection indicates closure, red dashed line shows the minimum FEC to qualify as a CR, 0.1). Initially the mouse closed his eye only in response to the air puff (top), but with continued training learned to close his eye in response to the light CS and in anticipation of the air puff (bottom, CR). C, Waterfall plots show each trial of a session from two well trained mice (blue indicates presentation of light CS, gray indicates presentation of air puff US). Training sessions consisted of 60 trials, with CS-only probe trials presented every fifth trial (arrows) to allow for analysis of CRs in the absence of reflexive US responses. Note the upward deflection of the black lines prior to the air puff presentation for many of the trials, indicative of a CR. Bottom graphs show probe trials from the example sessions, which were used to calculate the latencies and amplitude of CRs for each mouse/session (see text). Note the absence of apparent startle responses just after CS presentation and the ramping topography of eyelid closures such that the peaks of CRs were well timed to US presentation (which were not presented during probe trials). D, Example wheel-running behavior from one mouse during its first trace eyeblink conditioning session (pseudocolor plot, each row is one trial, “+” indicates trials in which mouse was running before CS onset and included in analysis), and the averaged momentary speed before and after CS presentation (middle graph, median ± IQR). Bottom graph shows the averaged momentary speed for six mice (black lines), indicating that mice did not decrease locomotion (freeze) in response to the light CS (paired t = −1.31, df = 5, p = 0.25, n.s.). E. Example eyelid responses from four mice subjected to a startle-eliciting tone (top and bottom rows) or blue light CS (middle row), sampling at either 200 or 1500 fps as indicated. Startle responses to the tone were clearly detected when sampling at 200 fps, and were never observed in response to the light CS, even when sampling at 1500 fps.
Mentions: Head-fixed mice were trained in custom-built sound-attenuating chambers. Animals were positioned on a cylindrical wheel that allowed them to run freely, according to the methods of Medina and colleagues (Fig. 1A; Chettih et al., 2011; Heiney et al., 2014). Training chambers contained an infrared light source for video illumination and a high-speed infrared camera (Prosilica GC 650 or GE 680, Allied Vision Technologies) to capture eyelid position at 200 or 250 fps, respectively, which was counterbalanced within training groups. Higher sampling rates than the maximum full-frame rate possible for each camera were achieved by specifying a subframe that surrounded the eye from the full frame and only sampling from that subframe during trials (Fig. 1B). Care was taken to ensure that the infrared light used to illuminate the fur surrounding the eye could not induce photothermal retinal damage by using a dispersed infrared LED array (FY-4748, Shenzhen Feyond Technology). The CS was delivered with a blue LED (HLMP-AB74-WXBDD, Avago Technologies; current = 6.2 mA at 2.78 V) mounted in view of both eyes 3–5 cm in front of and above the head. The air puff US was delivered using a 24 gauge stainless steel cannula positioned 2–4 mm lateral to the right eye (Fig. 1A). Each training chamber also included a loudspeaker to provide white noise during training (65 dB at the site of head fixation).

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