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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

Acquisition of trace 50–250 is dependent on a subregion of mPFC in mice. A, CR rates during acclimation and training sessions (left graph) in mPFC-lesioned (light red lines) and sham-operated control mice (light black lines). Black and red markers show median CR rates observed each session for control and lesioned mice, respectively. Note that although a subset of lesioned mice learned, the median value indicates that most lesioned mice showed few if any CRs after 15 training days. Most lesioned nonlearners were able to acquire normal CRs for a non-mPFC-dependent delay eyeblink-conditioning task, showing that the deficit was specific to trace conditioning and did not reflect a global learning impairment in these mice (right graph, example behavior from 2 lesioned and 1 control mice are shown, denoted by red and black lines, respectively). B, CR rates for all lesioned mice, color-coded as lesioned nonlearners (red lines; Effective) and lesioned learners (blue lines; Ineffective). Median CR rates between control mice (right graph, black markers) and mice with ineffective mPFC lesions (blue markers) were similar, in contrast to mice that did not learn the task (red markers). C, Lesion reconstructions for all mPFC lesioned mice, separated into Effective (red, top row; n = 10) and Ineffective (blue, bottom row; n = 6) mice (opacity was adjusted for each mouse such that the total number of mice = 100%). Note that mice with damage to at least 500 µm of the medial agranular (AGm) and anterior cingulate (AC) between bregma +0.75–1.75 were not able to acquire the task (top, red shaded regions within bracketed sections), whereas mice with lesions that spared this area were able to learn (bottom). D, Example behavior from probe trials from effective lesions (red), ineffective lesioned learners (blue), and control mice (black). Most lesioned mice showed no learning (red, top 2 graphs). One lesioned mouse that showed some learning but did not meet criterion is also shown (red, bottom graph). Mice with lesions restricted to the most rostral mPFC (anterior to bregma +1.75) showed normal learning (blue, middle left graphs), whereas lesions restricted to the most caudal mPFC (posterior to bregma +0.75) showed abnormal CRs with uncharacteristic double-peaked responses (blue, middle right graphs) relative to control mice (black, right graphs). One example from a control mouse that did not learn is also shown (bottom right graph). Br, Bregma; AGm, medial agranular cortex; P, prelimbic cortex; AC, anterior cingulate cortex.
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Figure 6: Acquisition of trace 50–250 is dependent on a subregion of mPFC in mice. A, CR rates during acclimation and training sessions (left graph) in mPFC-lesioned (light red lines) and sham-operated control mice (light black lines). Black and red markers show median CR rates observed each session for control and lesioned mice, respectively. Note that although a subset of lesioned mice learned, the median value indicates that most lesioned mice showed few if any CRs after 15 training days. Most lesioned nonlearners were able to acquire normal CRs for a non-mPFC-dependent delay eyeblink-conditioning task, showing that the deficit was specific to trace conditioning and did not reflect a global learning impairment in these mice (right graph, example behavior from 2 lesioned and 1 control mice are shown, denoted by red and black lines, respectively). B, CR rates for all lesioned mice, color-coded as lesioned nonlearners (red lines; Effective) and lesioned learners (blue lines; Ineffective). Median CR rates between control mice (right graph, black markers) and mice with ineffective mPFC lesions (blue markers) were similar, in contrast to mice that did not learn the task (red markers). C, Lesion reconstructions for all mPFC lesioned mice, separated into Effective (red, top row; n = 10) and Ineffective (blue, bottom row; n = 6) mice (opacity was adjusted for each mouse such that the total number of mice = 100%). Note that mice with damage to at least 500 µm of the medial agranular (AGm) and anterior cingulate (AC) between bregma +0.75–1.75 were not able to acquire the task (top, red shaded regions within bracketed sections), whereas mice with lesions that spared this area were able to learn (bottom). D, Example behavior from probe trials from effective lesions (red), ineffective lesioned learners (blue), and control mice (black). Most lesioned mice showed no learning (red, top 2 graphs). One lesioned mouse that showed some learning but did not meet criterion is also shown (red, bottom graph). Mice with lesions restricted to the most rostral mPFC (anterior to bregma +1.75) showed normal learning (blue, middle left graphs), whereas lesions restricted to the most caudal mPFC (posterior to bregma +0.75) showed abnormal CRs with uncharacteristic double-peaked responses (blue, middle right graphs) relative to control mice (black, right graphs). One example from a control mouse that did not learn is also shown (bottom right graph). Br, Bregma; AGm, medial agranular cortex; P, prelimbic cortex; AC, anterior cingulate cortex.

Mentions: We next tested whether the 250 ms trace interval was indeed forebrain-dependent under these training conditions. Sixteen naïve mice received mPFC lesions before training. Half received lesions targeted rostral to the genu of the corpus callosum (bregma +1.5–2.5) and half received lesions targeted caudal to the genu (bregma +0.5–1.5). Sixteen corresponding control mice for the two groups received craniotomies but no tissue was aspirated. For sham control mice, 10/16 mice learned to a criterion of >50% CR rate within 15 sessions. Three mice that failed to meet this criterion showed some learning (20–45% CR rate), and the remaining three showed few or no CRs (<5%). In contrast, only 6/16 lesioned mice were able to learn the task. One lesioned mouse that did not meet the 50% criterion showed some learning (31% CR rate), however the majority of lesioned mice showed little or no learning (9/16 mice, 0–15% CR rate; Fig. 6A, Trace 50–250, left graph; median CR rate is shown for each training day between lesioned and sham controls to highlight that most lesioned mice overlap at 0% CR rates). The data for mPFC lesioned mice are shown separately in Figure 6B (left graph) to highlight the difference in learning curves between lesioned nonlearners (“Effective”, red lines) versus lesioned learners (“Ineffective:, blue lines). The acquisition rates of mice with ineffective lesions appeared similar to that observed for control learners across training days (Fig. 6B, right graph, compare blue to black symbols; effective lesion data are also shown for comparison).


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 of trace 50–250 is dependent on a subregion of mPFC in mice. A, CR rates during acclimation and training sessions (left graph) in mPFC-lesioned (light red lines) and sham-operated control mice (light black lines). Black and red markers show median CR rates observed each session for control and lesioned mice, respectively. Note that although a subset of lesioned mice learned, the median value indicates that most lesioned mice showed few if any CRs after 15 training days. Most lesioned nonlearners were able to acquire normal CRs for a non-mPFC-dependent delay eyeblink-conditioning task, showing that the deficit was specific to trace conditioning and did not reflect a global learning impairment in these mice (right graph, example behavior from 2 lesioned and 1 control mice are shown, denoted by red and black lines, respectively). B, CR rates for all lesioned mice, color-coded as lesioned nonlearners (red lines; Effective) and lesioned learners (blue lines; Ineffective). Median CR rates between control mice (right graph, black markers) and mice with ineffective mPFC lesions (blue markers) were similar, in contrast to mice that did not learn the task (red markers). C, Lesion reconstructions for all mPFC lesioned mice, separated into Effective (red, top row; n = 10) and Ineffective (blue, bottom row; n = 6) mice (opacity was adjusted for each mouse such that the total number of mice = 100%). Note that mice with damage to at least 500 µm of the medial agranular (AGm) and anterior cingulate (AC) between bregma +0.75–1.75 were not able to acquire the task (top, red shaded regions within bracketed sections), whereas mice with lesions that spared this area were able to learn (bottom). D, Example behavior from probe trials from effective lesions (red), ineffective lesioned learners (blue), and control mice (black). Most lesioned mice showed no learning (red, top 2 graphs). One lesioned mouse that showed some learning but did not meet criterion is also shown (red, bottom graph). Mice with lesions restricted to the most rostral mPFC (anterior to bregma +1.75) showed normal learning (blue, middle left graphs), whereas lesions restricted to the most caudal mPFC (posterior to bregma +0.75) showed abnormal CRs with uncharacteristic double-peaked responses (blue, middle right graphs) relative to control mice (black, right graphs). One example from a control mouse that did not learn is also shown (bottom right graph). Br, Bregma; AGm, medial agranular cortex; P, prelimbic cortex; AC, anterior cingulate cortex.
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Figure 6: Acquisition of trace 50–250 is dependent on a subregion of mPFC in mice. A, CR rates during acclimation and training sessions (left graph) in mPFC-lesioned (light red lines) and sham-operated control mice (light black lines). Black and red markers show median CR rates observed each session for control and lesioned mice, respectively. Note that although a subset of lesioned mice learned, the median value indicates that most lesioned mice showed few if any CRs after 15 training days. Most lesioned nonlearners were able to acquire normal CRs for a non-mPFC-dependent delay eyeblink-conditioning task, showing that the deficit was specific to trace conditioning and did not reflect a global learning impairment in these mice (right graph, example behavior from 2 lesioned and 1 control mice are shown, denoted by red and black lines, respectively). B, CR rates for all lesioned mice, color-coded as lesioned nonlearners (red lines; Effective) and lesioned learners (blue lines; Ineffective). Median CR rates between control mice (right graph, black markers) and mice with ineffective mPFC lesions (blue markers) were similar, in contrast to mice that did not learn the task (red markers). C, Lesion reconstructions for all mPFC lesioned mice, separated into Effective (red, top row; n = 10) and Ineffective (blue, bottom row; n = 6) mice (opacity was adjusted for each mouse such that the total number of mice = 100%). Note that mice with damage to at least 500 µm of the medial agranular (AGm) and anterior cingulate (AC) between bregma +0.75–1.75 were not able to acquire the task (top, red shaded regions within bracketed sections), whereas mice with lesions that spared this area were able to learn (bottom). D, Example behavior from probe trials from effective lesions (red), ineffective lesioned learners (blue), and control mice (black). Most lesioned mice showed no learning (red, top 2 graphs). One lesioned mouse that showed some learning but did not meet criterion is also shown (red, bottom graph). Mice with lesions restricted to the most rostral mPFC (anterior to bregma +1.75) showed normal learning (blue, middle left graphs), whereas lesions restricted to the most caudal mPFC (posterior to bregma +0.75) showed abnormal CRs with uncharacteristic double-peaked responses (blue, middle right graphs) relative to control mice (black, right graphs). One example from a control mouse that did not learn is also shown (bottom right graph). Br, Bregma; AGm, medial agranular cortex; P, prelimbic cortex; AC, anterior cingulate cortex.
Mentions: We next tested whether the 250 ms trace interval was indeed forebrain-dependent under these training conditions. Sixteen naïve mice received mPFC lesions before training. Half received lesions targeted rostral to the genu of the corpus callosum (bregma +1.5–2.5) and half received lesions targeted caudal to the genu (bregma +0.5–1.5). Sixteen corresponding control mice for the two groups received craniotomies but no tissue was aspirated. For sham control mice, 10/16 mice learned to a criterion of >50% CR rate within 15 sessions. Three mice that failed to meet this criterion showed some learning (20–45% CR rate), and the remaining three showed few or no CRs (<5%). In contrast, only 6/16 lesioned mice were able to learn the task. One lesioned mouse that did not meet the 50% criterion showed some learning (31% CR rate), however the majority of lesioned mice showed little or no learning (9/16 mice, 0–15% CR rate; Fig. 6A, Trace 50–250, left graph; median CR rate is shown for each training day between lesioned and sham controls to highlight that most lesioned mice overlap at 0% CR rates). The data for mPFC lesioned mice are shown separately in Figure 6B (left graph) to highlight the difference in learning curves between lesioned nonlearners (“Effective”, red lines) versus lesioned learners (“Ineffective:, blue lines). The acquisition rates of mice with ineffective lesions appeared similar to that observed for control learners across training days (Fig. 6B, right graph, compare blue to black symbols; effective lesion data are also shown for comparison).

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