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5-HT2a receptor in mPFC influences context-guided reconsolidation of object memory in perirhinal cortex

  1. Juan Facundo Morici
  2. Magdalena Miranda
  3. Francisco Tomás Gallo
  4. Belén Zanoni
  5. Pedro Bekinschtein
  6. Noelia V Weisstaub  Is a corresponding author
  1. Facultad de Medicina, Universidad de Buenos Aires, CONICET, Argentina
  2. Universidad Favaloro, INECO, CONICET, Argentina
  3. Universidad de Buenos Aires, CONICET, Argentina
Research Article
Cite this article as: eLife 2018;7:e33746 doi: 10.7554/eLife.33746
6 figures, 1 video, 1 table and 5 additional files

Figures

Figure 1 with 2 supplements
Reconsolidation of OIC memory traces in PRH is susceptible to manipulation of 5-HT2aR.

(a) mPFC 5-HT2aR blockade alters object reconsolidation in PRH. Upper: Schematic representation of infusions and OIC paradigm in rats. Lower panels: Discrimination indexes during test phases, 24 and 48 hrs after the training sessions. Left: Infusion of MDL in mPFC impairs discrimination between contextually congruent and incongruent objects during test 1. Unpaired Student's t test (n = 12 per group), ***p<0.0001. Right: Infusion of EME in PRH immediately after test affects memory retention evaluated 24 hrs later (test 2). Infusion of MDL in mPFC before test 1 modified the memories affected by EME in PRH. Two-way ANOVA followed by Bonferroni post-hoc test, ***pinteraction <0.0001 (n = 6 per group). (b) Genetic deletion of 5-HT2aR influences reconsolidation in PRH. Upper: Schematic representation of infusions and OIC paradigm in mice. Lower panels: Discrimination indexes during test phases, 24 and 48 hrs after training sessions. Left: Discrimination index for Test 1. Genetic deletion of 5-HT2aR (KO) affects discrimination between contextually congruent and incongruent objects compared with wild type (WT) mice (Morici et al., 2015b). Unpaired Student's t test, **p=0.0023 (n = 12–14 per group). Right: Discrimination index for test 2. The constitutive and global absence of 5-HT2aR expression alters the memory traces affected by EME infusion in PRH. Two-way ANOVA followed by Bonferroni post-hoc test, **pinteraction <0.0029 (n = 6–7 per group). (c) mPFC 5-HT2aR blockade affects Zif268-dependent reconsolidation pattern of object memories in PRH. Upper panel: Schematic representation of infusions (ODNs in PRH and MDL/VEH in mPFC) and OIC paradigm. Lower panels: Discrimination indexes during test phases, 24 and 48 hrs after training sessions. Left: Discrimination index obtained during test 1, ***p<0.0001, unpaired Student's t test (n = 13–14 per group). Right: Infusion of Zif268 ASO in PRH after test 1 impaired memory retention evaluated during test 2. Previous infusion of MDL in mPFC modified the pattern of memory impairment induced by infusion of Zif268-ASO in PRH, *pinteraction = 0.049, Two-way ANOVA followed by Bonferroni post-hoc test (n = 6–7 per group). (d) Left: Representative spread of 1 nmol of biotinylated ODN, 90 min post injection in the PRH. Right: Zif268-ASO injection decreases Zif268 protein levels. Top: representative Zif268 immunoblots of PRH protein extracts prepared 90 min after ODN infusion; actin was used as a loading control. Bottom: Quantification of Zif268 immunoblots. Actin-normalized Zif268 protein levels are shown, relative to the mean of the Zif268-MSO in the PRH of non-trained animals. **p=0.0055, unpaired Student's t test (n = 4 per group); data represent mean ±SEM. A and B represent objects presented to the animals in each session. During test sessions, A represents the contextually congruent and B the contextually incongruent objects.

https://doi.org/10.7554/eLife.33746.002
Figure 1—figure supplement 1
Schematic representation of infusion site and coronal section showing the trace of the cannula.

This figure corresponds with the main Figures 2, 3, 4 and 5. Each dot placed in the figure of (a) mPFC, (b) PRH, (c) dCA1 and (d) vCA1 represents the spreading of the methyl blue infusion in the brains of different rats. (e) Representative coronal section of mouse brain showing the track of the cannula and PRH infusion sites.

https://doi.org/10.7554/eLife.33746.003
Figure 1—figure supplement 2
Absolute exploration times reflect the amnesic effect of EME infusion after test 1.

Exploratory time of objects A or B relative to the total exploratory time (A + C or B + D) obtained during test 2. EME affects the exploratory time compared with the vehicle group in a structure-dependent manner and reflects the EME effect evaluated with discrimination indexes. Data represent mean ±SEM. Paired t test, α <0.05, ***p<0.001, **p<0.01, *p<0.05. (a) EME was infused in the PRH after test 1. Differences in the exploratory time of object A but not object B were observed in this case. (b) EME was infused in the dHPC after test 1. Differences in the exploratory time of object B were observed. Interestingly in this case, a significant difference in the exploratory time for object A was also observed. The direction of the change is the opposite to what was seen for object B (decreased exploratory time of EME compared with the VEH group), suggesting that they have a better recognition of this object. Although we did not perform further experiments, this could be interpreted as if blocking the reconsolidation of object B increased the recognition of object A. This figure corresponds to the main Figure 4.

https://doi.org/10.7554/eLife.33746.004
Boundary conditions for reconsolidation in the OIC task.

(a) Upper panel: Schematic representation of infusions and behavioral paradigm. Rats were trained in the OIC paradigm but during test 1 were exposed to two copies of the contextually congruent object A. Immediately after test 1, rats were infused with VEH or EME in PRH and then exposed to the rest of the OIC task. Lower panel: Infusion of EME in PRH immediately after test 1 did not affect memory retention for any of the original objects. (n = 5 per group). (b) Upper panel: Schematic representation of infusions and behavioral paradigm. Rats were trained in the OIC paradigm. 24 hrs later, half the animals were exposed to a copy of the previously presented objects (A and B) and the other half were returned to their homecage. 24 hrs later memory retention was tested. Panel: Discrimination index for test 2 for object A. *pinteractionA <0.0216, Two-way ANOVA followed by Bonferroni post-hoc test. Right: Discrimination index for test 2 for object B (n = 5 per group). Horizontal stripes represent no re-exposure group. Data represent mean ±SEM. A and B represent objects presented to the animals in each session. During test sessions, A represents the contextually congruent and B the contextually incongruent objects.

https://doi.org/10.7554/eLife.33746.005
Contextual information drives object memory reconsolidation in PRH.

(a) Schematic representation of infusions and behavioral paradigm. Rats were trained in the OIC task. For test 1, half the animals continued with the OIC protocol. The other half were exposed to one of the contexts alone. Immediately after test 1, rats were infused with VEH or EME in PRH. During test 2, memories of the original objects (A and B) were evaluated. Lower panels: Discrimination index for test 2. Left: OIC group. *pdrug = 0.0197, pinteraction = 0.0593, Two-way ANOVA followed by Bonferroni post-hoc test. Right: Context-only group. ***pinteraction = 0.0003, Two-way ANOVA followed by Bonferroni post-hoc test (n = 6 per group). (b) Schematic representation of infusions and behavioral paradigm. Animals were trained in the OIC task and then divided into two groups. Half of the animals continued with the OIC task. The other half were given copies of objects A and B presented in a familiar way, but had not been previously associated with any of them. Immediately after test 1, rats were infused with VEH or EME in PRH. The black rectangle indicates a previously familiarized context that was not used during the training session, so not associated with the objects. Lower panels Discrimination index for test 2. Left: OIC group. ***p=0.0006, Two-way ANOVA followed by Bonferroni post-hoc test. Right: Not-associated group (n = 6 per group). (c) Upper: Schematic representation of infusions and behavioral paradigm. Rats were trained in the OIC task but during test 1 the contextually incongruent object was replace by a completely novel object transforming the paradigm into a SNOR task. Infusions into mPFC (VEH or MDL) and PRH (VEH or EME) were performed as indicated in the scheme. Lower panels: Discrimination index for test sessions. Left: Discrimination index for test 1. Right: Discrimination index for test 2. *** pinteraction <0.0001, (n = 4–6 per group), Two-way ANOVA followed by Bonferroni post-hoc test. Data represent mean ±SEM. A and B represent objects presented to the animals in each session. During test sessions, A represents the contextually congruent and B the contextually incongruent objects.

https://doi.org/10.7554/eLife.33746.006
mPFC 5-HT2aR blockade has no effect on reconsolidation of object memory traces in the dCA1.

(a) Upper panel: Schematic representation of infusions and OIC paradigm in rats. Lower panels: Discrimination indexes during test phases, 24 and 48 hrs after the training sessions. Lower left: Infusion of MDL in mPFC impairs discrimination between contextually congruent and incongruent objects during the test 1. ***p<0.0001, (n = 13–14 per group) unpaired Student's t test. Lower right: Infusion of EME in dCA1 immediately after test 1 affects memory retention evaluated 24 hrs later (test 2). Infusion of MDL in mPFC before test 1 did not modify the pattern of memories affected by EME. ***pinteraction <0.0001, (n = 6–7 per group), Two-way ANOVA followed by Bonferroni post-hoc test. (b) Upper panel: Schematic representation of infusions and behavioral paradigm. Rats were trained in the OIC paradigm but during test 1 were exposed to two copies of the contextually congruent object A. Immediately after test 1, rats were infused with VEH or EME in dCA1 and then exposed to the rest of the OIC task. Lower panel: Infusion of EME in dCA1 immediately after test 1 did not affect memory retention for any of the original objects (n = 5 per group). (c) mPFC 5-HT2aR blockade does not affect Zif268-dependent reconsolidation pattern of object memories in the dCA1. Upper panel: Schematic representation of infusions (ODNs in PRH and MDL/VEH in mPFC) and OIC paradigm. Lower panels: Discrimination indexes during test phases, 24 and 48 hrs after training sessions. Left: Discrimination index obtained during test 1 ***p<0.0001, (n = 14 per group), unpaired Student's t test. Right: Discrimination index obtained during test 2. *pinteraction = 0.03, ***p<0.0001, (n = 7 per group), Two-way ANOVA followed by Bonferroni post-hoc test. (d) Left: Representative spread of 1 nmol of biotinylated ODN, 90 min post injection in the dCA1. Right: Zif268-ASO injection decreases Zif268 protein levels. Top: representative Zif268 immunoblots of dCA1 protein extracts prepared 90 min after ODN infusion; actin was used as a loading control. Bottom: Quantification of Zif268 immunoblots. Actin-normalized Zif268 protein levels are shown, relative to the mean of the Zif268-MSO in the dCA1 of non-trained animals. **p=0.0019, (n = 5 per group), unpaired Student's t test. Data represent mean ±SEM. A and B represent objects presented in each session. During test sessions, A represents the contextually congruent and B the contextually incongruent objects.

https://doi.org/10.7554/eLife.33746.007
Interaction between mPFC and vCA1 is required for the control of memory retrieval and reconsolidation in the PRH.

(a) Activity of vCA1 is necessary for the correct resolution of OIC task. Upper panel: Schematic representation of infusions and behavioral paradigm. Animals were trained in the OIC task and paradigm. Bilateral infusions of MUS were made in vCA1 before test 1. Emetine was infused in PRH immediately after test 1. Lower panels. Discrimination index obtained during test sessions. Left: Test 1. ***p<0.0001, (n = 13–15 per group), unpaired Student's t test. Right: Test 2. ***pinteraction <0.0001, (n = 6–7 per group), Two-way ANOVA followed by Bonferroni post-hoc test. (b) Resolution of SNOR does not require vCA1 activity. Left panel: Schematic representation of infusions and SNOR version paradigm. Rats were trained in the SNOR task and infused with MUS immediately before test 1. Right panel: Discrimination index for test 1 (n = 5–6 per group). (c) vCA1 interacts with mPFC 5-HT2a to correctly solve the OIC task. Upper panel: Schematic representation of infusions and behavioral paradigm. Rats were trained in the OIC task, 15 min prior to test 1 MDL in the mPFC and MUS in vCA1 were infused in ipsi or contra-lateral hemispheres. For all the animals emetine was infused in PRH immediately after test 1. Lower panels: Discrimination indexes obtained during the test sessions. Left: Test 1. ***p<0.0001, (n = 16 per group), unpaired Student's t test. Right: Test 2. ***pinteraction <0.0001, (n = 8 per group), Two-way ANOVA followed by Bonferroni post-hoc test. Data represent mean ±SEM. A and B represent objects presented to the animals in each session. During test sessions, A represents the contextually congruent and B the contextually incongruent objects.

https://doi.org/10.7554/eLife.33746.008
Schematic representation of functional connectivity of some of the structures involved in the retrieval and reconsolidation of OIC.

mPFC and vCA1 interaction is required to control object memory trace retrieval and reconsolidation in PRH. The contextual information flows from the HPC (dCA1→vCA1) to mPFC. This information is used during retrieval by the mPFC to control the object memory traces expression and behavioral performance, as well as to influence the reconsolidation of object memories in the PRH. The arrows represent the flow of information suggested in this model. The discontinued lines represent the proposed interactions between structures (adapted from [Preston and Eichenbaum, 2013]).

https://doi.org/10.7554/eLife.33746.009

Videos

Video 1
Representative fragment of a test 1 session.

The rat was exposed to copies of objects A and B and allowed to freely explore them. Exploration of the objects was defined as directing the nose to the object at a distance of <2 cm and/or touching it with the nose. Turning around or sitting on the object was not considered exploratory behavior.

https://doi.org/10.7554/eLife.33746.010

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
strain, strain backgroundWistar rats, 129 Sv/Ev miceFacultad de Ciencias Exactas y Naturales, UBA Animal Facility and in house animal facility for the mice. Mice are original from Taconic
antibodyzif268, actinaSanta CruzSc189, sc1615
sequence-based reagentZif268, antisenseSigma80103076030020 HAO2389671
commercial assay or kitvectastin ABC kitVector labPK 6100
chemical compound, drugMDL 11,939Tocris#0870/10
chemical compound, drugEmetineSigma#7083-71-8
chemical compound, drugMuscimolSigma#2763-96-4
software, algorithmGraphPadGraphPad sofware

Additional files

Supplementary file 1

Exploration time for each object presented during test 2.

Mean ± SEM exploratory time for each object (A, B, C and D) presented during test 2. Raw exploratory times were analyzed separately by animals infused with VEH or MDL. Two-way ANOVA followed by Sidak's multiple comparisons were made, where one factor was the object (A vs. C and B vs. D) and the other factor was the treatment (e.g. emetine vs. vehicle). α <0.05, ****p<0.0001, ***p<0.001,**p<0.01, *p<0.05. For the experiment described in Figure 3a , the data were transformed because they did not follow a normal distribution.

https://doi.org/10.7554/eLife.33746.011
Supplementary file 2

Animals excluded based on the exclusion criteria.

Animals that explored any of the objects less than 5 s during any of test phases were excluded from the experiment.

https://doi.org/10.7554/eLife.33746.012
Supplementary file 3

Total exploration time during test 1.

Total exploratory time is presented for experiments in which a pharmacological or genetic manipulation is presented. No treatment effect is observed. Data represent mean ± SEM. Unpaired t test, α <0,05.

https://doi.org/10.7554/eLife.33746.013
Supplementary file 4

Exploration time for congruent and incongruent objects during test 1.

Mean ± SEM exploratory time. for each object during test 1. Object A represents the contextually congruent object; Object B represents the contextually incongruent object. Test 1. Two-way ANOVA with multiple comparisons, Sidak's multiple comparisons test α <0.05, ****p<0.0001, ***p<0.001,**p<0.01, *p<0.05.

https://doi.org/10.7554/eLife.33746.014
Transparent reporting form
https://doi.org/10.7554/eLife.33746.015

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