Retrograde and anterograde contextual fear amnesia induced by selective elimination of layer IV-Va neurons in the granular retrosplenial cortex (A29).

Differences in cytoarchitectural organization and connectivity distinguishes granular (or area 29, A29) and dysgranular (or area 30, A30) subdivisions of the retrosplenial cortex (RSC). Although increasing evidence supports the participation of RSC in contextual fear learning and memory, the contribution of each RSC subdivision remains unknown. Here we used orchiectomized rats and intraperitoneal (i.p.) injections of saline (control) or 5 mg/kg MK801, to trigger selective degeneration of pyramidal neurons in layers IV-Va of A29 (A29MK801 neurons). These treatments were applied 3 days before or two days after contextual fear conditioning, and contextual fear memory was evaluated by scoring freezing in the conditioned context five days after training. Afterwards, brains were fixed and c-Fos and Egr-1 expression were assessed as surrogates of neuronal activity elicited by the recall in A29, A30 and in limbic areas. We found that eliminating A29MK801 neurons after training reduces conditioned freezing to 43.1 ± 9.9% respect to control rats. This was associated with a significant reduction of c-Fos and Egr-1 expression in A30, but not in other limbic areas. On the other hand, eliminating A29MK801 neurons before training caused a mild but significant reduction of conditioned freezing to 79.7 ± 6.8%, which was associated to enhanced expression of c-Fos in A29, A30 and CA1 field of hippocampus, while Egr-1 expression in caudomedial (CEnt) entorhinal cortex was not depressed as in control animals. These observations show that severeness of amnesia differs according to whether A29MK801 neurons were eliminated before or after conditioning, likely because loss of A29MK801 neurons after conditioning disrupt memory engram while their elimination before training allow recruitment of other neurons in A29 for partial compensation of contextual fear learning and memory. These observations add further support for the critical role of A29MK801 neurons in contextual fear learning and memory by connecting limbic structures with A30.

Fear-context association during memory retrieval requires input from granular to dysgranular retrosplenial cortex.

The contribution of the granular (area 29, A29) and dysgranular (area 30, A30) subdivisions of the retrosplenial cortex (RSC) to contextual fear memory (CFM) retrieval remains elusive. Here, intact and orchiectomized (ORC) male rats received an intraperitoneal (I.P.) injection of saline (control) or 5 mg/Kg MK801 after training and memory formation. In ORC, but not in intact males, this MK801 treatment selectively induces overt loss of neurons in layers IV-Va of A29 (A29MK801 neurons) (Sigwald et al., 2016). Compared to ORC-saline, ORC-MK801 rats showed impaired CFM retrieval in an A-B-A design for contextual fear conditioning (CFC), however context recognition was not affected. In ORC-MK801 rats, neither novel object recognition nor object-in-context discrimination were impaired, further indicating that A29MK801 neurons are not required for contextual recognition. Elevated plus maze test showed that anxiety-like behavior was not affected in ORC-MK801 animals, suggesting that loss of A29MK801 neurons does not affect the emotional state that could impair freezing during test. Importantly, in a sensory preconditioning test, higher order CFM retrieval was abolished in ORC-MK801, but not in male-MK801. Collectively, these observations indicate that A29MK801 neurons are critically required for retrieving fear-context association. For dissecting the anatomofunctional contribution of A29MK801 neurons to CFM retrieval, expression of c-Fos and Egr-1 was used to map brain-wide neuronal activity. In control male rats CFC and CFM retrieval was associated with significant enhancement of both proteins in limbic structures and A30, but not in A29, suggesting that neurons in A30 and limbic structures encode and store the associative experience. Notably, in ORC but not in intact males, MK801 impairs CFM retrieval and expression of c-Fos and Egr-1 proteins in A30, without affecting their expression in limbic structures. Thus, the loss of A29MK801 neurons after CFM formation precludes activation of associative neurons in A30, impairing CFM recall. FluoroGold retrograde track-tracing confirmed that A29MK801 neurons project to A30. Silver staining provide evidence that MK801 in ORC rats induces axonal deafferentation of A29MK801 neuron in A30. Collectively, our experiments provide the first evidence that A30 neurons participate in encoding and storing CFM while A29 is required for their activation during recall.

Reactivation-Dependent Amnesia for Contextual Fear Memories: Evidence for Publication Bias.

Research on memory reconsolidation has been booming in the last two decades, with numerous high-impact publications reporting promising amnestic interventions in rodents and humans. However, our own recently-published failed replication attempts of reactivation-dependent amnesia for fear memories in rats suggest that such amnestic effects are not always readily found and that they depend on subtle and possibly uncontrollable parameters. The discrepancy between our observations and published studies in rodents suggests that the literature in this field might be biased. The aim of the current study was to gauge the presence of publication bias in a well-delineated part of the reconsolidation literature. To this end, we performed a systematic review of the literature on reactivation-dependent amnesia for contextual fear memories in rodents, followed by a statistical assessment of publication bias in this sample. In addition, relevant researchers were contacted for unpublished results, which were included in the current analyses. The obtained results support the presence of publication bias, suggesting that the literature provides an overly optimistic overall estimate of the size and reproducibility of amnestic effects. Reactivation-dependent amnesia for contextual fear memories in rodents is thus less robust than what is projected by the literature. The moderate success of clinical studies may be in line with this conclusion, rather than reflecting translational issues. For the field to evolve, replication and non-biased publication of obtained results are essential. A set of tools that can create opportunities to increase transparency, reproducibility and credibility of research findings is provided.

Selective neuronal degeneration in the retrosplenial cortex impairs the recall of contextual fear memory.

The retrosplenial cortex (RSC) is one of the largest cortical areas in rodents, and is subdivided in two main regions, A29 and A30, according to their cytoarchitectural organization and connectivities. However, very little is known about the functional activity of each RSC subdivision during the execution of complex cognitive tasks. Here, we used a well-established fear learning protocol that induced long-lasting contextual fear memory and showed that during evocation of the fear memory, the expression of early growth response gene 1 was up-regulated in A30, and in other brain areas implicated in fear and spatial memory, however, was down-regulated in A29, including layers IV and V. To search for the participation of A29 on fear memory, we triggered selective degeneration of neurons within cortical layers IV and V of A29 by using a non-invasive protocol that takes advantage of the vulnerability that these neurons have MK801-toxicity and the modulation of this neurodegeneration by testosterone. Application of 5 mg/kg MK801 in intact males induced negligible neuronal degeneration of A29 neurons and had no impact on fear memory retrieval. However, in orchiectomized rats, 5 mg/kg MK801 induced overt degeneration of layers IV-V neurons of A29, significantly impairing fear memory recall. Degeneration of A29 neurons did not affect exploratory or anxiety-related behavior nor altered unconditioned freezing. Importantly, protecting A29 neurons from MK801-toxicity by testosterone preserved fear memory recall in orchiectomized rats. Thus, neurons within cortical layers IV-V of A29 are critically required for efficient retrieval of contextual fear memory.