The dorsal subiculum is not necessary for step-through inhibitory avoidance acquisition and consolidation in rats.

Acknowledgement: The authors have no conflict of interest to declare.
This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP (process n° 2017/09837-1).
Márcio Braga de Melo contributed in conceptualization, data curation, formal analysis, funding acquisition, methodology. project administration, resources, investigation, validation, and visualization. Márcio Braga de Melo, Maria Gabriela Menezes Oliveira, and Vanessa Manchim Favaro contributed to writting, reviewing, and editing the original draft. Maria Gabriela Menezes Oliveira contributed in Conceptualization, Funding acquisition, Methodology, Project administration, Resources, and Supervision. Vanessa Manchim Favaro had contributed in conceptualization, methodology, project administration, supervision, and investigation.
The authors would like to thank José Bernardo da Costa, Juliana Carlota Kramer Soares, Thays Brenner dos Santos, Fabio Augusto Leonessa Alves, and Dimitri Daldegan Bueno for their valuable assistance with this work. We would also like to thank the Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP (process no. 2017/09837-1), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and the Associação Fundo de Incentivo à Pesquisa (AFIP) for their financial and institutional support for the present research.

The subiculum is usually considered, from an anatomical point of view, as one of the major outputs from the hippocampus, receiving direct projections from the CA1 (O’Mara et al., 2001; O’Mara, 2006; Witter & Amaral, 2004). In recent years, interest in the functional properties of the subiculum has increased, and several studies in respect of this region have been undertaken (Aggleton & Christiansen, 2015; Böhm et al., 2018; Matsumoto et al., 2019). The dorsal subiculum (DSub) has been shown to be engaged in the spatial representation processes involved in learning and memory (Böhm et al., 2018; O’Mara & Aggleton, 2019; O’Mara et al., 2009; Sun et al., 2019). This engagement may be related to the reciprocal connections between the DSub and the dorsal hippocampus (Commins et al., 2002; O’Mara et al., 2000; Xu et al., 2016), a region whose involvement in spatial processing is well established (O’Keefe & Nadel, 1978).

The role of the dorsal hippocampus in spatial representation is responsible for its involvement with contextual fear conditioning (CFC) (Rudy & O’Reilly, 2001; Rudy et al., 2002). In a previous study, we tested the hypothesis that the DSub also plays a similar role in CFC. Using posttraining infusions of the NMDA (N-methyl-d-aspartate) antagonist AP5 (2-amino-5-phosphonopentanoic acid) and the GABergic agonist muscimol, we confirmed the involvement of the DSub in CFC consolidation (Melo et al., 2020). CFC is an aversive learning paradigm that involves the association between an unconditioned stimulus (usually a footshock), and a conditioned stimulus (usually a determined environment) in classical Pavlovian conditioning (Pavlov, 1927; Phillips & LeDoux, 1992).

CFC can also be present in other behavioral paradigms, such as the step-through inhibitory avoidance (ST IA) task. This aversive learning task involves two major components: a classical conditioning component, as in CFC; and an instrumental learning component in which there is a punishment, usually a footshock, contingent to a response (Izquierdo et al., 2016; LeDoux et al., 2016; Skinner, 1974). The involvement of the hippocampus in inhibitory avoidance seems to depend on the protocol used, and whether the exposure of the animal to the context is weak or strong (Martel et al., 2010). The involvement of hippocampal NMDA receptors during the acquisition of ST IA has been shown behaviorally in several studies (Ebrahimi-Ghiri et al., 2018; Jamali-Raeufy et al., 2011; Khakpai et al., 2016). However, in these studies, the animals were preexposed to the apparatus before training, which could have increased the influence of the contextual component of the ST IA and consequently, the hippocampal participation in the task.

As in our previous study into the role of the DSub in CFC (Melo et al., 2020), other studies have shown that posttraining intra dorsal hippocampus infusions of muscimol (Zarrindast et al., 2002; Nazari-Serenjeh et al., 2011) and AP5 (Jafari-Sabet, 2006) led to impairments in ST IA consolidation. Furthermore, pretraining intra dorsal hippocampus infusions of muscimol (Yousefi et al., 2012) and AP5 (Khakpai et al., 2012, 2016) have also been shown to impair ST IA acquisition.

This suggests that the DSub may also be involved in ST IA through its engagement with spatial representation present in the classical conditioning component of this task, if the contextual component of the protocol is strong enough. Therefore, the present study aimed to investigate the effects of muscimol and AP5 infusions into the DSub on ST IA acquisition and consolidation.

Male Wistar rats aged 3–4 months were provided by the Centro de Desenvolvimento de Modelos Experimentais (CEDEME), which is part of the Universidade Federal de São Paulo (UNIFESP). They were allocated in groups of four per ventilated cage, provided with pine flakes for bedding, and maintained at 23 ± 2 °C under a 12/12-hr light/dark cycle (lights on at 7:00 hr), with food and water ad libitum. All methodological procedures were approved by the University Ethical Committee (CEUA no. 5032300517).

Stereotaxic surgery, the infusion procedure, and histology were conducted as described previously in other studies from our laboratory (De Oliveira Coelho et al., 2013; Santos et al., 2017), with slight modifications as follows. The surgery was conducted 7 days after the animals provided by CEDEME arrived at our bioterium. Once anesthetized by i.p. administration of ketamine hydrochloride (90 mg/kg) (Syntec®) and xylazine hydrochloride (10 mg/kg) (Syntec®), the animals were individually fixed in the stereotaxic frame (Insight®). Povidone-iodine (Riodeine®) was used as a topical antiseptic and 0.3 ml of 2% lidocaine hydrochloride with epinephrine (Xylestesin Cristália®) was subcutaneously applied to the rat´s head after the region was shaved. The DSub coordinates used were AP = −6.4 mm, ML = ±3.8 mm, DV = −3.4 mm (Melo et al., 2020; Paxinos & Watson, 2007) for the tip of the stylets (protruding 0.1 mm beyond the cannula tip) to hit the middle of the DSub. Acrylic resin was applied around the cannulas and screws were used as an anchor to protect the area. To avoid the entrance of material through the cannula, we inserted a protective metal wire into its interior. The animals received an intramuscular injection of 0.1 ml of a compound of benzathine benzylpenicillin, procaine benzylpenicillin, benzylpenicillin potassium, dihydrostreptomycin sulfate, streptomycin sulfate, sodium citrate, and vehicle of sterile distilled water (Pentabiótico Zoetis®) and a subcutaneous injection of 2% meloxicam (Maxicam Ourofino®) (2 mg/kg) every 24 hr for two consecutive days.

The behavioral procedures were initiated after a postsurgery recovery period of 10 days. The animals were regularly manipulated in the 4 days before the beginning of the behavioral experiment to habituate them to the investigator’s touch and presence and simulate the infusion protocol. On the day of the behavioral experiment, the animals received a bilateral infusion of muscimol (Sigma-Aldrich®) or AP5 (Tocris®) 5 min before the training (Souza & Carobrez, 2016; Nasehi et al., 2017) or immediately after training (Melo et al., 2020) by a technician blinded to the group allocation. The posttraining infusions occurred less than 30 s after the end of the training, which is the time it took to remove the animal from the conditioning chamber and attach the two infusion stylets (one per hemisphere) to the guide cannulas. The infusion pump and the training apparatus were located in the same room. Both drugs were dissolved in a 0.9% (1.0 mg/ml) saline solution and injected into the DSub with a volume of 0.2 μl/hemisphere and at a rate of 0.2 μl/min. The control group animals received the same volume of 0.9% saline solution. The coordinates, infusion volumes, and drug concentrations were the same as in our previous work manipulating the DSub (Melo et al., 2020), providing a “positive control” for the present study.

The ST IA apparatus used comprised an acrylic box with two chambers, each one measuring 22 × 21 × 22 cm and separated by an acrylic wall with a sliding guillotine door connecting the chambers. The walls of the compartment in which the animals received the aversive stimulus (footshock) were black, creating a dark environment, while the other compartment had white walls, creating a light environment. The ceiling of the apparatus was made of transparent acrylic with an attached camera to record the experiments and the floor formed by a metal grid (each grid measuring 0.4 cm in diameter and distant 1.2 cm from each other), connected to an electric shock generator to provide the footshocks (AVS, Projetos Especiais®). The apparatus was cleaned with a 20% ethanol solution between the training or test sessions of each animal.

After habituation, on the training day, each animal was placed in the light compartment and after 10 s, the sliding guillotine door was opened. The access door was closed as soon as the animal passed to the dark compartment, and it received a footshock (0.8 mA, 1 s). The animals were removed from the apparatus immediately after the presentation of the aversive stimulus. After 48 h, the animals were submitted to the avoidance test, which consisted of putting the animals in the light compartment again and opening the sliding guillotine door after 10 s. We measured, with a chronometer, the latency to enter the dark compartment, and the animal remained in this compartment for 5 min to assess freezing behavior, which was defined as the complete immobility of the animal´s body, without vibrissae movement or sniffing (Bouton & Bolles, 1980). The animals which remained in the light compartment after 300 s were removed by the investigator and placed in the dark compartment and freezing behavior was assessed for 5 min. The cumulative freezing time in each minute was then calculated. All the experiments were recorded and the behavioral parameters (latency and freezing times) were evaluated in videos by an investigator blinded to the group allocation.

We used Generalized Estimating Equations (GEE) to assess the group (AP5, muscimol, and saline) and session (training and test) effects on the latency to enter the dark chamber. To assess the group effect on freezing time at five different time points (every minute for 5 min after entering the dark compartment), we also used GEE analyses (Liang & Zeger, 1986). Sidak’s post hoc test was used whenever necessary. Generalized models, such as the GEE have already been used in other studies from our laboratory (Santos et al., 2020; Melo et al., 2020). These models are more suitable than the classical parametric tests, especially in cases of small samples, because they do not require assumptions such as normality and homogeneity, which are difficult to meet in samples like these (Muth et al., 2016; Nelder & Wedderburn, 1972; Pekár & Brabec, 2018). Furthermore, unlike nonparametric tests, which do not assume any known sample distribution, the generalized models enable the use of different known sample distributions chosen by the adherence indexes, providing more robust and reliable results (Wedderburn, 1974; Hardin & Hilbe, 2012; Garson, 2013). All the analyses were performed using IBM® SPSS Statistics 26 (2019, IBM Corp., Armonk, NY, USA). The assumed level of significance was set at 0.05.

Only the animals with correct bilateral cannula implantation in the DSub were considered in the statistical analysis. Figure 1C shows the correct implantation site in a sample. Those animals that did not have correct bilateral cannula implantation (pretraining: n = 4; posttraining n = 5) were excluded from the analysis. The final number of animals was 25 (AP5, n = 8; muscimol, n = 9; saline, n = 8) for pretraining and 25 (AP5, n = 9; muscimol, n = 8; saline, n = 8) for posttraining. Figure 1 A (pretraining) and Figure 1B (posttraining) show a representative scheme of the cannula implantation.

Figure 2 shows the effect of pretraining and posttraining muscimol or AP5 bilateral infusions into the DSub on ST IA latencies and freezing times. To evaluate the latency to cross into the dark compartment in the ST IA training and test sessions, the GEE was adjusted to γ distribution and an independent covariance matrix according to the score of Quasi-likelihood under Independence Model Criterion for pretraining (QIC = 32.99) and posttraining (QIC = 43.80). In the pretraining experiment, the GEE showed a null effect of group, Wald (2.22) = 0.395; p = .821, and interaction, Wald (2.22) = 0.433; p = .805. However, there was a significant effect of session, Wald (1.22) = 411.493; p < .0001, with animals showing higher latency in the test than the training session (205.37 ± 22.63 × 15.64 ± 1.90) with a large effect size (d = 2.41) (Figure 2 A). In the posttraining experiment, GEE also showed null effect of group, Wald (2.22) = 2.457; p = .293, and interaction, Wald (2.22) = 1.866; p = .393, but a significant effect of session, Wald (1.22) = 143.900; p < .0001, with animals showing higher latency in the test than the training session (131.71 ± 21.41 × 14.73 ± 1.31) with a large effect size (d = 1.57) (Figure 2C).

To evaluate the freezing time in the dark chamber in the ST IA test, GEE was adjusted to γ distribution and an unstructured covariance matrix according to the score of Quasi-likelihood under Independence Model Criterion for pretraining (QIC = 145.47) and posttraining (QIC = 134.41). GEE showed a significant effect of time factor, Wald (4.88) = 40.889; p < .0001, but not of group factor, Wald (2.22) = 0.74; p = .689, or the interaction, Wald (8.88) = 12.662; p = .124, in the mean freezing time in the dark chamber when the infusions were made pretraining (Figure 2B). Sidak post hoc test for time showed that minute 1 presented a higher mean freezing time than minutes 2, 3, 4, and 5 (p < .05) and minute 2 higher freezing time than minutes 3 and 4 (p < .05). In respect of the posttraining infusions, the GEE showed a significant effect of time factor, Wald (4.88) = 37.841; p < .0001, but not of group factor, Wald (2.22) = 5.083; p = .08, or the interaction, Wald (8.88) = 9.919; p = 0.271, in the mean freezing time in the dark chamber (Figure 2D). Sidak post hoc test for time showed that minute 1 presented higher mean freezing times than minutes 3, 4, and 5 (p < .05) and minute 2 higher freezing time than minutes 4 and 5 (p < .05).

The results showed that pre-or posttraining infusions of AP5 or muscimol into the DSub disrupted neither the latency to cross from the light to dark chamber, nor the freezing time in the dark chamber, that is, the manipulations did not affect ST IA acquisition and consolidation. Therefore, our initial hypothesis that the DSub involvement shown in respect of fear conditioning (Melo et al., 2020) would also be found in the ST IA was not supported; This led us to make the following observations.

First, although both CFC and ST IA are aversive learning behavioral paradigms that involve associations between stimuli and fear conditioning learning, there is a critical difference between these tasks that may determine the differential involvement of the DSub. The ST IA, but not the CFC, provides the animals with the possibility to escape from the punishment (unconditioned stimulus) (Roesler et al., 2006; Tinsley et al., 2004). This possibility can explain the low freezing time in the dark chamber (Figure 2B, D) because the previous avoidance experience may have changed the animal’s response to the conditioned context. For instance, the video records showed several animals that did not freeze spending time exploring the dark chamber, perhaps looking for opportunities to escape.

Second, it is possible that the instrumental component of our ST IA protocol is more prominent than the CFC one. To support this idea, another version of inhibitory avoidance, step-down avoidance, can have hippocampus-dependent and hippocampus-independent components according to the protocol utilized, which means that the participation of the hippocampus is not mandatory (Martel et al., 2010). Other studies show the involvement of hippocampal NMDA receptors during ST IA acquisition (Ebrahimi-Ghiri et al., 2018; Jamali-Raeufy et al., 2011; Khakpai et al., 2016), but in these studies the animals were preexposed to the device before training, which could have increased the effect of the contextual component of the ST IA, and, consequently, the hippocampal participation in the task. Furthermore, unpublished data from our laboratory show a different pattern of behavior response between CFC and ST IA, indicating that hippocampal NMDA receptors play a different role in each task (Favaro et al., 2021, submitted). Therefore, we may suppose that the DSub is not engaged in the ST IA task because the CFC component related to the hippocampal formation is reduced, whereas the instrumental component seems to be stronger.

Third, the anxiety components present in ST IA, but not in the CFC, could influence the differential engagement of the DSub. Rats have an innate preference to move from light to dark environments, but in the ST IA they are conditioned against this preference. Therefore, the fear introduced by the punishment must overcome the anxiety experienced to cross to the dark chamber (LeDoux et al., 2016). However, given the dorsal hippocampus and the DSub function in spatial representation (Aggleton & Christiansen, 2015; O’Mara et al., 2009; Rudy & O’Reilly, 2001; Rudy et al., 2002), these anxiety components seem unlikely to be the factors underlying the present null results.

Taken together, these possible interpretations provide a broad framework about the present results and raise some new questions, such as whether the DSub engagement would be necessary to inhibitory avoidance if other kinds of manipulation (pharmacological and/or optogenetic, etc.); other versions of the task (step-down, etc.); or other memory phases (retrieval and/or reconsolidation, etc.) were employed. However, to the best of our knowledge, there are no other studies exploring DSub participation in inhibitory avoidance. The only study involving the subiculum in this task is restricted to the ventral portion of this structure (Liu & Liang, 2009), which is more related to motivational aspects, stress, and anxiety (O’Mara, 2005, 2006; O’Mara et al., 2009). Therefore, more studies are necessary to address these questions.

Furthermore, the fact that our application of AP5 and muscimol in the DSub interfered only with CFC (Melo et al., 2020) and not with ST IA, show that common elements between the tasks, such as shock sensitivity, emotional reaction, and general learning, could not be responsible for the observed impairments in our previous work. More importantly, this dissociation suggests that the observed effects in CFC consolidation (Melo et al., 2020) are related to the involvement of the DSub with spatial aspects. It is unlikely that some technical issue is responsible for this dissociation between the tasks because all the investigators and procedures used in the present study were the same as those used in our previous (Melo et al., 2020) study, so any problem of this nature would have occurred in both of them.

In conclusion, the present findings add to the literature the new information that the DSub is not required for ST IA acquisition and consolidation, and that this could be due to a number of possible reasons which we describe above. Also, our data reinforce the proposition that the DSub is engaged in CFC consolidation through its involvement with spatial representation.

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Submitted: February 9, 2021 Revised: May 27, 2021 Accepted: June 3, 2021