Discussion
Although the potential significance of the direct hippocampal projections to retrosplenial cortex has long been appreciated (Sutherland & Hoesing, 1993; Vann et al., 2009), their importance for spatial memory has only been tested with classical context conditioning (Yamawaki et al., 2019a,b). The present study investigated the behavioural consequences of disrupting the direct projections from the dorsal subiculum to granular retrosplenial cortex, using five variations of a spatial working memory task, T-maze alternation. By combining iDREADDs injections into the dorsal subiculum with clozapine infusions into retrosplenial cortex, the present study sought to disrupt the direct projections from the dorsal subiculum to granular retrosplenial cortex. This manipulation impaired T-maze alternation on three of the five test conditions. No effect of clozapine was seen in the GFP control group.
Despite its apparent simplicity, T-maze alternation remains a complex task (Dudchenko, 2001). In the standard condition, animals have access to intra-maze cues, extra-maze (allocentric) cues, along with cues involving proprioception such as egocentric or directional information (Douglas, 1966; Dudchenko, 2001). The latter refers to using a sense of direction to alternate (e.g., East then West), which differs from egocentric strategies (Dudchenko & Davidson, 2002). The various T-maze conditions indicated that disruption of the dorsal subiculum projections to granular retrosplenial cortex impaired performance as soon as specific cue-types were put into conflict or selectively removed.
There was no apparent effect of retrosplenial disruption on the Standard or Start T-maze conditions, i.e., when all spatial strategies were available. The null result on the Start condition showed that iDREADDs activation did not affect the ability of the rats to adjust to changes in start position across the different trials. However, iDREADDs activation impaired spatial working memory on the Rotation, Opposite arm, and Dark alternation conditions. This pattern of deficits does not simply reflect task difficulty, as performance during the intervening infusion-free days, during the iDREADDs/saline condition, and by the GFP-control group (Figure 6) all remained extremely similar across all five conditions. The implication is that the clozapine infusions disrupted more than one type of task strategy, given the varying demands of the final three conditions (Figure 2). At the same time, a blanket disruption would most likely have also impaired the Standard and Start condition. This pattern of results points to the emergence of deficits when cue types are changed and restricted.
The temporal pattern of results (last three conditions impaired) showed that the chemogenetic effects did not disappear over time and training. This same temporal pattern does, however, raise the possible concern that post-operative testing may have resumed too soon, so that the virus was not fully transported. That possibility is, however, seen as most unlikely as pilot studies repeatedly show that by two weeks post-surgery there is extensive transport to granular retrosplenial cortex. In the present study, the first infusions were a minimum of three weeks post-surgery. In theory, by counterbalancing the sequence of the five behavioural conditions it would have been possible to address this issue. This was not, however, attempted. Each behavioural condition required different amounts of pre-training to establish appropriate performance levels prior to each set of drug infusions. This variation would have placed testing and testing intervals out of synchrony. The increase in individual variability would be exacerbated by the different transfer effects from each specific condition to the next condition.
While the present study lacks direct evidence as to how the clozapine infusions disrupted retrosplenial activity, other studies using comparable methodologies have demonstrated their effectiveness (Bubb et al., 2021; Yamawaki et al., 2019b). That the iDREADDS/clozapine combination disrupted neural processing can also be indirectly inferred from the performance disruptions seen on the last three conditions. Consistent with this assumption is how the pattern of behavioural deficits in the iDREADDS rats had obvious similarities with the effects of conventional lesions in the two target sites (Pothuizen et al., 2010; Potvin et al., 2007, 2010). A further potential concern is whether the clozapine infusions reached sites beyond retrosplenial cortex. While possible, any such site would also need to receive direct dorsal subiculum inputs to have any functional impact, so the likelihood is low. Furthermore, related cannula studies have concluded that infusions are well retained by retrosplenial cortex (Nelson et al., 2015; Yamawaki et al., 2019b).
As observed, the present results show clear parallels with prior behavioural studies testing either dorsal subiculum or retrosplenial cortex function. Permanent lesions of the dorsal subiculum were found to spare standard T-maze alternation in the light (Potvin et al., 2007). Again, radial-arm maze working memory did not appear affected after dorsal subiculum lesions, but impairments emerged when tested in the dark (Potvin et al., 2007) and when adjacent arms had to be distinguished (Potvin et al., 2009). Other dorsal subiculum lesion deficits include failing to select an object now placed in a novel position (Potvin et al., 2010), indicative of a deficit in location learning.
The present behavioural findings also resemble those from retrosplenial cortex lesions. Permanent lesions involving both granular and dysgranular retrosplenial cortex can have little or even no apparent effect on standard spatial alternation (Aggleton et al., 1995; Neave et al., 1994), i.e., as in the present study. More reliable spatial working memory deficits are found when, as in the present study, test conditions are suddenly changed, such as when intra-maze and extra-maze cues are made incongruent or when strategy switching is required (Nelson et al., 2015; Pothuizen et al., 2008; Vann & Aggleton, 2004; Vann et al., 2003). These examples include changing from the standard protocol to the ‘rotation’ condition, as well as when testing spatial alternation in the dark (Nelson et al., 2015).
Of especial relevance are those few studies that have made permanent lesions targeting just the granular retrosplenial cortex. Such lesions again appear to leave standard T-maze alternation intact but impair performance when intra-maze cues are removed by switching to adjacent, parallel mazes (Pothuizen et al., 2010). This profile closely resembles the current findings, even though the present iDREADDs manipulation was even more selective, targeting just one set of granular retrosplenial inputs (Figures 4, 5). Together, these findings underline the significance of the hippocampal (subiculum) efferents to granular retrosplenial cortex when spatial cue usage is restricted.
Findings from a very different type of behavioural task, contextual fear conditioning, also implicate both the hippocampus (including the dorsal subiculum) and retrosplenial cortex in learning about space (Anagnostaras et al., 2001; Keene & Bucci, 2008; Melo et al., 2020; Miller et al., 2014; Pan et al., 2022; Smith et al., 2012). Meanwhile, immediate-early gene analyses indicate that the two regions have complementary roles in spatial tasks (Czajkowski et al., 2020; Frankland & Bontempi, 2005). In addition, neuronal recordings suggest that the hippocampus may encode and help distinguish contexts, while the retrosplenial cortex may enable behaviourally significant cues to identify the current context (Smith et al., 2012) or help predict future navigational decisions (Miller et al., 2019).
An especially relevant study used chemogenetic methods similar to those in the present study to target hippocampal-retrosplenial projections during contextual fear conditioning. That study showed how the glutamatergic (vGlut1+ and vGlut2+) subiculum projections can differentially regulate the cellular functions of granular retrosplenial cortex (Yamawaki et al., 2019b). That same study also indicated that a major role of the vGlut1+ projections was in processing recent context memories, whilst the vGlut2+ projections assisted with the long-term retrosplenial storage of fear-inducing context memory (see also Czajkowski et al., 2014; De Sousa et al., 2019; Milczarek et al., 2018). In a related study, the sparse inhibitory CA1 projections to retrosplenial cortex were silenced, again in a contextual fear conditioning paradigm, and their actions contrasted with those of the anterior thalamic inputs to retrosplenial cortex (Yamawaki et al., 2019a). While both pathways are involved in the acquisition of contextual fear memory, they act in opposing ways. The inhibitory CA1 projections normally supressed, while the excitatory anterior thalamic projections normally enhanced the acquisition of context memories (Yamawaki et al., 2019a).
Further details of retrosplenial-anterior thalamic-hippocampal influences come from an optogenetic study showing how anterior thalamic and dorsal hippocampal projections recruit the same populations of pyramidal cells (layer III) within granular retrosplenial cortex (Brennan et al., 2021). These pyramidal cells are distinct from the cell populations influenced by the claustrum and anterior cingulate cortex (Brennan et al., 2021). Additionally, the timing of late neural spikes in layers II and III by the granular retrosplenial pyramidal neurons appears to be influenced by preceding activation of the subiculum (Gao et al., 2021). Together, these findings emphasise the reliance of the three regions on each other, suggesting that together the subiculum and anterior thalamic nuclei facilitate information processing in the retrosplenial cortex, which is gated by its inputs from CA1 (Aggleton & O’Mara, 2022; Yamawaki et al., 2019a). In addition, a recent study found that some granular retrosplenial neurons in layer V project directly to CA1 of the dorsal hippocampus in mice (Tsai et al., 2022). These projections may help retrieve remotely acquired contextual fear memory, demonstrating a bidirectional interdependence between regions (Tsai et al., 2022).
Finally, clear parallels exist between the present results and those of a previous experiment that also placed iDREADDs in the dorsal subiculum to examine spatial working memory (Nelson et al., 2020). Systemic activation of the iDREADDs did not influence Standard T-maze alternation, but impaired the same Rotation condition (Nelson et al., 2020), consistent with the present study. This same pattern of deficits (Standard - intact; Rotation - impaired) was then seen when just the subiculum projections to the anterior thalamic nuclei were disrupted (Nelson et al., 2020). These parallel effects with the present study again highlight the close anatomical (Bubb et al., 2017; Horikawa et al., 1988; Sripanidkulchai & Wyss, 1986) and functional (Aggleton & O’Mara, 2022; Kinnavane et al., 2019; Pothuizen et al., 2009; Sutherland & Rodriguez, 1989; Sutherland & Hoesing, 1993) relationships between the hippocampal formation, anterior thalamic nuclei, and retrosplenial cortex. Their common actions may reflect the way that many dorsal subiculum neurons collaterise to reach both granular retrosplenial cortex and the mammillary bodies (Kinnavane et al., 2018), the latter site relaying monosynaptically to the anterior thalamic nuclei (Umaba et al., 2021). Furthermore, the finding that the widespread disruption of multiple subiculum efferents has very similar effects to targeting just those reaching the anterior thalamic nuclei (Nelson et al., 2020) or reaching the retrosplenial cortex (present study) underlines the functional primacy of these particular interactions. Together, these results accord with the influential idea that retrosplenial cortex facilitates the ability to switch between spatial strategies (Byrne et al., 2007; Vann et al., 2009) and that this function is facilitated by direct inputs from the dorsal subiculum, along with anterior thalamic interactions.