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.