\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[ngerman,greek,english]{babel}



\usepackage{float}








\begin{document}

\title{Disrupting direct inputs from the dorsal subiculum to the granular
retrosplenial cortex impairs spatial memory in the rat}



\author[1]{Steliana Yanakieva}%
\author[1]{Bethany Frost}%
\author[1]{Eman Amin}%
\author[1]{Andrew Nelson}%
\author[2]{John Aggleton}%
\affil[1]{Cardiff University}%
\affil[2]{Cardiff University College of Biomedical and Life Sciences}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
The dorsal subiculum is the primary source of hippocampal projections to
the rat retrosplenial cortex. Although, both regions are implicated in
spatial memory and navigation, the significance of their direct
interconnections remains poorly understood. The present study
selectively disrupted dorsal subiculum projections to retrosplenial
cortex with inhibitory designer-receptors exclusively activated by
designer drugs (iDREADDs), activated locally by clozapine. iDREADDs were
injected in the dorsal subiculum in adult male rats (N=14), where they
were transported anterogradely to granular retrosplenial cortex. In a
separate control group, GFP expressing adeno-associated virus was
injected into the dorsal subiculum (N=8). Both groups received
behavioural sessions preceded either by intracerebral infusions of
clozapine or saline within retrosplenial cortex. Behavioural testing
involved reinforced T-maze alternation, with five test variations that
differentially taxed intra-maze, extra-maze, and egocentric strategies.
Disruption of the subiculum to retrosplenial projections impaired
spatial working memory whenever the test variant created a conflict
between cue-types, associated with a switch between different
strategies. These findings suggest that the direct projections from the
dorsal subiculum to the granular retrosplenial cortex help to maintain
the flexible integration of different spatial cue-types.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Effective spatial learning and related navigation are essential skills
for humans and animals alike. These are, however, complex, multisensory
processes that require the integration of external visual cues with
internally generated movement-related cues (Johnsen \& Rytter, 2021).
The mechanisms required to create a coherent representation of the
external environment have been intensively investigated, with the
hippocampal formation and parahippocampal region often providing the
start point (Eichenbaum, 2017; Moser et al., 2008; O'Keefe \& Nadel,
1979). Within the hippocampal formation, the subiculum may make specific
contributions given its diverse spatial cells and its significance as a
route for the hippocampus proper to influence distal sites (Aggleton \&
Christiansen, 2015; Kitanishi et al., 2021; Lever et al., 2009; O'Mara,
2005; Witter, 2006; Yamawaki et al., 2019a,b).

Both neurotoxic lesions of the hippocampus proper and lesions of the
subiculum impair location learning in the Morris Water Maze, suggesting
that together these hippocampal regions are necessary for successful
allocentric (world-centred) spatial learning (Morris et al., 1990).
Furthermore, the hippocampal formation is not uniform as is shows graded
anatomical and electrophysiological changes along its various axes. One
reflection is how the dorsal and ventral subiculum appear to be
functionally distinct in rodents. Based on a variety of evidence it
appears that the dorsal subiculum is the more critical for solving
spatial memory tasks (Bannerman et al., 2004; Burzynska et al., 2020;
Moser \& Moser, 1998; O'Mara, 2005; O'Mara et al., 2009; Strange et al.,
2014; Witter et al., 1990). Consistent with this view, permanent lesions
of the dorsal subiculum are sufficient to impair T-maze alternation
(Potvin et al., 2007), a measure of spatial working memory. The pattern
of deficits suggested that the dorsal subiculum is essential for
processing idiothetic cues for navigation (Potvin et al., 2007). In
addition, dorsal subiculum lesions impaired both object-location memory
(Potvin et al., 2010) and the ability to distinguish adjacent-arm trials
in the radial-arm maze, pointing to a role in pattern-separation (Potvin
et al., 2009). These same lesion studies also indicated that the dorsal
hippocampus proper and the dorsal subiculum can contribute differently
to spatial memory (Potvin et al., 2007, 2009, 2010).

There are dense subiculum projections to the retrosplenial cortex that
in rodents preferentially target the granular subdivision (area 29).
These same projections principally arise from the dorsal subiculum
(Kinnavane et al., 2018; Sugar et al., 2011; van Groen \& Wyss, 1992).
Like the hippocampus, retrosplenial cortex is repeatedly implicated in
spatial memory and navigation (Nelson et al., 2018; Nelson et al., 2015;
Harker \& Whishaw, 2004; Vann et al., 2009; Wolbers \& B\selectlanguage{ngerman}üchel, 2005) as
well as episodic memory (Hayashi et al., 2020; Maguire, 2001; Nestor et
al., 2003; Vann et al., 2009). Furthermore, recent studies suggest that
these subiculum projections may facilitate the flow of contextual
information to retrosplenial cortex, thereby enabling memory formation
(Gao et al., 2021; Yamawaki et al., 2019b).

The effects of retrosplenial cortex lesions on spatial tasks appear to
be most pronounced when rats must rely on flexible cue integration, such
as when intra-maze and extra-maze cues are opposed (Pothuizen et al.,
2008, 2010; Vann \& Aggleton, 2004; Vann et al., 2003) or when required
to choose between competing relevant and irrelevant spatial information
(Wesierska et al., 2009). Like the dorsal subiculum, retrosplenial
lesion deficits can also emerge when visual stimuli are removed from
spatial tasks (Cooper \& Mizumori, 2001; Elduayen \& Save, 2014). Given
the interconnectivity of retrosplenial cortex with motor, sensory, and
visual cortices (Miyashita \& Rockland, 2007; Sugar et al., 2011;
Yamawaki et al., 2016) this cortical area is well placed to integrate
information between different sensory modalities to help navigation
(Byrne et al., 2007; Mizumori et al., 2000; Powell et al., 2020).
However, it is unclear whether direct hippocampal -- retrosplenial
connections are required for this process.

While much is known about the effects of permanent retrosplenial lesions
on learning and memory, far less is known when just the hippocampal
inputs to this area are disrupted. These more targeted studies have, so
far, been confined to showing the importance of mouse subiculum and CA1
inputs to retrosplenial cortex for contextual fear conditioning
(Yamawaki et al., 2019a,b). The present study sought to examine more
flexible forms of spatial learning involving working memory.
Consequently, rats were trained on a T-maze alternation task, followed
by multiple cue conditions. To disrupt the direct projections from the
dorsal subiculum to retrosplenial cortex, inhibitory designer-receptor
exclusively activated by designer drugs (iDREADDs) injections in the
dorsal subiculum were combined with intracerebral infusions of a ligand
(clozapine) at the target site (retrosplenial cortex) to inactivate
those projections locally (Gomez et al., 2017; Manvich et al., 2018;
Roth, 2016, 2017).

\textbf{Materials and Methods}

\textbf{Experimental Design}

Either iDREADDs or a green fluorescent protein (GFP) expressing
adeno-associated virus (control) was injected into the dorsal subiculum
in two separate groups of rats. Shortly before each behavioural test,
both groups received intracerebral infusions targeted at retrosplenial
cortex. For some sessions clozapine was infused, on other sessions it
was saline. Animals were then tested on reinforced alternation in a
T-maze, using five successive variants that differently taxed the use of
available cues. The experiment was repeated with two separate cohorts of
rats, both treated in the same ways (Figure 1).

\textbf{Animals}

Two cohorts, respectively of 12 and 24 adult Lister Hooded male rats
(Envigo, UK), were trained prior to surgery on reinforced T-maze
alternation. The first cohort had 8 iDREADDs and 4 GFP-control animals.
The second cohort had 12 iDREADDs and 12 GFP-control animals, giving
totals of 20 in the iDREADDs group and 16 GFP- controls. At the time of
surgery all rats weighed between 236-360g. They were housed in pairs in
a temperature-controlled room, under a 12h light/dark cycle. For all
behavioural experiments, water was available \emph{ad libitum} . The
rats were put on a food-restricted diet whereby they were still able to
gain weight. None of the rats weighed less than 85\% of their
free-feeding weight.

All animals were randomly assigned to one of the virus conditions and
underwent the same surgical and behavioural procedures. The experimenter
was not, however, blind to the group membership of the animals. All
experimental procedures were carried out in accordance with the UK
Animals (Scientific Procedures) Act 1986 and were approved by the local
Cardiff University Ethics Committee.

\textbf{Surgery}

Prior to surgery, all rats were anaesthetised with an isoflurane-oxygen
mixture (5\% induction, 1.5-2.5\% maintenance). Then, each rat was
placed in a stereotaxic frame (David Kopf Instruments, CA, USA), so that
the skull was flat, with respect to the horizontal plane.
Chloramphenicol 0.5\% eye-gel was applied, meloxicam (0.06ml) was
administered subcutaneously for analgesic purposes, and lidocaine (0.1ml
of 20mg/ml solution) was applied topically to the incision site. Next, a
bilateral craniotomy was performed above the dorsal subiculum, and
either pAAV-CaMKIIa- hM4D(Gi)-mCherry (AAV5) (iDREADD)(Titer:
2.6x10\^{}13GC/ml, lot:v102676; Addgene, MA, USA) or pAAV-CaMKIIa-GFP
(AAV5)(Titer: 4.3x 10\^{}12GC/ml, lot: v5894, Addgene, MA,
USA)(GFP-control) virus was injected bilaterally into the dorsal
subiculum.

In both cohorts, 0.6\selectlanguage{greek}µ\selectlanguage{english}l of the viral construct was injected in the
anterior subiculum injection site and 0.4\selectlanguage{greek}µ\selectlanguage{english}l into the more posterior
site. The injection coordinates, with respect to bregma were as follows:
\emph{Anterior} . Cohort 1: AP: -5.9mm, ML: \selectlanguage{ngerman}±2.9mm, DV: -2.6mm; Cohort
2: AP: -5.9mm, ML: ±2.7mm, DV: -2.4mm; \emph{Posterior} . Cohort 1: AP:
-6.2mm, ML: ± 3.2mm, DV: -2.5mm; Cohort 2: AP: -6.2mm, ML: ± 3.0mm, DV:
-2.3mm), respectively. The very slight changes in coordinates reflected
individual preferences of two researchers, based on pilot experiments.
All injections were made vertically using a 10\selectlanguage{greek}µ\selectlanguage{english}l Hamilton Syringe
attached to a movable arm. A micro-syringe pump (World Precision
Instruments, Florida, USA) controlled the injection, with the flow rate
set at 150\selectlanguage{greek}η\selectlanguage{english}l/min. The injection needle was left \emph{in situ} for
further 5 minutes, before retracting it. The order of the iDREADDs and
GFP injections was randomized, so that animals were randomly allocated
to either group.

During the same surgeries, pairs of cannulas were implanted into the
left and right retrosplenial cortex. One cannula pair (1.5mm length x
1.2mm separation, 26-gauge, PlasticsOne, Virginia, USA) was implanted
into the anterior portion of the retrosplenial cortex (from bregma; AP:
-2.5 mm, ML: \selectlanguage{ngerman}± 0.6 mm, DV: -1.5mm), the other cannula pair (1.7mm length
x 1.4mm separation; 26-gauge, Plastic One, Virginia, USA) was implanted
into the posterior retrosplenial cortex (AP: -6.0mm, ML: ± 0.7mm, DV:
-1.7mm). The implantation coordinates for both cohorts remained the
same. The cannulas were held in place with bone cement (Zimmer Biomet,
Swindon, UK) and anchored to the skull with four screws (Precision
Technology Supplies, Uckfield, UK). Dummy cannulas were inserted into
the guide cannulas to prevent blocking and were secured in place with
aluminium dust caps. The analgesic Marcaine Polyamps (AstraZeneca, UK)
and antibiotic powder (Clindamycin, Pfizer, UK) were applied to the
surgical site. All animals were subcutaneously administered 5ml
glucose-saline solution for fluid replacement, prior to placing them in
a recovery chamber. Once the animals regained consciousness, they were
returned to their home cage and closely monitored.

\textbf{Apparatus for Behaviour}

Behavioural testing was conducted in an elevated (94cm), modifiable
cross-maze with clear Perspex walls and wooden floor. Each arm was 70cm
long and 10cm wide, with 17cm high walls. Inset food wells were
positioned at the end of each arm so that the food rewards could not be
seen from the choice point. An aluminium barrier was used to block one
arm to create a T-shape, while a second transferable barrier was used to
temporarily block access to one of the T-maze arms during the sample
run. Unless otherwise specified in the experimental condition, the
location of the start arm remained constant across experiments. The maze
was positioned in the centre of a room (280cm x 280cm x 20cm) with
salient visual cues on the walls. All room cues remained constant
throughout the experiments. For both pre-training and the five
experimental conditions the experimenter stood behind the start arm for
both the `sample' and `test' runs while the rat completed the trial. The
illumination in the room for all conditions, unless otherwise specified,
was 23-26lx.

\textbf{Behavioural Training Prior to Infusions}

Prior to surgery, all rats were habituated to the maze for four
sessions. During the first habituation session, multigrain hoops
(Crownfield, UK) were placed in the food wells in the choice-arms, and
the rats were placed in the maze in cage-pairs to explore the start arm
for 5 minutes. Then, they were placed in the choice-arms where they
could collect food rewards for a further 5 minutes. During sessions 2
and 3, the above procedure was repeated for each rat individually for 5
minutes. In session 4, the aluminium barrier was introduced at the
entrance of one arm and the rats allowed to explore for 5 minutes. The
same procedure was repeated but now the barrier blocked the other arm.
The food in the wells was continuously replaced. The rats were then run
on the `Standard' T-maze procedure (see below) for 5 to 9 days and the
animals for surgery were selected, based on their performance and
willingness to run.

At least seven days post-surgery, and after signs of full recovery, the
animals were retrained on the Standard T-maze task for 6 to 10 days,
until they reached at least 87.5\% on two consecutive sessions. The
infusion trials for Standard T-maze then followed, commencing at least
three weeks after surgery.

The rats were then trained on the next T-maze condition for 3 to 5 days,
followed by the infusion trials for that condition. An additional,
infusion-free training day was provided if there had been a gap of more
than two days between infusion sessions. An infusion-free training
session was also given on the day between the two clozapine infusions,
to help performance return to baseline (Figure 1). This testing regime
was repeated for the remaining behavioural conditions (Figures 1, 2).
The order of clozapine, saline, and injection-free sessions for the
various conditions was balanced between the two cohorts (Figure 1).

\textbf{Experimental Conditions (all with 8 trials per session)}

Each experimental condition consisted of a forced (i.e., `sample') run,
followed by a free (i.e., `test') run in the T-shaped maze. The correct
choice arm across the block of 8 trials was pseudorandomized so that the
same choice arm was not repeated more than twice consecutively. To start
each trial, both T-maze arms were baited with a quarter of a multigrain
hoop before the sample run, but access to one arm was blocked at its
base with an aluminium barrier.

To begin the sample run, the rat was released from the start position
and ran to the junction of the T-maze, where it turned into the
pre-selected arm and ate the reward. The rat was then immediately picked
up and the barrier at the choice point removed. Then, the animal was
carried to the start position and allowed to begin the test run. After
running down the stem of the maze, the rat could choose between the left
and right arms. The animal received a food reward only if it alternated,
i.e., selected the arm located opposite from the baited sample arm. A
test run was considered correct when the animal's back feet crossed
markings at the base of each side arm. The animal was picked up and held
until, the T-maze was reset, and the next trial commenced after 10-15s.
Each rat completed all 8 trials prior to running the next animal.

\emph{1. Standard T-maze (all spatial cue types available) (Figure 2A)
--} This condition was the same as that used in pre-training. The two
phases of each trial started at the same position.

2. \emph{Start T-maze (flexible learning, all cue types available)
(Figure 2B) --} The start position was changed after each trial between
the four arms of the maze. Importantly, the start arm remained
consistent for both the sample and test runs. In all other respects,
training followed the `Standard' procedure. Both the selection of the
start-arm and the correct test-arm were pseudorandomized, so that no
start-arm or test-arm was repeated more than two consecutive times.

3. \emph{Rotation T-maze (disrupted intra-maze cues} ) \emph{(Figure 2C)
--} The maze was rotated between the sample and the test run, by either
90°or 180° degrees with every trial. The arm on the test run, the degree
of rotation and the direction of the rotation were all pseudorandomized
so that the same manipulation was not repeated more than two consecutive
times. The location of the start position was consistent for all trials,
so that extra-maze and egocentric cues remained viable, while intra-maze
cues were nullified.

4. \emph{Opposite arm T-maze (disrupted egocentric cues) (Figure 2D)
--}The start position of the animal was rotated 180° between the sample
and the test runs. Therefore, each sample run began at the same start
arm and each test run began in the arm directly opposite. Critically,
the correct arm on the test run remained in the opposite room location
to the position of the baited arm in the sample run. No test arm was
used as the correct arm on more than two consecutive trials. For all
trials, the start position remained in the South (Figure 2D).

5. \emph{Dark T-maze (visual cues removed) (Figure 2E) --} The standard
T-maze protocol was repeated but now in the dark. The maze was baited,
and barriers put in place before each trial in dim illumination provided
by a 10W red light facing away from the maze. Then, the light was turned
off (\textasciitilde{}0.2lx) and the rat placed in the start position.
Once each trial was completed, the rat was picked up and held while the
maze was reset. Only Cohort 2 received the additional infusion-free
session.

\textbf{iDREADDs activation}

Each behavioural condition was run both after an infusion of clozapine
and after an infusion of saline, which served as a within-subject
control. Clozapine was infused on two separate occasions per condition,
reflecting the potential for greater within-subject variability. The
infusion order was counterbalanced between the two cohorts (Figure 1).
There was always an added infusion-free test day between the two
clozapine infusions for Conditions 1-4, i.e., apart from the Dark
condition.

Animals were first habituated to the infusion procedure, using saline,
prior to the commencement of behavioural testing with clozapine. On
infusion days, the animals were taken to a separate room in pairs and
lightly anaesthetized with an isoflurane-oxygen mixture (5\% induction,
1.5-2\% maintenance). Double infusion injectors (33-gauge, PlasticsOne,
Virginia, USA) were inserted into the guide cannula and either 1\selectlanguage{greek}µ\selectlanguage{english}l of
sterile saline or 1\selectlanguage{greek}µ\selectlanguage{english}l of clozapine (1mg/ml) was infused over 1 minute
using an infusion pump (11 Plus, Harvard Apparatus, UK). The injector
was held in place for a further minute and the dummy cannula replaced.
The infusions lasted no more than 4 minutes per animal, and the animal
was returned to its home cage. Animals rested for 15-20 minutes prior to
behavioural testing.

\textbf{Perfusions:}

Following completion of the experiment, animals were transcranially
perfused. All animals received a lethal dose of sodium phenobarbital
(2ml/kg, Euthatal, Marial Animal Health, UK) administered by
intraperitoneal injection. Once completely unresponsive, the animals
were transcardially perfused with 0.1M phosphate-buffered saline (PBS)
and 4\% paraformaldehyde in 0.1M PBS (PFA). The brains were further
post-fixed in PFA for at least 2 hours, and then placed in 25\% sucrose
solution for a minimum of 24h. A freezing microtome (8000 Sledge
Microtome, Bright Instruments) was used to cut the brain in 40\selectlanguage{greek}μ\selectlanguage{english}m coronal
sections, saved as four simultaneous series. The sections were stored in
cryoprotectant (30\% sucrose, 1\% polyvinyl pyrrolidone, 30\% ethylene
glycol in PBS) in a freezer at -200\selectlanguage{english}C until further processing.

\textbf{Histology:}

One series was mounted onto gelatin-coated slides before being stained
for Nissl using cresyl violet. The sections were then dehydrated through
increasing concentrations of alcohol (70\%; 90\%; 100\%; 100\%) and
washed in xylene. Then, the slides were cover-slipped with DPX (Sigma
Aldrich, Gillingham, UK) mounting medium. To enhance the fluorescence
signal of the mCherry (iDREADDs group) or GFP (control group),
additional series were washed three times in PBS and then blocked with
5\% normal goat serum (NGS) (Invitrogen, Inchinnan, UK) in Phosphate
Buffered Saline with Tritonx-1000 (PBST) for two hours. Both series were
then transferred in either a solution of rabbit polyclonal anti-mCherry
(Abcam, Cambridge, UK) or chicken polyclonal anti-GFP (Abcam, Cambridge,
UK) antibody at a dilution of 1:1000 in PBST with 1\% NGS and incubated
for 24 hours at room temperature. The sections were then washed three
times and transferred to a secondary antibody of either goat-anti-rabbit
(Dylight Alexa flour 594, Vector Laboratories, Peterborough, UK) or
Alexa Fluor 488 goat-anti-chicken (Invitrogen, Inchinnan, UK) at a
dilution of 1:200 in PBST for two hours. The sections were then washed
in PBS and mounted onto gelatin-coated slides and cover-slipped using
Fluromount (Sigma-Aldrich, Germany).

\textbf{Image Acquisition and Viral Expression Analysis:}

For each animal, cannula placement and viral expression were analysed
using a bright field and fluorescent microscope Leica DM5000B, equipped
with a DFC310 FX camera. The viral expression was assessed at the
injection site, as well as at dorsal subiculum efferent targets. These
targets included layers 2 and upper 3 of the retrosplenial cortex, along
with the anteroventral and anteromedial thalamic nuclei (Figures 4, 5).

\textbf{Statistical analyses:}

The principal behavioural measure was the mean percentage of correct
choices made across the blocks of 8 trials, for each experimental
condition. The behavioural data were analysed using multiple mixed-model
analyses of variance (ANOVAs), with the within-subject factor of drug
(saline vs clozapine) and between-subject factor of group (iDREADDs vs
GFP-control. Partial eta-squared
(\selectlanguage{greek}\emph{η\textsubscript{p}\textsuperscript{2}}\selectlanguage{english} ) is reported as a measure
of effect size.

All data were screened for outliers, the assumptions of normality,
homogeneity of variances and covariances using SPSS Statistics 27(SPSS
Inc., Chicago, Ill., USA). A single outlier score (37.5\%) was found for
just one animal for a single session (GFP group, Standard condition,
saline), and so this case remained in the analyses. Levene's test based
on medians assessed the homogeneity of variance, showed that the
assumption was violated on the opposite-arm saline condition (\emph{p} =
0.044) (Brown \& Forsythe, 1974). No violations to the assumption of
homogeneity of covariance were found (all
\emph{p\textsubscript{s}\textgreater{}} 0.024) (see Tabachnick \&
Fidell, 2013). Where there was a statistically significant interaction
term, simple main effect analyses were conducted using pooled error
terms in JASP 14.1 (JASP Team, 2022).

Multiple independent t-tests helped to compare control and baseline
scores, i.e., the pre-surgery training scores, post-surgery training
scores for each alternation condition prior to any infusions, as well as
for the infusion-free day scores between the clozapine infusions. These
analyses, applicable to Conditions 1-4, were to establish if the
performance of the iDREADDs and GFP-controls was statistically
comparable, prior to and between iDREADDs activations.

\textbf{ResultsHistological findings:}

Two criteria were required for inclusion in the experimental analyses.
First, the dorsal subiculum virus injections had to result in
appreciable bilateral label within granular retrosplenial cortex
(Figures 4, 5). Second, the infusion placements had to involve
retrosplenial cortex (Figure 3). Across both cohorts, a total of 6
iDREADDs and 8 GFP-control animals were excluded due to lack of viral
expression (unilateral or bilateral) in retrosplenial cortex (n=6),
off-target cannula placement (n=2) or both (n=6). Consequently, the
behavioural analyses derive from 14 iDREADDs and 8 GFP-control animals.
In four of these animals (n=3 iDREADDs; n=1 GFP) spread from the
anterior infusion cannulas may have reached the midcingulate cortex
(Vogt \& Paxinos, 2012) as well as retrosplenial cortex. In some cases,
the virus injection spread into the dentate gyrus, which does not
directly innervate the retrosplenial cortex.

\textbf{Pre-surgery training, post-surgery baseline analyses, and
non-infusion sessions}

A series of independent \emph{t} -tests considered whether there might
be pre-surgery (Standard condition only) or post-operative training
performance differences between the iDREADDs and GFP-control rats on the
five T-maze task conditions prior to any infusions. The two groups did
not differ significantly on the pre-surgery training nor on the baseline
training prior to commencement of infusion trials for the five test
conditions: all t\textsubscript{s} \textless{} 1.86,
p\textsubscript{s}\textgreater{} 0.078. A further set of \emph{t} -tests
took the scores from the non-infusion sessions that were interleaved
between the saline and clozapine sessions (Figure 1), for all but the
Dark condition. Again, there were no performance differences between the
iDREADDs and GFP-control animals on the infusion-free days for each of
the four conditions: all t\textsubscript{s} \textless{} 1.01,
p\textsubscript{s}\textgreater{} 0.29.

\textbf{Performance on test conditions}

In a preliminary analysis `Cohort' was included as a second
between-subject factor to establish if there were any differences
regarding the two cohorts. The only main effect of cohort was for the
Dark condition (p\textless{}0.05), and no other main effects or
interactions involving this factor were observed (all
F\textsubscript{s}\textless{} 4.00; all p\textsubscript{s}
\textgreater{} 0.06). Therefore, the cohort data were pooled and
analysed together for each separate condition, though the Dark condition
data received extra scrutiny.

In the following analyses, `Drug' refers to saline or clozapine (within
subject) while Group refers to iDREADDs or GFP infusions (between
subject).

\textbf{Standard T-maze:} There was a significant main effect of
Drug:\emph{F} \textsubscript{1,20} = 7.01, \emph{p} =
0.015,\selectlanguage{greek}\emph{η\textsubscript{p}\textsuperscript{2}}\selectlanguage{english} = 0.25, but not a
main effect of Group or Drug \selectlanguage{ngerman}× Group interaction:
\emph{F\textsubscript{s}}\textless{} 0.85, \emph{p\textsubscript{s}}
\textgreater{} 0.37,\selectlanguage{greek}\emph{η\textsubscript{ps}\textsuperscript{2}}\selectlanguage{english}
\textless{} 0.04 (Figure 6). This set of results showed that clozapine
did not exert a greater effect in the active viral group when compared
with the control viral group.

\textbf{Start T-maze:} There was a main effect of Drug:\emph{F}
\textsubscript{1,20} = 5.76, \emph{p} =
0.03,\selectlanguage{greek}\emph{η\textsubscript{p}\textsuperscript{2}}\selectlanguage{english} = 0.22, but no main
effect of Group or Drug \selectlanguage{ngerman}× Group interaction:
\emph{F\textsubscript{s}}\textless{} 3.48, \emph{p\textsubscript{s}}
\textgreater{} 0.08,\selectlanguage{greek}\emph{η\textsubscript{ps}\textsuperscript{2}}\selectlanguage{english}
\textless{} 0.15 (Figure 6). This pattern of results corresponded to
that seen for the Standard condition.

\textbf{Rotation T-maze:} There was a significant main effect of
Drug:\emph{F} \textsubscript{1,20} = 12.02, \emph{p} =
0.002,\selectlanguage{greek}\emph{η\textsubscript{p}\textsuperscript{2}}\selectlanguage{english} = 0.37 but also a
Drug \selectlanguage{ngerman}× Group interaction: \emph{F} \textsubscript{1,20} = 6.8, \emph{p}
= 0.016,\selectlanguage{greek}\emph{η\textsubscript{p}\textsuperscript{2}}\selectlanguage{english} = 0.25. Simple main
effects analyses revealed a significant decline in performance following
clozapine infusions within the iDREADDs group that was not seen in the
GFP control group: \emph{F} \textsubscript{1,13} =
25.36,\emph{p\textless{}} 0.0001 (Figure 6). All other tests were
non-significant: \emph{F\textsubscript{s}} \textless{}
2.75,\emph{p\textsubscript{s}} \textgreater{} 0.11.

\textbf{Opposite arm T-maze:} As in the Rotation condition, there was a
significant main effect of Drug: \emph{F} \textsubscript{1,20} =
7.7,\emph{p} = 0.01, \selectlanguage{greek}\emph{η\textsubscript{p}\textsuperscript{2}}\selectlanguage{english} =
0.278 and a Drug \selectlanguage{ngerman}× Group interaction: \emph{F} \textsubscript{1,20} =
4.55,\emph{p} = 0.045, \selectlanguage{greek}\emph{η\textsubscript{p}\textsuperscript{2}}\selectlanguage{english} =
0.18. Again, there was decline in performance following clozapine
infusions in the iDREADDs group that was not seen in the GFP control
group:\emph{F} \textsubscript{1,13} = 16.6, \emph{p} \textless{}0.001
(Figure 6). All other tests were non-significant:
\emph{F\textsubscript{s}}\textless{} 0.89, \emph{p\textsubscript{s}}
\textgreater{} 0.35.

\textbf{Dark T-maze:} The behavioural analyses revealed a significant
Drug \selectlanguage{ngerman}× Group interaction: \emph{F} \textsubscript{1,20} = 7.8, \emph{p}
= 0.011, \selectlanguage{greek}\emph{η\textsubscript{p}\textsuperscript{2}}\selectlanguage{english} = 0.28, however,
there was no main effect of Drug or Group:
\emph{F\textsubscript{s}}\textless{} 0.036, \emph{p\textsubscript{s}}
\textgreater{} 0.85. Follow-up simple main effects analyses showed that
again iDREADDs animals' performance declined following the clozapine
infusions relative to saline: \emph{F} \textsubscript{1,13} = 5.8,
\emph{p} = 0.025. All other tests were non-significant:
\emph{F\textsubscript{s}} \textless{} 2.84, \emph{p\textsubscript{s}}
\textgreater{} 0.10 (Figure 6). For this one condition, there was a
significant main effect of Cohort (\emph{F} \textsubscript{1,18} = 10.6,
p = 0.004). Overall, Cohort 1 had lower scores, possibly reflecting the
absence of an additional saline trial. Nevertheless, the key
intervention comparisons were within-subject (saline vs clozapine),
being effective across both Cohorts.

\textbf{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.

\textbf{References}

Aggleton, J. P., Neave, N., Nagle, S., \& Sahgal, A. (1995). A
comparison of the effects of medial prefrontal, cingulate cortex, and
cingulum bundle lesions on tests of spatial memory: Evidence of a double
dissociation between frontal and cingulum bundle
contributions.\emph{Journal of Neuroscience} , \emph{15} (11),
7270--7281. https://doi.org/10.1523/jneurosci.15-11-07270.1995

Aggleton, John P., \& Christiansen, K. (2015). The subiculum: The heart
of the extended hippocampal system. In \emph{Progress in Brain
Research}(1st ed., Vol. 219). Elsevier B.V.
https://doi.org/10.1016/bs.pbr.2015.03.003

Aggleton, John P., \& O'Mara, S. M. (2022). The anterior thalamic
nuclei: core components of a tripartite episodic memory
system.\emph{Nature Reviews Neuroscience} .
https://doi.org/10.1038/s41583-022-00591-8

Anagnostaras, S. G., Gale, G. D., \& Fanselow, M. S. (2001). Hippocampus
and contextual fear conditioning: Recent controversies and
advances.\emph{Hippocampus} , \emph{11} (1), 8--17.
https://doi.org/10.1002/1098-1063(2001)11:1\textless{}8::AID-HIPO1015\textgreater{}3.0.CO;2-7

Bannerman, D. M., Rawlins, J. N. P., McHugh, S. B., Deacon, R. M. J.,
Yee, B. K., Bast, T., Zhang, W. N., Pothuizen, H. H. J., \& Feldon, J.
(2004). Regional dissociations within the hippocampus - Memory and
anxiety. \emph{Neuroscience and Biobehavioral Reviews} , \emph{28} (3),
273--283. https://doi.org/10.1016/j.neubiorev.2004.03.004

Brown, M. B., \& Forsythe, A. B. (1974). Robust tests for the equality
of variances. \emph{Journal of the American Statistical Association}
,\emph{69} (346), 364--367.
https://doi.org/10.1080/01621459.1974.10482955

Bubb, E. J., Aggleton, J. P., O'Mara, S. M., \& Nelson, A. J. D. (2021).
Chemogenetics Reveal an Anterior Cingulate-Thalamic Pathway for
Attending to Task-Relevant Information. \emph{Cerebral Cortex}
,\emph{31} (4), 2169--2186. https://doi.org/10.1093/cercor/bhaa353

Bubb, E. J., Kinnavane, L., \& Aggleton, J. P. (2017).
Hippocampal--diencephalic--cingulate networks for memory and emotion: An
anatomical guide. \emph{Brain and Neuroscience Advances} , \emph{1} ,
239821281772344. https://doi.org/10.1177/2398212817723443

Burzynska, A. Z., Ganster, D. C., Fanning, J., Salerno, E. A., Gothe, N.
P., Voss, M. W., McAuley, E., \& Kramer, A. F. (2020). Occupational
Physical Stress Is Negatively Associated With Hippocampal Volume and
Memory in Older Adults. \emph{Frontiers in Human Neuroscience}
,\emph{14} (July), 1--9. https://doi.org/10.3389/fnhum.2020.00266

Byrne, P., Becker, S., \& Burgess, N. (2007). Remembering the past and
imagining the future: A neural model of spatial memory and
imagery.\emph{Psychological Review} , \emph{114} (2), 340--375.
https://doi.org/10.1037/0033-295X.114.2.340

Cooper, B. G., \& Mizumori, S. J. Y. (2001). Temporary inactivation of
the retrosplenial cortex causes a transient reorganization of spatial
coding in the hippocampus. \emph{Journal of Neuroscience} ,\emph{21}
(11), 3986--4001. https://doi.org/10.1523/jneurosci.21-11-03986.2001

Czajkowski, R., Jayaprakash, B., Wiltgen, B., Rogerson, T.,
Guzman-Karlsson, M. C., Barth, A. L., Trachtenberg, J. T., \& Silva, A.
J. (2014). Encoding and storage of spatial information in the
retrosplenial cortex. \emph{Proceedings of the National Academy of
Sciences of the United States of America} , \emph{111} (23), 8661--8666.
https://doi.org/10.1073/pnas.1313222111

Czajkowski, R., Zglinicki, B., Rejmak, E., \& Konopka, W. (2020).
Strategy-specific patterns of arc expression in the retrosplenial cortex
and hippocampus during t-maze learning in rats. \emph{Brain Sciences}
,\emph{10} (11), 1--9. https://doi.org/10.3390/brainsci10110854

De Sousa, A. F., Cowansage, K. K., Zutshi, I., Cardozo, L. M., Yoo, E.
J., Leutgeb, S., \& Mayford, M. (2019). Optogenetic reactivation of
memory ensembles in the retrosplenial cortex induces systems
consolidation. \emph{Proceedings of the National Academy of Sciences of
the United States of America} , \emph{116} (17), 8576--8581.
https://doi.org/10.1073/pnas.1818432116

Dudchenko, P. A., \& Davidson, M. (2002). Rats use a sense of direction
to alternate on T-mazes located in adjacent rooms. \emph{Animal
Cognition} , \emph{5} (2), 115--118.
https://doi.org/10.1007/s10071-002-0134-y

Dudchenko, P. a. (2001). \emph{How do rats actually solve the
T-maze}(pp. 115; 4: 850-860).

Douglas, R. J. (1966). Cues for spontaneous alternation.~\emph{Journal
of Comparative and Physiological Psychology, 62} (2),
171--183.~https://doi.org/10.1037/h0023668

Eichenbaum, H. (2017). The role of the hippocampus in navigation is
memory. \emph{Journal of Neurophysiology} , \emph{117} (4), 1785--1796.
https://doi.org/10.1152/jn.00005.2017

Elduayen, C., \& Save, E. (2014). The retrosplenial cortex is necessary
for path integration in the dark. \emph{Behavioural Brain Research}
,\emph{272} , 303--307. https://doi.org/10.1016/j.bbr.2014.07.009

Frankland, P. W., \& Bontempi, B. (2005). The organization of recent and
remote memories. \emph{Nature Reviews Neuroscience} , \emph{6} (2),
119--130. https://doi.org/10.1038/nrn1607

Gao, M., Noguchi, A., \& Ikegaya, Y. (2021). The subiculum sensitizes
retrosplenial cortex layer 2/3 pyramidal neurons. \emph{Journal of
Physiology} , \emph{94} (12), 3151--3167.
https://doi.org/10.1113/JP281152

Gomez, J. L., Bonaventura, J., Lesniak, W., Mathews, W. B., Sysa-Shah,
P., Rodriguez, L. A., Ellis, R. J., Richie, C. T., Harvey, B. K.,
Dannals, R. F., Pomper, M. G., Bonci, A., \& Michaelides, M. (2017).
Chemogenetics revealed: DREADD occupancy and activation via converted
clozapine. \emph{Science} , \emph{357} (6350), 503--507.
https://doi.org/10.1126/science.aan2475

Hayashi, T., Oguro, M., \& Sato, N. (2020). Involvement of the
retrosplenial cortex in the processing of the temporal aspect of
episodic-like memory in rats. \emph{Neuroscience Research} , \emph{154}
, 52--55. https://doi.org/10.1016/j.neures.2019.05.005

Horikawa, K., Kinjo, N., Stanley, L. C., \& Powell, E. W. (1988).
Topographic organization and collateralization of the projections of the
anterior and laterodorsal thalamic nuclei to cingulate areas 24 and 29
in the rat. \emph{Neuroscience Research} , \emph{6} (1), 31--44.
https://doi.org/10.1016/0168-0102(88)90004-1

Hunt, P. R., Neave, N., Shaw, C., \& Aggleton, J. P. (1994). The effects
of lesions to the fornix and dorsomedial thalamus on concurrent
discrimination learning by rats. \emph{Behavioural Brain Research}
,\emph{62} (2), 195--205. https://doi.org/10.1016/0166-4328(94)90028-0

JASP Team (2022). JASP (Version 0.16.3){[}Computer software{]}

Johnsen, S. H. W., \& Rytter, H. M. (2021). Dissociating spatial
strategies in animal research: Critical methodological review with focus
on egocentric navigation and the hippocampus. \emph{Neuroscience and
Biobehavioral Reviews} , \emph{126} (March), 57--78.
https://doi.org/10.1016/j.neubiorev.2021.03.022

Keene, C. S., \& Bucci, D. J. (2008). Neurotoxic Lesions of
Retrosplenial Cortex Disrupt Signaled and Unsignaled Contextual Fear
Conditioning. \emph{Behavioral Neuroscience} , \emph{122} (5),
1070--1077. https://doi.org/10.1037/a0012895

Kinnavane, L., Amin, E., Aggleton, J. P., \& Nelson, A. J. D. (2019). Do
the rat anterior thalamic nuclei contribute to behavioural
flexibility?\emph{Behavioural Brain Research} , \emph{359} (October
2018), 536--549. https://doi.org/10.1016/j.bbr.2018.10.012

Kinnavane, L., Vann, S. D., Nelson, A. J. D., O'Mara, S. M., \&
Aggleton, J. P. (2018). Collateral projections innervate the mammillary
bodies and retrosplenial cortex: A new category of hippocampal
cells.\emph{ENeuro} , \emph{5} (1), 1--14.
https://doi.org/10.1523/ENEURO.0383-17.2018

Kitanishi, T., Umaba, R., \& Mizuseki, K. (2021). Robust information
routing by dorsal subiculum neurons. \emph{Science Advances} ,\emph{7}
(11), 1--22. https://doi.org/10.1126/sciadv.abf1913

Lever, C., Burton, S., Jeewajee, A., O'Keefe, J., \& Burgess, N. (2009).
Boundary vector cells in the subiculum of the hippocampal
formation.\emph{Journal of Neuroscience} , \emph{29} (31), 9771--9777.
https://doi.org/10.1523/JNEUROSCI.1319-09.2009

Maguire, E. A. (2001). The retrosplenial contribution to human
navigation: A review of lesion and neuroimaging
findings.\emph{Scandinavian Journal of Psychology} , \emph{42} (3),
225--238. https://doi.org/10.1111/1467-9450.00233

Manvich, D. F., Webster, K. A., Foster, S. L., Farrell, M. S., Ritchie,
J. C., Porter, J. H., \& Weinshenker, D. (2018). The DREADD agonist
clozapine N-oxide (CNO) is reverse-metabolized to clozapine and produces
clozapine-like interoceptive stimulus effects in rats and
mice.\emph{Scientific Reports} , \emph{8} (1), 1--10.
https://doi.org/10.1038/s41598-018-22116-z

Melo, M. B. de, Favaro, V. M., \& Oliveira, M. G. M. (2020). The dorsal
subiculum is required for contextual fear conditioning consolidation in
rats. \emph{Behavioural Brain Research} , \emph{390} (March).
https://doi.org/10.1016/j.bbr.2020.112661

Milczarek, M. M., Vann, S. D., \& Sengpiel, F. (2018). Spatial Memory
Engram in the Mouse Retrosplenial Cortex. \emph{Current Biology}
,\emph{28} (12), 1975-1980.e6. https://doi.org/10.1016/j.cub.2018.05.002

Miller, A. M. P., Mau, W., \& Smith, D. M. (2019). Retrosplenial
Cortical Representations of Space and Future Goal Locations Develop with
Learning. \emph{Current Biology} , \emph{29} (12), 2083-2090.e4.
https://doi.org/10.1016/j.cub.2019.05.034

Miller, A. P., Vedder, L. C., Law, M. L., \& Smith, D. M. (2014). Cues,
context, and long-term memory: The role of the retrosplenial cortex in
spatial cognition. \emph{Frontiers in Human Neuroscience} ,\emph{8}
(AUG), 1--15. https://doi.org/10.3389/fnhum.2014.00586

Miyashita, T., \& Rockland, K. S. (2007). GABAergic projections from the
hippocampus to the retrosplenial cortex in the rat. \emph{European
Journal of Neuroscience} , \emph{26} (5), 1193--1204.
https://doi.org/10.1111/j.1460-9568.2007.05745.x

Mizumori, S. J. Y., Cooper, B. G., Leutgeb, S., \& Mizumori, S. J. Y.
(2000). A neural systems analysis of adaptive navigation.\emph{Molecular
Neurobiology} , \emph{21} (1--2), 57--82.
https://doi.org/10.1385/MN:21:1-2:057

Morris, R. G. M., Schenk, F., Tweedie, F., \& Jarrard, L. E. (1990).
Ibotenate Lesions of Hippocampus and/or Subiculum: Dissociating
Components of Allocentric Spatial Learning. \emph{European Journal of
Neuroscience} , \emph{2} (12), 1016--1028.
https://doi.org/10.1111/j.1460-9568.1990.tb00014.x

Moser, E. I., Kropff, E., \& Moser, M. B. (2008). Place cells, grid
cells, and the brain's spatial representation system. \emph{Annual
Review of Neuroscience} , \emph{31} , 69--89.
https://doi.org/10.1146/annurev.neuro.31.061307.090723

Moser, M. B., \& Moser, E. I. (1998). Functional differentiation in the
hippocampus. \emph{Hippocampus} , \emph{8} (6), 608--619.
https://doi.org/10.1002/(SICI)1098-1063(1998)8:6\textless{}608::AID-HIPO3\textgreater{}3.0.CO;2-7

Nelson, A. J. D., Hindley, E. L., Vann, S. D., \& Aggleton, J. P.
(2018). When is the rat retrosplenial cortex required for stimulus
integration? \emph{Behavioral Neuroscience} , \emph{132} (5), 366--377.
https://doi.org/10.1037/bne0000267

Nelson, A. J., Hindley, E. L., Pearce, J. M., Vann, S. D., \& Aggleton,
J. P. (2015). The effect of retrosplenial cortex lesions in rats on
incidental and active spatial learning. \emph{Frontiers in Behavioral
Neuroscience} , \emph{9} (FEB), 1--16.
https://doi.org/10.3389/fnbeh.2015.00011

Nestor, P. J., Fryer, T. D., Ikeda, M., \& Hodges, J. R. (2003).
Retrosplenial cortex (BA 29/30) hypometabolism in mild cognitive
impairment (prodromal Alzheimer's disease). \emph{European Journal of
Neuroscience} , \emph{18} (9), 2663--2667.
https://doi.org/10.1046/j.1460-9568.2003.02999.x

O'Keefe, J., \& Nadel, L. (1979). \emph{Hippocampus as cognitive map} .
487--533.

O'Mara, S. (2005). The subiculum: What it does, what it might do, and
what neuroanatomy has yet to tell us. \emph{Journal of Anatomy}
,\emph{207} (3), 271--282.
https://doi.org/10.1111/j.1469-7580.2005.00446.x

O'Mara, S. M., Sanchez-Vives, M. V., Brotons-Mas, J. R., \& O'Hare, E.
(2009). Roles for the subiculum in spatial information processing,
memory, motivation and the temporal control of behaviour. \emph{Progress
in Neuro-Psychopharmacology and Biological Psychiatry} , \emph{33} (5),
782--790. https://doi.org/10.1016/j.pnpbp.2009.03.040

Pan, T.-T., Liu, C., Li, D.-M., Zhang, T.-H., Zhang, W., Zhao, S.-L.,
Zhou, Q.-X., Nie, B.-B., Zhu, G.-H., Xu, L., \& Liu, H. (2022).
Retrosplenial Cortex Effects Contextual Fear Formation Relying on
Dysgranular Constituent in Rats. \emph{Frontiers in Neuroscience}
,\emph{16} (May), 1--10. https://doi.org/10.3389/fnins.2022.886858

Pothuizen, H. H. J., Aggleton, J. P., \& Vann, S. D. (2008). Do rats
with retrosplenial cortex lesions lack direction? \emph{European Journal
of Neuroscience} , \emph{28} (12), 2486--2498.
https://doi.org/10.1111/j.1460-9568.2008.06550.x

Pothuizen, H. H. J., Davies, M., Aggleton, J. P., \& Vann, S. D. (2010).
Effects of selective granular retrosplenial cortex lesions on spatial
working memory in rats. \emph{Behavioural Brain Research} ,\emph{208}
(2), 566--575. https://doi.org/10.1016/j.bbr.2010.01.001

Pothuizen, H. H. J., Davies, M., Albasser, M. M., Aggleton, J. P., \&
Vann, S. D. (2009). Granular and dysgranular retrosplenial cortices
provide qualitatively different contributions to spatial working memory:
Evidence from immediate-early gene imaging in rats. \emph{European
Journal of Neuroscience} , \emph{30} (5), 877--888.
https://doi.org/10.1111/j.1460-9568.2009.06881.x

Potvin, O., Dor\selectlanguage{ngerman}é, F. Y., \& Goulet, S. (2007). Contributions of the
dorsal hippocampus and the dorsal subiculum to processing of idiothetic
information and spatial memory. \emph{Neurobiology of Learning and
Memory} , \emph{87} (4), 669--678.
https://doi.org/10.1016/j.nlm.2007.01.002

Potvin, O., Doré, F. Y., \& Goulet, S. (2009). Lesions of the dorsal
subiculum and the dorsal hippocampus impaired pattern separation in a
task using distinct and overlapping visual stimuli. \emph{Neurobiology
of Learning and Memory} , \emph{91} (3), 287--297.
https://doi.org/10.1016/j.nlm.2008.10.003

Potvin, O., Lemay, F., Dion, M., Corado, G., Doré, F. Y., \& Goulet, S.
(2010). Contribution of the dorsal subiculum to memory for temporal
order and novelty detection using objects, odors, or spatial locations
in the rat. \emph{Neurobiology of Learning and Memory} , \emph{93} (3),
330--336. https://doi.org/10.1016/j.nlm.2009.11.007

Powell, A., Connelly, W. M., Vasalauskaite, A., Nelson, A. J. D., Vann,
S. D., Aggleton, J. P., Sengpiel, F., \& Ranson, A. (2020). Stable
Encoding of Visual Cues in the Mouse Retrosplenial Cortex.\emph{Cerebral
Cortex} , \emph{January 2001} , 1--14.
https://doi.org/10.1093/cercor/bhaa030

Roth, B. L. (2016). DREADDs for Neuroscientists. \emph{Neuron}
,\emph{89} (4), 683--694. https://doi.org/10.1016/j.neuron.2016.01.040

Roth, B. L. (2017). Use of DREADDS. \emph{Neuron} , \emph{89} (4),
683--694. https://doi.org/10.1016/j.neuron.2016.01.040.DREADDs

Smith, D. M., Barredo, J., \& Mizumori, S. J. Y. (2012). Complimentary
roles of the hippocampus and retrosplenial cortex in behavioral context
discrimination. \emph{Hippocampus} , \emph{22} (5), 1121--1133.
https://doi.org/10.1002/hipo.20958

Sripanidkulchai, K., \& Wyss, J. M. (1986). Thalamic projections to
retrosplenial cortex in the rat. \emph{Journal of Comparative Neurology}
, \emph{254} (2), 143--165. https://doi.org/10.1002/cne.902540202

Strange, B. A., Witter, M. P., Lein, E. S., \& Moser, E. I. (2014).
Functional organization of the hippocampal longitudinal
axis.\emph{Nature Reviews Neuroscience} , \emph{15} (10), 655--669.
https://doi.org/10.1038/nrn3785

Sugar, J., Witter, M. P., van Strien, N. M., \& Cappaert, N. L. M.
(2011). The retrosplenial cortex: Intrinsic connectivity and connections
with the (para)hippocampal region in the rat. An interactive
connectome.\emph{Frontiers in Neuroinformatics} , \emph{5} (July),
1--13. https://doi.org/10.3389/fninf.2011.00007

Sutherland, R. J., \& Rodriguez, A. J. (1989). The role of the
fornix/fimbria and some related subcortical structures in place learning
and memory. \emph{Behavioural Brain Research} , \emph{32} (3), 265--277.
https://doi.org/10.1016/S0166-4328(89)80059-2

Sutherland, Robert J., \& Hoesing, J. M. (1993). Posterior Cingulate
Cortex and Spatial Memory: A Microlimnology Analysis. \emph{Neurobiology
of Cingulate Cortex and Limbic Thalamus} , 461--477.
https://doi.org/10.1007/978-1-4899-6704-6\_17

Tabachnick, B. G. \& Fidell, L. S. (2018). Using Multivariate Statitics.
7\textsuperscript{th} ed. Pearson, 2018

Troy Harker, K., \& Whishaw, I. Q. (2004). A reaffirmation of the
retrosplenial contribution to rodent navigation: Reviewing the
influences of lesion, strain, and task. \emph{Neuroscience and
Biobehavioral Reviews} , \emph{28} (5), 485--496.
https://doi.org/10.1016/j.neubiorev.2004.06.005

Tsai, T. C., Yu, T. H., Hung, Y. C., Fong, L. I., \& Hsu, K. Sen.
(2022). Distinct Contribution of Granular and Agranular Subdivisions of
the Retrosplenial Cortex to Remote Contextual Fear Memory
Retrieval.\selectlanguage{english}\emph{The Journal of Neuroscience : The Official Journal of
the Society for Neuroscience} , \emph{42} (5), 877--893.
https://doi.org/10.1523/JNEUROSCI.1303-21.2021

Umaba, R., Kitanishi, T., \& Mizuseki, K. (2021). Monosynaptic
connection from the subiculum to medial mammillary nucleus neurons
projecting to the anterior thalamus and Gudden's ventral tegmental
nucleus. \emph{Neuroscience Research} , \emph{171} (xxxx), 1--8.
https://doi.org/10.1016/j.neures.2021.01.006

van Groen, T., \& Wyss, J. M. (1992). Connections of the retrosplenial
dysgranular cortex in the rat. \emph{Journal of Comparative Neurology}
,\emph{315} (2), 200--216. https://doi.org/10.1002/cne.903150207

Vann, S. D., \& Aggleton, J. P. (2004). Testing the importance of the
retrosplenial guidance system: Effects of different sized retrosplenial
cortex lesions on heading direction and spatial working
memory.\emph{Behavioural Brain Research} , \emph{155} (1), 97--108.
https://doi.org/10.1016/j.bbr.2004.04.005

Vann, S. D., Aggleton, J. P., \& Maguire, E. A. (2009). What does the
retrosplenial cortex do? \emph{Nature Reviews Neuroscience} ,\emph{10}
(11), 792--802. https://doi.org/10.1038/nrn2733

Vann, S. D., Wilton, L. A. K., Muir, J. L., \& Aggleton, J. P. (2003).
Testing the importance of the caudal retrosplenial cortex for spatial
memory in rats. \emph{Behavioural Brain Research} , \emph{140} (1--2),
107--118. https://doi.org/10.1016/S0166-4328(02)00274-7

Vogt, B. A., \& Paxinos, G. (2012). \emph{Cytoarchitecture of mouse and
rat cingulate cortex with human homologies} . \emph{1909} .
https://doi.org/10.1007/s00429-012-0493-3

Wesierska, M., Adamska, I., \& Malinowska, M. (2009). Retrosplenial
cortex lesion affected segregation of spatial information in place
avoidance task in the rat. \emph{Neurobiology of Learning and Memory}
,\emph{91} (1), 41--49. https://doi.org/10.1016/j.nlm.2008.09.005

Witter, M. P. (2006). Connections of the subiculum of the rat:
Topography in relation to columnar and laminar
organization.\emph{Behavioural Brain Research} , \emph{174} (2),
251--264. https://doi.org/10.1016/j.bbr.2006.06.022

Witter, M. P., Ostendorf, R. H., \& Groenewegen, H. J. (1990).
Heterogeneity in the Dorsal Subiculum of the Rat. Distinct Neuronal
Zones Project to Different Cortical and Subcortical
Targets.\emph{European Journal of Neuroscience} , \emph{2} (8),
718--725. https://doi.org/10.1111/j.1460-9568.1990.tb00462.x

Wolbers, T., \& Buchel, C. (2005). Dissociable retrosplenial and
hippocampal contributions to successful formation of survey
representations. \emph{Journal of Neuroscience} , \emph{25} (13),
3333--3340. https://doi.org/10.1523/JNEUROSCI.4705-04.2005

Yamawaki, N., Corcoran, K. A., Guedea, A. L., Shepherd, G. M. G., \&
Radulovic, J. (2019a). Differential Contributions of Glutamatergic
Hippocampal\selectlanguage{english}-Retrosplenial Cortical Projections to the Formation and
Persistence of Context Memories. \emph{Cerebral Cortex} , \emph{29} (6),
2728--2736. https://doi.org/10.1093/cercor/bhy142

Yamawaki, N., Li, X., Lambot, L., Ren, L. Y., Radulovic, J., \&
Shepherd, G. M. G. (2019b). Long-range inhibitory intersection of a
retrosplenial thalamocortical circuit by apical tuft-targeting CA1
neurons. \emph{Nature Neuroscience} , \emph{22} (April).
https://doi.org/10.1038/s41593-019-0355-x

Yamawaki, N., Radulovic, J., \& Shepherd, G. M. G. (2016). A
corticocortical circuit directly links retrosplenial cortex to M2 in the
mouse. \emph{Journal of Neuroscience} , \emph{36} (36), 9365--9374.
https://doi.org/10.1523/JNEUROSCI.1099-16.2016\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1. Schematic illustration of the behavioural training and
testing schedule post-surgery.} Animals received a sequence of
alternation sessions that involved preceding intracerebral infusions of
either clozapine or saline. There was also an additional infusion-free
session. After the four sessions, rats moved onto the next T-maze
condition, starting with training sessions followed by infusion
sessions. The order of infusions was counterbalanced across the two
cohorts as indicated.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Hosted file}

\verb`image3.emf`  available at \url{https://authorea.com/users/659511/articles/663433-disrupting-direct-inputs-from-the-dorsal-subiculum-to-the-granular-retrosplenial-cortex-impairs-spatial-memory-in-the-rat}

\textbf{Figure 2. Illustration of the various T-maze conditions.} The
figure shows examples of a single trial (sample and test run) for each
behavioural condition as follows: Standard T-maze; Start T-maze (with
randomized start positions); Rotation T-maze (with either
90\textsuperscript{o} or 180\textsuperscript{o} maze rotation in either
direction); Opposite arm T-maze (sample from South, test from North);
and Dark T-maze. Abbreviations: A, allocentric cues; E, egocentric cues;
I, intra-maze cues; D, directional cues; +, cue is available to solve
the maze; -, the cue does not solve the maze.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 3. Schematic representation of retrosplenial cannula
placement for each experimental animal.} Panel A shows coronal sections
(cresyl violet) with cannulation sites in the anterior (left) and
posterior (right) retrosplenial cortex. Panel B (below) is a schematic
representation of cannula placements adapted from the Paxinos and Watson
rat atlas (2004) for each animal in both the anterior (left) and
posterior (right) portions of the retrosplenial cortex. Squares denote
iDREADDs animals and triangles GFP-controls. In three iDREADDs and 1
GFP-control animal, the cannulas also affected the most posterior
portions of the cingulate cortex. The same implantation coordinates were
used for all animals, producing considerable overlap of cannula
placements. The numbers represent the approximate distance from bregma
in mm. All scale bars are 150\selectlanguage{greek}µ\selectlanguage{english}m. Abbreviations: Cg1/2, anterior
cingulate cortex; M2, secondary motor cortex, RSD, dysgranular
retrosplenial cortex; RSGc, granular retrosplenial cortex, area c; RSGb,
granular retrosplenial cortex, area b; RSGa, granular retrosplenial
cortex, area a; V2, secondary visual area.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 4. Virus expression in the iDREADDs group.} Panel A shows
the smallest (black) and largest (light grey) injection sites across the
dorsal subiculum. Numbers refer to the distance from bregma in mm. Panel
B shows an example of iDREADDs expression in the dorsal subiculum. Panel
C shows the robust expression of transported iDREADDs in layers I, II,
and upper III of the granular retrosplenial cortex. Panel D shows
anterograde transport from the dorsal subiculum to the anterior thalamic
nuclei. All scale bars are 150\selectlanguage{greek}µ\selectlanguage{english}m. AD, anterodorsal nucleus; AM,
anteromedial nucleus; AV, anteroventral nucleus, DS, dorsal subiculum,
RSD, dysgranular retrosplenial cortex; RSG, granular retrosplenial
cortex.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 5. Virus expression in the GFP-control group.} Panel A
shows the smallest (black) and largest (light grey) injection sites
across the dorsal subiculum. Numbers refer to the distance from bregma
in mm. Panel B shows an example of iDREADDs expression in the dorsal
subiculum. Panel C shows the robust expression of transported virus in
layers I, II and upper III of the granular retrosplenial cortex. Panel D
shows anterograde transport from the dorsal subiculum to the anterior
thalamic nuclei. All scale bars are 150\selectlanguage{greek}µ\selectlanguage{english}m. AD, anterodorsal nucleus; AM,
anteromedial nucleus; AV, anteroventral nucleus, DS, dorsal subiculum,
RSD, dysgranular retrosplenial cortex; RSG, granular retrosplenial
cortex.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

\textbf{Figure 6. Bar graphs depicting the mean and each animal's
individual percentage of correct alternation responses for both the
iDREADDs and GFP-control groups.} From top left to bottom right: 1)
Standard T-maze; 2) Start T-maze; 3) Rotation T-maze; 4) Opposite arm
T-maze; 5) Dark T-maze. Despite the within-group differences restricted
to the iDREADDs group, there were no between-group differences for the
iDREADDs group and the GFP-controls. Error bars indicate SEM.

* Denotes within-group statistically significant differences; the saline
condition is presented in white and the clozapine condition in grey.

\textbf{Graphical abstract}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

\textbf{Graphical abstract text}

The dorsal subiculum is the primary source of hippocampal projections to
the rat retrosplenial cortex. Disruption of the subiculum to
retrosplenial projections impaired spatial working memory whenever the
test variant created a conflict between cue-types, associated with a
switch between different strategies. These findings suggest that the
direct projections from the dorsal subiculum to the granular
retrosplenial cortex help to maintain the flexible integration of
different spatial cue-types.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure1/Figure1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure2/Figure2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure3/Figure3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure4/Figure4}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure5/Figure5}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure6/Figure6}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

