Sebastijan Kaplar
email: kaplar@uns.ac.rs

Miroslav Zarić
email: miroslavzaric@uns.ac.rs

Gordana Milosavljević

NewWave Workflow Engine

Keywords: Workflow, Workflow engine, Pharo, TaskIt

NewWave 1 is an open-source workflow engine written in Pharo. The driving idea behind the work presented in this paper is to create an extensible workflow management system, natively written in Pharo, to facilitate easy specification and implementation of application business logic. Workflows can then be executed in a controlled manner, with the ability to gather and process business data using highly customizable plugins. Although still in the development stage, NewWave engine already supports basic control-flow patterns such as sequence, parallel split and join with synchronization, exclusive split and join, several event types, etc. This makes the NewWave engine applicable for specific real-life workflow models.

Introduction

Process-aware business systems have gained, in the last decade, strong support throughout the landscape of business information systems. Most of these systems are relying on a possibility to decompose business functions as a sequence of actions that, when executed in a controlled order, are driving the business toward a successful outcome. Most of the business functions may be represented as workflow -an orchestrated and ordered execution of activities. Reduced to its core elements, workflow technology serves to control and coordinate, in a flexible way, the execution of sets of well-defined operations [START_REF] Puccini | Executable models for extensible workflow engines[END_REF]. Work scenarios are specified, executed, and controlled with the workflow system. This technology has found use in many different domains, including business processes, scientific applications, and e-learning. One of the main reasons for this expansion is that workflows employ concepts from the specific domains, which is why workflows are commonly used by domain experts without extensive technical background [START_REF] Puccini | Executable models for extensible workflow engines[END_REF]. Although identification of discrete actions and their proper ordering is usually the most important and the most obvious part of the workflow model, for successful execution, workflow model (and execution engine) must also have the capability to express process data and task assignment model in order to be truly functional. 1 https://github.com/skaplar/NewWave Pharo, a purely object-oriented language, has much potential to implement and support many business-oriented applications. Pharo is providing simple, yet powerful syntax, and is focused on simplicity and immediate feedback, promising developers almost instantaneous results. Pharo is also dynamically typed, and many Pharo related projects are aimed at supporting model-driven and dynamic approach throughout all phases of software development: specification, design, and implementation. However, there is a lack of widely adopted native support for workflow modeling and implementation. This fact impairs the ability to have a processdriven application dynamics. After deprecation of AARE [START_REF]AARE -Workflow Management System[END_REF], a workflow management system written in Pharo initially released in 2005, a gap was opened in its place. NewWave was created with the idea to reintroduce native support for workflows in Pharo, and to facilitate easy process handling by leveraging the TaskIt2 library.

Outline of this paper is the following. The motivation for creating a new workflow engine is described in Chapter 2. Chapter 3 describes the implementation details, while Chapter 4 contains conclusions and future work.

Motivation

Creating a workflow engine is a challenging task. There are many available options, on a variety of platforms, ranging from commercial solutions, partially commercial (where some advanced features are not available on free to use versions), as well as freeware solution (but usually with severely limited functionality). In Pharo language community, an idea to create an open-source workflow engine has emerged.

The Workflow Management Coalition (WfMC) has proposed a reference architecture, used as a blueprint for developing a fully functional workflow engine. While there is much work to implement all the standards and recommendations stated in the reference architecture, our goal is not to simply create yet another implementation of the workflow engine. The main idea is to design a flexible, extensible development environment which enables integration with other applications and tools, and also to be able to act independently, providing everything that a process-oriented application needs for its execution (dynamic generation of user interface forms for user tasks, database persistence of process definitions, active users, and active processes, etc.).

Other ideas include distributing NewWave over multiple Pharo images and enabling the orchestration and communication between those images where every image would be given a certain type of task to perform, and lastly to achieve dynamic process reconfiguration without violating the workflow system. Although AARE workflow engine is not supported anymore, there are two more projects, built for different purposes:

• BPM Flow [START_REF]BPM Flow Manual[END_REF] which is built at the backend of GemStone/S from GemTalkSystems. BPMFlow is an open-source implementation of BPM standard and it includes: back office application, front office application, business intelligence applications, and a number of features. Unfortunately, there is no similar solution in Pharo.

• A4BP (Assessment for Business Processes) is a platform whose purpose is to craft custom analysis and obtain quality metrics of imported Business Process models already created with external tools. The main idea is to provide a tool to navigate the entire business process definition including the relation between process and technological services related to that process [START_REF] Peralta | A Tool for Assessing Quality of Rescue Plans by Combining Visualizations of Different Business Process Perspectives[END_REF]. A4BP is based on the Pharo platform.

NewWave Workflow Engine

NewWave allows users to specify workflow composition. It enables specification of processes and activities as different types of tasks and ordering of those activities. Every workflow can be inspected with a simple yet informative visualization which is achieved using Roassal 3 . NewWave utilizes TaskIt, a library that enables the usage of tasks in Pharo, with abstractions to execute and synchronize concurrent tasks (Figure 1).

Figure 1. NewWave workflow engine dependencies

An activity is defined within the Business Process and represents work to be performed to achieve desired results. The activity usually takes some time to perform and requires one or 3 Roassal2 visualization engine -http://agilevisualization.com more resources, some input (i.e., a form to collect data) and will produce some output. An activation represents one instance of the activity in the running workflow. The activity can have multiple instances (activations) in the same workflow.

WfMC defines a Reference Model [START_REF]Workflow Reference Model[END_REF] which proposes standards in order to foster interoperability between different workflow systems. NewWave supports an array of widely accepted business workflow patterns which will be listed. Some of the patterns not listed can be achieved by combining supported patterns.

Sequence or sequential routing is a basic control-flow pattern, where activities are executed in a sequence, and the current activity is enabled after completion of previous [START_REF]Workflow Reference Model[END_REF].

Conditional routing or the XOR-split represents a decision in the workflow process where the branch is forked into two or more branches, and the control is passed to one of the outgoing branches based on a specific condition [START_REF]Workflow Reference Model[END_REF].

Parallel routing or the AND-split represents forking of a branch into two or more branches where each of them is executed concurrently [START_REF]Workflow Reference Model[END_REF].

Synchronization or the AND-join represents meeting two or more branches into one subsequent single branch, and the activity from that single branch is run only when all previous branches have been completed [START_REF]Workflow Reference Model[END_REF].

Types of tasks

NewWave supports different types of tasks that can be performed. Elementary type of task is BaseTask. BaseTask has only one instance variable, which is value, and it is used to store and retrieve data.

ScriptTask derives from BaseTask, and it has one additional instance property, which is a script. The script property holds the block of code that should be executed when called. This is the reason why the value from BaseTask is redefined, and it looks like

ScriptTask>>value ^ script value

By sending the message value to the script variable, the block is executed and the foreseen operation is performed.

CustomTask allows for complex tasks definition. It allows embedding custom code and different libraries to gather data from the user. CustomTask also uses announcers to notify the engine when the task is completed.

UserTask allows to define a simple form for gathering data from the user. It is possible to use the API, provided by NewWave. to add different types of fields which will be displayed to the user during the workflow execution.

Designing the workflow

NewWave, as it is still in the early development phase, has no graphical workflow editor/designer. Nevertheless, specification of the workflow, for now, can be accomplished directly in the Pharo environment. NewWave has an API that allows defining the desired workflow (see Listing 1, for example). After defining the workflow, it is possible to use Roassal to get simple visualization and inspect the designed workflow. An example of one such workflow is given in Figure 2.

Figure 2. Workflow visualization with Roassal in NewWave

Figure 2 depicts a workflow where some common elements are shown. As we can see, we have a StartEvent, followed by a BaseTask. After BaseTask is executed, ParallelSplit is executed, and two tasks are performed concurrently. Those tasks meet at a ParallelJoin which is used for synchronization. Finally, one more task is executed before the workflow is completed. Creating the described workflow is presented in Listing 1. For simplicity purposes, the creation of some elements is omitted.

Listing 1. Creating the workflow

It is also possible to inspect every element of the workflow using Roassal and Pharo builtin features. Figure 3. presents ParallelJoin, and it shows that it has two incoming flows, and one outgoing flow, which is also visible in Figure 2. Moreover, if it is needed, incoming and outgoing flows can also be inspected separately, for example, to show what elements are at the end of each flow.

Figure 3. Inspecting ParallelJoin

Workflow execution

Workflow execution in NewWave starts by sending a message startEngine to the WaveEngine (Figure 4). This event message activates the WaveExecutor, which starts executing the previously defined workflow. Workflow execution can be logged and followed from start to finish. During the execution, the user can interact with different types of tasks, entering data in the forms and deciding the path of execution based on offered choices.

WaveEngine

WaveEngine is one of the main components and represents the engine of the workflow. Engine controls and organizes the execution of the workflow, with all runtime information necessary to achieve effective control of the execution, such as work list, activations, control of the multiple executors, etc.

WaveExecutor

WaveEngine has one main WaveExecutor, which is responsible for executing the workflow. When necessary, for example, when ParallelSplit occurs, multiple WaveExecutors can be spawned and they exist until the end of their execution, for example, until ParallelJoin ends.

WaveExecutor has the task to provide a proper environment to execute the current element. To obtain the element for execution, WaveExecutor is relying on services of FlowHandler. FlowHandler, as its name suggests, handles the flow of execution. It keeps the information about the element that is currently executing. After one element execution is completed, FlowHandler determines, from the Workflow definition, the next element to be executed. It also keeps the information which elements were executed and in what particular order. This "execution history" is handy for retracing the steps of execution, following up on the workflow progress. Simplified architecture of the NewWave is shown in Figure 4. Tasks in NewWave, as it is a custom in most of the workflow engines, can be broadly classified as automatic or manual, and they can be combined through the workflow. Using the scheduling mechanism, tasks can be scheduled to perform at the desired time, with the option to repeat when necessary.

Activations in the NewWave

Activations API is a part of the NewWave engine. Every node (task or gate) in the engine has an activation associated with it. The activation becomes active when the engine tries to execute the current node. In that moment node is activated and information about it is recorded in the engine. When the execution finishes, the activation changes state to completed and updates the information in the engine, with all the additional data the engine needs.

This information can be used to query the activation for the nodes in the engine, to check in what state it is, or to get information about the executor instance running the node, and so on.

Using announcers in the NewWave

Pharo Announcement framework is a small but powerful event notification framework which provides the generic implementation of Observer pattern. Using announcements simplifies event system in NewWave and provides customization options for executing different types of events. For example, we can consider synchronization, where multiple branches converge into one. In order to continue workflow execution past join point, all previous parallel branches should have been completed. Every time a branch reaches the synchronization point, a JoinEvent gets announced, and it is used to check if the synchronization is completed. Announcement framework allows for customization so that the Announcement class can be subclassed (see Listing 2). JoinEventAnnouncer has two instance variables, a parameter, and an executor. The parameter represents a sequence that leads up to synchronization point, and the executor is the WaveExecutor instance running this execution. Since the Announcement allows events as objects, this particular behavior is used to notify the part of the engine which handles synchronization with the objects that are being executed. NewWave offers support for different kinds of events and allows embedding new types of events; the more common ones are shown in Figure 5. NewWave uses TaskIt, a library written in Pharo, that abstracts execution and synchronization of concurrent tasks. NewWave utilizes two mechanisms, in particular, the worker runner and futures. The worker runner is a task runner (instance of TKTWorker) that uses a single process to execute tasks from a queue, and with workers, NewWave can control the number of live processes as well as how tasks are distributed amongst them. Future is an object that represents a future value of the task's execution. When the task completes, the result is deployed into the corresponding future, thus allowing access to its value. However, it can not be accessed right away, and futures provide callbacks to access its value asynchronously.

Conclusion

NewWave project was started with the idea to reintroduce native support for workflows in Pharo. This paper presents the first steps towards the goal to create an extensible workflow management system to facilitate easy specification and execution of application business logic. Currently, NewWave supports:

• Control-flow patterns (sequential routing, parallel routing, conditional routing, synchronization)

• Different types of tasks (user tasks, custom tasks, script tasks)

• Scheduling • Different events (start event, end event, boundary event, custom engine events)

• Subprocesses • Specification of workflows using the NewWave API • Visualization and inspection of the specified workflows • Pharo Announcements framework.

• Rudimentary support for user interface forms for obtaining data for user tasks.

Future work involves: (1) development of graphical workflow designer, (2) better support for user interface forms for obtaining data from user tasks, (3) serialization of process definitions, active users, and active processes in a database, (4) importing BPMN (Business Process Model and Notation) diagrams, (6) support for the integration with various tools through plugin mechanism, and (7) distribution over multiple Pharo images and enabling the orchestration and communications between those images.

  startEvent addOutgoingEdge: task1. task1 addOutgoingEdge: split1. split1 addOutgoingEdge: task2. split1 addOutgoingEdge: task3. task2 addOutgoingEdge: parallelJoin. task3 addOutgoingEdge: parallelJoin. parallelJoin addOutgoingEdge: task4. task4 addOutgoingEdge: endEvent. engine := WaveEngine new. we := WaveExecutor initialNode: startEvent. engine addExecutor: we.

Figure 4 .

 4 Figure 4. Simplified engine architecture

  Announcement subclass: #JoinEventAnnouncer instanceVariableNames: 'parameter executor' classVariableNames: '' package: 'NewWave-Announcers' Listing 2. Describing the JoinEventAnnouncer

Figure 5 .

 5 Figure 5. Events supported in NewWave

TaskIt -https://github.com/sbragagnolo/taskit

ACKNOWLEDGMENTS

The authors would like to thank Stéphane Ducasse and Cédrick Béler for their kind help and support throughout the project.


Gisella Vetere

Frances Xia

Adam I Ramsaran

Lina M Tran

Sheena A Josselyn

Paul W Frankland

An inhibitory hippocampal-thalamic pathway modulates remote memory retrieval

Memories are supported by distributed hippocampal-thalamic-cortical networks, but the brain regions that contribute to network activity may vary with memory age. This process of reorganization is referred to as systems consolidation, and previous studies have examined the relationship between the activation of different hippocampal, thalamic, and cortical brain regions and memory age at the time of recall. While the activation of some brain regions increases with memory age, other regions become less active. Here we asked whether the active disengagement of one such brain region, the anterodorsal thalamic nucleus (ADn), is necessary for recall at remote time points, and in addition, which projection(s) mediate such inhibition. Specifically, we identified a sparse inhibitory projection from CA3 to the ADn that becomes more active during systems consolidation, such that it is necessary for contextual fear memory retrieval at remote, but not recent, time-points post-learning.

INTRODUCTION

While memories for events may initially depend upon the hippocampus, with time these memories become increasingly dependent upon the cortex for their expression [1][2][3][4] . Post-learning communication between the hippocampus and cortex, via the thalamus, is thought to drive this time-dependent reorganization process called systems consolidation 2,5-12 . While the contributions of hippocampal and cortical regions have been studied in detail, much less is known about how thalamic regions contribute to this memory reorganization process at different post-encoding delays.

Using brainwide mapping of immediate early gene induction, we previously identified networks of regions activated by recall of either recent or remote contextual fear memory 11,13 . Thalamic regions played a central role within these networks, with their retrieval-induced activation generally correlated with activity in cortical and hippocampal regions at both retention delays. However, one region, the anterodorsal thalamic nucleus (ADn), exhibited a distinct pattern.

Following recall of recent contextual fear memory, ADn activity was positively correlated with activity in other regions, including cortical and hippocampal subregions (Supplementary Figure 1a). In contrast, at the remote time point, this pattern switched. Following recall of remote contextual fear memory, ADn activity was negatively correlated with activity in other regions, including cortical and hippocampal subregions (Supplementary Figure 1b). The ADn has extensive connections with both the hippocampus and cortex [START_REF] Aggleton | Hippocampal-anterior thalamic pathways for memory: uncovering a network of direct and indirect actions[END_REF][START_REF] Child | Anterior nucleus of the thalamus: functional organization and clinical implications[END_REF][START_REF] Jankowski | The anterior thalamus provides a subcortical circuit supporting memory and spatial navigation[END_REF] , and lesioning or inhibiting ADn impairs performance in hippocampus-dependent memory tasks [START_REF] Aggleton | The effects of selective lesions within the anterior thalamic nuclei on spatial memory in the rat[END_REF][START_REF] Harvey | Posttraining Inactivation of the Anterior Thalamic Nuclei Impairs Spatial Performance on the Radial Arm Maze[END_REF][START_REF] Lopez | Differential role of the anterior and intralaminar/lateral thalamic nuclei in systems consolidation and reconsolidation[END_REF][START_REF] Mair | Impairment of radial maze delayed nonmatching after lesions of anterior thalamus and parahippocampal cortex[END_REF][START_REF] Mitchell | Lateral and anterior thalamic lesions impair independent memory systems[END_REF][START_REF] Van Groen | Role of the anterodorsal and anteroventral nuclei of the thalamus in spatial memory in the rat[END_REF] . Furthermore, ADn activity is tightly coupled with hippocampal oscillations that play important roles in memory processing [START_REF] Viejo | Precise coupling of the thalamic head-direction system to hippocampal ripples[END_REF] . In the network analysis 11,13 , the inverse correlation between ADn and other brain regions during remote memory recall raises an intriguing possibility. As other brain regions become more actively engaged during remote memory recall, ADn activity is actively inhibited by one or more brain regions, and such inhibition may be necessary for successful remote memory recall.

To explore the role of the ADn, and its connections, at recent and remote time points here we used contextual fear conditioning. We found that the ADn is necessary for the retrieval of recent, but not remote, contextual fear memory. Consistent with this, retrieval-induced activity of ADn decreases as a function of memory age. We then identified a novel long-range inhibitory pathway from the CA3 subfield of the hippocampus to the ADn. This CA3-ADn inhibitory pathway is recruited during memory consolidation, and becomes necessary for the retrieval of remote fear memory. Previous studies have shown that consolidation of event memories involves gradual recruitment of brain regions over time. Our current results suggest that active disengagement of at least the ADn is additionally necessary for successful memory retrieval at remote time points.

RESULTS

ADn activity is necessary for recall of recently acquired memory

We first assessed retrieval-induced activation of ADn as a function of memory age. To do this we trained wild-type (WT) mice in contextual fear conditioning, and tested their memory at either 1 or 28 days post-training (Fig. 1a). Ninety minutes following testing, mice were perfused and we analyzed the expression of the activity-regulated gene, Fos, in the ADn. c-Fos expression was additionally assessed in 3 control groups of mice: (1) home cage mice; (2) mice trained without a footshock and tested 1 or 28 days later, and (3) mice trained with an immediate shock and tested 1 or 28 days later. For all mice that were tested 1 or 28 days later, c-Fos levels were normalized to those levels in home cage controls (Fig. 1b-c). In comparison to control mice, memory retrieval at 1 day post-training significantly increased the expression of c-Fos in the ADn. In contrast, c-Fos levels were not increased in mice tested at the 28 day memory test point, and were equivalent to those in control mice. Consistent with a previous observation in rats [START_REF] Lopez | Differential role of the anterior and intralaminar/lateral thalamic nuclei in systems consolidation and reconsolidation[END_REF] , these results suggest that the ADn is actively engaged during retrieval of a recently-, but not remotely-acquired fear memory. At remote time-points, the ADn may either be not engaged or, alternatively, actively disengaged or inhibited.

To test whether the activity of ADn is necessary for retrieval of a recent memory, we next inhibited the ADn cells optogenetically during retrieval of a contextual fear memory. As ADn contains excitatory neurons [START_REF] Nakamura | Immunohistochemical study of the distribution of Ca(2+)/calmodulindependent protein kinase phosphatase in the rat central nervous system[END_REF][START_REF] Princivalle | Distribution of GABA(B) receptor protein in somatosensory cortex and thalamus of adult rats and during postnatal development[END_REF] (Supplementary Fig. 2), we micro-infused an adeno-associated virus (AAV) expressing the inhibitory opsin iC++ with a fluorescent reporter EYFP under the CaMKII promoter (AAV-CaMKII-iC++-EYFP) bilaterally in the ADn in WT mice (Fig. 1d). The iC++ opsin is a light-activated chloride channel that has improved chloride selectivity and conductivity compared to previous generations of inhibitory opsins using chloride pumps [START_REF] Berndt | Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity[END_REF] , and therefore allows efficient inhibition of targeted cells. Control mice received micro-infusion of a virus expressing only the fluorescent reporter (AAV-GFP). Four weeks following surgery, after allowing sufficient time for the virus to be expressed, mice were implanted with an optical fibre in ADn, and trained in contextual fear conditioning. Contextual fear memory was tested either 1 or 28 days post-training. During the test, photo-stimulation was delivered during the last 3 minutes of a 6 minute test session (Fig. 1e-f), allowing a within-animal comparison of the effect of inhibiting ADn cells during memory recall.

Inhibition of ADn cells during 1 day test impaired memory retrieval. Mice infused with the iC++ virus froze less during the light-ON period than OFF, and also less than the control mice microinfused with the GFP virus (Fig. 1e). In contrast, inhibition of ADn cells during the 28 day test had no effect on memory recall (Fig. 1f). In this case, mice infused with the iC++ virus froze at similar levels during the light-ON and light-OFF periods, and at equivalent levels to those in mice micro-infused with the control virus.

These results show that ADn is actively engaged and necessary for recent, but not remote, memory retrieval. These results extend previous experiments in which pharmacological inhibition of the anterior thalamus (including ADn) in rats impaired recall of recent, but not remote, contextual fear memory [START_REF] Lopez | Differential role of the anterior and intralaminar/lateral thalamic nuclei in systems consolidation and reconsolidation[END_REF] .

ADn receives inhibitory projections from CA3.

ADn may play no role in the remote recall of contextual fear conditioning. Alternatively, successful remote memory recall may involve active inhibition of ADn by one or more brain regions, and such inhibition may be necessary for successful remote memory retrieval. To explore this latter possibility, we next examined afferent input to the ADn. Retrobeads were unilaterally infused into the ADn, and retrogradely-labeled neurons were identified (Fig. 2a).

Consistent with previous findings [START_REF] Child | Anterior nucleus of the thalamus: functional organization and clinical implications[END_REF][START_REF] Jankowski | The anterior thalamus provides a subcortical circuit supporting memory and spatial navigation[END_REF][START_REF] Bienkowski | Integration of gene expression and brain-wide connectivity reveals the multiscale organization of mouse hippocampal networks[END_REF][START_REF] Shibata | Organization of anterior cingulate and frontal cortical projections to the anterior and laterodorsal thalamic nuclei in the rat[END_REF][START_REF] Vertes | Analysis of projections from the medial prefrontal cortex to the thalamus in the rat, with emphasis on nucleus reuniens[END_REF] , we found labeled neurons in layer VI of anterior cingulate cortex (ACC), retrosplenial cortex (RSP), pre-subiculum (PRE), indicating that these regions send projections to the ADn (Supplementary Fig. 3a-b). In addition, we found retrogradely labeled neurons in the ipsilateral stratum pyramidale of CA3 subfield of hippocampus (Fig. 2a-b).

To our knowledge, this CA3 to ADn projection has not been previously described. We therefore next sought to verify this connection using complementary tracing methods. We first microinfused the retrograde tracer cholera toxin subunit B (CTB; Fig. 2c-d, Supplementary Fig. 3c-d), unilaterally in the ADn. Consistent with our Retrobeads analysis, retrogradely-labeled neurons were found in the same regions, and specifically in the ipsilateral CA3 region of the hippocampus (Fig. 2d, Supplementary Fig. 3d). We next micro-infused the anterograde tracer Phytohaemagglutinin-L (PHA-L) unilaterally into the CA3 in a separate cohort of mice. We identified anterogradely labeled axons in the ipsilateral ADn, and also in ipsilateral CA1, and contralateral CA3 and CA1 (Fig. 2e-f, Supplementary Fig. 3e-f), as observed previously [START_REF] Finnerty | Functional connectivity from CA3 to the ipsilateral and contralateral CA1 in the rat dorsal hippocampus[END_REF][START_REF] Witter | Intrinsic and extrinsic wiring of CA3: indications for connectional heterogeneity[END_REF] .

Because the ADn might be actively inhibited during remote memory recall, we next used three complementary approaches in order to assess whether CA3 sends inhibitory projections to the ADn. First, we micro-infused the retrograde tracer CTB unilaterally into the ADn, and performed immunohistochemical staining of CA3 sections using the inhibitory cell marker, GAD67 (Fig. 3ab). We found sparse labeling of CTB+ cells in ipsilateral CA3 (2.5±0.7 CTB+ neurons / 10 5 mm 2 in CA3), with the majority of the CTB-labeled CA3 cells co-localized with GAD67.

Second, we used a viral tracing approach to label only the CA3-ADn projections and performed immunohistochemistry to investigate whether the projections are inhibitory. Specifically, we micro-infused AAVrg-Cre-EGFP unilaterally into ADn and AAV-DIO-mCherry bilaterally into CA3 (Fig. 2g-h). Using this approach, Cre is retrogradely transported from axon terminals in ADn to neuronal cell bodies, where Cre allows recombination and expression of mCherry exclusively in the CA3-ADn pathway. Consistent with our other tracing methods, we observed mCherrylabeled somas and axons in CA3 and ADn, respectively, specifically labeling the CA3-ADn projection neurons. The majority of the mCherry-labeled cells were observed in CA3 ipsilateral to the ADn infusion site -1.5 to -2.5 mm posterior to bregma, consistent with our other methods (Supplementary Fig. 3g-h). We further co-stained CA3 and ADn sections with GAD67 and VGAT, respectively, and found that many retrogradely-labeled (mCherry+) neurons in CA3 are inhibitory (GAD67+), and many (mCherry+) axons in ADn are inhibitory (VGAT+) (Fig. 3e-g).

Third, we used an anterograde approach to examine inhibitory projections to the ADn. Specifically, we micro-infused an AAV that expresses the fluorescent reporter (EYFP) in a Crerecombinase-dependent manner (AAV-DIO-EYFP) in mice expressing Cre-recombinase only in inhibitory (i.e., VGAT+) neurons (VGAT-Cre mice). We targeted cortical and hippocampal regions sending projections to the ADn, as identified by the analyses above (Fig. 2a-f, Supplementary Fig. 3a-d). These regions included CA3, retrosplenial cortex, pre-subiculum, and anterior cingulate cortex (Fig. 3c-d, Supplementary Fig. 3i-n). Dense terminal projection labeling in ADn was only observed following viral micro-infusions into CA3 (Fig. 3c-d). Together, these converging lines of evidence indicate that CA3 sends long-range, inhibitory projections to the ADn.

CA3 inhibitory cells projecting to the ADn become more active as memory ages

One possibility is that this CA3-ADn inhibitory projection is active during remote memory recall, and the resulting suppression of ADn activity is necessary for successful memory retrieval. To test this hypothesis, we analyzed the expression of c-Fos in these CA3 inhibitory cells that project to the ADn during memory retrieval at 1 or 28 days post-training. We micro-infused a retrograde AAV (AAV-Ef1a-DO_DIO-TdTomato_EGFP-WPRE-pA) in the ADn of VGAT-Cre mice (Fig. 4a). This virus is retrogradely transported from the axon terminals to neuronal cell bodies, where the double-floxed orientation (DO) allows the expression of the fluorescent tag, TdTomato, only in the cells lacking Cre (Cre-Off). At the same time, the double-floxed inverted open reading frame (DIO) allows the expression of another fluorescent tag, EGFP, only in VGAT+ cells (Cre-On) [START_REF] Saunders | Novel recombinant adeno-associated viruses for Cre activated and inactivated transgene expression in neurons[END_REF] . Therefore, using this approach, in CA3 we were able to simultaneously visualize both excitatory (TdTomato+) and inhibitory (EGFP+) ADn-projection neurons.

Following micro-infusion in the ADn, mice were fear conditioned and then tested 1 or 28 days later (Fig. 4a). Ninety minutes after the test, mice were perfused, and we analyzed the expression of c-Fos in the excitatory (TdTomato+) and inhibitory (EGFP+) projection neurons in the CA3. We found that the retrieval-induced c-Fos expression was elevated in inhibitory CA3 neurons at the 28 day time-point in comparison to the 1 day time-point (Fig. 4b-c). There was no significant change in the activity of excitatory CA3 cells that project to the ADn across retrieval delays (Fig. 4d). Nor was there any change in the overall CA3 c-Fos expression following recent vs. remote contextual fear memory recall (Fig. 4e), most likely reflecting the fact that neurons that project from CA3 to the ADn only represent a small fraction of all neurons in CA3. This result supports the hypothesis that the activity of CA3 inhibitory cells projecting to the ADn increases during remote memory recall.

The inhibitory pathway from CA3 to ADn is necessary for remote, but not recent, memory retrieval.

Since CA3 inhibitory neurons projecting to ADn become more active 28 days after training in comparison to 1 day, we hypothesized that CA3 may be inactivating the ADn during remote memory recall. To investigate this, we micro-infused the ADn with an AAV that expresses the fluorescent reporter (EYFP) with the inhibitory opsin iC++ in a Cre-recombinase-dependent manner (DIO) (AAV-DIO-iC++-EYFP) in VGAT-Cre mice. A different group of mice were micro-infused with a control virus that only contained the fluorescent tag (AAV-DIO-EYFP). We then implanted an optical fiber above the ADn (Fig. 5a-b). We trained the mice in contextual fear conditioning and then tested their memory 1 or 28 d later. During the memory test, the laser was OFF during the first 3 minutes, then turned on during the subsequent 3 minutes to inactivate the projection terminals of the CA3-ADn inhibitory cells.

At the 1-d delay, photo-stimulation did not alter freezing levels in either control or iC++ groups, suggesting that the CA3-ADn inhibitory pathway is not necessary for memory recall at this time point (Fig. 5c). At the 28-d delay, however, while both control and iC++-infused mice froze equally during the OFF period, photo-stimulation reduced freezing in the iC++ group (Fig. 5d), suggesting that selective silencing of this inhibitory CA3-ADn projection impairs recall of remote contextual fear memory. We observed similar results in an independent replication of this experiment (Supplementary Fig. 5a-c). Inhibition of the CA3-ADn inhibitory pathway does not alter locomotor activity in either novel or familiar environments (tested either in the open field or in the conditioning context; Supplementary Fig. 4a-f). These data indicate that the photostimulation-induced reductions in freezing observed during the remote contextual memory test were not confounded by non-specific locomotor effects.

We note that overall freezing levels were much lower in the 1 day test compared to the 28 day test. Reduced freezing at the recent delay was additionally observed in the independent replication of this experiment (Supplementary Fig. 5a-c). Because of reduced freezing levels in the 1 day test, it remains possible that potential effects of manipulating this inhibitory projection on recent memory are obscured by a floor effect. However, additional analyses suggest that freezing in the VGAT-Cre mice in the recent test reflects successful memory retrieval and that floor levels of freezing are much lower. First, freezing levels during the 1 day test were significantly higher than those during the pre-shock period during training (Supplementary Fig. 5e-f). Second, when a separate cohort of VGAT-Cre mice were trained without shocks they exhibited negligible freezing when tested 1 day later (Supplementary Fig. 5g), suggesting that 'floor' freezing is 0-1% in this strain. Third, behavioural (latent inhibition [START_REF] Kiernan | Effects of exposure to a to-be-shocked environment upon the rat's freezing response: evidence for facilitation, latent inhibition, and perceptual learning[END_REF][START_REF] Radulovic | Relationship between fos production and classical fear conditioning: effects of novelty, latent inhibition, and unconditioned stimulus preexposure[END_REF] , Supplementary Fig. 5h) and optogenetic (Fig. 5c-d, Supplementary Fig. 5b-e) interventions reduce freezing, even when control levels are 20-40%. Nonetheless, future experiments should verify that selective silencing of the inhibitory CA3-ADn projections impair remote memory in other mouse lines.

We also assessed how the activity of ADn changes when the CA3-ADn inhibitory projection was silenced following recent vs. remote memory recall. Consistent with our previous analyses (Fig. 1b), retrieval-induced c-Fos expression was reduced at the remote, compared to recent, timepoint in EYFP-infused mice. Importantly, retrieval-induced c-Fos expression was elevated in iC++-infused mice, only at the remote time point (Fig. 5e, Supplementary Fig. 5d). This result is consistent with our hypothesis that the CA3-ADn inhibitory projection becomes more active over time, such that it suppresses the activity of ADn during remote memory recall.

Together, our data show that the ADn becomes disengaged during memory consolidation. This disengagement is a result of recruitment of a novel inhibitory pathway from CA3 to ADn.

Importantly, activation of this CA3-ADn inhibitory pathway is necessary for successful remote fear memory recall. DISCUSSION Event memories are supported by distributed hippocampal-thalamic-cortical networks 11 , but the specific brain regions that contribute to memory expression may vary with memory age. While previous studies have examined the relationship between activation of brain regions and memory age at the time of recall [START_REF] Lopez | Differential role of the anterior and intralaminar/lateral thalamic nuclei in systems consolidation and reconsolidation[END_REF][START_REF] Do-Monte | A temporal shift in the circuits mediating retrieval of fear memory[END_REF][START_REF] Frankland | The involvement of the anterior cingulate cortex in remote contextual fear memory[END_REF][START_REF] Hall | Cellular imaging of zif268 expression in the hippocampus and amygdala during contextual and cued fear memory retrieval: selective activation of hippocampal CA1 neurons during the recall of contextual memories[END_REF][START_REF] Xu | A neural circuit for memory specificity and generalization[END_REF] , here we asked whether the active disengagement of one such brain region, the anterodorsal thalamic nucleus (ADn), is necessary for recall, and in addition, which projection(s) mediate such inhibition. Using a mouse contextual fear conditioning paradigm, we find that retrieval-induced activity of ADn is reduced at remote (28 days) compared to recent (1 day) time points following training. We identified a sparse, long-range projection from inhibitory CA3 neurons to the ADn, and find that retrieval-induced activity of these ADn projecting inhibitory neurons is increased from recent to remote memory recall. More importantly, inhibiting the CA3-ADn inhibitory pathway impairs memory recall, only at the remote time point. As far as we know, this is the first time that this inhibitory anatomical projection has been identified, likely due to its sparsity. Here, using seven different tracing methods (including singlestep, transneuronal, enzymatic, and viral anterograde and retrograde tracers), we provide convergent evidence for an inhibitory CA3-ADn projection. Despite its sparsity, we additionally show that this inhibitory CA3-ADn projection plays a critical role in the retrieval of remote contextual fear memory. While most of the GABAergic cells in the brain target local neurons, some examples of long-range inhibitory connections have also been previously reported. For example, there are bidirectional long-range GABAergic projections between the hippocampus (CA1, CA3, and DG) and entorhinal cortex [START_REF] Melzer | Long-range-projecting GABAergic neurons modulate inhibition in hippocampus and entorhinal cortex[END_REF] , as well as between hippocampus (CA1 and CA3) and medial septum [START_REF] Takacs | Quantitative ultrastructural analysis of basket and axo-axonic cell terminals in the mouse hippocampus[END_REF][START_REF] Toth | Calbindin D28k-containing nonpyramidal cells in the rat hippocampus: their immunoreactivity for GABA and projection to the medial septum[END_REF] . In addition, CA1 GABAergic cells also project to the subiculum and retrosplenial cortex [START_REF] Jinno | Neuronal diversity in GABAergic long-range projections from the hippocampus[END_REF] . Functionally, these long-range inhibitory connections can modulate memory function in a number of different ways, for example, by modulating theta oscillations in the hippocampus that is critical for spatial memory [START_REF] Melzer | Long-range-projecting GABAergic neurons modulate inhibition in hippocampus and entorhinal cortex[END_REF] , and facilitating interregional temporal coordination of activity [START_REF] Jinno | Neuronal diversity in GABAergic long-range projections from the hippocampus[END_REF][START_REF] Buzsaki | Temporal structure in spatially organized neuronal ensembles: a role for interneuronal networks[END_REF] . The CA3-ADn GABAergic connection that we identified here adds to the functional complexity of these long-range inhibitory connections. Our initial motivation to focus on the ADn was based on our previous study which examined c-Fos expression induced in 84 different brain regions by recall of recent and remote contextual fear in mice 11 . By examining how c-Fos expression co-varied across regions, this analysis identified collections of brain regions that were co-activated during recent and remote memory recall. One striking feature of these analyses was that whereas activity in the ADn was positively correlated with activity in other brain regions following recent memory recall, this pattern switched for remote memory recall, where activity in the ADn was negatively correlated with activity in other brain regions. Moreover, at the remote time point, the region with the strongest negative correlation with the ADn was CA3. It is interesting to note that at the recent time point, the correlation between CA3 and ADn was positive (r = 0.64, P = 0.085) in this earlier analysis, although not statistically significant 11 . This is consistent with our observation that the inhibitory CA3-ADn pathway is not active nor required for recent memory retrieval. Our discovery of an inhibitory pathway from CA3 to the ADn may account for the differential role of ADn in recent versus remote memory retrieval (Supplementary Fig. 1). The activity of this projection is dynamically modulated by fear retrieval, with the CA3-ADn GABAergic projection cells being most strongly activated by remote memory recall. This increase in retrieval-induced activity coincides with a decrease in retrieval-induced ADn activation with memory age. More importantly, we provide causal evidence that the inhibition of ADn activity via the CA3-ADn pathway is necessary for remote memory recall. Consistent with previous studies, we observed that CA3 is equivalently active at recent (day-old) and remote contextual fear memory recall (month-old) [START_REF] Lux | Imaging a memory trace over half a life-time in the medial temporal lobe reveals a time-limited role of CA3 neurons in retrieval[END_REF] . However, our results additionally suggest that there is time-dependent recruitment of inhibitory CA3 neurons, and, in particular those that project to ADn (Supplementary Fig. 6). Although we did not observe a statistically significant change in the activation level of the excitatory CA3-ADn projections over time, our data do not exclude the possibility that the excitatory projections also play a role in memory consolidation and retrieval. Analysis of activity-dependent gene expression indicates that recall of recent and remote contextual fear memory engages different networks, with remote memory in particular engaging broad hippocampal-thalamic-cortical networks 11 . The emerging importance of the CA3-ADn inhibitory pathway in recall of contextual fear memory may reflect the switch from the recent to the remote network state. One possible mechanism for this switch is via decrease in feedforward inhibition from DG to CA3 over time as memory ages [START_REF] Guo | Dentate granule cell recruitment of feedforward inhibition governs engram maintenance and remote memory generalization[END_REF][START_REF] Ruediger | Learning-related feedforward inhibitory connectivity growth required for memory precision[END_REF] . Specifically, feedforward inhibition from DG to CA3 is initially increased after fear learning, but is reduced at the remote time point. Reduced inhibition of CA3 could lead to enhanced activity in specific subpopulations of CA3 neurons, including the inhibitory CA3-ADn projection neurons that we studied here. Such a contribution of CA3 to remote memory expression is inconsistent with the idea that the hippocampus plays a time-limited role in expression of these types of memories [START_REF] Kim | Modality-specific retrograde amnesia of fear[END_REF] . In fact, more recent optogenetic [START_REF] Goshen | Dynamics of retrieval strategies for remote memories[END_REF][START_REF] Kitamura | Engrams and circuits crucial for systems consolidation of a memory[END_REF] and imaging [START_REF] Lux | Imaging a memory trace over half a life-time in the medial temporal lobe reveals a time-limited role of CA3 neurons in retrieval[END_REF] studies suggest that the hippocampus contributes to remote memory recall. These studies, together with our current data, are consistent with models of systems consolidation that emphasize a prolonged role for the hippocampus in memory retrieval, even at remote time-points [START_REF] Moscovitch | Episodic Memory and Beyond: The Hippocampus and Neocortex in Transformation[END_REF] .

MATERIALS AND METHODS

Mice

Micro-infusion of viral vectors and tracers

Various adeno-associated viral vectors (AAVs) and tracers were used, as specified in each experiment. All viral vectors were infused bilaterally at 1.2 µl per side and 0.1 µl/min, unless otherwise specified. Tracers were infused unilaterally at 0.5 µl volume and 0.1 µl/min (Retrobeads), 0.1 µl volume and 0.01 µl/min (CTB), or 0.05 µl volume and 0.01 µl/min (PHA-L). Specifically, in Figure 1, AAV8-CaMKII-iC++-EYFP virus was obtained from Stanford University vector core, and micro-infused bilaterally in the ADn (-0.8 mm AP, ± 0.2 mm ML, -3 mm DV, from bregma according to Paxinos and Franklin [2012] [START_REF] Paxinos | The mouse brain in stereotaxic coordinates[END_REF] ) of WT mice to allow optogenetic silencing of the ADn. The control group received ADn infusion of AAV(DJ)-CMV-GFP, where GFP is expressed under the CMV promoter (made in house). Four weeks following surgery, optical fibres were placed above ADn (-0.8 mm AP, ± 0 mm ML, -2.7 mm DV).

In Figure 2, Figure 3, and Supplementary Figure 3, the retrograde tracer Retrobeads (red) was obtained from Lumafluor (Durham, NC) and micro-infused unilaterally in the ADn of WT mice to retrogradely label regions that send monosynaptic projections to the ADn. To verify the results obtained using retrobeads, in separate cohorts of mice, we micro-infused another retrograde tracer cholera toxin subunit B (CTB, Thermo Fisher Scientific, Waltham, MA) into the ADn (Fig. 2, Supplementary Fig. 3). Mice were perfused 14 days following surgery inorder to visualize projection neurons. To verify these results with an anterograde tracer, we micro-infused Phytohaemagglutinin-L (PHA-L; Vector Laboratories, Burlingame, CA) into the CA3. Mice were perfused 3 weeks following surgery inorder to visualize projection neurons. We additionally verified the CA3-ADn projection by specifically labeling the projection using a dual-viral tracing approach. We micro-infused 0.5 µl of pENN-AAVrg-hSyn-H-eGFP-Cre-WPRE-SV40 (#105540-AAVrg, Addgene, Cambridge, MA) unilaterally into ADn and 0.6 µl of AAV(DJ)-hSyn-DIO-mCherry bilaterally into CA3. Mice were perfused 4 weeks following surgery inorder to visualize the CA3-ADn projection (mCherry+) neurons.

In Figure 4, AAVrg-EF1a-DO_DIO-TdTomato-EGFP was obtained from Addgene (Cambridge, MA), and micro-infused unilaterally in the ADn of VGAT-Cre mice to retrogradely label excitatory and inhibitory cells that project to the ADn. This construct allows Cre-dependent expression of EGFP, and expression of TdTomato in the absence of Cre [START_REF] Saunders | Novel recombinant adeno-associated viruses for Cre activated and inactivated transgene expression in neurons[END_REF] . The AAV is produced in a retrograde serotype to allow retrograde labeling or projection neurons to the site of injection.

In Figure 5, Supplementary Figure 4, and Supplementary Figure 5, AAV(DJ)-EF1a-DIO-iC++-EYFP was obtained from Stanford Neuroscience Gene Vector and Virus Core, and microinfused bilaterally in CA3 (-2.2 mm AP, ± 2.7 mm ML, -2.4 mm DV, from bregma according to The micro-infusion surgery protocol is similar to the previously described [START_REF] Richards | Patterns across multiple memories are identified over time[END_REF] . Specifically, mice were pretreated with atropine sulphate (0.1 mg/kg, intraperitoneal), then anesthetized with chloral hydrate (400 mg/kg, intraperitoneal). Mice were then placed on a stereotaxic frame, and holes were drilled in the skull at the targeted coordinates. Viral vector was micro-infused at 0.1 μl/min via glass pipettes connected to a Hamilton microsyringe with polyethylene tubing. After micro-infusion, the glass pipette was left in the brain for another 5 mins to allow sufficient time for the virus to diffuse. We have found that this infusion procedure produces high infection in the targeted region, without significant spread outside the region of interest [START_REF] Richards | Patterns across multiple memories are identified over time[END_REF][START_REF] Rashid | Competition between engrams influences fear memory formation and recall[END_REF] . For infusions of CTB and PHA-L tracers we followed a similar procedure but performed injections using a Nanoject III injector (Drummond Scientific, Broomall, PA) and left the pipette in the brain for 10-15 minutes following infusions to minimize spread of tracers outside of regions of interest. Mice were then treated with analgesic (ketoprofen, 5 mg/kg, subcutaneous) and 1 ml of 0.9% saline (subcutaneous).

For the optogenetic experiments, mice received optical fibre implant 4 weeks following viral infusion. Optical fibres were constructed in-house by attaching a 5 mm piece of 200-μm optical fibre (with a 0.37 numerical-aperture, NA) to a 1.25-mm zirconia ferrule (final fibre extended 3 mm beyond ferrule). Fibres were attached with epoxy resin into ferrules, cut and polished.

Optical fibres were stabilized to the skull with screws and black dental cement to minimize light leakage. For the 1 day groups, viral infusion was performed 5 weeks prior to training/testing. For the 28 day groups, viral infusion was performed 1 week prior to contextual fear training, and 4 weeks prior to testing. Thus, for both 1 day and 28 day groups, the interval between viral infusion and testing was 5 weeks.

Behavioural Experiments

Behavioural experiments for mice that did not undergo viral micro-infusion were carried out in 8 weeks old WT mice (Fig. 1a-b). Experiments for mice that were micro-infused with viral vectors started one week following optical fibre implant (five weeks after micro-infusion).

Contextual fear conditioning

WT mice were trained in a standard contextual fear conditioning paradigm, as previously described [START_REF] Wang | The precision of remote context memories does not require the hippocampus[END_REF] . Mice were first habituated to the conditioning chamber for 120 s, then given 3 shocks (0.5 mA each, 60 s apart), and remained in the chamber for another 60 s following the last shock. The amount of time mice spent freezing (% freezing, with minimum bout of 1 s) was monitored with overhead cameras, and calculated using automatic scoring software FreezeFrame (Actimetrics).

VGAT-Cre mice, when trained using the standard contextual fear conditioning protocol, froze less than the WT mice. Therefore, for the majority of experiments, VGAT-Cre mice were trained in an altered version of the contextual fear conditioning paradigm to ensure that they show successful fear memory retrieval (significantly higher freezing levels in comparison to pre-shock, Supplementary Fig. 5e-f). Specifically, VGAT-Cre mice were habituated to the conditioning chamber for 5 min one day prior to training. On the day of training, mice were habituated to the chamber for 120 s, then given 4 shocks (0.7 mA each, pseudo-random intervals), and remained in the context for another 30 s following the last shock (Supplementary Fig. 5a-d). For the independent replication, we extended the habituation period to 2 days (10 min, twice per day), as preliminary data showed that this extended habituation protocol slightly reduced variability in freezing levels. All other training and testing protocols were otherwise identical.

Either 1 or 28 days following training, mice were placed back into the training context for 5 mins, without shock. The amount of time mice spent freezing (% freezing) was monitored with overhead cameras, and manually scored by experiments blinded to the group assignment of mice. For the optogenetic experiments, the test session was 6 mins in duration, where the laser was OFF during the first 3 mins, then turned ON during the last 3 mins when continuous direct current blue laser photo-stimulation (473 nm, 10 mW power) was applied.

To control for the possibility that optogenetic manipulation of the CA3-ADn pathway alters locomotion in a novel environment, VGAT-Cre mice were micro-infused with AAV-DIO-iC++-EYFP or control virus (AAV-DIO-EYFP) in the CA3, then implanted with optical fibres in the ADn four weeks later. One week after optical fibre implant, mice were placed in the centre of an open square arena (45 cm x 45 cm x 20 cm height) and allowed to explore for 6 mins [START_REF] Arruda-Carvalho | Conditional deletion of alpha-CaMKII impairs integration of adult-generated granule cells into dentate gyrus circuits and hippocampus-dependent learning[END_REF] , where the laser was OFF during the first 3 mins, then turned ON during the last 3 mins (Supplementary Fig. 4a-b). To control for the possibility that optogenetic manipulation of the CA3-ADn pathway alters locomotion in a familiar environment, VGAT-Cre mice were micro-infused with AAV-DIO-iC++-EYFP or control virus (AAV-DIO-EYFP) in the CA3, then implanted with optical fibres in the ADn four weeks later. One week after optical fibre implant, mice were placed in the centre of an open square arena (45 cm x 45 cm x 20 cm height) and allowed to explore for 6 mins (open field pre-exposure) [START_REF] Arruda-Carvalho | Conditional deletion of alpha-CaMKII impairs integration of adult-generated granule cells into dentate gyrus circuits and hippocampus-dependent learning[END_REF] on day 1. Twenty-four hours later, mice were placed back into the same open field arena and allowed to explore for 6 mins (open field test) where the laser was OFF during the first 3 mins, then turned ON during the last 3 mins (Supplementary Fig. 4c-e). We additionally assessed the effect of optogenetic inhibition of the CA3-ADn pathway on locomotion in a second familiar environment, the fear conditioning chamber (without shocks). VGAT-Cre mice were habituated to the fear conditioning chamber for 6 min without shocks, 1 day after open field test (fear chamber pre-exposure). Twenty-four hours later, mice were placed back into the same conditioning chamber to explore for 6 mins (fear test) where the laser was OFF during the first 3 mins, then turned ON during the last 3 mins (Supplementary Fig. 4c,f). The location of the mouse was tracked using an overhead camera. The total distance traveled was assessed using ImageJ and freezing levels were assessed using FreezeFrame.

Immunohistochemistry

Immunofluorescence staining was conducted as previously described [START_REF] Restivo | Development of Adult-Generated Cell Connectivity with Excitatory and Inhibitory Cell Populations in the Hippocampus[END_REF] . Specifically, at the end of behaviour experiments, mice were transcardially perfused with 1x PBS followed by 4% paraformaldehyde. For the c-Fos experiment, mice were perfused 90 mins after behaviour test or training. Brains were fixed overnight at 4 o C, and transferred to 30% sucrose solution for 48 hours. Brains were sectioned coronally using a cryostat (Leica CM1850), and 50 µm sections were obtained for the ADn or hippocampus.

For ɑCaMKII and GAD67 immunostaining, free-floating sections were blocked with PBS containing 2.5% bovine serum albumin and 0.3% Triton-X for 30 mins. Afterwards, sections were incubated in PBS containing mouse anti-ɑCaMKII primary antibody (1:200 dilution; Sigma Cat# C265 RRID:AB_258808) or mouse anti-GAD67 (1:500 dilution; EMD Millipore Cat# MAB5406 RRID: AB_2278725) primary antibody for 24 hours at 4 ºC. Sections were washed with PBS (3 times), then incubated with PBS containing goat anti-mouse ALEXA Fluor 488

(1:500 dilution, Thermo Fisher Scientific Cat# A-11001 RRID:AB_2534069) secondary antibody for 24 hours at 4 ºC. Sections were washed with PBS, mounted on gel-coated slides, and coverslipped with Vectashield fluorescent mounting medium (Vector Laboratories). Images were obtained using a confocal laser scanning microscope (LSM 710; Zeiss) with a 20X objective.

For c-Fos immunostaining, free-floating sections were blocked with PBS containing 2.5% bovine serum albumin and 0.3% Triton-X for 30 mins. Afterwards, sections were incubated in PBS containing rabbit polyclonal anti-c-Fos primary antibody (1:1000 dilution; Santa Cruz Biotechnology Cat# sc-52 RRID:AB_2106783) for 48 hours at 4 o C. Sections were washed with PBS (3 times), then incubated with PBS containing goat anti-rabbit ALEXA Fluor 633 (for c-Fos, 1:500 dilution, Thermo Fisher Scientific Cat# A-21070 RRID:AB_2535731) secondary antibody for 24 hours at 4 o C. Sections were washed with PBS, mounted on gel-coated slides, and coverslipped with Vectashield fluorescent mounting medium (Vector Laboratories). Images were obtained using a confocal laser scanning microscope (LSM 710; Zeiss) with a 20X objective.

For cell counting experiments in ADn, every section in ADn or every second section in CA3 was assessed for c-Fos+ cells. Approximately 4-6 sections/mouse were counted and averaged, with 6-10 mice/group. To evaluate the activity in ADn or CA3 following recent vs. remote memory test, the number of c-Fos+ cells in ADn or CA3 (total number of c-Fos+ cells / 10,000 um 2 ) was counted. To evaluate the activity of CA3 to ADn projection neurons, c-Fos co-localization in TdTomato+ cells (excitatory connections; total numbers of c-Fos+ and TdTomato+ cells / um 3 ), or c-Fos co-localization in EGFP+ cells (inhibitory connections; total numbers of c-Fos+ and EGFP+ cells / um 3 ) were calculated. All results were then normalized to values obtained in home cage control groups (3-8 animals) in respective cell populations.

To visualize CTB retrogradely-labeled cells, brain sections were blocked as described above and then incubated with rabbit anti-CTB (1:1000 dilution; Abcam Cat# ab34992 RRID: AB_726859) and mouse anti-GAD67 (1:500 dilution; EMD Millipore Cat# MAB5406 RRID: AB_2278725) primary antibodies for 48 hours at 4 ºC. Sections were washed with PBS (3 times), then incubated with PBS containing goat anti-rabbit ALEXA Fluor 633 (for CTB, 1:500 dilution, Thermo Fisher Scientific Cat# A-21070 RRID:AB_2535731) and goat anti-mouse ALEXA Fluor 488 (for GAD67, 1:500 dilution, Thermo Fisher Scientific Cat# A-11001 RRID:AB_2534069) secondary antibodies for 24 hours at 4 ºC. Sections were washed with PBS, mounted on gel-coated slides, and coverslipped with Vectashield fluorescent mounting medium (Vector Laboratories). Images were obtained using a confocal laser scanning microscope (LSM 710; Zeiss) with 20X and 40X objectives. To visualize PHA-L anterogradelylabeled cells, brain sections were co-stained with PHA-L antibody. Specifically, brain sections were blocked as described above and incubated with rabbit anti-PHA (E+L) primary antibody (1:1000 dilution, Vector Laboratories Cat# AS-2300-1 RRID: AB_2313686) for 24 hours at 4 ºC.

Sections were washed with PBS (3 times), then incubated with PBS containing goat anti-rabbit ALEXA Fluor 488 (1:500 dilution, Thermo Fisher Scientific Cat# A-11008 RRID:AB_143165) secondary antibody for 24 hours at 4 ºC. Sections were washed with PBS, mounted on gelcoated slides, and coverslipped with Vectashield fluorescent mounting medium (Vector Laboratories). Images were obtained using a confocal laser scanning microscope (LSM 710; Zeiss) with 20X and 40X objectives.

To specifically visualize CA3-ADn projection neurons, brain sections containing CA3 from mice infused with AAVrg-Cre-EGFP and AAV(DJ)-hSyn-DIO-mCherry (unilaterally in ADn and bilaterally in CA3, respectively) were blocked as described above and then incubated with rabbit anti-RFP (for mCherry, 1:1000 dilution, Rockland Cat# 600-401-379 RRID: AB_2209751) and mouse anti-GAD67 (1:500 dilution; EMD Millipore Cat# MAB5406 RRID: AB_2278725) for 48 hours at 4 ºC. Sections were washed with PBS (3 times), then incubated with PBS containing goat anti-rabbit ALEXA Fluor 568 (for mCherry, 1:500 dilution, Thermo Fisher Scientific Cat# A-11011 RRID:AB_143157) and goat anti-mouse ALEXA Fluor 647 (for GAD67, 1:500 dilution, Thermo Fisher Scientific Cat# A-21235 RRID: AB_2535804) secondary antibodies for 24 hours at 4 ºC. To visualize the projection terminals of the same neurons, sections containing the ADn from the same brains were blocked and then incubated with mouse anti-RFP (1:1000 dilution, Rockland Cat# 200-301-379 RRID: AB_2611063) and rabbit anti-VGAT (1:500 dilution, EMD Millipore Cat# AB2257 RRID: AB_1587623) primary antibodies for 48 hours at 4 ºC. Sections were washed with PBS (3 times), then incubated with PBS containing goat anti-mouse ALEXA Fluor 568 (for mCherry, 1:500 dilution, Thermo Fisher Scientific Cat# A-11004 RRID:AB_2534072) and goat anti-rabbit ALEXA Fluor 633 (for VGAT, 1:500 dilution, Thermo Fisher Scientific Cat# A-21070 RRID:AB_2535731) secondary antibodies for 24 hours at 4 ºC.

Sections were washed with PBS, mounted on gel-coated slides, and coverslipped with Vectashield fluorescent mounting medium (Vector Laboratories). Images were obtained using a confocal laser scanning microscope (LSM 710; Zeiss) with 20X, 40X, and 100X objectives.

Statistical analysis

No statistical tests were used to pre-determine sample size, but the sample sizes used are similar to those generally used within the field. Data were analyzed using parametric two-way repeated measures ANOVA, or two-sample Student's unpaired t-test. For comparisons to a hypothetical mean of 1, one-sample t-test was used. Where appropriate, ANOVA was followed by post hoc pairwise comparisons with Bonferroni correction. If data were significantly nonnormal (with ɑ = 0.05), Kruskal-Wallis test or Mann-Whitney test (between-group comparisons) and Wilcoxon signed-rank test (within-group comparisons) were used accordingly. All tests were two-sided. Statistical analyses were performed using Graphpad Prism V8.

ACKNOWLEDGMENTS

  All procedures were approved by the Canadian Council for Animal Care (CCAC) and the Animal Care Committees at the Hospital for Sick Children and the University of Toronto. Experiments were conducted on 8-12 week old male and female F1 hybrid (C57BL/6NTac x 129S6/SvEvTac) wild-type (WT) mice, or VGAT-Cre knock-in transgenic mice where Cre-recombinase was targeted to the VGAT locus, without disrupting endogenous VGAT expression. The VGAT-Cre mice were obtained from Jackson Lab. Heterozygous VGAT-Cre mice were maintained on a C57BL/6NTac x 129S6/SvEvTac genetic background.All mice were weaned at 21 days, and group housed with 2-5 mice per cage in a temperaturecontrolled room with 12 h light/dark cycle (light on during the day). All experiments were performed between 8 am and 12 pm. Mice were given ad libitum access to food and water. Mice were randomly assigned to experimental groups. The experimenters were aware of the experimental group assignment, as the same experimenters conducted the training and testing of all mice, but were blinded during behavioural assessment and cell counting experiments.Mice were excluded from analysis based on post-experimental histology: only mice with robust expression of the viral vectors (as specified in each experiment) specifically in the targeted region were included.

  Paxinos and Franklin [2012] 51 ) of VGAT-Cre mice. In this inhibitory opsin viral vector, the double-floxed inverted open reading frame of iC++ fused to EYFP can be expressed from the EF1a promoter after Cre-mediated recombination. Four weeks following surgery, optical fibres were placed above ADn to allow optogenetic silencing of CA3-ADn projections. The control group received infusion of AAV-hSyn-DIO-EGFP (Addgene) in CA3 of VGAT-Cre mice, where the double-floxed inverted open reading frame of the EGFP fluorescence tag can be expressed from the human synapsin (hSyn) promoter after Cre-mediated recombination.

Fig. 1 |

 1 Fig. 1 | ADn activity is necessary for recall of recently acquired contextual fear memory.

Fig. 2 |Fig. 3 |

 23 Fig. 2 | Identification of anatomical projections to the ADn. WT mice were micro-infused

Fig. 4 |

 4 Fig. 4 | CA3 inhibitory cells projecting to the ADn become more active at remote time

Fig. 5 |

 5 Fig. 5 | CA3 inhibitory cells projecting to the ADn are necessary for the recall of remote

This work was supported by Canadian Institutes of Health Research (CIHR) grants to PWF (FDN143227) and SAJ (MOP74650). FX was supported by fellowships from NSERC and Biological Sleep and Rhythms (CIHR) scholarships. GV was supported by the Biological Sleep and Rhythms (CIHR) fellowship. AIR was supported by fellowships from NSERC and NIMH (1 F31 MH120920-01). LMT was supported by fellowships from The Hospital for Sick Children RestraComp and NSERC scholarships. PWF and SAJ are senior fellows in the Child Brain & Development Program and the Brain, Mind & Consciousness programs, respectively, at the Canadian Institute for Advanced Research (CIFAR).

Data availability

The data generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

c-Fos analysis

To examine how the activity of ADn changes from recent (1 day) to remote (28 days) memory retrieval (Fig. 1a-b), we trained WT mice in contextual fear conditioning and tested them either 1 or 28 days later. Ninety minutes post-test, mice were perfused, and their brains used for c-Fos staining (see below). We analyzed c-Fos expression in the ADn in home cage control mice, immediate-shock controls (1 shock at 0.6mA upon exposure to fear conditioning chamber, tested at 1 or 28 days post-training), no-shock controls (5 min exploration in fear conditioning chamber, tested at 1 or 28 days post-training), and those that underwent contextual fear conditioning (tested at 1 or 28 days post-training). For quantification and visualizations, c-Fos levels in all groups were normalized to averaged c-Fos in home cage control mice.

To examine how the activity of the excitatory and inhibitory projection neurons from CA3 to ADn changes from recent to remote memory retrieval, VGAT-Cre mice were infused with the retrograde tracer AAVrg-EF1a-DO_DIO-TdTomato-EGFP. Five weeks following infusion, mice were trained in contextual fear conditioning paradigm, and tested 1 day later. For the 28 day group, mice were trained in contextual fear conditioning 1 week following viral infusion, and tested 4 weeks later. Ninety minutes post-test, all mice were perfused, and their brains used for c-Fos staining. We analyzed c-Fos expression in the CA3-ADn projection neurons (excitatory and inhibitory), as well as total c-Fos in CA3, in home cage control mice, and those that underwent recent or remote memory test (Fig. 4).

Open field

AUTHOR CONTRIBUTIONS

GV, FX, AIR, LMT, SAJ and PWF designed the experiments. GV, FX, AIR and LMT conducted all of the experiments and data analysis. PWF, FX, GV, AIR and LMT wrote the paper.

COMPETING INTERESTS

The authors declare no competing financial interests.

FIGURES AND FIGURE LEGENDS