Emmanuel Procyk

Vincent Fontanier

Matthieu Sarazin

Bruno Delord

Clément Goussi

Charlie R E Wilson

The midcingulate cortex and temporal integration

Keywords: Cingulate, Timescales, Anterior Cingulate, Midcingulate, Adaptation, Performance monitoring, Inhibition, Metastability

The ability to integrate information across time at multiple timescales is a vital element of adaptive behaviour, because it provides the capacity to link events separated in time, extract useful information from previous events and actions, and to construct plans for behaviour over time. Here we make the argument that this information integration capacity is a central function of the midcingulate cortex (MCC), by reviewing the anatomical, intrinsic network, neurophysiological, and behavioural properties of MCC. The MCC is the region of the medial wall situated dorsal to the corpus callosum and sometimes referred to as dACC. It is positioned within the densely connected core network of the primate brain, with a rich diversity of cognitive, somatomotor and autonomic connections. The MCC shows strong local network inhibition which appears to control the metastability of the regionan established feature of many cortical networks in which the neural dynamics move through a series of quasi-stationary states. We propose that the strong local inhibition in MCC leads to particularly long dynamic state durations, and so less frequent transitions. Apparently as a result of these anatomical features and synaptic and ionic determinants, the MCC cells display the longest neuronal timescales amongst a range of recorded cortical areas. We conclude that the anatomical position, intrinsic properties, and local network interactions of MCC make it a uniquely positioned cortical area to perform the integration of diverse information over time that is necessary for behavioural adaptation.

On time and behaviour

Integrating information across time at multiple timescales is a key feature of higher order cognition. Information integration across time permits us to link events and actions separated in time; to draw out useful evidence from a history of behavioural and environmental reactions; and to construct serial, time-extended plans that provide both retrospective and prospective contexts to behaviour. This capacity is thus linked to the fact that the environment itself is structured along multiple timescales. This capacity appears phenomenologically in the structure of behaviours of animal species that have advanced adaptive abilities and problem-solving skills. Primates, for instance, can plan extended routes to seek resources; and they can decide, reorganize, and use detours in the face of intervening events and knowledge acquired from past events [START_REF] Janmaat | Primates Take Weather into Account when Searching for Fruits[END_REF][START_REF] Noser | Mental maps in chacma baboons (Papio ursinus): using inter-group encounters as a natural experiment[END_REF]. Certain primates and birds also use the history of interactions with objects to build tools [START_REF] Gruber | Travel fosters tool use in wild chimpanzees[END_REF]. In laboratory settings the ability of monkey species to extract adaptive performance rules over a long task history is well established [START_REF] Harlow | The formation of learning sets[END_REF]. These capacities can be considered together as capacities of behavioural adaptation. In its essence, the adaptation of behaviour requires integration of multiple information sources over multiple time scales, to integrate action, outcomes, and episodic information. Adapting to real life situations is mostly non-markovian because the history of behavioural interaction is crucial.

In a highly dynamical system like the brain, performing functions and calculations at multiple concurrent timescales requires specialized algorithms and machinery. This is therefore a difficult task requiring a complex neurobiological implementation, but in highly adaptive species the development of such capacities is clearly worthwhile [START_REF] Pearson | Decision Making: The Neuroethological Turn[END_REF]. Several lines of research suggest that certain frontal areas have specific roles in the elaboration of behaviours over long timescales [START_REF] Passingham | The Neurobiology of the Prefrontal Cortex: Anatomy, Evolution, and the Origin of Insight[END_REF]. In this chapter we collect evidence from multiple domains suggesting that the midcingulate cortex (MCC) of primates contributes to the integration of information over long timescales. We propose that the physiological properties of the MCC network provide the dynamical properties necessary for this temporal integration, and that in turn these properties permit the multiple functions that have been ascribed to MCC, i.e. learning, feedback adaptation, and regulation of exploratory behaviour.

The first lines of evidence for a role of frontal medial cortex regions in temporal integration come from studies focused on adaptive behaviour. Indeed, efficient adaptation relies on extracting knowledge, structures as well as changes, from a series of interactions with the environment. In experimental setups this corresponds learning from outcomes and feedback after a series of choices. In human and non-human primates a large-scale network encompassing frontal and posterior associative cortical areas contribute to adaptive behaviours [START_REF] Duncan | The multiple-demand (MD) system of the primate brain: mental programs for intelligent behaviour[END_REF][START_REF] Mitchell | A Putative Multiple-Demand System in the Macaque Brain[END_REF][START_REF] Premereur | Functional MRI in Macaque Monkeys during Task Switching[END_REF].

Int Rev Neurobiol. 2021;158:395-419. doi: 10.1016/bs.irn.2020.12.004. Epub 2021 Jan 29. PMID: 33785153 Moreover, fMRI consistently reveals activations in one region in the medial wall in relation to outcome monitoring and the apparent regulation of cognitive control [START_REF] Passingham | Attention to action[END_REF][START_REF] Jueptner | Anatomy of motor learning. I. Frontal cortex and attention to action[END_REF][START_REF] Bush | Dorsal anterior cingulate cortex: a role in reward-based decision making[END_REF][START_REF] Kouneiher | Motivation and cognitive control in the human prefrontal cortex[END_REF][START_REF] Amiez | Modulation of feedback related activity in the rostral anterior cingulate cortex during trial and error exploration[END_REF]. The region, often named dACC in the primate literature, corresponds to the anatomically defined midcingulate cortex (MCC, see figure). Studies sometimes show extensions of activation into preSMA or SMA and in more or less anterior parts of the MCC, and the dissociation of these activations remains an important task for the field [START_REF] Amiez | The Location of Feedback-Related Activity in the Midcingulate Cortex Is Predicted by Local Morphology[END_REF]. MCC activation is observed when subjects actively seek information and/or rewards, for example when they are exploring or foraging. The MCC seems sensitive to the volatility of the environment, for example to the temporal stability of outcomes, as well as to the propensity of subjects to make choices based on information integrated over multiple trials [START_REF] Behrens | Learning the value of information in an uncertain world[END_REF][START_REF] Meder | Simultaneous representation of a spectrum of dynamically changing value estimates during decision making[END_REF]. The activation of this region in particular during exploratory decisions reveals its particular role in encoding values and prospective information relevant to adaptation [START_REF] Kolling | Neural Mechanisms of Foraging[END_REF]2016b). Studies suggest, then, that evaluation and temporal integration of information in or through the MCC might serve to guide and sustain selected behaviours in the face of current and changing features of the environment (Kolling et al., 2016b). Testing in patients for clinical investigations has provided compelling data on the behavioural effect but also on the subjective feeling induced by electrical microstimulation of the MCC [START_REF] Talairach | The cingulate gyrus and human behaviour[END_REF][START_REF] Parvizi | The will to persevere induced by electrical stimulation of the human cingulate gyrus[END_REF][START_REF] Caruana | 33785153 elicited by electrical stimulation of the human cingulate cortex[END_REF]. This stimulation regularly leads localized effects on natural goal-directed behaviours like exploratory scanning of the environment with the eyes or the head, tactile search, kneading or palpation directed toward the body or towards nearby objects [START_REF] Talairach | The cingulate gyrus and human behaviour[END_REF][START_REF] Caruana | 33785153 elicited by electrical stimulation of the human cingulate cortex[END_REF]. Patients also report general feelings of an urge to act. These results could be interpreted as reflecting MCC contribution to generate the incentive for behaviours directed towards information search. The effects of microstimulation on search-oriented behaviours concern mostly the anterior part of the MCC, the equivalent of CMAr in monkeys.

Studies in human subjects reveal a wide array of correlative roles for the MCC, but there is amongst these a clear case for a role for the MCC in the temporal integration of diverse signals necessary for adaptive behaviour. Our proposition here is that the best way to understand this role, and the way in which seemingly diverse responses emerge from the same region, is to consider in detail the mechanisms within MCC and its position within the anatomical hierarchy. The rest of this chapter therefore considers the evidence, largely from animal studies, for mechanisms of temporal integration in MCC.

The neurobiological source of temporal integration in MCC

Multiple neuroanatomical and physiological features suggest that the MCC holds a special position within the executive and more global networks, and that it is well placed and constructed to integrate diverse information in the temporal domain.

Anatomical specificities of the MCC.

In most monkey recording studies referred to in this chapter, the regions of interest have been the cortex lying within the cingulate sulcus, most often the dorsal bank, and at rostro-caudal levels anterior to the genu of the arcuate sulcus [START_REF] Procyk | Midcingulate Motor Map and Feedback Detection: Converging Data from Humans and Monkeys[END_REF]. We refer to this region as MCC. The entire MCC region is subject to debate in part because of the multiple versions of labelling used to describe its subdivisions. Regions' names as well as cytoarchitectonic labels are used in different ways across multiple papers, reviews, and even brain atlases. Major labelling issues are the definitions of 'ACC' or '24c', which have evolved. Here we provide a definition of the term MCC we use.

The MCC is an anatomical entity defined by B evidenced in humans and monkeys, with putative equivalents in rodents, and it appears to have a separate functional identity [START_REF] Vogt | Human cingulate cortex: surface features, flat maps, and cytoarchitecture[END_REF][START_REF] Vogt | Cytoarchitecture of mouse and rat cingulate cortex with human homologies[END_REF]Vogt, 2016).

Cytoarchitecture. In humans, the MCC includes the functional subdivision frequently referred to as dACC. It is positioned dorsal to the corpus callosum and, in humans, posterior to the level of the genu of the corpus callosum. MCC cytoarchitecture can be contrasted to the cytoarchitecture of the ACC (anterior cingulate cortex) which is rostral to MCC, and to the cytoarchitecture of the PCC (posterior cingulate cortex). MCC is composed of agranular cortex, which like ACC lacks a granular layer IV [START_REF] Palomero-Gallagher | Receptor architecture of human cingulate cortex: Evaluation of the four-region neurobiological model[END_REF]. In Vogt's nomenclature, MCC includes cytoarchitectonic areas a24c',b', and a', areas p24c',b' and a' as well as area 24d. The posterior part of MCC, containing area 24d, has the largest pyramidal cells which contribute to cingulate spinal projections and form the caudal cingulate motor areas in monkeys. Debates regarding comparative assessments in monkeys and humans relate to the dorsal and anterior parts of the MCC. Anteriorly, the limit with ACC is not clearly identified and could contain an intermediate (rACC) region. The dorsal limit of the sulcus, in terms of architecture and function is also debated [START_REF] Sallet | Neuroanatomical Basis of Motivational and Cognitive Control: A Focus on the Medial and Lateral Prefrontal Cortex[END_REF]. Histological work from multiple groups show that indeed the dorsal limit position might vary along the rostral-caudal axis and potentially vary with species and individuals. More precise work is required to solve these issues. In this chapter we include the dorsal bank of the cingulate sulcus because the cytoarchitecture, the connectivity patterns and the extension of cingulate motor areas (see below) suggest an anatomo-functional link to the rest of MCC [START_REF] Petrides | Comparative architectonic analysis of the human and the macaque frontal cortex[END_REF][START_REF] Sallet | Neuroanatomical Basis of Motivational and Cognitive Control: A Focus on the Medial and Lateral Prefrontal Cortex[END_REF][START_REF] Procyk | Midcingulate Motor Map and Feedback Detection: Converging Data from Humans and Monkeys[END_REF].

Receptor mapping. In humans and non-human primates, the MCC region and its subdivisions are also characterized by specific patterns of receptor distributions. We cite here two remarkable features. The anterior MCC contains higher levels of dopamine receptor D1 binding in the superficial layers, whereas area 24d has virtually none in superficial and deep layers. Also of note is the relative level of GABAergic receptor mappings: anterior MCC contains relatively lower levels of GABA-B receptors and slightly higher levels of GABA-A, compared to posterior MCC. The distribution of GABA receptors differs also clearly between anterior MCC and ACC [START_REF] Bozkurt | Distributions of transmitter receptors in the macaque cingulate cortex[END_REF][START_REF] Palomero-Gallagher | Cytology and receptor architecture of human anterior cingulate cortex[END_REF]2009).

MCC in the anatomical hierarchy.

The MCC also sits at a remarkable anatomical position, first within the large-scale network formed by cortical areas, second within the somato-motor systems, and third in relation to the autonomic system. The large-scale cortical network is, in primates, a system with heterogeneous densities of inter-area connectivity [START_REF] Markov | Cortical high-density counterstream architectures[END_REF]. It forms a so-called bow-tie organization including a core network with high density (>90%) of connections between areas, linked to side systems by low density connectivity patterns [START_REF] Markov | Cortical high-density counterstream architectures[END_REF]. The structure has some similarities with the early model of global workspace architecture that contained a central general workspace network connected to hierarchically distant modules processing specific information [START_REF] Dehaene | A neuronal model of a global workspace in effortful cognitive tasks[END_REF]. Within the core network lies the MCC and its interconnections with the lateral prefrontal cortex, as well as other cortical regions that were shown to form the Multiple-demand network [START_REF] Duncan | The Structure of Cognition:Attentional Episodes in Mind and Brain[END_REF]. As we shall see, MCC's position within this architecture, combined with more local intrinsic properties, appears crucial for its functional identity.

The MCC displays other anatomical features that, we think, must be considered to comprehend its functional specificity and contributions to cognition. First the MCC has anatomical links with the sensorimotor system, and contains, in monkeys and humans, two sensorimotor maps named CMAr and CMAc in monkeys (M3 and M4 in [START_REF] Morecraft | Organization of face representation in the cingulate cortex of the rhesus monkey[END_REF]) and RCZa and RCZp in humans [START_REF] Picard | Motor areas of the medial wall: a review of their location and functional activation[END_REF][START_REF] Amiez | Neuroimaging Evidence of the Anatomo-Functional Organization of the Human Cingulate Motor Areas[END_REF]. These maps are somatotopically organized and are connected to corresponding fields in supplementary premotor, lateral premotor, motor and spinal regions, with a rostro-caudal gradient displaying more primary and spinal connections posteriorly and more premotor and prefrontal connections anteriorly [START_REF] Dum | The origin of corticospinal projections from the premotor areas in the frontal lobe[END_REF][START_REF] He | Topographic organization of corticospinal projections from the frontal lobe: motor areas on the medial surface of the hemisphere[END_REF][START_REF] Dum | Motor areas in the frontal lobe of the primate[END_REF][START_REF] Loh | Rostro-Caudal Organization of Connectivity between Cingulate Motor Areas and Lateral Frontal Regions[END_REF]. Face, eye, upper and lower limbs seem to be represented, with one face representation positioned at the most anterior limit of the MCC. The most rostral and dorsal MCC region has been shown active in multiple studies involving behavioural adaptation in one way or another. The region mentioned in the human brain imaging literature is often referred to as dACC or dmPFC, with terms associated to significantly variable locations of activation within the human brain. In fact, the anterior and posterior subdivisions of the MCC (aMCC and pMCC) seem to be activated differently during cognitive tasks and more precise descriptions of brain activations, potentially on a subject by subject basis, are required to really grasp the functional specificity within this region [START_REF] Amiez | The Location of Feedback-Related Activity in the Midcingulate Cortex Is Predicted by Local Morphology[END_REF]. We have suggested recently that the somatotopic organization of MCC reflects fields devoted to the detection and evaluation of domain-specific feedback relevant for adaptations [START_REF] Procyk | Midcingulate Motor Map and Feedback Detection: Converging Data from Humans and Monkeys[END_REF][START_REF] Loh | Cognitive control of orofacial motor and vocal responses in the ventrolateral and dorsomedial human frontal cortex[END_REF]. Because of the specificity of connectivity Int Rev Neurobiol. 2021;158:395-419. doi: 10.1016/bs.irn.2020.12.004. Epub 2021 Jan 29. PMID: 33785153 patterns of MCC maps it is possible that they contribute to different functional uses of feedback, i.e. contributing to trigger different adaptive cognitive or behavioural reactions.

Finally, the MCC is, within the medial frontal cortex of primates, one main source and target of connections with the autonomic system. Recent investigations have shown that, contrary to rodents, the primate medial frontal cortex is 3 to 4 synapses away from an organ like the adrenal medulla [START_REF] Dum | Motor, cognitive, and affective areas of the cerebral cortex influence the adrenal medulla[END_REF]2019). Neurons transsynaptically labelled after injection in the adrenal medulla are notably located within the somato-motor fields of the CMAr and CMAc. A review of human brain activations testing covariations of brain activity with autonomic measures showed that the MCC might have subdivisions related to the sympathetic and parasympathetic systems respectively [START_REF] Amiez | Midcingulate somatomotor and autonomic functions[END_REF].

In sum, the most rostral subdivisions of MCC that contain somatomotor fields also have connections with the lateral prefrontal cortex and with the sympathetic system. Such overlap of cognitive, somatomotor and autonomic connectivity is, we propose, a crucial property of the MCC. This connectivity and position within the cortical core bring rich and diverse inputs to the MCC, permitting it to perform functions that extend beyond any specific modality of information type, in particular favouring an integrative function. Our argument here is that a particularly important part of this role is the integration across time that can be carried out with this diversity of information. Central to this claim is the idea that the intrinsic properties of the cortical cells themselves, the construction of the local circuits, and in particular the nature of local inhibition, allow MCC to perform temporal integration over longer timescales than other cortical regions, and to do so with diverse information provided by its position in the cortical hierarchy.

Intrinsic cortical features of MCC.

The cell physiological properties of MCC can provide clues to its dynamical specificities and position in the temporal hierarchy. Specific physiological data in monkeys for a region at the border between ACC and MCC show that the nature of local inhibition might be an important feature in MCC [START_REF] Medalla | Strength and Diversity of Inhibitory Signaling Differentiates Primate Anterior Cingulate from Lateral Prefrontal Cortex[END_REF]. Spontaneous inhibitory postsynaptic currents are more frequent, long and large in cingulate layer III neurons compared to other prefrontal regions [START_REF] Medalla | Strength and Diversity of Inhibitory Signaling Differentiates Primate Anterior Cingulate from Lateral Prefrontal Cortex[END_REF]. Data from human MCC shows that GABA-B receptors are dense, especially in its posterior part, pMCC [START_REF] Zilles | Multiple Transmitter Receptors in Regions and Layers of the Human Cerebral Cortex[END_REF]. Robust inhibition and long inhibitory time constants should contribute to the extension of the time window for signal summation and thus extend local temporal receptive fields. In artificial hierarchical networks, environmental uncertainty can be dynamically captured by variations of the E/I tone [START_REF] Pettine | Hierarchical Network Model Excitatory-Inhibitory Tone Shapes Alternative Strategies for Different Degrees of Uncertainty in Multi-Attribute Decisions[END_REF]. In humans, dynamical integration of environmental uncertainty is circumscribed to the MCC [START_REF] Behrens | Learning the value of information in an uncertain world[END_REF]. A recent study also linked subjects' ability to integrate information in time and their glutamate and GABA concentrations in the MCC [START_REF] Scholl | Excitation and inhibition in anterior cingulate predict use of past experiences[END_REF]. In general terms, inhibition is a strong determinant of network activity [START_REF] Mongillo | Inhibitory connectivity defines the realm of excitatory plasticity[END_REF]. The diversity of synaptic inhibition can flexibly structure network dynamics by gating pyramidal inputs depending on the current task demand [START_REF] Womelsdorf | Dynamic circuit motifs underlying rhythmic gain control, gating and integration[END_REF][START_REF] Tremblay | GABAergic Interneurons in the Neocortex: From Cellular Properties to Circuits[END_REF][START_REF] Wang | Macroscopic gradients of synaptic excitation and inhibition in the neocortex[END_REF]. For example, MCC inhibitory activity has been proposed as a mechanism for transient network disengagement in response to errors [START_REF] Rothé | Coordination of High Gamma Activity in Anterior Cingulate and Lateral Prefrontal Cortical Areas during Adaptation[END_REF][START_REF] Shen | Anterior Cingulate Cortex Cells Identify Process-Specific Errors of Attentional Control Prior to Transient Prefrontal-Cingulate Inhibition[END_REF]. Inhibitory controlled circuits are more robust to excitatory volatility [START_REF] Mongillo | Inhibitory connectivity defines the realm of excitatory plasticity[END_REF]. This robustness might be particularly important for areas receiving converging multimodal inputs, and that need to integrate that information across time. Indeed, there is a macroscopic gradient of synaptic inhibition from sensory cortices to associative areas [START_REF] Wang | Macroscopic gradients of synaptic excitation and inhibition in the neocortex[END_REF]. MCC slow inhibitory decay times generate low frequency rhythmic oscillatory activity [START_REF] Kopell | Are Different Rhythms Good for Different Functions?[END_REF][START_REF] Medalla | Strength and Diversity of Inhibitory Signaling Differentiates Primate Anterior Cingulate from Lateral Prefrontal Cortex[END_REF] supporting communication between distant cortical areas [START_REF] Hahn | Communication through Resonance in Spiking Neuronal Networks Brunel N[END_REF]. MCC excitatory and inhibitory spike trains synchronize to different frequency bands [START_REF] Voloh | Cell-Type Specific Burst Firing Interacts with Theta and Beta Activity in Prefrontal Cortex During Attention States[END_REF]. Such cell type specific oscillations could be a means for flexible long-range tuning of coherent network activities according to the current demand. A recent study reported that MCC stimulation elicits recurrent activity in the LPFC [START_REF] Nácher | Asymmetric effective connectivity between primate anterior cingulate and lateral prefrontal cortex revealed by electrical microstimulation[END_REF], an activity pattern that has been associated with working memory temporal maintenance [START_REF] Mongillo | Inhibitory connectivity defines the realm of excitatory plasticity[END_REF][START_REF] Wasmuht | Intrinsic neuronal dynamics predict distinct functional roles during working memory[END_REF]. In this framework, MCC activity and in particular inhibition would contribute to network synchronisation and structuration of local and distant cortical areas.

We propose therefore that MCC inhibitory tone determines the activity of local units and leads to more stable or slowly evolving network dynamics favouring multimodal associations in the temporal domain. And these properties appear to be particularly exaggerated within MCC relative to other areas of association cortex, placing MCC not just at the heart of the dense core of associative cortical areas [START_REF] Markov | Cortical high-density counterstream architectures[END_REF], but also as new analyses and modelling suggest, at the pinnacle of a hierarchy of cortical timescales. Based on anatomical data and large-scale network modelling, XJ Wang and colleagues have recently shown that the anatomical organization and hierarchies within the large-scale cortical network was accompanied by temporal hierarchies [START_REF] Murray | A hierarchy of intrinsic timescales across primate cortex[END_REF][START_REF] Chaudhuri | A Large-Scale Circuit Mechanism for Hierarchical Dynamical Processing in the Primate Cortex[END_REF]. Individual neuron spiking shows some level of autocorrelation that reflects its propensity to keep a firing mode. The autocorrelation decay, referred to as spiking timescale [START_REF] Murray | A hierarchy of intrinsic timescales across primate cortex[END_REF], measured at the population level in different areas increases from posterior, sensory, areas to anterior frontal regions, forming a temporal hierarchy. Within the set of areas investigated in this study MCC displayed the longest timescale (about 300ms), compared to LPFC (190ms) or area MT (65 ms) [START_REF] Murray | A hierarchy of intrinsic timescales across primate cortex[END_REF][START_REF] Cavanagh | Reconciling persistent and dynamic hypotheses of working memory coding in prefrontal cortex[END_REF]. Medial frontal regions also show longer timescales in rats [START_REF] Murakami | Distinct Sources of Deterministic and Stochastic Components of Action Timing Decisions in Rodent Frontal Cortex[END_REF]. Such differences in timescale might provide areas with different information processing capability, and in particular the capacity to integrate information over long temporal scales, like rewards or feedback encountered across trials of a cognitive task [START_REF] Bernacchia | A reservoir of time constants for memory traces in cortical neurons[END_REF]. Indeed very recent work proposes that cortical dynamics are modulated by a range of timescales, potentially generated by independent mechanisms, some of which directly link to relevant task parameters, whilst others like the general intrinsic timescale remain independent [START_REF] Spitmaan | Multiple timescales of neural dynamics and integration of taskrelevant signals across cortex[END_REF]. Large scale modelling leads to the proposition that the specific timescale of cortical areas emerges both from the large-scale pattern of inter-areal connectivity and from intrinsic properties of areas, including local recurrent excitatory connectivity which was found to increase in density in parallel to the temporal hierarchy [START_REF] Chaudhuri | A Large-Scale Circuit Mechanism for Hierarchical Dynamical Processing in the Primate Cortex[END_REF]. Other important features that appear to change across the cortical hierarchy and may relate to these changes in timescale include the ratio T1w/T2w in structural MRI scans, and the level of tonic inhibition [START_REF] Wang | Macroscopic gradients of synaptic excitation and inhibition in the neocortex[END_REF]. We explore Indeed the interplay between the macroscopic gradient of synaptic inhibition and timescale hierarchy (possibly via disinhibition; [START_REF] Wang | Macroscopic gradients of synaptic excitation and inhibition in the neocortex[END_REF] in the final section.

The outcome of this work is that the MCC is situated at the core of a cortical network, with the longest neuronal timescale of recorded cortical areas, relatively strong local network inhibition, and a rich diversity of cognitive, somatomotor and autonomic connections. We argue that these are the building blocks for a system whose neurophysiological role will be to integrate diverse signals over time, a role particularly relevant in the context of behavioural adaptation. In the following sections we discuss the neurophysiological correlates of behaviour in MCC and relate them to the properties described above. We propose that the diversity of neural responses in MCC makes particular sense in the light of this overarching function of temporal integration for adaptation

Neurophysiological and causal correlates of temporal integration in MCC

Neurophysiological studies in rodent and non-human primate models provide details of the neural processes implemented by MCC and reveal neural correlates of the history of outcomes and values in a task, as well as the switching between behavioural states. These point to a role for MCC neurons in functions dedicated to gathering information over time for subsequent regulation of behaviour, a role that a limited number of intervention studies appear to confirm. Similar properties have been observed in the rat medial frontal cortex and while the anatomical correspondence in the rat brain is still in discussion, we will refer here to relevant data from medial frontal cortex (MFC) areas IL, PL and AC [START_REF] Vogt | Cytoarchitecture of mouse and rat cingulate cortex with human homologies[END_REF][START_REF] Mars | Whole brain comparative anatomy using connectivity blueprints[END_REF][START_REF] Schaeffer | Divergence of rodent and primate medial frontal cortex functional connectivity[END_REF].

History of outcomes and values.

MCC and MFC single unit activity encodes for reward based action selection in simple tasks in rats, monkeys and humans [START_REF] Shima | Role for cingulate motor area cells in voluntary movement selection based on reward[END_REF][START_REF] Williams | Human anterior cingulate neurons and the integration of monetary reward with motor responses[END_REF][START_REF] Sul | Distinct roles of rodent orbitofrontal and medial prefrontal cortex in decision making[END_REF], but also for the enactment of serial actions where each action is coded relative to future outcomes [START_REF] Procyk | Anterior cingulate activity during routine and non-routine sequential behaviors in macaques[END_REF][START_REF] Procyk | Characterization of serial order encoding in the monkey anterior cingulate sulcus[END_REF][START_REF] Shidara | Anterior cingulate: single neuronal signals related to degree of reward expectancy[END_REF]. Intervention studies in the MCC are rare, but those that exist confirm that MCC is directly contributing to the regulation of decisions based on rewards [START_REF] Shima | Role for cingulate motor area cells in voluntary movement selection based on reward[END_REF][START_REF] Amiez | Reward encoding in the monkey anterior cingulate cortex[END_REF][START_REF] Kennerley | Optimal decision making and the anterior cingulate cortex[END_REF]. [START_REF] Shima | Role for cingulate motor area cells in voluntary movement selection based on reward[END_REF], for example, showed that neurons in the cingulate motor areas within MCC signalled a reduction of reward that indicated that a change in action should be subsequently chosen, but only when the action was indeed successfully changed. Such activity is necessarily a response to a recent reward history over several trials, rather than a single outcome. The alteration of this MCC activity by local muscimol injection leads to a deterioration in the adaptation of action selection based on the diminishing reward size [START_REF] Shima | Role for cingulate motor area cells in voluntary movement selection based on reward[END_REF]. These responses in MCC are therefore driven by more information than the immediate action to be committed -they include information about the temporal context of that action. This temporal context is of course mostly of use to ensure that the action is well adapted, and importantly MCC activity also encodes feedback of actions or choices that is relevant to adaptation, i.e. in situations where action and feedback are the focus of attention and where it can reduce uncertainty. It is long established that cells in MCC respond to both rewards and errors [START_REF] Amiez | Anterior cingulate error-related activity is modulated by predicted reward[END_REF], and that responses to these events are modulated by their predictability and potentially their behavioural relevance [START_REF] Quilodran | Behavioral Shifts and Action Valuation in the Anterior Cingulate Cortex[END_REF]. In addition, MCC neurons represent the expected magnitude or the probability of an upcoming reward on trial-and-error learning tasks [START_REF] Kennerley | Double dissociation of value computations in orbitofrontal and anterior cingulate neurons[END_REF]. So whilst initial reports made a straight link between immediate reward and cell activity, it is now clear that the recent history of reward alters the firing of these cells (Seo and Lee, 2007;[START_REF] Kawai | Roles of the Lateral Habenula and Anterior Cingulate Cortex in Negative Outcome Monitoring and Behavioral Adjustment in Nonhuman Primates[END_REF], and that this effect will occur with varying timescales across neurons [START_REF] Bernacchia | A reservoir of time constants for memory traces in cortical neurons[END_REF]. For example, [START_REF] Kawai | Roles of the Lateral Habenula and Anterior Cingulate Cortex in Negative Outcome Monitoring and Behavioral Adjustment in Nonhuman Primates[END_REF] showed that MCC cells, and in particular putative inhibitory neurons [START_REF] Kawai | Preferential Representation of Past Outcome Information and Future Choice Behavior by Putative Inhibitory Interneurons Rather Than Putative Pyramidal Neurons in the Primate Dorsal Anterior Cingulate Cortex[END_REF], not only maintained the information about the last outcome but also stored outcome experiences from several past trials. MCC cells in this protocol also signalled a subsequent shift in strategy, activity that is discussed in more detail below.

The ability of MCC activity to reflect outcome history has an impact on behaviour. Monkeys with bilateral MCC sulcus lesions have difficulties in integrating and building reward-action history in an uncertain context and in adapting choice behaviour [START_REF] Kennerley | Optimal decision making and the anterior cingulate cortex[END_REF]. Effectively the lesion appears to reduce the span over which recent outcomes are used to adjust decisions. MCC appears to have a central role in updating action values based on the outcome history, helping to improve behavioural performance towards a specific goal [START_REF] Kennerley | Optimal decision making and the anterior cingulate cortex[END_REF]Seo et al., 2007). In general therefore these deficits seem to involve a loss of exploratory behavioursthe observed effects appear to combine altered valuation of outcomes and a reduced capacity to integrate the implications of those outcomes over time, the consequence on adaptive behaviour being quite consistent.

Rodent pharmacological inactivation and chemogenetic perturbation also show that medial frontal regions in rats have a causal role in integrating outcome history and in regulating explore/exploit behaviours [START_REF] Tervo | Anterior Cingulate Cortex Directs Exploration of Alternative Strategies[END_REF]. Indeed, the distributed ensemble dynamics might be tuned by monoaminergic afferents. Noradrenergic (NA) inputs are proposed to contribute to abrupt network reconfiguration and to promote transitions between exploitation and exploration [START_REF] Bouret | Network reset: a simplified overarching theory of locus coeruleus noradrenaline function[END_REF], and we discuss these state transitions in more detail below. NA inputs to the MCC would allow the dynamical modulation of learning rates [START_REF] Silvetti | Dorsal anterior cingulate-brainstem ensemble as a reinforcement meta-learner O'Reilly J[END_REF]. Consistent with those views, selective NA input enhancement in the rat MCC artificially increases behavioural stochasticity and leads to an increase in response variability. In turn, NA input suppression restores the ability to weight previously received feedback for choice regulation [START_REF] Tervo | Behavioral Variabilitythrough Stochastic Choice and Its Gating by Anterior Cingulate Cortex[END_REF].

In addition rare studies can provide information on the effect of cingulotomy in human patients, supporting a role for ongoing behavioural adaptation based on context from prior trials, both in the context of decisions to act [START_REF] Williams | Human anterior cingulate neurons and the integration of monetary reward with motor responses[END_REF] and in more cognitive contexts where previous trials might provide interfering information [START_REF] Sheth | Human dorsal anterior cingulate cortex neurons mediate ongoing behavioural adaptation[END_REF]. Wider lesions to the cingulate region have been reported to lead to global reductions of action and speech, often characterized as akinetic mutism [START_REF] Németh | Akinetic mutism associated with bicingular lesions: clinicopathological and functional anatomical correlates[END_REF][START_REF] Devinsky | Contributions of anterior cingulate cortex to behaviour[END_REF]. Such deficits have been proposed consistent with much of the above evidence if we consider the role of the MCC as acting to motivate time-extended behaviours, rather than driving trial-by-trial adaptations [START_REF] Holroyd | Motivation of extended behaviors by anterior cingulate cortex[END_REF].

Recent work proposes how the MCC goes about using outcome history to drive upcoming behaviour: it is proposed that MCC neurons encode expected outcomes as a latent state representation of actual outcomes [START_REF] Hyman | A Novel Neural Prediction Error Found in Anterior Cingulate Cortex Ensembles[END_REF]. The interaction between expected and actual outcome representations on trials where they do not match may drive a dynamical shift that we often record as an error signal. MCC cells certainly do show error signals -they respond to errors in the form of absent rewards [START_REF] Niki | Prefrontal and cingulate unit activity during timing behavior in the monkey[END_REF]. Again this error activity is modulated in a manner that reflects the temporal context, for example how much or how close a reward was before the error [START_REF] Amiez | Anterior cingulate error-related activity is modulated by predicted reward[END_REF], and indeed cells in MCC also encode both the overall task value in terms of reward, as well as the actual reward obtained [START_REF] Amiez | Reward encoding in the monkey anterior cingulate cortex[END_REF][START_REF] Sallet | Expectations, gains, and losses in the anterior cingulate cortex[END_REF]. This activity has a causal impact on behaviour as alteration of local MCC activity by local muscimol injection leads to a loss of efficiency in exploration for more rewarded stimuli in a choice task [START_REF] Amiez | Reward encoding in the monkey anterior cingulate cortex[END_REF].

These comparative feedback responses translate to the population level, where error-and feedback-related potentials (such as the error-related negativity [ERN] and feedback potentials [FRPs]) have been widely shown to differentiate outcome valences when recorded over the medial part of the frontal lobe in electroencephalography (EEG) [START_REF] Gehring | A neural system for error detection and compensation[END_REF][START_REF] Falkenstein | Action monitoring, error detection, and the basal ganglia: an ERP study[END_REF][START_REF] Miltner | Eventrelated brain potentials following incorrect feedback in a time-estimation task: evidence for a "generic" neural system for error detection[END_REF], electrocorticography (EcoG, [START_REF] Wilson | Prefrontal Markers and Cognitive Performance Are Dissociated during Progressive Dopamine Lesion[END_REF], and the local field potential (LFP) [START_REF] Gemba | Error" potentials in limbic cortex (anterior cingulate area 24) of monkeys during motor learning[END_REF]. Of course these are not standalone signals, rather they appear to provide information about the value of the feedback in terms of behavioural adaptation, be it for directly driving adaptation on subsequent trials [START_REF] Shima | Role for cingulate motor area cells in voluntary movement selection based on reward[END_REF][START_REF] Quilodran | Behavioral Shifts and Action Valuation in the Anterior Cingulate Cortex[END_REF][START_REF] Khamassi | Behavioral Regulation and the Modulation of Information Coding in the Lateral Prefrontal and Cingulate Cortex[END_REF][START_REF] Monosov | Anterior cingulate is a source of valence-specific information about value and uncertainty[END_REF], or for motivating more extended behaviours beyond simple trial-to-trial adaptation [START_REF] Walsh | Modulation of the feedback-related negativity by instruction and experience[END_REF][START_REF] Holroyd | Motivation of extended behaviors by anterior cingulate cortex[END_REF]. It is this Int Rev Neurobiol. 2021;158:395-419. doi: 10.1016/bs.irn.2020.12.004. Epub 2021 Jan 29. PMID: 33785153 concept of time-extended behaviours that is of particular relevance here, therefore, and it is this time-extended information that is necessary to signal the necessity to change state or strategy in a task.

Neural and behavioural state switches.

Monkey MCC and rat MFC show phasic neural changes at behaviourally relevant events together with global neural state changes between different behavioural states or strategies separated by switches, like exploratory behaviour and repetitive behaviour [START_REF] Karlsson | Network Resets in Medial Prefrontal Cortex Mark the Onset of Behavioral Uncertainty[END_REF][START_REF] Enel | Reservoir Computing Properties of Neural Dynamics in Prefrontal Cortex O'Reilly JX[END_REF]. For example MCC neurons signal behavioural shifts on subsequent trials [START_REF] Kawai | Roles of the Lateral Habenula and Anterior Cingulate Cortex in Negative Outcome Monitoring and Behavioral Adjustment in Nonhuman Primates[END_REF], whilst neural signals anticipating and signalling patch leaving or strategy switching in sequential foraging tasks are observed in phasic unitary and high-gamma activities in both primates and rodents [START_REF] Quilodran | Behavioral Shifts and Action Valuation in the Anterior Cingulate Cortex[END_REF][START_REF] Rothé | Coordination of High Gamma Activity in Anterior Cingulate and Lateral Prefrontal Cortical Areas during Adaptation[END_REF][START_REF] Karlsson | Network Resets in Medial Prefrontal Cortex Mark the Onset of Behavioral Uncertainty[END_REF]. [START_REF] Powell | Representational changes of latent strategies in rat medial prefrontal cortex precede changes in behaviour[END_REF] showed that on tasks where a reward criterion change was imposed, rats showed state transitions of MFC activity after they had learned about that contingency change, but before their behaviour changed. In contrast, when rats were permitted to change strategies themselves (i.e. without imposed criterion changes), similar state transitions occurred before changes in behaviour, therefore in a manner predictive of this un-imposed strategy switch [START_REF] Powell | Representational changes of latent strategies in rat medial prefrontal cortex precede changes in behaviour[END_REF]. This implies that the MFC is signalling the need for a change in strategy, rather than passively reflecting such a change.

Across a range of studies, a picture emerges where the neural dynamics of MCC correlate with the state of uncertainty inherent to exploratory situations and the control or monitoring of information seeking behaviour [START_REF] Stoll | Specific frontal neural dynamics contribute to decisions to check[END_REF][START_REF] White | A neural network for information seeking[END_REF]. In addition, MCC activity has strong correlation with valuation processesa multiplicity of signals reflect dynamical information accumulation in the MCC [START_REF] Kennerley | Double dissociation of value computations in orbitofrontal and anterior cingulate neurons[END_REF]Hunt et al., 2018). These signals are absent from OFC and LPFC activities. This integrated information is therefore used to make the decision to maintain or change the current course of action [START_REF] Quilodran | Behavioral Shifts and Action Valuation in the Anterior Cingulate Cortex[END_REF][START_REF] Mansouri | Conflictinduced behavioural adjustment: a clue to the executive functions of the prefrontal cortex[END_REF][START_REF] Khamassi | Behavioral Regulation and the Modulation of Information Coding in the Lateral Prefrontal and Cingulate Cortex[END_REF][START_REF] Stoll | Specific frontal neural dynamics contribute to decisions to check[END_REF].

Hayden et al 2011 found that neural activity during foraging scaled with the value of leaving the current exploited option and reflected an integrate-to-threshold process built across trials and leading to patch leaving [START_REF] Hayden | Neuronal basis of sequential foraging decisions in a patchy environment[END_REF]. Accumulation processes are observed in other frontal areas such as the FEF but they are continuous and ramp within trials, i.e. over a short timescale [START_REF] Hanes | Neural control of voluntary movement initiation[END_REF]. It is tempting to think that the specific neural dynamics observed during exploration or foraging provide the MCC with neural state properties that favour integration of information across time, i.e. over long timescales.

The way these correlated activity states are expressed over time is an important feature of encoding in MCC and across the frontal cortex. It may be that the capacity to shift neural state is an intrinsic feature of these cortical architectures, perhaps independent of external stimuli or constraints, which is then tuned to the needs of any ongoing task. An interesting and open question in the field is therefore how these properties are harnessed for the functional uses we ascribe to them, and a starting point is to consider how general this dynamic shifting of neural state is in MCC.

For example, we described above that information about feedback is encoded in the MCC, but this encoding occurs dynamically within the time course of a trial, starting just before outcome delivery until the next decision is made [START_REF] Stoll | Specific frontal neural dynamics contribute to decisions to check[END_REF][START_REF] Hunt | Triple dissociation of attention and decision computations across prefrontal cortex[END_REF]. The term dynamic here refers to the idea that information encoding changes over time, such that a decoder that can decode some behavioural variable from neural data at one period in time is no longer able to decode the same information later in time, while the variable is still encoded by the neural activity. In this sense the encoding is dynamic. The question of whether representations in MCC are dynamic or stable over time depends on the timescale in question, but it is increasingly clear that frontal cortical regions inherently combine elements of stable and dynamic coding. For example values in MCC are represented by mixed activity regimes composed of a stable subspace and a dynamic ensemble [START_REF] Enel | Stable and dynamic representations of value in the prefrontal cortex[END_REF]. This configuration grants the system a level of flexibility -a substrate of sustained information which can be combined with time-sensitive representations. The combination of both representations is likely crucial for the organization of behaviours in time and resembles the properties of delay activity much discussed regarding lateral prefrontal cortex [START_REF] Cavanagh | Reconciling persistent and dynamic hypotheses of working memory coding in prefrontal cortex[END_REF]Miller et al., 2018).

It is becoming increasingly clear that there is a distinct organization in this combination of stable and dynamic coding regimes. For example, [START_REF] Stoll | Specific frontal neural dynamics contribute to decisions to check[END_REF] used cross-temporal decoding of MCC activity to show semistable codes of feedback and decisions that carry over inter trial periods during foraging tasks. This appears to be particularly the case when, as in this study, decisions rely on the history of previous choices. MCC activity in this case can be considered as a set of trajectories organized in a sequence of discrete quasi-stationary states. This organization of activities in time is referred to as metastability [START_REF] Durstewitz | Abrupt transitions between prefrontal neural ensemble states accompany behavioral transitions during rule learning[END_REF][START_REF] Camera | Cortical computations via metastable activity[END_REF]. States can be organized around key perceptual Int Rev Neurobiol. 2021;158:395-419. doi: 10.1016/bs.irn.2020.12.004. Epub 2021 Jan 29. PMID: 33785153 and motor events or structured by ensemble activity regimes independently from external cues. Metastability has been observed in other cortical areas and is proposed to form a neuronal substrate for specific internal representations of the environment [START_REF] Rich | Decoding subjective decisions from orbitofrontal cortex[END_REF][START_REF] Camera | Cortical computations via metastable activity[END_REF]. State stability is believed to reflect internal representations and the deployment of selective attention required for the current task demand [START_REF] Engel | Selective modulation of cortical state during spatial attention[END_REF]. Transitions are more frequent when representations of internal rules are susceptible to change and are marked by an abrupt shift of dynamics in distributed neuronal ensemble [START_REF] Durstewitz | Abrupt transitions between prefrontal neural ensemble states accompany behavioral transitions during rule learning[END_REF].

How might these metastable states be used to promote the functions of MCC? There are likely to be several answers to this question, again depending on the temporal scale of investigation. Value signalling in the MCC reflects temporal integration of experienced rewards [START_REF] Amiez | Reward encoding in the monkey anterior cingulate cortex[END_REF][START_REF] Hunt | Triple dissociation of attention and decision computations across prefrontal cortex[END_REF] while also monitoring counterfactual options [START_REF] Hayden | Fictive reward signals in the anterior cingulate cortex[END_REF]Kolling et al., 2016a), and network transitions occur in periods when internal states are being updated and actively monitored. So, state transitions may well represent shifts in belief based on the same weighting of prior evidence and current outcomes. For example such state transitions would be signalling a shift to a more exploratory strategy of choice [START_REF] Karlsson | Network Resets in Medial Prefrontal Cortex Mark the Onset of Behavioral Uncertainty[END_REF]. Indeed, in foraging tasks, MCC trajectories describe two pseudo attractor spaces reflecting the structure of the task that alternates between phases of behavioural exploration and exploitation. Here state-space transitions are triggered by the monitoring of specific events such as feedback that reflect the need to adapt strategy [START_REF] Quilodran | Behavioral Shifts and Action Valuation in the Anterior Cingulate Cortex[END_REF][START_REF] Enel | Reservoir Computing Properties of Neural Dynamics in Prefrontal Cortex O'Reilly JX[END_REF].

Inhibitory control of MCC metastable states

Our most recent work provides direct evidence on how local intrinsic properties and timescales of MCC cells cause metastability, suggesting how such dynamical properties contribute to the role of MCC in adaptive behaviour [START_REF] Fontanier | Local inhibitory control of frontal network metastability underlies the temporal signature of cognitive states[END_REF]. We studied the temporal signatures, specifically the auto correlogram time constant and latency, of single neurons in monkey MCC and lateral prefrontal cortex (LPFC), recorded during a decision-making task. As suggested by Murray and colleagues [START_REF] Murray | A hierarchy of intrinsic timescales across primate cortex[END_REF], cells demonstrated a longer intrinsic timescale in the MCC than LPFC. We then showed that the temporal signatures were highly structured, showing anatomical organization within the MCC, and specific cell-type differences in the auto correlogram features between fast and regular spiking neurons.

Importantly the timescales of certain neurons showed functional adaptation to the timescale of behaviourally relevant information in the task at hand. In MCC and for regular spiking neurons only, we observed that on average timescales of the neurons lengthened when the monkeys were engaged in the task, compared to when they were pausing from the task. In addition, we showed that both the neuron-type and the timescale of that neuron within that type were determinant of the contribution of that neuron to encoding important behavioural features of the task, like reward feedback provided after choices (particularly putative interneurons for negative feedback), or value related information used across multiple trials [START_REF] Fontanier | Local inhibitory control of frontal network metastability underlies the temporal signature of cognitive states[END_REF].

We then used these data to inform research using biophysically constrained network models to link back to the inhibitory properties discussed above. This work revealed that specific cellular conductances in the modelled cells (specifically intrinsic afterhyperpolarization (AHP) potassium and synaptic inhibitory GABA-B conductances) were critical determinants of the specificity of dynamics of MCC and LPFC. These features were sufficient to provide a causal account for temporal signatures. Intriguingly, these conductances drove the cells to the recorded timescales by organizing activity into metastable states, with inhibition controlling state stability and transitions. Our model predicted therefore that the state duration in this metastable system would scale non-linearly with the timescales of different cortical regions, and we provided strong evidence to support this prediction from the monkey neurophysiological data, even reaching behavioural timescales. Our proposition on the basis of this work, therefore, is that inhibitory-controlled metastability constitutes the central dynamical process of MCC network function, locally specifying the temporal organization underlying cognitive processes operated by frontal areas.

Conclusion

The MCC, the region of the medial wall situated dorsal to the corpus callosum and sometimes referred to as dACC, is positioned within the densely connected core network of the primate brain, and has a rich diversity of cognitive, somatomotor and autonomic connections. The MCC shows strong local network inhibition in particular driven by GABA-B activity. This inhibition appears to control the metastability of the regionan established feature of many cortical networks in which the neural dynamics move through a series of quasi-stationary states with jump-like modulations between them. In Int Rev Neurobiol. 2021;158:395-419. doi: 10.1016/bs.irn.2020.12.004. Epub 2021 Jan 29. PMID: 33785153 particular it is proposed that the strong local inhibition in MCC leads to particularly long dynamic state durations, and so less frequent transitions, with the duration of these states even approaching the timescale of behavioural phenomena. Apparently as a result of these anatomical features and synaptic and ionic determinants, the MCC cells display the longest neuronal timescales amongst a range of recorded cortical areas.

We have argued that the anatomical position, intrinsic properties, and local network interactions of MCC make it a uniquely positioned cortical area to perform the integration of diverse information over time that is necessary for behavioural adaptation. The functional role of the MCC is therefore defined by these properties and we consider it a single central function. The MCC will perform this integrative function on different information types that arrive, given its rich connectivity. Performing the same integrative function on different incoming types of information will lead to a diversity of behavioural correlates. Whilst one reading of the literature might be that these diverse behavioural correlates mean that MCC has many different cognitive functions, we underline that the evidence presented above suggests that the operation performed by MCC itself is a single underlying function common to different cognitive labels.

This proposal leaves of course many important questions to be addressed. The neurobiological source of these temporal features, and the contribution of the place within the cortical hierarchy remain to be further examined. We also need to better understand the extent to which the variations in metastability and timescale are causal of the functions of MCC and other regions, or whether in fact those features result from adaptation to the context. Are these intrinsic properties that are applied to different functional uses, or are these properties tuned to fit the task in hand? Studies during task acquisition will be crucial here. Regions like the MCC do not, of course, act independently but as part of a densely connected network, for example showing interaction with LPFC. As such there remain questions to be answered about how regions with differing timescales and differing dynamic regimes can interact in a manner that permits computations that are relevant for cognition.

The purpose of this chapter was to elaborate a synthesis of data from multiple levels of investigation, in order to seek a coherent multi-scale view of the function of the midcingulate cortex. While many pieces of information are still required from anatomical, biophysical, neurophysiological and behavioural experiments, the recent data suggest that the synaptic and cellular properties, the local network configuration as well as the embedding of MCC in specific large scale functional networks provide this region with capabilities relevant to the regulation and adaptation of behaviour over long time scales.

Figure 1 .

 1 Figure 1. Schematic representation of the cingulate regions MCC and ACC in human (left) and monkey (right). In both species the MCC contains cingulate motor areas, CMAr, CMAd and CMV in monkeys, and RCZa and RCZp in humans.


Md Inja-Mamun Haque
email: injamamunhaque@du.ac.bd

A S M Rubayet

Ul Alam

Nafia Akter

Mohammad Anwar Siddique
email: anwar.siddique@du.ac.bd

M Anwar

Mahmud Hasan
email: mhasan@du.ac.bd

Md. Inja-Mamun A S M Haque

Rubayet Ul

Molecular characterization of 'tubifex worms' based on 16S rRNA and cytochrome c oxidase subunit I

Keywords: Tubifex, Phylogeny, Cytochrome Oxidase, 16S rRNA, Bangladesh

Tubifex worms' encompass extensive cryptic diversity that requires molecular characterization to confirm the species commonly found in Bangladesh including their lineages. Therefore, this study focused on the molecular characterization and phylogeny analysis at lineages level by sequencing the fragment of 16S rRNA and cytochrome c oxidase subunit I (COI) genes through standard cloning and sequencing techniques followed by phylogenetic characterization. All six isolates, belong to the genus Limnodrilus that is confirmed into two different species (L. hoffmeisteri and L. claparedianus-cervix) of the tubifex. The phylogenetic analysis estimated that the resident worms are under the same clade although transmissions from other countries might likely occur through different aquatic routes and birds. The drawbacks were the use of a complex colony rather than a single worm and taking representative isolates instead of all the positive amplicons for sequencing. However, this study is the first report of genetical confirmation of the genus Limnodrilus at lineage level from Bangladesh.

Introduction

Fresh water oligochaetes, usually known as 'tubifex worms', denotes a mixture of worms that may comprise the subfamilies of Tubificidae, and three tubificid genera, namely, Tubifex, Limnodrilus, and Aulodrilus, have been identified morphologically in Bangladesh and are widely used as a live feed in fish industry [START_REF] Mariom | Identification of genera of tubificid worms in Bangladesh through morphological study[END_REF]. Liu et al. (2017a) reported that hair and pectinate chaetae are the common morphological features in the genera of the subfamily Tubificinae, although may be lacking in many other tubifex worms including the common bioturbator benthic Limnodrilus, which is represented by few species [START_REF] Erséus | 18S rDNA phylogeny of the Tubificidae (Clitellata) and its constituent taxa: dismissal of the Naididae[END_REF]. Remarkably, the morphology of the hard structure (the anterior bificid chaetae and the shape of the penis sheaths) have extensively deliberated to denote the Limnodrilus species which includes at least 17 documented morphospecies, and the penis sheaths show great morphological variation within and among taxa [START_REF] Erséus | Phylogenetic analysis of Tubificidae (Annelida, Clitellata) based on 18S rDNA sequences[END_REF][START_REF] Martin | Global diversity of oligochaetous clitellates ("Oligochaeta"; Clitellata) in freshwater[END_REF]. Therefore, great effort was given to differentiate the interspecies alterations to the intra-species distinction of these features within the genus [START_REF] Hallett | Countering morphological ambiguities: development of a PCR assay to assist the identification of Tubifex tubifex oligochaetes[END_REF]Vivien et al., 2016a).

The recent studies on molecular systematics revealed that the taxon L. hoffmeisteri corresponds to a complex of species instead of a single species [START_REF] Beauchamp | Molecular phylogeny of tubificid oligochaetes with special emphasis on Tubifex tubifex (Tubificidae)[END_REF]Liu et al., 2017a). Overall, the complexities in morphological studies and the development of molecular genetic assays combinedly provided the basis to utilize the molecular approaches in identifying and classifying morphologically similar worms (Vivien et al., 2016b).

Literatures report the morphometric identification of 'tubifex worms' commonly found in Bangladesh [START_REF] Ali | A systematic study of freshwater Oligochaeta from Dacca city, Bangladesh[END_REF][START_REF] Mariom | Identification of genera of tubificid worms in Bangladesh through morphological study[END_REF], information related to molecular-based identification and phylogenetic analysis of these worms is lacking. In addition, there is no report on the species diversity of tubifex in Bangladesh. Therefore, the objective of the present study was to identify and analyze the molecular characterization of the commonly found tubifex genera in Bangladesh through state-of-the-art biological techniques.

Materials and Methods

Acquisition, handling and preparation of tubifex sample

'Tubifex worms' (100 g) were collected from the drains (muddy sediments) of Jashore (Sample JA) and Mymensingh (Sample MM) regions of Bangladesh where most of the catfish hatcheries operate. Specimens of tubifex were also sampled from the muddy area of Manikganj, Dhaka (Sample DK) which were used as ornamental fish feed in ornamental fish market of Katabon, Dhaka. The live worms were sorted out by sieving (0.2 mm sieve mesh size) into fine or sandy sediment, placed in jars with water, and stored on ice for transportation to the laboratory for further study.

DNA extraction and Polymerase Chain Reaction (PCR) amplification of mixed sample

The total genomic DNA was extracted from the mixed samples of tubifex by Maxwell ® 16 automated nucleic acid extraction system (Promega, Madison, WI, USA) using Maxwell ® 16 tissue DNA purification kit (Promega, Madison, WI, USA; Catalog-AS1030) according to manufacturer's protocol. NanoDrop™ 2000 was used to determine the concentration and optical purity of the extracted DNA. A portion (658 base pairs) of the mitochondrial gene (COI) was amplified using LCO 1490 (5' GGT CAA CAA ATC ATA AAG ATA TTG G 3') and HCO 2198 (5' TAA ACT TCA GGG TGA CCA AAA AAT CA 3') primers [START_REF] Folmer | DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates[END_REF]. The universal primers [START_REF] Palumbi | The simple fool's guide to PCR: version 2.0[END_REF] 16sar (5'CGC CTG TTT ATC AAA AAC AT 3') and 16sbr (5' CCG GTY TGA ACT CAG ATC AYG T3') were used to amplify the large subunit ribosomal RNA gene fragment (520 base pairs). Each PCR was performed in a total volume of 25 µl containing 12.5 µl GoTaq 2× Hot Start Colorless Master Mix (Promega, USA), 1 µl (200nM) of each primer, 2.5 µl of template (extracted genomic DNA) and 8 µl of nuclease free water. The thermal profile comprised an initial denaturation at 95 0 C for 5 min, followed by 35 cycles of 95 0 C for 40 s, annealing at 45 0 C for 45 s and extension at 72 0 C for 60 s, with a final elongation step at 72 0 C for 8 min. The PCR-amplified products (amplicons) were scrutinized by 1% agarose gels (containing ethidium bromide) and purified by using the Wizard® SV Gel and PCR Clean-Up System (Promega, USA).

Cloning and sequencing

Escherichia coli DH5α was used to prepare competent cell by chemical method. The amplified PCR products were cloned into pCR™ 4-TOPO® vector using TOPO® TA Cloning® Kit (Invitrogen, Life Technologies, Carlsbad, CA, USA). Transformation of the chemically competent Escherichia coli DH5α was prepared with recombinant plasmid constructed by cloning reaction. The day after transformation (usually 18 hours after plating transformation reaction on LB agar containing kanamycin), the colonies that appeared on the plates were investigated for transformants by colony PCR by using the primers (T3: 5´ATTAACCCTCACTAAAGGGA 3´ and T7: 5´ TAATACGACTCACTATAGGG 3´) provided in the TOPO® TA Cloning® Kit (Invitrogen, Life Technologies, Carlsbad, CA, USA).

The amplification profile of the colony PCR was 35 cycles of 94 ºC for 1 minute, 48 ºC for 1 minute and 72 ºC for 1 minute 30 seconds with an initial single denaturation step at 94 ºC for 10 minutes and a single final extension at 72 ºC for 25 minutes. To reduce the number of samples having possible redundant sequencing, Restriction Fragment Length Polymorphism (RFLP) technique (9 µl nuclease free water, 1 µl 10× buffer, 1 µl enzyme and 5 µl PCR product) was applied by using the enzyme Pst1 and Hinf1 for amplicons of COI and16S rRNA gene, respectively.

From the representative group based on RFLP, the constructed plasmid from the colony was extracted from the transformants using PureYield™ Plasmid Miniprep System (Promega, Madison, WI, USA) conferring to producer's guidelines. The extracted plasmid (after being confirmed that is the recombinant one) was separated by gel electrophoresis in 2 % low melting agarose (L.M.T) slab gel (UltraPure TM L.M.P. Agarose, Spain) in a Tris-acetate EDTA (TAE) buffer. The precise band position [Vector (3956 bp) + Insert (658 or 520 bp)] was pooled by gel purification using Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA).

The purified cloned products were subjected to an automated cycle sequencing reaction using BigDye ® Terminator v3.1 cycle sequencing kit (Applied Biosystems ®, USA) according to manufacturer's guidelines, analysis of the data was accomplished in ABI Genetic Analyzer (Applied Biosystems ®, USA).

Sequence Submission and Phylogenetic Reconstruction

The raw sequence data from the six different colonies of different samples were assembled using SeqMan version 7.0 (DNASTAR, Inc., Madison, WI, USA) and the assembled sequences were compared with other entries from NCBI GenBank (Benson et al., 2005) using BLAST [START_REF] Altschul | Basic local alignment search tool[END_REF] search to reveal the identification of tubifex. Using the sequence data, we tested for model selection and reconstructed phylogenetic trees for 16S rRNA genes and COI gene fragments with maximum likelihood methods using generalized time-reversible model by bootstrapping 1000 times.

Results and Discussion

The morphological identification of aquatic oligochaete often misguided the taxonomist for the two main reason. Firstly, a greater portion of specimens is immature and cannot be identified. Secondly, some common morphospecies encompasses a high level of cryptic diversity. In addition, a cluster of 'tubifex worms' forms a small colony (Baldo and Ferraguti 2005;[START_REF] Vivien | Cytochrome c oxidase barcodes for aquatic oligochaete identification: development of a Swiss reference database[END_REF][START_REF] Vivien | Molecular barcoding of aquatic oligochaetes: implications for biomonitoring[END_REF]. Moreover, it is difficult to detach one worm from a colony, and there is a chance to lost the tiny body fragment which may misguide in morphological identification. As a result, the molecular technique was applied as a suitable approach to identify the tubifex genera and their lineages. Hence, we proceed with the cloning method to divulge the different genera/lineages randomly distributed in the samples by targeting two genes (16S rRNA and COI) of the organisms.

RFLP of the maximum possible colonies was performed to differentiate strains after successful cloning reaction. For COI gene-based identification randomly selected three isolates (from two RFLP groups) were sequenced (BAN/DK/KB-9/2017, BAN/MM/MS-9/2017 and BAN/ JA/JS-8/2017; GenBank Accession no: MN294954-MN294956) and another three isolates (from one RFLP groups) namely BAN/JA/JS-1/2017, BAN/DK/KB-1/2017 and BAN/MM/MS-1/2019 (GenBank Accession no: MN267043-MN267045) were sequenced for 16S rRNA gene.

Cytochrome c oxidase subunit I gene-based identification found that while BAN/JA/JS-8/2017 strain had G, BAN/MM/MS-9/2017 and BAN/DK/KB-9/2017 had A in 470 nucleotide position. However, the predicted organism, using both BLAST and phylogenetic reconstruction tools in all three cases, is Limnodrilus hoffmeisteri complex unlabelled lineage (Figure 1). This tree confirmed that most groups (A, B and C) were under the genus Limnodrilus, and within group C, the unlabeled lineage resides in between other lineages. Another barcode (16S rRNA gene) typically used for identification of invertebrates, predicted the organism as-Limnodrilus hoffmeisteri complex lineage IX (isolate-BAN/DK/KB-1/2017) and Limnodrilus sp. claparedianus-cervix (isolate-BAN/JA/JS-1/2017 and BAN/MM/MS-1/2017) by BLASTing and molecular phylogeny (Figure 2). The phylogenetic tree showed the position of the isolates within group C, in two different clades-one within lineage IX of L. hoffmeisteri complex and another belongs to Limnodrilus sp. claparedianus-cervix. Recently, to overcome the difficulties related to morphology-based identification of oligochaetes, standard cytochrome c oxidase I (COI) barcode, including other barcoding markers, has been used as a genetic tool for rapid identification of oligochaete diversity based on mixed specimen samples (Vivien et al., 2016b).

However, species delimitation based on only mitochondrial COI gene may not reveal species margins as precisely as an integrative tactic exploring a combination of numerous data sources, hence mitochondrial 16S ribosomal DNA and mitochondrial marker (COI), in combination, have been applied in contemporary species identification and description.

Before the molecular genetics era, the combination of allozyme data (by electrophoresis) with morphometric characteristics isolated four Limnodrilus species, L. hoffmeisteri, L. udekemianus, L. claparedianus, and L. profundicola by Schmelz (1996). The monophyletic status of the genus Limnodrilus has confirmed that L. udekemianus is a species distinct from the clade comprising of L. claparedianus, L. hoffmeisteri, L. cervix and L. maumeensis (Weider 1992;Liu et al., 2017a). Although the L. hoffmeisteri sensu stricto was partly defined and characterized by COI (cytochrome c oxidase subunit I) barcoding (Liu et al., 2017b;Sjolin et al., 2005) , the small mitochondrial 16S rDNA data sets in earlier molecular studies advocated that the common and broadly dispersed clitellate morphospecies are complexes in cryptic species [START_REF] Erséus | Cryptic speciation in clitellate model organisms[END_REF][START_REF] James | DNA barcoding reveals cryptic diversity in Lumbricus terrestris L., 1758 (Clitellata): resurrection of L. herculeus (Savigny, 1826)[END_REF][START_REF] Martinsson | Cryptic diversity in the well-studied terrestrial worm Cognettia sphagnetorum (Clitellata: Enchytraeidae)[END_REF][START_REF] Martinsson | Cryptic speciation and limited hybridization within Lumbricus earthworms (Clitellata: Lumbricidae)[END_REF], and the morpho-taxon L. hoffmeisteri is likely to encompass cryptic lineages [START_REF] Marton | The susceptibility of diverse species of cultured oligochaetes to the fish parasite Myxobolus pseudodispar Gorbunova (Myxozoa)[END_REF]. The large Limnodrilus sensu stricto clade entails three groups (A-C), in which, group C covers all species of L. hoffmeisteri complex (Liu et al., 2017a) with its numerous siblings (spp. I-X). The complex C is alienated into two well-supported lineages, one enclosing L. hoffmeisteri species I-IV and L. claparedianus, the other covering L. hoffmeisteri species V-X (including IX= L. hoffmeisteri sensu stricto), L. maumeensis and L. claparedianuscervix which is the morphologically intermediate between L. claparedianus and L.cervix (Liu et al., 2017a). The lineage identified as L. claparedianus, L. maumeensis and L. claparedianuscervix are species (Liu et al., 2017b). The morphospecies L. hoffmeisteri (group C) is the most broadly circulated cosmopolitan taxon [START_REF] Brinkhurst | Aquatic Oligochaeta of the world[END_REF]. Based on both molecular characterization using genetic markers (mitochondrial COI, nuclear ITS, and 16S rDNA) and morphological evidence, L. hoffmeisteri has been designated as a species complex rather than a single species (Liu et al., 2017a).

The genus Limnodrilus includes 17 morphospecies and 10 genospecies (nine separate species and a single complex) [START_REF] Pinder | A preliminary guide to the identification of microdrile Oligochaeta of Australian inland waters[END_REF]Liu et al., 2017a). According to Liu et al. (2017b), species to date categorized with Limnodrilus formed a non-monophyletic group with two detached clades (one of them consist of three speciemens of L. rubripenis and other comprise all other Limnodrilus spp.). The Limnodrilus individuals were morphologically indistinguishable and different clades suggest the existence a of genetic barrier for gene flow [START_REF] Crottini | The world in a river? A preliminary analysis of the 16S rDNA variability of Tubifex species (Clitellata: Tubificidae) from the Lambro River[END_REF][START_REF] Gustafsson | Genetic variation in the popular lab worm Lumbriculus variegatus (Annelida: Clitellata: Lumbriculidae) reveals cryptic speciation[END_REF].

While two different species of Limnodrilus have been identified within samples JA and MM, we have identified only one distinct lineage of L. hoffmeisteri for sample DK. However, all of the isolates are under Group C which is quite distinct from other groups of Limnodrilus.

According to [START_REF] Timm | A guide to the freshwater Oligochaeta and Polychaeta of Northern and Central Europe[END_REF] asexual mode of reproduction of ;tubifex worms' dominates in tropical water which might be the possible cause of the absence of other tubifex species.

Active immigration is a tremendously deliberate process, hence, passive transport may play a vital role in the pan-distribution of L. hoffmeisteri complex species and other Limnodrilus species [START_REF] Koel | Avian piscivores as vectors for Myxobolus cerebralis in the Greater Yellowstone ecosystem[END_REF]. Several transport mechanisms are known to cause large scale massive dispersal of aquatic fauna, among this anemochorous dispersal of cocoon or cyst, zoochorous transport (most likely exploiting bird migration) or antropic use of the species [START_REF] Milbrink | Distribution and dispersal capacity of the Ponto-Caspian tubificid oligochaete Potamothrix moldaviensis Vejdovský et Mrázek, 1903 in the Baltic Sea Region[END_REF][START_REF] Crottini | The world in a river? A preliminary analysis of the 16S rDNA variability of Tubifex species (Clitellata: Tubificidae) from the Lambro River[END_REF]. [START_REF] Naveed | Preliminary studies on aquatic Oligochaeta in and around Chennai, Tamil Nadu, India[END_REF] demonstrated that the cosmopolitan species L. hoffmeisteri is collected by aquarium owners and is widely used as live feed for ornamental fishes worldwide. Therefore, the use of these worms as aquaria food in different firms may have played a key role in the distribution of these species complex.

The route of entry of L. hoffmeisteri complex lineage IX, L. hoffmeisteri complex unlabelled lineage, and L. claparedianus-cervix into Bangladesh would be from Japan, China, Malaysia, and Thailand; India and China, respectively as other countries related to those groups are related to European countries, USA and Canada. (Liu et al., 2017a).

The main drawback of this study might be grouping of the isolates from transformants using RFLP wherein some of the isolates might get missed. Nevertheless, it is an established technique to choose the representative from many samples. Another drawback was using a mixed colony instead of a single worm which would help us avoid the cloning step also. Nevertheless, this study is successful as fulfilling the main objective of the study characterizing the tubifex at the lineage level using gene data.

Conclusion

The findings of this study, for the first time, confirmed the existence of two genetically different, unnamed taxa (species) of Limnodrilus in Bangladesh. However, within a single sample, multiple species were spotted which refers to the complex colony formation. However, our key goal was to identify tubifex by using molecular data in this study, and in the future work, the single colony will be isolated before sorting out from the core sample.

Authorship contribution statement

Md. Inja-Mamun Haque collected the worm samples and did molecular works including manuscript preparation. A. S. M. Rubayet Ul Alam carried out the studies (molecular and bioinformatics analysis) and participated in the preparation of the manuscript. Nafia Akter partially did molecular works on bench. Mohammad Anwar Siddique helped to develop the molecular methodology. Munawar Sultana and M. Anwar Hossain investigated the whole work and helped to review and prepare the manuscript. Mahmud Hasan designed the study, acquire fund and participated in preparing and revising the manuscript. All authors read and approved the final manuscript. For a better representation of the tree, we compressed the branches.

Caption of the Figure

Fig. 1 .

 1 Fig. 1. Phylogenetic tree showing the relationships among different Limnodrilus spp. based on

Fig. 2 .

 2 Fig. 2. Phylogenetic tree showing the relationships among different Limnodrilus spp. based on

Acknowledgements

The corresponding author wish to thank Ministry of Education, Govt. of the Peoples Republic of Bangladesh for their financial support (Grant no: LS-13102/2015) to conduct this study.


Nadia Benmoussa
email: nadia.benmoussa@yahoo.fr

Anne-Laure Muller

Amanda Fanous

Saudamini Deo

P Conan

P Charlier

Infantile malignant osteopetrosis (« Leontiasis ossea ») in a mid-19 th c. French skull 2-Authors

Keywords: Paleopathology, Skull base, osteopetrosis, radiology, retrospective diagnosis, leontiasis ossea

Introduction: Autosomal recessive osteopetrosis (ARO) is a rare genetic disorder that is also called "malignant osteopetrosis" because it typically manifests within the first few months after birth and later manifests severe complications (blindness, deafness, facial palsy, dental caries, hypocalcemia) and medullary haematopoetic disorders. The aim of this study is to describe lesions and conclude hypothetical clinical symptoms by performing a CT-scan analysis on an early 19 th century human skull preserved in the Dupuytren museum collection (Paris).

Materials and Methods

The dried skull was examined and described using classical methods of paleopathology. We conducted additional research using historical data on the skull and a CT scan was performed.

Results: Screening of the archives from the museum allowed us to estimate the skull's origin as somewhere between 1850 and 1900, with an estimated age of six years. Several ducts and foramens of the skull and the facial bones were obstructed as a result of this disease, clinically corresponding to diffuse neurological (and also vascular) deficits. This devastating pathology led to many symptoms stemming from brain tissue damage and cranial nerve deficits, leading to the inevitable death of this child. These symptoms include headache, intracranial hypertension with vomiting, blindness, exophthalmos, deafness, oral breathing, eating disorder (suffocation), facial palsy, balance disorder (excessive weight, vestibulo-cochlear and cerebellar involvement).

Conclusion

The study of this specimen from the 19 th century confirms that the six-year old patient suffered from malignant osteopetrosis. CT-scan allowed confirmation of the diagnosis, as well as a complete description of the disease, and highlight a possible associated anaemia.

Introduction

Osteopetrosis is a rare genetic disorder that is characterised by increased bone density caused by abnormal osteoclast activity. The overall incidence of osteopetrosis is difficult to estimate but autosomal recessive osteopetrosis (ARO) is observed in 1 in 250,000 births, and autosomal dominant osteopetrosis (ADO) is observed in 1 in 20,000 births [START_REF] Stark | Osteopetrosis[END_REF]. ARO is also called «malignant osteopetrosis » because it typically manifests within the first few months after birth and develops severe complications (blindness, deafness, facial palsy, dental caries, hypocalcemia) as well as medullary haematopoetic disorders (anemia, pancytopaenia) [START_REF] Stark | Osteopetrosis[END_REF].

In the context of a paleopathological study of the Dupuytren museum collection (Paris) conducted by our scientific team, we have identified an early 19 th century human skull presenting with an increased bone density (« Leontiasis ossea »). The aim of this study is to describe lesions and conclude hypothetical clinical symptomatology using CT-scan analysis and allow us to reach the conclusive diagnosis and description of infantile malignant osteopetrosis.

Materials and Methods

The dried skull was examined and described using classical methods of paleopathology (2-5): measurements of the lesions were performed with metric tools and expressed in centimetres.

The brain volume was determined by using small seeds and lentils. In collaboration with the Dupuytren Museum (held by the Pierre et Marie Curie University, Paris, France) we conducted additional research using historical data on the skull. Informed consent was not required for this study, given its purely historical nature.

A CT-scan was carried out using helical acquisition with a General Electric OPTIMA 64band scanner. Thickness of cuts was 0.625. 100 kV. mA adapted by the software -machine.

Reconstruction was done in bone filter, and reading was performed on a dedicated General Electric console. MPR analysis with axial, coronal, and sagittal cuts and reconstructions in volume rendering were performed.

Results

Paleopathological analysis

The skull in question is displayed in the museum without any associated description.

Screening of the archives from the museum allowed us to estimate its origin as between 1850 and 1900 but we were not able to find any clinical information about this « patient ». The specimen was reassembled with lateral iron fastener, which allowed us to directly examine the inside of the skull (Fig 1). The frontal view shows that there is stenosis or occlusions of all the orifices of the face: piriform orifices, foramen and vascular foramen and orbiatal cavities.

There is also osteolysis (apico-dentary cyst) in the left mandibular. We also observed preserved morphology of the inner ear and ossicular ankylosis of the middle ear (Fig 7c). All these lesions are compatible with a diagnosis of diffuse osteopetrosis [START_REF] Stark | Osteopetrosis[END_REF][START_REF]Osteopetrosis | Genetic and Rare Diseases Information Center (GARD) -an NCATS Program[END_REF]. Tumoral etiology in regard to the periosteal appositions seems unlikely given the extent of disease and the absence of modification of the underlying bone in relation to the general structure of the adjacent bone [START_REF] Cotten | Imagerie musculosquelettique: pathologies générales, chapitre dysplasie osteocondansantes[END_REF]. Instead, chronic anemia is a more likely possibility.

Clinical symptoms

We have hypothesized some clinical symptoms, given the lesions observed on the skull. This pathology has resulted in many symptoms related to brain damage and cranial nerve injuries and the death of this child: headache, intracranial hypertension with vomiting, blindness, exophthalmos, deafness, oral breathing, eating disorder (suffocation), facial palsy, balance disorder (excessive weight, vestibulo-cochlear and cerebellar involvement). (1).

Discussion

It is not possible to conclude the precise origin of this disease without complementary genomic study. This would require a bone sample for DNA extraction and screening of the currently known mutations, which were not possible for conservation reasons.

Infantile malignant osteopetosis or autosomal dominant malignant osteopetry seems to be the most likely diagnosis, especially in comparison to other possible diagnoses (1) :

-Autosomal dominant osteopetrosis usually starts at an adult age, which excludes this diagnosis.

-Osteogenic dysplasias related to the SOST gene are Van Buchem disease and sclerostosis. Van Buchem disease is excluded because it usually manifests during puberty [START_REF]Osteopetrosis | Genetic and Rare Diseases Information Center (GARD) -an NCATS Program[END_REF].

-Sclerosteosis can be proposed as a differential diagnosis because it occurs around the age of 5 and is potentially lethal.

-Craniometaphyseal dysplasias characterized by progressive diffuse hyperostosis of cranial bones evident clinically as wide nasal bridge, paranasal bossing, widely spaced eyes with an increase in bizygomatic width, and prominent mandible. Long skull shape (dolichocephaly) resulting from fronto-occipital hyperostosis has been reported in a number of individuals and overgrowth of the lower jaw (mandibular hyperostosis) and recessed midface (midface retrusion) are often observed [START_REF] Reichenberger | Craniometaphyseal Dysplasia, Autosomal Dominant[END_REF]. In this case, there is no dolichocephaly or midface retrusion.

-Cranio-dyaphyseal dysplasia is a rare bone sclerosis disease with variable phenotypic expression. It is due to a mutation of the SOST gene. Sclerosis with widespread massive hyperostosis of the skull and facial bones leads to severe deformities. The clinical course is characterized by a thickening of the cranial vault and a decrease in the cranial volume by narrowing of the foramina of the face and the skull, with possible brain compression by unlimited bone deposits. At the level of the diaphyses of the long bones, hyperostosis with sclerosis is observed and sometimes there is a lack of modelling of the metaphyses. Most of the cases described are sporadic but there has been a reported case of autosomal recessive inheritance in a mother and her daughter, in whom the degree of hyperostosis and sclerosis was more severe than in other known patients, with significant related metaphyses [START_REF] Brueton | Craniodiaphyseal dysplasia[END_REF]. Owing to the lack of any other bones from the same skeleton, we cannot confirm or exclude this hypothesis.

Conclusion

The study of this specimen from the 19 th century confirms that this six-year-old patient suffered from malignant osteopetrosis. CT-scan analysis allowed us to reach the diagnosis and,complete a global description of the disease as well as bring to light a possible associated anaemia. In the 21 st century, such cases are often lethal in the first decade of life and the only curative treatment is stem cell transplantation [START_REF] Gillani | Malignant Infantile Osteopetrosis[END_REF]. Skeletons from medical anatomical museums must continue to be studied to allow a deeper understanding of the early reported cases of rare diseases. DNA analysis should be permitted to help conclude the mutation origin and confirm the diagnosis. Furthermore, a new mutation may be discovered. In our opinion this study highlights the need for paleopathological approach using state-of-the-art biomedical tools for retrospective diagnosis. A deeper understanding of rare diseases through these historical approaches may allow a better understanding of current established procedures in a multitude of medical disciplines [START_REF] Benmoussa | A 1842 skull from Dupuytren's museum of Paris: an original artifact of Joseph Gensoul first maxillectomy technique[END_REF][START_REF] Benmoussa | HPV 16 in squamous cell carcinoma of 19th century tonsils[END_REF].

Figures

  The skull's estimated age is six years because tooth 16 and tooth 26 are under the bony arch, where a beginning of furcation formation at the level of tooth 26 can be observed (Fig 2). There is also a dental alveolar cyst at the level of tooth 73 (Fig 3). Brain volume has been measured at 890 cm 2 . The total weight of the skull (with mandible) is 3.5 kg. There is marked thickening of the inner and outer layers of the calvaria with absence of diploe and sunburst periosteal reaction. (Fig 4). Several ducts and foramens of the skull base and of the facial bones were obstructed as a result of this diseased, clinically corresponding to diffuse neurological (and also vascular) deficits (Fig 2-4-5). Careful inspection of the cranial convolutions as well as the meningeal vessels and the sagittal sinus clearly indicates chronic cranial hyper-tension. Porous arch appearance is visible on both parietal bones in their extended posterior segment measuring 11x14x2 cm (Fig 6): this finding is compatible with chronic anemia (with hyper medullar production) and/or a malignant transformation of the bony pathology (osteosarcoma). There is no trace of surgery (cut marks) except those due to the post-mortem skull preparation. CT scan CT-scan clearly displays bone thickening of the entire skull vault, bones of the face, and base of the skull (right frontal = 31.5mm ; left frontal = 18.6 mm ; right temporal = 21.9mm ; left temporal = 19.3mm; right vertex 39.4 mm; left vertex = 27 mm), with densification, loss of bone marrow, major narrowing of the foramens of the face (right optique duct = 1.1 mm , left = 2.4mm) (Fig 7a), venous sinus imprint on the internal table of the cranial vault, disappearance of the fissures. A periosteal « grass fire » aspect is visible in both parietal regions (predominant on the right) (Fig 7b), extending from the vertex to the occiput (137 mm of height and 92 mm of width).

Figure 1 :

 1 Figure 1 : Frontal (a) and lateral (b) views from the pathological skull : The frontal view

  Figure 1 : Frontal (a) and lateral (b) views from the pathological skull : The frontal view

Figure 2 :

 2 Figure 2 : Basal view from the pathological skull : several ducts and foramens of the skull

Figure 3 :Figure 4 :

 34 Figure 3 : Local teeth infection from the pathological skull : dental alveolar cyst at the level

Figure 5 :

 5 Figure 5 : Obstruced right orbite from the pathological skull (exophtalmia).

Figure 6 :

 6 Figure 6 : Upper view (a) and enlargement (b) from the pathological skull.

Figure 7 :

 7 Figure 7 : a Narrow of the right and left optique canal b Marked thickening of the inner and