Fani Koukouli
email: fanikoukouli@icloud.com

Jean-Pierre Changeux
email: changeux@noos.fr

Karl Zilles

Do nicotinic receptors modulate high order cognitive processing?

Keywords: Nicotinic receptors, conscious processing, prefrontal cortex, anaesthesia, neuropsychiatric disorders, nicotine addiction

Recent studies provided strong evidence that deficits in cholinergic signalling cause disorders of cognition and affect conscious processing. Technical advances that combine molecular approaches, in vivo recordings in awake behaving animals, human brain imaging and genetics have strengthened our understanding of the roles of nicotinic acetylcholine receptors (nAChRs) in the modulation of cognitive behavior and network dynamics. Here, we review the emergent role of nAChRs in high order cognitive processes and discuss recent work implicating cholinergic circuits in cognitive control, including conscious processing.

Acetylcholine signalling in the brain and neuronal nicotinic receptors

The cholinergic system is a well-organized network of neurons that uses the neurotransmitter acetylcholine (ACh) for intercellular communication and signal transduction. One of the primary roles of ACh in the brain is to coordinate the response of brain networks to internal and external inputs [START_REF] Picciotto | Acetylcholine as a neuromodulator: cholinergic signaling shapes nervous system function and behavior[END_REF] and shape global neuronal activity from multiple brain territories. In particular, ACh plays a key role in orchestrating cortical circuit dynamics during wakefulness and sleep. ACh acts through two classes of receptors: the metabotropic muscarinic receptors and the ionotropic nicotinic receptors. Here, we will focus exclusively on ionotropic nAChR modulation in the brain.

Almost all regions of the mammalian brain are innervated by cholinergic inputs with abundant populations of neurons and glial cells expressing nAChRs. The genes encoding for nAChRs are grouped into two families with nine neuronal α-subunits (α2-α10) and three βsubunits (β2-β4) which make different combinations of pentameric oligomers with distinct pharmacological, kinetic and physiological properties. Among the homopentameric nAChRs, receptors composed of one α-subunit category, the α7-containing subtypes account for the greatest proportion of high affinity α-bungarotoxin binding sites in the mammalian nervous systems (yet with significant contributions of α8-α9). α7-containing subtypes are highly expressed in the cortex, hippocampus and limbic areas, whereas lower expression is found in the thalamus and basal ganglia [START_REF] Zoli | Diversity of native nicotinic receptor subtypes in mammalian brain[END_REF]. Among the heteropentameric nAChRs, the α4β2 containing nAChR, which has high affinity for nicotine [START_REF] Gotti | Brain nicotinic acetylcholine receptors: native subtypes and their relevance[END_REF], is the most frequent receptor subtype both in the rodent and human brain. There are many similarities between the distribution of nAChRs in the rodent and primate brain. The most notable differences in nAChRs distribution between these species are those of α2 containing nAChRs [START_REF] Gotti | Brain nicotinic acetylcholine receptors: native subtypes and their relevance[END_REF]. There is also expression of subunits that do not directly contribute to the agonist-binding site, such as the accessory α5 subunit, but play a critical role in the functional properties of the receptor [START_REF] Ramirez-Latorre | Functional contributions of α5 subunit to neuronal acetylcholine receptor channels[END_REF].

There is a wide variety and distribution of nAChRs in the brain, implying important regional and functional contributions of each nicotinic subunit. The construction of genetically modified mice with deletion of specific nAChR subunits offered exquisite tools to unravel the defined contribution to brain functions of particular subunit types within multi-subunit oligomers [START_REF] Changeux | Nicotine addiction and nicotinic receptors: lessons from genetically modified mice[END_REF][START_REF] Picciotto | Abnormal avoidance learning in mice lacking functional highaffinity nicotine receptor in the brain[END_REF][START_REF] Picciotto | Acetylcholine receptors containing the β2 subunit are involved in the reinforcing properties of nicotine[END_REF][START_REF] Maskos | Nicotine reinforcement and cognition restored by targeted expression of nicotinic receptors[END_REF].

Nicotinic receptors have been found to be key players in processes such as wakefulness, sleep, and anesthesia, and to contribute to major disorders of the central nervous system, such as Alzheimer's disease, Parkinson's disease and schizophrenia as highlighted by studies using biochemical, molecular, imaging and genetic tools. The goal of this review is to discuss recent advances in the understanding of how the diverse nAChRs contribute to cognitive functions and modulate them, both in humans and animal models, ultimately shedding light on the role of nAChRs in the orchestration of higher brain functions.

Conscious awareness, wakefulness and sleep

"Qualitative richness, situatedness, intentionality, integration and brain dynamics" are considered as key features of conscious processing [START_REF] Pennartz | Indicators and Criteria of Consciousness in Animals and Intelligent Machines: An Inside-Out Approach[END_REF]. Several nested levels of conscious processing have been recognized which, in humans, develop postnatally and include the awareness of body, self and the relation with physical, social and cultural worlds [START_REF] Lou | Towards a cognitive neuroscience of self-awareness[END_REF][START_REF] Lagercrantz | The Emergence of Human Consciousness: From Fetal to Neonatal Life[END_REF].

Disturbances of conscious processing are frequently observed in major diseases such as dementia with Lewy bodies, Parkinson's disease, Alzheimer's disease and schizophrenia [START_REF] Ballard | Fluctuations in attention: PD dementia vs DLB with parkinsonism[END_REF][START_REF] Berkovitch | Disruption of Conscious Access in Schizophrenia[END_REF][START_REF] Koukouli | Nicotinic receptors in mouse prefrontal cortex modulate ultraslow fluctuations related to conscious processing[END_REF]. Many theories of conscious processing have been suggested and recently reviewed [START_REF] Mashour | Conscious Processing and the Global Neuronal Workspace Hypothesis[END_REF]. Among them, the Global Neuronal Workspace (GNW) hypothesis [START_REF] Dehaene | A neuronal model of a global workspace in effortful cognitive tasks[END_REF] postulates a global computational space composed of widely distributed excitatory neurons with long-range axons, forming a reciprocally connected network. This network was initially suggested to include the dorsolateral prefrontal, inferior parietal and cingulate cortices on top of a set of specialized and modular perceptual, motor, memory, evaluative, and attentional processors. This bidirectional connectivity creates the conditions for "ignition", which is the triggering of a sudden collective and reverberant coordinated activity that mediates global broadcasting [START_REF] Mashour | Conscious Processing and the Global Neuronal Workspace Hypothesis[END_REF][START_REF] Dehaene | Experimental and theoretical approaches to conscious processing[END_REF]. At the neuronal level, the GNW hypothesis posits that large pyramidal cells in cortical layers II/III and pyramidal cells in deeper layer V, together with their thalamic relationships, play a central role in ignition [START_REF] Dehaene | Experimental and theoretical approaches to conscious processing[END_REF].

The tuning of sleep-wake cycles is an elaborate process involving the participation of several neurotransmitters and brain structures. Sleep is the only period in which consciousness tones down under physiological conditions, although it has been argued that sleep can still be associated with the presence or absence of conscious experiences [START_REF] Siclari | The neural correlates of dreaming[END_REF]. This synergy between the presence and absence of experiences within two types of sleep, non-rapid eye movement sleep (NREM) and rapid eye movement sleep (REM), still poses several questions. Recently, reductions of low-frequency activity in the posterior confluence of sensory and association cortex were associated with dreaming during REM and NREM sleep, but simultaneously, higher frequency gamma activity was detected in frontal and prefrontal cortex [START_REF] Siclari | The neural correlates of dreaming[END_REF].

The cholinergic system is considered a key player, together with norepinephrine, in modulating REM sleep and contributes to cortical arousal with high levels of ACh release in the forebrain during REM [START_REF] Vazquez | Basal forebrain acetylcholine release during REM sleep is significantly greater than during waking[END_REF] and low levels of neuromodulators during NREM sleep [START_REF] Vyazovskiy | Triggering Slow Waves During NREM Sleep in the Rat by Intracortical Electrical Stimulation: Effects of Sleep/Wake History and Background Activity[END_REF][START_REF] Tononi | Sleep and the Price of Plasticity: From Synaptic and Cellular Homeostasis to Memory Consolidation and Integration[END_REF].

Recently, machine learning approaches combined with in vivo neurochemical data demonstrated that concentrations of neurotransmitters such as ACh and norepinephrine in prefrontal cortex can predict the levels of cortical and behavioural arousal [START_REF] Zhang | Neurotransmitter networks in mouse prefrontal cortex are reconfigured by isoflurane anesthesia[END_REF]. Low levels of ACh during NREM sleep has been suggested to enhance synapse formation and episodic/motor memory consolidation mechanisms [23,[START_REF] Inayat | Low acetylcholine during early sleep is important for motor memory consolidation[END_REF].

The contribution of nAChRs to the organization of sleep is supported by a study showing that β2-containing nAChRs modulate the transient phasic activity in NREM, the onset of REM and its duration, and the REM promoting effects of stress [START_REF] Léna | Beta2-containing nicotinic receptors contribute to the organization of sleep and regulate putative micro-arousals in mice[END_REF]. In addition, nicotine intake leads to brain desynchronization and increases arousal and attention [26]. In non-smokers, acute nicotine administration is linked to low REM sleep and slow-wave sleep [START_REF] Jaehne | Effects of nicotine on sleep during consumption, withdrawal and replacement therapy[END_REF]. In rats, acute nicotine administration increased waking but chronic delivery of nicotine led to higher levels of REM sleep [START_REF] Salin-Pascual | Changes in sleep after acute and repeated administration of nicotine in the rat[END_REF]. Treatment with mecamylamine, a non-selective, non-competitive antagonist of nAChRs reversed the sleeping patterns of nicotine treated rats back to normal [START_REF] Salin-Pascual | Changes in sleep after acute and repeated administration of nicotine in the rat[END_REF]. Three constitutive mutations of the α4 subunit, and one mutation of the β2 subunit of the human nAChR, were linked to a hereditary epileptic syndrome known as autosomal nocturnal frontal lobe epilepsy causing seizures that usually occur at night during sleeping, causing awakening [START_REF] Fusco | The nicotinic receptor β2 subunit is mutant in nocturnal frontal lobe epilepsy[END_REF][30][START_REF] Sutor | Neuronal nicotinic acetylcholine receptors and autosomal dominant nocturnal frontal lobe epilepsy: a critical review[END_REF]. A contribution of nAChRs was also identified in the differential modulation of sleep and wakefulness [START_REF] Léna | Beta2-containing nicotinic receptors contribute to the organization of sleep and regulate putative micro-arousals in mice[END_REF][START_REF] Cohen | beta 2 nicotinic acetylcholine receptor subunit modulates protective responses to stress: A receptor basis for sleep-disordered breathing after nicotine exposure[END_REF]33] and observed deficits in awakening suggested to contribute, among other possibilities, to sudden infant death syndrome [START_REF] Cohen | beta 2 nicotinic acetylcholine receptor subunit modulates protective responses to stress: A receptor basis for sleep-disordered breathing after nicotine exposure[END_REF][33][START_REF] Slotkin | Fetal nicotine or cocaine exposure: which one is worse?[END_REF].

Evidence exists for the contribution of nAChRs to the modulation of wakefulness and sleep which deserves additional investigation including studies focused on recordings in humans.

Anesthesia

In humans, general anesthesia (GA) is used in patients that undergo surgery for providing analgesia and "loss of consciousness" (LOC), despite reports of anesthesia occasionally causing disconnected states of conscious processing [START_REF] Huang | Brain imaging reveals covert consciousness during behavioral unresponsiveness induced by propofol[END_REF] or dream-like states [START_REF] Sanders | Is consciousness fragile?[END_REF]. Mashour and colleagues suggested that LOC is the primary therapeutic action of general anesthesia [START_REF] Mashour | Conscious Processing and the Global Neuronal Workspace Hypothesis[END_REF]. There is evidence that GA affects feedback connectivity originating in the frontal areas [START_REF] Moon | General relationship of global topology, local dynamics, and directionality in large-scale brain networks[END_REF][38][START_REF] Wollstadt | Breakdown of local information processing may underlie isoflurane anesthesia effects[END_REF], a finding consistent with detailed models of the GNW [START_REF] Dehaene | Experimental and theoretical approaches to conscious processing[END_REF].

Even if the mechanism of GA is not fully understood, their direct, stereospecific binding to ligand gated ion channels (LGICs) is considered today as the main mode of action [40]. The superfamily of LGICs includes the inhibitory gamma-aminobutyric acid (GABA), glycine and excitatory 5-hydroxytryptamine (5-HT3) receptors, with nAChRs being the privileged, though non-exclusive, target for GA in vertebrates causing allosteric modulation of GABAA receptors.

In the case of the bacterial receptor, GLIC (from Gloeobacter) [START_REF] Nury | X-ray structures of general anaesthetics bound to a pentameric ligand-gated ion channel[END_REF], propofol and desflurane separately bind to allosteric modulatory sites [START_REF] Nury | X-ray structures of general anaesthetics bound to a pentameric ligand-gated ion channel[END_REF][START_REF] Sauguet | Crystallographic studies of pharmacological sites in pentameric ligand-gated ion channels[END_REF].

Most general anesthetics inhibit neuronal nAChRs utilizing possible mechanisms including allosteric stabilisation of the closed channel, direct channel blockade, and enhanced desensitization [START_REF] Changeux | Conscious processing: implications for general anesthesia[END_REF]. Each nAChR combination exhibits different sensitivity to general anesthetics, even though they share high sequence homology [START_REF] Mowrey | Alpha 4 beta 2 neuronal nicotinic acetylcholine receptors in the central nervous system are inhibited by isoflurane and propofol, but alpha 7-type nicotinic acetylcholine receptors are unaffected[END_REF]. The nAChRs oligomers containing α4β2 and α3β4 subunits are the most inhibited nAChRs by volatile anesthetics and ketamine with clinically relevant IC50 values [45]. The α4β2 receptor is affected by isoflurane more potently than the most sensitive GABAA or glycine receptor, whereas the α7 nAChR is not significantly affected by volatile anesthetics or ketamine [46,[START_REF] Zhang | Volatile general anaesthetic actions on recombinant nACh alpha 7, 5-HT3 and chimeric nACh alpha 7-5-HT3 receptors expressed in Xenopus oocytes[END_REF].

Functional disconnection of the brain due to anesthesia has been studied using neuroimaging techniques in humans and non-human primates. Functional MRI of awake and sedated spontaneous activity in the entire brain of monkeys revealed that under GA the mechanistic relationships between prefrontal, posterior parietal and cingulate cortices -assumed to compose the GNW -are altered [START_REF] Barttfeld | Signature of consciousness in the dynamics of resting-state brain activity[END_REF][START_REF] Uhrig | Resting-state Dynamics as a Cortical Signature of Anesthesia in Monkeys[END_REF]. In addition, electrocorticography of subjects administered propofol and ketamine in human and nonhuman primates revealed disrupted functional connectivity across primary sensory, motor cortices, and altered beta oscillations, which are associated with feedback processing [START_REF] Mashour | Conscious Processing and the Global Neuronal Workspace Hypothesis[END_REF]. Recently, it was demonstrated that wakelike behaviour in rodents could be restored using stimulation of the prefrontal cortex by the cholinergic agonist carbachol in animals that had continuous administration of the general anesthetic sevoflurane, suggesting that cholinergic mechanisms in prefrontal cortex can differentially modulate conscious processing in anesthetized animals [START_REF] Pal | Differential Role of Prefrontal and Parietal Cortices in Controlling Level of Consciousness[END_REF][START_REF] Pal | Level of Consciousness Is Dissociable from Electroencephalographic Measures of Cortical Connectivity, Slow Oscillations, and Complexity[END_REF].

Recordings performed in humans demonstrated that ongoing spontaneous brain activity forms constant reproducible patterns and exhibits spontaneous ultraslow fluctuations of very low frequency (<0.1 Hz) [START_REF] Moutard | Spontaneous Fluctuations and Non-linear Ignitions: Two Dynamic Faces of Cortical Recurrent Loops[END_REF][START_REF] Biswal | Interhemispheric correlations of slow spontaneous neuronal fluctuations revealed in human sensory cortex[END_REF][54][55] (Figure 1A). These ultra-slow neuronal activity patterns are considered as signatures of conscious access [START_REF] Dehaene | Ongoing spontaneous activity controls access to consciousness: a neuronal model for inattentional blindness[END_REF], and are also observed in animal models [START_REF] Koukouli | Nicotinic receptors in mouse prefrontal cortex modulate ultraslow fluctuations related to conscious processing[END_REF], yet the neuronal mechanisms of these fluctuations are a matter of debate. In vivo recordings in animal models revealed that ultraslow-fluctuations (USFs) persist under general anesthesia with no difference between the percentage of populations that exhibit USFs in the awake state compared to the anesthetized state. However, isoflurane causes a reduction of the synchronously firing populations, thus general anesthesia shows strong inhibitory effects on the generation of neuronal synchrony [START_REF] Koukouli | Nicotinic receptors in mouse prefrontal cortex modulate ultraslow fluctuations related to conscious processing[END_REF] (Figure 1B). It was demonstrated, by recording the ongoing neuronal spontaneous activity of mouse prelimbic cortex, both in the awake and anesthetized (isoflurane) state, that nAChRs play an important role in the generation of USFs, but differently during quiet wakefulness and anesthesia [START_REF] Koukouli | Nicotinic receptors in mouse prefrontal cortex modulate ultraslow fluctuations related to conscious processing[END_REF] (Figure 1C). USFs and neuronal synchronicity are disrupted in genetically modified mice that lack the β2-nAChRs, but not with α7 nAChR knockout. Furthermore, pharmacological blockade of β2 nAChRs suffices to disrupt USFs in awake mice [START_REF] Koukouli | Nicotinic receptors in mouse prefrontal cortex modulate ultraslow fluctuations related to conscious processing[END_REF] (Figure 1C). In addition, light anesthesia using isoflurane was able to disrupt neuronal synchronicity while maintaining the generation of USFs [START_REF] Koukouli | Nicotinic receptors in mouse prefrontal cortex modulate ultraslow fluctuations related to conscious processing[END_REF]. This is in accordance with recent observations supporting that the anesthetized brain is more dynamic than previously thought with persistent metastable oscillations and patterns that reflect intrinsic dynamics [START_REF] Li | Dynamic Cortical Connectivity during General Anesthesia in Healthy Volunteers[END_REF][START_REF] Vlisides | Dynamic Cortical Connectivity during General Anesthesia in Surgical Patients[END_REF].

In conclusion, important progress in the understanding of the molecular mechanisms of LOC by general anesthetics have been achieved, including defining a contribution of AChRs in the modulation of cortical dynamics and conscious processing.

nAChR modulation of high order cognitive processing

The prefrontal cortex is an important component of the GNW which receives dense cholinergic innervation from the basal forebrain and is thought to play a critical role in high order cognitive processes that demand high levels of convergence and integration. Experiments in which cholinergic innervation was removed revealed deficits in attention, whereas stimulation of cholinergic projections led to enhanced attentional performance [START_REF] St Peters | Enhanced control of attention by stimulating mesolimbiccorticopetal cholinergic circuitry[END_REF][START_REF] Granon | Deciding between conflicting motivations: what mice make of their prefrontal cortex[END_REF]. This finding is consistent with the observation that humans and animal models with lesions to the prefrontal cortex (or prelimbic area) show deficits in attention, working memory and motor behavior. Note that in rodents, the prelimbic area is considered functionally homologous to the dorsolateral prefrontal cortex in humans and non-human primates [START_REF] Uylings | Do rats have a prefrontal cortex?[END_REF]62].

Transgenic mice with deletion of specific nAChR subunits have been an important tool in examining the contribution of nAChRs to cognitive processes [START_REF] Changeux | Nicotine addiction and nicotinic receptors: lessons from genetically modified mice[END_REF][START_REF] Picciotto | Acetylcholine receptors containing the β2 subunit are involved in the reinforcing properties of nicotine[END_REF][START_REF] Picciotto | Abnormal avoidance learning in mice lacking functional highaffinity nicotine receptor in the brain[END_REF]. Mice lacking the β2nicotinic subunits (β2-KO) display similar patterns of social interaction deficits as those caused by lesions in the prelimbic cortex, whereas β2-subunit rescue in β2-KO mice reversed the deficit, indicating that β2-containing nAChRs are necessary for social interactions [64]. A role of β2-nAChRs in motor strategies has been identified as well, with more prominent contribution to exploration than navigation [65]. Of note, exploration typically requires high level cognitive processing, whereas navigation, at least in some of its forms, is more of an automatic behaviour.

Genetic deletion of the β2subunit also resulted in an impairment of attentional performance in a 5-choice serial reaction time task [START_REF] Guillem | Nicotinic acetylcholine receptor β2 subunits in the medial prefrontal cortex control attention[END_REF]. Re-expressing β2-subunits in neurons of the prelimbic cortex restored attentional performance in the same task. By contrast, re-expression of β2subunits in the adjacent anterior cingulate medial prefrontal cortex did not restore the attentional performance deficits, suggesting that nicotinic modulation of anterior cingulate neurons through β2-containing nAChRs does not modulate attentional behaviour [START_REF] Guillem | Nicotinic acetylcholine receptor β2 subunits in the medial prefrontal cortex control attention[END_REF]. The same experiments performed in α7-KO mice showed no effect on attentional performance, suggesting that this function does not depend on the α7-nicotinic subunit, although previous studies yielded conflicting results [START_REF] Young | Nicotine improves sustained attention in mice: evidence for involvement of the alpha7 nicotinic acetylcholine receptor[END_REF].

In rodents, monkeys and humans, the expression of nAChRs in prefrontal cortex is layer dependent and cell type specific [68] (Figure 2A). Recently, it was shown, in awake mice, that endogenous ACh distinctly recruits specific interneurons within a hierarchy that controls pyramidal neuron activity in layers II/III of the prelimbic cortex, through differential expression of nAChRs [69] (Figure 2B). Specifically, α7-KO and β2-KO awake mice exhibit pyramidal cell hyperactivity in layer II/III of prelimbic cortex whereas α5-KO mice exhibit hypofrontality.

Targeted local re-expression of these subunits in defined categories of inhibitory neurons restored the activity to control levels [69]. In addition, a key role of α5-subunits in attentional performance and social interactions was identified [69,[START_REF] Bailey | The nicotinic acetylcholine receptor alpha5 subunit plays a key role in attention circuitry and accuracy[END_REF]. These data highlight the importance of the balance between excitatory and inhibitory neurotransmission (E/I) in the modulation of cognitive functions.

The effect of nicotine administration on mental disorders has been documented in several clinical studies. For instance, nicotine is shown to improve attention in schizophrenia patients as well as in nicotine-addicted individuals under craving [26,[START_REF] Hong | A CHRNA5 allele related to nicotine addiction and schizophrenia[END_REF][START_REF] Barr | The Effects of Transdermal Nicotine on Cognition in Nonsmokers with Schizophrenia and Nonpsychiatric Controls[END_REF]. In addition, nicotine decreased depression symptoms, compulsion and anxiety in individuals with attention-deficit hyperactivity disorder or obsessive-compulsive disorder [START_REF] Bekker | Acute effects of nicotine on attention and response inhibition[END_REF][START_REF] Salín-Pascual | Changes in compulsion and anxiety symptoms with nicotine transdermal patches in non-smoking obsessive-compulsive disorder patients[END_REF]. A meta-analysis of 41 placebo controlled studies that included nicotine administration in non-smokers and satiated smokers showed that nicotine had positive effects on short-term episodic memory, working memory performance and motor ability [75]. Another study demonstrated that nicotine can improve working memory in abstinent smokers but not in non-smokers [START_REF] Grundey | Double dissociation of working memory and attentional processes in smokers and non-smokers with and without nicotine[END_REF]. Imaging studies have been instrumental in identifying brain regions and large-scale networks where nicotine exhibit enhancing effects upon attention, working memory, fine motor skills and episodic memory functions [START_REF] Valentine | Cognitive Effects of Nicotine: Recent Progress[END_REF].

Administration of a α4β2 agonist to monkeys produced improved working memory performance [START_REF] Prendergast | Central nicotinic receptor agonists ABT-418, ABT-089, and (-)-nicotine reduce distractibility in adult monkeys[END_REF]. Recently, ACh stimulation of α4β2 nAChR enhanced the firing of neurons in the prefrontal cortex of primates performing a working memory task [START_REF] Sun | Nicotinic α4β2 Cholinergic Receptor Influences on Dorsolateral Prefrontal Cortical Neuronal Firing during a Working Memory Task[END_REF]. Together, these studies show a unified contribution of nAChRs expressed in the prefrontal cortex to enhance attention, working memory, social behavior and conscious processing in general.

Role of nAChRs in human diseases

Nicotinic receptors have been found to be involved in several disorders of the central nervous system, including Alzheimer's disease, Parkinson's disease and schizophrenia, which we discuss in this section.

Alzheimer's disease

According to the cholinergic hypothesis of Alzheimer's disease (AD), a reduction of cholinergic innervation is responsible (or contributes significantly) to cognitive decline in AD.

Building upon this model, inhibitors of acetylcholine-esterase that ameliorate cholinergic signalling [START_REF] Bartus | The cholinergic hypothesis of geriatric memory dysfunction[END_REF][START_REF] Hampel | The cholinergic system in the pathophysiology and treatment of Alzheimer's disease[END_REF] have been tested as treatments for AD, but they have recently been found of little efficiency in improving mild cognitive impairments and mild AD dementia [START_REF] Long | Alzheimer Disease: An Update on Pathobiology and Treatment Strategies[END_REF][START_REF] Han | Cholinesterase Inhibitors May Not Benefit Mild Cognitive Impairment and Mild Alzheimer Disease Dementia[END_REF].

Using post-mortem human samples, it was shown that the α7 -subunit co-localizes with Aβ1-42 in amyloid plaques of brain slices obtained from patients with sporadic AD, and immunoprecipitation experiments showed that this interaction is specific for the α7-subunit [START_REF] Wang | beta-Amyloid(1-42) binds to alpha7 nicotinic acetylcholine receptor with high affinity. Implications for Alzheimer's disease pathology[END_REF][START_REF] Wang | Amyloid peptide Abeta(1-42) binds selectively and with picomolar affinity to alpha7 nicotinic acetylcholine receptors[END_REF]. Using knock-out mice for the α7-receptors, researchers attempted to identify a role of the α7subunit in AD mouse models, but came to conflicting conclusions regarding the overall pathophysiological contribution of these receptors [START_REF] Dziewczapolski | Deletion of the alpha 7 nicotinic acetylcholine receptor gene improves cognitive deficits and synaptic pathology in a mouse model of Alzheimer's disease[END_REF][START_REF] Hernandez | Loss of alpha7 nicotinic receptors enhances beta-amyloid oligomer accumulation, exacerbating early-stage cognitive decline and septohippocampal pathology in a mouse model of Alzheimer's disease[END_REF]. Specifically, in one of these studies, an AD mouse model that expresses the human amyloid precursor protein with the Swedish and Indiana mutations (associated with early onset of AD) was crossed with α7-KO mice. Although the AD mice displayed spatial memory deficits, the α7-KO AD mice were protected against memory impairments [START_REF] Dziewczapolski | Deletion of the alpha 7 nicotinic acetylcholine receptor gene improves cognitive deficits and synaptic pathology in a mouse model of Alzheimer's disease[END_REF]. However, in a separate study using a different AD mouse model, cognitive deficits were found to be more severe in AD mice lacking the α7-nicotinic subunit [START_REF] Hernandez | Loss of alpha7 nicotinic receptors enhances beta-amyloid oligomer accumulation, exacerbating early-stage cognitive decline and septohippocampal pathology in a mouse model of Alzheimer's disease[END_REF]. It has also been demonstrated that non-α7-nAChRs, such as β2-containing nAChRs, bind Aβ. Electrophysiological and behavioral studies support this interaction [88,89], however, to date it still remains unknown whether Aβ inhibits or activates nAChR and how this interaction may lead to AD pathology.

Overall, nAChRs might be key players in the aetiology of AD pathology, and more investigations in both animal models and humans are urgently needed to potentially discover efficient treatments.

Parkinson's disease

A hallmark of Parkinson's disease (PD) is the loss of nigrostriatal dopaminergic neurons.

Presynaptic and post-synaptic nAChRs modulate dopamine release, and both in vitro and in vivo animal model studies revealed a crucial role of β2-containing nAChRs in striatal dopaminergic signalling control [START_REF] Quik | Nicotinic receptors as CNS targets for Parkinson's disease[END_REF]. Rodent and monkey PD models exhibit a decrease in the levels of nAChR expression in the striatum accompanied by dopaminergic degeneration. Specifically, a loss of α6α4β2β3 is observed during mild nigrostriatal lesioning, a decrease in α6β2β3 receptors is seen when the damage is higher and low levels of α4β2-containing receptors when the degeneration is extremely severe [START_REF] Quik | Nicotinic receptors as CNS targets for Parkinson's disease[END_REF]. Similarly, decreased expression of nAChR is found in the caudate, putamen and substantia nigra of humans affected by PD and this decrease is linked to the level of nigrostriatal damage [START_REF] Tizabi | Nicotinic Receptor Intervention in Parkinson's Disease: Future Directions[END_REF].

Nicotine is found to reduce levodopa-induced dyskinesias, a major side effect of levodopa therapy, improves motor abilities in PD patients and protects against nigrostriatal damage [START_REF] Schneider | Effects of SIB-1508Y, a novel neuronal nicotinic acetylcholine receptor agonist, on motor behavior in parkinsonian monkeys[END_REF][START_REF] Decamp | Effects of nicotinic therapies on attention and executive functions in chronic low-dose MPTP-treated monkeys[END_REF], pointing at nicotine and nicotinic agonists as promising candidates as part of a treatment regimen for PD.

Schizophrenia

Schizophrenia is a neuropsychiatric disorder that is characterized by both positive symptoms (e.g. hallucinations and delusions) and negative ones (e.g. flattened affect) that usually begin in late adolescence or early adulthood [94]. A distinct role of nAChR subunits in schizophrenia has been initially shown by identifying decreased levels of α7-nAChRs in postmortem hippocampus [START_REF] Freedman | Evidence in postmortem brain tissue for decreased numbers of hippocampal nicotinic receptors in schizophrenia[END_REF], cortex [96] and in the thalamus of schizophrenia patients [START_REF] Court | Neuronal nicotinic receptors in dementia with Lewy bodies and schizophrenia: alpha-bungarotoxin and nicotine binding in the thalamus[END_REF]. Also, there is evidence that the expression of α4β2containing-nAChRs, as determined by 3H-nicotine binding, is reduced in the hippocampus of these patients [START_REF] Breese | Abnormal regulation of high affinity nicotinic receptors in subjects with schizophrenia[END_REF].

The prevalence of smoking among schizophrenia patients is three to five times higher than in the general population [99] and schizophrenia patients extract 50% more nicotine per cigarette than other smokers, by inhaling more deeply [100]. The aetiology of this prevalence of smoking among schizophrenia patients is not yet understood and several mechanisms have been suggested. Among them, it has been proposed that nicotine serves as a form of self-medication to alleviate the negative symptoms of the disease, enhance cognitive deficits or reduce the side effects of antipsychotic treatments. At variance with this interpretation, it was suggested that cigarette smoking might be itself a cause of psychosis [101]. Large, longitudinal, prospective studies are needed to investigate the relationship between smoking and psychosis in order to be able to elucidate whether cigarette smoking has a causal role or not in the development of In conclusion, clinical trials of nicotinic agonists but also positive allosteric modulators of defined nAChR oligomers are of crucial importance to develop potential treatments of psychiatric disorders such as schizophrenia.

Genetic studies on the contribution of nAChR to human diseases

Genome-wide association studies (GWAS) identified susceptible loci in specific diseases by screening millions of genetic factors in cohorts of thousands of patients and controls. This strategy may help the development of personalized medicine approaches based on the evaluation of genetic risks. Genetic variations in nAChRs subunits have been found to be strongly associated with complex neuropsychiatric diseases such as schizophrenia and a number of

Mendelian disorders (Box 1).

Loss of control in Nicotine addiction

Subjects addicted to drugs such as nicotine or cocaine reveal neural circuit impairments associated with disruptions of the dopaminergic system [111]. Drug addiction causes a marked dysregulation of brain circuits involved in reward, motivation, memory, judgment and selfawareness, including circuits in frontal cortical regions. These deficits cause a compulsive self-administration of drug with a loss of control (altered inhibition) and disrupted cognitive operations that impair judgment and favor relapse [START_REF] Changeux | Nicotine addiction and nicotinic receptors: lessons from genetically modified mice[END_REF]111,112]. Behavioural differences and increased dopamine release in response to nicotine leads to greater likelihood of addiction occurring in adolescents compared with adults, and long-lasting dysregulation of attention and sensory processing [113,114]. Several molecular changes are taking place as a consequence of chronic nicotine exposure such as upregulation of α7-nAChRs, increased presynaptic α7-nAChR transmission, upregulation of β2-nAChRs, presynaptic downregulation of β2-nAChRs and increased dopaminergic neuron activity [START_REF] Changeux | Nicotine addiction and nicotinic receptors: lessons from genetically modified mice[END_REF]. An effect of nicotine and ACh on axon excitability was also described, indicating that nAChRs are probably located at nodes of Ranvier in particular along myelinated thalamocortical axons [115,116], revealing a non-synaptic action of nicotinic agents. Human individuals addicted to nicotine exhibit cognitive deficits similar to those of patients with an impaired ventromedial prefrontal cortex [117].

Functional MRI studies in rodents have shown that, consistent with the enhancing effect of nicotine, acute injections of nicotine increased the activation of prelimbic, frontal motor and somatosensory cortices as well as the nucleus accumbens, ventral tegmental area, substantia nigra and thalamus [118]. Chronic administration of nicotine produces pyramidal cell hyperactivity in the prefrontal cortex of awake mice [69]. Moreover, mice expressing a human polymorphism in the α5 nicotinic subunit (rs16969968_α5SNP), which is associated with nicotine addiction and schizophrenia, exhibit reduced pyramidal cell activity in layer II/III of prelimbic cortex compared to wild-type control mice due to alterations in the balance between excitation and inhibition [69] (Figure 2B). Interestingly, chronic administration of nicotine through an osmotic minipump was able to restore the hypofrontality associated with the α5SNP, suggesting an explanation of why individuals with schizophrenia may use tobacco smoking as a form of self-medication [69] (Figure 2B). Similarly, humans with the α5SNP exhibit reduced resting state functional connectivity and altered prefrontal cortex circuitry [119] (Figure 3 and

Box 1).

Neuroimaging techniques reveal that in smokers, α4β2-containing nAChRs are upregulated compared to non-smoking individuals. Furthermore abstinence for 3 to 12 weeks can normalize the receptor levels to that of non-smokers [120,121]. Yet the possible relationship of these changes to long-term loss of inhibitory control is still not understood.

Tobacco smoking is robustly linked to epigenetic modifications. In smokers, DNA methylation changes take place at several genomic loci, mainly at cytosine-phosphate-guanine sites [122,123]. At most of the loci, smoking causes loss of methylation to the extent that epigenome profiling of DNA methylation patterns associated with tobacco smoking can serve as molecular biomarkers for major diseases such as lung cancer [124]. Exposure to nicotine during prenatal and early postnatal periods is claimed to cause abnormal development of the brain and favors the onset of mental disorders [125]. This is of particular importance since in some countries, up to about 10% of women smoke during pregnancy, thereby exposing their offspring to nicotine during critical neurodevelopmental periods [125]. Early nicotine exposure produces long lasting impairments in neuronal plasticity such as increased spine density and branching in the frontal, medial and caudal cerebral cortex [126]. Recently, it was demonstrated that activation of heteropentameric nAChRs by early nicotine exposure causes alterations in histone methylation in the cerebral cortex, which in turn increases the expression of genes involved in synapse maintenance and dendritic remodeling together with behavioral alterations such as hypersensitive passive avoidance performance [126].

These studies reveal a dual action of nicotine as a drug; a positive, therapeutic role as a cognitive stimulant and a negative effect as an addictive drug causing loss of control in drug consumption and other cognitive impairments.

Concluding remarks

The neuronal and molecular mechanisms involved in modulating high order cognitive functions, and more specifically, conscious processing, remain one of the most intriguing issues of research in neuroscience. Yet substantial progress has been achieved over the last decades in the identification of the molecular components of the circuit engaged in cognition and its dysfunctions, both in animal models and in humans. In particular, deciphering the modulatory mechanisms of awareness, sleep, anesthesia, LOC and mental disorders has contributed to our improved understanding of the conscious processes involved.

It is our opinion that through the use of a broad range of methods, including genetically modified animals and brain imaging in humans, the evidence is clear that nAChRs do modulate high order cognitive functions, including conscious processing. Thanks to investigations of nAChRs -among other receptors -a "pharmacology of consciousness" becomes scientifically accessible thereby opening multiple novel strategies for the diagnosis, prevention and therapies of neuropsychiatric diseases (see Outstanding Questions). Box 1. Human genetic studies identify variants of nAChRs linked to major psychiatric disorders.

The CHRNA5/CHRNA3/CHRNB4 gene cluster

The 15q25 gene cluster, which contains the CHRNA5/CHRNA3/CHRNB4 genes, coding for the α5-, α3and β4-nAChR subunits, was associated with risk for heavy smoking and nicotine addiction [127-132] and several single nucleotide polymorphisms (SNPs) were identified [130][131][132][133]. The non-synonymous SNP rs16969968, found in exon 5 of the α5-human gene, involves a substitution of an aspartic acid by an asparagine in position 398 (D398N). The α5SNP is present in about 35% of Europeans and 50% of Middle Eastern populations and has been linked to nicotine dependence, lower ratings of aversive effect, delayed smoking cessation, early lung cancer and cognitive improvement after nicotine intake [127,134]. Recently, the same genetic locus was associated with schizophrenia in a major GWAS study [135]. The α5SNP is linked to schizophrenia in both Caucasian and African-American patients and is associated with decreased intrinsic resting functional connectivity (rsFC) in humans [119] (Figure 3). Mice with this polymorphism exhibit schizophrenia related cognitive deficits and hypofrontality that is reversed after chronic nicotine administration [69]. In addition to nicotine dependence and schizophrenia, the 15q25 gene cluster has been associated with alcohol, cocaine and opioid dependence, suggesting a potential association with substance use disorders in general [128]. However, the same rs16969968_α5SNP has been linked to lower prevalence of cocaine use disorder in humans [128].

Interestingly, rare (<5%) missense variants at conserved sites in CHRNB4 are associated with decreased risk of developing nicotine dependence and decrease the number of cigarettes consumed daily by smokers [136].

The human CHRNA7 and CHRFAM7A genes

The human CHRNA7 gene has 10 exons and is located on the long arm of chromosome 15q13-q14), which is a highly unstable area of the human genome that is linked to schizophrenia. A polymorphism in the 50 -regulatory region (not duplicated) of CHRNA7 (rs3087454) was found to be strongly associated with schizophrenia [137,138]. However, there is no convincing evidence associating the CHRNA7 gene with smoking [139-141].

In humans, gene duplication in exons 5-10 in CHRNA7 during evolution produced a hybrid gene, CHRFAM7A. The duplication is human specific, found neither in primates nor in rodents, but a similar sequence has been identified in gorillas [142]. Its product, referred as dupa7, lacks part of the N-terminal extracellular ligand-binding domain and it is linked to many neuropsychiatric conditions. Dupa7 is considered a dominant-negative regulator of ion channel function [143]. It was suggested that increased expression of the CHRFAM7A gene in patients with schizophrenia might contribute to failure to regulate nicotine binding to high affinity nicotinic receptors. In addition, a 2 bp deletion was identified in exon 6 of the CHRFAM7A gene [144]. This deletion is more common in Caucasians than African-Americans and is a more potent negative regulator of α7-nAChR function than the normal copy and it was linked to both bipolar disorder and A) The expression of nicotinic receptors in prefrontal cortex is layer dependent and neuronal type specific. The α7and β2-nicotinic subunits are highly expressed and the α5-nicotinic subunit is expressed at a lower extent. Almost no pyramidal neurons (P) in layer II/III of prefrontal cortex express nAChRs, whereas the pyramidal neurons of layer V mouse prefrontal cortex exhibit fast nAChR currents that are mediated by α7-containing nAChRs. Both human and mouse layer VI pyramidal neurons express α5and β2-containing nAChRs. Modified from

  psychosis [102]. Schizophrenia patients develop multiple sensory processing deficits including auditory sensory processing (P50) impairments, pre-pulse inhibition (PPI) reductions, and eye-tracking deficiencies [103]. P50 gating impairments have been linked to the chromosome 15q13-14 site of the CHRNA7 gene [104]. Interestingly, it has been shown that in schizophrenia patients nicotine normalizes the P50 gating deficits and that treatment of mice with α7-nAChR agonists corrects the sensory gating anomalies [103,105,106]. Activation of α7-nAChRs by nicotine or nicotinic agonists increases glutamate release and in turn normalized P50 gating deficits [107]. Moreover, patients with schizophrenia exhibit reduced PPI and eye-tracking deficits, although nicotine intake increases PPI and eye-tracking performance in these patients [108-110]. Finally, studies in animal models, but also in humans, found that nicotine improves cognitive functions such as working memory, attention and social interactions, which are impaired in schizophrenia patients [105].

  schizophrenia [143]. The CHRFAM7A gene can vary in copy number with individuals with one copy or none and deletions of the CHRNA7 and CHRFAM7A are strongly associated with schizophrenia [145,146]. In addition, rare deletions and duplication of the same area have been linked to autism, epilepsy, bipolar disorder and attention deficit hyperactivity disorder [147]. A recent study that used induced pluripotent cells and neural progenitor cells derived from individuals with heterozygous 15q13.3 deletions and duplications demonstrated that calcium signal cascades that are dependent on α7-nAChRs are downregulated suggesting that individuals with deletions and duplications of 15q13.3 exhibit cognitive deficits [148]. The CHRNA2, CHRNA4 and CHRNB2 genes Missense mutations or indels in CHRNA2, CHRNA4 and CHRNB2 genes have been linked to autosomal dominant nocturnal frontal lobe seizures which usually occur during non-rapid eye movement sleep [149].

Figure 1 .

 1 Figure 1. Ultra-slow fluctuations (USFs) as a signature of conscious processing.

Figure 2 .

 2 Figure 2. Nicotinic receptor modulation of PFC circuit dynamics involved in schizophrenia

[

  150] based on [69,151-154]. B) In layer II/III of mouse prelimbic cortex, α5-containing nAChRs are expressed by vasoactive intestinal polypeptide (VIP) interneurons, somatostatin (SOM) interneurons express α7and β2-containing nAChRs, whereas parvalbumin (PV) fast spiking neurons express α7-nAChRs [69,151,152]. α5-containing nAChRs control the VIPmediated disinhibition via SOM interneuron inhibition of pyramidal neurons in layers II/III in prelimbic cortex [69]. C) The α5SNP, a human polymorphism linked to nicotine addiction and schizophrenia, decreases the activity of VIP interneurons. This leads to increased SOM interneuron activity that, in turn, reduces pyramidal neuron activity, resulting in hypofrontality, a feature of schizophrenia. D) Chronic nicotine administration desensitizes β2-containing nAChRs on SOM interneurons. As a result, SOM inhibition of pyramidal neurons is reduced and the activity is normalized. Modified from [69,155].

Figure 3 .

 3 Figure 3. Brain imaging of resting states by MRI of human nAChR genetic variants. A) The α5SNP (Asp3898Asn) is associated with a dorsal anterior cingulate (dACC)-ventral

Figure 3

 3 Figure 3

Tassonyi, E. et al. (2002) The role of nicotinic acetylcholine receptors in the mechanisms of anesthesia. Brain Res. Bull. 57, 133-50

Acknowledgements

This work was supported by the European Union's Horizon 2020 Human Brain Project SGA2 and SGA3 and the Kavli Institute for Brain and Mind, University of California San Diego. We thank Uwe Maskos for valuable scientific discussions and Kurt Sailor for comments on the manuscript.


Thierry Adoukonou
email: adoukonouthierry@yahoo.fr

Mendinatou Agbétou

Arlos Sowanou

Oyéné Kossi

Pervenche Fotso

Corine Houéhanou

Jean-Michel Vallat

Dismand Houinato

Pierre-Marie Preux

Philippe Lacroix

Adoukonou Thierry

Stroke care and outcomes in the Department of Neurology in Parakou, Benin: Retrospective cohort study

Keywords: stroke, mortality, Benin

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Stroke is a leading cause of death and has a high economic burden throughout the world [START_REF] Rajsic | Economic burden of stroke: a systematic review on post-stroke care[END_REF]. Sub-Saharan African (SSA) countries have undergone an epidemiological transition and have a high burden of stroke and other noncommunicable diseases. This geographical area is characterized by a lack of stroke specialists, stroke diagnostic facilities, and low availability of new stroke treatments [START_REF] Urimubenshi | Stroke care in Africa: A systematic review of the literature[END_REF][START_REF] Adoukonou | Management of stroke in sub-Saharan Africa: current issues[END_REF]. Stroke was characterized by the high proportion of hemorrhage due to hypertension and small vessel disease in this geographical area [START_REF] Adoukonou | Management of stroke in sub-Saharan Africa: current issues[END_REF].

Because of the scarcity of diagnostic tools and stroke specialists many stroke patients had unknown stroke subtypes in sub-Saharan Africa. Hypertension can explain more than 80% of all cases of stroke, and prevention needs to focus on stroke management. The management of the acute phase of stroke has not been compared in detail in those in western countries. The lack of facilities, lack of health insurance, misconceptions about stroke symptoms, cultural considerations and paucity of stroke specialists can explain the difference between high income countries and low and middle income countries [START_REF] Adoukonou | Management of stroke in sub-Saharan Africa: current issues[END_REF]. Stroke mortality is also high in low income countries compared with those reported in western countries.

Many studies have reported the in-hospital mortality of stroke in SSA with a range of 7.2% to 45% [START_REF] Cisse | Minimal Setting Stroke Unit in a Sub-Saharan African Public Hospital[END_REF][START_REF] Fekadu | Burden, clinical outcomes and predictors of time to in hospital mortality among adult patients admitted to stroke unit of Jimma university medical center: a prospective cohort study[END_REF][START_REF] Komolafe | Stroke mortality in a teaching hospital in South Western Nigeria[END_REF]. We found an in-hospital case fatality of 16.8% in 85 cases [START_REF] Adoukonou | Short term (3) months prognosis of stroke in Parakou[END_REF]. Many factors have been reported to influence the outcome of stroke, particularly late admission, the acute complications of stroke and vascular risk factors. We aimed to describe the stroke pattern and in-hospital case fatality in a large cohort in Parakou University Hospital.

Methods

Registration and ethics

This section is written according to the STROCSS statement guidelines Those hospitals both received stroke patients but only the University hospital has neurologists and the beds dedicated for stroke patients. At the University hospital there are two neurologists, two cardiologists, one Intensive Care Unit (with three specialists), two neurosurgeons and a physiotherapy unit. The neurology department has two neurologists and 10 beds with two dedicated to stroke. The two dedicated stroke beds are scoped beds with 24-hour multi-parameter monitoring (blood pressure, oxygen saturation, pulsation, heart rhythm, temperature) with access to oxygen and a mucus aspirator, and the beds have anti-decubitus mattresses. The room with two beds is intended for subjects in the acute phase of stroke. However, patients past that phase, as well as those admitted after 3 days and who are stable, are hospitalized in the other beds of the service.

Protocols drawn up in accordance with international recommendations are available and accessible in the service. There is a 24-hour nurse and two caregivers for all 10 beds. The medical staff is made up of two neurologists, including a professor specializing in stroke, two general practionners and two physiotherapists. All service personnel were trained beforehand regarding stroke management.

Modern treatments such as thrombolysis and thrombectomy are not available, and despite the availability of rt-PA in the hospital for the prior six months, no intravenous thrombolysis had been performed. Osmotic drugs, intravenous antihypertensive drugs, low weight molecular heparin, acetaminophen, aspirin, clopidogrel, and statins are some of the drugs that are available. Few people in Benin have health insurance, which means that many patients cannot benefit from additional diagnostic or etiological examinations.

Cohort group: All stroke patients admitted between January 1, 2013 to December 31, 2019 were included and followed-up until hospital discharge.

Participants:

Inclusion criteria: All stroke cases met the inclusion criteria. The diagnosis of stroke was based on the World Health Organization (WHO) criteria [START_REF] Aho | Cerebrovascular disease in the community: results of a WHO collaborative study[END_REF] or on brain CT-scan results. Stroke was defined as "rapidly developing signs of focal (or global) disturbance of cerebral function, leading to death or lasting longer than 24 hours, with no apparent cause other than vascular" [START_REF] Aho | Cerebrovascular disease in the community: results of a WHO collaborative study[END_REF]. The patients with transient ischemic attack, those who died prior to evaluation and others with subarachnoid hemorrhage were excluded.

Sampling: All stroke cases were included and were recruited systematically.

Intervention: Sociodemographic data, vascular risk factors, and clinical data were recorded upon admission. The vascular risk factors were defined according to WHO stepwise approach [START_REF]Le Manuel de Surveillance STEPS de l'OMS: l'approche STEPWISE de l'OMS pour la surveillance des facteurs de risque des maladies chroniques Suisse[END_REF] The initial neurologic impairment assessment was based on the National Institute of Health Stroke Scale (NIHSS). We recorded the systolic and diastolic blood pressure and temperature. Some patients had laboratory tests (blood sugar, acid uric, blood cell count, cholesterol, triglycerides) and cardiac explorations (electrocardiogram or echocardiography) or ultrasound of neck vessels for the etiologic investigation. We systematically recorded the stroke subtype as ischemic, hemorrhagic, and unknown (without CT scan); the medical complications that occurred during acute phases were pneumonia, swallowing disorders, urinary tract infection, and seizure. The diagnosis of each complication was made by a neurologist based on the clinical presentation and the results of biological tests and other examinations.

The swallowing test was systematically carried out by the doctor upon admission to the service. A nasogastric tube was placed and monitored daily for those with swallowing disorders. Diagnostic examinations (CT scan, cardiac explorations, biological tests, etc.) are routinely prescribed to all patients depending on the type of stroke.

However, in the absence of insurance and medical coverage, only patients with the financial resources can carry them out.

Management was performed according to the international recommendations regarding the local protocol for each stroke subtype.

The patients with good improvement following an acute phase were released to home but those who worsened were sent to the intensive care unit. Our physiotherapy department cannot admit patients for hospitalization; the main issues were death, transfer to another unit or home discharge.

Outcome:

We recorded the outcome of each patient and the length of hospital stay. The primary outcome was the stroke case fatality on discharge.

Statistical methods:

The data were analyzed using Statistical Package for Social Sciences (SPSS) version 21.0 software (IBM) and the usual parameters were used to describe the variables. We compared the different types of stroke according to other variables and the associated variables for in-hospital death. We used the chi-2 test (or Fisher test) for the percentages and the Student t-test to compare the quantitative variables. A p value of 0.05 was considered to be significant.

Results

Four hundred twenty-three stroke cases were recorded with an incidence of 51.5% (423/822) of all hospitalizations during the study period. Among the 423 strokes cases, 372 were included in this study (51 patient records were unusable or lost of their medical sheet) with a participation rate of 87.9% (cf. figure 1). The patients were 16 to 97 years of age with a mean of 58.2+/-14.2 years. The sex ratio was 1:3 (210 were male). The median time to admission since the onset of symptoms was 48 hours with an interquartile interval of [20-96 hours]. Only 10.3% of patients were admitted to hospital within three hours. The characteristics of the patients are summarized in Table 1 according to vascular risk factors and clinical and biological data. The greatest vascular risk factor was hypertension (69.1%) followed by alcoholism (23.9%) and diabetes mellitus (16.9%). There was no difference in the frequency of vascular risk factors in the three stroke types. Ischemic stroke cases accounted for 40.3%, hemorrhage for 30.4% and unknown subtype for 29.3%. The patients with ischemic stroke were older and had lower admission blood pressure than others.

In fact, the ischemic stroke patients had a mean blood pressure of 151.3+/-31.7 mm Hg and 94.0+/-19.4 mm Hg, respectively, for systolic and diastolic. Those values were significantly lower than those in hemorrhagic and unknown stroke subtype (p=0.002 for systolic and 0.0001 for diastolic). Admission blood glucose was higher in patients with unknown stroke, although the proportion of diabetics was the same in all three groups (Table 1). The patients with intracerebral hemorrhage stayed in hospital longer than the other patients.

The NIHSS ranged from 0 to 29 with a mean of 9.4+/-5.7. In patients with ischemic stroke the locations were middle cerebral artery (67.4%); anterior cerebral artery (14.2%), posterior cerebral artery (4.2%), vertebro-basilar territory (5.0%) and multifocal sites (9.2%). However, deep location (77.5%) followed by lobar location (19.1%) and brainstem (3.4%) was the most frequent site of intracerebral hemorrhage. Many acute complications were recorded during hospitalization: 9.1% had pneumoniae, 10.2% swallowing disorders, 20

(5.4%) patients had seizures and 31 patients (8.3%) had urinary tract infection.

Among the patients only 121 (32.5%) had EKG; 31 (8.3%) had cardiac echography and 34 had ultrasound echography of neck vessels (9.1%). Four cases of atrial fibrillation were detected from the EKG data of ischemic stroke patients. The outcomes are listed in Table 2. Upon discharge, patients with ischemic stroke were taking more antiplatelet drugs and statins than the other patients, but only 132 (88.0%) of them had antiplatelet. Four patients had anticoagulant on discharge after antiplatelet in the acute phase. The use of an antiplatelet drug differed for others due to the hemorrhagic transformation of the infarction in the acute phase. Surprisingly, one patient with hemorrhagic stroke used one 75 mg aspirin per day on discharge due to the cardiac prescription (he accumulated four vascular risk factors such as hypertension, diabetes, previous myocardial infarction and a minor hemorrhage on brain CT scan). The antihypertensive drugs were prescribed more to patients with intracerebral hemorrhage (ICH). Twenty-three deaths were recorded with an in-hospital mortality of 6.2% during the hospitalization period. Disorders of consciousness, high neurological impairment, fever on admission, swallowing disorders and hyperleucocytosis were the associated factors for death (Table 3). Upon discharge, among the 349 survivors, 27.6% were independent (Rankin ≤2), 23 (6.5%) had RANKIN 0, 40 (11.4%) had RANKIN 1; 54 (15.4%) had RANKIN 3, 133 (38.2%) had RANKIN 4 and 62 (18.7%) had 5. The median RANKIN was 4 and the interquartile interval was [START_REF] Urimubenshi | Stroke care in Africa: A systematic review of the literature[END_REF][START_REF] Adoukonou | Management of stroke in sub-Saharan Africa: current issues[END_REF][START_REF] Cisse | Minimal Setting Stroke Unit in a Sub-Saharan African Public Hospital[END_REF].

Key results: Hypertension was the greatest risk factor and ischemic stroke accounted for 40.3% of all strokes. Mean patient age was 58.2+/-14.2 years. The in-hospital mortality in this setting was 6.2% and was significantly associated with the high neurological impairment, fever on admission, swallowing disorders and hyperleucocytosis.

Discussion

We aimed to describe stroke characteristics and in-hospital mortality at the University Hospital of Parakou over a 6-year period (2013 to 2019).

Stroke was the leading cause of hospitalization in neurology and accounted for half (51.5%) of all hospitalizations. This finding is consistent with all studies on the increasing prevalence and incidence of stroke worldwide, especially in sub-Saharan Africa [START_REF]The top 10 causes of death [Internet[END_REF]. Nevertheless, the exclusion of 51 patients can induce bias, although in the registry of the death, no patients died during hospitalization. We can postulate that if we take them into account the case fatality would be lower than what we reported. On the other hand, many patients did not present to the hospital and the data we reported here cannot be generalized to all patients in the city. We think that it is very important to build an urban registry of stroke in this city to appreciate the real stroke mortality.

Contrary to European literature [START_REF] Béjot | Epidemiology of stroke in Europe and trends for the 21st century[END_REF], in our study, ICH is more common (ischemic stroke 40.32% versus ICH 30.37%) in SSA. Mapouré et al. reported 52% ischemic stroke and 48% ICH. This is probably linked to the early incidence and severity of arterial hypertension in the black African population, responsible for lipohyalinosis of the small arteries with fibrinoid necrosis, which in turn promotes the formation of micro-aneurysms which occasionally break up under high blood pressure [START_REF] Cane | Hypertension artérielle chez les patients d'ascendance africaine[END_REF]. On other hand, most of the patients with hypertension were unaware of their status before stroke onset, and may be why the patients with ICH had higher blood pressure on admission than did the other patients. The management of high blood pressure often requires longer time frames to obtain optimal values and can explain the length of hospital stay of the patients with ICH. Those patients were prescribed additional antihypertensive drugs on discharge. Hypertension is the most common risk factor (69.1%) irrespective of the type of stroke (68.7% ischemia versus 68.1% hemorrhage), followed by alcoholism and diabetes. This observation is comparable to the results of other African authors. Indeed, these countries have vascular similarities with our observations. The overall mortality was 6.2%. Mortality rates are rather disparate in the various African and Western publications. Mapouré et al. in Douala reported inhospital mortality of 26.8%, Touré et al. reported 24.84% in Senegal in 2010 [START_REF] Mapoure | Predictors of In-Hospital Mortality for Stroke in Douala, Cameroon[END_REF][START_REF] Touré | Predictors of stroke mortality at the department of neurology, Fann university teaching hospital, Dakar-Senegal[END_REF]. Other sub-Saharan African authors have described a mortality of 9.5 and 15.5% in patients followed-up exclusively in a Neurology clinic, as in our study [START_REF] Ndouongo | Epidemiological features and outcome of stroke at Libreville Hospital (Gabon)[END_REF][START_REF] Komolafe | Profile of stroke in Nigerians: a prospective clinical study[END_REF]. Western studies that included both ischemic and hemorrhagic strokes reported in-hospital mortality rates of between 4.8% [START_REF] Baptista | Prediction of in-Hospital mortality after first-ever stroke: the Lausanne Stroke Registry[END_REF] and 16% [START_REF] Langhorne | Medical complications after stroke: a multicenter study[END_REF].

A multicentric Asian study similar to the current study reported 12.5% [START_REF] Wong | Risk factors for early death in acute ischemic stroke and intracerebral hemorrhage: a prospective hospital based study in Asia[END_REF].

These differences could be explained by the quality of the global healthcare system: systematic screening of cardiovascular conditions, the delays in management, and mostly the universal access to healthcare [START_REF] Cane | Hypertension artérielle chez les patients d'ascendance africaine[END_REF].

Accessibility to the tools of exploration for stroke remains a real problem with wide social inequalities and health insurance benefiting mostly the wealthy.

Among the patients: 109 (29.3%) unknown stroke, only 121 (32.5%) had an EKG; 31 (8.3%) benefited from cardiac echography and 34 ultrasound echography of neck vessels (9.1%). This undoubtedly affects the prognosis of the patients who do not receive optimal and specific treatment. In secondary prevention only 23.7% of ischemic stroke were taking statins. In our current practice the use of statins depends on the level of LDL-cholesterol. In fact, only patients with LDL-cholesterol higher than 1 g/l require statins. The low rate of patients on statins can be explained by the lack of biological tests (no insurance).

Swallowing disorders, pneumonia and urinary tract infections were the most common complications in the deceased. Similar results have been reported by Mapouré and Langhorne et al. [START_REF] Mapoure | Predictors of In-Hospital Mortality for Stroke in Douala, Cameroon[END_REF][START_REF] Langhorne | Medical complications after stroke: a multicenter study[END_REF]. Inhalation pneumonia complicated swallowing disorders and is favored by the feeding of patients by their families often without authorization from the nursing staff. Urinary tract infections often complicated prolonged decubitus and co-morbidities such as diabetes. Touré et al. also reported that long hospital stays were a predictor of mortality [START_REF] Touré | Predictors of stroke mortality at the department of neurology, Fann university teaching hospital, Dakar-Senegal[END_REF]. The hyperleucocytosis was also associated to the in-hospital mortality. Patients with pneumonia and urinary tract infections had infection and the usual biological marker of infectious diseases is hyperleucocytosis.

The existence of consciousness disorders was a predictor of mortality in our study. Touré et al., and Mapouré et al. made the same observation [START_REF] Mapoure | Predictors of In-Hospital Mortality for Stroke in Douala, Cameroon[END_REF][START_REF] Touré | Predictors of stroke mortality at the department of neurology, Fann university teaching hospital, Dakar-Senegal[END_REF]. Consciousness disorders not only induce cardio-respiratory distress, but also promote pneumonia. Other poor predictors of mortality were poor neurological status assessed by the NIHSS, fever on admission, and leukocytosis probably stemming from infectious complications which shows the importance of actively seeking out infectious complications, and preventing and treating them early to avoid deleterious development.

Strengths and limitations

The low rate of in-hospital mortality was probably due to availability of a stroke specialist (TA) and the systematic detection of swallowing disorders and other acute stroke complications. However, the low percentage of patients benefiting from CT scan and the low rate of etiological explorations may present limitations to our findings.

Conclusion

The in-hospital mortality of stroke was comparable to other reports in African countries. It could be explained by acute complications such as pneumonia due to swallowing disorders. The main strategy to reduce the inhospital stroke mortality is the detection and the treatment of the acute complications.

Declarations

Conflict of interest: No conflict of interest

Funding: No funding.

Provenance and peer review

Not commissioned, externally peer reviewed.

[ 8 ]

 8 . This study was approved by the Local Ethical Committee of Biomedical Research of the University of Parakou (Number 0300/CLERB-UP/P/SP/R/SA). Confidentiality of data was guaranteed in this study and it has been registered under the unique identifying number researchregistry5687 and is available at https://www.researchregistry.com/browse-the-registry#home/ Design: This was a single center retrospective cohort study. Setting: The study took place in the Neurology Department of the University Hospital of Parakou. Many people in Benin do not have health insurance and do not present at the hospital. The city of Parakou is located 425 km north of Cotonou in Benin. The population is estimated at 255,478 inhabitants. The city has two large hospitals (a military hospital and a University teaching hospital).

Table 1 :

 1 Main characteristics of stroke patients, Parakou, 2020

	All patients	Ischaemic stroke	ICH	Unknown stroke	p
	(N=372)	(N=150)	(N=113)	(N=109)

Table 2 :

 2 Discharge characteristics of stroke patients, Parakou 2020

		All patients	Ischaemic	ICH	Unknown stroke	p
		(N=372)	stroke (N=150)	(N=113)	(N=109)
	Outcome data
	Length of hospital stay (days)	9.0+/-7.3	9.6+/-6.9	11.8+/-8.1	5.4+/-5.4	0.0001
	In-hospital death	23 (6.2)	11 (7.3)	7 (6.2)	5 (4.6)	0.663
	Treatment at hospital Discharge (%)
	Antihypertensive drugs	327 (87.9)	127 (84.7)	109 (96.5)	90 (82.6)	0.074
	Antiplatelet	163 (43.8)	132 (88.0)	1 (0.9)	30 (27.5)	0.0001
	Antidiabetic drugs	36 (9.7)	15 (10.0)	7 (6.2)	15 (13.8)	0.424
	Statins	50 (13.5)	36 (24.0)	9 (7.9)	16 (14.7)	0.015
	Physiotherapy	228 (61.3)	103 (68.7)	85 (75.2)	40 (36.7)	0.0001

Table 3 :

 3 In-hospital mortality of stroke patients, Parakou 2020

	Alive (N=349)	Dead (N=23)	p