Progress in unravelling the cellular and molecular basis of mammalian circadian regulation over the past decade has provided us with new avenues through which we can explore central nervous system disease. Deteriorations in measurable circadian output parameters, such as sleep/wake deficits and dysregulation of circulating hormone levels, are common features of most central nervous system disorders. At the core of the mammalian circadian system is a complex of molecular oscillations within the hypothalamic suprachiasmatic nucleus. These oscillations are modifiable by afferent signals from the environment, and integrated signals are subsequently conveyed to remote central neural circuits where specific output rhythms are regulated. Mutations in circadian genes in mice can disturb both molecular oscillations and measurable output rhythms. Moreover, systematic analysis of these mutants indicates that they can express an array of abnormal behavioural phenotypes that are intermediate signatures of central nervous system disorders. Furthermore, the response of these mutants to psychoactive drugs suggests that clock genes can modify a number of the brain’s critical neurotransmitter systems. This evidence has led to promising investigations into clock gene polymorphisms in psychiatric disease. Preliminary indications favour the systematic investigation of the contribution of circadian genes to central nervous system disease. The correct functioning of the endogenous circadian clock enables organisms to anticipate daily environmental changes and temporally modify behavioural and physiological functions appropriately. All organisms maintain a large number of physiological variables  under control of the circadian clock. There are well-known consequences of disrupted circadian function outside of the brain; metabolism, reproduction, and even longevity can be adversely affected when the means of determining time of day are altered at a molecular level (for review see The suprachiasmatic nucleus (SCN) of the hypothalamus is the centre for the circadian system in the mammalian brain Aside from generating robust rhythms, the circadian system must be sensitive to environmental cues and be able to adjust its phase to synchronise with the prevailing day/night rhythm, a process known as entrainment. In mammals, the signalling of environmental light exposure, carried by dedicated retinal afferents, is the most important entrainment cue of the SCN clock For molecular circadian rhythms to have an impact on physiology and behaviour, they must be converted into functionally relevant outputs. This is achieved by complex spatiotemporal regulation of gene expression that impacts on cell/tissue-specific function. The orchestrated expression of a number of so-called clock-controlled genes (CCGs) then dictates the temporal regulation of clock-output/functional rhythms in brain regions/systems and peripheral tissues. These may take the form of altered hormone release, changes in action potential firing rate, modified rates of cellular metabolism, etc. All of the above indicates that the generation of daily rhythms in behavioural and physiological functions, together with the ability to integrate a range of environmental inputs, relies on a complex circadian system. And evidently this system relies on the action of many genes. Moreover, novel genetic factors that influence circadian parameters continue to be identified in mutagenesis screens Molecular-genetic modifications of the circadian system have a wide range of effects depending on the particular gene affected and also the nature of the mutation . InvestiClock (MGI:1861634), Bmal1 (MGI:2180361), and Cry1 (MGI:1270841)/Cry2 (MGI:2181206) Npas2 (MGI:109232), Dbp (MGI:94866), and Prok2 (MGI:1354178) affect elements of sleep homeostasis Per1 (MGI:2182834)/Per2 (MGI:1934020) double mutants show no effects on sleep homeostatic mechanisms, although robust rhythms of sleep and wakefulness are lost in constant conditions Disturbances in sleep parameters are associated with a spectrum of neurological and psychiatric disorders. Sleep patterns are affected not only by circadian timing mechanisms but also by independent homeostatic mechanisms that determine the amount of sleep required Npas2 can deputise for Clock in maintaining molecular rhythms in the SCN and is in fact the normal binding partner for Bmal1 in forebrain clocks Npas2 show deficits in the acquisition of cued and contextual fear-conditioning paradigms Npas2 null mice is secondary to major, centralised circadian disruption, as these mice show only a subtle activity phenotype. A pleiotropic, non-clock effect of Npas2 in memory formation is one possible explanation. Alternatively, the loss of Npas2 may abrogate particular extra-SCN brain clock(s) that regulate memory formation. Evidence for the participation of other clock genes in memory regulation is sparse. Although no specific memory deficits have been found in Per1/Per2 mutant mice Drosophila Per mutants (FBgn0003068) are defective in long-term memory formation timeless (FBgn0014396), clock (FBgn0023076), and cycle (FBgn0023094) mutants, indicating that an association between clock genes and memory regulation is not universal and a degree of functional segregation exists within the system.The process of memory formation in clock mutant mice has received limited attention. Clock. Homozygous mutants have a spectrum of behavioural abnormalities including low anxiety, mania, and hyperactivity Clock mutation on core molecular circadian oscillations or are secondary to an alteration in the dynamics of communication between CNS clocks dysregulated by the altered function of the core oscillator. In support of the second possibility, behavioural disturbances in homozygotes are associated with increased dopaminergic activity There is increasing evidence that a dysfunctional circadian system could be a primary cause of altered emotional behaviour. The majority of this evidence in mice comes from studies on the ENU-induced mutation Clock mutant mice Drosophila homologue, shaggy (FBgn0003371), can lengthen the circadian period Bmal1 (MGI:1096381) The observations that the mood-stabilising agent, lithium, can reverse behavioural disturbances in Drosophila mutants including period, clock, cycle, and doubletime (FBgn0002413) Per1 mutants show no behavioural sensitisation to cocaine, and, in a conditioned place preference task, show no preference to cocaine Per2 mutants have a hypersensitised response to cocaine, and the place preference response is strong Clock also show a hypersensitive response to cocaine and increased place preference Eaat1 (MGI:99917) is reduced in mouse Per2 mutants, leading to decreased uptake of glutamate by astrocytes and increased extracellular glutamate levels Per2 mutant mice were reversed by agents believed to act through reducing abnormally high glutamatergic activity. Overall, the interactions between circadian clocks and addiction mechanisms seem to be complex. The possibility of pleiotropy in clock genes cannot be discounted, although these effects could be dissected using conditional mutants. Nevertheless, there is an increasing body of work detailing the interaction between the circadian system and brain neurotransmitter systems. As described above, clock gene expression can be modulated by a number of afferent neurotransmitter effects, and, conversely, the functions of efferent neurotransmitter systems/brain nuclei are modifiable by altered clock gene expression Additional evidence for behavioural disturbances in clock gene mutants is supported by the fact that they appear to affect the sensitisation to, and preference for, drugs of abuse. This was first evidenced in a number of Undoubtedly, circadian parameters are consistently disrupted in a spectrum of CNS disorders . In manyClock mutant mice In neurodegenerative disease and aging , disturbMagel2), an SCN-enriched transcript within the PWS deletion region, have a reduced circadian activity amplitude with increased daytime activity relative to controls (MGI:3760092) Circadian disturbances are more likely to contribute to the aetiology of a number of syndromic disorders . These dASMT, OMIM *300015), is implicated as a susceptibility gene for ASD PTEN) have been reported in autistic individuals with macrocephaly. In mice carrying a conditional deletion of Pten (MGI:2182005), abnormal phenotypes include macrocephaly, increased susceptibility to seizures, social interaction deficits, anxiety, and a significantly longer free-running period Other behavioural disorders with circadian and sleep-related disturbances include autism spectrum disorders (ASD) (OMIM %209850) The contribution of the circadian regulatory system, arising from conflicts between internal biological clocks and environmental (solar) and social clocks, is evident in affective disorders. All major affective disorders  include circadian phase disturbances in sleep, activity, temperature, and hormone levels (for reviews see PER3 (OMIM *603427) gene is associated with extreme evening preference and DSPS PER3 encoding a putative phosphorylation domain. The significance of these observations has been highlighted by the identification of two distinct mutations in familial ASPS pedigrees. The first PER2 (OMIM *603426), is within the binding domain for casein kinase I epsilon  and results in hypophosphorylated PER2 in vitro. Transgenic mice expressing this mutation had an advanced phase of activity in a light–dark schedule and a short free-running period of activity CKIδ (OMIM *600864) itself The contribution of circadian gene dysregulation to CNS disorders has only recently been explored through the analysis of clock gene polymorphisms. Circadian gene polymorphisms may simply underlie a morning or evening preference in human populations, but extremes in these behaviours may also underlie the more complex phenotypes associated with psychiatric disorders. A quantitative scoring system for diurnal preference, the Horne-Ostberg questionnaire PER2, BMAL1 (OMIM *602550), and NPAS2 (OMIM *603347) with additive effects from combined risk genotypes. An earlier study had also identified significant associations between diurnal preference and SAD with a significant effect of NPAS2 471 Leu on disease susceptibility Conflicts between internal biological clocks and environmental (solar) and social clocks should be evident in individuals with extreme diurnal preferences, and this could have the potential to disrupt homeostatic processes and behaviours affected by them. In fact, Xu et al. PER1 (OMIM *602260), PER3, NPAS2, BMAL1, CLOCK (OMIM *601851), and TIM (OMIM *603887) all showed specific associations with a number of disorders. The promise of these pilot studies should encourage more systematic studies in investigating the contribution of clock gene variants to the onset and severity of CNS disorders.Investigations into clock gene associations with other affective disorders are in their infancy. Although limited, there are a number of indications warranting further investigation into associations with bipolar disorder, schizoaffective disorder, schizophrenia, and autism Biological rhythms are undoubtedly disrupted in a spectrum of CNS disorders. Establishing the contribution of clock genes to cause and effect in these disorders has proven difficult. Nevertheless, these investigations continue to be helped through the systematic characterisation of behavioural phenotypes in mouse circadian mutants, the identification of new genetic factors that contribute to circadian and behavioural function, and the investigations of clock gene polymorphisms in all CNS disease areas. Continued investigation into these three areas should lead to new insights into the causes and progression of neurological and psychiatric disorders.


While optimal pharmacotherapy would directly target the neuropathology of schizophrenia neither the underlying neurobiological substrates of the behavioral symptoms nor the cognitive deficits have been clearly established. Abnormalities in the neurotransmitters dopamine, serotonin, glutamate, and GABA are commonly implicated in schizophrenia; however, it is not uncommon for alterations in the brain cholinergic system  to also be reported. Further, there is now considerable evidence in the animal literature to suggest that both first and second generation antipsychotics  can alter the levels of several cholinergic markers in the brain as well as impair memory-related task performance. Given the well-established importance of central cholinergic neurons to information processing and cognition, it is important that cholinergic function in schizophrenia be further elucidated and that the mechanisms of the chronic effects of antipsychotic drugs on this important neurotransmitter system be identified. A better understanding of these mechanisms would be expected to facilitate optimal treatment strategies for schizophrenia as well as the identification of novel therapeutic targets. In this review, the following topics are discussed: 1) the central cholinergic system in schizophrenia 2) effects of antipsychotic drugs on central cholinergic neurons 3) important neurotrophins in schizophrenia, especially those that support central cholinergic neurons; 4) novel strategies to optimize the therapeutics of schizophrenia  Schizophrenia is a chronic neuropsychiatric illness that affects approximately 1% of the world’s population. Core features of the disease reviewed, include 2) receptor subtype in the CNS, whereas the mechanism of newer, second generation compounds is commonly attributed to a weaker affinity at D2 receptors combined with antagonist activity at other sites, such as the 5HT2A serotonin receptor [reviewed,An important limitation to strategies for novel drug development is the relatively poor understanding of the neuropathology of schizophrenia. In postmortem brains of schizophrenic patients, evidence of reduced neuronal size, neuropil loss (particularly synaptic elements), decreased cortical,volume, and abnormal synaptic organization is substantial . In addireviewed,. It shouNotwithstanding the neurotransmitter anomalies described above, it is also not widely appreciated that alterations in markers for the neurotransmitter, acetylcholine have been commonly reported in schizophrenia , 91. SucIt has been known for many years that anticholinergic compounds when administered in high doses can induce psychosis. For example, muscarinic antagonists such as scopolamine or atropine can evoke a psychotic state termed “anticholinergic syndrome” or “antimuscarinic psychosis” which shares many features with endogenous schizophrenia , 105. Otvia magnetic resonance spectroscopy in thalamic, anterior cingulate, and caudate nucleus regions of antipsychotic-naïve patients [For more than 30 years alterations in the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT), in schizophrenia have been reported see ref . Examplepatients , 95, pos1 and M2) in schizophrenia. These mAChR subtypes are known to play important roles in several signaling pathways that modulate neuronal excitability, synaptic plasticity, and learning and memory [2/M4 receptor binding in the orbital frontal cortex and putamen [1/M4 receptor binding in the striatum, hippocampal formation, prefrontal cortex, and anterior cingulate cortex [2/M4 receptors as well as cholinergic interneurons in the striatum of schizophrenic brains have also been reported [There is also considerable evidence of muscarinic acetylcholine receptor (mAChR) alterations  and low-affinity (homomeric α7) nAChRs are ligand  gated ion channels that have been implicated in a variety of learning and memory processes [4β2 nAChR subtype of high-affinity nAChR predominates in density and distribution compared with other subtypes [4β2 nAChR binding in several brain regions including the striatum, hippocampus, and cortex compared with controls [7 nAChR deficits in schizophrenia. α7 nAChRs comprise approximately 10% of the total number of nAChRs in mammalian brain, but modulate a variety of calcium-dependent events in neurons including neurotransmitter release [7 nAChR alterations in schizophrenia includes postmortem deficits in the hippocampus and frontal cortex [7 neuronal nAChR gene). Polymorphisms in the core promoter of the α7 neuronal nAChR gene  are associated altered P50 evoked responses to repeated auditory stimuli  in schizophrenia [reviewed,7 nAChR deficits may also contribute to other abnormalities in schizophrenia including deficits in smooth pursuit eye movements, sustained attention, and cognition [reviewed,7 nAChR expression could potentially serve as a biomarker for schizophrenia [There is also credible evidence to support the argument that both high and low affinity nicotinic acetylcholine receptors (nAChRs) are diminished in schizophrenia. High-affinity  and subchronic (21 days) exposure to haloperidol decreased the levels of BDNF in the prefrontal cortex, hippocampus, and amygdala. Decreased BDNF concentrations  and decrTaking into consideration the information provided above, an interesting question arises as to what extent antipsychotic drugs affect the central cholinergic system. The conventional antipsychotics, thioridazine and chlorpromazine, and the atypical agents, clozapine and olanzapine bind all of the known muscarinic acetylcholine receptor subtypes with relatively high affinity reviewed,, althoug7) and muscarininc (M2) acetylcholine receptors [2 receptors on cholinergic neurons (leading to cholinergic dysregulation) and, adverse antipsychotic effects on neurotrophins that support cholinergic neurons such as nerve growth factor and brain derived growth factor  cholinergic responses in the striatum of animals. This effect has been implicated as a potential mechanism of the adverse motor effects of conventional antipsychotic in humans. Specifically, an increase in the activity of cholinergic interneurons in the striatum induced by conventional antipsychotics initially appears to correlate with the extrapyamidal side effects in humans, whereas more prolonged periods of exposure correlate with decreases in cholinergic activity below baseline . 1/M4 preferring receptor agonist, xanomeline have been reported to improve behavioral symptoms in Alzheimer's disease patients [mAChR-mutant mice indicate that M1 and/or M4 receptors (in particular) might serve as important therapeutic targets for schizophrenia [reviewed,1/M4 preferring muscarinic agonist, xanomeline  demonstrates an antipsychotic-like profile in the rat [4 mAChR  and the M1 mAChR (TBPB) have been developed and shown to have robust effects in several animal models commonly used to predict antipsychotic activity [reviewed,Reports of cholinergic deficits in schizophrenia (described above), have given rise to cholinergic strategies for improving both the psychotic symptoms and cognitive deficits in schizophrenia. More than 50 years ago Pfeiffer and Jenney  reportedpatients . This inpatients , 19. Amoreviewed,. Accordi the rat  and it h the rat ). Most rreviewed,, 82. Morreviewed,. Most rereviewed,. The treAs we have reviewed previously  there ma7 nAChRs in association with haloperidol and risperidone). Such findings may indicate that a more patient specific  therapeutic approach (using specific types of cholinergic agents) that takes into account the unique medication history (and current antipsychotic therapy) of the patient may be necessary to improve the symptoms of schizophreniaAn important factor that could certainly influence responses to the adjunctive agent is the unique medication history of the study subject. Specifically, factors such as the history of antipsychotic drug exposure, the particular antipsychotic agent currently being administered, and how long the patient has been exposed to a particular antipsychotic, could be especially important to the adjunctive treatment response. In our chronic animal studies , 92, we The therapeutics of schizophrenia are particularly challenging given the heterogeneity of disease symptoms and our relatively poor understanding of the neuropathology. In order to effectively address the core features of the illness , novel strategies need to be designed that take in to account a number of import patient-specific factors. These factors include the specific symptom profile, the unique medication history of the particular patient, and concurrent exposure to other drugs . Reports of alterations in the brain cholinergic system in schizophrenic patients combined with animal data suggesting that chronic exposure to antipsychotics can lead to cholinergic alterations, provides the impetus for investigating both primary and adjunctive cholinergic strategies to the pharmacotherapy of schizophrenia.


Vestibulo-ocular reflex (VOR) gain adaptation, a longstanding experimental model of cerebellar learning, utilizes sites of plasticity in both cerebellar cortex and brainstem. However, the mechanisms by which the activity of cortical Purkinje cells may guide synaptic plasticity in brainstem vestibular neurons are unclear. Theoretical analyses indicate that vestibular plasticity should depend upon the correlation between Purkinje cell and vestibular afferent inputs, so that, in gain-down learning for example, increased cortical activity should induce long-term depression (LTD) at vestibular synapses.in vitro. Control EPSC recordings were followed by an induction protocol where membrane hyperpolarizing pulses, mimicking IPSPs evoked by flocculus inputs, were paired with single vestibular nerve stimuli. A robust LTD developed at vestibular synapses when the afferent EPSPs coincided with membrane hyperpolarisation, while EPSPs occurring before or after the simulated IPSPs induced no lasting change. Furthermore, the iSTDP rule also successfully predicted the effects of a complex protocol using EPSP trains designed to mimic classical conditioning.Here we expressed this correlational learning rule in its simplest form, as an anti-Hebbian, heterosynaptic spike-timing dependent plasticity interaction between excitatory (vestibular) and inhibitory (floccular) inputs converging on medial vestibular nucleus (MVN) neurons . To test this rule, we stimulated vestibular afferents to evoke EPSCs in rat MVN neurons in vivo.These results, in strong support of theoretical predictions, suggest that the cerebellum alters the strength of vestibular synapses on MVN neurons through hetero-synaptic, anti-Hebbian iSTDP. Since the iSTDP rule does not depend on post-synaptic firing, it suggests a possible mechanism for VOR adaptation without compromising gaze-holding and VOR performance  Adaptation of the vestibulo-ocular reflex (VOR) has been extensively used to test theories of cerebellar function A possible answer to this question has been suggested by a recent computational analysis of VOR adaptation This theoretical analysis provides a rationale for an additional brainstem site of plasticity, and also suggests a learning rule by which the flocculus may regulate the strength of vestibular afferent inputs to brainstem neurons, to alter the gain of the VOR . A positin vitro. We also tested the rule with the more complex induction protocol developed by Pugh and Raman Although there is evidence for plasticity in the intrinsic excitability of vestibular neurons in vivo.Our results show that repeated coincidence of EPSCs and membrane hyperpolarisation within a narrow temporal window causes robust LTD of the afferent synapses, strongly supporting the theoretical predictions. The effects of the more complex induction protocol were also well predicted by the iSTDP rule. In contrast to proposed mechanisms for plasticity in the DCN that require the silencing of the postsynaptic cell followed by rebound depolarization, it is possible that in the vestibular nuclei vestibular synapse strength may be modulated by iSTDP interactions, independently of postsynaptic firing. Since MVN neurons are directly involved in VOR execution, this would provide a possible mechanism for VOR adaptation without compromising gaze-holding and VOR performance decrease in vestibular synaptic weight. Similarly for gain-up adaptation a negative correlation should lead to a long-term increase in vestibular synaptic weight  in its simplest form as an anti-Hebbian, hetero-synaptic, input-spike-timing dependent plasticity  that the learning rate The correlational learning rule (2) given in terms of firing rates can be extended to apply to general spiking inputs by taking the sum over all spike pairs of the individual weight changesin vivo learning in MVN neurons with stochastic inputs, to simulate the iSTDP rule superimposed on neuronal spike firing rates similar to those seen in vivo. In both cases simulations were implemented in MATLAB™ using the Fast Fourier Transform to implement the convolution in equation (6)  efficiently using the convolution theorem The iSTDP rule can therefore be used to predict the effects of any arbitrary combination of excitatory and inhibitory input spikes. We exploited this fact to investigate two particular input sequences: (i) the pause-rebound conditioning protocol developed by Pugh and Raman all frequencies of head movement, whereas both experimental evidence and theoretical analysis indicate that learning rates in VOR gain adaptation are very markedly affected by head-movement frequency. Indeed it has been argued that only because of these frequency effects is brainstem plasticity required at all The general form of the learning rule in equation (1) operates equally effectively over bandpassed correlational learning rule, which operates only at frequencies between a lower limit  and an upper limit  The underlying problem is that, while VOR performance is accurate at frequencies up to at least 20 Hz To obtain different learning rates at different frequencies, the learning rule in equation (1) can be replaced by a more general correlational learning rule of the formApplying this generalized rule to spike trains lues see , Fig. 2.Since the bandpass constraints do not fix the shape of the profile uniquely, we have used a difference of Gaussians (DoG) filter as an example of a time-symmetric filter . Slices were cut using a Vibratome 3000 , transferred to aCSF (composition (mM): NaCl, 140; KCl, 2.5; HEPES, 10; glucose, 11; NaH2PO4, 1.2; MgCl2, 1.3; CaCl2, 2.4, equilibrated with 100% oxygen, pH 7.3) for 1 hour at 36°C, and then maintained at room temperature for at least a further hour before transfer to the recording chamber.Experiments were performed on 250–300 µm coronal slices of the brainstem containing the rostral part of the MVN and the central stump of the VIIIth nerve from Lister Hooded rats aged P18–38 , except where the aim was to examine the effect of age when slices from animals aged P13–17  were used. All procedures were approved by the Ethical Review Panel, University of Edinburgh, and were carried out in compliance with the UK Animals (Scientific Procedures) Act 1986 (project licence 6003334). Animals of either sex were decapitated under isofluorane anaesthesia, and the brains quickly removed into ice-cold modified aCSF (composition (mM): NaCl, 87; KCl, 1.2; HEPES, 10; glucose, 25; sucrose, 75; KH2, 2; K2ATP, 5).Slices were maintained in bath solution  for at least 20 min before recording. A bipolar stimulating electrode was placed at the lateral border of the MVN, in the region of the root of the VIIIth nerve Data were recorded using an Axopatch 200B amplifier, sampled at 20 kHz and filtered at 5 kHz (in current clamp) or 2 kHz (voltage clamp). All neurons included here had input impedances >100 MOhm and spike heights of >+40 mV, and showed an early fast post-spike after-hyperpolarsation (AHP) followed by a delayed slow AHP  protocol a 20 msec hyperpolarizing current injection, mimicking the time-course of an inhibitory post-synaptic potential (IPSP) in the MVN neuron, was repeatedly paired with a single vestibular nerve stimulus which was applied at various times relative to the membrane hyperpolarisation -2-amino-5-phosphonopentanoic acid (D-APV) and kynurenic acid .Data are presented as mean ± S.E.M. EPSC amplitudes (pA) were binned into 1 min periods, and normalised to the average EPSC amplitude in the pre-induction period. To determine the changes in EPSC amplitude after exposure to an induction protocol, a mean of 5 normalised bins recorded immediately prior to induction and a mean recorded 25 min post-induction were compared using a two-tailed paired t-test. To compare the effects of different induction protocols, a mean of 5 post-induction bins from each protocol were compared using a two-tailed unpaired t-test. The series of iST induction protocols was analysed by one-way ANOVA with Dunnett's multiple comparison post test with the Ts = −150 induction protocol designated as the control. P<0.05 was considered significant.The term STDP does not by itself specify whether the relevant timing is between two sets of input spikes to a neuron, or between input spikes and postsynaptic action potentials. However, in practice it is so closely associated with the latter alternative that the term iSTDP (input-Spike Timing Dependent Plasticity) is used here for a proposed learning rule in which synaptic weight change depends on the relative timing of excitatory and inhibitory input spikes (see METHODS). The term “input timing dependent plasticity” (ITDP) has been used by Dudman et al in vitro results requires the rule to be in input-spike-time dependent plasticity form. The derivation of this iSTDP form for the linear case is described in Methods, and the outcome illustrated in Theoretical analyses of VOR gain-adaptation have suggested a correlational learning rule for plasticity in the vestibular nuclei . HoweverThe simplest version of the iSTDP rule is that if vestibular and cerebellar spikes arrive at an MVN neuron at the same time, substantial LTD is induced at the vestibular synapse. When the spikes arrive at different times, much weaker LTP is induced. The green traces in However, it is known that VOR adaptation learning is frequency dependent. At high frequencies learning-rate decreases above ∼2 Hz, reaching low levels at 10 Hz The blue curve in To test the above correlational rule experimentally, we explored the interaction between membrane hyperpolarisation and vestibular afferent EPSCs using the iST induction protocol consisting of a 20 msec hyperpolarizing current injection, mimicking the time-course of an inhibitory post-synaptic potential (IPSP) in the MVN neuron, paired with a single vestibular nerve stimulus that was applied at various times relative to the membrane hyperpolarisation ; Methodss = 0 msec, 1000 presentations at 0.5 sec intervals, s = 20 msec, Repeated pairing of the vestibular nerve stimulus with the onset of the hyperpolarizing current pulse (Ts = 40 or 60 msec), or in advance of the hyperpolarizing pulse (Ts = −150 msec) induced only short-term depression of EPSC amplitude, similar to that seen with Ts = 0 msec  and then, after a observation period of 15 minutes post-induction, the vestibular nerve stimulus was applied at Ts = 20 msec . The comparison shown in end of the 20ms membrane hyperpolarising pulse. This is presumably due to the biological constraints imposed by the underlying mechanisms, so that for example changes in membrane potential do not occur instantaneously following an inhibitory or excitatory input at The theoretical iSTDP profiles show maximum LTD for an ISI The LTD obtained here with the iST protocol differs from the LTP found in experimental studies of DCN plasticity, albeit using a more complex protocol In young adult MVN neurons  tested with the PR protocol (30 presentations every 5 sec), the amplitude of the vestibular nerve-evoked EPSC was significantly reduced compared to control and remained depressed for the duration of the recordings . VestibuOne possible explanation for the finding that the PR protocol induces depression of the vestibular synapses in MVN neurons, in contrast to the potentiation of mossy fibre synapses that occurs in DCN neurons s = 20 msec) induction protocol also occurred in juvenile MVN neurons. In contrast to young adult cells, in juvenile cells this protocol induced a small potentiation of the vestibular EPSC amplitude, which did not reach significance . A significant feature of this result is the dependence of the iSTDP learning rule upon the local membrane potential of the post-synaptic cell at the time of arrival of the EPSCs, not upon action potential firing of the post-synaptic cell. However, since the post-synaptic cell depolarization in the modified PR protocols is accompanied by spiking, this raises the possibility that post-synaptic spikes may also affect plasticity at the vestibular synapse. The PR protocols are ambiguous in this regard, since they necessarily confound post-synaptic depolarization with post-synaptic spiking. Resolving this confound experimentally, to determine whether post-synaptic spike firing may also have a role in regulating vestibular synapse strength, is an important issue that is difficult to address directly. This is particularly the case if the iSTDP interactions between excitatory and inhibitory inputs take place in the distal dendrites of MVN cells, some distance removed from the soma . In the in vitro may be close to their maximum values, which would create a nonlinearity and mean that in vitro LTP could only be elicited after prior LTD. This possibility is shown in simulation in in vitro experimental conditions is actually predicted by the iSTDP learning rule because of the absence of cerebellar inhibitory synaptic inputs to the MVN neurons in the slice preparation. Thus while in vivo the ongoing simple-spike firing of Purkinje cells would provide a continuing inhibitory synaptic input to the MVN neurons, inducing a steady level of LTD in the vestibular synapses, the lack of this inhibitory input in vitro may allow the vestibular synapse strength to drift towards the maximal value.Our iSTDP model features both LTP and LTD, and its success in predicting the effects of the complex PR protocols in MVN neurons depend upon both. However in these experiments the LTP for these protocols is not observed directly, but is ‘implicit’ in the model. Nevertheless the linear model predicts explicit LTP for protocol PR-3 , raisingin vivoSince the nature of the plasticity induced by the iSTDP rule depends upon accurate spike timing, it is important to show that it is equivalent to a rate-coded learning rule for the stochastic, asynchronous spiking inputs received by MVN neurons However The fact that these results illustrate how the model can work well in principle points to the necessity for further experimental work, to establish whether the implicit LTP or LTD reduction observed here is in fact driven by membrane depolarization as predicted by the model, and to clarify the role of post-synaptic spiking. The assumption of linearity used here, which means that the iSTDP profile against a null background also applies at other firing rates, is widely made in spike-timing dependent plasticity models versus asymmetrical, see Methods).The robustness of the modelling results shown in Secondly the equivalence of the spiking and firing rate formulations of the learning rule was checked. The frequency and depth of input modulation, and baseline tonic firing rate of stochastic firing rate coded signals were varied . The effThirdly, the effects of unbalanced iSTDP profiles were examined. An unbalanced profile is one in which the area under the LTD part of the profile is not equal to that under the LTP part. In the simplest case, where the inputs to the vestibular nucleus are unaffected by changes to its output, the main effect of an imbalance is that tonic firing rates produce a constant rate of weight change, eventually driving the synaptic weight to its upper or lower limit .In the present experiments, membrane hyperpolarizing currents were injected via the recording electrode on the MVN cell soma in order to induce LTD at the vestibular synapses. In reality, Purkinje synaptic terminals may primarily influence vestibular afferent synapses in their immediate vicinity. Convergence of vestibular afferents and Purkinje terminals onto individual dendritic compartments The iSTDP profiles for vestibular synaptic plasticity resemble the anti-Hebbian STDP profiles A further advantage of the iSTDP learning rule is that it successfully predicts the effects of more complex induction protocols on vestibular synaptic plasticity , 6 and 7in vitro were close to their maximum values . In the rodent MVN, only a proportion (∼5–20%) of neurons are FTNs in vitro, vestibular synapses may drift to be close to their maximal strength in slices  indicates activation of both NMDA receptors and low-voltage activated Ca2+ channels, so that Ca2+ influx from both sources is likely to be necessary for LTD. NMDA currents are active around the resting potential in MVN cells, and contribute to their resting discharge and the vestibular nerve EPSC in vitro, has been reported by Grassi et al 2+ block and allow significant inward Ca2+ current even at relatively hyperpolarized membrane potentials Input-spike-timing dependent LTD at the vestibular synapse is prevented by the NMDA receptor antagonist D-APV, indicating that it requires NMDA receptor activation and Cain vivo.In conclusion, these results suggest the cerebellum alters the strength of vestibular synapses on MVN neurons through hetero-synaptic, anti-Hebbian iSTDP. The iSTDP rule predicts the LTD of vestibular synapses when excitatory and inhibitory inputs interact within a precise temporal window, and also predicts the effects of more complex trains of excitatory and inhibitory inputs. Since the iSTDP rule does not depend on post-synaptic firing, it suggests a possible mechanism for VOR adaptation without compromising gaze-holding and VOR performance


The development of the mammary gland is spatially regulated by the interaction of the mammary epithelium with the extracellular matrix (ECM). Cells receive cues from the ECM through a family of adhesion receptors called integrins, consisting of α- and β-chain dimers. Integrins assist cells in sensing their appropriate developmental context in response to both hormones and growth factors. Here we argue that cell adhesion to the ECM plays a key role in specific developmental checkpoints, particularly in alveolar survival, morphogenesis and function. Specific ablation of αβ1-integrins in the luminal epithelium of the mammary gland shows that this sub-type of receptors is required for proliferation, accurate morphological organisation, as well as milk secretion. Downstream, small Rho GTPases mediate cellular polarisation and differentiation. Current challenges in studying the integration of signals in checkpoints of mammary gland development are discussed. The spaIn the juvenile mammal, the mammary gland consists of primitive ducts, with limited side branching. Upon stimulus by ovarian hormones during puberty, these ducts elongate and branch further into the fat pad surrounding the gland. With pregnancy, the ends of the branches in the ductal system become mature acinar structures, also known as terminal ductal-lobular units (TDLUs) or alveoli . StromalThe BM is established by stromal–epithelial interactions leading to secretion of specific ECM components, and assembly of the 100 nm thick matrix at the basal surface of the epithelium . MECs adThis review will concentrate on the known effects of ECM on MEC behaviour, particularly focussing on the BM, and the specific integrin signalling events underlying them. We will discuss how integrin-mediated adhesion serves as a checkpoint for different aspects of MEC function through integration with other signals received during mammary gland development.2The ECM consists of multiple components, depending on the stroma and epithelium within the specific tissue. The mammary BM contains collagen IV (Col IV) and Laminin I , as welWhile some of the studies to dissect ECM function have used purified ECM components, others rely on BM isolated from the Engelbreth–Holm–Swarm tumour , which is thought to mimic effectively the mammary BM . There aIntegrins bind ECM proteins when in an extended active conformation, and simultaneously promote the formation of multi-protein adhesion complexes at the plasma membrane . AdhesioILK binds to β-chains of various integrin dimers and its activation leads to many important events downstream of integrin signalling . ILK is Another kinase interacting directly with the integrin β-chains is Syk . This kiThe interaction of cells with ECM leads to the formation of focal adhesions, an event that involves FAK. This enzyme is linked to activated integrins through the proteins talin and paxillin . In MECsA pressing question is to decipher the molecular details of how integrins control fate decisions of mammary epithelium, and elucidating the role of specific adhesion complex components will be implicit in this analysis.3Perhaps the most general role ascribed to integrin-mediated adhesion is cell survival. In virtually all adherent cells, including MECs, cell detachment from the ECM leads to a type of programmed cell death (or apoptosis) called anoikis .c is released to the cytoplasm. Cytochrome c, together with Apaf-1, activates the cysteine protease family of caspases, which cleaves a wide range of protein targets ultimately resulting in cell death  may contribute directly to involution. A complementary view suggests that involution occurs as a result of changes in the composition of the mammary BM , we view cell–BM interactions as a positive checkpoint to suppress apoptosis, ensuring that mammary epithelial cells are normally only maintained within ducts and alveoli. This is one of the mechanisms guaranteeing that development occurs normally, leading to the formation of intricate epithelial structures that underlie the function of the tissue. A corollary is that if this apoptosis checkpoint becomes activated autonomously, MEC may acquire the potential to survive within inappropriate ECM locations, such as within stroma or lymph nodes; this is a prerequisite for the more subversive aspects of breast cancer, when tumours become malignant.In summary, adhesion to the ECM is an absolute requirement for cell survival. However, since survival is ECM-specific  and neuregulin (ErbB4) are required for proper alveolar development . Using aIGF-I is locally induced within the mammary stroma by systemic growth hormone and is capable of mimicking the effects of insulin in supporting, together with the ECM, survival of MECs . AdhesioTGFβ has a well-documented role in inhibition of MEC proliferation and modulating epithelial plasticity . EctopicHGF and its receptor c-Met are involved in branching of mammary ducts in a PI3K-dependent manner . HGF indGrowth factor signalling determines whether MEC proliferate, undergo apoptosis, or form branched structures. However, only cell interactions with the appropriate ECM context, mediated by integrins, allow the proper developmental decision. Thus, although growth factors have a wide variety of effects on cells, ECM adhesion permits a much more specific set of outcomes than might have been predicted from studies using 2D culture on plastic alone.These collective studies therefore indicate that adhesion provides a key checkpoint to determine the proliferative fate of MEC. Subversion of this checkpoint has disastrous consequences, sometimes leading to cancer. The expression of an integrin dimer, the α6β4 laminin receptor, is altered in breast cancer, contributing to poor patient prognosis . Integri6Acinar structures in vivo  are made of a dual layer of cells: an internal layer of luminal epithelial cells encompassed in a mesh-like layer of myoepithelial cells . Both prThe cellular events that are particular to MECs forming alveoli are only partially understood. In other epithelial systems, cellular polarity and lumen formation on purified BM are closely linked . PolaritSystemic glucocorticoids also contribute to acinar architecture C. WithouPolarised acinar structures are key components of the mammary gland, allowing it to deliver milk to infants. We suggest that integrin signalling through small GTPases provide an essential polarisation check point. Without these integrators of adhesion function, actin polymerisaton is absent and acinar structures do not form properly. The JNK pathway is also required for this process, thus only coordinated signals from the ECM and glucocorticoids result in lumen formation.7Prolactin drives both alveolar proliferation and differentiation during pregnancy, and the subsequent secretion of milk into the alveolar lumen . However−/− MECs had reduced Rac activity, and constitutively active Rac1, was sufficient to rescue the β1−/− MEC phenotype and differentiation  Which of the precise integrin dimers mediate each of the functions discussed above: survival, proliferation, morphogenesis and differentiation? (2) How do the integrin-mediated signals result in proper terminal differentiation and milk secretion, and in the process of involution that follows it? (3) Which precise components of the integrin-containing adhesion complex are involved with the different checkpoints? (4) What is the role of integrins on maintaining the stem cell niche? (5) How does local ECM remodelling by membrane-bound proteases affect integrin signalling and thereby cell fate? (6) What is the influence of integrins on other cell types within the mammary gland, including stromal cells and myoepithelial cells? (7) To what extent to the less-well studied ECM ligands and receptors influence integrin signalling?To achieve this level of understanding more research into the molecular mechanisms of signal integration is required. For example, anecdotal evidence shows that FAK and ILK are probably involved as scaffold proteins in integrin signaling as much as they are as kinases  and may The issue of signalling events underlying acinar morphogesis is particularly intriguing. Evidence regarding the involvement of Rho GTPases begins to shed light on the processes that may be involved . NonetheCre-Lox gene deletion and the use of shRNA, have now become tractable for dissecting these questions both in culture and in vivo, better models will be of key importance. Particular challenges will be the development of 3D primary cell culture models for studying in vivo-like branching morphogenesis, the stem cell niche, alveolar formation from ducts, and luminal–myoepithelial–stromal cell interactions, as well as animal models for live cell imaging and genetic targeting of both stem cells and stromal cells.Although genetic systems, such as In this review we have attempted to demonstrate the interdependency of tissue architecture and cell fate decisions in mammary gland development and function. Continuing efforts in the area of signalling integration will undoubtedly be of outmost importance.


More than half a century after the discovery of the molecular basis of Sickle Cell Disease (SCD), the causes of the phenotypic heterogeneity of the disease remain unclear. This heterogeneity manifests with different clinical outcomes such as stroke, vaso-occlusive episodes, acute chest syndrome, avascular necrosis, leg ulcers, priapism and retinopathy. These outcomes cannot be explained by the single mutation in the beta-globin gene alone but may be attributed to genetic modifiers and environmental effects. Recent advances in the post human genome sequence era have opened the door for the identification of novel genetic modifiers in SCD. Studies are showing that phenotypes of SCD seem to be modulated by polymorphisms in genes that are involved in inflammation, cell–cell interaction and modulators of oxidant injury and nitric oxide biology. The discovery of genes implicated in different phenotypes will help understanding of the physiopathology of the disease and aid in establishing targeted cures. However, caution is needed in asserting that genetic modifiers are the cause of all SCD phenotypes, because there are other factors such as genetic background of the population, environmental components, socio-economics and psychology that can play significant roles in the clinical heterogeneity. Hemoglobinopathies are the world’s most widespread genetic diseases.Experiments showed that decreased NO bioavailability, due to scavenging of NO by cell free hemoglobin, a product of the hemolytic process associated with SCD, played a significant role in the vascular patho-biology of SCD.The elucidation of genetic variants and mechanisms responsible for the phenotypic variability in SCD will have important clinical implications for genetic counseling and clinical management. Targeted follow up of patients and utilization of individual therapeutic strategies and preventive cures will produce better outcomes for patients with SCD.In this review, we summarize some of the recent genomic findings focused on non globin genetic modifiers, shown to be potentially implicated in different clinical outcomes of SCD.The main Hb function is to transport oxygen to tissues. Adult Hb is composed of a major HbA alpha2beta2) and a minor HbA2 , with traces of fetal HbF . Each of the different globin chains is controlled by distinct genes; two genes exist for the alpha and gamma chains and one for each of the other chains.beta2 andSince the clinical phenotypes of homozygous HbS are extremely variable, it is now clear that even if SCA is a monogenic disorder, at the phenotypic level it is most likely to be a multigenic disease. Among risk factors for early mortality are stroke, painful crisis and infection.Plasmodium falciparum malaria,Genetics is a major determinant of childhood survival in malaria endemic countries. The past few years have seen significant progress towards showing some of the best-known malaria resistance genes that determine the structure or function of RBCs, such as Gerbich blood group antigen negativity, polymorphisms of the complement receptor genes (most notably CR1), Southeast Asian ovalocytosis, pyruvate kinase deficiency; HbE, the sickle cell trait (SCT) and alpha-thalassemia.Plasmodium falciparum malaria infection. This could explain its high gene frequency in geographic malaria-endemic regions. However, several patients with SCA have coincidental alpha-thalassemia and the presence of both SCA and alpha-thalassemia mutations seems to act as a negative epistatic factor.13Alpha-thalassemia is due to mutations of the alpha-globin genes (chromosome 16pter-p13.3) and it has been shown that the presence of alpha-thalassemia has a protective role against Alpha-thalassemia reduces the concentration of HbS and therefore of HbS polymerization. Thus it is expected that this will prevent vaso-occlusive events that are consequences of hemolysis, including stroke, leg ulcer, priapism, and pulmonary hypertension. Complications more dependent on blood viscosity, such as painful episodes, acute chest syndrome (ACS) and avascular necrosis will usually be more prevalent when alpha-thalassemia coexists with SCD mutation.16Glucose-6-phosphate dehydrogenase (G6PD) deficiency (Chromosome Xq28) is commonly found in HbS populations. Although this deficiency does not appear to have a direct effect on the SCD phenotype,The consequences of the sickle mutation and its downstream effects are clearly variable. Complications due to chronic hemolytic anemia, episodic vaso-occlusion with resultant painful episodes and chronic organ damage lead to very variable phenotypes of SCD. It is very difficult to determine the exact factors mediating the severity of the disease. It seems at least that all hematologists agree that they can definitely define the very mild or the “asymptomatic” patients as an obvious phenotype.5It is critical to carefully characterize phenotypes in order to study complex gene interactions. Studies of sickle cell patients from different populations will very likely yield important information due to the differences in genetic backgrounds of these populations and potential implications on the disease phenotype. In the past few years, many centers have focused on the study of genetic modifiers of SCD. Selected findings are summarized here. HbF concentration is high at birth and declines slowly during the first three decades of life and the final level in adults is variable. It has been well established that in most cases, HbF has an ameliorative effect on SCD patients because the gamma-chains of HbF do not polymerize with HbS. This potential anti-sickling effect resulted in the development of a major therapeutic product for amelioration of SCD effects. To date, the only drug that is therapeutically used in this way is the S-phase specific chemotherapeutic agent, hydroxyurea (HU) which increases HbF production.26Among SCA patients, HbF concentrations vary from 0.1% to 30% with an average of about 8%. Many laboratories have made efforts to elucidate the reasons behind differences in HbF levels between patients.32XmnI restriction enzyme.XmnI variant on chromosome 11. As the overall heritability of the cellular HbF trait in Europeans is 89%,Several genetic determinants have been shown to contribute to the heterogeneity of baseline HbF levels in SCD. Haplotypes of the beta-globin gene cluster are among the most extensively studied.Studies in 137 SCA patients treated with HU showed an association between SNPs and the change in HbF level after 2 years of treatment with HU. Using a candidate gene approach, they found SNPs in TOX (thymocyte selection-associated high mobility group box) gene within the 8q12.1 linkage peaks, and SNPs within the 6q22.33 region on mitogen-activated protein kinase kinase kinase 5 (MAP3K5) gene. They reported SNPs in other genes such as hydroxyacid oxidase 2 (HAO2), nitric oxide synthase 1 (NOS1), fms-related tyrosine kinase 1 (FLT1), arginase, type II (ARG2), nitric oxide synthase 2A (NOS2A) and phosphodiesterase 7B (PDE7B) that were associated with the HbF response to HU.48The interactions of NO with Hb result in the formation of a vast biological sink of NO activity that modulates microvascular tone throughout the body. NO reacts with oxygenated Hb and reduced Hb to produce nitrate plus met-Hb and iron-nitrosyl-Hb, respectively; NO has several mechanisms of action, but it appears to play a major role in both the regulation of vascular muscle tone at the cellular level as well as in platelet aggregation (clumping).5Using a newly developed Bayesian modeling approach, the same team used two independent groups of patients, one group of 1,518 adults and children from the Cooperative Study of Sickle Cell Disease (CSSCD), and validated the results in a second independent group of 211 adults from the Multicenter Study of Hydroxyurea (MSH) in SCA.23Although the significance of all these data seems attractive, a genome-wide screening using new high throughput technologies is suggested to find how all the polymorphisms in genes regulating HbF expression, HU metabolism and erythroid progenitor proliferation might modulate patient response to HU. Also, it is important to note that, some SNPs and genes found to be associated in the CSSCD population were not found to be associated in the MSH population. Because these two populations were of African American origins, it is possible that some modifier genes may not have originated from an African ancestry. This shows the importance of studying different isolated populations composed of large cohorts of individuals to conduct association studies.Multiple genes are very likely to affect the response of HU and HbF levels. Their interactions and the predictive values of their polymorphisms will help to elucidate their complex mechanisms.Stroke is a devastating complication of SCD, which occurs in about 11% of patients under 20 years of age.57Studies have shown that increased TCD  velocities of blood flow (>200 cm/sec) in major intracranial arteries, is a predictor of stroke risk in children with SCD.In a study of seven candidate genes in 42 children with large vessel cerebral artery stenosis, SNPs in the transforming growth factor-β receptor 3 (TGFBR3) and adenine cyclase 9 (ADCY9) were associated with this phenotype, when compared with 71 controls.59NcoI polymorphisms with small vessel stroke.65Other studies, investigating candidate genes in about 230 children from the CSSCD population found associations of stroke with several genes including interleukin-4 receptor (IL-4R) S503P, tumor necrosis factor super-family, member 2  −308G and adrenergic beta-2 receptor (ADRB2) Q27E polymorphisms with large vessel stroke and vascular cell adhesion molecule 1 (VCAM1) −1594T>C, low density lipoprotein receptor (LDLR) Another studyOther studies attempted to associate stroke risk with SNPs in the angiotensinogen (AGT), cystathionine-beta-synthase (CBS), cholesteryl ester transfer protein (CETP), integrin, beta 3 (ITGB3), apolipoprotein C-III (APOC3), 5,10-methylenetetrahydrofolate reductase (MTHFR), serpin peptidase inhibitor, clade E (PAI1), intercellular adhesion molecule 1 (ICAM1) and thrombospondin receptor (CD36) genes,To further define the genetic basis of stroke, the association of SNPs in candidate genes of different functional classes with the likelihood of having a stroke was examined. A total of 113 patients with SCA and a confirmed history of, or incident complete, non-hemorrhagic stroke, documented by imaging studies were compared with 493 control patients. Polymorphisms in four candidate genes, KL, TGFBR3, ANXA2 and BMP6, were associated with stroke.No association with cerebro-vascular disease was found when multiple coagulation factors were measured in children transfused for stroke, at risk for stroke and in untransfused controls.74To examine the interaction among genes and their variant SNPs and to develop a prognostic model for stroke in SCA, a Bayesian network was developed to analyze 235 SNPs in 80 candidate genes in 1398 unrelated subjects with SCA. SNPs on 11 genes and four clinical variables, including alpha-thalassemia and HbF, interacted in a complex network of dependency to modulate the risk of stroke. This network of interactions included three genes, BMP6, TGFBR2, TGFBR3 with a functional role in the TGF-beta pathway and one gene (SELP) associated with stroke in the general population. The model was validated in a different population by predicting the occurrence of stroke in 114 unrelated individuals with 98% accuracy, predicting the correct outcome for all seven stroke subjects, and for 105 of 107 non-stroke subjects. This gave a 100% true positive rate and 98. 14% true negative rate, and an overall predictive accuracy of 98%.In the CSSCD study it was shown that alpha-thalassemia, age, high hematocrit and frequent vaso-occlusive events, are risk factors for the development of avascular necrosis (AVN) of the femoral head in SCD.Increased susceptibility to ACS has been associated with the T-786C SNP in the NOS3 gene in females.86Priapism, a persistent, usually painful, erection that lasts for more than four hours and occurs without sexual stimulation, occurs in 30%–45% of male patients with SCD. The possible influence of genetic risk factors on the incidence of priapism is not well understood. A study made on 44 candidate genes, in 148 patients with SCA and a confirmed history of priapism, versus 529 control SCA patients who never developed priapism, revealed a polymorphic association with the KL gene.Cutaneous leg ulcers are common in SCA. In the United States, 2.5% of patients with all common genotypes of SCD have leg ulcers.93In a study made on the CSSCD cohort, and after screening 215 SNPs in more than 100 candidate genes, associations were found with SNPs in KL, TEK and several genes in the TGF-beta/BMP signaling pathway by genotypic association analyses. KL directly or indirectly promotes endothelial NO production and the TEK receptor tyrosine kinase is involved in angiogenesis. The TGF-beta/BMP signaling pathway modulates wound healing and angiogenesis, among its other functions. They suggest that hemolysis-driven phenotypes, such as leg ulcers, could be improved by agents that reduce sickle erythrocyte density or increase NO bioavailability.94Twin and family linkage studies suggested a genetic component to the development of gallstones.103Carriers of the 7/7 genotype had bilirubin levels greater than 51,3 μmol/l despite full-dose hydroxyurea therapy,Infection and bacteremia are common in SCD and severe bacterial infections are the major causes of morbidity and mortality in SCA. A recent study on the CSSCD cohort, showed significant associations with SNPs in insulin-like growth factor 1 receptor (IGF1R) and genes of the TGF-beta/BMP pathway , SMAD6 and SMAD3) suggesting that both could play important roles in immune function in SCA and their polymorphisms may help identify a “bacteremia-prone” phenotype.108Another study on 144 sub-Saharan African SCD patients, 73 of whom had at least one severe bacterial infection history and 71 had none, showed a bi-allelic polymorphism (Arg107Gly) distribution of a human leukocyte antigen–E (HLA-E) locus. The HLA-E*0101/E*0101 genotype was more frequent among the group with infections than their counterparts (47% vs. 21%). This genetic association is of relevance for the involvement of HLA-E molecules in host response to pathogens.111The glomerular filtration rate (GFR) in SCA is supra-normal in childhood but falls quickly with age, often resulting in renal failure. The renal failure is common among older patients with homozygous SCA and contributes to death of adult patients.94ACVRL1), bone morphogenetic protein receptor 2 (BMPR2), and BMP6, the beta-1 adrenergic receptor (ADRB1).115Genetic studies concerning this phenotype are very limited. One recent study measured with a tricuspid regurgitation jet of >2.5 m/sec, showed evidence of association on genes of the TGF-beta superfamily, including activin A receptor type II–like 1  RBCs, unlike unaffected (AA) RBCs, adhere avidly to components of the vascular wall, and this abnormal adhesion is believed to contribute to the painful vaso-occlusive crises that occur in patients with SCA.P = 0.037 and P = 0.0002 respectively). These two genes are activated by adrenaline and may affect SS RBC adhesion to laminin. They theorize that these two genetic polymorphisms of signalling pathways on SS RBC adhesion may lead to pathophysiological consequences as well as disease variability in SCD. Future investigations are needed to confirm a role for adrenaline-mediated in vivo activation of SS RBC adhesion in vaso-occlusion.A recent study suggested that polymorphisms in the beta2-adrenergic receptors (ADRB2) and adenylate cyclase 6 (ADCY6) may influence SCD severity through the signalling pathway of RBC adhesion to laminin.Acute painful episodes are a major complication of SCD and can be predictive of early death in adults. The only genetic modulations known to date about pain in SCA are the protective role of HbF and the deleterious or neutral role of alpha-thalassemia.Other complications can be associated with genetic predisposition. Although only few studies exist on the risk factors of some of these phenotypes, many of them remain to be studied. Some potential sub-phenotypes that can be considered genetically associated with the disease, and thus needing further genetic association studies are: Mortality, dactylitis, aplastc crisis, acute splenic sequestration, chronic hypersplenism, megaloblastic change, iron and folate deficiency, bone pain, pregnancy complications, osteomyelitis, acute pulmonary sequestration, pulmonary fat embolism, deep jaundice, iris atrophy and eye complications and chronic transfusion complications.Due to the advances in alternative controls and therapies such as HU treatment, transfusions, prophylaxis and antibiotics to prevent infection, new born screening programs, prevention and gene counseling, and improving of socio-economical features, the survival rate of SCA patients is clearly increasing.Fetal Hb and hydroxyurea response section, the Senegal and Asian-Indian haplotypes have a general milder clinical and hematological phenotype compared with the other African haplotypes . Individuals with Bantu haplotypes have the most severe phenotype and individuals with the Benin haplotype usually have intermediate features.34The beta-globin gene is located on the short arm of Chromosome 11. Variations in non-coding nucleotides flanking the globin genes have been used to track the origin of the sickle cell mutation and its flow to other geographical regions in the world. Using restriction endonucleases, haplotype blocks of 8 SNPs spanning ∼30,000 bases of the beta-globin locus could be determined and was used to study the ancestral origin of the sickle cell mutation in individuals from different parts of Africa and Asia. In Africa, the HbS gene was associated with four haplotypes representing regions where independent mutations occurred. These haplotypes include the Benin, the Senegal, the Bantu  and the Cameroon.While the mutation prevalence is the highest in the Mediterranean, Africa and Asia, the migration of the populations from these areas has increased globally.1Comprehensive control programs in recent years have succeeded in limiting the numbers of new births and prolonging life in affected individuals. Such programs have been successful in a minority of countries and have little global impact. Over 300,000 infants with major syndromes are born every year and the majority dies undiagnosed, untreated or under-treated. Countries may be divided into three general categories according to the services available: A. Endemic Mediterranean countries. In these countries, long-established prevention programs have achieved 80 to 100% prevention through specialized clinics which provide optimum treatment. B. Areas of the developed, industrialized world where prevalence is increasing because of migration. These countries have the means to provide adequate control but have problems in reaching immigrant groups with different cultural background. C. Countries of the developing world where the provision of services is hampered by economic difficulties, other health priorities due to high infant mortality from infectious diseases, and religious/cultural constraints.129In The National Institutes of Health (NIH) has established funds to monitor clinical trials and human genetic studies. However, genetic studies require a large number of patient DNA and clinical information. To collect information on patients and protect the personal privacy of each individual, the institutional review board (IRB), which is an independent committee of physicians, statisticians, community advocates and others, insure that the clinical trial or the genetic studies conducted by the investigators are ethical, and all the rights of the studied participants are protected. In the past however, minorities and populations from developing countries were exploited and abused in several clinical trials which has created resentment and suspicion of researchers among these populations. Regaining the trust of minorities and establishment of collaborative studies with developing countries to participate in large scale genetic screenings remains a problematic and challenging task (US DHHS 1994). Recruiting volunteer participants for medical research is critical to developing medical innovation toward improved patient health. A sophisticated and clear collaborative policy will definitely help regain trust in medical research, the backbone of medical cure.SCD is one of more than 10,000 human diseases that are caused by defects in single genes. Screening for a diverse group of serious and common disorders in the prenatal setting presents a great challenge.Although it is well known that 85%–95% of human genetic variation is due to variation among individuals within a population, whereas 5%–15% is attributable to variation among populations,One example of the importance of comparing the population SNPs is the MTHFR gene described above and its implication in the avascular necrosis. This gene has been found associated with this phenotype in a large number of patients of the CSSCD population but could not be confirmed in the Kuwaiti population.The differences between populations do not only refer to a pure genetic paradigm, but also refer to geographic, social, dietetic, traditional and socio-economical matter. The outcome of the hemoglobin sickling on patients eating different diet will be different. Even though very few studies has been made on water intake and it’s real patho-physiological influence on the phenotype, doctors advise children with SCA to drink water and fluids and avoid dehydration. Very few studies have been done on the effect of diet on SCD patients. Some studies done in our laboratory, showed correlations between the metabolic demands of increased erythropoiesis and cardiac energy consumption with the excess protein and energy metabolism in children with SCA.133SCD is a blood disorder. Blood does not only carry oxygen and carbon dioxide, but also has primordial roles in distribution of water, metabolites, energy, hormones and enzymes to different parts of the body. It is logical that the diet, environmental factors and lifestyle in general, as well as psychological and socio-economical factors will definitely influence the outcome of the disease. It is important to note that, when we genetically screen patients for polymorphisms, we take into consideration the environmental factors that could influence the outcome of the disease.For thousands of years, in sub-Saharan Africa, malaria transmission resulted in natural selection which resulted in preferential killing of healthy and SCA children while individuals with SCT were protected. This resulted in high frequency of SCD patients in malaria endemic regions. The global frequency of hemoglobinopathies shows a remarkably consistent match of hemoglobinopathies with malaria endemic region. The eradication of malaria in some countries, such as in the Mediterranean countries, has contributed to the lower frequency of HbS in these regions. Interestingly, SCD is a genetic disorder while malaria is due to a parasitic infection. It is therefore important to continue the fight to completely eradicate malaria in all the countries.Plasmodium falciparum malaria has been correlated with polymorphisms in more than 30 human genes on patients from Africa and south-east Asia and India.134Susceptibility/resistance to Genome-wide association analysis based on 10,000 single-nucleotide polymorphisms in a Ghanaian population identified several regions which showed evidence for linkage disequilibrium to parasitological and clinical phenotypes. Among them a prominent signal on Chromosome 10p15 obtained with malaria fever episodesIt is extremely important to utilize new high throughput technologies now available commercially to determine how best to translate such progress into improved patient care. Nowadays, a single DNA chip microarray can be used to screen a whole genome-wide SNPs panel per individual. New technologies have enabled genome-wide association studies to be conducted with hundreds of thousands of genotyped SNPs. Several different first-generation genome-wide panels of SNPs have been commercialized. The total amount of common genetic variation is still unknown; however, the coverage of commercial panels can be evaluated against reference population samples genotyped by the International HapMap project. Less information is available about coverage in samples from other populations. The high cost of these technologies is a major limitation not only for the gene polymorphism studies, but also for the practical exploitation of the biomarker-based therapies. But, despite the high cost, the new technologies became more “per-sample” cost effective and can allow the determination of a whole patient’s genotype in one single experiment. It also gives us the copy number of the gene expression. This advancement of biomedical technologies enables the efficient and reliable screening of patients with different phenotypes and also appears to be the key for establishing biomakers that will allow better diagnosis and treatment of patients that have a susceptibility SNP implicated in the clinical manifestation of the disease. These technologies have been proven to be effective in revealing associations between SNPs and many disease phenotypes, even in non monogenic disorders such as infectious diseases and in different populations including Caucasians, Asians and Africans.139On the other hand, a recent twin study, introduced a controversy in the paradigm of epistatic genetic control of disease.Coordinating efforts and cooperative studies with different laboratories and hospitals around the world, setting up a common database that is freely shared, and helping developing countries to set up new born screening centers for collecting data will facilitate the search for a cure for SCD.These kind of genomic investigations are very important for the detection of genomic variations leading to different phenotypes in SCD patients. However, functional studies will be required to correlate the results with the phenotypes. These studies are limited to the extent to which it is a genetic study using new genomic tools and the complexity of genetic interactions and environmental factors require further gene expression confirmations such RNA and protein levels.The understanding of genomic variations in SCD patients will be key in the use of possible biomarkers for diagnostic of the severity of SCD and that is critical for targeted therapeutics delivery and personalized treatments.


The common presence of an underlying inflammatory component suggests that inflammation may contribute to the pathogenesis of the systemic effects of alveolar hypoxia. While the role of alveolar macrophages in the immune and defense functions of the lung has been long known, recent evidence indicates that activation of alveolar macrophages causes inflammatory disturbances in the systemic microcirculation. The purpose of this review is to describe observations in experimental animals showing that alveolar macrophages initiate a systemic inflammatory response to alveolar hypoxia. Evidence obtained in intact animals and in primary cell cultures indicate that alveolar macrophages activated by hypoxia release a mediator(s) into the circulation. This mediator activates perivascular mast cells and initiates a widespread systemic inflammation. The inflammatory cascade includes activation of the local renin-angiotensin system and results in increased leukocyte-endothelial interactions in post-capillary venules, increased microvascular levels of reactive O2 species; and extravasation of albumin. Given the known extrapulmonary responses elicited by activation of alveolar macrophages, this novel phenomenon could contribute to some of the systemic effects of conditions featuring low alveolar PO2.Diseases featuring abnormally low alveolar PO On the other hand, alveolar macrophage-induced systemic inflammation has been documented in humans and in animal experiments. The objective of this review is to describe a novel phenomenon, namely the systemic inflammation initiated by alveolar macrophages activated by a reduction of alveolar PO2. Investigation of the links between alveolar macrophages, alveolar hypoxia, and systemic inflammation could provide insights into the pathogenesis of the systemic effects of conditions associated with alveolar hypoxia.Reduced alveolar PO2. While the pathogenesis of this diverse group of conditions is varied, the presence of systemic markers of inflammation has been demonstrated either in clinical cases or in animal models. Examples of systemic consequences of alveolar hypoxia in which an inflammatory component has been proposed are the cachexia and muscle wasting of chronic obstructive pulmonary disease [Systemic effects are frequently observed in pulmonary or extrapulmonary diseases associated with low alveolar PO disease -4, the i disease , the car disease -9, the m disease , acute l disease -13 and p disease , the sys disease ,16 and t disease -19. Whil2 for periods ranging from 1 to 8 h show extravasation of albumin and increased pulmonary expression of HIF-1α, NF-κB, and pro-inflammatory cytokines; these markers are attenuated by elimination of alveolar macrophages [There is evidence that alveolar hypoxia induces lung inflammation, and that alveolar macrophages play an important role in the modulation of this phenomenon. Rats breathing 10% Orophages -23. Hyporophages . Activatrophages ,25. Furtrophages ,27.In addition to the well known pulmonary effects of alveolar macrophage activation with hypoxia and other stimuli, there is mounting evidence that activation of alveolar macrophages may have substantial extrapulmonary effects. An example is the systemic microvascular response to particulate matter inhalation. Epidemiological studies have demonstrated a correlation between environmental air pollution and cardiovascular morbidity , and hum2 activates alveolar macrophages and initiates a systemic inflammatory cascade, demonstrating the presence of a link between alveolar hypoxia, alveolar macrophages and systemic inflammation.The results discussed below will show that reduction of alveolar PO2 show a rapid inflammatory response in mesentery, skeletal muscle and pial microcirculations within minutes of the onset of hypoxia [2 species (ROS) [2 value [2 substrate availability in hypoxia [Rats breathing 10% O hypoxia -38. Thises (ROS) , mast cees (ROS) , increases (ROS) -37,40, aes (ROS) . Increases (ROS) ,41. The [2 value . Both RO[2 value ,41. Incr[2 value . This su hypoxia ,44. Howe hypoxia .Mast cell degranulation is a necessary event which provides the chemotactic gradient for the increased leukocyte-endothelial adhesive interactions of hypoxia . Prevent2 of the environment surrounding the mast cells, but rather by an agent(s) released by alveolar macrophages into the circulation. The evidence leading to this hypothesis is as follows:A series of observations suggests that the key initial event of the inflammatory response, the activation of mast cells, is not triggered by the reduced PO2 (CmvPO2), estimated using a phosphorescence decay method [in vivo equilibration of the cremaster with 5% CO2/95% N2 [2. Although CmvPO2 decreased to levels comparable to those seen in rats breathing 10% O2, neither of these interventions produced mast cell degranulation or leukocyte endothelial adherence in cremaster post-capillary venules. On the other hand, cremaster mast cell degranulation and leukocyte adherence occurred when the animals breathed 10% O2 and CmvPO2 was maintained at a higher than normal level [Cremaster microvascular POy method , was sely method , or by i2/95% N2 ,37 in thal level ,37. One 2 for 5 min produced mast cell degranulation, leukocyte endothelial adherence, and extravasation of albumin when applied to the cremaster muscle of normoxic rats [in vitro with hypoxic gas mixtures did not produce inflammation [If a putative mediator released from a distant site is transported by the systemic circulation, it would be expected that plasma obtained from hypoxic animals would elicit inflammation in normoxic tissues. Plasma obtained from conscious rats breathing 10% Oxic rats . The infxic rats , did notammation .2 was reduced, alveolar macrophages, given their location and their systemic effects, were thought of as a likely source of the circulating mediator of the systemic inflammation of alveolar hypoxia. A role for alveolar macrophages in this phenomenon was demonstrated by three lines of evidence [2 induced mast cell degranulation and leukocyte endothelial adherence when applied topically onto the normoxic cremaster. A non-specific effect was ruled out by the observation that supernatant of alveolar macrophages cultured in normoxia had no inflammatory effect. The inflammation initiated by hypoxic alveolar macrophage supernatant shares common pathways with that secondary to alveolar hypoxia in intact animals, since both are attenuated by blockade of the RAS [Since systemic inflammation occurred only when alveolar POevidence : first,  the RAS ,47.Non-selective Ang II antagonists and ACE inhibitors attenuate the inflammation induced by alveolar hypoxia in intact rats, pointing to a participation of the RAS in the inflammatory cascade initiated by alveolar hypoxia. Several lines of evidence obtained in skeletal muscle and mesentery microcirculations indicate that mast cell degranulation is responsible for the activation of the RAS: first, while cromolyn blocks the inflammatory effects of hypoxic rat plasma and of hypoxic alveolar macrophage supernatant, it does not block the inflammatory response to topical Ang II . This isSupernatant of hypoxic alveolar macrophages elicits degranulation of mast cells followed by activation of the RAS in two different microvascular beds: mesentery and skeletal muscle. The similarity of these responses is consistent with the notion of a widespread inflammation originated by an alveolar macrophage-borne mediator carried by the circulatory system.2 in intact organisms initiates the sequence of events presented schematically in Figure The results presented so far suggest that reduction of alveolar PO2 values that would encompass conditions from normoxia to severe hypoxia in vivo. In this respect it should be remembered that alveolar macrophages are normally exposed to PO2 values close to 100 Torr, while resident tissue macrophages (and tissue mast cells) may be in "normoxic" conditions in PO2 environments lower than 30–35 Torr [2 ranging from 5 to 65 Torr) resulted in a respiratory burst evidenced by a release of H2O2 into the supernatant, the magnitude of which was inversely related to the medium PO2. The H2O2 release reached a peak at 15 min of hypoxia and returned to pre-hypoxic values by 30 min of exposure [2 > 100 Torr did not induce a respiratory burst.Indirect evidence that alveolar macrophages are activated by hypoxia is provided by the observation that supernatant of hypoxic alveolar macrophages initiates an inflammatory response in skeletal muscle  and in m–35 Torr . Equilibexposure . Equilib2O2 when medium PO2 was reduced to values as low as ~5 Torr. The dissimilar effects of hypoxia on activation of the two types of macrophages are paralleled by the different effects of their supernatants applied onto the normoxic mesentery: while supernatant of alveolar macrophages exposed to hypoxia for > 30 min elicited mast cell degranulation and leukocyte endothelial adherence, supernatant of peritoneal macrophages equilibrated with even lower PO2 had no inflammatory effects [In contrast with alveolar macrophages under hypoxia, peritoneal macrophages did not release H effects 2. That this is the case was suggested by the lack of cremaster mast cell degranulation in the experiments in which CmvPO2 was selectively reduced while alveolar and arterial PO2 were maintained within normoxic values [2 to ~5 Torr failed to elicit degranulation in primary cultures of peritoneal mast cells. However, degranulation occurred when mast cells of the same culture were immersed in supernatant of alveolar macrophages which had been exposed to a PO2 of ~65 Torr for 30 min. In contrast, immersion of peritoneal mast cells in supernatant of hypoxic peritoneal macrophages (PO2 ~5 Torr) did not elicit degranulation. These results provide a direct link between alveolar macrophages and mast cells and demonstrate that alveolar macrophages release a mast cell secretagogue when stimulated by hypoxia. The results also show that reduced tissue PO2 and activation of resident tissue macrophages are not necessary to initiate the systemic inflammation of alveolar hypoxia.According to the sequence of events illustrated in Figure c values ,37. Thes2. In contrast, no changes in MCP-1 supernatant concentration were observed when primary cultures of peritoneal macrophages or peritoneal mast cells were exposed to hypoxia [in vitro in response to hypoxia and hypoxia/reoxygenation [The hypothesis that the systemic inflammation of alveolar hypoxia is initiated by an alveolar macrophage-borne mediator is strengthened by the findings that monocyte chemoattractant protein-1 (MCP-1), a mast cell secretagogue, is released by primary cell cultures of alveolar macrophages exposed to hypoxia . MCP-1,  hypoxia . MCP-1 f hypoxia . MCP-1 igenation ,54, inflgenation  and actigenation -58. Furtgenation ,25,59. Tgenation ,59.2. The opposite phenomenon, namely acute lung injury initiated by remote events, has been known for some time and is exemplified by the acute lung injury which may develop after non-thoracic trauma, hemorrhage, sepsis, or ischemia/reperfusion[This review addresses a novel phenomenon, the systemic inflammation elicited in response to activation of alveolar macrophages, the stimulus in this case being a reduction of alveolar POperfusion. A frequperfusion. Both thperfusion, and mesperfusion. Mesenteperfusion. Furtherperfusion. Recent perfusion,66.2O2 in response to phorbol myristate acetate and produce more TNFα than those recovered from control animals[Although there is evidence that alveolar macrophage activation plays an important role in the development of acute lung injury induced by intestinal I/R, its exact role is not clear. Alveolar macrophages recovered from broncho alveolar lavage of rats with intestinal ischemia reperfusion release more Hl animals. Selectil animals. Hemorrhl animals, as welll animals.Thus, while the lungs can be the target of remote ischemic or hypoxic processes, they also can be the source of agents that may induce inflammatory responses in peripheral tissues. While the specific cellular pathways and the modes of translocation of inflammatory agents vary in different conditions, these examples point out to an important phenomenon, namely the production of inflammatory responses initiated from remote sites, an issue of important clinical significance.2.In summary, strong evidence supports the hypothesis that the systemic inflammation of alveolar hypoxia is initiated by the release of a circulating mediator from activated alveolar macrophages. The data represents an example of a growing body of evidence regarding systemic effects of alveolar macrophage activation. While the sequence of events described in this review has firm experimental support, several areas remain unclear. These include the mechanism of activation of the alveolar macrophages, the possible contribution of additional alveolar macrophage-borne mediators, the mechanism of activation of the RAS, and whether Ang II is the only effector of the microvascular response. It should be kept in mind that exposure of an intact organism to environmental hypoxia is a complex stimulus which sets in motion a number of processes with different time courses. Other mechanisms are likely to participate later in the development of the systemic inflammation. Nevertheless, given the widespread nature of the inflammatory response, and the prevalence of inflammation in many conditions associated with alveolar hypoxia, further understanding of this phenomenon should provide insights into the role of inflammation in conditions associated with reduced alveolar POThe authors declare that they have no competing interests.JC performed the experiments in intravital microscopy and primary cell lines described in reference 48 and edited the manuscript; JGW edited the manuscript and NCG wrote the manuscript. All authors read and approved the final version of the manuscript.