Andrew Morton

Key Physiological Parameters Dictate Triggering of Activity-Dependent Bulk Endocytosis in Hippocampal Synapses

To maintain neurotransmission in central neurons, several mechanisms are employed to retrieve synaptically exocytosed membrane. The two major modes of synaptic vesicle (SV) retrieval are clathrin-mediated endocytosis and activity-dependent bulk endocytosis (ADBE). ADBE is the dominant SV retrieval mode during intense stimulation, however the precise physiological conditions that trigger this mode are not resolved. To determine these parameters we manipulated rat hippocampal neurons using a wide spectrum of stimuli by varying both the pattern and duration of stimulation. Using live-cell fluorescence imaging and electron microscopy approaches, we established that stimulation frequency, rather than the stimulation load, was critical in the triggering of ADBE. Thus two hundred action potentials, when delivered at high frequency, were sufficient to induce near maximal bulk formation. Furthermore we observed a strong correlation between SV pool size and ability to perform ADBE. We also identified that inhibitory nerve terminals were more likely to utilize ADBE and had a larger SV recycling pool. Thus ADBE in hippocampal synaptic terminals is tightly coupled to stimulation frequency and is more likely to occur in terminals with large SV pools. These results implicate ADBE as a key modulator of both hippocampal neurotransmission and plasticity.

Quantitative differences in developmental profiles of spontaneous activity in cortical and hippocampal cultures.

BackgroundNeural circuits can spontaneously generate complex spatiotemporal firing patterns during development. This spontaneous activity is thought to help guide development of the nervous system. In this study, we had two aims. First, to characterise the changes in spontaneous activity in cultures of developing networks of either hippocampal or cortical neurons dissociated from mouse. Second, to assess whether there are any functional differences in the patterns of activity in hippocampal and cortical networks.ResultsWe used multielectrode arrays to record the development of spontaneous activity in cultured networks of either hippocampal or cortical neurons every 2 or 3 days for the first month after plating. Within a few days of culturing, networks exhibited spontaneous activity. This activity strengthened and then stabilised typically around 21 days in vitro. We quantified the activity patterns in hippocampal and cortical networks using 11 features. Three out of 11 features showed striking differences in activity between hippocampal and cortical networks: (1) interburst intervals are less variable in spike trains from hippocampal cultures; (2) hippocampal networks have higher correlations and (3) hippocampal networks generate more robust theta-bursting patterns. Machine-learning techniques confirmed that these differences in patterning are sufficient to classify recordings reliably at any given age as either hippocampal or cortical networks.ConclusionsAlthough cultured networks of hippocampal and cortical networks both generate spontaneous activity that changes over time, at any given time we can reliably detect differences in the activity patterns. We anticipate that this quantitative framework could have applications in many areas, including neurotoxicity testing and for characterising the phenotype of different mutant mice. All code and data relating to this report are freely available for others to use.

A Neuronal Transcriptome Response Involving Stress Pathways is Buffered by Neuronal microRNAs

A single microRNA (miRNA) can inhibit a large number of mRNA transcripts. This widespread regulatory function has been experimentally demonstrated for a number of miRNAs. However, even when a multitude of targets is confirmed, function of a miRNA is frequently interpreted through a prism of a handful arbitrarily selected “interesting” targets. In this work we first show that hundreds of transcripts with target sites for two miRNAs expressed endogenously in neurons, miR-124 and miR-434-3p, are coordinately upregulated in a variety of neuronal stresses. This creates a landscape where these two miRNAs can exert their widespread inhibitory potential on stress-induced transcripts. Next, we experimentally demonstrate that overexpression of these two miRNAs indeed significantly inhibits expression of hundreds of stress-induced transcripts, thus confirming that these transcripts are enriched in true targets of examined miRNAs. A number of miRNAs were previously shown to have important roles in the regulation of stress responses, and our results suggest that these roles should be understood in light of a wide spread activation of miRNA targets during stresses. Importantly, a popular cationic lipid transfection reagent triggers such induction of miRNA targets. Therefore, when a transfection paradigm is employed to study miRNA function, the results of such studies should be interpreted with consideration for the inadvertent induction of miRNA targets.

Canalization of genetic and pharmacological perturbations in developing primary neuronal activity patterns

The function of the nervous system depends on the integrity of synapses and the patterning of electrical activity in brain circuits. The rapid advances in genome sequencing reveal a large number of mutations disrupting synaptic proteins, which potentially result in diseases known as synaptopathies. However, it is also evident that every normal individual carries hundreds of potentially damaging mutations. Although genetic studies in several organisms show that mutations can be masked during development by a process known as canalization, it is unknown if this occurs in the development of the electrical activity in the brain. Using longitudinal recordings of primary cultured neurons on multi-electrode arrays from mice carrying knockout mutations we report evidence of canalization in development of spontaneous activity patterns. Phenotypes in the activity patterns in young cultures from mice lacking the Gria1 subunit of the AMPA receptor were ameliorated as cultures matured. Similarly, the effects of chronic pharmacological NMDA receptor blockade diminished as cultures matured. Moreover, disturbances in activity patterns by simultaneous disruption of Gria1 and NMDA receptors were also canalized by three weeks in culture. Additional mutations and genetic variations also appeared to be canalized to varying degrees. These findings indicate that neuronal network canalization is a form of nervous system plasticity that provides resilience to developmental disruption. This article is part of the Special Issue entitled ‘Synaptopathy – from Biology to Therapy’.

Synaptic vesicle exocytosis and increased cytosolic calcium are both necessary but not sufficient for activity-dependent bulk endocytosis

Activity‐dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle (SV) endocytosis mode in central nerve terminals during intense neuronal activity. By definition this mode is triggered by neuronal activity; however, key questions regarding its mechanism of activation remain unaddressed. To determine the basic requirements for ADBE triggering in central nerve terminals, we decoupled SV fusion events from activity‐dependent calcium influx using either clostridial neurotoxins or buffering of intracellular calcium. ADBE was monitored both optically and morphologically by observing uptake of the fluid phase markers tetramethylrhodamine‐dextran and horse radish peroxidase respectively. Ablation of SV fusion with tetanus toxin resulted in the arrest of ADBE, but had no effect on other calcium‐dependent events such as activity‐dependent dynamin I dephosphorylation, indicating that SV exocytosis is necessary for triggering. Furthermore, the calcium chelator EGTA abolished ADBE while leaving SV exocytosis intact, demonstrating that ADBE is triggered by intracellular free calcium increases outside the active zone. Activity‐dependent dynamin I dephosphorylation was also arrested in EGTA‐treated neurons, consistent with its proposed role in triggering ADBE. Thus, SV fusion and increased cytoplasmic free calcium are both necessary but not sufficient individually to trigger ADBE.

The Best Things Come in Small Packages– Vesicular Delivery of Weak Base Antipsychotics

In this issue of Neuron reveal that weak base antipsychotic drugs inhibit presynaptic function in an activity-dependent manner via their release from synaptic vesicles.

Book Review: ‘Peace, Peace’: Daniel Philpott & Gerard Powers (eds), Strategies of Peace: Transforming Conflict in a Violent World (Oxford & New York: OUP, 2010. £18.99. pp. 380. ISBN: 978-0-19-539590-7)

course of which traditional UN ‘peacekeeping’ (with the consent of the parties) has been complemented by ‘peacemaking’ (without such consent) and then ‘peacebuilding’ (i.e. diplomacy to prevent a conflict and consolidation of peace after one). However, they also itemise and analyse the failures, including the tendency for the peace not to last, as in Angola, Rwanda and elsewhere. The core of their analysis is that, though this peace work has developed fruitfully under the leadership of Boutros Boutros-Ghali and Kofi Annan, it is still inadequate, because its guiding concepts have been too exclusively derived from the Western liberal tradition of human rights, democracy and free markets. This has been expressed in its typical measures, such as mediation, disarmament, humanitarian aid, refugee resettlement, election monitoring and the establishment of free political institutions, markets and media. They see these as necessary but not sufficient. They wish to see the modern liberal tradition which informs them, with its emphasis on political institutions, laws and policies, complemented by more local and more cultural, religious and tribal ones, with their rather different emphasis on underlying emotions, attitudes and beliefs. They therefore advocate a much broader range of activities, designed both to heal the wounds of the conflict and to right the wrongs that led to it, and therefore requiring a much longer timescale and a much wider range of actors across the whole civil society at all its levels, including local leaders, non-governmental organisations and religious bodies, along with the typical present actors, namely war leaders, governments and international organisations. They elaborate on these deeper-going and longer-lasting processes, which are clearly based on the belief that there is no peace without reconciliation and no reconciliation without justice. These include measures both of distributive justice to overcome inequalities and of restorative justice such as ‘trials, truth commissions and reparations’ and ‘apology, forgiveness and rituals of reconciliation’, as well as educational and other measures to heal bad memories and reconcile competing ones. Their criticism of existing peacebuilding, however, is not only that it is too limited in its activities, actors and timescales, but also that it is too fragmented. They call therefore for a more holistic approach, which will be more integrated, with all This reviewer was left with one frustration and one reservation about Believing the Creed. The frustration came with the appendix, which promises Ogden’s reflections on fifteen ‘modern formulations of belief’. Only three of the modern formulations are printed in full in the text; some are quoted in part; for the rest, the reader is referred to websites or other books. This makes the author’s comments on those documents largely redundant. The reservation concerns the intended readership. An inquirer looking for a history of Christian believing would not find it in the text, or in the somewhat eclectic bibliography: she would need to follow up the reference to Frances Young’s The Making of the Creeds in the first footnote in the acknowledgements (p. ix). Sometimes very basic explanations are given (for instance, defining the Magnificat (p. 82) and even the Old Testament (p. 47)); elsewhere the argument makes considerable demands on a reader with some theological training. It is possible, therefore, that this thoughtful book will not achieve all that was intended, and that would be a shame.

Book Review: E Pluribus Unum - in Ethics?: John E. Hare, God and Morality: A Philosophical History (Oxford: Wiley-Blackwell, 2009. £18.99/ €21.90. pp. vi + 309. ISBN: 978-1-4051-9598-0)

constituted as relations of opposition within the divine essence. The attention of the rest of the book is mainly focussed on the varieties of intellectual ‘machinery’ developed by the Franciscans in order to entrench and defend their position. Prime among these was the scripturally motivated and Augustinian ‘psychological model’. Going beyond Aquinas’ merely analogical use of this, Franciscans such as Duns Scotus asserted the Son and Holy Spirit to be actual emanations of divine intellect and will. Furthermore, with the use of his intermediate ‘formal distinction’ to ground emanational Trinitarian theology, Friedman rightly puts forward Scotus as the high point of what he calls ‘Trinitarian explanation’. Some of his fourteenth century successors, motivated by a desire to maintain divine simplicity in the face of mounting intra-Trinitarian distinctions, reacted against such explanation. Friedman therefore concludes with a brief survey of their theologies, asserting against Gilson’s hypothesis of fourteenth century fideism as a kneejerk reaction to high scholasticism, the distinctive and positive aspects of their ‘search for simplicity’.

Book Review: Doing Hope: Ellen Ott Marshall, Though the Fig Tree Does Not Blossom: Toward a Responsible Theology of Christian Hope (Nashville, TN: Abingdon Press, 2006. £9.99. pp. xx + 137. ISBN 0—687—46480—3)

marshall’s main message is that hope is ‘responsible hope’, a ‘workhorse’ not a ‘showhorse’. in other words, it is a practice, an enacted disposition. having hope is doing hope. far from being a diffuse and detached mood, it is ‘a way of generating and sustaining moral agency’. it ‘has a job to do’. this job, this responsibility is ‘ongoing negotiation’ with two realities of life in the world, ‘promise’ and ‘peril’, ‘beauty’ and ‘tragedy’, ‘goodness’ and ‘cruelty’, ‘resiliency’ and ‘fragility’. it involves being accountable to both of those conflicting realities and never giving up the hard labour of negotiating them, knowing that there is no easy balance between them, only taxing tension. to construe this thesis as the substitution of a ‘practical’ for a ‘theological’ approach to hope could not be more wrong; for the essence of hope is the affirmation of ‘God’s presence within every aspect of creation’. such affirmation of God’s pervasive agency is, however, in no way an abdication of human agency; for in a proper understanding of the divinehuman relation, God’s action, far from excluding human action, invites and empowers it. Not that this leads to an easy-going and euphoric ‘can-do spirit’ that sails through, but on the contrary to a ‘relentless persistence’ in the sober and careful auditing of both life-giving and death-dealing potential. this book is the story of a conversation in search of an answer to the existential question: how to cope with the real losses and limits of life, how avoid both empty optimism and dark despair, and uncover possibility. marshall finds her own experience, including several miscarriages, resonating with habakkuk’s agony over Babylon’s violent overthrow of Judah, in which he could say ‘o Lord how long shall i cry for help, and you will not listen?’ yet somehow could also say ‘though the fig tree does not blossom, yet will i rejoice in the Lord’. her conversation with habakkuk is then enlarged to include firstly Aristotle and Aquinas and secondly a range of twentieth-century theologies – social gospel, liberation, process and feminist. While grateful to the former, she finds in the latter a more nuanced understanding of God’s power as ‘relational’ and of hope as a broadening of vision of the world rather than an averting of gaze from it. this short and vivid book succeeds in being simultaneously a moving spiritual autobiography, an insightful theological essay and a helpful ethical guide to the practice of hope.

Resurrection as Revolution

THIS new edition of a book first published in 1968 is a fitting memorial to an outstanding Dominican philosopher-theologian who died in 2001. Influential speaker and writer, though his books were few, Herbert McCabe made central doctrines directly accessible, under titles like God Matters. On the face of it, this relatively short book (the print is large) is about ethics; but this moral philosopher also gives us what might be called anthropology, soteriology, ecclesiology and eschatology, though such a staccato categorization does not do justice to the ligato line of his thought. Ethics is understood as not just about the difference between right and wrong but as involvement at ever-increasing depth in the significance of human action. Its three essential elements are law, love and language, this last understood as the communicativeness which distinguishes humanity in particular from animality in general; for the defining unity of humanity is not only biological (we are interfertile) but also linguistic (we are intercommunicative) and this transforms our animality into something at once more free and more fragile. Thus all human action is meaningful communication; it says as well as does; and any dualism of soul and body is ruled out. The close relation between law and love is carefully analysed. Moral law is understood as natural law, i.e. the law of our nature insofar as we participate in the total human community and enable that community to act within our action. It represents our deep and long desires, which may conflict with our short and shallow ones. Such a nature-based ethical theory challenges the view that going from is to ought is a ‘naturalistic fallacy’. However, law in this sense of moral law is only a necessary and not a sufficient condition of good action. It gives us merely a moral apprenticeship. Its rules are only guides, rules of thumb, not absolute; for they designate only patches of any action, not their total pattern. Hence the second term, love. This is a ‘growing’ word, which does not mean that it is vague (its prohibitions are clear) but that it is only understood when we encounter and respond actively to it, thus advancing from the patches toward the pattern. So the law-love relation is a dialectical one. Love, though not separate from law, cannot be anticipated by it, but in retrospect makes sense of it as a pointer to love. This transition from law to love introduces us to one of McCabe’s key ideas, ‘revolutionary continuity’. Revolution is not intelligible in terms of what precedes it (as reform is), but what precedes it is intelligible in terms of it; thus continuity is only visible in retrospect. Revolution appears again under the third of the three terms, language or communication. Since communication means ‘actively sharing in a common world’, with all communication being action and all action communication, any broadening of communication is a broadening of community. The trouble is that we do not yet have a universal medium of communication; hence we are not yet a fully human community. Sin is our comfortable enslavement to narrow parodies of humanity, little gods of tribe, time and place, which are merely collective solipsisms. This makes us reluctant to be liberated by communication, because, being revolutionary, it is unrecognizable in advance of participation in it. Blessedly however the word of God, which is the self-communication and self-realization of God, is shared with humanity through incarnation, allowing the meaning of human history to become nothing other than sharing the life of God, who is the ultimate language. This is our way out of our sinful slavery to the little gods through which we assert ourselves and dominate others. B O O K O F T H E M O N T H