Connectivity and Circuitry in a Dish Versus in a Brain
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Chinchalongporn et al. Alzheimer's Research & Therapy (2015) 7:44 DOI 10.1186/s13195-015-0129-y REVIEW Open Access Connectivity and circuitry in a dish versus in a brain Vorapin Chinchalongporn1,2,3,4, Peter Koppensteiner1,2,3,5, Deborah Prè1,2,3, Wipawan Thangnipon4, Leonilda Bilo1,2,3,6 and Ottavio Arancio1,2,3* Abstract In order to understand and find therapeutic strategies for neurological disorders, disease models that recapitulate the connectivity and circuitry of patients’ brain are needed. Owing to many limitations of animal disease models, in vitro neuronal models using patient-derived stem cells are currently being developed. However, prior to employing neurons as a model in a dish, they need to be evaluated for their electrophysiological properties, including both passive and active membrane properties, dynamics of neurotransmitter release, and capacity to undergo synaptic plasticity. In this review, we survey recent attempts to study these issues in human induced pluripotent stem cell-derived neurons. Although progress has been made, there are still many hurdles to overcome before human induced pluripotent stem cell-derived neurons can fully recapitulate all of the above physiological properties of adult mature neurons. Moreover, proper integration of neurons into pre-existing circuitry still needs to be achieved. Nevertheless, in vitro neuronal stem cell-derived models hold great promise for clinical application in neurological diseases in the future. Introduction attractive possibility of studying newly generated human The complexity of the human central nervous system neurons, previous studies have revealed problems regard- and its inaccessibility to direct studies make its modeling ing the maturation of the stem cell-derived neurons, as necessary in order to investigate physiological and well as the survival of implanted iPSC-derived cells, the di- pathological processes occurring in it. Animal disease rected differentiation into certain cell types [2] and the models have been introduced to study pathophysio- tumorigenic potential of incompletely differentiated iPSCs logical processes and eventually develop new treatments. [2, 3]. Such limitations will have to be overcome before However, the use of animal models has drawbacks, in- newly generated human neurons become clinically useful. cluding high costs of maintenance and difficulties to In this review, we will first discuss the characteristics of fully mimic the characteristics of a human neurological the development of both basic electrophysiological prop- disease. In vitro models using patient-derived cells are cur- erties in maturing neurons and their synaptic activity, as rently emerging to study neuropathologies and test pos- well as integration of individual neurons into synaptic cir- sible treatments, as the in vitro system is more scalable, cuitry. The passive and active membrane properties and controllable and cheaper. In particular, recently developed the presence of spontaneous postsynaptic currents are techniques to generate human induced pluripotent stem strong indicators of neuronal maturation and can be used cells (iPSCs) [1] allow the investigation of cells derived to evaluate the potential therapeutic viability of the differ- from patients. This technological development has led to ent protocols. Next, we will evaluate the derangement of the need to confirm functionality of the newly generated synaptic properties underlying disease processes. Finally, neurons with respect to electrophysiological properties of we will discuss recent studies on stem cell-derived human individual neurons, their ability to express pathophysio- neurons and how they recapitulate physiopathological logically relevant phenotypes, and their capability to func- features of brain neurons. tionally integrate into the brain’s circuitry. Despite the The physiological role of synaptic * Correspondence: [email protected] neurotransmission 1Department of Pathology & Cell Biology, Columbia University, New York, NY Electrophysiological markers of neuronal development 10032, USA and stem cell conversion 2Taub Institute for Research on Alzheimer’s Disease and the Aging Brain P&S Bldg, Room 12-420D, Columbia University, New York, NY 10032, USA A central characteristic of neurons is their ability to send Full list of author information is available at the end of the article and receive signals by means of action potential (AP) © 2015 Chinchalongporn et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chinchalongporn et al. Alzheimer's Research & Therapy (2015) 7:44 Page 2 of 18 formation and propagation with subsequent synaptic via the release of thrombospondins, hevin and secreted neurotransmission. The underlying neuronal properties protein acidic and rich in cysteine [13, 14]. The expression permitting intercellular signaling are progressively chan- of thrombospondins coincides with the early postnatal ging during early network formation as well as during period of synaptogenesis while hevin and secreted protein differentiation of stem cells into neurons. Indeed, the de- acidic and rich in cysteine are also expressed in astrocytes velopmental stage of neurons can be assessed electro- in the adult central nervous system [15, 16]. In their role physiologically by measuring their passive and active of regulating the synaptic development and maintenance, membrane properties as well as synaptic currents. Pas- the astrocytes act by releasing glutamate or ATP [17, 18] sive membrane properties commonly investigated in and cell contact molecules such as ephrins [19,20]. Con- studies monitoring neuronal development include input sistently, co-cultures of neural progenitor cells (NPCs) de- resistance (Rin), membrane capacitance (Cm), and the rived from human iPSCs together with astrocytes show membrane time constant (τ) as well as the resting mem- significantly faster rates of neuronal maturation [10]. Add- brane potential (RMP). With progressive neuronal devel- itionally, microglial cells, in coordination with the comple- opment, Rin and τ values have been found to decrease ment system, are involved in synapse degradation, termed whereas Cm values increase and the RMP shows a nega- synaptic pruning [21–23]. Although several studies util- tive shift [4, 5]. These passive membrane properties ren- izing iPSC-derived neurons used glial-conditioned der immature neurons highly excitable, as high Rin and τ medium obtained from astrocyte-rich cultures, the lack values together with depolarized RMPs enable AP gener- of microglial cells in iPSC-derived neuronal cultures ation in response to weak membrane currents. Thereby, might negatively impact synaptic development in such the electrophysiological profile of immature neurons cells. Additionally, since microglia cells require comple- might function to compensate for the rather low fre- ment system activation to exert their function on synaptic quencies of synaptic neurotransmission in early develop- pruning, the addition of complement system proteins ing networks by increasing the chance of AP generation might be required. upon presynaptic transmitter release. Similar to passive membrane properties, measurement of active membrane Synaptic plasticity and its molecular machinery properties underlying AP formation and propagation allows Strong, repetitive synaptic activity leads to synaptic plasti- for analysis of the electrophysiological profile of developing city. Long-term potentiation (LTP) and long-term depres- neurons and is particularly helpful in distinguishing pyram- sion (LTD) are two forms of synaptic plasticity that have idal glutamatergic from inhibitory interneurons via their been widely investigated and are linked to learning and distinct AP shapes and firing patterns [6]. memory (reviewed in [24]). During induction of LTP at Synaptic activity is another fundamental feature that the CA3–CA1 hippocampal synapse, large amounts of characterizes neurons. The activity in early developing glutamate are released from the presynaptic terminal and networks differs from that of mature networks by a bind to the N-methyl-D-aspartate receptors (NMDARs) number of factors, including the excitatory–inhibitory and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropio- shift of γ-aminobutyric acid (GABA), the occurrence of nic acid receptors located at the postsynaptic membrane. giant depolarizing potentials (GDPs) and progressively While during the resting state NMDARs are blocked by increasing frequencies of both GABAergic and glutamater- Mg2+ ions, when a strong depolarization is induced by gic spontaneous neurotransmission, indicative of develop- glutamate binding to α-amino-3-hydroxy-5-methyl-4-iso- mental synaptogenesis [7]. Importantly, spontaneous xazolepropionic acid receptors the Mg2+ block is removed synaptic activity after birth serves as a guidance signal and Ca2+ ions enter the cell via NMDARs. The calcium in- for synaptogenesis in immature neurons (reviewed in flux activates a cascade of second messenger events that [8]). Although the progression of synaptic neurotrans- trigger nuclear transcription factors