The spinal cord exhibits a complex cytoarchitecture and neuronal circuitry organization that challenges in vitro replication of its functional integrity. Here we employed a previously published protocol to generate spinal cord organoids (SCOs) from human-induced pluripotent stem cells (hiPSCs). We characterized phenotypic changes in functional neuronal properties in SCO neurons at early developmental stages (15-32 days in culture) using a quantitative electrophysiological approach. Using the whole-cell patch-clamp technique to evaluate electrophysiological properties, we found that SCO neurons exhibited progressive maturation, as evidenced by hyperpolarized resting membrane potentials, increased inward current amplitude, refined action potential kinetics, and the early emergence of mature-type firing patterns. In particular, we show that the spike frequency adaptation phenomenon, which prevails in motor neurons, appears at early stages of SCO neuron development. Immunohistochemical assessment confirmed the expression of key transcription factors in motor neurons (ISLET1 and HB9) and immature spinal interneurons (LHX1/5 and PAX2). Collectively, our findings demonstrate that neurons in hiPSC-derived SCOs exhibit physiological differentiation, which is important for using SCOs to investigate human spinal cord development and advance translational research in CNS disorders and cell replacement therapies.

Introduction and methodsWe assessed the functional development of synapses from defined vestibulo-ocular projection neurons to motoneurons (MNs) in the oculomotor nuclear complex in the chicken embryo using optical recording of postsynaptic responses with the calcium-sensitive probe Calcium Green Dextran Amine (CGDA). The vestibulo-ocular projection neuron groups were defined according to the hodological nomenclature established by Díaz et al. (1) and encompassed the ipsilateral rostral, ipsilateral caudal and contralateral caudal vestibulo-ocular (iR-VO, iC-VO and cC-VO) groups. These groups provide differential input to the inferior rectus (IR), superior oblique (SO), superior rectus (SR) and inferior oblique IO) MN pools. The cC-VO group includes abducens interneurons (abd INs) which innervate the medial rectus (MR) MN pool. Since the SO MN pool, which projects out the trochlear nerve, was not labeled, recordings were limited to the IR, SR, IO and MR MN pools.ResultsSingle pulse stimulation of all the presynaptic axons collectively in wholemount preparations of the brain stem elicited robust calcium responses in all four of the CGDA-labeled postsynaptic MN pools. Responses were recorded from the dorsal surface in different regions of interest (ROIs) deployed along the mediolateral axis, which allowed us to distinguish responses in IR, MR+IO and SR MNs. Responses were first detected in a fraction of preparations at d7 of development, and from d8 in all preparations. Response magnitudes increased through d9, and then diminished through d11. Pharmacological experiments showed that responses included both glutamatergic (AMPA receptor and NMDA receptor-mediated) and GABAergic (GABAA receptor-mediated) components. All three components were present when the first responses appeared, but their proportional contribution changed during development. Covert NMDA-sensitive responses could be revealed by superfusing unresponsive d7 preparations with Mg2+-free Ringer, indicating that the very first responses to develop were mediated by NMDA receptors. GABAergic responses involved the activation of voltage-gated calcium channels and were therefore likely to be depolarizing, but nevertheless had an inhibitory effect on glutamatergic responses. Paired pulse and train stimulation at d9-d11 demonstrated substantial facilitation of the aggregate response.DiscussionThese data provide new information about the functional development of vestibulo-ocular reflex (VOR) circuitry and set the stage for testing the role of specific receptors and downstream signaling in establishing the specific synaptic connections that characterize the VOR.

In the recently published article Motor neurons are dispensable for the assembly of a sensorimotor circuit for gaze stabilization, Goldblatt et al (2024) provide strong evidence that vestibulo-ocular projection neurons in the zebrafish acquire their functional identities in vestibulo-ocular reflex circuitry despite the absence of their motoneuron targets. This is touted as disproving a longstanding hypothesis, purported to have originated from me (Glover 2003) and formalized by Hans Straka (Straka 2010), in which target extraocular motoneurons retrogradely specify vestibulo-ocular neuron identities. Goldblatt et al (2024) present this work as a major paradigm shift in how sensorimotor circuit development is conceived, a premise that is promoted by an accompanying commentary entitled Neuronal development: Rethinking sensorimotor circuits (Zwart 2024).There's just one problem with this narrative. It is simply not correct. I have never proposed the hypothesis that motoneurons retrogradely specify vestibulo-ocular neuron identities, and Hans Straka (now deceased) never formalized or promulgated it. Quite the contrary: the hypothesis that I proposed 3 decades ago (Glover 1994) is that vestibulo-ocular projection neurons are specified according to their positions in the hindbrain neuroepithelium, through the actions of anteroposterior and dorsoventral expression domains of key transcription factors. They are not retrogradely specified after contacting their target motoneurons, but rather have already obtained their identities prior to projecting their axons to the motoneurons. Numerous publications from my laboratory since then have provided evidence to support the pre-specification of vestibulo-ocular neurons (see below). Thus, the results obtained by Goldblatt et al (2024), rather than "comprehensively overturning" it, support the working model of vestibulo-ocular reflex pathway development that originally sprang from research in my laboratory (Figures 1).How then did Goldblatt et al (2024) and Zwart (2024) manage to mix this up? To understand this, some historical background is in order.In 1989 I presented an abstract at a meeting of the Society for Neuroscience demonstrating that vestibulospinal and vestibulo-ocular projection neurons with distinct axonal projection pathways are arranged in the early chicken embryo in coherent groups in a checkerboard-like pattern in register with the hindbrain rhombomeres and longitudinal domains intersecting these (Glover, 1989). Over the next few years, further investigation showed that these groups maintain their coherence through subsequent development (Petursdottir 1990), are synaptically linked to specific target motoneuron pools in the trochlear and oculomotor nuclei (Jansen 1991), that their lineages can be traced to specific rhombomeric and sub-rhombomeric domains (Diaz et al 1998), that the pattern arises before the vestibulo-ocular axons reach the motoneurons and the axons grow along specific pathways to the site of the target motoneurons unerringly (Glover andPetursdottir 1991, Glover 1994) and establish initially specific termination patterns (Rinde and Glover 1995), and that manipulations of activity known to disrupt topographic patterns of visual and somatosensory projections do not affect the connections from vestibulo-ocular axons to oculomotor motoneurons (reviewed in Glover 2003). These findings led to the idea of the "vestibular hodological mosaic", in which positional determinants specify the axon trajectories and functional identities of vestibular projection neurons (the idea was originally presented in Glover 1994, and the term "hodological mosaic" was coined in Glover 2000). Continued work in my laboratory and in collaboration with others demonstrated the same developmental organization of vestibular projection neurons in the mouse and used mouse transgenics to test the role of Hox genes (specifically HoxB1) in establishing it (Pasqualetti et al 2007, DiBonito et al 2015). In parallel, Hans Straka and colleagues showed that the same organization pertains in larval frogs (Straka et al 2001). More recently, the hypothesis that the distinct projection pathways and synaptic targets of vestibular projection neurons involves an early molecular specification was tested and supported by RNA sequencing of different vestibulospinal neuron groups (Lunde et al 2019). And in a recently submitted study (Glover et al 2024) we show that vestibulo-ocular circuitry emerges with appropriate afferent selectivity as soon as the reflex arc is completed and despite all vestibulo-ocular neurons engaging in synchronous waves of activity, favoring some form of cellular recognition over patterned activity as the underlying substrate.Over the years, these studies and their implications have been summarized in numerous review articles (Glover 1994, Glover 1996, Glover 2000, Diaz and Glover 2002, Glover 2003, Straka 2010, Straka et al 2013, Diaz and Puelles 2019, Glover 2020, Diaz and Glover 2022), and in an upcoming review will be placed in a broader hodological context (Glover 2024). In all of these reviews, the following proposal is laid out clearly: vestibulo-ocular projection neurons are specified by their early positions, independently of and presaging their later selective synaptic contacts with motoneurons. Here from Glover (1996):"The correlation of cluster domains with unique fields of regulatory gene expression at early stages suggests a mechanistic link between the position of a vestibular interneuronfoot_0 and the differentiation of its axon trajectory, termination pattern, and neurotransmitter phenotype."Correcting the misinterpretation in Goldblatt et al (2024) Where then does the notion of retrograde specification from target motoneurons arise? Goldblatt et al (2024) and Zwart (2024) appear to have latched on to a very specific (and interesting) feature of vestibulo-ocular reflex circuit development, originally documented in the chicken embryo. Once the axons of the already distinct vestibulo-ocular neuron groups have grown along their specific pathways to the site of their target motoneurons, they pause before extending axon collaterals into the individual motoneuron pools (Glover and Rinde 1995;Glover 2003). In the interim, the motoneurons make synaptic contacts with their target muscles. This led me to suggest that the motoneuron identities are not yet established, or not yet made available to the waiting presynaptic vestibulo-ocular axons, at the time the latter arrive on the scene. Thus, motoneuron identities may be retrogradely specified by contact with muscle or other features in the periphery (as appears to be the case for at least some motoneurons in the spinal cord, reviewed in Jessell et al 2011), forcing the already specified vestibulo-ocular axons to wait patiently before they can consummate the predetermined synaptic relationship (Figure 2). Thus, when Straka (2010) in his review states that "….the synaptic connectivity of the VOR pathway is established in reversed order to the signaling direction…" and "This suggests that VOR wiring is accomplished by a specification process that retrogradely transmits postsynaptic target identities to presynaptic neurons…", the sequence in which synapses are made is retrograde, but it is the specification of motoneuron identities and the transmission of these to the presynaptic vestibulo-ocular axons that is at play, not the specification of vestibulo-ocular neuron identities. Although the meaning of the second sentence might be misconstrued, uncertainty on this point can be quickly dispelled by a cursory perusal of the extensive literature cited above.In their discussion, Goldblatt et al (2024) place considerable weight on the alternative notion that vestibular projection neuron identities arise through some sort of "intrinsic specification", such as through the action of developmental patterning genes. What they fail to point out is that this is exactly the idea I proposed long ago.Goldblatt et al (2024) present an excellent set of high-quality data. Their conclusion is clear: vestibulo-ocular neuron identities and upstream circuit connectivity do not depend on interactions with their postsynaptic motoneurons. But despite "comprehensively overturning" what they couch as "the strongest version of the retrograde specification model", that model is still their invention, not mine or Hans Straka's. The "current" model -extant since I proposed it 30 years ago -states that the functional identities of vestibulo-ocular projection neurons (and likely all vestibular projection neurons) are specified long before their axons reach their motoneuron targets. This model remains intact. Indeed, it has been strengthened, not overturned, by Goldblatt et al (2024). There has thus been no paradigm shift, and no need to "rethink sensorimotor circuits", at least not in the context of the vestibulo-ocular reflex.Figure 1. A) The hypothesis proposed by Glover (1994), reviewed in Glover (2003) and Straka (2010) as cited by Goldblatt et al (2024). The functional identities of vestibulo-ocular neurons within the vestibulo-ocular reflex circuit are specified by positional information imposed by rhombomeric and longitudinal domains within the hindbrain neuroepithelium (upper panel). This allows the vestibuloocular neurons to make appropriately selective connections with extraocular motoneurons and receive appropriately selective afferent inputs (lower panel). Note that according to this model, elimination of MNs would not prevent the formation of appropriately selective afferent inputs. B) The hypothesis put forth by Goldblatt et al (2024), which they incorrectly attribute to Glover (2003) and Straka (2010). The functional identities of vestibulo-ocular neurons are initially unspecified (upper panel), but become so after contacting target MNs and receiving retrograde signals from them (middle panel). Once specified, the vestibulo-ocular neurons can then receive appropriately selective afferent input (lower panel).Figure 2. Proposal made by Glover (2003) to explain why pre-specified vestibulo-ocular axons pause before innervating their target MNs. Axons from pre-specified vestibulo-ocular neurons reach the outskirts of the oculomotor and trochlear nuclei before MN identities are evident, and must therefore wait there before extending axon collaterals into the MN pools (upper panel). Retrograde signals from muscle are required before MN identities are specified or made available to presynaptic partners (middle panel). Once this occurs, the pre-specified vestibulo-ocular axons can proceed with collateral extension and synapse formation on their appropriate MN targets (lower panel).

Pluripotent stem cells provide opportunities for treating injuries and previously incurable diseases. A major concern is the immunogenicity of stem cells and their progeny. Here, we have dissected the molecular mechanisms that allow natural killer (NK) cells to respond to human pluripotent stem cells, investigating a wide selection of activating and inhibitory NK-cell receptors and their ligands. Reporter cells expressing the activating receptor NKG2D responded strongly to embryonic stem (ES) cell lines and induced pluripotent stem (iPS) cell lines, whereas reporter cells expressing the activating receptors NKp30, NKp46, KIR2DS1, KIR2DS2, and KIR2DS4 did not respond. Human ES and iPS cells invariably expressed several ligands for NKG2D. Expression of HLA-C and HLA-E was lacking or low, insufficient to trigger reporter cells expressing the inhibitory receptors KIR2DL1, -2DL2, or -2DL3. Similar results were obtained for the pluripotent embryonic carcinoma cell lines NTERA-2 and 2102Ep, and also iPS-cell-derived neural progenitor cells. Importantly, neural progenitor cells and iPS-cell-derived motoneurons also expressed B7H6, the ligand for the activating receptor NKp30. In line with these observations, IL-2-stimulated NK cells showed robust cytotoxic responses to ES and iPS cells as well as to iPS-cell-derived motoneurons. No significant differences in cytotoxicity levels were observed between KIR/HLA matched and mismatched combinations of NK cells and pluripotent targets. Together, these data indicate that pluripotent stem cells and their neural progeny are targets for NK-cell killing both by failing to sufficiently express ligands for inhibitory receptors and by expression of ligands for activating receptors.

Hodological patterning refers to developmental mechanisms that link the location of neurons in the brain or spinal cord to specific axonal trajectories that direct connectivity to synaptic targets either within the central nervous system or in the periphery. In vertebrate motor circuits, hodological patterning has been demonstrated at different levels, from the final motor output of somatic and preganglionic autonomic neurons targeting peripheral motoneurons and ganglion cells, to premotor inputs from spinal and brainstem neuron populations targeting the somatic motoneurons and preganglionic autonomic neurons, to cortical neurons that delegate movement commands to the brainstem and spinal neurons. In many cases molecular profiling reveals potential underlying mechanisms whereby selective gene expression creates the link between location and axon trajectory. At the cortical level, somatotopic organization suggests a potential underlying hodological patterning, but this has not been proven. This review describes examples of hodological patterning in motor circuits and covers current knowledge about how this patterning arises.

[This corrects the article DOI: 10.1016/j.ibneur.2022.09.011.].

Translational medicine for neurodegenerative diseases can advance through the use of in vitro models incorporating human neural cells derived from patient-specific induced pluripotent stem cells (iPSCs). Previously, we investigated whether motor neuron (MN) progenitors derived from human iPSCs could differentiate to MNs within 3D-printed scaffolds. While extensive neurite arborization was observed on the scaffold surface, no neurite outgrowth occurred within the scaffold interior. Here we show the extensive growth of the neurites from iPSC-derived neural progenitors, imbedded into the gelatin scaffolds during 30 days of experimental time. We present a bioink formulation that softens the scaffold while preserving its 3D structure, thereby facilitating neurite outgrowth throughout the scaffold. MN differentiation, evidenced by extensive neurite arborization and the expression of choline acetyltransferase (ChAT) was verified in 3D images deep within the scaffold structure. Notably, the degree of MN differentiation appeared to depend on two factors: the delivery of MN differentiation factors via mesoporous silica particles (MSPs) embedded in the bioink and the method used to generate MN progenitors prior to 3D printing. We provide a detailed protocol for 3D printing human iPSC-derived MN progenitors, enabling their differentiation and survival within gelatin scaffolds. This protocol could be expanded to incorporate additional cell types, allowing the creation of more complex and standardized 3D neural tissues. Such advancements could facilitate investigations into the pathophysiology of motor neuron diseases and the development of new therapeutic strategies.

A comparative overview is provided of Ca2+ signaling and its potential mechanistic roles during development in tunicates. As background, the review presents an introduction to tunicate taxonomy, and then a general overview of Ca2+ signaling and methods for recording and measuring Ca2+ signals. It then covers the dynamics and implicated mechanisms of Ca2+ signals during different phases of development from oocyte to larva. These include signals arising in the unfertilized oocyte, signals associated with fertilization and meiosis, intercellular signals occurring from early cleavage stages through gastrulation, intercellular signals during organogenesis, and signals associated with early behavior. Comparisons are made among different tunicate species and where relevant to other chordate species. In many tunicate species, Ca2+ currents across the oocyte membrane are present prior to fertilization, and in the appendicularian Oikopleura dioica regular Ca2+ transients have been recorded optically prior to fertilization. Ca2+ signals at this stage have been implicated in pre-fertilization oocyte maturation events. The fertilization transient is the most well-studied Ca2+ signal and is triggered by factors from the sperm, including pivotally a phospholipase C (PLC) isoform that catalyzes the generation of IP3, which elicits release of Ca2+ from the endoplasmic reticulum. Post-fertilization signals are similarly dependent on IP3 signaling and are regulated by cyclin-dependent kinase 1 (Cdk1), and thereby linked to the meiotic divisions required for zygote formation. Ca2+ signals associated with early cleavages through gastrulation arise in blastomeres of the muscle lineage and spread from these in a coordinated fashion to other blastomeres through gap junctions. Post-gastrulation Ca2+ signals begin to show tissue-specificity in their temporal pattern as organogenesis proceeds, likely associated with loss of general gap junction transmission. Once neurulation has occurred, Ca2+ signals arise first in the nervous system and are transmitted synaptically to muscle, while Ca2+ signals arising spontaneously in the epidermis follow a separate temporal pattern. Species differences in the spatiotemporal characteristics of pre- and postgastrulation Ca+2 signals are discussed.

Governance infrastructures streamline scientific and ethical provenance verification of human pluripotent stem cell (SC) lines. Yet, scientific developments (e.g., SC-derived embryo models, organoids) challenge research governance approaches to stored biospecimens, questioning the validity of informed consent (IC) models. Likewise, e-health platforms are driving major transformations in data processing, prompting a reappraisal of IC. Given these developments, participatory research platforms are identified as effective tools to promote longitudinal engagement, interactive decision-making, and dynamic governance. Learning from European initiatives piloting dynamic IC for biobanking and SC research, this Perspective explores the benefits and challenges of implementing dynamic IC and governance for SC.

The use of allogeneic induced pluripotent stem cell (iPSC)-derived cell therapies for regenerative medicine offers an affordable and realistic alternative to producing individual iPSC lines for each patient in need. Human Leukocyte Antigens (HLA)-homozygous iPSCs matched in hemi-similarity could provide cell therapies with reduced immune rejection covering a wide range of the population with a few iPSC lines. Several banks of HLA-homozygous iPSCs (haplobanks) have been established worldwide or are underway, to provide clinical grade starting material for cell therapies covering the most frequent HLA haplotypes for certain populations. Harmonizing quality standards among haplobanks and creating a global registry could minimize the collective effort and provide a much wider access to HLA-compatible cell therapies for patients with less frequent haplotypes. In this review we present all the current haplobank initiatives and their potential benefits for the global population.

Key developmental pathways and gene networks underlie the formation of sensory cell types and structures involved in chemosensation, vision and mechanosensation, and of the efferents these sensory inputs can activate. We describe similarities and differences in these pathways and gene networks in selected species of the three main chordate groups, lancelets, tunicates, and vertebrates, leading to divergent development of olfactory receptors, eyes, hair cells and motoneurons. The lack of appropriately posited expression of certain transcription factors in lancelets and tunicates prevents them from developing vertebrate-like olfactory receptors and eyes, although they generate alternative structures for chemosensation and vision. Lancelets and tunicates lack mechanosensory cells associated with the sensation of acoustic stimuli, but have gravisensitive organs and ciliated epidermal sensory cells that may (and in some cases clearly do) provide mechanosensation and thus the capacity to respond to movement relative to surrounding water. Although functionally analogous to the vertebrate vestibular apparatus and lateral line, homology is questionable due to differences in the expression of the key transcription factors Neurog and Atoh1/7, on which development of vertebrate hair cells depends. The vertebrate hair cell-bearing inner ear and lateral line thus likely represent major evolutionary advances specific to vertebrates. Motoneurons develop in vertebrates under the control of the ventral signaling molecule hedgehog/sonic hedgehog (Hh,Shh), against an opposing inhibitory effect mediated by dorsal signaling molecules. Many elements of Shh-signaling and downstream genes involved in specifying and differentiating motoneurons are also exhibited by lancelets and tunicates, but the repertoire of MNs in vertebrates is broader, indicating greater diversity in motoneuron differentiation programs.

Hans Straka died in the morning of December 11, 2022 at his home in Munich, unexpected and much too early. He was a dedicated biologist, loved the mountains and was connected to home (Oberammergau, active participant in the Passion Play). His scientific journey took him from Munich via Paris and New York back to Munich and his many academic accomplishments ranged from a membership of the Editorial board of the Journal of Neurophysiology and of the Journal of Neuroscience. He was associate editor for Frontiers in Neuro-otology and for the volume "The Senses" he edited the part on Vestibular Function in 2020. In 2009 he became Professor of Systemic Neurosciences at the Department of Biology in Munich. Apart from his many academic accomplishments, however, Hans was a close friend to those of us who were fortunate enough to get to know him better.

Hans Straka died in the morning of December 11, 2022 at his home in Munich, unexpected and much too early. He was a dedicated biologist, loved the mountains and was connected to home (Oberammergau, active participant in the Passion Play). His scientific journey took him from Munich via Paris and New York back to Munich and his many academic accomplishments ranged from a membership of the Editorial board of the Journal of Neurophysiology and of the Journal of Neuroscience. He was associate editor for Frontiers in Neuro-otology and for the volume "The Senses" he edited the part on Vestibular Function in 2020. In 2009 he became Professor of Systemic Neurosciences at the Department of Biology in Munich. Apart from his many academic accomplishments, however, Hans was a close friend to those of us who were fortunate enough to get to know him better.

To gain insight into the evolution of motor control systems at the origin of vertebrates, we have investigated higher-order motor circuitry in the protochordate Oikopleura dioica. We have identified a highly miniaturized circuit in Oikopleura with a projection from a single pair of dopaminergic neurons to a small set of synaptically coupled GABAergic neurons, which in turn exert a disinhibitory descending projection onto the locomotor central pattern generator. The circuit is reminiscent of the nigrostriatopallidal system in the vertebrate basal ganglia, in which disinhibitory circuits release specific movements under the modulatory control of dopa-mine. We demonstrate further that dopamine is required to optimize locomotor performance in Oikopleura, mirroring its role in vertebrates. A dopamine-regulated disinhibitory locomotor control circuit reminiscent of the vertebrate nigrostriatopallidal system was thus already present at the origin of ancestral chordates and has been maintained in the face of extreme nervous system miniaturization in the urochordate lineage.

EDITORIAL article Front. Neurol., 31 March 2023Sec. Neuro-Otology Volume 14 - 2023 | https://doi.org/10.3389/fneur.2023.1191086

Human pluripotent stem cells (human embryonic stem cells, hESCs, and human induced pluripotent stem cells, hiPSCs) were originally cultured on different types of feeder cells for maintenance in an undifferentiated state in long-term culture. This approach has been supplanted to a large extent by feeder-free culture protocols, but these involve more costly reagents and can promote a transition to a primed state, which restricts the cells' differentiation capacity. In both feeder and feeder-free conditions, the harvesting of hESC or hiPSC colonies for passaging is a necessary procedure for expanding the cultures. To provide an easy and high-yield procedure for passaging hESCs/hiPSCs cultured on feeder cells, we have established a harvesting method using dis-adhesion elicited by the calcium chelator ethylenediaminetetraacetic acid (EDTA). We have assessed the yield and quality of the resultant passaged cells by comparing this approach to the original mechanical harvesting approach, in which colonies are isolated with a scalpel under a microscope (mechanical harvesting was chosen as a comparator to avoid the reagent variability associated with enzymatic harvesting). In one set of experiments, two different hESC lines were maintained on a feeder cell layer of human foreskin fibroblasts. Each line was subjected to multiple passages using EDTA-based or mechanical harvesting and assessed for colony size and morphology, cell density, stemness marker expression, differentiation to the three germ layers in embryoid bodies, and genomic aberrations. In another set of experiments, we used EDTA-based harvesting on two different hiPSC lines and obtained similar results. EDTA-induced dis-adhesion saved time and gave a higher yield of colonies of a more favorable size and more uniform morphology compared to mechanical harvesting. It was also faster than enzymatic harvesting and not prone to enzyme batch variability. The EDTA-induced dis-adhesion method also facilitates the transfer of hESC/hiPSC lines from feeder cell-based culture to feeder-free conditions if desired for downstream use and analysis.

The cellular pathology of schizophrenia and the potential of antipsychotics to target underlying neuronal dysfunctions are still largely unknown. We employed glutamatergic neurons derived from induced pluripotent stem cells (iPSC) obtained from schizophrenia patients with known histories of response to clozapine and healthy controls to decipher the mechanisms of action of clozapine, spanning from molecular (transcriptomic profiling) and cellular (electrophysiology) levels to observed clinical effects in living patients. Glutamatergic neurons derived from schizophrenia patients exhibited deficits in intrinsic electrophysiological properties, synaptic function and network activity. Deficits in K+ and Na+ currents, network behavior, and glutamatergic synaptic signaling were restored by clozapine treatment, but only in neurons from clozapine-responsive patients. Moreover, neurons from clozapine-responsive patients exhibited a reciprocal dysregulation of gene expression, particularly related to glutamatergic and downstream signaling, which was reversed by clozapine treatment. Only neurons from clozapine responders showed return to normal function and transcriptomic profile. Our results underscore the importance of K+ and Na+ channels and glutamatergic synaptic signaling in the pathogenesis of schizophrenia and demonstrate that clozapine might act by normalizing perturbances in this signaling pathway. To our knowledge this is the first study to demonstrate that schizophrenia iPSC-derived neurons exhibit a response phenotype correlated with clinical response to an antipsychotic. This opens a new avenue in the search for an effective treatment agent tailored to the needs of individual patients.

Studies by His from 1868 to 1904 delineated the critical role of the dorsal roof plate in the development of the hindbrain choroid plexus, and of the rhombic lips in the development of hindbrain auditory centers. Modern molecular studies have confirmed these observations and placed them in a mechanistic context. Expression of the transcription factor Lmx1a/b is crucial to the development of the hindbrain choroid plexus, and also regulates the expression of Atoh1, a transcription factor that is essential for the formation of the cochlear hair cells and auditory nuclei. By contrast, development of the vestibular hair cells, vestibular ganglion and vestibular nuclei does not depend on Lmx1a/b. These findings demonstrate a common dependence on a specific gene for the hindbrain choroid plexus and the primary auditory projection from hair cells to sensory neurons to hindbrain nuclei. Thus, His' conclusions regarding the origins of specific hindbrain structures are borne out by molecular genetic experiments conducted more than a hundred years later.

The vestibular column is located in the hindbrain between the sensory auditory (dorsal) and trigeminal (ventral) columns, spanning rhombomeres r1 (or r2) to r9. It contains the vestibular nuclear complex that receives sensory innervation from the labyrinthine end organs in the inner ear. Gene expression studies and experimental manipulations of developmental genes, particularly Hox genes and other developmental patterning genes, are providing insight into the morphological and functional organization of the vestibular nuclear complex, particularly from a segmental standpoint. Here, we will review studies of the classical vestibular nuclei and of vestibular projection neurons that innervate distinct targets in relation to individual rhombomeres and the expression of specific genes. Studies in different species have demonstrated that the vestibular complex is organized into a hodological mosaic that relates axon trajectory and target to specific hindbrain rhombomeres and intrarhombomeric domains, with a molecular underpinning in the form of transcription factor signatures, which has been highly conserved during the evolution of the vertebrate lineage.

We recently described calcium signaling in the appendicularian tunicate Oikopleura dioica during pre-gastrulation stages, and showed that regularly occurring calcium waves progress throughout the embryo in a characteristic spatiotemporal pattern from an initiation site in muscle lineage blastomeres. Here, we have extended our observations to the period spanning from gastrulation to post-hatching stages. We find that repetitive Ca2+ waves persist throughout this developmental window, albeit with a gradual increase in frequency. The initiation site of the waves shifts from muscle cells at gastrulation and early tailbud stages, to the central nervous system at late tailbud and post-hatching stages, indicating a transition from muscle-driven to neurally driven events as tail movements emerge. At these later stages, both the voltage gated Na+ ​channel blocker tetrodotoxin (TTX) and the T-type Ca2+ channel blocker and nAChR antagonist mecamylamine eliminate tail movements. At late post-hatching stages, mecamylamine blocks Ca2+ signals in the muscles but not the central nervous system. Post-gastrulation Ca2+ signals also arise in epithelial cells, first in a haphazard pattern in scattered cells during tailbud stages, evolving after hatching into repetitive rostrocaudal waves with a different frequency than the nervous system-to-muscle waves, and insensitive to mecamylamine. The desynchronization of Ca2+ waves arising in different parts of the body indicates a shift from whole-body to tissue/organ-specific Ca2+ signaling dynamics as organogenesis occurs, with neurally driven Ca2+ signaling dominating at the later stages when behavior emerges.