Directed and acyclic synaptic connectivity in the human layer 2-3 cortical microcircuit.

The computational capabilities of neuronal networks are fundamentally constrained by their specific connectivity. Previous studies of cortical connectivity have mostly been carried out in rodents; whether the principles established therein also apply to the evolutionarily expanded human cortex is unclear. We studied network properties within the human temporal cortex using samples obtained from brain surgery. We analyzed multineuron patch-clamp recordings in layer 2-3 pyramidal neurons and identified substantial differences compared with rodents. Reciprocity showed random distribution, synaptic strength was independent from connection probability, and connectivity of the supragranular temporal cortex followed a directed and mostly acyclic graph topology. Application of these principles in neuronal models increased dimensionality of network dynamics, suggesting a critical role for cortical computation.

Dissociation of structural and functional connectomic coherence in glioma patients.

With diffuse infiltrative glioma being increasingly recognized as a systemic brain disorder, the macroscopically apparent tumor lesion is suggested to impact on cerebral functional and structural integrity beyond the apparent lesion site. We investigated resting-state functional connectivity (FC) and diffusion-MRI-based structural connectivity (SC) (comprising edge-weight (EW) and fractional anisotropy (FA)) in isodehydrogenase mutated (IDHmut) and wildtype (IDHwt) patients and healthy controls. SC and FC were determined for whole-brain and the Default-Mode Network (DMN), mean intra- and interhemispheric SC and FC were compared across groups, and partial correlations were analyzed intra- and intermodally. With interhemispheric EW being reduced in both patient groups, IDHwt patients showed FA decreases in the ipsi- and contralesional hemisphere, whereas IDHmut patients revealed FA increases in the contralesional hemisphere. Healthy controls showed strong intramodal connectivity, each within the structural and functional connectome. Patients however showed a loss in structural and reductions in functional connectomic coherence, which appeared to be more pronounced in IDHwt glioma patients. Findings suggest a relative dissociation of structural and functional connectomic coherence in glioma patients at the time of diagnosis, with more structural connectomic aberrations being encountered in IDHwt glioma patients. Connectomic profiling may aid in phenotyping and monitoring prognostically differing tumor types.

Expression of SOLOIST/MRTFB i4, a novel neuronal isoform of the mouse serum response factor coactivator myocardin-related transcription factor-B, negatively regulates dendritic complexity in cortical neurons.

Megakaryoblastic leukemia 2 (MKL2)/myocardin-related transcription factor-B (MRTFB), a serum response factor (SRF) coactivator, is an important regulator of gene expression and neuronal morphology. Here, we show that different mouse MRTFB splice isoforms, including a novel fourth MRTFB isoform named spliced neuronal long isoform of SRF transcriptional coactivator (SOLOIST)/MRTFB isoform 4 (MRTFB i4), play distinct roles in this process. SOLOIST/MRTFB i4 has a short exon that encodes 21 amino acid residues ahead of the first RPXXXEL (RPEL) motif in MRTFB isoform 3. Quantitative PCR revealed that SOLOIST/MRTFB i4 and isoform 1 were enriched in the forebrain and neurons, and up-regulated during brain development. Conversely, isoform 3 was detected in various tissues, including both neurons and astrocytes, and was down-regulated in the developing brain. Reporter assays supported the SRF-coactivator function of SOLOIST/MRTFB i4 as well as isoform 1. Acute expression of MRTFB isoform 1, but not isoform 3 or SOLOIST/MRTFB i4, in neuronal cells within 24 hr drastically increased endogenous immediate early gene [c-fos, egr1, and activity-regulated cytoskeleton-associated protein] expression, but not endogenous actinin α1, ß-actin, gelsolin, or srf gene expression measured by qPCR. Over-expression of SOLOIST/MRTFB i4 reduced the dendritic complexity of cortical neurons, whereas over-expression of isoform 1 increased this complexity. Co-expression of isoform 1 and SOLOIST/MRTFB i4 in cortical neurons revealed that isoform 1 competitively counteracted down-regulation by SOLOIST/MRTFB i4. Our findings indicate that MRTFB isoforms have unique expression patterns and differential effects on gene expression and dendritic complexity, which contribute to shaping neuronal circuits, at least in part.

Miniaturized microscope with flexible light source input for neuronal imaging and manipulation in freely behaving animals.

Simultaneous imaging and manipulation of a genetically defined neuronal population can provide a causal link between its activity and function. Here, we designed a miniaturized microscope (or 'miniscope') that allows fluorescence imaging and optogenetic manipulation at the cellular level in freely behaving animals. This miniscope has an integrated optical connector that accepts any combination of external light sources, allowing flexibility in the choice of sensors and manipulators. Moreover, due to its simple structure and use of open source software, the miniscope is easy to build and modify. Using this miniscope, we demonstrate the optogenetic silencing of hippocampal CA1 neurons using two laser light sources-one stimulating a calcium sensor (i.e., jGCaAMP7c) and the other serving as an optogenetic silencer (i.e., Jaws). This new miniscope can contribute to efforts to determine causal relationships between neuronal network dynamics and animal behavior.

Unified Classification of Molecular, Network, and Endocrine Features of Hypothalamic Neurons.

Peripheral endocrine output relies on either direct or feed-forward multi-order command from the hypothalamus. Efficient coding of endocrine responses is made possible by the many neuronal cell types that coexist in intercalated hypothalamic nuclei and communicate through extensive synaptic connectivity. Although general anatomical and neurochemical features of hypothalamic neurons were described during the past decades, they have yet to be reconciled with recently discovered molecular classifiers and neurogenetic function determination. By interrogating magnocellular as well as parvocellular dopamine, GABA, glutamate, and phenotypically mixed neurons, we integrate available information at the molecular, cellular, network, and endocrine output levels to propose a framework for the comprehensive classification of hypothalamic neurons. Simultaneously, we single out putative neuronal subclasses for which future research can fill in existing gaps of knowledge to rationalize cellular diversity through function-determinant molecular marks in the hypothalamus.

Post mortem single-cell labeling with DiI and immunoelectron microscopy unveil the fine structure of kisspeptin neurons in humans.

Kisspeptin (KP) synthesizing neurons of the hypothalamic infundibular region are critically involved in the central regulation of fertility; these cells regulate pulsatile gonadotropin-releasing hormone (GnRH) secretion and mediate sex steroid feedback signals to GnRH neurons. Fine structural analysis of the human KP system is complicated by the use of post mortem tissues. To gain better insight into the neuroanatomy of the somato-dendritic cellular compartment, we introduced the diolistic labeling of immunohistochemically identified KP neurons using a gene gun loaded with the lipophilic dye, DiI. Confocal microscopic studies of primary dendrites in 100-µm-thick tissue sections established that 79.3% of KP cells were bipolar, 14.1% were tripolar, and 6.6% were unipolar. Primary dendrites branched sparsely, contained numerous appendages (9.1 ± 1.1 spines/100 µm dendrite), and received rich innervation from GABAergic, glutamatergic, and KP-containing terminals. KP neuron synaptology was analyzed with immunoelectron microscopy on perfusion-fixed specimens. KP axons established frequent contacts and classical synapses on unlabeled, and on KP-immunoreactive somata, dendrites, and spines. Synapses were asymmetric and the presynaptic structures contained round and regular synaptic vesicles, in addition to dense-core granules. Although immunofluorescent studies failed to detect vesicular glutamate transporter isoforms in KP axons, ultrastructural characteristics of synaptic terminals suggested use of glutamatergic, in addition to peptidergic, neurotransmission. In summary, immunofluorescent and DiI labeling of KP neurons in thick hypothalamic sections and immunoelectron microscopic studies of KP-immunoreactive neurons in brains perfusion-fixed shortly post mortem allowed us to identify previously unexplored fine structural features of KP neurons in the mediobasal hypothalamus of humans.

Imaging Neural Architecture in Brainbow Samples.

The fluorescent protein revolution has made the light microscope the most widely used tool for studying biological structure from the single-molecule to whole organism scales. However, traditional approaches are limited in their ability to resolve components in highly complex structures, such as the brain. In recent years, this limitation has been circumvented by the development of multicolor labeling methods, termed Brainbow. Brainbow tools rely on site-specific recombinases to make stochastic "choices" between different combinations of fluorescent proteins so that structures in close proximity to one another can be resolved based on their color profile. These new approaches, however, call for more refined methods of sample preparation and imaging optimized for multispectral imaging, which are presented here. The most robust approach for generating useful Brainbow data combines immunohistology with multispectral laser scanning confocal microscopy. This chapter, therefore, focuses on this particular technique, though the imaging principle discussed here is applicable to other Brainbow approaches as well.

Morphology and hydro-sensory role of superficial neuromasts in schooling behaviour of yellow-eyed mullet (Aldrichetta forsteri).

The lateral line system is a mechanosensory organ found in all fish species and located on the skin or in subdermal canals. The basic functional units are superficial and canal neuromasts, which are involved in hydrodynamic sensing and cohesion in schooling fish. Yellow-eyed mullet (Aldrichetta forsteri) are an obligate schooling species found commonly in shallow coastal areas of New Zealand and Australia. Schooling is a fundamental part of their behavioural repertoire, yet little is known about the structure or functionality of the lateral line in this species. We used scanning electron microscopy to characterise the morphology of trunk superficial neuromasts. We then took a multi-sensory approach and conducted behavioural experiments comparing school structure in groups of fish with and without fully functioning lateral lines, under photopic and scotopic conditions. A highly developed hydro-sensing system exists on the trunk of yellow-eyed mullet consisting of superficial neuromasts containing hundreds of hair cells aligned, with respect to their most sensitive axis, in a rostrocaudal direction. Without functioning superficial neuromasts, schooling behaviour was disrupted under both photopic and scotopic conditions and the ability to detect stationary objects decreased. Results highlight the importance of this component of the lateral line system to schooling behaviour.

Temporal Rewiring of Striatal Circuits Initiated by Nicotine.

Drug addiction has been conceptualized as maladaptive recruitment of integrative circuits coursing through the striatum, facilitating drug-seeking and drug-taking behavior. The aim of this study was to define temporal neuroadaptations in striatal subregions initiated by 3 weeks of intermittent nicotine exposure followed by protracted abstinence. Enhanced rearing activity was assessed in motor activity boxes as a measurement of behavioral change induced by nicotine (0.36 mg/kg), whereas electrophysiological field potential recordings were performed to evaluate treatment effects on neuronal activity. Dopamine receptor mRNA expression was quantified by qPCR, and nicotine-induced dopamine release was measured in striatal subregions using in vivo microdialysis. Golgi staining was performed to assess nicotine-induced changes in spine density of medium spiny neurons. The data presented here show that a brief period of nicotine exposure followed by abstinence leads to temporal changes in synaptic efficacy, dopamine receptor expression, and spine density in a subregion-specific manner. Nicotine may thus initiate a reorganization of striatal circuits that continues to develop despite protracted abstinence. We also show that the response to nicotine is modulated in previously exposed rats even after 6 months of abstinence. The data presented here suggests that, even though not self-administered, nicotine may produce progressive neuronal alterations in brain regions associated with goal-directed and habitual performance, which might contribute to the development of compulsive drug seeking and the increased vulnerability to relapse, which are hallmarks of drug addiction.

Rescue of the Functional Alterations of Motor Cortical Circuits in Arginase Deficiency by Neonatal Gene Therapy.

UNLABELLED: Arginase 1 deficiency is a urea cycle disorder associated with hyperargininemia, spastic diplegia, loss of ambulation, intellectual disability, and seizures. To gain insight on how loss of arginase expression affects the excitability and synaptic connectivity of the cortical neurons in the developing brain, we used anatomical, ultrastructural, and electrophysiological techniques to determine how single-copy and double-copy arginase deletion affects cortical circuits in mice. We find that the loss of arginase 1 expression results in decreased dendritic complexity, decreased excitatory and inhibitory synapse numbers, decreased intrinsic excitability, and altered synaptic transmission in layer 5 motor cortical neurons. Hepatic arginase 1 gene therapy using adeno-associated virus rescued nearly all these abnormalities when administered to neonatal homozygous knock-out animals. Therefore, gene therapeutic strategies can reverse physiological and anatomical markers of arginase 1 deficiency and therefore may be of therapeutic benefit for the neurological disabilities in this syndrome. SIGNIFICANCE STATEMENT: These studies are one of the few investigations to try to understand the underlying neurological dysfunction that occurs in urea cycle disorders and the only to examine arginase deficiency. We have demonstrated by multiple modalities that, in murine layer 5 cortical neurons, a gradation of abnormalities exists based on the functional copy number of arginase: intrinsic excitability is altered, there is decreased density in asymmetrical and perisomatic synapses, and analysis of the dendritic complexity is lowest in the homozygous knock-out. With neonatal administration of adeno-associated virus expressing arginase, there is near-total recovery of the abnormalities in neurons and cortical circuits, supporting the concept that neonatal gene therapy may prevent the functional abnormalities that occur in arginase deficiency.

Traumatic Brain Injury Causes Aberrant Migration of Adult-Born Neurons in the Hippocampus.

Traumatic brain injury (TBI) promotes neural stem/progenitor cell (NSC) proliferation in an attempt to initiate innate repair mechanisms. However, all immature neurons in the CNS are required to migrate from their birthplace to their final destination to develop into functional neurons. Here we assessed the destination of adult-born neurons following TBI. We found that a large percentage of immature neurons migrated past their normal stopping site at the inner granular cell layer (GCL), and became misplaced in the outer GCL of the hippocampal dentate gyrus. The aberrant migration of adult-born neurons in the hippocampus occurred 48 hours after TBI, and lasted for 8 weeks, resulting in a great number of newly generated neurons misplaced in the outer GCL in the hippocampus. Those misplaced neurons were able to become mature and differentiate into granular neurons, but located ectopically in the outer GCL with reduced dendritic complexity after TBI. The adult-born neurons at the misplaced position may make wrong connections with inappropriate nearby targets in the pre-existing neural network. These results suggest that although stimulation of endogenous NSCs following TBI might offer new avenues for cell-based therapy, additional intervention is required to further enhance successful neurogenesis for repairing the damaged brain.

MIM-Induced Membrane Bending Promotes Dendritic Spine Initiation.

Proper morphogenesis of neuronal dendritic spines is essential for the formation of functional synaptic networks. However, it is not known how spines are initiated. Here, we identify the inverse-BAR (I-BAR) protein MIM/MTSS1 as a nucleator of dendritic spines. MIM accumulated to future spine initiation sites in a PIP2-dependent manner and deformed the plasma membrane outward into a proto-protrusion via its I-BAR domain. Unexpectedly, the initial protrusion formation did not involve actin polymerization. However, PIP2-dependent activation of Arp2/3-mediated actin assembly was required for protrusion elongation. Overexpression of MIM increased the density of dendritic protrusions and suppressed spine maturation. In contrast, MIM deficiency led to decreased density of dendritic protrusions and larger spine heads. Moreover, MIM-deficient mice displayed altered glutamatergic synaptic transmission and compatible behavioral defects. Collectively, our data identify an important morphogenetic pathway, which initiates spine protrusions by coupling phosphoinositide signaling, direct membrane bending, and actin assembly to ensure proper synaptogenesis.

Area-specific reestablishment of damaged circuits in the adult cerebral cortex by cortical neurons derived from mouse embryonic stem cells.

Pluripotent stem-cell-derived neurons constitute an attractive source for replacement therapies, but their utility remains unclear for cortical diseases. Here, we show that neurons of visual cortex identity, differentiated in vitro from mouse embryonic stem cells (ESCs), can be transplanted successfully following a lesion of the adult mouse visual cortex. Reestablishment of the damaged pathways included long-range and reciprocal axonal projections and synaptic connections with targets of the damaged cortex. Electrophysiological recordings revealed that some grafted neurons were functional and responsive to visual stimuli. No significant integration was observed following grafting of the same neurons in motor cortex, or transplantation of embryonic motor cortex in visual cortex, indicating that successful transplantation required a match in the areal identity of grafted and lesioned neurons. These findings demonstrate that transplantation of mouse ESC-derived neurons of appropriate cortical areal identity can contribute to the reconstruction of an adult damaged cortical circuit.

Gene dosage in the dysbindin schizophrenia susceptibility network differentially affect synaptic function and plasticity.

Neurodevelopmental disorders arise from single or multiple gene defects. However, the way multiple loci interact to modify phenotypic outcomes remains poorly understood. Here, we studied phenotypes associated with mutations in the schizophrenia susceptibility gene dysbindin (dysb), in isolation or in combination with null alleles in the dysb network component Blos1. In humans, the Blos1 ortholog Bloc1s1 encodes a polypeptide that assembles, with dysbindin, into the octameric BLOC-1 complex. We biochemically confirmed BLOC-1 presence in Drosophila neurons, and measured synaptic output and complex adaptive behavior in response to BLOC-1 perturbation. Homozygous loss-of-function alleles of dysb, Blos1, or compound heterozygotes of these alleles impaired neurotransmitter release, synapse morphology, and homeostatic plasticity at the larval neuromuscular junction, and impaired olfactory habituation. This multiparameter assessment indicated that phenotypes were differentially sensitive to genetic dosages of loss-of-function BLOC-1 alleles. Our findings suggest that modification of a second genetic locus in a defined neurodevelopmental regulatory network does not follow a strict additive genetic inheritance, but rather, precise stoichiometry within the network determines phenotypic outcomes.

The orexinergic neurons receive synaptic input from C1 cells in rats.

The C1 cells, located in the rostral ventrolateral medulla (RVLM), are activated by pain, hypoxia, hypoglycemia, infection, and hypotension and elicit cardiorespiratory stimulation, adrenaline and adrenocorticotropic hormone (ACTH) release, and arousal. The orexin neurons contribute to the autonomic responses to acute psychological stress. Here, using an anatomical approach, we consider whether the orexin neurons could also be contributing to the autonomic effects elicited by C1 neuron activation. Phenylethanolamine N-methyl transferase-immunoreactive (PNMT-ir) axons were detected among orexin-ir somata, and close appositions between PNMT-ir axonal varicosities and orexin-ir profiles were observed. The existence of synapses between PNMT-ir boutons labeled with diaminobenzidine and orexinergic neurons labeled with immunogold was confirmed by electron microscopy. We labeled RVLM neurons with a lentiviral vector that expresses the fusion protein ChR2-mCherry under the control of the catecholaminergic neuron-selective promoter PRSx8 and obtained light and ultrastructural evidence that these neurons innervate the orexin cells. By using a Cre-dependent adeno-associated vector and TH-Cre rats, we confirmed that the projection from RVLM catecholaminergic neurons to the orexinergic neurons originates predominantly from PNMT-ir catecholaminergic (i.e., C1 cells). The C1 neurons were found to establish predominantly asymmetric synapses with orexin-ir cell bodies or dendrites. These synapses were packed with small clear vesicles and also contained dense-core vesicles. In summary, the orexin neurons are among the hypothalamic neurons contacted and presumably excited by the C1 cells. The C1-orexin neuronal connection is probably one of several suprabulbar pathways through which the C1 neurons activate breathing and the circulation, raise blood glucose, and facilitate arousal from sleep.

The structure of the caudal wall of the zebrafish (Danio rerio) swim bladder: evidence of localized lamellar body secretion and a proximate neural plexus.

In this study, we present a morphological description of the fine structure of the tissues composing the caudal tip of the adult zebrafish swim bladder and an immunochemical survey of the innervation at this site. The internal aspect of the caudal tip is lined by an epithelium specialized to secrete surfactant into the lumen as evinced by the exocytosis of lamellar bodies. The sole innervation to this region consists of a neural plexus, present on the external surface, of nitric oxide synthase-positive (nNOS) neuronal cell bodies that are contacted by axon terminals, some containing neuropeptide Y and vasoactive intestinal polypeptide. As the specialized epithelium and neural plexus are coincident and of common extent, we suggest that the morphological relationship between the two elements allows the nervous system to affect surfactant processing, possibly through a paracrine mechanism.

Pyramidal neurons derived from human pluripotent stem cells integrate efficiently into mouse brain circuits in vivo.

The study of human cortical development has major implications for brain evolution and diseases but has remained elusive due to paucity of experimental models. Here we found that human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), cultured without added morphogens, recapitulate corticogenesis leading to the sequential generation of functional pyramidal neurons of all six layer identities. After transplantation into mouse neonatal brain, human ESC-derived cortical neurons integrated robustly and established specific axonal projections and dendritic patterns corresponding to native cortical neurons. The differentiation and connectivity of the transplanted human cortical neurons complexified progressively over several months in vivo, culminating in the establishment of functional synapses with the host circuitry. Our data demonstrate that human cortical neurons generated in vitro from ESC/iPSC can develop complex hodological properties characteristic of the cerebral cortex in vivo, thereby offering unprecedented opportunities for the modeling of human cortex diseases and brain repair.

Estradiol promotes spine growth and synapse formation without affecting pre-established networks.

Estrogens regulate dendritic spine density, but the mechanism and significance of this effect for brain networks remain unknown. We used repetitive imaging over several days to investigate how 17ß-estradiol affected the turnover and long-term behavior of dendritic spines in CA1 cells of hippocampal slice cultures. We find that 17ß-estradiol and serum in the culture medium tightly regulated spine density by promoting an increase in the rate of new spine formation and their transformation into synapses, without affecting spine elimination or stability. New spines formed during a transient 17ß-estradiol application were preferentially eliminated upon removal of the hormone, in contrast with pre-existing spines that remained unaffected. Our results reveal that 17ß-estradiol transiently regulates the complexity of hippocampal circuits without causing major alterations of pre-existing networks.

[C-terminal fragment of tetanus toxin: its use in neuronal network analysis and its potential as non-viral vector]. / Utilisation du fragment C-terminal de la neurotoxine tétanique pour visualiser et analyser des connexions neuronales et pour le transfert d'une activité biologique associée.

The atoxic C-terminal fragment of tetanus neurotoxin or TTC fragment presents similar retrograde and transsynaptic properties to that of holotoxin. Detection of this fragment is easier when it is associated with a fluorescent marker or with beta-galactosidase activity by genetic fusion or chemical conjugation. Thus, these tracers have been used to study and analyse the synaptic connections of a neural network. In this article, we shortly review the various methods used with this aim including: injection of the fusion protein, adenovirus in vivo expression and transgenesis. Since neural activity is essential for neuronal TTC binding and internalization, the functionality of connections can be also evaluated. Moreover, modifications of the retrograde transport can be detected by using this fragment. Thus, TTC fragment is an excellent tracer to analyse the connectivity and functionality of a neural network. The TTC fragment was also soon proposed as potential therapeutic vector to transport and to deliver a biological activity or gene in a neural network. With this aim, the efficiency of a translocation domain to induce the cytosolic release of the associated activity has been evaluated. The use of the TTC fragment to target specifically a neurotrophic factor to neurons and thus avoid secondary effects has been tested with interesting results.

Immunocytochemical observation of ghrelin-containing neurons in the rat arcuate nucleus.

Ghrelin is a novel peptide that stimulates the release of growth hormone from the pituitary and is involved in hypothalamic feeding regulation. A pre-embedding immunostaining technique was used to study the ultrastructure and synaptic relationships of ghrelin-containing neurons in the rat arcuate nucleus (ARC). Ghrelin-like immunoreactive (ghrelin-LI) neurons were found in the ARC, and were especially abundant in its ventral part. At the electron microscopic level, ghrelin-LI neurons received afferent synapses from many unknown axon terminals. Ghrelin-LI products in the immunoreactive cell bodies, processes, and axon terminals were detected mainly in dense granular vesicles about 110 nm in diameter. Ghrelin-LI presynaptic axon terminals often made synapses with unknown immunonegative neurons. These results suggest that ghrelin acts to regulate food intake through synaptic connections in hypothalamic neuronal networks.