Search

After complete spinal cord injuries (SCI), spinal segments below the lesion maintain inter-segmental communication via the intraspinal propriospinal network. However, it is unknown whether selective manipulation of these circuits can restore locomotor function in the absence of brain-derived inputs. By taking advantage of the compromised blood-spinal cord barrier following SCI, we optimized a set of procedures in which AAV9 vectors administered via the tail vein efficiently transduce neurons in lesion-adjacent spinal segments after a thoracic crush injury in adult mice. With this method, we used chemogenetic actuators to alter the excitability of propriospinal neurons in the thoracic cord of the adult mice with a complete thoracic crush injury. We showed that activating these thoracic neurons enables consistent and significant hindlimb stepping improvement, whereas direct manipulations of the neurons in the lumbar spinal cord led to muscle spasms without meaningful locomotion. Strikingly, manipulating either excitatory or inhibitory propriospinal neurons in the thoracic levels leads to distinct behavioural outcomes, with preferential effects on standing or stepping, two key elements of the locomotor function. These results demonstrate a strategy of engaging thoracic propriospinal neurons to improve hindlimb function and provide insights into optimizing neuromodulation-based strategies for treating SCI., After complete spinal cord injury, spinal segments below the lesion maintain inter-segmental communication via the intraspinal propriospinal network. Here, the authors show that neurons in these circuits can be chemogenetically modulated to improve locomotor function in mice after spinal cord injury.

The damage or loss of retinal ganglion cells (RGCs) and their axons accounts for the visual functional defects observed after traumatic injury, in degenerative diseases such as glaucoma, or in compressive optic neuropathies such as from optic glioma. By using optic nerve crush injury models, recent studies have revealed the cellular and molecular logic behind the regenerative failure of injured RGC axons in adult mammals and suggested several strategies with translational potential. This review summarizes these findings and discusses challenges for developing clinically applicable neural repair strategies.

Predators use vision to hunt, and hunting success is one of evolution's main selection pressures. However, how viewing strategies and visual systems are adapted to predation is unclear. Tracking predator-prey interactions of mice and crickets in 3D, we find that mice trace crickets with their binocular visual fields and that monocular mice are poor hunters. Mammalian binocular vision requires ipsi- and contralateral projections of retinal ganglion cells (RGCs) to the brain. Large-scale single-cell recordings and morphological reconstructions reveal that only a small subset (9 of 40+) of RGC types in the ventrotemporal mouse retina innervate ipsilateral brain areas (ipsi-RGCs). Selective ablation of ipsi-RGCs (

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer's disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access. This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

Neuronal identity has long been thought of as immutable, so that once a cell acquires a specific fate, it is maintained for life.1 Studies using the overexpression of potent transcription factors to experimentally reprogram neuronal fate in the mouse neocortex2,3 and retina4,5 have challenged this notion by revealing that post-mitotic neurons can switch their identity. Whether fate reprogramming is part of normal development in the central nervous system (CNS) is unclear. While there are some reports of physiological cell fate reprogramming in invertebrates,6,7 and in the vertebrate peripheral nervous system,8 endogenous fate reprogramming in the vertebrate CNS has not been documented. Here, we demonstrate spontaneous fate re-specification in an interneuron lineage in the zebrafish retina. We show that the visual system homeobox 1 (vsx1)-expressing lineage, which has been associated exclusively with excitatory bipolar cell (BC) interneurons,9-12 also generates inhibitory amacrine cells (ACs). We identify a role for Notch signaling in conferring plasticity to nascent vsx1 BCs, allowing suitable transcription factor programs to re-specify them to an AC fate. Overstimulating Notch signaling enhances this physiological phenotype so that both daughters of a vsx1 progenitor differentiate into ACs and partially differentiated vsx1 BCs can be converted into ACs. Furthermore, this physiological re-specification can be mimicked to allow experimental induction of an entirely distinct fate, that of retinal projection neurons, from the vsx1 lineage. Our observations reveal unanticipated plasticity of cell fate during retinal development.

Summary Axon loss determines persistent disability in multiple sclerosis patients. Here, we use in vivo calcium imaging in a multiple sclerosis model to show that cytoplasmic calcium levels determine the choice between axon loss and survival. We rule out the endoplasmic reticulum, glutamate excitotoxicity, and the reversal of the sodium-calcium exchanger as sources of intra-axonal calcium accumulation and instead identify nanoscale ruptures of the axonal plasma membrane as the critical path of calcium entry., Graphical Abstract, Highlights • Cytoplasmic calcium accumulations predict axon degeneration in neuroinflammation • Release from the axoplasmic reticulum does not explain calcium accumulations • Calcium influx from the extracellular space drives axon degeneration • Nanoscale ruptures allow entry of calcium across the axonal plasma membrane, Witte et al. identify cytoplasmic calcium accumulations as a key driver of axon degeneration in a model of multiple sclerosis. Calcium accumulates in the axoplasm because nanoscale ruptures of the axonal plasma membrane provide an entry path for extracellular calcium.

Many human spinal cord injuries are anatomically incomplete, but exhibit complete paralysis. It is unknown why spared axons fail to mediate functional recovery in these cases. To investigate this, we undertook a small-molecule screen in mice with staggered bilateral hemisections, in which the lumbar spinal cord is deprived of all direct brain-derived innervation but dormant relay circuits remain. We discovered that a KCC2 agonist restored stepping ability, which could be mimicked by selective expression of KCC2, or hyperpolarizing DREADDs, in the inhibitory interneurons between and around the staggered spinal lesions. Mechanistically, these treatments transformed this injury-induced dysfunctional spinal circuit to a functional state, facilitating the relay of brain-derived commands towards the lumbar spinal cord. Thus, our results identify spinal inhibitory interneurons as a roadblock limiting the integration of descending inputs into relay circuits after injury, and suggest KCC2 agonists as promising treatments for promoting functional recovery after spinal cord injury.

After axotomy, neuronal survival and growth cone re-formation are required for axon regeneration. We discovered that doublecortin-like kinases (DCLKs), members of the doublecortin (DCX) family expressed in the adult retinal ganglion cells (RGCs), play critical roles in both processes, through distinct mechanisms. Over-expression of DCLK2 accelerated growth cone re-formation in vitro and enhanced the initiation and elongation of axon re-growth after optic nerve injury. These effects depended on both the microtubule (MT)-binding domain and the serine-proline-rich (S/P-rich) region of DCXs in-cis in the same molecules. While the MT-binding domain is known to stabilize MT structures, we show that the S/P-rich region prevents F-actin destabilization in injured axon stumps. Additionally, while DCXs synergize with mTOR to stimulate axon regeneration, alone they can promote neuronal survival possibly through regulating the retrograde propagation of injury signals. Multifunctional DCXs thus represent potential targets for promoting both survival and regeneration of injured neurons.

Conventionally, neuronal development is regarded to follow a stereotypic sequence of neurogenesis, migration, and differentiation. We demonstrate that this notion is not a general principle of neuronal development by documenting the timing of mitosis in relation to multiple differentiation events for bipolar cells (BCs) in the zebrafish retina using in vivo imaging. We found that BC progenitors undergo terminal neurogenic divisions while in markedly disparate stages of neuronal differentiation. Remarkably, the differentiation state of individual BC progenitors at mitosis is not arbitrary but matches the differentiation state of post‐mitotic BCs in their surround. By experimentally shifting the relative timing of progenitor division and differentiation, we provide evidence that neurogenesis and differentiation can occur independently of each other. We propose that the uncoupling of neurogenesis and differentiation could provide neurogenic programs with flexibility, while allowing for synchronous neuronal development within a continuously expanding cell pool.

Proper functioning of sensory systems requires the generation of appropriate numbers and proportions of neuronal subtypes that encode distinct information. Perception of color relies on signals from multiple cone photoreceptor types. In cone-dominated retinas, each cone expresses a single opsin type with peak sensitivity to UV, long (L) (red), medium (M) (green), or short (S) (blue) wavelengths. The modes of cell division generating distinct cone types are unknown. We report here a mechanism whereby zebrafish cone photoreceptors of the same type are produced by symmetric division of dedicated precursors. Transgenic fish in which the thyroid hormone receptor β2 (trβ2) promoter drives fluorescent protein expression before L-cone precursors themselves are produced permitted tracking of their division in vivo. Every L cone in a local region resulted from the terminal division of an L-cone precursor, suggesting that such divisions contribute significantly to L-cone production. Analysis of the fate of isolated pairs of cones and time-lapse observations suggest that other cone types can also arise by symmetric terminal divisions. Such divisions of dedicated precursors may help to rapidly attain the final numbers and proportions of cone types (L > M, UV > S) in zebrafish larvae. Loss- and gain-of-function experiments show that L-opsin expression requires trβ2 activity before cone differentiation. Ectopic expression of trβ2 after cone differentiation produces cones with mixed opsins. Temporal differences in the onset of trβ2 expression could explain why some species have mixed, and others have pure, cone types.

Corticospinal neurons (CSNs) represent the direct cortical outputs to the spinal cord and play important roles in motor control across different species. However, their organizational principle remains unclear. By using a retrograde labeling system, we defined the requirement of CSNs in the execution of a skilled forelimb food-pellet retrieval task in mice. In vivo imaging of CSN activity during performance revealed the sequential activation of topographically ordered functional ensembles with moderate local mixing. Region-specific manipulations indicate that CSNs from caudal or rostral forelimb area control reaching or grasping, respectively, and both are required in the transitional pronation step. These region-specific CSNs terminate in different spinal levels and locations, therefore preferentially connecting with the premotor neurons of muscles engaged in different steps of the task. Together, our findings suggest that spatially defined groups of CSNs encode different movement modules, providing a logic for parallel-ordered corticospinal circuits to orchestrate multistep motor skills.

Astroglia secrete factors that promote synapse formation and maintenance. In culture, glial contact has also been shown to facilitate synaptogenesis. Here, we examined whether glial contact is important for establishing circuitsin vivoby simultaneously monitoring differentiation of glial cells and local synaptogenesis over time. Photoreceptor circuits of the vertebrate retina are particularly suitable for this study because of the relatively simple, laminar organization of their connectivity with their target neurons, horizontal cells and bipolar cells. Also, individual photoreceptor terminals are ensheathed within the outer plexiform layer (OPL) by the processes of one type of glia, Müller glia cells (MGs). We conductedin vivotime-lapse multiphoton imaging of the rapidly developing and relatively transparent zebrafish retina to ascertain the time course of MG development relative to OPL synaptogenesis. The emergence of synaptic triads, indicative of functional photoreceptor circuits, and structural association with glial processes were also examined across ages by electron microscopy. We first show that MG processes form territories that tile within the inner and outer synaptic layers. We then demonstrate that cone photoreceptor synapses are assembled before the elaboration of MG processes in the OPL. Using a targeted cell ablation approach, we also determined whether the maintenance of photoreceptor synapses is perturbed when local MGs are absent. We found that removal of MGs had no appreciable effect on the stability of newly formed cone synapses. Thus, in contrast to other CNS circuits, contact from glia is not necessary for the formation or immediate stabilization of outer retinal synapses.

Liver allografts are spontaneously accepted in the liver transplantation mouse model; however, the basis for this tolerance and the conditions that abrogate spontaneous tolerance to liver allografts are incompletely understood. We examined the role of CpG oligodeoxynucleotide (ODN) in triggering the liver inflammatory reaction and allograft rejection. Bioluminescence imaging quantified the activation of nuclear transcriptional factor kappaB (NF-kappaB) at different time points post-transplantation. Intrahepatic lymphocyte subsets were analyzed by immunofluorescence assay and flow cytometry. The results showed that liver allografts survived for more than 100 days without a requirement for any immunosuppressive therapy. Donor-matched cardiac allografts were permanently accepted, whereas third-party cardiac grafts were rejected with delayed kinetics; this confirmed donor-specific tolerance. NF-kappaB activation in the liver allografts was transiently increased on day 1 and diminished by day 4; in comparison, it was elevated up to 10 days post-transplantation in the cardiac allografts. When CpG ODN was administered at a high dose (50 microg per mouse x 1) to the recipients on day 7 post-transplantation, it induced an acute liver inflammatory reaction with elevated NF-kappaB activation in both allogeneic and syngeneic liver grafts. Multiple doses of CpG ODN (10 microg per mouse x 3) elicited acute rejection of the liver allografts with significant T cell infiltration in the liver allografts, reduced T regulatory cells, and enhanced interferon gamma-producing cells in the intrahepatic infiltrating lymphocytes. These data demonstrate that CpG ODN initiates an inflammatory reaction and abrogates spontaneous tolerance in the liver transplantation mouse model. Liver Transpl 15:915-923, 2009. (c) 2009 AASLD.

BACKGROUND The purpose of the current study was to evaluate the role of bioluminescence imaging (BLI) in the determination of nuclear factor (NF)-kappaB activation in cardiac allograft rejection and ischemia-reperfusion injury. METHODS To visualize NF-kappaB activation, luciferase transgenic mice under the control of a mouse NF-kappaB promoter (NF-kappaB-Luc) were used as donors or recipients of cardiac grafts. Alternatively, NF-kappaB-Luc spleen cells were adoptively transferred into Rag2 -/- mice with or without cardiac allografts. BLI was performed posttransplantation to detect luciferase activity that represents NF-kappaB activation. RESULTS The results show that luciferase activity was significantly increased in the cardiac allografts when NF-kappaB-Luc mice were used as recipients as well as donors. Luciferase activity was also elevated in the wild-type cardiac allografts in Rag2 -/- mice that were transferred with NF-kappaB-Luc spleen cells. CD154 monoclonal antibody (mAb) therapy inhibited luciferase activity and induced long-term survival of cardiac allografts. toll-like receptor-9 ligand, CpG DNA, enhanced luciferase activity and abrogated tolerance induction by CD154 mAb. Luciferase activity was also increased in ischemia-reperfusion injury of the cardiac grafts. CONCLUSION BLI using Luc-NF-kappaB mice is a noninvasive approach to visualize the activation of NF-kappaB signaling in mouse cardiac allograft rejection and ischemia-reperfusion injury. CD154 mAb can inhibit NF-kappaB activation, which is reversed by toll-like receptor engagement.

SummaryTargeting of axons and dendrites to particular synaptic laminae is an important mechanism by which precise patterns of neuronal connectivity are established. Although axons target specific laminae during development, dendritic lamination has been thought to occur largely by pruning of inappropriately placed arbors. We discovered by in vivo time-lapse imaging that retinal ganglion cell (RGC) dendrites in zebrafish show growth patterns implicating dendritic targeting as a mechanism for contacting appropriate synaptic partners. Populations of RGCs labeled in transgenic animals establish distinct dendritic strata sequentially, predominantly from the inner to outer retina. Imaging individual cells over successive days confirmed that multistratified RGCs generate strata sequentially, each arbor elaborating within a specific lamina. Simultaneous imaging of RGCs and subpopulations of presynaptic amacrine interneurons revealed that RGC dendrites appear to target amacrine plexuses that had already laminated. Dendritic targeting of prepatterned afferents may thus be a novel mechanism for establishing proper synaptic connectivity.

After traumatic spinal cord injury, local damage responses have an important effect on regenerating axons. In lower organisms (i.e., zebrafish and newt), glial cells and other non-neuronal cell types proliferate, migrate, and differentiate to form a bridge between the two ends of a transected spinal cord. This glial bridge supports axon regeneration across the lesion site, enabling functional recovery. However, in mammals, a glial scar composed of mixed cell types and extracellular matrix forms to seal such a wound. Although early studies emphasized the inhibitory nature of this scar, recent studies have revealed that in mammals, as in lower organisms, it can also serve as a bridge to facilitate axon regeneration ( 1 – 3 ). Yet, how this glial reaction is regulated remains largely unknown. On page 630 of this issue, Mokalled et al. ( 4 ) report that connective tissue growth factor a (CTGFa) is crucial for directing glial bridging and subsequent axon regeneration in a zebrafish model of spinal cord injury. Although it remains to be determined whether this is the case in mammals, this finding could inform the design of neural repair strategies.

A major hurdle for functional recovery after both spinal cord injury and cortical stroke is the limited regrowth of the axons in the corticospinal tract (CST) that originate in the motor cortex and innervate the spinal cord. Despite recent advances in engaging the intrinsic mechanisms that control CST regrowth, it remains to be tested whether such methods can promote functional recovery in translatable settings. Here we show that post-lesional AAV-assisted co-expression of two soluble proteins, namely insulin-like growth factor 1 (IGF1) and osteopontin (OPN), in cortical neurons leads to robust CST regrowth, and the recovery of CST-dependent behavioral performance after both T10 lateral spinal hemisection and a unilateral cortical stroke. In these mice, a compound able to increase axon conduction, 4-aminopyridine-3-methanol, promotes further improvement in CST-dependent behavioral tasks. Thus, our results demonstrate a potentially translatable strategy for restoring cortical dependent function after injury in the adult.

Background Neuregulin‐1β ( NRG ‐1β) is a growth factor critical for cardiac development and repair with therapeutic potential for heart failure. We previously showed that the glial growth factor 2 ( GGF 2) isoform of NRG ‐1β improves cardiac function in rodents after myocardial infarction ( MI ), but its efficacy in a large animal model of cardiac injury has not been examined. We therefore sought to examine the effects of GGF 2 on ventricular remodeling, cardiac function, and global transcription in post‐ MI swine, as well as potential mechanisms for anti‐remodeling effects. Methods and Results MI was induced in anesthetized swine (n=23) by intracoronary balloon occlusion. At 1 week post‐ MI , survivors (n=13) received GGF 2 treatment (intravenous, biweekly for 4 weeks; n=8) or were untreated (n=5). At 5 weeks post‐ MI , fractional shortening was higher (32.8% versus 25.3%, P =0.019), and left ventricular ( LV ) end‐diastolic dimension lower (4.5 versus 5.3 cm, P =0.003) in GGF 2‐treated animals. Treatment altered expression of 528 genes, as measured by microarrays, including collagens, basal lamina components, and matricellular proteins. GGF 2‐treated pigs exhibited improvements in LV cardiomyocyte mitochondria and intercalated disk structures and showed less fibrosis, altered matrix structure, and fewer myofibroblasts (myoFbs), based on trichrome staining, electron microscopy, and immunostaining. In vitro experiments with isolated murine and rat cardiac fibroblasts demonstrate that NRG ‐1β reduces myoFbs, and suppresses TGF β‐induced phospho‐ SMAD 3 as well as α SMA expression. Conclusions These results suggest that GGF 2/ NRG ‐1β prevents adverse remodeling after injury in part via anti‐fibrotic effects in the heart.

Microtubule dynamics in neurons play critical roles in physiology, injury and disease and determine microtubule orientation, the cell biological correlate of neurite polarization. Several microtubule binding proteins, including end-binding protein 3 (EB3), specifically bind to the growing plus tip of microtubules. In the past, fluorescently tagged end-binding proteins have revealed microtubule dynamics in vitro and in non-mammalian model organisms. Here, we devise an imaging assay based on transgenic mice expressing yellow fluorescent protein-tagged EB3 to study microtubules in intact mammalian neurites. Our approach allows measurement of microtubule dynamics in vivo and ex vivo in peripheral nervous system and central nervous system neurites under physiological conditions and after exposure to microtubule-modifying drugs. We find an increase in dynamic microtubules after injury and in neurodegenerative disease states, before axons show morphological indications of degeneration or regrowth. Thus increased microtubule dynamics might serve as a general indicator of neurite remodelling in health and disease., Microtubule dynamics in neurons play critical roles in physiology, injury and disease. Here the authors develop a transgenic mouse line expressing a fluorescently tagged version of the microtubule binding protein EB3, and using a range of imaging techniques, study microtubule dynamics under normal and injury conditions in living mice.

Therapeutic strategies for spinal cord injury (SCI) commonly focus on regenerating disconnected axons. An alternative approach would be to maintain continuity of damaged axons, especially after contusion. The viability of such neuropreservative strategies depends on the degree to which initially injured axons can recover. Here we use morphological and molecular in vivo imaging after contusion SCI in mice to show that injured axons persist in a metastable state for hours. Intra-axonal calcium dynamics influence fate, but the outcome is not invariably destructive in that many axons with calcium elevations recover homeostasis without intervention. Calcium enters axons primarily through mechanopores. Spontaneous pore resealing allows calcium levels to normalize and axons to survive long term. Axon loss can be halted by blocking calcium influx or calpain, even with delayed initiation. Our data identify an inherent self-preservation process in contused axons and a window of opportunity for rescuing connectivity after nontransecting SCI.

Mitochondrial redox signals have a central role in neuronal physiology and disease. Here we describe a new optical approach to measure fast redox signals with single-organelle resolution in living mice that express genetically encoded redox biosensors in their neuronal mitochondria. Moreover, we demonstrate how parallel measurements with several biosensors can integrate these redox signals into a comprehensive characterization of mitochondrial function. This approach revealed that axonal mitochondria undergo spontaneous 'contractions' that are accompanied by reversible redox changes. These contractions are amplified by neuronal activity and acute or chronic neuronal insults. Multiparametric imaging reveals that contractions constitute respiratory chain-dependent episodes of depolarization coinciding with matrix alkalinization, followed by uncoupling. In contrast, permanent mitochondrial damage after spinal cord injury depends on calcium influx and mitochondrial permeability transition. Thus, our approach allows us to identify heterogeneity among physiological and pathological redox signals, correlate such signals to functional and structural organelle dynamics and dissect the underlying mechanisms.

Neuronal circuits of the vertebrate retina are organized into stereotyped laminae. This orderly arrangement makes the retina an ideal model system for imaging studies aimed at understanding how circuits assemble during development. In particular, live-cell imaging techniques are readily applied to the developing retina to monitor dynamic changes over time in cell structure and connectivity. Such imaging studies have collectively revealed novel strategies by which retinal neurons contact their presynaptic and postsynaptic partners to establish synaptic connections. We describe here the procedures developed in our laboratory for confocal and multiphoton live-cell imaging of the developing retina using in vivo preparations. Zebrafish larvae are an ideal specimen for in vivo imaging experiments as they can be made to remain transparent throughout development. Isolated retinal cells can be readily labeled by DNA injection into the one-cell staged embryo, or via transplantation of fluorescently labeled cells from stable transgenics.

In many retinal diseases, the malfunction that results in photoreceptor loss occurs only in either rods or cones, but degeneration can progress from the affected cell type to its healthy neighbors. Specifically, in human and mouse models of Retinitis Pigmentosa the loss of rods results in the death of neighboring healthy cones. Significantly less is known about cone-initiated degenerations and their affect on neighboring cells. Sometimes rods remain normal after cone death, whereas other patients experience a loss of scotopic vision over time. The affect of cone death on neighboring cones is unknown. The zebrafish is a cone-rich animal model in which the potential for dying cones to kill neighboring healthy cones can be evaluated. We previously reported that the zebrafish cone phosphodiesterase mutant (pde6cw59) displays a rapid death of cones soon after their formation and a subsequent loss of rods in the central retina. In this study we examine morphological changes associated with cone deathin vivoinpde6cw59fish. We then use blastulae transplantations to create chimeric fish with a photoreceptor layer of mixed wild-type (WT) andpde6cw59cones. We find that the death of inoperative cones does not cause neighboring WT cone loss. The survival of WT cones is independent of transplant size and location within the retina. Furthermore, transplanted WT cones persist at least several weeks after the initial death of dysfunctional mutant cones. Our results suggest a potential for the therapeutic transplantation of healthy cones into an environment of damaged cones.

Rate coefficients have been measured for the Smiles rearrangement of a series of 4- and 5-substituted 2-hydroxy-2′-nitrodiphenyl sulphones in 70%(v/v) dioxane–water at 60.0 °C, and for three substrates at several temperatures. The activation parameters for these substrates have been evaluated, as has the dependence on solvent composition [aqueous dioxane and dimethyl sulphoxide (DMSO)] of the rates for these substrates. The effects of substitution have been assessed by means of the Hammett equation using meta- and para-σ values for the 4- and 5-substituents, respectively, to give ρca.–2.0. All the evidence indicates reaction via a spiro Meisenheimer intermediate with the formation of the intermediate being rate determining. Rate coefficients have been also measured for the Smiles rearrangement of a series of 5-substituted 2-hydroxy-2′,4′-dinitrodiphenyl sulphones in 70%(v/v) dioxane–water at 24.8 °C and for three typical substrates at several temperatures. The activation parameters for these substrates have been evaluated, as has the dependence of rate on aqueous dioxane and aqueous DMSO solvent composition. In aqueous DMSO spiro Meisenheimer complexes are observed as the DMSO content increases. Above 50 mol% DMSO these intermediates are very rapidly and completely formed. The effects of substitution in 70%(v/v) dioxane–water have been assessed by means of the Hammett equation using meta-σ values to give ρca. 1.7. The evidence indicates that, in aqueous dioxane, reaction occurs via the spiro intermediate, with rate-determining decomposition of the Meisenheimer intermediate, and, in aqueous DMSO, the spiro intermediate becomes effectively the ‘initial’ state. The rate coefficients for the formation of the symmetrical spiro complex from 2-hydroxy-2′,4′-dinitrodiphenyl ether in DMSO–water and DMSO-tert-butyl alcohol containing base have been measured. The rate coefficients for the rearrangements of 2-amino-2′,4′-dinitrodiphenyl sulphide and 2-[(2-aminophenyl)thio]-3-nitropyridine have been measured in the same systems. Under these conditions, the ‘initial’ state is the spiro complex and the rate-determining step is the decomposition of the intermediate to form the product.

Summary Symmetric cell divisions have been proposed to rapidly increase neuronal number late in neurogenesis, but how critical this mode of division is to establishing a specific neuronal layer is unknown. Using in vivo time-lapse imaging methods, we discovered that in the laminated zebrafish retina, the horizontal cell (HC) layer forms quickly during embryonic development upon division of a precursor cell population. The precursor cells morphologically resemble immature, postmitotic HCs and express HC markers such as ptf1a and Prox1 prior to division. These precursors undergo nonapical symmetric division at the laminar location where mature HCs contact photoreceptors. Strikingly, the precursor cell type we observed generates exclusively HCs. We have thus identified a dedicated HC precursor, and our findings suggest a mechanism of neuronal layer formation whereby the location of mitosis could facilitate rapid contact between synaptic partners.

Cellular mechanisms underlying the precision by which neurons target their synaptic partners have largely been determined based on the study of projection neurons. By contrast, little is known about how interneurons establish their local connections in vivo. Here, we investigated how developing amacrine interneurons selectively innervate the appropriate region of the synaptic neuropil in the inner retina, the inner plexiform layer (IPL). Increases (ON) and decreases (OFF) in light intensity are processed by circuits that are structurally confined to separate ON and OFF synaptic sublaminae within the IPL. Using transgenic zebrafish in which the majority of amacrine cells express fluorescent protein, we determined that the earliest amacrine-derived neuritic plexus formed between two cell populations whose somata, at maturity, resided on opposite sides of this plexus. When we followed the behavior of individual amacrine cells over time, we discovered that they exhibited distinct patterns of structural dynamics at different stages of development. During cellular migration, amacrine cells exhibited an exuberant outgrowth of neurites that was undirected. Upon reaching the forming IPL, neurites extending towards the ganglion cell layer were relatively more stable. Importantly, when an arbor first formed, it preferentially ramified in either the inner or outer IPL corresponding to the future ON and OFF sublaminae, and maintained this stratification pattern. The specificity by which ON and OFF amacrine interneurons innervate their respective sublaminae in the IPL contrasts with that observed for projection neurons in the retina and elsewhere in the central nervous system.

Spinal cord injury (SCI) induces a progressive pathophysiology affecting cell survival and neurological integrity via complex and evolving molecular cascades whose interrelationships are not fully understood. The present experiments were designed to: (1) determine potential functional interactions within transcriptional expression profiles obtained after a clinically relevant SCI and (2) test the consistency of transcript expression after SCI in two genetically and immunologically diverse rat strains characterized by differences in T cell competence and associated inflammatory responses. By interrogating Affymetrix U34A rat genome GeneChip microarrays, we defined the transcriptional expression patterns in midcervical contusion lesion sites between 1 and 90 d postinjury of athymic nude (AN) and Sprague Dawley (SD) strains. Stringent statistical analyses detected significant changes in 3638 probe sets, with 80 genes differing between the AN and SD groups. Subsequent detailed functional categorization of these transcripts unveiled an overall tissue remodeling response that was common to both strains. The functionally organized gene profiles were temporally distinct and correlated with repair indices observed microscopically and by magnetic resonance microimaging. Our molecular and anatomical observations have identified a novel, longitudinal perspective of the post-SCI response, namely, that of a highly orchestrated tissue repair and remodeling repertoire with a prominent cutaneous wound healing signature that is conserved between two widely differing rat strains. These results have significant bearing on the continuing development of cellular and pharmacological therapeutics directed at tissue rescue and neuronal regeneration in the injured spinal cord.

Many neurons receive synapses in stereotypic proportions from converging but functionally distinct afferents. However, developmental mechanisms regulating synaptic convergence are not well understood. Here we describe a heterotypic mechanism by which one afferent controls synaptogenesis of another afferent, but not vice versa. Like other CNS circuits, zebrafish retinal H3 horizontal cells (HC) undergo an initial period of remodelling, establishing synapses with ultraviolet and blue cones while eliminating red and green cone contacts. As development progresses, the HCs selectively synapse with ultraviolet cones to generate a 5:1 ultraviolet-to-blue cone synapse ratio. Blue cone synaptogenesis increases in mutants lacking ultraviolet cones, and when transmitter release or visual stimulation of ultraviolet cones is perturbed. Connectivity is unaltered when blue cone transmission is suppressed. Moreover, there is no cell-autonomous regulation of cone synaptogenesis by neurotransmission. Thus, biased connectivity in this circuit is established by an unusual activity-dependent, unidirectional control of synaptogenesis exerted by the dominant input.

Neuronal circuits of the vertebrate retina are organized into stereotyped laminae. This orderly arrangement makes the retina an ideal model system for imaging studies aimed at understanding how circuits assemble during development. In particular, live-cell imaging techniques are readily applied to the developing retina to monitor dynamic changes over time in cell structure and connectivity. Such imaging studies have collectively revealed novel strategies by which retinal neurons contact their presynaptic and postsynaptic partners to establish synaptic connections. We describe here the procedures developed in our laboratory for confocal and multiphoton live-cell imaging of the developing retina using in vitro retinal explants. Retinas can be removed from the eye and kept in culture conditions for several days with limited disruption to the retinal circuit. The explanted retina is amenable to a variety of labeling techniques and provides a large, flat, unobstructed surface that is ideal for optical imaging experiments. This protocol describes procedures for mounting and imaging the isolated mouse retina. The same general procedure, with only minor modification (composition of culture medium), has been used to image retinas from a variety of vertebrates (e.g., chick, ferret, and rabbit).