Physiologic maturation is both extrinsically and intrinsically regulated in progenitor-derived neurons.

During development, newly-differentiated neurons undergo several morphological and physiological changes to become functional, mature neurons. Physiologic maturation of neuronal cells derived from isolated stem or progenitor cells may provide insight into maturation in vivo but is not well studied. As a step towards understanding how neuronal maturation is regulated, we studied the developmental switch of response to the neurotransmitter GABA, from excitatory depolarization to inhibitory hyperpolarization.

Novel Regulatory Mechanisms for the SoxC Transcriptional Network Required for Visual Pathway Development.

What pathways specify retinal ganglion cell (RGC) fate in the developing retina? Here we report on mechanisms by which a molecular pathway involving Sox4/Sox11 is required for RGC differentiation and for optic nerve formation in mice , and is sufficient to differentiate human induced pluripotent stem cells into electrophysiologically active RGCs. These data place Sox4 downstream of RE1 silencing transcription factor in regulating RGC fate, and further describe a newly identified, Sox4-regulated site for post-translational modification with small ubiquitin-related modifier (SUMOylation) in Sox11, which suppresses Sox11's nuclear localization and its ability to promote RGC differentiation, providing a mechanism for the SoxC familial compensation observed here and elsewhere in the nervous system. These data define novel regulatory mechanisms for this SoxC molecular network, and suggest pro-RGC molecular approaches for cell replacement-based therapies for glaucoma and other optic neuropathies.

Transplanted neurons integrate into adult retinas and respond to light.

Retinal ganglion cells (RGCs) degenerate in diseases like glaucoma and are not replaced in adult mammals. Here we investigate whether transplanted RGCs can integrate into the mature retina. We have transplanted GFP-labelled RGCs into uninjured rat retinas in vivo by intravitreal injection.

Innexin and pannexin channels and their signaling.

Innexins are bifunctional membrane proteins in invertebrates, forming gap junctions as well as non-junctional membrane channels (innexons). Their vertebrate analogues, the pannexins, have not only lost the ability to form gap junctions but are also prevented from it by glycosylation. Pannexins appear to form only non-junctional membrane channels (pannexons).

Arachidonic acid closes innexin/pannexin channels and thereby inhibits microglia cell movement to a nerve injury.

Pannexons are membrane channels formed by pannexins and are permeable to ATP. They have been implicated in various physiological and pathophysiological processes. Innexins, the invertebrate homologues of the pannexins, form innexons.

Tissue engineering the retinal ganglion cell nerve fiber layer.

Retinal degenerative diseases, such as glaucoma and macular degeneration, affect millions of people worldwide and ultimately lead to retinal cell death and blindness. Cell transplantation therapies for photoreceptors demonstrate integration and restoration of function, but transplantation into the ganglion cell layer is more complex, requiring guidance of axons from transplanted cells to the optic nerve head in order to reach targets in the brain. Here we create a biodegradable electrospun (ES) scaffold designed to direct the growth of retinal ganglion cell (RGC) axons radially, mimicking axon orientation in the retina.

Neuroglial ATP release through innexin channels controls microglial cell movement to a nerve injury.

Microglia, the immune cells of the central nervous system, are attracted to sites of injury. The injury releases adenosine triphosphate (ATP) into the extracellular space, activating the microglia, but the full mechanism of release is not known. In glial cells, a family of physiologically regulated unpaired gap junction channels called innexons (invertebrates) or pannexons (vertebrates) located in the cell membrane is permeable to ATP.

Optical analysis of circuitry for respiratory rhythm in isolated brainstem of foetal mice.

Respiratory rhythms arise from neurons situated in the ventral medulla. We are investigating their spatial and functional relationships optically by measuring changes in intracellular calcium using the fluorescent, calcium-sensitive dye Oregon Green 488 BAPTA-1 AM while simultaneously recording the regular firing of motoneurons in the phrenic nerve in isolated brainstem/spinal cord preparations of E17 to E19 mice. Responses of identified cells are associated breath by breath with inspiratory and expiratory phases of respiration and depend on CO(2) and pH levels.

ATP and NO dually control migration of microglia to nerve lesions.

Microglia migrate rapidly to lesions in the central nervous system (CNS), presumably in response to chemoattractants including ATP released directly or indirectly by the injury. Previous work on the leech has shown that nitric oxide (NO), generated at the lesion, is both a stop signal for microglia at the lesion and crucial for their directed migration from hundreds of micrometers away within the nerve cord, perhaps mediated by a soluble guanylate cyclase (sGC). In this study, application of 100 microM ATP caused maximal movement of microglia in leech nerve cords.

Innexins form two types of channels.

Injury to the central nervous system triggers glial calcium waves in both vertebrates and invertebrates. In vertebrates the pannexin1 ATP-release channel appears to provide for calcium wave initiation and propagation. The innexins, which form invertebrate gap junctions and have sequence similarity with the pannexins, are candidates to form non-junctional membrane channels.

Atypical embryonic synapses fail to regenerate in adulthood.

Functional recovery following central nervous system (CNS) injury in adult animals may depend on the reestablishment of the precise pattern of connections made during development. When the nervous system is injured during embryonic development, functional recovery may involve the formation of atypical connections. Can such atypical synapses regenerate in adults, particularly in a nervous system known for its capacity for repair? When the S interneuron in one segmental ganglion of the leech Hirudo is killed during development, two neighboring S cells extend their axons into the ganglion and restore function by making electrical synapses with the usual synaptic targets of the killed S cell.

Reduced axon sprouting after treatment that diminishes microglia accumulation at lesions in the leech CNS.

The role of mammalian microglia in central nervous system (CNS) repair is controversial. Microglia accumulate at lesions where they act as immune cells and phagocytize debris, and they may secrete neurotrophins, but they also produce molecules that can be cytotoxic, like nitric oxide (NO). To determine the importance of microglial accumulation at lesions on growth of severed CNS axons in the leech (Hirudo medicinalis), in which axon and synapse regeneration are notably successful even when isolated in tissue culture medium, microglial migration to lesions was reduced.

A 3-synapse positive feedback loop regulates the excitability of an interneuron critical for sensitization in the leech.

Sensitization of reflexive shortening in the leech has been linked to serotonin (5-HT)-induced changes in the excitability of a single interneuron, the S cell. This neuron is necessary for sensitization and complete dishabituation of reflexive shortening, during which it contributes to the sensory-motor reflex. The S cell does not contain 5-HT, which is released primarily from the Retzius (R) cells, whose firing enhances S-cell excitability.

Repair and regeneration of functional synaptic connections: cellular and molecular interactions in the leech.

A major problem for neuroscience has been to find a means to achieve reliable regeneration of synaptic connections following injury to the adult CNS. This problem has been solved by the leech, where identified neurons reconnect precisely with their usual targets following axotomy, re-establishing in the adult the connections formed during embryonic development. It cannot be assumed that once axons regenerate specific synapses, function will be restored.

Methylene blue blocks cGMP production and disrupts directed migration of microglia to nerve lesions in the leech CNS.

Migration and accumulation of microglial cells at sites of injury are important for nerve repair. Recent studies on the leech central nervous system (CNS), in which synapse regeneration is successful, have shown that nitric oxide (NO) generated immediately after injury by endothelial nitric oxide synthase (eNOS) stops migrating microglia at the lesion. The present study obtained results indicating that NO may act earlier, on microglia migration, and aimed to determine mechanisms underlying NO's effects.

Progressive recovery of learning during regeneration of a single synapse in the medicinal leech.

The leech escape reflex-shortening of the body-can change with nonassociative conditioning, including sensitization, habituation, and dishabituation. Capacity for sensitization, which is an enhancement of the reflex, is lost when a single S-interneuron is ablated, but the reflex response itself remains. In the present experiments, the S-interneuron's axon in the living leech was filled with 6-carboxyfluorescein (6-CF) dye and cut with an argon laser microbeam (lambda = 488 nm).

Axotomy of single fluorescent nerve fibers in developing mammalian spinal cord by photoconversion of diaminobenzidine.

A technique has been developed for cutting single nerve fibers in mammalian spinal cord. In the presence of diaminobenzidine (DAB), a laser microbeam was applied to carbocyanine (Dil) stained sensory fibers in cultured spinal cords of the newly born opossum Monodelphis domestica. Digital images of fluorescent fibers were acquired with an intensified video CCD-camera coupled to an image processor.

Differential effects of serotonin enhance activity of an electrically coupled neural network.

Networks of electrically coupled neurons play an important role in coordinating activity among widely distributed neurons in the CNS. Such networks are sensitive to neuromodulation; but how modulation of individual cells affects activity of the entire network is not well understood. In the CNS of the medicinal leech, the S interneuron (S-cell) forms a network of electrically coupled neurons where each S-cell is linked to its two neighboring S-cells by electrical synapses.