David R. Copenhagen

The identification of vesicular glutamate transporter 3 suggests novel modes of signaling by glutamate

Quantal release of the principal excitatory neurotransmitter glutamate requires a mechanism for its transport into secretory vesicles. Within the brain, the complementary expression of vesicular glutamate transporters (VGLUTs) 1 and 2 accounts for the release of glutamate by all known excitatory neurons. We now report the identification of VGLUT3 and its expression by many cells generally considered to release a classical transmitter with properties very different from glutamate. Remarkably, subpopulations of inhibitory neurons as well as cholinergic interneurons, monoamine neurons, and glia express VGLUT3. The dendritic expression of VGLUT3 by particular neurons also indicates the potential for retrograde synaptic signaling. The distribution and subcellular location of VGLUT3 thus suggest novel modes of signaling by glutamate.

Molecular analysis of system N suggests novel physiological roles in nitrogen metabolism and synaptic transmission

The amino acid glutamine has a central role in nitrogen metabolism. Although the molecular mechanisms responsible for its transport across cell membranes remain poorly understood, classical amino acid transport system N appears particularly important. Using intracellular pH measurements, we have now identified an orphan protein related to a vesicular neurotransmitter transporter as system N. Functional analysis shows that this protein (SN1) involves H+ exchange as well as Na+ cotransport and, under physiological conditions, mediates glutamine efflux as well as uptake. Together with the pattern of SN1 expression, these unusual properties suggest novel physiological roles for system N in nitrogen metabolism and synaptic transmission.

Visual Stimulation Is Required for Refinement of ON and OFF Pathways in Postnatal Retina

ON and OFF pathways separately relay increment and decrement luminance signals from retinal bipolar cells to cortex. ON-OFF retinal ganglion cells (RGCs) are activated via synaptic inputs onto bistratified dendrites localized in the ON and OFF regions of the inner plexiform layer. Postnatal maturational processes convert bistratifying ON-OFF RGCs to monostratifying ON and OFF RGCs. Although visual deprivation influences refinement of higher visual centers, no previous studies suggest that light regulates either the development of the visual-evoked signaling in retinal ON and OFF pathways, nor pruning of bistratified RGC dendrites. We find that dark rearing blocks both the maturational loss of ON-OFF responsive RGCs and the pruning of dendrites. Thus, in retina, there is a previously unrecognized, pathway-specific maturation that is profoundly affected by visual deprivation.

Development of Precise Maps in Visual Cortex Requires Patterned Spontaneous Activity in the Retina

The visual cortex is organized into retinotopic maps that preserve an orderly representation of the visual world, achieved by topographically precise inputs from the lateral geniculate nucleus. We show here that geniculocortical mapping is imprecise when the waves of spontaneous activity in the retina during the first postnatal week are disrupted genetically. This anatomical mapping defect is present by postnatal day 8 and has functional consequences, as revealed by optical imaging and microelectrode recording in adults. Pharmacological disruption of these retinal waves during the first week phenocopies the mapping defect, confirming both the site and the timing of the disruption in neural activity responsible for the defect. Analysis shows that the geniculocortical miswiring is not a trivial or necessary consequence of the retinogeniculate defect. Our findings demonstrate that disrupting early spontaneous activity in the eye alters thalamic connections to the cortex.

Ephrin-As and neural activity are required for eye-specific patterning during retinogeniculate mapping

In mammals, retinal ganglion cell (RGC) projections initially intermingle and then segregate into a stereotyped pattern of eye-specific layers in the dorsal lateral geniculate nucleus (dLGN). Here we found that in mice deficient for ephrin-A2, ephrin-A3 and ephrin-A5, eye-specific inputs segregated but the shape and location of eye-specific layers were profoundly disrupted. In contrast, mice that lacked correlated retinal activity did not segregate eye-specific inputs. Inhibition of correlated neural activity in ephrin mutants led to overlapping retinal projections that were located in inappropriate regions of the dLGN. Thus, ephrin-As and neural activity act together to control patterning of eye-specific retinogeniculate layers.

Concomitant activation of two types of glutamate receptor mediates excitation of salamander retinal ganglion cells

1. Cells in the ganglion cell layer of salamander retinal slices were voltage clamped using patch pipettes. Light elicited transient excitatory postsynaptic currents (EPSCs) in on‐off ganglion cells and sustained EPSCs in on ganglion cells. Light‐evoked inhibitory postsynaptic currents in these cells could be blocked by 100 microM‐bicuculline methobromide and 500 nM‐strychnine. 2. In the presence of external Cd2+, at a concentration that blocked light‐evoked synaptic inputs, N‐methyl‐D‐aspartate (NMDA) and the non‐NMDA‐receptor agonists, quisqualate and kainate, gated conductances in both on‐off and on ganglion cells. The current‐voltage (I‐V) curve for the conductance elicited by NMDA had a negative slope between ‐40 and ‐70 mV and a reversal potential near 0 mV. The I‐V curves for the non‐NMDA‐receptor‐mediated conductances were nearly linear and also had reversal potentials near 0 mV. 3. I‐V curves were measured at an early time point near the peak of transient EPSCs and at a later time point during the decay phase of the responses. The late I‐V curve had a negative slope below ‐40 mV. The early I‐V curve had a positive slope over the entire voltage range but the slope was greater at positive than at negative potentials. The evoked current reversed near 0 mV at both time points. 4. The region of negative slope of the late I‐V curve was eliminated when Mg2+ was removed from the external saline. A slowly decaying component of transient EPSCs was eliminated in 20 microM‐DL‐2‐amino‐7‐phosphonoheptanoate (AP7), an NMDA‐receptor antagonist. 5. Application of 1 microM‐6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), a non‐NMDA‐receptor antagonist at this concentration, blocked a fast component of transient EPSCs. 6. Our results demonstrate that the synaptic inputs to on‐off ganglion cells have two components: a slower NMDA‐receptor‐mediated component having a time‐to‐peak of 110 +/‐ 45 ms and an e‐fold decay time of 209 +/‐ 35 ms at ‐31 mV (mean +/‐ S.D., n = 5), and a faster non‐NMDA‐receptor‐mediated component having a time‐to‐peak of 28 +/‐ 10 ms and an e‐fold decay time of 43 +/‐ 20 ms at ‐31 mV (n = 8). 7. A similar analysis of sustained EPSCs of on ganglion cells showed that these currents resulted from sustained activation of both NMDA and non‐NMDA receptors.

Neuronal Pentraxins Mediate Synaptic Refinement in the Developing Visual System

Neuronal pentraxins (NPs) define a family of proteins that are homologous to C-reactive and acute-phase proteins in the immune system and have been hypothesized to be involved in activity-dependent synaptic plasticity. To investigate the role of NPs in vivo, we generated mice that lack one, two, or all three NPs. NP1/2 knock-out mice exhibited defects in the segregation of eye-specific retinal ganglion cell (RGC) projections to the dorsal lateral geniculate nucleus, a process that involves activity-dependent synapse formation and elimination. Retinas from mice lacking NP1 and NP2 had cholinergically driven waves of activity that occurred at a frequency similar to that of wild-type mice, but several other parameters of retinal activity were altered. RGCs cultured from these mice exhibited a significant delay in functional maturation of glutamatergic synapses. Other developmental processes, such as pathfinding of RGCs at the optic chiasm and hippocampal long-term potentiation and long-term depression, appeared normal in NP-deficient mice. These data indicate that NPs are necessary for early synaptic refinements in the mammalian retina and dorsal lateral geniculate nucleus. We speculate that NPs exert their effects through mechanisms that parallel the known role of short pentraxins outside the CNS.

Visual Deprivation Alters Development of Synaptic Function in Inner Retina after Eye Opening

Visual deprivation impedes refinement of neuronal function in higher visual centers of mammals. It is often assumed that visual deprivation has minimal effect, if any, on neuronal function in retina. Here we report that dark rearing reduces the light-evoked responsiveness of inner retinal neurons in young mice. We also find that 1 to 2 weeks after eye opening, there is a surge (>4-fold) in the frequency of spontaneous excitatory and inhibitory synaptic events in ganglion cells. Dark rearing reversibly suppresses this surge, but recovery takes >6 days. Frequency changes are not accompanied by amplitude changes, indicating that synaptic reorganization is likely to be presynaptic. These findings indicate there is a degree of activity-dependent plasticity in the mammalian retina that has not been previously described.

Compartmentalization of calcium extrusion mechanisms in the outer and inner segments of photoreceptors.

Differential localization of calcium channel subtypes in divergent regions of individual neurons strongly suggests that calcium signaling and regulation could be compartmentalized. Region-specific expression of calcium extrusion transporters would serve also to partition calcium regulation within single cells. Little is known about selective localization of the calcium extrusion transporters, nor has compartmentalized calcium regulation within single neurons been studied in detail. Sensory neurons provide an experimentally tractable preparation to investigate this functional compartmentalization. We studied calcium regulation in the outer segment (OS) and inner segment/synaptic terminal (IS/ST) regions of rods and cones. We report these areas can function as separate compartments. Moreover, ionic, pharmacological, and immunolocalization results show that a Ca-ATPase, but not the Na+/K+, Ca2+ exchanger found in the OSs, extrudes calcium from the IS/ST region. The compartmentalization of calcium regulation in the photoreceptor outer and inner segments implies that transduction and synaptic signaling can be independently controlled. Similar separation of calcium-dependent functions is likely to apply in many types of neuron.

A direct and melanopsin-dependent fetal light response regulates mouse eye development

Vascular patterning is critical for organ function. In the eye, there is simultaneous regression of embryonic hyaloid vasculature (important to clear the optical path) and formation of the retinal vasculature (important for the high metabolic demands of retinal neurons). These events occur postnatally in the mouse. Here we have identified a light-response pathway that regulates both processes. We show that when mice are mutated in the gene (Opn4) for the atypical opsin melanopsin, or are dark-reared from late gestation, the hyaloid vessels are persistent at 8 days post-partum and the retinal vasculature overgrows. We provide evidence that these vascular anomalies are explained by a light-response pathway that suppresses retinal neuron number, limits hypoxia and, as a consequence, holds local expression of vascular endothelial growth factor (VEGFA) in check. We also show that the light response for this pathway occurs in late gestation at about embryonic day 16 and requires the photopigment in the fetus and not the mother. Measurements show that visceral cavity photon flux is probably sufficient to activate melanopsin-expressing retinal ganglion cells in the mouse fetus. These data thus show that light—the stimulus for function of the mature eye—is also critical in preparing the eye for vision by regulating retinal neuron number and initiating a series of events that ultimately pattern the ocular blood vessels.