XB-ART-12566J Neurosci August 15, 1999; 19 (16): 7066-76.
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Nitric oxide in the retinotectal system: a signal but not a retrograde messenger during map refinement and segregation.
The role of nitric oxide (NO) as a mediator of synaptic plasticity is controversial in both the adult and developing brain. NO generation appears to be necessary for some types of NMDA receptor-dependent synaptic plasticity during development but not for others. Our previous work using several NO donors revealed that Xenopus laevis retinal ganglion cell axons stop growing in response to NO exposure. We demonstrate here that the same response occurs in tectal neuron processes bathed in the NO donor S-nitrosocysteine (SNOC) and in RGC growth cones to which SNOC is very locally applied. We show that NO synthase (NOS) activity is present in the Rana pipiens optic tectum throughout development in a dispersed subpopulation of tectal neurons, although effects of NO on synaptic function in a Rana pipiens tectal slice were varied. We chronically inhibited NOS in doubly innervated Rana tadpole optic tecta using L-N(G)-nitroarginine methyl ester in Elvax. Despite significant NOS inhibition as measured biochemically, eye-specific stripes remained normally segregated. This suggests that NOS activity is not downstream of NMDA receptor activation during retinotectal synaptic competition because NMDA receptor activation is necessary for segregation of retinal afferents into ocular dominance stripes in the doubly innervated tadpole optic tectum. We conclude that NO has some signaling function in the retinotectal pathway, but this function is not critical to the mechanism that refines the projection and causes eye-specific stripes.
PubMed ID: 10436061
PMC ID: PMC6782861
Article link: J Neurosci
Species referenced: Xenopus laevis
Genes referenced: mhc2-dab (provisional) nos1 nos3 tecta.2
Antibodies: Nos1 Ab3
Article Images: [+] show captions
|Fig. 1. Local application of the NO donor SNOC causes RGC axonal growth cone collapse. A1, A phase image of a pipette filled with FITC–dextran control solution positioned near an RGC axonal growth cone is shown. A2, This is the same field as A1 but was imaged using fluorescence optics. Pulses of positive pressure applied to the pipette release small amounts of the FITC–dextran solution. B1, Pipettes were filled with solutions containing the NO donor SNOC and positioned near actively extending growth cones. Positive pressure to the pipette caused release of NO donor solution onto the growth cone.B2, This is the same field as B1 but 5 min after the onset of SNOC application. Puffs of the NO donor solution caused motility inhibition and collapse of the lamellipodium in this growth cone and most of the others examined. Scale bar, 10 μm.|
|Fig. 2. Both NADPH–diaphorase and immunohistochemistry reveal NOS in neurons in tadpole optic tectum. Dorsal isup, and lateral is to the right.A, Several NADPH–diaphorase-positive neurons in the densely packed layer 6, the main retinorecipient cell layer, can be seen sending apical dendrites dorsally that penetrate the layer 9 neuropil. In addition, three neurons in layer 4 and a single neuron in layer 8 are labeled. Blood vessels (bv) are also labeled in this tissue. P indicates the pial membrane layer.B, A coronal section from another tectum was immunostained with a commercial polyclonal type I NOS antibody. Layers 6 and 7 are shown. Several neurons with a morphology similar to those in NADPH—diaphorase-stained tissue can be seen. Scale bar: A, 80 μm; B, 50 μm.|
|Fig. 3. Chronic treatment of normal, developingRana pipiens tadpole optic tecta with L-NAME but not D-NAME in Elvax inhibits tectal NOS activity.L-NAME is a NOS inhibitor, and D-NAME is the inactive isomer used as a control. The normal groups (N) were untreated, the L-NAME groups (L) were assayed at 2, 4, and 6 weeks of treatment, and the D-NAME groups (D) were assayed at 2 and 4 weeks of treatment. Degree of blockade did not correlate with treatment time. Mean ± SEM.|
|Fig. 4. Chronic L-NAME treatment does not desegregate stripes in the doubly innervated optic tectum of three-eyed tadpoles. In A and B, the supernumerary optic nerve was labeled with HRP, and the brain was reacted with DAB. The labeled tectal lobe was cut at the rostral and caudal poles and then was flat-mounted. Rostral is up, and medial is to the right. (The black curves in some images are outside of the tissue and are the edge of an air bubble within the slide.) A1,A2, Doubly innervated tecta from two untreated tadpoles show normal eye-specific stripes. The forks, fusions, and breaks in the stripes are all normal. B1–B3, L-NAME treatment for 4 weeks does not alter stripe segregation. Three different tectal lobes from treated animals are shown; all stripes appear normal. Note that stripe sharpening, seen as very abrupt boundaries of each stripe and which occurs with chronic NMDA treatment (Cline and Constantine-Paton, 1990), is also not observed afterL-NAME treatment. A good example of the puffs characteristic of caudal-most areas in many doubly innervated tecta can be seen in B3. The small black dots inB1 and B2 are pieces of pigmented membrane that escaped removal during dissection. C, A coronal section from an L-NAME-treated three-eyed tadpole optic tectum reveals that stripe segregation occurred throughout the thickness of the neuropil, indicating that segregation for all RGC types was normal under conditions of chronic NOS inhibition. Dorsal isup, and lateral is to the right. Scale bar: A, B, 500 μm; C, 400 μm.|
|Fig. 5. Normal eye-specific stripes are also preserved in tadpole tecta with chronic NOS inhibition when the supernumerary retina innervates complementary areas of the two tectal lobes. Projections were labeled as in Figure 4 Aand B. Medial (M) is in themiddle of the figure, and lateral is in the direction of the arrows, toward the outside of the figure for each tectal lobe. In both A and B, the stripes are complementary. In areas with double innervation, eye-specific stripes form. A1, A2, Both tectal lobes (left and right) from a single animal chronically treated with D-NAME, the inactive isomer, are shown. The supernumerary eye innervated and caused segregation in the medial region of the left tectal lobe and the lateral region of the right tectal lobe. B1, B2, Both tectal lobes (left and right) from a single animal chronically treated with L-NAME, which inhibits NOS, are shown. The supernumerary eye innervated and caused segregation along the medial and lateral border of the left tectal lobe and within the central region of the right tectal lobe. In both sets of tecta, regions of low-density supernumerary eye innervation also show evidence of segregation. Scale bar: A, 400 μm;B, 500 μm.|
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