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Synaptic inputs to identified color-coded amacrine and ganglion cells in the turtle retina.

Previous studies have proposed models of the specific synaptic circuitry responsible for color processing in the turtle retina. To determine the accuracy of these models of the circuits underlying color opponency in the inner retina of the turtle (Pseudemys scripta), we have studied the physiology, morphology, and synaptic connectivity of identified amacrine and ganglion cells. These cells were first characterized electrophysiologically and were then stained with horseradish peroxidase. Postembedding electron immunocytochemistry for gamma-aminobutyric acid (GABA) and glycine was used to reveal the neurochemical identity of their synaptic inputs. The red-ON/green, blue-OFF small-field ganglion cell, classified as G24, branched primarily in strata S1, S4, and S5 of the inner plexiform layer (IPL). Ganglion cell G24 showed a complex receptive field organized into a red-ON center surrounded by an inhibitory region, which, in turn, was surrounded by a second excitatory region. Only the center responses were color opponent. The red-OFF/green, blue-ON large-field, stellate amacrine cell, classified as A23b, stratified exclusively in stratum S2, near the S2/S3 border. The color-coded center was surrounded by a luminosity, red-sensitive surround. Synaptic input to G24 and A23b was dominated by amacrine cells (89% and 87%, respectively). G24 received significant input from amacrine cell profiles with GABA (13% of total) as well as glycine (11% of total) immunoreactivity, mostly in the proximal stratum S5 of the IPL (64% and 67% of the total GABA- and glycine-immunoreactive input, respectively). Bipolar cell synaptic input was also found predominantly in S4 and S5 (89%). In contrast, we found no glycine-immunoreactive input to A23b, and the density of the GABA-immunoreactive amacrine cell synaptic input revealed a central (15%) to peripheral (3%) gradient within the dendritic tree. The results of the present study support the previous models of the synaptic circuitry responsible for color-opponent signal processing in the inner retina of the turtle.

Animals↗

Stimulation with N-methyl-D-aspartate or kainic acid increases cyclic guanosine monophosphate-like immunoreactivity in turtle retina: involvement of nitric oxide synthase.

In brain and retina, stimulation with excitatory amino acids (EAA) can generate nitric oxide (NO) and increase levels of cyclic guanosine monophosphate (cGMP). Because nitric oxide synthase (NOS) has been found in retinas of all species examined to date, an NO signal-transduction pathway is likely to be present in all retinas. We tested the hypothesis that stimulation of ionotropic glutamate receptors in turtle retina would result in increases in cGMP through an NOS/NO/cGMP pathway. Following in vitro incubations of turtle eye cups with the glutamate receptor agonists, N-methyl-D-aspartate (NMDA) or kainic acid (KA), we quantified the increases in cGMP-like immunoreactivity (cGMP-LI) by using enzyme-linked immunosorbant assay (ELISA) and localized the increased cGMP-LI by using an antibody against cGMP. Stimulation with NMDA or KA increased cGMP-LI in bipolar and amacrine cells as well as in some somata in the ganglion cell layer. Either KA or NMDA produced statistically significant increases in total retinal cGMP-LI by ELISA. To test the involvement of NO, we used the NOS inhibitors 7-nitroindazole and L-nitroarginine. Both inhibitors blocked virtually all of the KA- or NMDA-stimulated increases in cGMP-LI. These results indicate that activation of ionotropic glutamate receptors can increase cGMP in select retinal neurons. Differences between the agonist-evoked increases of retinal cGMP-LI suggest that there can be specificity in the activation of the NOS/NO/cGMP signal-transduction pathway by glutamate. This suggests that, in addition to short-term electrical changes, activation of ionotropic glutamate receptors also may produce longer term modulatory or metabolic effects involving NO/cGMP.

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Characterization of disease-specific brain fluorophores in ceroid-lipofuscinosis.

By isolating and identifying the molecular components of the storage material in the ceroid-lipofuscinoses, it should be possible to elucidate the metabolic basis for these diseases. Using brains of English setter dogs afflicted with a form of this disorder, the autofluorescent storage granules have been isolated and subjected to extraction with chloroform-methanol. A significant amount of autofluorescent material was solubilized by this procedure. Spectral analysis of the extracts indicated that the disease-specific fluorophores have corrected fluorescence excitation maxima at 335-340 nm and emission maxima at 530 nm. Analysis of the extracts with thin layer chromatography showed the presence of several disease-related fluorophores. In addition, the amounts of several nonfluorescent lipids appeared to be enhanced as a result of ceroid-lipofuscinosis. A cerebral cortex sample from a human subject who died with the late infantile form of ceroid-lipofuscinosis was subjected to the same analytical procedures. The chloroform-methanol extract from the human brain had a fluorescence spectrum very similar to the extracts from the brains of affected dogs. TLC analysis showed a single fluorophore from the human brain sample. This fluorophore had the same retention time as the most prominent fluorophore present in the samples from affected dogs. These findings are consistent with the possibility that the defects underlying the human and canine ceroid-lipofuscinoses are similar. The analytical procedures established in these experiments are useful in evaluating the various models of ceroid-lipofuscinosis and may ultimately serve as the basis for distinguishing among different forms of these disorders.(ABSTRACT TRUNCATED AT 250 WORDS)

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The physiological cross-sectional area of motor units in the cat tibialis anterior.

The physiological cross-sectional area (CSA) of a motor unit (MU), taken as the sum of fiber areas measured on a single section through the approximate midlength of the MU, has been compared with the physiological CSA more strictly defined as the sum of the maximal areas to be found anywhere along the length of each of the MU fibers. The CSA at intervals along the fiber length was measured in fibers selected from four glycogen-depleted, isolated MUs in the cat tibialis anterior (TA), and profiles of the summed areas made. In one MU, measurements were also taken on all the MU's fibers at less frequent intervals. The profiles demonstrate that the summed CSA based on each fiber's maximum CSA may exceed that derived from observation on any single section by as much as 20%. As a consequence, values that have been reported for specific tension (force per unit area) of MUs in the TA and probably other muscles may have been overestimated, especially for those MUs of fast type. Estimates were also made of the share of the MU's total force transmitted directly to the tendons of origin and insertion via endings of the blunt musculotendinous type as distinct from tapering intrafascicular endings acting through in-series connective tissue and non-MU fibers. In two MUs of slow type in which most fibers ran from tendon to tendon, "partial tapering" extending over 1 cm of the fiber length accounted for a third of the total physiological CSA, and indicated yet another mode for relay of the MU's force to the tendon.

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Tapering of the intrafascicular endings of muscle fibers and its implications to relay of force.

The geometric shape of the filamentous, intrafascicular type of muscle fiber ending was reconstructed as a basis for understanding the pattern in relay of the fiber's force to the muscle tendon. Single motor units (MUs) identified physiologically as being fast and slow, respectively, were isolated in cat tibialis muscles and glycogen-depleted for recognition in cross sections of the muscle frozen at its Lo. Serial measurements of cross-sectional area (CSA) using an image processing system were made along 14 intrafascicular endings of MU fibers and an additional seven, non-depleted fibers identified histochemically as slow. Comparison of coefficients of variation for the linear relation of the CSAs and of the equivalent diameters with length along the taper indicated that in both fast and slow fibers the areas bore a closer relationship, that is, the taper had the equivalent of a parabolic, rather than a conical outline. The implications of these two conformations to relay of the fiber's contractile force to surrounding structures are displayed graphically.

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Nitric oxide stimulates gamma-aminobutyric acid release and inhibits glycine release in retina.

Nitric oxide (NO) modulates the uptake and/or release of neurotransmitters through a variety of cellular mechanisms. However, the pharmacological and biochemical processes underlying these neurochemical effects of NO often remain unclear. In our study, we used immunocytochemical methods to study the effects of NO, cyclic guanosine monophosphate (cGMP), and peroxynitrite on the uptake and release of gamma-aminobutyric acid (GABA) and glycine in the turtle retina. In addition, we examined the involvement of glutamate receptors, calcium, and the GABA transporter in this GABA uptake and release. We also tested for interactions between the GABAergic and glycinergic systems. In general, we show that NO stimulated GABA release and inhibited glycine release. The NO-stimulated GABA release involved calcium-dependent or calcium-independent synaptic release or reversal of the GABA transporter. Some effects of NO on GABA release involved glutamate, cGMP, or peroxynitrite. NO promoted glycine uptake and inhibited its release, and this inhibition of glycine release was influenced by GABAergic modulation. These findings indicate that NO modulates the levels of the inhibitory transmitters GABA and glycine through several specific biochemical mechanisms in different retinal cell types and layers. Thus it appears that some of the previously described reciprocal interactions between GABA and glycine in the retina function through specific NO signaling pathways.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

Spinal projection to the dorsolateral nucleus of the caudal basilar pons in the cat.

In the cat, a spinal projection to a restricted area of the basilar pontine grey has been revealed with use of anterograde degeneration technique (Fink-Heimer). The area was ipsilateral to the spinal lesion, restricted to the far caudal limit of the pons, and included the dorsal and the dorsolateral subdivisions of the pontine nuclei (PN). Comparisons following high cervical, midthoracic and upper lumbar spinal lesions did not reveal any somatotopic organization. Only a few spinopontine fibers had origins below segmental level L4. Lesions of various quadrants of the cord indicated that the spinopontine fibers ascended through the dorsolateral funiculus, and not through either the dorsal or the ventral funiculi. Comparison with the degeneration effects of cerebral cortical lesions showed that the spinal projection to the PN overlapped to some extent with the projection from the first sensorimotor and second somatosensory cortices. In the rat no comparable spinopontine projection was found. It is suggested that the spinopontine pathway might forward information to the cerebellum from visceral sensory receptors or perhaps from pools of spinal interneurons.

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Multiple classes of photoreceptors and neurons in the frontal organ of Rana pipiens.

An attempt has been made to characterize the photoreceptors and neurons of the frontal organ of Rana pipiens and the synaptic contacts of these cells, in order to provide morphological correlates for previous physiological findings. Neurons, glial cells, and photoreceptors with two different appearances can be seen in normal anatomical preparations. The photoreceptors make ribbon synaptic contacts onto both neurons and other photoreceptors. Retrograde transport of either cobalt or horseradish peroxidase through the frontal nerve results in labeling of neurons which are often located near the exit point of the frontal nerve from the frontal organ. Some of the dendritic processes of these neurons ramify among the neurons themselves, while others ramify among the photoreceptors. In addition, the frontal organ contains two photoreceptor types which synaptically contact both neurons and other photoreceptors. In some frontal organs labeled with cobalt (but never in peroxidase-labeled preparations) certain photoreceptors were also labeled. The labeled photoreceptors were probably the result of transcellular cobalt movement from labeled neurons, and not the result of some photoreceptors having axons in the frontal nerve. These results are discussed in conjunction with physiological evidence that chromatic interactions occur within single photoreceptors, each of which contains a single visual pigment with two physiologically active states.

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