Synaptic activity of frog retinal photoreceptors. A peroxidase uptake study

The uptake of horseradish peroxidase (HRP) into membranous structures, detectable by light and electron microscopy, is used here to monitor the synaptic activity of photoreceptors of isolated frog retinas maintained in the dark or under various illumination conditions. The major findings are: (a) Neurotransmission from photoreceptor terminals seems to involve the same types of endocytic membrane-retrieval processes that occur at other nerve terminals. Presumably, the endocytic processes compensate for exocytic events associated with neurotransmission. The retrieved membrane is "recycled" to form vesicles. Some of these accumulate near the synaptic ribbons, perhaps indicating reutilization for exocytosis. On the other hand, some retrieved membrane evidently is degraded via multivesicular bodies that appear to undergo "retrograde" transport from the receptor synapses to the myoid regions. (b) Photoreceptor terminals take up much HRP in the dark. Steady illumination markedly decreases uptake by rods. Uptake by cones is notably reduced only at illumination intensities higher than those that have maximal effects on rods. (c) The decrease in rod HRP uptake with light is reversible when retinas are allowed to adapt to the dark, if the light exposures used were at intensities that bleach very little visual pigment. Such "recovery" is not observed after light exposures that bleach a considerable amount of visual pigment. The cones recover their dark levels of HRP uptake even after light exposures that bleach considerable amounts of visual pigment. The changes in HRP uptake that we observe parallel expectations for photoreceptor synaptic neurotransmission derived from indirect physiological evidence.     

Circulation and turnover of synaptic vesicle membrane in cultured fetal mammalian spinal cord neurons

Intact neurons in cultures of fetal rodent spinal cord explants show stimulation-dependent uptake of horseradish peroxidase (HRP) into many small vesicles and occasional tubules and multivesicular bodies (MVB) at presynaptic terminals. Presynaptic terminals were allowed to take up HRP during 1 h of strychnine-enhanced stimulation of synaptic transmitter release and then "chased" in tracer-free medium either with strychnine or with 10 mM Mg++ which depresses transmitter release. Tracer-containing vesicles are lost from terminals under both chase conditions; the loss is more rapid (4-8 h) with strychnine than with 10 mM Mg++ (8-16 h). There is a parallel decrease in the numbers of labeled MVB's at terminals. Loss of tracer with 10 mM Mg++ does not appear to be due to the membrane rearrangements (exocytosis coupled to endocytosis) that presumably lead to initial tracer uptake; terminals exposed to HRP and Mg++ for up to 16 h show little tracer uptake into vesicles. Nor is the decrease likely to the due to loss of HRP enzyme activity; HRP is very stable in solution. During the chases there is a striking accumulation of HRP in perikarya that is far more extensive in cultures initially exposed to tracer with strychnine than 10 mM Mg++ regardless of chase conditions. Much of the tracer ends up in large dense bodies. These findings suggest that synaptic vesicle membrane turnover involves retrograde axonal transport of membrane to neuronal perikarya for further processing, including lysosomal degradation. The more rapid (4-8 h) loss of tracer-containing vesicles with strychnine may reflect vesicle membrane reutilization for exocytosis.     

Ca2+-dependent recycling of synaptic vesicles at the frog neuromuscular junction

Frog cutaneous pectoris muscles were treated with low doses of crude black widow spider venom (BWSV) or purified alpha-latrotoxin, and neuromuscular transmission, quantal secretion, changes in ultrastructure and uptake of horseradish peroxidase (HRP) were studied. When these agents were applied to muscles bathed in a Ca2+-free solution with 1 mM EGTA and 4 mM Mg2+, the rate of quantal secretion rose to high levels but quickly subsided; neuromuscular transmission was totally and irreversibly blocked within 1 h. The terminals became swollen and were depleted of vesicles; HRP was not taken up. When BWSV was applied to other muscles bathed in a solution with 1.8 mM Ca2+ and 4 mM Mg2+, the rate of secretion rose to high levels and then declined to intermediate levels that were sustained throughot the hour of exposure. Neuromuscular transmission was blocked in fewer than 50% of these fibers. The ultrastructure of these terminals was normal and they contained large numbers of synaptic vesicles. If HRP had been present, most of the synaptic vesicles were labeled with reaction product. These observations suggest that Ca2+ plays an important role in endocytosis at the frog neuromuscular junction.     

Dopaminergic Regulation of Cone Retinomotor Movement in Isolated Teleost Retinas: I. Induction of Cone Contraction Is Mediated by D2 Receptors

In the retinas of lower vertebrates, retinal photoreceptors and melanin pigment granules of the retinal pigment epithelium (RPE) undergo characteristic movements in response to changes in light intensity and to signals from an endogenous circadian clock. To identify agents responsible for mediating light and/or circadian regulation of these retinomotor movements, we investigated the effects of hormones and neurotransmitters on cone, rod, and RPE movements in the green sunfish, Lepomis cyanellus. We report here that 3,4-dihydroxyphenylethylamine (dopamine) mimics the effect of light by inducing light-adaptive retinomotor movements in all three cell types. In isolated dark-cultured retinas, dopamine induced light-adaptive cone contraction with a half-maximal effect at 10(-8) M. This effect of dopamine was inhibited by antagonists with a potency order characteristic of D2 receptor mediation. The dopamine uptake blocker benztropine also induced light-adaptive cone contraction in isolated dark-cultured retinas, suggesting that there is continuous dopamine release in the dark but that concomitant uptake normally prevents activation of cone contraction. That dopamine plays a role in light regulation of cone movement is further suggested by the observation that light-induced cone contraction was partially inhibited by sulpiride, a selective D2 dopamine antagonist, or by Co2+, a blocker of synaptic transmission. Sulpiride also promoted dark-adaptive cone elongation in isolated light-adapted retinas, suggesting that continuous dopamine action is required in the light to maintain the light-adapted cone position. Dopamine can act directly on D2 receptors located on rod and cone inner/outer segments: dopamine induced light-adaptive retinomotor movements in isolated distal fragments of dark-adapted photoreceptors cultured in the dark. Together our results indicate that dopamine induces light-adaptive retinomotor movements in cones, rods, and RPE cells by activating D2 receptors. We suggest that, in vivo, dopamine plays a role in both light and circadian regulation of retinomotor movements.     

Ultrastructural Analysis of the Terminals of Various Somatosensory Pathways in the Ventrobasal Complex of the Raft Thalamus: An Electron-Microscopic Study Using Wheatgerm Agglutinin Conjugated to Horseradish Peroxidase as an Axonal Tracer

We used wheatgerm agglutinin conjugated to horseradish peroxidase (WGA:HRP) as an anterograde tracer to label the terminals of the lemniscal, spinothalamic, and trigeminothalamic pathways in the ventrobasal complex of the rat thalamus (VB). The use of benzidine dihydrochloride (BDHC) as the chromogen allowed us to view the labeled profiles with the electron microscope and permitted us to compare the morphology of the terminals from the various pathways. We found that all the labeled somatosensory pathways terminate in the VB in the form of large terminals that contain round synaptic vesicles and make numerous asymmetrical synaptic contacts, usually with dendritic protrusions and proximal dendrites. The present results demonstrate that pathways conveying noxious and non-noxious somatosensory information terminate upon thalamic neurons with synaptic terminals having similar morphological features.     

The uptake of horseradish peroxidase by cortical synapses in rat brain

Summary Horseradish peroxidase (HRP) was introduced directly into the cerebral cortex of adult rats, which were allowed to survive for 60 min before perfusion fixation. After the tissue had been incubated to demonstrate HRP at the LM and EM levels, blocks of cortical tissue were taken at varying distances from the injection site. These eight blocks of tissue constituted a time sequence for HRP diffusion.Qualitative examination of the presynaptic terminals showed that the most commonly encountered profiles are the plain synaptic vesicles, many of which accumulate tracer. In some terminals labelled vesicles are lined-up in tubular fashion. Other profiles commonly labelled are coated vesicles, tubular and vacuolar cisternae, and plain and coated pinocytotic vesicles.Quantitative analyses based on the number of terminals containing labelled profiles demonstrate an early rise in the rate of labelling of both plain synaptic vesicles and coated vesicles, after which synaptic vesicle labelling rises slowly towards a plateau. By contrast, there is a late parallel increase in the rate of labelling of coated vesicles and cisternae. A more detailed analysis, based on the actual numbers of labelled and total profiles within each presynaptic terminal, highlight early and late periods of rapid labelling for plain synaptic vesicles, coated vesicles and cisternae. A further aspect of HRP incorporation studied, concerns its uptake into four delineated regions of the presynaptic terminal.Our data indicate that membrane uptake into the presynaptic terminal is accomplished mainly via coated vesicles, although plain synaptic vesicles may also be involved. Coated vesicles, in turn, appear to give rise directly to plain synaptic vesicles, with some coalescing to produce vacuolar cisternae. The latter are involved in a two-way interchange of membrane with tubular cisternae, plain synaptic vesicles and coated vesicles. An additional source of plain synaptic vesicles are the tubular cisternae. Exocytosis of plain synaptic vesicles constitutes the mechanism by which transmitter is released from the presynaptic terminal.Supported by the Nuffield Foundation. We are grateful to Mr. M. Austin for help with the photography     

EVIDENCE FOR RECYCLING OF SYNAPTIC VESICLE MEMBRANE DURING TRANSMITTER RELEASE AT THE FROG NEUROMUSCULAR JUNCTION

When the nerves of isolated frog sartorius muscles were stimulated at 10 Hz, synaptic vesicles in the motor nerve terminals became transiently depleted. This depletion apparently resulted from a redistribution rather than disappearance of synaptic vesicle membrane, since the total amount of membrane comprising these nerve terminals remained constant during stimulation. At 1 min of stimulation, the 30% depletion in synaptic vesicle membrane was nearly balanced by an increase in plasma membrane, suggesting that vesicle membrane rapidly moved to the surface as it might if vesicles released their content of transmitter by exocytosis. After 15 min of stimulation, the 60% depletion of synaptic vesicle membrane was largely balanced by the appearance of numerous irregular membrane-walled cisternae inside the terminals, suggesting that vesicle membrane was retrieved from the surface as cisternae. When muscles were rested after 15 min of stimulation, cisternae disappeared and synaptic vesicles reappeared, suggesting that cisternae divided to form new synaptic vesicles so that the original vesicle membrane was now recycled into new synaptic vesicles. When muscles were soaked in horseradish peroxidase (HRP), this tracerfirst entered the cisternae which formed during stimulation and then entered a large proportion of the synaptic vesicles which reappeared during rest, strengthening the idea that synaptic vesicle membrane added to the surface was retrieved as cisternae which subsequently divided to form new vesicles. When muscles containing HRP in synaptic vesicles were washed to remove extracellular HRP and restimulated, HRP disappeared from vesicles without appearing in the new cisternae formed during the second stimulation, confirming that a one-way recycling of synaptic membrane, from the surface through cisternae to new vesicles, was occurring. Coated vesicles apparently represented the actual mechanism for retrieval of synaptic vesicle membrane from the plasma membrane, because during nerve stimulation they proliferated at regions of the nerve terminals covered by Schwann processes, took up peroxidase, and appeared in various stages of coalescence with cisternae. In contrast, synaptic vesicles did not appear to return directly from the surface to form cisternae, and cisternae themselves never appeared directly connected to the surface. Thus, during stimulation the intracellular compartments of this synapse change shape and take up extracellular protein in a manner which indicates that synaptic vesicle membrane added to the surface during exocytosis is retrieved by coated vesicles and recycled into new synaptic vesicles by way of intermediate cisternae.     

Effect of barium and tetraethylammonium on membrane circulation in frog retinal photoreceptors

We studied the influence of altered ionic conditions on the recycling of synaptic vesicle membrane in frog retinal photoreceptors using horseradish peroxidase to monitor synaptic activity and trace the fate of internalized membrane. The addition of 1.2 mM barium or 20 mM tetraethylammonium to isolated retinas maintained in Ringer's solution, changes the usual balance of membrane circulation in the rod cells; the cone cells are much less affected. Retrieval of synaptic vesicle membrane in the rods, which normally regenerates small vesicles, becomes mediated predominantly by large sacs and vacuoles ("cisternae"). Because these cisternae can be labeled with peroxidase, they appear to arise from endocytized membrane. Morphometric analysis suggests strongly that the cisternae are formed of circulating synaptic vesicle membrane. The effects of barium and tetraethylammonium can be inhibited by high extracellular potassium, by high intensity light, and by 5 mM cobalt. They seem likely to depend on potassium channels, though additional more complex mediation may also be involved. The alterations in membrane retrieval that we find are of interest in terms of the multiple pathways of membrane cycling now being uncovered. They open potential experimental approaches to the controls of this circulation. In addition, the findings extend our previous ones demonstrating that rod cells and cone cells differ in their responses to divalent cations in ways that seem likely to be of physiological importance.     

Dopaminergic Regulation of Cone Retinomotor Movement in Isolated Teleost Retinas: II. Modulation by γ-Aminobutyric Acid and Serotonin

In the accompanying paper we reported that 3,4-dihydroxyphenylethylamine (dopamine) induced light-adaptive retinomotor movements in teleost photoreceptors and that this effect was mediated by D2 dopamine receptors located on the photoreceptors themselves. In this study, we investigated the effects on cone retinomotor movement of three agents that have been reported by others to modulate retinal dopamine release: gamma-aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT, serotonin), and melatonin. We report here that the GABA antagonists bicuculline and picrotoxin induced light-adaptive cone contraction in dark-adapted green sunfish retinas cultured in constant darkness; thus they mimic the effect of light or exogenously applied dopamine. Since their effects were blocked by either the D2 dopamine antagonist sulpiride or by Co2+, it seems likely that these agents act by enhancing retinal dopamine release. The GABA agonist muscimol produced effects opposite to those of GABA antagonists. Muscimol inhibited light-induced cone contraction in previously dark-adapted retinas and induced dark-adaptive cone elongation in light-adapted retinas. These results suggest that in green sunfish retinas, as has been reported for other retinas, GABA inhibits dopamine release. 5-HT induced light-adaptive cone contraction in dark-adapted retinas; thus 5-HT also mimics the effect of light or exogenously applied dopamine. The effect of 5-HT was blocked by sulpiride, Co2+, or the 5-HT antagonist mianserin. These results suggest that 5-HT induces cone contraction by stimulating dopamine release. Melatonin neither inhibited dopamine-induced cone contraction in retinas cultured in the dark nor induced cone elongation in retinas cultured in the light. Our results suggest that both GABA and 5-HT (but not melatonin) affect cone retinomotor movements in green sunfish by modulating dopamine release: GABA by inhibiting and 5-HT by stimulating dopamine release. We report in the companion paper that dopamine induced contraction in isolated cone fragments. Together these observations strongly suggest that dopamine serves as the final extracellular messenger directly inducing light-adaptive cone retinomotor movement, and that GABA and 5-HT affect these movements by modulating dopamine release.     

Differential sensitivity of rods and cones in Xenopus retina to hemicholinium-3

Summary Hemicholinium-3 (HC-3), a drug which prevents synthesis of acetylcholine in neurons, when injected intraperitoneally in doses as low as 2×5 mg/kg produces marked ultrastructural changes and damage in rod but not in cone photoreceptors. In rods it causes a reduction in cytoplasmic back-ground density, swelling of the endoplasmic reticulum, ballooning of the outer membrane of the nucleus, leaching of the nucleoplasm and clumping of the nuclear chromatin. In dark-adapted rods HC-3 produces some loss of cytoplasmic synaptic vesicles but no reduction in numbers of those vesicles which lie adjacent to the synaptic ribbons. In light-adapted rods the drug does not cause such an apparent reduction of synaptic vesicles but does induce a considerable reduction in numbers of vesicles associated with the ribbons. These structural changes are discussed in the light of what is known about the pharmacology of HC-3 and neurotransmitter release from vertebrate photoreceptors.The authors wish to thank SRC for a grant in support of this work     

Light-dependent ion influx into toad photoreceptors

To measure the influx of Na+ and other ions through the light-dependent permeability of photoreceptors, we superfused the isolated retina of the toad, Bufo marinus, with a low-Ca2+ (10(-8) M), low-Cl- Ringer's solution containing 0.5 mM ouabain. Under these conditions, the membrane potential of the rod is near zero and there is no light-induced potential change either in the rod or in more proximal neurons. The photoreceptors, however, continue to show a light-dependent increase in membrane resistance, which indicates that the light-sensitive channels still close with illumination. Dark-adapted retinas show a larger 22Na+ accumulation than do light-adapted retinas. The extra accumulation of 22Na+ into dark-adapted retinas can be removed if the retinas are washed in darkness with low-Ca2+ Ringer's solutions, or if the ionophore gramicidin D is present in the perfusate. The additional accumulation in dark retinas corresponds to a flux of at least 10(9) Na+ per receptor per second, which is close to the value of the photoreceptor dark current. The light-dependent uptake of 22Na+ can be prevented by exposing the retinas to Ca2+ during the incubation period, but is restored if the phosphodiesterase inhibitor IBMX is added to the perfusate. A significant light-dependent ion accumulation can be observed for the cations K+, Rb+, Cs+, and Tl+, in addition to Na+, but not for methylamine, choline, or tetraethylammonium.     

Conical tomography of a ribbon synapse: structural evidence for vesicle fusion

To characterize the sites of synaptic vesicle fusion in photoreceptors, we evaluated the three-dimensional structure of rod spherules from mice exposed to steady bright light or dark-adapted for periods ranging from 3 to 180 minutes using conical electron tomography. Conical tilt series from mice retinas were reconstructed using the weighted back projection algorithm, refined by projection matching and analyzed using semiautomatic density segmentation. In the light, rod spherules contained ～470 vesicles that were hemi-fused and ～187 vesicles that were fully fused (omega figures) with the plasma membrane. Active zones, defined by the presence of fully fused vesicles, extended along the entire area of contact between the rod spherule and the horizontal cell ending, and included the base of the ribbon, the slope of the synaptic ridge and ribbon-free regions apposed to horizontal cell axonal endings. There were transient changes of the rod spherules during dark adaptation. At early periods in the dark (3-15 minutes), there was a) an increase in the number of fully fused synaptic vesicles, b) a decrease in rod spherule volume, and c) an increase in the surface area of the contact between the rod spherule and horizontal cell endings. These changes partially compensate for the increase in the rod spherule plasma membrane following vesicle fusion. After 30 minutes of dark-adaptation, the rod spherules returned to dimensions similar to those measured in the light. These findings show that vesicle fusion occurs at both ribbon-associated and ribbon-free regions, and that transient changes in rod spherules and horizontal cell endings occur shortly after dark onset.     

Rod cells dissociated from mature salamander retina: ultrastructure and uptake of horseradish peroxidase

To test the effects of isolation on adult neurons, we investigated the fine structure and synaptic activity of rod cells dissociated from the mature salamander retina and maintained in vitro. First, freshly isolated rod cells appeared remarkably similar to their counterparts in the intact retina: the outer segment retained its stack of membranous disks and the inner segment contained its normal complements of organelles. Some reorganization of the cell surface, however, was observed: (a) radial fins, present at the level of the cell body, were lost; and (b) the apical and distal surfaces of the inner and outer segments, respectively became broadly fused. Second, the synaptic endings or pedicles retained their presynaptic active zones: reconstruction of serially sectioned pedicles by using three-dimensional computer graphics revealed that 73% of the synaptic ribbons remained attached to the plasmalemma either at the cell surface or along its invaginations. Finally, tracer experiments that used horseradish peroxidase demonstrated that dissociated rod cells recycled synaptic vesicle membrane in the dark and thus probably released transmitter by exocytosis. Under optimal conditions, a maximum of 40% of the synaptic vesicles within the pedicle were labeled. As in the intact retina, uptake of horseradish peroxidase was suppressed by light. Thus, freshly dissociated receptor neurons retained many of their adult morphological and physiological characteristics. In long-term culture, the photoreceptors tended to round up; however, active zones were present even 2 wk after removal of the postsynaptic processes.     

A Survey of the Cytology and Synaptic Organization of the Insular Trigeminal—Cuneatus Lateralis Nucleus in the Rat,Including an Identification of Spinal Afferent Inputs

The cytology and synaptic organization of the insular trigeminal—cuneatus lateralis (iV-Cul) nucleus was examined in the rat. In addition, the ultrastructural morphology and synaptic connectivity of anterogradely labeled spinal afferent axons terminating in iV-Cul were examined following injection of horseradish peroxidase (HRP) into the cervical spinal cord. The uniformity of the ultrastructural features of iV-Cul neurons supports the presence of a homogeneous neuronal population. The most prominent feature of the iV-Cul neuropil is the presence of numerous interdigitating astrocytic processes, which extensively isolate neuronal somata and processes. iV-Cul contains a heterogeneous population of axonal endings that can be separated into three categories, depending upon whether they contain predominantly spherical-shaped agranular synaptic vesicles (R endings), predominantly pleomorphic-shaped agranular synaptic vesicles (P endings), or a heterogeneous population of dense-core vesicles (DC endings). The R endings represent the majority of axonal endings in iV-Cul and establish asymmetrical axodendritic and axospinous synaptic contacts, primarily along the distal portions of the dendritic tree. P endings establish symmetrical axosomatic, axodendritic, and axospinous synaptic contacts and exhibit a more generalized distribution along the somadendritic tree. DC terminals establish asymmetrical axodendritic synaptic contacts with distal dendritic processes and are the least frequently observed endings in the iV-Cul neuropil. Numerous synaptic glomeruli, exhibiting a single large central R bouton that establishes multiple axodendritic or axospinous synapses, characterize the iV-Cul neuropil. Axoaxonic synapses are conspicuously absent from the iV-Cul neuropil and glomeruli. The anterograde HRP labeling of spinal afferent axons that terminate in iV-Cul indicates that the terminals along these fibers are of the R type and that they are engaged predominantly in synaptic glomeruli. The results of this study indicate that the synaptic organization of iV-Cul is distinctly different from that of neighboring somatosensory nuclei, and supports the recent suggestion that this nucleus should be considered a separate precerebellar spinal relay nucleus in the lateral medulla.     

A survey of the cytology and synaptic organization of the insular trigeminal-cuneatus lateralis nucleus in the rat, including an identification of spinal afferent inputs

The cytology and synaptic organization of the insular trigeminal-cuneatus lateralis (iV-Cul) nucleus was examined in the rat. In addition, the ultrastructural morphology and synaptic connectivity of anterogradely labeled spinal afferent axons terminating in iV-Cul were examined following injection of horseradish peroxidase (HRP) into the cervical spinal cord. The uniformity of the ultrastructural features of iV-Cul neurons supports the presence of a homogeneous neuronal population. The most prominent feature of the iV-Cul neuropil is the presence of numerous interdigitating astrocytic processes, which extensively isolate neuronal somata and processes. iV-Cul contains a heterogeneous population of axonal endings that can be separated into three categories, depending upon whether they contain predominantly spherical-shaped agranular synaptic vesicles (R endings), predominantly pleomorphic-shaped agranular synaptic vesicles (P endings), or a heterogeneous population of dense-core vesicles (DC endings). The R endings represent the majority of axonal endings in iV-Cul and establish asymmetrical axodendritic and axospinous synaptic contacts, primarily along the distal portions of the dendritic tree. P endings establish symmetrical axosomatic, axodendritic, and axospinous synaptic contacts and exhibit a more generalized distribution along the somadendritic tree. DC terminals establish asymmetrical axodendritic synaptic contacts with distal dendritic processes and are the least frequently observed endings in the iV-Cul neuropil. Numerous synaptic glomeruli, exhibiting a single large central R bouton that establishes multiple axodendritic or axospinous synapses, characterize the iV-Cul neuropil. Axoaxonic synapses are conspicuously absent from the iV-Cul neuropil and glomeruli. The anterograde HRP labeling of spinal afferent axons that terminate in iV-Cul indicates that the terminals along these fibers are of the R type and that they are engaged predominantly in synaptic glomeruli. The results of this study indicate that the synaptic organization of iV-Cul is distinctly different from that of neighboring somatosensory nuclei, and supports the recent suggestion that this nucleus should be considered a separate precerebellar spinal relay nucleus in the lateral medulla.     

The Intrinsic Organization of the Ventroposterolateral Nucleus and Related Reticular Thalamic Nucleus of the Rat: A Double-Labeling Ultrastructural Investigation with γ-Aminobutyric Acid Immunogold Staining and Lectin-Conjugated Horseradish Peroxidase

An electron-microscopic investigation of the synaptic organization of the rat's ventroposterolateral nucleus (VPL) and of a reticular thalamic nucleus (RTN) area related to somatosensory thalamic nucleus was performed. In a group of 11 rats, wheatgerm agglutinin conjugated to horseradish peroxidase (WGA:HRP) was injected either in the first somatosensory area of cortex (SI) or in the dorsal column nuclei (DCN). The retrogradely and/or anterogradely transported enzyme was visualized using paraphenylenediamine-pyrocatechol (PPD-PC) as substrate. In a second series of six experiments, an immunocytochemical procedure using a specific anti-γ-aminobutyric acid (anti-GABA) was employed. Postembedding localization of GABA was performed for ultrastructural observation by means of the colloidal gold immunostaining procedure. Thin sections of recognized VPL and RTN areas from WGA:HRP-injected animals were further processed for immunocytochemistry in order to localize simultaneously, at the electron-microscopic level, the transported enzyme and GABA.

The results obtained with this procedure demonstrated that HRP-labeled terminals from DCN contacted the soma and proximal dendrites of VPL neurons, while the terminals labeled after SI cortical injections were predominantly localized to the distal portion of the dendrites. The same cortical injection also determined the presence of labeled synaptic boutons contacting the soma, and both proximal and distal dendrites of RTN neurons. GABA-immunolabeled terminals were observed in VPL in a number larger than those observed with other methods, since not only typical F terminals were labeled but also terminals containing round and/or pleomorphic vesicles. GABA-ergic terminals contacted the soma and the proximal and distal dendrites of VPL neurons, while in RTN cells they made synaptic contact mainly with the soma and proximal dendrites. In the double-labeling experiments, terminals containing both HRP and specific immunogold GABA staining were never observed.

The present data provide a direct demonstration of the presence of a strong inhibitory input from RTN upon VPL neurons and of the existence of autoinhibition within RTN neurons.     

Internalization of lectins in neuronal GERL

Conjugates of ricin agglutinin and phytohemagglutinin with horseradish peroxidase (HRP) were used for a cytochemical study of internalization of their plasma membrane "receptors" in cultured isolated mouse dorsal root ganglion neurons. Labeling of cells with lectin-HRP was done at 4 degrees C, and internalization was performed at 37 degrees C in a culture medium free of lectin-HRP. 15-20 min after incubation at 37 degrees C, lectin-HRP receptor complexes were seen in vesicles or tubules located near the plasma membrane. After 1-3 h at 37 degrees C, lectin-HRP-receptor complexes accumulated in vesicles and tubules corresponding to acid phosphatase-rich vesicles and tubules (GERL) at the trans aspect of the Golgi apparatus. A few coated vesicles and probably some dense bodies contained HRP after 3-6 h of incubation at 37 degrees C. Soluble HRP was not endocytosed under the conditions of this experiment or when it was present in the incubation medium at 37 degrees C. Internalization of lectin-HRP-receptor conjugates was decreased or inhibited by mitochondrial respiration inhibitors but not by cytochalasin B or colchicine. These studies indicate that lectin- labeled plasma membrane moieties of neurons are endocytosed primarily in elements of GERL.     

Glycine input induces the synaptic facilitation in salamander rod photoreceptors

Glycinergic synapses in photoreceptors are made by centrifugal feedback neurons in the network, but the function of the synapses is largely unknown. Here we report that glycinergic input enhances photoreceptor synapses in amphibian retinas. Using specific antibodies against a glycine transporter (GlyT2) and glycine receptor β subunit, we identified the morphology of glycinergic input in photoreceptor terminals. Electrophysiological recordings indicated that 10 μM glycine depolarized rods and activated voltage-gated Ca²⁺ channels in the neurons. The effects facilitated glutamate vesicle release in photoreceptors, meanwhile increased the spontaneous excitatory postsynaptic currents in Off-bipolar cells. Endogenous glycine feedback also enhanced glutamate transmission in photoreceptors. Additionally, inhibition of a Cl⁻ uptake transporter NKCC1 with bumetanid effectively eliminated glycine-evoked a weak depolarization in rods, suggesting that NKCC1 maintains a high Cl⁻ level in rods, which causes to depolarize in responding to glycine input. This study reveals a new function of glycine in retinal synaptic transmission.     

The inositide cycle in bovine photoreceptor membranes

Various enzymic steps in the inositide cycle were investigated in purified bovine retinal rod outer segments (ROS). Incubation of ROS with [gamma-32P]ATP resulted in a rapid labeling of phosphatidic acid and phosphatidylinositol 4-phosphate (PIP), while little radio-tracer was recovered from phosphatidylinositol 4,5-bisphosphate (PIP2). This can be explained by the relatively low activity of PIP-kinase activity in ROS as compared to the remainder of the retina. Similarly, relatively little phosphodiesteratic activity degraded PIP2 and PIP in ROS when 32P labeled phosphoinositides in synaptic membranes (heat-treated to inactivate endogenous enzymes) were used. Although light exposure of ROS did cause rapid rhodopsin phosphorylation, no enzymic steps of the cycle were changed, even when ROS were obtained from retinas excised from cows dark-adapted by unilateral eye patching the day prior to kill. These studies do not support the view that light is an agonist of the inositide cycle in mammalian photoreceptors.