Axon Terminals Of Rod Bipolar Cells Research Articles

Retinal neurodegenerative changes in the adult insulin receptor substrate-2 deficient mouse

Insulin receptor substrate-2 (Irs2) mediates peripheral insulin action and is essential for retinal health. Previous investigations have reported severe photoreceptor degeneration and abnormal visual function in Irs2-deficient mice. However, molecular changes in the Irs2−/− mouse retina have not been described. In this study, we examined retinal degenerative changes in neuronal and glial cells of adult (9- and 12-week old) Irs2−/− mice by immunohistochemistry. 9-week old Irs2−/− mice showed significant thinning of outer retinal layers, concomitant to Müller and microglial cell activation. Photoreceptor cells displayed different signs of degeneration, such as outer/inner segment atrophy, redistribution of rod- and cone-opsins and spatial disorganization of cone cells. This was accompanied by synaptic changes at the outer plexiform layer, including the retraction of rod-spherules, reduction of photoreceptor synaptic ribbons and synaptic remodeling in second order neurons (i.e. loss and sprouting of dendritic processes in rod bipolar and horizontal cells). By 12 weeks of age, the thickness of inner retinal layers was severely affected. Although inner plexiform layer stratification remained unchanged at this stage, rod bipolar cell axon terminals were significantly depleted. Significant loss of Brn3a+ retinal ganglion cells occurred in 12-week old Irs2−/− mice, in contrast to younger ages. Adult Irs2−/− mice showed clear hallmarks of neurodegeneration and disruption of the inner retina with increasing age. Pharmacological stimulation of Irs2 signaling pathway may provide additional neuroprotection in certain degenerative retinopathies.

Characterisation of diabetic retinal neuropathy in Ins2Akita mice

AbstractPurpose To characterize diabetic retinal neuropathy in the Ins2Akita mouse.Methods Male heterozygous Ins2Akita mice (2‐8 months of hyperglycemia) and C57BL/6J age‐matched siblings were used in this study. Retinal function was assessed by electroretinography (ERG) and thickness measured by optical coherence tomography. Eyes were then processed for immunostaining of different neuronal cells, including photoreceptors, bipolar, horizontal, amacrine and ganglion cells.Results Ins2Akita mice exhibited altered scotopic ERG after 6‐8 months of hyperglycemia. The reduction of outer and inner retinal thickness was observed after 2 months of diabetes. Despite no striking abnormalities were found in photoreceptors, a 40% reduction of photoreceptor synaptic ribbons was observed in Ins2Akita mice compared to age‐matched controls. This was accompanied by a total depletion of horizontal cell dendritic plexus and disarrangement of rod and cone bipolar cell processes at the outer plexiform layer. In the inner retina, the number of rod and cone bipolar cells remained similar to controls, but abnormal GABAergic amacrine cell stratification and 25% reduction of rod bipolar cell axon terminals were observed. Brn3a staining revealed a 20% loss of retinal ganglion cells, which exhibited abnormal aggregates of light neurofilament. In addition, a significant retinal vascular degeneration evidenced by around 50% reductions of vascular plexus was observed in 8 month hyperglycemic Ins2Akita mice. No evidence of retinal neovascularisation was detected.Conclusion Ins2Akita mice present a variety of retinal neuropathies including the reduction in different types of neuronal cells and disruption of synaptic structures. The mice also develop severe retinal vascular degeneration but no neovascularisation.

Developmental Regulation and Activity-Dependent Maintenance of GABAergic Presynaptic Inhibition onto Rod Bipolar Cell Axonal Terminals

Presynaptic inhibition onto axons regulates neuronal output, but how such inhibitory synapses develop and are maintained invivo remains unclear. Axon terminals of glutamatergic retinal rod bipolar cells (RBCs) receive GABAA and GABAC receptor-mediated synaptic inhibition. We found that perturbing GABAergic or glutamatergic neurotransmission does not prevent GABAergic synaptogenesis onto RBC axons. But, GABA release is necessary for maintaining axonal GABA receptors. This activity-dependent process is receptor subtype specific: GABAC receptors are maintained, whereas GABAA receptors containing α1, but not α3, subunits decrease over time in mice with deficient GABA synthesis. GABAA receptor distribution on RBC axons is unaffected in GABAC receptor knockout mice. Thus, GABAA and GABAC receptor maintenance are regulated separately. Although immature RBCs elevate their glutamate release when GABA synthesis is impaired, homeostatic mechanisms ensure that the RBC output operates within its normal range after eye opening, perhaps to regain proper visual processing within the scotopic pathway.

Spikes in Mammalian Bipolar Cells Support Temporal Layering of the Inner Retina

In the mammalian retina, 10-12 different cone bipolar cell (BC) types decompose the photoreceptor signal into parallel channels, providing the basis for the functional diversity of retinal ganglion cells (RGCs). BCs differing in their temporal properties appear to project to different strata of the retina's inner synaptic layer, based on somatic recordings of BCs and excitatory synaptic currents measured in RGCs. However, postsynaptic currents in RGCs are influenced by dendritic morphology and receptor types, and the BC signal can be transformed at the axon terminals both through interactions with amacrine cells and through the generation of all-or-nothing spikes. Therefore, the temporal properties of the BC output have not been analyzed systematically across different types of mammalian BCs. We recorded calcium signals directly within axon terminals using two-photon imaging and show that BCs can be divided into ≥eight functional clusters. The temporal properties of the BC output were directly reflected in their anatomical organization within the retina's inner synaptic layer: faster cells stratified closer to the border between ON and OFF sublamina. Moreover, ≥three fastest groups generated clear all-or-nothing spikes. Therefore, the systematic projection pattern of BCs provides distinct temporal "building blocks" for the feature extracting circuits of the inner retina.

The Mode of Retinal Presynaptic Inhibition Switches with Light Intensity

Excitatory amino acid transporters (EAATs) terminate signaling in the CNS by clearing released glutamate. Glutamate also evokes an EAAT-mediated Cl(-) current, but its role in CNS signaling is poorly understood. We show in mouse retina that EAAT-mediated Cl(-) currents that were evoked by light inhibit rod pathway signaling. EAATs reside on rod bipolar cell axon terminals where GABA and glycine receptors also mediate light-evoked inhibition. We found that the mode of inhibition depended on light intensity. Dim light evoked GABAergic and glycinergic inhibition with rapid kinetics and a large spatial extent. Bright light evoked predominantly EAAT-mediated inhibition with slow kinetics and a small spatial extent. The switch to EAAT-mediated signaling in bright light supplements receptor-mediated signaling to expand the dynamic range of inhibition and contributes to the transition from rod to cone signaling by suppressing rod pathway signaling in bright light conditions.

The effect of glutamate-gated chloride current on the excitability of a Purkinje cell: a modeling study

Purkinje neurons express, in high abundance, a glutamate gated chloride channel commonly known as the Excitatory Amino Acid Transporter subtype 4 (EAAT4). EAAT4 belongs to the family of glutamate transporters, which in mammalian nervous system is responsible for clearing synaptic glutamate [1]. Studies of these transporters in heterogeneous expression systems demonstrated that in addition to glutamate transport, the binding of glutamate to the transporter activates a chloride current through the transporter with properties liken that of a channel and which is functionally independent from the transport process [2]. The role of the chloride channel in glutamate transporters is only known for the EAAT subtype 5 (EAAT5). On rod bipolar cell axon terminals, EAAT5 activation by glutamate results in membrane hyperpolarization, which consequently inhibits terminal glutamate release [3]. Whether the chloride channel of EAAT4 has a physiological role in Purkinje neurons remains unknown. A synaptic model was developed to determine conditions in which the chloride channel of EAAT4 could influence Purkinje neuron function and whether these conditions are physiologically relevant. The model comprises a single compartment with uniform distribution of AMPA receptors and EAAT4. The EAAT4 model is based on a 16-state kinetic model of EAAT2 [4] using reaction rates of EAAT4 measured in [5]. AMPA receptor conductance and absolute permeability of EAAT4 were parameterized for adjusting their respective current amplitudes. The model reproduced the EPSC and EAAT4 channel currents evoked in a Purkinje neuron by parallel fiber stimulation under conditions similar to those used in [5]. The result serves as a basis for investigating the effects of the EAAT4 chloride current on Purkinje neuron excitability and intracellular chloride concentration.

Localization and possible function of P2Y 4 receptors in the rodent retina

Extracellular ATP acts as a neurotransmitter in the retina, via the activation of ionotropic P2X receptors and metabotropic P2Y receptors. The expression of various P2X and P2Y receptor subtypes has been demonstrated in the retina, but the localization of P2Y receptors and their role in retinal signaling remains ill defined. In this study, we were interested in determining the localization of the P2Y 4 receptor subtype in the rat retina, and using the electroretinogram (ERG) to assess whether activation of these receptors modulated visual transmission. Using light and electron microscopy, we demonstrated that P2Y 4 receptors were expressed pre-synaptically in rod bipolar cells and in processes postsynaptic to cone bipolar cells. Furthermore, we show that the expression of P2Y 4 receptors on rod bipolar cell axon terminals is reduced following dark adaptation, suggesting receptor expression may be dependent on retinal activity. Finally, using the electroretinogram, we show that intravitreal injection of uridine triphosphate, a P2Y receptor agonist, decreases the amplitude of the rod PII, supporting a role for P2Y receptors in altering inner retinal function. Taken together, these results suggest a role for P2Y 4 receptors in the modulation of inner retinal signaling.

The absence of the clathrin-dependent endocytosis in rod bipolar cells of the FVB/N mouse retina

The high rate of exocytosis at the ribbon synapses is balanced by following compensatory endocytosis. Unlike conventional synaptic terminals where clathrin-mediated endocytosis (CME) is a predominant mechanism for membrane retrieval, CME is thought to be only a minor mechanism of endocytosis at the retinal ribbon synapses, but CME is present there and it works. We examined the clathrin expression in the FVB/N rd1 mouse, which is an animal model of retinitis pigmentosa. The broadly distributed pattern of clathrin immunoreactivity in the inner plexiform layer was similar in both the control and FVB/N mouse retinas, but the immunoreactive punta within the rod bipolar axon terminals located in the proximal IPL were decreased in number and reduced in size at postnatal days 14 and they came to disappear at postnatal days 21. This preferential decrease of the clathrin expression at ribbon synapses in the rod bipolar cell axon terminals of the FVB/N mouse retina demonstrates another plastic change after photoreceptor degeneration and this suggests that clathrin may be important for normal synaptic function at the rod bipolar ribbon synapses in the mammalian retina.

Neuronal expression of P2X 3 purinoceptors in the rat retina

P2X 3 purinoceptors are involved in fast, excitatory neurotransmission in the nervous system, and are expressed predominantly within sensory neurons. In this study, we examined the cellular and synaptic localization of the P2X 3 receptor subunit in the retina of the rat using immunofluorescence immunohistochemistry and pre-embedding immunoelectron microscopy. In addition, we investigated the activity of ecto-ATPases in the inner retina using an enzyme cytochemical method. The P2X 3 receptor subunit was expressed in the soma of a subset of GABA immunoreactive amacrine cells, some of which also expressed protein kinase C-α. In addition, punctate immunoreactivity was observed within both the inner and outer plexiform layers of the retina. Double labeling studies showed that P2X 3 receptor puncta were associated with both rod and cone bipolar cell axon terminals in the inner plexiform layer. Ultrastructural studies indicated that P2X 3 receptor subunits were expressed on putative A17 amacrine cells at sites of reciprocal synaptic input to the rod bipolar cell axon terminal. Moreover, we observed P2X 3 immunolabeling on amacrine cell processes that were associated with cone bipolar cell axon terminals and other conventional synapses. In the outer retina, P2X 3 immunoreactivity was observed on specialized junctions made by putative interplexiform cells. Ecto-ATPase activity was localized to the inner plexiform layer on the extracellular side of all plasma membranes, but was not apparent in the ganglion cell layer or the inner nuclear layer, suggesting that ATP dephosphorylation occurs exclusively in synaptic regions of the inner retina. These data provide further evidence that purines participate in retinal transmission, particularly within the rod pathway.

The glutamate transporter EAAT5 works as a presynaptic receptor in mouse rod bipolar cells

Membrane neurotransmitter transporters control the concentration of their substrate in the synaptic clefts, through the thermodynamic coupling of uptake to the movement of Na(+) and other ions. In addition, excitatory amino acid transporters (EAAT) have a Cl(-) conductance which is gated by the joint binding of Na(+) and glutamate, but thermodynamically uncoupled to the flux of glutamate. This conductance is particularly large in the retina-specific EAAT5 isoform. In the mouse retina, we located EAAT5 in both cone and rod photoreceptor terminals and in axon terminals of rod bipolar cells. In these later cells, application of glutamate on the axon terminal evoked a current that reversed at E(Cl), was insensitive to bicuculline, TPMPA, strychnine, dl-AP5, CNQX and MCPG, but blocked by the glutamate transporter inhibitor dl-tBOA. Furthermore, short depolarizations of the bipolar cells evoked a dl-tBOA and Cd(2+)-sensitive current whose amplitude was comparable to the glutamate-evoked current. Its kinetics indicated that EAAT5 was located close to the glutamate release site. For 2 ms depolarizations evoking maximal responses, the EAAT5-mediated current carried between 2 and 8 times more charge as an average inhibitory GABA or glycine postsynaptic current received spontaneously from amacrine cells, with 10 mm or 0.5 mm intracellular EGTA, respectively. In conditions for which reciprocal inhibition could be monitored, the charge carried by the EAAT5 current was 1.5 times larger than the one carried by the inhibitory postsynaptic currents received from amacrine cells. These results indicate that EAAT5 acts as a major inhibitory presynaptic receptor at mammalian rod bipolar cell axon terminals. This feedback mechanism could control glutamate release at the ribbon synapses of a non-spiking neuron and increase the temporal contrast in the rod photoreceptor pathway.

P2X2 receptors on ganglion and amacrine cells in cone pathways of the rat retina

Extracellular ATP is known to mediate fast, excitatory neurotransmission through activation of ionotropic P2X receptors. In this study, the localization of the P2X(2) receptor (P2X(2)R) subunit was studied in rat retina by using immunofluorescence immunohistochemistry and preembedding immunoelectron microscopy. The P2X(2)R was observed in large ganglion cells as well as in a subset of amacrine cells. Double labeling revealed that 96% of all P2X(2)R-immunoreactive amacrine cells showed gamma-aminobutyric acid (GABA) immunoreactivity. Subsets of P2X(2)R-immunoreactive amacrine cells expressed nitric oxide synthase and substance P; however, no colocalization was observed with choline acetyltransferase, vasoactive intestinal peptide, or tyrosine hydroxylase. Nearest-neighbor analysis confirmed that P2X(2)Rs were expressed by a heterogeneous population of amacrine cells. The synaptic connectivity of P2X(2)R amacrine cells was also investigated. It was interesting that P2X(2)R-immunoreactive amacrine cell dendrites stratified in the sublaminae of the inner plexiform layer occupied by cone, but not rod bipolar cell axon terminals. Immunoelectron microscopy revealed that P2X(2)-immunoreactive amacrine cell processes were associated with cone bipolar cell axon terminals as well as other conventional synapses in the inner plexiform layer. Taken together, these data provide further evidence for the involvement of extracellular ATP in neuronal signaling in the retina, particularly within cone pathways.

Morphological characterization of the retina of the CNGA3(-/-)Rho(-/-) mutant mouse lacking functional cones and rods.

To assess the structural changes in the retina caused by a functional blockade of rods and cones and to document the time course of their degeneration. Double knockout mice were generated by cross-breeding CNGA3(-/-) mice with Rho(-/-) mice. Retinas of mutant and wild-type mice from 3 weeks up to 12 months of age were studied by confocal light and electron microscopy. The retinas were immunostained with cell-type-specific markers and with antibodies against synapse-associated proteins and transmitter receptors. In 3-week-old CNGA3(-/-)Rho(-/-) mice, retinal layers showed normal structural organization, and photoreceptors established normal synaptic contacts. Until postnatal week (Pw)7, presynaptic markers and postsynaptic glutamate receptors were well expressed at the photoreceptor terminals. Photoreceptor degeneration started at Pw4, progressing to an almost complete loss by 3 months. Rod spherules showed an increase in the number of synaptic ribbons and postsynaptic elements during this early stage of degeneration, and horizontal cell processes grew into the outer nuclear layer. At later stages of retinal degeneration, the inner plexiform layer (IPL) was also affected. Rod bipolar cell axon terminals showed morphologic alterations, but the stratification pattern of cone bipolar cell axons and amacrine cell processes appeared unaffected. Transmitter receptors (GlyRalpha3, GABA(A) alpha2, GluR2/3) showed no obvious changes in the distribution and density of their synaptic clusters throughout the IPL at postnatal month 12. The normal structural and synaptic organization of the mutant retina at Pw3 suggests that photoreceptor light responses are not essential for the development of the retinal circuitry. However, functional photoreceptors are necessary for the maintenance of rods and cones and their contacts in the OPL, because they degenerate almost completely by 3 months after birth. Degenerative changes can also be observed in the IPL; however, they appear to have a slower time course and by 12 months of age the IPL circuitry appears to be surprisingly intact.

Y2 receptor expression and inhibition of voltage-dependent ca 2+ influx into rod bipolar cell terminals

Neuropeptide Y (NPY) is a potent inhibitory neuropeptide expressed by amacrine cells in the rat retina. NPY modulates the release of multiple neurotransmitters in mammalian retina, yet the mechanisms mediating this regulation are not well defined. To further understand the action of NPY in the retina, Y receptor coupling to voltage-dependent Ca 2+ channels was investigated using Ca 2+ imaging with fura-2 AM to measure [Ca 2+] i increases in rod bipolar cell terminals. Y receptor expression was studied in rat retinal tissue with reverse transcription-polymerase chain reaction (RT-PCR). NPY inhibited the depolarization-evoked Ca 2+ influx into rod bipolar cell axon terminals and caused a dose-dependent reduction and an average maximal inhibition of 72% at 1 μM, which was reversed upon washout. K +-evoked Ca 2+ increases were also inhibited by the selective Y2 receptor agonists, C2-NPY and NPY(13–36), at concentrations of 1 μM, but not by the selective Y1 receptor agonist, [Leu 31Pro 34]NPY, selective Y4 receptor agonist, rPP, or the selective Y5 receptor agonist, [ d-Trp32]-NPY. Y receptor expression was determined using RT-PCR for all known Y receptor subtypes. Y2 receptor mRNA, as well as Y1, Y4, and Y5 receptor mRNAs, are present in the rat retina. Like the rod bipolar cell, other studies in central neurons have shown that the Y2 receptor is expressed predominantly as a presynaptic receptor and that it modulates transmitter release. Together, these findings suggest that NPY activates presynaptic Y2 receptors to inhibit voltage-dependent Ca 2+ influx into rod bipolar cell terminals, and establishes one mechanism by which NPY may reduce l-glutamate release from the rod bipolar cell synapse.

Glycine receptors and glycinergic synaptic input at the axon terminals of mammalian retinal rod bipolar cells.

We investigated the properties of glycine receptors and glycinergic synaptic inputs at the axon terminals of rod bipolar cells (RBCs) in rats by patch-clamp recording. Glycine currents recorded from isolated axon terminals were larger than those from isolated somata/dendrites; this was confirmed by puffing glycine onto these two regions in retinal slices. The current density at terminal endings was more than one order of magnitude higher than the density at somatic/dendritic regions. Glycine currents from isolated terminals and isolated somata/dendrites showed similar sensitivity to picrotoxinin blockade. Single-channel opening recorded from isolated terminals and somata/dendrites displayed a similar main-state conductance of ~46 pS. Application of glycine effectively suppressed depolarization-evoked increases in intracellular Ca2+ at the terminals. In the presence of GABAA and GABAC antagonists, strychnine-sensitive chloride currents were evoked in RBCs in retinal slices by puffing kainate onto the inner plexiform layer. No such currents were observed if the recorded RBCs did not retain axon terminals or if Ca2+ was replaced by Co2+ in the extracellular solution. The currents displayed discrete miniature-like events, which were partially blocked by tetrodotoxin. Consistent with early studies in the rabbit and mouse, this study demonstrates that glycine receptors are highly concentrated at the axon terminals of rat RBCs. The pharmacological and physiological properties of glycine receptors located in the axon terminal and somatic/dendritic regions, however, appear to be the same. This study provides evidence for the existence of functional glycinergic synaptic input at the axon terminals of RBCs, suggesting that glycine receptors may play a role in modulating bipolar cell synaptic transmission.

Adaptation-dependent plasticity of rod bipolar cell axon terminal morphology in the rat retina.

We chose synaptic terminals of rat rod bipolar cells as a model system to study activity-related changes in the overall morphology and the fine structure of synaptic sites. Using confocal laser scanning microscopy in conjunction with three-dimensional reconstruction and electron microscopy, we examined the effect of light and dark adaptation on axon terminals identified by protein kinase C (PKC) immunoreactivity. Rod bipolar cell axon terminals consisted of 2-3 polymorphic boutons situated close to the ganglion cell layer and a single ovoid swelling located more distally. Both components of the terminal complex showed adaptation-dependent differences in the distribution of PKC immunoreactivity and in their morphology. In light-adapted rod bipolar cell axon terminals, PKC immunoreactivity was homogeneously distributed throughout the cytoplasm, whereas terminals from dark-adapted animals showed PKC immunoreactivity preferentially localised in the submembrane compartment and a reduced staining of the more central cytoplasm. In three-dimensional reconstructions of optical sections and at the ultrastructural level, the shape of light-adapted axon terminals was round and smooth and exhibited more convexly curved synaptic membranes. In contrast, dark-adapted terminals had irregular contours, numerous dimples and a concave synaptic curvature. No spinules of bipolar cell terminals were observed in dark-adapted material. These observations are discussed in the context of activity-related morphological plasticity of central nervous system synapses and of the functions of PKC in the cycle of vesicle fusion and retrieval at the tonically active ribbon synapses of the rod bipolar axon terminal.

Expression of the somatostatin subtype 2A receptor in the rabbit retina.

In the retina, somatostatin influences neuronal activity likely by acting at one or more somatostatin subtype (sst) receptors. Somatostatin and somatostatin-binding sites are distributed predominantly to the inner retina. The present study has investigated the cellular expression of one of the sst receptors, the sst2A receptor isoform, in the rabbit retina. These studies have used a new polyclonal antibody directed to the predicted C-terminus of mouse sst2A(361-369) receptor. Antibody specificity was tested by preadsorption of the primary antibody with a peptide corresponding to sst2A(361-369). sst2A Receptor immunoreactivity was localized mainly to the plasma membrane of rod bipolar cells and to sparsely occurring, wide-field amacrine cells. Immunostaining in rod bipolar cells was strongest in the axon and axon terminals in lamina 5 of the inner plexiform layer (IPL) and was weakest in the cell body and dendrites. Double-labeling experiments using a monoclonal antibody against protein kinase C (PKC; alpha and beta), a rod bipolar cell-selective marker, showed complete colocalization. In horizontal sections of retina, immunostained bipolar cell bodies had a dense distribution, which is in agreement with the reported distribution of rod bipolar cell bodies. Immunoreactive amacrine cell bodies were located at the border of the inner nuclear layer and the IPL, and thin varicose processes ramified mainly in laminae 2 and 4 of the IPL. These observations indicate that somatostatin influences visual information processing in the retina 1) by acting presynaptically on rod bipolar cell axon terminals and b) by influencing the activity of sparsely occurring amacrine cells.