scholarly journals Two Functional Classes of Rod Bipolar Cells in the Healthy and Degenerated Optogenetically Treated Murine Retina

2022 ◽  
Vol 15 ◽  
Author(s):  
Giulia Schilardi ◽  
Sonja Kleinlogel

Bipolar cells have become successful targets for optogenetic gene therapies that restore vision after photoreceptor degeneration. However, degeneration was shown to cause changes in neuronal connectivity and protein expression, which may impact the quality of synthetically restored signaling. Further, the expression of an optogenetic protein may alter passive membrane properties of bipolar cells affecting signal propagation. We here investigated the passive membrane properties of rod bipolar cells in three different systems, the healthy retina, the degenerated retina, and the degenerated retina expressing the optogenetic actuator Opto-mGluR6. We found that, based on the shape of their current-voltage relations, rod bipolar cells in healthy and degenerated retinas form two clear functional groups (type 1 and type 2 cells). Depolarizing the membrane potential changed recorded current-voltage curves from type 1 to type 2, confirming a single cell identity with two functional states. Expression of Opto-mGluR6 did not alter the passive properties of the rod bipolar cell. With progressing degeneration, dominant outward rectifying currents recorded in type 2 rod bipolar cells decreased significantly. We demonstrate that this is caused by a downregulation of BK channel expression in the degenerated retina. Since this BK conductance will normally recover the membrane potential after RBCs are excited by open TRPM1 channels, a loss in BK will decrease high-pass filtering at the rod bipolar cell level. A better understanding of the changes of bipolar cell physiology during retinal degeneration may pave the way to optimize future treatment strategies of blindness.

1992 ◽  
Vol 67 (3) ◽  
pp. 508-529 ◽  
Author(s):  
N. Spruston ◽  
D. Johnston

1. Perforated patch-clamp recordings were made from the three major classes of hippocampal neurons in conventional in vitro slices prepared from adult guinea pigs. This technique provided experimental estimates of passive membrane properties (input resistance, RN, and membrane time constant, tau m) determined in the absence of the leak conductance associated with microelectrode impalement or the washout of cytoplasmic constituents associated with conventional whole-cell recordings. 2. To facilitate comparison of our data with previous results and to determine the passive membrane properties under conditions as physiological as possible, recordings were made at the resting potential, in physiological saline, and without any added blockers of voltage-dependent conductances. 3. Membrane-potential responses to current steps were analyzed, and four criteria were used to identify voltage responses that were the least affected by activation of voltage-dependent conductances. tau m was estimated from the slowest component (tau 0) of multiexponential fits of responses deemed passive by these criteria. RN was estimated from the slope of the linear region in the hyperpolarizing direction of the voltage-current relation. 4. It was not possible to measure purely passive membrane properties that were completely independent of membrane potential in any of the three classes of hippocampal neurons. Changing the membrane potential by constant current injection resulted in changes in RN and tau 0; subthreshold depolarization produced an increase, and hyperpolarization a decrease, in both RN and tau 0 for all three classes of hippocampal neurons. 5. Each of the three classes of hippocampal neurons also displayed a depolarizing "sag" during larger hyperpolarizing voltage transients. To evaluate the effect of the conductances underlying this sag on passive membrane properties, 2-5 mM Cs+ was added to the physiological saline. Extracellular Cs+ effectively blocked the sag in all three classes of hippocampal neurons, but the effect of Cs+ on RN, tau 0, and the voltage dependence of these parameters was unique for each class of neurons. 6. CA1 pyramidal neurons had an RN of 104 +/- 10 (SE) M omega and tau 0 of 28 +/- 2 ms at a resting potential of -64 +/- 2 mV (n = 12). RN and tau 0 were larger at more depolarized potentials in these neurons, but the addition of Cs+ to the physiological saline reversed this voltage dependence. 7. CA3 pyramidal neurons had an RN of 135 +/- 8 M omega and tau 0 of 66 +/- 4 ms at a resting potential of -64 +/- 1 mV (n = 14).(ABSTRACT TRUNCATED AT 400 WORDS)


1989 ◽  
Vol 62 (4) ◽  
pp. 924-934 ◽  
Author(s):  
M. J. Correia ◽  
B. N. Christensen ◽  
L. E. Moore ◽  
D. G. Lang

1. Hair cells were enzymatically dissociated from the neuroepithelium (cristae ampullares) of the semicircular canals of white king pigeons (Columba livia). Those hair cells determined to be type II by an anatomic criterion, the ratio of the minimum width of the neck to the width of the cuticular plate, were studied with the use of the whole cell patch-clamp technique. 2. The mean +/- SD zero-current membrane potential, Vz, was found to be -54 +/- 12 mV for anterior crista hair cells (n = 71), -62 +/- 14 mV for posterior crista hair cells (n = 14), and -55 +/- 12 mV for lateral (horizontal) crista hair cells (n = 18). The mean +/- SD value of Vz for hair cells from all cristae (n = 103) was -56 +/- 13 mV. 3. Active and passive membrane properties were calculated in the time domain, in voltage- or current-clamp mode, from responses to voltage or current pulses and, in the frequency domain, by fitting a membrane model to admittance magnitude and phase data resulting from current responses to sum-of-sines voltages at different d.c. levels of voltage-clamp membrane potential. 4. The average value +/- SE of input resistance (Rin), over the range from -100 to -60 mV, was found to 1.5 +/- 0.3 G omega from a mean-voltage-as-a-function-of-current plot, V-I, (n = 7) and a mean of 1.4 +/- 0.3 G omega from individual (n = 15) current-as-a-function-of-voltage plots, I-V. A lower mean value 0.8 +/- 0.4 G omega was obtained for the input resistance from frequency-domain calculations for a different set of cells (n = 21). Also, in two different sets of cells, average input capacitance (Cin) was determined to be 12 +/- 3 pF (n = 7) from time-domain estimates and 14 +/- 3 pF (n = 21) from frequency-domain estimates. The (Rin)(Cin) product was 11 ms based on frequency-domain estimates and 17 ms from time-domain estimates. 5. I-V curves for hair cells voltage clamped at -60 mV showed some anomalous rectification for hyperpolarizations between -60 and -120 mV but no detectable N-shape for depolarizations between -50 and 90 mV. The I-V relation showed increasing slope with depolarization through the resting potential (Vz) and increased linearly between -40 and 80 mV; the best-fit straight-line maximum slope conductance for six cells over this range was 17.4 +/- 0.3 nS.(ABSTRACT TRUNCATED AT 400 WORDS)


2008 ◽  
Vol 25 (4) ◽  
pp. 523-533 ◽  
Author(s):  
QUN-FANG WAN ◽  
ALEJANDRO VILA ◽  
ZHEN-YU ZHOU ◽  
RUTH HEIDELBERGER

AbstractTo better understand synaptic signaling at the mammalian rod bipolar cell terminal and pave the way for applying genetic approaches to the study of visual information processing in the mammalian retina, synaptic vesicle dynamics and intraterminal calcium were monitored in terminals of acutely isolated mouse rod bipolar cells and the number of ribbon-style active zones quantified. We identified a releasable pool, corresponding to a maximum of ≈35 vesicles/ribbon-style active zone. Following depletion, this pool was refilled with a time constant of ≈7 s. The presence of a smaller, rapidly releasing pool and a small, fast component of refilling was also suggested. Following calcium channel closure, membrane surface area was restored to baseline with a time constant that ranged from 2 to 21 s depending on the magnitude of the preceding Ca2+ transient. In addition, a brief, calcium-dependent delay often preceded the start of onset of membrane recovery. Thus, several aspects of synaptic vesicle dynamics appear to be conserved between rod-dominant bipolar cells of fish and mammalian rod bipolar cells. A major difference is that the number of vesicles available for release is significantly smaller in the mouse rod bipolar cell, both as a function of the total number per neuron and on a per active zone basis.


1996 ◽  
Vol 76 (4) ◽  
pp. 2192-2199 ◽  
Author(s):  
L. P. Del Mar ◽  
R. S. Scroggs

1. The membrane properties of dorsal root ganglion (DRG) cells expressing the lactoseries carbohydrate antigen Gal beta 1-4GlcNAc-R were studied and compared with those of DRG cells lacking this antigen. Acutely dissociated rat DRG cells that expressed Gal beta 1-4GlcNAc-R on their outer cell membranes were detected with the use of a primary monoclonal mouse IgM antibody (A5), directed against Gal beta 1-4GlcNAc-R, and a fluorescent secondary antibody (fluorescein-conjugated goat anti-mouse IgM). We found 12.8 micrograms/ml of A5 to be a saturating concentration of primary antibody that labeled approximately 19% of the DRG cells. A battery of membrane properties including action potential (AP) duration; sensitivity to capsaicin; expression of H current (IH), A current (IA), and Ca2+ current subtypes (L, N, and T); and inhibition of high-threshold Ca2+ currents by serotonin (5HT) or 8-hydroxy-2-(di-N-propylamino)-tetralin (8-OH-DPAT) was measured in DRG cells labeled (A5+) and unlabeled (A5-) by a saturating concentration of A5. 2. There was a significant difference in the number of capsaicin-sensitive DRG cells and a significant difference in the magnitude of the capsaicin-induced inward current in A5+ versus A5- DRG cells. Of 35 A5+ cells tested, 33 were sensitive to 1 microM capsaicin, which produced an inward current averaging 4 +/- 0.46 (SE) nA (n = 33). In contrast, only 12 of 33 A5- cells were sensitive to 1 microM capsaicin, which produced an inward current averaging 1.2 +/- 0.52 nA (n = 12). 3. There were also significant differences between A5+ and A5- cells regarding average AP duration, N- and T-type Ca2+ current amplitude, and number of cells that expressed IH and IA. A5+ cells had significantly larger N-type Ca2+ currents and expressed IA more frequently than A5- cells. Conversely, A5- cells had significantly longer AP duration and larger T-type Ca2+ currents, and expressed IH more frequently compared with A5+ cells. 4. A5+ and A5- cells differed regarding the inhibition of high-threshold Ca2+ currents by maximal concentrations of 5HT1A agonists (10 microM 5HT or 1 microM 8-OH-DPAT). Inhibition of Ca2+ currents in A5+ cells by 1 microM 8-OH-DPAT (n = 15) or 10 microM 5HT (n = 18) averaged 4 +/- 0.9%. In contrast, inhibition of Ca2+ currents in A5- cells by 10 microM 5HT (n = 33) averaged 20 +/- 3.8%. 5. Cells for which sufficient data were collected were categorized as type 1, 2, 3, or 4 on the basis of sensitivity to capsaicin and expression of IH, IA, and T-type Ca2+ current amplitude, and the distribution of A5+ and A5- cells among the various groups was observed. The categories were defined as follows: type 1 (capsaicin sensitive, no IH or IA); type 2 (capsaicin sensitive, significant IA); type 3 (capsaicin insensitive, T-type Ca2+ currents < 1 nA, significant IH but no IA); and type 4 (capsaicin insensitive, T-type Ca2+ currents > 2.4 nA). On the basis of this criteria, 6 of 15 type 1 cells and all type 2 cells (n = 19) were A5+. All type 3 cells (n = 8) and all type 4 cells (n = 11) were A5-. 6. As indicated above, the expression of the Gal beta 1-4GlcNAc-R antigen differentiated two subgroups of DRG cells in the type 1 category (A5+, n = 6 and A5-, n = 9). These two groups varied regarding the sensitivity of Ca2+ currents to maximally effective concentrations of 5HTIA agonists. In type 1 A5+ DRG cells, high-threshold Ca2+ currents were not significantly inhibited by 1 microM 8-OH-DPAT (average inhibition = 1.2 +/- 0.8%, n = 6). However, in type 1 A5- cells, high-threshold Ca2+ currents were reduced 47 +/- 6.0% (n = 9) by 10 microM 5HT. 7. The several significant differences in membrane properties between A5+ and A5- DRG cells suggest that the Gal beta 1-4GlcNAc-R antigen is expressed by a distinct subset of DRG cells, consisting predominately of type 1 and type 2 cells. The observation that most A5+ DRG cells were capsaicin sensitive suggests that the Gal beta 1-4GlcNAc-R antigen is expressed primarily by n


2003 ◽  
Vol 89 (1) ◽  
pp. 382-389 ◽  
Author(s):  
Akira Miura ◽  
Masahito Kawatani ◽  
William C. De Groat

Excitatory pathways from the dorsal commissure (DCM) to L6–S1 parasympathetic preganglionic neurons (PGN) were examined using whole-cell patch-clamp recording techniques in spinal cord slices from neonatal rats. PGN were identified by retrograde axonal transport of a fluorescent dye injected into the intraperitoneal space. Excitatory postsynaptic currents (EPSCs) were evoked in PGN by stimulation of DCM in the presence of bicuculline methiodide (10 μM) and strychnine (1 μM) to block inhibitory pathways. Electrical stimulation of DCM evoked two types of inward currents. In the majority of PGN ( n = 66), currents (mean amplitude, 47.9 ± 4.7 pA) occurred at a short and relatively constant latency (3.8 ± 0.1 ms) and presumably represent monosynaptic EPSCs (Type 1). However, in other neurons ( n = 20), a different type of EPSC (Type 2) was noted, consisting of a fast monosynaptic component followed by a prolonged inward current with superimposed fast transients presumably representing excitatory inputs mediated by polysynaptic pathways. Type 1 EPSCs were pharmacologically dissected into two components. A fast component was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5μM) and a slowly decaying component was blocked by 2-amino-5-phosphonovalerate (APV, 50 μM). The fast component of Type 1 EPSCs had a linear current-voltage relationship and reversed at a membrane potential of −7.6 ± 1.3 mV ( n = 5). The fast component of Type 2 EPSCs was also blocked by 5 μM CNQX and the remaining slower component was blocked by 50 μM APV. When the DCM was stimulated in the presence of 50 μM APV, the time to peak and decay time constant in Type 1 EPSCs were 1.9 ± 0.2 and 4.1 ± 0.8 ms, respectively. Examination of the NMDA receptor-mediated component of the EPSCs in the presence of 5 μM CNQX revealed a current-voltage relationship that had a region of negative slope conductance (from −20 to −80 mV), which was abolished in Mg2+-free external solution. The time to peak and decay time constant of this component were 14.2 ± 2.0 and 91.0 ± 12.4 ms, respectively. Type 1 EPSCs in some PGN responded in an all-or-none manner and presumably represented unitary synaptic responses; whereas Type 2 EPSCs always exhibited a graded stimulus intensity–response relationship. Paired-pulse facilitation (50-ms interstimulus intervals; 141 ± 5.6% increase, n = 8) of EPSCs was observed. These results indicate that PGN receive monosynaptic and polysynaptic glutamatergic excitatory inputs from neurons and/or axonal pathways in the DCM.


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