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Arthroscopic distal clavicle excision (DCE) is a reliable procedure to treat acromioclavicular joint arthritis. Typically, only 1 to 2 cm of distal clavicle should be removed. Resection of too much bone can lead to instability of the joint or lack of support to the shoulder. We describe 2 patients who had excessive clavicular bone removed arthroscopically, leading to irreparable clavicular pain and dysfunction. The 2 female patients, ages 56 and 60 years, presented to our clinic with continued pain after DCE. Both had pain intractable with nonoperative treatment and loss of range of motion of the shoulder. Radiographs revealed a distal clavicle defect of 7.5 cm in 1 patient. The second patient had a 2-cm distal clavicular defect with an adjacent 2-cm clavicle bone fragment between the defect and residual clavicle shaft. Both underwent surgery with subtotal claviculectomy for pain control. During surgery, 1 patient had a subclavian vein requiring vascular repair. After 1 year of follow-up, both patients had reduced but residual pain and restricted range of motion. Only 1 patient could rejoin her preinjury occupation. Neither patient could continue with preinjury recreational sports. Excessive removal of the distal clavicle during DCE can result in continued pain and disability of the shoulder. Methods to visualize the anatomy of the distal clavicle and its articulation to the acromion should be considered when performing this operation arthroscopically. Reoperation to remove subtotal clavicle has good clinical outcomes but may lead to serious complications due to the proximity to major neurovascular structures. [ Orthopedics . 2024;47(1):e57-e60.].

Anatomical total shoulder arthroplasty in its modern form where it reproduces the normal shoulder has been utilized clinically for more than half a century. As the technology and the designs have changed to recreate the humeral and glenoid sides of the joint, the sophistication of design has resulted in the growing number of cases annually worldwide. This increase is due in part to the increasing number of indications that the prosthesis can treat with successful results. On the humeral side, there have been design changes to better reflect the proximal humeral anatomy, and humeral stems are increasingly placed safely without cement. Platform systems which allow conversion of a failed arthroplasty to a reverse configuration without stem extraction is another design change. Similarly, there has been increasing utilization of short stem and stemless humeral components. Extensive experience with shorter stem and stemless devices, however, has yet to demonstrate the purported advantages of these devices, as recent studies have demonstrated equivalent blood loss, fracture rates, operative times, and outcome scores. Easier revision with these shorter stems remains to be definitively established, with only one study comparing the ease of revision between stem types. On the glenoid side, hybrid cementless glenoids, inlay glenoids, cementless all-polyethylene glenoids, and augmented glenoids have all been investigated; however, the indications for these devices remain unclear. Lastly, innovative surgical approaches to implanting shoulder arthroplasty and the use of patient specific guides and computerized planning, while interesting concepts, still await validation before they are utilized on a widespread basis. While reverse shoulder arthroplasty has been increasingly used to reconstruct the arthritic shoulder, anatomic glenohumeral replacement maintains a significant role in the armamentarium of the shoulder surgeon., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

In patients with severe glenohumeral osteoarthritis (OA) and preserved rotator cuff function who have failed nonoperative treatment, anatomic total shoulder arthroplasty (TSA) has historically been the preferred surgical treatment. Shoulder arthroplasty in the setting of glenoid bone loss setting is technically demanding. Many techniques have been described to deal with glenoid bone loss including eccentric reaming, bone grafting, augmented glenoid baseplates, and patient-specific implants. Still, the decision to perform anatomic TSA or reverse total shoulder arthroplasty (RTSA) is often unclear, especially as the use of RTSA increases and evolves, making historical studies less useful when considering modern implant designs. RTSA has been advocated as a solution for patients with severe glenoid bone loss with intact rotator cuff function. Moreover, in appropriately selected patients, good outcomes can be achieved without the use of bone grafting or augmented baseplates. In cases of severe glenoid bone loss, RTSA can be performed with reaming the glenoid flat such that the baseplate rests on native glenoid bone. We have previously reported excellent prosthetic survival with this technique at 5-year follow-up. The purpose of this article is to highlight our suggested treatment algorithm for glenohumeral OA with glenoid bone loss and intact rotator cuff. Specifically, we focus on situations where RTSA may be preferred as opposed to anatomic TSA, and our suggested approach to managing bone loss intraoperatively in this complex patient population., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://aoj.amegroups.com/article/view/10.21037/aoj-22-53/coif). The series “Controversies in Shoulder Surgery and Algorithmic Approach to Decision Making” was commissioned by the editorial office without any funding or sponsorship. The authors have no other conflicts of interest to declare., (2023 Annals of Joint. All rights reserved.)

A novel fluorapatite/glucan composite ("FAP/glucan") was developed for the treatment of bone defects. Due to the presence of polysaccharide polymer (β-1,3-glucan), the composite is highly flexible and thus very convenient for surgery. Its physicochemical and microstructural properties were evaluated using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), mercury intrusion, mechanical testing and compared with the reference material, which was a hydroxyapatite/glucan composite ("HAP/glucan") with hydroxyapatite granules (HAP) instead of FAP. It was found that FAP/glucan has a higher density and lower porosity than the reference material. The correlation between the Young's modulus and the compressive strength between the materials is different in a dry and wet state. Bioactivity assessment showed a lower ability to form apatite and lower uptake of apatite-forming ions from the simulated body fluid by FAP/glucan material in comparison to the reference material. Moreover, FAP/glucan was determined to be of optimal fluoride release capacity for osteoblasts growth requirements. The results of cell culture experiments showed that fluoride-containing biomaterial was non-toxic, enhanced the synthesis of osteocalcin and stimulated the adhesion of osteogenic cells.

Obesity predisposes to human type 2 diabetes, the most common cause of diabetic retinopathy. To determine if high-fat diet-induced diabetes in mice can model retinal disease, we weaned mice to chow or a high-fat diet and tested the hypothesis that diet-induced metabolic disease promotes retinopathy. Compared with controls, mice fed a diet providing 42% of energy as fat developed obesity-related glucose intolerance by 6 months. There was no evidence of microvascular disease until 12 months, when trypsin digests and dye leakage assays showed high fat-fed mice had greater atrophic capillaries, pericyte ghosts, and permeability than controls. However, electroretinographic dysfunction began at 6 months in high fat-fed mice, manifested by increased latencies and reduced amplitudes of oscillatory potentials compared with controls. These electroretinographic abnormalities were correlated with glucose intolerance. Unexpectedly, retinas from high fat-fed mice manifested striking induction of stress kinase and neural inflammasome activation at 3 months, before the development of systemic glucose intolerance, electroretinographic defects, or microvascular disease. These results suggest that retinal disease in the diabetic milieu may progress through inflammatory and neuroretinal stages long before the development of vascular lesions representing the classic hallmark of diabetic retinopathy, establishing a model for assessing novel interventions to treat eye disease., (© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Metabotropic glutamate receptors (mGluRs) are located in both plexiform layers in the retina and may modulate transmission between photoreceptors and bipolar cells and between bipolar and ganglion cells. We investigated whether mGluR activation modulates excitatory synaptic input to bipolar cells and ganglion cells in the salamander retinal slice preparation. The group III mGluR agonist L-2-amino-4-phosphonobutyric acid (AP4) inhibited monosynaptic excitatory postsynaptic currents (EPSCs) in ganglion cells evoked by electrical stimuli, whereas group I and group II agonists had no significant effect. AP4 reduced the frequency but not the amplitude of ganglion cell miniature EPSCs, suggesting a presynaptic action at bipolar cell terminals. AP4 also reduced ganglion cell EPSCs evoked by the offset of a light stimulus, suggesting that group III mGluRs modulate release from OFF bipolar cells. Comparison of light-evoked EPSCs in OFF bipolar cells and ganglion cells indicated that AP4 reduced ganglion cell EPSCs by acting primarily at bipolar cell terminals, and to a lesser extent at photoreceptor terminals. The group II/III mGluR antagonist (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG) blocked the effect of AP4 at bipolar cell terminals, consistent with localization of group III mGluRs at these sites. However, CPPG did not increase EPSCs at light offset, indicating that activation of group III mGluRs by synaptic glutamate does not play a large role in modulating transmission from bipolar cells to ganglion cells.

Patterned neuronal activity is implicated in the refinement of connectivity during development. Calcium-imaging studies of the immature ferret visual system demonstrated previously that functionally separate ON and OFF retinal ganglion cells (RGCs) develop distinct temporal patterns of spontaneous activity as their axonal projections undergo refinement. OFF RGCs become spontaneously more active compared with ON cells, resulting in a decrease in synchronous activity between these cell types. This change in ON and OFF activity patterns is suitable for driving the activity-dependent refinement of their axonal projections. Here, we used whole-cell and perforated-patch recording techniques to elucidate the mechanisms that underlie the developmental alteration in the ON and OFF RGC activity patterns. First, we show that before the refinement period, ON and OFF RGCs have similar spike patterns; however, during the period of segregation, OFF RGCs demonstrate significantly higher spike rates relative to ON cells. With increasing age, OFF cells require less depolarization to reach their action potential threshold and fire more spikes in response to current injection compared with ON cells. In addition, spontaneous postsynaptic currents and potentials are greater in magnitude in OFF cells than ON cells. In contrast, before axonal refinement, there are no differences in the intrinsic excitability or synaptic drive onto ON and OFF cells. Together, our results show that developmental changes in ON and OFF RGC excitability and in the strength of their synaptic drives act together to reshape the spike patterns of these cells in a manner appropriate for the refinement of their connectivity.

Ionotropic GABA receptors can mediate presynaptic and postsynaptic inhibition. We assessed the contributions of GABA(A) and GABA(C) receptors to inhibition at the dendrites and axon terminals of ferret retinal bipolar cells by recording currents evoked by focal application of GABA in the retinal slice. Currents elicited at the dendrites were mediated predominantly by GABA(A) receptors, whereas responses evoked at the terminals had GABA(A) and GABA(C) components. The ratio of GABA(C) to GABA(A) (GABA(C):GABA(A)) was highest in rod bipolar cell terminals and variable among cone bipolars, but generally was lower in OFF than in ON classes. Our results also suggest that the GABA(C):GABA(A) could influence the time course of responses. Currents evoked at the terminals decayed slowly in cell types for which the GABA(C):GABA(A) was high, but decayed relatively rapidly in cells for which this ratio was low. Immunohistochemical studies corroborated our physiological results. GABA(A) beta2/3 subunit immunoreactivity was intense in the outer and inner plexiform layers (OPL and IPL, respectively). GABA(C) rho subunit labeling was weak in the OPL but strong in the IPL in which puncta colocalized with terminals of rod bipolars immunoreactive for protein kinase C and of cone bipolars immunoreactive for calbindin or recoverin. These data demonstrate that GABA(A) receptors mediate GABAergic inhibition on bipolar cell dendrites in the OPL, that GABA(A) and GABA(C) receptors mediate inhibition on axon terminals in the IPL, and that the GABA(C):GABA(A) on the terminals may tune the response characteristics of the bipolar cell.

We studied the role of GABA in adaptive changes in a lateral inhibitory system in the tiger salamander retina. In dark-adapted retinal slice preparations picrotoxin caused a slow enhancement of glycine-mediated IPSCs in ganglion cells. The enhancement of glycinergic IPSCs developed slowly over the course of 5-20 min, even though picrotoxin blocked both GABA(A) and GABA(C) receptors within a few seconds. The slow enhancement of glycinergic IPSCs by picrotoxin was much weaker in light-adapted preparations. The slow enhancement of glycinergic inhibitory inputs was not produced by bicuculline, indicating that it involved GABA(C) receptors. The responses of ganglion cells to direct application of glycine were not enhanced by picrotoxin, indicating that the enhancement was not caused by an action on glycine receptors. In dark-adapted eyecup preparations picrotoxin caused a slow enhancement of glycinergic IPSPs and transient lateral inhibition produced by a rotating windmill pattern, similar to the effect of light adaptation. The results suggest that the glycinergic inhibitory inputs are modulated by an unknown substance whose synthesis and/or release is inhibited in dark-adapted retinas by GABA acting at GABA(C) receptors.

Amacrine cells that respond transiently to maintained illumination are thought to mediate transient inhibitory input to ganglion cells. The excitation of these transient amacrine cells is thought to be limited by inhibitory feedback to bipolar cells. We investigated the possibility that desensitizing AMPA and/or kainate (KA) receptors on amacrine cells might also limit the duration of amacrine cell excitation. To determine how these receptors might affect amacrine cell input and output, we made whole-cell recordings from amacrine and ganglion cells in the salamander retinal slice. The specific AMPA receptor antagonist GYKI-53655 blocked non-NMDA receptor-mediated amacrine cell excitatory postsynaptic currents (EPSCs) and kainate puff-elicited currents, indicating that AMPA, and not KA, receptors mediated the responses. Cyclothiazide, an agent that reduces AMPA receptor desensitization, increased the amplitude and duration of amacrine cell EPSCs. To measure the output of transient amacrine cells, we recorded glycinergic inhibitory postsynaptic currents (IPSCs) from ganglion cells, and found that these were also enhanced by cyclothiazide. Thus, prolongation of amacrine cell AMPA receptor activation enhanced amacrine cell output. Current responses elicited by puffing glycine onto ganglion cell dendrites were not affected by cyclothiazide, indicating that the enhancement of glycinergic IPSCs was not due to a direct effect on glycine receptors. These data suggest that rapid AMPA receptor desensitization and/or deactivation limits glycinergic amacrine cell excitation and the resulting inhibitory synaptic output.

EPSCs of retinal ganglion cells decay more slowly than do those of most other CNS neurons, in part because of the long time course of glutamate release from bipolar cells. Here we investigated how glutamate clearance and AMPA receptor desensitization affect ganglion cell EPSCs in the salamander retinal slice preparation. Inhibition of glutamate uptake greatly prolonged ganglion cell EPSCs evoked by light or monosynaptic electrical stimuli but had little effect on spontaneous miniature EPSCs (mEPSCs). This suggests that single quanta of glutamate are cleared rapidly by diffusion but multiple quanta can interact to lengthen the postsynaptic response. Some interaction between quanta is likely to occur even when glutamate uptake is not inhibited. This seems to depend on quantal content, because reducing glutamate release with low Ca2+, paired-pulse depression, or weak stimuli shortened the EPSC decay. High quantal content glutamate release may lead to desensitization of postsynaptic receptors. We reduced the extent of AMPA receptor desensitization by holding ganglion cells at positive potentials. This increased the amplitude of the late phase of evoked EPSCs but did not affect the decay rate after the first 50 msec of the response. In contrast, the holding potential had little effect on mEPSC kinetics. Our results suggest that desensitization limits the late phase of AMPA receptor-mediated EPSCs, whereas glutamate uptake controls the duration of both AMPA and NMDA receptor-mediated responses.

This study addresses how gamma-aminobutyric acid-A(GABAA) and GABAC receptors confer distinct temporal properties to neuronal synaptic responses. The retina is a model system for the study of postsynaptic contributions to synaptic responses because GABAergic amacrine cells synapse onto neurons, which have different combinations of GABAA and GABAC receptors. It is not known, however, how GABAA versus GABAC receptors influence the time course of retinal synaptic responses or what proportion of inhibitory input is mediated by each receptor type. We examined the time courses of synaptic responses mediated by GABA receptors in ganglion and bipolar cells by recording currents evoked by activating amacrine cells with a stimulating electrode in the salamander retinal slice. The pharmacologically isolated, GABAergic synaptic currents were long-lasting in bipolar cells and relatively brief in ganglion cells. The receptors that mediated these temporally distinct synaptic responses exhibited different pharmacological properties. In ganglion cells, GABAergic synaptic currents were abolished by the GABAA receptor antagonists bicuculline or SR95531. In bipolar cells, the GABAC receptor antagonist 3-aminopropyl[methyl]phosphonic acid (3-APMPA) largely blocked GABAergic synaptic responses; the remaining response was blocked by bicuculline or SR95531. The GABAA receptor component of the bipolar cell response was relatively brief compared with the GABAC receptor component. Puffing GABA onto ganglion cell dendrites or bipolar cell terminals yielded similar pharmacological and kinetic results, indicating that transmitter release differences did not determine the response time courses. Moreover, the GABAC receptors on bipolar cells may be different from those reported in rat or fish retina because imidazole-4-acetic acid (I4AA), which acts as an antagonist in these preparations, acts as an agonist in salamander. Our data show that the prolonged synaptic responses in bipolar cells were mediated predominantly by GABAC receptors, whereas transient synaptic responses in ganglion cells were mediated by GABAA receptors.

Competition for postsynaptic targets during development is thought to be driven by differences in temporal patterns of neuronal activity. In the ferret visual system, retinal ganglion cells that are responsive either to the onset (On) or to the offset (Off) of light exhibit similar patterns of spontaneous bursting activity early in development but later develop different bursting rhythms during the period when their axonal arbors segregate to occupy spatially distinct regions in the dorsal lateral geniculate nucleus. Here, we demonstrate that GABAergic transmission plays an important, although not exclusive, role in regulating the bursting patterns of morphologically identified On and Off ganglion cells. During the first and second postnatal weeks, blocking GABAA receptors leads to a decrease in the bursting activity of all ganglion cells, suggesting that GABA potentiates activity at the early ages. Subsequently, during the period of On-Off segregation in the geniculate nucleus, GABA suppresses ganglion cell bursting activity. In particular, On ganglion cells show significantly higher bursting rates when GABAergic transmission is blocked, but the bursting rates of Off ganglion cells are not affected systematically. Thus, developmental differences in the bursting rates of On and Off ganglion cells emerge as GABA becomes inhibitory and as it consistently and more strongly inhibits On compared with Off ganglion cells. Because in many parts of the CNS GABAergic circuits appear early in development, our results also implicate a potentially important and possibly general role for local inhibitory interneurons in creating distinct temporal patterns of presynaptic activity that are specific to each developmental period.

Transient lateral inhibition (TLI), the suppression of responses of a ganglion cell to light stimuli in the receptive field center by changes in illumination in the receptive field surround, was studied in light-adapted mud puppy and tiger salamander retinas using both eyecup and retinal slice preparations. In the eyecup, TLI was measured in on-off ganglion cells as the ability of rotating, concentric windmill patterns of 500-1200 micron inner diameter to suppress the response to a small spot stimulus in the receptive field center. Both the suppression of the spot response and the hyperpolarization produced in ganglion cells by rotation of the windmill were blocked in the presence of 2 microM strychnine or 500 nM tetrodotoxin (TTX), but not by 150 microM picrotoxin. In the slice preparation in which GABA-mediated currents were blocked with picrotoxin, IPSCs elicited by diffuse illumination were blocked by strychnine and strongly reduced by TTX. The TTX-resistant component was probably attributable to illumination of the receptive field center. TTX had a much greater effect in reducing the glycinergic inhibition elicited by laterally displaced stimulation versus nearby focal electrical stimulation. Strychnine enhanced light-evoked excitatory currents in ganglion cells, but this was not mimicked by TTX. The results suggest that local glycinergic transient inhibition does not require action potentials and is mediated by synapses onto both ganglion cell dendrites and bipolar cell terminals. In contrast, the lateral spread of this inhibition (at least over distances >250 micron) requires action potentials and is mainly onto ganglion cell dendrites.

Rapidly triggered excitotoxic cell death is widely thought to be due to excessive influx of extracellular Ca2+, primarily through the N-methyl-D-aspartate subtype of glutamate receptor. By devising conditions that permit the maintenance of isolated retina in the absence of Ca2+, it has become technically feasible to test the dependence of excitotoxic neurodegeneration in this intact neural system on extracellular Ca2+. Using biochemical, Ca2+ imaging, and electrophysiological techniques, we found that (1) rapidly triggered excitotoxic cell death in this system occurs independently of both extracellular Ca2+ and increases in intracellular Ca2+; (2) this cell death is highly dependent on extracellular Cl-; and (3) lethal Cl- entry occurs by multiple paths, but a significant fraction occurs through pathologically activated gamma-aminobutyric acid and glycine receptors. These results emphasize the importance of Ca2+-independent mechanisms and the role that local transmitter circuitry plays in excitotoxic cell death.

The GABAC receptor subtypes on bipolar cells of rats and cold-blooded vertebrates differ in their pharmacological properties and probably have different molecular compositions. With the exception of the rat, native GABAC receptors in mammals had not been studied. In ferret, whole-cell, voltage-clamp recordings were made from bipolar cells in the retinal slice preparation to determine which subtype of GABAC receptor predominated. Puff-evoked GABA currents in bipolar cells were partially reduced by the GABAA receptor antagonist bicuculline, indicating that both GABAA and GABAC receptors mediated the responses. By contrast, GABA currents of ganglion cells were always completely blocked by bicuculline, indicating that GABAA receptors predominated on these cells. Small-amplitude GABA currents of bipolar cells evoked by short-duration puffs were less sensitive to bicuculline than large-amplitude currents evoked by long-duration puffs. This indicates that GABAC receptors mediated proportionately more of the small-amplitude, puff-evoked responses and GABAA receptors mediated more of the large-amplitude, puff-evoked responses. In bipolar cells, the bicuculline-resistant component of the GABA current was entirely blocked by 3-APMPA (3-aminopropyl-(methyl)phosphonic acid), a GABAC receptor antagonist. Picrotoxin, which is relatively ineffective at rat GABAC receptors, completely blocked GABA currents in ferret bipolar cells, indicating that GABAC receptors on ferret bipolar cells resemble those in lower vertebrates rather than those in the rat retina. These results suggest that there may be a diversity of GABAC receptor subtypes on mammalian bipolar cells.

AMPA/kainate (KA) receptors mediate a component of ganglion cell excitatory postsynaptic currents (EPSCs). We investigated whether desensitization at these receptors contribute to the shape of transient EPSCs in ON-OFF ganglion cells. Whole-cell, voltage-clamp recordings were made from ganglion cells in the retinal slice or in isolation. EPSCs were evoked by either stimulating the slice with light or puffing K+ at the outer plexiform layer (OPL). The AMPA/KA receptor-mediated component of the EPSCs was isolated by including NMDA receptor antagonists in the bath. Strychnine and picrotoxin blocked inhibitory inputs. In isolated ganglion cells, cyclothiazide (10 microM), which blocks desensitization in non-NMDA receptors, enhanced both the amplitude and the duration of currents evoked by puffs of AMPA or glutamate. EPSCs evoked by K(+)-puffs in the OPL were also enhanced by cyclothiazide (30 microM). When AMPA/KA receptors were blocked with NBQX (10 microM), no enhancement of the EPSCs by cyclothiazide was observed, indicating that cyclothiazide did not act presynaptically. Cyclothiazide also enhanced the amplitude and duration of both the ON and OFF light-evoked (L-) EPSCs recorded in ON-OFF ganglion cells. Current-voltage relationships showed the enhancement was not voltage dependent. When control and enhanced responses where normalized, it was observed that the rate of desensitization of both the ON and OFF L-EPSCs was decreased by cyclothiazide. Cyclothiazide selectively enhanced the AMPA/KA receptor-mediated component of ganglion cells EPSCs, suggesting that desensitization of AMPA/KA receptors shape transient L-EPSCs.

The actions of glycine on the NMDA receptor-mediated synaptic responses of ganglion cells were studied in the tiger salamander retinal slice. Ganglion cell excitatory postsynaptic currents (EPSCs) were elicited either by exciting bipolar cells with potassium puffs or by light stimulation, and were measured using whole-cell patch-clamp techniques. Increasing bath glycine concentrations to 10 microM had little effect on the amplitude of the puff-evoked EPSCs, indicating either that synaptic glycine concentrations were saturating or that the added glycine was buffered by uptake mechanisms. However, 5,7-dichlorokynurenic acid (5,7-DCK), an antagonist for the glycine site on the NMDA receptor, reduced the ganglion cell responses to NMDA puffs, and reduced the potassium puff- and light-elicited EPSCs. The IC50 values for 5,7-DCK became larger with increasing glycine concentrations, but not with increasing NMDA concentrations, indicating that 5,7-DCK acted at the glycine site. The IC50 values for 5,7-DCK were increased with stronger potassium puffs or light stimuli, suggesting that synaptic glycine levels increased with the strength of the stimuli. EPSCs measured in ON-OFF ganglion cells at light ON and OFF were reduced by 5,7-DCK. For dim light stimuli, the IC50 values were lower for the OFF EPSCs compared to the ON EPSCs, indicating that glycine concentrations were different at the times of OFF and ON activity. Estimates of synaptic glycine concentrations suggest that for dim light stimuli, concentrations at the OFF synapses were not saturated, but concentrations at the ON synapses were saturated.

We investigated the mechanisms underlying the modulation of transmitter release from bipolar cells. Three currents, measured under whole-cell patch clamp in the tiger salamander retinal slice, were used to identify the sites of modulation and to establish their pharmacological profile. (1) A light-elicited inhibitory current was measured in bipolar cells that could be blocked by picrotoxin. This input probably arrives via GABAergic amacrine cells since there is high GABA sensitivity at the bipolar cell terminals and little GABA sensitivity at the dendrites. (2) Voltage-gated barium currents were elicited by depolarizing voltage ramps in bipolar cells. These currents most likely flowed through the calcium channels that are associated with transmitter release at the bipolar terminal. Bath-applied GABA suppressed the barium currents. (3) Puffs of potassium at bipolar dendrites depolarized bipolar cells and elicited an excitatory synaptic current measured in amacrine and ganglion cells. The excitatory synaptic currents, which reflect bipolar cell transmitter release, were also blocked by bath-applied GABA. For all three currents, the effects of GABA could be reversed by picrotoxin, but not by bicuculline or SR95531. The pharmacological profile of the receptors mediating GABA suppression of the barium currents and of excitatory synaptic transmission is characteristic of GABAC receptors (Cutting et al., 1991; Polenzani et al., 1991; Shimada et al., 1992). GABA receptors at bipolar terminals gate a chloride conductance, and most were found to have the pharmacological properties of GABAC receptors (Lukasiewicz et al., 1994). By contrast, the GABA receptors on ganglion cells have been found to be the GABAA subtype (Lukasiewicz and Werblin, 1990; Lukasiewicz et al., 1994). These results suggest that GABA acts presynaptically at GABAC receptors at the bipolar cell terminals. The GABAC receptors open chloride channels that can modulate the release of excitatory transmitter. In some experiments, bicuculline or SR95531 reversed a component of the GABA suppression of synaptic transmission. This indicates that GABAA receptors may also play a role in modulating transmission between bipolar and ganglion cells.

We studied the pharmacology of the GABA receptors on bipolar cell terminals in the retinal slice preparation. Whole-cell patch-clamp recordings were made from the somas of bipolar cells and GABA was puffed near their terminals, after synaptic transmission was blocked. GABA puffs evoked a large chloride current that was reduced by picrotoxin, but in many cells this current was insensitive to blockade by the competitive GABAA receptor antagonists bicuculline and SR95531. Pentobarbital, an enhancer of GABAA receptor-mediated responses, did not significantly increase the magnitude of the current responses to GABA puffed at the bipolar cell terminals. To confirm the effectiveness of GABAA antagonists and pentobarbital in the slice preparation, we measured GABA currents in ganglion cells. In contrast to bipolar cells, the ganglion cell GABA responses were strongly reduced by both bicuculline and SR95531. In addition, pentobarbital strongly enhanced the action of GABA at the ganglion cells. The isomeric GABA agonists cis- and transaminocrotonic acid (CACA and TACA), elicited picrotoxin-sensitive currents in both bipolar and ganglion cells. TACA was more effective than CACA at both cell types. In bipolar cells, TACA and CACA currents were relatively resistant to bicuculline blockade, but in ganglion cells both currents were reduced by bicuculline. GABA receptors on bipolar terminals appear to be pharmacologically different from the GABA receptors found on ganglion cell dendrites. The bipolar cell terminal GABA receptor pharmacology is similar to the pharmacology reported for the rho 1 GABA receptor subunit that was isolated from retina and expressed in Xenopus oocytes (Cutting et al., 1991; Polenzani et al., 1991; Shimada et al., 1992). This receptor, which is both bicuculline and pentobarbital insensitive, has been called the GABAC receptor (Johnston, 1986; Shimada et al., 1992). However, some bipolar cells were somewhat sensitive to blockade by bicuculline, suggesting that these cells had both GABAA and GABAC receptors on their bipolar terminals.

Amacrine cells of the vertebrate retina comprise multiple neurochemical types. Yet details of their electrophysiological and morphology properties as they relate to neurotransmitter content are limited. This issue of relating light responsiveness, dendritic projection, and neurotransmitter content has been addressed in the retinal slice preparation of the tiger salamander. Amacrine cells were whole-cell clamped and stained with Lucifer yellow (LY), then processed to determine their immunoreactivity (IR) to GABA, glycine, dopamine or tyrosine hydroxylase (TOH), and glucagon antisera. Widefield, ON-OFF amacrine cells were glycine-IR. The processes of these cells extended laterally in the inner plexiform layer (IPL) from 250-600 microns. They were either multistratified in the IPL or monostratified near the IPL midline. Three multistratified ON-OFF narrowfield glycine-IR cells also were found. Four types of ON amacrine cells were found to be GABA-IR; all types had their processes concentrated in the proximal IPL (sublamina b). Type I cells were narrowfield (approximately 100 microns) with a compact projection. Type II cells were widefield (220-300 microns) with a sparse projection. Type III cells had an asymmetrical projection and varicose processes. Type IV cells were pyriform and monostratified in sublamina b. One narrowfield ON-OFF amacrine cell, with processes broadly distributed in the middle of the IPL, was GABA-IR. This cell appeared similar to an ON-OFF cell that was glycine-IR and may comprise a type in which GABA and glycine colocalize. Another class of amacrine cell, with processes forming a major plexus along the distal border of the IPL and a lesser plexus in the proximal IPL, produced slow responses at light ON and OFF; these cells were dopamine/TOH-IR. A narrowfield class of transient ON-OFF amacrine cell, with processes ramifying throughout both sublaminae a and b of the IPL, were glucagon-IR; these cells appeared to be dye-coupled at the soma. We have shown that, with respect to GABA, glycine, dopamine, and glucagon, salamander amacrine cells fall into rather discrete groups on the basis of ramification patterns in the IPL and responses to photic stimulation. The physiological, structural, and neurochemical diversity of amacrine cells is indicative of multiple and complex roles in retinal processing.

In response to focal stimuli, ganglion cell dendrites receive excitation over a relatively narrow extent of the inner plexiform layer (IPL). This excitation is embedded in 2 wider lateral inhibitory regions. Here we estimate the lateral dimensions of the inhibitory regions. Ganglion cells were whole-cell patch-clamped and dendrites were identified and located in retinal slices using Lucifer yellow in the pipettes. The spatial distribution of ganglion cell dendritic sensitivity was measured with puffs of transmitter substances applied at different distances along the dendrites. All ganglion cell dendrites were sensitive to glutamate, GABA, and glycine across their full extent. The responses to puffs decreased with lateral distance from the soma and were well fit by Gaussians. The responses to puffs of potassium showed a similar decrement with distance. Since potassium channels are probably uniformly distributed along the dendrites, the similarity in profiles suggests that receptor density is also uniform along the dendrites. The spatial distribution of responses of ganglion cells to excitatory and inhibitory synaptic inputs was measured by depolarizing local populations of bipolar terminals (and subsequently local populations of amacrine cells) with transretinal current (TRC). TRC-stimulating electrodes were displaced laterally, with respect to the ganglion cell soma, to generate response profiles. We estimated the dimensions of the inhibitory and excitatory signals received by the ganglion cells by removing the contributions of their dendrites, the stimulus, and other interneurons from the response profiles. The excitatory signal extended less than 100 microns, the approximate dimensions of the ganglion cell dendrites, and corresponds roughly to the width of the bipolar inputs. The GABAergic signal extended, on average, 253 microns and glycinergic signal extended, on average, 315 microns. These inhibitory signal dimensions correspond to the width of classes of amacrine cell processes measured in other studies.

The neural circuitry underlying movement detection was inferred from studies of amacrine cells under whole-cell patch clamp in retinal slices. Cells were identified by Lucifer yellow staining. Synaptic inputs were driven by "puffing" transmitter substances at the dendrites of presynaptic cells. Spatial sensitivity profiles for amacrine cells were measured by puffing transmitter substances along the lateral spread of their processes. Synaptic pathways were separated and identified with appropriate pre- and postsynaptic pharmacological blocking agents. Two distinct amacrine cell types were found: one with narrow spread of processes that received sustained excitatory synaptic current, the other with very wide spread of processes that received transient excitatory synaptic currents. The transient currents found only in the wide-field amacrine cell were formed presynaptically at GABAB receptors. They could be blocked with baclofen, a GABAB agonist, and their time course was extended by AVA, a GABAB antagonist. Baclofen and AVA had no direct affect upon the wide-field amacrine cell, but picrotoxin blocked a separate, direct GABA input to this cell. The narrow-field amacrine cell was shown to be GABAergic by counterstaining with anti-GABA antiserum after it was filled with Lucifer yellow. Its narrow, spatial profile and sustained synaptic input are properties that closely match those of the GABAergic antagonistic signal that forms transient activity (described above), suggesting that the narrow-field amacrine cell itself is the source of the GABAergic interaction mediating transient activity in the inner plexiform layer (IPL). Other work has shown a GABAB sensitivity at some bipolar terminals, suggesting a population of bipolars as the probable site of interaction mediating transient action. The results suggest that two local populations of amacrine cell types (sustained and transient) interact with the two populations of bipolar cell types (transient forming and nontransient forming). These interactions underlie the formation of the change-detecting subunits. We suggest that local populations of these subunits converge to form the receptive fields of movement-detecting ganglion cells.

Horizontal cells in the mudpuppy eyecup responded to continuous superfusion with L-glutamate, L-aspartate, kainate and quisqualate with a transient depolarization and reduction of the light evoked responses. However, in isolated retina preparations, in which these substances were applied to the photoreceptor side of the retina, the effects were sustained as long as the agonists were present. These results suggest that the transient action of these agonists in eyecup preparations was due to the rapid development of an intraretinal diffusion barrier, and are consistent with the hypothesis that photoreceptors release an excitatory amino acid transmitter.