| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1Departments of Ophthalmology, 2Physiology and Neuroscience, and 3Biochemistry, New York University School of Medicine, New York, New York
Submitted 15 March 2005; accepted in final form 18 May 2005
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
3.5 Hz and an average maximal amplitude of 120 pA. Application of TEA, a potassium channel blocker, increased the amplitude of oscillatory currents by >70% but reduced their frequency by 17%. The TEA effects did not appear to result from direct actions on starburst cells, but rather a modulation of their synaptic inputs. Oscillatory currents were inhibited by 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX), an antagonist of AMPA/kainate receptors, indicating that they were dependent on a periodic glutamatergic input likely from presynaptic bipolar cells. The oscillations were also inhibited by the calcium channel blockers cadmium and nifedipine, suggesting that the glutamate release was calcium dependent. Application of AP4, an agonist of mGluR6 receptors on on-center bipolar cells, blocked the oscillatory currents in starburst cells. However, application of TEA overcame the AP4 blockade, suggesting that the periodic glutamate release from bipolar cells is intrinsic to the inner plexiform layer in that, under experimental conditions, it can occur independent of photoreceptor input. The GABA receptor antagonists picrotoxin and bicuculline enhanced the amplitude of oscillations in starburst cells prestimulated with TEA. Our results suggest that this enhancement was due to a reduction of a GABAergic feedback inhibition from amacrine cells to bipolar cells and the resultant increased glutamate release. Finally, we found that some ganglion cells and other types of amacrine cell also displayed rhythmic activity, suggesting that oscillatory behavior is expressed by a number of inner retinal neurons. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
; Llinás et al. 1994). In the visual system, rhythmicity of cortical activity, which has been proposed to bind local features within an image, appears to reflect not just intracortical processing, but oscillatory signaling received through the lateral geniculate nucleus (LGN) of the thalamus (Castelo-Branco et al. 1998; Doty and Kimura 1963; Ghose and Freeman 1992, 1997; Neuenschwander and Singer 1996). In retina, rhythmic ganglion cell discharges first appear prenatally in the form of spontaneous propagating waves, which underlie activity-dependent development of circuits both within retina and the LGN (Goodman and Shatz 1993; Meister et al. 1991; Wong 1993). In the adult, precise oscillatory discharge patterns have been described for ganglion cells in a number of species under conditions of constant ambient light as well as in response to changes in luminance (Ariel et al. 1983; Doty and Kimura 1963; Kuffler 1953; Neuenschwander et al. 1999; Steinberg 1966). These oscillations show a wide range of frequencies and appear dependent on stimulus size and contrast.
It is now clear that oscillatory activity within the retina is not restricted to ganglion cells. The oscillatory potentials (OPs) of the electroretinogram (ERG) consist of both fast and slow rhythmic components and thereby indicate prominent and widespread oscillations within the retina (Steinberg et al. 1983; Wachtmeister 1998). Although originally thought to be part of the major ERG wave components, it is now clear that OPs reflect postsynaptic activity as evidenced by their sensitivity to glutamate and dopamine (Jaffe et al. 1987; Wachtmeister 1981). Moreover, OPs are attenuated or abolished by the inhibitory transmitters GABA and glycine and are enhanced by the amacrine cell peptide somatostatin (Wachtmeister 1980, 1983). Together, these drug effects suggest that the OPs reflect rhythmic activity generated within the proximal retina. This idea is supported by the finding that bipolar cell axon terminals display calcium-dependent spontaneous membrane oscillations (Burrone and Lagnado 1997; Ma and Pan 2003; Zenisek and Matthews 1998), which may lead to pulsatile transmitter release and rhythmic activity of postsynaptic amacrine and ganglion cells. Indeed, the oscillatory activity displayed by certain amacrine cells in fish retina is thought to be synaptically driven (Djamgoz et al. 1989; Sakai and Naka 1990, 1992). In contrast, subthreshold oscillations in wide-field amacrine cells in the fish retina (Solessio et al. 2002) as well as rhythmic spiking of dopaminergic amacrine cells in mouse (Feigenspan et al. 1998) survive cell isolation, indicating an intrinsic generating mechanism.
Here, we report spontaneous, rhythmic activity recorded from the starburst amacrine cells, a unique subtype that releases both acetylcholine and GABA and thereby functions as both an excitatory and inhibitory retinal interneuron (Agardh and Ehinger 1983; Brecha et al. 1988; O'Malley and Masland 1989). Our pharmacological data indicate that this oscillatory activity is derived from pulsatile, calcium-dependent glutamate release from presynaptic bipolar cell axon terminals. These oscillations appear to reflect a synaptic generating mechanism intrinsic to the proximal retina as they can be induced experimentally in the absence of photoreceptor signaling.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
All animal procedures complied with National Institutes of Health guidelines for the ethical use of animals. Kv3.1/3.2 double knockout mice were generated and bred in the laboratory of Dr. Bernardo Rudy (Ozaita et al. 2004). ICR wild-type and Kv3.1/3.2 double knockout mice (25–60 days old) were deeply anesthetized with an intraperitoneal injection of pentobarbital (0.08g/g body wt). Lidocaine hydrochloride (20 mg/ml) was applied locally to the eyelids and surrounding tissue. A flattened retinal-scleral eyecup preparation developed for rabbit by Hu et al. (2000) was adopted and modified for the mouse. Briefly, the eye was removed under dim red illumination and hemisected anterior to the ora serrata. Animals were killed immediately after enucleation by cervical dislocation. The lens and vitreous humor were removed, and the resultant eyecup preparation was placed on the base of a submersion-type recording chamber. Several radial incisions were made peripherally, and the retina was flattened in the chamber vitreal side up. The chamber was mounted on a microscope stage within a Faraday cage and superfused (1–2 ml/min) with an oxygenated mammalian Ringer solution composed of (in mM) 120 NaCl, 5 KCl, 25 NaHCO3, 0.8 Na2HPO4, 0.1 NaH2PO4, 1 MgSO4, 2 CaCl2, and 10 D-glucose. A pH of 7.4 was maintained by bubbling with 95% O2-5% CO2 at room temperature of 20–22°C.
Electrophysiological recordings
Recordings were made in the whole cell patch mode with an Axopatch 200B amplifier (Axon Instruments, Burlingame, CA). Cells were visualized with near infrared light (>775 nm) at x80 magnification with a Nuvicon tube camera (Dage-MTI, Michigan City, IN) and differential interference optics (DIC) on a fixed-stage microscope (BX51WI; Olympus, Tokyo, Japan). Currents were recorded under voltage clamp, filtered at 1 kHz, sampled at 20 kHz, and stored directly on the computer's hard drive using a Digidata 1200 A/D interface (Axon Instruments). For the characterization of voltage responses, neurons were recorded in the fast current-clamp mode of the amplifier. The resting potential of neurons was adjusted to –70 mV with small injections of DC. pCLAMP (v. 8.02; Axon Instruments) was used for data acquisition with data analysis performed off-line using Minianalysis (v. 6.0.1; Synaptosoft, Decatur, GA) and Origin (v. 6.1; OriginLab, Northampton, MA) software packages. The average oscillatory current was calculated by averaging all oscillations that occurred during a 1-min-long recording. The activation phase (baseline to peak) and the relaxation phase (peak to baseline) of the average current were fitted with first-order exponentials using Clampfit (v. 8.02; Axon Instruments) to calculate the time constants.
Patch electrodes (3–5 M
) were pulled from standard wall borosilicate glass tubing (World Precision Instruments, Sarasota, FL) with a Flaming/Brown type micropipette puller (Sutter Instruments, Novato, CA). Pipettes were filled with a K-gluconate internal solution composed of (in mM) 144 K-gluconate, 3 MgCl2, 0.2 EGTA, 10 HEPES, 4 ATP-Mg, and 0.5 GTP-Tris, pH 7.3 with KOH, and biocytin (0.2% wt/vol, Sigma, St. Louis, MO). All recordings were made under ambient dim light.
Biocytin labeling
Neurons were labeled by allowing biocytin to diffuse from the micropipette during patch recordings. After electrophysiological experiments were completed, retinas were fixed in a cold (4°C) solution of 4% paraformaldehyde in 0.1 M phosphate buffer (pH = 7.3) overnight. Retinas were then washed in phosphate buffer and soaked in a solution of 0.18% hydrogen peroxide in methyl alcohol for 1 h. This treatment completely abolished the endogenous peroxidase activity. Retinas were then washed in phosphate buffer and reacted with the Elite ABC kit (Vector Laboratories, Burlingame, CA) and 1% Triton X-100 in sodium phosphate-buffered saline (9% saline, pH = 7.5). Retinas were subsequently processed for peroxidase histochemistry using 3,3'-diaminobenzidine (DAB) as the chromagen, dehydrated and flat-mounted for light microscopy.
Statistical analyses
Data were analyzed using Student's t-test statistic and are presented as means ± SE.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Recordings were made from on-center starburst amacrine cells the somata of which were displaced to the ganglion cell layer (GCL) and the dendritic arbors of which stratified within sublamina b of the inner plexiform layer (IPL). We were unable to specifically label starburst amacrine cells in our mouse preparation prior to recordings, and so we typically targeted small, round somata in the GCL. After electrophysiological recordings, biocytin was injected into all cells to confirm their identity by post hoc histology. Overall, 60% of our recordings were from starburst cells, with the remainder from other amacrine cell types as well as small ganglion cells.
Starburst amacrine cells in the mouse showed the typical symmetric dendritic morphology described in other species (Bloomfield and Miller 1986; Famiglietti 1983; Tauchi and Masland 1984). This included four to five primary dendrites that first branched into thin, wavy, intermediate segments that divided further into a dense plexus of distal branches showing numerous varicosities (Fig. 1A). We found that starburst amacrine cells displayed robust and stereotypic electrophysiological properties. This included an average membrane capacitance of 22.61 ± 0.40 pF and input resistance of 195.6 ± 5.9 M (n = 70). By comparison, the non-starburst cells in our sample showed a significantly larger average membrane capacitance (33.67 ± 1.04 pF) and membrane input resistance (340.5 ± 26.4 M, n = 20) despite having apparently similar soma sizes. Another characteristic feature of starburst cell physiology was revealed by their membrane voltage response to extrinsic current steps. Membrane depolarization triggered by pulses larger than +50 pA tended to saturate, so it was not possible to depolarize the membrane to potentials more positive than –20 mV (Fig. 1B). It has been suggested that this may reflect the presence of Kv3 potassium channels in the soma and proximal dendrites, which create a shunt when activated and thereby limit the extent of depolarization (Ozaita et al. 2004). Under our recording conditions, starburst cells showed no spontaneous or evoked spiking, consistent with previous studies using whole cell recording techniques (Peters and Masland 1996; Taylor and Wässle 1995). Large depolarizing current pulses did evoke a characteristic small, transient response component (Fig. 1B). However, this component never reached potentials more positive than 0 mV and was not blocked by application of TTX.
|
Under control conditions, oscillations had a mean frequency of 3.52 ± 0.14 Hz and an average maximal amplitude of 121.36 ± 8.74 pA (n = 33). Interestingly, the oscillations present in mouse starburst cells resemble those described in a subset of displaced amacrine cells in the adult ferret retina (Aboelela and Robinson 2004), although the frequency of the oscillations in the ferret was considerably lower.
Effects of TEA on oscillatory currents in starburst cells
During the course of a recent study, we used TEA to examine the role of Kv3 potassium channels in starburst cell activity (Ozaita et al. 2004). A surprising result was that low doses of TEA (1 mM) produced a large and reproducible increase in the amplitude of oscillatory activity (Fig. 1C). The average maximal amplitude of oscillations was increased >70% from 125.2 ± 14.4 pA in control Ringer to 214.8 ± 33.2 pA in TEA (n = 9) (Fig. 2B). In addition, TEA reduced the frequency of oscillations by 17%, from an average 3.48 ± 0.16 Hz in Ringer to 2.88 ± 0.17 Hz (Fig. 2A).
|
relaxation = 20.7 ± 2.3 ms in control and 14.8 ± 1.3 ms in TEA; Fig. 2D). Overall, TEA affected both the amplitude and kinetics of starburst cell oscillations. It should also be noted that TEA decreased the frequency of miniature synaptic events. When miniature events were counted outside oscillations, it was found that TEA significantly diminished their number by >35% (from 2.0 ± 0.28 Hz in control to 1.29 ± 0.17 Hz in TEA, n = 9, P < 0.01) without affecting their amplitude.
Starburst cells in the mouse retina display a high density of voltage-gated Kv3 channels that are responsible for large outward potassium currents (Ozaita et al. 2004). Therefore the effects of TEA on the oscillatory currents could reflect direct actions on the Kv3 channels in starburst cells. To test this idea, we examined the effects of TEA on oscillations in starburst cells from Kv3.1–Kv3.2 double knockout (DKO) animals. As shown in Fig. 2E, TEA remained effective in increasing the amplitude of oscillatory currents in starburst cells from DKO animals; the average maximum amplitude in TEA was 215.2 ± 79.4 pA (n = 4). These data indicate that TEA effects on the oscillations in wild-type animals were not due to a blockade of Kv3 channels in starburst cells.
Oscillatory currents in starburst amacrine cells are synaptically mediated
In the next series of experiments, we examined whether the oscillatory currents in starburst cells were dependent on the excitatory synaptic drive from presynaptic bipolar cells. Bipolars cells form glutamatergic synapses onto starburst amacrine cells at which AMPA/kainate ionotropic receptors are localized (Brandstätter and Hack 2001; Brandstätter et al. 1998; Firth et al. 2003; Morgans 2000; Thoreson and Witkovsky 1999; Yang 2004). Application of CNQX, a specific blocker of AMPA/kainate receptors (Mayer and Armstrong 2004), reversibly blocked the oscillatory currents and miniature synaptic events (Fig. 3A). On average, CNQX (10 µM) reduced the oscillation frequency by 81% under control conditions and reduced the frequency of oscillations prestimulated by TEA by 76% (Fig. 3B). In addition, CNQX decreased the maximal amplitude of oscillations in control retinas by 90% and the oscillations enhanced by TEA by 89% (Fig. 3C). Clearly, these data indicate that oscillatory currents in starburst amacrine cells are dependent on the glutamatergic drive, likely from bipolar cells to starburst cells.
|
Because bipolar cells have different calcium channels, the activation of which participates in the modulation of glutamate release, we examined the effects of calcium blockers on the spontaneous oscillations in starburst cells (Berntson et al. 2003; Pan 2000, 2001; Tachibana 1999). Application of cadmium ions produced a nearly complete blockade of both basal and TEA-enhanced oscillatory currents, leaving only the miniature synaptic events (Fig. 4A). The frequency of basal oscillations as well as those enhanced with TEA was inhibited by >98% by CdCl2 (Fig. 4B). The maximal amplitude of basal oscillations and those enhanced by TEA were reduced by cadmium to a similar degree, 85 and 90%, respectively (n = 4; Fig. 4C). Oscillatory currents enhanced by TEA were also largely inhibited by application of nifedipine (30 µM), a specific blocker of L-type calcium channels. Nifedipine blocked >74% of the oscillations' amplitude without significantly affecting their frequency (n = 2, data not shown). Taken together, these data are consistent with a calcium-dependent glutamate release underlying the oscillatory activity of starburst amacrine cells.
|
The mGluR6 metabotropic glutamate receptors are expressed in the retina at the synapse between photoreceptors and on-center bipolar cells (Nomura et al. 1994; Ueda et al. 1997). When activated, these receptors lead to a closure of cation channels that result in a hyperpolarization of on-center bipolar cells and a reduction in their excitatory drive of proximal neurons (Gerber 2003; Nakajima et al. 1993; Tian and Slaughter 1994, 2003; Thomsen 1997). Application of AP4, an agonist of these receptors, produced a sustained blockade of the basal oscillatory currents until it was washed out (Fig. 5A). In contrast, after enhancement of the oscillations with TEA, application of AP4 produced only a transient inhibition (Fig. 5A). Within 6 min of AP4 application, the oscillations were totally recovered while still in the presence of the drug. At its maximal effect, AP4 reduced the average frequency of oscillations enhanced by TEA by 83% and maximal amplitude by 76% (n = 4; Fig. 5, B and C). Again, the maximal inhibition by AP4 occurred within 3 min, but recovery was complete by 6 min (Fig. 5D). In contrast, we examined the effects of AP4 for time periods of up to 10 min and never saw a reversal of the blockade of basal oscillations until we returned to control conditions.
|
The excitatory drive from bipolar cells to starburst amacrine cells can be modified by GABAergic feedback inhibition from amarine cells (Freed et al. 2003; Matsui et al. 2001; Pan 2001; Shen and Slaughter 2001; Völgyi et al. 2002; Wässle et al. 1998). In addition, starburst cell activity may be altered by direct inhibition from neighboring amacrine cells. We therefore examined the effects of GABA receptor blockers on the oscillatory currents. Application of picrotoxin (PTX, 50 µM), a mixed GABAA and GABAC receptor antagonist, had no effect on basal oscillations under control conditions (Fig. 6A). However, PTX did dramatically increase the amplitude of the oscillations already enhanced with TEA (Fig. 6B). As shown in Fig. 6C, PTX increased the amplitude of the average current without affecting significantly the kinetics of the activation and relaxation phases. Interestingly, PTX produced a reduction in the mean frequency of oscillations by 37%, concomitant with the 72% increase in average maximal amplitude (n = 5; Fig. 6D). Application of the GABAA receptor antagonist, bicuculline (BMI, 10 µM), produced effects very similar to those of PTX, including a reduction in the average frequency (32%) and an increase in the average maximal amplitude (32%) of the oscillatory currents (n = 3; Fig. 6E).
|
As aforementioned, we encountered other types of amacrine and ganglion cells during the course of experimentation. Many of these also showed spontaneous oscillations, but with characteristics different from those of starburst cells. For example, a type of wild-field amacrine cell we encountered in the mouse displayed oscillatory currents that were broader, more frequent, and with more constant amplitudes than those displayed by starburst cells (Fig. 7, A and B). In general, ganglion cell oscillations were more irregularly shaped and with slightly lower amplitudes than those recorded in the starburst cells (Fig. 7C). Figure 7D illustrates the different kinetics of the average oscillatory current recorded in wide-field amacrine cells from those recorded in starburst or ganglion cells. These data indicate that oscillatory currents, although highly variable across cells, are expressed by a number of neurons in the inner retina.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
; Meister et al. 1991; Zhou 1998). However, these oscillations, which occur every 1–2 min, rely on cholinergic circuitry as well as excitatory, glycinergic circuitry that subsequently becomes inhibitory in the adult. Moreover, these oscillations disappear within several days postnatally and are thus quite distinct from the oscillatory currents reported here. In contrast, our results indicate that the oscillatory activity in the adult results from periodic, calcium-dependent release of glutamate from presynaptic bipolar cell axon terminals. Taken together, these data suggest strongly that the oscillatory activity we recorded in starburst cells does not reflect propagating spontaneous waves as seen during development. Oscillations are dependent on glutamatergic input from bipolar cells
The finding that application of CNQX largely blocked the oscillatory currents suggests that they are dependent on activation of AMPA/kainate receptors postsynaptic to bipolar cell terminals. Cadmium ions and nifedipine also blocked the oscillatory currents, indicating that calcium channels are involved in the regulation of periodic glutamate release from bipolar cells. The persistent, pulsatile release of glutamate from presynaptic terminals has been found to be secondary to oscillations in intracellular calcium that are maintained by a calcium-induced calcium release process in various types of neuron including retinal bipolar cells (Aniksztejn et al. 1995; Burrone et al. 2002; Cherubini et al. 1991; Llobet et al. 2003; Pasti et al. 2001). In bipolar cells, calcium ion entry via calcium channels organized in clusters could trigger the release of calcium from internal stores that ultimately control the exocytosis of glutamate vesicles (Burrone et al. 2002; Llobet et al. 2003). Also, a resonant mechanism combining calcium channel activation/inactivation and calcium-activated potassium channel activation/deactivation could underlie a cyclic entry of calcium into the synaptic terminal (Vigh et al. 2003). Calcium influx through T- and L-types of voltage-dependent calcium channels has been shown to underlie the spontaneous membrane oscillations in rat bipolar cells (Ma and Pan 2003). A similar generating mechanism has been suggested for the oscillatory activity of CA3 neurons in the hippocampus (Aniksztejn et al. 1995). Here, spontaneous oscillatory currents were synaptically driven by presynaptic glutamatergic inputs and were dependent on calcium entry likely via high-voltage-activated calcium channels (Bacci et al. 1999).
Our finding that oscillations in starburst cell activity is dependent on bipolar cell input is consistent with observations in fish retina that oscillations in some amacrine cells arise from the activity of local circuits (Djamgoz et al. 1989; Sakai and Naka 1990, 1992). In contrast, Solessio et al. (2002) described intrinsic oscillatory activity in isolated wide-field amacrine cells of the fish that are generated from the interplay between calcium and potassium currents. Intrinsic rhythmic spike activity has also been described in isolated dopaminergic amacrine cells in the mouse (Feigenspan et al. 1998). Together, these data indicate that both synaptic circuitry and intrinsic membrane properties can play roles in generating the oscillatory activity of different amacrine cell types across a number of species.
Actions of TEA are presynaptic to starburst amacrine cells
Low concentrations of TEA produced a robust enhancement of the oscillatory currents in starburst cells. This suggests the involvement of TEA-sensitive potassium channel subfamilies, such as BK, Kv1, and/or Kv3 (Rudy et al. 1999). It has been shown recently that starburst cells express Kv3 channels (Ozaita et al. 2004; Tian et al. 2003), raising the possibility that the TEA effects on oscillatory activity were due to direct actions on starburst cells. However, two of our findings argue against this. First, in our voltage-clamp experiments, starburst cells were held at –70 mV, below the threshold of activation of Kv3 channels (Rudy et al. 1999). Second, we found that the oscillatory activity recorded from starburst cells in Kv3.1/3.2 double knockout mice was still enhanced by TEA. Together, these data suggest strongly that TEA enhancement of the oscillations reflected actions on the large conductance calcium-activated (BK) and/or Kv1.1 potassium channels found in bipolar cell axon terminals (Gribkoff et al. 2001; Pinto and Klumpp 1998; Sakaba et al. 1997). In this scenario, application of TEA would block the potassium channels in bipolar cell terminals leading to a membrane depolarization and activation of voltage-gated calcium channels. The increase in internal calcium ion concentration triggered a calcium-induced release of glutamate. In addition to this increased release, perhaps TEA synchronized the release of multiple vesicles from one or more active zones (Sharma and Vijayaraghavan 2003; Singer et al. 2004), leading to a larger overall release of glutamate from the synaptic endings. Although TEA increased the amplitude of the average current, it also accelerated both the activation and the relaxation phases consistent with a modification of the release mechanism. The finding that TEA slightly decreased the frequency of oscillations while increasing their amplitude suggests that larger vesicles were released less frequently, consistent with a modification of release synchronization. Further, the finding that TEA decreased the frequency of miniature synaptic events suggests a modification of the recruitment of a finite pool of synaptic vesicles. Perhaps TEA enhancement of the oscillations reflected the fusion of individual vesicles or an increased simultaneous release of individual vesicles as clusters.
Oscillations in starburst cells can occur independent of photoreceptor synaptic drive
Application of AP4, a specific agonist of the mGluR6 receptors (Nakajima et al. 1993; Thomsen 1997) at photoreceptor to on-center bipolar cell synapse, eliminated the oscillatory currents in starburst cells. Interestingly, whereas AP4 produced a sustained inhibition of basal oscillations, its effect on TEA-enhanced oscillations was only transient. This suggests that the sustained depolarization induced by TEA could overcome the hyperpolarization of bipolar cells induced by AP4 and thereby trigger periodic glutamate release. Moreover, these data show that the underlying pulsatile glutamate release can occur, at least under experimental conditions, when photoreceptor synaptic drive is absent. This suggests that the machinery underlying periodic glutamate release and the resultant starburst cell oscillations is located at the bipolar cell level and can occur without periodic glutamate release from photoreceptor terminals. It is important to note here that our recordings were made from starburst-b amacrine cells for which morphological and physiological data indicate innervation strictly via the ON retinal pathway (Bloomfield and Miller 1986; Famiglietti 1983; Peters and Masland 1996; Taylor and Wässle 1995). Thus the oscillatory activity under conditions of ON pathway blockade with AP4 cannot be due to an emergent photoreceptor signaling to the starburst cells via the OFF pathway.
Feedback inhibition affects starburst cell oscillations
Both GABAA and GABAC receptors are localized to mammalian bipolar cell axon terminals (Bloomfield and Dacheux 2001; Pan 2001; Wässle et al. 1998), which mediate the feedback inhibition from amacrine cells that regulates glutamate release (Euler and Masland 2000; Matsui et al. 2001; Shen and Slaughter 2001; Völgyi et al. 2002; Zhang et al. 2002). Our finding that both picrotoxin and bicuculline increased the amplitude of the TEA-enhanced oscillatory currents in starburst cells is consistent with an enhanced release of glutamate from bipolar cell terminals due to a reduced feedback inhibition. Like TEA, the enhanced oscillatory currents produced by the GABA blockers was concomitant with a reduced frequency, again suggesting improved coordination of glutamate release from a finite vesicle pool in bipolar cell terminals. Interestingly, we found that GABA blockers had little effect on the amplitude of basal oscillations. Perhaps, under our basal experimental conditions, the glutamate release from bipolar cells is insufficient to generate a significant GABAergic feedback inhibition of the bipolar cell terminal. In essence, the GABA blockers are ineffective because there is no inhibition to block. In this scenario, only after glutamate release is increased by TEA is there a sizeable feedback inhibition generated to modulate further release. In any event, these results indicate that the oscillatory activity of starburst cells is plastic as it can be modulated by the established feedback circuitry in the IPL.
Significance of oscillations in the retina
Our results add to a growing list of reports of oscillatory activity within the retina. At a cellular level, it has been long known that ganglion cell spike discharges show both spontaneous and light-evoked oscillations (Ariel et al. 1983; Doty and Kimura 1963; Kuffler 1953; Neuenschwander et al. 1999; Steinberg 1966). It is now clear that spontaneous oscillatory activity is not restricted to the ganglion cells but occurs in both bipolar cells and amacrine cells as well (Burrone and Lagnado 1997; Djamgoz et al. 1989; Ma and Pan 2003; Zenisek and Matthews 1998). These rhythmic activities are reflected in the prominent oscillatory potentials of the ERG (Wachtmeister 1998). Our results indicate not only a variety of cells in the inner mouse retina with oscillatory activity, but a variety of kinetics as well. These data are consistent with the wide range of frequencies found for the oscillatory activity reported for ganglion cells in a number of preparations (Neuenschwander et al. 1999). Thus the inner retina appears to contain groups of independent oscillators, possibly differentiated by neuronal subtype.
Clearly, the subthreshold oscillatory activity of bipolar and amacrine cells can produce periodic release of neurotransmitter that, in turn, will produce oscillatory activity in postsynaptic cells, particularly ganglion cells (Arai et al. 2004). Indeed, our data indicate that it is the periodic release of glutamate that underlies the robust oscillations in starburst amacrine cells. Interestingly, the periodic, spontaneous, and light-evoked discharges of neighboring ganglion cells have been found to be highly synchronized (Arnett and Spraker 1981; Hu and Bloomfield 2003; Mastronarde 1983; Meister et al. 1995; Neuenschwander et al. 1999). It is thought that electrical coupling and/or synchronized synaptic release play a role. This suggests that the subthreshold oscillations in presynaptic amacrine cells may also be synchronized, possibly due to inputs from common bipolar cells. It will be of interest to determine whether the oscillatory currents in neighboring starburst amacrine cells are synchronous as well.
Synchronous subthreshold oscillations distributed among a network of cells, when combined with stimulus-driven inputs, will result in light-evoked synchronous activity as cells will tend to reach spike threshold together. Synchronous activity of retinal neurons, as suggested elsewhere in the CNS, can serve to increase stimulus efficacy, encode additional information and/or bind information about local visual features (reviewed by Singer et al. 1997). It has also been shown that oscillatory activity, possibly propagated via electrical synapses, can distribute signals over long distances, thereby coordinating the activity of large cell networks (Neuenschwander and Singer 1996). Overall, these data suggest that the spontaneous oscillatory activity of inner retinal neurons may serve to organize ensembles of cells that can coordinate via synchronous activity when activated by appropriate visual stimuli.
At the cortical level, it has been posited that synchronized oscillations in cell assemblies act to bind temporal and spatial visual features within the image (Singer and Gray 1995). These rhythmic cortical discharges reflect, at least in part, synchronous oscillations within the LGN (Alonso et al. 1996). Moreover, the temporal precision of the geniculate cell spiking appears to be due to the synchronized oscillatory activity derived from the retina (Neuenschwander and Singer 1996). In this scheme, the widespread, spontaneous oscillatory activity created in the inner retina appears to be the initial step in entraining stimulus-induced rhythmicity conserved throughout the visual system.
|
GRANTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: S. Bloomfield, Dept. Ophthalmology, NYU School of Medicine, 550 First Ave., New York, NY 10016 (E-mail: blooms01{at}med.nyu.edu)
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Agardh E and Ehinger B. Retinal GABA neuron labelling with [3H]isoguvacine in different species. Exp Eye Res 36: 215–229, 1983.[CrossRef][ISI][Medline]
Alonso JM, Usrey WM, and Reid RC. Precisely correlated firing in cells of the lateral geniculate nucleus. Nature 383: 815–819, 1996.[CrossRef][Medline]
Aniksztejn L, Sciancalepore M, Ben Ari Y, and Cherubini E. Persistent current oscillations produced by activation of metabotropic glutamate receptors in immature rat CA3 hippocampal neurons. J Neurophysiol 73: 1422–1429, 1995.
Arai I, Yamada Y, Asaka T, and Tachibana M. Light-evoked oscillatory discharges in retinal ganglion cells are generated by rhythmic synaptic inputs. J Neurophysiol 92: 715–725, 2004.
Ariel M, Daw NW, and Rader RK. Rhythmicity in rabbit retinal ganglion cell responses. Vision Res 23: 1485–1493, 1983.[CrossRef][ISI][Medline]
Arnett D and Spraker TE. Cross-correlation analysis of the maintained discharge of rabbit retinal ganglion cells. J Physiol 317: 29–47, 1981.
Bacci A, Verderio C, Pravettoni E, and Matteoli M. Synaptic and intrinsic mechanisms shape synchronous oscillations in hippocampal neurons in culture. Eur J Neurosci 11: 389–397, 1999.[CrossRef][ISI][Medline]
Berntson A, Taylor WR, and Morgans CW. Molecular identity, synaptic localization, and physiology of calcium channels in retinal bipolar cells. J Neurosci Res 71: 146–151, 2003.[CrossRef][ISI][Medline]
Bloomfield SA and Dacheux RF. Rod vision: pathways and processing in the mammalian retina. Prog Retin Eye Res 20: 351–384, 2001.[CrossRef][ISI][Medline]
Bloomfield SA and Miller RF. A functional organization of ON and OFF pathways in the rabbit retina. J Neurosci 6: 1–13, 1986.[Abstract]
Brandstatter JH and Hack I. Localization of glutamate receptors at a complex synapse. The mammalian photoreceptor synapse. Cell Tissue Res 303: 1–14, 2001.[CrossRef][ISI][Medline]
Brandstatter JH, Koulen P, and Wässle H. Diversity of glutamate receptors in the mammalian retina. Vision Res 38: 1385–1397, 1998.[CrossRef][ISI][Medline]
Brecha N, Johnson D, Peichl L, and Wässle H. Cholinergic amacrine cells of the rabbit retina contain glutamate decarboxylase and gamma-aminobutyrate immunoreactivity. Proc Natl Acad Sci USA 85: 6187–6191, 1988.
Burrone J and Lagnado L. Electrical resonance and Ca2+ influx in the synaptic terminal of depolarizing bipolar cells from the goldfish retina. J Physiol 505: 571–584, 1997.[CrossRef][ISI][Medline]
Burrone J, Neves G, Gomis A, Cooke A, and Lagnado L. Endogenous calcium buffers regulate fast exocytosis in the synaptic terminal of retinal bipolar cells. Neuron 33: 101–112, 2002.[CrossRef][ISI][Medline]
Castelo-Branco M, Neuenschwander S, and Singer W. Synchronization of visual responses between the cortex, lateral geniculate nucleus, and retina in the anesthetized cat. J Neurosci 18: 6395–6410, 1998.
Cherubini E, Ben-Ari Y, Ito S, and Krnjevic K. Persistent pulsatile release of glutamate induced by N-methyl-D-aspartate in neonatal rat hippocampal neurones. J Physiol 436: 531–547, 1991.
Djamgoz MB, Downing JE, and Wagner HJ. Amacrine cells in the retina of a cyprinid fish: functional characterization and intracellular labelling with horseradish peroxidase. Cell Tissue Res 256: 607–622, 1989.[ISI][Medline]
Doty RW and Kimura DS. Oscillatory potentials in the visual system of cats and monkeys. J Physiol 168: 205–218, 1963.
Euler T and Masland RH. Light-evoked responses of bipolar cells in a mammalian retina. J Neurophysiol 83: 1817–1829, 2000.
Famiglietti EV. On and off pathways through amacrine cells in mammalian retina: the synaptic connections of "starburst" amacrine cells. Vision Res 23: 1265–1279, 1983.[CrossRef][ISI][Medline]
Feller MB. Spontaneous correlated activity in developing neural circuits. Neuron 22: 653–656, 1999.[CrossRef][ISI][Medline]
Feigenspan A, Gustincich S, Bean BP, and Raviola E. Spontaneous activity of solitary dopaminergic cells of the retina. J Neurosci 18: 6776–6789, 1998.
Firth SI, Li W, Massey SC, and Marshak DW. AMPA receptors mediate acetylcholine release from starburst amacrine cells in the rabbit retina. J Comp Neurol 466: 80–90, 2003.[CrossRef][ISI][Medline]
Freed MA, Smith RG, and Sterling P. Timing of quantal release from the retinal bipolar terminal is regulated by a feedback circuit. Neuron 38: 89–101, 2003.[CrossRef][ISI][Medline]
Gerber U. Metabotropic glutamate receptors in vertebrate retina. Doc Ophthalmol 106: 83–87, 2003.[CrossRef][ISI][Medline]
Ghose GM and Freeman RD. Oscillatory discharge in the visual system: does it have a functional role? J Neurophysiol 68: 1558–1574, 1992.
Ghose GM and Freeman RD. Intracortical connections are not required for oscillatory activity in the visual cortex. Vis Neurosci 14: 963–979, 1997.[ISI][Medline]
Goodman CS and Shatz CJ. Developmental mechanisms that generate precise patterns of neuronal connectivity. Cell 72: 77–98, 1993.
Gribkoff VK, Starrett JE Jr, and Dworetzky SI. Maxi-K potassium channels: form, function, and modulation of a class of endogenous regulators of intracellular calcium. Neuroscientist 7: 166–177, 2001.[Abstract]
Hu EH and Bloomfield SA. Gap junctional coupling underlies the short-latency spike synchrony of retinal alpha ganglion cells. J Neurosci 23: 6768–6777, 2003.
Hu EH, Dacheux RF, and Bloomfield SA. A flattened retina-eyecup preparation suitable for electrophysiological studies of neurons visualized with trans-scleral infrared illumination. J Neurosci Methods 103: 209–216, 2000.[CrossRef][ISI][Medline]
Jaffe EH, Urbina M, Ayala C, Drujan Y, and Drujan BD. Dopamine and noradrenaline content in fish retina: modulation by serotonin. J Neurosci Res 18: 345–351, 1987.[CrossRef][ISI][Medline]
Kuffler SW. Discharge patterns and functional organization of mammalian retina. J Neurophysiol 16: 37–68, 1953.
Llinás RR. The intrinsic electrophysiological properties of mammalian neurons: insights into central nervous system function. Science 242: 1654–1664, 1988.
Llinás R, Ribary U, Joliot M, and Wang XJ. In: Temporal Coding in the Brain, edited by Buzsaki G, Llinás R, Singer W, Berthoz A, and Christen Y. Berlin: Springer, 1994, p. 251–272.
Llobet A, Cooke A, and Lagnado L. Exocytosis at the ribbon synapse of retinal bipolar cells studied in patches of presynaptic membrane. J Neurosci 23: 2706–2714, 2003.
Ma YP and Pan ZH. Spontaneous regenerative activity in mammalian retinal bipolar cells: roles of multiple subtypes of voltage-dependent Ca2+ channels. Vis Neurosci 20: 131–139, 2003.[CrossRef][ISI][Medline]
Mastronarde DN. Correlated firing of cat retinal ganglion cells. Spontaneously active inputs to X and Y cells. J Neurophysiol 49: 303–324, 1983.
Matsui K, Hasegawa J, and Tachibana M. Modulation of excitatory synaptic transmission by GABA(C) receptor-mediated feedback in the mouse inner retina. J Neurophysiol 86: 2285–2298, 2001.
Mayer ML and Armstrong N. Structure and function of glutamate receptor ion channels. Annu Rev Physiol 66: 161–181, 2004.[CrossRef][ISI][Medline]
Meister M, Lagnado L, and Baylor DA. Concerted signaling by retinal ganglion cells. Science 270: 1207–1210, 1995.
Meister M, Wong RO, Baylor DA, and Shatz CJ. Synchronous bursts of action potentials in ganglion cells of the developing mammalian retina. Science 252: 939–943, 1991.
Morgans CW. Neurotransmitter release at ribbon synapses in the retina. Immunol Cell Biol 78: 442–446, 2000.[CrossRef][Medline]
Nakajima Y, Iwakabe H, Akazawa C, Nawa H, Shigemoto R, Mizuno N, and Nakanishi S. Molecular characterization of a novel retinal metabotropic glutamate receptor mGluR6 with a high agonist selectivity for L-2-amino-4-phosphonobutyrate. J Biol Chem 268: 11868–11873, 1993.
Neuenschwander S, Castelo-Branco M, and Singer W. Synchronous oscillations in the cat retina. Vision Res 39: 2485–2497, 1999.[CrossRef][ISI][Medline]
Neuenschwander S and Singer W. Long-range synchronization of oscillatory light responses in the cat retina and lateral geniculate nucleus. Nature 379: 728–732, 1996.[CrossRef][Medline]
Nomura A, Shigemoto R, Nakamura Y, Okamoto N, Mizuno N, and Nakanishi S. Developmentally regulated postsynaptic localization of a metabotropic glutamate receptor in rat rod bipolar cells. Cell 77: 361–369, 1994.[CrossRef][ISI][Medline]
O'Malley DM and Masland RH. Co-release of acetylcholine and gamma-aminobutyric acid by a retinal neuron. Proc Natl Acad Sci USA 86: 3414–3418, 1989.
Ozaita A, Petit-Jacques J, Volgyi B, Ho CS, Joho RH, Bloomfield SA, and Rudy B. A unique role for Kv3 voltage-gated potassium channels in starburst amacrine cell signaling in mouse retina. J Neurosci 24: 7335–7343, 2004.
Pan ZH. Differential expression of high- and two types of low-voltage-activated calcium currents in rod and cone bipolar cells of the rat retina. J Neurophysiol 83: 513–527, 2000.
Pan ZH. Voltage-activated Ca2+ channels and ionotropic GABA receptors localized at axon terminals of mammalian retinal bipolar cells. Vis Neurosci 18: 279–288, 2001.[CrossRef][ISI][Medline]
Pasti L, Zonta M, Pozzan T, Vicini S, and Carmignoto G. Cytosolic calcium oscillations in astrocytes may regulate exocytotic release of glutamate. J Neurosci 21: 477–484, 2001.
Peters BN and Masland RH. Responses to light of starburst amacrine cells. J Neurophysiol 75: 469–480, 1996.
Pinto LH and Klumpp DJ. Localization of potassium channels in the retina. Prog Retin Eye Res 17: 207–230, 1998.[CrossRef][ISI][Medline]
Rudy B, Chow A, Lau D, Amarillo Y, Ozaita A, Saganich M, Moreno H, Nadal MS, Hernandez-Pineda R, Hernandez-Cruz A, Erisir A, Leonard C, and Vega-Saenz de Miera E. Contributions of Kv3 channels to neuronal excitability. Ann NY Acad Sci 868: 304–343, 1999.
Sakaba T, Ishikane H, and Tachibana M. Ca2+ -activated K+ current at presynaptic terminals of goldfish retinal bipolar cells. Neurosci Res 27: 219–228, 1997.[CrossRef][ISI][Medline]
Sakai HM and Naka KI. Dissection of the neuron network in the catfish inner retina. V. Interactions between NA and NB amacrine cells. J Neurophysiol 63: 120–130, 1990.
Sakai HM and Naka K. Response dynamics and receptive-field organization of catfish amacrine cells. J Neurophysiol 67: 430–42, 1992.
