Morphology of dissociated cells
Viewed in Hoffman optics, horizontal cells were larger in size, more flattened in appearance than bipolar cells and other retinal neurons which surrounded them. Unlike in situ stains (Connaughton et al., 2004
; Song et al., 2008
), axons were not found and may have been sheared off. In zebrafish different L- or C-type physiologies can not as yet be associated with morphological features in culture, so we have followed the previous convention of dividing plated cells into two size types (McMahon, 1994
; Connaughton & Dowling, 1998
). There are the somewhat smaller round radiate types with 5-7 dendrites (HA
), and larger types with irregularly shaped cell bodies and 2 or 3 stout dendrites (HB
) (Connaughton & Dowling, 1998
). The cell bodies of HA
types (28/80, 35%) were 11.4 ± 2.2 × 9.4 ± 1.6 μm diameter (major and minor axes). Cell bodies of the more numerous HB
type cells (52/80, 65%) were 14.5 ± 2.4 × 11.2 ± 1.5 μm diameter. These dimensions are similar to those previously reported (Connaughton & Dowling, 1998
). Horizontal cells represented only a small fraction of dissociated cells. Even in our selected fields only about 3% (80/2396) were horizontal cells.
Bipolar cell bodies were flask shaped with short dendrites emerging from the top. Some (42 /223, 19%) retained axons with terminal boutons. Bipolar cell bodies, where measured, were 8.2 ± 1.3 × 7.1 ± 1.0 μm (N=155), smaller than horizontal cell bodies. Bipolar cell bodies were convex and appeared to protrude farther into the medium than the mesa-like horizontal cell counterparts. Morphologically identifiable bipolar cells were 12% (298/2396) of plated and studied cells.
The remaining plated cells were rounded in appearance, and similar to bipolar cells in size. With diolistic staining of zebrafish slices, a shotgun approach without known bias, 48% of stains were bipolar cells (Connaughton et al., 2004
). While the type-specific efficiencies of cell dissociation and diolistic staining may differ somewhat, still it seems reasonable to suppose that a substantial fraction the remaining dissociated cells were also bipolar cells.
GABA and glutamate after-hyperpolarizations in horizontal cells
In previous studies (Nelson et al., 2003
), horizontal cells responded to glutamate treatment with waveforms composed of depolarizations and/or after-hyperpolarizations (dep/AHP). The AHP component was traced to glutamate stimulation of Na+
ATPase activity. GABA elicits a similar pattern of response. In , a horizontal cell, because of depolarized initial resting potential, does not react immediately to glutamate application, but hyperpolarizes on withdrawal of glutamate (AHP). This AHP response restores membrane potential. When GABA is then applied to the same cell a GABA-induced depolarization is seen. With GABA removal, a second, weaker AHP occurs after a short delay. In a third treatment in the same cell, the combination of GABA and glutamate produces an even more robust depolarization followed by AHP. GABA does not act to antagonize the glutamate response, as might be expected of an inhibitory neurotransmitter, but rather appears synergistic. In a 4th
treatment on this same cell glutamate alone evokes another dep/AHP response. In the 5th
and final treatment, gramicidin is applied. This irreversibly permeabilizes the membrane to cations, depolarizing the cell to ‘0’ membrane potential. In a total of 27 horizontal cells found with dep/AHP responses to glutamate, 13 dep/AHP responses were also evoked by GABA. No GABA-induced hyperpolarizations were seen in this group.
Fig. 1 GABA, glutamate (glut) and the combination evoke depolarization/after-hyperpolarization (dep/AHP) in a dissociated horizontal cell. Arrows point to AHP components, which follow stimulation by any of these three treatments. The asterisk marks the peak (more ...)
GABA and muscimol responses in horizontal and bipolar cells
Horizontal and bipolar cells that are depolarized by GABA are less affected by muscimol. In is an example of a bipolar-cell subtype that responds to GABA with a dep/AHP response. Muscimol (1st treatment), an agonist for GABAA,C ionotropic receptors, evokes a smaller dep/AHP response from this cell than GABA (2nd treatment). More typically, however, bipolar cells hyperpolarize in response to either of these agonists (, 1st and 2nd treatments). This latter pattern suggests GABAA,C receptors are expressed on bipolar cell dendrites and caused hyperpolarization. This common, hyperpolarizing, bipolar-cell GABA response is further developed in a companion paper (Connaughton et al., in preparation).
Fig. 2 Bipolar cells can be ether depolarized or hyperpolarized by GABA. A. A bipolar type with dep/AHP response to GABA responds with much lower amplitude to muscimol (Musc). The arrow points to the GABA AHP component. A combination of bicuculline (Bic) and (more ...)
The probability of GABA or muscimol evoking hyperpolarizing or dep/AHP responses in horizontal and bipolar cells is summarized in . About 38% of horizontal cells responded to GABA with dep/AHP responses (27 /71; ), about evenly distributed between HA (12) and HB (15) types. Depolarizing components were found in 18 of these HA or HB types, while the remainder only responded with the AHP component. For cells with depolarizing components, the median amplitude (both HA and HB) was 0.17 log units, about 22 mV. GABA-evoked hyperpolarizations in horizontal cells (3%, 2 /71) were almost non existent as compared to dep/AHP responses, or as compared to bipolar cell hyperpolariztions (67%, 194/288). A large group of horizontal cells did not respond to GABA at all (59%, 42/71). Nonetheless these latter cells responded robustly to gramicidin, indicating an intact and functional plasma membrane.
Fig. 3 Statistics for GABA and muscimol response types in horizontal and bipolar cells. A. GABA, but not muscimol, evokes dep/AHP responses in horizontal cells. B. GABA and muscimol evoke hyperpolarizing responses in most bipolar cells, but GABA evokes dep/AHP (more ...)
With muscimol treatments only 8% of horizontal cells responded with dep/AHP (3/40), while ~90% did not respond. Muscimol was less effective than GABA in evoking dep/AHP in horizontal cells (p < 0.001, Chi Square test). In horizontal cells there was no pattern of responsiveness either to baclofen (a GABAB agonist) or glycine, with 86% (12/14) not responding to baclofen, and 93% (14/15) not responding to glycine (data not shown).
Bipolar cell patterns of response to GABA and muscimol differed from those of horizontal cells (). For GABA, 194 of 288 bipolar cell regions (67%) hyperpolarized. Similarly for muscimol 56 of 91 bipolar cell regions (62%) hyperpolarized. GABA-evoked dep/AHP responses were seen in 5% of bipolar cell regions (13/288), much less common than bipolar-cell hyperpolarizations, or horizontal-cell dep/AHP responses (p < 0.001, Chi Square test). Nonetheless the 13 examples of this response in bipolar cells are sufficient to group as a subtype and to characterize. Eight of the 13 GABA-excited bipolar cell regions were recorded from axotomized cells. This suggests, at least, a soma-dendritic origin for bipolar cell dep/AHP responses, as is also true for hyperpolarizing responses (Connaughton et al., in preparation). Muscimol evoked dep/AHP responses occurred in 2% of bipolar cells (2 /91). While this fraction was not significantly less than for GABA (p ≥ 0.05, Chi Square test), it is clear from individual records () that for GABA-excited bipolar cells, as for horizontal cells, the actions of GABA and muscimol are indeed distinct, and that muscimol was the weaker agonist for the dep/AHP response.
Actions of ionotropic GABA antagonists
Antagonists of ionotropic GABA receptors did not block GABA-induced dep/AHP responses in any type of dissociated zebrafish retinal neuron. This suggests the dep/AHP mechanism does not involve ionotropic GABA receptors. For bipolar cells, the combination of bicuculline and picrotoxin failed to block a GABA-induced dep/AHP response (, 3rd treatment). In the same microscope field, these same antagonists effectively blocked a GABA-induced bipolar-cell hyperpolarization (, 3rd treatment). In a horizontal cell depolarizes in response to GABA, and an AHP response is seen (1st and 2nd treatments). Although the AHP component is reduced, in the presence of the bicuculline and picrotoxin mixture (2nd treatment), the depolarizing component persists.
Fig. 4 Picrotoxin and bicuculline do not block GABA-induced depolarizatons in horizontal cells. GABA depolarizes this horizontal cell and induces AHP (arrow) producing a dep/AHP response. The depolarization persists in the presence of bicuculline (Bic) and picrotoxin (more ...)
Bicuculline and picrotoxin insensitivity of the dep/AHP response was the rule not only for horizontal cells and bipolar cells, but for all dissociated cells where this response was seen. Taken as a group, which includes 3 horizontal cells and 3 bipolar cells, 31 of 32 dep/AHP responses to GABA (10 or 20 μM) failed to be either fully or partially blocked by picrotoxin (25 or 50 μM, data not shown). In the dep/AHP group as a whole, some depolarizing responses (38%, 12/32) were enhanced by picrotoxin, but no enhancements occurred among horizontal cells (0/3). Among cells with measured initial GABA depolarizing components in the picrotoxin-treated group, the median control and recovery response was 0.14 log units (n = 58). The median picrotoxin treated GABA response was 0.21 log units (n = 29). The increase is significant (p < 0.01, Mann Whitney U test). This suggests an enhancement of depolarizing GABA responses by picrotoxin in some retinal neurons.
Bicuculline (25 or 50 μM) was not an effective antagonist of horizontal cell dep/AHP. Of 6 such responses seen in horizontal cells only 1 was partially blocked and none of 3 bipolar cell dep/AHP was blocked (data not shown). Enhancement was seen in 1 of the 6 horizontal cell dep/AHP responses. Bicuculline was ineffective at blocking dep/AHP responses in other dissociated retinal neurons. Six of 51 responses (12%) were blocked or partially blocked. Response enhancement occurred in 41% of cases (21/51). Among bicuculline treated cells with initial GABA depolarizations, the median control and recovery response was 0.10 log units (n = 92). The median bicuculline treated GABA depolarization was 0.19 log unit (n = 46, data not shown). This increase was significant (p < 0.01, Mann Whitney U test). As with picrotoxin, the change was not in the direction of response blockade, but of response enhancement.
Together with weak muscimol sensitivity ( & ), the ineffectiveness of ionotropic GABA antagonists, applied either alone or in combination, suggests that depolarizing GABA responses in dissociated zebrafish retinal neurons, including some horizontal cells and a subtype of bipolar cells, do not result from the actions of iontropic GABA receptors. In the same experiments, however, the hyperpolarizing responses evoked by GABA in bipolar cells were blocked by picrotoxin (but not bicuculline) with a complete or partial block observed in all cases (; see also Connaughton et al., in preparation).
Dose dependence of GABA dep/AHP responses
GABA-induced dep/AHP responses were evoked by treatments using less than 1 μM GABA (). Five concentrations were applied to each cell, each increasing by a factor of 2, as seen in . While the initial concentration in was 0.25 μM GABA, initial concentrations in other experiments varied from 0.125 to 1.0 μM GABA. The half-amplitude concentration was estimated by eye for 25 cells. This median was 0.5 μM. This group included 6 identified horizontal cells with a median half-amplitude concentration of 1.5 μM. The horizontal cell half amplitudes did not differ from the group as a whole (p ≥ 0.05, Mann Whitney U test). No bipolar cells were identified in this treatment group.
Fig. 5 Dose-response properties of dep/AHP responses. A. GABA treatments, increasing from 0.25 μM to 4.00 μM, evoke increasing amplitudes of depolarization. The half amplitude dose is about 0.5 μM for this HA type horizontal cell. B. (more ...)
The dep components of the dep/AHP responses were then measured and pooled so that median responses could be calculated for each concentration tested in this set of experiments. The pooled data were fit to a Hill model (). An apparent k value (half saturation) of 0.61 μM was found with an n value of 1.15.
Sodium and chloride requirement of GABA dep/AHP responses
The sodium and chloride requirements of dep/AHP responses were investigated by lowering ionic concentrations in the perfusate. In GABA induces a dep/AHP response in a dissociated neuron (1st treatment). With replacement of NaCl by LiCl in the extracellular medium, both the depolarization and the AHP components were lost (2nd treatment). This blockade was reversible. In 15 cells with dep/AHP responses, all were blocked by lithium for sodium substitution. In 8 cases a small hyperpolarizing GABA response was revealed during the substitution. The median depolarization in control and recovery treatments was 0.15 log units (n = 30). The median response in the absence of [Na]o was −0.025 log units (n = 15). The change was significant (p < 0.001, Mann Whitney U test). One of the 15 cells in this group was an identified bipolar cell.
Fig. 6 Ionic dependence of dep/AHP responses. A. GABA evoked dep/AHP response is blocked by substituting lithium chloride for sodium chloride (LiCl for NaCl) in the extracellular perfusate. The effect was reversible. B. GABA evoked a dep/AHP response is blocked (more ...)
Lithium substitution did not block GABA-induced hyperpolarizations, but did reduce response amplitudes (data not shown). The median hyperpolarization in control and recovery was −0.155 log units (n = 48). The median hyperpolarization in the [Na]o free medium was −0.095 (n = 24). The amplitude reduction was significant (p < 0.01, Mann Whitney U test). This group included 7 identified bipolar-cell AOI's.
Lowering extracellular chloride was achieved by substituting sodium isethionate for sodium chloride. This reduced [Cl−]o from 130 mM to 10 mM. As seen in , this treatment abolished both depolarizing and AHP components of the dep/AHP response. In this example the depolarizing GABA response does not recover, but the AHP component is restored. Fourteen dissociated neurons with GABA-induced dep/AHP responses were treated with low [Cl−]o. In 13 cases the dep/AHP response was blocked or partially blocked. In one case the response was enhanced. The result is significant in that a GABA depolarization resulting from opening ionotropic channels in cells with high [Cl−]i might be expected to increase in amplitude when [Cl−]o was lowered. The opposite was observed. The median control depolarization was 0.13 log units. In low [Cl−]o the median response changed to 0.00 log units. The change is significant (p < 0.001, Mann Whitney U test). The group included one identified horizontal cell and one bipolar cell.
Association of GABA and muscimol responses
Dep/AHP response enhancement, sometimes caused by GABA antagonists, might result from blockade of an underlying but hidden, hyperpolarizing, ionotropic GABA mechanism mixed together with the dep/AHP mechanism. Indeed lithium blockade of the dep/AHP mechanism left a small, residual, median GABA hyperpolarization. This could be evidence for two mechanisms on the same neuron: a dep/AHP mechanism and an ionotropic GABA mechanism. In one example of a bipolar type with dep/AHP response to GABA (, st treatment), muscimol induced a hyperpolarizing response (2nd treatment). Glycine also evoked hyperpolarization in this cell (4th treatment). In 3 of 5 bipolar cell AOI's depolarized by GABA, a subsequent dose of muscimol evoked a hyperpolarizing response. In 8 horizontal cells depolarized by GABA, no muscimol-evoked hyperpolarizing responses were seen. Neither of these results appears to be preferential association of the two mechanisms as, in any event, ~60% of bipolar cells are hyperpolarized by muscimol (), while virtually no horizontal cells are (). In 187 other cells with GABA evoked dep/AHP, there were 54 cases (29%) of muscimol inducing a hyperpolarizing response. This is 7 short of the 61 that might have been expected by independent association (based on the frequency of each response in this population), but the difference is not significant (p ≥ 0.05, Chi Square test). Taken together these results suggest that depolarizing GABA responses and hyperpolarizing muscimol responses on bipolar cells and other retinal neurons are separate mechanisms, and can associate independently. The exception to this pattern appears to be horizontal cells, where hyperpolarizing, ionotropic, GABA receptors are excluded, but the dep/AHP mechanism is preferentially expressed.
Fig. 7 Mixed GABA mechanisms in a bipolar cell depolarized by GABA. A bipolar cell depolarized by GABA is hyperpolarized by muscimol (Musc). Glycine (Gly) also hyperpolarizes this cell. Baclofen (Bac) has little effect. The bipolar cell image revealed a long (more ...)
Muscimol induced dep/AHP was much less common than GABA induced dep/AHP, nonetheless, where present, the mechanism appears to be shared. In the present study there were 900 dissociated cells, excluding identified horizontal and bipolar types, where GABA treatment was followed by muscimol treatment. GABA dep/AHP responses were found in 187 of these. Among these 187, muscimol dep/AHP occurred in 27 cases. But only 9 cases were expected by independent association based on the separate frequency of occurrence of GABA and muscimol dep/AHP responses in this population. The excess is significant (p < 0.01, Chi square test), suggesting that in the general population of retinal neurons there is an increased probability of muscimol dep/AHP responses among cells with a GABA dep/AHP response, albeit the frequency was very low.