Participation of the histamine receptor encoded by the gene hclB (HCLB) in visual sensitivity control: an electroretinographic study in Drosophila melanogaster

Drosophila stocks

Two hclB mutant lines with the genotypes st hclB^T1/TM3, Sb and st hclB^T2/TM3, Sb were generated by mutagenesis with ethyl methanesulfonate. They originated from the Zuker’s collection (Department of Neurosciences, University of California, San Diego, CA). The amino acid substitution (P293S) in hclB^T1 mutants affects a highly conserved residue within the second transmembrane domain of the protein, whereas hclB^T2 is a null mutant (W111*) [21]. The flies were kept on yeast-molasses medium at 25 °C with a 12 h:12 h light-dark regime. Because of lack of viable homozygotes from the hclB^T1 mutants for all experiments, we used 3–7-day-old hemizygous females produced by crossing of two mutant lines with flies Df(3R)E79/MRS, where the deficiency (86F1–87B9) eliminated the hclB chromosomal region. Hemizygous control flies were obtained in a similar way to the wild-type flies Oregon R (Bloomington Drosophila stock center, Bloomington, IN). Here, we follow the nomenclature proposed by [8] for two genes: hclA (ort) and hclB. These are also known as Dm HA-Cl I and Dm HA-Cl II [11], HisCl 2 and HisCl 1 [10], and HisCl-α1 and HisCl-α2 [9].

Electroretinogram recording

ERG was chosen for assessment of sensitivity at the level of photoreceptor-to-LMC synapses because as mentioned above, it represents the activity of both photoreceptors and LMCs [18-20]. Furthermore, as a mass response, it has the advantage of representing the activity of many populations of LMCs with different sensitivities. Because of the high gain of the photoreceptor-to-LMC synapses [23,24], which varies significantly among individual synapses [24], many LMCs with high contrast gain and narrow dynamic range of their responses cover as a population the overall dynamic range of visual responses in particular conditions of ambient illumination.

Flies were briefly anesthetized with CO₂ and immobilized into Eppendorf pipettes with cut ends, allowing the fly heads to protrude. The heads were additionally fixed to the pipette tips using a small droplet of low-melting wax. The ERGs were recorded by means of glass microelectrodes with a tip diameter of 15–20 μm, filled with Ringer solution (in mmol/l: NaCl 130, KCl 4.7, CaCl₂ 1.9; MgCl₂ 4, HEPES 1.3; pH 7.14). The recording electrode was positioned at the corneal surface and the reference electrode was placed onto the head carapace. Conductive electrode gel was used for contact improvement. The ERG responses were amplified at a bandpass of 0–1,000 Hz using a low noise WPI ISO-DAM preamplifier (World Precision Instruments, Sarasota, FL). They were digitized at 5 kHz and analyzed using the WPI LAB-Trax4 Data acquisition system (Data-Trax software, World Precision Instruments).

Light stimulation

Diffuse light from two green LUXEON^®V light-emitting diodes (LEDs; LXHL-PMo2; Lumileds Future Electronics, Pointe Claire, Quebec, Canada) with a dominant wavelength of 530 nm were used for test and background illumination. The parameters and the mode of presentation of the test stimuli were controlled by a custom-made LED controller (Stimuled 01, Department of Electronic Engineering, Technical University of Sofia, Bulgaria). The test stimulus intensity was varied over a range of 5.5 log units, the maximal intensity being 7.23 log quanta s⁻¹ μm⁻² at the plane of the eye. The intensities of the backgrounds used were 4.66, 5.66, and 6.66 log quanta s⁻¹ μm⁻². When the intensity-response (V/logI) functions of the ERG responses were tested, each of the eyes was first dark adapted for 2 min and a V/log I function was obtained, with stimulus intensity being increased in 0.5 log unit steps. Then, after 2 min adaptation to each of the backgrounds, V/log I functions were obtained again under the backgrounds. To prove that the functional state of the flies was not changed by the end of the experiments, the eyes were dark adapted again and control V/log I curves were obtained; these did not differ significantly from the first dark adapted curves. To obtain V/logI functions, two types of intermittent stimuli were used: 1) short stimuli with 0.3 s ON and 1.2 s OFF periods; the 0.3 s stimuli were the shortest that allowed for reliable separation and assessment of ON and OFF ERG transients; and 2) long stimuli with 2 s ON and 8 s OFF periods. To test the dynamics of dark sensitivity recovery after short-term light adaptation, 0.3 s ON/1.2 s OFF intermittent stimuli with intensity of 3.73 or 4.73 log quanta s⁻¹ μm⁻² were continuously presented in the dark. Short adapting backgrounds with 2 to 20 s duration and intensities of 5.66, 6.16, or 6.66 log quanta s⁻¹ μm⁻² were added periodically and the amplitudes of the ERG responses preceding and following the light adapting pulses were compared.

Data analysis

The intensity-response functions of the ERG responses were fitted by the least square method to Naka-Rushton equation, V/V_max=Iⁿ/(Iⁿ+σⁿ), where V represents ERG response amplitude; V_max, response maximal amplitude; I, test stimulus intensity; σ, stimulus intensity required to produce 0.5 V_max response (I₅₀); and n, an exponent related to the steepness of the intensity-response function and hence to response dynamic range. The response dynamic range was estimated as the intensity span of the responses with 5 to 95% V_max amplitude. The absolute sensitivity of the ERG responses was assessed by their threshold, specifically the stimulus intensity necessary for obtaining a criterion amplitude of 0.5 mV. The response relative sensitivity was assessed by the σ (I₅₀) value. For statistical evaluation of the data, two-way ANOVA (ANOVA) with the Bonferroni test (alpha=0.05) was used.

Intensity-response functions of the electroretinogram responses

Temporal characteristics of the electroretinogram responses

The temporal characteristics of the ERG transients were also changed in the two hclB mutants. The ON and OFF transients had slower time course (see Figure 1 and Figure 4A –inset). The peak latencies of the ON and OFF transients were longer in the mutant as compared to the wild-type flies (10⁻⁹<p<0.05 for different stimulation conditions, n=10 for all groups of flies in each of the light stimulation conditions; Figure 4A). The difference was small under dark adaptation. Under light adaptation, a well expressed difference was obtained in responses to 2 s stimuli. The changes in the temporal characteristics of the mutant ERG transients were also tested by offline filtering of the ERG records. When low-pass filtered, the responses of the mutant flies were not dramatically reduced, while those of the wild-type flies were strongly diminished (Figure 4B). Conversely, the mutant fly responses were more sensitive to high-pass filtering (result not shown). The difference between the mutant and wild-type flies was more pronounced under light adaptation (Figure 4B, right).

Time course of dark sensitivity recovery after short-term light adaptation

The time course of sensitivity recovery of the ERG responses after termination of light adapting stimuli with a few seconds’ duration was assessed by comparing the amplitudes of the ERG responses to 0.3 s stimuli in the periods, preceding and following presentation of adapting stimuli with 2 to 20 s duration and three intensities, specifically Ib1=5.66 log quanta s⁻¹ μm⁻², Ib2=6.16 log quanta s⁻¹ μm⁻², and Ib3=6.66 log quanta s⁻¹ μm⁻². The 0.3 s test stimulus duration was the shortest that allowed for reliable separation between the ON and OFF transients. The following two test stimulus intensities were used: 3.73 and 4.73 log quanta s⁻¹ μm⁻². The first was below the threshold of the ERG receptor component, so that the two ERG transients were recorded in isolation. The second elicited both the receptor component and transients, thus allowing for comparison of sensitivity changes of the receptor and LMC responses. The difference between the hclB mutants and the wild-type flies was best manifested when 2 s adapting stimuli were used. Depending on the particular combinations of test and adapting stimuli (the number [n] of the flies in the groups varied between 10 and 20), the following results were obtained: 1) In most cases, in the postadaptational period, the ON and OFF transients of the wild-type flies showed instant sensitivity recovery (Figure 5A). 2) The recovery took several seconds when the dimmer stimulus was combined with a very bright background (not shown). 3) When the brighter test stimulus was combined with Ib1 and Ib2, postadaptational potentiation was observed (Figure 5B,C). The ON transient amplitude increased up to 120% of the preadaptational value, on average, while only a few percent increase of the OFF transient amplitude was observed. The amplitude of the ERG receptor component was significantly diminished during the first few seconds of the postadaptational period in all conditions tested, so in wild-type flies, a clear discrepancy was observed between the dynamics of sensitivity changes of the receptor and postreceptoral ERG components. In the hclB mutants, the ON and OFF transient sensitivity recovery was significantly delayed in all conditions tested (two-way ANOVA, with the Bonferroni test, 10⁻¹⁵<p<10⁻⁴ for different combinations of test and adapting stimuli; Figure 5). Some small postadaptational potentiation was only occasionally seen. The dynamics of sensitivity recovery was similar between the receptor and postreceptoral ERG components.

Contribution of the influences mediated through the histamine receptor encoded by the gene hclB to the light adaptation process

The light adaptation process allows the visual system to work under a wide range of ambient illumination (~10 log units), regardless of the restricted range of visual cell responses. It changes visual sensitivity to prevent response saturation while keeping the contrast sensitivity constant (independent of the ambient illumination) [25,26]. This is achieved by a shift of the visual operating range along the stimulus intensity axis (demonstrated by the almost parallel shift of the response V/logI curves). This mechanism already operates at the photoreceptor level, but postreceptoral mechanisms are also involved. The process of light adaptation is also characterized by changes in the temporal characteristics of the transmitted signals such that the redundant (low-frequency) information is filtered, which increases temporal resolution and optimizes encoding the dynamic properties of visual stimuli [24,27,28]. This raises the following question: Are the histamine-gated HCLB receptors involved in the above mentioned processes? Our results show that 1) under light adaptation, the ON transients of the hclB mutants saturate earlier. Their dynamic range is narrowed (in our experiments by 0.5 log units for 0.3 s stimuli and by 1 log unit for 2 s stimuli). This means that, under light adaptation, the functional HCLB receptors contribute to avoid saturation and widen the dynamic range of the laminar ON responses. The same is not true, however, for the OFF responses, whose dynamic range is not significantly changed in the hclB mutants. 2) In the hclB mutants, the relative sensitivity of the ON responses to long stimuli is decreased (the V/logI curves are shifted to the right) under dark adaptation as well as under moderate-intensity backgrounds, while the relative sensitivity of the responses obtained under bright backgrounds is not changed. Therefore, the intensity span of the V/logI curve shift along the intensity axis during light adaptation is narrowed in the mutant flies. This implies that the functional HCLB receptors have some contribution to this shift. This effect is particularly true when long light stimuli are presented. Again, a similar effect is not observed with respect to the OFF response. 3) The hclB mutant flies have slower kinetics of their ON and OFF transients, indicated by the delayed peak latencies of these responses. This result is consistent with the data, obtained at a cellular level, showing slower kinetics of the mutant LMCs’ responses to light flashes [12]. The slower kinetics of the mutant ON and OFF transients is also indicated by the fact that they are less sensitive to low-pass filtering and more sensitive to high-pass filtering. The effect of hclB mutation on the peak latency is most prominent in responses to long stimuli under light adaptation. The effect of filtering is also more pronounced under light than dark adaptation. This implies that the HCLB receptors contribute to the filtering of low-frequency components of visual signals during light adaptation (redundancy reduction). On the basis of the results presented above, a conclusion may be drawn that the histamine-gated HCLB receptors have some contribution to the process of light adaptation, which is more pronounced with respect to the ON response. Concerning OFF response, the effects of light adaptation on temporal characteristics are mainly affected.

Diverse mechanisms may underlie the sensitivity changes mediated by the histamine receptor encoded by the gene hclB

What are the possible mechanisms involved in the HCLB-mediated modulation of visual responses mentioned above? The HCLB receptor is expressed in the epithelial glia [12,13], which separates the individual cartridges in the lamina, participating in the creation of a high-resistance barrier [14-16]. Along with LMCs, the epithelial glia is a postsynaptic element in the tetrad synapses, which are the most numerous synaptic contacts of R1-R6 type photoreceptors in the first optic neuropile [14,16,17]. Epithelial glia is an astrocyte-type glia, so it is involved in regulation of electrolyte homeostasis of the extracellular space, as well as in neurotransmitter clearance and recycling [29-32]. As the tetrad synapses are invaginated synapses, they are characterized by a narrow synaptic cleft and high extracellular electrical resistance. This facilitates changes in neurotransmitter and electrolyte concentrations and the creation of electrical field potentials. Several mechanisms might thus be involved in the HCLB receptor–mediated modulation of synaptic transmission at the photoreceptor-to-LMC synapse. Some of these have been discussed elsewhere [12,32]. 1) HCLB receptors may effectively compete with HCLA receptors for histamine, released by photoreceptors upon light stimulation. They have been shown to be even more sensitive to histamine than the homomeric HCLA receptors, expressed in LMCs [10,12]. 2) Histamine binding to HCLB may activate histamine uptake by epithelial glia and thus change the histamine concentration in the synaptic cleft. 3) As HCLB receptors are Cl¯ channels, the ion currents through these channels may shunt the postsynaptic Cl¯ currents in LMCs. This is possible if the Cl¯ equilibrium potential is more negative than the glial membrane potential and the histamine-gated HCLB channel opening results in inward Cl¯ currents. The membrane potential of glial cells is highly dependent on K⁺ permeability and is thus strongly negative. Indeed, an intracellular potential of about –90 mV has been recorded in laminar glia with respect to the intercellular space of the retina [33]. However, if we take into account the strongly negative value of the extracellular field potential in the laminar cartridges (−20 to −40 mV [28,33]), the transmembrane potential of the epithelial glia in the laminar cartridges may well be within the range of −50 to −70 mV and positive in reference to E_Cl¯. This assumption is supported by the fact that laminar glial cells produce slow hyperpolarization of a few mV during light illumination [33]. 4) Inward Cl^¯ currents through HCLB channels may be a source of the depolarizing shift of the extracellular field potential in the cartridges during illumination, similar to the postsynaptic currents in the LMCs, a mechanism proposed in [28]. It has been suggested by several authors [15,16,24,28,34,35] that changes in the laminar extracellular field potential may be strongly involved in the process of light adaptation because of the resultant changes in membrane potential of photoreceptor axon terminals and thus the neurotransmitter release. They also may take part in the modulation of temporal characteristics of the signal transmitted through the photoreceptor-to-LMC synapse by subtracting low-frequency components of the signal. This mechanism is similar to that proposed for the horizontal cell-to-photoreceptor feedback in vertebrate retina [36,37]. 5) Cl^¯ currents through HCLB channels can change the extracellular Cl^¯ concentration, and thus the Cl^¯ electrochemical gradient and postsynaptic currents in the LMCs and amacrine cells. The effect of inward Cl^¯ currents in LMCs and glial cells is opposed by the effect of Cl^¯ extrusion mechanisms in these cells. As shown in [38], these ion fluxes may not be balanced, which may modulate visual responses of LMCs. This mechanism will be discussed further below.

Effects on the dynamics of sensitivity changes after short-term light adaptation, mediated through the histamine receptor encoded by the gene hclB

Our results are the first to show the involvement of the histamine receptor HCLB in the control of dynamics of dark sensitivity recovery after short-term light adaptation. They clearly demonstrate that HCLB receptors speed up the sensitivity recovery after cessation of light-adapting stimuli with a duration of a few seconds. They may also participate in potentiation of the LMC responses in the postadaptational period. In our experiments, the dynamics of sensitivity changes was tested in light adaptation conditions similar to those reported previously in the blowfly [38]. The authors of this research followed the changes in the resting potential and responses of the photoreceptors and LMCs to light flashes after cessation of an adapting illumination with 100 ms to 20 s duration. They found a small depolarization and potentiation of the LMCs’ light responses in the postadaptational period lasting up to 20 s (postadaptational potentiation). During the postadaptational period, photoreceptors were shown to undergo hyperpolarization and an increase of their light response amplitude. However, this process had a slower time course (Figure 5 in their paper) and in the first few seconds of the postadaptational period, the potentiation of the LMCs responses was not in concert with the changes in photoreceptor responses. A mechanism was proposed and supported by the results of ionophoretic injections of Cl^¯ whereby the postadaptational potentiation is at least partially due to the imbalance between the reduced inward Cl^¯ current through histamine-gated Cl^¯ channels in LMCs after cessation of light stimulation and the increased outward Cl^¯ current created by activation of Cl^¯ extrusion mechanisms (because of Cl^¯ accumulation during the preceding adaptation). The resulting change in E_Cl¯ was supposed to underlie, at least partially, the potentiation of the LMCs’ responses. The histamine-gated Cl^¯ channels that are expressed on LMCs and are thus supposed to participate in the above mentioned mechanism are HCLA receptors [12,13]. It seems plausible, however, that a similar mechanism may take place in the epithelial glia, which are also postsynaptic elements in tetrad synapses, with the participation of the HCLB receptors. Indeed, our results support this hypothesis. In wild-type flies, we found a postadaptational potentiation of the ERG ON and OFF transients, which depended in the same manner on background intensity and duration and which followed a similar time course to that described for LMCs [38]. At the same time, the ERG receptor component was diminished and recovered in a few seconds. The discrepancy between the sensitivity changes in the ERG receptor and laminar components in the wild-type flies observed in our experiments was similar to the discrepancy between the sensitivity changes in photoreceptor and LMC responses during the first few seconds of the postadaptational period (but not later on) seen in [38]. Potentiation was absent or only rarely seen in the hclB mutants; the dark sensitivity recovery was delayed and the postadaptational changes in the amplitude of the ERG transients were similar to those of the photoreceptor component.

The delayed sensitivity recovery in the hclB mutants might also be a possible explanation of the difference in the degree of potentiation of their ERG transients, depending on the type of stimulation, namely, 0.3 s ON/1.2 s OFF or 2 s ON/8 s OFF, the potentiation being more pronounced in the second case. Although in both cases the ON/OFF period duration ratio was the same, the delayed dynamics of sensitivity changes in the hclB mutants resulted in better recovery and greater sensitivity when longer stimuli were presented at longer interstimulus intervals.

Comparison of the influences mediated through the histamine receptor encoded by the gene hclB on the ON and OFF responses

The results of this study, as well as those of [12], show that mutations in hclB produce more pronounced effects on the ERG ON as compared to OFF transient. A question arises as to why the OFF responses of the second-order neurons, represented by the ERG OFF transient, are less affected by the HCLB receptor/channel activation. A possible explanation may be that in LMCs, the closure of the histamine-gated Cl^¯ channels is not the only mechanism for OFF response generation. It has been shown that a conductance increase rather than decrease was observed during LMC OFF response generation [39,40], and that mutations in certain genes can separately affect the ON and OFF responses of LMCs [19].

Concluding remarks

The existence of two types of receptors—a neuronal (HCLA) and a glial one (HCLB)—for the photoreceptor neurotransmitter histamine is an important mechanism for precise control of synaptic transmission in the photoreceptor synapse in the optic lamina of Drosophila. Although not directly involved in forward transmission of the visual signal, the glial receptor HCLB can significantly affect synaptic gain and response dynamics through changes in the synaptic environment, such as the electrical isolation of the synapse, neurotransmitter concentration, or Cl^¯ gradients. Comparing the ERG responses of hclB mutants, which lack functional HCLB receptors, to those of wild-type flies, we were able to characterize some functional consequences of the HCLB receptor-mediated modulation of synaptic transmission. The results of our study demonstrate that the effects of histamine, mediated by the glial receptor HCLB, contribute to the process of light adaptation, which helps avoid saturation and produce dynamic responses to a wide range of stimulus intensities under different levels of ambient illumination. The HCLB-mediated effects also speed up the sensitivity recovery after short-term light adaptation (such as after looking at a bright object in the visual scene) and contribute to the mechanism of postadaptational potentiation. By speeding up the time course of visual responses, HCLB receptors help improve the temporal resolution of the visual system and reduce the redundant (low-frequency) information, which is important for information encoding. As a whole, the results of this study support the concept of the important contribution of glial cells in synaptic transmission and information processing in the central nervous system.