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. Author manuscript; available in PMC: 2019 Jan 1.
Published in final edited form as: J Neurosci Methods. 2017 Oct 16;293:321–328. doi: 10.1016/j.jneumeth.2017.10.013

Comparative one- and two-photon uncaging of MNI-glutamate and MNI-kainate on hippocampal CA1 neurons

Stefan Passlick 1,*, Graham CR Ellis-Davies 1
PMCID: PMC5705412  NIHMSID: NIHMS915574  PMID: 29051090

Abstract

Background

The light-induced release of neurotransmitters from caging chromophores provides a powerful means to study the underlying receptors in a physiologically relevant context. Surprisingly, most caged neurotransmitters, including the widely used 4-methoxy-7-nitroindolinyl (MNI)-glutamate, show strong antagonism against GABA-A receptors. Kainate has been shown to exhibit a higher efficacy at glutamate receptors compared to glutamate itself. Thus, uncaging of kainate might allow the application of the caged compound at lower, less antagonistic concentrations.

New methods

This study provides a detailed comparison of MNI-glutamate and MNI–kainate uncaging by different modes of one- and two-photon irradiation on hippocampal CA1 pyramidal neurons in acute brain slices.

Results/Comparison with existing methods

Unexpectedly, the data revealed that currents in response to MNI-glutamate uncaging were larger compared to MNI-kainate with local one-photon laser uncaging at the soma and two-photon uncaging at the same spines. Furthermore, the direct comparison demonstrates the influence of type of caged agonist and light delivery conditions used for uncaging on the amplitude and kinetic properties of the current response.

Conclusion

These findings highlight the importance of experimental design for uncaging experiments and provide a basis for future studies employing one- and two-photon uncaging to understand glutamate-dependent processes. It further provides the first example of two-photon uncaging of kainate at single spines in acute brain slices.

Keywords: uncaging, glutamate, kainate

Introduction

Ionotropic glutamate receptors mediate the majority of the fast excitatory signaling in the central nervous system. The classic approaches to study these receptors in brain slices have been application of agonists via a perfusion system or via local pressure application. The common drawbacks of these techniques are the speed of drug delivery and the precision of the application. The speed is crucial considering the fast activation and deactivation kinetics of glutamate receptors being much faster than the modes of application. The precision of application is crucial considering the targeted localization of glutamate receptors to specific domains of the cells, e.g. postsynaptic densities. Caged compounds on the other hand facilitate the study of these receptors on a more physiologically relevant time scale (Ellis-Davies, 2007). The caged compound MNI-Glu has been widely used to study the physiology of glutamate-dependent processes. Since MNI-Glu is also sensitive to two-photon light, glutamate may be applied with sub-millisecond stimuli precisely to single spines evoking excitatory postsynaptic currents almost indistinguishable from currents evoked by endogenous synaptic release (Matsuzaki et al., 2001). Surprisingly, MNI-Glu is a strong antagonist at GABA-A receptors at concentrations necessary for two-photon uncaging, limiting its use to study excitation under conditions of normal GABAergic tone (Matsuzaki et al., 2010). One way to circumvent this side effect is to replace glutamate with a higher efficacy agonist allowing the application of the caged compound at lower concentrations being less antagonistic towards GABA-A receptors. Kainate is a partial agonist with a lower affinity at AMPA-type glutamate receptors and a higher affinity at kainate-type glutamate receptors compared to the full agonist glutamate (Patneau et al., 1993; Traynelis et al., 2010). However, several studies demonstrated that when coexpressed with their native regulatory proteins such as transmembrane AMPA regulatory proteins (TARPs) and cornichons, the efficacy of kainate at AMPA receptors is strongly increased exceeding that of glutamate (Jackson and Nicoll, 2011; Kato et al., 2010; Tomita et al., 2005). These results were substantiated by recordings from hippocampal neurons, where outside-out patches showed smaller currents in response to glutamate than to kainate (Tomita et al., 2005).

To explore whether uncaging kainate might allow to reduce the concentration of caged compound and therefore the antagonism towards GABA-A receptors, we performed a detailed study using different modes of one- and two-photon uncaging of MNI-Glu compared to MNI-kainate (-KA) combined with electrophysiological recordings from hippocampal CA1 neurons in acute brain slices prepared from young adult mice. While one-photon uncaging of MNI-KA on cerebellar Purkinje neurons was reported by the inventors of the optical probe (Palma-Cerda et al., 2012), there has been no report of two-photon uncaging of kainate.

Our results show that one- and two-photon uncaging of both compounds elicit glutamatergic currents at the soma and spines of hippocampal CA1 neurons. The kinetic properties of these currents were strongly influenced by the type of light source and the agonist used. Surprisingly, we found that the amplitudes in response to MNI-KA uncaging were significantly smaller compared to MNI-Glu at the soma and when tested at the same spines.

Results

Full-field, one-photon uncaging of MNI-glutamate and MNI–kainate

One- and two-photon uncaging of glutamate has been widely used to study a variety of processes (Ellis-Davies, 2007). In contrast, uncaging of kainate has gained much less attention (Palma-Cerda et al., 2012).

We compared the current responses of hippocampal CA1 neurons to photorelease of MNI-Glu and –KA with different modes of one- and two-photon uncaging in acute mouse brain slices. One-photon uncaging may be conducted either with full-field illumination when using flash lamps or LEDs as light sources or more localized when employing one-photon lasers. These modes of light delivery can yield very different responses from cells and may therefore be used to answer diverse questions.

In the first set of experiments, MNI-Glu or -KA were applied separately at the same concentration (690 µM) via the bath perfusion system to acute brain slices and whole-cell currents in response to increasing durations (2–10 ms) of full-field LED uncaging at 365 nm (5 mW) were recorded (Fig. 1). For both compounds, current amplitudes increased with uncaging duration (Fig. 1c) with the slopes of the linear regression lines for these relationships being indistinguishable between glutamate and kainate uncaging (Fig. 1c; MNI-Glu: 45.4 +/− 5.1, n = 6 cells, MNI-KA: 56.6 +/− 8.4, n = 3 cells, F = 1.47, p = 0.23, linear regression analysis). In line with this, the amplitudes in response to 4 and 8 ms uncaging of MNI-Glu and –KA showed no significant differences for each uncaging duration (Fig. 1d; amplitude (in pA): MNI-Glu: 4 ms: 104.7 +/− 17.2, 8 ms: 319.5 +/− 57.7; MNI-KA: 4 ms: 62.3 +/− 10.2, 8 ms: 340.3 +/− 75.2, p > 0.05 for 4 and 8 ms, One-Way ANOVA with post-hoc Tukey’s test). The kinetic responses in contrast were severely different between the compounds tested (Fig. 1b, e). While MNI-Glu uncaging currents showed the characteristic fast rise and decay of excitatory currents, MNI-KA responses typically consisted of a fast rise to an initial peak amplitude (Ipeak in Fig. 1b), which was followed by a very slow decay. The rise time of MNI-KA responses (calculated for the initial fast rise, Ifast, Fig. 1b) was slower compared to MNI-Glu at 4 ms uncaging duration while it was indistinguishable at 8 ms (Fig. 1e, left; rise time (in ms): MNI-Glu: 4 ms: 4.05 +/− 0.30, 8 ms: 5.71 +/− 0.04; MNI-KA: 4 ms: 6.28 +/− 0.15, 8 ms: 5.83 +/− 0.15, p < 0.05 for 4 ms, One-Way ANOVA with post-hoc Tukey’s test). The decay time was >1000 times slower for MNI-KA compared to - Glu responses evoked by the same stimulus (Fig. 1e, right; decay time (in s): MNI-Glu: 4 ms: 0.0144 +/− 0.0013, 8 ms: 0.0199 +/− 0.0025; MNI-KA: 4 ms: 21.9340 +/− 4.2111, 8 ms: 26.4032 +/− 4.1707, p < 0.05 for 4 and 8 ms, One-Way ANOVA with post-hoc Tukey’s test).

Fig. 1. Full-field, one-photon uncaging of MNI-glutamate and MNI– kainate.

Fig. 1

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(a) Schematic of the full-field uncaging approach using a 365 nm LED.

(b) Representative current recordings from two hippocampal CA1 pyramidal neurons in response to MNI-Glu (yellow) or -kainate (-KA, blue) uncaging (365 nm LED, 6 ms), respectively. Right: same recordings as on left on expanded time scale. Note the fast rise to initial peak (Ifast) but extremely slow decay of the MNI-KA response.

(c) Current amplitudes of all cells (light yellow/blue points) plotted against uncaging duration for MNI-Glu (left, n = 6 cells) and -KA (middle, n = 3 cells). Linear regression lines for individual cells (left, middle; light yellow/blue lines) and for each compound (right; dark yellow/blue lines) were fitted. Linear regression analysis indicated no significant difference between the compounds (F = 1.46, p = 0.23). MNI-KA amplitudes were measured for initial fast peak (Ifast; b, right).

(d) Summary bar graph of the amplitudes in response to MNI-Glu and –KA uncaging with 4 and 8 ms duration, respectively. Data were not significantly different for both durations (One-Way ANOVA with post-hoc Tukey’s test).

(e) Summary bar graphs of rise and decay time of currents recorded in response to MNI-Glu and –KA uncaging with 4 and 8 ms duration, respectively. The rise time in response to MNI-KA was increased for 4 ms but indistinguishable for 8 ms compared to MNI-Glu (left). The decay time was markedly slower for MNI-KA compared to -Glu uncaging for both uncaging durations (right, One-Way ANOVA with post-hoc Tukey’s test). Note, y-axis had to be modified to show decay time for both compounds in same graph (right). For MNI-KA, rise time was measured for initial fast peak (Ifast; b, right).

All recordings were made in the presence of 1 µM TTX. Caged compounds were bath applied at 690 µM, respectively. Uncaging power was 5 mW in all experiments. ns indicates not significant, * p < 0.05.

In summary, while the amplitudes were indistinguishable between MNI-Glu and –KA uncaging, the decay times of the currents were distinctly slower with MNI-KA when using full-field one-photon LED uncaging.

Localized one-photon laser uncaging of MNI-glutamate and MNI–kainate

Continuous wave lasers allow much more localized one-photon uncaging compared to full-field illumination. To compare the uncaging of MNI-Glu versus –KA under these conditions, either compound was bath-applied at a similar concentration (660–670 µM) and whole-cell currents in response to increasing durations (1–5 ms) of one-photon uncaging with a 410 nm continuous wave laser (1.7 mW) were recorded (Fig. 2). For each experiment, the laser was directed to 4 randomly selected points at the soma of a patched, dye-filled hippocampal CA1 neuron. The current amplitudes increased linearly with uncaging duration for both compounds (Fig. 2c). However, in contrast to full-field one-photon uncaging (Fig. 1c), the slopes of the linear regression lines for these relationships, were larger for glutamate compared to kainate uncaging (Fig. 2c; MNI-Glu: 42.9 +/− 2.5, n = 20 curves from 5 cells, MNI-KA: 26.0 +/− 1.7, n = 24 curves from 6 cells, F = 32.11, p < 0.0001, linear regression analysis). This result was corroborated by comparing the amplitudes in response to 2 and 4 ms uncaging of MNI-Glu and –KA. For both uncaging durations, currents for MNI-Glu were significantly larger compared to MNI-KA (Fig. 1d; amplitude (in pA): MNI-Glu: 2 ms: 78.4 +/− 7.4, 4 ms: 164.9 +/− 14.5; MNI-KA: 2 ms: 36.6 +/− 4.6, 4 ms: 94.6 +/− 9.5, p < 0.05 for 2 and 4 ms, One-Way ANOVA with post-hoc Tukey’s test). However, there was no significant difference between 2 ms MNI-Glu and 4 ms MNI-KA amplitudes (p > 0.05, One-Way ANOVA with post-hoc Tukey’s test). To determine the relative quantum yield (QY) of photolysis of MNI-KA when compared to MNI-Glu (QY = 0.065, Corrie et al., 2016), the change in absorption spectrum during photolysis with a 365 nm LED in physiological buffer (pH 7.3) was monitored. The time course was indistinguishable for MNI-Glu and –KA indicating a similar QY of photolysis for both caged compounds (Fig. S1). In contrast to full-field one-photon uncaging, the differences in rise and decay time kinetics between MNI-Glu and –KA currents were much less pronounced (Fig. 1b, e, and 2b, e). The rise time for currents evoked by 2 and 4 ms uncaging of MNI-Glu and –KA were not significantly different for each duration (Fig. 2e, left; rise time (in ms): MNI-Glu: 2 ms: 2.41 +/− 0.14, 4 ms: 3.43 +/− 0.11; MNI-KA: 2 ms: 3.01 +/− 0.44, 4 ms: 3.80 +/− 0.30, p > 0.05 for 2 and 4 ms, One-Way ANOVA with post-hoc Tukey’s test). However, rise times compared between 2 ms MNI-Glu and 4 ms MNI-KA, where current amplitudes are indistinguishable (Fig. 2d), were significantly increased for MNI-KA (Fig. 2e, left; p < 0.05, One-Way ANOVA with post-hoc Tukey’s test). Decay times were not significantly different between the two compounds for 2 ms uncaging duration currents but significantly longer for 4 ms MNI-KA currents compared to 2 and 4 ms MNI-Glu currents (Fig. 2e, right; decay time (in ms): MNI-Glu: 2 ms: 10.02 +/− 0.50, 4 ms: 12.72 +/− 0.54; MNI-KA: 2 ms: 12.91 +/− 1.09, 4 ms: 24.93 +/− 2.45, p > 0.05 for 2 ms, p < 0.05 for 4 ms and 2 ms MNI-Glu vs 4 ms MNI-KA, One-Way ANOVA with post-hoc Tukey’s test).

Fig. 2. Localized one-photon laser uncaging of MNI-glutamate and – kainate.

Fig. 2

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(a) Schematic of the localized one-photon uncaging approach using a continuous wave 410 nm laser. For each cell, currents were recorded from 4 random locations at the soma.

(b) Representative current recordings from two hippocampal CA1 pyramidal neurons in response to MNI-Glu (yellow) or –kainate (-KA, blue) uncaging (410 nm, 1.7 mW), respectively. Left: 2 ms, middle: 4 ms, right: 2 ms (MNI-Glu) and 4 ms (MNI-KA).

(c) Current amplitudes of all locations (light yellow/blue points) plotted against uncaging duration for MNI-Glu (left, n = 20 curves from 5 cells) and -KA (middle, n = 24 curves from 6 cells). Linear regression lines for individual curves (left, middle; light yellow/blue lines) and for each compound (right; dark yellow/blue lines) were fitted. Linear regression analysis indicated a significant difference between the compounds (F = 32.11, p < 0.0001).

(d) Summary bar graph of the amplitudes in response to MNI-Glu and –KA uncaging with 2 and 4 ms duration, each. MNI-KA uncaging current amplitudes were significantly reduced compared to MNI-Glu for both uncaging durations while the amplitudes of 2 ms MNI-Glu versus 4 ms MNI-KA were not significantly different (One-Way ANOVA with post-hoc Tukey’s test).

(e) Summary bar graphs of rise and decay time of currents recorded in response to MNI-Glu and –KA uncaging with 2 and 4 ms duration, each. The rise time was not significantly different between the compounds for each uncaging duration (left). In contrast, it was increased for MNI- KA at 4 ms compared to MNI-Glu at 2 ms under which conditions the amplitudes are indistinguishable (e). The decay time was increased for 4 ms MNI-KA compared to 2 and 4 ms MNI-Glu (right, One-Way ANOVA with post-hoc Tukey’s test).

Recordings were made in the presence of 1 µM TTX. Caged compounds were bath applied at 660–670 µM, respectively. Uncaging power was 1.7 mW in all experiments. ns indicates not significant, * p < 0.05.

In summary, with one-photon laser uncaging, amplitudes of MNI-KA-evoked currents were significantly smaller than those evoked by MNI-Glu uncaging. However, differences in kinetic properties were much milder compared to full-field one-photon uncaging.

Two-photon uncaging of MNI-glutamate and –kainate at the same spines

Due to its two-photon-sensitivity, the MNI caging chromophore allows the diffraction-limited photorelease of caged molecules. In the case of MNI-Glu, this enables the release of glutamate at single spines, mimicking the endogenous synaptic neurotransmitter release (Matsuzaki et al., 2001). While the former has been employed by many labs, two-photon uncaging of kainate has not been reported so far.

In order to directly compare the two-photon uncaging of MNI-Glu and –KA at individual spines, both compounds were consecutively applied (3.8 mM, respectively) to the same neuron by means of a dual local application system (Fig. 3a) (Civillico et al., 2012; Kantevari et al., 2016). For each cell, 3–4 spines on an oblique or basal dendrite of a patched, dye-filled hippocampal CA1 neuron were selected (Fig. 3b) and whole-cell currents in response to two-photon uncaging (720 nm) with increasing laser power (0–35 mW, 2 ms) were recorded for both compounds at the same spines (Fig. 3). The current amplitudes increased linearly with the squared laser power for both compounds (Fig. 3d), as expected for two-photon uncaging. Similar to one-photon laser uncaging (Fig. 2c), the slopes of the linear regression lines for these relationships, were larger for glutamate compared to kainate uncaging (Fig. 3d; MNI-Glu: 1.32 +/− 0.11; MNI-KA: 0.79 +/− 0.04, n = 14 curves from 4 cells, F = 22.75, p < 0.0001, linear regression analysis). In accordance with that, amplitudes in response to 20 mW two-photon uncaging at the same spines were significantly larger with MNI-Glu compared to –KA (Fig. 3e; amplitude (in pA): MNI-Glu: 20 mW: 38.8 +/− 4.5; MNI-KA: 20 mW: 14.4 +/− 1.6, p < 0.05, repeated measure One-Way ANOVA with post-hoc Tukey’s test). However, amplitudes were indistinguishable when comparing 20 mW MNI-Glu with 30 mW MNI-KA at the same spines (Fig. 3e; MNI-KA: 30 mW: 30.5 +/− 2.6, p > 0.05, repeated measure One-Way ANOVA with post-hoc Tukey’s test). Comparison of the kinetic properties of synaptic uncaging currents revealed no differences in rise time between 20 mW MNI-Glu and 20 or 30 mW MNI-KA at the same spines (Fig. 3f, left; rise time (in ms): MNI-Glu: 20 mW: 3.01 +/− 0.22; MNI-KA: 20 mW: 2.71 +/− 0.36, 30 mW: 3.36 +/− 0.30, p > 0.05, repeated measures One-Way ANOVA with post-hoc Tukey’s test). In contrast, the decay times were significantly faster for MNI-KA compared to MNI-Glu at 20 mW (Fig. 3f, right; decay time (in ms): MNI-Glu: 20 mW: 7.02 +/− 0.78; MNI-KA: 20 mW: 3.92 +/− 0.58, p < 0.05, repeated measures One-Way ANOVA with post-hoc Tukey’s test). However, there was no significant difference in decay time kinetics between 20 mW MNI-Glu and 30 mW MNI-KA, where the amplitudes were indistinguishable at the same spines (Fig. 3e, Fig. 3f, right; decay time (in ms): 30 mW: 7.14 +/− 0.93, p > 0.05, repeated measures One-Way ANOVA with post-hoc Tukey’s test).

Fig. 3. Two-photon uncaging of MNI-glutamate and – kainate on the same spines.

Fig. 3

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(a) Schematic of the two-photon uncaging approach using a pulsed laser tuned to 720 nm. MNI-Glu and –kainate (-KA) were applied from a dual local application system to the same neuron. For each cell, currents were recorded from 3–4 spines on an oblique or basal dendrite.

(b) Representative fluorescent two-photon image of a basal dendrite of a patch-clamped, dye-filled hippocampal CA1 pyramidal neuron. Two-photon uncaging of MNI-Glu and –KA was effected at 4 spines (stars).

(c) Representative current recordings from the same spine on a hippocampal CA1 pyramidal neuron in response to MNI-Glu (yellow) or -KA (blue) uncaging (720 nm), respectively. Left: 25 mW, right: 15 mW (MNI-Glu) and 25 mW (MNI-KA).

(d) Current amplitudes (light yellow/blue points) plotted against photolytic input for MNI-Glu (left) and –KA (middle) for all spines (n = 14 spines from 4 cells; all spines tested with both compounds). Linear regression lines for individual spines (left, middle; light yellow/blue lines) and for each compound (right; dark yellow/blue lines) were fitted. Linear regression analysis indicated a significant difference between the compounds (F = 22.75, p < 0.0001). p2 = power (in mW), t = 2 ms (uncaging duration), c = 3.8 mM (concentration), τ = 170 fs (pulse width of uncaging laser at 720 nm).

(e) Summary bar graph of the amplitudes in response to MNI-Glu and –KA uncaging on the same spines with 20 mW and 30 mW (MNI-KA only). MNI-Glu uncaging current amplitudes at 20 mW were significantly larger compared to MNI-KA at the same power while they were indistinguishable compared to currents evoked by 30 mW (repeated measures One-Way ANOVA with post-hoc Tukey’s test).

(f) Summary bar graphs of rise and decay time of currents recorded in response to MNI-Glu and –KA uncaging on the same spines with 20 and 30 mW (MNI-KA only). The rise time was not significantly different between the conditions tested (left). The decay time was decreased for 20 mW MNI-KA compared to 20 mW MNI-Glu and 30 mW MNI-KA (right). In contrast, there was no significant difference between 20 mW MNI-Glu and 30 mW MNI-KA under which conditions the amplitudes were similar at the same spine (e) (repeated measures One-Way ANOVA with post-hoc Tukey’s test).

Caged compounds were locally applied at 3.8 mM, respectively. Uncaging duration was 2 ms in all experiments. Pairs of data points in (e) and (f) were not connected for clarity. ns indicates not significant, * p < 0.05.

In summary, current amplitudes in response to two-photon uncaging of MNI-Glu were larger compared to MNI-KA at the same spines. While there was no difference in rise times, decay times were faster for MNI-KA with the same power at the same spines.

Discussion

The present study provides a detailed comparison of the caged compounds MNI-Glu and MNI–kainate (–KA) with different modes of one- and two-photon uncaging. Unexpectedly, the data shows that uncaging MNI-Glu evokes larger currents at the soma and spines of hippocampal CA1 neurons compared to MNI-KA. This was demonstrated in separate experiments for one-photon laser uncaging and in dual application experiments for two-photon uncaging allowing the direct assessment of the efficacy of two caged compounds on the same spines. Furthermore, the data reveals how different types of light delivery in combination with the type of agonist shape the evoked currents.

MNI-glutamate uncaging yields larger current responses compared to MNI-kainate

The aim of this study was to identify a caged glutamate receptor agonist with a higher efficacy at glutamate receptors compared to caged glutamate itself. This would allow the application of this compound at a lower concentration, thereby reducing the GABA-A receptor antagonism inherent to many caged glutamate compounds.

Several studies demonstrated that the efficacy of the partial agonist kainate at AMPA receptors was increased compared to the full agonist glutamate when AMPA receptors were co-expressed with their native regulatory proteins such as TAPRs or cornichons (Jackson and Nicoll, 2011; Kato et al., 2010; Tomita et al., 2005). In pyramidal neurons of the hippocampal CA1 region, the majority of AMPA receptors were shown to be in a complex with these regulatory proteins (Kato et al., 2010; Schwenk et al., 2009). Pyramidal neurons of the CA1 region also express certain subunits of kainate receptors (Bureau et al., 1999). However, in contrast to neurons of the CA3 region, kainate receptors do not seem to localize to synapses since stimulation experiments failed to produce any responses mediated by these receptors (Carta et al., 2014).

Based on these findings, caged kainate seemed to be a good candidate to evoke AMPA receptor-mediated currents with a higher efficacy on hippocampal CA1 neurons compared to caged glutamate. Surprisingly, the data presented here shows that currents evoked by one- and two- photon uncaging of MNI-KA were significantly smaller compared to MNI-Glu at the soma and spines of hippocampal CA1 neurons (Figs. 2d, 3e). Since the time course of photolysis, and therefore quantum yield of uncaging, was similar for both caged compounds (Fig. S1), this discrepancy cannot be explained by a less effective release of kainate compared to glutamate. Current amplitudes of ionotropic receptors are influenced by a variety of factors. On the one hand, responses to the same agonist may be very different depending on the receptor subunit composition and its association with other proteins (Jackson and Nicoll, 2011; Traynelis et al., 2010). On the other hand, the speed of application strongly influences the amplitude and kinetic properties of these receptors due to their specific activation, deactivation and desensitization characteristics (Dingledine et al., 1999). One distinct difference between glutamate and kainate is that the desensitization of AMPA receptor currents is significantly weaker for kainate (Dingledine et al., 1999). However, desensitization only influences the current response in prolonged presence of the agonist while short pulses (≤ 1 ms) are dominated by the deactivation kinetics (Jonas and Spruston, 1994). Furthermore, using ultra-fast perfusion systems it was shown that AMPA receptors in hippocampal CA1 neurons deactivate relatively slowly (τ ≈ 2.5 ms) (Colquhoun et al., 1992; Jonas and Spruston, 1994).

Most studies comparing the efficacy of glutamate and kainate at AMPA receptors analyzed the amplitude of steady-state currents, where the fast desensitization in response to glutamate significantly affects the amplitude measured (Kato et al., 2010). Furthermore, studies investigating the kinetics of AMPA receptors suggested that in contrast to glutamate, the majority of receptors bound to kainate may go into the desensitized state without opening (Levchenko-Lambert et al., 2011; Plested and Mayer, 2009). Moreover, kainate action on AMPA receptors has mainly been studied in cultured cells where diffusion and reuptake of agonist are very different compared to native tissue in brain slices. This, in combination with the fast application of the two agonists used in the present study, might explain the comparably smaller amplitudes in response to kainate compared to glutamate.

Different sources of light significantly affect the uncaging current response

In the present study, uncaging of glutamate and kainate was tested with three different light sources. The most striking difference between the compounds was found with full-field LED uncaging. While current amplitudes were indistinguishable between the compounds (Fig. 1d), the decay was drastically longer for MNI-KA compared to MNI-Glu (Fig. 1e, right). In contrast, the differences in kinetics were much more subtle for one-photon laser uncaging and two-photon uncaging.

The most obvious difference between these modes of uncaging is the precision of light application. Laser uncaging releases the caged compound in a very defined area of the cell where the local concentration of compound quickly diminishes due to diffusion. In contrast, full-field illumination bathes a large portion of the cell in agonist. While in case of glutamate, this may be compensated to some extend by astrocytic glutamate transporters removing the excess glutamate from the extracellular space, kainate acts as an antagonist at these transporters preventing its clearance (Arriza et al., 1994). Beyond this, our knowledge of the endogenous uptake and clearance of kainate in native tissue is very limited. The prolonged exposure of the AMPA receptors to kainate due to lack of reuptake by astroglia in combination with the reduced desensitization compared to glutamate might explain the striking difference in decay time with full-field illumination which was not as pronounced with laser uncaging.

Furthermore, bath-application of kainate evoked slow ionotropic kainate receptor-mediated currents in CA1 pyramidal neurons, while electrical stimulation failed to detect synaptic kainate receptors (Bureau et al., 1999; Carta et al., 2014). This might indicate that while two-photon uncaging at spines mainly activates synaptic AMPA receptors, part of the slow current response of full-field LED uncaging might be mediated by kainate receptors with slower current kinetics. Our result is also in line with another study employing full-field uncaging of MNI-KA on cerebellar Purkinje neurons expressing kainate receptors, where extremely long decay times were observed (Palma-Cerda et al., 2012). The usefulness of MNI-KA for full-field illumination is therefore limited.

However, we showed for the first time that two-photon currents may be elicited at single spines by MNI-KA uncaging. Due to the higher affinity of kainate receptors to kainate compared to glutamate (Traynelis et al., 2010), MNI-KA may be useful to study the subcellular localization of kainate receptors by two-photon functional mapping on neurons exhibiting synaptic kainate receptors (Fièvre et al., 2016).

In the case of one-photon laser uncaging, the decay time in response to MNI-KA uncaging was increased compared to MNI-Glu for longer durations of uncaging while short durations were indistinguishable (Fig. 2e, right). This is in line with the observation that desensitization only has a significant effect on current decay in the continued presence of agonist (Dingledine et al., 1999; Jonas and Spruston, 1994). It further emphasizes that not only the precision of light delivery but also the duration of light exposure significantly influences the evoked current response for specific agonists. Beyond this, the aforementioned selective reuptake of glutamate in contrast to kainate by astroglia might also contribute to the increased decay time with longer durations of one-photon laser uncaging of MNI-KA compared to -Glu.

Conclusion

The present study highlights the significance of choice of agonist, light source and application duration conditions in uncaging experiments with one- and two-photon irradiation. Due to the surprisingly lower efficacy of MNI-KA compared to MNI-Glu, MNI-KA may not be used as a more effective caged compound to reduce the GABA-A receptor antagonism. However, this study demonstrates for the first time that two-photon uncaging currents may be elicited with MNI-KA at single spines. This might allow the functional mapping of the localization of kainate receptors on a fine scale giving new insights into the function of these enigmatic receptors.

Materials and Methods

Brain slice preparation

All experiments were conducted according to approved institutional IACUC protocols. Male and female C57BL/6J mice (4–5 weeks old) were anesthetized with isoflurane and the brain quickly removed. The brain was cut in horizontal orientation into 350 µm thick slices in ice-cold cutting solution containing (in mM): 60 NaCl, 2.5 KCl, 1.25 NaH2PO4, 7 MgCl2, 0.5 CaCl2, 26 NaHCO3, 10 glucose, 100 sucrose, 3 sodium pyruvate, 1.3 sodium ascorbate equilibrated with 95% O2/5% CO2. The brain slices were then incubated for 15–20 minutes at 33°C in artificial cerebrospinal fluid (ACSF) before being stored at room temperature. ACSF (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2, 26 NaHCO3, 10 glucose, 3 sodium pyruvate, 1.3 sodium ascorbate (95% O2/ 5% CO2).

Electrophysiological recordings

Hemisected brain slices were transferred to the recording chamber of a BX-61 microscope (Olympus) and superfused with ACSF at room temperature. Experiments in Fig. 1 and 2 were conducted in the presence of the voltage-gated Na+ channel blocker TTX (1 µM). Pyramidal neurons of the hippocampal CA1 region were visualized with a 60× objective (Olympus, 1.0 numerical aperture) and IR-DIC optics. Whole-cell recordings were made with an EPC-9 or -10 amplifier (HEKA Instruments, Bellmore, NY, USA) in voltage-clamp mode (Vhold = −60 mV). Patch pipettes were filled with an internal solution containing (in mM): 135 K-gluconate, 4 MgCl2, 10 HEPES, 5 EGTA, 4 Na2-ATP, 0.4 Na2-GTP, 10 Na2 phosphocreatine, 0.05 Alexa 594, pH 7.35. Currents were recorded at 20 kHz and filtered at 3 kHz with Patchmaster/Pulse (HEKA).

Two-photon imaging and one-/two-photon uncaging

Dye-filled patch-clamped neurons were imaged with a Prairie Technologies Ultima dual galvanometer two-photon laser scanning system (Middleton, WI, USA) equipped with two pulsed Chameleon lasers (Ultra II and Vision II, Coherent, Palo Alto, CA, USA). Alexa 594 was excited with the imaging laser tuned to 820 nm. Two-photon laser intensity was independently modulated by electro-optical pockels cells (Conoptics, Danby, CT, USA).

Full-field one-photon uncaging (Fig. 1) was carried out with a UV LED (365 nm LED, Thorlabs, NJ, USA) mounted on the epifluorescence port of the microscope. The light was directed to the objective by a standard long-pass dichroic (488 nm, Semrock, Rochester, NY, USA) in the fluorescence turret. Light power was controlled via the LED driver (LEDD1B, Thorlabs) by external voltage modulation. Localized one-photon uncaging (Fig. 2) was carried out with a continuous wave 410 nm laser (CUBE, Coherent), two-photon uncaging (Fig. 3) with a pulsed Chameleon laser (Coherent) tuned to 720 nm. Uncaging lasers were controlled by the second, independent galvanometer mirror system and were aligned to the imaging laser.

Light power for all light sources was measured with a photometer (S120VC, Thorlabs) at the exit of the objective prior to the experiment. All time-correlated imaging, uncaging and electrophysiology experiments were controlled and triggered via Prairie View Software (Prairie Technologies).

The caged compound MNI-Glutamate was synthesized as previously described (Papageorgiou & Corrie, 2000; Matsuzaki et al., 2001), MNI-Kainate was purchased from Tocris (Minneapolis, MN, USA). For experiments in Fig. 1 and 2, each caged compound was applied separately via a recirculating perfusion system (7–10 ml). For experiments in Fig. 3, both compounds were applied to the same cell successively via a home build dual local application system based on the principle described by Civillico and colleagues (2012) but using two capillary glass tubing glued together at the tip (diameter 50 µM, Polymicro Technologies, Lisle, IL, USA) from two separate reservoirs. The compound was dissolved in ACSF +100 µM Alexa 488 to visualize the puffing and to ensure that caged compound was released from both channels and reached the dendrite used for uncaging.

The concentration of caged compound was determined separately for each experiment with a UV spectrophotometer (Cary 50 Bio, Varian, Palo Alto, CA, USA) based on the known extinction coefficient of MNI (ε = 4300). This is an important step for direct comparisons of caged compounds since the weight of small amounts of purchased compounds may not be accurate yielding unprecise concentrations when preparing solutions solely based on the indicated weight.

Data analysis and statistics

Analysis of power/duration/current relationships was carried out using FitMaster (HEKA), Excel (Microsoft, Redmond, WA, USA) and IGOR Pro (WaveMetrics, Lake Oswego, OR, USA). Rise times (10–90%) and decay times (37%) were measured with NeuroMatic (Jason Rothman) for IGOR Pro (WaveMetrics).

Data is presented as mean +/− SEM.

Data in Fig. 1c, 2c and 3d were tested with linear regression analysis for all data points for each compound. Data in Fig. 1d–e and 2d–e were tested with an One-Way ANOVA with post-hoc Tukey’s Test, data in Fig. 3e–f with a repeated measures One-Way ANOVA with post-hoc Tukey’s Test. Significance level was set to p < 0.05. Statistical testing was carried out using Prism (GraphPad, La Jolla, CA, USA) and Excel (Microsoft).

Supplementary Material

supplement

Highlights.

  • Detailed comparison of one- and two-photon uncaging of MNI-Glu and MNI-kainate.

  • Influence of type of agonist, light source and uncaging conditions on cellular response.

  • MNI-Glu uncaging induces larger current responses compared to MNI-kainate.

  • Two-photon uncaging of MNI-kainate at single spines of CA1 neurons in brain slices.

Acknowledgments

This work was supported by the NIH (GM053395 and NS069720 to Graham C.R. Ellis-Davies) and the Deutsche Forschungsgemeinschaft (DFG, Research Fellowship to Stefan Passlick).

Footnotes

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Competing interests.

The authors are aware of no conflicts of interests.

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Supplementary Materials

supplement
NIHMS915574-supplement.pdf (313.9KB, pdf)

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