Abstract
The zebrafish larva has been a valuable model system for genetic and molecular studies of development. More recently, biologists have begun to exploit the surprisingly rich behavioral repertoire of zebrafish larvae to investigate behavior. One prominent behavior exhibited by zebrafish early in development is a rapid escape reflex (the C-start). This reflex is mediated by a relatively simple neural circuit, and is therefore an attractive model behavior for neurobiological investigations of simple forms of learning and memory. Here, we describe two forms of short-lived habituation of the C-start in response to brief pulses of auditory stimuli. A rapid form, persisting for ≥1 min but <15 min, was induced by 120 pulses delivered at 0.5–2.0 Hz. A more extended form (termed “short-term habituation” here), which persisted for ≥25 min but <1 h, was induced by spaced training. The spaced training consisted of 10 blocks of auditory pulses delivered at 1 Hz (5 min interblock interval, 900 pulses per block). We found that these two temporally distinguishable forms of habituation are mediated by different cellular mechanisms. The short-term form depends on activation of N-methyl-d-aspartate receptors (NMDARs), whereas the rapid form does not.
Introduction
A major goal of modern neuroscience is to characterize the physical changes within the nervous system that underlie learning and memory. Significant progress has been made in mammalian systems toward identifying potential neuronal substrates of memory [1]–[4], and molecular techniques are now available for labeling specific neurons that participate in the memory engram for some types of learning [5], [6]. Despite these advances, cataloging all of the cellular and molecular processes that mediate sophisticated forms of learning in the enormously complex mammalian brain is, at present, a quixotic enterprise. To more readily achieve the goal of linking neuronal modifications to learned behavioral changes, we have chosen to study elementary learning in an inframammalian vertebrate, the zebrafish.
The zebrafish has several attributes that make it particularly attractive as a model organism for biological investigations of behavior. Among these are rapid development, high fecundity, and ease of genetic manipulation [7], [8]. Another significant advantage of the zebrafish is that it is transparent in the larval stage, making it ideally suited for optical and optogenetic investigations of neuronal function [9]–[12]. Finally, although a vertebrate with complex vertebrate behavior [13], zebrafish exhibit some simple behaviors that are regulated by relatively simple neural circuits, circuits that are highly amenable to neurophysiological analyses [14], [15]. One such behavior is the startle response. This rapid escape response (the C-start) is mediated by a well-defined neural circuit in the brainstem and spinal cord; a major component of this circuit is a small number of hindbrain neurons, the most prominent of which are the large, bilaterally paired Mauthner (M) cells [7], [16]–[19]. In adult goldfish, a close relative of the zebrafish, the C-start circuit is highly plastic [20]–[24].
In the present study we examined habituation of the C-start in the larval zebrafish. Habituation is a nonassociative form of learning during which an organism decreases its responsiveness to a repeated stimulus [25], [26]. An evolutionarily ancient form of learning, habituation is present in organisms ranging from Cnidarians [27] to humans [28]. But despite its simplicity and apparent ubiquity, at present we possess only a rudimentary understanding of the neurobiology of habituation [29], [30].
Short-term habituation of the C-start in zebrafish larvae was first described by Eaton and colleagues in 1977 [31]; during the intervening decades, however, there has been no in-depth investigation of this form of learning. A recent study by Best and colleagues [32] examined habituation of escape-related movements by larval zebrafish in response to auditory stimuli. But these investigators did not use high-speed videography to record the movement of the fish. This is mechanistically problematic because zebrafish can generate an escape response through non-M-cell neural circuits [18], [33], [34]; unless one makes direct electrophysiological or optical recordings of the M-cell's action potential, the only reliable method for distinguishing between the M-cell-mediated and non-M-cell-mediated escape responses is latency of response onset: the M-cell mediated escape (the C-start) has a significantly shorter onset latency (<12 ms) than does the non-M-cell-mediated response (mean ∼28 ms) [33], [34] (but see Ref. [18]). Best and colleagues did not attempt to distinguish between the short-latency and long-latency escapes in their behavioral study, and therefore could not know whether or not the responses of the animals were the consequence of M-cell firing.
We have performed a comprehensive parametric study of short-lasting habituation of the C-start in zebrafish larvae, and have begun to analyze its underlying mechanisms. Here we present evidence that there are distinct forms of short-lasting habituation of the C-start in the larvae. In addition, we demonstrate a critical role for N-methyl-d-aspartate receptors (NMDARs) [3], [35], [36] in one of these forms.
Methods
Ethics Statement
All experimental procedures in this study were approved by the Chancellor's Animal Research Committee (ARC) at the University of California at Los Angeles (#2005-053-21).
Animals and behavioral apparatus
Zebrafish eggs were collected after standard breeding protocols, and placed into E3 solution (5 mM NaCl, 0.33 mM MgCl2, 0.33 mM CaCl2, 0.17 mM KCl, 10−5% methylene blue, pH 7.2) in an incubator (28.5°) to allow the embryos to develop. Wild-type TL zebrafish, obtained from the core facility at the University of California, Los Angeles (UCLA), were used for all experiments. To image the zebrafish escape response, we used one of two high-speed cameras. For the experiments investigating pharmacological manipulation of rapid habituation (below), a Casio Exilim ExFH25 (Casio America, Dover, NJ) was used, and images were recorded at 240 frames/s. For all other experiments, we employed the TroubleShooter TS100MS (Fastec Imaging, San Diego, CA), and recorded at 1000 frames/s. In order to achieve sufficient visual contrast to detect the escape responses (Fig. 1A) in the high-speed recordings of their behavior, the larvae had to be illuminated with intense light. Therefore, the larvae were put into individual wells, each containing 3 ml of E3 solution, and the wells were placed on a light box (Gagne Inc., Johnson City NY). A speaker, positioned next to the wells, was used to deliver auditory/vibrational (AV) stimulation (Fig. 1B). The fish were permitted to acclimate to the wells for 1 h prior to the start of the experiments. 1 ms-long (200 Hz ramp wave), 109 dB auditory pulses, produced with a function generator (NS-R2001; Leader, Cypress, CA) and amplifier (Insignia, Richfield, MN), were used to elicit startle responses. A response to an auditory pulse was scored as a startle response when the animal began the characteristic C-bend within 25 ms after the stimulus.
Behavioral protocols and statistical analyses
In the experiments involving drug treatment, the fish were housed in individual wells with drug or control solutions for 24 h prior to the start of the experiment. Each fish was given paramecia for the first 23 h of this period, after which the fresh experimental solutions were introduced into the wells. In the experiments with dl-2-amino-5-phosphonopentanoic acid (APV), the drug was dissolved directly into E3. Animals were maintained in drug-containing or control wells throughout the experiment. APV was obtained from Sigma (St. Louis, MO).
Rapid habituation. After a 1-h acclimation period, zebrafish were given habituation training (120 pulses at 1 Hz, 109 dB). In one set of experiments C-starts were measured throughout the training period. In a second set of experiments the same training protocol was used, but habituation memory was tested 10 s, 1 min, and (in some experiments) 15 min after training. To assess the significance of group differences, the number of responses that occurred during the training period, or on the posttest, were compared using a two-tailed, unpaired t-test.
Short-term habituation. A more persistent form of habituation was elicited by giving larvae spaced training. After a 1-h acclimation period, three pretests (5 min ISI, 109 dB) were performed. Five min after the final pretest, the larvae were given extended habituation training, which consisted of 10 blocks (900 pulses, 1 Hz) of spaced (5 min interblock interval) AV stimulation. Fifteen min after the last block of stimuli, the zebrafish received three posttests (5 min ISI, 109 dB). To determine the amount of habituation produced by the training, we calculated a habituation index (HI). The HI was the posttest response rate (average of the three posttests) minus the pretest response rate (average of the three pretests). Two-tailed, unpaired t-tests, one-way ANOVAs, and repeated measures ANOVAs were used for the statistical comparisons. Student Newman Keuls tests were used for post-hoc analyses when the results of the ANOVA were significant.
Results
Initially, we determined the developmental age at which larval zebrafish were able to perform an escape response to AV stimuli. Although morphological contacts between the primary auditory nerve and the M-cell form early in development [37], [38], and sound-evoked postsynaptic currents are observed as early as 40 h post-fertilization [38], we failed to observe escape responses at 3 d post-fertilization (dpf) (Fig. 2A). We first elicited escape responses at 4 dpf, but the auditory threshold at which these responses were elicited was significantly higher than that for older fish (5–6 dpf, p<0.05), and the responses were more variable (Fig. 2B). The mean threshold (dB) for eliciting an escape was 105.83±5.19 dB in 4 dpf larval zebrafish and 91±1.31 dB in 5–6 dpf zebrafish. Accordingly, we restricted our experiments to larvae ages 6–8 dpf. Similar to the results of a previous study that investigated escape behavior in zebrafish larvae [32], we found that the best stimulus frequency for eliciting an escape response was 200 Hz (Fig. 2C) (one-way ANOVA, p<0.05), although escape responses could be elicited by a wide range of frequencies (50–1000 Hz).
Characterization of rapid habituation
To determine the experimental protocol that most effectively elicited rapid habituation, we stimulated larval zebrafish with 120 auditory pulses at different frequencies and tested the animals' responsiveness at 10 s and 1 min after the last pulse. AV stimuli at frequencies ranging from 0.5–2 Hz were most effective in habituating the escape response, as assayed 10 s after training (Fig. 3A). Interestingly, lower (0.0167–0.1 Hz) and higher (10–60 Hz) stimulation frequencies produced less habituation. We also included a control group (Test alone group) that received the same testing protocol as the Trained group, but was not given habituation training. We compared a Test alone group (mean response rate of 71.0±0.10) to a Trained group (mean response rate of 0.04±0.04) that received 1-Hz habituation training. The difference between the two groups was highly significant on the 10-s posttest (p<0.0001, Fig. 3B). We found that a relatively broad range of stimulus frequencies could produce habituation at 1 min after training. In fact, as shown in Figure 3C, all low-frequency stimulation protocols (0.0167–2 Hz) yielded similar amounts of habituation; the higher frequency stimulation protocols, however, were less effective at inducing habituation. The mean response rate on the 1-min posttest was 0.38±0.10 in the Trained group and 0.67±0.10 in the Test alone group. A planned comparison between these two groups for the 1-min posttest indicated that the training produced significant habituation (p<0.05, Fig. 3D).
We next sought to determine the time course of the habituation memory. We measured the escape response at 10 s, 1 min and 15 min after habituation training (120 pulses delivered at 1 Hz). We found that the C-start was habituated for at least 1 min posttraining (Fig. 3E), and afterwards returned to pretest levels (defined as the animal's responsiveness to the first pulse of habituation training) within 15 min (repeated measures ANOVA, p<0.05). The mean response rates were: pretest, 0.83±0.11 (n = 12): 10 sec, 0.00±0.00 (n = 12); 1 min, 0.25±0.13 (n = 12); and 15 min, 0.67±0.14 (n = 12). To ascertain the most effective number of auditory pulses for eliciting rapid habituation, we measured habituation at the 1-min posttest after varying the number of auditory pulses from 20–900. We found that 120 pulses of auditory stimuli were the most effective at eliciting habituation; no additional habituation was achieved with further stimulation (900 pulses) (one-way ANOVA, p<0.05; Fig. 3F). The mean response rates after training with varying numbers of pulses were: 20 pulses, 0.71±0.10 (n = 24); 60 pulses, 0.38±0.10 (n = 24); 120 pulses, 0.08±0.06 (n = 24); and 900 pulses, 0.21±0.09 (n = 24).
The role of NMDAR-mediated transmission in rapid habituation
We exploited the ability of larval zebrafish to passively absorb drugs from their environment [39] in order to test the involvement of NMDAR-dependent activity in rapid habituation of the C-start. We incubated zebrafish larvae in the NMDAR antagonist APV (100–200 µM in E3) for 24 h; control larvae were incubated in E3 alone. The fish were then trained with 120 auditory pulses at 1 Hz. Blockade of NMDARs had no effect on the responses of the fish during training (p>0.05, Fig. 4A). Furthermore, a comparison of the responsiveness of APV-treated (0.16±0.09) and control animals (0.24±0.10) at 1 min posttraining also showed no significant difference between the two groups (p>0.5, Fig. 4B). We have confidence in this negative result because we found that the same concentration of APV did block more persistent habituation (below).
Demonstration of short-term habituation
Having identified a form of habituation of the C-start that lasted≥1 min, we sought protocols that would elicit longer-lasting habituation. Accordingly, we used spaced training, which has been previously shown to be more effective in eliciting prolonged habituation than massed training [40]. We found that auditory pulses delivered at 1 Hz in 10 spaced blocks of stimuli (900 pulses per block, 5 min interblock interval; Fig. 5A) produced the greatest habituation (Fig. 5B). (Massed training, 9000 pulses at 1 Hz, failed to elicit short-term memory; data not shown.) Although training at a stimulus rate of 2 Hz also elicited significant habituation, training at a lower frequency (0.5 Hz) and higher frequencies (10 and 50 Hz) were ineffective. To confirm these results, and to measure the persistence of the memory induced by spaced training, we compared the effect of 1-Hz spaced training protocol as assessed 15–25 min after training (Trained15–25 min posttests group), or 60–70 min after training (Trained60–70 min posttests group), with that of the test stimulation alone (Test alone group). The Trained15–25 min posttests group exhibited significantly lower responsiveness (HI = −0.54±0.13) than did either the Trained60–70 min posttests group (HI = −0.03±0.08) or the Test alone group (HI = 0.00±0.09, p<0.05, Fig. 5C). These results indicate that short-term habituation training produced memory that lasted for ≥25 min, but <1 h.
The role of NMDAR-mediated transmission in short-term habituation
To determine whether NMDAR-mediated activity was required for short-term habituation, larval zebrafish were incubated in either 100 µM APV or control (E3) solution for 24 h prior to the start of an experiment. Following incubation in APV, larvae that received spaced training at 1 Hz did not exhibit habituation (HI = −0.04±0.10) on the 15–25-min posttests, in contrast to the control larvae incubated in E3 alone (HI = −0.33±0.09, p<0.05; Fig. 5D). To confirm that exposure to APV did not affect the health or responsiveness of the zebrafish, we examined zebrafish incubated for 24 h in either APV or E3, but not given habituation training. We found no significant difference in the responsiveness of the two groups of untrained fish (p>0.9, Fig. 5E). Thus, NMDAR activity is required for short-term (25 min-to-1-h) habituation of the C-start.
Discussion
The present study is a systematic investigation of habituation of the M-cell-mediated escape response in larval zebrafish, a form of learning originally demonstrated by Eaton and colleagues [31] over forty years ago. The relative simplicity of the neural circuitry that underlies the C-start, together with other significant biological advantages of the zebrafish system [7], [15], [41], [42], should significantly facilitate the analysis of the cellular and molecular modifications that mediate habitation and, potentially, other elementary forms of learning and memory. (See Ref. [43] for a related approach.) Note that, despite significant advances during the past few decades in our understanding of the biology of learning and memory, we still lack a complete mechanistic scheme for any form of learning and memory [29].
Although there is evidence of synaptic connectivity between the 8th nerve auditory input and the M-cell as early as 40 hpf [37], [38], we did not observe an escape response until 4 dpf, as previously reported [44]. Also similar to the findings of Best and colleagues [32], who measured auditory-elicited escape movements, we found that a 200 Hz tone was the most effective in evoking the C-start in larval zebrafish. However, we observed C-starts to a range of auditory frequencies (50–1000 Hz).
We characterized two forms of habituation of the C-start in larval (6–8 dpf) zebrafish.
One form, a rapid form lasting between 1 min and 15 min, could be reliably elicited by low-frequency auditory stimulation. Nonetheless, we observed significant variability in the amount of habituation elicited in our experiments. At present, we do not understand the source of this variability. We were unable to block the rapid form of habituation of the C-start with the NMDAR antagonist APV, although the same dose of the antagonist was able to block the more extended form of habituation (Fig. 5D, E).
It is possible that changes in electrical transmission underlie rapid habituation of the C-start. For example, transient modifications of gap junction conductance and/or gap junction number, may reduce the strength of the 8th nerve input to the M-cell—which contains a significant electrical component [45]— and thereby modify the threshold for escape. Rapid, reversible uncoupling of electrical synapses is a well-described phenomenon in the central nervous system [46]–[49], and functional uncoupling of electrical synapses has been proposed as a mechanism for behavioral switching [50], [51]. Another neuronal mechanism that could explain our results is ephaptic inhibition of the M-cell through extracellular currents created by inhibitory neurons within the axon cap [52], [53]. As shown by Weiss et al. [53], the electrical field effect generated by such currents can inhibit the generation of M-cell action potentials, and thereby regulate the threshold for the elicitation of the C-start.
Feed-forward chemical inhibition is another potential cellular mechanism for rapid habituation of the escape response. However, Weiss and colleagues [53] have reported that feed-forward chemical inhibition of the M-cell occurs several milliseconds after gap junction-mediated transmission from the primary afferents of the 8th nerve in adult goldfish. If similar temporal kinetics characterize feed-forward chemical inhibition in larval zebrafish, such a delay would make it unlikely that chemical inhibition contributes to rapid habituation of the C-start. However, changes in tonic chemical inhibition could play a role.
We also demonstrated a more persistent form of habituation of the C-start response, one lasting between 25 min and 1 h. We intend in future experiments to determine, what role, if any, inhibition plays in this short-term habituation. Activity-dependent potentiation of chemical inhibitory synapses in the auditory pathway to the M-cell has been well documented [45], and has been previously linked to behavioral plasticity in fish [22]; possibly, this form of synaptic plasticity contributes to short-term habituation of the C-start. In support of this idea, we found evidence for a role for NMDARs in short-term habituation, although we do not know the synaptic location of these NMDARs. Another possibility is that habituating stimuli cause NMDAR-dependent potentiation of inhibitory electrical synapses. For example, homosynaptic potentiation of synapses made by auditory fibers of the 8th nerve onto feed-forward inhibitory interneurons could increase inhibition of the M-cell, either through chemical or ephaptic inhibition (Fig. 6). (Interestingly, in the goldfish brain the NR1 subunit of the NMDAR has been reported to be present in postsynaptic densities juxtaposed to the club endings of 8th nerve excitatory axons, which synapse onto the lateral dendrites of the M-cell close to gap junction plaques [55].) Finally, NMDAR-dependent [54] of the excitatory auditory inputs, either chemical or electrical (or both) [24], to the M-cell, could play a role in habituation of the C-start. Future experiments, involving calcium imaging or, possibly, electrophysiological recording, from M-cells in semi-intact zebrafish larvae, should be able to clarify the synaptic mechanisms of this simple form of learning.
In summary, the present study demonstrates that short-lasting habituation of the zebrafish C-start reflex exhibits multiple forms that are both temporally and mechanistically distinct. It also provides the basis for cellular and molecular investigations of a simple form of learning in a genetically tractable vertebrate organism. We believe that such investigations may well yield novel insights into the fundamental biological mechanisms that regulate vertebrate learning and memory.
Addendum
While this manuscript was under review, Wolman et al. [56] published results related to ours. In particular, they characterized a form of habituation similar to our rapid habituation. Consistent with our findings, Wolman and colleagues found that 1-Hz stimulation was effective in eliciting this form of habituation. However, these investigators reported that the noncompetitive NMDAR antagonists MK-801 and ketamine enhanced the baseline response rate and blocked rapid habituation; their results contrast with those from our experiments in which we found the competitive NMDAR antagonist, APV, to be without effect on either the baseline response rate or rapid habituation (Fig. 4). To determine the source of these contrasting effects of the NMDAR antagonists, we tested the effect of MK-801 (Methods S1). We found that incubating the larvae in 100 µM MK801 for 15–45 min enhanced the baseline response rate and disrupted rapid habituation of the C-start (Fig. S1), consistent with Wolman et al.'s findings. By contrast, we replicated our original result for APV using Wolman et al.'s incubation protocol; specifically, we found that APV (≤200 µM) had no effect on the baseline response rate or on rapid habituation of the C-start (Fig. S2). There are several possible explanations for the differential effects of the NMDAR antagonists on rapid habituation. First, APV may not completely antagonize NMDARs in larval zebrafish, although notice that we did observe an effect of APV on short-term habituation of the C-start (Fig. 5). Second, the non-competitive antagonists MK-801 and ketamine may have nonspecific effects on the M-cell neurocircuitry [57]. Future experiments using genetic manipulation of NMDARs may be able to resolve the discrepancy between the results with MK-801 and ketamine, and those with APV.
Supporting Information
Acknowledgments
We thank Brent Bill, Petronella Kettunen, and Frank Krasne for their helpful comments on the manuscript.
Footnotes
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by the National Science Foundation (0923143). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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