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REPORT
Department of Psychology and Brain Research Institute, University of California, Los Angeles, California
Submitted 1 September 2005; accepted in final form 19 December 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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) and the crayfish (Krasne 1969; Zucker 1972; Zucker et al. 1971). However, these early studies raised the question of why animals with encephalized nervous systems should abdicate to low-level circuitry the decision to habituate to stimuli that might provide life-saving warnings of danger.
The circuitry that mediates reflexive escape by the crayfish to stimulation of the abdomen resides entirely within segmental ganglia of the abdomen (Fig. 1A) (Krasne 1969; Zucker 1972; Zucker et al. 1971). Escape responses, which are triggered by firing of the lateral giant escape command neurons (LGs), habituate when the mechanosensitive primary afferents of the reflex are repeatedly activated (Krasne and Teshiba 1995; Wine et al. 1975). In acute experiments, transmitter release from the initial, cholinergic synapses of the circuit readily diminishes with repeated activation of afferents, and this was originally thought to be the sole mechanism of behavioral habituation (Krasne and Roberts 1967; Zucker 1972). Subsequently, however, it was found that a descending inhibitory pathway, which originates in the brain and to a lesser extent in other more rostral ganglia, can tonically inhibit the LGs (Krasne and Wine 1975; Vu and Krasne 1992, 1993; Vu et al. 1993) (see Fig. 1A); this putatively GABAergic (Vu and Krasne 1993; but see Heitler et al. 2001) descending inhibitory pathway also plays a major role in habituation (Krasne and Teshiba 1995).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Experimental design
The crayfish were given four sessions, at one per day, of habituation training; each session consisted of a sequence of single shocks through the stimulating electrodes. In the Trained Intact group, the nerve cord remained intact during training; in the Trained Cut group the cord was severed between abdomen and thorax before training to remove descending influences, including those causing tonic inhibition. During training, both groups received sequences of exactly comparable stimulus voltages. At the end of training, the cords of the Trained Intact group were cut, and 3 days of test sessions were begun the next day. Thus both groups lacked input from higher ganglia during testing.
Stimulus levels and assessment of habituation
Habituation was assessed as a rise in the sensory threshold for eliciting LG firing. Threshold was determined and stimulating levels were chosen in different ways during training and testing.
During testing, stimulus threshold was "tracked" by increasing the stimulus voltage by a prespecified increment whenever the LG failed to fire and decreasing it whenever the LG did fire, thus obtaining a very accurate running measure of LG sensory threshold.
Tracking could not be used during training because, in the Trained Intact group, tonic inhibition was expected to drive thresholds up with the result that Trained Intact animals would on average have received stronger stimuli during training than did Trained Cut animals, and that might in itself have led to differences in amount of habituation that developed. Therefore during training, all animals got comparable sequences of stimuli. During the bulk of training, the stimulus voltage was set at six times the voltage needed to fire interneuron A at the start of the session (interneuron A maintains a quite constant sensory firing threshold at the stimulus frequencies we used). Scaling of stimulus voltage to interneuron A threshold (
A) was done to compensate for differences in stimulation effectiveness caused by variation in stimulating electrode placement. To assess LG threshold during training, the fixed voltage stimulation was periodically interrupted by presentation of a series of ascending stimulus levels (a stimulus "sweep") in which stimulus voltage x A was the same for all animals of both groups. Sweeps were also given during testing.
Training epochs
Training and test sessions were composed of several epochs of stimulation at different rates separated by sweeps. As in previous work from this laboratory, several rates were used to reduce the possibility of encountering ceiling or floor effects in threshold that could make it impossible to get a quantitative assessment of the excitability of the escape reflex. At the start of each session, 30 stimuli were delivered at 1/2 min (epoch I) followed by 60 stimuli at 1/min (epoch II). This was done throughout the experiment, during both training and testing. During training, Epoch II was followed by 45 stimuli at 2/min in an effort to promote habituation (epoch III); data from epoch III were not analyzed.
Interneuron A threshold tracking
Throughout the experiment, the threshold of interneuron A was tracked on trials given halfway between LG training/test trials. Information on the threshold of interneuron A allowed us to confirm that the stimulating electrodes were still working properly when, because of habituation, LGs no longer fired.
Pretesting and data selection
Before assignment to an experimental condition, each animal was run using the test session protocol but with the addition of an epoch III, until threshold had reached a criterion of 4.0 x A, at which time the session was terminated, and the animal was assigned randomly to an experimental condition. If this criterion was not met within three sessions, the animal was discarded. Animals whose interneuron A threshold exceeded 300 (equipment-specific units) at any time during the experiment were also discarded.
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RESULTS |
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, 2005; Krasne 1969; Krasne and Teshiba 1995; Wine et al. 1975; Zucker 1972), a small amount of habituation occurred during training in the cut group, but a great deal more occurred in the intact animals (Figs. 2 and 3). In the Trained Cut group, LG thresholds started low [
2.5 x interneuron A threshold (A)]. As training progressed, they rose, on average, 20%; however, in occasional animals, either no increases or quite large increases (>100%) were seen. Furthermore, the LGs commonly fired on most training trials (as in Fig. 2). In contrast, in the Trained Intact group, LG thresholds sometimes started low (as in Fig. 2), but often they started high because of already ongoing tonic inhibition; thus average threshold was initially 4 x A (Fig. 3, initial session). By the end of training, LG thresholds were commonly high (Figs. 2 and 3), and in many cases, the LGs fired only occasionally or not at all (as in Fig. 2, training session 4). At the end of training, the stimulus threshold of the LG in Trained Intact animals was >4.0 x A (our criterion level for habituation) in 86% of Trained Intact compared with 16% of the Trained Cut animals (P < 0.01, 2-tailed Fisher exact probability test).
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5 x A), although a little lower than before cutting, was markedly higher than that of the animals that had been trained with their nerve cords cut (3 x A; Figs. 2 and 3). This was true even though Trained Intact animals had had exactly the same training regimen as the Trained Cut animals and were no longer subject to descending tonic inhibition. Because the average initial threshold of the Trained Cut animals was 2.5 x A, and that of the Trained Intact animals, in the absence of descending inhibition, would be presumed to have been the same, average threshold in the absence of descending inhibition rose 20% in the Trained Cut versus 100% in the Trained Intact animals. During testing, 9/16 (56%) of the Trained Intact group but only 1/17 (6%) of the Trained Cut group had thresholds >4.0 x A (P < 0.003, 2-tailed Fisher test).
It was those intact individuals whose thresholds were highest by the end of training that were most likely to have high thresholds when their nerve cords were subsequently severed (threshold at test correlated r = 0.66 with threshold at the end of training, P < 0.01), Indeed, all but one of the nine Trained Intact animals whose threshold rose to 
7.0 x A (89%) showed postcut thresholds >4.0 x A.
One possible explanation for these results is that the absence of habituation during testing of the Trained Cut animals was caused by their nerve cords having been severed for a longer period of time at testing than those of the Trained Intact animals. This seems unlikely, however, because the Trained Cut animals had lower thresholds than Trained Intact animals throughout the entire experiment. Nevertheless, to evaluate this possibility, we delayed testing in a small group of Trained Intact animals (Trained Intact-Delayed Test group). These animals had had their nerve cords severed for the same amount of time on test day 2 as had the average of the Trained Cut animals. Despite this, they behaved similarly to the regular Trained Intact animals: 75% of them had normalized thresholds >4.0 x A compared with 6% for the Trained Cut animals (P < 0.001, 2-tailed Fisher test).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). This study shows that habituation training with higher centers operative additionally induces habituation-mediating changes that are intrinsic to abdominal ganglia and that these changes require the higher centers for their development but not their maintenance. The development of the intrinsic changes seems to occur when the descending, putatively GABAergic, inhibition that causes most of the threshold increase during training turns on strongly. Whether the inhibition itself is the factor that is crucial for inducing the alterations in the abdominal cord that persist after removal of descending inhibition will need to be resolved by future work.
Also unknown is the nature of the alterations produced. One possibility is an increase in the susceptibility to intrinsic depression of the synapses of primary afferents on escape reflex interneurons. It is also possible that synapses directly on the LGs become depressed or subject to activity-produced depression, because there is evidence that these synapses are plastic under certain circumstances (Araki and Nagayama 2003; Tsai et al. 2005). Yet another possibility is an augmentation of some form of inhibition of local (as opposed to rostral) origin; there is in fact evidence that there may be a source of tonic inhibition that is intrinsic to abdominal ganglia (Krasne, unpublished data). Given the possible role of inhibition of the LGs in inducing these changes and the potential plasticity of synapses directly on the LGs, it is of interest to note that long-term depression (LTD) of mossy fiber-to-deep nucleus synapses in the cerebellum, which is hypothesized to be part of the mechanism of extinction of conditioned eye-blink responses, develops when excitatory inputs to deep nucleus cells are stimulated during inhibition of them by cortical Purkinje cells (Perrett and Mauk 1995).
The role of descending inhibition, or other accompanying input, in inducing intrinsic alterations of abdominal ganglion circuitry may be seen as an instance of neuromodulation. There are many examples of activity-dependent alterations of neuronal properties being contingent on neuromodulation (Brembs et al. 2002; Hawkins et al. 1983; Segal and Auerbach 1997; Watabe et al. 2000). However, what we find especially interesting here is the nature of the possible interplay between higher- and lower-level circuitry. It seems that the onset of habituation is predominantly caused by descending inhibition that turns on in response to input from the low-level escape-mediating circuitry and is directed back to that circuitry, in part to control it, but also to modify it. Furthermore, the onset of the inhibition is far from obligatory and so could be contingent on relatively complex processing by the higher centers. Once the descending influence turns on, it induces or helps induce low-level changes that allow the inhibition's effects to persist or be mimicked even after the cord is cut. This makes continual active inhibition unnecessary. In effect, higher centers are reprogramming or helping to reprogram lower level controllers, which can operate autonomously on a routine basis until higher centers perhaps again intervene to cause further reprogramming. Evidence for reprogramming of lower centers by higher ones has also been seen in the modification of spinal stretch reflexes in mammals (Chen and Wolpaw 2002; Chen et al. 2001, 2003). One can imagine the use of such an arrangement at various levels of any hierarchically organized nervous system.
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GRANTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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Address for reprint requests and other correspondence: F. Krasne, Dept. of Psychology, Univ. of California, 1285 Franz Hall, Box 951563, Los Angeles, CA 90095-1563 (E-mail: krasne{at}psych.ucla.edu)
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REFERENCES |
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Zucker RS, Kennedy D, and Selverston AI. Neuronal circuit mediating escape responses in crayfish. Science 173: 645–650, 1971.
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; n = 17), 1 day before the 1st test session (Trained Intact group, 
; n = 16), or 8 days before the 1st test session (Trained Intact-Delayed Test group, 
; n = 4). Ordinate, LG threshold/interneuron A threshold. Error markers, SE.