Daniel Tomsic

Escape behavior and neuronal responses to looming stimuli in the crab Chasmagnathus granulatus (Decapoda: Grapsidae).

SUMMARY Behavioral responses to looming stimuli have been studied in many vertebrate and invertebrate species, but neurons sensitive to looming have been investigated in very few animals. In this paper we introduce a new experimental model using the crab Chasmagnathus granulatus, which allows investigation of the processes of looming detection and escape decision at both the behavioral and neuronal levels. By analyzing the escape response of the crab in a walking simulator device we show that: (i) a robust and reliable escape response can be elicited by computer-generated looming stimuli in all tested animals; (ii) parameters such as distance, speed, timing and directionality of the escape run, are easy to record and quantify precisely in the walking device; (iii) although the magnitude of escape varies between animals and stimulus presentations, the timing of the response is remarkably consistent and does not habituate at 3 min stimulus intervals. We then study the response of neurons from the brain of the crab by means of intracellular recordings in the intact animal and show that: (iv) two subclasses of previously identified movement detector neurons from the lobula (third optic neuropil) exhibit robust and reliable responses to the same looming stimuli that trigger the behavioral response; (v) the neurons respond to the object approach by increasing their rate of firing in a way that closely matches the dynamics of the image expansion. Finally, we compare the neuronal with the behavioral response showing that: (vi) differences in the neuronal responses to looming, receding or laterally moving stimuli closely reflect the behavioral differences to such stimuli; (vii) during looming, the crab starts to run soon after the looming-sensitive neurons begin to increase their firing rate. The increase in the running speed during stimulus approach faithfully follows the increment in the firing rate, until the moment of maximum stimulus expansion. Thereafter, the neurons abruptly stop firing and the animal immediately decelerates its run. The results are discussed in connection with studies of responses to looming stimuli in the locust.

Context Shift and Protein Synthesis Inhibition Disrupt Long-Term Habituation after Spaced, but Not Massed, Training in the CrabChasmagnathus

An opaque screen moving overhead elicits an escape response in the crab Chasmagnathus that after a few presentations habituates for a long period (long-term habituation, LTH). Previous results suggested that spaced (15 trials separated by 171 s) and massed training (300 trials without rest interval) were correlated with two different memory components of LTH. The present experiments were aimed at further studying the mechanisms subserving these components. Results indicate that LTH acquired by spaced but not by massed training is blocked either by a training-to-testing context shift or by cycloheximide (15-25 microg) pre- or posttraining injection and that LTH after spaced training persists for longer time (5 days) than after massed training (2 days). A model based on these results that distinguishes two LTH-memory components is proposed: a (context-signal) LTH yielded by spaced training, dependent of context, sensitive to cycloheximide (CYX), and long lasting; and a (signal) LTH yielded by massed training, dependent only on the signal invariance, insensitive to CYX, and shorter lasting.

Context-us association as a determinant of long-term habituation in the crabChasmagnathus

An opaque screen moving overhead elicits an escape response in the crabChasmagnathus that habituates for a long period after just a few presentations. A series of experiments was performed to determine whether the crab’s long-term habituation (LTH) is mediated by an association between contextual cues and the eliciting stimulus.Chasmagnathus failed to exhibit LTH when a visual cue in the experimental environment was changed between training and testing. In addition, long-term exposure to the context in the absence of the eliciting stimulus impaired LTH, both when the exposure preceded the habituation training (latent inhibition) and when the exposure came after the training (extinction). Long exposure to the context alone prior to training also produced a decrease in responsiveness to the eliciting stimulus, which confirmed previous results. However, both effects of long exposure were only manifested when the crabs spent a period of time between exposure and testing out of the experimental context. The results of this paper are interpreted as supporting the view thatChasmagnathus LTH can be understood largely by Wagner’s associative theory of habituation.

Cycloheximide inhibits context memory and long-term habituation in the crab Chasmagnathus

A shadow moving over head elicits an escape response in the crab Chasmagnathus that habituates promptly and for a long period. The effect of the protein synthesis inhibitor cycloheximide (CY) on this long-term memory was analyzed. Two hours after injection, 10 micrograms CY inhibited [14C]-amino amino acid incorporation into cerebral plus thoracic ganglia by 88% and 20 micrograms by 92%, but no inhibition was found at 24 h. A single injection of 10-20 micrograms CY given 30 min before training, failed to affect the short-term habituation. Similar doses impaired both context memory (CM) and long-term habituation (LTH) when tested at 72 and 120 h but only CM at 24 h. Such a disparity was explained by an unspecific depressing effect upon the response, attributed to an interaction between CY and training. The hypothesis was confirmed, because CY injected immediately after training disclosed amnestic effect at 24 h on both CM and LTH. A similar effect was proven when animals were injected at 2 h but not at 6 h after training. Results from experiments with pretraining and pretesting injections put aside a state-dependence or retrieval deficit effects of the drug. Taken together, findings of this article argue strongly for de novo protein synthesis as a mechanism of LTH and for the close relation between CM and LTH.

Organization of optic lobes that support motion detection in a semiterrestrial crab

There is a mismatch between the documentation of the visually guided behaviors and visual physiology of decapods (Malacostraca, Crustacea) and knowledge about the neural architecture of their visual systems. The present study provides a description of the neuroanatomical features of the four visual neuropils of the grapsid crab Chasmagnathus granulatus, which is currently used as a model for investigating the neurobiology of learning and memory. Visual memory in Chasmagnathus is thought to be driven from within deep retinotopic neuropil by large‐field motion‐sensitive neurons. Here we describe the neural architecture characterizing the Chasmagnathus lobula, in which such neurons are found. It is shown that, unlike the equivalent region of insects, the malacostracan lobula is densely packed with columns, the spacing of which is the same as that of retinotopic units of the lamina. The lobula comprises many levels of strata and columnar afferents that supply systems of tangential neurons. Two of these, which are known to respond to movement across the retina, have orthogonally arranged dendritic fields deep in the lobula. They also show evidence of dye coupling. We discuss the significance of commonalties across taxa with respect to the organization of the lamina and medulla and contrasts these with possible taxon‐specific arrangements of deeper neuropils that support systems of matched filters. J. Comp. Neurol. 493:396–411, 2005.

Massed and spaced training build up different components of long-term habituation in the crabChasmagnathus

The crabChasmagnathus granulatus reacts to a shadow passing overhead with an escape response that habituates after 30 trials and for 5 days at least. The effect of a wide range of different intertrial intervals (ITIs) (0, 9, 27, 45, 81, 135, and 171 sec) on theChasmagnathus long-term habituation (LTH) was evaluated at 24 h. Memory retention was estimated separately at two phases of a six-trial testing session: at first trial (the initial testing phase) and at the subsequent block of five trials (the retraining phase). A training of 30 trials with an ITI equal to or longer than 27 sec induced LTH at both testing phases, however, with a 0- or a 9-sec ITI, training wholly failed to build up LTH. When the number of trials was increased, a massed training (ITI=0 or 9 sec) induced LTH at retraining but not at initial testing. Thus, massed training produces LTH only at retraining, whereas spaced training (ITI ≥ 27 sec) produces LTH at both initial phase and retraining. An ITI shift from training to testing diminished or abolished retention at retraining regardless of the direction of the shift, thus suggesting that crabs acquire a memory of the trial-spacing at training. According to these results, it is postulated that LTH consists of two memory components: one produced by spaced training and expressed at both initial testing and retraining, and one yielded by massed training and expressed only at retraining. The possibility that the two components of LTH were differentially affected by cycloxemide and context shift is discussed.

Morphine and GABA : effects on perception, escape response and long-term habituation to a danger stimulus in the crab Chasmagnathus

Prior results (37) showed that morphine pretreatment reduces the escape response of the crab Chasmagnathus to a shadow passing overhead and prevents the acquisition of a long-term habituation. These results were explained by a reduction in the danger signalled by the stimulus, and to test this hypothesis methods other than morphine injection were used herein to abolish response during training. GABA pretreatment induced a dose-dependent reduction in responsiveness to the danger stimulus, and instances of autotomy were shown with doses larger than 12 micrograms/g. A response was rarely displayed with a 9 micrograms GABA/g dose given 5 min before training, but long-term memory was acquired. In one experiment, both morphine and GABA pretreatment produced similar mild response inhibition during training, but morphine, not GABA impaired long-term habituation. Morphine administered immediately after training had no amnesic effect. These results support the hypothesis that morphine effects may be explained by transient disruption between the stimulus and its danger meaning, ruling out alternative explanations such as response inhibition or amnesia due to either storage or retrieval failure.

Acute administration of a permeant analog of cAMP and a phosphodiesterase inhibitor improve long-term habituation in the crab Chasmagnathus

A shadow passing overhead acts as a danger stimulus and elicits an escape response in the crab Chasmagnathus that habituates promptly and for a long period. Robust retention is shown at 24 h after 15 trials of shadow presentation or at 120 h after 30 trials, but no significant retention is disclosed at 24 h after 5 trials or at 72 h after 15. A cocktail of the cAMP membrane permeable analog 8-(4-chlorophenylthio)-cAMP (CPT-cAMP), plus the phosphodiesterase inhibitor isobutyl methylxanthine (IBMX), was given by systemic administration. Pretraining injection of the cocktail (25 or 50 microM, 15 min before a 5-trial session) failed to affect short-term habituation, but induced significant retention when tested at 24 h. This facilitatory effect was not shown when a lower dose (5 microM) was used. A post-training injection of 25 microM, immediately after a 5- or 15-trial session, induced retention when tested at 24 or 72 h, respectively. Thus, the administration of CPT-cAMP + IBMX during acquisition of a habituated response or immediately after, improves long-term habituation, a result supporting the view that an increase in the cAMP level is one of the steps in long-term memory consolidation.

Physiology and morphology of visual movement detector neurons in a crab (Decapoda: Brachyura).

Abstract. Although visually elicited behaviors have been extensively studied in crabs, their investigation at the neurophysiological level is scant. The present study is a physiological and morphological description of intracellularly recorded and dye injected visual movement detector neurons that respond to the same stimulus that elicits the escape response in the crab Chasmagnathus granulatus. The neurons were investigated in intact animals. The response of movement detector neurons to the danger stimulus (an object moving above the animal) consists of a strong discharge of action potentials frequently superimposed on noisy graded potentials, whereas the response to stationary changes in illumination is weak or undetectable. The response to the moving stimulus is relatively independent of the background intensity and of the contrast between target and background. Repeated presentations of the moving stimulus produce rapid habituation of the neural response. Some of the neurons also respond to mechanical stimulation. These physiological results coincide with those from early studies on visual movement detector fibers of crustaceans achieved by extracellular recordings. However, there are no previous morphological studies of these neurons. Intracellular injection with Lucifer Yellow revealed that these neurons in Chasmagnathus arborize extensively in the internal medulla and in the lateral protocerebrum. They have their somata located in the cell body cluster laying beneath the internal medulla. Their axons project centripetally across the protocerebral tract.

Brain Modularity in Arthropods: Individual Neurons That Support “What” But Not “Where” Memories

Experiments with insects and crabs have demonstrated their remarkable capacity to learn and memorize complex visual features (Giurfa et al., 2001; Pedreira and Maldonado, 2003; Chittka and Niven, 2009). Such abilities are thought to require modular brain processing similar to that occurring in vertebrates (Menzel and Giurfa, 2001). Yet, physiological evidence for this type of functioning in the small brains of arthropods is still scarce (Liu et al., 1999, 2006; Menzel and Giurfa, 2001). In the crab Chasmagnathus granulatus, the learning rate as well as the long-term memory of a visual stimulus has been found to be reflected in the performance of identified lobula giant neurons (LGs) (Tomsic et al., 2003). The memory can only be evoked in the training context, indicating that animals store two components of the learned experience, one related to the visual stimulus and one related to the visual context (Tomsic et al., 1998; Hermitte et al., 1999). By performing intracellular recordings in the intact animal, we show that the ability of crabs to generalize the learned stimulus into new space positions and to distinguish it from a similar but unlearned stimulus, two of the main attributes of stimulus memory, is reflected by the performance of the LGs. Conversely, we found that LGs do not support the visual context memory component. Our results provide physiological evidence that the memory traces regarding “what” and “where” are stored separately in the arthropod brain.