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

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.

Visual interneurons of the crab Chasmagnathus studied by intracellular recordings in vivo.

Comparative physiology of visual systems has become an important field of investigation. However, despite the fact that Crustacea represents a major phylogenetic group, research on the physiology of vision of these animals is scant and almost limited to the crayfish. We developed a preparation to study in vivo the visual nervous system of a semiterrestrial crab through intracellular recordings. The response to a pulse of light was investigated in 206 interneurons from 38 animals. Seventy-eight of these neurons could be classified by functional criteria as sustaining cells, dimming cells, nonspiking hyperpolarizing cells and nonspiking depolarizing cells. Quantitative description is provided for the first two groups and qualitative description is given for the last two. The remaining neurons presented a broad range of different types of phasic responses to light. Although semiterrestrial crabs are behaviorally more reactive to visual stimuli than the crayfish, the general physiological properties of identified lamina and medullary neurons of Chasmagnathus resemble those of the crayfish. The results described here represent the first attempt to study the visual system of crabs with intracellular recordings and constitute the beginning of a project aimed to investigate the neuronal functions underlying behavioral responses elicited by visual stimuli.

Background illumination effects upon in vitro conditioning in Hermissenda.

In the marine snail Hermissenda, associative learning can be accomplished by paired presentations of light and vestibular stimulation. It is generally assumed that associative learning depends upon the intensity or salience of the conditioned or unconditioned stimulus (CS and US, respectively). Accordingly, during Hermissenda conditioning a stronger dark adaptation is expected to render the CS (the light) more salient and hence facilitate association. We studied the influence of background illumination level using an in vitro pairing procedure in Hermissenda. This procedure allows one to assess the effect of conditioning upon a single cell, the B photoreceptor, which is implicated in this learning process. After 15 min of adaptation to a dim background light, B photoreceptors maintained a basal rate of firing, while after adaptation to complete darkness, they stopped firing. Paired and unpaired groups received 10 training trials in either a completely dark or a dim light environment. Although a trial to trial cumulative increase in excitability was found in the paired group trained in darkness, only the paired group trained under dim background light showed a higher input resistance and cell excitability 10 min after training. These results suggest that the background dim illumination was not needed for the induction but played a role in the maintenance of the pairing effect. Possible mechanisms for such a modulatory effect are discussed.

Context shift and protein synthesis inhibition disrupt long-term habituation after spaced, but not massed, training in the crab Chasmagnathus.

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.

Intracellular correlates of acquisition and long-term memory of classical conditioning in Purkinje cell dendrites in slices of rabbit cerebellar lobule HVI.

Intradendritic recordings in Purkinje cells from a defined area in parasaggital slices of cerebellar lobule HVI, obtained after rabbits were given either paired (classical conditioning) or explicitly unpaired (control) presentations of tone and periorbital electrical stimulation, were used to assess the nature and duration of conditioning-specific changes in Purkinje cell dendritic membrane excitability. We found a strong relationship between the level of conditioning and Purkinje cell dendritic membrane excitability after initial acquisition of the conditioned response. Moreover, conditioning-specific increases in Purkinje cell excitability were still present 1 month after classical conditioning. Although dendritically recorded membrane potential, input resistance, and amplitude of somatic and dendritic spikes were not different in cells from paired or control animals, the size of a potassium channel-mediated transient hyperpolarization was significantly smaller in cells from animals that received classical conditioning. In slices of lobule HVI obtained from naive rabbits, the conditioning-related increases in membrane excitability could be mimicked by application of potassium channel antagonist tetraethylammonium chloride, iberiotoxin, or 4-aminopyridine. However, only 4-aminopyridine was able to reduce the transient hyperpolarization. The pharmacological data suggest a role for potassium channels and, possibly, channels mediating an IA-like current, in learning-specific changes in membrane excitability. The conditioning-specific increase in Purkinje cell dendritic excitability produces an afterhyperpolarization, which is hypothesized to release the cerebellar deep nuclei from inhibition, allowing conditioned responses to be elicited via the red nucleus and accessory abducens motorneurons.

Long-term habituation (LTH) in the crab Chasmagnathus: a model for behavioral and mechanistic studies of memory.

A decade of studies on long-term habituation (LTH) in the crab Chasmagnathus is reviewed. Upon sudden presentation of a passing object overhead, the crab reacts with an escape response that habituates promptly and for at least five days. LTH proved to be an instance of associative memory and showed context, stimulus frequency and circadian phase specificity. A strong training protocol (STP) (> or = 15 trials, intertrial interval (ITI) of 171 s) invariably yielded LTH, while a weak training protocol (WTP) (< or = 10 trials, ITI = 171 s) invariably failed. STP was used with a presumably amnestic agent and WTP with a presumably hypermnestic agent. Remarkably, systemic administration of low doses was effective, which is likely to be due to the lack of an endothelial blood-brain barrier. LTH was blocked by inhibitors of protein and RNA synthesis, enhanced by protein kinase A (PKA) activators and reduced by PKA inhibitors, facilitated by angiotensin II and IV and disrupted by saralasin. The presence of angiotensins and related compounds in the crab brain was demonstrated. Diverse results suggest that LTH includes two components: an initial memory produced by spaced training and mainly expressed at an initial phase of testing, and a retraining memory produced by massed training and expressed at a later phase of testing (retraining). The initial memory would be associative, context specific and sensitive to cycloheximide, while the retraining memory would be nonassociative, context independent and insensitive to cycloheximide.

Long-term habituation (LTH) in the crab Chasmagnathus: a model for behavioral and mechanistic studies of memory

A decade of studies on long-term habituation (LTH) in the crab Chasmagnathus is reviewed. Upon sudden presentation of a passing object overhead, the crab reacts with an escape response that habituates promptly and for at least five days. LTH proved to be an instance of associative memory and showed context, stimulus frequency and circadian phase specificity. A strong training protocol (STP) (( 15 trials, intertrial interval (ITI) of 171 s) invariably yielded LTH, while a weak training protocol (WTP) (( 10 trials, ITI = 171 s) invariably failed. STP was used with a presumably amnestic agent and WTP with a presumably hypermnestic agent. Remarkably, systemic admministration of low doses was effective, which is likley to be due to the lack of an endothelial blood-brain barrier. LTH was blocked by inhibitors of protein and RNA synthesis, enhanced by protein kinase A (PKA) activators and reduced by PKA inhibitors, facilitated by angiotensin II and IV and disrupted by saralasin. The presence of angiotensins and related compounds in the crab brain was demonstrated. Diverse results suggest that LTH includes two components: an inital memory produced by spaced training and mainly expressed at an inital phase of testing, and a retraining memory produced by massed training and expressed at a later phase of testing (retraining). The inital memory would be associative, context specific and sensitive to cycloheximide, while the retraining memory would be nonassociative, context independent and insensitive to cycloheximide.

Reversible antisense inhibition of Shaker-like Kv1.1 potassium channel expression impairs associative memory in mouse and rat.

Long-term memory is thought to be subserved by functional remodeling of neuronal circuits. Changes in the weights of existing synapses in networks might depend on voltage-gated potassium currents. We therefore studied the physiological role of potassium channels in memory, concentrating on the Shaker-like Kv1.1, a late rectifying potassium channel that is highly localized within dendrites of hippocampal CA3 pyramidal and dentate gyrus granular cells. Repeated intracerebroventricular injection of antisense oligodeoxyribonucleotide to Kv1.1 reduces expression of its particular intracellular mRNA target, decreases late rectifying K+ current(s) in dentate granule cells, and impairs memory but not other motor or sensory behaviors, in two different learning paradigms, mouse passive avoidance and rat spatial memory. The latter, hippocampal-dependent memory loss occurred in the absence of long-term potentiation changes recorded both from the dentate gyrus or CA1. The specificity of the reversible antisense targeting of mRNA in adult animal brains may avoid irreversible developmental and genetic background effects that accompany transgenic "knockouts".

Dendritic excitability microzones and occluded long-term depression after classical conditioning of the rabbit's nictitating membrane response.

We made intradendritic recordings in Purkinje cells (n = 164) from parasaggital slices of cerebellar lobule HVI obtained from rabbits given paired presentations of tone and periorbital electrical stimulation (classical conditioning, n = 27) or explicitly unpaired presentations of tone and periorbital stimulation (control, n = 16). Purkinje cell dendritic membrane excitability, assessed by the current required to elicit local dendritic calcium spikes, increased significantly in slices from animals that received classical conditioning. In contrast, membrane potential, input resistance, and amplitude of somatic and dendritic spikes were not different in slices from animals given paired or explicitly unpaired stimulus presentations. The location of cells with low thresholds for local dendritic calcium spikes suggested that there are specific sites for learning-related changes within lobule HVI. These areas may correspond to learning "microzones" and are consistent with locations of learning-related in vivo changes in Purkinje cell activity. Application of 4-aminopyridine, an antagonist of the rapidly inactivating potassium current IA, reduced the threshold for dendritic spikes in slices from naive animals to levels found in slices from trained animals. In cells where thresholds for eliciting parallel fiber-stimulated Purkinje cell excitatory postsynaptic potentials (EPSPs) were measured, levels of parallel fiber stimulation required to elicit a 6-mV EPSP as well as a 4-mV EPSP (n = 30) and a Purkinje cell spike (n = 56) were found to be significantly lower in slices from paired animals than unpaired controls. A classical conditioning procedure was simulated in slices of lobule HVI by pairing a brief, high-frequency train of parallel fiber stimulation (8 pulses, 100 Hz) with a brief, lower frequency train of climbing fiber stimulation (3 pulses, 20 Hz) to the same Purkinje cell. Following paired stimulation of the parallel and climbing fibers, Purkinje cell EPSPs underwent a long-term (> 20 min) reduction in peak amplitude (-24%) in cells (n = 12) from animals given unpaired stimulus presentations but to a far less extent (-9%) in cells (n = 20) from animals given in vivo paired training. Whereas 92% of cells from unpaired animals showed pairing-specific depression, 50% of cells from paired animals showed no depression and in several cases showed potentiation. Our data establish that there are localized learning-specific changes in membrane and synaptic excitability of Purkinje cells in rabbit lobule HVI that can be detected in slices 24 h after classical conditioning. Long-term changes within Purkinje cells that effect this enhanced excitability may occlude pairing-specific long-term depression.

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.

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.

Habituation and sensitization to an electrical shock in the crab Chasmagnathus. Effect of background illumination.

The escape response of the crab Chasmagnathus granulatus, elicited by an electrical leg-shock, wanes as a consequence of repeated stimulation, and the decrease persists after a 24-h rest interval. Results concerning stimulus specificity in within- and between-sessions habituation strongly indicate that neither motor fatigue nor sensory adaptation nor damage can account for the response waning, which thus meets the major criteria of habituation. A comparison between the escape response habituation to leg-shock and that to a shadow passing overhead discloses clearcut differences. The shock curve shows an initial hump positively related to stimulus intensity, suggesting that a shock-induced sensitization along with habituation subserves the response curve. The curve asymptote is remarkably high and, unlike the initial waning portion, not greatly affected by treatment changes, hinting that the response may be the combination of an escape response and a basal one (an undirected burst of activity). The waning portion is higher when shocks are given in dark than when given in light, but this effect is not considered as an extra eliciting-stimulus-induced sensitization, since the reactivity enhancement depends necessarily on the darkness-shock concomitance, regardless of prior exposure to darkness. Accordingly, the hypothesis that crab habituates not only to a shock but to a complex stimulus (shock + background illumination) is put forward and 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.

Central effect of morphine pretreatment on short- and long-term habituation to a danger stimulus in the crab Chasmagnathus.

Morphine is believed to inhibit the crab's escape response to a danger stimulus due to central drug action. To test alternative explanations of such an effect in terms of afferent and/or efferent impairment, experiments were conducted using the crab's optokinetic response as indicator. Doses of morphine with maximal detrimental effect on the escape response (75-100 micrograms/g) showed no effect on the optokinetic response, both by measuring the crab's eyestalk displacement and by recording its body rotation, supporting the hypothesis of a morphine central action on the danger-induced escape response. As regards the effect on habituation, a 75 micrograms morphine/g injection administered 30 min before the first trial produced a parallel shift of the short-term (within-session) habituation curve, suggesting a modulatory central drug action that would mimic a putative endogenous opioid action. A 100 morphine micrograms/g dose injected 30 min before training sharply reduced reactivity during training and impaired the acquisition of long-term (between-session) habituation. It may be speculated that the decrease in the danger meaning of the stimulus due to morphine explains both effects in terms of a stimulation impairment during training.