Early-light embryonic stimulation suggests a second route, via gene activation, to cerebral lateralization in vertebrates.

Genetic factors determine the asymmetrical position of vertebrate embryos allowing asymmetric environmental stimulation to shape cerebral lateralization. In birds, late-light stimulation, just before hatching, on the right optic nerve triggers anatomical and functional cerebral asymmetries. However, some brain asymmetries develop in absence of embryonic light stimulation. Furthermore, early-light action affects lateralization in the transparent zebrafish embryos before their visual system is functional. Here we investigated whether another pathway intervenes in establishing brain specialization. We exposed chicks' embryos to light before their visual system was formed. We observed that such early stimulation modulates cerebral lateralization in a comparable vein of late-light stimulation on active retinal cells. Our results show that, in a higher vertebrate brain, a second route, likely affecting the genetic expression of photosensitive regions, acts before the development of a functional visual system. More than one sensitive period seems thus available to light stimulation to trigger brain lateralization.

Sensory generalization and learning about novel colours by poultry chicks.

In nature animals constantly encounter novel stimuli and need to generalize from known stimuli. The animal may then learn about the novel stimulus. Hull (1947) suggested that as they learn animals distinguish knowledge based on direct experience from inference by generalization and in support of this view suggested that if a subject is directly trained to a stimulus subsequent extinction of responses is slower than when the response is based on generalization. Such an effect is also predicted by Bayesian models that relate the rate of learning to uncertainty in the estimate of stimulus value. We find support for this prediction when chicks learn about a novel colour (orange) if the initial evaluation is based on similarity to known colours (red, yellow). Specifically, if an expected food reward is absent the rate of extinction of the response to the novel stimulus exceeds that for the familiar colours. Interestingly, the change in relative preference for novel and familiar stimuli occurs after a delay of an hour. This type of delay has not, to our knowledge, been reported in previous studies of single-trial learning, but given its importance of generalization in natural behaviour this type of learning may have wide relevance.

Time-window for sensitivity to cooling distinguishes the effects of hypothermia and protein synthesis inhibition on the consolidation of long-term memory.

The effects of hypothermia on memory formation have been examined extensively, and while it is clear that post-training cooling interferes with the process of consolidation, the nature of the temperature sensitive processes disrupted in this way remain poorly defined. Post-training manipulations that disrupt consolidation tend to be effective during specific time-windows of sensitivity, the timing and duration of which are directly related to the mechanism through which the treatment induces amnesia. As such, different treatments that target the same basic processes should be associated with similar time-windows of sensitivity. Using this rationale we have investigated the possibility that cooling induced blockade of long-term memory (LTM) stems from the disruption of protein synthesis. By varying the timing of post-training hypothermia we have determined the critical period during which cooling disrupts the consolidation of appetitive long-term memory in the pond snail Lymnaea. Post-training hypothermia was found to disrupt LTM only when applied immediately after conditioning, while delaying the treatment by 10 min left the 24 h memory trace intact. This brief (<10 min) window of sensitivity differs from the time-window we have previously described for the protein synthesis inhibitor anisomycin, which was effective during at least the first 30 min after conditioning [Fulton, D., Kemenes, I., Andrew, R. J., & Benjamin, P. R. (2005). A single time-window for protein synthesis-dependent long-term memory formation after one-trial appetitive conditioning. European Journal of Neuroscience, 21, 1347-1358]. We conclude that hypothermia and protein synthesis inhibition exhibit distinct time-windows of effectiveness in Lymnaea, a fact that is inconsistent with the hypothesis that cooling induced amnesia occurs through the direct disruption of macromolecular synthesis.

The zebrafish as a model for behavioral studies.

Zebrafish genetics is developing at a rapid pace, and this opens up new approaches to understanding genetic control. This short review discusses recent results obtained in behavioral studies in this species, and also shows some promising ways of combining behavioral studies with modern genetic techniques. The zebrafish could provide behavioral models for understanding the neural and genetic control of species-specific behavioral patterns associated with feeding and predator avoidance. Careful experimentation may also reveal the genetic underpinning of simple cognitive processes such as habituation and memory, or lateralized control of behavior.

fsi zebrafish show concordant reversal of laterality of viscera, neuroanatomy, and a subset of behavioral responses.

Asymmetries in CNS neuroanatomy are assumed to underlie the widespread cognitive and behavioral asymmetries in vertebrates. Studies in humans have shown that the laterality of some cognitive asymmetries is independent of the laterality of the viscera; discrete mechanisms may therefore regulate visceral and neural lateralization. However, through analysis of visceral, neuroanatomical, and behavioral asymmetries in the frequent-situs-inversus (fsi) line of zebrafish, we show that the principal left-right body asymmetries are coupled to certain brain asymmetries and lateralized behaviors. fsi fish with asymmetry defects show concordant reversal of heart, gut, and neuroanatomical asymmetries in the diencephalon. Moreover, the neuroanatomical reversals in reversed fsi fish correlate with reversal of some behavioral responses in both fry and adult fsi fish. Surprisingly, two behavioral asymmetries do not reverse, suggesting that at least two separable mechanisms must influence functional lateralization in the CNS. Partial reversal of CNS asymmetries may generate new behavioral phenotypes; supporting this idea, reversed fsi fry differ markedly from their normally lateralized siblings in their behavioral response to a novel visual feature. Revealing a link between visceral and brain asymmetry and lateralized behavior, our studies help to explain the complexity of the relationship between the lateralities of visceral and neural asymmetries.

A single time-window for protein synthesis-dependent long-term memory formation after one-trial appetitive conditioning.

Protein synthesis is generally held to be essential for long-term memory formation. Often two periods of sensitivity to blockade of protein synthesis have been described, one immediately after training and another several hours later. We wished to relate the timing of protein synthesis-dependence of behavioural long-term memory (LTM) formation to an electrophysiological correlate of the LTM memory trace. We used the snail Lymnaea because one-trial appetitive conditioning of feeding using a chemical conditioned stimulus leads to a stable LTM trace that can be monitored behaviourally and then electrophysiologically in preparations made from the same animals. Anisomycin (an inhibitor of translation) injected 10 min after training blocked behavioural LTM formation. Actinomycin D (an inhibitor of transcription) was also effective at 10 min. When anisomycin, at doses shown to be effective in blocking central nervous system protein synthesis, was injected at 1, 2, 3, 4, 5 and 6 h after training there was no effect on recall. These results indicate that there is a single period of sensitivity to protein synthesis inhibition in Lymnaea lasting for between 10 min and 1 h after training with no evidence for a second window of sensitivity. An electrophysiological correlate of LTM was found to be sensitive to anisomycin injected 10 min after training. It is unusual to find only one period of protein synthesis-dependence in detailed time-course studies of LTM, and this suggests that the consolidation processes involving protein synthesis are relatively rapid in one-trial appetitive conditioning and complete within 1 h of training.

Light experience and the development of behavioural lateralisation in chicks. II. Choice of familiar versus unfamiliar model social partner.

In late-stage embryos of domestic fowl, exposure of the right eye to light entering through the shell induces asymmetry of the thalamofugal visual pathway, together with differences in performance according to whether the right or left eye (RE, LE) is in use (Behav. Brain Res. 38 (1990) 211). Nevertheless, at least some of the main specialisations of the right and left eye systems (RES, LES) are not dependent on such exposure. Higher ability of LES to assess and respond to novelty is present in dark-incubated (Da) chicks. This is probably also true of RES ability to control response, and specifically to inhibit shift to an alternative response (i.e. to a novel stimulus). We imprinted chicks on red table-tennis balls with a horizontal, white strip on their equator. At test, they chose between this and a ball with a vertical, white strip. Da chicks showed clear choice with the LE, but not with the RE. Unexpectedly, light-incubated (Li) chicks failed to show LE/RE differences in choice. Exploratory pecks at a novel feature were greatly reduced in Li. Two effects of light exposure on RES are likely. The first is greater use of RES in the home-cage, affecting what is learned about the companion ball. This may make RES more competent in assessing ball properties, and so explain the enhanced choice by RE, that abolished the RE/LE difference in Li. Secondly, the ability of RES to inhibit shift to an alternative response is enhanced. Light exposure and being female similarly opposed shift to the novel feature, but probably via different mechanisms. The effects of exposure are discussed as an example of the generation of a range of behavioural phenotypes, which are sustained within a single population by varying or frequency-dependent selection.

Separate processing mechanisms for encoding of geometric and landmark information in the avian hippocampus.

Domestic chicks bilaterally or unilaterally lesioned to the hippocampus were trained to search for food hidden beneath sawdust by ground-scratching in the centre of a large enclosure, the correct position of food being indicated by a local landmark in the absence of any extra-enclosure visual cues. At test, the landmark was removed or displaced at a distance from its original position. Results showed that sham-operated chicks and chicks with a lesion of the left hippocampus searched in the centre, relying on large-scale geometric information provided by the enclosure, whereas chicks with a lesion of either the right hippocampus or both hippocampi were completely disoriented (landmark removed) or searched close to the landmark shifted from the centre (landmark displaced). These results indicate that encoding of geometric features of an enclosure occurs in the right hippocampus even when local information provided by a landmark would suffice to localize the goal; encoding based on local information, in contrast, seems to occur outside the hippocampus. These findings provide evidence that the left and right avian hippocampi play different roles in spatial cognition, a phenomenon which had been documented previously only for the human hippocampus.

Hemispheric lateralization of memory stages for discriminated avoidance learning in the chick.

Memory formation following the one trial discriminated bead task in the chick falls into three stages (short-term, intermediate and long-term memory) that are defined by susceptibility to different classes of drugs. The stages show sharply timed offsets of sensitivity and loss at specific times after inhibition. Recall of the memory in the chick shows cyclical changes that differ in period between left and right hemispheres, and is marked by a series of brief windows of enhanced recall that recur with periods of about 16 and 25 min in the left and right hemispheres respectively. The timing of these 'retrieval events' corresponds, to a large extent, with the timing of the memory stages seen in the visual discrimination task. Here we examine the effects of left or right hemisphere injection of the main agents (glutamate, ouabain and anisomycin) that have been used to characterize the three stages of memory. We show that memory in the left hemisphere is largely responsible for performance at test and that processes involved in its consolidation generate the phases of memory.

The use of viewing posture to control visual processing by lateralised mechanisms.

Chicks were trained and tested to run to a dish and feed from it under one of four conditions. In three of these, the dish was covered by a light lid that the chicks readily learned to remove. The square lid (SL) had slightly protruding corners, so that it could be removed by a blow of the bill at a range of positions. A round lid (UL), which fitted snugly, could best be removed by inserting the bill into a medial U-shaped indentation. A round lid (STR), which fitted all the way round to the edge of the dish, could be removed by grasping and tugging a centrally placed piece of string. The final dish had no lid (NOL). The dish and the layout of the arena were otherwise identical under all conditions. Chicks trained and tested with lids predominantly fixated the dish during approach with the right eye and showed leftward deviation from the direct line of approach (which facilitated right eye use). NOL chicks fixated with the left eye and deviated rightwards. The right eye is thus used when a motor plan has to be set up and sustained. The use of the left eye is expected when topographical information has to be used. Here, the approach was so simple and practised that the assumption of left eye viewing is likely to be a default condition. It would facilitate detection of any change in layout. A standard set of head positions were used, particularly by SL and NOL chicks, showing that the head was aligned with some reference point, perhaps the centre of the dish. These fell into two series (used by both eyes), and in both the peaks of frequent use were 10 degrees apart. One (20 degrees, 10 degrees ) was probably generated by head saccades ending with the bill pointing directly at the dish (0 degrees ). The other (35 degrees, 25 degrees, 15 degrees ) is best explained by slight divergence of the optic axes. The 25 degrees right eye position was consistently used by STR chicks at the beginning of approach. The STR condition requires the most difficult manipulation, and the manipulandum is most obvious from a distance. This is consistent with right eye use during establishment of a motor plan. Head postures consistent with divergence were also used when close to the dish under conditions where choice between targets was necessary. This was clear in the NOL condition, where the chick could see the food grains as it approached. Here, it is likely that both eyes are used in independent search. If so, it may be that divergence is used as a strategy during establishment of a motor plan (as in STR chicks) to increase the independence of the right eye system.

Critical time-window for NO-cGMP-dependent long-term memory formation after one-trial appetitive conditioning.

The nitric oxide (NO)-cGMP signaling pathway is implicated in an increasing number of experimental models of plasticity. Here, in a behavioral analysis using one-trial appetitive associative conditioning, we show that there is an obligatory requirement for this pathway in the formation of long-term memory (LTM). Moreover, we demonstrate that this requirement lasts for a critical period of approximately 5 hr after training. Specifically, we trained intact specimens of the snail Lymnaea stagnalis in a single conditioning trial using a conditioned stimulus, amyl-acetate, paired with a salient unconditioned stimulus, sucrose, for feeding. Long-term associative memory induced by a single associative trial was demonstrated at 24 hr and shown to last at least 14 d after training. Tests for LTM and its dependence on NO were performed routinely 24 hr after training. The critical period when NO was needed for memory formation was established by transiently depleting it from the animals at a series of time points after training by the injection of the NO-scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide (PTIO). By blocking the activity of NO synthase and soluble guanylyl cyclase enzymes after training, we provided further evidence that LTM formation depends on an intact NO-cGMP pathway. An electrophysiological correlate of LTM was also blocked by PTIO, showing that the dependence of LTM on NO is amenable to analysis at the cellular level in vitro. This represents the first demonstration that associative memory formation after single-trial appetitive classical conditioning is dependent on an intact NO-cGMP signaling pathway.