Ontogeny of oxytocin-like immunoreactivity in the cuttlefish, Sepia officinalis, central nervous system.

In vertebrate species, the neuropeptide oxytocin (OT) has been implicated in neural and behavioral development. Although several OT-like peptides have been characterized in invertebrate species, the ontogenesis of the OT-like system has not yet been described in these species. Thus, the aim of the present study was to perform an immunohistochemical investigation of the spatiotemporal distribution of OT-like elements in the central nervous system (CNS) of a decapod cephalopod mollusc, the cuttlefish, Sepia officinalis, during the first 3 months of postembryonic development. On the day of birth, OT-like immunoreactivity was detected throughout the whole CNS. Some nervous structures (e.g. the magnocellular lobes) exhibited a stained pattern in newborns similar to that reported in our previous study in adult cuttlefish whereas other lobes (e.g. the vertical lobe complex) showed maturation during the first weeks of life. Finally, at the age of 60 days, the general pattern of staining in the CNS was comparable to the adult distribution. The putative roles of the OT-like system with regard to the development of some behaviors in juvenile cuttlefish are discussed. The present study provides a neurochemical basis for the investigation of postnatal development of complex behaviors in cephalopods and suggests, for the first time in an invertebrate species, important organizational effects for the OT-like system in the course of the first weeks of life.

Vasopressin/oxytocin-related peptides influence long-term memory of a passive avoidance task in the cuttlefish, Sepia officinalis.

The vasopressin (VP)/oxytocin (OT)-related peptides constitute a large superfamily found in a wide range of both vertebrate and invertebrate species. While intensive literature reports that these neuropeptides influence behavior, especially learning and memory, in numerous species from diverse vertebrate groups, their roles in behavioral regulation have never been studied in invertebrates. Here, we investigated the role of two VP/OT superfamily peptides, octopressin (OP) and cephalotocin (CT), on long-term memory (LTM) formation of a passive avoidance task in a cephalopod mollusc, the cuttlefish, Sepia officinalis. Subadult cuttlefish were intravenously injected, in a dose range of 3-60 microg/kg, 1h after the training phase (consolidation design); retention performance was tested 24h post-training. We found that administration of OP at low dose (3 microg/kg) enhanced LTM, whereas a dose of 60 microg/kg attenuated it. No effect of OP on LTM was observed for the 15 microg/kg dose. Conversely, an enhancement of retention performance was observed at all doses of CT tested. This study is the first to demonstrate the behavioral effects of VP/OT superfamily peptides in an invertebrate species. The valuable role of VP/OT-like peptides on memory processes offers new evolutionary perspectives on peptidergic transmission and neuromodulation.

Distribution of oxytocin-like and vasopressin-like immunoreactivities within the central nervous system of the cuttlefish, Sepia officinalis.

We have investigated the distribution of oxytocin/vasopressin (OT/VP) superfamily peptides in the central nervous system (CNS) of the cuttlefish, Sepia officinalis, by using antibodies raised against mammalian OT and VP. Several populations of OT-like and VP-like immunoreactive cell bodies and fibers were widely distributed in cerebral structures involved in learning processes (vertical lobe complex, optic lobes), behavioral communication (peduncle, lateral basal and chromatophore lobes), feeding behavior (inferior frontal, brachial and buccal lobes), sexual activity (dorsal basal, subpedunculate, olfactory lobes), and metabolism (visceral lobes). The two most remarkable findings of this study were the occurrence of OT-like immunoreactivity in many amacrine cells of the vertical lobe and the dense accumulation of VP-like immunoreactive cell bodies in the subpedunculate 1 lobe. No double-immunolabeled cell bodies or fibers were found in any lobes of the CNS, indicating, for the first time in a decapod cephalopod mollusc, the existence of distinct oxytocinergic-like and vasopressinergic-like systems. The widespread distribution of the immunoreactive neurons suggests that these OT-like and VP-like peptides act as neurotransmitters or neuromodulators.

Does kainic acid induce partial brain lesion in an invertebrate model: sepia officinalis? Comparison with electrolytic lesion.

The present study investigates the feasibility of excitotoxic lesions in the cuttlefish in the mapping of brain functions in Cephalopods. Adult animals were injected locally with a neurotoxin, kainic acid. The brain region receiving the neurotoxin was the vertical lobe, a key brain structure for learning and memory processes. Brain damage induced by these injections was evaluated using different histological stainings: hematoxilin-eosin, Fink-Heimer and DAPI. The results were compared with histological changes after electrolytic lesion of the vertical lobe. Neurodegeneration was revealed in and around the injection site: an intense area of proliferative cells, degenerating terminal axon ramifications and cell death. In comparison with electrolytic lesion, excitotoxic lesion displays important advantages, since fibres of passage are not destroyed by kainic acid injection, which induces only a restricted lesion and so is an appropriate method of investigating the role of the vertical lobe or other brain regions in a Cephalopod model, Sepia officinalis.

Orientation in the cuttlefish Sepia officinalis: response versus place learning.

Several studies have demonstrated that mammals, birds and fish use comparable spatial learning strategies. Unfortunately, except in insects, few studies have investigated spatial learning mechanisms in invertebrates. Our study aimed to identify the strategies used by cuttlefish (Sepia officinalis) to solve a spatial task commonly used with vertebrates. A new spatial learning procedure using a T-maze was designed. In this maze, the cuttlefish learned how to enter a dark and sandy compartment. A preliminary test confirmed that individual cuttlefish showed an untrained side-turning preference (preference for turning right or left) in the T-maze. This preference could be reliably detected in a single probe trial. In the following two experiments, each individual was trained to enter the compartment opposite to its side-turning preference. In Experiment 1, distal visual cues were provided around the maze. In Experiment 2, the T-maze was surrounded by curtains and two proximal visual cues were provided above the apparatus. In both experiments, after acquisition, strategies used by cuttlefish to orient in the T-maze were tested by creating a conflict between the formerly rewarded algorithmic behaviour (turn, response learning) and the visual cues identifying the goal (place learning). Most cuttlefish relied on response learning in Experiment 1; the two strategies were used equally often in Experiment 2. In these experiments, the salience of cues provided during the experiment determined whether cuttlefish used response or place learning to solve this spatial task. Our study demonstrates for the first time the presence of multiple spatial strategies in cuttlefish that appear to closely parallel those described in vertebrates.

Distribution of neurokinin A-like and serotonin immunoreactivities within the vertical lobe complex in Sepia officinalis.

Immunohistochemistry, using antibodies raised against mammalian neurokinin A (NKA) and serotonin (5-HT), was applied in double-staining experiments to map these molecules within the vertical lobe complex (inferior frontal, superior frontal, post-frontal, vertical, subvertical and precommissural lobes). NKA-like and 5-HT immunoreactivities were detected in all the lobes of the vertical lobe complex but were never colocalized in cell bodies or fibres. Except for the cell layers of the superior frontal lobe, both types of labelled cell bodies were observed in all the lobes. Both types of immunoreactive fibres were detected in all the neuropils and interestingly revealed clear subdivisions within some lobes, e.g., 5-HT-IR fibres were more abundant in the peripheral part of the vertical lobe whereas NKA-IR ones were widely observed in both the peripheral and central parts. In cephalopods, the vertical lobe complex is involved in learning and memory; thus, our results strongly suggest that one or more NKA-like and 5-HT molecules may function as neurochemical messengers in these cognitive processes.

Food imprinting, new evidence from the cuttlefish Sepia officinalis.

Imprinting provides precocial offspring with an efficient means to optimize their subsequent behaviours. We discovered food imprinting using a sophisticated invertebrate model: the cuttlefish. We showed that early juveniles preferred the prey to which they have been visually familiarized, when the amount of information was sufficient and only if such familiarization occurred during a short sensitive period. We also demonstrated that the effects of visual food imprinting overcame those of the first food ingested. Our study shows that visual imprinting is a critical process in animals, surpassing more direct reward experiences that occur outside the critical exposure period.

Effects of dorsal and ventral vertical lobe electrolytic lesions on spatial learning and locomotor activity in Sepia officinalis.

This study aims to analyze the effects of electrolytic lesion, restricted to either the ventral or the dorsal parts of the vertical lobe (VL), on the behavior of cuttlefish (Sepia officinalis). Two behavioral tests were performed on sham-operated and lesioned cuttlefish: assessment of locomotor activity in an open field and determination of spatial learning abilities in a T maze. The results showed that ventral lesions of the VL led to marked impairment in the acquisition of spatial learning, whereas dorsal lesions of the VL increased locomotor activity in the open field and impaired long-term retention of spatial learning. This study establishes for the first time the existence of distinct functions in the ventral and the dorsal parts of the VL in cephalopods.

Developmental study of multiple memory stages in the cuttlefish, Sepia officinalis.

The inhibition of predatory behavior observed during the "prawn-in-the-tube" procedure has been extensively used in studies of cuttlefish learning. The present study examines the effect of age on the conditioning of this response in the cuttlefish, Sepia officinalis. Data show that a significant increase in acquisition performance occurs between 15 and 21 days of age. The retention curves in 8- and 15-day-old cuttlefish show a monotonic memory process, presumably reflecting the presence of only short-term memory. In 21-day-old cuttlefish, there are two distinct processes which could be a labile short-term memory, and a subsequent intermediate memory. These mnesic systems seem to become more effective over the course of post-embryonic development. Moreover, the retention curves obtained in the oldest cuttlefish (30- and 90-day-old) bear a close resemblance to that observed in adults. These behavioral findings will allow further work on the cellular and molecular mechanisms of learning and memory in the cuttlefish.

The "prawn-in-the-tube" procedure in the cuttlefish: habituation or passive avoidance learning?

This study examines whether or not habituation contributes to the regulation of the inhibition of predatory behavior observed during the "prawn-in-the-tube" training procedure. When presented with prawns that are visible behind glass but untouchable, cuttlefish promptly learn to inhibit their capture attempts. The first three experiments demonstrated that the acquired response in the course of training cannot be dishabituated. The fourth experiment demonstrated that the repeated application of a brief visual prawn stimulus, one that is terminated before the cuttlefish can strike, decreases attack latencies. Taken together, the results of this study establish that habituation does not play a significant role in this learning task. In fact, the present findings strengthen the results of previous studies suggesting that passive avoidance learning contributes to the regulation of the inhibition of predatory behavior.

In vitro evidence and age-related changes for nicotinic but not muscarinic acetylcholine receptors in the central nervous system of Sepia officinalis.

Binding putative muscarinic ([3H]-NMS and [3H]-QNB) or nicotinic ([3H]-cytisine) acetylcholine receptors was quantitatively studied through the use of in vitro binding experiments on either membrane preparations or brain sections of juvenile (3 months), mature (15 months) or senescent (23 months) cuttlefish. No specific binding could be detected with muscarinic receptor ligands under any of the experimental conditions employed (ligand concentrations, buffers, ionic charges, types of tissue, i.e., brain sections or membrane preparations). On the other hand, [3H]-cytisine demonstrated a specific and saturable binding with a single class of high affinity binding sites (Kd of 2.6-34.6 nM; Bmax of 128-1682 fmol/mg tissue equivalent, depending on the central structure). This binding was found to be heterogeneous throughout the central regions (optic lobe>pedal lobe; superior frontal lobe>...precommissural lobe; vertical lobe>...anterior basal lobe; subvertical lobe; inferior frontal lobe; median basal lobe). These results question the existence of muscarinic-like receptors in the cuttlefish brain, or at least of a pharmacological dissimilarity from vertebrate muscarinic receptors. In contrast, nicotinic-like receptors are widely present; interestingly, their density was found to be significantly reduced in most nervous central lobes of senescent cuttlefish when compared with mature animals. The most significant decrease (-71%) was found in the anterior part of the superior frontal lobe, which is involved in visual learning; this might be related to the changes, previously demonstrated, in cholinergic neurons in this lobe in the course of aging.

Early experience and postembryonic maturation of body patterns in cuttlefish (Sepia officinalis).

This study investigates effects of the environment on the maturation of body patterns in cuttlefish (Sepia officinalis). Cuttlefish were reared either individually on a uniform background, which the authors have termed uniform-solitary conditions (Group A), or grouped on variegated backgrounds, which the authors have termed varied-social conditions (Group B). At Days 1, 15, 30, and 60, juveniles were placed individually in perceptually different testing conditions, either on small, variegated stones or on a uniform pale gray background. During development in both testing conditions, juveniles from Group B concealed themselves differently from those from Group A. Thus, it appears that the response to the background is subject to individual experience. Some hypotheses are discussed relating to the effect of early experience on the maturation of body patterns.

Effect of early feeding experience on subsequent prey preference by cuttlefish, Sepia officinalis.

Food preferences were investigated in cuttlefish during the first 3 months' posthatching, using choice tests between crabs, shrimps, and young fish. The results showed that without previous feeding experience, cuttlefish preferred shrimps on Day 3. This suggests an innate food preference; however, it was possible to induce a preference for an originally nonpreferred prey item in 3-day-old and naïve cuttlefish, demonstrating the flexibility of this initial behavioral preference in response to previous individual experience. This preference suggests a learning process involving a form of long-term memory, demonstrated for the first time in juvenile cuttlefish. Until Day 30, juvenile cuttlefish fed exclusively shrimps chose shrimps. This preference probably depends on their previous feeding experience. Finally, it appears that from Day 60, cuttlefish reared on the same restricted diet have a tendency to switch their preference to novel prey items, which diversify their diet.

Changes in cholinergic enzyme activities in the cuttlefish brain during memory formation.

Cephalopods are promising invertebrate models to investigate the neurobiology of learning and memory due to their advanced behavioral abilities. In the present study, acetylcholine synthesis and catabolism were studied in various central nervous system (CNS) regions of cuttlefish subjected to associative learning procedures with different retention delays, with the aim of characterizing the involvement of cholinergic neurotransmission in learning and memory processes in this species. In the first experiment, the acquisition procedure consisted of a single continuous trial. As a response to a short retention delay (60 min), when compared to control or to a very short retention delay (2 min), cholinergic enzymatic activities decreased in structures involved in learning and memory (vertical-subvertical lobes) and in lower motor centers (brachial and pedal lobes). In the second experiment, the same learning paradigm was used but with repeated trials. In this case, a long-term retention (24 h) induced an increase in acetylcholine catabolism in the pedal and optic lobes (lower motor and higher centers, respectively) when compared to control. These results suggest the occurrence of different regulatory mechanisms of cholinergic enzymes as a response to memory formation with a long- or a short-term retention delay.

Time-dependent effects of cycloheximide on long-term memory in the cuttlefish.

When shown prawns in a glass tube, cuttlefish promptly learn to inhibit their predatory behavior and retain this ability for a long time. The cellular and molecular mechanisms of this long-term memory (LTM) are not yet known. In this study, we analyzed the dependency of LTM on de novo brain protein synthesis. Cycloheximide (CXM), a protein synthesis inhibitor, is injected intravenously immediately, 1 h, 3 h, 4 h or 6 h after the training. Retention is tested 24 h posttraining. The injections of CXM revealed one period of memory sensitivity to pharmacological intervention. CXM administered immediately or 6 h after training has no effect on LTM. Conversely, injections given between 1 and 4 h posttraining resulted in amnesia. Taken together, findings of this study establish for the first time in Sepia officinalis that de novo protein synthesis is an essential and time-dependent event for LTM formation of this form of associative learning.