Among invertebrates, cephalopods provide a powerful tool to explore cellular and molecular mechanisms underlying learning and memory. Indeed, cephalopods show particularly good learning and memory abilities indicating the emergence of complex cognitive processes [1–3]. Furthermore, they possess a sophisticated central nervous system (CNS), subdivided into numerous lobes interconnected by tracts and commissures, which has been the subject of extensive anatomical and histological research [4–8]. Behavioural experiments have investigated brain functions in these animals [9–14]. Surprisingly, cellular and molecular studies of behavioural plasticity have not yet been done in cephalopods.
Cytochrome oxidase (CO) is a mitochondrial enzyme crucial for oxidative phosphorylation which is commonly used as an endogeneous marker of neuronal activity in mammals [15–17]. In contrast to markers of glucose utilisation (2-deoxyglucose and fluoro-deoxyglucose) which assess short-term metabolic changes, CO histochemistry allows to reveal neuronal activity over long periods. Using this technique, some studies have shown, for instance, changes relating to development [17,18], ageing , disease  and learning . In this study, CO histochemistry was used to determine metabolic changes in the CNS of the cuttlefish, Sepia officinalis, after instrumental conditioning.
MATERIALS AND METHODS
Adult cuttlefish (110–280 mm dorsal mantle length) of both sexes were caught off Luc-sur-Mer (France), and housed in individual tanks with circulating seawater at 15°C. Conditioned animals from the first experiment (n = 10) were sacrificed just after the training procedure, whereas those from the second experiment (n = 11) were sacrificed 24 h after the training procedure. For each experiment, a control group was composed of 10 and 11 unconditioned cuttlefish, respectively.
A detailed description of the learning protocol is given in a previous paper . Briefly, five shrimp were enclosed in a transparent glass tube. The training procedure included eight trials, lasting 3 min each, separated by 30 min. For each trial, the number of capture attempts (tentacle strikes) was counted. Under these conditions, adult cuttlefish quickly learnt to inhibit their predatory behaviour . A retention test was run 24 h after the training procedure for conditioned animals of the second experiment to determine the level of memory recall.
Cuttlefish were killed by decapitation. Brains were rapidly removed and fixed in 4% paraformaldehyde dissolved in 0.1 M phosphate buffer, pH 7.6, for 1 h at 4°C. The tissue was washed in 0.1 M phosphate buffer (three times for 15 min each), and incubated for about 12 h in 30% sucrose in 0.1 M phosphate buffer (until the brains sank to the bottom). The brains were covered with Tissue-Tek fluid, frozen, and horizontal sections were cut at 10 μm in a cryostat and mounted on slides coated with a chrome alum/gelatin mixture and stored at −80°C until processing.
CO histochemistry was performed according to the protocol of Wong-Riley . Slides were incubated in the dark for 3 h at 37°C. Sections of conditioned and control animals were incubated under the same conditions at the same time. Fresh incubation medium consisted of 50 mg diaminobenzidine, 20 mg horse heart cytochrome c (Sigma Chemical Co., St. Louis, USA), 4 g sucrose, and 18 mg catalase per 90 ml in 0.1 M phosphate buffer. After incubation, the slides were immersed for 5 min in 10% sucrose in 0.1 M phosphate buffer, and washed in 0.1 M phosphate buffer (twice for 5 min), dehydrated in alcohol, transferred to toluene and embedded in DePex. Additional sections incubated after removal of diaminobenzidine from the incubation medium showed no visible CO reaction.
A Leitz-Aristoplan universal microscope was used for examination under light illumination. The optical density (OD) in the CO stained sections was analysed using an image-processing system (BIOCOM). The mean grey level of eight sections of each analysed structure was calculated for conditioned and control groups. Briefly, the CNS of the cuttlefish comprises a central mass, containing a supra- and a suboesophageal mass, flanked by two large optic lobes. We focused our attention on some structures of the vertical lobe system (inferior frontal, subvertical, superior frontal and vertical lobes) located dorsally in the supraoesophageal mass, which seem to be involved in learning and memory . In each lobe, the cortical and neuropil areas were analysed separately. The nomenclature of these regions and mapping of Sepia brain follow previous anatomical data .
To evaluate the acquisition performances within conditioned groups, the number of tentacle strikes observed during the first trial was compared with the number observed during the last trial of the training procedure. To evaluate 24 h retention of the conditioned animals of the second experiment, the number of tentacle strikes observed during the first trial of the training procedure was compared with the number observed during the 3 min of the retention test. The statistical significance of differences between the two time periods was evaluated using a Wilcoxon signed ranks test for matched samples . To reduce variation in the intensity of CO staining from one experiment to another, OD in conditioned animals was expressed as a percentage of OD observed in control animals. The statistical significance of differences between the two groups was evaluated using multiple comparisons based on Mann-Whitney U-tests . Spearman rank correlation coefficients analyses were undertaken between acquisition (experiment 1) or retention performances (experiment 2) and CO activity . We only considered the brain regions in which CO staining differed significantly between conditioned and control animals.
Conditioned cuttlefish from the two experiments showed significant acquisition during the training procedure (experiment 1: Z = −2.809, n = 10, p < 0.05; experiment 2: Z = −2.937, n = 11, p < 0.005;Fig. 1a,b). Retention at 24 h was evident for conditioned animals of the second experiment (Z = −2.807, n = 11, p < 0.05;Fig. 1b).
Among the different lobes analysed, only the superior frontal lobe showed modifications in the level of CO labelling between conditioned and control groups (Table 1). This lobe may be divided into anterior (ASF: anterior superior frontal lobe) and posterior (PSF: posterior superior frontal lobe) parts. Each of these parts may be further divided into a cortical area, an external neuropil area and an internal neuropil area according to the CO staining pattern.
In the first experiment, conditioned cuttlefish exhibited an increase in CO staining in the cortex and external neuropil areas of the PSF (cortex area: U = 56, n = 10, p < 0.01; external neuropil area: U = 63, n = 10, p < 0.05;Fig. 2a, Table 1), but not in the internal neuropil area. Moreover, there was no difference in ASF nor in other lobes (inferior frontal, subvertical and vertical lobes) between conditioned and control groups.
In the second experiment, the cortex and external neuropil areas of the ASF in conditioned animals showed a decrease of the labelling (cortex area: U = 10, n = 11, p < 0.005; external neuropil area: U = 10, n = 11, p < 0.005;Fig. 2b, Table 1), whereas no significant difference was observed in either internal neuropil area or PSF. No modification in CO staining pattern was noted for the other lobes analysed between conditioned and control groups.
Correlations between CO activity and behaviour:
In experiment 1, no significant correlation was found between CO activity in the PSF and acquisition performances (cortex area: rs = −0.024, n = 10, p > 0.05; external neuropil area: rs = −0.343, n = 10, p > 0.05). In the same way, there was no correlation between CO activity in the ASF and retention performances in experiment 2 (cortex area: rs = −0.041, n = 11, p > 0.05; external neuropil area: rs = −0.254, n = 11, p > 0.05).
Our study showed that CO histochemistry can be successfully used in the cephalopod nervous system. The data obtained demonstrated that learning induced changes in CO staining in a specific brain region of the cuttlefish: the superior frontal lobe. Moreover, the latter exhibited two different patterns of labelling according to the delay after learning. Indeed, CO staining was increased in the PSF just after training and decreased in the ASF at 24 h post-training. These physiological findings strengthen anatomical data relating to the superior frontal lobe. Young  reported that the superior frontal lobe shows two distinct parts, with different inputs and outputs. The anterior part has larger cells and sends fibres to the subvertical lobe and perhaps beyond. The posterior part sends all its axons to the vertical lobe.
Previous studies have demonstrated that CO activity is correlated with neuronal activity [15–17]. In our study, the increase of CO activity in the PSF may reflect changes of neuronal activity in this area during consolidation processes of the memory for a restricted period. Conversely, the decrease of CO activity in the ASF may reveal decrease of neuronal activation during long term storage of memory. The visual learning process in cephalopods has been the subject of numerous studies. These studies suggested, after surgical lesions, that the vertical lobe system may play an important role in acquisition processes as well as in retention processes . Our results are consistent with data obtained previously and provide a first metabolic evidence for the involvement of the superior frontal lobe in learning and memory in Sepia.
Previous anatomical data suggested that ASF receives its inputs from various areas of the vertical lobe system (inferior frontal, subvertical and vertical lobes) and from optic lobes . The decrease of neuronal activation observed in the ASF might in part reveal changes of neuronal activity of the afferent structures. In this context, the absence of metabolic changes between conditioned and control animals in the other lobes of the vertical lobe system is surprising. A possible explanation could be that our technique used to measure the CO labelling does not fit all types of tissues. Indeed, a large heterogeneity is observed in the arrangement of tissues. For instance, the vertical lobe shows a cortex with numerous small neurons and less than one percent of large neurons; its neuropil is composed of scattered fibres. Our method, based on averages of the OD in each structure (cortex or neuropil), could dilute some local changes of CO activity. For future investigations, a measure of the CO labelling in each category of cells would be probably more appropriate for these structures to define precisely any local changes in CO activity.
Our study demonstrates that CO histochemistry, performed mostly in vertebrates [15,21,24] and more recently in invertebrates , can be applied successfully to the cuttlefish brain. Instrumental conditioning modifies metabolic neuronal responses in CO activity of the superior frontal lobe. Moreover, two specific patterns of CO staining were found in two distinct parts of this lobe, in agreement with the known anatomical data . These results strongly argue for an involvement of the superior frontal lobe in learning and memory. This technique is a useful tool to provide a functional mapping of learning-induced plasticity in the central nervous system of the cuttlefish.
This research was supported by a grant from the Ministère de la Recherche et de la Technologie to V.A. We are grateful to Mrs Chantal Marais for her technical assistance and Dr Sigurd von Boletzky for helping to correct the English translation.
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Keywords:© 2001 Wolters Kluwer Health | Lippincott Williams & Wilkins
Brain; Cephalopod; Cuttlefish; Cytochrome oxidase; Learning; Memory; Superior frontal lobe