Cephalopod brains: promising preparations for ...

CEPHALOPOD NEUROBIOLOGY N.J. Abbott, R. Williamson and L. Maddock (eds.) Oxford: Oxford University Press, 1995, pp. 399-413

24 Cephalopod brains: promising preparations for , :: brain physiology BERND

U. BUDELMANN, RODDY

THEODORE WILLIAMSON

H. BULLOCK,

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Summary The brains of cephalopods are the most sophisticated brains of all invertebrates and their gross anatomy and neuronal pathways are well known. Also much is known in cephalopods about learning and memory functions. Yet physiological recordings from cephalopod brains are scanty. Recently, however, three preparations have been developed and are now available for experiments on cephalopod brain physiology: (i) a brain slice preparation that allows intracellular recordings from identified brain neurones, (ii) an intact animal preparation that permits multiple electrode recordings of spikes and compound field potentials from unanaesthetized and unrestrained cuttlefish, and (iii) mapping of metabolic brain activity with C4C]deoxyglucose. These preparations are complementary and allow a variety of physiological experiments to be done. With further improvements of the techniques and in combination with the morphological information that already exists on pathways in the cephalopod brain, these new preparations are promising tools for cephalopod brain physiology. They may even serve as supplementary or alternative invertebrate preparations for vertebrate research.

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For more than 50 years the cephalopod brain and nervous system have played an important role in neurobiological research. Beyond question, cephalopods have the most complex of any invertebrate brains and a tremendous body of precise information already exists about the embryology, the gross anatomy, and the neuronal pathways of their brains (e.g. Boycott 1961; Young 1965, 1971, 1974, 1976, 1977a,b, 1979; Woodhams 1977; Messenger 1979; Budelmann and Young 1985, 1987; Plan 1987; Marquis 1989; Gleadall 1990). In addition to these morphological data, much is known in cephalopods abo~t motor behaviour, learning, and memory functions (e.g. Sanders and Young 1940; Boycott 1961; Muntz 1963; Messenger 1973; Chichery 1983; Young 1983, 1991; Budelmann and Young 1984; Gilly et al. 1991; for further references, see Wells 1978; Mangold 1989). This wealth of information, however, contrasts with the fact that electrophysiological recordings from cephalopod brains are scanty. Two reasons may

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account for this. One is the difficulty in adequately immobilizing cephalopods for brain recordings, and the other is the poor survival rate of the animals when anaesthetized or perfused. Because of these difficulties only two papers (Laverack 1980; Bullock 1984) and several short notes (Mislin 1955; Bullock and Uter 1976; Robertson et al. 1991; Bleckmann et al. 1991; Miyan and Messenger, Chapter 25 of this volume) have been published that describe electrophysiological recordings from cephalopod brains, either in isolated brain preparations or in heavily restrained animals. Several recordings, however, have been successfully made from the optic lobes (MacNichol and Love 1960a,b; Lettvin and Maturana 1961, 1962; Boycott et al. 1965; Corner and Schade 1967; Hartline and Lange 1974; Lange and Hartline 1974; Stephen 1974; Patterson and Silver 1983; Saidel et al. 1983). Recently, substantial progress has been made. Three new preparations have been described in detail: (1) a brain slice preparation that allows intracellular recordings from cells of any area of the brain (Williamson and Budelmann 1991); (2) an intact animal preparation that permits multiple electrode recordings of spike and compound field potentials in different brain areas of unanaesthetized and unrestrained cuttlefish that are capable of behaving (Bullock and Budelmann 1991); and (3) mapping of metabolic brain activity with [14C]deoxyglucose W4C]DG; Novicki et al. 1992). These three preparations are complementary and allow a variety of physiological experiments to be done. This chapter will briefly describe the preparations and discuss the impact they may have on future research on cephalopod and vertebrate brain functions.

Brain slice preparation For many years thin brain slices have been successfully used in many types of research on the physiology of the vertebrate brain. In contrast, a slice preparation of the cephalopod brain has only recently been developed (Williamson and Budelmann 1991). Although the preparations used an octopod species (Octopus bimaculoides), slices from any decapod species should function just as well. To obtain the brain slices, the animals were killed by decapitation (without prior anaesthesia) and the brain quickly exposed under artificial sea water (ASW) and removed from its cartilaginous capsule. The ASW contained 10 mM KCl, 470 mM NaCl, 55 mM MgCI2, 11 mM CaCI2, 10 mM glucose, and was buffered to pH 7.6 with MOPS. It was chilled to about 4°C. The low temperature during all processing of the brain tissue is probably an essential factor tor good survival of the brain. Removal of the left and right optic lobes permits brain slices to be cut in any desired direction (transverse, sagittal, horizontal, or oblique). Williamson and Budelmann (1991) used a tangential sagittal slice, up to 1.5 mm thick, cut by hand with a razor blade,

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! Fig. 24.1. Brain slice preparation of Octopus vulgaris. (a) Posterior view of the whole brain (with the two optic lobes removed), showing the direction of the cut and the brain slice (stippled). (b) Transverse section through the anterior lateral pedal lobe [as outlined in (a)], showing cobalt-labelled oculomotor neurones stained via the anterior oculomotor nerve. Scale bar: 200 urn. (c) Brain slice seen laterally. Intracellular recordings were made with glass microelectrodes from oculomotor neurones in the anterior lateral pedal lobe [ct. (b)]. Stimulation of the anterior oculomotor nerve was with a polyethylene suction electrode, stimulation of the sensory cells in the macula epithelium was via a bipolar pair electrode, and stimulation of the optic tract was via a concentric needle electrode. (From Williamson and Budelmann 1991; reprinted with the permission of Elsevier Scientific Publishers.) and containing the most lateral -one-quarter (by volume) of the brain of a 100-150 g octopus [Fig. 24.1(a)]. The beauty of such a relatively thick slice preparation is that it leaves intact many of the axons that run from the somata of the neurones to other parts of the brain [Fig. 24.1(b)]. Depending upon the angle of sectioning, slices can even be obtained to which one, or more, nerves are still attached [Fig. 24.1(c)]. This permits intracellular recordings from brain neurones while stimulating a nerve, or recordings from one or several nerves while stimulating brain neurones. For recordings, the brain slice was transferred to a small dish and sandwiched between two nylon grids. The dish was continuously perfused with cooled (12-14°C) oxygenated ASW, which had access to both sides of the slice. Under such conditions the preparation remained viable and stable for 3-4 h, and intracellular recordings could be held for up to 20 min. Using such a slice preparation, Williamson and Budelmann (1991) obtained the first intracellular recordings from identified neurones in a cephalopod

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Fig. 24.2. Intracellular recordingsfrom neurones in the perikaryallayer of the anterior lateral pedal lobe of the Octopus brain. (a) Recordings from two neurones showing membrane resting potential fluctuations similar to junctional evoked postsynaptic potentials (upper trace) and inhibitory postsynapticpotentials (lower trace). (b) Recording showing an action potential evoked by injecting a depolarizingpulse into the neurone. (c) Recordings from an oculomotor neurone to electrical stimulation of the anterior oculomotor nerve to give an antidromicspike (response latency 8 ms), of the optic tract to represent a visual input (response latency 27 ms), and of sensory cellsin the statocyst macula epithelium to represent an equilibrium receptor input (response latency 20 ms). (From Williamsonand Budelmann 1991; reprinted with the permission of Elsevier ScientificPublishers.) brain. The neurones investigated were from the 'oculomotor centre' (anterior lateral pedal lobe ). They showed membrane resting potentials of about -40 mV and spontaneous action potentials of up to 20 mV in amplitude. Some neurones showed membrane potential fluctuations similar to junctional excitatory and inhibitory postsynaptic potentials [Fig 24.2(a)]. . Williamson and Budelmann (1991) used a tangential sagittal section of the brain to verify physiologically the convergence of a visual and an equilibrium receptor input on to oculomotor neurones in the anterior lateral pedal lobe that had earlier been described morphologically (Budelmann and Young 1984). Figure 24.2( c) shows intracellular recordings from oculomotor neurones (identified as such by antidromic stimulation of one of the oculomotor nerves) that could be depolarized-with some latency-by stimulating the optic tract (latency betwe~n 10 and 32 ms) as well as sensory cells in the statocyst macula epithelium (latency up to 20 ms). Thick brain slice preparations thus are suitable tools to examine the prop-

Boycott (1961), Chi chery and Chanelet (1976), and Chichery (1983) were the first to describe an intact unrestrained cephalopod preparation for experiments on brain function, although they used the implanted electrodes for stimulation, not for recording. Bullock and Budelmann (1991) recently developed a preparation that allows such recordings in animals that are unanaesthetized, unrestrained, and capable of behaving. They used large specimens (15-18 em mantle length) of the European cuttlefish, Sepia officinalis. Only these large specimens have a layer of cartilage above the vertical lobe of the brain that is thick enough (about 5 mm) for a mechanically stable electrode implantation. Several days prior to the experiment, 10-15 mm of the rostrally protruding tip of the cuttlebone was cut off. This exposes the anterior part of the nuchal cartilage (which overlies the neck and the posterior end of the head) and avoids damage to the implanted electrodes if the animal retracts its head underneath the cuttlebone. On the day of the experiment, the animal was gently transferred (by scooping up, not netting) from the holding tank to the laboratory and, after a resting period of about 20 min, was eased into a small experimental tank. Such gentle handling is essential to avoid any stress prior to the experiment, and thus to avoid any startle or other nervous reactions. The experimental tank contained a 15 mm layer of gravel and 70 mm depth of sea water and was continuously aerated with a mixture of 95 per cent oxygen and 5 per cent carbon dioxide. The total volume of sea water in the tank was three to four times the volume of the animal. A fenced partition .of the tank was just large enough to allow the animal some forward and backward, and some lateral movements, but no turns. With the animal in the experimental tank the electrodes were implanted into the brain in two steps, involving no surgery or anaesthesia. In a first step, a 15 mm length of a 25 gauge hypodermic needle was pushed vertically down, or in any desired direction, through the anterior end of the nuchal cartilage, or the skin slightly anterior to it; it was then pushed through the muscle layer and the dorsal cranial cartilage above the vertical lobe to a depth of 10 mm so that 5 mm of the needle still protruded above the nuchal cartilage, or above the skin. Because the muscle layer and the cranial cartilage are together 10 mm thick, the 15 mm needle did not penetrate into the brain (Fig. 24.3). Four such hypodermic needles were usually placed, aiming at selected parts of the brain. This aiming needs some experience and a good knowledge of the

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Fig.24.3. Diagrams of sections through the brain of an adult cuttlefish (Sepia officinalis, mantle length 17-18 em), showing the dimensions of the supra- and suboesophageal brain, and the cartilage and muscle layers above the brain. (a) Lateral view; (b) anterior view. For technique of electrode positioning, see text. gross anatomy of the cuttlefish brain. In a second step, commercial platinum/ iridium recording electrodes of straight, thin, sharpened wire were inserted through the hypodermic needles and beyond into the brain to a predetermined depth, according to the dimensions of the brain (Fig. 24.3). These dimensions, of course, vary according to the animal's size. The depth (D) of penetration of the electrode into the brain can easily be calculated as D = LE - (LH + LEO), with LE being the total length of the electrode, LH the length of the hypodermic needle, and LEO the length of the electrode outside the needle (Fig. 24.3). Surprisingly, throughout the insertion of the hypodermic needles and of the electrodes, and afterwards for up to 12 hours of recordings, the animals lay quietly in their confined space. They moved forwards from time to time, but always slowly without strong jetting; inking never occurred;' With the electrodes implanted, the animals could then be exposed to various stimuli, such as visual, acoustic, electrical, and mechanical. To localize exactly where the tips of the electrodes had been during the recordings, the animals were anaesthetized in the experimental tank at the

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end of the experiment by slowly adding an overdose of ethanol. Subsequently, the electrodes were replaced by needles of the same diameter and length, pushed through the hypodermic needles down to the depth at which the tips of the electrodes had been during the recordings. With all these needles in place, the head Of the animal was then carefully removed and fixed in formalin. After fix-ation the brain was dissected out, with the needles in place, and cut parallel to the needles, sagittally or transversely, into about 1 mm thick slices. These slices are thin enough to become transparent and show the borderlines between the various lobes when illuminated from below. Stereomicroscopical analysis of the set of sections allows an accurate localization of the tip of the needles (= tip of the electrodes) within the brain, and even within a given brain lobe. Bullock and Budelmann (1991) recorded compound field potentials and spikes during ongoing, spontaneous activity, and after sensory stimulation from seven different supra- and suboesophageal areas of the brain. The overall aspect of the compound field potentials has been found to be highly variable from locus to locus and from moment to moment (Fig. 24.4); thus, cephalopods seem to have more steeply differentiated electric fields than mammals. Slow waves have been the major feature, as in the vertebrate electrocorticogram, but they are more prominent than in gastropod or arthropod brain activity (Bullock 1945; Bullock and Basar 1988). Spikes, if present, were mostly large and occurred with a frequency of a few, to a few dozen times per second. In cephalopods, potentials evoked by sensory stimuli, in general, were remarkably similar to those known in mammals and other vertebrates. Visual evoked potentials to a single flash were of long duration (100-125 ms, or more; Fig. 24.5). Potentials to trains of flashes showed an upper following frequency of usually more than 20 Hz; i.e. they had a fusion frequency of about 50 ms (Fig. 24.6). At the end of a train, sometimes a slow wave occurred, representing a special form of central 'off' potential. It resembled the 'omitted stimulus potential' of fishes and belongs by definition to the 'Event related potentials' (Fig. 24.7; Bullock et al. 1990).

Mapping of metabolic brain activity with [14C]DG For several years, the [14C]DG technique has been successfully used for mapping metabolic activity in vertebrate brains (e.g. Sokoloff et al. '1977; Sokoloff 1987), and has been applied to a few invertebrate preparations as well (e.g. arthropods: Buchner et al. 1984, Bausenwein 1990; gastropod molluscs: Sejnowski et al. 1980; Chase 1985). Since the cephalopod brain metabolizes glucose (Cory and Rose 1969), Novicki et al. (1992) successfully adapted the [14C)DG technique to cephalopod preparations. They used animals, unilaterally visually deprived by lens or eye removal, or by cutting

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Fig. 24.5. Visual evoked potentials in the brain of the cuttlefish Sepia, recorded in two loci to a 20 ms light flash (lower trace), one in the dorsal precommissurallobe (upper trace) and one from the border between the anterior part of the median basal lobe and precommissural lobe, near the mid-line, possibly in the optic commissure (middle trace). Single sweeps, filtered from 1 to 100 Hz. (From Bullock and Budelmann 1991; reprinted with the permission of Springer-Verlag.)

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Fig. 24.4. Ongoing background activity (EEG) in the brain of the cuttlefish Sepia officinalis without intentional stimulation, recorded at two time scales in different loci of the brain. (a) Two simultaneous recordings, one dorsally in the posterior part of the anterior basal lobe, close to the subverticallobe (upper trace), and one in the middle of the anterior basal lobe (lower trace) . (b) Two simultaneous recordings in the anterior basal lobe, one dorsally in its posterior part (upper trace) and one ventrally (lower trace), digitized at 100 Hz, filtered from 10 to 500 Hz. (c) Two simultaneous recordings, one in the dorsal precornmissural lobe (upper trace), and one from the border between the anterior part of the median basal lobe and the precommissural lobe, near the mid-line, possibly in the optic commissure (lower trace), digitized at 300 Hz, filtered from 1 to 100 Hz. (From Bullock and Budelmann 1991; reprinted with the permission of Springer-Verlag.) the optic nerves. In such deprived octopods the dorsal aorta was cannulated with a fine polyethylene tubing (Andrews and Tansey 1981) to inject 4C]DG, dissolved in sterile ASW. Cuttlefish were injected directly into the blood sinus on the ventral .side of the head. Shortly after injection of [14C]DG, the animals received a variety of visual stimuli for 45 min, before they were killed by decapitation. The brain was then quickly processed for cryostat sectioning. The sections were exposed for about 9 days to a calibrated X-ray film and the

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14C' concentrations were measure d b y means of a charge-coup led-device camera and an image analysis system. Novicki et al. (1992) showed that the autoradiographs of the optic lobe sections of the animals injected with [14C]DG had a significantly lower density in the lobe of the visually deprived side, indicating a lower [14C]DG uptake and thus a lower metabolic activity in the deprived lobe. Differences between the non-deprived and the deprived side were also seen at the level of the superior frontal-vertical lobe system, which is involved in visual memory (Young 1965). Thus, Novicki et al. (1992) were able to demonstrate that the [14C]DG autoradiographic technique can be profitably applied to octopod and decapod cephalopods.

Concluding remarks For many years the three preparations described above have been very valuable and powerful tools for physiological experiments on vertebrate brain function. They have now been successfully adapted to physiological research on cephalopod brains, which at the level of gross anatomy and behaviour are undoubtedly the best understood of all the invertebrate brains. Thus, these new preparations finally allow us to make use of the wealth of morphological information we have from the outstanding anatomical and behavioural work of 1. Z. Young and others over the past 50 years on the pathways, connections, and functions of the various lobes of the octopus, cuttlefish, and squid brains.

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