MR microscopy of cobalt-labeled nerve cells and pathways in an invertebrate brain (Sepia officinalis, Cephalopoda).

This article describes a novel application of contrast-enhanced MR microscopy to trace nerve cells and pathways through small invertebrate brains. Using the cuttlefish Sepia officinalis (Cephalopoda) as a model, the cells and pathways of one of the brain nerves were labeled with paramagnetic cobalt(II) ions by conventional centripetal cobalt iontophoresis. In MR microscopy, the cobalt-labeled cell bodies and pathways became hypointense in 9.4 T spin echo images. Their course and distribution were identical with those seen with conventional histological techniques after cobalt sulphide precipitation (with or without subsequent silver intensification). Magn Reson Med 45:575-579, 2001.

Inhibitory effect of cyclic guanosine 3',5'-monophosphate (cGMP) on the afferent resting activity in the cephalopod statocyst.

The effects of exo- and endogenous cGMP on the resting activity (RA) of afferent crista fibers were studied in isolated preparations of the statocysts of the cuttlefish Sepia officinalis and the squid Sepioteuthis lessoniana. Bath application of the membrane-permeable cGMP analogs 8-bromo-cGMP (B-cGMP) and N(2),2'-o-dibutyryl 3', 5'-cyclic guanosine monophosphate (dB-cGMP), and of the selective inhibitor of cGMP-phosphodiesterase zaprinast (ZAP), caused an inhibition of RA. The inhibitory effects of B-cGMP and dB-cGMP remained when the preparation was pre-treated with: (i) the guanylate cyclase inhibitors 1H-[1,2,4]oxadiazolo[4,3, -a]quinoxalin-1-one (ODQ) or cystamine (CYS); (ii) the adenylate cyclase inhibitors nicotinic acid (NIC-A), 2',3'dideoxyadenosine (DDA), or MDL-12330A (MDL); (iii) the guanylate cyclase inhibitor methylene blue (M-BLU) and the adenylate cyclase inhibitor MDL combined; or (iv) the nitric oxide (NO) synthase inhibitors N(G)-nitric-L-arginine methyl ester HCl (L-NAME) or N(G)-nitro-L-arginine (L-NOARG). These data indicate that cGMP, as an intracellular messenger, has a tonic inhibitory effect on the RA of afferent crista fibers in cephalopod statocysts.

Effects of nitric oxide donors on the afferent resting activity in the cephalopod statocyst.

The effects of bath applications of the nitric oxide (NO) donors sodium nitroprusside (SNP), diethylamine sodium (DEA), 3-morpholinosydnonimine (SIN-1), and S-nitroso-N-acetyl-penicillamine (SNAP) on the resting activity (RA) of afferent crista fibers were studied in isolated statocysts of the cuttlefish Sepia officinalis. The NO donors had three different effects: inhibition, excitation, and excitation followed by an inhibition. The SNAP analog N-acetyl-DL-penicillamine (xSNAP; with no NO moiety) had no effect. When the preparation was pre-treated with the NO synthase inhibitor N(G)-nitric-L-arginine methyl ester HCl (L-NAME), the NO donors were still effective. When the preparation was pre-treated with the guanylate cyclase inhibitors methylene blue (M-BLU) or cystamine (CYS), NO donors had only excitatory effects, whereas their effects were inhibitory only when pre-treatment was with the adenylate cyclase inhibitors nicotinic acid (NIC-A), 2',3'-dideoxyadenosine (DDA), or MDL-12330A. When pre-treatment was with a guanylate and an adenylate cyclase inhibitor combined, NO donors had no effect; in that situation, the RA of the afferent fibers remained and the preparation still responded to bath applications of GABA. Selective experiments with statocysts from the squid Sepioteuthis lessoniana and the octopod Octopus vulgaris gave comparable results. These data indicate that in cephalopod statocysts an inhibitory NO-cGMP and an excitatory NO-cAMP signal transduction pathway exist, that these two pathways are the key pathways for the action of NO, and that they have only modulatory effects on, and are not essential for the generation of, the RA.

The effect of early experience on learning and memory in cuttlefish.

The effect of early experience on the growth and ontogeny of memory in cuttlefish (Sepia officinalis) was studied using an associative learning protocol. Five groups of cuttlefish were reared in different conditions (standard conditions, SC; impoverished conditions, IC; enriched conditions, EC; impoverished then enriched conditions, I/EC; enriched then impoverished conditions, E/IC) from birth to the 3rd month of postembryonic life. Acquisition and retention of the learning task were assessed at 1 and 3 months. Growth was slower and maturation of memory abilities occurred later in cuttlefish from Group IC than in cuttlefish from Group EC, with the maturation rate of memory in cuttlefish from Group SC intermediate between these two groups. Retention performances of cuttlefish from Groups I/EC and E/IC indicated that the environment of rearing during the 2nd and/or 3rd months of life was crucial for the development of memory.

Effects of L-arginine on the afferent resting activity in the cephalopod statocyst.

The effects of bath application of the nitric oxide (NO) precursor L-arginine (L-ARG) on the resting activity (RA) of afferent crista fibers were studied in isolated statocysts of the cuttlefish Sepia officinalis under various experimental conditions. L-ARG (threshold 10(-7) M) had three different effects: inhibition, excitation, and excitation followed by an inhibition; only the inhibitory effect of L-ARG was dose-dependent. D-Arginine (D-ARG) had no effect. When the preparation was pre-treated with NO synthase inhibitors (N(G)-Nitric-L-arginine methyl ester HCl (L-NAME), N(G)-Nitro-L-arginine (L-NOARG)), both the inhibitory and the excitatory effects of L-ARG significantly decreased at higher concentrations (10(-5 to -4) M), or were completely blocked at lower concentrations (10(-7 to -6) M), of L-ARG. When the preparation was pre-treated with guanylate cyclase inhibitors (1H-[1,2, 4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), methylene blue (M-BLU), cystamine (CYS)), L-ARG had only excitatory effects, whereas its effects were only inhibitory when the preparation was pre-treated with adenylate cyclase inhibitors 2',3'-dideoxyadenosine (DDA), MDL-12330A (MDL), nicotinic acid (NIC-A)). L-ARG had no effects when the pre-treatment was with a guanylate cyclase inhibitor and an adenylate cyclase inhibitor combined; in that situation, the RA of the afferent fibers remained. These data indicate that in cephalopod statocysts, a cGMP and a cAMP signal transduction pathway (presumably via the generation of NO) are responsible for the effects of L-ARG on the RA of crista afferent fibers. They also indicate that the L-ARG-cGMP pathway is the dominant pathway and is inhibitory, and that both pathways have only modulatory effects on, but are not essential for, the generation of the RA.

Structure and function of the Nautilus statocyst.

The two equilibrium receptor organs (statocysts) of Nautilus are avoid sacks, half-filled with numerous small, free-moving statoconia and half with endolymph. The inner surface of each statocyst is lined with 130,000-150,000 primary sensory hair cells. The hair cells are of two morphological types. Type A hair cells carry 10-15 kinocilia arranged in a single ciliary row; they are present in the ventral half of the statocyst. Type B hair cells carry 8-10 irregularly arranged kinocilia; they are present in the dorsal half of the statocyst. Both type of hair cells are morphologically polarized. To test whether these features allow the Nautilus statocyst to sense angular accelerations, behavioural experiments were performed to measure statocyst-dependent funnel movements during sinusoidal oscillations of restrained Nautilus around a vertical body axis. Such dynamic rotatory stimulation caused horizontal phase-locked movements of the funnel. The funnel movements were either in the same direction (compensatory funnel response), or in the opposite direction (funnel follow response) to that of the applied rotation. Compensatory funnel movements were also seen during optokinetic stimulation (with a black and white stripe pattern) and during stimulations in which optokinetic and statocyst stimulations were combined. These morphological and behavioural findings show that the statocysts of Nautilus, in addition to their function as gravity receptor organs, are able to detect rotatory movements (angular accelerations) without the specialized receptor systems (crista/cupula systems) that are found in the statocysts of coleoid cephalopods. The findings further indicate that both statocyst and visual inputs control compensatory funnel movements.

Proprioceptive hair cells on the neck of the squid Lolliguncula brevis: a sense organ in cephalopods for the control of head-to-body position.

Decapod cephalopods, such as cuttlefishes and squids, have a distinct neck region that allows movements (roll, pitch and yaw) of the head relative to the body. This paper describes the structure, innervation and central pathways of proprioceptive hair cells on the neck of the squid Lolliguncula brevis that sense such movements and control head-to-body position. These hair cells exist on the dorsal side of the neck underneath the nuchal cartilage, close to the animal's midline on either side of the nuchal crest. On each side, the hair cells can be divided into an anterior and a posterior group of 25-35 and 70-80 cells, respectively. An individual hair cell carries up to 300 kinocilia of equal length (about 30 microns), arranged in up to seven rows. The hair cells of the left and right anterior group are morphologically polarized in the medial direction, whereas the hair cells of the left and right posterior group are polarized in the anterior direction. The hair cells are primary sensory cells. They are innervated by a branch of the postorbital nerve and project ipsilaterally into the ventral part of the ventral magnocellular lobe. Efferent synaptic contacts are present at the base of the hair cells. In behavioural tests the influence of the neck hair cells on head position control was investigated. During imposed body rolls, a unilateral deafferentation of the cells caused an asymmetric change of the compensatory head roll response and elicited a head roll offset to the operated side. Bilateral deafferentation of the cells elicited a downward head pitch offset. This offset was superimposed on the compensatory head pitch response during imposed body pitch. These morphological and behavioural findings show that the neck hair cells and the associated nuchal cartilage structures of Lolliguncula brevis form a neck receptor organ that, together with statocyst and visual inputs, controls the position of the animal's head and body.

A dorsal light reflex in a squid.

A dorsal light reflex is described in the squid Lolliguncula brevis. When illuminated from the side in visually homogeneous surroundings, a free-swimming squid rolls the dorsal side of its head and trunk 10-20 degrees towards the light. With the trunk restricted in a holder, the squid rolls its head 4-5 degrees towards the light; this reaction increases by about 50% when the statocysts are bilaterally removed and increases further when the neck receptor organ is also destroyed. The results indicate a multi-modal interaction of visual, statocyst and proprioceptive inputs during postural control.

Induction of a cellular defense reaction is accompanied by an increase in sensory neuron excitability in Aplysia.

The complexity of vertebrate immune and nervous systems makes detailed cellular analysis of neuroimmune interactions a challenging prospect. The immune systems of invertebrates, although much less complex than their vertebrate counterparts, share basic cellular defense responses to wounded self or nonself. We have developed a simple model system to study neuroimmune interactions using an invertebrate preparation well suited to detailed cellular analysis. Loose ligation of peripheral nerves in Aplysia induced a cellular defense reaction evidenced by the accumulation of large numbers of amebocytes at the ligation site. From 5 to 30 d after ligation, the excitability of the soma of sensory neurons having axons in ligated nerves was increased compared to contralateral sensory neurons with axons in nonligated nerves. Spike threshold and afterhyperpolarization were reduced, and spike amplitude and duration were increased. Spike accommodation was also decreased such that sensory neurons on the ligated side fired more spikes to a 1 sec intracellular depolarizing pulse than control sensory neurons. These effects are unlikely to be accounted for by ligation-induced injury of sensory axons since both morphological and electrophysiological evidence indicated that the axons in ligated nerves were healthy and able to conduct action potentials. Amebocytes activated by the presence of nonself may release factors that lead to a central sensitization of sensory neurons with axons in close proximity to the amebocytes.

The effect of L-glutamate on the afferent resting activity in the cephalopod statocyst.

The effects of bath application of L-glutamate and of excitatory amino acid agonists and antagonists on the resting activity of afferent crista fibers were studied in isolated preparations of the statocyst of the cuttlefish, Sepia officinalis. L-Glutamate (threshold 10(-5) M) and its agonists quisqualate and kainate (thresholds 10(-6) M) increased the resting activity in a dose-dependent manner. Glutamine (threshold 10(-5) M) was also excitatory, while D-glutamate had no effect. Also, no obvious excitatory effects were seen for NMDA and L-aspartate, nor was any antagonistic effect seen for the selective NMDA-receptor antagonist D-2-amino-5-phosphonovaleric acid (D-AP-5). The spider toxin Argiotoxin636 (threshold 10(-11) M), 2-amino-4-phosphonobutyric acid (AP-4), glutamic acid diethyl ester (GDEE), gamma-D-glutamylaminomethyl-sulfonic acid (GAMS), and kynurenic acid decreased the resting activity and effectively blocked or reversed the effect of L-glutamate and its non-NMDA agonists. Preliminary experiments with statocysts from the squid Sepioteuthis lessoniana and the octopod Octopus bimaculoides gave comparable results. All data show that in cephalopod statocysts L-glutamate, via non-NMDA receptors, has an excitatory effect on the activity of afferent fibers, an effect consistent with its possible function as a hair cell transmitter.

Directional sensitivity of hair cell afferents in the Octopus statocyst.

Changes in threshold sensitivity of hair cell afferents of the macula and crista of the Octopus statocyst were analyzed when the hair cells were stimulated with sinusoidal water movements from different directions. The experiments indicate that cephalopod statocyst hair cells are directionally sensitive in a way that is similar to the responses of the hair cells of the vertebrate vestibular and lateral line systems, with the amplitude of the response changing according to the cosine of the angle by which the direction of the stimulus (the deflection of the ciliary bundle) deviates from the direction of the hair cell's morphological polarization.

The oculomotor system of decapod cephalopods: eye muscles, eye muscle nerves, and the oculomotor neurons in the central nervous system.

Fourteen extraocular eye muscles are described in the decapods Loligo and Sepioteuthis, and thirteen in Sepia; they are supplied by four eye muscle nerves. The main action of most of the muscles is a linear movement of the eyeball, only three muscles produce strong rotations. The arrangement, innervation and action of the decapod eye muscles are compared with those of the seven eye muscles and seven eye muscle nerves in Octopus. The extra muscles in decapods are attached to the anterior and superior faces of the eyes. At least, the anterior muscles, and presumably also the superior muscles, are concerned with convergent eye movements for binocular vision during fixation and capture of prey by the tentacles. The remaining muscles are rather similar in the two cephalopod groups. In decapods, the anterior muscles include conjunctive muscles; these cross the midline and each presumably moves both eyes at the same time during fixation. In the squids Loligo and Sepioteuthis there is an additional superior conjunctive muscle of perhaps similar function. Some of the anterior muscles are associated with a narrow moveable plate, the trochlear cartilage; it is attached to the eyeball by trochlear membranes. Centripetal cobalt fillings showed that all four eye muscle nerves have fibres that originate from somata in the ipsilateral anterior lateral pedal lobe, which is the oculomotor centre. The somata of the individual nerves show different but overlapping distributions. Bundles of small presumably afferent fibres were seen in two of the four nerves. They do not enter the anterior lateral pedal lobe but run to the ventral magnocellular lobe; some afferent fibres enter the brachio-palliovisceral connective and run perhaps as far as the palliovisceral lobe.

[14C]deoxyglucose labelling of functional activity in the cephalopod central nervous system.

For the first time, the [14C]deoxyglucose radioautographic technique has been successfully used to map physiological activity in cephalopod brains. In unilaterally blinded octopus and cuttlefish, the optic lobe of the deprived side showed a decreased uptake of the labelled tracer. This suggests that the uptake is related to functional activity. The potential of the [14C]deoxyglucose technique as a powerful tool in studying the functional organization of cephalopod brains is discussed.

Peripheral and central nervous responses evoked by small water movements in a cephalopod.

Potentials were recorded from the epidermal head lines and from the CNS of young cuttlefish, Sepia officinalis, in response to weak water movements. 1. Within the test range 0.5-400 Hz a sinusoidal water movement elicits up to 4 components of response if the electrode is placed on a headline: (i) a positive phasic ON response; (ii) a tonic frequency-following microphonic response; (iii) a slow negative OFF response; and (iv) compound nerve impulses. 2. The amplitude of both the ON wave and the microphonic potential depends on stimulus frequency, stimulus amplitude and stimulus rise time. Frequencies around 100 Hz and short rise times are most effective in eliciting strong potentials. The minimal threshold was 0.06 microns peak-to-peak water displacement at 100 Hz (18.8 microns/s as velocity). 3. Change of direction of tangential sphere movement (parallel vs. across the head lines) has only a small effect on the microphonic and the summed nerve potentials. 4. Frequency and/or amplitude modulations of a carrier stimulus elicit responses at the onset and offset of the modulation and marked changes in the tonic microphonic response. 5. Evoked potentials can be recorded from the brain while stimulating the epidermal lines with weak water movements. The brain potentials differ in several aspects from the potentials of the head lines and show little or no onset or offset wave at the transitions of a frequency and amplitude modulation.

Sensory evoked potentials in unanesthetized unrestrained cuttlefish: a new preparation for brain physiology in cephalopods.

Up to five microelectrodes inserted through short hypodermic needles in the cranial cartilage of Sepia officinalis recorded potentials while the cuttlefish moved freely in a small enclosure. Compound field potentials and unit spikes were seen during ongoing, spontaneous activity and after sensory stimulation. Ongoing activity resembles that reported for octopus, with maximum power usually below 20 Hz. Amplitude varies greatly but has not been seen to shut off or turn on abruptly and globally as in octopus. Evoked potentials, focally large after flashes of light consist of several waves; the first is largest, positive and peaks at ca. 35 ms (called P35), followed by ca. P75, P95, N110 and smaller waves or oscillations lasting more than 0.5 s. The Upper Following Frequency (highest flashing rate the potentials can follow 1:1), without averaging, is greater than 15 flashes/s (20-22 degrees C); at 20/s the 1:1 following lasts for 1 or 2 s. The Lower Fusion Frequency of averaged responses is less than 30/s. Gentle tapping of the tank wall evokes local, brief, fast potentials. No responses have been found to loud air-borne clicks and tone bursts with principal energy at 300 Hz or to electric fields in the bath at 50-100 microV/cm. In a few loci relatively large slow Omitted Stimulus Potentials have been seen following the end of a train of flashes at more than 5/s; these are by definition event related potentials and a special, central form of OFF response.

Convergent inputs to Octopus oculomotor neurones demonstrated in a brain slice preparation.

A brain slice preparation was developed to permit recordings to be made from neurones in the anterior lateral pedal lobe of the octopus CNS. These are the first intracellular recordings obtained from identified neurones in the cephalopod CNS. The neurones had membrane resting potentials of around -40 mV and action potentials up to 20 mV in amplitude. Spontaneous synaptic potentials could be seen in some cells. All of the oculomotor neurones, identified by stimulation of the anterior oculomotor nerve to evoke an antidromic potential, received an excitatory input from the visual system, shown by electrical stimulation of the optic tract, and some also received an excitatory input from the statocyst, as shown by electrical stimulation of the macula nerve.

Brain pathways of the chromatophore system in the squid Lolliguncula brevis.

Brain pathways controlling the chromatophores of the squid Lolliguncula brevis are described using cobalt iontophoresis. The results show several input and output pathways of the anterior and posterior chromatophore and lateral basal lobes. These connections allow coordination and modification of the chromatophore motor program throughout the motor pathway. Unlike other cephalopod species, there seems to be no direct input from the optic lobes to the lateral basal lobes in L. brevis. This species displays only a few simple patterns; therefore the underlying neural pathways for chromatophore control may be different from those of other cephalopods with more extensive patterning repertoires.

Physiological color change in squid iridophores. II. Ultrastructural mechanisms in Lolliguncula brevis.

Evidence is presented that changes in the optical properties of active iridophores in the dermis of the squid Lolliguncula brevis are the result of changes in the ultrastructure of these cells. At least two mechanisms may be involved when active cells change from non-iridescent to iridescent or change iridescent color. One is the reversible change of labile, detergent-resistant proteinaceous material within the iridophore platelets, from a contracted gel state (non-iridescent) to an expanded fluid or sol state when the cells become iridescent. The other is a change in the thickness of the platelets, with platelets becoming significantly thinner as the optical properties of the iridophores change from non-iridescent to iridescent red, and progressively thinner still as the observed iridescent colors become those of shorter wavelengths. Optical change from Rayleigh scattering (non-iridescent) to structural reflection (iridescent) may be due to the viscosity change in the platelet material, with the variations in observed iridescent colors due to changes in the dimensions of the iridophore platelets.

Physiological color change in squid iridophores. I. Behavior, morphology and pharmacology in Lolliguncula brevis.

Cephalopods generally are thought to have only static iridophores, but this report provides qualitative and quantitative evidence for active control of certain iridescent cells in the dermis of the squid Lolliguncula brevis. In vivo observations indicate the expression of iridescence to be linked to agonistic or reproductive behavior. The neuromodulator acetylcholine (ACh) induced dramatic opitcla changes in active iridophores in vitro, whereas ACh had little effect on passive iridophores elsewhere in the mantle skin. Bath application of physiological concentrations of ACh (10(-7)M to 10(-6)M) to excised dermal skin layers transformed the active iridophores from a non-reflective diffuse blue to brightly iridescent colors, and this reaction was reversible and repeatable. The speed of change to iridescent in vitro corresponded well to the speed of changes in the living animal. Pharmacological results indicate the presence of muscarinic receptors in this system and that Ca++ is a mediator for the observed changes. Although ACh is present in physiological quantities in the dermal iridophore layer, it is possible that ACh release is not controlled directly by the nervous system because electrophysiological stimulation of major nerves in the periphery resulted in no iridescence in L. brevis; nor did silver staining or transmission electron microscopy reveal neuronal elements in the iridophore layer. Thus, active iridophores may be controlled by ACh acting as a hormone.

A lateral line analogue in cephalopods: water waves generate microphonic potentials in the epidermal head lines of Sepia and Lolliguncula.

Many cephalopods have lines of ciliated cells on their head and arms. In the cuttlefish Sepia and the squid Lolliguncula, electrophysiological recordings clearly identify these epidermal lines as an invertebrate analogue to the mechanoreceptive lateral lines of fish and aquatic amphibians and thus as another example of convergent evolution between a sophisticated cephalopod and vertebrate sensory system. Stimulation of the epidermal lines with local water displacements, generated by a vibrating sphere, causes receptor potentials that have many features known from lateral line microphonic potentials. The minimal threshold of the head lines is 0.2 micron peak-to-peak water displacement (calculated at the skin surface) at 75-100 Hz.