The vertical lobe of cephalopods: an attractive brain structure for understanding the evolution of advanced learning and memory systems.

In this review we show that the cephalopod vertical lobe (VL) provides a good system for assessing the level of evolutionary convergence of the function and organization of neuronal circuitry for mediating learning and memory in animals with complex behavior. The pioneering work of JZ Young described the morphological convergence of the VL with the mammalian hippocampus, cerebellum and the insect mushroom body. Studies in octopus and cuttlefish VL networks suggest evolutionary convergence into a universal organization of connectivity as a divergence-convergence ('fan-out fan-in') network with activity-dependent long-term plasticity mechanisms. Yet, these studies also show that the properties of the neurons, neurotransmitters, neuromodulators and mechanisms of long-term potentiation (LTP) induction and maintenance are highly variable among different species. This suggests that complex networks may have evolved independently multiple times and that even though memory and learning networks share similar organization and cellular processes, there are many molecular ways of constructing them.

Stereotypical reaching movements of the octopus involve both bend propagation and arm elongation.

The bend propagation involved in the stereotypical reaching movement of the octopus arm has been extensively studied. While these studies have analyzed the kinematics of bend propagation along the arm during its extension, possible length changes have been ignored. Here, the elongation profiles of the reaching movements of Octopus vulgaris were assessed using three-dimensional reconstructions. The analysis revealed that, in addition to bend propagation, arm extension movements involve elongation of the proximal part of the arm, i.e., the section from the base of the arm to the propagating bend. The elongations are quite substantial and highly variable, ranging from an average strain along the arm of -0.12 (i.e. shortening) up to 1.8 at the end of the movement (0.57 ± 0.41, n = 64 movements, four animals). Less variability was discovered in an additional set of experiments on reaching movements (0.64 ± 0.28, n = 30 movements, two animals), where target and octopus positions were kept more stationary. Visual observation and subsequent kinematic analysis suggest that the reaching movements can be broadly segregated into two groups. The first group involves bend propagation beginning at the base of the arm and propagating towards the arm tip. In the second, the bend is formed or present more distally and reaching is achieved mainly by elongation and straightening of the segment proximal to the bend. Only in the second type of movements is elongation significantly positively correlated with the distance of the bend from the target. We suggest that reaching towards a target is generated by a combination of both propagation of a bend along the arm and arm elongation. These two motor primitives may be combined to create a broad spectrum of reaching movements. The dynamical model, which recapitulates the biomechanics of the octopus muscular hydrostatic arm, suggests that achieving the observed elongation requires an extremely low ratio of longitudinal to transverse muscle force (<0.0016 for an average strain along the arm of around 0.5). This was not observed and moreover such extremely low value does not seem to be physiologically possible. Hence the assumptions made in applying the dynamic model to behaviors such as static arm stiffening that leads to arm extension through bend propagation and the patterns of activation used to simulate such behaviors should be modified to account for movements combining bend propagation and arm elongation.

An octopus-bioinspired solution to movement and manipulation for soft robots.

Soft robotics is a challenging and promising branch of robotics. It can drive significant improvements across various fields of traditional robotics, and contribute solutions to basic problems such as locomotion and manipulation in unstructured environments. A challenging task for soft robotics is to build and control soft robots able to exert effective forces. In recent years, biology has inspired several solutions to such complex problems. This study aims at investigating the smart solution that the Octopus vulgaris adopts to perform a crawling movement, with the same limbs used for grasping and manipulation. An ad hoc robot was designed and built taking as a reference a biological hypothesis on crawling. A silicone arm with cables embedded to replicate the functionality of the arm muscles of the octopus was built. This novel arm is capable of pushing-based locomotion and object grasping, mimicking the movements that octopuses adopt when crawling. The results support the biological observations and clearly show a suitable way to build a more complex soft robot that, with minimum control, can perform diverse tasks.

Serotonin is a facilitatory neuromodulator of synaptic transmission and "reinforces" long-term potentiation induction in the vertical lobe of Octopus vulgaris.

The modern cephalopod mollusks (coleoids) are considered the most behaviorally advanced invertebrate, yet little is known about the neurophysiological basis of their behaviors. Previous work suggested that the vertical lobe (VL) of cephalopods is a crucial site for the learning and memory components of these behaviors. We are therefore studying the neurophysiology of the VL in Octopus vulgaris and have discovered a robust activity-dependent long-term potentiation (LTP) of the synaptic input to the VL. Moreover, we have shown that the VL and its LTP are involved in behavioral long-term memory acquisition. To advance our understanding of the VL as a learning neural network we explore the possible involvement of neuromodulation in VL function. Here we examine whether the well studied serotonergic modulation in simple models of learning in gastropods mollusks is conserved in the octopus VL. We demonstrate histochemically that the VL is innervated by afferent terminals containing 5-HT immunoreactivity (5-HT-IR). Physiologically, 5-HT has a robust facilitatory effect on synaptic transmission and activity-dependent LTP induction. These results suggest that serotonergic neuromodulation is a part of a reinforcing/reward signaling system conserved in both simple and complex learning systems of mollusks. However, there are notable functional differences. First, the effective concentration of 5-HT in the VL is rather high (100 microM); secondly, only neuropilar regions but not cell bodies in the VL are innervated by terminals containing 5-HT-IR. Thirdly, repetitive or long exposures to 5-HT do not lead to a clear long-term facilitation. We propose that in the octopus VL, while the basic facilitatory properties of molluscan 5-HT system are conserved, the system has adapted to convey signals from other brain areas to reinforce the activity-dependent associations at specific sites in the large connections matrix in the VL.

Stress-induced alternative splicing of acetylcholinesterase results in enhanced fear memory and long-term potentiation.

Stress insults intensify fear memory; however, the mechanism(s) facilitating this physiological response is still unclear. Here, we report the molecular, neurophysiological and behavioral findings attributing much of this effect to alternative splicing of the acetylcholinesterase (AChE) gene in hippocampal neurons. As a case study, we explored immobilization-stressed mice with intensified fear memory and enhanced long-term potentiation (LTP), in which alternative splicing was found to induce overproduction of neuronal 'readthrough' AChE-R (AChE-R). Selective downregulation of AChE-R mRNA and protein by antisense oligonucleotides abolished the stress-associated increase in AChE-R, the elevation of contextual fear and LTP in the hippocampal CA1 region. Reciprocally, we intrahippocampally injected a synthetic peptide representing the C-terminal sequence unique to AChE-R. The injected peptide, which has been earlier found to exhibit no enzymatic activity, was incorporated into cortical, hippocampal and basal nuclei neurons by endocytosis and retrograde transport and enhanced contextual fear. Compatible with this hypothesis, inherited AChE-R overexpression in transgenic mice resulted in perikaryal clusters enriched with PKCbetaII, accompanied by PKC-augmented LTP enhancement. Our findings demonstrate a primary role for stress-induced alternative splicing of the AChE gene to elevated contextual fear and synaptic plasticity, and attribute to the AChE-R splice variant a major role in this process.

Control of octopus arm extension by a peripheral motor program.

For goal-directed arm movements, the nervous system generates a sequence of motor commands that bring the arm toward the target. Control of the octopus arm is especially complex because the arm can be moved in any direction, with a virtually infinite number of degrees of freedom. Here we show that arm extensions can be evoked mechanically or electrically in arms whose connection with the brain has been severed. These extensions show kinematic features that are almost identical to normal behavior, suggesting that the basic motor program for voluntary movement is embedded within the neural circuitry of the arm itself. Such peripheral motor programs represent considerable simplification in the motor control of this highly redundant appendage.

Neuromuscular system of the flexible arm of the octopus: physiological characterization.

The octopus arm is an outstanding example of an efficient boneless and highly flexible appendage. We have begun characterizing the neuromuscular system of the octopus arm in both innervated muscle preparations and dissociated muscle cells. Functionally antagonistic longitudinal and transverse muscle fibers showed no differences in membrane properties and mode of innervation. The muscle cells are excitable but have a broad range of linear membrane properties. They are electrotonically very compact so that localized synaptic inputs can control the membrane potential of the entire muscle cell. Three distinct excitatory neuronal inputs to each arm muscle cell were identified; their reversal potentials were extrapolated to be about -10 mV. These appear to be cholinergic as they are blocked by hexamethonium, D-tubocurarine, and atropine. Two inputs have low quantal amplitude (1-7 mV) and slow rise times (4-15 ms), whereas the third has a large size (5-25 mV) and fast rise time (2-4 ms). This large synaptic input is most likely due to exceptionally large quantal events. The probability of release is rather low, suggesting a stochastic activation of muscle cells. All inputs demonstrated a modest activity-dependent plasticity typical of fast neuromuscular systems. The pre- and postsynaptic properties suggest a rather direct relation between neuronal activity and muscle action. The lack of significant electrical coupling between muscle fibers and the indications for the small size of the motor units suggest that the neuromuscular system of the octopus arm has evolved to ensure a high level of precise localization in the neural control of arm function.

Patterns of arm muscle activation involved in octopus reaching movements.

The extreme flexibility of the octopus arm allows it to perform many different movements, yet octopuses reach toward a target in a stereotyped manner using a basic invariant motor structure: a bend traveling from the base of the arm toward the tip (Gutfreund et al., 1996a). To study the neuronal control of these movements, arm muscle activation [electromyogram (EMG)] was measured together with the kinematics of reaching movements. The traveling bend is associated with a propagating wave of muscle activation, with maximal muscle activation slightly preceding the traveling bend. Tonic activation was occasionally maintained afterward. Correlation of the EMG signals with the kinematic variables (velocities and accelerations) reveals that a significant part of the kinematic variability can be explained by the level of muscle activation. Furthermore, the EMG level measured during the initial stages of movement predicts the peak velocity attained toward the end of the reaching movement. These results suggest that feed-forward motor commands play an important role in the control of movement velocity and that simple adjustment of the excitation levels at the initial stages of the movement can set the velocity profile of the whole movement. A simple model of octopus arm extension is proposed in which the driving force is set initially and is then decreased in proportion to arm diameter at the bend. The model qualitatively reproduces the typical velocity profiles of octopus reaching movements, suggesting a simple control mechanism for bend propagation in the octopus arm.

Organization of octopus arm movements: a model system for studying the control of flexible arms.

Octopus arm movements provide an extreme example of controlled movements of a flexible arm with virtually unlimited degrees of freedom. This study aims to identify general principles in the organization of these movements. Video records of the movements of Octopus vulgaris performing the task of reaching toward a target were studied. The octopus extends its arm toward the target by a wave-like propagation of a bend that travels from the base of the arm toward the tip. Similar bend propagation is seen in other octopus arm movements, such as locomotion and searching. The kinematics (position and velocity) of the midpoint of the bend in three-dimensional space were extracted using the direct linear transformation algorithm. This showed that the bend tends to move within a single linear plane in a simple, slightly curved path connecting the center of the animal's body with the target location. Approximately 70% of the reaching movements demonstrated a stereotyped tangential velocity profile. An invariant profile was observed when movements were normalized for velocity and distance. Two arms, extended together in the same behavioral context, demonstrated identical velocity profiles. The stereotyped features of the movements were also observed in spontaneous arm extensions (not toward an external target). The simple and stereotypic appearance of the bend trajectory suggests that the position of the bend in space and time is the controlled variable. We propose that this strategy reduces the immense redundancy of the octopus arm movements and hence simplifies motor control.

Spatially resolved dynamics of cAMP and protein kinase A subunits in Aplysia sensory neurons.

Cyclic adenosine monophosphate (cAMP)-dependent protein kinase, labeled with fluorescein and rhodamine on the catalytic and regulatory subunits, respectively, was injected into Aplysia sensory neurons either in culture or in intact cell clusters. Energy transfer between the subunits, a measure of cytosolic cAMP concentration ([cAMP]), and compartmentation of the dissociated subunits were monitored by confocal fluorescence microscopy. Bath application of serotonin produced a much greater elevation of [cAMP] in the processes than in the central bodies of the neurons. The resulting gradients must drive a sizable centripetal flux of cAMP because direct microinjection of cAMP showed that it diffused readily. Perinuclear increases in [cAMP] slowly caused the translocation of the freed catalytic subunit into the nucleus to an extent proportional to the percentage of its dissociation from the regulatory subunit.

Modulation of a transient K+ current in the pleural sensory neurons of Aplysia by serotonin and cAMP: implications for spike broadening.

To study the contribution of cAMP to the spike broadening produced by serotonin (5-HT) in the pleural sensory neurons of the tail withdrawal reflex, we utilized two phosphodiesterase-resistant cAMP analogs: the Sp diastereomer of cyclic adenosine 3',5'-monophosphothioate (Sp-cAMP[S]), which activates protein kinase A, and the antagonist Rp diastereomer of cyclic adenosine 3',5'-monophosphothioate (Rp-cAMP[S]), agonist Sp-cAMP[S] was injected into the sensory neurons, it caused spike broadening comparable to that induced by 5-HT. In turn, the cAMP antagonist Rp-cAMP[S] blocked approximately 50% of the 5-HT-induced spike broadening. We next examined the K+ currents that are modulated by 5-HT and determined how these currents are affected by cAMP. Confirming Baxter and Byrne [(1989) J. Neurophysiol. 62, 665-679], we found that 5-HT modulated two currents, an S-type K+ current (IKS) as well as a transient and voltage-dependent K+ current (IKV). Rp-cAMP[S] blocked the reduction by 5-HT of the early phase of IKV in parallel with, and to the same degree (60%), as this inhibitor blocked the IKS and spike broadening. These results support the idea that in the pleural sensory neurons cAMP mediates a significant part of the spike broadening that accompanies short-term facilitation produced by 5-HT and that cAMP can produce spike broadening by modulating both IKV and IKS.

Roles of PKA and PKC in facilitation of evoked and spontaneous transmitter release at depressed and nondepressed synapses in Aplysia sensory neurons.

Two second messenger pathways, one that uses the cAMP-dependent protein kinase A (PKA), the other that uses protein kinase C (PKC), have been found to contribute to the short-term presynaptic facilitation of the connections between the sensory neurons in Aplysia and their target cells, the interneurons and motor neurons of the gill-withdrawal reflex. To study their relative contributions as a function of the previous history of the neuron's activity, we have examined the effects of inhibiting PKA (using Rp-cAMPS) and PKC (using H7) on the short-term facilitation of spontaneous release as well as of the evoked release induced by serotonin at nondepressed, partially depressed, and highly depressed synapses. Our results suggest that whereas activation of PKA is sufficient to trigger the facilitation of nondepressed synapses, activation of both PKA and PKC is required to facilitate depressed synapses, with the contribution of PKC becoming progressively more important as synaptic transmission becomes more depressed.

Effects of intra-axonal injection of Ca2+ buffers on evoked release and on facilitation in the crayfish neuromuscular junction.

Ca2+ buffers were injected into the excitatory axon of the crayfish opener muscle. The magnitude and time course of evoked release and of facilitation were measured. EGTA (on-rate about 10(6) M-1S-1) had no effect on evoked release but reduced facilitation. BAPTA and nitr-5, buffers with similar Kd's but faster on-rates, reduced both evoked release and facilitation. However, these buffers had no effect on the time course of evoked release. These results show that fast Ca2+ buffers reduce the Ca2+ transient associated with evoked release and also the level of residual Ca2+ involved in facilitation. However, Ca2+ buffering is not the mechanism which controls the time course of release.

Injection of the cAMP-responsive element into the nucleus of Aplysia sensory neurons blocks long-term facilitation.

In both vertebrates and invertebrates, long-term memory differs from short-term in requiring protein synthesis during training. Studies of the gill and siphon withdrawal reflex in Aplysia indicate that similar requirements can be demonstrated at the level of sensory and motor neurons which may participate in memory storage. A single application of serotonin, a transmitter that mediates sensitization, to individual sensory and motor cells in dissociated cell cultures leads to enhanced transmitter release from the sensory neurons that is independent of new macromolecular synthesis. Five applications of serotonin cause a long-term enhancement, lasting one or more days, which requires translation and transcription. Prolonged application or intracellular injection into the sensory neuron of cyclic AMP, a second messenger for the action of serotonin, also produce long-term increases in synaptic strength, suggesting that some of the gene products important for long-term facilitation are cAMP-inducible. In eukaryotic cells, most cAMP-inducible genes so far studied are activated by the cAMP-dependent protein kinase (A kinase), which phosphorylates transcription factors that bind the cAMP-responsive element TGACGTCA. The cAMP-responsive element (CRE) binds a protein dimer of relative molecular mass 43,000, the CRE-binding protein (CREBP), which has been purified and shown to increase transcription when phosphorylated by the A kinase. Here we show that extracts of the Aplysia central nervous system and extracts of sensory neurons contain a set of proteins, including one with properties similar to mammalian CREBPs, that specifically bind the mammalian CRE sequence. Microinjection of the CRE sequence into the nucleus of a sensory neuron selectively blocks the serotonin-induced long-term increase in synaptic strength, without affecting short-term facilitation. Taken together, these observations suggest that one or more CREB-like transcriptional activators are required for long-term facilitation.

Second messengers involved in the two processes of presynaptic facilitation that contribute to sensitization and dishabituation in Aplysia sensory neurons.

Presynaptic facilitation of transmitter release contributes to behavioral sensitization and dishabituation, two simple forms of learning in Aplysia. This enhancement of transmitter release can be simulated by the facilitatory transmitter serotonin and has been shown to result from two types of mechanisms. The first facilitating process involves broadening of the presynaptic action potential in the sensory neurons of the reflex and is maximally effective when the synapse has not been depressed by repeated stimulation, as during sensitization. The second process is independent of changes in spike duration and can enhance release even when the synapse is quite depressed, as during dishabituation. Earlier work suggests that the first process is mediated by an increase in the intracellular level of cyclic AMP in the sensory neurons. We show here that release of free cyclic AMP from a photolyzable analogue introduced into sensory neurons can enhance release even at depressed synapses, indicating that cyclic AMP can activate the second as well as the first process. In addition, we find that phorbol esters, activators of protein kinase C, enhance release at depressed synapses. This is consistent with the report in the accompanying paper [Sacktor, T. C. and Schwartz, J. H. (1990) Proc. Natl. Acad. Sci. USA 87, 2036-2039] that serotonin and sensitizing stimuli translocate protein kinase C from cytoplasm to membrane. Our findings suggest that the cyclic AMP-dependent phosphorylation system can mediate more than one facilitatory process and that both cyclic AMP-dependent kinase and protein kinase C may be involved in facilitation of depressed synapses.

Phorbol ester enhances synaptic transmission at crustacean neuromuscular junctions.

Effects of phorbol ester (PE) (4 beta-phorbol-12,13-dibutyrate) on transmitter release were studied in the deep extensor neuromuscular system of the prawn, Macrobrachium rosenbergii. Our findings show that PE enhances transmitter release as indicated by an increase in the quantal content. PE had no post-synaptic effects. The increase in release is accompanied by a slight decline in twin pulse facilitation, suggesting a minor increase in Ca2+ entry. The fact that the increase in Ca2+ entry has a minor contribution to the PE effect is supported by the following observations: the duration of facilitation was not affected by PE, and 3,4-diaminopyridine (3,4-DAP), which by itself increased release, did not reduce the effect of PE. The time course of release was measured from synaptic delay histograms, upon which PE had no effect. This finding indicates that protein kinase C (PKC) is probably not involved in the rate limiting step of the process of secretion. The log/log plot of the initial part of the delay histogram is not affected by PE, suggesting a lack of effect on cooperativity of the release process. Increased release by loading the presynaptic terminal with Ca2+ either by pretreatment with Ca2+ ionophore or by frequent stimulation prevented further increase in release by PE. We conclude that the main effect of PE is confined to stages of release that are secondary to the first elevation in presynaptic Ca2+. PKC in this system probably plays a role in long term modulation of release, and it can be activated in processes leading to presynaptic Ca2+ accumulation.

Membrane depolarization evokes neurotransmitter release in the absence of calcium entry.

The discovery that Ca2+ is necessary for the release of neurotransmitter, the primary means by which nerve cells communicate, led to the calcium hypothesis of neutransmitter release, in which release is initiated after an action potential only by an increase in intracellular Ca2+ concentration near the release sites and is terminated (1-2 ms) by the rapid removal of Ca2+. Since then, the calcium-voltage hypothesis has been proposed, in which the depolarization of the presynaptic terminals has two functions. First, in common with the calcium hypothesis, the Ca2+ conductance is increased, thereby permitting Ca2+ entry. Second, a conformational change is induced in a membrane molecule that renders it sensitive to Ca2+, and then binding of Ca2+ to this active form triggers release of neurotransmitter. When the membrane is repolarized, the molecule is inactivated and release is terminated, regardless of the local Ca2+ concentration at that moment. This hypothesis, in contrast to the calcium hypothesis, accounts for the insensitivity of the time course of release to experimental manipulations of intracellular Ca2+ concentration. Furthermore, it explains rapid termination of release after depolarization, even though Ca2+ concentration may still be high. Here we describe experiments that distinguish between these two hypotheses and find that our results support the calcium voltage hypothesis.