Julien Bacqué-Cazenave

Anxiety-like behavior in crayfish is controlled by serotonin

The crayfish that was afraid of the dark We tend to assume that complex emotions, such as anxiety, only occur in mammals or other cognitively complex vertebrates. But a heightened sense of awareness and the avoidance of novel or dangerous environments could be helpful for any animal species. Fossat et al. show that crayfish exposed to a stressful electric field refuse to enter dark arms in a light/dark maze, even after the electric field has been removed. The animals calmed down when they were injected with an anxiolytic drug used to treat anxiety in humans, and they entered the dark as normal. The stressed animals had increased levels of the neurotransmitter serotonin in the brain, and injections of serotonin induced anxiety-like behavior in control animals. Thus, these invertebrates display a primitive form of anxiety that shares a mechanism with the more complex emotions displayed by vertebrates. Science, this issue p. 1293 Crayfish respond to stress with an apparent fear of the dark that can be abolished with an anxiolytic drug. Anxiety, a behavioral consequence of stress, has been characterized in humans and some vertebrates, but not invertebrates. Here, we demonstrate that after exposure to stress, crayfish sustainably avoided the aversive illuminated arms of an aquatic plus-maze. This behavior was correlated with an increase in brain serotonin and was abolished by the injection of the benzodiazepine anxiolytic chlordiazepoxide. Serotonin injection into unstressed crayfish induced avoidance; again, this effect was reversed by injection with chlordiazepoxide. Our results demonstrate that crayfish exhibit a form of anxiety similar to that described in vertebrates, suggesting the conservation of several underlying mechanisms during evolution. Analyses of this ancestral behavior in a simple model reveal a new route to understanding anxiety and may alter our conceptions of the emotional status of invertebrates.

Serotonin, but not dopamine, controls the stress response and anxiety-like behavior in the crayfish Procambarus clarkii

ABSTRACT In the animal kingdom, biogenic amines are widespread modulators of the nervous system that frequently interact to control mood. Our previous investigations in crayfish (Procambarus clarkii) have established that stress induces changes in brain serotonin (5-HT) concentrations that are responsible for the appearance of anxiety-like behavior (ALB). Here, we further analyze the roles of 5-HT and another biogenic amine, dopamine (DA), on the crayfish response to stress. We show that the intensity of crayfish ALB depends on the intensity of stressful stimulation and is associated with increased concentrations of 5-HT in the brain. These 5-HT levels were significantly correlated, before, as well as after stress, with those of DA, which were approximately 3- to 5-times less abundant. However, whereas the degree of ALB was clearly correlated with brain 5-HT concentrations, it was not significantly correlated with DA. Moreover, in contrast to injections of 5-HT, DA injections were not able to elicit a stress response or ALB. In addition, 5-HT and DA levels were not modified by treatment with the anxiolytic chlordiazepoxide, confirming that suppression of ALB by this GABA-A receptor ligand acts downstream and is independent of changes in crayfish bioamine levels. Our study also provides evidence that the anxiogenic effect of 5-HT injections can be prevented by a preliminary injection of 5-HT antagonists. Altogether, our results emphasize that the rises in brain concentrations of 5-HT, but not DA, play a role in controlling the induction and the intensity of crayfish ALB. Summary: After stress, crayfish brain 5-HT levels increase, which induces anxiety-like behavior (ALB) but the level of dopamine does not change, and in contrast to 5-HT, injection of dopamine does not trigger a metabolic response or ALB.

Social harassment induces anxiety-like behaviour in crayfish

Social interactions leading to dominance hierarchies often elicit psychological disorders in mammals including harassment and anxiety. Here, we demonstrate that this sequence also occurs in an invertebrate, the crayfish Procambarus clarkii. When placed in the restricted space of an aquarium, crayfish dyads generally fight until one of the opponents suddenly escapes, thereafter clearly expressing a submissive behaviour. Nevertheless, the winner frequently keeps on displaying excessive aggressive acts, having deleterious consequences in losers and interpreted as harassment behaviour. We indeed observed that, contrary to winners, losers expressed anxiety-like behaviour (ALB) in correlation with the stress intensity they suffered during the harassment period mainly. Injections of an anxiolytic abolished ALB, confirming its homology with anxiety. A serotonin (5-HT) antagonist had the same effect, suggesting a role for 5-HT, whose brain concentrations increased much more in losers than in winners. Our findings suggest that the bases of harassment and of its anxiogenic consequences have emerged very early during evolution, and emphasize crayfish as an unexpected but potentially fruitful model for the study of these social disorders.

The effect of sensory feedback on crayfish posture and locomotion: I. Experimental analysis of closing the loop.

The effect of proprioceptive feedback on the control of posture and locomotion was studied in the crayfish Procambarus clarkii (Girard). Sensory and motor nerves of an isolated crayfish thoracic nerve cord were connected to a computational neuromechanical model of the crayfish thorax and leg. Recorded levator (Lev) and depressor (Dep) nerve activity drove the model Lev and Dep muscles to move the leg up and down. These movements released and stretched a model stretch receptor, the coxobasal chordotonal organ (CBCO). Model CBCO length changes drove identical changes in the real CBCO; CBCO afferent responses completed the feedback loop. In a quiescent preparation, imposed model leg lifts evoked resistance reflexes in the Dep motor neurons that drove the leg back down. A muscarinic agonist, oxotremorine, induced an active state in which spontaneous Lev/Dep burst pairs occurred and an imposed leg lift excited a Lev assistance reflex followed by a Lev/Dep burst pair. When the feedback loop was intact, Lev/Dep burst pairs moved the leg up and down rhythmically at nearly three times the frequency of burst pairs when the feedback loop was open. The increased rate of rhythmic bursting appeared to result from the positive feedback produced by the assistance reflex.

The effect of sensory feedback on crayfish posture and locomotion: II. Neuromechanical simulation of closing the loop

Neuromechanical simulation was used to determine whether proposed thoracic circuit mechanisms for the control of leg elevation and depression in crayfish could account for the responses of an experimental hybrid neuromechanical preparation when the proprioceptive feedback loop was open and closed. The hybrid neuromechanical preparation consisted of a computational model of the fifth crayfish leg driven in real time by the experimentally recorded activity of the levator and depressor (Lev/Dep) nerves of an in vitro preparation of the crayfish thoracic nerve cord. Up and down movements of the model leg evoked by motor nerve activity released and stretched the model coxobasal chordotonal organ (CBCO); variations in the CBCO length were used to drive identical variations in the length of the live CBCO in the in vitro preparation. CBCO afferent responses provided proprioceptive feedback to affect the thoracic motor output. Experiments performed with this hybrid neuromechanical preparation were simulated with a neuromechanical model in which a computational circuit model represented the relevant thoracic circuitry. Model simulations were able to reproduce the hybrid neuromechanical experimental results to show that proposed circuit mechanisms with sensory feedback could account for resistance reflexes displayed in the quiescent state and for reflex reversal and spontaneous Lev/Dep bursting seen in the active state.

Spatial segregation of excitatory and inhibitory effects of 5-HT on crayfish motoneurons

Altering neuronal membrane properties, including input resistance, is a key modulatory mechanism for changing neural activity patterns. The effect of membrane currents generated by either synaptic or voltage-dependent channels directly depends on neuron input resistance. We found that local application of serotonin to different regions of identified motoneurons (MNs) of the postural/walking network of isolated crayfish produced different changes in input resistance. Puff-applied 5-HT in the periphery of the initial segment produced exclusively inhibitory responses. In contrast, when 5-HT was puff-applied on the central arbor of the same depressor (Dep) MN, exclusively depolarizing responses were obtained. Both inhibitory and excitatory responses were direct because they persisted in low-calcium saline. We found numerous close appositions between 5-HT-immunoreactive processes and the initial segment of dextran-rhodamine-filled Dep MNs. In contrast, almost no close apposition sites were found in Dep MN arbor. It seems that the 5-HT controls the level of excitability of postural network MNs by two mechanisms acting at two different sites: inhibitory responses (consistent with an action involving opening of K(+) channels) occur in the initial segment region and may involve classic synaptic transmission, whereas depolarizing responses (consistent with an action involving closing of K(+) channels) occur on MN branches via apparent paracrine effects.

Alteration of size perception: serotonin has opposite effects on the aggressiveness of crayfish confronting either a smaller or a larger rival

ABSTRACT We injected serotonin (5-HT) into adult male crayfish before pairing them with size-matched non-injected competitors, and observed dyadic agonistic interactions. Paradoxically, 5-HT elicited opposite behavioral responses if the injected animal was opposed by a smaller or larger rival: the level of aggressiveness of the injected crayfish was higher when facing a larger rival but lower when facing a smaller rival. Our results indicate that the effects of 5-HT on aggressiveness are dependent on the perception of the relative size difference of the opponent. In both cases, however, 5-HT significantly delayed the decision to retreat. We conclude that 5-HT does not primarily act on aggressiveness but rather on the brain centers that integrate risk assessment and/or decision making, which then modulate the aggressive response. Our findings support a reinterpretation of the role of 5-HT in crustacean agonistic behavior that may be of interest for studies of other animals. Highlighted Article: Serotonin in crayfish is able to alter the perception of a rival, leading to paradoxical consequences for aggressiveness.

Control of motor activity in crayfish by the steroid hormone 20-hydroxyecdysone via motoneuron excitability and sensory-motor integration

SUMMARY We studied the effects of the molting hormone 20-hydroxyecdysone (20E) on leg sensory-motor networks of the red swamp crayfish, Procambarus clarkii. The hormone was injected in isolated crayfish and network activity was analyzed 3 days after injection using electrophysiology on an in vitro preparation of the leg locomotor network. This 20E treatment deeply reduced motor activity, by affecting both intrinsic motoneuron (MN) properties and sensory-motor integration. Indeed, we noticed a general decrease in motor nerve tonic activities, principally in depressor and promotor nerves. Moreover, intracellular recordings of depressor MNs confirmed a decrease of MN excitability due to a drop in input resistance. In parallel, sensory inputs originating from a proprioceptor, which codes joint movements controlled by these MNs, were also reduced. The shape of excitatory post-synaptic potentials (PSPs) triggered in MNs by sensory activity of this proprioceptor showed a reduction of polysynaptic components, whereas inhibitory PSPs were suppressed, demonstrating that 20E acted also on interneurons relaying sensory to motor inputs. Consequently, 20E injection modified the whole sensory-motor loop, as demonstrated by the alteration of the resistance reflex amplitude. These locomotor network changes induced by 20E were consistent with the decrease of locomotion observed in a behavioral test. In summary, 20E controls locomotion during crayfish premolt by acting on both MN excitability and sensory-motor integration. Among these cooperative effects, the drop of input resistance of MNs seems to be mostly responsible for the reduction of motor activity.

Temporal relationship of ocular and tail segmental movements underlying locomotor-induced gaze stabilization during undulatory swimming in larval xenopus

In larval xenopus, locomotor-induced oculomotor behavior produces gaze-stabilizing eye movements to counteract the disruptive effects of tail undulation during swimming. While neuronal circuitries responsible for feed-forward intrinsic spino-extraocular signaling have recently been described, the resulting oculomotor behavior remains poorly understood. Conveying locomotor CPG efference copy, the spino-extraocular motor command coordinates the multi-segmental rostrocaudal spinal rhythmic activity with the extraocular motor activity. By recording sequences of xenopus tadpole free swimming, we quantified the temporal calibration of conjugate eye movements originating from spino-extraocular motor coupled activity during pre-metamorphic tail-based undulatory swimming. Our results show that eye movements are produced only during robust propulsive forward swimming activity and increase with the amplitude of tail movements. The use of larval isolated in vitro and semi-intact fixed head preparations revealed that spinal locomotor networks driving the rostral portion of the tail set the precise timing of the spino-extraocular motor coupling by adjusting the phase relationship between spinal segment and extraocular rhythmic activity with the swimming frequency. The resulting spinal-evoked oculomotor behavior produced conjugated eye movements that were in phase opposition with the mid-caudal part of the tail. This time adjustment is independent of locomotor activity in the more caudal spinal parts of the tail. Altogether our findings demonstrate that locomotor feed-forward spino-extraocular signaling produce conjugate eye movements that compensate specifically the undulation of the mid-caudal tail during active swimming. Finally, this study constitutes the first extensive behavioral quantification of spino-extraocular motor coupling, which sets the basis for understanding the mechanisms of locomotor-induced oculomotor behavior in larval frog.