Combining voltage-sensitive dye, carbon fiber array, and extracellular nerve electrodes using a 3-D printed recording chamber and manipulators.

BACKGROUND: The analyses of neuronal circuits require high-throughput technologies for stimulating and recording many neurons simultaneously with single-neuron precision. Voltage-sensitive dyes (VSDs) have enabled the monitoring of membrane potentials of many (10-100 s) neurons simultaneously. Carbon fiber electrode (CFE) arrays allow for stimulation and recording of many neurons simultaneously, including intracellularly. NEW METHOD: Combining CFE with VSD leverages the advantages of both technologies, allowing for stimulation of single neurons while recording the activity of the entire network. 3-D printing technology was used to develop a chamber to simultaneously perform VSD imaging, CFE array recording, and extracellular recording from individual glass electrodes. RESULTS: Aplysia buccal ganglia were stained with VSD and imaged while also recording using a CFE array and extracellular nerve electrodes. Coincident spiking activity was recorded by VSD, CFE, and extracellular nerve electrodes. Current injection with CFE electrodes could activate and inhibit individual neurons as detected by VSD and nerve recordings. COMPARISON TO EXISTING METHODS: The large size of traditional manipulators limits the number of electrodes used and the number of neurons recorded during an experiment. Here we present a method to build a 3-D printed recording chamber that includes a 3-axis micromanipulator to position a CFE array and eight 2-axis manipulators to position eight extracellular electrodes. CONCLUSIONS: 3-D printing technology can be used to build a custom recording chamber and micromanipulators. Combining these technologies allows for the direct modulation of the activity of neurons while recording the activity of 100 s of neurons simultaneously.

Specific Plasticity Loci and Their Synergism Mediate Operant Conditioning.

Despite numerous studies examining the mechanisms of operant conditioning (OC), the diversity of OC plasticity loci and their synergism have not been examined sufficiently. In the well-characterized feeding neural circuit of Aplysia, in vivo and in vitro appetitive OC increases neuronal excitability and electrical coupling among several neurons leading to an increase in expression of ingestive behavior. Here, we used the in vitro analog of OC to investigate whether OC reduces the excitability of a neuron, B4, whose inhibitory connections decrease expression of ingestive behavior. We found OC decreased the excitability of B4. This change appeared intrinsic to B4 because it could be replicated with an analog of OC in isolated cultures of B4 neurons. In addition to changes in B4 excitability, OC decreased the strength of B4's inhibitory connection to a key decision-making neuron, B51. The OC-induced changes were specific without affecting the excitability of another neuron critical for feeding behavior, B8, or the B4-to-B8 inhibitory connection. A conductance-based circuit model indicated that reducing the B4-to-B51 synapse, or increasing B51 excitability, mediated the OC phenotype more effectively than did decreasing B4 excitability. We combined these modifications to examine whether they could act synergistically. Combinations including B51 synergistically enhanced feeding. Taken together, these results suggest modifications of diverse loci work synergistically to mediate OC and that some neurons are well suited to work synergistically with plasticity in other loci.SIGNIFICANCE STATEMENT The ways in which synergism of diverse plasticity loci mediate the change in motor patterns in operant conditioning (OC) are poorly understood. Here, we found that OC was in part mediated by decreasing the intrinsic excitability of a critical neuron of Aplysia feeding behavior, and specifically reducing the strength of one of its inhibitory connections that targets a key decision-making neuron. A conductance-based computational model indicated that the known plasticity loci showed a surprising level of synergism to mediate the behavioral changes associated with OC. These results highlight the importance of understanding the diversity, specificity and synergy among different types of plasticity that encode memory. Also, because OC in Aplysia is mediated by dopamine (DA), the present study provides insights into specific and synergistic mechanisms of DA-mediated reinforcement of behaviors.

Active propagation of dendritic electrical signals in C. elegans.

Active propagation of electrical signals in C. elegans neurons requires ion channels capable of regenerating membrane potentials. Here we report regenerative depolarization of a major gustatory sensory neuron, ASEL. Whole-cell patch-clamp recordings in vivo showed supralinear depolarization of ASEL upon current injection. Furthermore, stimulation of animal's nose with NaCl evoked all-or-none membrane depolarization in ASEL. Mutant analysis showed that EGL-19, the α1 subunit of L-type voltage-gated Ca2+ channels, is essential for regenerative depolarization of ASEL. ASEL-specific knock-down of EGL-19 by RNAi demonstrated that EGL-19 functions in C. elegans chemotaxis along an NaCl gradient. These results demonstrate that a natural substance induces regenerative all-or-none electrical signals in dendrites, and that these signals are essential for activation of sensory neurons for chemotaxis. As in other vertebrate and invertebrate nervous systems, active information processing in dendrites occurs in C. elegans, and is necessary for adaptive behavior.

Tyraminergic modulation of agonistic outcomes in crayfish.

Octopamine, a biogenic amine, modulates various behaviors, ranging from locomotion and aggression to learning and memory in invertebrates. Several studies recently demonstrated that tyramine, the biological precursor of octopamine, also affects behaviors independent of octopamine. Here we investigated the involvement of tyramine in agonistic interaction of the male crayfish Procambarus clarkii. When male crayfish fight, larger animals (3-7% difference in body length) are more likely to win. By contrast, direct injection of tyramine or octopamine counteracted the physical advantage of larger animals. Tyramine or octopamine-injected naive large animals were mostly beaten by untreated smaller naive animals. This pharmacological effect was similar to the loser effect in which subordinate larger animals are frequently beaten by smaller animals. Furthermore, loser effects were partly eliminated by either injection of epinastine, an octopamine blocker, or yohimbine, a tyramine blocker, and significantly diminished by injection of a mixture of both blockers. We also observed that tyramine levels in the subesophageal ganglion were remarkably increased in subordinate crayfish after losing a fight. These results suggest that tyramine modulates aggressive levels of crayfish and contributes to the loser effect in parallel with octopamine.

Cyclic AMP-regulated opposing and parallel effects of serotonin and dopamine on phototaxis in the Marmorkrebs (marbled crayfish).

Phototactic behaviours are observed from prokaryotes to amphibians and are a basic form of orientation. We showed that the marbled crayfish displays phototaxis in which the behavioural response reversed from negative to positive depending on external light conditions. Animals reared in a 12-L/12-D light cycle showed negative phototaxis during daytime and positive phototaxis during night-time. Animals reared under constant light conditioning showed negative phototaxis during day- and night-time, while animals reared under constant dark conditioning showed positive phototaxis during day- and night-time. Injection of serotonin leads to a reversal of negative to positive phototaxis in both light/dark-reared and light/light-reared animals while injection of dopamine induced reversed negative phototaxis in dark/dark-reared animals. Four hours of dark adaptation were enough for light/dark-reared animals to reverse phototaxis from negative to positive. Injection of a serotonin 5HT1 receptor antagonist blocked the reverse phototaxis while serotonin 5HT2 receptor antagonists had no effects. Similarly, dark/dark-reared animals reversed to showing negative phototaxis after 4 h of light adaptation. Injection of a dopamine DA1 receptor antagonist blocked this reverse phototaxis, while dopamine DA2 receptor antagonists had no effects. Injection of a cAMP analogue into light/dark-reared animals blocked reverse phototaxis after dark adaptation, while adenylate cyclase inhibitor in dark/dark-reared animals blocked reverse phototaxis after light adaptation. These results strongly suggest that serotonin mediates positive phototaxis owing to decreased cAMP levels, while dopamine-mediated negative phototaxis occurs due to increased cAMP levels. Supporting this, the ratio of serotonin to dopamine in the brain was much higher in dark/dark-reared than light/dark-reared animals.

Role of cAMP signalling in winner and loser effects in crayfish agonistic encounters.

For territorial animals, establishment of status-dependent dominance order is essential to maintain social stability. In agonistic encounters of the crayfish Procambarus clarkii, a difference of body length of 3-7% is enough for larger animals to become dominant. Despite a physical disadvantage, small winners of the first pairings were more likely to win subsequent conflicts with larger inexperienced animals. In contrast, the losers of the first pairings rarely won subsequent conflicts with smaller naive animals. Such experiences of previous winning or losing affected agonistic outcomes for a long period. The winner effects lasted more than 2 weeks and the loser effect lasted about 10 days. Injection of 5HT1 receptor antagonist into the dominant animals 15-30 min after establishment of dominance order blocked the formation of the winner effects. In contrast, injection of adrenergic-like octopamine receptor antagonist into subordinate animals blocked the formation of the loser. 5HT1 receptors are negatively coupled to adenylyl cyclase and adrenergic-like octopamine receptors are positively coupled. Consistent with this, dominant animals failed to show the winner effect when injected with pCPT-cAMP, a cAMP analogue, and subordinate animals failed to show a loser effect when injected with adenylyl cyclase inhibitor SQ 22536. These results suggest that an increase and decrease of cAMP concentration is essential in mediating loser and winner effects, respectively. Furthermore, formation of the loser effect was blocked by injection of protein kinase A (PKA) inhibitor H89, suggesting long-term memory of the loser effect is dependent on the cAMP-PKA signalling pathway.

Serotonergic modulation of social status-dependent behavioural plasticity of the crayfish avoidance reaction.

Small crayfish usually showed escape-like dart responses to mechanical stimulation of the tailfan. Following agonistic bouts with conspecifics, dominant crayfish showed a defensive-like turn response to the same sensory stimulus. During the dart response, both uropods closed and animals walked forwards with the abdomen extended, while during the turn response the uropod on the stimulated side opened and animals turned towards the stimulus source with the abdomen frequently flexed. Using an isolated nerve cord preparation, we found that the spike activities of both the abdominal postural and uropod motor neurones in response to sensory stimulation of the exopodite reversed in dominant animals. In naive and subordinate animals, extensor motor neurones were excited and antagonistic flexor motor neurones inhibited in response to sensory stimulation. Furthermore, the spike frequency of uropod closer motor neurones increased while that of the antagonistic opener motor neurones decreased. By contrast, in more than half of the dominant animals, flexor and opener motor neurones showed excitatory responses while extensor and closer motor neurones showed inhibitory responses to the same sensory stimulation. This reverse of activity of the abdominal postural and uropod motor neurones was also observed when serotonin of more than 12.5 µM in concentration was applied.

Aminergic control of social status in crayfish agonistic encounters.

Using pairings of male crayfish Procambarus clarkii with a 3-7% difference in size, we confirmed that physically larger crayfish were more likely to win encounters (winning probability of over 80%). Despite a physical disadvantage, small winners of the first pairings were more likely to win their subsequent conflicts with larger naive animals (winning probability was about 70%). By contrast, the losers of the first pairings rarely won their subsequent conflicts with smaller naive animals (winning probability of 6%). These winner and loser effects were mimicked by injection of serotonin and octopamine. Serotonin-injected naive small crayfish were more likely to win in pairings with untreated larger naive crayfish (winning probability of over 60%), while octopamine-injected naive large animals were beaten by untreated smaller naive animals (winning probability of 20%). Furthermore, the winner effects of dominant crayfish were cancelled by the injection of mianserin, an antagonist of serotonin receptors and were reinforced by the injection of fluoxetin, serotonin reuptake inhibitor, just after the establishment of social order of the first pairings. Injection of octopamine channel blockers, phentolamine and epinastine, by contrast, cancelled the loser effects. These results strongly suggested that serotonin and octopamine were responsible for winner and loser effects, respectively.