Investigating Serotonin Receptor Expression in Single Homologous Neurons Underlying Independently Evolved and Species-Specific Behaviors

Serotonin (5-HT) receptors modulate neuronal and synaptic properties, altering the functional output of neural circuits. Changing the functions of a neural circuit can alter behavior. Over evolutionary time, species differences in neuromodulation could allow for species-specific behaviors to evolve. To investigate this idea, this dissertation compared neuromodulatory receptor gene expression underlying species-specific swimming behaviors in sea slugs. The sea slug Tritonia diomedea (Mollusca, Gastropoda, Nudipleura, Nudibranchia), performs a rhythmic dorsal-ventral (DV) escape swim behavior. The behavior is controlled by a central pattern generator (CPG), composed of a small number of large, identifiable neurons. During swimming, 5-HT enhances the synaptic strength of a neuron in the swim CPG, called C2. In contrast, the nudibranch Hermissenda crassicornis does not swim in this manner. It has C2 homologues, and 5-HT is present, however, 5-HT does not modulate C2 synaptic strength. Pleurobranchaea californica, a Nudipleura species belonging to a sister clade of Nudibranchia, swims with DV flexions, although in this species swimming varies within individuals. 5-HT enhances Pleurobranchaea C2 homologue synaptic strength in swimming animals, only. Phylogenetic analysis showed that Tritonia and Pleurobranchaea independently evolved DV-swimming. Thus, there is a correlation between independently evolved swimming and serotonergic modulation of C2 homologues. It was hypothesized that 5-HT receptor differences in C2 neurons underlie species-specific swimming and modulation. To test this hypothesis, 5-HT receptor genes were identified in each species. A total of seven receptor subtypes, from five gene families, were found to be expressed in the brains of each species. Using single-cell quantitative PCR (qPCR), 5-HT receptor expression profiles were determined in C2 homologues. Genes known as 5-HT2a and 5-HT7 were expressed in C2 homologues from Tritonia and swimming Pleurobranchaea, only. Single-neuron transcriptome sequencing verified these results. The expression profiles of neuromodulatory receptor genes in single, homologous neurons correlated with species-specific swimming and modulation. The results illustrate how differences in neuromodulatory gene expression may alter the functional output of homologous neural structures, shedding light on a means by which neuromodulation can alter the brain to facilitate the evolution of species-specific behaviors. Evolution, Mollusc, Neuromodulation, Serotonin, Receptor, Behavior, Next-Generation Sequencing, Transcriptomic

Higher-order associative processing in Hermissenda suggests multiple sites of neuronal modulation.

Two important features of modern accounts of associative learning are (1) the capacity for contextual stimuli to serve as a signal for an unconditioned stimulus (US) and (2) the capacity for a previously conditioned (excitatory) stimulus to block learning about a redundant stimulus when both stimuli serve as a signal for the same US. Here, we examined the process of blocking, thought by some to reflect a cognitive aspect of classical conditioning, and its underlying mechanisms in the marine mollusc Hermissenda. In two behavioral experiments, a context defined by chemosensory stimuli was made excitatory by presenting unsignalled USs (rotation) in that context. The excitatory context subsequently blocked overt learning about a discrete conditioned stimulus (CS; light) paired with the US in that context. In a third experiment, the excitability of the B photoreceptors in the Hermissenda eye, which typically increases following light-rotation pairings, was examined in behaviorally blocked animals, as well as in animals that had acquired a normal CS-US association or animals that had been exposed to the CS and US unpaired. Both the behaviorally blocked and the normal learning groups exhibited increases in neuronal excitability relative to unpaired animals. However, light-induced multiunit activity in pedal nerves was suppressed following normal conditioning but not in blocked or unpaired control animals, suggesting that the expression of blocking is mediated by neuronal modifications not directly reflected in B-cell excitability, possibly within an extensive network of central light-responsive interneurons

Ubiquitous molecular substrates for associative learning and activity-dependent neuronal facilitation.

Recent evidence suggests that many of the molecular cascades and substrates that contribute to learning-related forms of neuronal plasticity may be conserved across ostensibly disparate model systems. Notably, the facilitation of neuronal excitability and synaptic transmission that contribute to associative learning in Aplysia and Hermissenda, as well as associative LTP in hippocampal CA1 cells, all require (or are enhanced by) the convergence of a transient elevation in intracellular Ca2+ with transmitter binding to metabotropic cell-surface receptors. This temporal convergence of Ca2+ and G-protein-stimulated second-messenger cascades synergistically stimulates several classes of serine/threonine protein kinases, which in turn modulate receptor function or cell excitability through the phosphorylation of ion channels. We present a summary of the biophysical and molecular constituents of neuronal and synaptic facilitation in each of these three model systems. Although specific components of the underlying molecular cascades differ across these three systems, fundamental aspects of these cascades are widely conserved, leading to the conclusion that the conceptual semblance of these superficially disparate systems is far greater than is generally acknowledged. We suggest that the elucidation of mechanistic similarities between different systems will ultimately fulfill the goal of the model systems approach, that is, the description of critical and ubiquitous features of neuronal and synaptic events that contribute to memory induction

Postsynaptic Ca2+, but not cumulative depolarization, is necessary for the induction of associative plasticity in Hermissenda

The neuronal modifications that underlie associative memory in Hermissenda have their origins in a synaptic interaction between the visual and vestibular systems, and can be mimicked by contiguous in vitro stimulation of these converging pathways. At the offset of vestibular stimulation (i.e., hair cell activity), the B photoreceptors are briefly released from synaptic inhibition resulting in a slight depolarization (2–4 mV). If contiguous pairings of light-induced depolarization and presynaptic vestibular activity occur in close temporal succession, this depolarization “accumulates” and has been hypothesized to culminate in a sustained rise in intracellular Ca2+ and a resultant Ca(2+)-mediated phosphorylation of K+ channels as well as an associated increase in input resistance. Here we demonstrate that this cumulative depolarization is neither necessary nor sufficient for the biophysical modifications of the B cell membrane indicative of memory formation. Consistent with several recent reports of one-trial learning in Hermissenda, one pairing of light with mechanical stimulation of the vestibular hair cells resulted in a rise in neuronal input resistance across the B cell membrane that was attenuated by a prepairing iontophoretic injection of the Ca2+ chelator EGTA (25 mM), indicating that this potentiation was Ca2+ dependent. However, the use of a single pairing negates the possibility of an accumulation of depolarization across trials. In a subsequent experiment, B photoreceptors underwent a cumulative depolarization, and a coincident rise in input resistance, during multiple pairings of light and hair cell stimulation. However, if the B photoreceptor was voltage clamped at its initial resting potential before and after each pairing, thus eliminating the cumulative depolarization, the rise in resistance not only persisted, but was enhanced. Moreover, if unpaired light presentations were followed by a current-induced depolarization (to mimic cumulative depolarization), no increase in input resistance was detected. To assess directly the effect of a cumulative depolarization on the voltage-dependent Ca2+ current, an analysis of the inward current on the B cell soma membrane was conducted. It was determined that (1) the inward current may undergo a partial inactivation during sustained depolarization, (2) the peak current was depressed during repetitive depolarizations, and (3) the peak current underwent a steady- state inactivation, such that it was reduced when elicited from holding potentials more positive than -60 mV. The analysis of this current suggests that pairings of light and presynaptic activity would reduce voltage-dependent Ca2+ influx when those pairings are conducted at depolarized membrane potentials, such as during cumulative depolarization

A Comparative Analysis of the Neural Basis for Dorsal-Ventral Swimming in the Nudipleura

Despite having similar brains, related species can display divergent behaviors. Investigating the neural basis of such behavioral divergence can elucidate the neural mechanisms that allow behavioral change and identify neural mechanisms that influence the evolution of behavior. Fewer than three percent of Nudipleura (Mollusca, Opisthobranchia, Gastropoda) species have been documented to swim. However, Tritonia diomedea and Pleurobranchaea californica express analogous, independently evolved swim behaviors consisting of rhythmic, alternating dorsal and ventral flexions. The Tritonia and Pleurobranchaea swims are produced by central pattern generator (CPG) circuits containing homologous neurons named DSI and C2. Homologues of DSI have been identified throughout the Nudipleura, including in species that do not express a dorsal-ventral swim. It is unclear what neural mechanisms allow Tritonia and Pleurobranchaea to produce a rhythmic swim behavior using homologous neurons that are not rhythmic in the majority of Nudipleura species. Here, C2 homologues were also identified in species that do not express a dorsal-ventral swim. We found that certain electrophysiological properties of the DSI and C2 homologues were similar regardless of swim behavior. However, some synaptic connections differed in the non-dorsal-ventral swimming Hermissenda crassicornis compared to Tritonia and Pleurobranchaea. This suggests that particular CPG synaptic connections may play a role in dorsal-ventral swim expression. DSI modulates the strength of C2 synapses in Tritonia, and this serotonergic modulation appears to be necessary for Tritonia to swim. DSI modulation of C2 synapses was also found to be present in Pleurobranchaea. Moreover, serotonergic modulation was necessary for swimming in Pleurobranchaea. The extent of this neuromodulation also correlated with the swimming ability in individual Pleurobranchaea; as the modulatory effect increased, so too did the number of swim cycles produced. Conversely, DSI did not modulate the amplitude of C2 synapses in Hermissenda. This indicates that species differences in neuromodulation may account for the ability to produce a dorsal-ventral swim. The results indicate that differences in synaptic connections and neuromodulatory dynamics allow the expression of rhythmic swim behavior from homologous non-rhythmic components. Additionally, the results suggest that constraints on the nervous system may influence the neural mechanisms and behaviors that can evolve from homologous neural components

Evolution of swimming behaviors in nudibranch molluscs: A comparative analysis of neural circuitry

Behaviors are a product of underlying neural circuits, yet there is a paucity of mechanistic information about how nervous systems contribute to the repeated evolution of similar behaviors. Theoretical studies have predicted that the same behavioral output can be generated by neural circuits with different properties. Here, we test the theory in biological circuits by comparing the central pattern generator (CPG) circuits underlying swimming behaviors in nudibranchs (Mollusca, Gastropoda, Euthyneura, Nudipleura). In comparative studies of neural circuits, neurotransmitter content can serve as landmarks or molecular markers for neuron types. Here, we created a comprehensive map of GABA-immunoreactive neurons in six Nudipleura species. None of the known swim CPG neurons were GABA-ir, but they were located next to identifiable GABA-ir neurons/clusters. Despite strong conservation of the GABA-ergic system, there were differences, particularly in the buccal ganglia, which may represent adaptive changes. We applied our knowledge of neurotransmitter distribution along with morphological traits to identify the neuron type Si1 in Flabellina, a species that swims via whole body left-right (LR) flexions and in Tritonia, a dorsal-ventral (DV) swimming species. Si1 is a CPG member of the LR species Melibe, whereas its homologue in the LR species Dendronotus is not. In Flabellina, Si1 was part of the LR CPG and despite having similar synaptic connections as Flabellina and Melibe, Si1 in Tritonia was not part of its DV swim CPG. Side by side circuit comparison of Flabellina, Melibe and Dendronotus revealed different combinations of circuit architecture and modulation resulting in different circuit configurations for LR swimming. This includes differences in the role and activity pattern of Si1, sensitivity to curare and the effect of homologues of C2, a DV CPG neuron, on the LR motor pattern. These results collectively reveal three different circuit variations for generating the same behavior. It suggests that the neural substrate from which behaviors arise is phylogenetically constrained. While this neural substrate can be configured in multiple different ways to generate the same outcome, the possibilities are finite and, as seen here, similar structural and functional neural motifs are used in the evolution of these circuits

Homologous Neurons and their Locomotor Functions in Nudibranch Molluscs

These studies compare neurotransmitter localization and the behavioral functions of homologous neurons in nudibranch molluscs to determine the types of changes that might underlie the evolution of species-specific behaviors. Serotonin (5-HT) immunohistochemistry in eleven nudibranch species indicated that certain groups of 5 HT-immunoreactive neurons, such as the Cerebral Serotonergic Posterior (CeSP) cluster, are present in all species. However, the locations and numbers of many other 5 HT-immunoreactive neurons were variable. Thus, particular parts of the serotonergic system have changed during the evolution of nudibranchs. To test whether the functions of homologous neurons are phylogenetically variable, comparisons were made in species with divergent behaviors. In Tritonia diomedea, which crawls and also swims via dorsal-ventral body flexions, the CeSP cluster includes the Dorsal Swim Interneurons (DSIs). It was previously shown that the DSIs are members of the swim central pattern generator (CPG); they are rhythmically active during swimming and, along with their neurotransmitter 5-HT, are necessary and sufficient for swimming. It was also known that the DSIs excite efferent neurons used in crawling. DSI homologues, the CeSP-A neurons, were identified in six species that do not exhibit dorsal-ventral swimming. Many physiological characteristics, including excitation of putative crawling neurons were conserved, but the CeSP A neurons were not rhythmically active in any of the six species. In the lateral flexion swimmer, Melibe leonina, the CeSP-A neurons and 5-HT, were sufficient, but not necessary, for swimming. Thus, homologous neurons, and their neurotransmitter, have functionally diverged in species with different behaviors. Homologous neurons in species with similar behaviors also exhibited functional divergence. Like Melibe, Dendronotus iris is a lateral flexion swimmer. Swim interneuron 1 (Si1) is in the Melibe swim CPG. However, its putative homologue in Dendronotus, the Cerebral Posterior ipsilateral Pedal (CPiP) neuron, was not rhythmically active during swim-like motor patterns, but could initiate such a motor pattern. Together, these studies suggest that neurons have changed their functional relationships to neural circuits during the evolution of species-specific behaviors and have functionally diverged even in species that exhibit similar behaviors

Lymnaea stagnalis as model for translational neuroscience research: from pond to bench

The purpose of this review is to illustrate how a reductionistic, but sophisticated, approach based on the use of a simple model system such as the pond snail Lymnaea stagnalis (L. stagnalis), might be useful to address fundamental questions in learning and memory. L. stagnalis, as a model, provides an interesting platform to investigate the dialog between the synapse and the nucleus and vice versa during memory and learning. More importantly, the "molecular actors" of the memory dialogue are well-conserved both across phylogenetic groups and learning paradigms, involving single- or multi-trials, aversion or reward, operant or classical conditioning. At the same time, this model could help to study how, where and when the memory dialog is impaired in stressful conditions and during aging and neurodegeneration in humans and thus offers new insights and targets in order to develop innovative therapies and technology for the treatment of a range of neurological and neurodegenerative disorders

Long-term sensitization in the leech: role of proteins of about 11 kDa

In questa tesi sono stati analizzati i meccanismi molecolari alla base della sensitizzazione a lungo termine (LT), nella sanguisuga Hirudo medicinalis. H. medicinalis rappresenta un buon modello sperimentale per studiare i vari e complessi aspetti della funzionalità del sistema nervoso (SN), apprendimento e memoria inclusi, poiché associa alla semplicità anatomica comportamenti complessi organizzati in circuiti neurali semplici. È stato utilizzato come atto comportamentale l’induzione al nuoto, ovvero l’induzione di un ciclo di nuoto in risposta ad un lieve stimolo elettrico (stimolo test) applicato sulla cute della porzione caudale dell’animale che aveva precedentemente subito la disconnessione microchirurgica del primo ganglio segmentale dal ganglio cefalico. Sottoponendo gli animali ad uno specifico protocollo di addestramento è stato possibile indurre sensitizzazione a breve (BT) e a lungo termine (LT). In una sessione di sensitizzazione, gli animali sono sottoposti a 4 stimoli test applicati ad intervalli random per valutare la risposta basale, quindi subiscono 15 spazzolate sul dorso (brushing) al termine delle quali viene nuovamente presentato lo stimolo test per tre volte ad intervalli di 5 minuti. Le risposte registrate dopo quest’ultimi risultarono potenziate rispetto alla risposta basale e tale potenziamento perdurava per almeno 40 minuti. Se gli animali, dopo essere stati sottoposti il primo giorno a una seduta di sensitizzazione, ricevevano nei 4 giorni successivi stimoli nocicettivi ripetuti, rappresentati da serie di 15 spazzolate ripetute per 4 volte a distanza di 10 minuti, il sesto giorno durante una seconda sessione di sensitizzazione mostravano un incremento della risposta post brushing molto maggiore rispetto a quella registrata il primo giorno e tale potenziamento durava almeno 24 ore, indicando che era avvenuta sensitizzazione LT. Animali di controllo, sottoposti a sensitizzazione il primo e il sesto giorno ma solo manipolati a temperatura ambiente per la durata dell’applicazioni degli stimoli nocicettivi ripetuti durante i 4 giorni intermedi, il 6° giorno mostravano risposte agli stimoli test presentati dopo brushing del tutto sovrapponibili a quelle registrate il 1° giorno. La somministrazione di cicloesimide, un inibitore della sintesi proteica, bloccava l’instaurarsi di sensitizzazione LT, suggerendo che la formazione ex-novo di proteine nel SN sia un meccanismo fondamentale per il consolidamento delle tracce mnemoniche nell’apprendimento LT. L’analisi elettroforetica mediante SDS-PAGE condotta su pellet e surnatante ottenuti sonicando e in seguito centrifugando catene gangliari e ganglio caudale isolati da animali addestrati LT e di controllo, ha rilevato un diverso profilo proteico negli omogenati di tessuto nervoso ottenuti dagli animali sensitizzati LT rispetto agli animali di controllo. Pellet e surnatante ottenuti sia da animali sensitizzati LT che da animali di controllo sono stati iniettati in animali naïve (sottoposti soltanto alla disconnessione microchirurgica del primo ganglio segmentale dal ganglio cefalico), precedentemente sottoposti ad una sessione di sensitizzazione. In una seconda sessione di sensitizzazione eseguita un’ora dopo l’iniezione, solo gli animali che avevano ricevuto il pellet derivato da animali sensitizzati LT hanno mostrato un potenziamento della risposta allo stimolo test dopo brushing, simile a quello ottenuto dopo ripetute stimolazioni nocicettive. Analogamente a quanto precedentemente osservato dopo sensitizzazione LT, tale potenziamento perdurava per 24 h e non si aveva se il pellet era stato ottenuto da omogenato di tessuto gangliare di animali sensitizzati LT e iniettati quotidianamente con cicloesimide 10 μM. Campioni di pellet ottenuto da omogenati derivati da catene gangliari di animali sensitizzati LT e di animali di controllo sono stati analizzati tramite cromatografia size-exclusion che permette la separazione delle molecole organiche in base al loro peso molecolare. L’analisi ha evidenziato spettri di assorbanza delle proteine a 280 nm diversi tra pellet derivati dall’omogenato di animali sensitizzati LT e di animali di controllo. In seguito alla costruzione di una curva di taratura tramite iniezione nella colonna cromatografica di proteine a peso molecolare noto, sono stati evidenziati, in 7 campioni distinti, picchi di assorbanza corrispondenti a vari pesi molecolari (> 50 kDa a 5.42 min; circa 17,5 kDa a 7.89 min; circa 11 kDa a 9.18 min.) presenti esclusivamente nel pellet di animali sensitizzati LT e non in quello dei controlli. Sono state raccolte varie frazioni in un range di 5-12 minuti corrispondenti ai diversi picchi di assorbanza osservati e sono state iniettate in animali naïve precedentemente sottoposti ad una sessione di sensitizzazione. In una seconda sessione di sensitizzazione condotta un’ora dopo l’iniezione, gli animali trattati con la frazione di pellet ottenuto da animali sensitizzati LT corrispondente al peso molecolare di circa 11 kDa e indicata come frazione 8, hanno mostrato un potenziamento della risposta post brushing analoga a quello osservato dopo training LT, mentre le altre frazioni iniettate non hanno indotto effetto. Questi risultati hanno dunque evidenziato il coinvolgimento di proteine di circa 11kDa nell’instaurarsi dell’apprendimento LT. Questo dato è stato convalidato anche dall’analisi cromatografica a fase inversa effettuata sulla frazione 8 di animali sensitizzati LT, in cui sia gli spettri di assorbanza a 280 nm sia gli spettri di fluorescenza hanno indicato la presenza di tre picchi presenti nella frazione 8 degli animali LT e non nei controlli. Infine, l’analisi FTRI ha mostrato spettri nell’infrarosso tipici di proteine. È attualmente in corso l’analisi proteomica per identificare la sequenza nucleotidica di queste proteine

Early calcium increase triggers the formation of olfactory long-term memory in honeybees

<p>Abstract</p> <p>Background</p> <p>Synaptic plasticity associated with an important wave of gene transcription and protein synthesis underlies long-term memory processes. Calcium (Ca²⁺) plays an important role in a variety of neuronal functions and indirect evidence suggests that it may be involved in synaptic plasticity and in the regulation of gene expression correlated to long-term memory formation. The aim of this study was to determine whether Ca²⁺is necessary and sufficient for inducing long-term memory formation. A suitable model to address this question is the Pavlovian appetitive conditioning of the proboscis extension reflex in the honeybee <it>Apis mellifera, </it>in which animals learn to associate an odor with a sucrose reward.</p> <p>Results</p> <p>By modulating the intracellular Ca²⁺concentration ([Ca²⁺]i) in the brain, we show that: (i) blocking [Ca²⁺]i increase during multiple-trial conditioning selectively impairs long-term memory performance; (ii) conversely, increasing [Ca²⁺]i during single-trial conditioning triggers long-term memory formation; and finally, (iii) as was the case for long-term memory produced by multiple-trial conditioning, enhancement of long-term memory performance induced by a [Ca²⁺]i increase depends on <it>de novo </it>protein synthesis.</p> <p>Conclusion</p> <p>Altogether our data suggest that during olfactory conditioning Ca²⁺is both a necessary and a sufficient signal for the formation of protein-dependent long-term memory. Ca²⁺therefore appears to act as a switch between short- and long-term storage of learned information.</p