Helisoma neurones in the construction of circuits in vitro

The work described here used the invertebrate neurone culture methodology to study neurones from the pond snail Helisoma trivolvis. Isolated neurones were used to form small circuits, the input/output properties of which were investigated electrophysiologically. The activity of the circuits were then described with respect to the intrinsic properties of the individual neurones and the synaptic connections between them. Neurite extension from isolated neurones could be altered by making changes to the culture medium in which the neurones were maintained. Electrophysiological recordings made from pairs of neurones in culture revealed electrical, chemical and mixed connections. The purely chemical connections and the chemical component of the mixed connections were inhibitory in nature. Connections were not detected between all of the pairs. In many cases no connections at all were recorded between neurone pairs. Connections could also be obtained between neurone pairs if the two neurones were placed in culture on consecutive days. Connections were still obtained when three neurones were placed together to form circuits, although such connections were found to be weak. The three neurone circuits formed were unable to produce any rhythmic output, due both to the weak synaptic connections present and intrinsic membrane properties of the neurones. This study shows that invertebrate neurone culture is a viable way to study small circuits of neurones

The development of novel methods for the targeting and manipulation of neural circuits in vivo

Neural networks are at the core of the brain’s ability to compute complex responses to our external environment. Clinically, network dysfunction is emerging as a key component of several psychiatric and neurodegenerative disorders such as Alzheimer’s disease or schizophrenia. However, our ability to precisely and safely manipulate neural networks for research and deliver network-specific therapy remains limited. To address this problem, our lab recently developed a monosynaptically restricted Self-Inactivating Rabies virus (SiR) which enables the targeting of neural circuits without cytotoxicity. To expand the scope of SiR we further developed the technology in two directions: A) By incorporating the CRISPR/CAS9 gene-editing machinery into the SiR genome to successfully edit endogenous loci in vitro and in vivo. B) By designing an improved second generation SiR virus (SiR 2.0) which applies the same SiR technology to a challenge rabies strain (CVS-N2C). SiR 2.0 demonstrates increased neurotropism, increased trans-synaptic transfer efficiency and markedly decreased immunogenicity compared to the SiR 1.0 vector. These advancements expand the scope of SiR viruses to be used in the genome-editing of circuits in vivo. A combined SiR 2.0 CAS9 virus, in physiology, allows us to investigate the roles of genes within circuits in the brain function of live animals. For therapy, it paves the way for the rabies virus’ potential use to edit disease-related genes in dysfunctional circuits. Despite the circuit-basis of many neurological disorders, existing gene therapy vectors are not circuit specific. In addition, the practical difficulties of delivering therapeutic agents at high doses into the central nervous system exacerbates our inability to achieve high therapeutic loads into affected circuits. In contrast, a SiR 2.0 CAS9 virus would, following injection into peripheral organs, trans-synaptically spread into desired circuits of the central nervous system that are affected in neurological disease (e.g. networks demonstrating pathological protein propagation in neurodegenerative disorders) and edit disease-related genes. Lastly, our interest in network-level pathological protein propagation also led us to investigate the biology behind this observation. Due to additional evidence that a significant number of other proteins in physiology also show interneuronal movement, we hypothesised that perhaps this is an overlooked phenomena in neurobiology which could have key implication

Computational models in the age of large datasets.

Technological advances in experimental neuroscience are generating vast quantities of data, from the dynamics of single molecules to the structure and activity patterns of large networks of neurons. How do we make sense of these voluminous, complex, disparate and often incomplete data? How do we find general principles in the morass of detail? Computational models are invaluable and necessary in this task and yield insights that cannot otherwise be obtained. However, building and interpreting good computational models is a substantial challenge, especially so in the era of large datasets. Fitting detailed models to experimental data is difficult and often requires onerous assumptions, while more loosely constrained conceptual models that explore broad hypotheses and principles can yield more useful insights.Charles A King TrustThis is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.conb.2015.01.00

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

Distinct Neuromuscular Patterns from a Single Motor Network

My thesis aimed to elucidate several aspects of motor circuit regulation and its impact on movement. It is well established that a single motor network can produce different output patterns in response to different inputs. However, in most model systems it remains challenging to identify the neurons comprising these networks and determine their role(s) in network operation, including whether each network neuron retains its role(s) when the network generates different output patterns. Also, most work on these circuits has occurred in the isolated nervous system, so little is known about how muscles respond to distinct neural outputs. I therefore aimed to address the cellular and synaptic mechanisms underlying these unresolved issues using the decapod crustacean stomatogastric nervous system. My work focused on a rhythmically active, network-driven motor circuit (central pattern generator [CPG] circuit) called the gastric mill (chewing) CPG in the crab stomatogastric ganglion. This circuit generates the gastric mill rhythm when activated by modulatory projection neurons (e.g. MCN1, CPN2) located in the commissural ganglia, and it is regulated by identified sensory feedback. I addressed and confirmed the hypothesis that, in the isolated nervous system, different extrinsic inputs can drive different gastric mill motor patterns. This enabled me to determine, for the first time in a network-driven motor circuit, that different motor patterns generated by the same motor circuit are paced by the same set of rhythm generator neurons. I further hypothesized and confirmed that these distinct motor patterns are retained at the level of at least some target muscles, and hence likely underlie different behavioral patterns. Lastly, I obtained data supporting the hypothesis that different extrinsic inputs distinctly modify the influence of a sensory feedback pathway on the relevant projection neurons (MCN1, CPN2), enabling the same sensory system to have different effects on different gastric mill rhythms. These results provide among the most detailed comparisons of how motor patterns generated by a single sensorimotor system are selected and regulated. The results thereby provide evidence for several novel cellular and synaptic mechanisms that expand our appreciation of the number of degrees of freedom available to even small sensorimotor systems

Dynamics and function of nicotinic acetylcholine receptors in the nervous system

Tese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2011Fundação para a Ciência e a Tecnologia (FCT, POCI 2010/FSE e Fundação Calouste Gulbenkian

Homeostatic compensation and neuromodulation maintain synchronized motor neuron activity in the crustacean cardiac ganglion

Dissertation supervisor: Dr. David J. Schulz.Includes vita.Animals rely on the nervous system to produce appropriate behavior throughout their lives. In sending commands to the musculature for rhythmic motor behaviors such as breathing or walking, neural networks must be stable enough to send a reliable level of drive with the proper temporal coordination. Networks must also be flexible enough to meet changing environmental demands. A network's output ultimately arises from the intrinsic excitability of its constituent neurons and the synaptic connections between them. Interestingly, neurons and networks are able to produce highly conserved output from highly variable underlying intrinsic and synaptic properties. To explore the consequences of this variability, we have used the crustacean cardiac ganglion (CG) which consists of 9 neurons: 4 pacemaker cells that give excitatory input to 5 Large Cell motor neurons (LCs) which are responsible for driving the simultaneous contraction of the musculature that makes up the walls of the animal's single-chambered heart (Alexandrowicz, 1934; Hartline, 1967; Anderson and Cooke, 1971). The intact network can be dissected from the animal in physiological saline and it continues to produce robust, reliable, and rhythmic output (Welsh and Maynard, 1951; Cooke, 2002). LCs have virtually identical synchronized activity, but their intrinsic ionic conductances can be highly variable (Ransdell et al., 2013a). In Chapter 1, we exploit this variability by pharmacologically blocking a subset of their conductances to make LCs hyperexcitable and desynchronize their activity. We find that homeostatic compensation restores synchronized activity and excitability within one hour. This happens via two synergistic mechanisms: the membrane properties of each cell are re-tuned to converge on similar voltage activity, and increased conductance of the gap junctions between the cells helps to buffer away differences in their voltage activity. A separate but related study asked whether naturalistic perturbations of network activity would also result in desynchronization. Neuromodulation provides flexibility in the output of neural networks by altering a subset of their conductances. We hypothesized that this could also cause desynchronization. We found that modulation with serotonin and dopamine both increased the excitability of the CG. Interestingly, serotonin desynchronized the CG, but dopamine did not. We found that dopaminergic modulation directly increases gap junctional conductance. By co-applying these modulators, we found dopamine was able to prevent serotonin from desynchronizing the network without occluding its effects. It was also able to prevent the desynchronization caused by ion channel blockers. Finally, to fully understand the output of LCs, we must recognize that their activity arises not only from their intrinsic properties, but also from their synaptic drive from pacemaker cells. To address how variable this can be from one animal to the next, we analyze the activity of 131 animals taken over the course of approximately 5 years. We use this to address the fundamental question of how variable networks underlying a particular behavior can be across animals. We recognize two distinct classes of pacemaker inputs to LCs, and characterize bursting patterns for both types of pacemaker spike and LC output. We conclude that LCs from different animals receive different temporal patterns of pacemaker drive, which may have important functional implications. We also compare animals from winter and summer months, and find that temperature-independent seasonal effects may explain some of the variance in our data.Includes bibliographical references

A Combinatorial Premotor Neural Code: Transformation Of Sensory Information Into Meaningful Rhythmic Motor Output By A Network Of Heterogeneous Modulatory Neurons

The goal of the following research was to investigate the contributions of neural networks in selecting distinct variants of rhythmic motor activity. We used the premotor commissural ganglion (CoG) in the stomatogastric nervous system of the Jonah crab to understand how this network effectively controls the rhythms produced in downstream motor circuits. Prior research determined that individual CoG neurons are necessary to mediate sensory-induced variation in the effected motor patterns. However, single premotor neuron inputs to the STG are not sufficient to recreate the patterns induced by the selective activation of sensory pathways. Thus, it was hypothesized that the CoG-mediated effects on these sensorimotor transformations must be explained at the level of CoG population activity. We embraced the exploratory nature of this study by approaching it in three phases. First, we established voltage-sensitive dye imaging in the stomatogastric nervous system, as a technique that reports the simultaneous activity of many neurons with single-neuron resolution. In short, this form of imaging was effective at reporting both slow and fast changes in membrane potential, and provided an effective means of staining fine neural structures through neural sheaths, structures that often act as barriers to many substances. Then, we characterized the distribution of somata in the CoG, and found that soma location was not fixed in its location from animal to animal, but that clustering of CoG somata did occur near their different nerve pathway origins. Finally, we used the voltage-sensitive dye-imaging technique to investigate the CoG population under many different sensory conditions, and found that two different sensory modalities, one chemosensory and one mechanosensory pathway, differentially affected the balance of excited and inhibited (network activation) neurons found in the CoGs. Moreover, differences in the composition of CoG participants between modalities was not extremely robust. However, it differed enough so that both CoG participation and activation were drivers of the observed changes in the downstream pyloric motor network, providing support for a premotor combinatorial code for motor pattern selection