Modulation of feeding and its role in appetitive memory consolidation in Lymnaea stagnalis

Feeding in the pond snail (Lymnaea stagnalis) is heavily regulated by modulatory neurons throughout the brain. Following appetitive conditioning many of these identified neurons show changes in electrophysiological properties that contribute to the conditioned feeding response. The aim of this thesis was to further characterise modulatory feeding neurons and the neural plasticity that occurs within them during appetitive memory consolidation. Previous research had shown that the cerebral giant cell (CGC) is persistently depolarised 16-24 hours following appetitive conditioning and onwards. A Hodgkin-Huxley model of the CGC predicted this nonsynaptic plasticity to occur via an increase in the maximum conductance of the persistent sodium current (INaP), delayed rectifier current (IK) and high voltage activated calcium current (IHVA). To test this hypothesis these three modelled conductances were introduced into the membrane of the CGC of untrained snails, using dynamic clamp. Little effect on electrophysiological properties was initially seen due to an unexpectedly large cell surface area. To correct for this underestimation the response of the membrane to sine wave current injection was used to calculate the total cell surface area prior to dynamic clamp. When correctly scaled conductances were applied to the cell, there was still no significant change in the resting membrane potential (RMP). This suggests that the changes in ionic conductances causing CGC nonsynaptic plasticity during long term memory are more complex than previously realised and may involve other membrane conductances. A simpler attempt to depolarise the CGC with pattern clamp was able to cause significant RMP depolarisation, but also led to ectopic spike initiation in the proximal axon. Sub-optimal classical conditioning of Lymnaea with dilute sucrose (US) results in memory expression which is temporarily undetectable both at 30 minutes and 2 hours after training. The neural mechanisms involved during these memory lapses are unknown. Therefore, two key modulatory neurons, the CGC and the pleural buccal interneuron (PlB) were recorded during lapse and non-lapse time points after sub-optimal conditioning. These neurons were targeted as they can upregulate and downregulate the entire feeding system, and modulatory cells have previously been shown to play a role in memory consolidation. When conditioned preparations were compared to unconditioned preparations there was no significant difference in spontaneous firing frequency or CS-induced firing frequency in the CGC or PlB. This suggests that neither of these identified modulatory neurons play a role at 1 hr (non-lapse), 2 hr (lapse), and 3 hr (non-lapse) following sub-optimal appetitive conditioning. To better understand the neural mechanisms of appetitive memory storage it is also important to characterise the feeding network. Particularly little is known about the control of the modulatory feeding neurons which project to the buccal ganglia, where the feeding CPG and motoneurons are located. Therefore, a search was made for a neuron presynaptic to modulatory projection cells. Such a cell was identified in the parietal ganglion and was termed parietal dorsal 4 (PD4). Intracellular dye filling revealed an expansive morphology with a large axon projecting through the pleural ganglion, cerebral ganglion, and lastly the buccal ganglion. The axon was also seen to branch extensively and cross both the cerebral and buccal commissure. Furthermore, a peripheral projection was observed from a nerve of the visceral ganglion. This extensive morphology makes this neuron an ideal candidate to regulate control of the feeding system as a whole. When artificially activated by depolarisation PD4 caused monosynaptic excitation of both the ipsilateral and contralateral CGC. Delayed excitation was also observed on the ipsilateral CV1a and inhibition on the ipsilateral PlB. However, no direct connection was found between PD4 and identified dorsal buccal feeding neurons. Depolarisation of PD4 could occasionally cause feeding cycles to occur but could also inhibit spontaneous fictive feeding. This differential effect is likely due to polysynaptic depolarisation of the inhibitory CPG neuron N3t, which becomes dominant when there are high frequency action potentials in the CGC. In a semi-intact preparation PD4 did not respond to sucrose applied to the lips, demonstrating it is not involved in the unconditioned feeding response. PD4 was depolarised to threshold by extracellular stimulation of multiple nerves including the lip, tentacle, anal/intestinal and parietal nerves. Activation of PD4 also caused inhibition of the caudodorsal cells (CDCs) and excitation of the ring neuron (RN), suggesting that the neuron may be involved in controlling modulation of the feeding network during egg laying behaviour. In summary, this thesis examines important conceptual gaps in the understanding of appetitive memory consolidation in Lymnaea. It also provides the characterisation of an entirely new cell with widespread influence over the feeding system

Modulation of defensive reflex conditioning in snails by serotonin

We studied the role of serotonin in the mechanisms of learning in terrestrial snails. To produce a serotonin deficit, the "neurotoxic" analogues of serotonin, 5,6- or 5,7-dihydroxytryptamine (5,6/5,7-DHT) were used. Injection of 5,6/5,7-DHT was found to disrupt defensive reflex conditioning. Within two weeks of neurotoxin application, the ability to learn had recovered. Daily injection of serotonin before a training session accelerated defensive reflex conditioning and daily injections of 5-HTP in snails with a deficiency of serotonin induced by 5,7-DHT restored the snail's ability to learn. We discovered that injections of the neurotoxins 5,6/5,7-DHT as well as serotonin, caused a decrease in the resting and threshold potentials of the premotor interneurons LPa3 and RPa3

Relationship Between Learning-Related Synaptic and Intrinsic Plasticity Within Lateral Amygdala

A central question in neuroscience is to determine the mechanisms that govern formation, storage and modulation of memories. Determining these mechanisms would allow us to facilitate new memory formation as in the case of aging-related cognitive decline or weaken preexisting pathological memories such as traumatic memories and cue-induced drug craving. Pharmacological and genetic manipulation of intrinsic neuronal excitability has been demonstrated to impact the strength of memory formation, allocation of memories, and modulation of memories through retrieval and reconsolidation-dependent processes. In addition to experimental manipulations of intrinsic excitability, intrinsic plasticity, a change in neuronal intrinsic excitability, can be brought about by behavioral means such as learning. Indeed, learning-related intrinsic plasticity has been observed in many brain structures following acquisition of a variety of learning paradigms. Despite its ubiquitous nature, little is known about the functional significance of learning-induced intrinsic plasticity. Using the well-characterized lateral amygdala-dependent auditory fear conditioning as a behavioral paradigm, the current experiments investigated the time course and relationship between intrinsic and synaptic plasticity. We found that learning-related changes in amygdala intrinsic excitability were transient and were no longer evident 10 days following fear conditioning. We also found that fear learning related synaptic plasticity was evident up to 24hr following fear conditioning but not 4 days later. Finally, we demonstrate that the intrinsic excitability changes are evident in many of the same neurons that are undergoing synaptic facilitation immediately following fear conditioning. These data demonstrate that learning related intrinsic and synaptic changes are transient and co-localized to the same neurons. These data demonstrate that memory encoding neurons are more excitable, thus more likely to capture new memories for a time after the learning event

Mechanisms of Olfactory Plasticity in Caenorhabditis Elegans

Animals live in constantly changing environments with fluctuating resource availability and hazardous threats. By gathering information from past experiences, individuals modify their behavioral response to adapt to the changing environment, a phenomenon known as “experience-dependent plasticity”. This ability to change is a crucial for survival, and how an organism achieves this adaptive plasticity is a question of much interest. Research in the field has yielded insight into how changes in connectivity within the brain can drive changes in behavior. Understanding the neural mechanisms of plasticity not only satisfies intellectual curiosity, but also provides a basis for understanding pathological conditions that come from excessive or insufficient plasticity. With a well-characterized nervous system, stereotyped behaviors, and an armory of molecular and genetic tools, C. elegans is well-suited for the study of experience-dependent plasticity. Using an olfactory adaptation paradigm in which animals lose attraction to butanone after it is paired with starvation, I here describe neuronal and molecular mechanisms that are associated with and necessary for plasticity in C. elegans. In Chapter 2, I report my findings on circuit mechanisms of butanone adaptation, identifying neurons that are required for adaptation and changes in neuronal activity associated with adaptation. I show that an interneuron is required for adaptive changes in the olfactory sensory neuron. In particular, I show that nuclear translocation of a protein kinase, a process known to be necessary for adaptation, requires activity of the interneuron. This feedback from downstream neurons is transformed into changes in sensory properties. Using pharmacogenetic tools that allowed me to disrupt different parts of the circuit with temporal precision, I identified a group of neurons whose activity is required during adaptation. Finally, I performed functional calcium imaging of animals before and after adaptation, and determined that changes in neuronal responses to butanone can be detected at multiple sites within the circuit, starting as early as the as the sensory neurons. In Chapter 3, I describe the analysis of two genes, a G-protein β subunit and a K+ channel, that have different roles in adaptation. I used whole-genome sequencing and genetic mutations to identify the genes that are required for butanone adaptation, then characterized the odor-specificity of each gene. This analysis provides the basis for future work that should examine the molecular context in which these genes act and the impact they have on circuit mechanisms of adaptation

Excitabilidad intrínseca y su plasticidad en el hipocampo de rata

Durante las primeras semanas del desarrollo postnatal se producen cambios en las propiedades electrofisiológicas y morfológicas (Pokorny & Yamamoto 1981a; Pokorny & Yamamoto 1981b; Schwartzkroin & Kunkel 1982; Liao et al. 1999), así como en la conectividad sináptica (Hsia et al. 1998; Groc et al. 2003) de las neuronas piramidales de CA1. Además, aumenta el comportamiento exploratorio que depende de la actividad hipocampal en las ratas (Langston et al. 2010). La propagación de la información dentro de un circuito neuronal como el hipocampal, depende de la conectividad sináptica dentro del circuito y de las propiedades intrínsecas de cada neurona. Cambios en la actividad del circuito pueden provocar plasticidades en: la eficacia sináptica, la integración de la señal de entrada y la generación de una señal de salida (Daoudal & Debanne 2003; Debanne et al. 2003; Remy et al. 2010). Diversos estudios han demostrado la existencia de plasticidades de la excitabilidad intrínseca en respuesta a variaciones de la actividad previa (Fan et al. 2005; O’Leary et al. 2010; Cudmore et al. 2010; Campanac et al. 2013). El calcio es un ion fundamental en el mantenimiento de la excitabilidad neuronal. La disminución de su concentración extracelular se relaciona con crisis epilépticas (Heinemann et al. 1977) y con un comportamiento epileptiforme en estudios in vitro (Bikson et al. 2002; Isaev et al. 2012; Aivar et al. 2014). Además, el calcio está implicado en gran parte de las plasticidades sinápticas e intrínsecas descritas (Katz & Miledi 1968; Lynch et al. 1983; Zucker & Regehr 2002; Fan et al. 2005; Grubb & Burrone 2010)..