Non-invasive, Focused Ultrasound-Facilitated Gene Delivery for Optogenetics

Optogenetics, a widely used technique in neuroscience research, is often limited by its invasive nature of application. Here, we present a noninvasive, ultrasound-based technique to introduce optogenetic channels into the brain by temporarily opening the blood-brain barrier (BBB). We demonstrate the efficiency of the method developed and evaluate the bioactivity of the non-invasively introduced channelrhodopsin channels by performing stimulation in freely behaving mice

Excess Synaptojanin 1 Contributes to Place Cell Dysfunction and Memory Deficits in the Aging Hippocampus in Three Types of Alzheimer’s Disease

The phosphoinositide phosphatase synaptojanin 1 (SYNJ1) is a key regulator of synaptic function. We first tested whether SYNJ1 contributes to phenotypic variations in familial Alzheimer’s disease (FAD) and show that SYNJ1 polymorphisms are associated with age of onset in both early- and late-onset human FAD cohorts. We then interrogated whether SYNJ1 levels could directly affect memory. We show that increased SYNJ1 levels in autopsy brains from adults with Down syndrome (DS/AD) are inversely correlated with synaptophysin levels, a direct readout of synaptic integrity. We further report age-dependent cognitive decline in a mouse model overexpressing murine Synj1 to the levels observed in human sporadic AD, triggered through hippocampal hyperexcitability and defects in the spatial reproducibility of place fields. Taken together, our findings suggest that SYNJ1 contributes to memory deficits in the aging hippocampus in all forms of AD

Komplexe Formen des Lernens bei der Honigbiene: eine Verhaltens- und neuronale Analyse & Honigbienen und Schlaf: die Rolle des Schlafes beim Lernen und für das Gedächtnis

1\. Title page 3 2\. Table Of Contents 1 3\. Introduction 3 4\. Chapter 1: Forward and backward second-order Pavlovian conditioning in honeybees. 16 5\. Chapter 2: Context-dependent learning in honeybees: a behavioral and neural analysis. 26 6\. Chapter 3: Sleep in honeybees: its role in learning and memory. 23 7\. Conclusion and Bibliography 27 8\. Summary 1 9\. Zusammenfassung 2 10\. Acknowledgements 2This thesis describes olfactory learning in honeybees (Apis mellifera). The first two chapters describe complex forms of learning; Second-order conditioning (SOC) and Context-dependent learning (CDL). The third chapter focuses on sleep behavior. 1) To show SOC we used classical conditioning of the proboscis extension response (PER) in honeybees using odors as conditioning stimulus (CS) and sugar as US. We showed that bees can learn an odor (C) even without an US, but only if C was paired with a previously rewarded odor (A). In other words, bees respond to the new odor C after 5 times A-US pairing followed by 5 times C-A pairing (A-US -> C-A). By changing the sequence of odors (A-US -> A-C) we could not demonstrate an effective SOC. Furthermore not only the sequence of odors, but also the sequence of training is important. Fewer bees show PER to C, after the sequence C-A -> A-US in comparison to the sequence A-US -> C-A These results helped us to understand conditions critical for SOC. 2) In context dependent learning experiment we could simultaneously test several combinations of contexts and study properties of the brain by extracellular recordings. We showed that bees can learn to differentiate contexts in relatively short time (<1h). Bees showed better learning when two contexts were presented together as opposed to single context. Extracellular recordings from alpha lobe of mushroom bodies showed that the neuronal firing changed sharply between hot and cold temperature contexts. Neurons responded more strongly to the rewarded context as compared to unrewarded context, but the response towards rewarded odor was weaker compared to other odors. 3) Sleep-like behavior in bees has been observed before but its role in learning and memory has not been studied. Described here is a technique which allowed monitoring of sleep combined with olfactory learning tasks. We found that, like many animals, bees also slept more during night compared to daytime and the the patterns of antennal movements (sleep indicators) were different during sleep and wake period. Learning experiments showed that bees that were conditioned to odors slept lesser compared to unconditioned bees. Sleep depriving bees with shaking did not affect acquisition learning but affected extinction learning.Diese Arbeit beschäftigt sich mit dem olfaktorischen Lernen in der Honigbiene Apis mellifera. In den ersten beiden Kapitel dieser Arbeit werden komplexe Formen des Lernens beschrieben: Second-Order-Konditionierung (SOC)in Kapitel eins und kontextabhängiges Lernen in Kapitel zwei. Das dritte Kapitel konzentriert sich auf das Schlafverhalten. 1) Zum Nachweis der SOC nutzten wir die Klassische Konditionierung des Rüssel- Streck-Verhaltens (Proboscis- Extension-Response PER)der Honigbiene. Dabei wird ein Duft, als konditionierter Stimulus (CS), mit Zuckerwasser, als unkonditionierten Stimulus (US), gepaart. Wir konnten zeigen, dass Bienen einen Duft (C) auch ohne US - Präsentation erlernen können, allerdings nur wenn C zuvor mit einem belohnten Duft (A) gepaart wurde. Anders ausgedrückt, die fünfmaliger Paarung von A-US, gefolgt von der fünfmaligen C-A Paarung (A-US -> C-A), führt dazu, dass die Bienen auf den neuen Duft C mit dem Strecken des Proboscis reagieren. Wurde jedoch die Sequenz der Düfte vertauscht (A-US -> A-C) konnte keine erfolgreiche SOC erzielt werden. Neben der Sequenz der Düfte, ist auch die Sequenz des Trainings wichtig. Vertauscht man die Trainingssequenzen von A-US -> C-A zu C-A -> A-US reagieren weniger Bienen auf C mit einer konditionierten Antwort. Diese Ergebnisse helfen uns die entscheidenden Bedingungen bei der SOC zu verstehen. 2) In den Experimenten zum kontextabhängigen Lernen konnten wir verschiedene Kombinationen von Kontexten präsentieren und zeitgleich extrazellulär ableiten. Wir konnten zeigen, dass die Bienen in relativ kurzer Zeit (< 1 Std.) lernen können verschiedene Kontexte zu diskriminieren. Präsentierte man zwei Kontexte zusammen anstelle eines einzelnen Kontextes zeigten die Bienen ein besseres Lernverhalten. Extrazellulär Ableitungen vom Alphalobus des Pilzkörpers zeigten, dass die Erregungsmuster der Neurone sich in Abhängigkeit des Temperaturkontextes (heiß-kalt) stark veränderten. Die Neuronen antworteten stärker im belohnten Kontext verglichen mit dem unbelohnten Kontext, jedoch waren die Antworten auf den belohnten Duft schwächer im vergleich zu anderen Düften. 3) Schlaf-ähnliches Verhalten wurde schon zuvor bei Bienen beschrieben, die Auswirkungen auf das Lernen und die Gedächtnisbildung wurde allerdings noch nicht untersucht. Hier wird eine Technik beschrieben, die es ermöglicht nach der Klassischen Konditionierung eines Duftes, das Schlafverhalten zu beobachten und zu quantifizieren. Unsere Ergebnisse zeigen, dass Bienen, wie viele andere Tiere, mehr während der Nacht schlafen als während des Tages. Die Bewegungen der Antennen (als ein Schlafindikator) führen während der Schlafperiode öfter rhythmische symmetrische Bewegungen aus, im 98 Vergleich zu den Wachperioden. So konnten wir zeigen, dass Bienen, die auf einen Duft konditioniert wurden weniger schliefen im Vergleich zu unkonditionierten Bienen. Wurden die Bienen durch Schütteln am Schlaf gehindert, ist das Erwerbslernen nicht betroffen, jedoch das Extinktionslernen

Forward and backward second-order Pavlovian conditioning in honeybees

Second-order conditioning (SOC) is the association of a neutral stimulus with another stimulus that had previously been combined with an unconditioned stimulus (US). We used classical conditioning of the proboscis extension response (PER) in honeybees (Apis mellifera) with odors (CS) and sugar (US). Previous SOC experiments in bees were inconclusive, and, therefore, we attempted to demonstrate SOC in the following three experiments: (Experiment 1) After differential conditioning (pairing odor A with US and presenting odor B without US), the bees experienced two pairs of partially overlapping odors, either a new odor C followed by a previously reinforced odor A (C-A) or a new odor C followed by a previously nonreinforced odor B (C-B). (Experiment 2) After differential conditioning, bees were presented with C-A or A-C. (Experiment 3) Bees were first presented with C-A or A-C before differential conditioning and were tested with odor C. We observed: (Experiment 1) 40% of the bees showed PER to the C-A presentation, but only 20% showed PER to the C-B presentation. (Experiment 2) 40% of the bees showed PER to the C-A presentation, while only 20% showed PER to the reversed sequence A-C. Experiments 1 and 2 showed that a previously reinforced odor can be a secondary reinforcer for excitatory SOC only with forward-pairing. (Experiment 3) PER toward C was lower (15%) in bees presented with A-C than with C-A (25%). This showed that backward SOC is not as effective as forward SOC. These results help to delineate different conditions that are critical for the phenomenon of SOC

Focused Ultrasound Facilitated Adenoviral Delivery for Optogenetic Stimulation

Abstract-Optogenetics is a recently developed technique that has been widely implemented in the field of neuroscience. Channelrhodopsins (ChR), i.e., proteins that function as optically activated ion channels, are introduced into neurons either by viral transduction or transgenic manipulations. Recent advances in focused ultrasound (FUS) offer an alternative, non-invasive approach to carrying out viral transduction in the brain. In this study, we propose a FUS-based, non-invasive adenoviral delivery scheme that is suitable for optogenetic applications. Wild-type mice were used in this study, and each animal was sonicated using a single-element FUS transducer (center frequency 1.5 MHz). The acoustic parameters used for each sonication are: peak-rarefactional pressure 0.55 MPa, pulse length 10 ms, pulse repetition frequency 5 Hz, and a duration of 120 s. Viral vectors (both AAV1 and AAV9) were mixed with in-house manufactured polydisperse microbubbles prior to intravenous injections. Animals were survived for 12 days after sonication and electrode (with optic fiber) was implanted for electrophysiology recording. Upon optical stimulation, neuronal depolarization was observed in mice that received FUS-facilitated AAV transduction. In addition, fluorescence imaging revealed abundant ChR expression in the FUS sonicated brain regions. In conclusion, the successful non-invasive AAV delivery can provide an alternate and safer route for Channelrhodopsin neuronal transduction for optogenetic stimulation