Expansion Light Sheet Microscopy Resolves Subcellular Structures in Large Portions of the Songbird Brain

Expansion microscopy and light sheet imaging (ExLSM) provide a viable alternative to existing tissue clearing and large volume imaging approaches. The analysis of intact volumes of brain tissue presents a distinct challenge in neuroscience. Recent advances in tissue clearing and light sheet microscopy have re-addressed this challenge and blossomed into a plethora of protocols with diverse advantages and disadvantages. While refractive index matching achieves near perfect transparency and allows for imaging at large depths, the resolution of cleared brains is usually limited to the micrometer range. Moreover, the often long and harsh tissue clearing protocols hinder preservation of native fluorescence and antigenicity. Here we image large expanded brain volumes of zebra finch brain tissue in commercially available light sheet microscopes. Our expansion light sheet microscopy (ExLSM) approach presents a viable alternative to many clearing and imaging methods because it improves on tissue processing times, fluorophore compatibility, and image resolution

Tissue Clearing and Light Sheet Microscopy: Imaging the Unsectioned Adult Zebra Finch Brain at Cellular Resolution

The inherent complexity of brain tissue, with brain cells intertwining locally and projecting to distant regions, has made three-dimensional visualization of intact brains a highly desirable but challenging task in neuroscience. The natural opaqueness of tissue has traditionally limited researchers to techniques short of single cell resolution such as computer tomography or magnetic resonance imaging. By contrast, techniques with single-cell resolution required mechanical slicing into thin sections, which entails tissue distortions that severely hinder accurate reconstruction of large volumes. Recent developments in tissue clearing and light sheet microscopy have made it possible to investigate large volumes at micrometer resolution. The value of tissue clearing has been shown in a variety of tissue types and animal models. However, its potential for examining the songbird brain remains unexplored. Songbirds are an established model system for the study of vocal learning and sensorimotor control. They share with humans the capacity to adapt vocalizations based on auditory input. Song learning and production are controlled in songbirds by the song system, which forms a network of interconnected discrete brain nuclei. Here, we use the CUBIC and iDISCO+ protocols for clearing adult songbird brain tissue. Combined with light sheet imaging, we show the potential of tissue clearing for the investigation of connectivity between song nuclei, as well as for neuroanatomy and brain vasculature studies

Adult neuroplasticity under the influence of vocal motor skill practice and sex hormones : new findings and new tools

Remarkable plasticity is one of the hallmarks of the central nervous system. The vertebrate brain remains plastic throughout the animal’s life, and constantly reshapes itself in response to the animal’s environment, experiences, and physiology. Adult neuroplasticity can be exploited to help patients recover from stroke and traumatic brain injury, while abnormal plasticity is associated with many pathologies, including Alzheimer’s and Parkinson’s diseases. Thus, understanding the mechanisms behind adult neuroplasticity has the potential to help develop better clinical applications to help patients suffering from such disorders. However, adult neuroplasticity processes are rather complex, and can be influenced by many factors, such as stress, seasonal variations, hormonal effects, learning, and memory formation. Two such factors are motor learning and sex hormones. We use adult female canaries as a model system to investigate the processes involved in brain circuit changes in structure and function brought about by sex hormone-induced vocal motor learning experience. Adult female canaries can be induced to produce song, a complex motor skill, by testosterone implantation, which will also trigger the reshaping of the brain regions responsible for song learning and production. We implanted female canaries with testosterone and examined the development of their vocal motor skills over many months, followed by a period when singing practice was abolished by implant removal, and a subsequent period of sex hormone-induced vocal motor skill relearning. We found manifold lasting changes brought about by a first testosterone-induced vocal motor skill learning experience. Singing experience elicited the formation of vocal motor skill memory, as even after long periods when practice was abolished, the rate of vocal motor skill relearning was up to 7x faster than the initial learning. This was accompanied by the optimization of the song premotor circuits, with brain nucleus HVC undergoing a remarkable dendritic spine pruning, posing the excitatory synapses within this nucleus as a likely neural correlate for vocal motor skill memory. Once consolidated, this circuit remained stable and resisted further singing practice and sex hormone-induced changes. Furthermore, a first singing experience also prompted changes in sex hormone responsiveness. Female canaries can only be induced to sing by implantation with DHT, a testosterone metabolite, if they had previous testosterone-induced singing experience. This last finding hints at experienced-induced changes in the activation of hormonal receptor-activated signaling cascades, which might be key to preserve previously optimized circuits. Finally, our findings left open questions that needed detailed large volume investigations of the songbird brain circuits with cell type specificity, which were previously unavailable for songbird brain tissue. Thus, we invested in developing such tools and successfully applied tissue clearing and expansion microscopy in combination with light sheet imaging to songbird brain tissue for the first time. Combined with the use of viral vectors for fluorescent protein expression, these techniques make the exploration of large tissue volumes at high resolution possible, enabling detailed connectomics studies, and can also be applied to examine brain wide gene expression. We thus expect these techniques to help answer the questions left unresolved by the findings in this thesis. With the new findings and tools presented in this we work, I hope to contribute to further the understanding on the important mechanisms controlling motor learning- and sex hormone-induced adult neuroplasticity.publishe

Expansion Light Sheet Microscopy Resolves Subcellular Structures in Large Portions of the Songbird Brain

Expansion microscopy and light sheet imaging (ExLSM) provide a viable alternative to existing tissue clearing and large volume imaging approaches. The analysis of intact volumes of brain tissue presents a distinct challenge in neuroscience. Recent advances in tissue clearing and light sheet microscopy have re-addressed this challenge and blossomed into a plethora of protocols with diverse advantages and disadvantages. While refractive index matching achieves near perfect transparency and allows for imaging at large depths, the resolution of cleared brains is usually limited to the micrometer range. Moreover, the often long and harsh tissue clearing protocols hinder preservation of native fluorescence and antigenicity. Here we image large expanded brain volumes of zebra finch brain tissue in commercially available light sheet microscopes. Our expansion light sheet microscopy (ExLSM) approach presents a viable alternative to many clearing and imaging methods because it improves on tissue processing times, fluorophore compatibility, and image resolution

Accelerated redevelopment of vocal skills is preceded by lasting reorganization of the song motor circuitry

Complex motor skills take considerable time and practice to learn. Without continued practice the level of skill performance quickly degrades, posing a problem for the timely utilization of skilled motor behaviors. Here we quantified the recurring development of vocal motor skills and the accompanying changes in synaptic connectivity in the brain of a songbird, while manipulating skill performance by consecutively administrating and withdrawing testosterone. We demonstrate that a songbird with prior singing experience can significantly accelerate the re-acquisition of vocal performance. We further demonstrate that an increase in vocal performance is accompanied by a pronounced synaptic pruning in the forebrain vocal motor area HVC, a reduction that is not reversed when birds stop singing. These results provide evidence that lasting synaptic changes in the motor circuitry are associated with the savings of motor skills, enabling a rapid recovery of motor performance under environmental time constraints

Data from: Accelerated redevelopment of vocal skills is preceded by lasting reorganization of the song motor circuitry

Complex motor skills take considerable time and practice to learn. Without continued practice the level of skill performance quickly degrades, posing a problem for the timely utilization of skilled motor behaviors. Here we quantified the recurring development of vocal motor skills and the accompanying changes in synaptic connectivity in the brain of a songbird, while manipulating skill performance by consecutively administrating and withdrawing testosterone. We demonstrate that a songbird with prior singing experience can significantly accelerate the re-acquisition of vocal performance. We further demonstrate that an increase in vocal performance is accompanied by a pronounced synaptic pruning in the forebrain vocal motor area HVC, a reduction that is not reversed when birds stop singing. These results provide evidence that lasting synaptic changes in the motor circuitry are associated with the savings of motor skills, enabling a rapid recovery of motor performance under environmental time constraints

Tissue Clearing and Light Sheet Microscopy: Imaging the Unsectioned Adult Zebra Finch Brain at Cellular Resolution

The inherent complexity of brain tissue, with brain cells intertwining locally and projecting to distant regions, has made three-dimensional visualization of intact brains a highly desirable but challenging task in neuroscience. The natural opaqueness of tissue has traditionally limited researchers to techniques short of single cell resolution such as computer tomography or magnetic resonance imaging. By contrast, techniques with single-cell resolution required mechanical slicing into thin sections, which entails tissue distortions that severely hinder accurate reconstruction of large volumes. Recent developments in tissue clearing and light sheet microscopy have made it possible to investigate large volumes at micrometer resolution. The value of tissue clearing has been shown in a variety of tissue types and animal models. However, its potential for examining the songbird brain remains unexplored. Songbirds are an established model system for the study of vocal learning and sensorimotor control. They share with humans the capacity to adapt vocalizations based on auditory input. Song learning and production are controlled in songbirds by the song system, which forms a network of interconnected discrete brain nuclei. Here, we use the CUBIC and iDISCO+ protocols for clearing adult songbird brain tissue. Combined with light sheet imaging, we show the potential of tissue clearing for the investigation of connectivity between song nuclei, as well as for neuroanatomy and brain vasculature studies