Structural and functional alterations of cortical neurons in Alzheimer’s disease transgenic mice assessed by two-photon in vivo imaging

Alzheimer’s disease (AD), the most common form of dementia, has been proposed to result from the degeneration of synapses, putatively caused by assemblies of the amyloid-β peptide (Aβ). The spatiotemporal dynamics of this synaptopathy, its potential reversibility as well as its consequences on the function of single neurons and neuronal circuits, however, are not fully understood to date. In order to address these questions, I assessed structural and functional alterations of neurons in the neocortex in a transgenic mouse model of Alzheimer’s disease, namely APP/PS1 (APPswe, PS1L166P) mice, using in vivo two-photon imaging. Chronic imaging of dendrites and axons over the course of four weeks revealed not only a reduction in dendritic spine density close to amyloid plaques (proteinaceous extracellular deposits typical of AD), but I also identified synaptic instability as a main aspect contributing to AD pathology. Importantly, while synapse loss was confined to the immediate plaque vicinity (up to 15µm from the histological plaque border), synaptic instability was evident in a much larger region surrounding plaques (50 µm) and affected both, pre- and postsynaptic compartments. As the prevailing hypothesis in AD holds that Aβ conveys these detrimental effects on synapses one therapeutic approach is based on the pharmacological inhibition of Aβ generation. I thus assessed the impact of a novel selective γ-secretase inhibitor (GSI), a compound that prevents the last cleavage step necessary for the release of Aβ from the longer transmembrane amyloid precursor protein (APP). Notably, the GSI used here primarily interferes with the processing of APP and still allows for processing of other γ-secretase substrates, and hence should largely reduce side effects seen with earlier generations of GSIs before. Daily treatment with the GSI reduced the deposition of Aβ as evidenced by the initial reduction in the number of new plaques and a sustained decrease in the growth of these newly deposited plaques. Importantly, it also ameliorated the plaque-associated synaptic instability, without displaying overt adverse effects on dendritic spines in WT mice. These data represent the first in vivo evidence that selective pharmacological inhibition of the γ-secretase mediated APP cleavage can have beneficial effects on synaptic pathology in AD. Given the widespread impact of Aβ assemblies on neuronal structures, I then asked to which extent these structural alterations affect the function of neurons. To address this question, I recorded neuronal response properties in the primary visual cortex of behaving APP/PS1 mice, employing in vivo two-photon calcium imaging using the genetically encoded calcium indicator GCaMP6m. In order to probe the impact of AD related pathology on specific aspects of information processing, which rely on multiple neuronal circuits, I characterized visually driven and motor-related activity, as well as signals based on mismatches between actual and expected visual input. My data reveal a massive reduction in responsiveness under almost all conditions tested, which is line with the profound impact on neuronal structure. Stimulus selectivity, like orientation or direction tuning, were not altered in APP/PS1 mice, indicating that the main effect is caused by a change in response gain. Along with the massive decrease in feedforward signals, I observed an increase in spontaneous, hence uncorrelated neuronal activity in AD transgenic mice. Both features jointly affected the coding accuracy of the network, and I propose that this combination may represent a common characteristic leading to impaired information processing in AD. Surprisingly, I found that responses elicited after a discordance of actual and expected visual flow during running, i.e. a visuomotor mismatch, were selectively spared in APP/PS1 mice, suggesting a particular resilience of this very signal. Together, both studies demonstrate that global widespread structural changes of neurons in the AD brain are accompanied by a severe impact on information processing, most prominently seen in a strong reduction of feedforward signals. My data, thus, provide a correlate of impaired cognition in AD at the level of single neurons and neural circuits

Structural and functional alterations of cortical neurons in Alzheimer’s disease transgenic mice assessed by two-photon in vivo imaging

Alzheimer’s disease (AD), the most common form of dementia, has been proposed to result from the degeneration of synapses, putatively caused by assemblies of the amyloid-β peptide (Aβ). The spatiotemporal dynamics of this synaptopathy, its potential reversibility as well as its consequences on the function of single neurons and neuronal circuits, however, are not fully understood to date. In order to address these questions, I assessed structural and functional alterations of neurons in the neocortex in a transgenic mouse model of Alzheimer’s disease, namely APP/PS1 (APPswe, PS1L166P) mice, using in vivo two-photon imaging. Chronic imaging of dendrites and axons over the course of four weeks revealed not only a reduction in dendritic spine density close to amyloid plaques (proteinaceous extracellular deposits typical of AD), but I also identified synaptic instability as a main aspect contributing to AD pathology. Importantly, while synapse loss was confined to the immediate plaque vicinity (up to 15µm from the histological plaque border), synaptic instability was evident in a much larger region surrounding plaques (50 µm) and affected both, pre- and postsynaptic compartments. As the prevailing hypothesis in AD holds that Aβ conveys these detrimental effects on synapses one therapeutic approach is based on the pharmacological inhibition of Aβ generation. I thus assessed the impact of a novel selective γ-secretase inhibitor (GSI), a compound that prevents the last cleavage step necessary for the release of Aβ from the longer transmembrane amyloid precursor protein (APP). Notably, the GSI used here primarily interferes with the processing of APP and still allows for processing of other γ-secretase substrates, and hence should largely reduce side effects seen with earlier generations of GSIs before. Daily treatment with the GSI reduced the deposition of Aβ as evidenced by the initial reduction in the number of new plaques and a sustained decrease in the growth of these newly deposited plaques. Importantly, it also ameliorated the plaque-associated synaptic instability, without displaying overt adverse effects on dendritic spines in WT mice. These data represent the first in vivo evidence that selective pharmacological inhibition of the γ-secretase mediated APP cleavage can have beneficial effects on synaptic pathology in AD. Given the widespread impact of Aβ assemblies on neuronal structures, I then asked to which extent these structural alterations affect the function of neurons. To address this question, I recorded neuronal response properties in the primary visual cortex of behaving APP/PS1 mice, employing in vivo two-photon calcium imaging using the genetically encoded calcium indicator GCaMP6m. In order to probe the impact of AD related pathology on specific aspects of information processing, which rely on multiple neuronal circuits, I characterized visually driven and motor-related activity, as well as signals based on mismatches between actual and expected visual input. My data reveal a massive reduction in responsiveness under almost all conditions tested, which is line with the profound impact on neuronal structure. Stimulus selectivity, like orientation or direction tuning, were not altered in APP/PS1 mice, indicating that the main effect is caused by a change in response gain. Along with the massive decrease in feedforward signals, I observed an increase in spontaneous, hence uncorrelated neuronal activity in AD transgenic mice. Both features jointly affected the coding accuracy of the network, and I propose that this combination may represent a common characteristic leading to impaired information processing in AD. Surprisingly, I found that responses elicited after a discordance of actual and expected visual flow during running, i.e. a visuomotor mismatch, were selectively spared in APP/PS1 mice, suggesting a particular resilience of this very signal. Together, both studies demonstrate that global widespread structural changes of neurons in the AD brain are accompanied by a severe impact on information processing, most prominently seen in a strong reduction of feedforward signals. My data, thus, provide a correlate of impaired cognition in AD at the level of single neurons and neural circuits

Exciting Complexity: The Role of Motor Circuit Elements in ALS Pathophysiology

Amyotrophic lateral sclerosis (ALS) is a fatal disease, characterized by the degeneration of both upper and lower motor neurons. Despite decades of research, we still to date lack a cure or disease modifying treatment, emphasizing the need for a much-improved insight into disease mechanisms and cell type vulnerability. Altered neuronal excitability is a common phenomenon reported in ALS patients, as well as in animal models of the disease, but the cellular and circuit processes involved, as well as the causal relevance of those observations to molecular alterations and final cell death, remain poorly understood. Here, we review evidence from clinical studies, cell type-specific electrophysiology, genetic manipulations and molecular characterizations in animal models and culture experiments, which argue for a causal involvement of complex alterations of structure, function and connectivity of different neuronal subtypes within the cortical and spinal cord motor circuitries. We also summarize the current knowledge regarding the detrimental role of astrocytes and reassess the frequently proposed hypothesis of glutamate-mediated excitotoxicity with respect to changes in neuronal excitability. Together, these findings suggest multifaceted cell type-, brain area- and disease stage- specific disturbances of the excitation/inhibition balance as a cardinal aspect of ALS pathophysiology

Projektbericht (Mediumfassung)

Der Mediumbericht fasst die einzelnen Teile des Berichts in der Langfassung zusammen. Zunächst wird anhand von statistischen Zahlen und Fakten nachgewiesen, dass das männliche Ernährermodell noch immer sehr prägend für die Existenzsicherung von Frauen und Männern in Deutschland ist. Dies wird darauf zurückgeführt, dass im bundesdeutschen Recht diverse Schnittstellen des ehelichen Unterhaltsrechts mit dem Arbeits-, Sozial- und Steuerrecht existieren, die das Ernährermodell voraussetzen, faktisch auch auf Unverheiratete ausdehnen und gleichzeitig perpetuieren. Da aber auch der Gleichberechtigungsgrundsatz für Frauen und Männer gilt und ein Staatsziel sogar die "tatsächliche Durchsetzung der Gleichberechtigung" fordert, sind normative Wertungswidersprüche entstanden, die nach einer Reform der Schnittstellen und der Gesamtkonzeption der Berücksichtigung von Unterhalt und finanzieller Paarsolidarität in verschiedenen Regelungsbereichen verlangen. Anschließend an die Darstellung und Kritik der Schnittstellenregelungen wird am Beispiel der Anrechnung von Partnereinkommen und -vermögen gemäß SGB II ("Hartz IV") die subjektive Seite der sozialrechtlichen Einstandspflicht beleuchtet, indem Ergebnisse einer Befragung von Betroffenen referiert werden. Ein abschließendes Kapitel resümiert den Reformbedarf und skizziert die nötigen politischen Entwicklungen und Maßnahmen zur Überwindung der noch immer starken Stellung des männlichen Ernährermodells in Deutschland.200

Early molecular layer interneuron hyperactivity triggers Purkinje neuron degeneration in SCA1.

Toxic proteinaceous deposits and alterations in excitability and activity levels characterize vulnerable neuronal populations in neurodegenerative diseases. Using in vivo two-photon imaging in behaving spinocerebellar ataxia type 1 (Sca1) mice, wherein Purkinje neurons (PNs) degenerate, we identify an inhibitory circuit element (molecular layer interneurons [MLINs]) that becomes prematurely hyperexcitable, compromising sensorimotor signals in the cerebellum at early stages. Mutant MLINs express abnormally elevated parvalbumin, harbor high excitatory-to-inhibitory synaptic density, and display more numerous synaptic connections on PNs, indicating an excitation/inhibition imbalance. Chemogenetic inhibition of hyperexcitable MLINs normalizes parvalbumin expression and restores calcium signaling in Sca1 PNs. Chronic inhibition of mutant MLINs delayed PN degeneration, reduced pathology, and ameliorated motor deficits in Sca1 mice. Conserved proteomic signature of Sca1 MLINs, shared with human SCA1 interneurons, involved the higher expression of FRRS1L, implicated in AMPA receptor trafficking. We thus propose that circuit-level deficits upstream of PNs are one of the main disease triggers in SCA1

Early molecular layer interneuron hyperactivity triggers Purkinje neuron degeneration in SCA1

Toxic proteinaceous deposits and alterations in excitability and activity levels characterize vulnerable neuronal populations in neurodegenerative diseases. Using in vivo two-photon imaging in behaving spino-cerebellar ataxia type 1 (Sca1) mice, wherein Purkinje neurons (PNs) degenerate, we identify an inhibitory cir-cuit element (molecular layer interneurons [MLINs]) that becomes prematurely hyperexcitable, compro-mising sensorimotor signals in the cerebellum at early stages. Mutant MLINs express abnormally elevated parvalbumin, harbor high excitatory-to-inhibitory synaptic density, and display more numerous synaptic connections on PNs, indicating an excitation/inhibition imbalance. Chemogenetic inhibition of hyperexcit-able MLINs normalizes parvalbumin expression and restores calcium signaling in Sca1 PNs. Chronic inhibi-tion of mutant MLINs delayed PN degeneration, reduced pathology, and ameliorated motor deficits in Sca1 mice. Conserved proteomic signature of Sca1 MLINs, shared with human SCA1 interneurons, involved the higher expression of FRRS1L, implicated in AMPA receptor trafficking. We thus propose that circuit-level def-icits upstream of PNs are one of the main disease triggers in SCA1

Probing cerebellar circuit dysfunction in rodent models of spinocerebellar ataxia by means of in vivo two-photon calcium imaging

Summary: Purkinje neuron degeneration characterizes spinocerebellar ataxia type 1, yet the comprehension of the impact on the broader cerebellar circuit remains incomplete. We here detail simultaneous in vivo two-photon calcium imaging of diverse neuronal populations in the cerebellar cortex of Sca1 mice while they are navigating a virtual environment. We outline surgical procedures and protocols to chronically record from identical neurons, and we detail data post-processing and analysis to delineate disease-related alterations in neuronal activity and sensorimotor-driven response properties.For complete details on the use and execution of this protocol, please refer to Pilotto et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Cortical Hyperexcitability in the Driver’s Seat in ALS

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by the degeneration of cortical and spinal motor neurons. With no effective treatment available to date, patients face progressive paralysis and eventually succumb to the disease due to respiratory failure within only a few years. Recent research has revealed the multifaceted nature of the mechanisms and cell types involved in motor neuron degeneration, thereby opening up new therapeutic avenues. Intriguingly, two key features present in both ALS patients and rodent models of the disease are cortical hyperexcitability and hyperconnectivity, the mechanisms of which are still not fully understood. We here recapitulate current findings arguing for cell autonomous and non-cell autonomous mechanisms causing cortical excitation and inhibition imbalance, which is involved in the degeneration of motor neurons in ALS. Moreover, we will highlight recent evidence that strongly indicates a cardinal role for the motor cortex as a main driver and source of the disease, thus arguing for a corticofugal trajectory of the pathology

Cortical circuit dysfunction in a mouse model of alpha-synucleinopathy in vivo

Blumenstock et al. report brain state-dependent hyperreactivity in somatosensory cortex months after striatal seeding of alpha-synuclein preformed fibrils. A concerted reduction of GAD67 positive interneurons argues for excitation/inhibition imbalance as a driver of cortical network dysfunction. Considerable fluctuations in cognitive performance and eventual dementia are an important characteristic of alpha-synucleinopathies, such as Parkinson's disease and Lewy Body dementia and are linked to cortical dysfunction. The presence of misfolded and aggregated alpha-synuclein in the cerebral cortex of patients has been suggested to play a crucial role in this process. However, the consequences of a-synuclein accumulation on the function of cortical networks at cellular resolution in vivo are largely unknown. Here, we induced robust a-synuclein pathology in the cerebral cortex using the striatal seeding model in wild-type mice. Nine months after a single intrastriatal injection of a-synuclein preformed fibrils, we observed profound alterations of the function of layer 2/3 cortical neurons in somatosensory cortex by in vivo two-photon calcium imaging in awake mice. We detected increased spontaneous activity levels, an enhanced response to whisking and increased synchrony. Stereological analyses revealed a reduction in glutamic acid decarboxylase 67-positive inhibitory neurons in the somatosensory cortex of mice injected with preformed fibrils. Importantly, these findings point to a disturbed excitation/inhibition balance as a relevant driver of circuit dysfunction, potentially underlying cognitive changes in alpha-synucleinopathies