Aberrant hippocampal Ca2+ micro-waves following synapsin-dependent adeno-associated viral expression of Ca2+ indicators.

Genetically encoded calcium indicators (GECIs) such as GCaMP are invaluable tools in neuroscience to monitor neuronal activity using optical imaging. The viral transduction of GECIs is commonly used to target expression to specific brain regions, can be conveniently used with any mouse strain of interest without the need for prior crossing with a GECI mouse line and avoids potential hazards due to the chronic expression of GECIs during development. A key requirement for monitoring neuronal activity with an indicator is that the indicator itself minimally affects activity. Here, using common adeno-associated viral (AAV) transduction procedures, we describe spatially confined aberrant Ca2+ micro-waves slowly travelling through the hippocampus following expression of GCaMP6, GCaMP7 or R-CaMP1.07 driven by the synapsin promoter with AAV-dependent gene transfer, in a titre-dependent fashion. Ca2+ micro-waves developed in hippocampal CA1 and CA3, but not dentate gyrus (DG) nor neocortex, were typically first observed at 4 weeks after viral transduction, and persisted up to at least 8 weeks. The phenomenon was robust, observed across laboratories with various experimenters and setups. Our results indicate that aberrant hippocampal Ca2+ micro-waves depend on the promoter and viral titre of the GECI, density of expression as well as the targeted brain region. We used an alternative viral transduction method of GCaMP which avoids this artifact. The results show that commonly used Ca2+-indicator AAV transduction procedures can produce artefactual Ca2+ responses. Our aim is to raise awareness in the field of these artefactual transduction-induced Ca2+ micro-waves and we provide a potential solution.

Microglial motility is modulated by neuronal activity and correlates with dendritic spine plasticity in the hippocampus of awake mice.

Microglia, the resident immune cells of the brain, play a complex role in health and disease. They actively survey the brain parenchyma by physically interacting with other cells and structurally shaping the brain. Yet, the mechanisms underlying microglial motility and significance for synapse stability, especially in the hippocampus during adulthood, remain widely unresolved. Here, we investigated the effect of neuronal activity on microglial motility and the implications for the formation and survival of dendritic spines on hippocampal CA1 neurons in vivo. We used repetitive two-photon in vivo imaging in the hippocampus of awake and anesthetized mice to simultaneously study the motility of microglia and their interaction with dendritic spines. We found that CA3 to CA1 input is sufficient to modulate microglial process motility. Simultaneously, more dendritic spines emerged in mice after awake compared to anesthetized imaging. Interestingly, the rate of microglial contacts with individual dendritic spines and dendrites was associated with the stability, removal, and emergence of dendritic spines. These results suggest that microglia might sense neuronal activity via neurotransmitter release and actively participate in synaptic rewiring of the hippocampal neural network during adulthood. Further, this study has profound relevance for hippocampal learning and memory processes.

Next generation genetically encoded fluorescent sensors for serotonin.

We developed a family of genetically encoded serotonin (5-HT) sensors (sDarken) on the basis of the native 5-HT1A receptor and circularly permuted GFP. sDarken 5-HT sensors are bright in the unbound state and diminish their fluorescence upon binding of 5-HT. Sensor variants with different affinities for serotonin were engineered to increase the versatility in imaging of serotonin dynamics. Experiments in vitro and in vivo showed the feasibility of imaging serotonin dynamics with high temporal and spatial resolution. As demonstrated here, the designed sensors show excellent membrane expression, have high specificity and a superior signal-to-noise ratio, detect the endogenous release of serotonin and are suitable for two-photon in vivo imaging.

Elevated expression of complement C4 in the mouse prefrontal cortex causes schizophrenia-associated phenotypes.

Accumulating evidence supports immune involvement in the pathogenesis of schizophrenia, a severe psychiatric disorder. In particular, high expression variants of C4, a gene of the innate immune complement system, were shown to confer susceptibility to schizophrenia. However, how elevated C4 expression may impact brain circuits remains largely unknown. We used in utero electroporation to overexpress C4 in the mouse prefrontal cortex. We found reduced glutamatergic input to pyramidal cells of juvenile and adult, but not of newborn C4-overexpressing (C4-OE) mice, together with decreased spine density, which mirrors spine loss observed in the schizophrenic cortex. Using time-lapse two-photon imaging in vivo, we observed that these deficits were associated with decreased dendritic spine gain and elimination in juvenile C4-OE mice, which may reflect poor formation and/or stabilization of immature spines. In juvenile and adult C4-OE mice, we found evidence for NMDA receptor hypofunction, another schizophrenia-associated phenotype, and synaptic accumulation of calcium-permeable AMPA receptors. Alterations in cortical GABAergic networks have been repeatedly associated with schizophrenia. We found that functional GABAergic transmission was reduced in C4-OE mice, in line with diminished GABA release probability from parvalbumin interneurons, lower GAD67 expression, and decreased intrinsic excitability in parvalbumin interneurons. These cellular abnormalities were associated with working memory impairment. Our results substantiate the causal relationship between an immunogenetic risk factor and several distinct cortical endophenotypes of schizophrenia and shed light on the underlying cellular mechanisms.

Hippocampal hyperactivity in a rat model of Alzheimer's disease.

Neuronal network dysfunction is a hallmark of Alzheimer's disease (AD). However, the underlying pathomechanisms remain unknown. We analyzed the hippocampal micronetwork in transgenic McGill-R-Thy1-APP rats (APPtg) at the beginning of extracellular amyloid beta (Aß) deposition. We established two-photon Ca2+ -imaging in vivo in the hippocampus of rats and found hyperactivity of CA1 neurons. Patch-clamp recordings in brain slices in vitro revealed increased neuronal input resistance and prolonged action potential width in CA1 pyramidal neurons. We did neither observe changes in synaptic inhibition, nor in excitation. Our data support the view that increased intrinsic excitability of CA1 neurons may precede inhibitory dysfunction at an early stage of Aß-deposition and disease progression.

Loss of Ryanodine Receptor 2 impairs neuronal activity-dependent remodeling of dendritic spines and triggers compensatory neuronal hyperexcitability.

Dendritic spines are postsynaptic domains that shape structural and functional properties of neurons. Upon neuronal activity, Ca2+ transients trigger signaling cascades that determine the plastic remodeling of dendritic spines, which modulate learning and memory. Here, we study in mice the role of the intracellular Ca2+ channel Ryanodine Receptor 2 (RyR2) in synaptic plasticity and memory formation. We demonstrate that loss of RyR2 in pyramidal neurons of the hippocampus impairs maintenance and activity-evoked structural plasticity of dendritic spines during memory acquisition. Furthermore, post-developmental deletion of RyR2 causes loss of excitatory synapses, dendritic sparsification, overcompensatory excitability, network hyperactivity and disruption of spatially tuned place cells. Altogether, our data underpin RyR2 as a link between spine remodeling, circuitry dysfunction and memory acquisition, which closely resemble pathological mechanisms observed in neurodegenerative disorders.

Memory trace interference impairs recall in a mouse model of Alzheimer's disease.

In Alzheimer's disease (AD), hippocampus-dependent memories underlie an extensive decline. The neuronal ensemble encoding a memory, termed engram, is partially recapitulated during memory recall. Artificial activation of an engram can restore memory in a mouse model of early AD, but its fate and the factors that render the engram nonfunctional are yet to be revealed. Here, we used repeated two-photon in vivo imaging to analyze fosGFP transgenic mice (which express enhanced GFP under the Fos promoter) performing a hippocampus-dependent memory task. We found that partial reactivation of the CA1 engram during recall is preserved under AD-like conditions. However, we identified a novelty-like ensemble that interfered with the engram and thus compromised recall. Mimicking a novelty-like ensemble in healthy mice was sufficient to affect memory recall. In turn, reducing the novelty-like signal rescued the recall impairment under AD-like conditions. These findings suggest a novel mechanistic process that contributes to the deterioration of memories in AD.

Chronic 2P-STED imaging reveals high turnover of dendritic spines in the hippocampus in vivo.

Rewiring neural circuits by the formation and elimination of synapses is thought to be a key cellular mechanism of learning and memory in the mammalian brain. Dendritic spines are the postsynaptic structural component of excitatory synapses, and their experience-dependent plasticity has been extensively studied in mouse superficial cortex using two-photon microscopy in vivo. By contrast, very little is known about spine plasticity in the hippocampus, which is the archetypical memory center of the brain, mostly because it is difficult to visualize dendritic spines in this deeply embedded structure with sufficient spatial resolution. We developed chronic 2P-STED microscopy in mouse hippocampus, using a 'hippocampal window' based on resection of cortical tissue and a long working distance objective for optical access. We observed a two-fold higher spine density than previous studies and measured a spine turnover of ~40% within 4 days, which depended on spine size. We thus provide direct evidence for a high level of structural rewiring of synaptic circuits and new insights into the structure-dynamics relationship of hippocampal spines. Having established chronic super-resolution microscopy in the hippocampus in vivo, our study enables longitudinal and correlative analyses of nanoscale neuroanatomical structures with genetic, molecular and behavioral experiments.

Dysfunction of Somatostatin-Positive Interneurons Associated with Memory Deficits in an Alzheimer's Disease Model.

Alzheimer's disease (AD) is characterized by cognitive decline and neuronal network dysfunction, but the underlying mechanisms remain unknown. In the hippocampus, microcircuit activity during learning and memory processes is tightly controlled by O-LM interneurons. Here, we investigated the effect of beta-amyloidosis on O-LM interneuron structural and functional connectivity, combining two-photon in vivo imaging of synaptic morphology, awake Ca2+ imaging, and retrograde mono-transsynaptic rabies tracing. We find severely impaired synaptic rewiring that occurs on the O-LM interneuron input and output level in a mouse model of AD. Synaptic rewiring that occurs upon fear learning on O-LM interneuron input level is affected in mice with AD-like pathology. This process requires the release of acetylcholine from septo-hippocampal projections. We identify decreased cholinergic action on O-LM interneurons in APP/PS1 mice as a key pathomechanism that contributes to memory impairment in a mouse model, with potential relevance for human AD.