Biology subjects

Hilscher, M. M.

Publications and source records attributed to Hilscher, M. M..

8 recordsLinked to original sources

bioRxiv2023

Single cell-resolution in situ sequencing elucidates spatial dynamics of multiple sclerosis lesion and disease evolution

Multiple sclerosis (MS) is a neurological disease characterised by multifocal lesions and smouldering pathology. While single-cell analyses have provided insights into neuropathology, cellular processes underlying MS remain poorly understood. We modelled the cellular dynamics of MS by examining temporal and regional rates of disease progression in the experimental autoimmune encephalomyelitis (EAE) mouse model. By performing single-cell spatial expression profiling using In situ sequencing, we annotated disease neighbourhoods during lesion evolution and found centrifugal propagation of active lesions. We demonstrated that disease-associated (DA) glia are dynamic and induced independently of lesions, preceding their formation. Single-cell spatial analysis of human archival MS spinal cord confirmed differential distribution of DA-glia, enabled deconvolution of active and inactive lesions into sub-compartments, and identification of new lesion areas. By establishing a spatial resource of mouse and human MS neuropathology at a single-cell resolution, our study unveils the intricate cellular dynamics underlying MS disease evolution.

cell biology↗

bioRxiv2023

Unraveling the role of Slc10a4 in auditory processing and sensory motor gating: implications for neuropsychiatric disorders?

BackgroundPsychiatric disorders, such as schizophrenia, are complex and challenging to study, partly due to the lack of suitable animal models. However, the absence of the Slc10a4 gene, which codes for a monoaminergic and cholinergic associated vesicular transporter protein, in knockout mice (Slc10a4 -/-), leads to the accumulation of extracellular dopamine. This makes them a potential animal model for schizophrenia, a disorder known to be associated with altered dopamine signaling in the brain. MethodsThe locomotion, auditory sensory filtering and prepulse inhibition (PPI) of Slc10a4 -/- mice were quantified and compared to wildtype (WT) littermates. Intrahippocampal electrodes were used to record auditory event-related potentials (aERPs) for quantifying sensory filtering in response to paired-clicks. The channel above aERPs phase reversal was chosen for reliably comparing results between animals, and aERPs amplitude and latency of click responses were quantified. WT and Slc10a4 -/- mice were also administered subanesthetic doses of ketamine to provoke psychomimetic behavior. ResultsBaseline locomotion during auditory stimulation was similar between Slc10a4 -/- mice and WT littermates. In WT animals, normal auditory gating was observed after i.p saline injections, and it was maintained under the influence of 5 mg/kg ketamine, but disrupted by 20 mg/kg ketamine. On the other hand, Slc10a4 -/- mice did not show significant differences between N40 S1 and S2 amplitude responses in saline or low dose ketamine treatment. Auditory gating was considered preserved since the second N40 peak was consistently suppressed, but with increased latency. The P80 component showed higher amplitude, with shorter S2 latency under saline and 5 mg/kg ketamine treatment in Slc10a4 -/- mice, which was not observed in WT littermates. Prepulse inhibition was also decreased in Slc10a4 -/- mice when the longer interstimulus interval of 100 ms was applied, compared to WT littermates. ConclusionThe Slc10a4 -/- mice responses indicate that cholinergic and monoaminergic systems participate in the PPI magnitude, in the temporal coding (response latency) of the auditory sensory gating component N40, and in the amplitude of aERPs P80 component. These results suggest that Slc10a4 -/- mice can be considered as potential models for neuropsychiatric conditions.

neuroscience↗

bioRxiv2021

Spatial cell type mapping of the oligodendrocyte lineage in the mouse juvenile and adult CNS with in situ sequencing

Oligodendrocytes show transcriptional heterogeneity but the regional and functional implications of this heterogeneity are less clear. Here, we apply in situ sequencing (ISS) to simultaneously probe the expression of 124 marker genes of distinct oligodendrocyte populations, providing comprehensive maps of corpus callosum, cingulate, motor and somatosensory cortex in the brain, as well as gray (GM) and white matter (WM) regions in the spinal cord, at juvenile and adult stages. We systematically compare abundances of these populations and investigate the neighboring preference of distinct oligodendrocyte populations. As previously described, we observed that oligodendrocyte lineage progression is more advanced in the juvenile spinal cord compared to the brain. Additionally, myelination is ongoing in the adult corpus callosum while it is mostly completed in the cortex. Interestingly, we found a medial-to-lateral gradient of oligodendrocyte lineage progression in the juvenile cortex, which could be linked to arealization, as well as a deep-to-superficial gradient with mature oligodendrocytes preferentially accumulating in the deeper layers of the cortex. We observed differences in abundances and population dynamics over time between GM and WM regions in the brain and spinal cord, indicating regional differences within GM and WM. We also found that oligodendroglia populations neighboring preferences are altered from the juvenile to the adult CNS. Thus, our ISS dataset reveals spatial heterogeneity of the oligodendrocyte lineage progression in the brain and spinal cord, which could be relevant to further investigate functional heterogeneity of oligodendroglia.

neuroscience↗

bioRxiv2021

Activity of CaMKIIa+ dorsal cochlear nucleus neurons are crucial for tinnitus perception but not for tinnitus induction

The dorsal cochlear nucleus (DCN) is a region known to integrate somatosensory and auditory inputs and is identified as a potential key structure in the generation of phantom sound perception, especially noise-induced tinnitus. Yet, how altered homeostatic plasticity of the DCN induces and maintains the sensation of tinnitus is not clear. Here, we chemogenetically decrease activity of a subgroup of DCN neurons, Ca2+/Calmodulin kinase 2 (CaMKII) positive DCN neurons, using Gi-coupled human M4 Designer Receptors Exclusively Activated by Designer Drugs (hM4Di DREADDs), to investigate their role in noise-induced tinnitus. Mice were exposed to loud noise (9-11kHz, 90dBSPL, 1h, followed by 2h of silence) and auditory brainstem responses (ABRs) and gap prepulse inhibition of acoustic startle (GPIAS) were recorded two days before and two weeks after noise exposure to identify animals with a significantly decreased inhibition of startle, indicating tinnitus but without permanent hearing loss. Neuronal activity of CaMKII+ neurons expressing hM4Di in the DCN was lowered by administration of clozapine-N-oxide (CNO). We found that acutely decreasing firing rate of CaMKII+ DCN units decrease tinnitus-like responses (p = 0.038, n = 11 mice), compared to the control group that showed no improvement in GPIAS (control virus; CaMKII-YFP + CNO, p = 0.696, n = 7 mice). Extracellular recordings confirmed CNO to decrease unit firing frequency of CaMKII-hM4Di+ mice and alter best frequency and tuning width of response to sound. However, these effects were not seen if CNO had been previously administered during the noise exposure (n = 6 experimental and 6 control mice). Our results suggest that CaMKII-hM4Di positive cells in the DCN are not crucial for tinnitus induction but play a significant role in maintaining tinnitus perception in mice.

neuroscience↗

bioRxiv2021

Comprehensive in situ mapping of human cortical transcriptomic cell types

The ability to spatially resolve the cellular architecture of human cortical cell types over informative areas is essential to understanding brain function. We combined in situ sequencing gene expression data and single-nucleus RNA-sequencing cell type definitions to spatially map cells in sections of the human cortex via probabilistic cell typing. We mapped and classified a total of 59,816 cells into all 75 previously defined subtypes to create a first spatial atlas of human cortical cells in their native position, their abundances and genetic signatures.

neuroscience↗

bioRxiv2020

Using cortical neuron markers to target cells in the dorsal cochlear nucleus

The dorsal cochlear nucleus (DCN) is the first auditory region that integrates somatosensory and auditory inputs. The region is of particular interest for auditory research due to the large incidence of somatic tinnitus and increased aberrant activity in other forms of tinnitus. Yet, the lack of useful genetic markers for in vivo manipulations hinders the elucidation of the DCN contribution to tinnitus pathophysiology. In this work, we assessed whether adeno-associated viral vectors (AAV) containing the calcium/calmodulin-dependent protein kinase 2 alpha (CaMKII) promoter and our mouse line of nicotinic acetylcholine receptor alpha 2 subunit (Chrna2)-Cre can be used to target specific DCN populations. The CaMKII promoter is usually applied in studies of principal neurons of neo and paleocortex while Chrna2-cre mice express Cre recombinase in cortical dendrite inhibiting interneurons. We found that CaMKII cannot be used to specifically target excitatory fusiform DCN neurons. EYFP expression driven by the CaMKII promoter was stronger in the fusiform layer but labelled cells showed a diverse morphology indicating that they belong to different classes of DCN neurons. Light stimulation after driving Channelrhodopsin2 (ChR2) by the CaMKII promoter generated spikes in some units but firing rate decreased when light stimulation coincide with sound presentation. Expression and activation of eArch3.0 (CaMKII driven) in the DCN produced spike inhibition in some units but, most importantly, sound-driven spikes were delayed by concomitant light stimulation. We explored the existence of Cre+ cells in the DCN of Chrna2-Cre mice by hydrogel embedding technique (CLARITY). There were almost no Cre+ cell bodies in the DCN; however, we observed profuse projections arising from the ventral cochlear nucleus (VCN). Anterograde labeling Cre dependent AAV injected in the VCN revealed two main projections: one arising in the ipsilateral superior olive and the contralateral medial nucleus of the trapezoid body (bushy cells) and a second bundle terminating in the DCN, suggesting the latter to be excitatory Chrna2+ T-stellate cells). Stimulating ChR2 expressing terminals (light applied on the DCN) of VCN Chrna2+ cells increased firing of sound responding and nonresponding DCN units. This work shows that molecular tools intensively used in cortical studies may be useful for manipulating the DCN especially in tinnitus studies.

neuroscience↗

bioRxiv2020

Hybridization-based In Situ Sequencing (HybISS): spatial transcriptomic detection in human and mouse brain tissue

Visualization of the transcriptome in situ has proven to be a valuable tool in exploring single-cell RNA-sequencing data, providing an additional dimension to investigate spatial cell typing and cell atlases, disease architecture or even data driven discoveries. The field of spatially resolved transcriptomic technologies is emerging as a vital tool to profile gene-expression, continuously pushing current methods to accommodate larger gene panels and larger areas without compromising throughput efficiency. Here, we describe a new version of the in situ sequencing (ISS) method based on padlock probes and rolling circle amplification. Modifications in probe design allows for a new barcoding system via sequence-by-hybridization chemistry for improved spatial detection of RNA transcripts. Due to the amplification of probes, amplicons can be visualized with standard epifluorescence microscopes with high-throughput efficiency and the new sequencing chemistry removes limitations bound by sequence-by-ligation chemistry of ISS. Here we present hybridization-based in situ sequencing (HybISS) that allows for increased flexibility and multiplexing, increased signal-to-noise, all without compromising throughput efficiency of imaging large fields of view. Moreover, the current protocol is demonstrated to work on human brain tissue samples, a source that has proven to be difficult to work with image-based spatial analysis techniques. Overall, HybISS technology works as a target amplification detection method for improved spatial transcriptomic visualization, and importantly, with an ease of implementation.

molecular biology↗

bioRxiv2019

Identification of spatial compartments in tissue from in situ sequencing data

Spatial organization of tissue characterizes biological function, and spatially resolved gene expression has the power to reveal variations of features with high resolution. Here, we propose a novel graph-based in situ sequencing decoding approach that improves recall, enabling precise spatial gene expression analysis. We apply our method on in situ sequencing data from mouse brain sections, identify spatial compartments that correspond with known brain regions, and relate them with tissue morphology.

neuroscience↗