Data from: Structural and functional features of medium spiny neurons in the BACHDΔN17 mouse model of Huntington’s disease

Luebke, Jennifer 1 ; Goodliffe, Joseph1 ; Rubakovic, Anna1 ; Pathak, Dhruba1 ; Chang, Wayne1

Published Jul 18, 2024; Updated Feb 27, 2025 on Dryad. https://doi.org/10.5061/dryad.v6wwpzh42

Data files

Jul 18, 2024 version files 9.58 GB

Oct 07, 2024 version files 9.58 GB

Behavior_Data.zip
4.74 GB
Electrophysiology_Data.zip
4.84 GB
Morphology_Data_10-3-2024_upload.zip
108.86 KB
README.md
6.40 KB

Feb 27, 2025 version files 9.58 GB

Behavior_Data_04112025.zip
4.74 GB
Electrophysiology_Data_04112025.zip
4.84 GB
Morphology_Data_04112025.zip
109.72 KB
README.md
7 KB

Abstract

In the BACHD mouse model of Huntington’s disease (HD), deletion of the N17 domain of the Huntingtin gene (BACHDΔN17, Q97) has been reported to lead to nuclear accumulation of mHTT and exacerbation of motor deficits, neuroinflammation and striatal atrophy (Gu et al., 2015). Here we characterized the effect of N17 deletion on dorsolateral striatal medium spiny neurons (MSNs) in BACHDΔN17 (Q97) and BACWTΔN17 (Q31) mice by comparing them to MSNs in wildtype (WT) mice. Mice were characterized on a series of motor tasks and subsequently whole cell patch clamp recordings with simultaneous biocytin filling of MSNs in in vitro striatal slices from these mice were used to comprehensively assess their physiological and morphological features. Key findings include that: Q97 mice exhibit impaired gait and righting reflexes but normal tail suspension reflexes and normal coats while Q31 mice do not differ from WT; intrinsic membrane and action potential properties are altered -but differentially so- in MSNs from Q97 and from Q31 mice; excitatory and inhibitory synaptic currents exhibit higher amplitudes in Q31 but not Q97 MSNs, while excitatory synaptic currents occur at lower frequency in Q97 than in WT and Q31 MSNs; there is a reduced total dendritic length in Q31 -but not Q97- MSNs compared to WT, while spine density and number did not differ in MSNs in the three groups. The findings that Q31 MSNs differed from Q97 and WT neurons with regard to some physiological features and structurally suggest a novel role of the N17 domain in the function of WT Htt. The motor phenotype seen in Q97 mice was less robust than that reported in an earlier study (Gu et al., 2015), and the alterations to MSN physiological properties were largely consistent with changes reported previously in a number of other mouse models of HD. Together this study indicates that N17 plays a role in the modulation of the properties of MSNs in both mHtt and WT-Htt mice, but does not markedly exacerbate HD-like pathogenesis in the BACHD model.

Description of the data and file structure

Our raw data for motor behavior (video files: each_individual_subject.MOV); rubric for scoring of the motor behavior (behavior rubric for scoring.xlsx); DN17 Behavior Data for Figure 1.xlsx Note that in the behavior by phenotype datasheet values for the different groups are color-coded as follows: black- WT; blue- Q31; red- Q97.

Our raw data for electrophysiological assessments including Synaptic Events raw data (.ABF and .IBW files generated by MiniAnalysis from .DAT files for excitatory -EPSC- and inhibitory -IPSC- synaptic currents); raw basic membrane property data (Electrophysiology raw data files; .DAT files generated by PatchMaster for ALL physiological data recorded from each cell for each subject); and related excel files with numeric data (.xlsx)

Our raw data analyses spreadsheets for morphological variables quantified from reconstructed medium spiny neurons from which recordings were obtained (dendrite data.xlsx; spine data.xlsx)

DESCRIPTIONS

Behavior_Data

This folder contains:

raw data for motor behavior (video files: each_individual_subject.MOV)

rubric for scoring of the motor behavior (behavior rubric for scoring.xlsx): behavior on tests of tail suspension, gait and righting on a scale from 0 (normal behavior) to 4 (rigid paralysis, no forward motion, righting >10s)

DN17 Behavior Data for Figure 1.xlsx: information in database includes 3 sheets DN17 Subjects & Weights; Behavior by Genotype; Behavior by Sex.

Variables quantified and shown in the spreadsheet include: Animal identifier number; Sex; Date (of sacrifice); Body (weight in grams); Brain (weight in grams); Tail (suspension score); Gait (forward motion score); Righting (time); Coat appearance and; Overall Score

“null” represents a missing value in the database that is either not applicable (e.g. asymptote previously achieved) or not available (e.g. subject did not respond to a given test)

Electrophysiology_Data

This folder contains:

Synaptic events raw data (.abf and .ibw for excitatory -EPSC- and inhibitory IPSC- synaptic currents);

raw basic membrane property data (Electrophysiology raw data files; .dat files for each cell from each subject);

Excel files with numeric data for all synaptic current properties (DN17 EPSC Data and DN17 IPSC Data in .xlsx format) and basic membrane properties (DN17 Ephys in xlsx format); each row includes all raw electrophysiological data for a cell from a given subject (sheet 1 shows raw data from all cells included in the study) other sheets show analyses by animal genotype, by firing rates (spike counts), by medium spiny neuron type (D1 versus D2 type); tables and charts shown in the electrophysiology figures are also provided.

Variables included are:

For Synaptic Event Data- Time (total time recorded in ms); and MEAN- Amplitude (of events in pA); Rise (time of events in ms); Decay (time of events in ms); Area (of events in pA/ms); Baseline (value in pA); Noise (value in pA); Group (0 indicates single type f synaptic event); Channel (0 indicates one acquisition channel);10-90Rise (rise time in ms); HalfWidth (event half-with in ms); Rise50 (event rise time in ms); Peak Dir (direction of current -1 is inward, 1 is outward); Burst#BurstE# (bursting behavior, 0 indicates none);10-90Slope (indicates rise time pA/ms); Rel Time (relative time from onset of first event in ms).

For Basic Membrane Electrophysiology Data- Intrinsic Membrane Properties: GFP+/- (indicates whether the cell was D1 or D2 based on presence or absence of GFP); GFP Check (indicates confirmation of GFP status); Drug (no drugs were applied so value is always “null”; Biocytin filled (indicates whether neuron was filled during recording and thus subsequently used for morphometrics);Tau (membrane time constant in ms); Rn (input resistance in MOhm); Vr (resting membrane potential in mV). Action Potential (AP) Properties: Thrshld (AP threshold in mV); Amp (AP amplitude in mV); Dur@1/2 (AP duration at half amplitude in ms); Rise (AP rise time in ms); Fall (AP fall time in ms); Rheobase (AP rheobase in pA).

“null” represents a missing value in the database that is either not applicable (e.g. asymptote previously achieved) or not available (e.g. cell did not respond to a given stimulus)

Morphology_Data

Our raw data for each individual neuron reconstruction are posted as .SWC files at NeuroMorpho.org and noted in the paper: https://neuromorpho.org/bylab.jsp#top (each cell is identified by name, e.g. Q31-D1-Aug28IR3c-whole-cell)

This folder contains:

Excel databases containing raw data for each cell from each subject are given in two databases- Dendrites, and Spines showing all quantitative information for these morphological features as shown in the morphology figures by genotype.

Variables included are:

For Dendritic Data- *for each cell that was reconstructed we provide: *Total dendritic length (in microns); branch order count; branch order average length (in microns); Sholl analysis (concentric 20 micron rings from 0-20 to 260-280 microns)

For Spine Data- *DENSITY spreadsheet- for each cell that was reconstructed we provide: *Spine Density Counts, shown in columns B to H; Spines (count); none Spine (unclassified spine type count, typically 0); thin Spine (count); stubby Spine (count); mushroom Spine (count); filopodia Spine (count); Length Total (of dendrites on which spines were assessed, in microns)- followed by calculated density in columns J to X. Mean values are given in columns Z to AI. SHOLL spreadsheet- for each cell that was reconstructed we provide: number and density of spines as a function of distance from soma in concentric 10 micron rings (varies depending on cell).

“null” represents a missing value in the database that is not applicable (e.g. asymptote previously achieved or end of process achieved in Sholl rings)

Version changes

22 Feb 2025 : These include changing the n of subjects in two databases and addition of one variable in the morphology database. For the behavior database we changed the n to 6 and uploaded a revised database indicating this simple change. We also added total node data for figure 5 has been uploaded to the dryad morphology database.

11 April 2025: These include removal of t test statistical outcomes that were preliminary in the excel databases (note that actual statistical analyses reported in the paper with MatLab analyses are found in the Electrophysiology folder).

Sharing/Access information

Links to other publicly accessible locations of the data:

NeuroMorpho.Org

http://neuromorpho.org/, Luebke archive includes the morphological reconstruction of the neurons studied in Goodliffe et al., 2020.

Behavior Data Videos:

Mice were weighed and assessed for quality of their coat prior to being assessed on three different tests of motor function including: tail suspension, self-righting, and gait assessment [49]. Coat quality was rated on a scale of 0 (normal, shiny)- 4 (matted, unkempt, yellowing). During testing, mice were videotaped and subsequently videos were analyzed by three researchers blinded to gender and genotype. For tail suspension, mice were suspended by their tails, one third of the length away from the base of the tail. Mice were suspended and videotaped for 15 seconds. The tail suspension task was scored on a scale of 0 (normal splaying of toes and legs)- 4 (curled toes, both legs close to body) For assessment of self-righting ability, mice were placed onto their backs and then released. This was repeated twice and videotaped for 20 seconds each time. Self-righting was scored as 0 (mouse rights self under 5 seconds)- 4 (mouse takes more than 5 seconds to right itself). For assessment of gait, mice were placed in an 18 inch x 18 inch x 5-inch arena and videotaped for 60 seconds. Mice were assigned a gait score from 0 (normal limb movement, feet under body)- 4 (limbs splay when walking, unsteady) based on the scoring system previously reported [49]. Total score was obtained by summing the scores on tail suspension, self-righting, gait, and coat quality.

Electrophysiology .DAT files:

Whole-cell patch clamp recordings were obtained from visually identified MSNs in the dorsolateral quadrant of the striatum [53, 55]. Electrodes were pulled on a Flaming and Brown horizontal pipette puller (model P87, Sutter Instrument) and filled with potassium methanesulfonate (KMS) internal solution, concentrations inmMas follows: (KCH3SO3 122, MgCl2 2, EGTA 5, NaHEPES 10, Na2ATP 5 and 1% biocytin). Electrodes in Ringer’s solution had a resistance of 4–6MO. Electrophysiology data was obtained using PatchMaster software (HEKA Elektronik) and EPC-9/EPC-10 amplifiers (HEKA Elektronik). Assessment of intrinsic membrane and action potential properties. Passive membrane properties (resting membrane potential–Vr-, input resistance–Rn- and membrane time constant –τ-) and action potential firing properties were assessed under current clamp [50–53]. Vr was measured as the voltage in the absence of current injection. A series of 200 ms or 2s hyperpolarizing and depolarizing current steps was applied for the rest of the measures. The voltage responses to each step were measured at steady state and plotted on a voltage-current graph: Rn was calculated as the slope of the best-fit line through the linear portion of the plot. Membrane time constant was measured by fitting a single exponential function to the membrane voltage response to the -10 pA hyperpolarizing step. Rheobase was determined with a 10 s depolarizing current dual ramp (0–100 pA, 0–200 pA; 3.03 kHz sampling frequency). Single AP properties, including threshold and amplitude, were measured on the second evoked AP in a 200 ms current-clamp series in which the current step elicited 3 or more action potentials. An expanded timescale and the linear measure tool were used in FitMaster analysis software (HEKA Elektronik). Finally, a series of 2 s hyperpolarizing and depolarizing steps (-200 to +450 pA, using 25 or 50 pA increments, 12.5kHz sampling frequency) was used to assess repetitive AP firing. Firing rate in response to current steps was analyzed with a generalized linear model, using the genotype, MSN type, rheobase, input resistance, injected current level and their respective interactions as independent variables. Assessment of spontaneous excitatory postsynaptic currents. AMPA receptor-mediated spontaneous excitatory currents (sEPSCs) were recorded for 2 min at a holding potential of -80 mV (6.67 kHz sampling frequency). MiniAnalysis software (Synaptosoft) was used to assess synaptic current properties including: frequency, amplitude, area, time to rise and time to decay. For assessment of kinetics, the rise and decay of averaged traces were each fit to a single- exponential function. For all synaptic current measurements, the event detection threshold was set at the maximum root mean squared noise level (5 pA). Morphology XLS files (reconstructions are posted at NeuroMorpho.org: Streptavidin-Alexa labeling of biocytin filled neurons Following recordings, brain slices were sandwiched between filter paper in fixative (4% paraformaldehyde) overnight at 4˚C. Next, slices were washed in 0.1Mphosphate buffered saline (PBS) 3x 5 minutes. Slices were then incubated in 0.1% Tx-100/PBS for 2 h at room temperature (RT), then incubated in Streptavidin-Alexa 568 (1:500, 0.1% Tx-100/PBS) for 2d at 4˚C, followed by subsequent washes in 0.1MPBS and stored in anti-freeze solution (30% glycerol, 30% ethylene glycol in 0.05Mphosphate buffer) [50–53].

Confocal imaging

For verification of eGFP labeling of biocytin-filled cells, slices were placed in an inverted well slide and temporarily coverslipped for an initial imaging of the soma using a 40x oil immersion objective on a Leica SPE confocal microscope. Cell somata were scanned in their entirety in two channels, 568 and 488, to detect filled cells and the presence or absence of somal eGFP, respectively. Cells were classified as D2/eGFP+ if the 488 and 568 signals overlapped along the x-, y-, and z- planes, D1/eGFP- cells lacked eGFP signal in their soma. Brain slices that contained cells met rigid criteria for morphometric analysis (zero to minimal dendritic varicosities, high signal-to-noise ratio indicative of a well filled cell, few cut branches) were either mounted in Prolong Antifade (Life Technologies) after streptavidin-Alexa staining or used for immunohistochemistry. MSNs were scanned for dendritic morphometric analyses in their entirety using a Leica SPE confocal microscope with a 40x oil immersion objective obtaining a voxel size of 0.27 x 0.27 x 0.5 μm (as described previously [50,51]). Images were deconvolved with AutoQuant Software and 8-bit images imported into NeuronStudio for reconstruction and quantitative analysis. For assessment of spines, 3 dendrites from each cell were chosen for imaging of spines. Each dendrite was selected by dividing the entire dendritic arbor into equal thirds and selecting the dendrite that were located perpendicular to the z-plane. In order to obtain the necessary resolution for spine sub-typing, dendrites were imaged with a 63x oil emersion objective (1.4 NA) with a 2.5 zoom using a Leica SPE laser scanning confocal microscope. The resulting voxel size was 0.034 x 0.034 x 0.17 μm. Each dendrite was imaged in its entirety from the soma to the distal dendrite ending. Z-stacks were deconvolved using Auto-Quant software and 8-bit images imported into Neurolucida 360 for reconstruction, spine sub-typing, and analysis. Spine sub-types were classified based on spine head diameter and spine head distance from the dendritic shaft. Spines were classified as: thin (diameter .0.6 μm), mushroom (diameter > 0.6 μm), stubby (spines lacking a neck), or filopodia (length > 3 μm) [50,52].

Data from: Structural and functional features of medium spiny neurons in the BACHDΔN17 mouse model of Huntington’s disease

Data files

Abstract

README: Data from: Structural and functional features of medium spiny neurons in the BACHDΔN17 mouse model of Huntington’s disease

Description of the data and file structure

DESCRIPTIONS

Version changes

Sharing/Access information

Methods

Works referencing this dataset