DOI: 10.5061/dryad.v6wwpzh42
title: Data from: Structural and functional features of medium spiny neurons in the BACHDΔN17 mouse model of Huntington’s disease

description: 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: <em>Behavior Data Videos: </em>
   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.
   <em>Electrophysiology .DAT
 files:</em>  
 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].
   <em>Confocal
 imaging</em>   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 &gt; 0.6 μm), stubby (spines lacking a neck), or
 filopodia (length &gt; 3 μm) [50,52].
description: # 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  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 &gt;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](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/](http://neuromorpho.org/), Luebke archive includes the morphological reconstruction of the neurons studied in Goodliffe et al., 2020.