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Altering cortical input unmasks synaptic phenotypes in the YAC128 cortico-striatal co-culture model of… Schmidt, Mandi E; Buren, Caodu; Mackay, James P; Cheung, Daphne; Dal Cengio, Louisa; Raymond, Lynn A; Hayden, Michael R Jun 27, 2018

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METHODOLOGY ARTICLE Open AccessAltering cortical input unmasks synapticphenotypes in the YAC128 cortico-striatalco-culture model of Huntington diseaseMandi E. Schmidt1, Caodu Buren2,3, James P. Mackay2, Daphne Cheung1, Louisa Dal Cengio1,Lynn A. Raymond2 and Michael R. Hayden1*AbstractBackground: Huntington disease (HD) is a fatal neurodegenerative disorder caused by a CAG expansion in thehuntingtin (HTT) gene, leading to selective and progressive neuronal death predominantly in the striatum. MutantHTT expression causes dysfunctional cortico-striatal (CS) transmission, loss of CS synapses, and striatal medium spinyneuron (MSN) dendritic spine instability prior to neuronal death. Co-culturing cortical and striatal neurons in vitropromotes the formation of functional CS synapses and is a widely used approach to elucidate pathogenicmechanisms of HD and to validate potential synapto-protective therapies. A number of relevant in vivo synapticphenotypes from the YAC128 HD mouse model, which expresses full-length transgenic human mutant HTT, arerecapitulated in CS co-culture by 21 days in vitro (DIV). However, striatal spine loss, which occurs in HD patients andin vivo animal models, has been observed in YAC128 CS co-culture in some studies but not in others, leading todifficulties in reproducing and interpreting results. Here, we investigated whether differences in the relativeproportion of cortical and striatal neurons alter YAC128 synaptic phenotypes in this model.Results: YAC128 MSNs in 1:1 CS co-culture exhibited impaired dendritic length and complexity compared to wild-type,whereas reducing cortical input using a 1:3 CS ratio revealed a dramatic loss of YAC128 MSN dendritic spines. Chimericexperiments determined that this spine instability was primarily cell autonomous, depending largely on mutant HTTexpression in striatal neurons. Moreover, we found that spontaneous electrophysiological MSN activity correlatedclosely with overall dendritic length, with no differences observed between genotypes in 1:3 co-cultures despitesignificant YAC128 spine loss. Finally, limiting cortical input with a 1:3 CS ratio impaired the basal survival of YAC128neurons at DIV21, and this was partially selective for dopamine- and cAMP-regulated phosphoprotein 32-positive MSNs.Conclusions: Our findings reconcile previous discordant reports of spine loss in this model, and improve the utility andreliability of the CS co-culture for the development of novel therapeutic strategies for HD.Keywords: Huntington disease, huntingtin, synapse, spine, dendrite, corticostriatal co-culture, YAC128, DARPP32BackgroundHuntington disease (HD) is a devastating neurodegener-ative disorder caused by a CAG repeat expansion inexon 1 of the huntingtin (HTT) gene [1]. The disease ischaracterized neuropathologically by progressive striatalatrophy and cortical degeneration, leading to impairedcognitive, psychiatric, and motor function [2]. Althoughovert disease onset occurs during mid-life, human andanimal studies have collectively demonstrated thatcortico-striatal (CS) synaptic dysfunction occurs early inHD and likely contributes to later neuronal loss [2–5].Medium spiny neurons (MSNs) make up the vast ma-jority of the striatal neuronal population, and receive ahigh level of glutamatergic input from the cortex [6, 7].MSNs are the earliest and most-affected neuronal popu-lation in HD, undergoing significant loss of dendriticstructure and spines with disease progression in humansand animal models [8–13]. Dysregulated glutamate* Correspondence: mrh@cmmt.ubc.ca1Centre for Molecular Medicine and Therapeutics, BC Children’s HospitalResearch Institute, University of British Columbia, 950 West 28th Avenue,Vancouver V5Z 4H4, CanadaFull list of author information is available at the end of the article© Hayden et al. 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Schmidt et al. BMC Biology  (2018) 16:58 https://doi.org/10.1186/s12915-018-0526-3release at CS synapses in addition to intrinsic MSN prop-erties are hypothesized to ultimately cause selective vul-nerability of this cell type [14–17]. However, due to theplasticity of neural connections, CS synaptic dysfunctionas well as MSN spine and synapse loss may be therapeut-ically reversible before neuronal death occurs [4].The CS neuronal co-culture is a commonly-used in vitromodel which consists of cortical and striatal neurons platedhomogenously, generally at either a 1:1 or 1:3 cortical:stria-tal ratio [18]. This method partially recapitulates in vivocircuitry and MSN development, and permits functionalCS synapses to be studied in relative isolation from othermodulatory neurotransmitters or neuronal inputs [19, 20].Previous characterization has been performed in 1:1embryonic CS co-cultures from wild-type (WT) andYAC128 mice (expressing a yeast artificial chromosomecontaining the full-length human mutant HTT (mHTT)gene encoding 125–128 glutamines [21, 22]) [23, 24].These studies demonstrated altered extrasynapticN-methyl D-aspartate (NMDA) receptor function inYAC128 co-cultured MSNs, accompanied by enhancedsusceptibility to excitotoxicity as well as reduced CS ex-citatory synapse activity by 21 days in vitro (DIV), aphenotype undetectable in vivo until 6–7 months of age[15, 25]. Morphology was also evaluated by transfectingMSNs with yellow fluorescent protein (YFP) at the timeof plating and, although this analysis showed stunteddendritic complexity in 1:1 co-cultured YAC128 MSNscompared to WT, no difference in spine numbers wasobserved [23]. This is in stark contrast to studies fromanother group, in which staining for dopamine- andcAMP-regulated phosphoprotein 32 (DARPP32), amarker of mature MSNs, was used for morphologicalanalysis instead of transfected YFP to show dramaticspine loss in 1:3 CS co-cultured postnatal YAC128MSNs [13, 26]. The methodological factors underlyingthe ability to observe this highly relevant HD phenotyperemain unknown. DARPP32+ WTMSNs in 1:3 co-cultureexhibit less dendritic complexity and fewer spines andsynapses than in 1:1 co-culture, indicating that reducingcortical input impairs WT MSN development in vitro[18]. However, the impact of altering cortical input in thecontext of HD has not been evaluated.In the present study, we have explored whether spineloss is a reproducible feature of HD in this model andinvestigated the potential methodological factors con-tributing to the emergence of this phenotype.ResultsReducing cortical input elucidates robust YAC128 MSNspine loss in CS co-cultureWe first sought to evaluate the effect of altered corticalinput on HD-like phenotypes in vitro by culturing WTand YAC128 MSNs with cortical neurons side-by-side atboth 1:1 and 1:3 CS ratios, using identical total celldensities. We utilized DARPP32 immunofluorescencestaining for MSN morphological analysis in order to re-main consistent with the methodology used by Wu et al.[13], as well as to avoid the requirement for YFP nucleo-fection, which we found to reduce the general health ofneuronal cultures. Striatal DARPP32 is decreased inseveral models of HD, including YAC128 mice [21, 22,27–31]. To confirm that potentially altered YAC128DARPP32 expression levels would not interfere withaccurate structural analysis, we measured immunofluores-cence staining intensity in each culture condition. Weco-stained for the dendritic marker microtubule-associatedprotein 2 (MAP2) and imaged both channels at identicallaser intensities across samples. We observed no differ-ences in dendritic DARPP32 intensity normalized toMAP2 intensity (Fig. 1a, b), indicating that MSNDARPP32 expression does not obviously differ betweengenotypes and that this is an appropriate method for den-dritic and spine analysis in this model.Using this approach, we observed a subtle reductionin MSN total spine density (90% of WT) and anon-significant decrease in mature mushroom spinedensity (88% of WT) in DIV21 1:1 YAC128 cultures(Fig. 1c, Di, Dii). Remarkably, limiting excitatory inputusing a 1:3 ratio dramatically enhanced this pheno-type, such that the number of total and maturemushroom spines in YAC128 MSNs were reduced toapproximately 78% and 63% of WT 1:3 levels, respect-ively (Fig. 1c, Di, Dii). We did not observe significant dif-ferences in the density of immature (stubby, thin, andfilopodia) spine types (Fig. 1c, Diii), suggesting a selectiveimpairment in the stability of functionally mature spines.A previous study using injection of Lucifer yellowfluorescent dye into striatal neurons in brain slices foundYAC128 MSN spine loss at 12 months of age, but not at6 months [13]. We confirmed this finding using a simpleGolgi stain method and observe that spine density valuesand the degree of YAC128 total spine loss at 12 monthsin vivo (71% of WT) are accurately recapitulated in 1:3CS co-cultures (Fig. 1e, f and Additional file 1).To further investigate the relationship between MSNspine density and cortical input, we compared two add-itional CS ratios (1:2 and 1:5) side-by-side with 1:1 and1:3 conditions. In this set of experiments, there were nosignificant genotypic differences in either total or maturemushroom spine densities using a 1:1 ratio. We ob-served a negative correlation between total and maturemushroom spine densities versus the proportion ofstriatal cells at the time of plating in both genotypes(Fig. 1Gi, Gii). Interestingly, there was a significant inter-action between genotype and CS ratio, with the pheno-type becoming more severe with increasing proportionof striatal cells at plating. This indicates that YAC128Schmidt et al. BMC Biology  (2018) 16:58 Page 2 of 12Fig. 1 YAC128 MSNs co-cultured with cortical neurons at a 1:3 CS ratio recapitulate in vivo spine loss. WT and YAC128 (Y128) co-cultures weregenerated at either a 1:1 or 1:3 CS ratio and processed at DIV21 for DARPP32 and MAP2 immunocytochemistry, imaging, and spine analysis inNeuronStudio. (a) Sample images of DARPP32- and MAP2-stained dendrites in CS co-culture (scale bar = 5 μm). (b) Quantification of DARPP32staining intensity normalized to MAP2 intensity reveals no differences between genotypes or conditions [n = 30(3); two-way ANOVA withBonferroni post-hoc analysis]. (c) Sample images of DARPP32-stained spines on secondary or tertiary dendrites in co-cultured MSNs at higherexposure (scale bar = 5 μm). The differences in numbers of (Di) total and (Dii) mature mushroom, but not (Diii) immature spines, are exacerbatedin 1:3 co-cultured YAC128 MSNs [n = 32(4); two-way ANOVA with Bonferroni post-hoc analysis; *p < 0.05, ***p < 0.001]. (e) Representative Golgistaining of striatal MSNs in vivo (scale bar = 5 μm). (f) Golgi analysis confirms that reduced MSN total spine number occurs by 12 months of agein the YAC128 striatum, to a similar degree as in 1:3 co-cultures [n = 4–5 6-month-old animals and 3 12-month-old animals per genotype;two-way ANOVA with Bonferroni post-hoc analysis; **p < 0.01]. Individual data values for graph in F are available in Additional file 1. A linearcorrelation exists between (Gi) total and (Gii) mushroom spines versus the proportion of striatal cells at plating. A significant interaction occursbetween striatal proportion and genotype [n = 30(3); two-way ANOVA with Bonferroni post-hoc analysis; *p < 0.05, **p < 0.01, ***p < 0.001]Schmidt et al. BMC Biology  (2018) 16:58 Page 3 of 12MSN spine stability is progressively more sensitive thanWT to reduced amounts of cortical input.Finally, we evaluated the impact of altering the totalcell number per well (150,000, 170,000 or 230,000 in24-well plates), keeping the CS ratio consistent at 1:3.We did not find an effect of initial plating density on thepresence or severity of the YAC128 MSN spine pheno-type at DIV21 (Additional file 2: Figure S1).YAC128 spine instability is predominantly MSN intrinsicAn impaired developmental increase in miniature excita-tory post-synaptic current (mEPSC) frequency fromDIV14 to DIV21 in 1:1 co-cultured YAC128 MSNs com-pared to WT was previously reported [23]. Chimericco-cultures (WT striatal MSNs plated with YAC128 cor-tical neurons, or vice versa) exhibited an intermediatephenotype, indicating that altered excitatory functionalconnectivity is partially dependent on mHTT expressionin both pre- and post-synaptic compartments [23]. Weutilized a similar strategy to determine the relativecontribution of each cell type to MSN spine stability in1:3 co-cultures. We discovered that the difference intotal spine numbers between WT and YAC128 was en-tirely dependent on mHTT expression in the MSN(Fig. 2a, Bi). When we specifically evaluated maturemushroom spines, we found a small contribution fromcortical mHTT expression, with chimeric cultures dem-onstrating a trend to a more intermediate mushroomspine density (Fig. 2a, Bii). When assessed by t test, WTMSNs co-cultured with YAC128 cortical neurons hadfewer mushroom spines and a greater number ofimmature spines than those co-cultured with WT corticalneurons, despite similar total spine densities (Fig. 2a, b).Thus, cortical mHTT expression alters the ratio of ma-ture/immature spines in WT neurons. These results sug-gest that mHTT expression primarily, but not exclusively,in the MSN impairs mechanisms of spine development orstability in response to reduced cortical input.Decreasing cortical input masks the YAC128 MSNdendritic complexity phenotype in CS co-cultureInterestingly, in comparison to MSN spine density, wediscovered an opposite effect of CS ratio on MSN den-dritic structure by Sholl analysis. A robust impairmentin total dendritic length and complexity was observed inDIV21 1:1 co-cultured YAC128 MSNs compared to WT(Fig. 3a, Bi, Bii), in agreement with previous results [23].However, when a 1:3 CS ratio was utilized, WT MSNdendritic development became impaired, resulting in amuch smaller genotypic difference between WT andYAC128 (Fig. 3a, Bi, Bii). Thus, differential elucidationof YAC128 MSN dendritic or spine phenotypes can beachieved by manipulation of the CS ratio.YAC128 MSN dendritic and spine phenotypes aredevelopmental in CS co-cultureWe next sought to determine at what time-point theidentified structural phenotypes are present in CSco-culture. When our DIV21 results were plotted overtime along with DIV14 and DIV18 data from the samecultures, we observed that most of the identifiedYAC128 spine and dendrite alterations were present byFig. 2 YAC128 spine instability is predominantly MSN intrinsic. WT, YAC128, and chimeric co-cultures generated at a 1:3 CS ratio were processedat DIV21 for DARPP32 immunocytochemistry, imaging, and spine analysis. (a) Sample images of DARPP32-stained spines in pure or chimericco-cultured MSNs (scale bar = 5 μm). (Bi) Total spine density values in chimeric cultures are similar to pure cultures of the same MSN genotype.(Bii) Mature mushroom and (Biii) immature spine numbers are affected by both striatal (STR) and cortical (CTX) mHTT expression [n = 32(4);one-way ANOVA with Bonferroni post-hoc analysis; **p < 0.01, ***p < 0.001]. Student’s t test was used to compare WT STR/WT CTX and WT STR/Y128 CTX [n = 32(4); Student’s t test; #p < 0.05]Schmidt et al. BMC Biology  (2018) 16:58 Page 4 of 12DIV18 and all could be attributed to impaired develop-ment of YAC128 MSNs after DIV14, at which time therewere no discernable phenotypes (Additional file 3: FigureS2 and Additional file 4: Figure S3).CS plating ratio influences electrophysiologicalphenotypes in YAC128 MSNsTo determine the functional impact of altering CS ratio,whole-cell patch-clamp electrophysiology was used torecord mEPSCs and basal membrane capacitance fromMSNs in 1:1 and 1:3 co-cultures at DIV14 and DIV21.Previously published data showed an increase in mEPSCfrequency from DIV14 to DIV21 in 1:1 co-cultures,which was blunted in YAC128 MSNs [23]. We observeda similar trend in the current study, although there wasno significant genotypic difference between WT andYAC128 at DIV21 (Fig. 4a, Bi). However, when a 1:3 ra-tio was utilized, there was only a small increase inmEPSC frequency from DIV14 to DIV21 for both WTand YAC128 such that there was no longer a trend to adifference between genotypes at DIV21 (Fig. 4a, Bii).This is consistent with a prior study which found re-duced mEPSC frequency in DIV18 1:3 co-cultured WTMSNs compared to 1:1 [18]. Membrane capacitance, ameasure of overall MSN size, increased with time in allculture conditions (Fig. 4Ci, Cii). However, the increasein 1:1 WT MSNs was more dramatic than in 1:1YAC128 MSNs, elucidating a significant genotypic dif-ference at DIV21, which was not observed in 1:3co-cultures (Fig. 4Ci, Cii). This correlates well with ourobservation of a greater difference in dendritic arbor sizeand complexity between genotypes using a 1:1 CS ratio.These findings indicate that the previously publishedYAC128 mEPSC frequency and capacitance phenotypesare also CS ratio-dependent and that overall MSN func-tional connectivity correlates more closely with dendriticdevelopment than with spine density.Reducing cortical input promotes neuronal death inYAC128 CS co-culturePreviously, WT neurons (both cortical and striatalDARPP32+ MSNs) exhibited reduced basal survival atDIV18 when co-cultured at a 1:3 CS ratio versus 1:1[18]. We used a similar approach to compare neur-onal survival in DIV21 WT and YAC128 neurons atboth CS ratios. We found significantly reduced sur-vival of all neurons (MAP2+) as well as DARPP32+MSNs in YAC128 1:3 co-cultures compared to WT1:3 (Fig. 5a, Bi, Bii), despite being initially plated atidentical live cell density. When we calculated theproportion of surviving MAP2+ neurons that werealso DARPP32+, we found that neuronal loss inYAC128 1:3 co-cultures was partially selective forthis cell population (Fig. 5a, Biii). This reveals anFig. 3 YAC128 MSNs in 1:1 CS co-culture demonstrate reduced dendritic length and complexity. WT and YAC128 co-cultures were generated ateither a 1:1 or 1:3 CS ratio and processed at DIV21 for DARPP32 immunocytochemistry, imaging, and dendritic analysis. (a) Sample images ofMSN dendritic traces generated in NeuronStudio (scale bar = 15 μm). (Bi) Total length of the dendritic trace and (Bii) complexity by Sholl analysisare significantly reduced in 1:1 YAC128 MSNs compared to WT. Post-hoc statistical significance for Sholl analysis is shown only for WT 1:1 vs.YAC128 1:1 (*) or WT 1:3 vs. YAC128 1:3 (#) comparisons [n = 32(4); two-way ANOVA with Bonferroni post-hoc analysis; *p < 0.05,**p < 0.01, ***p < 0.001]Schmidt et al. BMC Biology  (2018) 16:58 Page 5 of 12Fig. 5 Neuronal survival is compromised in YAC128 1:3 CS co-cultures. DIV21 WT and YAC128 co-cultures were fixed at DIV21 and stained forMAP2 and DARPP32 (D32). (a) Sample fields of view at 20X objective (scale bar = 100 μm). The numbers of (Bi) MAP2+ and (Bii) DARPP32+neurons per field of view are reduced in YAC128 1:3 co-cultures. (Biii) The proportion of DARPP32+ neurons (# DARPP32+ divided by # MAP2+)surviving at DIV21 is also significantly lower in YAC128 1:3 co-cultures [n = 30 fields of view from three independent cultures; two-way ANOVAwith Bonferroni post-hoc analysis; *p < 0.05, ***p < 0.001]Fig. 4 YAC128 MSNs co-cultured at 1:1 exhibit an impaired increase in membrane capacitance with maturation. (a) Representative recordingtraces from WT and YAC128 MSNs in 1:1 or 1:3 co-culture at DIV14 and 21. (Bi, Bii) mEPSC frequency and (Ci, Cii) membrane capacitance (Cm)tend to increase with maturation, but a significant genotypic difference was only observed for Cm at DIV21 in 1:1 cultures [n = 12–29(3); two-wayANOVA with Bonferroni post-hoc analysis; *p < 0.05]Schmidt et al. BMC Biology  (2018) 16:58 Page 6 of 12additional CS ratio-dependent co-culture phenotypethat may be useful for future studies of mutantHTT-induced neuronal death.In vitro DiOlistic labeling reveals increased thin spinesand reduced mushroom spine head size in mono-culturedYAC128 cortical neuronsAlthough striatal MSNs are the most severely-affectedcell type in HD, there is evidence that mHTT causesneuronal and synaptic dysfunction in other brain regionsas well, including the cortex and thalamus [5, 32, 33].Thus, it may be desirable to utilize modified culturemodels for the study of these neuronal populations. Forexample, a YAC128 thalamo-striatal co-culture modelwas recently used to demonstrate mHTT-inducedthalamo-striatal synaptic dysfunction [32].We attempted to combine a previously reported invitro 1,1′-dioctadecyl-3,3,3′,3’-tetramethylindocarbocya-nine perchlorate (DiI) DiOlistic dye labeling protocol[34] with immunocytochemistry for glutamatergicmarkers in order to perform spine analysis on corticalneurons in CS co-culture. However, permeabilization ofDiI-stained cells for internal staining resulted in the re-lease of DiI from cell membranes and poor filling ofspines. Instead, we generated WT and YAC128 pure cor-tical monocultures for DiI spine analysis at DIV21. Wedid not observe any differences in total, mushroom, orstubby spine densities between genotypes, althoughthere was an increased number of thin spines inYAC128 cortical neurons (Additional file 5: Figure S4).Interestingly, we observed a significant 7% reduction inthe diameter of YAC128 mushroom spines (Additionalfile 5: Figure S4), indicating that subtle dysfunction incortical neurons may also exist in vitro, which couldcontribute to CS synaptic alterations.DiscussionOptimization of the CS co-culture for elucidation ofYAC128 synaptic phenotypesThe CS co-culture has become an attractive methodo-logical option for the isolated study of both physiologicaland pathogenic mechanisms of CS synaptic function.This model allows direct assessment of neuronal morph-ology and synaptic transmission and can be used toquickly answer specific questions that are difficult toinvestigate using in vivo animal models. MutantHTT-expressing YAC128 CS co-cultures recapitulatemany relevant in vivo synaptic phenotypes by 21 days invitro [23], highlighting the practicality of this model as aprimary tool for therapeutic target validation.Spine instability, hypothesized to contribute to neuronaldysfunction in HD and other neurodegenerative disorders,has been observed in YAC128 MSNs in CS co-culture insome studies, but not in others [13, 23, 26]. Recently,altering CS plating ratio was found to affect a number offunctional and morphological characteristics of WTMSNs[18], leading us to hypothesize that modifying cortical in-put in YAC128 CS co-cultures may elucidate or exacer-bate synaptic phenotypes, thus enhancing the utility ofthis culture system for HD research. In the present study,we have clearly shown that modifying CS ratio inco-culture differentially elucidates YAC128 MSN synapticphenotypes (summarized in Table 1). For future studies oftherapeutic strategies to modify neurite growth or stabilityin HD, a 1:1 CS ratio is recommended. Conversely, forevaluation of potential neuroprotective or spine-stabilizingtherapies, a 1:3 CS ratio is ideal, as this accurately recapit-ulates YAC128 age-associated in vivo MSN spine loss andneuronal death.Intrinsic versus extrinsic effects of mHTT on MSN spinestabilityOur result showing that decreasing the proportion of cor-tical neurons in CS co-culture promotes spine instabilityin YAC128 MSNs raises the interesting possibility thatspine loss with disease progression in vivo is partially dueto reduced cortical input. Indeed, studies support the hy-pothesis that progressive CS disconnect in HD results inloss of cortical excitatory and trophic support to MSNsover time and striatal degeneration [3, 35]. However, inseeming contradiction, our experiments using chimericcultures demonstrate that YAC128 MSN spine instabilityis primarily cell autonomous (Fig. 2). We propose thatmHTT expression in MSNs renders spines intrinsicallymore sensitive to low levels of cortical support, causingthis phenotype to only emerge in the presence of reducedcortical input. There is evidence that depletion of endo-plasmic reticulum calcium stores and consequent en-hanced store-operated calcium entry in YAC128 MSNscontributes to spine loss in CS co-culture [13]. It is pos-sible that reducing glutamatergic input with a 1:3 CS ratioexacerbates endoplasmic reticulum store depletion inYAC128 MSNs by limiting normal activity-induced extra-cellular calcium influx, which might subsequently pro-mote more dramatic spine loss.A recent study investigated the contribution of corticalor striatal mHTT to synaptic dysfunction by crossingregion-specific Cre-expressing mice to the BACHDTable 1 Optimal CS ratios to elucidate YAC128 MSNphenotypes in co-cultureYAC128 MSN Phenotype Optimal CS ratioSpine loss 1:3Impaired neuronal survival 1:3Decreased dendritic length/complexity 1:1Reduced mEPSC frequency 1:1Reduced membrane capacitance 1:1Schmidt et al. BMC Biology  (2018) 16:58 Page 7 of 12mouse model (expressing a bacterial artificial chromo-some containing the full-length human mutant hunting-tin gene with 97 mixed CAA-CAG repeats [PMID:18550760]) [36]. It was discovered that mHTT expres-sion predominantly in the cortex was required for al-tered synaptic protein levels and reduced spontaneousEPSC frequency in the striatum of aged BACHD mice,while impaired evoked NMDA current was dependenton mHTT expression in both the striatum and cortex[36]. A follow-up study found improvement in striatalactivity patterns and behavioral phenotypes in responseto mHTT reduction in the cortex of BACHD mice [37].Although our results in the present study showed thattotal spine density was determined entirely by mHTTexpression in striatal neurons, we did observe a small ef-fect of cortical expression on mushroom spine numbers.In particular, WT MSNs co-cultured with WT corticalneurons possessed similar total spine density as thoseco-cultured with YAC128 cortical neurons, but weobserved fewer mushroom spines and a greater numberof immature spines in MSNs from the chimeric cultures(Fig. 2). Since mature and immature spines would beexpected to have different functional properties, this indi-cates that cortical mHTT expression may contribute toaltered CS synaptic readouts. In further support of thishypothesis, we also report subtle spine morphologyalterations in monocultured YAC128 cortical neurons(Additional file 5: Figure S4).Spine and dendritic alterations in HD patients and animalmodelsEarly reports using Golgi staining of postmortem HD pa-tient brain samples demonstrated both proliferative anddegenerative morphological alterations in striatal MSNs[8, 38]. These included an increase in the number and sizeof dendritic spines as well as altered dendritic branchingin early stage (grade 2) HD [8]. In advanced HD brains,smaller dendritic arbors, spine loss, and dendritic swell-ings were observed [8]. It is hypothesized that early prolif-erative changes could reflect activation of compensatorymechanisms in response to synaptic dysfunction, whicheventually become overwhelmed with disease progressionand age. This is supported by observations of increasedglutamate transmission onto striatal neurons at early timepoints in the YAC128 and BACHD mouse models,followed by reduced transmission at later ages [15, 39].Multiple mouse models of HD recapitulate the struc-tural degeneration observed in advanced HD brains.Both MSNs and cortical pyramidal neurons in R6/1 mice(N-terminal HTT fragment mouse model of HD with116 CAG repeats [40]) exhibit reduced spine density andspine length at symptomatic ages, and a later study alsoreported thinner apical dendrites in the somatosensorycortex [12, 41]. Similarly, symptomatic R6/2 mice(N-terminal HTT fragment mouse model of HD with144–150 CAG repeats [40]) demonstrate MSN spine lossin addition to thinner dendritic shafts [9, 42]. Studies infull-length mHTT models, including mHTT knock-inand BACHD mice, have also shown loss of dendriticspines in HD MSNs [43, 44]. Although we and othersobserved YAC128 MSN total spine loss at 12 months ofage, but not at 6 months (Fig. 1f ) [13], a 15% reductionin secondary and tertiary dendrite spine density at3 months of age has been reported [11], as well as di-minished excitatory CS activity at 6–7 months [15, 25].Thus, an effect of mHTT expression on spines andsynapses is present in YAC128 mice but may be toosubtle at early ages to be detected reliably by structuralanalysis in vivo.Developmental synaptic phenotypes in YAC128 CS co-cultureWe found that all of the identified DIV21 phenotypeswere due to impaired development of YAC128 MSNsafter DIV14 (Additional file 3: Figure S2 and Additionalfile 4: Figure S3). In vivo, MSN spines and dendrites de-velop normally in WT and YAC128 animals whenassessed by Golgi staining at 1 month of age [17]. Thus,our observation of developmental phenotypes in CSco-culture suggests that impaired synaptic function oc-curs early in vitro, before MSNs have reached a maturestate. This is in agreement with previous work showingan impaired developmental increase in mEPSC fre-quency and stunted dendritic development after DIV14using YFP transfection in co-cultured YAC128 MSNs[23]. However, our results are discordant with a recentstudy showing degenerative spine loss from DIV14 toDIV21 in YAC128 CS co-cultured MSNs [13]. Differ-ences in culture methodology might explain why Wu etal. [13] observed a degenerative phenotype and we didnot. If our culturing conditions were inherently morestressful to the neurons, their maturation by DIV14 mayhave been impaired, such that synaptic dysfunction oc-curred before spines or dendrites were fully developed.Alternatively, the use of postnatal cultures in Wu et al.[13] might have promoted earlier maturation of MSNsby DIV14, either due to the later developmental age usedor the presence of a greater number of supporting glialcells in the postnatal brain [45]. The existence of YAC128dendritic and spine phenotypes at DIV18 but not atDIV14 is advantageous as it allows for in vitro testing ofboth preventative therapies (i.e., from DIV14–21) or strat-egies aimed at phenotype reversal (i.e., from DIV18–21).Functional impact of altering cortical input in CS co-cultureOur electrophysiological results demonstrate that a 1:1CS ratio is critical for the emergence of a YAC128mEPSC frequency or membrane capacitance phenotype,which tend to correlate with total dendritic lengthSchmidt et al. BMC Biology  (2018) 16:58 Page 8 of 12(summarized in Table 1). Surprisingly, 1:3 co-culturedYAC128 MSNs had similar mEPSC frequencies to 1:3 WTMSNs, despite exhibiting significantly impaired spine stabil-ity. This finding raises the possibility that YAC128 corticalor striatal neurons in 1:3 cultures undergo compensatoryupregulation of spontaneous CS activity, potentially by in-creasing cortical glutamate release. It is also plausible thatsome of the additional spines on WT 1:3 MSNs possessNMDA receptor-containing silent synapses, which wouldnot be active in our electrophysiological recording condi-tions, and thus may not result in an increased mEPSC fre-quency compared to YAC128 [46]. Alternatively, YAC1281:3 MSNs could conceivably contain a higher number ofactive shaft synapses, which likely constitute a large propor-tion of synapses in cultured neurons [47], and may be de-tected by electrophysiological recording, but would not beidentifiable through spine analysis. One caveat in our inter-pretation of these results is that identification of MSNs forelectrophysiological recording in CS co-culture requires astriatal YFP transfection step at the time of plating [23, 24],which could reduce overall culture health and thus impactthe level of spontaneous activity observed. Furthermore, itis possible that YFP transfection and DARPP32 stainingdisproportionately identify MSN populations of differentsubtypes or maturity, leading to inconsistencies when com-paring data obtained with each method.Selective, age-associated loss of DARPP32+ MSNs in theYAC128 mouse modelPrevious analysis of DARPP32+ MSN survival in WT CSco-cultures demonstrated that, despite a 50% higherstriatal plating density in 1:3 versus 1:1 cultures, thenumber of DARPP32+ cells at DIV18 was similar,suggesting selective vulnerability of this cell type [18]. Inthe present study, the density and proportion of WTDARPP32+ MSNs in 1:3 conditions at DIV21 increasedby 27% and 21%, respectively, compared to 1:1, althoughthis was still less than the expected 50% increase (Fig. 5).It is possible that DARPP32 expression was higher afterlonger maturation to DIV21 in our study, potentially im-proving the sensitivity of this readout compared to theDIV18 study. Interestingly, YAC128 DARPP32+ MSNsin 1:3 CS co-culture exhibit reduced survival comparedto WT when assessed at DIV21 (Fig. 5). This correlateswell with our previously established findings of striatalvolume loss and reduced DARPP32+ MSN cell countsin 12-month old YAC128 brains [22, 27–29], as well asdecreased DARPP32 protein and mRNA levels at10 months of age [21]. These in vivo alterations are as-sociated with behavioral impairments that are less severeor not observable at earlier ages [22, 48]. Thus, we haveenhanced our in vitro CS co-culture model to recapitu-late age-associated MSN loss without the use of anyacute stressors, such as glutamate, to induce cell death.This will prospectively be useful for preclinical testing ofneuroprotective therapeutic approaches in a more repre-sentative model of chronic disease.ConclusionsWe have optimized the CS co-culture system for broaderand more reliable use in HD research and show that in-trinsic MSN spine stability is highly sensitive to corticalinput, thus providing both a clear explanation for incon-sistent results from previous studies and a strategy togenerate reproducible and disease-relevant findings inthe future. The ability to observe a consistent spinephenotype in vitro is likely to be useful for preclinicalHD drug development, because spine loss in YAC128MSNs is dynamic, such that it can be modulated overrelatively short periods of time [13, 26]. This provides asensitive experimental readout for future studies ofmHTT-induced synaptic dysfunction. Furthermore, thetechniques we have utilized for morphological analysisare accessible, easy to establish and can be used to gen-erate results quickly compared to in vivo studies. Ultim-ately, our findings demonstrate that the CS co-culturesystem is amenable to modifications that allow differen-tial elucidation of HD-like phenotypes in vitro, and pro-vide a useful tool for future studies on mechanisms ofsynaptic dysfunction in HD.MethodsNeuronal cultureTimed pregnancies were set up by mating wild-typeFVB/N female mice with YAC128 (line 53) males. AtE17.5, embryos were removed from anesthetizedmothers and brains were extracted and stored in aHibernate solution (Hibernate-E supplemented withL-glutamine and B27; Gibco) overnight while excess em-bryonic tissue was genotyped. Cortical and striatal tis-sues from both male and female embryos were dissectedseparately the following day in ice-cold Hank’s BalancedSalt Solution, gently dissociated with a P1000 pipette,and incubated in 0.05% trypsin-EDTA (Gibco) at 37 °Cfor 8 min. Cells were further dissociated with a shortDNase treatment followed by resuspension in completeneurobasal medium (NBM; supplemented with B27,penicillin-streptomycin, and L-glutamine; Gibco). Neuronsfrom appropriate genotypes were combined at a 1:1, 1:2,1:3, or 1:5 cortico:striatal ratio and plated on 12 mm glasscoverslips (Marienfeld Superior) in 24-well plates at a finaldensity of 170,000 cells per well in 1 mL of completeNBM. Prior to plating, coverslips were treated overnightwith 6 N hydrochloric acid, washed thoroughly with sterilewater and 70% ethanol, transferred to culture plates, andcoated with sterile-filtered 50 μg/mL of poly-D-lysinehydrobromide (Sigma; P7886) in water overnight at roomtemperature. Coverslips were washed four times withSchmidt et al. BMC Biology  (2018) 16:58 Page 9 of 12sterile water and allowed to air dry before plating. Forelectrophysiological experiments, YFP was transfected intostriatal neurons at the time of plating to allow for MSNidentification. Approximately 2 million striatal neuronswere suspended in 100 μL of electroporation solution(Mirus Bio) prior to the final plating step, mixed with 2 μgof DNA (YFP on a β-actin promoter; a gift from A.M.Craig, University of British Columbia), and nucleofected(Amaxa Nucleofector, Lonza Bio, program 05). Cells werediluted and plated in 500 μL of 10% fetal bovine serum/DMEM. Media was replaced with 500 μL of completeNBM after 4 h, and topped up to 1 mL the following day.All cultures were supplemented with fresh NBM complete(20% well volume) every 3–7 days until fixation of cover-slips at DIV14, 18, or 21.ImmunocytochemistryNeurons on coverslips were fixed in 4% paraformalde-hyde (PFA)/phosphate buffered saline (PBS) for 15 minat room temperature (RT), incubated in ice-cold metha-nol for 5 min at −20 °C, permeabilized in 0.03%Triton-X/PBS for 5 min at RT, and blocked for 30 minat RT in 0.2% gelatin/PBS. Coverslips were incubatedwith primary antibody against DARPP32 (rat anti-DARPP32;R&D Systems Cat# MAB4230; RRID:AB_2169021; 1:500)and MAP2 (mouse anti-MAP2; Invitrogen Cat# MA5–12823; RRID:AB_10982160; 1:200) in blocking buffer over-night at 4 °C, washed in PBS, stained with secondary anti-bodies against rat IgG (Alexa Fluor 568 goat anti-rat IgG;Invitrogen Cat# A-11077; RRID:AB_141874; 1:500) oragainst mouse IgG (Alexa Fluor 488 goat anti-mouse IgG;Invitrogen Cat# A-11001; RRID:AB_2534069; 1:500) for1.5 h at RT, washed in PBS, and mounted on slides usingProlong Gold Antifade Reagent with DAPI (Invitrogen). Forspine and dendrite analysis, fluorescence images were ac-quired using a Leica TCS SP8 confocal laser scanning micro-scope at 63X objective magnification. Samples from differentgroups were interleaved and the researcher was blinded toexperimental conditions during imaging and analysis. Imagestacks of Z-step size of 60 μm were converted to 2D inImage J using the maximum intensity Z-projection function.Images were then background subtracted with a rolling ballradius of 35 pixels and de-speckled. Images were importedinto NeuronStudio (Version 0.9.92) for semi-automatedSholl analysis as well as spine characterization using a mini-mum of three representative secondary or tertiary dendriticsegments per cell. For analysis of DARPP32 and MAP2staining intensity and cell survival counts, random fields ofview were imaged at 20X objective magnification usingidentical laser intensities across samples. The number ofMAP2+ or DARPP32+ with healthy nuclei in each field ofview were counted, and staining intensity was measuredwithin multiple secondary or tertiary dendrite regions fromeach neuron selected for analysis.DiOlistic labeling of cortical neuronsCortical neurons were labeled in vitro with DiI stain(Invitrogen Cat# D282) as previously described [34], withminor alterations. Briefly, DIV21 cortical cultures werefixed in 2% PFA/PBS for 15 min at RT. Then, 15–20 DiIcrystals were sprinkled on top of coverslips, and a smallvolume of PBS was added to prevent cells from drying out.Coverslips were incubated in the dark for 10 min at RT,followed by thorough PBS washing to remove crystals andincubation in the dark for an additional 6 h in PBS. Cover-slips were rinsed again in PBS and mounted on slides.Imaging and spine analysis were performed as describedabove with an excitation wavelength of 549 nm.Golgi-Cox stainingSix- or 12-month-old mice were perfused with 2% PFA/2% glutaraldehyde/PBS, post-fixed in the same solutionovernight at 4 °C and processed as previously described[49], with minor alterations. Briefly, brains were washedin PBS, incubated in Golgi-Cox solution (1% potassiumdichromate, 1% mercuric chloride, 0.8% potassium chro-mate) for 5 days, and transferred to 30% sucrose/PBS.Then, 100 μm sections were cut on a vibratome andmounted on slides, which were dried overnight, washedin ddH2O, incubated in 20% ammonium hydroxide for10 min, washed in ddH2O, passed through ascendinggrades of alcohol, and placed in xylene for 5 min. Cover-slips were mounted on top of sections with Cytosealmounting medium (Thermo Scientific). Transmittedlight images were acquired with a Leica TCS SP8 con-focal laser scanning microscope and a 63X objectivelens. Images were imported into NeuronStudio andspines on dendritic segments from at least 15–20 neu-rons per animal were semi-automatically analyzed.ElectrophysiologyWhole-cell patch-clamp electrophysiology was conductedas previously described [23]. Briefly, an Axon InstrumentAxopatch B200B amplifier and pClamp 10.2 software(Molecular Devices) were used to collect data under thevoltage-clamp mode. Culture coverslips were perfused ina recording chamber with external recording solutioncontaining picrotoxin and tetrodotoxin [23]. mEPSCswere recorded in YFP-positive neurons at a holding mem-brane potential of –70 mV with the recording pipettesfilled with K-gluconate internal solution [23]. Membranecapacitances were measured within 2 min of patchingeach cell, and at least 30 synaptic events were analyzedper cell with Clampfit 10.2 or 10.7.Data analysisAll data is presented as mean ± SEM. Statistical analysisand graph generation were performed using GraphPadPrism 5, and figures were created in Adobe PhotoshopSchmidt et al. BMC Biology  (2018) 16:58 Page 10 of 12CS5. n values for all experiments are recorded as thetotal number of cells analyzed, with the number of inde-pendent cultures in parentheses. Student’s t test or one-or two-way ANOVA statistical tests with Bonferronipost-hoc analysis were used for all experiments.Additional filesAdditional file 1: Individual data values. Individual values for data withn < 6. (XLSX 12 kb)Additional file 2: Figure S1. Initial plating density does not impact thepresence or severity of YAC128 MSN spine instability. WT and YAC128co-cultures were generated at a 1:3 CS ratio and plated at three differenttotal cell numbers per well (150,000, 170,000, or 230,000 in 24-well plates).Coverslips were fixed at DIV21 and processed for DARPP32 immunocyto-chemistry, imaging, and spine analysis. (A) Sample images of DARPP32-stained spines on secondary or tertiary MSN dendrites (scale bar = 5 μm).There was no effect of initial plating density on YAC128 (Bi) total or (Bii)mature mushroom spine density phenotypes [n = 20(2); two-wayANOVA with Bonferroni post-hoc analysis; *p < 0.05, **p < 0.01,***p < 0.001]. (TIF 423 kb)Additional file 3: Figure S2. Reduced spine density in co-culturedYAC128 MSNs is a developmental phenotype. WT and YAC128 co-cultureswere generated at either a 1:1 or 1:3 CS ratio and processed at DIV14, 18, or21 for DARPP32 immunocytochemistry, imaging, and spine analysis. (A)Sample images of DARPP32-stained spines on secondary or tertiary MSNdendrites (scale bar = 5 μm). A developmental increase in (Bi) total and (Bii)mature mushroom spine numbers is impaired after DIV14 in co-culturedYAC128 MSNs compared to WT [n = 32(4); two-way ANOVA with Bonferronipost-hoc analysis; *p < 0.05, **p < 0.01, ***p < 0.001]. (TIF 988 kb)Additional file 4: Figure S3. Reduced dendritic length and complexityin co-cultured YAC128 MSNs are developmental phenotypes. WT andYAC128 co-cultures were generated at either a 1:1 or 1:3 CS ratio andprocessed at DIV14, 18, and 21 for DARPP32 immunocytochemistry,imaging, and dendritic analysis. (A) Sample images of MSN dendritictraces generated in NeuronStudio (scale bar = 15 μm). A developmentalincrease in (Bi, Bii, Biii) dendritic complexity by Sholl analysis and (Biv)total dendritic length are impaired after DIV14 in co-cultured YAC128MSNs compared to WT. Post-hoc statistical significance for Sholl analysisis shown only for WT 1:1 vs. YAC128 1:1 (*) or WT 1:3 vs. YAC128 1:3 (#)comparisons [n = 32(4); two-way ANOVA with Bonferroni post-hocanalysis; *p < 0.05, **p < 0.01, ***p < 0.001]. (TIF 875 kb)Additional file 5: Figure S4. Increased thin spine density and reducedmushroom spine head diameter in DIV21 YAC128 cortical neurons. WTand YAC128 pure cortical cultures were fixed at DIV21 and subjected toin vitro DiI DiOlistic dye labeling for spine analysis. (A) Sample images ofDiI-stained spines on cortical dendrites (scale bar = 5 μm). No significantdifferences in (Bi) total, (Bii) mushroom, or (Biii) stubby spine densitieswere observed in YAC128 cortical neurons. (Biv) Increased thin spinedensity and (Bv) reduced mushroom spine head diameter were measuredin YAC128 cortical neurons compared to WT [n = 30(3); Student’s t test;*p < 0.05]. (TIF 288 kb)AbbreviationsCS: cortico-striatal; DARPP32: dopamine- and cyclic AMP-regulated phospho-protein 32; DiI: 1,1′-dioctadecyl-3,3,3′,3’-tetramethylindocarbocyanineperchlorate; DIV: days in vitro; HD: Huntington disease; HTT: huntingtin;MAP2: microtubule-associated protein 2; mEPSC: miniature excitatorypostsynaptic current; mHTT: mutant huntingtin; MSN: medium spiny neuron;NBM: neurobasal medium; NMDA: N-methyl D-aspartate; PBS: phosphatebuffered saline; PFA: paraformaldehyde; RT: room temperature; WT: wild-type;YFP: yellow fluorescent proteinAcknowledgementsThe authors thank Qingwen Xia, Mark Wang, Sheng Yu, Lisa Anderson,Mahsa Amirabbasi, and Yun Ko for their technical support. We also thankShaun Sanders, Dale Martin, Fanny Lemarié, Amirah Aly, Nicholas Caron,Amber Southwell, Philip Ly, and Daniel Ryskamp for their intellectual inputon the project.FundingMES was supported by a Vanier Canada Graduate Scholarship. CB wasfunded by the University of British Columbia 4-Year Fellowship. LAR receivedfunding from a Canadian Institutes of Health Research (CIHR) Foundationgrant (FDN-143210). MRH received funding from a CIHR operating grant(MOP-84437) and a CIHR Foundation grant (FDN-154278).Availability of data and materialsData sharing is not applicable to this article as no datasets were generatedor analyzed during the current study.Authors’ contributionsMES designed, performed, and analyzed all imaging experiments, assistedwith the design and interpretation of electrophysiological data, prepared allof the figures, and wrote the manuscript. CB and JPM designed, performed,analyzed, and interpreted all electrophysiological experiments, providedintellectual input, and edited the manuscript. DC and LDC assisted inoptimizing and performing neuronal culture, immunofluorescence, and Golgistaining experiments. LAR and MRH supervised the project, providedintellectual input, and edited the manuscript. All authors read and approvedthe final manuscript.Ethics approval and consent to participateAll experiments were performed according to protocols approved by theUniversity of British Columbia Animal Care Committee (Protocol numberA16-0130).Competing interestsMRH was an employee of Teva Pharmaceuticals, Inc. during this study. Tevadid not play a role in the design, collection, analysis, or interpretation of datain this study. All other authors declare no competing interests.Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.Author details1Centre for Molecular Medicine and Therapeutics, BC Children’s HospitalResearch Institute, University of British Columbia, 950 West 28th Avenue,Vancouver V5Z 4H4, Canada. 2Department of Psychiatry and DjavadMowafaghian Centre for Brain Health, University of British Columbia,4834-2255 Wesbrook Mall, Vancouver V6T 1Z3, Canada. 3Present address: TheHospital for Sick Children, 555 University Avenue, Toronto M5G 1X8, Canada.Received: 12 January 2018 Accepted: 8 May 2018References1. Huntington’s Disease Collaborative Research Group. A novel genecontaining a trinucleotide repeat that is expanded and unstable onHuntington’s disease chromosomes. The Huntington’s Disease CollaborativeResearch Group. Cell. 1993;72:971–83.2. Raymond LA, André VM, Cepeda C, Gladding CM, Milnerwood AJ, LevineMS. 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