Selective Loss of Smaller Spines in Schizophrenia

Abstract

Objective

Deceased density of dendritic spines in adult schizophrenia subjects has been hypothesized to result from increased pruning of excess synapses in adolescence. In vivo imaging studies have confirmed that synaptic pruning is largely driven by the loss of large/mature synapses. Thus, increased pruning throughout adolescence would likely result in a deficit of large spines in adulthood. Here, we examined the density and volume of dendritic spines in deep layer 3 auditory cortex of 20 schizophrenia and matched control subjects as well as aberrant voltage-gated calcium channel subunit protein expression linked to spine loss.

Methods

Primary auditory cortex deep layer 3 spine density and volume was assessed in 20 pairs of schizophrenia and matched comparison subjects in an initial and replication cohort (12 and 8 pairs) by immunohistochemistry-confocal microscopy. Targeted Mass Spectrometry was used to quantify postsynaptic density and voltage-gated calcium channel protein expression. The effect of increased voltage-gated calcium channel subunit protein expression on spine density and volume was assessed in primary rat neuronal culture.

Findings

Only the smallest spines are lost in deep layer 3 primary auditory cortex of schizophrenia, while larger spines are retained. Levels of the tryptic peptide ALFDFLK, found in the schizophrenia risk gene CACNB4, inversely correlated with the density of smaller, but not larger, spines in schizophrenia subjects. Consistent with this observation, CACNB4 overexpression resulted in a lower density of smaller spines in primary neuronal cultures.

Conclusion

These findings require a rethinking of the over pruning hypothesis, demonstrate a link between small spine loss and a schizophrenia risk gene, and should spur more in-depth investigations of the mechanisms that govern new/small spine generation and stabilization under normal conditions as well as how this process is impaired in schizophrenia.

Introduction

In 1982 Feinberg hypothesized that increased pruning of existing synapses during adolescence may contribute to the onset of schizophrenia¹, the incidence of which peaks toward the end of this developmental stage. Since then, increased synaptic pruning has become a major hypothesized pathogenic mechanism in schizophrenia, supported by the linking of two well established observations. First, progressive grey matter loss has been observed in patients around the time of disease onset². Second, significant reductions in the density of dendritic spines on pyramidal neurons, the major post-synaptic sites of excitatory, glutamatergic synapses in the cerebral cortex, have consistently been observed in schizophrenia subjects in multiple brain regions within the frontal and temporal neocortex³. As dendritic structural features are critical for signal processing^4–7 spine loss may then directly contribute to schizophrenia symptoms^3,8.

Dendritic spines on cortical pyramidal neurons are highly plastic throughout all stages of development, with shifting rate constants of formation and loss governing the net spine population in each developmental epoch. Spine, and synapse, formation is most robust perinataly, leading to a peak in synapse numbers at 2–4 years of age. These excess synapses are then “pruned”, most intensely during adolescence, to adult levels. Throughout adulthood large fractions of synapses are stably maintained, the physical incarnation of accumulated skills and memories^9–12. There is some turnover, as skills and memories are lost, gained, or modified^13–15. In vivo imaging studies have confirmed the long held view that adolescent pruning of dendritic spines is largely driven by elevated rates of elimination of mature/stable spines, while spine formation rates are similar to those seen in adulthood¹⁴.

In vivo imaging studies have also found that mature/stable spines are larger while new/transient spines are smaller^13,15. Thus, the increase in synaptic pruning proposed to drive spine loss in schizophrenia should result in a deficit of larger spines in the adult cortex. Alternatively, recent genetic studies have implicated NMDA, BDNF, and mTor signaling, pathways which are involved in spine formation and stabilization^16,17. If spine loss in schizophrenia is driven by a decrease in the rate of new spine formation and stabilization, a decrease in the number of smaller spines would be expected. To test these alternate hypotheses we used the co-localization of the postsynaptic density (PSD) protein spinophilin and the cytoskeleton protein filamentous-actin (F-actin) to assess, for the first time, both the density and volume of dendritic spines in schizophrenia.

Methods

Human Subjects

Tissue from two cohorts (Table 1, Supplemental Table 1) comprised of subjects diagnosed with schizophrenia or schizoaffective disorder (together referred to as schizophrenia) and controls matched on the basis of sex, and as closely as possible for age, post-mortem interval (PMI), and handedness^18–21. We have previously reported on spine density, but not spine density by spine volume, in this cohort²².

Table 1. Summary of Subject Characteristics.

There were no diagnostic group differences in age, sex, postmortem interval, storage time, or in the distribution of handedness between the diagnostic groups. A, ambidextrous; F, female; L, left-handed; M, male; PMI, postmortem interval; R, right-handed; U, unknown. This cohort was previously reported in Shelton et al. 2015²².

	Control	Schizophrenia	p
N	20	20
Mean Age (SD)	45.8 (11.3)	46.9 (13.7)	0.71
Range	19–65	25–71
Sex (F/M)	7/13	7/13
Handedness (R/L/A/U)	19/1/00	11/5/1/3
PMI (SD)	16.4 (16.7)	17.0 (7.9)	0.64
Storage Time, mos (SD)	131.8 (38.2)	124.4 (35.9)	0.69
Suicide, N (%)		4 (20%)
Schizoaffecive, N (%)		6 (20%)
Alcohol/Substance abuce ATOD, N (%)		5 (25%)
Antipsychotic AT OD, N (%)		17 (85%)

Human Tissue Processing

At the University of Pittsburgh brain bank the right and left hemispheres of a postmortem brain are processed differently. Tissue blocks from the left hemisphere are fixed in paraformaldehyde, while blocks from the right hemisphere are fast-frozen in isopentane. Thus, tissue from the left hemisphere was utilized for Immunohistochemistry while tissue from the right hemisphere was utilized for targeted mass spectrometry.

Immunohistochemistry

Tissue from the 20 pairs was divided into an initial and a replication cohort: Cohort 1 (n = 12 pairs) and Cohort 2 (n = 8 pairs) (Supplemental Table 2). These two cohorts were assayed independently. Tissue with in each cohort was processed together over a series of immunohistochemical runs. In order to visualize dendritic spines, we used two markers in combination: a polyclonal antibody directed against spinophilin (Millipore AB5669, Billerica, MA), a protein that is highly enriched in spine heads, and the f-actin binding mushroom toxin phalloidin (Invitrogen A12380, Carlsbad, CA), which is also highly enriched in dendritic spines.

Image Collection and Processing

Matched pairs from each cohort were imaged during the same session by an experimenter blinded to diagnostic or antipsychotic exposure group. All images were taken using a confocal microscope equipped with a 60X oil supercorrected objective (equipment details found in the Supplemental Methods). Tissue thickness was measured at each sampling site and did not differ by diagnostic group (F_1,18.5=0.05, p=0.83) or cohort (F_1,18.6=2.35, p=0.14). Image stacks were taken beginning 12.5 µm below the tissue surface closest to the coverglass, stepping up 0.25 µm with each image until the tissue surface was reached. This produced an image stack comprised of 50 individual planes, each 512 × 512 pixels in size. Exposure times for 488nm and 568 nm excitation wavelengths were set to optimize the spread of the intensity histogram for each cohort, then were kept consistent for all subjects within cohorts.

Images were processed using Slidebook software version 5.027 with keystrokes automated by Automation Anywhere software (Automation Anywhere, Inc. San Jose, CA). Camera background was subtracted from channels 488 and 568 prior to processing. Underlying gray level values were extracted from the mask objects. See Supplemental Methods for more detail on image processing.

Calculation of Spine Density, Number, and Area

While both spinophilin-IR and phalloidin binding are strongly localized to spines, each has some off target label. Therefore, identification of putative dendritic spines required co-localization of spinophilin-IR and phalloidin label (Fig. 2), operationalized as phalloidin mask objects that overlapped (≥ 1 voxel) with a spinophilin-IR mask object Spine density (N_v) in cohort 1 was calculated as previously described with minor modification:

$$N_v:=\frac{{\overline{t}}_{w{Q}^{-}}}{h}\cdot \frac{{{\displaystyle \sum }}^{}\left(Q_i^{-}\cdot w_i\right)}{BA\cdot a\cdot {{\displaystyle \sum }}^{}\left(P_i\cdot w_i\right)}$$

Figure 2. Correlation of ALFDFLK with spine density by bin.

The CACNB peptide ALFDFLK was quantified by liquid chromatograph – selected reaction monitoring mass spectrometry. Expression of this peptide was plotted against the density of spine objects from each size bin, e.g. panel A (0–0.15 µM³) reports the correlation of ALFDFLK with the density of spine objects 0–0.15 µM³ in both control (black open circle) and schizophrenia (grey filled circle) subjects. ALFDFLK expression was significantly inversely correlated with the density of small, but not medium or large, spines across all subjects (solid black line). This effect was similar for the control (black dashed line) and schizophrenia (grey dashed line) cohorts when separated.

Where a is the area of the counting frames, $Q_i^{-}$ is the count of dendritic spines within the ith block, $P_i$ is the count of the associated points hitting the region of interest in the ith block, h= disector height (see supplemental methods for additional details), BA is the cryostat block advance (50 μm for cohort 1 and 60 μm for cohort 2, ${\overline{t}}_{w{Q}^{-}}$ is the block-and-number-weighted mean section thickness calculated using this formula:

$${\overline{t}}_{w{Q}^{-}}:=\frac{{{\displaystyle \sum }}^{}\left(t_j\cdot q_j^{-}\cdot w_i\right)}{{{\displaystyle \sum }}^{}\left(q_j^{-}\cdot w_i\right)}$$ (2)

where $t_j$ is the local section thickness measured centrally in the jth sampling frame and $q_j^{-}$ is the corresponding count of dendritic spines in the jth frame. $w_i$ is the block weight—i.e. either 1 or 1/3. Because for cohort 2, sections adjacent to the mapping sections were sampled, calculation of $N_v$ were as above but omitting the block weighting.

Targeted Mass Spectrometry

Tissue homogenates were prepared from fresh frozen human A1 grey matter described above and in Supplemental Methods. Total protein was extracted using SDS extraction buffer (0.125 M Tris – HCl (pH 7), 2% SDS, and 10% glycerol) at 70°C. Using bicinchoninic acid assay (Micro BCA™ Protein Assay, Pierce) protein concentration was measured. A pooled technical replicate sample composed of homogenate aliquots from all subjects was also prepared. 20 ug of total protein from the gray matter homogenate or pooled sample was mixed with Lysine ¹³C₆ Stable Isotope Labeled Neuronal Proteome Standard²³ (¹³C₆ STD; 20 ug) for on gel trypsin digestion. Samples were organized in a block distribution. Each block was run on a single 10 well 4–12% BisTris gel with two SeeBlue® Plus2 Pre-stained Protein Standards (Invitrogen, Carlsbad, CA). On-gel trypsin digestion was performed as previously described²³ with samples being run 4 cm into the gel and divided into two fractions (above and below 65kd). LC-SRM/MS assay development and implementation was performed a previously described^23,24, see Supplemental Methods for details.

Primary Neuronal Culture

Primary cortical neuronal cultures were prepared from embryonic day 17 (E17) Sprague Dawley rats (Envigo and Charles River Laboratories). Neurons were plated at 450,000 cells/well in 12-well plates. On day in vitro (DIV) 12 the neurons were transfected with CACNB4/Myc (OriGene; Catalog #: RR204310) and GFP (gift of Ryan Logan, University of Pittsburgh, PA). Thus, in the same plate some neurons were transfected with both constructs and some with only one. Neurons were fixed on DIV 15 for imaging with mouse anti-c-Myc antibody (1:1000, monoclonal 9E10; Santa Cruz Biotechnology, Inc.) or with mouse anti-CACNB4 (1:100, monoclonal S10-7; antibodies-online Inc.), and goat anti-c-Myc (1:100, polyclonal; Novus Biologicals). Image acquisition was performed on an Olympus (Center Valley, PA) BX51 WI upright microscope equipped with an Olympus spinning disk confocal (SDCM) using an Olympus PlanAPO N 10X 0.40 NA air objective and a 1.42 numerical aperture 60X oil supercorrected objective.

Neurons were first categorized as either CACNB4-overexpressing or GFP-only controls based on c-Myc intensity. GFP-positive neurons on coverslips stained for both c-Myc and CACNB4 were imaged at 10x. Exposure times for the 488 channel were optimized whereas the 568 and 647 channels were shot at fixed exposures of 447 ms and 3000 ms, respectively.

Spine Counting and Masking

The TIFF files of all dendrites were imported into StereoInvestigator, (MicroBrightField, Inc.) for counting. Protrusions from the dendritic shaft were manually assigned to one of the following categories, blind to experimental condition: short mushroom spine, long mushroom spine, short stubby spine, long stubby spine, or filopodia. Criteria for assignment were previously described²⁵. Briefly, mushrooms spines had distinct heads while stubby spines did not. Long spines had a length greater than maximal width and the rest were short. Filopodia were longer than 2 μm, thinner than 0.3 μm, and lacked a distinct head. For a given dendrite, the number of protrusions in each category was recorded and the densities per µm of dendrite length for all spines, mushroom and stubby subtypes, and filopodia were calculated. A subset of the 60x dendritic images used for spine counting was randomly selected, blind to condition, for spine area analysis. In total, 35 GFP+ only dendrites and 38 Myc+/GFP+ dendrites were analyzed. Details on spine area calculation can be found in Supplemental Methods.

Statistical Analysis

Human spine density by volume

The data was analyzed through a linear mixed model with the pair effect taken into account. Spine density was assumed to be normally distributed. In the model, pair, cohort, diagnosis, spine size category, cohort*spine size category and diagnosis*spine size category were the fixed effects. Subject was treated as a normal random effect to account for the repeated measures within each subject. Insignificant interaction terms were not included in the final model. The Kenward-Roger method was used to adjust for the denominator degrees of freedom. The analysis was implemented in SAS 9.4 with the PROC MIXED procedure. Categorical confound effects were also assessed, see Supplemental Methods.

Human Spine Density Continuous Confound Effects

For the continuous confound effect analysis, the percent change of spine density within each pair was calculated ((C-S)/C) ×100% and a simple linear regression analysis was done upon each of the two confound variables separately (age of onset and duration of disease). The slope of the percent change of spine density on each confound variable was estimated and the p-value to test whether the slope was significantly different from 0 was provided in the table.

Protein Level Correlations with Spine Density by Size

Person’s Correlation was used to determine the relationship between peptide expressions and spine density by volume.

Results

Spine density by volume

As F-actin fills the spine²⁶, it can be used to estimate spine volume in immunohistochemical preparations of human postmortem brain tissue (Figure 1A, B & C). Thus, we estimated spine volumes in these subjects (Table 1, Supplemental Table 1) and calculated the density of spines by volume. The distribution of spine sizes we observed in the primary auditory cortex (A1) from control subjects (Figure 1D) was similar to previous reports from filled cells in human cerebral cortex²⁷. Contrary to our hypothesis, we found that significant decreases in spine density were limited to the smallest spines (Figure 1D). Densities of medium and large spines were unaltered (Figure 1D). This effect was not associated with any potential confounding variables (Supplemental Table 2). Mean and total F-actin content per spine were unaffected in spines of all volumes (Figure 1E & F). The unchanged F-actin content per spine of small spines in schizophrenia indicates that the observed reduction in density was not due to decreased sensitivity of detection. This effect was not significantly associated with any confounding variables such as sex, medication history, or duration of disease (Supplemental Table 2).

Figure 1. Spine attributes by volume.

A. Phalloidin-labeled and spinophilin-immunoreactive puncta in deep layer 3 of primary auditory cortex. (Top) Phalloidin-labeled puncta (red) and spinophilin-immunoreactive puncta (green) colocalize throughout deep layer 3 of Brodmann area 41 and are found along microtubule-associated protein 2 (MAP2) immunoreactive processes (blue) suggesting spine structures along dendrites. (Bottom) Images are a magnification of the inset in the top panel highlighting the relationship between phalloidin-labeled (1) and spinophilin-immunoreactive objects (2) and their colocalization in presumptive spine structures (3). The volume of phalloidin objects, co-localized with spinophilin, was used to estimate spine volume. B. and C. show representative phalloidin-labeled presumptive spines (60×) from schizophrenia and a matched control, respectively. D. 20 pairs of subjects were assayed as an initial and an independent replication cohort: Cohort 1 (n = 12 pairs) and Cohort 2 (n = 8 pairs) (Supplemental Table 1). The results were equally significant between the two groups and are presented here as a single cohort for simplicity. The density of spines of different volumes (0.15 µM³ increments, with the final bin including all objects with volume > 1.35 µM³) for control and schizophrenia subjects from both cohorts are shown. The density of spines in the bins of smaller volumes differed significantly between control and schizophrenia. P-values are Bonferroni corrected. E. The mean phalloidin intensity (calculated as the total phalloidin intensity of the spine object divided by spine object volume) of spines of different volumes from control and schizophrenia subjects are shown. F. The sum phalloidin intensity of spines of different volumes from control and schizophrenia subjects are shown. Mean and sum phalloidin intensity was consistent between control and schizophrenia across all of the spine volume categories.

Voltage Gated Calcium Channel protein expression

PSD and synaptic calcium signaling has been implicated in schizophrenia. Targeted mass spectrometry was used to quantify PSD and synaptic calcium signaling proteins in a subset of the subjects in which spine density was measured. The expression of one tryptic peptide, ALFDFLK, was inversely correlated with the density of only smaller spines. (Figure 2). It is important to note here that two subjects, one control and one SZ, had very high small spine densities (although the spine density for medium and large volume spines for these two individuals were similar to other subjects). Exclusion of these two outliers did not alter the association of reduced small spine density with schizophrenia. In addition, when these outliers were removed we observed an even greater degree of correlation of ALFDFLK levels with small spine density (Bin 1. r = −0.55, p = 0.0025, Bin 2. r = −0.56, p = 0.0021, Bin 3. r = -0.51, p = 0.0051). ALFDFLK levels were not significantly increased in schizophrenia compared to controls (schizophrenia/control = 1.08, p = 0.2). When subjects were separated by diagnosis the correlation of ALFDFLK with small spine density was similar in control and SZ subjects (Figure 2). In contrast, a module of PSD proteins we had previously identified as correlated with spine density in schizophrenia²⁴, did not demonstrate selectivity for small spines (data not shown).

CACNB4 overexpression alters spine density

To determine if altered CACNB4 expression could contribute to small spine loss, CACNB4 was overexpressed in primary neuronal culture (Figure 3A & B). Total spine density was significantly decreased (Figure 3D). The density of filopodia, immature spine precursors, was unaltered (Figure 3E), suggesting the reduced spine density observed was not the result of deficits in filopodia assembly. Further analysis of spine density by size (Figure 3C) revealed that loss was limited to smaller spines (Figure 3F).

Figure 3. CACNB4 overexpression decreases small spine density in primary neuronal culture.

E17 rat cortex primary neuronal cultures were transfected on day 12 with either GFP or GFP/CACNB4-Myc. A. shows representative 10x confocal images of transfected neurons. Endogenous CACNB4 expression was not observed in these cultures, in line with previous reports that CACNB4 is not expressed at this stage of development in the cortical cultures. CACNB4 transfection induced robust somatodendritic expression (A.) as well as expression in spines (B.). C. Shows processing for determination of dendritic spine volumes in another representative dendritic segment. Note that the filopodia is not masked. Spine and filopodia density was evaluated in three independent experiments. In each experiment spine density was significantly decreased in CACNB4 expressing neurons (D.). N (number of dendrites assayed in each experiment) is reported in each bar. In addition to reaching significance in each independent experiment, spine density was also significantly decreased across all three experiments (p = 6.5E⁻⁵). E. compares filopodia densities between GFP+ and GFP+/CACNB4-Myc+ neurons from all experiments, which were equivalent. Spine density was then assessed for spines of different sizes (F.). A significant reduction in density selective for small spines was observed (two-way ANOVA). The reported P-value is Bonferroni corrected.

Discussion

We observed that spine decreases in deep layer 3 of the primary auditory cortex in schizophrenia was limited to spines of smaller volumes. The decrease in small spines most likely reflects a reduction in the number of new/transient spines. The appearance of new/transient spines and their probability of stabilization is regulated by local calcium signaling generated by voltage-gated calcium channels (VGCCs)^28,29. Glutamate uncaging can also induce spinogenesis^12,30 and the presence of a PSD is essential, but not sufficient, for new spine stabilization at a bouton³¹. GWAS¹⁷ and rare variant¹⁶ analyses of schizophrenia have identified VGCCs and glutamate signaling from the PSD to the F-actin cytoskeleton in schizophrenia risk. It is important to note that the majority of small/new spines, even those that sample a bouton and acquire a PSD, are transient³¹. Thus, even in healthy circuits the early stages of spine stabilization are tentative and the genetic factors that converge upon and destabilize PSD and VGCC activity would be likely to have their greatest effect in these early, less robust stages of the spine lifecycle.

An important consideration regards the potential effects of postmortem interval. We note that the cohorts in the spine density analysis were matched for postmortem interval as closely as possible. Furthermore, we did not observe any correlation between PMI and small spine density (Supplemental Table 2), strongly suggesting that PMI does not impact our findings in schizophrenia vs control. Nevertheless, it is possible that postmortem interval could alter the relationship between spine size and spine class (i.e. stable/mature versus new/transient), affecting our interpretation of the nature of the reduction in small spines in schizophrenia.

We sought to determine if a link between PSD and/or VGCC signaling proteins and small spine loss was present in this cohort, finding that the expression of one tryptic peptide, ALFDFLK, was inversely correlated with the density of only smaller spines (Figure 1G). The amino acid sequence ALFDFLK is unique to the four calcium channel beta subunits (CACNB1-4). Of these subunits, CACNB2 and CACNB4 have been implicated in schizophrenia pathogenesis by GWAS¹⁷ and rare variant¹⁶ analyses. Of these two, CACNB4 is the predominant isoform expressed in the adult temporal lobe^32,33. In line with this observation in human tissue, we found that the over-expression of CACNB4 decreased the density of smaller spines in primary neuronal culture. However, as ALFDFLK is present in other CACNB variants, it will be important to trace this effect to specific proteins.

CACNB4, like all VGCC beta subunits, modulates the trafficking and biophysical properties of the alpha pore forming subunits³⁴. While the association of common and rare alleles of several VGCC subunits with schizophrenia risk is well established^17,35, a consensus on whether they result in a loss or gain of function, and on how they may exert an effect on synapses is currently lacking³⁵. Our findings suggest that CACNB4 overexpression depresses the calcium currents that drive spine formation and/or stabilization. Previous studies provide some potential mechanisms and precedent for the observed effect. CACNB’s are required for voltage-dependent inactivation of calcium channels by a number of second messengers³⁶. Moreover, CACNB4 KO mice display evidence of increased calcium currents and increased excitability, manifest as epilepsy, suggesting that the dominant effect of CACNB4 expression may be to reduce calcium currents³⁶. CACNB4 is also noteworthy among the beta subunits in that it traffics from the synapse to the nucleus impacting gene expression³⁷, providing a second mechanism by which VGCC activity could impact spine plasticity in addition to the regulation of local calcium signaling.

Despite identifying a significant correlation between ALFDFLK and small spine density, ALFDFLK levels were not significantly increased in our schizophrenia subjects. However, our in vitro data firmly demonstrates that increased CACNB4 expression alone can drive small spine loss. This combination of findings is consistent with the current model for schizophrenia risk in which multiple genetic variants, epigenetic, and environmental influences are believed to act in concert. No single gene/protein is believed to account for more than a small share of the population risk. As CACNB4 interacts with and regulates multiple VGCC subunits, many of which are also implicated in schizophrenia¹⁷, it likely acts in concert with, or parallel to, other VGCC genetic risk factors.

In the adult cortex current evidence indicates that new/transient spines act as the substrate for reorganization of cortical networks in response to experience³⁸. They accomplish this rewiring both by providing for the formation of new persistent synapses^13,15,39 and by contributing to the destabilization of persistent synaptic spines through bouton competition^31,40. Thus, a decrease in the rate of spine formation and/or their subsequent stabilization would result not only in their depletion but also in a decreased turnover rate of large spines by decreasing competitive loss, potentially explaining the observed conservation of large spines in schizophrenia despite a decrease in the small spines from which they are formed. The predicted cognitive consequences of new/transient spine reductions, in a general sense, would be impairments known to affect individuals with schizophrenia: deficits in new learning⁴¹. Recently, auditory training exercises have shown promise for remediating auditory processing deficits in schizophrenia patients⁴². Impairments in the generation or stabilization of new/transient spines in some subjects with schizophrenia may limit the available gains through such an approach. Thus, identification of therapeutics promoting small spine generation and stabilization might enhance the benefits of such training, providing a measurable outcome of drug efficacy.

In closing, our finding that A1 layer 3 spine loss in schizophrenia is limited to smaller spines strongly suggests that adult spine deficits result from a failure to generate and/or stabilize new/transient spines. This finding requires a rethinking of the over pruning hypothesis and should spur a more in-depth investigation of the mechanisms that govern spinogenesis, spine stabilization, and their role in schizophrenia. As a first step, we have identified one such mechanism, CACNB4 expression levels, which could contribute in part to the loss of small spines in schizophrenia. Future studies to confirm the effect of CACNB4, and other CACNB subunits, in vivo are warranted, and might include direct observation of spine dynamics. In addition, the pleiotropic genetic associations of calcium channel subunits with schizophrenia risk highlight the continued need to expand investigations beyond single proteins to the larger signaling network to enhance understanding of how genetic risk is translated into synaptic pathology.