Differences in synaptic vesicle pool behavior between male and female hippocampal cultured neurons

A strong focus on sex-related differences has arisen recently in neurobiology, but most investigations focus on brain function in vivo, ignoring common experimental models like cultured neurons. A few studies have addressed morphological differences between male and female neurons in culture, but very few works focused on functional aspects, and especially on presynaptic function. To fill this gap, we studied here functional parameters of synaptic vesicle recycling in hippocampal cultures from male and female rats, which are a standard model system for many laboratories. We found that, although the total vesicle pools are similar, the recycling pool of male synapses was larger, and was more frequently used. This was in line with the observation that the male synapses engaged in stronger local translation. Nevertheless, the general network activity of the neurons was similar, and only small differences could be found when stimulating the cultures. We also found only limited differences in several other assays. We conclude that, albeit these cultures are similar in behavior, future studies of synapse behavior in culture should take the sex of the animals into account.


Scientific Reports
| (2021) 11:17374 | https://doi.org/10.1038/s41598-021-96846-y www.nature.com/scientificreports/ of a readily retrievable pool of vesicle membranes 14 , and another ~ 25% within the presynaptic boutons (e.g. 15 ). The rest of the vesicles form a so-called reserve pool, whose involvement in functional reactions is unclear [16][17][18] , and whose function may be mostly related to collecting soluble cofactor proteins and providing them for exoand endocytosis reactions [19][20][21][22] . Sex-related differences are completely unknown for synaptic vesicle pools, especially as these pools can be studied most effectively in cultured neurons, where sex-related issues have been only little investigated so far. Here we used several well-established tools to investigate vesicle pools, and found that they are different between male and female neurons, at least for hippocampal neurons of ~ 15 days in vitro, which are the most common culture model for synapse investigations. The total vesicle pools were similar, but the recycling pool was larger for synapses in male neurons, and was more frequently used. This correlated well with the fact that local translation, which has been often connected to synaptic function 23 , was higher in synapses of male neurons.

Results and discussion
We prepared cultures from the hippocampi of newborn male and female rats 24 , separately, and allowed them to mature and to establish synaptic connections until at least day in vitro (DIV) 15. The cultures had similar cell numbers, and similar proportions of neurons and glia (Supplementary Figure S1). The overall morphology, which has been analyzed in detail in the past 10 , appeared similar, and the cellular volumes were similar (Supplementary Figure S2).
To determine functional differences between these cultures, we first compared their synaptic activity, relying on a well-established assay, in which the intravesicular domain of synaptotagmin 1 (Syt1) is detected by fluorescently-conjugated antibodies (Fig. 1a), which are then endocytosed 25,26 . A 45-min incubation with the antibodies is sufficient to saturate the entire pool of recycling vesicles 15 , thereby providing a measure of its size. To also provide a measure of the activity of the synapses, we added a fluorescently-conjugated nanobody that detects the antibodies 27 for 15 min. The nanobody detects the vesicles that recycle during the respective 15 min, and thus provides a direct measurement of synaptic exo-and endocytosis.
Both labels could be detected readily in the cultures (Fig. 1b), and in both cases the male neurons showed significantly higher intensities (Fig. 1c,d). Blocking synaptic activity with the Na + channel blocker tetrodotoxin (TTX), which halts the action potential generation, enabled us to test differences between the spontaneously recycling pools of vesicles 28 . As expected 15 , this treatment strongly reduced the labeling. At the same time, it removed any differences between the two types of cultures, suggesting that, as for the total vesicle pools, there is little difference between the spontaneously recycling pools of male and female neurons.
To test the surface pool of vesicle molecules, we performed this assay while keeping the neurons on ice. This only enables the detection of the surface pool of Syt1 molecules, which are representative for the surface vesicle pool 14,15 . This resulted in a low labeling that was similar for male and female cultures (Supplementary Figure S3).
To investigate the synaptic activity in more detail, we performed the same assay using excitatory and inhibitory synaptic markers, VGAT (Vesicular Gamma Aminobutyric Acid Transporter) and VGLUT1 (Vesicular Glutamate Transporter 1), respectively (Fig. 2a). We did not find any differences between the VGAT (Fig. 2b) or VGLUT1 (Fig. 2c) abundances in the male and female cultures. The culture network also contains similar amounts of inhibitory and excitatory synapses (Fig. 2d). However, the synaptic vesicles were recycled again more in male cultures, regardless of the synapse type (Fig. 2e,f).
Overall, these results suggest that the main difference between male and female neurons, in terms of vesicle pools, is the size and usage of the recycling pool, suggesting that male neurons use their vesicles more often. As activity strongly correlates with the synaptic turnover 29 , we then tested whether the sex-related activity differences were mirrored by differences in local turnover. We first used a metabolic labeling assay 30 , in which the cultures were incubated with a methionine substitute, L-Homopropargylglycine (HPG), for 4 h. The HPG molecules become incorporated in the newly-synthesized proteins, in the place of methionine, and can be then revealed by click chemistry (Supplementary Figure S4a). No differences could be found, either in synapses or elsewhere (Supplementary Figure S4b-e), implying that protein production (and presumably turnover) is overall similar in the male and female cells.
To follow up on these findings, we compared the protein production within the synaptic compartments 23 . We performed a similar assay, in which the cells were incubated with the antibiotic puromycin, which incorporates itself into the growing polypeptide chain during translation, and releases it from the ribosome (Fig. 3a). This assay indicates with high specificity the synthesis of new protein chains in the synapses (as opposed to their transport to synapses from other locations, which cannot be excluded in the case of HPG labeling). We found that puromycin labeling was similar for the male and female cell bodies, but was significantly higher for the male synapses, for both the pre-and postsynaptic sides (Fig. 3b-e).
The functional differences in synaptic activity, as well as translation differences, pointed out possible differences in the spontaneous firing rate. To take a closer look at the calcium activity, we relied on Ca 2+ imaging. We transfected the cultures at DIV10 with a genetically-encoded Ca 2+ indicator, NeuroBurst (Fig. 4a). At DIV17 we imaged the neurons to determine their spontaneous activity patterns, focusing on the evident Ca 2+ dynamics in the somas (Fig. 4b). Both the frequency and the intensity of the activity events appeared similar (Fig. 4c). For a more thorough comparison, we measured the areas under the peaks of the normalized calcium intensity curves, which provides a summed "activity score" for each cell 31 . This measurement suggests that female and male neurons exhibit similar base-line NeuroBurst expression as well as similar levels of spontaneous activity (Fig. 4d,e). Furthermore, we analyzed several parameters of the calcium signals, such as event frequency, length and intensity (Supplementary Figure S5), without finding any significant differences between the two sexes. Therefore, the "physiological" activity of the neurons seems to be similar for both male and female cultures. To verify their capacity to respond to stimuli that surpass the physiological levels, we subjected the cells to a Scientific Reports | (2021) 11:17374 | https://doi.org/10.1038/s41598-021-96846-y www.nature.com/scientificreports/ prolonged depolarization, using a high K + /low Na + buffer (Fig. 5a). The peak Ca 2+ increase was similar for both male and female cultures, but the male cultures took longer to reach a Ca 2+ plateau, possibly due to differences in the speed of the Ca 2+ channel inactivation 32 , or to differences in Ca 2+ buffering dynamics (Fig. 5b,c). The dynamics observed here are typical for such prolonged high K + stimulation [33][34][35] .
To study the neuronal dynamics by a different method, we then used a voltage-sensitive membrane dye (FluoVolt, Thermo Fisher) (Supplementary Figure S6). The recordings were performed in a similar fashion to the Ca 2+ imaging from   www.nature.com/scientificreports/ These observations suggest that male neurons contain larger pools of active vesicles, and are more active, at the single-synapse level. To test whether this relates to different levels of key synaptic proteins, we immunostained vesicle markers (VAMP2, synaptophysin, VGLUT1), active zone markers (bassoon), cytoskeletal components (tubulin, MAP2), postsynaptic receptors (GluR2, Glun2B (Glun2A domain was substantially less abundant in the culture and therefore not selected for the survey)), postsynaptic density components (PSD95, Homer1), important synaptic regulators (α-synuclein, RBM3) and calcium binding proteins (calbindin, calmodulin and calreticulin). Only three proteins showed different intensities, bassoon, synaptophysin and calbindin (Supplementary Figure S7), and the overall differences were small. The number of objects (such as synapses, active zones, PSDs) was similar for all investigated proteins (Supplementary Figure S8). Cultures from other regions, such as cortex and hypothalamus, do show sex-dependent differences in the abundance of calbindin 36,37 . These small differences in protein abundance might have a contribution to the intracellular calcium regulation, and then to calcium dynamics and synaptic activity.
To confirm these observations at the whole-culture level, we proceeded with-omics analysis at transcript (Supplementary Figure S9a,b) and protein (Supplementary Figure S9c) levels. Beside the expected differences in Y-chromosome linked transcripts, we only found a slight tendency for higher amounts of transcripts relating to To report local translation rates, we performed a "puromycin assay". During translation, the ribosome moves along the mRNA, enabling the growth of the poly-peptide chain (1). Puromycin interferes with the translation by binding to the ribosome, and stops the translation by incorporating itself into the premature polypeptide chain (2). It will release the premature poly-peptide chain (3). An immunostaining against puromycin shows the amount of translation at a certain location (4). To calculate the background of the puromycin treatment, we used another antibiotic, anisomycin, which prevents the incorporation of puromycin to a poly-peptide chain (not shown in the cartoon).  Figure S9d), and the mutual hits between these two datasets are very few (only a handful, shown in Supplementary Table S1). This implies that a gene enrichment or network analysis is difficult to perform on these data. Overall, these observations lead to the conclusion that male synapses have larger recycling vesicle pools, which they employ more often than female synapses. The male neurons also d engage in higher levels of local translation, although this does not result in differences at the level of network activity, presumably due to compensatory mechanisms. One such possible mechanism would be higher synaptic activity in both excitatory and inhibitory neurons in male cultures, which thereby leads to similar overall network firing levels, albeit many other scenarios could be envisioned.
Our observations lead to a two-pronged conclusion. First, the limited differences noted here imply that most experiments using cultures formed of mixed male and female neurons are likely to be unaffected by sex-related differences, which is an important finding for the field. Second, the significant differences found for synaptic activity and local translation imply that studies of synapses should take the animal sex into account, and To compare the spontaneous calcium activity between the two sexes, we calculated the area under the curve of the intensity graphs, which we termed "activity score", over 6 independent culture preparations that have 6 coverslips for each sex (N = 6, n = 36 independent experiments). The graph presents the activity score (mean ± SEM) and each symbol represents the mean activity score of a coverslip. Maximum 10 neurons were selected per coverslip. The statistical comparison between the two sexes was performed by the Mann-Whitney test, not significant (ns). (e) To examine the NeuroBurst expression levels, we calculated the fluorescence intensity in the soma versus the background. The graph presents the baseline intensity over background (mean ± SEM) and each dot indicates the mean of a coverslip. The statistical comparison between the two sexes was performed by the unpaired t-test, not significant (ns).

Methods
Animals. Rats (Rattus norvegicus. wild type, Wistar) for the primary neuron cultures were obtained from the animal facility of the University Medical Center Göttingen. Animals were handled according to the specifications of the University of Göttingen and of the local authority, the State of Lower Saxony (Landesamt für Verbraucherschutz, LAVES, Braunschweig, Germany). All experiments and procedures were approved by the local authority, the Lower Saxony State Office for Consumer Protection and Food Safety (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit). All methods are reported in accordance with ARRIVE guidelines and were carried out in accordance with relevant guidelines and regulations. The experimental procedure was approved by the relevant institutional entity, the Tierschutzbüro of the University Medical Center Göttingen (approval number T 09/08).
Hippocampal cultures. Newborn female and male rats were sacrificed for the primary dissociated hippocampal culture preparation 24 . The sex determination was performed by animal facility personnel according to the animal morphology 38 . Hippocampi were dissected and washed in HBSS (Thermo Fisher, US). For dissociation, dissected hippocampi were incubated for 1 h with the enzyme (1.6 mM cysteine, 100 mM CaCl 2 , 50 mM EDTA, and 25 units papain in 10 ml DMEM (Thermo Fisher, US)). 5 ml of DMEM, containing 10% fetal calf serum (FCS), 0.5% albumin, and 0.5% trypsin inhibitor, was used for enzyme inactivation. After an enzymatic treatment, cells were dissociated further by pipetting. 80,000 neurons were plated on 1.8 cm in diameter glass coverslips, which were coated with poly-L-lysine (PLL, Sigma-Aldrich, Germany   Puromycin assay. 1 µg/ml of puromycin (ant-pr-1, InvivoGen, US) was added into the cultures. After 10 min of incubation, coverslips were washed with Tyrode buffer. The fixation was followed with 4% PFA. Anisomycin (0.13 μM, A5862, Sigma-Aldrich, Germany) was used in order to detect the background signal of the assay. For the control group, it was added 10 min before the actual puromycin treatment. After the fixation, cells were immunostained, as described in the immunostaining section, against Synaptophysin (101004, Synaptic Systems, Germany), Homer1 (160011, Synaptic Systems, Germany), and puromycin (MABE343, Merck Millipore, Germany).
Image analysis. Matlab (MathWorks, US) was used for image analysis, and plots were prepared in GraphPad Prism version 8.00 (GraphPad Software, US). All analysis was performed using simple thresholding procedures, based on empirically-derived thresholds, followed by the analysis of signal intensities within the thresholddefined ROIs. The ROIs were defined in the synaptic marker channels (Syph, Homer 1) for synaptic experiments, and not in the channels representing the measurements of interest (Syt1, puromycin, HPG). License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.