Studying synaptic efficiency by post-hoc immunolabelling
© Ramírez-Franco et al.; licensee BioMed Central Ltd. 2013
Received: 16 April 2013
Accepted: 10 October 2013
Published: 18 October 2013
In terms of vesicular recycling, synaptic efficiency is a key determinant of the fidelity of synaptic transmission. The ability of a presynaptic terminal to reuse its vesicular content is thought to be a signature of synaptic maturity and this process depends on the activity of several proteins that govern exo/endocytosis. Upon stimulation, individual terminals in networks of cultured cerebellar granule neurons exhibit heterogeneous exocytic responses, which reflect the distinct states of maturity and plasticity intrinsic to individual synaptic terminals. This dynamic scenario serves as the substrate for processes such as scaling, plasticity and synaptic weight redistribution. Presynaptic strength has been associated with the activity of several types of proteins, including the scaffolding proteins that form the active zone cytomatrix and the proteins involved in presynaptic exocytosis.
We have combined fluorescence imaging techniques using the styryl dye FM1-43 in primary cultures of cerebellar granule cells with subsequent post-hoc immunocytochemistry in order to study synaptic efficiency in terms of vesicular release. We describe a protocol to easily quantify these results with minimal user intervention.
In this study we describe a technique that specifically correlates presynaptic activity with the levels of presynaptic markers. This method involves the use of the styryl dye FM1-43 to estimate the release capacity of a synaptic terminal, and the subsequent post-hoc immunolabelling of thousands of individual nerve terminals. We observed a strong correlation between the release capacity of the nerve terminal and the levels of the RIM1α but not the Munc13-1 protein in the active zone.
Our findings support those of previous studies and point out to RIM1α as a crucial factor in determining synaptic efficiency. These results also demonstrate that this technique is a useful tool to analyse the molecular differences underlying the heterogeneous responses exhibited by neuronal networks.
Presynaptic active zones (AZ) are specialized axonal sites of fusion that mediate neurotransmitter release into chemical synapses. A complex network of proteins is assembled at axonal sites that generate the so-called cytomatrix at the active zone (CAZ). These proteins interact with other proteins located either at the presynaptic plasma membrane or at vesicular membranes that regulate Ca2+-dependent fusion of synaptic vesicles. The different stages of the synaptic vesicular cycle (docking, priming, exocytosis and compensatory endocytosis) are orchestrated by distinct subsets of proteins, and the amount and interaction of these different proteins are thought to be crucial in determining presynaptic strength. The capacity of a given synapse to efficiently reuse synaptic vesicles has been proposed as a hallmark of maturation, and differences in vesicular reuse appear to underlie the enormous variability of responses observed in cultured neuronal networks . Remodelling of the active zone through changes in protein content or post-translational modifications has been linked with several crucial mechanisms involved in synaptic physiology, including presynaptic potentiation/depression, homeostatic synaptic scaling, synaptic silencing and synaptic weight redistribution [2–7].
In the present study, we focused on RIM1α as this protein is a key organizer of the active zone and it interacts directly or indirectly with all other known active zone proteins, including Rab3A and Munc13 . Indeed, the RIM proteins are required for synaptic vesicle priming and both short- and long-term synaptic plasticity [9–12]. These RIM’s tether Ca2+ channels to the presynaptic active zone  and activate vesicle priming by reversing the autoinhibitory homodimerization of Munc13 . Moreover, the RIM1/2 content is linearly associated with release probability and the size of the active zone . Consistent with this central role, Rim deletion prevents neurotransmitter release . In addition to RIM1α, we also analyzed Munc13-1, given the key role of Munc13 proteins in priming synaptic vesicles to a fusion-competent state  and in short-term potentiation of transmitter release [15–17].
Post-hoc immunocytochemistry and immunohistochemistry have previously been used to study synaptic and neuronal function [1, 18–20], yet to date, no detailed method to perform and analyse these experiments has been described. Here, we present a method that combines the assessment of presynaptic function in primary cultures of cerebellar granule neurons by monitoring synaptic vesicle recycling using the styryl dye FM1-43 with subsequent post-hoc immunocytochemistry. In addition, we describe a semi-automated protocol to easily quantify the data obtained, which enables the levels of immunoreactivity (IR) to be correlated with synaptic efficiency.
FM1-43 live cell imaging
The following antibodies were used in the present study: a mouse monoclonal anti-Munc13-1, (1:1000; ref. 126 111 Synaptic Systems); a rabbit polyclonal anti-RIM1 (1:400; ref. 140 003 Synaptic Systems); and a guinea pig polyclonal anti-CB1R (1:300; ref. CB1-GP-Af530, Frontier Institute Co., Ltd.), the latter used exclusively as a presynaptic marker to obtain a positive linear relationship between the channels. To check for the specificity of the antibodies we performed western blot experiments as previously described . Both antibodies recognized a single band with the expected molecular weight for both proteins (Additional file 2: Figure S2). The western blot protocol is described in Additional file 3.
1 Fixation and blocking: After the removal of the coverslip, rinse it thoroughly in PBS at 37°C and fix by immersion in 4% paraformaldehyde for 15 minutes. Wash twice in PBS and permeabilize the cells for 6 minutes in PBS-0.2% Triton X-100 if immunodetection of intracellular epitopes is required. Wash for 5 minutes in PBS and using a P100 pipette, gently apply 90–100 μl of blocking solution (PBS + 0.05% Triton ×100, 5% goat serum, 5% donkey serum) onto the coverslip and incubate for 1 h at 37°C. It is helpful to leave the coverslips on a Parafilm-covered surface during the blocking and subsequent incubations to avoid spillage of the solution from the coverslip; 2 Labelling with primary and secondary antibodies: Incubate o/n at 4°C with the desired primary antibodies diluted in PBS containing 0.05% Triton X-100 and 2.5% donkey serum. Wash 3 times in PBS containing 0.1% Triton X-100 and twice in PBS alone. Subsequently, incubate the cells with the specific Alexa-conjugated secondary antibodies for 1 h at 37°C. Wash 5 times in PBS and rinse the covers in MilliQ water to remove excess salts.
FM1-43 experiment and post-hocimmunocytochemical analysis
Results and discussion
The intensity of RIM1α, unlike that of Munc13-1, is a bona fideindicator of synaptic efficiency
Although for decades primary neuronal cultures have been used to study various aspects of synaptic physiology, including pre- and postsynaptic function [6, 13, 22, 32, 33], synaptopathies and intersynaptic trafficking [34–37], the relationship between certain parameters of synaptic activity and protein content remains unclear. While it is widely accepted that different proteins carry out specific activities (e.g., exo- and endocytic proteins mediate exo- and endocytic processes, respectively), few studies have demonstrated a quantitative correlation between synaptic function and protein content. Several aspects of presynaptic function have recently been correlated with protein levels at a given release site , and interference with the dynamics of protein synthesis/degradation has been shown to modulate synaptic strength [4, 6, 38]. For example, RIM1α levels are linearly related to the release probability (Pr) of local axon collaterals of CA3 , and presynaptic efficiency can be bidirectionally modulated by modifying RIM1α levels . To determine the extent to which differences in the magnitude of responses between individual nerve terminals are caused by variations in protein content, we developed a method in which post-hoc immunolabelling is combined with (but not limited to) optical tracking of the synaptic vesicle (SV) cycle with FM1-43, this is a powerful tool to study the correlation between protein content and synaptic function, in this case vesicular release, a relationship that has remained undefined for decades in studies of synaptic function. Using the extensive body of published data relating to the exocytotic steps of the SV cycle (for review see ), we investigated whether the levels of two key proteins involved in the formation of exocytic complexes (Munc13-1 and RIM1α) are correlated with presynaptic activity, as measured by vesicular release.
Our results demonstrate that synaptic levels of RIM1α are positively correlated with FM1-43 unloading, which is a direct measure of vesicular reuse and release (Figure 3E and 3F). In this context we have found that those synaptic boutons whose RIM levels are higher than 2 times the mean IR value yielded by the whole population of boutons, are more efficient in terms of FM1-43 release. The opposite effect was found in the subpopulation of boutons with RIM1α levels lower than 0.5 times the mean IR value (Unloaded fraction; Whole population: 33.95 ± 0.27%, N = 4, n = 3666; RIM1α IR > 2: 36.41 ± 0.53%, N = 4, n = 502, **p < 0,01; RIM1α IR < 0.5: 30.18 ± 0, 47%, N = 4, n = 1300, **p > 0,01; ANOVA followed by Bonferroni’s Test for means comparison). These results are consistent with previous findings [4, 7, 13, 29] and they validate the use of this technique. Based on these findings, it is possible to classify the functional responses of the FM1-43 experiment blindly into categories of increasing efficiency by sorting the ROIs according to the intensity of RIM1α IR. RIM1α is a pivotal protein in the arrangement of presynaptic active zones  and it participates in a complex interaction network along with other presynaptic proteins, such as the vesicular protein Rab3  and the priming factor Munc13-1 . Another important role of RIM1α is to tether calcium channels to presynaptic active zones via its PDZ domain [13, 29, 41], a function that may be critical in coupling exocytosis to calcium influx. RIM1α also undergoes PKA-dependent phosphorylation  and ubiquitin-dependent degradation via the E3 ubiquitin-ligase SCRAPPER . Low levels of RIM are associated with low mEPSC frequencies and low calcium sensitivity , while high levels are linked with an increased Pr . These data support the hypothesis that RIM levels can dictate the release properties of different synapses, serving as a source of variability among populations of synapses.
In the present study, we present a method that can be used to correlate a functional parameter of synaptic physiology (SV release) with the levels of different proteins present at a given release site. The main advantage of this technique is the reduced user intervention during data processing, as manual selection and drawing of the different ROIs is avoided. In most studies assessing synaptic function using image techniques, ROIs are user-defined and as such, they represent a source of potential bias. This bias is not only due to the poor sensitivity of the human eye compared with processing software but also, because the ROI shape does not exactly match that of the synaptic boutons, resulting in the inclusion of dead pixels in the ROIs defined, which can in turn affect the numerical data obtained. Moreover, manual selection usually renders fewer ROIs, which is not optimal for high-level statistical tests. In our protocol we employed an automated routine  to generate a mask that includes several hundred ROIs per experiment, the shape of which exactly matches that of the FM1-43 puncta and the IR puncta in the post-hoc images. This routine also generates a set of images in which the background is subtracted automatically and that can be used for analysis. The validity of this method is corroborated by the correlation observed between RIM1α levels and the effectiveness of vesicular release, consistent with previous data [4, 7, 38]. Moreover, the responses are blindly sorted according to their IR intensity, thereby eliminating another source of bias. This protocol is not limited to the assessment of synaptic activity using FM1-43, and it can be used to correlate semiquantitative IR data with the calcium influx, measures of the exo/endocytic cycle with pHluorins, or electrophysiological recordings. This technique could also be further improved by incorporating super-resolution microscopy techniques such as STED [47, 48] or STORM .
Altogether, our results indicate that nerve terminal content of RIM1α strongly correlates with the release capacity of the nerve terminal measured with FM1-43, while no such a correlation was found with Munc13-1. This finding point out to RIM1α as a crucial factor in determining synaptic efficiency and demonstrate the usefulness of this technique to analyse the molecular differences underlying the heterogeneous responses exhibited by neuronal networks.
We thank María del Carmen Zamora for her excellent technical assistance. This work was financed by grants from the Spanish MINECO (BFU2010-16947 to JS-P and BFU2012-32105 to MT), the ‘Instituto de Salud Carlos III’ RD06/0026 and the ‘Comunidad de Madrid’ (CAM-I2M2 2011-BMD-2349 to J S-P and MT). We thank Dr. M Sefton for editorial assistance.
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