1. Introduction
Synaptic transmission is a fundamental step in neuronal communication and the main place for neuromodulation. In presynaptic boutons, the opening of high threshold voltage-gated calcium channels (VGCCs) is a central step in the action potential-driven transmitter release [
1,
2]. Synaptic strength and synchronous of release depend on the subtype, number, activity, and topography of VGCCs [
3,
4]. Action potential-triggered vesicle release mainly depends on Cav2.1 (P/Q-type) and Cav2.2 (N-type) VGCCs as determined by postsynaptic excitatory postsynaptic currents (EPSCs) [
5,
6], but in some synapses only P/Q type VGCCs seem to be relevant for fast synaptic vesicle release [
7]. Additional Ca
2+ influx at the presynapse employs Cav2.3 (R-type) and Cav1.2/3 (L-type) channels [
8,
9], although the latter is believed to have a limited impact on vesicle release and, thus, eIPSC amplitude [
1,
7,
10]. The high-voltage activated Cav2 channels show faster activation and inactivation making them suitable for fast transmission of neuronal action potential activity, whereas Cav1 channels are primarily involved in slower processes like hormone secretion and Ca
2+ signaling to gene transcription [
11]. Accordingly, biochemical and functional studies have identified numerous molecular interactions between VGCC subunits and various partners that serve, for example, to couple Ca
2+ channels to the release machinery (reviewed in [
11,
12]). Interestingly, these interactions of VGCC subunits include not only intracellular pathways but also crosstalk to extracellular or cell surface molecules [
13,
14,
15,
16,
17,
18,
19,
20].
We discovered many years ago that neurexins (Nx), a polymorphic family of synaptic cell surface molecules [
21,
22], are involved in the regulation of VGCC-dependent neurotransmitter release from excitatory and inhibitory synapses [
23]. Neurexins are encoded by three genes in vertebrates, each of which contains independent promoters that drive transcription of longer α-Nx and shorter β-Nx. A truncated γ-isoform is transcribed in neurexin-1 [
24] and more variants arise from up to six conserved splice sites [
25,
26]. Extracellularly, α-Nx proteins mostly comprise six laminin-Nx-sex-hormone-binding (LNS) domains with interspersed epidermal growth factor (EGF)-like repeats. Shorter β-Nx differ by expressing a β-specific, 37-residue-long N-terminal domain before splicing into the last (sixth) LNS domain of the respective gene [
21,
22]. Since LNS6 and subsequent sequences are identical in α- and β-Nx, they share properties such as a C-terminal PDZ recognition motif required for intracellular trafficking [
27,
28], a heparan sulfate glycan moiety [
29], and physiological ectodomain cleavage [
30]. α- and β-Nx also share binding partners such as neuroligins [
31,
32,
33], leucine-rich repeat transmembrane neuronal proteins (LRRTMs) [
34,
35,
36], α -dystroglycan [
37,
38], latrophilins [
39], and cerebellins [
40,
41].
The functional link between Nx and VGCCs was initially observed in a constitutive deletion mouse model (knockout) of all α-Nx [
23,
42,
43], and later confirmed in conditional knockout neurons lacking all β-Nx [
44,
45]. Surprisingly, investigations of conditional knockout neurons lacking all Nx variants detected reduced total Ca
2+ transients only in somatostatin- but not parvalbumin-positive interneurons of the medial prefrontal cortex [
46], and failed to see reduced Ca
2+ influx into the parvalbumin-positive excitatory calyx of Held synapses in the brainstem [
47]. A possible explanation of this discrepancy might be that the functional link between Nx and VGCCs involves specific combinations of Nx variants and VGCC subtypes which may differ between brain regions and subpopulations of synapses. In support, we found recently that the reduced Ca
2+ influx into boutons of excitatory hippocampal neurons in α-Nx triple knockout mice predominantly involved Cav2.1 (P/Q-type) VGCCs [
17] and could be rescued by overexpression of the Nx1α variant which is abundant in hippocampal neurons [
48].
To further explore this important aspect in our current study, we compared directly in the same model system whether and how deletions of one or all Nx isoforms can affect different synaptic VGCC subtypes. Therefore, we generated a conditional Nx1α knockout mouse model and compared presynaptic Ca
2+ influx in primary hippocampal cultures of control to conditional knockout neurons lacking either the single Nx1α variant (Nx1α cKO, created for this study) or all Nx isoforms (Nx123 cKO [
46,
47]). We particularly focused on how the deletions affect single action potential-evoked Ca
2+ influx through different VGCC subtypes, using transfected synGCaMP7b [
49] as Ca
2+ indicator and pharmacological isolation by sequential addition of subtype-specific blockers [
9] which together allowed quantification even at the level of individual presynaptic boutons. We report here that Nx variants likely alter the contribution of most VGCC subtypes to presynaptic Ca
2+ transients, including P/Q-type (CaV2.1), N-type (CaV2.1), L-type (CaV1.2/3) and R-type (CaV2.3) channels. Strikingly, the deletions of a single Nx1α or all Nx variants resulted in a different pattern of VGCC subtypes affected. These findings may indicate that Nx variants modulate Ca
2+ influx in a partially overlapping, partially unique way, depending on the actual presence and/or relative amount of Nx variants and VGCC subtypes in a particular synapse population or even in individual terminals.
4. Discussion
The present study revealed an unexpectedly complex modulation of presynaptic Ca
2+ influx by Nx based on a comparison of Ca
2+ transients through specific VGCC subtypes. In hippocampal neurons of Nx123 cKO mice, the presynaptic Ca
2+ influx was reduced upon conditional knockout of all Nx variants (
Figure 2 D,F). Interestingly, this reduction was stronger than that previously seen in neurons lacking all α-Nx but not β-Nx [
17] or in neurons lacking all β-Nx but not α-Nx [
44,
45]. But even our novel deletion of the ASD candidate gene Nx1α alone induced a reduced total presynaptic Ca
2+ influx (
Figure 2 E,F), suggesting that already the lack of a single Nx variant affects synaptic efficiency. Obviously, the removal of more and more Nx variants gradually induces a stronger reduction of total presynaptic Ca
2+ influx. While these data confirm our initial hypothesis of a general dose effect of Nx on synaptic function [
23], we surprisingly found that deletions of Nx may produce different and complex patterns of affected VGCC subtypes.
In the complete Nx123 cKO, the reduced total presynaptic Ca
2+ influx was mainly due to a reduced influx through P/Q-type channels (
Figure 3). This is in line with similar data from hippocampal neurons of a constitutive knockout of all α-Nx variants [
17] and from an analysis of the calyx of the Held synapses, in which Nx were shown to be crucial for clustering of P/Q-type channels in the active zone [
47]. In addition, our investigation of Nx123 cKO produced a tendency toward the reduction of N-type channel contribution while increasing the contribution of L-type and R-type VGCCs. Since this shift would imply a change from channels directly coupled to vesicle release to channel subtypes with a mere supportive role in fast synaptic release [
1,
6], the transition away from P/Q- and N-type to L- and R-type likely predicts a more dramatic influence on synaptic release than the moderate reduction of the Ca
2+ transients suggest. In fact, a large release defect has been previously described with a reduction of postsynaptic EPSCs in αTKO neurons (lacking all α-Nx) by more than 50% compared to controls [
42].
A different pattern of VGCC modulation was seen in the case of the single Nx1α cKO. The deletion of Nx1α alone did not change the Ca
2+ influx through the P/Q-type channel, but, unexpectedly, elevated the contribution of N-type channels. Since the total Ca
2+ influx was moderately reduced in this deletion model, the increased Ca
2+ influx through N-type channels was likely compensated by a reduced L-type channel contribution (
Figure 4). As P/Q-type and N-type VGCCs are the main Ca
2+ channels for presynaptic transmitter release and deletion of Nx1α induces a shift in the relative contribution from L-type to N-type channels, transmitter release in Nx1α-deficient synapses should be normal or the probability of release increased, unlike in the complete deletion of Nx. Thus, the overall organization of the presynaptic active zone and clustering of P/Q-type channels observed earlier in the Calyx of Heldt synapse in absence of all Nx [
47] most likely does not depend on the Nx1α variant because Nx1α knockout did not affect P/Q-type channel-driven Ca
2+ influx as shown here in hippocampal neurons. However, this finding is in contrast to observations in neurons from a constitutive knockout of all α-Nx variants, in which overexpressed Nx1α partially rescued the amount of Ca
2+ influx through P/Q-type channels [
17]. This discrepancy indicates that Nx1α is not alone responsible for the modulation of the P/Q-type channel [
17], but that other α-Nx variants can compensate for the deletion, e.g. in concert with α2δ auxiliary subunits of VGCCs. Together, these results are consistent with the view that Nx regulate presynaptic Ca
2+ influx and that individual Nx variants may have partially overlapping, partially non-redundant effects on the distribution or function of different VGCC subtypes.
To further explore the possibility that α-Nx are also involved in additional signaling pathways targeting presynaptic VGCCs as suggested previously for the GABA
B receptor pathway [
43,
69], we investigated retrograde signaling via the endocannabinoid system (ECS). In this retrograde pathway, postsynaptically synthesized endocannabinoids (e.g., 2-AG or AEA) diffuse to the presynapses to stimulate presynaptic CB1-receptor, inhibiting the activity of VGCCs [
64,
65]. We therefore compared neurons containing or lacking Nx and revealed a lower relative CB1-receptor activation by 2-AG in absence of all Nx variants. This is in line with an increased tonic endocannabinoid signaling as it has been proposed for reduced β-Nx levels according to a study of the ECS in β-Nx-deficient neurons [
44]. Our current results now suggest that the role of α-Nx in this regulation may even be stronger than β-Nx, based on a direct comparison of neurons lacking all Nx123 cKO versus β-Nx cKO neurons. While these results present an important extension of the role of Nx in regulating presynaptic VGCCs, the measured changes in presynaptic Ca
2+ transients could not elucidate the precise mechanism of how Nx modulate the ECS. However, at least three hypotheses are conceivable. First, a postsynaptic regulation of 2-AG synthesis as postulated in [
44] is possible since Nx engages in transsynaptic interactions and can cluster receptors in the postsynaptic membrane, for example, AMPAR [
48,
70,
71] and GABA
AR [
72]. Naturally, such a potential postsynaptic influence on 2-AG synthesis could hardly be attributed to β-Nx alone as α-Nx share the same binding partners, supporting our observation here. This scenario would imply that α-Nx have an additional effect on VGCCs by modulating the ECS postsynaptically. Second, Nx could modulate the effect of the ECS system via the presynaptic organization of VGCCs and/or the localization of CB1-receptors. In support, it was shown that α-Nx is presumably involved in the overall organization at the active zone [
47], and an altered distribution or activity of either CB1-receptors or the VGCC subtypes themselves may explain the effect of Nx reported here. Third, since the activity of the CB1-receptor is regulated by on-demand production and degradation of 2-AG [
68], it cannot be ruled out that Nx might influence presynaptic 2-AG degradation as an additional alternative. Future research will have to distinguish between these possibilities.
Neurexins belong to the candidate genes for autism spectrum disorders (ASD) and mutations in
Nx1 are among the most frequently found variants in ASD cohort studies [
73,
74]. To confirm the face validity of mouse models for ASD, numerous studies have addressed higher brain functions in deletion models of molecules related to synaptic function by behavioral profiling (reviewed in [
75]). We and others reported, for example, that Nx1α KO mice display a decrease in social investigation and an increase in aggressive behavior [
57], augmented repetitive behaviors [
56], and altered social memory and novelty behavior even at the heterozygous level [
58,
76]. Such relatively mild impairments could well be explained by the subtle changes in VGCC subtype contribution as shown here in Nx1α deficient neurons since a decrease in spontaneous EPSC release and evoked field potentials have been reported in a constitutive KO of Nx1α [
56]. However, more studies correlating mutations to alterations of behavior and synaptic function, most notably of Ca
2+ influx and release, are needed to determine the specific contribution of individual Nx variants to the pathomechanism of ASD.
Author Contributions
Conceptualization, M.M. and J.B.; methodology, M.M., J.B. and C.R.; software, J.B. and I.K.; formal analysis, J.B., I.K., C.R., D.R. and M.A.; investigation, I.K., J.B., D.R., C.R. and M.A.; resources, M.M.; data curation, J.B. and C.R.; writing—original draft preparation, J.B., I.K. and M.M.; writing—review and editing, M.M.; visualization, J.B., I.K. and C.R.; supervision, M.M.; project administration, M.M.; funding acquisition, M.M. All authors have read and agreed to the published version of the manuscript.
Figure 1.
Conditional deletion of the single Nx1α variant. (A) Immunoblots of Nx in Nx123 cKO cells tested with 3 different antibodies: pan-Nx1 (A1, Millipore #AB161-I), Nx1α (A2, Frontier Institute #AB_2571817) and Nx123 (A3, SySy #175003). (B) Wild type allele of the 5’ end of the Nx1α gene including the first coding exon (indicated in red) is illustrated. After successful homologous recombination of the wild-type allele with the targeting vector (not depicted), the knock-in allele that resulted is indicated. The 5’ loxP site is introduced via the BamH1 (‘B’) site upstream of the first coding exon. Downstream of the first coding exon and at the EcoR1 site (‘E’), the 3’ loxP site and NeoR (Neomycin resistance) gene are inserted (blunt end cloning). Via the addition of a Cre-recombinase, the knock-in allele is converted into the knockout allele. The region between the loxP sites of the Nx1α gene including the first coding exon is excised. Further restriction sites: S = Spel, N = Nhel. (C) Immunoblots of Nx1α cKO and WT neurons with anti-Nx123 (SySy #175003). (D) Quantification of αNx normalized to ΔCre condition (100%) for Nx123 cKO neurons and Nx1α cKO neurons. Data are based on n independent immunoblot experiments (WT: 3, 2; Nx123: 4, 4; Nx1α: 3, 3); columns were compared with an unpaired t-test. n.s. = non-significant: p > 0.05, ** p < 0.01, ***p < 0.001.
Figure 1.
Conditional deletion of the single Nx1α variant. (A) Immunoblots of Nx in Nx123 cKO cells tested with 3 different antibodies: pan-Nx1 (A1, Millipore #AB161-I), Nx1α (A2, Frontier Institute #AB_2571817) and Nx123 (A3, SySy #175003). (B) Wild type allele of the 5’ end of the Nx1α gene including the first coding exon (indicated in red) is illustrated. After successful homologous recombination of the wild-type allele with the targeting vector (not depicted), the knock-in allele that resulted is indicated. The 5’ loxP site is introduced via the BamH1 (‘B’) site upstream of the first coding exon. Downstream of the first coding exon and at the EcoR1 site (‘E’), the 3’ loxP site and NeoR (Neomycin resistance) gene are inserted (blunt end cloning). Via the addition of a Cre-recombinase, the knock-in allele is converted into the knockout allele. The region between the loxP sites of the Nx1α gene including the first coding exon is excised. Further restriction sites: S = Spel, N = Nhel. (C) Immunoblots of Nx1α cKO and WT neurons with anti-Nx123 (SySy #175003). (D) Quantification of αNx normalized to ΔCre condition (100%) for Nx123 cKO neurons and Nx1α cKO neurons. Data are based on n independent immunoblot experiments (WT: 3, 2; Nx123: 4, 4; Nx1α: 3, 3); columns were compared with an unpaired t-test. n.s. = non-significant: p > 0.05, ** p < 0.01, ***p < 0.001.

Figure 2.
Presynaptic Ca2+ transients recorded from individual active boutons with synGCaMP7b. (A) Example picture of fluorescence intensity of synGCaMP7b before stimulation (left, F0, shown in magenta), representing the baseline fluorescence; fluorescence intensity changes after stimulation with 3 AP, isolated by subtraction (middle, ΔF, shown in green). The green fluorescence dots lighting up indicate active boutons. Both images merged represent the effect image (right) that allows the identification of active boutons that are not disturbed by high baseline fluorescence of other sources like Cre-EGFP-fluorescent cell nuclei (asterisk). (B) Enlarged perspective (yellow box in A), showing the change in fluorescence (ΔF, green) as well as the cell process morphology indicated by co-transfected RFP (red). (C) ROIs (red circles) were placed on active boutons for the quantification of presynaptic Ca2+ transients. (D) Averaged synGCaMP7b fluorescence changes from Nx123 cKO neurons with Nx (Cremut, n = 14 cells/1045 boutons) or without all Nx variants (dashed line, Cre, 13/916) show Ca2+ transients following a single AP stimulation. (E) Neurons lacking only Nx1α (Cre, 8/681) and equivalent controls (Cremut, 12/1074) showed comparable fluorescence alterations as those seen in N123 cKO. (F) Comparing peak values of Ca2+ transients (mean ± SEM) in Cremut and Cre cells from both mouse lines in response to a single AP stimulation. Nx123 cKO: Cremut 48 cells/3964 boutons, Cre 58/4582, and Nx1α cKO: Cremut 21/1672, Cre 25/2051. The mean values of all boutons of a single cell are shown as dots and used for statistics. Columns were compared with an unpaired t-test. *: p < 0.05, **** p < 0.0001.
Figure 2.
Presynaptic Ca2+ transients recorded from individual active boutons with synGCaMP7b. (A) Example picture of fluorescence intensity of synGCaMP7b before stimulation (left, F0, shown in magenta), representing the baseline fluorescence; fluorescence intensity changes after stimulation with 3 AP, isolated by subtraction (middle, ΔF, shown in green). The green fluorescence dots lighting up indicate active boutons. Both images merged represent the effect image (right) that allows the identification of active boutons that are not disturbed by high baseline fluorescence of other sources like Cre-EGFP-fluorescent cell nuclei (asterisk). (B) Enlarged perspective (yellow box in A), showing the change in fluorescence (ΔF, green) as well as the cell process morphology indicated by co-transfected RFP (red). (C) ROIs (red circles) were placed on active boutons for the quantification of presynaptic Ca2+ transients. (D) Averaged synGCaMP7b fluorescence changes from Nx123 cKO neurons with Nx (Cremut, n = 14 cells/1045 boutons) or without all Nx variants (dashed line, Cre, 13/916) show Ca2+ transients following a single AP stimulation. (E) Neurons lacking only Nx1α (Cre, 8/681) and equivalent controls (Cremut, 12/1074) showed comparable fluorescence alterations as those seen in N123 cKO. (F) Comparing peak values of Ca2+ transients (mean ± SEM) in Cremut and Cre cells from both mouse lines in response to a single AP stimulation. Nx123 cKO: Cremut 48 cells/3964 boutons, Cre 58/4582, and Nx1α cKO: Cremut 21/1672, Cre 25/2051. The mean values of all boutons of a single cell are shown as dots and used for statistics. Columns were compared with an unpaired t-test. *: p < 0.05, **** p < 0.0001.

Figure 3.
Deletion of all Nx variants decreased presynaptic Ca2+ influx primarily via P/Q-type VGCC. Pharmacologically isolated VGCC subtype contribution to the Ca2+ influx measured during single AP stimulation in Nx123 cKO neurons with synGCaMP7b by sequential addition of specific blockers: ω-agatoxin IVA (AgTX, 0.1 μM; P/Q-type); ω-conotoxin GVIA (CTX, 2 μM; N-type); nifedipine (Nif, 20 μM; L-type); SNX-482 (SNX, 0.5 μM; R-type). (A) Averaged traces of control neurons (Cremut, 12 cells/869 boutons, left) and neurons lacking all neurexin variants (Cre, 13/916, right). (B) Ca2+ transients that specifically reflect Ca2+ influx through the given VGCC subtypes are isolated by subtraction from the traces in A, comparing Nx123 cKO Cremut (continuous lines) and Nx123 cKO Cre (dashed lines). (C) Mean ± SEM of relative VGCC subtype contribution (%) calculated for each bouton (ROI, relative to total Ca2+ influx) in Nx123 cKO neurons. The number of examined boutons/cells is shown in the P/Q columns and applies to all VGCC subtypes. Columns were compared with Kruskal-Wallis test, n.s.: p > 0.05, **: p < 0.01, ****: p < 0.0001. (D) Each presynaptic bouton’s P/Q-type contribution was determined, and the spreading is depicted in a histogram that contrasts the distribution of Nx123 cKO neurons with and without Nx, showing that without Nx, the number of boutons with more than 50% P/Q-type Ca2+ influx is almost lost (Cremut, 584/12; Cre, 501/13). The same analysis is shown in (E) for the N-type and (F) for the L-type portion in individual boutons.
Figure 3.
Deletion of all Nx variants decreased presynaptic Ca2+ influx primarily via P/Q-type VGCC. Pharmacologically isolated VGCC subtype contribution to the Ca2+ influx measured during single AP stimulation in Nx123 cKO neurons with synGCaMP7b by sequential addition of specific blockers: ω-agatoxin IVA (AgTX, 0.1 μM; P/Q-type); ω-conotoxin GVIA (CTX, 2 μM; N-type); nifedipine (Nif, 20 μM; L-type); SNX-482 (SNX, 0.5 μM; R-type). (A) Averaged traces of control neurons (Cremut, 12 cells/869 boutons, left) and neurons lacking all neurexin variants (Cre, 13/916, right). (B) Ca2+ transients that specifically reflect Ca2+ influx through the given VGCC subtypes are isolated by subtraction from the traces in A, comparing Nx123 cKO Cremut (continuous lines) and Nx123 cKO Cre (dashed lines). (C) Mean ± SEM of relative VGCC subtype contribution (%) calculated for each bouton (ROI, relative to total Ca2+ influx) in Nx123 cKO neurons. The number of examined boutons/cells is shown in the P/Q columns and applies to all VGCC subtypes. Columns were compared with Kruskal-Wallis test, n.s.: p > 0.05, **: p < 0.01, ****: p < 0.0001. (D) Each presynaptic bouton’s P/Q-type contribution was determined, and the spreading is depicted in a histogram that contrasts the distribution of Nx123 cKO neurons with and without Nx, showing that without Nx, the number of boutons with more than 50% P/Q-type Ca2+ influx is almost lost (Cremut, 584/12; Cre, 501/13). The same analysis is shown in (E) for the N-type and (F) for the L-type portion in individual boutons.

Figure 4.
Single Nx1α deletion changed the VGCC subtype contribution to presynaptic Ca
2+ influx. VGCC subtype contribution on Ca
2+ influx after single AP stimulation was measured in Nx1α cKO neurons with synGCaMP7b by sequential addition of specific blockers as described in
Figure 3. (
A) Averaged traces of control neurons (Nx1α cKO Cre
mut, 13 cells/1154 boutons;
left) and neurons lacking only Nx1α (Cre, 11/956,
right). (
B) Ca
2+ transients that specifically reflect Ca
2+ influx through the sequentially blocked VGCC subtypes were isolated by subtraction from the traces in A and compared between Nx1α cKO Cre
mut (continuous lines) and Nx1α cKO Cre (dashed lines). (
C) Mean ± SEM of relative Ca
2+ contribution (%) per VGCC subtype calculated for individual boutons (ROIs) in Nx1α cKO neurons. The number of examined boutons/cells is shown in the P/Q columns and applies to all VGCC subtypes. Columns were compared with Kruskal-Wallis test, n.s.: p > 0.05, **: p < 0.01, ****: p < 0.0001. (
D) The P/Q-type portion of Ca
2+ transients was calculated for each synaptic bouton and the spreading is shown in a histogram comparing the variability in neurons with and without Nx1α (Cre
mut, 755/13; Cre, 552/11), in (
E) for the N-type and (
F) for L-type.
Figure 4.
Single Nx1α deletion changed the VGCC subtype contribution to presynaptic Ca
2+ influx. VGCC subtype contribution on Ca
2+ influx after single AP stimulation was measured in Nx1α cKO neurons with synGCaMP7b by sequential addition of specific blockers as described in
Figure 3. (
A) Averaged traces of control neurons (Nx1α cKO Cre
mut, 13 cells/1154 boutons;
left) and neurons lacking only Nx1α (Cre, 11/956,
right). (
B) Ca
2+ transients that specifically reflect Ca
2+ influx through the sequentially blocked VGCC subtypes were isolated by subtraction from the traces in A and compared between Nx1α cKO Cre
mut (continuous lines) and Nx1α cKO Cre (dashed lines). (
C) Mean ± SEM of relative Ca
2+ contribution (%) per VGCC subtype calculated for individual boutons (ROIs) in Nx1α cKO neurons. The number of examined boutons/cells is shown in the P/Q columns and applies to all VGCC subtypes. Columns were compared with Kruskal-Wallis test, n.s.: p > 0.05, **: p < 0.01, ****: p < 0.0001. (
D) The P/Q-type portion of Ca
2+ transients was calculated for each synaptic bouton and the spreading is shown in a histogram comparing the variability in neurons with and without Nx1α (Cre
mut, 755/13; Cre, 552/11), in (
E) for the N-type and (
F) for L-type.

Figure 5.
Endocannabinoid-evoked CB1 receptor activation reduces presynaptic Ca2+ transients in an Nx-dependent manner. (A) Several presynaptic boutons of a synGCaMP7b transfected Nx123 cKO Cre neuron are shown in an exemplary ΔF image during 3 AP stimulation. (B) The identical presynaptic boutons as in A after 5 min of CB1-receptor activation with 2 µM 2-AG, again during a 3 AP stimulation. (C) Repetitive stimulation (1 AP every 30 s) shows a reduction of Ca2+ transients in response to the application of 2-AG, averaged (mean ± SEM) from neurons of Nx123 Cremut (13 neurons, continuous line) and Nx123 Cre (16, dotted line), displayed as relative changes normalized to the mean of four stimulations before 2-AG application. (D) Similar recordings as in C for β-Nx cKO Cremut (10, continuous line) and β-Nx cKO Cre (9, dotted line). (E) Boxplot (quartiles and median) of Ca2+ ΔF/F0 for Nx123 Cremut (960 ROIs/13 cells) and Nx123 Cre (1168/16), respectively, before and after 2-AG application. (F) Relative change (%) of presynaptic Ca2+ transients by activation of CB1-receptor with 2-AG. Cells were measured under both conditions (control and 5 min of 2-AG) and reduction was calculated for each bouton separately, plotted as mean ± SEM in Nx123 cKO (blue) and β-Nx cKO (red) neurons (Cremut and Cre). Outliers were detected and removed with ROUT-method (Q=1) and columns were compared with an unpaired t-test, * p < 0.05 , **** p < 0.0001; numbers (included ROIs/cells) are given in the columns.
Figure 5.
Endocannabinoid-evoked CB1 receptor activation reduces presynaptic Ca2+ transients in an Nx-dependent manner. (A) Several presynaptic boutons of a synGCaMP7b transfected Nx123 cKO Cre neuron are shown in an exemplary ΔF image during 3 AP stimulation. (B) The identical presynaptic boutons as in A after 5 min of CB1-receptor activation with 2 µM 2-AG, again during a 3 AP stimulation. (C) Repetitive stimulation (1 AP every 30 s) shows a reduction of Ca2+ transients in response to the application of 2-AG, averaged (mean ± SEM) from neurons of Nx123 Cremut (13 neurons, continuous line) and Nx123 Cre (16, dotted line), displayed as relative changes normalized to the mean of four stimulations before 2-AG application. (D) Similar recordings as in C for β-Nx cKO Cremut (10, continuous line) and β-Nx cKO Cre (9, dotted line). (E) Boxplot (quartiles and median) of Ca2+ ΔF/F0 for Nx123 Cremut (960 ROIs/13 cells) and Nx123 Cre (1168/16), respectively, before and after 2-AG application. (F) Relative change (%) of presynaptic Ca2+ transients by activation of CB1-receptor with 2-AG. Cells were measured under both conditions (control and 5 min of 2-AG) and reduction was calculated for each bouton separately, plotted as mean ± SEM in Nx123 cKO (blue) and β-Nx cKO (red) neurons (Cremut and Cre). Outliers were detected and removed with ROUT-method (Q=1) and columns were compared with an unpaired t-test, * p < 0.05 , **** p < 0.0001; numbers (included ROIs/cells) are given in the columns.
