The synaptic connection from medial habenula (MHb) to interpeduncular nucleus (IPN) is critical for emotion-related behaviors, and uniquely expresses R-type Ca2+ channels (Cav2.3) and auxiliary GABAB receptor (GBR) subunits, the K+-channel tetramerization domain-containing proteins (KCTDs). Activation of GBRs facilitates or inhibits transmitter release from MHb terminals depending on the IPN subnucleus, but the role of KCTDs is unknown. We therefore examined the localization and function of Cav2.3, GBRs, and KCTDs in this pathway in mice. We show in heterologous cells that KCTD8 and KCTD12b directly bind to Cav2.3 and that KCTD8 potentiates Cav2.3 currents in the absence of GBRs. In the rostral IPN, KCTD8, KCTD12b and Cav2.3 co-localize at the presynaptic active zone. Genetic deletion indicated a bidirectional modulation of Cav2.3-mediated release by these KCTDs with a compensatory increase of KCTD8 in the active zone in KCTD12b-deficient mice. The interaction of Cav2.3 with KCTDs therefore scales synaptic strength independent of GBR activation.

The molecular anatomy of synapses defines their characteristics in transmission and plasticity. Precise measurements of the number and distribution of synaptic proteins are important for our understanding of synapse heterogeneity within and between brain regions. Freeze–fracture replica immunogold electron microscopy enables us to analyze them quantitatively on a two-dimensional membrane surface. Here, we introduce Darea software, which utilizes deep learning for analysis of replica images and demonstrate its usefulness for quick measurements of the pre- and postsynaptic areas, density and distribution of gold particles at synapses in a reproducible manner. We used Darea for comparing glutamate receptor and calcium channel distributions between hippocampal CA3-CA1 spine synapses on apical and basal dendrites, which differ in signaling pathways involved in synaptic plasticity. We found that apical synapses express a higher density of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and a stronger increase of AMPA receptors with synaptic size, while basal synapses show a larger increase in N-methyl-D-aspartate (NMDA) receptors with size. Interestingly, AMPA and NMDA receptors are segregated within postsynaptic sites and negatively correlated in density among both apical and basal synapses. In the presynaptic sites, Cav2.1 voltage-gated calcium channels show similar densities in apical and basal synapses with distributions consistent with an exclusion zone model of calcium channel-release site topography.

15The connection from medial habenula (MHb) to interpeduncular nucleus is critical for aversion-16 and addiction-related behaviors. This pathway is unique in selective expression of R-type 17 voltage-gated Ca 2+ channels (Cav2.3) in its terminals, and robust potentiation of release via 18 presynaptic GABA B receptors (GBRs). To understand the mechanism underlying this peculiar 19 GBR effect, we examined the presynaptic localization and function of Cav2.3, GBR, and its 20 auxiliary subunits, K + -channel tetramerization domain-containing (KCTD) proteins. We found 21 selective co-expression of KCTD12b and Cav2.3 at the presynaptic active zone. GBR-mediated 22 potentiation remained intact in KCTD12b KO mice but lasted significantly shorter. This 23 impairment was associated with increased release and an insertion of KCTD8 into the active 24 zone. In heterologous cells, we found direct binding of KCTD8 and KCTD12b to Cav2.3, and 25 potentiation of Cav2.3 currents by KCTD8. The unexpected interaction of Cav2.3 with KCTDs 26 therefore provides a means to scale synaptic strength independent of GBR activation. 27 28 domain-containing (KCTD) subunits of GBRs may play a role in the GBR-mediated potentiation 52 of neurotransmitter release, as KCTD subunits show distinct expression patterns in MHb 53 neurons. 54There are four auxiliary KCTD subunits of GBRs, KCTD8, 12, 12b and 16. These 55 subunits form hetero-and homo-pentamers that modulate GBR signaling kinetics (Fritzius and 56 Bettler, 2019;Schwenk et al., 2010;Zheng et al., 2019). The expression patterns of KCTD 57 subunits in the MHb are unique: With the exception of a weak expression in the cerebellum and 58 superior colliculus, KCTD8 is exclusively and strongly expressed in MHb and, to a lesser extent, 59 IPN neurons. Furthermore, KCTD12b is exclusively expressed in the ventral part of the MHb 60 (Metz et al., 2011). In contrast, KCTD12 is weakly expressed in the ventral part of the MHb 61 while KCTD16 is expressed in most brain areas but not the MHb. Both KCTD12 and 12b induce 62 a rapid desensitization of GBR effector channels by uncoupling the βγ subunits (Gβγ) of the 63 guanine nucleotide-binding protein (G-protein) from effector channels (Turecek et al., 2014). 64 KCTD12 and 12b can form hetero-pentamers with each other but also with KCTD8 and 16 65 (Fritzius and Bettler, 2019). Based on proteomics studies, GBRs and their auxiliary KCTD 66 subunits co-precipitate with release machinery proteins of the presynaptic active zone, and 67 KCTD8 and KCTD16 were found to co-purify with presynaptic Cav2.2 Ca 2+ channels (Müller et 68 al., 2010; Schwenk et al., 2016). However, the functional consequences of these interactions and 69 whether other voltage-gated Ca 2+ channels interact with KCTDs remain unknown. 70Here, we studied the nano-anatomy of Cav2.3, GBR and KCTDs and their roles in the 71 modulation of neurotransmission from the MHb to the IPN. Our results demonstrate that Cav2.3 72 is located in the presynaptic active zone of habenular terminals and require...

GABAB receptor (GBR) activation inhibits neurotransmitter release in axon terminals in the brain, except in medial habenula (MHb) terminals, which show robust potentiation. However, mechanisms underlying this enigmatic potentiation remain elusive. Here, we report that GBR activation induces a transition from tonic to phasic release accompanied by a 4-fold increase in readily releasable pool (RRP) size in MHb terminals, mirrored by a similar increase in the docked vesicle number at the presynaptic active zone (AZ). The tonic and phasic release vesicles have distinct coupling distances. We identified two vesicle-associated molecules, synaptoporin and CAPS2, selectively involved in tonic and phasic release, respectively. Synaptoporin mediates augmentation of tonic release and CAPS2 stabilizes readily releasable vesicles during phasic release. A newly developed Flash and Freeze-fracture method revealed selective recruitment of CAPS2 to the AZ during phasic release. Thus, we propose a novel two-pool mechanism underlying the GBR-mediated potentiation of release from MHb terminals.

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