Tissue‐ and cell‐specific expression of a splice variant in the II‐III cytoplasmic loop of Cacna1b

Presynaptic CaV2.2 (N‐type) channels are fundamental for transmitter release across the nervous system. The gene encoding CaV2.2 channels, Cacna1b, contains alternatively spliced exons that result in functionally distinct splice variants (e18a, e24a, e31a, and 37a/37b). Alternative splicing of the cassette exon 18a generates two mRNA transcripts (+e18a‐Cacna1b and ∆e18a‐Cacna1b). In this study, using novel mouse genetic models and in situ hybridization (BaseScope™), we confirmed that +e18a‐Cacna1b splice variants are expressed in monoaminergic regions of the midbrain. We expanded these studies and identified +e18a‐Cacna1b mRNA in deep cerebellar cells and spinal cord motor neurons. Furthermore, we determined that +e18a‐Cacna1b is enriched in cholecystokinin‐expressing interneurons. Our results provide key information to understand cell‐specific functions of CaV2.2 channels.

Presynaptic Ca V 2.2 (N-type) channels are fundamental for transmitter release across the nervous system. The gene encoding Ca V 2.2 channels, Cacna1b, contains alternatively spliced exons that result in functionally distinct splice variants (e18a, e24a, e31a, and 37a/37b). Alternative splicing of the cassette exon 18a generates two mRNA transcripts (+e18a-Cacna1b and Δe18a-Cacna1b). In this study, using novel mouse genetic models and in situ hybridization (BaseScope TM ), we confirmed that +e18a-Cacna1b splice variants are expressed in monoaminergic regions of the midbrain. We expanded these studies and identified +e18a-Cacna1b mRNA in deep cerebellar cells and spinal cord motor neurons. Furthermore, we determined that +e18a-Cacna1b is enriched in cholecystokinin-expressing interneurons. Our results provide key information to understand cell-specific functions of Ca V 2.2 channels.
Presynaptic Ca V 2.2 (N-type) channels are key mediators of transmitter release across the nervous system. The calcium that enters through Ca V 2.2 channels in response to action potentials triggers transmitter release [1,2]. Cacna1b is a multi-exon gene that encodes the Ca V a 1 pore-forming subunit of Ca V 2.2 channels. The Cacna1b pre-mRNA contains more than 40 exons that are spliced during mRNA maturation [3]. Most Cacna1b exons are constitutively included in the final Cacna1b mRNA; however, some are selectively included through alternative splicing (e18a, e24a, e31a, and 37a/37b) [4]. Alternative splicing of the Cacna1b pre-mRNA originates splice variants that are translated into Ca V 2.2 channels with distinct biophysical properties and pharmacological profiles [5,6]. Ca V 2.2 splice variants with differences in activation, inactivation, and current density [7][8][9], as well as in their response to neurotransmitters and drugs such as GABA and morphine, have been previously characterized [10,11]. Thus, alternative splicing of the Cacna1b pre-mRNA is thought to provide functional diversification to cells that utilize Ca V 2.2 channels to release neurotransmitter.
Among the alternative spliced exons in the Cacna1b, pre-mRNA is the cassette exon 18a (e18a) [12]. Alternative splicing of e18a generates two splice variants, +e18a-Cacna1b (e18a is included) and Δe18a-Cacna1b (e18a is skipped) (Fig. 1A). E18a encodes 21 amino acids within the 'synprint' region of the II-III cytoplasmic loop of Ca V 2.2 (LII-III), an essential region for interaction of Ca V 2.2 channels with presynaptic proteins [13][14][15]. Functionally, +e18a-Ca V 2.2 channels are more resistant to cumulative inactivation induced by repetitive stimulation and closed-state inactivation than Δe18a-Ca V 2.2 channels [9]. Furthermore, +e18a-Ca V 2.2 channels exhibit larger calcium currents relative to Δe18a-Ca V 2.2 channels in both mammalian expression systems and neurons [16]. Despite all of this information, very little is known about the functional role of +18a-Cacna1b and Δe18a-Cacna1b splice variants in the nervous system.
To understand the functional role of +e18a-Cacna1b and Δe18a-Cacna1b splice variants, it is necessary to determine their tissue and cell-type expression. Previous studies have shed light on this. In the adult nervous system, the abundance of +e18a-Cacna1b mRNA differs among tissues. Higher levels of +e18a-Cacna1b mRNA are observed in dorsal root ganglia, superior cervical ganglia, and spinal cord relative to whole brain [6,16]. Interestingly, the relative abundance between +e18a-Cacna1b and Δe18a-Cacna1b mRNAs also differs among brain subregions. Less than 10% of Cacna1b splice variants contain e18a in whole cerebral cortex and hippocampus, whereas 30-60% of Cacna1b splice variants contain e18a in the thalamus, cerebellum, hypothalamus, and midbrain [12]. Within the midbrain,~80% of Cacna1b splice variants contain e18a in monoaminergic regions including the ventral tegmental area (VTA), substantia nigra (SN), dorsal raphe nuclei (DRN), and locus coeruleus (LC) [17].
The expression of +e18a-Cacna1b is also cell-specific. +e18a-Cacna1b mRNA is enriched in tyrosine hydroxylase-expressing cells of the SN pars compacta (SNc) and VTA [17]. +e18a-Cacna1b has also been identified in magnocellular neurosecretory cells of hypothalamus and capsaicin-responsive neurons of dorsal root ganglia [7,18]. The molecular mechanisms underlying the tissue-and cell-specific expression of +e18a-Cacna1b and Δe18a-Cacna1b mRNAs are beginning to be elucidated. The RNA-binding protein, Rbfox2, is a splicing factor that represses e18a by binding an intronic region upstream of this exon [16,19]. Rbfox2 expression and activity depend on the cell type; thus, this would help to explain the cell-specific expression of +e18a-Cacna1b and Δe18a-Cacna1b [20,21]. 2) pre-mRNA in WT and Δe18aonly mice. In WT mice, e18a is spliced to generate two mRNA transcripts, +e18a-and Δe18a-Cacna1b, which in turn are translated into two different Ca V 2.2 channels, +e18a-and Δe18a-Ca V 2.2. The Cacna1b gene was modified to remove e18a and its flanking intronic regions to generate eΔ18a-only mice, which generate only the Δe18a-Ca V 2.2 splice variant. (B) Top, Schematic of Cacna1b pre-mRNA. Arrows indicate the approximate location of RT-PCR primers flanking e18a and qPCR primers in constitutive exons 45 and 46. Black box shows the approximate location of qPCR probe spanning exon junction [45][46]. Bottom left, RT-PCR from whole-brain samples of WT and Δe18a-only mice. Bottom right, comparison of whole-brain Cacna1b mRNA levels between WT and Δe18a-only mice. Data are shown as mean (filled symbols) AE SEM and individual values for biological replicates (empty symbols).
In this study, we utilized a novel version of in situ hybridization (ISH) (BaseScope TM ) and genetic mouse models, to precisely determine the expression of +e18a-Cacna1b in the central nervous system. We generated a mouse model that lacks expression of +e18a-Cac-na1b, thereby expresses only the Δe18a-Cacna1b splice variant (Δe18a-only). This mouse was used to control for probe specificity in our BaseScope TM experiments. With these new approaches and tools, we confirmed that +e18a-Cacna1b mRNA is expressed in monoaminergic regions (SN, VTA, DRN, and LC). Furthermore, we identified new cell populations that express +e18a-Cacna1b mRNA such as cells of the deep cerebellar nuclei (DCN) and spinal cord motor neurons. Finally, using fluorescence-activated cell sorting (FACS) of genetically identified cell populations coupled to RT-PCR, we found that +e18a-Cacna1b mRNA is more abundant in cholecystokinin-expressing interneurons (CCK + INs) relative to Ca2+/calmodulin-dependent protein kinase IIa-expressing projection neurons [CaMKIIa + pyramidal neurons (PNs)].

Housing conditions
A combination of adult males or females was used in all of our experiments, and no association with sex was found in the amount of e18a in Cacna1b in whole brain. Mice were housed with food and water ad libitum in temperature-controlled rooms with a 12-h light/dark cycle. All experimental procedures followed the guidelines of the Institutional Animal Care Committee of the University of New Hampshire.

Genotyping
Conventional toe biopsy was performed on P7-P9 pups. Genomic DNA was extracted using Phire Animal Tissue Direct Kit II (Thermo Fisher Scientific, Waltham, MA, USA, F140WH) according to the manufacturer's instructions. Next, PCR was performed with AmpliTaq GoldÒ 360 Master Mix (Thermo Fisher Scientific) using the following conditions: a hot start of 95°C for 10 min, followed by 35 cycles (95°C, 30 s; 60°C, 30 s; and 72°C, 1 min), and final step of 72°C for 7 min. Primers and expected products are shown in Table 1. Primers were added together to genotype each mouse line.

In situ hybridization (BaseScope TM )
Mice were deeply anesthetized with Euthasol (Virbac, Centurion, South Africa, 200-071). Next, mice were transcardially perfused with 19 PBS for 10 min and subsequently with 10% neutral buffered formalin (NBF,~4% formaldehyde) fixative solution (Sigma, St. Louis, MO, USA, HT501128) for 10 min, and then again with 19 PBS for 10 min. Both PBS and NBF were kept on ice during the perfusion. Brain and spinal cord were immediately removed and postfixated in 10% NBF at 4°C for 24 h. After washing with 19 PBS, tissue was sequentially dehydrated in 15% and 30% sucrose : PBS solutions for at least 18 h in each sucrose concentration, or until tissue sank to the bottom of the tube. Next, tissue was cryopreserved in optimal cutting temperature compound, or OCT (Fisher, 4585), with isopentane prechilled in dry ice. Twelve micrometer cryosections from brain and spinal cord were collected (Shandon, ThermoFisher Scientific, 77200222) and placed in prechilled 15-mm Netwell TM inserts (Corning, NY, USA, 3478). Sections were allowed to free-float in 19 PBS and mounted on positively charged microscope slides (VWR, 48311-703) using a paintbrush. After air-drying for 10-20 min, sections were incubated at 60°C in a drying oven for 30 min to aid tissue adhesion to the slides. Before ISH, sections were postfixed in 10% NBF at 4°C for 15 min and then dehydrated with 50%, 70%, and two rounds of 100% ethanol for 5 min in sequential steps. Slides were airdried for an additional 5 min before incubating with RNA-scopeÒ hydrogen peroxide solution for 10 min (ACD, Newark, CA, USA, 322381). Next, sections were washed with Milli-Q water and transferred to RNAscopeÒ Target Retrieval solution, preheated to 99°C for 15 min (ACD, 322000). Sections were briefly washed with Milli-Q water, transferred to 100% ethanol for 3 min, and then placed in drying oven at 60°C for 30 min. Sections were isolated with a hydrophobic barrier pen (ACD, 310018) and airdried overnight at room temperature. Following incubation with RNAscopeÒ Protease III for 30 min (ACD, 322381) at 40°C in the ACD HybEZ TM Hybridization System (ACD, 310010), sections were exposed to a probe spanning e18 and e18a (BaseScope TM Mm-Cacna1b-e18e18a) (ACD, 701151) for 2 h at 40°C in the ACD HybEZ TM Hybridization System. BaseScope TM Detection Reagents AMP 0-AMP 6 and FastRed (ACD, 322910) were applied according to the manufacturer's instructions and washed using RNAscopeÒ Wash Buffer (ACD, 310091). To visualize nuclei, sections were counterstained in Gill's Hematoxylin I for 2 min at RT (American Master Tech, Lodi, CA, USA, HXGHE). Next, sections were washed with tap water three times, briefly transferred to 0.02% ammonia water, and washed again with tap water. Finally, sections were dried at 60°C for 15 min and mounted using VectaMount TM mounting medium (Vector Laboratories, Burlingame, CA, USA, H-5000). Phase-contrast images were acquired using an Olympus IX-81 microscope.

Fluorescence-activated cell sorting
Adult CCK;Dlx5/6;tdT and CaMKIa;tdT mice were deeply anesthetized with isoflurane. After decapitation, brains were quickly removed and placed on a petri dish with Earl's balanced salt solution (EBSS) (Sigma, E3024) containing 21 UÁmL À1 of papain. Rapid dissection (< 45 s) of cerebral cortex and hippocampus was performed. Then, tissue was dissociated using a modified version of Worthington Papain Dissociation SystemÒ (Worthington Biochemical Corporation, Lakewood, NJ, USA, LK003150). After incubating with papain for 45 min at 37°C on a rocking platform, tissue was triturated with three sequential diameter fire-polished glass pipettes. Next, cell suspensions were centrifuged at 300 g for 5 min. After discarding supernatants, pellets were resuspended in 3 mL of EBSS containing 0.1% of ovomucoid protease inhibitor and 0.1% BSA (Worthington, LK003182) to quench papain. Cell suspension was centrifuged at 270 g for 6 min and resuspended in EBSS (3 mL). To isolate tdT-expressing cells, we performed FACS in a Sony SH800 flow cytometer using a 561 nm laser to excite and a 570-to 630-nm filter for event selection. At least 300 000 events were collected directly into TRIzol TM LS Reagent (Thermo Fisher Scientific, 10296028). Collection was performed keeping 1 : 3 (v/v) sorted cell suspension: TRIzol TM LS ratio. Cell suspension was kept on ice throughout the sorting session.

RT-PCR and RT-qPCR
Total RNA from tissue was extracted with RNeasy Mini Kit columns (Qiagen, Hilden, Germany, 74134) according to the manufacturer's instructions. Total RNA from sorted cells was extracted using TRIzol LS and isopropanol precipitation with the addition of 30 lg of GlycoBlueÒ Coprecipitant (Thermo Fisher Scientific, AM9516) to facilitate visualization of RNA pellet. 1 lg (tissue) or 300 ng (sorted Table 1. Primers to perform genotyping of mouse lines and expected products.  These sets of primers quantify +e18aand Δe18a-Cacna1b splice variants simultaneously. PCR products were run in 3% agarose gel stained with ethidium bromide, and densitometric analysis was performed using IMAGEJ [24]. This quantification method has been validated before [16]. To confirm band identity, the two bands were cloned and sequenced. To quantify total mRNA levels for Cacna1b, glutamate decarboxylase-2 (Gad-2) and cannabinoid receptor 1 (Cnr1), we performed TaqManÒ real-time PCR assays (Thermo Fisher Scientific) with the following probes:

Statistical analysis
Two-tailed unpaired Student's t-test was performed in Excel (Microsoft).

Results and Discussion
Validation of a mouse model to detect +e18a-Cacna1b mRNA in the central nervous system To determine the localization of +e18a-Cacna1b mRNAs in the central nervous system (CNS), we performed a modality of ISH (BaseScope TM ). We used a mouse line with targeted deletion of e18a (Δe18a-only) to control for probe specificity. In this mouse model, DNA sequences between e18 and e19 (e18-e18a intron, e18a, and e18a-e19 intron) were removed using homologous recombination (Fig. 1A, [16]). To confirm deletion of e18a sequence, we performed RT-PCR in whole-brain samples from WT and Δe18a-only mice. We utilized primers flanking e18a to quantify both +18aand Δe18a-Cacna1b transcripts. Amplicons for each splice variant were resolved on gel electrophoresis based on size (Fig. 1B). In WT whole-brain samples, we observed two bands,~290 and~230 bp, corresponding to +18aand Δ18a-Cacna1b transcripts, respectively (Fig. 1B, bottom left panel, lanes 1-3). As expected, the upper band was absent in samples from Δe18a-only mice (Fig. 1B, bottom left panel, lanes 4-6). Next, we tested whether the targeted deletion of e18a alters the overall Cacna1b mRNA levels. We quantified total Cacna1b mRNA using RT-qPCR with a probe that spans the splice junction between two constitutive exons, e45 and e46 (Fig. 1B, top panel).
We found no significant differences in the total amount of Cacna1b mRNA between WT and Δe18aonly mice (Fold change relative to control AE SEM: WT = 1 AE 0.12, n = 7; e18a-null = 0.89 AE 0.18, n = 7. P = 0.61. Two-tailed, unpaired, Student's t-test. Fig. 1B, right lower panel). Our results show that the e18a sequence was successfully eliminated from the Cacna1b gene and that this deletion does not alter the total Cacna1b mRNA levels in whole brain. Therefore, this model is ideal to control for the specificity of probes directed to e18a, thereby allowing the localization of +e18a-Cacna1b splice variants in CNS tissue.

+e18a-Cacna1b mRNA is expressed in substantia nigra and ventral tegmental area
In the CNS, expression of Cacna1b is restricted to neurons; however, the cell-specific expression of the Cac-na1b splice variants has not been fully determined [4]. Previous studies using microdissections and RT-PCR showed that +e18a-Cacna1b mRNA is abundantly expressed in SN and VTA [17]. Furthermore, +e18a-Cacna1b mRNAs colocalize with tyrosine hydroxylase mRNA in both of these brain areas [17], suggesting that +e18a-Cacna1b mRNA is enriched in dopaminergic neurons. Here, we confirmed these findings using BaseScope TM with a probe designed against the e18a sequence. Briefly, 'Z' probes containing a short complementary region bind to the e18a sequence. This binding leads to the assembly of a signal amplification system, thereby allowing the detection of short RNA sequences ( Fig. 2A). In our experiments, we used brain sections from Δe18a-only mice to control for probe specificity. counterstaining with hematoxylin (Hem) (Fig. 2A). Each dot detects a single mRNA molecule. We performed BaseScope TM in midbrain sections of WT and Δe18a-only mice. We compared our BaseScope TM images (Fig. 2B, top panels) to conventional ISH images for the dopaminergic marker, Slc6a3 dopamine transporter, from SN and VTA found in the Allen Mouse Brain Atlas (Fig. 2B, bottom panels) [26]. We observed that staining for e18a follows a pattern similar to Slc6a3 in the midbrain. No signal for e18a was detected on sections from Δe18a-only mice (Fig. 2B, bottom panels). These results confirm previous studies showing that e18a is expressed in SN and VTA regions. It is important to note that we observed +e18a-Cacna1b expression in other cells near VTA and SNc; the identity of these cells is currently unknown. Some cells expressing +e18a-Cacna1b were also observed in SN pars reticulata. To our knowledge, antibodies specific for +e18a-Ca V 2.2 channels are unavailable. This would help to convincingly show that +e18a-Ca V 2.2 channels are present in dopaminergic neurons. However, our use of BaseScope TM with adequate negative controls sheds light on the localization of e18a in the midbrain. Previous results have shown that dopamine release from VTA and SNc heavily relies on Ca V 2.2 channels [27,28]. Our studies and mouse models combined with previous functional studies of +e18aand Δe18a-Ca V 2.2 channels in mammalian systems and neurons will enable to propose further studies to unveil a potential role of e18a splicing on dopamine release.
Expression of +e18a-Cacna1b mRNA in dorsal raphe nuclei and locus coeruleus DRN and LC contain neurons that release serotonin and norepinephrine, respectively. DRN is located ventral to the cerebral aqueduct (AQ) (Fig. 3A, top right  panel). Prior studies showed that +e18a-Cacna1b mRNA is expressed in DRN and LC [17]. We next performed BaseScope TM in these brain areas. To guide our analysis, we compared images for conventional ISH staining for the serotoninergic marker, Slc6a4 (serotonin transporter), from the Allen Mouse Brain Atlas (Fig. 3A, top left panel [26]) to our BaseScope TM staining. Signal for e18a was observed ventral to AQ and followed a pattern similar to Slc6a4, suggesting that +e18a-Cacna1b splice variants are expressed in DRN (Fig. 3A, middle and bottom left panels). Sections of Δe18a-only mice in a similar area show little to no signal for e18a (Fig. 3A, middle and bottom right panels). Our results show that e18a is present in the DRN; however, further studies are needed to determine the cell populations within the DRN that express +e18a-Cacna1b mRNA. Ca V 2.2 channels are involved in the release of serotonin [27], and Ca V 2.2null mice show enhanced aggression that has been linked to the control of serotonin neurons excitability [29]. Our results open the possibility that splicing e18a is linked to the activity of the serotonin system.
To determine whether +e18a-Cacna1b mRNA is expressed in LC, we stained sections containing the fourth ventricle (V4) and the central lobule of the cerebellum II (CENT2) (Fig. 3B, top panel). We found that the signal for +e18a-Cacna1b mRNA is located lateral to V4 (Fig. 3B, middle and bottom panels). This signal is similar to the pattern of expression for Slc6a2 (norepinephrine transporter, Net) observed in ISH images from the Allen Mouse Brain Atlas [26] (Fig. 3B, top panel). Our results suggest that e18a is present in LC, as previously reported [17].

Distribution of +e18a-Cacna1b mRNA in cerebellum
In cerebellum, Ca V 2.2 channels control the release of neurotransmitter from climbing fibers and parallel fibers synapsing onto Purkinje cells [30][31][32]. Ca V 2.2 channels are also critical for the intrinsic firing of DCN neurons by coupling to calcium-dependent potassium channels [33]. In cerebellum,~20% of Cacna1b splice variants contain e18a [16]. However, the distribution of +e18a-Cacna1b splice variant in the cerebellum is unknown. Using the well-defined anatomy of cerebellum as landmark, we determined the expression of e18a in this area. In cerebellar cortex, little e18a signal was observed in the Purkinje cell layer, as well as the molecular and granular layers (m.l. and g.l.) (Fig. 4A, left panel, inset 1). However, in the DCN, we observed several cell bodies stained for e18a (Fig. 4A, left panel, inset 2 and 3). Very little signal was detected in cerebellar sections from Δe18a-only mice (Fig. 4B, right panel and insets 4-6). Our results provide a framework to test whether splicing of e18a influences the firing properties of neuronal populations present in DCN.

Expression of +e18a-Cacna1b in spinal cord
Ca V 2.2 channels have been previously reported in spinal cord at nociceptive afferents, interneurons, and motor neurons [34]. In spinal cord, we have previously shown that e18a-containing splice variants represent 55% of the Cacna1b pool of transcripts [12]. We next determined the pattern of expression of +e18a-Cacna1b in spinal cord. We found very little signal for e18a in laminae I-III (Fig. 5A, insets 1 and 2). In contrast, large cell bodies in ventral areas (lamina IX) of spinal cord contained > 6 red dots, indicating the presence of e18a (Fig. 5A, inset 2). Note the absence of e18a signal in spinal cord sections of Δe18a-only mice (Fig. 5B, insets 3 and 4). It is well known that dorsal laminae of spinal cord primarily contain neurons that receive sensory inputs from dorsal root ganglia, whereas ventral areas contain circuits that control motor neuron activity. Given that motor neurons in lamina IX have larger cell bodies relative to interneurons [35,36], our results suggest that e18a is expressed in motor neurons. However, further studies are needed to determine whether +e18a-Cacna1b mRNA is more abundant in motor neurons relative to interneurons of spinal cord. Previous studies have shown that some neuromuscular junctions rely on Ca V 2.2 channels to release acetylcholine (phrenic nerve-diaphragm), whereas others utilize almost exclusively Ca V 2.1 (sciatic nerve-tibialis muscle) [37,38]. The cell-specific expression of +e18a-Cacna1b mRNA could provide an explanation for this neuromuscular junction-specific role of Ca V 2.2 channels. Complementary Z probes were designed to target the junction e18 and e18a. These two independent Z probes bind to the Cacna1b mRNA in tandem, thereby allowing the assembly of an amplification complex. Subsequent signal amplification leads to the development of red coloration. Sections were counterstained with Hem. (B) Top panels, BaseScope TM images of VTA and SN from WT mouse brain (left). Insets of VTA and SN (middle and right, respectively). Blue indicates all nuclei stained with Hem. Red dots indicate the presence of +e18a-Cacna1b mRNA molecules. Middle panels, BaseScope TM images from areas similar to top panels in Δe18a-only mice. Bottom panels, ISH images for Slc6a3 in SN and VTA from brain-map.org (left). Black squares represent sections within SN and VTA that were magnified for clarity (insets 1-6). Images credit: Allen Institute. Scale bar = 400 lm.

Expression of +e18a-Cacna1b splice variants in hippocampus
The functional role of Ca V 2.2 channels in controlling neurotransmission has been extensively described in hippocampal synapses. Interestingly, the distribution of Cacna1b splice variants in the hippocampus is unknown. Interneurons reside in stratum radiatum (s.r.), whereas PNs are arranged along stratum pyramidale (s.p.) of both cornu ammonis 1 (CA1) and cornu ammonis 3 (CA3) regions. Using these anatomical landmarks, we determined the expression of e18a in CA1 and CA3 regions. Signal for +e18a-Cacna1b mRNA was detected in nuclei of s.r. and s.p. Interestingly, only a subpopulation of s.r. nuclei showed strong signal for e18a in both regions (Fig. 6A,B, left panels). In dentate gyrus (DG), interneurons are located in the hilus (h.), whereas granular cells (GC) are localized in the granular cell layer (g.c.l). Here, we observed signal for e18a in several nuclei located in h. and g.c.l. (Fig. 6C). In sections from Δe18a-only mice, signal for e18a was absent throughout all regions of hippocampus analyzed ( Fig. 6A-C, right panels). We also compared the percentage of cells that show more than one single dot in the DG of hippocampal sections from WT and Δ18aonly mice; less than one dot per cell is likely to be background [39]. We found that~30% of cells in DG from WT contained more than a single dot, compared tõ 5% the ones from Δ18a-only mice (% cells with more than one dot AE SEM. WT = 29.69 AE 1.23, n = 5; D18a-only = 4.81 AE 0.17, n = 4, P < 0.00001, Student's t-test). These results further support the specificity of the e18a-Cacna1b probe. Based on anatomical landmarks for hippocampus, our results suggest that e18a is broadly distributed among PN, GC, and a subpopulation of interneurons in the hippocampus.
With our current results, we are unable to discard the possibility that e18a-Cacna1b is also present in other types of GABAergic interneurons in cortex and hippocampus. However, we focus on CCK + INs because 100% of GABA release relies on Ca V 2.2 channels; therefore, the functional significance of e18a-Cacna1b has high potential [42]. Furthermore, this type of interneurons are linked to mood disorders [47,48], express relatively high levels of the cannabinoid receptor protein (CB1R) [49], and contribute to the behavioral tolerance of tetrahydrocannabinol [50]. Given that CB1R agonists downregulate Ca V 2.2 channels to inhibit GABA release in CCK + INs, our findings suggest an anatomical link between e18a splicing in Cacna1b and the effects of CB1R agonists of transmitter release. CCK + INs in hippocampus show robust asynchronous release and spontaneous release that relies on Ca V 2.2 channels [42]. Due to the slower inactivation rates and positive shift in voltage-dependent inactivation of +18a-Ca V 2.2 channels relative to Δ18a-Ca V 2.2 channels, it is possible that inclusion of e18a in this neuronal type enhances asynchronous and spontaneous release.