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- Open Access
Mapping of the spontaneous deletion in the Ap3d1 gene of mocha mice: fast and reliable genotyping
© Holm et al; licensee BioMed Central Ltd. 2008
- Received: 19 August 2008
- Accepted: 25 November 2008
- Published: 25 November 2008
The mocha mouse carries a spontaneous deletion in the Ap3d1 gene, encoding the delta 1 subunit of the adaptor related protein complex 3, (Ap3d1), and subsequently lack the expression of functional AP-3. This leads to a deficiency in vesicle transport and storage, which affects neurotransmitter vesicle turnover and release in the central nervous system. Since the genomic sequence of the Ap3d1 gene of mocha mouse is not known, precise mapping of the deletion as well as reliable genotyping protocols are lacking.
We sequenced the Ap3d1 gene (HGNC GeneID: 8943) around the deletion site in the mocha mouse and revealed a 10639 bp deletion covering exon 2 to 6. Subsequently, new PCR primers were designed yielding a reliable genotyping protocol of both newborn and adult tissue. To examine the genotypes further, hippocampal neurons were cultured from mocha and control mice. Patch-clamp recordings showed that mocha neurons had a higher input resistance, and that autaptic EPSC in mocha cultures depressed faster and stronger as compared with control cultures.
Our study reports the sequence of the deleted part of the Ap3d1 gene in mocha mice, as well as a reliable PCR-based genotyping protocol. We cultured hippocampal neurons from control and mocha mice, and found a difference in input resistance of the neurons, and in the synaptic short-term plasticity of glutamatergic autapses showing a larger synaptic depression than controls. The described procedures may be useful for the future utilization of the mocha mouse as a model of defective vesicle biogenesis. Importantly, as genotyping by eye color is complicated in newborn mice, the designed protocol is so fast and reliable that newborn mice could rapidly be genotyped and hippocampal neurons dissociated and cultured, which is normally best done at P0-P2.
- Hippocampal Neuron
- Synaptic Vesicle
- Input Resistance
- Genomic Deletion
- Synaptic Depression
For the study of neuronal function, a range of useful mouse models exist where spontaneous genomic alterations have occurred. In order to efficiently utilize these models, a precise insight into the altered sequence must be available, and reliable genotyping protocols must be set up .
Adaptor related proteins (APs) are a group of heterotetrameric complexes thought to be involved in endocytosis and vesicle biogenesis in neuronal and non-neuronal tissues . Of the four adaptor related proteins described so far (AP-1 to AP-4), AP-3 is expressed in brain and preferentially located in endosomal membranes. AP-3 is composed of a δ, β, μ and ρ subunit, all thought to be necessary for AP-3 to participate in late endocytotic steps and vesicle budding from endosomes. Therefore, it has been speculated that AP-3 participates in synaptic vesicle biogenesis and thereby in the regulation of synaptic transmission in the CNS. Indeed, two recent studies have found that fast synaptic transmission is impaired in AP-3 deficient neurons [3, 4].
The mocha mouse carries a deletion in the AP-3 δ subunit and homozygous mocha mice display epileptiform activity , auditory changes , pigmentation dysfunction, and storage deficiency in platelets , while heterozygotes are phenotypically similar to wild types. It is possible that the CNS phenotype is caused by impaired synaptic vesicle formation and recycling. As pigmentation is affected by the mutation, genotyping of Ap3d1 deficient mocha mice may be done by inspection of eye color due to different degrees of eye pigmentation . However, this procedure is challenging, especially in younger animals, and not completely reliable.
The exact sequence of the altered gene in mocha mice is unknown. Therefore, we have sequenced the genomic deletion and used this to design an efficient genotyping protocol, which facilitated culturing and electrophysiological studies of primary mocha neurons.
Sequencing of the gene deletion in mocha mice and developing a new genotyping protocol
Primers used for sequencing and genotyping
5'- CCT CAA GCC CGT CAG AAG- 3'
5'- TTG AAC TCG GAG ACA TCC TCT GC- 3'
5'- CTG GGT GGC ATT TAG TTA CTT GAG GC- 3'
5'- CTT CTG GGC TGG CTG GAT GTA AG- 3'
5'- CCA ATA CCA ACC AAC CAA CAA CC- 3'
5'- TGA CAT TAA CAG CCG CAG AC- 3'
5'- CCT GCT TCT GAG TGC TTG GG- 3'
5'- GGC TGC TTC TGG ACT GTT CG- 3'
5'- CTT CTC CAC CCT CTT TCC ACC C- 3'
Cultured hippocampal neurons from mocha mice show increased input resistance
To analyze for functional differences, microcultures of hippocampal neurons from genotyped newborn control and mocha mice were prepared. Cell capacitance was measured electrophysiologically, showing no significant differences (22.4 ± 1.4 vs. 19.4 ± 1.4 pF, P = 0.12) between control (n = 18) and mocha neurons (n = 21), indicating equal cell size.
Hippocampal mocha neurons show increased short-term synaptic depression
This study had three goals namely 1) to sequence the genomic deletion in mocha mice, 2) to set up a reliable PCR genotyping protocol for unambiguous identification of genotypes of all offspring from heterozygous breeding, and 3) to perform initial functional studies in cultured mocha neurons of membrane properties and synaptic plasticity. Reaching all three goals, the protocol reported here will facilitate further functional studies of mocha neuronal and non-neuronal cells in a variety of settings, including cell culture.
The sequence of the genomic deletion in mocha mice
As the heterotetrameric AP-3 protein complexes are known to be involved in vesicle sorting and fusion, it is comprehensible that AP-3 deficiency leads to vesicle storage and transport deficiencies in various tissues . While AP-3 is composed of δ, β3, μ3 and ρ3 subunits, the mocha mutation affects the δ subunit of AP-3, leading to a lack of expression of the whole complex. Although mice deficient for each individual subunit exist, the mocha mutation leads to the most severe phenotype, which has been explained by the finding that the δ subunit is common to all AP-3 complexes .
Since the exact sequence of the genomic deletion of mocha mice was previously not known, we sequenced the mocha gene and found that a 10639 bp long segment of the Ap3d1 gene was deleted including exons 2–6. Interestingly, we found that the site of deletion was flanked by a repeated sequence 5'CATCT (Fig. 1) in the wild type sequence. Genomic deletions can be the result of recombination between SINE repeat elements, however, as these elements usually are 2–300 bp long with a high homology between the recombining repeats, this is probably not the cause of the mocha deletion, since only the CATCT sequence is repeated. Therefore, we speculate that the spontaneous deletion has arised possibly by some kind of "replication slip" of the DNA polymerase from one repeat to the other repeated sequence most likely during replication of the genomic DNA. Afterwards, the deletion has been conserved since heterozygous and to some degree homozygous mice are able to reproduce.
Functional alterations in cultured hippocampal neurons of mocha mice
Patch-clamp recordings showed that the mocha neurons have a larger input resistance. A likely explanation for this is a decrease in membrane conductance associated with a reduced surface expression of functional ion channels. Since AP-3 is known to be involved in the transport and targeting of ion channels, such as the chloride channel ClC-3 , it is possible that the cultured mocha neurons display a certain degree of missorting of ion channels. The exact nature and extent of such an ion channel missorting may be the topic for further investigations.
Although there are still no functional data to support this, it is possible that an increased membrane resistance would make the mocha neurons more responsive to excitatory synaptic input, which could in fact make them hyperexcitable. Noting that mocha mice display an epileptiform phenotype , it could be interesting to investigate whether the increased input resistance also exists in vivo.
The other finding of our paper was that glutamatergic autapses on cultured mocha neurons display a stronger synaptic depression of EPSCs in response to brief stimulus trains. Synaptic depression usually depends on depletion of the readily releasable pool of synaptic vesicles and on the refilling of that pool. Thus, the Ap3d1 deficiency appears to lead to alterations in the exocytosis and possibly turnover of synaptic vesicles in cultured neurons. Short-term synaptic plasticity of excitatory neurotransmission has recently been studied by others, who found that field EPSPs (excitatory postsynaptic potentials) actually depressed less in adult mocha mouse brain slices in response to repetitive stimulation . This could be due to differences in vesicle turnover and release probability at excitatory synapses between adult slices and postnatal hippocampal cultures. While excitatory synapses in culture usually display a higher release probability and show a substantial synaptic depression , it is likely that their synaptic vesicle endo- and exocytotic processes are governed by the development stage and culture conditions. Despite this, hippocampal cultures may be very useful in future studies of the involvement of Ap3d1 in vesicle release using combinations of patch-clamp and imaging techniques, such as FM1-43 or SynaptopHluorin signals, that can report both vesicle endo-and exocytosis . Since AP-3 may both participate in the synchronous  and asynchronous release of neurotransmitter , and controls the targeting of other presynaptic proteins, such as TI-VAMP (tetanus neurotoxin-insensitive vesicle-associated membrane protein) [3, 12], Vglut1 (vesicular glutamate transporter 1)  and VGAT (vesicular GABA transporter) , the deletion in the Ap3d1 gene of mocha mice is likely to have an impact on the function of many different synapses.
Mouse supplier and breeding
Mice were obtained from The Jackson Laboratory (Bar Harbor, Maine, USA) strain name: STOCK gr +/+ Ap3d1 mh /J, stock number: 000279 and kept in a university facility on a 12/12 hours light/dark schedule with unlimited access to food and water. All animal procedures were performed according to the European Communities Council Directive of 24 November 1986 (86/609/EEC).
HotShot genomic DNA extraction
A modified HotShot protocol  was used to purify DNA. 50 μl of alkaline lysis buffer (25 mM NaOH, 0.2 mM Na-EDTA, pH 12.3) was added to a 2 mm clip of the mouse tail on ice. Lysis was carried out in a PCR machine (Peltier Thermal Cycler, PTC-200, MJ Research, Waltham, MA, USA) for 30 min at 65°C followed by 16 min at 98°C. Afterwards the samples were neutralized by adding 50 μl of a neutralizing buffer (40 mM Tris-HCl, pH 5.0) on ice.
PCR for the mapping of the deletion
2 μl purified DNA (diluted 1:5 in TE buffer after HotShot) was used for the PCR performed in 25 μl consisting of 3.5 mM MgCl2, 0.3 mM dNTP, 0.5 μM of forward and reverse primer (Table 1) and 0.05 units/μl of HotStarTaq polymerase (Qiagen, Hilden, Germany). The PCR was performed as follows: The HotStarTaq enzyme was activated at 95°C for 15 min followed by 30 cycles of a 3-step protocol (95°C for 30 sec, 60°C for 30 sec and 72°C for 10 min) and a final elongation step at 72°C for 10 min. The reaction was visualized on a 2% agarose gel in TBE buffer.
All DNA sequencing was done by Eurofins MWG Operon (Ebersberg, Germany) and analyzed using VectorNTI (Invitrogen, Paisley, United Kingdom).
The PCR was performed as above with the following changes: 0.5 μM of primer "Common R", 0.5 μM of primer "WT F", 0.5 μM of primer "Mocha F" was used together with a shorter elongation step in each cycle at 72°C for 1 min. The reaction was visualized on a 2% agarose gel in TBE buffer.
Hippocampal cell culture
Single hippocampal neurons from newborn (P1-P2) mice were cultured on microislands, using an established protocol . Briefly, coverslips with stamps of ~200 micrometer large collagen islands covered with Poly-D-lysine and a feeder layer of astrocytes were prepared . Brains were dissected out, meninges and vascular tissue washed off and hippocampi were enzymatically dissociated in papain in DMEM for 1 hour at 37°C and plated at low density on microislands. Neurons were allowed to mature for 14–21 days before recording, and only microislands containing a single neuron were used.
Current- or voltage-clamp whole-cell patch-clamp recordings were made using 3–4 MΩ patch-pipettes (P-97 puller, Sutter Instrument Co, Novato, CA, USA) and filled with (in mM): 135 K-Gluconate, 10 HEPES, 1 EGTA, 4.6 MgCl2, 4 Na-ATP, 15 creatine phosphate, 50 U/ml phosphocreatine kinase (295 mOsm kg-1, pH 7.3). The extracellular solution contained (in mM); 140 NaCl, 2.4 KCl, 10 HEPES, 10 glucose, 4 CaCl2, and 4 MgCl2 (300 mOsm, pH 7.4). Input resistance was estimated in current-clamp by injecting 100 ms long current steps and measuring the resulting voltage deflection. To record autaptic EPSCs, neurons were voltage-clamped at -70 mV using an EPC-10 amplifier (HEKA Elektronik, Lambrecht, Germany) and stimulated by stepping the membrane potential to 0 mV for 3 ms. This evoked a single break-away action-potential that propagated to the autaptic nerve terminals and gave rise to a short latency EPSC in the same cell. Series resistances were below 15 MΩ and compensated by 70–80%. Currents were low-pass filtered at 5 kHz and acquired at 10 kHz using Pulse (HEKA).
We are indebted to Dr. Pablo García-Junco Clemente and Dr. Rafael Fernández-Chacón, University of Seville, Spain for expert advice on the microisland cell culture.
The study was supported by The Danish Medical Research Council (to MMH and KJ) and the Lundbeck Foundation (Denmark). We are grateful to Vibeke Nielsen, Dianna Haun Nielsen and Brita Holst Jensen for excellent technical assistance.
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