Mechanism underlying unaltered cortical inhibitory synaptic transmission in contrast with enhanced excitatory transmission in CaV2.1 knockin migraine mice

Familial hemiplegic migraine type 1 (FHM1), a monogenic subtype of migraine with aura, is caused by gain-of-function mutations in CaV2.1 (P/Q-type) calcium channels. In FHM1 knockin mice, excitatory neurotransmission at cortical pyramidal cell synapses is enhanced, but inhibitory neurotransmission at connected pairs of fast-spiking (FS) interneurons and pyramidal cells is unaltered, despite being initiated by CaV2.1 channels. The mechanism underlying the unaltered GABA release at cortical FS interneuron synapses remains unknown. Here, we show that the FHM1 R192Q mutation does not affect inhibitory transmission at autapses of cortical FS and other types of multipolar interneurons in microculture from R192Q knockin mice, and investigate the underlying mechanism. Lowering the extracellular [Ca2+] did not reveal gain-of-function of evoked transmission neither in control nor after prolongation of the action potential (AP) with tetraethylammonium, indicating unaltered AP-evoked presynaptic calcium influx at inhibitory autapses in FHM1 KI mice. Neither saturation of the presynaptic calcium sensor nor short duration of the AP can explain the unaltered inhibitory transmission in the mutant mice. Recordings of the P/Q-type calcium current in multipolar interneurons in microculture revealed that the current density and the gating properties of the CaV2.1 channels expressed in these interneurons are barely affected by the FHM1 mutation, in contrast with the enhanced current density and left-shifted activation gating of mutant CaV2.1 channels in cortical pyramidal cells. Our findings suggest that expression of specific CaV2.1 channels differentially sensitive to modulation by FHM1 mutations in inhibitory and excitatory cortical neurons underlies the gain-of-function of excitatory but unaltered inhibitory synaptic transmission and the likely consequent dysregulation of the cortical excitatory–inhibitory balance in FHM1.


Introduction
Voltage-gated P/Q-type calcium channels (Ca V 2.1) play a prominent role in controlling neurotransmitter release at brain excitatory and inhibitory synapses (Pietrobon, 2005(Pietrobon, , 2010. Mutations in the gene encoding the Ca V 2.1α1 subunit cause several neurological disorders including familial hemiplegic migraine type 1 (FHM1), a rare monogenic form of migraine with aura (Ophoff et al., 1996;Pietrobon, 2010). Migraine is a common disabling disorder caused by a primary brain dysfunction that leads to episodic activation of the trigeminovascular pain pathway and headache (Pietrobon and Moskowitz, 2013). There is increasing evidence that the headache mechanisms can be triggered by cortical spreading depression (CSD), the phenomenon underlying migraine aura (Pietrobon and Moskowitz, 2013). The molecular and cellular mechanisms of the primary brain dysfunction(s) leading to susceptibility to CSD and to the migraine attack remain a major open issue. Important insights into these mechanisms have been obtained from the analysis of functional consequences of FHM1 mutations, revealing that these mutations produce: (i) gain-of-function of human recombinant and native neuronal mouse Ca V 2.1 channels, mainly due to channel activation to lower voltages and increased channel open probability; and (ii) facilitation of induction and propagation of experimental CSD, due to increased glutamate release at cortical pyramidal cell synapses [ (Pietrobon, 2010;Pietrobon and Moskowitz, 2013) and references therein]. In striking contrast with enhanced glutamatergic neurotransmission, the inhibitory neurotransmission at connected pairs of layer 2/3 fastspiking (FS) interneurons and pyramidal cells was unaltered in FHM1 knockin (KI) mice, despite being initiated by Ca V 2.1 channels (Tottene et al., 2009). The differential effect of FHM1 mutations at excitatory and inhibitory synapses suggests altered regulation of the cortical excitatory-inhibitory balance in FHM1 and supports the view of migraine as a disorder of brain excitatory-inhibitory balance (Pietrobon and Moskowitz, 2013;Vecchia and Pietrobon, 2012).
The lack of effect of FHM1 mutations on GABA release at FS interneuron synapses is puzzling and the underlying mechanism remains unknown. Several possible mechanisms were suggested, including: (i) saturation of the presynaptic calcium sensor; (ii) short duration of the action potential (AP) leading to unaltered AP-evoked presynaptic Ca 2+ (Ca) influx despite shifted activation of mutant Ca V 2.1 channels to lower voltages; and (iii) interneuron-specific Ca V 2.1 channels whose gating properties are not affected by the FHM1 mutation (Fioretti et al., 2011;Inchauspe et al., 2010;Tottene et al., 2009;Xue and Rosenmund, 2009). Another interesting question is whether other inhibitory synapses, besides the FS interneuron-pyramidal cell synapse, are unaffected by FHM1 mutations.
Here, we show that the FHM1 R192Q mutation does not affect inhibitory transmission at autapses of cortical FS and other types of multipolar interneurons in microculture, and we investigate the mechanisms underlying this lack of effect despite a dominant role of P/Q channels in controlling GABA release. Our findings support the conclusion that the unaltered inhibitory transmission at multipolar (mainly FS) interneuron autapses is due to the expression of specific Ca V 2.1 channels whose gating is barely affected by the FHM1 mutation, and not to near saturation of the presynaptic calcium sensor or short duration of the AP.

Preparation of cortical neurons in microculture
Cortical neurons were isolated from P0-2 homozygous KI mice carrying the Ca V 2.1 FHM1 R192Q mutation (van den  and wild-type (WT) C57Bl6J mice with the same genetic background following the procedure of Levi et al. (1984). The neurons were cultured on glial microislands (at the density of 6000-25,000 cells/ml) essentially as reported in Brody and Yue (2000) for hippocampal neurons, except for the following details: astrocytes culture medium was basal Eagle's medium (BME) plus 10% fetal bovine serum, 25 mM KCl, 2 mM glutamine and 50 μg/ml gentamicin; neuronal medium was Neurobasal A plus 2% B27 Supplement, 0.5 mM glutamine and 1% PSN Antibiotic mix (all from Gibco); only half volume of astrocytes medium was replaced with neuronal medium to allow the astrocytes to condition the medium before neuron plating. Every 4 days half volume of neuronal medium was changed with a fresh one.
Single cortical neurons grown on glial microislands form synaptic connections onto themselves (autapses). These autaptic connections are by definition monosynaptic, offer an unusually homogeneous population of synapses producing large synaptic responses and solution exchanges can be fast and complete (Bekkers and Stevens, 1991).
All experimental procedures were carried out in accordance with the Italian Animal Welfare Act and with the Use Committee guidelines of the University of Padova and were approved by the local authority veterinary service.

Immunofluorescence assays
Cells grown on glial microislands for 12 days were washed with phosphate buffered saline (PBS), fixed for 20 minutes at room temperature with 4% paraformaldehyde, 4% sucrose in PBS, quenched (0.38% glycine, 0.24% NH 4 Cl in PBS, twice for 10 minutes each) and permeabilized with 5% acetic acid in ethanol for 20 minutes at −20°C. After saturation with 3% bovine serum albumin (BSA) and 10% goat serum in PBS for 20 minutes, samples were incubated with the primary antibodies (diluted in 3% BSA, 10 % goat serum in PBS) for 60 minutes at room temperature. Mouse monoclonal anti-glutamic acid decarboxylase (GAD-67; from Millipore, Billerica, MA, USA) was used at dilution of 1:500; rabbit polyclonal anti-parvalbumin (PV; from Abcam, Cambridge, UK) was used at dilution of 1:300; rat monoclonal antisomatostatin (SOM; from Millipore) was used at a dilution of 1:100. The specific primary antibodies were detected with Alexa-conjugated secondary antibodies raised in goat (anti-mouse 488, anti-rabbit 647, anti-rat 568; from Life Technologies, Monza, Italy), which were incubated for 60 minutes at room temperature after washing with 3% BSA in PBS; working dilution 1:200 in 3% BSA, 10% goat serum. Coverslips were mounted in mowiol (Sigma). Images were acquired with a DMI6000 inverted epifluorescence microscope (Leica, Germany) equipped with a HCX PL APO 60× oil immersion objective NA 1.4. Differential interference contrast microscopy was used to increase contrast for brightfield imaging of the cultures. Images were acquired with an Orca-Flash4 digital camera (Hamamatsu, Japan).

Electrophysiological recordings and data analysis
Whole-cell patch-clamp recordings were made at room temperature following standard techniques. Electrical signals were recorded through an Axopatch-200B patch-clamp amplifier and digitized using a Digidata 1440A interface and pClamp software (Axon Instruments). Compensation (typically 60-80%) for series resistance was used (5-6 MΩ after compensation).
Microislands containing several glial cells and a single neuron with irregular soma morphology and multiple asymmetrical processes emanating from it (multipolar interneuron; Fig. 1) were selected for recording of evoked postsynaptic currents (PSCs) in voltage-clamp mode (sampling 5 KHz; filter 1 KHz) after 10 to 14 days (DIVs) in culture. Given the well known large diversity of cortical interneurons (Petilla Interneuron Nomenclature Group, 2008;Rudy et al., 2011), to limit the heterogeneity, cells characterized by fusiform ovoid or spindleshaped soma with symmetrical processes (Fig. 1) were not considered (to exclude bitufted and bipolar interneurons). Immunofluorescence using antibodies specific for PV and/or SOM revealed that almost all PVexpressing (PV+) interneurons had morphology similar to that of the cells selected for recordings (multipolar interneurons: Fig. 1C) whereas 61% of SOM-expressing (SOM+) interneurons had fusiform ovoid or spindle-shaped soma; the remaining 39% had multipolar morphology similar to that of some of the cells selected for recordings (Fig. 1D).
APs in the unclamped processes were induced by a 2 ms voltage pulse to +20 mV every 10 s from a holding potential of −80 mV. The evoked PSCs were measured at −90 mV.
Although the GABA A -mediated inhibitory postsynaptic currents (IPSCs) recorded at −90 mV are inward currents (given the predicted and measured E rev of −69 and −63 mV, respectively), they were easily distinguished from glutamate receptor-mediated excitatory postsynaptic currents on the basis of their slower time course and their complete inhibition by 20 μM bicuculline (Ascent Scientific-Abcam). The currents recorded in the presence of bicuculline were subtracted to all records to obtain the evoked IPSCs (displayed in the figures after blanking 1-3 ms around each stimulus artefact for clarity). After IPSC stabilization (typically 3 minutes after break-in), 5-10 sweeps were averaged to obtain the IPSC amplitude. A liquid junction potential (LJP) of −8 mV should be added to all voltages to obtain the correct membrane potentials (Neher, 1992). Patch-clamp pipettes had resistances of 1.8-2.5 MΩ.
The relative number of neurons recorded from WT and KI mice at different DIVs was closely matched.
The firing properties of neurons in microculture were studied with current clamp recordings (sampling 50 KHz; filter 10 KHz), in which baseline current injection was usually applied to maintain the resting potential near −70 mV and either long (1 s) depolarizing current pulses of increasing amplitude or short (20 ms) depolarizing current pulses were applied to elicit multiple and single action potentials (APs), respectively.
Acute thalamocortical slices of the barrel cortex were prepared from P11-13 mice as described in Tottene et al. (2009), and the firing properties of layer 2/3 FS interneurons were measured by applying (1 s long) suprathreshold current injections of increasing amplitude as in Tottene et al. (2009).
Action potential half width (AP hw ) was measured as the width of the AP at half maximal height (defined as the amplitude from AP voltage threshold to AP peak), considering the first AP in the train at rheobase current injection. The after-hyperpolarization (AHP) amplitude was measured as the difference between AP voltage threshold and the depth of the AHP. The frequency of APs was measured from the average of the last three interspike intervals, and the accommodation ratio as the ratio of the first to the mean of the last three interspike intervals after 600 ms during current pulses at 2-3 times the rheobase.
Data are given as mean ± SEM; stars indicate a statistically significant difference from control assessed by the unpaired or paired Student's t test, unless otherwise specified (*, p b 0.05; **, p b 0.01; ***, p b 0.001).

Results
Single cortical neurons grown on glial microislands form synaptic connections onto themselves (autapses) with properties very similar under Nomarski optics of a microisland containing several glial cells and a single multipolar neuron with irregular soma morphology and multiple asymmetrical processes emanating from it (which is magnified in the inset), similar to those selected for recordings. Scale bar: 20 μm. Right panel: microisland containing several glial cells and two neurons, one representative of the neurons with ovoid soma morphology and symmetrical processes that were not considered for recordings and one representative of the multipolar neurons (that were used for recordings when they were the only neuron in the microisland). B. Representative pictures of different types of interneurons in microculture immunostained for GAD67: the three panels on the left show different multipolar interneurons representing the morphologies that were selected for recordings; the two panels on the right show interneurons with fusiform, ovoid or spindle-shaped soma and symmetrical processes that were not considered for recording. Scale bar: 10 μm. C. Representative pictures of interneurons in microculture immunostained for both GAD67 and parvalbumin (PV) in dual labeling experiments. The PV + interneurons have multipolar morphologies similar to those selected for recordings. Scale bar: 10 μm. D. Representative pictures of interneurons in microculture immunostained for both GAD67 and somatostatin (SOM) in dual labeling experiments. Sixty-one percent of SOM + interneurons had fusiform, ovoid or spindle shaped soma (right panel); 39% of SOM + interneurons had multipolar morphology similar to that of some of the cells selected for recordings (left panel). In triple labeling experiments, none of the neurons were positive for both PV and SOM. Scale bar: 10 μm.
to those of synapses between neurons (Bekkers and Stevens, 1991). We investigated inhibitory autaptic neurotransmission in microcultures of cortical neurons from neonatal wild-type (WT) and R192Q knockin (KI) mice. Given the well known large diversity of cortical interneurons (Petilla Interneuron Nomenclature Group, 2008;Rudy et al., 2011), to limit the heterogeneity, the recordings were performed only on cells characterized by irregular soma morphology with multiple asymmetrical processes emanating from it (multipolar interneurons) (cf Methods and Fig. 1). The recorded cells were enriched in fast-spiking (FS) interneurons relative to other types of interneurons because current clamp recordings revealed the high-frequency non-accommodating firing typical of FS interneurons in 62% (24 out of 39) of the cells (Fig. 2A). These interneurons had a mean firing frequency of 53 ± 3 Hz (with current injections 2 to 3 times the rheobase) and an accommodation ratio of 1.01 ± 0.06 (n = 22). Typically FS interneurons display brief action potentials (APs) with large amplitude fast afterhyperpolarization (AHP); the AP half width (AP hw ) is age-and temperature-dependent decreasing with both age and temperature (Ali et al., 2007;Goldberg et al., 2011). The FS interneurons in our DIV 10-13 microcultures had on average an AP hw of 1.03 ± 0.04 ms and an AHP of 25 ± 1 mV (n = 24) at room temperature ( Fig. 2A), which are consistent with the AP properties of FS interneurons in acute cortical slices from juvenile mice. In fact, we obtained similar values of AP duration and AHP from room temperature current clamp recordings of FS interneurons in acute thalamocortical slices of P11-13 WT (AP hw = 0.87 ± 0.07 ms; AHP = 21 ± 1 mV; n = 11) and R192Q KI mice (AP hw = 1.00 ± 0.06 ms; AHP = 22 ± 1 mV; n = 11).
Possibly, the fraction of FS interneurons derived from current clamp recordings is an underestimation because three interneurons classified as non-FS showed a single (or maximum two) AP of short duration (0.83 ± 0.16 ms) with a very deep AHP (35 ± 2 mV) at all current injections. To our knowledge similar firing properties have not been reported for interneurons in cortical slices and might be a consequence of the autaptic connections in microculture; if these cells are considered as modified FS interneurons, then the fraction of FS interneurons in the recorded cells increases to 69%.
Large inhibitory postsynaptic currents (IPSCs) were evoked in single cortical multipolar interneurons by brief depolarizing voltage steps eliciting an AP in the unclamped axonal processes (Fig. 2B). As previously shown for inhibitory synaptic transmission between layer 2/3 FS interneurons and pyramidal cells in cortical slices (Tottene et al., 2009), autaptic inhibitory neurotransmission in multipolar interneurons in microculture was unaltered in FHM1 KI mice. The average amplitude of the IPSC evoked in WT and KI interneurons was similar ( Fig. 2B: 1.2 ± 0.1 nA, n = 63, in WT versus 1.1 ± 0.1 nA, n = 53, in KI; p = 0.64), despite the fact that P/Q-type Ca channels strongly contribute to control GABA release at the inhibitory autapses, as shown by the large fraction of the IPSC inhibited by the specific blocker ω-AgaIVA (200 nM) in most cells (Fig. 2C). Consistent with and further supporting the lack of effect of the FHM1 mutation on inhibitory autaptic transmission, ω-AgaIVA inhibited a similar fraction of the IPSC at WT and KI autapses (62 ± 6%, n = 17, in WT; 69 ± 8%, n = 12, in KI; p = 0.319, Wilcoxon test). As a likely reflection of the heterogeneity of the recorded multipolar interneurons and/or the heterogeneity of the Ca V channels controlling release at FS interneuron synapses in different cortical areas (Ali and Nelson, 2006;Zaitsev et al., 2007;Kruglikov and Rudy, 2008;Tottene et al., 2009), the fractional inhibitions by ω-AgaIVA in one KI and one WT interneuron (b 20%) were outside the normal distribution; if one excludes these values, the average inhibition by ω-AgaIVA of autaptic inhibitory transmission increases, remaining similar in WT and KI mice (65 ± 5%, n = 16, in WT; 75 ± 5%, n = 11, in KI; p = 0.204). Thus, the conclusion that the FHM1 mutation does not affect inhibitory neurotransmission at the synapses between layer 2/3 FS interneurons and pyramidal cells can be extended to the inhibitory autapses of cortical FS interneurons and, likely, to inhibitory autapses of other types of multipolar interneurons.
Given the low coefficient of variation of the unitary IPSPs and the close to zero percentage of failures at the connection between layer 2/ 3 FS interneurons and pyramidal cells, which is consistent with a high probability of AP-evoked GABA release at the active zones, Tottene et al. (2009) suggested saturation of the presynaptic Ca sensor as a possible explanation for the unaltered inhibitory neurotransmission in FHM1 KI mice despite the dominant role of P/Q-type Ca channels at these FS interneuron synapses. To investigate whether saturation of the presynaptic Ca sensor underlies the unaltered neurotransmission at the inhibitory autapses of cortical multipolar (mostly FS) interneurons, we measured the dependence of the evoked IPSC on the concentration of extracellular Ca ions ([Ca 2+ ] out ) in WT and FHM1 KI inhibitory autapses.
In comparison with the [Ca 2+ ] out -dependence of the evoked EPSC at the excitatory autapses of WT cortical pyramidal cells (Tottene et al., 2009), the [Ca 2+ ] out -dependence of the evoked IPSC at the inhibitory autapses of WT multipolar interneurons was shifted to lower [Ca 2+ ] out (EC 50 : 1.00 ± 0.09 mM, n = 9, for inhibitory autapses versus 1.65 ± 0.1 mM, n = 7, for excitatory autapses; p = 0.0003) (Fig. 3A). As a consequence of the increased AP-evoked Ca influx through presynaptic P/Q-type Ca channels, the evoked EPSC at cortical pyramidal cell autapses of R192Q KI mice (at 2 mM Ca) was 1.7 times larger than that at WT autapses (Tottene et al, 2009), which corresponds to the maximal increment predicted by the Ca-dose response curve (in which the maximal EPSC is 1.7 times larger than the EPSC at 2 mM [Ca 2+ ] out ) (Fig. 3A). In contrast, at the inhibitory autapses the maximal value of the evoked IPSC is only 1.19 times larger than the IPSC at 2 mM [Ca 2+ ] out , which is consistent with near saturation of the Ca 2+ sensor at the [Ca 2+ ] out used in our experiments (Fig. 3A). This is confirmed by the ratio between the IPSCs evoked at 4 and 2 mM [Ca 2+ ] out in 23 multipolar interneurons, Ca4/Ca2 = 1.12 ± 0.03, which is similar to the value of  1.14 ± 0.04 from the 9 cells in which we obtained the complete Ca-dose response curve. Assuming that the FHM1 mutation does increase AP-evoked Ca influx through P/Q-type Ca channels at the inhibitory autapses, one expects, at most, a 19% increase of the evoked IPSC in R192Q KI mice. Such a small increase might have escaped detection due to experimental variability. However, if indeed near saturation of the Ca sensor underlies the similar evoked IPSCs at WT and mutant autapses, one expects to reveal gain-of-function of inhibitory autaptic transmission by lowering [Ca 2+ ] out , and one predicts that the Ca 2+ dependence of the IPSC should be shifted to lower [Ca 2+ ] out in R192Q KI compared to WT mice [as found at excitatory pyramidal cell autapses by Tottene et al. (2009)].
In contrast with this prediction, the evoked IPSC at WT and mutant autapses showed a similar dependence on [Ca 2+ ] out (EC 50 : 0.91 ± 0.08 mM, n = 8, for KI autapses versus 1.0 ± 0.09 mM, n = 9, for WT multipolar interneuron autapses, p = 0.44) and similar amplitudes at each [Ca 2+ ] out (Fig. 3B), implying similar AP-evoked Ca influx at the active zones of the inhibitory autapses of WT and R192Q KI mice. We therefore conclude that the unaltered inhibitory autaptic transmission in the FHM1 mouse model is due to unaltered AP-evoked Ca influx through presynaptic P/Q-type Ca channels at multipolar (mostly FS) interneuron autapses rather than to near saturation of the presynaptic Ca sensor.
Unaltered AP-evoked Ca current at calyx of Held synaptic terminals of R192Q KI mice, despite a shifted activation to lower voltages of mutant calyx presynaptic Ca V 2.1 channels similar to that of mutant channels in cortical pyramidal cells, has been recently reported (Inchauspe et al., 2010). It has been shown that the (about four times) shorter duration of the AP in Calyx terminals than in pyramidal cells could largely explain the differential effect of the FHM1 mutation on AP-evoked Ca current at cortical pyramidal cell and Calyx synapses (Inchauspe et al., 2010), leading to the suggestion that the short duration of the AP in FS interneurons could similarly result in unaltered AP-evoked Ca current at FS interneuron synapses and thus explain the unaltered synaptic transmission at FS interneuron-pyramidal cell synapses reported by Tottene et al. (2009).
To investigate whether the relatively short duration of the AP explains the lack of effect of the FHM1 mutation on AP-evoked Ca influx at the multipolar (mainly FS) interneuron inhibitory autapses, we prolonged the duration of the AP using tetraethylammonium (TEA, 10 mM) and measured evoked IPSCs at 1, 2 and 4 mM [Ca 2+ ] out in the presence of TEA in WT and R192Q KI multipolar interneurons. If a gain-of-function of AP-evoked Ca influx due to activation of mutant presynaptic Ca V 2.1 channels at lower voltages than WT channels is prevented by the relatively short AP duration, one expects that prolongation of the AP should uncover the gain-of-function, resulting in a shift to lower voltages of the [Ca 2+ ] out -dependence of the IPSC and a corresponding increase of the IPSC at 1 mM [Ca 2+ ] out (far from saturation) in KI compared to WT inhibitory autapses.
Evoked IPSCs in the presence of TEA at 1, 2 and 4 mM [Ca 2+ ] out were measured in WT and R192Q KI multipolar interneurons using the protocol shown in Fig. 5A. The similar values of the average WT and KI IPSCs (normalized to the value at 4 mM [Ca 2+ ] out in each cell: 0.72 ± 0.04, n = 14, in WT; 0.77 ± 0.04, n = 11, in KI at 1 mM [Ca 2+ ] out , p = 0.34) (Fig. 5B) show that prolongation of the AP does not uncover gain-offunction of inhibitory neurotransmission at multipolar interneuron autapses. We therefore conclude that the unaltered AP-evoked Ca influx through presynaptic P/Q-type Ca channels at multipolar (mostly FS) interneuron autapses and the unaltered inhibitory autaptic transmission in the FHM1 mouse model is not due to the relatively short AP duration in the interneurons. Fioretti et al. (2011) have shown that the gating properties of the specific Ca V 2.1 channels expressed in capsaicin-sensitive trigeminal ganglion neurons of R192Q KI mice are not affected by the FHM1 mutation. Accordingly, CGRP release from the peripheral terminals of trigeminal dural afferents was unaltered in the FHM1 KI mice. It seems then possible that the expression in cortical multipolar interneurons of specific Ca V 2.1 channels whose gating properties are not affected by the FHM1 mutation may similarly underlie the unaltered inhibitory transmission at the interneuron autapses of R192Q KI mice. To investigate this possibility, we measured the whole-cell P/Q-type Ca current density as a function of voltage in single cortical multipolar interneurons grown on glial microislands. The P/Q-type Ca 2+ current component was obtained as the amount of current inhibited by either ω-conotoxin MVIIC (MVIIC, 3 μM) or ω-AgaIVA (200 nM), applied after sequential additions of nimodipine (5 μM) and ω-conotoxin GVIA (GVIA, 1 μM) to inhibit L- In five of these interneurons current-and voltage-clamp experiments were performed on the same cell, thus allowing correlations between firing properties, increase in AP duration by TEA and increase in IPSC consequent to the AP prolongation. Four of these multipolar interneurons were FS or "modified FS" interneurons and showed a larger increase of IPSC by TEA (65 ± 13%, in correlation with an average prolongation of the AP by 45 ± 7%; n = 4) compared to the non-FS multipolar interneuron (23%, in correlation with a smaller prolongation of the AP of 18%). and N-type Ca channels, respectively (Fig. 6A). In the large majority of WT multipolar interneurons (29 out of 33: 88%) the P/Q-type Ca current was the largest pharmacological component amounting to 36 ± 2% of the total Ca current (53 ± 2% of the non L-type Ca current) (Fig. 6B). Twelve percent of multipolar interneurons did not have a measurable P/Q-type Ca current, a fraction comparable to that of the interneurons showing a small (b 20%) contribution of P/Q-type Ca channels to inhibitory autaptic neurotransmission.
Fitting of the current-voltage (I-V) relationships of the P/Q-type Ca current in individual WT and KI multipolar interneurons gave mean half activation voltages of − 9.8 ± 1.1 mV (n = 12) and − 13.1 ± 0.8 mV (n = 14), respectively (p = 0.02) (Fig. 6C). The small shift to more negative voltages of the P/Q-type Ca current activation in KI compared to WT interneurons (much smaller than that measured in cortical pyramidal cells, shown for comparison in Fig. 6D) did not result in significantly larger P/Q-type Ca current densities in KI interneurons (p = 0.532, one-way ANOVA for repeated measures test; Fig. 6C), in contrast with the significantly larger P/Q-type Ca current densities in KI compared to WT pyramidal cells (Tottene et al, 2009). The L-, N-and R-type Ca current densities were also similar in WT and KI multipolar interneurons (at 0 mV, L-type: 11 ± 1 pA/pF versus 11.4 ± 0.9 pA/pF; N-type: 4.3 ± 0.7 pA/pF versus 6.3 ± 0.8 pA/pF; R-type: 6.1 ± 0.5 pA/ pF versus 7 ± 2 pA/pF; P/Q-type: 11.7 ± 0.8 pA/pF versus 12.5 ± 1.2 pA/pF; n = 17 for WT and n = 18 for KI; p = 0.92, 0.07, 0.55 and 0.6, respectively), thus confirming the absence of compensatory changes found in other types of neurons (Fioretti et al., 2011;Tottene et al., 2009;van den Maagdenberg et al., 2004).
We conclude that the current density and the gating properties of the Ca V 2.1 channels expressed by multipolar interneurons are barely affected by the FHM1 mutation, thus providing an explanation for the unaltered AP-evoked Ca influx through P/Q-type Ca channels at the multipolar interneuron autapses and the unaltered autaptic inhibitory neurotransmission in FHM1 KI mice.

Discussion
Our analysis of inhibitory neurotransmission in microcultures of cortical neurons from WT and FHM1 R192Q KI mice has shown that the FHM1 mutation does not affect inhibitory transmission at autapses of cortical FS (and other types of multipolar) interneurons, despite a dominant role of P/Q-type Ca channels in controlling GABA release. This confirms and extends our previous finding of unaltered inhibitory synaptic transmission between layer 2/3 FS interneurons and pyramidal cells in acute slices of R192Q KI mice (Tottene et al., 2009).
We have investigated several mechanisms that were suggested as possible explanations for the unaltered inhibitory transmission at FS interneuron synapses despite a dominant role of P/Q-type channels in controlling GABA release, namely: (i) saturation of the presynaptic Ca sensor (Tottene et al., 2009); (ii) short duration of the AP in FS interneurons leading to unaltered AP-evoked presynaptic Ca influx despite shifted activation of mutant Ca V 2.1 channels to lower voltages (Inchauspe et al., 2010;Inchauspe et al., 2012;Tottene et al., 2009); and (iii) interneuron-specific Ca V 2.1 channels whose gating properties are not (or barely) affected by the FHM1 mutation (Fioretti et al., 2011;Tottene et al., 2009;Xue and Rosenmund, 2009).
We have shown that the unaltered inhibitory transmission at multipolar interneuron autapses in the FHM1 mouse model is due to unaltered AP-evoked Ca influx through presynaptic Ca V 2.1 channels and not to near saturation of the presynaptic Ca sensor, since lowering [Ca] out did not reveal a gain-of-function of evoked transmission (Fig. 3). Gain-of-function of inhibitory transmission at low [Ca] out was not uncovered even after prolongation of the AP with TEA (Fig. 5), indicating that the unaltered AP-evoked Ca influx through presynaptic Ca V 2.1 channels is not due to the relatively short duration of the AP in multipolar (mostly FS) interneurons. In contrast with the shifted activation to lower voltages of mutant P/Q-type Ca channels in pyramidal cells and other CNS excitatory neurons (Inchauspe et al., 2010;Tottene et al., 2009;van den Maagdenberg et al., 2004), the gating properties of the P/Q-type Ca channels expressed in cortical multipolar interneurons in microculture were barely affected by the FHM1 mutation. These findings support the conclusion that the lack of effect of the FHM1 mutation on inhibitory neurotransmission at multipolar (mainly FS) interneuron autapses may be explained by the expression in these interneurons of specific Ca V 2.1 channels whose gating is barely affected by the FHM1 mutation.
Most likely, this explains also the lack of effect of the FHM1 mutation on inhibitory synaptic transmission at FS interneuron synapses in cortical slices. In fact, given the age dependence of AP duration in FS interneurons (Goldberg et al., 2011), at the age (P11-13) when unaltered inhibitory transmission at unitary layer 2/3 FS interneuron-pyramidal cell synapses was described (Tottene et al., 2009), the AP duration measured in layer 2/3 FS interneurons in cortical slices (AP hw : 0.94 ± 0.05 ms, n = 22, at room temperature) is similar to that measured in multipolar interneurons in microculture (AP hw : 1.03 ± 0.04 ms; p = 0.140). At Calyx of Held synapses, APs of similar duration (about 1 ms half duration, in the presence of TEA) did reveal gain-of-function of AP-evoked Ca current and excitatory transmission due to the shifted activation gating of presynaptic Ca V 2.1 channels (in contrast with the lack of gain-of-function with the much shorter control APs of 0.44 ms half duration) (Inchauspe et al., 2010;Inchauspe et al., 2012). Similarly, in cortical pyramidal cells, gain-of-function of the AP-evoked Ca current was observed using as voltage stimulus APs of 0.93 ms half duration that were recorded at physiological temperature (Inchauspe et al., 2010).
Inhibition in the cortex is generated by a variety of different types of GABAergic interneurons, that can be subdivided in three non overlapping populations on the basis of the expression of PV, SOM and 5HT3a receptors (5HT3aR); each population is heterogeneous in terms of morphology and firing properties of the interneurons composing it, although to a different degree . The large majority of PV + interneurons (the largest population of interneurons in the neocortex) shows FS non-adapting firing and irregular soma with multipolar dendritic morphology. In contrast, a large fraction of Martinotti cells, the SOM + interneurons that target the apical dendrites of pyramidal cells, and a fraction of 5HT3aR + interneurons show ovoid, fusiform or spindle-shaped soma with bitufted or bipolar dendritic morphology and various types of (regular or irregular) adapting firing . To limit the heterogeneity, we measured autaptic neurotransmission and Ca currents only in interneurons with irregular soma and multiple asymmetrical processes emanating from it (multipolar interneurons) and not in interneurons with ovoid, fusiform or spindleshaped soma and symmetrical processes. As expected, most (62-70%) multipolar interneurons were FS interneurons, but not all. The nature of the non-FS multipolar interneurons we recorded from remains unclear since both SOM + and 5HT3aR + interneurons comprise multipolar interneurons with regular adapting or irregular firing similar to that shown by non-FS interneurons in microculture. However, in three DIV 12 microcultures, only 33 out of a total of 483 GAD-positive neurons were SOM + (6.8%) and 13 of these SOM + interneurons were multipolar (data not shown); the small fraction of SOM  . 6. The gating properties of the Ca V 2.1 channels expressed in multipolar interneurons are barely affected by the FHM1 mutation. A. Peak whole-cell Ba 2+ current versus time recorded from a DIV 9 multipolar interneuron in microculture during depolarizations at 0 mV every 10 s from holding potential of −70 mV before and after sequential application of the indicated drugs (applied for the time indicated by the horizontal bars). Insets: on the left, representative P/Q-type current trace obtained as the difference between traces at times a and b; on the right, representative traces at increasing voltage from −40 to 0 mV, taken during current-voltage (I-V) measurements at times a and b. Scale bars: 50 ms, 100 pA. B. Contribution of the L-, N-, P/Q-, and R-type Ca current components in DIV 6-9 WT multipolar interneurons (n = 17), as obtained from the percentage of whole-cell Ba 2+ current (at 0 mV) inhibited by nimodipine, GVIA, MVIIC or ω-AgaIVA (Aga) and remaining (in the presence of nimodipine after subsequent applications of GVIA and Aga), respectively. Only interneurons with P/Qtype Ca current for which all four pharmacological components could be measured were included (n = 17). Out of a total of 33 recorded multipolar interneurons, four (12%) did not show any P/Q-type Ca current; in these interneurons an increased fractional contribution of N-type Ca current (42 ± 4%) compensated for the lack of P/Q-type current; the fractional contribution of the L-and R-type components were 38 ± 4% and 20 ± 3%, respectively, similar to those in P/Q-expressing interneurons. C. P/Q-type current density as a function of voltage in WT (n = 12) and KI (n = 14) multipolar interneurons in microculture. Lines are fits of the equation I = G(V − E rev )(1 + exp[(V 1/2 − V)/k]) −1 with V 1/2 = −10.3 ± 0.1 mV (k = 5.3 ± 0.04 mV) and V 1/2 = −13.1 ± 0.2 mV (k = 5.2 ± 0.1 mV) for WT and KI, respectively. D. Normalized P/Q-type current densities as a function of voltage in WT (n = 12) and KI (n = 14) multipolar interneurons in microculture and in WT (n = 10) and KI (n = 9) dissociated cortical pyramidal cells (Tottene et al., 2009). Fitting the I-V curves for cortical pyramidal cells gave V 1/2 = −8.0 ± 1 mV and V 1/2 = −16 ± 1 mV for WT and KI, respectively (Tottene et al., 2009). + interneurons suggests that 5HT3aR + interneurons might represent the majority of the recorded non-FS multipolar interneurons.
In any case, the lack of effect of the FHM1 mutation on autaptic neurotransmission and Ca current in the heterogeneous population of multipolar interneurons in microculture point to the possibility that unaltered inhibitory synaptic transmission in FHM1 is a general property not limited to FS interneuron synapses. Indeed, we have preliminary evidence that the R192Q mutation does not affect synaptic transmission at unitary inhibitory connections between layer 2/3 SOM + interneurons and pyramidal cells (N. Pilati, M. Sessolo and D. Pietrobon, unpublished findings).
Overall, our findings suggest that expression of different Ca V 2.1 channels in inhibitory and excitatory cortical neurons may underlie the differential effect of the FHM1 mutation on inhibitory and excitatory synaptic transmission. While the molecular mechanisms underlying the differential modulation of the gating properties of the specific Ca V 2.1 channels expressed in excitatory and inhibitory cortical neurons remain unknown, possible mechanisms include the expression of different splicing variants of the α1 subunit (Adams et al., 2009) and/or the expression of different auxiliary subunits (Mullner et al., 2004) and/or other modulatory proteins (Catterall and Few, 2008). In this respect, it is interesting that inhibitory modulation by SNAP25 of the Ca current was observed in glutamatergic but not GABAergic hippocampal neurons in primary culture (Condliffe and Matteoli, 2011;Condliffe et al., 2010), and reduction of SNAP25 by siRNA knockdown or in SNAP25 -/+ mice resulted in reduced evoked inhibitory synaptic transmission [ascribed to the function of SNAP25 as component of the SNARE complex (Boyken et al., 2013;Tafoya et al., 2006)] but, in contrast, enhanced evoked excitatory synaptic transmission due to increased probability of glutamate release, which is consistent with reduced inhibition of presynaptic Ca channels by SNAP25 at excitatory synapses (Antonucci et al., 2013).
The evidence of cortical neuron subtype-specific alterations of Ca V 2.1 channels by FHM1 mutations presented here [and cf also the previous evidence of trigeminal ganglion neuron subtype-specific alterations in Fioretti et al. (2011)] has an important implication for familial migraine (and in general for channelopathies) mechanisms in that it may help to explain why a mutation in a Ca channel that is widely expressed in the nervous system (Westenbroek et al., 1995) produces the specific neuronal dysfunctions leading to migraine. It has been suggested that, as a consequence of the differential effect of the FHM1 mutations on synaptic transmission and short-term plasticity at different cortical synapses (noting the lack of effect at inhibitory synapses), the neuronal circuits that dynamically maintain a tight balance between excitation and inhibition during cortical activity are very likely altered in FHM1. Functional alterations in these circuits may underlie the abnormal processing of sensory information typical of migraineurs in the interictal period and, in certain conditions (e.g. in response to specific migraine triggers), lead to disruption of the excitatory/inhibitory balance and to neuronal hyperactivity, that may result in elevations of extracellular K + above the critical value for ignition of CSD (Pietrobon and Moskowitz, 2013;Tottene et al., 2009;Vecchia and Pietrobon, 2012).
Despite recent drug developments, there is a great need for more efficacious and specific prophylactic migraine medications. The finding of neuron subtype-specific alterations of Ca V 2.1 channel functioning by FHM1 mutations poses a challenge to future drug discovery aimed at counteracting excessive glutamatergic synaptic transmission in FHM and other migraine variants by targeting glutamatergic synapses without compromising inhibitory (and other normal) synapses (Vecchia and Pietrobon, 2012;Xue and Rosenmund, 2009).

Conclusions
In contrast with the increased current density and the left-shifted activation gating of the Ca V 2.1 channels in cortical pyramidal cells from R192Q FHM1 knockin mice, the current density and activation gating of the Ca V 2.1 channels expressed in cortical multipolar (mainly FS) interneurons in microculture are barely affected by the FHM1 mutation. This may explain the unaltered AP-evoked Ca influx and the consequent unaltered inhibitory neurotransmission at the autapses of these interneurons, that contrasts with the enhanced AP-evoked Ca influx and enhanced excitatory neurotransmission at cortical pyramidal cell synapses. Neither saturation of the presynaptic Ca sensor nor the relatively short duration of the interneuron AP can explain the unaltered AP-evoked Ca influx and the unaltered neurotransmission at the inhibitory autapses. Overall, our findings suggest that expression of different Ca V 2.1 channels in inhibitory and excitatory cortical neurons underlies the differential effect of FHM1 mutations on inhibitory and excitatory synaptic transmission and the likely consequent dysregulation of the cortical excitatory-inhibitory balance in FHM1.