Abnormal cortical synaptic transmission in CaV2.1 knockin mice with the S218L missense mutation which causes a severe familial hemiplegic migraine syndrome in humans

Familial hemiplegic migraine type 1 (FHM1) is caused by gain-of-function mutations in CaV2.1 (P/Q-type) Ca2+ channels. Knockin (KI) mice carrying the FHM1 R192Q missense mutation show enhanced cortical excitatory synaptic transmission at pyramidal cell synapses but unaltered cortical inhibitory neurotransmission at fast-spiking interneuron synapses. Enhanced cortical glutamate release was shown to cause the facilitation of cortical spreading depression (CSD) in R192Q KI mice. It, however, remains unknown how other FHM1 mutations affect cortical synaptic transmission. Here, we studied neurotransmission in cortical neurons in microculture from KI mice carrying the S218L mutation, which causes a severe FHM syndrome in humans and an allele-dosage dependent facilitation of experimental CSD in KI mice, which is larger than that caused by the R192Q mutation. We show gain-of-function of excitatory neurotransmission, due to increased action-potential evoked Ca2+ influx and increased probability of glutamate release at pyramidal cell synapses, but unaltered inhibitory neurotransmission at multipolar interneuron synapses in S218L KI mice. In contrast with the larger gain-of-function of neuronal CaV2.1 current in homozygous than heterozygous S218L KI mice, the gain-of-function of evoked glutamate release, the paired-pulse ratio and the Ca2+ dependence of the EPSC were all similar in homozygous and heterozygous S218L KI mice, suggesting compensatory changes in the homozygous mice. Furthermore, we reveal a unique feature of S218L KI cortical synapses which is the presence of a fraction of mutant CaV2.1 channels being open at resting potential. Our data suggest that, while the gain-of-function of evoked glutamate release may explain the facilitation of CSD in heterozygous S218L KI mice, the further facilitation of CSD in homozygous S218L KI mice is due to other CaV2.1-dependent mechanisms, that likely include Ca2+ influx at voltages sub-threshold for action potential generation

An inhibitory gate for state transition in cortex

Large scale transitions between active (up) and silent (down) states during quiet wakefulness or NREM sleep regulate fundamental cortical functions and are known to involve both excitatory and inhibitory cells. However, if and how inhibition regulates these activity transitions is unclear. Using fluorescence-targeted electrophysiological recording and cell-specific optogenetic manipulation in both anesthetized and non-anesthetized mice, we found that two major classes of interneurons, the parvalbumin and the somatostatin positive cells, tightly control both up-to-down and down-to-up state transitions. Inhibitory regulation of state transition was observed under both natural and optogenetically-evoked conditions. Moreover, perturbative optogenetic experiments revealed that the inhibitory control of state transition was interneuron-type specific. Finally, local manipulation of small ensembles of interneurons affected cortical populations millimetres away from the modulated region. Together, these results demonstrate that inhibition potently gates transitions between cortical activity states, and reveal the cellular mechanisms by which local inhibitory microcircuits regulate state transitions at the mesoscale

Two-Photon Bidirectional Control and Imaging of Neuronal Excitability with High Spatial Resolution In Vivo

Summary: Sensory information is encoded within the brain in distributed spatiotemporal patterns of neuronal activity. Understanding how these patterns influence behavior requires a method to measure and to bidirectionally perturb with high spatial resolution the activity of the multiple neuronal cell types engaged in sensory processing. Here, we combined two-photon holography to stimulate neurons expressing blue light-sensitive opsins (ChR2 and GtACR2) with two-photon imaging of the red-shifted indicator jRCaMP1a in the mouse neocortex in vivo. We demonstrate efficient control of neural excitability across cell types and layers with holographic stimulation and improved spatial resolution by opsin somatic targeting. Moreover, we performed simultaneous two-photon imaging of jRCaMP1a and bidirectional two-photon manipulation of cellular activity with negligible effect of the imaging beam on opsin excitation. This all-optical approach represents a powerful tool to causally dissect how activity patterns in specified ensembles of neurons determine brain function and animal behavior. : Forli et al. developed an all-optical method to image and bidirectionally manipulate brain networks with high spatial resolution and minimal crosstalk in the intact mammalian brain. They validate the method across cell types and layers in the mouse neocortex. Keywords: optogenetics, two-photon excitation, digital holography, patterned illumination, two-photon imagin

EXCITATORY AND INHIBITORY SYNAPTIC TRANSMISSION AT CORTICAL SYNAPSES IN CaV2.1 KNOCK-IN MICE CARRYING FAMILIAL HEMIPLEGIC MIGRAINE MUTATIONS

Missense mutations in the human CACNA1A gene, which encodes the pore-forming a1 subunit of the CaV2.1 (P/Q-type) Ca2+ channel, cause familial hemiplegic migraine type 1 (FHM1), a rare subtype of migraine with aura (Ophoff et al., 1996). Apart from the characteristic hemiparesis, the headache, autonomic and aura symptoms of typical attacks of FHM1 are similar to those of the common forms of migraine with aura (Pietrobon, 2007; Pietrobon and Striessnig, 2003). CaV2.1 channels are located in presynaptic terminals and somatodendritic membranes throughout the brain, where they play a dominant role in controlling neurotransmitter release. FHM1 mutations produce gain of function of human recombinant CaV2.1 channels, mainly due to a shift of channel activation to more negative voltages and an increase of the open probability and single channel influx over a broad voltage range (Hans et al., 1999; Tottene et al., 2002, 2005; Pietrobon, unpublished data). Accordingly, homozygous knock-in (KI) mice carrying the R192Q FHM1 mutation show an increased P/Q-type Ca2+ current density in cerebellar granule cells and cortical pyramidal cells (Tottene et al., 2009; van den Maagdenberg et al., 2004). Interestingly, both the induction and the propagation of cortical spreading depression (CSD: the phenomenon underlying migraine aura, and a possible trigger of migraine headache) are facilitated in homozygous R192Q (RQ/RQ) FHM1 KI mice in vivo (Pietrobon, 2005, 2007; van den Maagdenberg et al., 2004). To investigate the mechanisms underlying the facilitation of CSD, in my laboratory we have recently studied excitatory cortical synaptic transmission in neuronal microcultures (this was part of my PhD project) and at connected pairs of pyramidal cells and multipolar fast-spiking (FS) interneurons in acute thalamocortical slices of RQ/RQ KI mice (Tottene et al., 2009). We found gain of function of excitatory neurotransmission due to increased action potential (AP)-evoked Ca2+ influx through presynaptic P/Q-type Ca2+ channels and increased probability of glutamate release at pyramidal cell synapses. Using an in vitro model of CSD, we provided direct evidence of a causative link between enhanced glutamate release at pyramidal cell synapses and facilitation of experimental CSD in RQ/RQ KI mice. In striking contrast, inhibitory neurotransmission at connected pairs of multipolar FS interneurons and pyramidal cells in thalamocortical slices of RQ/RQ KI mice was unaltered, despite being initiated by P/Q-type Ca2+ channels (Tottene et al., 2009). The synapse-specific effect of FHM1 mutations supports the view of migraine as an episodic disorder of brain excitability, with disruption of the excitation-inhibition balance and hyperactivity of cortical circuits in response to specific migraine triggers as the basis for episodic vulnerability to CSD ignition in migraine. In patients, the R192Q mutation causes typical FHM attacks; in contrast, the S218L mutation causes a dramatic hemiplegic migraine syndrome that is associated with seizures, coma and severe cerebral oedema often triggered by mild head trauma (Kors et al., 2001). In comparison with R192Q, mutation S218L produces a larger shift of activation of recombinant human CaV2.1 channels towards more negative membrane potentials and, accordingly, a larger gain of function of neuronal Ca2+ influx at low voltages and a larger facilitation of CSD induction and propagation in homozygous S218L (SL/SL) FHM1 KI mice compared to RQ/RQ KI mice in vivo (Tottene et al., 2002, 2005; van den Maagdenberg et al., 2004, 2010). Moreover, the effect of the S218L mutation on the neuronal CaV2.1 Ca2+ current and facilitation of CSD is allele dosage-dependent. To investigate the mechanisms underlying the greater facilitation of CSD in SL/SL KI mice compared to RQ/RQ KI mice, I studied glutamatergic synaptic transmission in cortical pyramidal cells grown on glial microislands from heterozygous S218L (SL/WT) KI mice. I found gain of function of cortical excitatory neurotransmission due to increased action potential-evoked Ca2+ influx through presynaptic P/Q-type Ca2+ channels and increased probability of glutamate release at cortical pyramidal cell synapses of SL/WT KI mice. In fact, in single cortical pyramidal cells forming autapses from mutant mice the amplitude of the evoked excitatory postsynaptic current (EPSC) and the contribution of P/Q-type Ca2+ channels to synaptic transmission were both increased. Moreover, saturation of the EPSC occurred at lower Ca2+ concentration and the paired pulse ratio (PPR) was decreased. The changes in EPSC amplitude, Ca2+ dependence of the EPSC and PPR in SL/WT KI mice were quantitatively similar to those measured in RQ/RQ KI mice. Therefore, the S218L mutation produces a larger increase in presynaptic Ca2+ influx and glutamate release at cortical pyramidal cell synapses than the mild R192Q FHM1 mutation. Given our evidence of a causative link between enhanced glutamate release and CSD facilitation (Tottene et al, 2009), this may explain the greater susceptibility to CSD induced by the S218L mutation, and possibly its dramatic clinical phenotype. I also found that cortical excitatory transmission in both FHM1 KI mice was less susceptible to presynaptic inhibition by activation of G protein-coupled GABAB receptors. In fact, the fraction of the EPSC inhibited by the GABAB receptor agonist baclofen was lower in SL/WT KI and RQ/RQ KI mice than in wild-type (WT) mice. As a consequence, excitatory neurotransmission was further facilitated in the presence of baclofen. Heterozygous S218L KI and homozygous R192Q KI mice showed a similar reduction in presynaptic inhibition. The data suggest that hyperactivity of cortical circuits due to both enhanced CaV2.1-dependent glutamate release and reduced presynaptic inhibition of glutamate release during G protein-coupled neuromodulation may render the cortex of FHM patients vulnerable to CSD ignition in response to migraine triggers. Another aim of my PhD project was to investigate the mechanism underlying the different effect of the R192Q FHM1 mutation on excitatory and inhibitory cortical synaptic transmission found in Tottene et al. (2009). I investigated inhibitory autaptic neurotransmission in single cortical multipolar fast-spiking interneurons grown on glial microislands from WT and RQ/RQ KI mice. The average amplitude of the AP-evoked inhibitory postsynaptic current (IPSC) was similar in multipolar interneurons of WT and RQ/RQ KI mice, despite a dominant role of P/Q Ca2+ channels in controlling GABA release at these synapses. I found that AP-evoked Ca2+ influx nearly saturates the presynaptic Ca2+ sensor at multipolar interneuron autapses in WT mice. However, the unaltered IPSC amplitude in RQ/RQ KI mice is not due to saturation of the Ca2+ sensor, because a similar IPSC at WT and RQ/RQ KI autapses was found also at low external Ca2+ concentrations. Indeed, I found a similar Ca2+ dependence of the IPSC at WT and RQ/RQ KI multipolar interneuron autapses. These findings suggest that the unaltered cortical inhibitory neurotransmission in RQ/RQ KI mice is largely due to the lack of significant increase of action potential-evoked Ca2+ influx through mutant presynaptic P/Q-type Ca2+ channels at multipolar interneuron synapse, possibly as a consequence of the short action potential and/or the expression of a splice variant of the CaV2.1 a1 subunit little affected by the R192Q mutation in FS interneurons. The unaltered AP-evoked Ca2+ influx through mutant presynaptic CaV2.1 channels at multipolar FS interneuron synapses is probably a common effect of all FHM1 mutations, as I also found unaltered inhibitory synaptic transmission at multipolar FS interneuron autapses in heterozygous S218L KI mice.L’emicrania emiplegica familiare di tipo 1 (FHM1), un raro sottotipo di emicrania con aura, è causata da mutazioni missense nel gene umano CACNA1A che codifica per la subunità α1 dei canali del calcio CaV2.1 (tipo P/Q) (Ophoff et al., 1996). Il mal di testa e i sintomi neurologici dell’aura che caratterizzano i tipici attacchi di FHM1 sono simili a quelli delle forme comuni di emicrania, eccetto per il sintomo dell’emiparesi (Pietrobon, 2007; Pietrobon and Striessnig, 2003). I canali CaV2.1 sono espressi nei terminali presinaptici e nelle membrane somatodendritiche di tutti i neuroni del cervello, dove svolgono un ruolo fondamentale nel controllo del rilascio di neurotrasmettitore. Le mutazioni FHM1 determinano un guadagno di funzione della corrente Ca2+ dei canali ricombinanti umani CaV2.1; in particolare causano un aumento dell’influsso di Ca2+ a livello di singolo canale in un ampio intervallo di potenziali vicini alla soglia di attivazione del canale, dovuto a un’aumentata probabilità d’apertura del canale, causata per lo più dallo spostamento della curva di attivazione del canale verso potenziali più negativi (Hans et al., 1999; Tottene et al., 2002, 2005; Pietrobon, unpublished data). In accordo con tali risultati, i topi omozigoti knock-in (KI) recanti la mutazione FHM1 R192Q (RQ/RQ) presentano, in granuli di cervelletto e in cellule piramidali corticali, un aumento della densità di corrente Ca2+ di tipo P/Q (Tottene et al., 2009; van den Maagdenberg et al., 2004). Questi topi mostrano, inoltre, una facilitazione dell’induzione e propagazione, in vivo, della cortical spreading depression (CSD: il fenomeno neurologico che causa l’aura e il possibile trigger del mal di testa emicranico) (Pietrobon, 2005, 2007; van den Maagdenberg et al., 2004). Per capire i meccanismi che determinano la facilitazione della CSD nei topi KI, in laboratorio abbiamo recentemente studiato la trasmissione sinaptica eccitatoria in neuroni corticali in microcoltura e in neuroni piramidali e interneuroni multipolari fast-spiking (FS) connessi tra loro sinapticamente in fettine acute talamo-corticali di topi KI RQ/RQ (Tottene et al., 2009). Lo studio della neurotrasmissione in microcolture di neuroni corticali ha rappresentato parte del mio progetto di Dottorato. I risultati hanno dimostrato un guadagno di funzione della neurotrasmissione eccitatoria dovuto a un aumentato influsso di Ca2+ evocato da potenziale d’azione, attraverso i canali del Ca2+ presinaptici di tipo P/Q, e a un’aumentata probabilità di rilascio di glutammato alle sinapsi delle cellule piramidali. Inoltre, usando un modello in vitro di CSD, abbiamo dimostrato una correlazione causale tra l’aumentato rilascio di glutammato alle sinapsi delle cellule piramidali e la facilitazione sperimentale della CSD nei topi KI RQ/RQ. Abbiamo costatato che la neurotrasmissione inibitoria alle sinapsi tra gli interneuroni multipolari FS e le cellule piramidali nelle fettine talamo-corticali di topi KI RQ/RQ era invece inalterata, sebbene la trasmissione sinaptica fosse controllata dai canali del Ca2+ di tipo P/Q (Tottene et al., 2009). Il suddetto effetto sinapsi-specifico delle mutazioni FHM1 consolida la visione dell’emicrania come un disordine episodico dell’eccitabilità cerebrale. Infatti, la distruzione del bilancio tra eccitazione e inibizione e la conseguente iperattività dei circuiti neuronali possono costituire, in risposta a specifici triggers, la causa degli episodi di suscettibilità all’innesco della CSD nell’emicrania. Mentre nei pazienti la mutazione R192Q causa tipici attacchi di FHM, la mutazione S218L causa una grave sindrome di emicrania emiplegica associata a convulsioni epilettiche, coma e grave edema cerebrale, dovuti spesso a traumi alla testa di lieve entità (Kors et al., 2001). La mutazione S218L, rispetto alla mutazione R192Q, determina un maggior spostamento verso potenziali più negativi della curva di attivazione dei canali umani ricombinanti CaV2.1 (Tottene et al., 2002, 2005). Inoltre, in accordo con il suddetto effetto, determina un maggior guadagno di funzione dell’influsso di Ca2+ nei neuroni a potenziali negativi e una maggiore facilitazione dell’induzione e propagazione della CSD in vivo nei topi omozigoti FHM1 KI S218L (SL/SL) rispetto ai topi KI RQ/RQ (van den Maagdenberg et al., 2004, 2010). L’effetto della mutazione S218L sulla corrente CaV2.1 Ca2+ neuronale e sulla facilitazione dell’induzione e propagazione della CSD risulta essere dipendente dal dosaggio allelico. Al fine di investigare i meccanismi che determinano la maggior facilitazione della CSD in topi KI SL/SL rispetto ai topi KI RQ/RQ, ho studiato la trasmissione sinaptica glutamatergica in neuroni piramidali corticali di topi eterozigoti KI S218L (SL/WT) cresciuti su microisole di cellule gliali in modo da formare sinapsi su se stessi (autapsi). Ho trovato un guadagno di funzione della neurotrasmissione corticale eccitatoria dovuto a un aumentato influsso di Ca2+ evocato da potenziale d’azione, attraverso i canali del Ca2+ presinaptici di tipo P/Q e a un’aumentata probabilità di rilascio di glutammato alle sinapsi delle cellule piramidali corticali dei topi KI SL/WT. Infatti, l’ampiezza della corrente postsinaptica eccitatoria evocata (EPSC) e il contributo dei canali Ca2+ di tipo P/Q alla trasmissione sinaptica erano entrambi aumentati alle sinapsi dei neuroni piramidali corticali in microcoltura. Inoltre ho dimostrato che la saturazione del sensore del Ca2+ avveniva a minor concentrazioni esterne di Ca2+ e che la paired pulse ratio (PPR) era diminuita. I cambiamenti nell’ampiezza dell’EPSC, nella Ca2+ dipendenza dell’EPSC e nella PPR, trovati nei topi KI SL/WT erano quantitativamente simili a quelli rilevati nei topi KI RQ/RQ. La mutazione S218L determina quindi un aumento maggiore dell’influsso di Ca2+ presinaptico e del rilascio di glutammato alle sinapsi corticali delle cellule piramidali rispetto alla mutazione R192Q che causa un fenotipo lieve di FHM1. Vista la correlazione causale tra l’aumentato rilascio di glutammato e la facilitazione sperimentale della CSD (Tottene et al., 2009), questo risultato potrebbe spiegare la maggior suscettibilità alla CSD indotta dalla mutazione S218L e il suo fenotipo grave. I dati hanno anche dimostrato che la trasmissione corticale eccitatoria in entrambi i topi FHM1 KI era meno suscettibile all’inibizione presinaptica conseguente all’attivazione dei recettori GABAB accoppiati a proteine G. Infatti, la frazione dell’EPSC inibito dal baclofen, agonista dei recettori GABAB, era minore nei topi KI SL/WT e KI RQ/RQ rispetto ai topi selvatici (WT) e, conseguentemente, la neurotrasmissione eccitatoria era ulteriormente facilitata in presenza di modulazione. I topi eterozigoti KI S218L e omozigoti KI R192Q presentavano una riduzione dell’inibizione presinaptica simile. I dati suggeriscono che l’iperattività dei circuiti corticali dovuta sia all’aumentato rilascio di glutammato dipendente dai canali CaV2.1, sia alla ridotta inibizione presinaptica del rilascio di glutammato durante neuromodulazione (attraverso l’attivazione di proteine G) possono rendere la corteccia dei pazienti FHM vulnerabile all’innesco della CSD in risposta a triggers emicranici. Un altro scopo del mio progetto di Dottorato consisteva nel determinare il meccanismo che causa l’effetto diverso della mutazione FHM1 R192Q sulla trasmissione sinaptica corticale eccitatoria e inibitoria, come descritto in Tottene et al. (2009). Ho studiato la neurotrasmissione inibitoria autaptica in singoli interneuroni corticali multipolari fast-spiking cresciuti su microisole di cellule gliali da topi WT e topi KI RQ/RQ. L’ampiezza media della corrente postsinaptica inibitoria (IPSC) evocata da potenziale d’azione era simile negli interneuroni multipolari dei topi WT e KI RQ/RQ, nonostante l’importante ruolo svolto dai canali Ca2+ di tipo P/Q nel controllo del rilascio di GABA a queste sinapsi. Ho trovato che il sensore del Ca2+ presinaptico, alle autapsi degli interneuroni multipolari nei topi WT, viene quasi saturato dall’influsso di Ca2+ evocato da potenziale d’azione. La mancanza del guadagno di funzione della neurotrasmissione inibitoria nei topi RQ/RQ KI non è comunque causata dalla saturazione del sensore Ca2+, poiché l’ampiezza dell’IPSC in interneuroni di topi WT e KI RQ/RQ era simile anche a basse concentrazioni di Ca2+ esterno. Ho costatato, infatti, una simile dipendenza dell’IPSC dalle concentrazioni esterne di Ca2+ alle autapsi degli interneuroni multipolari WT e KI RQ/RQ. Tali risultati suggeriscono che l’inalterata neurotrasmissione corticale inibitoria nei topi KI RQ/RQ sia dovuta soprattutto ad un aumento non significativo dell’influsso di Ca2+, evocato da potenziale d’azione attraverso i canali presinaptici CaV2.1 alle sinapsi degli interneuroni multipolari fast-spiking. Una possibile causa potrebbe essere la forma più corta del potenziale d’azione degli interneuroni FS e/o l’espressione, in questi interneuroni, di una variante di splicing della subunità α1 CaV2.1 poco influenzata dalla mutazione R192Q. L’inalterato influsso di Ca2+ evocato da potenziale d’azione attraverso i canali mutati presinaptici CaV2.1, alle sinapsi degli interneuroni multipolari FS, è probabilmente un effetto comune a tutte le mutazioni FHM1, poiché ho trovato inalterata anche la trasmissione sinaptica inibitoria alle autapsi degli interneuroni corticali multipolari FS nei topi eterozigoti KI S218L

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 Ca(V)2.1 (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 fastspiking (FS) intemeurons and pyramidal cells is unaltered, despite being initiated by Ca(V)2.1 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 intemeurons 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 RI 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 Ca(V)2.1 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 Ca(V)2.1 channels in cortical pyramidal cells. Our findings suggest that expression of specific Ca(V)2.1 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

Abnormal cortical synaptic transmission in Ca(V)2.1 knockin mice with the S218L missense mutation which causes a severe familial hemiplegic migraine syndrome in humans

Familial hemiplegic migraine type 1 (FHM1) is caused by gain-of-function mutations in Ca(V)2.1 (P/Q-type) Ca2+ channels. Knockin (KI) mice carrying the FHM1 R192Q missense mutation show enhanced cortical excitatory synaptic transmission at pyramidal cell synapses but unaltered cortical inhibitory neurotransmission at fast-spiking interneuron synapses. Enhanced cortical glutamate release was shown to cause the facilitation of cortical spreading depression (CSD) in R192Q KI mice. It, however, remains unknown how other FHM1 mutations affect cortical synaptic transmission. Here, we studied neurotransmission in cortical neurons in microculture from KI mice carrying the S218L mutation, which causes a severe FHM syndrome in humans and an allele dosage dependent facilitation of experimental CSD in KI mice, which is larger than that caused by the R192Q mutation. We show gain-of-function of excitatory neurotransmission, due to increased action-potential evoked Ca2+ influx and increased probability of glutamate release at pyramidal cell synapses, but unaltered inhibitory neurotransmission at multipolar interneuron synapses in S218L KI mice. In contrast with the larger gain-of-function of neuronal Ca(V)2.1 current in homozygous than heterozygous S218L KI mice, the gain-of-function of evoked glutamate release, the paired-pulse ratio and the Ca2+ dependence of the excitatory postsynaptic current were similar in homozygous and heterozygous S218L KI mice, suggesting compensatory changes in the homozygous mice. Furthermore, we reveal a unique feature of S218L KI cortical synapses which is the presence of a fraction of mutant Ca(V)2.1 channels being open at resting potential. Our data suggest that, while the gain-of-function of evoked glutamate release may explain the facilitation of CSD in heterozygous S218L KI mice, the further facilitation of CSD in homozygous S218L KI mice is due to other Ca(V)2.1-dependent mechanisms, that likely include Ca2+ influx at voltages sub-threshold for action potential generation

Enhanced excitatory transmission at cortical synapses as the basis for facilitated spreading depression in CaV2.1 knockin migraine mice

Migraine is a common disabling brain disorder. A subtype of migraine with aura (familial hemiplegic migraine type 1: FHM1) is caused by mutations in Ca(V)2.1 (P/Q-type) Ca(2+) channels. Knockin mice carrying a FHM1 mutation show increased neuronal P/Q-type current and facilitation of induction and propagation of cortical spreading depression (CSD), the phenomenon that underlies migraine aura and may activate migraine headache mechanisms. We studied cortical neurotransmission in neuronal microcultures and brain slices of FHM1 mice. We show gain of function of excitatory neurotransmission due to increased action-potential-evoked Ca(2+) influx and increased probability of glutamate release at pyramidal cell synapses but unaltered inhibitory neurotransmission at fast-spiking interneuron synapses. Using an in vitro model of CSD, we show a causative link between enhanced glutamate release and CSD facilitation. The synapse-specific effect of FHM1 mutations points to disruption of excitation-inhibition balance and neuronal hyperactivity as the basis for episodic vulnerability to CSD ignition in migraine

Temporal sharpening of sensory responses by layer V in the mouse primary somatosensory cortex

The timing of stimulus-evoked spikes encodes information about sensory stimuli. Here we studied the neural circuits controlling this process in the mouse primary somatosensory cortex. We found that brief optogenetic activation of layer V pyramidal cells just after whisker deflection modulated the membrane potential of neurons and interrupted their long-latency whisker responses, increasing their accuracy in encoding whisker deflection time. In contrast, optogenetic inhibition of layer V during either passive whisker deflection or active whisking decreased accuracy in encoding stimulus or touch time, respectively. Suppression of layer V pyramidal cells increased reaction times in a texture discrimination task. Moreover, two-color optogenetic experiments revealed that cortical inhibition was efficiently recruited by layer V stimulation and that it mainly involved activation of parvalbumin-positive rather than somatostatin-positive interneurons. Layer V thus performs behaviorally relevant temporal sharpening of sensory responses through circuit-specific recruitment of cortical inhibition