CaSR modulates sodium channel-mediated Ca2+-dependent excitability

Increasing extracellular [Ca2+] ([Ca2+]o) strongly decreases intrinsic excitability in neurons but the mechanism is unclear. By one hypothesis, [Ca2+]o screens surface charge reducing voltage-dependent sodium channel (VGSC) activation and by another [Ca2+]o activates Calcium-sensing receptor (CaSR) closing the sodium-leak channel (NALCN). Here we report that action potential (AP) firing rates increased in wild-type (WT), but not CaSR null mutant (Casr-/-) neocortical neurons, following the switch from physiological to reduced Ca2+-containing Tyrode. However, after membrane potential correction, AP firing increased similarly in both genotypes inconsistent with CaSR regulation of NALCN. Activation of VGSCs was the dominant contributor to the increase in excitability after the [Ca2+]o change. VGSC conductance-voltage relationships were hyperpolarized by decreasing [Ca2+]o for Casr-/- neurons indicating CaSR contributes to [Ca2+]o-dependent excitability via VGSCs. Regulation of VGSC gating by [Ca2+]o is the key mechanism mediating [Ca2+]o-dependent changes in neocortical neuron excitability and CaSR influences neuronal excitability by its effects on VGSC gating.

Furthermore under pathological conditions, larger decreases in [Ca 2+ ]o occur, resulting in even greater changes in circuit activity, and implicating [Ca 2+ ]o-dependent excitability in the pathogenesis of brain injury (Ayata and Lauritzen, 2015).
Classical studies proposed that the mechanism underlying [Ca 2+ ]o-dependent excitability centers on voltage-gated sodium channel (VGSC) sensitivity to extracellular Ca 2+ . External Ca 2+ was proposed to interact with local negative charges on the extracellular face of the membrane or ion channels thereby increasing the potential field experienced by VGSCs and reducing the likelihood of VGSC activation at the resting membrane potential (Frankenhaeuser and Hodgkin, 1957;Hille, 1968). Reductions in [Ca 2+ ]o decrease the screening of the surface potential, reverse the membrane stabilization and facilitate VGSC activation (Frankenhaeuser and Hodgkin, 1957;Hille, 1968). This surface potential screening model accounted for [Ca 2+ ]odependent excitability in nerves and muscle without a need for additional molecular players and was widely accepted (Hille, 2001). However, the theory was challenged by new data demonstrating that activation of the sodium leak channel (NALCN), a non-selective cation channel, by the intracellular proteins, UNC79 and UNC80 (Lu et al., 2009;Lu et al., 2010) was necessary for [Ca 2+ ]o-dependent excitability to occur in hippocampal neurons. Following the deletion of NALCN or UNC79, [Ca 2+ ]o-dependent excitability was completely lost suggesting the increased excitability resulted from the activation of the non-rectifying NALCN which depolarized neurons and increased the likelihood of action potential generation independent of changes in VGSC function (Lu et al., 2010). The calcium-sensing receptor (CaSR), a G-protein coupled receptor (GPCR), was hypothesized to detect and transduce the [Ca 2+ ]o changes and signal to the downstream multistep pathway (Lu et al., 2010). CaSR is well-positioned as a candidate [Ca 2+ ]o detector because at nerve terminals it detects [Ca 2+ ]o and regulates a non-selective cation channel (Smith et al., 2004;Chen et al., 2010) and because it transduces changes in [Ca 2+ ]o into NALCN activity following heterologous co-expression of CaSR, NALCN, UNC79, and UNC80 (Lu et al., 2010). Interest in the UNC79-UNC80-NALCN pathway has also risen, due to its essential role in the maintenance of respiration (Lu et al., 2007), the regulation of circadian rhythms (Lear et al., 2013;Flourakis et al., 2015), and because mutations of UNC80 and NALCN cause neurodevelopmental disorders, characterized by development delay and hypotonia (Al-Sayed et al., 2013;Perez et al., 2016).
Here we address the question of whether the G-protein mediated NALCN pathway or VGSCs transduce the [Ca 2+ ]o-dependent effects on excitability. We test if CaSR is a modulator of neuronal excitability via its action on a nonselective cation channel, determine the impact of CaSR expression on factors of intrinsic neuronal excitability, and examine the relative contributions of [Ca 2+ ]o-regulated changes on VGSC and NALCN gating. In recordings from neocortical neurons, isolated by pharmacological block of excitatory and inhibitory inputs, we determine that neuronal firing is increased by decreasing external divalent concentrations and that this is almost entirely attributable to [Ca 2+ ]o-dependent shifts in VGSC gating. Surprisingly, CaSR deletion substantially shifted VGSC gating, but had no effect on NALCN sensitivity to [Ca 2+ ]o. Taken together our experiments indicate that acute [Ca 2+ ]o-dependent increases in neuronal excitability result from changes in VGSC and NALCN gating and that CaSR contributes by an, as yet, uncharacterized action on VGSCs.

CaSR and [Ca 2+ ]o-dependent neuronal excitability
The elimination of [Ca 2+ ]o-dependent excitability in neurons by deletion of UNC79 or NALCN challenged the long-standing hypothesis that local or diffuse surface charge screening of VGSCs mediated these effects (Lu et al., 2010). But how were changes in external divalent ion concentrations transduced to UNC79 and NALCN? We tested if CaSR provided the link, by comparing neuronal excitability in wild-type (WT) and nestin cre-recombinase expressing CaSR null-mutant (creCasr -/-) neurons that were genotyped by PCR (see Methods) (Chang et al., 2008). Quantification by RT-qPCR indicated >98% reduction in the Casr expression levels in neocortical cultures produced from creCasr -/mice compared to cre-positive WT (creWT; Figure   1-supplement). Current clamp recordings were performed to measure the intrinsic spontaneous action potential firing rate from pharmacologically-isolated, cultured, neocortical neurons (glutamatergic and GABAergic activity blocked by 10 µM CNQX, 50 µM APV, and 10 µM Gabazine respectively). After establishing the whole-cell configuration, we measured the action potential firing rates of conventional WT (conWT), creWT, and creCasr -/neurons in increase in spontaneous action potential firing in both WT (con-and cre-) neurons within 15 seconds of the solution change that was substantially attenuated in the creCasr -/neuron ( Figure   1A, middle row). Changing back to physiological external divalent concentrations reversed this effect within 10 s ( Figure 1A,

CaSR-independent mechanisms contribute to [Ca 2+ ]o-dependent signaling
If CaSR-regulated NALCN-dependent depolarization is entirely responsible for the extracellular divalent-sensitive changes in neuronal excitability (Lu et al., 2010) Figure 1E; P=0.0075 and =0.156, respectively). Similarly, in the reciprocal experiment in which the membrane potential in was depolarized to match that at low divalent concentration, the decrease in external divalent concentration increased action potential frequency ( Figure 1F, Suppl. Table 3; F (1,35)=15.17, P=0.0004, 2-way RM ANOVA), and this was significant in creWT but not creCasr -/neurons ( Figure 1F, P = 0.004). Ineffective matching of the membrane potential following solution changes did not account for the persistence of [Ca 2+ ]o-dependent excitability (insets, Figure 1E,F). These data indicate a mechanism in addition to CaSR-NALCN-mediated depolarization must contribute to the extracellular divalent-sensitive changes in neuronal excitability.

Does CaSR modulate RMP and [Ca2+]o-dependent depolarization?
We tested if there was an endogenous difference in the excitability of creWT and creCasr -/neurons to explain the retained sensitivity to divalents by measuring the action potential threshold. Action potentials were elicited in and T0.2 using minimal current injection (50-250 pA) and the threshold measured as the point at which dV/dt reached 20 mV/ms ( Figure 1G, membrane potential-corrected as in Figure 1 E). The action potential threshold was hyperpolarized from -48.6 ± 0.7 mV to -54.3 ± 1.1 mV with the switch from to T0.2 in creWT neurons ( Figure 1H) which would have increased excitability. However, the same effect was observed in creCasr -/neurons (-50.9 ± 0.86 mV to -55.4 ± 2.1 mV; F (1,27)=56.48, P < 0.0001, 2-way RM ANOVA, Suppl. Table 4). The lack of effect of CaSR on AP threshold (F (1, 27) = 2.284, P = 0.142) in these experiments, indicated the reduced divalent sensitivity of creCasr -/-( Figure 1E,F) was not simply due to altered action potential threshold.
We next asked if differences in RMP in and the response of RMP to T0.2 could contribute to the different divalent-dependent excitability of creWT and creCasr -/neurons.
Overall these data support the idea that CaSR played a substantial role in mediating divalent dependent changes in excitability, but that WT neurons also possessed CaSR-independent mechanisms to fully account for the [Ca 2+ ]o-dependent excitability. After establishing a stable current-clamp recording in we injected a standing current (Ia) until the resting membrane potential was -70 mV. We then recorded for 50 s before switching the bath solution to T0.2. As before, there was a small depolarization followed by an increase in action potential frequency in creWT neurons ( Figure   2A,B). To test if this increase in excitability was fully attributable to [Ca 2+ ]o-dependent depolarization we adjusted the standing current (Ib) until the membrane potential was -70 mV and then measured the action potential frequency ( Figure 2C). In the exemplar, AP firing was reduced by the hyperpolarization but remained higher in T0.2 at -70 mV than in at -70 mV ( Figure 2A-C) confirming CaSR-mediated depolarization was not acting alone to increase the excitability (c.f. Figure 1I). The creCasr -/neurons responded similarly to T0.2 and hyperpolarization ( Figure 2A-C) indicating the effect was not mediated by CaSR. We compared the average effects of at -70 mV with Ia, T0.2 with Ia, and T0.2 at -70 mV with Ib on creWT and creCasr -/genotypes ( Figure 2D, Suppl. Table 6) using a 2-way RM ANOVA. Extracellular divalent concentration and membrane potential substantially affected action potential frequency (F (3, 87) = 17.97, P < 0.0001). CaSR deletion did not impact the response to extracellular divalent concentration when creWT and creCasr -/neuron recordings were started at a membrane potential of -70 mV l (F (1, 29) = 0.2005, P = 0.6577). Post-hoc testing showed that excitability was increased in T0.2 compared with regardless which of the two holding currents were used ( Figure 2D; Suppl. Table 7). The depolarization of Casr -/neurons in response to T0.2 was unexpected ( Figure 1I) since initially CaSR-NALCN signaling was hypothesized as the mechanism for [Ca 2+ ]o-dependent excitability (Lu et al., 2010). After injection of Ia to set the membrane potential to -70 mV, the switch from to T0.2 significantly depolarized the membrane potential ( Figure 2E; Suppl. Table 8, Two-way RM ANOVA, F (1, 29) = 29.22, P < 0.0001) as did CaSR deletion (F (1, 29) = 4.874, P = 0.0353). Post-hoc testing indicate that the membrane potential in T0.2 was more depolarized in the creCasr -/than in creWT neurons ( Figure 2B,E; -65.6 ±1.6 mV vs -59.4 ± 2.4 mV, P = 0.0083). Taken together, these experiments indicate CaSR-NALCN signaling was not contributing to the difference in [Ca 2+ ]o-dependent excitability between creWT and creCasr -/neurons but that these differences may be due to genotypedependent differences in RMP or intrinsic excitability. ]o-dependent excitability by examining action potential threshold in neurons held at a membrane potential of -70 mV. AP threshold was hyperpolarized by 8 mV on average following the change from to T0.2 in creWT and creCasr -/neurons ( Figure 2F,G, Suppl. Table 9A; F (1, 25) = 51.66, P< 0.0001). Furthermore, the AP threshold was relatively depolarized in the creCasr -/neurons in and T0.2 (5.3 ± 2.0 mV (P = 0.020) and 5.5 ± 2.0 mV (P = 0.017) respectively), indicating creWT neurons possessed increased excitability and increased sensitivity to decreases in external divalent concentration ( Figure 2F,G). The action potential half-width recorded under the same conditions, was also sensitive to deletion of CaSR and reduction of [Ca 2+ ]o ( Figure 2H,I Suppl. Table 9B). ANOVA indicated that the switch to T0.2 from broadened AP half-width (F (1,28) = 19.7, P = 0.0001) but this effect was restricted to creWT (P = 0.0004) and not creCasr-/-(P = 0.117) neurons. The genotype and [Ca 2+ ]o interacted to both affect AP peak voltage ( Figure 2I, Suppl. Table 9C; F (1, 28) = 6.76, P = 0.015) with the peak potential being reduced by T0.2 in the creWT (P < 0.0001) but not creCasr-/-neurons (P = 0.34).

Voltage-gated sodium channels contribute to [Ca 2+ ]o-dependent excitability
We examined the properties of VGSCs and voltage-gated potassium channels (VGPCs) to determine the reason for the altered AP threshold. VGSCs were isolated in neocortical neurons and the current-voltage characteristics examined. Families of VGSC currents were activated in neurons after 2-4 weeks in culture. Maximum VGSC currents were elicited at -30 mV and averaged -8.0 ± 0.8 nA (n=7) and -8.8 ± 2.8 nA (n=6) in creWT and creCasr -/neurons respectively. The current-voltage curve shifted in a hyperpolarizing direction with the switch from to T0.2 but extensive neuronal processes limited the quality of the voltage-clamp and prevented useful analysis. We next examined VGSC gating in nucleated outside-out patches (Sather et al., 1992;Almog et al., 2018) to ensure better voltage control. VGSC currents were elicited by voltage steps from -80 mV (10 mV increments to 40 mV). In T0.2, the VGSC inactivation (see below) resulted in smaller currents that were more sensitive to depolarization (bold traces elicited by steps to -50 mV, Figure 3A) as previously observed (Frankenhaeuser and Hodgkin, 1957;Campbell and Hille, 1976;Armstrong and Cota, 1991). [Ca 2+ ]o sensitivity was confirmed in the normalized current-voltage plot for both creWT (blue, n= 8) and creCasr -/-(red, n= 11) neurons ( Figure 3A,C). VGSC current inactivation was studied using a test pulse to -20 mV, each of which was preceded by a conditioning step (100 ms) to between -140 mV and -20 mV.

VGSCs are the dominant contributor to [Ca 2+ ]o-dependent currents
To compare the contributions of VGSCs and NALCN to the [Ca 2+ ]o-dependent depolarization seen in neocortical neurons ( Figure 2) we measured the size of the currents elicited at -70 mV in neurons following the switch from to T0.2. We used conWT neurons to avoid potential confounding Cre-dependent effects (Qiu et al., 2011). Since NALCN is resistant to the VGSC blocker tetrodotoxin (TTX) (Lu et al., 2007;Swayne et al., 2009) but Gd 3+ (10 µM) inhibits NALCN and VGSCs (Elinder and Arhem, 1994;Li and Baumgarten, 2001;Lu et al., 2009), we were able to pharmacologically separate the contributions of VGSCs and NALCN to the basal current following the switch from to T0.2 (-31 ± 3 pA, n=13; Figure Figure 5G). However, the Gd 3+ -sensitive current reversed at -60 mV and outward currents were elicited by steps to -60 and -50 mV that exhibited a voltagedependent activation and inactivation ( Figure 5H, right-hand). This is consistent with the Gd 3+sensitive current consisting of the sum of NALCN and an outward voltage-dependent current.
Assuming conservatively that all of the Gd 3+ -sensitive current at -100 mV could be attributed to NALCN and employing the channel's linear voltage-dependence and zero mV reversal potential (Lu et al., 2007;Lu et al., 2010), then the amplitude of NALCN currents could be estimated over the voltage range -100 to 0 mV (broken black line, Figure 5G

VGSCs are the dominant contributor to [Ca 2+ ]o-dependent excitability
The complex architecture of neocortical neurons restricted our ability to clamp the membrane potential following the activation of large, rapid VGSC currents. Thus we re-examined the contribution of VGSCs and NALCN to the depolarizations that mediate [Ca 2+ ]o-dependent excitability in current clamp recordings from conWT neurons. Consistent with earlier experiments (Figure 2), switching from to T0.2 depolarized the membrane potential from -70 mV by 7.2 ± 1.5 mV (n = 12) and increased spontaneous action potential firing in pharmacologically isolated neurons ( Figure 6A Table 15) and more sensitive to TTX than Gd 3+ or following co-application (P = 0.022 and 0.0028 respectively, Supp.  Figure 6C) and we observed a similar pattern in Casr -/neurons ( Figure 6C).  (Figures 5,6).
The fractions of the [Ca 2+ ]o-dependent currents and depolarizations that were sensitive to TTX were surprisingly large compared to those that were Gd 3+ -sensitive ( Figures 5D,6C) indicating the relative importance of VGSC-and NALCN-mediated contributions to [Ca 2+ ]odependent excitability respectively. The resistance of NALCN to TTX (Lu et al., 2007;Swayne et al., 2009) reassures that the relatively large TTX-sensitive component is due to selective block of VGSC. Persistent subthreshold VGSC currents have been shown to determine spiking rates in other central neurons (Taddese and Bean, 2002;Gorelova and Seamans, 2015) and so the increased VGSC currents we observed in T0.2 are well-positioned to explain the increased action potential frequency ( Figure 6). We are unable to determine from these experiments which neuronal compartment is most affected by the change in [Ca 2+ ]o (Gorelova and Seamans, 2015).
However, the physiological impact of VGSC-mediated [Ca 2+ ]o-dependent excitability will be enormous overall because of the dynamic nature of [Ca 2+ ]o in vivo where it decreases from basal (1.1-1.2 mM) by 30-80% (Nicholson et al., 1978;Ohta et al., 1997;Pietrobon and Moskowitz, 2014). The overall computational effects of physiological decrements in [Ca 2+ ]o will be complex because the increased action potential generation due to changes on VGSCs ( Figure 3,6) will be confounded by the impact of reduced Ca 2+ entry through VACCs (Hess et al., 1986;Weber et al., 2010), reduced synaptic transmission (Neher and Sakaba, 2008), and altered CaSR-mediated signaling at the nerve terminal (Phillips et al., 2008;Vyleta and Smith, 2011).  (Figure 2) whereas the response to current injections was the measure of excitability in hippocampal neurons (Lu et al., 2010) could the differences reflect different experimental protocols. We cannot exclude this possibility however, it is unclear how NALCN facilitated acute [Ca 2+ ]o-dependent excitability during depolarizing injections from matched membrane potentials since the current injections were expected to produce similar or smaller depolarizations given NALCN is relatively voltage insensitive (Lu et al., 2009). Could the increased action potential number seen in WT compared to NALCN deficient neurons (Lu et al., 2010) reflect other indirect actions of a persistent leak current (Sokolov et al., 2007) or compensatory changes in ion channel activity (Jun et al., 1999)? It is also surprising that deletion of NALCN or UNC-79 completely ablated [Ca 2+ ]o-dependent excitability in hippocampal neurons (Lu et al., 2010) (Philippart and Khaliq, 2018) but GPCRs other than CaSR may be involved (Kubo et al., 1998;Tabata and Kano, 2004). Further characterization of the UNC79-UNC80-NALCN signaling pathway is essential given the major changes in neurological function that have been described following mutations of NALCN or upstream co-molecules such as UNC79 and UNC80 (Stray-Pedersen et al., 2016;Bourque et al., 2018;Kuptanon et al., 2019).
In a small fraction of the neocortical neurons (Figures 5,6) there was a modest inward current or depolarization with the application of low calcium once VGSCs had been blocked. In a few cases they were sensitive to 10 µM Gd 3+ consistent with a NALCN-mediated effect and  (Figures 2 and 6) it was clear that Casr -/neurons were substantially less sensitive to changes [Ca 2+ ]o ( Figure 1A,B). The reduced [Ca 2+ ]o sensitivity in these neurons is attributable to the combination of altered VGSC gating ( Figure   3E,F) and the hyperpolarized RMP ( Figure 1I). Although CaSR did not affect the amplitude of the shift in V0.5 following the switch to T0.2, V0.5 in was hyperpolarized by loss of CaSR ( Figure   3EF). Could CaSR stimulation activate G-proteins and regulate the basal V0.5 for VGSC currents ( Figure 3E,F)? In neocortical neurons, G-protein activation hyperpolarized VGSC gating and this was blocked by GDPβS (Mattheisen et al., 2018) which is inconsistent with the effect we observed here. Other possible explanations are that CaSR regulates VGSC subunit expression or post translational modification (Cantrell et al., 1996;Zhang et al., 2019). Loss of CaSR could hyperpolarize the neocortical neurons ( Figure 1I) by increasing the function of depolarizing components or inhibiting hyperpolarizing elements, but it is unclear which of the many ion channels or pumps mediate this effect on RMP (Tavalin et al., 1997;Talley et al., 2001;Bean, 2007;Harnett et al., 2015;Hu and Bean, 2018).
Overall our studies indicate that [Ca 2+ ]o-dependent excitability in neurons is largely attributable to actions of extracellular calcium on the VGSC function. Given the dynamic nature of brain extracellular calcium, this mechanism is likely to impact neuronal signaling greatly under physiological and pathological conditions. CaSR-dependent reduction of VGSC sensitivity to membrane potential adds further complexity to extracellular calcium signaling and identifies another potential mechanism by which CaSR stimulation increases neuronal death following stroke and traumatic brain injury (Kim et al., 2013;Hannan et al., 2018).

Genotyping and CaSR mutant mice
ConWT animals were obtained from a 20 year old colony consisting of a stable strain of C57BL/6J and 129x1 mice. The creCasr-/-mice were generated by breeding floxed Casr (Chang et al., 2008) and nestin Cre mice (B6.Cg-Tg (Nes-cre)1Kln/J, The Jackson Laboratory) as described previously (Sun et al., 2018). The lox sites were positioned to delete Casr exon 7 which resulted in the loss of Casr expression (Chang et al., 2008) and the nestin promoter was designed to ensure floxing occurred in neuronal and glial precursors. The creWT mice were generated by crossing mice that did not contain the flox Casr mutation but did express the nestin Cre mutation. The creCasr-/-and creWT mice were all generated using a background C57BL/6J and 129S4 strain. Tail DNA extraction was performed using the Hot Shot Technique with a 1-2 hour boil (Montero-Pau et al., 2008). The presence or absence of the flox Casr mutation and Cre mutation were confirmed by PCR for each mouse. MoPrimers used for cre PCR were: Nes-Cre 1: GCAAAACAGGCTCTAGCGTTCG, Nes-Cre 2: CTGTTTCACTATCCAGGTTACGG; run on a 1% agarose gel. Primers for lox PCR were: P3U: TGTGACGGAAAACATACTGC, Lox R: GCGTTTTTAGAGGGAAGCAG; run on a 1.5% agarose gel (Chang et al., 2008). Successful deletion of Casr in the neocortical cultures was confirmed by measuring mRNA expression levels with the QuantStudio12K Flex Real-time PCR System (Applied Biosystems)and the TaqMan mouse probe set to Casr (Mm00443377_m1) with ActB (Mm04394036_g1) as the endogenous control (Supplementary Figure 1).

Neuronal Cell Culture
Neocortical neurons were isolated from postnatal day 1-2 mouse pups of either sex as described previously (Phillips et al., 2008). All animal procedures were approved by V.A.
Portland Health Care System Institutional Animal Care and Use Committee in accordance with the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animals were decapitated following induction of general anesthesia with isoflurane and then the cerebral cortices were removed. Cortices were incubated in trypsin and DNase and then dissociated with a heat polished pipette. Dissociated cells were cultured in MEM plus 5% FBS on glass coverslips. Cytosine arabinoside (4 µM) was added 48 hours after plating to limit glial division.
Cells were used, unless otherwise stated after ≥14days in culture.

Electrophysiological Recordings
Cells were visualized with a Zeiss IM 35 inverted microscope. Whole-cell voltage-and currentclamp recordings were made from cultured neocortical neurons using a HEKA EPC10 USB amplifier. Except where stated in the text, extracellular Tyrodes solution contained (mM) 150 NaCl, 4 KCl, 10 HEPES, 10 glucose, 1.1 MgCl2, 1.1 CaCl2, pH 7.35 with NaOH. Calcium and magnesium were modified as described in the Figure legends Synaptic transmission was blocked by the addition of (in µM) 10 CNQX, 10 Gabazine, and 50 APV to the bath solution.

Data Acquisition and Analysis
Whole-cell voltage-and current-clamp recordings were made using a HEKA EPC10 USB amplifier, filtered at 2.9 kHz using a Bessel filter, and sampled at 20 kHz during injection protocols and 10 kHz during continuous acquisition. Analysis was performed using Igor Pro (Wavemetrics, Lake Oswego, OR) and Minianalysis (Synaptosoft). Data values are reported as mean ± SEM. Statistical tests were performed using GraphPad Prism (6) and P-values < 0.05, 0.01, 0.001, and 0.0001 were indicate with *, **, ***, and ****. All post-hoc tests were Sidak compensated for multiple comparisons. Data were log-transformed to improve normalization in Figure 2D. To ensure non-zero values, minimize bias, and allow logarithmic transformation, each AP frequency measurement was increased by 0.02 as the duration of the recording at -70 mV was 50 s.

Solution Application
Solutions were applied by gravity from a glass capillary (1.2 mm outer diameter) placed ~1 mm from the neuron under study. Solutions were switched manually using a low dead volume manifold upstream of the glass capillary. CNQX and Gabazine were supplied by Abcam. KB-R7943 Mesylate was supplied by Tocris. Creatine Phosphate was supplied by Santa Cruz

Biotech. Cinacalcet was supplied by Toronto Research Chemicals and Tetrodotoxin by Alomone
Other reagents were obtained from Sigma-Aldrich. Inset shows average membrane potential after the current injection.   Table 9C).      Each solution applied to conWT (n=12) and creCasr-/-(n = 9) neurons.