The loose-patch clamp method was first introduced to study surface membrane ionic currents in relatively large-diameter amphibian skeletal muscle (SkM) fibers (Stühmer et al., 1983). It used pipettes with relatively large tip diameters, compared to those used in conventional patch clamp, on native in situ muscle fibers without requiring enzyme pre-treatment or membrane rupture. The larger pipette areas meant that the pipettes did not form the tight seal made by conventional patch clamps electrodes with the cell membrane. This permits significant current to pass through the lower resulting seal resistances between the membrane and pipette tip. The latter is compensated for by using specific recording and current delivery electronics distinct from those used in conventional tight patch clamping. This can deliver larger compensation currents. Nevertheless, this approach permits repeated electrode applications and withdrawals. Measurements can be made from the same or successive membrane patches on the same cell. The procedure avoids membrane disruption, potentially altering intracellular content. These advantages have proven valuable in investigations of channel localization (Almers et al., 1983) and lateral mobility (Stühmer and Almers, 1982; Roberts et al., 1986). This reversibility of the loose-patch seal additionally allows pair-wise comparisons from the same patch (Stühmer et al., 1983) before and following experimental maneuvers involving the application or withdrawal of pharmacological or osmotic agents (Chin et al., 2004). This overcomes problems of variability between patches or cells (Liu et al., 2021). It also permits studies in successively different cells using the same electrode with consistent geometric and electrical properties (Milton and Caldwell, 1990; Roberts and Almers, 1992). These advantages have been used in extensions of this technique in murine cardiac muscle (Valli et al., 2018a; Ahmad et al., 2019).

The latter advantages proved useful in pharmacological studies of the feedback effects of ryanodine receptor, RyR1- or RyR2-mediated intracellular store Ca²⁺ release upon skeletal or cardiac muscle Na⁺ channel, Nav1.4 or Nav1.5, function in intact in situ native loose-patch clamped murine skeletal and cardiac myocytes. These led to discoveries of mechanisms by which the Ca²⁺ signaling initiated by feedforward, excitation–contraction coupling exerts feedback effects on Nav excitability (Salvage et al., 2021; Salvage et al., 2023).

Such studies investigated the effects of RyR-specific agonists and antagonists expected to alter rates of Ca²⁺ release from their sarcoplasmic reticular (SR) stores relative to background release levels. These properties were demonstrated by spectrofluometric cytosolic [Ca²⁺]_i measurements in resting mammalian murine skeletal muscle SR or fiber preparations (Fryer and Neering, 1989; Pagala and Taylor, 1998). Here, the direct RyR modulator caffeine (Herrmann-Frank et al., 1999) at 0.5 mM persistently (3–10 min) increased [Ca²⁺]_i (to ∼300 nM) from its resting concentrations (typically ∼106 ± 2 nM: (Fryer and Neering, 1989; Head, 1993; Dettbarn et al., 1994). Contrastingly, higher, ≥1.0 mM, caffeine challenge induced early [Ca²⁺]_i peaks followed by persistent below-resting [Ca²⁺]_i reductions within 80–90 s (Dettbarn et al., 1994; Pagala and Taylor, 1998). The latter was attributed to Ca²⁺-induced sarcoplasmic reticular Ca²⁺ transporter (SERCA) activation, followed by a sustained slow (∼seconds) RyR inactivation, reducing channel open probabilities. This inactivation property was independently demonstrated in RyR1s reconstituted in lipid bilayers exposed to steady [Ca²⁺]_i at levels similarly initially activating RyR (∼10–100 µM) (Laver and Curtis, 1996). The same 0.5 mM and 2 mM caffeine concentrations, respectively, decreased and increased peak inward current. Conversely, the RyR1 inhibitor (Zhao et al., 2001) and muscle-relaxant (Ellis et al., 1973) dantrolene (10 μM) and sarcoplasmic reticular (SR) Ca²⁺ depletion produced by the SERCA inhibitor cyclopiazonic acid (CPA; 10 μM) (Matthews et al., 2019; Sarbjit-Singh et al., 2020; Liu et al., 2021) increased peak inward current and abrogated caffeine’s effects.

These findings identified caffeine as increasing and dantrolene and CPA as decreasing RyR1-mediated SR store Ca²⁺ release into a T-SR Ca²⁺ domain (Bardsley et al., 2021) with corresponding effects on Nav activation. Concordant findings emerged in reports from murine atrial and ventricular cardiomyocytes (Valli et al., 2018a; Ahmad et al., 2019). These findings were consistent with molecular structural data bearing on Ca²⁺ action, either direct or through Ca²⁺-calmodulin, in the region of the Nav1.4 or Nav1.5 C-terminal domains (Salvage et al., 2021).

Intracellular Ca²⁺ signaling also operates in central nervous system (CNS) neurons, modulating diverse physiological processes, particularly cell excitability (Berridge, 1998). In hippocampal cornu ammonis-1 (CA1) pyramidal neurons, it is implicated in age-related cognitive decline through altered synaptic plasticity and neuronal excitability (Foster, 2007; Kumar et al., 2009; Burke and Barnes, 2010; Oh et al., 2010), impaired neurogenesis in experimental Alzheimer’s disease models (Mattson and Chan, 2003), and toxic effects, potentially leading to cell death at high concentrations (Stanika et al., 2012). In common with striated muscle, neurons maintain low background intracellular [Ca²⁺]_i ∼100 nM levels (Xu et al., 2014) through cytosolic exchanges with the extracellular environment and intracellular, mitochondrial, and endoplasmic reticular (ER) reservoirs. In striated SkM or cardiac muscle, Nav1.4 or Nav1.5-mediated action potential generation (Adrian and Peachey, 1973) increases [Ca²⁺]_i through RyR1- or RyR2-mediated intracellular SR Ca²⁺ store release. This involves direct, allosteric, or extracellular Ca²⁺ entry-dependent coupling of transitions in the tubular membrane, voltage-sensing, or Ca²⁺-conducting Cav1.1 or Cav1.2 channels, to the activation of RyR1 or RyR2 SR Ca²⁺ release channels, respectively (Huang et al., 2011). These interactions occur at triad or dyad junctions (Martin et al., 2003) between surface tubular and SR membranes (Chawla et al., 2001) that may form Ca²⁺ domains within their enclosed T-SR space, enclosing both RyR and Nav1.4 or 1.5 (Bardsley et al., 2021). The resulting elevated bulk [Ca²⁺]_i then initiates muscle contraction. It also exerts negative feedback actions on its initiating RyR-mediated Ca²⁺ release (Pagala and Taylor, 1998). SERCA1 or SERCA2 and plasma membrane Ca²⁺ transporters (PMCA) then return the released Ca²⁺ into the ER for sequestration by high concentrations of specialized low-affinity, high-capacity buffer molecules, such as calsequestrin, or to the extracellular space (Clapham, 2007). This restores the baseline low cytosolic [Ca²⁺]_i. Surface membrane excitation is finally terminated by voltage-gated SkM Kv1 and/or cardiac muscle Kv4 K⁺ channel action.

Hippocampal pyramidal CA1 neurons similarly possess surface voltage-gated Na⁺, Ca²⁺, and K⁺, and intracellular Ca2⁺ release RyR channels. They also show endoplasmic reticular (ER)–plasma membrane (PM) junctions (EPJs) with clustered and functionally coupled L-type Ca²⁺ (LTCCs) and RyR channels. However, differing channel subtypes are involved. Of surface membrane ion channels, CA1 pyramidal neurons express Nav 1.1, 1.2, and 1.6 amongst Na⁺ channels (Wang et al., 2012), and Cav1.3, Cav2.3, and Cav3.x, activated by different magnitudes of membrane depolarization, amongst Ca²⁺ channels (Armstrong and Matteson, 1985). Among intracellular RyR-Ca²⁺ release channels, anti-RyR antibody labeling methods (Furuichi et al., 1994; Giannini et al., 1995) indicated expression of all three established RyR1-3 subtypes (McPherson and Campbell, 1993) in neuronal cells. More recent studies in RyR3^−/− mouse CA1 cells suggested important functional roles of Ca²⁺-induced Ca²⁺ release by highly expressed RyR3 in potentiating slow afterhyperpolarizing current, sIAHP (Tedoldi et al., 2020). In addition, Cav1.3–RyR3 interactions are promoted by Kv2.1 K⁺ channels present within the resulting Kv2.1–LTCC–RyR triads (Johnson et al., 2018; Vierra et al., 2019). Recently, in a mouse model expressing green fluorescent protein (GFP)-tagged RyR2, a specific GFP probe demonstrated high RyR2 expression in soma and dendrites, but not dendritic spines or presynaptic terminals, of CA1 pyramidal neurons or dentate gyrus granular neurons (Hiess et al., 2022).

Among K⁺ channels, the delayed rectifier Kv2.1, in addition to modifying neuronal excitability (Martina et al., 1998) to extents dependent on metabolic state (Misonou et al., 2005), enhances LTCC opening at polarized membrane potentials. Conversely, Cav3.1 and Cav1.2 channels were implicated in modulating Kv4.2 in hippocampal dendrites (Anderson et al., 2010; Murphy et al., 2022). Finally, their parallel increases and decreases in amplitude in intact neurons could be a basis for a dependence of transient outward I _K upon entry of extracellular Na⁺ (Marrero and Lemos, 2003). The latter findings parallel features of Na⁺-dependent K⁺ current activation in a different K⁺ channel subtype (Bhattacharjee and Kaczmarek, 2005; Hage and Salkoff, 2012). In this connection, the RyR antagonist dantrolene was recently proposed as a novel treatment for Alzheimer’s disease (Liang and Wei, 2015).

These similarities and differences in muscle and neuronal interactions between surface and intracellular membranes predict corresponding functional similarities and differences between them. Furthermore, native CA1 neurons in hippocampal slices may be amenable to study by similar pharmacological and biophysical methods as skeletal muscle. Ca²⁺ imaging methods had similarly demonstrated, respectively, enhanced and reduced Ca²⁺ transient amplitudes following challenge by low and high caffeine concentrations, and these effects were similarly occluded by the pharmacological depletion of intracellular Ca²⁺ stores (Sandler and Barbara, 1999). We here introduce loose-patch clamp methods (Stühmer et al., 1983) to investigate corresponding effects in native in situ CA1 pyramidal neurons in hippocampal coronal slices for the first time. This approach complements conventional tight patch electrode techniques previously used in both on- and whole-cell electrophysiological single-cell current-clamp (Liu et al., 2001; Minlebaev et al., 2013; Ghasemi et al., 2018; Vazetdinova et al., 2022) or voltage-clamp studies of neuronal cells in hippocampal tissue slices (Kodirov, 2023) or following isolation (Mathias et al., 1990; Klee et al., 1995). However, on the one hand, the whole-cell conventional patch clamp configuration permitting membrane current measurement over the entire somal cell membrane involves cell membrane rupture to electrically access the intracellular environment (French et al., 1990). The dialysis of often Ca²⁺-chelating ethylene glycol-bis (β-aminoethyl ether)-N, N, N′, N′-tetra acetic acid (EGTA)-containing pipette solution into the intracellular environment could then itself perturb intracellular Ca²⁺ dynamics (Leech & Holz IV, 1994; Perkins, 2006). Furthermore, neither this nor perforated patch variants permit multiple experimental seal formations and detachments with the same pipette before and following pharmacological challenges. Finally, in one report, it was not possible to control potential during the early sodium current transient, likely due to currents generated at a distance from the soma in the axon or dendrites (French et al., 1990). On the other hand, with the small pipette diameters, on-cell techniques leave intact membrane confine readings to relatively small localized membrane areas (Magee and Johnston, 1995; Perkins, 2006).

Intracellular Ca²⁺ homeostasis has been extensively studied in connection with skeletal (SkM) and cardiac muscle excitation–contraction coupling. In this process, Nav1.4- or Nav1.5-mediated action potential generation (Adrian and Peachey, 1973) elevates [Ca²⁺]_i from the low background ∼100 nM to contractile activating levels through RyR1- or RyR2-mediated SR Ca²⁺ store release (Chawla et al., 2001). The latter involves direct allosteric or Ca²⁺-induced coupling following Cav1.1- or Cav1.2-mediated voltage-sensing or Ca²⁺-entry (Huang et al., 2011). The action was localized to potential Ca²⁺ domains within triad or dyad (T-SR) junctions between surface tubular and SR membranes (Bardsley et al., 2021).

Ca²⁺ homeostasis is similarly central to neuronal physiology. In hippocampal CA1 pyramidal neurons, it may modulate cell excitability and synaptic function clinically implicated in age-related cognitive decline (Mattson and Chan, 2003; Foster, 2007; Kumar et al., 2009; Burke and Barnes, 2010; Oh et al., 2010), extending to cell death (Stanika et al., 2012). In common with skeletal and cardiac muscles, CA1 pyramidal neurons possess voltage-gated Na⁺, Ca²⁺, and K⁺, as well as RyR-Ca²⁺ release channels and endoplasmic reticular (ER)–plasma membrane (PM) junctions (EPJs). However, they contain different Nav, Cav, Kv, and RyR subtypes. Anti-RyR antibody labeling methods (Furuichi et al., 1994; Giannini et al., 1995) suggest the expression of all three established RyR1-3 subtypes (McPherson and Campbell, 1993). Studies in RyR3^−/− mouse CA1 cells implicate highly expressed RyR3 potentiating slow afterhyperpolarizing current, sIAHP. Murine models expressing GFP-tagged RyR2 demonstrated significant expression of RyR2 in CA1 pyramidal neurons (Hiess et al., 2022). They show Cav1.3–RyR3 interactions promoted by Kv2.1 within Kv2.1-LTCC-RyR triads (Johnson et al., 2018; Vierra et al., 2019) and interactions between Cav3.1 and Cav1.2 and Kv4.2 in hippocampal dendrites and between the resulting altered intracellular Ca²⁺ and K_Ca late in action potential membrane repolarization (Verkhratsky and Shmigol, 1996; Vergara et al., 1998). Their parallel increases and decreases in amplitude suggested interactions between Na⁺ influx and transient outward I _K in intact neurons (Marrero and Lemos, 2003), resembling features reported of a further K⁺ channel subtype (Bhattacharjee and Kaczmarek, 2005; Hage and Salkoff, 2012). Finally, RyR activity could influence hippocampal neuronal firing, overall activity (Tedoldi et al., 2020), and synaptic transmission and plasticity (Johenning et al., 2015). RyR dysregulation has been implicated in long-term potentiation and depression (Maffei, 2018), key to hippocampal memory formation (Hiess et al., 2022). It has been linked to a wide range of neurological disorders, ranging from epilepsy (Lehnart et al., 2008) to Alzheimer’s disease (Chiantia et al., 2023).

Recent reports described the feedback effects of both background and evoked RyR-mediated intracellular store Ca²⁺ release upon Na⁺ channel function in intact loose-patch clamped skeletal and cardiac myocytes. These findings fulfilled predictions of structural evidence for either direct or Ca²⁺ calmodulin-mediated Ca²⁺ action on Nav C-terminal domains (Salvage et al., 2021; Salvage et al., 2023). Caffeine at 0.5 and 2 mM, respectively, decreased and increased I _Na. This was consistent with its RyR agonist action at low (0.5 mM) and ultimate RyR inhibitory effects at high (>1.0 mM) concentrations. In the latter situation, caffeine induces a rapid transient [Ca²⁺]_i increase followed by its decrease below even resting levels, attributed to a combination of an initial SERCA activation and subsequent sustained RyR inactivation (See Introduction; (Pagala and Taylor, 1998; Vogalis et al., 2001)).

The RyR blocker dantrolene and SERCA inhibitor cyclopiazonic acid then themselves increased I _Na, suggesting the effects of a reduced background Ca²⁺ on I _Na. These findings fulfilled expectations from actions on I _Na of blocking RyR-mediated Ca²⁺ release (Sarbjit-Singh et al., 2020) or of ER Ca²⁺ store depletion by SERCA pump inhibition (Seidler et al., 1989; Liu et al., 2021). Both actions would reduce the resting release of SR store Ca²⁺, increasing the background [Ca²⁺] within a T-SR Ca²⁺ domain (Bardsley et al., 2021). These agents also abrogated caffeine actions when the agents were applied in combination, consistent with common actions on SR store Ca²⁺ release (Matthews et al., 2019; Sarbjit-Singh et al., 2020; Liu et al., 2021). Together, these findings suggested the existence of inhibitory feedback effects of released Ca²⁺ upon Na⁺ channel function and that this took place within microdomains of elevated [Ca²⁺] within the T-SR junctions. Here, resting [Ca²⁺]_i was both elevated by a background RyR-mediated Ca²⁺ release and further elevated by RyR-agonists (Bardsley et al., 2021).

We here investigate parallel effects within native in situ CA1 pyramidal neurons in murine hippocampal coronal slices using similar pharmacological manipulations. Continued use of a loose-patch clamping approach permitted the study of ionic currents using successive low-resistance seals, leaving cell surface membranes intact and changes in intracellular Ca²⁺ homeostasis unperturbed before and after pharmacological challenges (Matthews et al., 2019; Sarbjit-Singh et al., 2020; Liu et al., 2021). This loose-patch clamping involves specific circuitry required to correct for the much greater leaks and consequent pipette currents. Thus, the true membrane current is much smaller than the leak current, which must be corrected before feeding into the actual patch clamp circuit. Such corrections are potentially limited by the ability of the Ag/AgCl junction for sustained current delivery through the loose-patch electrode. Nevertheless, the approach permits effective overall assessments for, and examination of, current contributions and their comparison before and following physiological interventions in intact cells without disrupting intracellular contents. Conventional high-resistance tight-patch seals can contrastingly involve membrane disruption and possible alterations in [Ca²⁺]_i by the use of pipettes containing Ca²⁺-sequestrating ethylene glycol-bis (β-aminoethyl ether)-N, N, N′, N′-tetra acetic acid with its own Ca²⁺ buffering capacity.

The loose-patch recordings demonstrated transient inward followed by transient, then prolonged outward currents in response to depolarizing steps, agreeing with previous reports with the same technique in other neuronal cell types that had I _Na and I _K in these deflections (Marrero and Lemos, 2003). As in the previous reports, Na⁺ replacement experiments abolished peak inward and reduced transient outward currents. In the latter respect, these features resembled those of outward Na⁺-activated K⁺ (Slack and Slick, KCNT1 and KCNT2) currents (Bhattacharjee and Kaczmarek, 2005; Hage and Salkoff, 2012). They did not affect prolonged outward currents. Further controls successively used both low (10 μM) and high nifedipine concentrations (100 μM), respectively, selectively or non-selectively blocking high and both high- and low-voltage-activated Ca²⁺ channel activity (Curtis and Scholfield, 2001; Perez-Reyes, 2003). Neither blocking conditions affected either inward, or transient or prolonged outward, current. Thus, the inward currents did not include detectable Cav contributions. These controls also exclude regulatory contributions attributable to any associated influxes of extracellular Ca²⁺ upon the observed inward and outward currents.

The experiments then explored the effects on voltage-gated membrane currents of manipulations directed instead at RyR-mediated release of SR store Ca²⁺, using protocols that directly paralleled the similar loose-patch clamp I _Na investigations in SkM. These findings, for the first time, suggest a similarity between hippocampal CA1 pyramidal neurons and skeletal and cardiac myocytes in that caffeine-induced increases or decreases in RyR-mediated ER Ca²⁺ release either decreased or increased inward current. Consistent with previous observations, they also demonstrate accompanying changes in transient but not prolonged outward current. Here, low (0.5 mM) RyR-agonist caffeine concentrations, expected to increase RyR-mediated store Ca²⁺ release into the cytosol, reduced both inward and transient outward currents, resembling previously reported I _Na alterations in SkM (Sarbjit-Singh et al., 2020; Liu et al., 2021), as well as previously suggested interactions between inward I _Na and transient outward I _K in intact neurons (Marrero and Lemos, 2003). The prolonged outward currents contrastingly remained unchanged. Such a mechanism could provide negative regulatory feedback on Nav activity, following excessive ER Ca²⁺ release in murine CA1 pyramidal neurons.

In other respects, results in CA1 pyramidal neurons contrasted with SkM findings. First, a higher (2 mM) caffeine concentration, instead of increasing inward current, left both inward and transient outward currents unchanged. Second, applications of the respective RyR and SERCA blockers dantrolene (10 μM) and CPA (1 μM) by themselves, in contrast to previous SkM findings (Sarbjit-Singh et al., 2020; Liu et al., 2021), altered neither inward nor outward currents. Third, both dantrolene and CPA, nevertheless, reversed the effects of caffeine on peak inward current when either of these agents was given in combination with caffeine. In agreement with previous findings in SkM, following pretreatment, either dantrolene or CPA applied in combination abrogated the effects of both 0.5 mM and 2 mM caffeine, leaving inward and outward currents indistinguishable from control results obtained in the absence of any pharmacological agent. This was consistent with all three agents acting directly or indirectly on the ER store Ca²⁺ release. In considering the possible involvement of specific RyR isoforms in these findings, recent in vitro reports provide evidence that dantrolene acts on RyR1 and RyR3 but not RyR2 (Zhao et al., 2001). However, this contrasts with its effective in vivo action with anti-arrhythmic consequences in cardiac cells, known to express RyR2 (Kobayashi et al., 2009; Kobayashi et al., 2010; Avula et al., 2018; Greco et al., 2022). Previous reports also indicate that it abrogates the effects of pharmacological challenge on I _Na (Salvage et al., 2015; Li et al., 2017; Valli et al., 2018b) and arrhythmic phenotype in intact cardiac tissue (Hartmann et al., 2017; Nofi et al., 2020). The present findings themselves also describe its abrogation of the effect of caffeine challenge in reducing I _Na. Finally, besides interactions between Ca²⁺ and delayed rectifier Kvs (Misonou et al., 2005), CA1 pyramidal cells possess intermediate-conductance Ca²⁺-dependent K⁺ (I _KCa) channels whose activation by intracellular [Ca²⁺] elevation by membrane Cav action is amplified by RyR-mediated store Ca²⁺ release (Verkhratsky and Shmigol, 1996; Vergara et al., 1998). The latter may underlie slow afterhyperpolarization (sAHP), following trains of synaptic input or postsynaptic stimuli (King et al., 2015; Tedoldi et al., 2020).

However, such sAHP strongly contributes to the prolonged outward current component, reaching maximum amplitude over longer time courses of several hundred milliseconds (Lancaster and Adams, 1986). Nevertheless, such mechanisms could complement established longer timescale, similarly Ca²⁺-related, K⁺ channel regulation in skeletal (Maqoud et al., 2017) and cardiac muscles (Yang et al., 2022; Liu et al., 2023).

Both the present and previously reported studies were made in, and were, therefore, applicable to, intact CA1 and skeletal muscle cells with unperturbed Ca²⁺ homeostatic and surface membrane mechanisms. This involved the applied investigational agents partitioning across cell surface membranes to access their respective intracellular targets. In addition, larger RyR-mediated Ca²⁺ elevations could additionally act on inhibitory RyR Ca²⁺ binding sites, giving the previously reported bimodal [Ca²⁺]_i effects (Laver and Curtis, 1996; Pagala and Taylor, 1998; Vogalis et al., 2001) and be affected by cytosolic Ca²⁺ buffering and SERCA-mediated Ca²⁺ SR reuptake. Such a scheme was corroborated by comparing results from three separate agents, namely, caffeine, dantrolene, and CPA, each directed at distinct aspects of Ca²⁺ homeostasis. Together, the findings thus suggest, for the first time, the direct effects of ER-mediated modulation of intracellular calcium on inward and transient outward surface membrane currents. They merit future explorations as to whether the present findings reflect a potential non-canonical mechanism regulating membrane excitability through actions on Nav triggering or the resulting neuronal firing rates. The loose-patch technique could also be used to investigate voltage-gated ionic currents and their modulation not only in the cell body but also in the dendrites of hippocampal neurons. This could provide information about the functional compartmentalization of Nav and Kv channel cellular functions. Finally, the technique could be extended to follow successive stages of development in animals at different age groups.