Anesthetics and antidepressants affect brain function by modifying the coordinated activity of both neurons and glia. Ketamine was initially used as an anesthetic and is also known to induce psychosis, but because of its rapid antidepressant actions, it is of great interest for better understanding depression and developing novel antidepressants (Zunszain et al . 2013 ). Unlike classical antidepressants, ketamine exerts fast as well as sustained effects (Henderson 2016 ), indicative of a fundamentally different mechanism of action that may acutely alter synaptic efficacy (Kavalali and Monteggia 2015 ). A well‐described molecular mechanism of the antidepressant action of ketamine is the blockade of NMDA receptors (Autry et al . 2011 ). However, as ketamine readily permeates membranes and can accumulate within cells, evidence suggests that ketamine also acts intracellularly and, as a weak base, is expected to accumulate in acidic vesicles (Lester et al . 2015 ). In addition, ketamine affects presynaptic exo‐/endocytotic machinery by altering soluble NSF‐attachment protein receptor complex, synaptotagmin‐1, syntaxin‐1A (Muller et al . 2013 ), synapsin‐1 (Akinfiresoye and Tizabi 2013 ; Muller et al . 2013 ), vesicle‐fusing‐ATPase, synapsin‐1, and syndapin‐1 protein levels in the brain (Wesseling et al . 2014 ). Moreover, ketamine suppresses Ca 2+ transients in astrocytes (Thrane et al . 2012 ; Stenovec et al . 2015 ) and inhibits exocytotic release from astrocytic vesicles (Stenovec et al . 2015 ), indicating additional, non‐neuronal mechanisms of action. Considering the vesicle‐associated actions of ketamine and astrocytes as cellular targets for antidepressants (Czeh and Di Benedetto 2013 ) including ketamine (Mitterauer 2012 ; Stenovec et al . 2015 ), we investigated the influence of ketamine on single vesicle interactions with the plasma membrane in cultured rat astrocytes.

Events were analyzed with the CellAn program (Celica) in MATLAB (MathWorks, Natick, MA, USA). An event was defined as a step in Im where signal‐to‐noise ratio was at least 3 : 1 and no concomitant projection to the current trace was observed. An event was considered irreversible if a step in Im was not followed by a step of similar amplitude (± 15%) in the opposite direction within 15 s; whereas an event was considered reversible if an on‐step was followed by an off‐step of similar amplitude within 15 s. Reversible events following in succession were termed bursts. Each burst was composed of at least four successive steps of similar amplitude and alternating direction that followed their predecessor within 15 s. Individual events in bursts were detected by a custom written MATLAB program that identified individual steps consisting of a steep Im increase with a slope of 0.16 fF/ms, lasting at least 10–20 ms; all detected events were validated by visual re‐examination.

Vesicle capacitance ( C v ) was calculated: C v = [( Re 2 + Im 2 )/ Im ]/ω; where ω denotes angular frequency (ω = 2π f ) (Lindau 1991 ). As C m is proportional to the plasma membrane area, the vesicle surface area and thus diameter ( d ) can be determined (assuming spherical geometry): C v = C spec πd 2 ; where C spec is specific membrane capacitance (10 fF/μm 2 ) (Trachtenberg et al . 1972 ). Reversible events in Im that exhibited measurable projections to Re were used to calculate fusion pore conductance: G p = ( Re 2 + Im 2 )/ Re , from which fusion pore diameter was calculated: G p = (πr 2 )/(ρλ); ρ is estimated saline resistivity (100 Ωcm), and r and λ estimated fusion pore radius and length (15 nm) (Spruce et al . 1990 ).

Discrete steps in membrane capacitance ( C m ) were recorded using the compensated cell‐attached patch‐clamp technique with a dual‐phase lock‐in patch‐clamp amplifier (SWAM CELL, Celica, Ljubljana, Slovenia; Rituper et al . 2013 ) with fire‐polished glass pipettes of 1.5–4 MΩ coated with Sylgard ® 184. The cell membrane was voltage‐clamped at a holding potential (0 mV) to which a sine wave voltage (111 mV r.m.s.) was applied at a frequency ( f ) of 6400 Hz. The phase setting of the lock‐in amplifier was adjusted to nullify changes in the real ( Re ) part of the admittance signal in response to 10 fF calibration steps (triggered manually every ~ 15 s) in the imaginary ( Im ) part (Neher and Marty 1982 ; Jorgacevski et al . 2010 ). Signals were low‐pass filtered at 100 Hz for the Re and Im signals and 10 Hz for the membrane current (4‐pole Bessel filter, −3 dB) and digitized at an acquisition rate of 200 Hz.

Primary astrocyte cultures were prepared from the cortex of 2–3‐day‐old Wistar female rats (Medical Experimental Centre of the Institute of Pathology, University of Ljubljana, Slovenia) as described (Schwartz and Wilson 1992 ); all animal procedures were in accordance with International Guiding Principles for Biomedical Research Involving Animals developed by the Council for International Organizations of Medical Sciences and Animal Protection Act (Official Gazette of the RS 38/13). Cells were maintained in high‐glucose Dulbecco's modified Eagle's medium (Invitrogen) with 10% fetal bovine serum, 1 mM sodium pyruvate, 2 mM l‐glutamine, and 25 μg/mL penicillin/streptomycin in 5% CO 2 /humidified air (95%) atmosphere at 37°C. Cells were plated onto poly‐l‐lysine‐coated coverslips and experiments were performed after 1–3 days. Ketamine hydrochloride (KTM; Tocris Bioscience, Bristol, UK) was added to the culture medium 30 min or 24 h prior to electrophysiological measurements to reach a final concentration of 25 μM, or 30 min prior to reach final concentrations of 0.025, 0.25, 2.5, or 25 μM.

The increase in burst occurrence is correlated with a decrease in irreversible off‐steps. (a) A schematic representation of an irreversible off‐step (full fission) and (b) a burst of off‐ and on‐steps (closing and opening of the fusion pore) observed in C m recordings ( below ). (c) Irreversible off‐step in Im in a control cell. (d) Burst of reversible steps starting with an off‐step in Im in a ketamine (KTM)‐treated cell. *Denote calibration pulses. (e‐j) The correlation between the percentage of bursts and other event types within each cell plotted for increasing 25 μM KTM incubation periods (e, g, i) or [KTM] (30 min) (f, h, j). Controls are depicted in white followed by increasing KTM incubation periods (acute application, 30 min, 24 h incubations) or [KTM] (0.025, 0.25, 2.5, 25 μM) represented in incrementing gray scale intensity. Linear functions ( black lines ) fit to data in e): irreversible off‐steps ( circles ): y = 0.74–0.79 x ( R = 0.93), g) irreversible on‐steps ( squares ): y = 0.13–0.13 x ( R = 0.48), (i) reversible solitary events ( triangles ): y = 0.13–0.08 x ( R = 0.38), f): irreversible off‐steps: y = 0.71–0.72 x ( R = 0.99), h) irreversible on‐steps: y = 0.15–0.18 x ( R = 0.87), j) reversible solitary events: y = 0.13–0.10 x ( R = 0.52). In both (e, f) the regression line slopes for irreversible off‐steps are significantly different ( p < 0.001, one‐way ancova ) from the slopes of irreversible on‐steps (g, h) and reversible solitary events (i, j). * p < 0.05,** p < 0.01,*** p < 0.001.

A possible intermediate state between a transient fusion pore and full fusion/fission (Fig. 4 a) is likely represented by opening and closing of the fusion pore (Fig. 4 b). To gain insights into the occurrence of bursts, the proportions of different types of events were determined. In controls, irreversible off‐steps were regularly observed (Fig. 4 c), whereas KTM reduced their occurrence. In KTM‐treated cells, bursts that started with a reversible off‐step (Fig. 4 d), indicative of incomplete fission, were very frequently observed. The proportions of different events (irreversible on‐steps, irreversible off‐steps, and reversible solitary events; see Fig. 1 ) were correlated with the proportion of bursts to examine whether bursts occurred at the expense of other events. All plotted data were fit with the linear function: y = y 0 + a × x , where a denotes the slope and x the relative burst proportion. The apparent Pearson correlation coefficient ( R ) between irreversible on‐steps or reversible solitary events and bursts was relatively poor; after longer incubations ( R = 0.48, Fig. 4 g; R = 0.38; Fig. 4 i) and increasing [KTM] ( R = 0.87, Fig. 4 h; R = 0.52; Fig. 4 j) with insignificant slopes ( p > 0.05). In contrast, the correlation between the decrease in irreversible off‐steps and increase in bursts was high in cells exposed to KTM for longer periods ( R = 0.93; Fig. 4 e) and increasing [KTM] ( R = 0.99; Fig. 4 f). The proportion of bursts increased from 5 ± 2% in controls to a maximum of 49 ± 6% ( p < 0.001) with longer incubations (Fig. 4 e), and to 51 ± 6% ( p < 0.001) with increasing [KTM] (Fig. 4 f). Correspondingly, the proportion of irreversible off‐steps decreased from 68 ± 5% in controls to a minimum of 33 ± 5% ( p < 0.001) with longer incubations (Fig. 4 e), and to 31 ± 5% ( p < 0.001) with increasing [KTM] (Fig. 4 f). Moreover, the slopes of irreversible off‐steps versus bursts were significantly different ( p < 0.001) for both increasing incubation periods and [KTM] (Fig. 4 e and f) compared with irreversible on‐steps (Fig. 4 g and h) or reversible solitary events (Fig 4 i and j). These data suggest that the KTM‐dependent increase in bursts is occurring at the expense of irreversible off‐steps, indicating that KTM stabilizes the fusion pore in a state that is unlikely to proceed to full fission (Fig. 4 b).

Ketamine (KTM)‐evoked burst occurrence increases in a concentration‐dependent manner. (a) The relative proportion of cells exhibiting bursts (black) and the burst frequency per cell (b) increase with higher [KTM]. The duration of bursts (c) and number of events per burst (e) exhibit a similar [KTM]‐dependency, with a significant increase at 2.5 μM KTM. The probability of a cell being engaged in bursting activity (d) and the frequency of reversible events per cell (f) increase with higher [KTM]. Numbers below the box plots denote the number of cells (a, b, d, f) or bursts (c, e) analyzed. * p < 0.05,** p < 0.01,*** p < 0.001.

A maximum in KTM‐evoked burst occurrence was observed already after 30 min (Fig. 2 d), thus experiments addressing KTM dose‐dependency were performed with this incubation period. In control conditions, 19% of cells exhibited bursts, which increased to 33%, 47%, 82%, and 95% with increasing [KTM] (Fig. 3 a). The frequency of bursts per cell also increased as a function of increasing [KTM] (Fig. 3 b). The median duration of bursts in controls was 19 s (4, 51) and increased significantly only after treatment with 2.5 μM KTM to 91 s (21, 319) ( p < 0.05) (Fig. 3 c). Similarly, the number of events per burst was significantly larger only after treatment with 2.5 μM KTM; with a median of 319 (123, 1649) ( p < 0.01) compared to 11 (3, 233) in controls (Fig. 3 e). Both the probability of observing bursts (Fig. 3 d) and the frequency of reversible events per cell (Fig. 3 f) drastically increased with increasing [KTM] and reached peaks at 2.5 μM KTM; these values were not significantly different from values obtained with 25 μM KTM, indicating that the KTM‐evoked effect plateaued at ~ 2.5 μM, an already subanesthetic dose (Tassonyi et al . 2002 ).

Ketamine (KTM)‐evoked burst occurrence increases with longer incubation periods. (a) The relative proportion of cells exhibiting bursts (black) and the burst frequency per cell (b) increase with longer 25 μM KTM incubations. Longer KTM incubations do not affect the duration of individual bursts (c) or the number of events per burst (e). (d) Burst probability (fraction of time a cell was engaged in bursting activity during a recording) and the frequency of reversible events per cell (f) increase with longer KTM incubations. The increase is already notable upon acute KTM application. Numbers below the box plots denote the number of cells (a, b, d, f) or bursts (c, e) analyzed.* p < 0.05,*** p < 0.001.

In controls, 19% of cells exhibited at least one burst. Following prolonged KTM incubations (acute, 30 min, 24 h), the percentage of burst‐exhibiting cells increased to 73%, 93% and 92%, respectively (Fig. 2 a). Upon acute KTM application, the mean time necessary for the onset of a burst was 8.8 ± 4.2 min ( n = 8), with a minimal time of 45.5 s. The frequency of bursts increased after acute KTM treatment and the increase became statistically significant after 30 min and 24 h incubations ( p < 0.001) (Fig. 2 b). Prolonged KTM treatment did not affect the duration of bursts (Fig. 2 c) or the number of reversible events per burst (Fig. 2 e). However, KTM significantly increased the probability of burst occurrence, which was calculated as the fraction of time a cell was engaged in bursting activity during a recording (Fig. 2 d). The frequency of reversible events per cell increased significantly already after acute KTM application as well as after 30 min or 24 h incubations (Fig. 2 f). These results demonstrate that the effects of ketamine on the fusion pore are immediate and persist also after 24 h, indicating their fast‐acting and long‐lasting action.

For reversible events exhibiting a projection to the Re trace, G p (fusion pore conductance) can be estimated (Spruce et al . 1990 ). KTM decreased G p during bursts [Fig. 1 f ( left ), Fig. 1 g ( left )]. In controls, the median G p was 75 (Q1:32, Q2:215) pS and decreased to 53 (33, 65), 48 (37, 69), and 40 (33, 61) pS ( p < 0.05), after acute, 30 min, and 24 h incubations, respectively. Different [KTM] also caused a significant decrease in G p , from 75 (32, 215) pS in controls to 46 (30, 74), 37 (24, 52) ( p < 0.05), 56 (41, 78), and 48 (37, 69) pS with increasing [KTM]. In contrast, the pore conductance of solitary reversible events was not altered by KTM [Fig. 1 f ( right ), Fig. 1 g ( right )], suggesting a different mechanism intrinsic to reversible events in bursts compared to solitary reversible events (see Discussion ).

The amplitude of discrete C m steps reflects the size of vesicles engaging in fusion pore activity. The median vesicle diameter was not affected by increasing incubation periods (acute, 30 min, 24 h) with 25 μM KTM (Fig. 1 e, left ) nor KTM concentrations ([KTM], 0.025–25 μM), incubated for 30 min (Fig. 1 e, right ), indicating that KTM does not influence the size of vesicles exhibiting fusion pore activity.

Discrete C m steps observed in cultured rat astrocytes. (a) Irreversible on‐ ( left ) and off‐step ( right ) in Im representing full vesicle fusion with and fission from the plasma membrane. (b) Solitary reversible event representing transient fusion pore opening without ( left ) or with ( right ) a projection to Re, indicating formation of a relatively wide ( left ) or narrow ( right ) fusion pore. C v and G p ( inset ) calculated from the event amplitudes ( framed ). (c) Reversible events in bursts observed in a control cell ( left ) and ketamine (KTM)‐treated cell ( right ). Reversible events either exhibit ( left ) or do not exhibit ( right ) projections to Re . (d) An entire burst evoked by KTM that exhibits no concomitant projections to the patch current trace ( I DC ). Changes in I DC are not projected to Im ( right ) *Denote calibration pulses that are not projected to Re or I DC . (e) Superimposed frequency distributions and box plots ( inset ) of vesicle diameters, measured in cells exposed to increasing 25 μM KTM incubations ( left ) or [KTM] (30 min) ( right ). Note that the median size of vesicles exhibiting fusion pore activity is not affected by KTM. (f and g) KTM decreases G p of reversible events in bursts ( left ), but not of reversible solitary events ( right ), indicating that KTM narrows fusion pore diameter during bursts. Numbers at box plots represent the number of bursts or solitary events analyzed. * p < 0.05.

Capacitance measurements revealed several distinct types of events. Irreversible events consisted of on‐steps indicating full fusion or off‐steps indicating full fission (Fig. 1 a); solitary reversible events consisted of a fusion pore opening, which was followed by its closing within 15 s (Fig. 1 b); and bursts of reversible events consisted of at least four successive on‐ and off‐steps indicating repetitive reopening of the fusion pore (Fig. 1 c and d). On average, 1462 ± 187 steps/burst ( n = 303) were observed in control and KTM‐treated cells.

High‐resolution capacitance measurements were performed with which fusion pore activity of single vesicles was studied on cells from 15 different primary astrocyte cultures. Out of the 177 cells observed, 168 exhibited discrete on‐ and/or off‐steps, representing unitary exocytotic and endocytotic events, respectively, and were included in the analysis, with a total recording time of 47.8 h and average time of 17.1 ± 0.8 min/recording.

Discussion

This study is the first to reveal a novel effect of ketamine on fusion pore activity. Ketamine caused vesicles to lapse into a state of repetitive fusion pore opening and closing. Although similar bursting activity has been observed under different conditions before (Lollike et al. 1998; Henkel et al. 2000; Thiel et al. 2009; Bandmann et al. 2010; Jorgacevski et al. 2010; Calejo et al. 2013), the detailed mechanisms governing this process are poorly understood. It is believed that repetitive fusion pore activity indicates that the fusion pore is energetically favorable and thus a stable structure (Jorgacevski et al. 2010). Unlike solitary reversible fusion events, ketamine‐evoked reversible events in bursts were characterized by low conductance, narrower fusion pores, consistent with recent findings that ketamine inhibits peptide release from single vesicles in astrocytes (Stenovec et al. 2015), possibly because of the release unproductive narrow fusion pore configuration. It is possible that ketamine directly affects the structure of the fusion pore, as evidence suggests that it accumulates inside acidic vesicles in the protonated form (Lester et al. 2015) and may therefore electrostatically change the local anisotropicity of the fusion pore (Kabaso et al. 2011).

Electrostatic interactions can affect the transitions between discrete fusion pore states (Calejo et al. 2012; Gucek et al. 2015), and repetitive pore reopening is likely driven by cation concentration changes in microdomains proximal to the fused vesicle (Kabaso et al. 2011). Electrostatic interactions resulting from altered cation currents across the membrane may affect the fusion pore via NMDA channels, molecular targets of ketamine that are expressed in astrocytes (Lalo et al. 2006; Lee et al. 2010). Although ketamine is commonly known as an NMDAR antagonist, ketamine also targets hyperpolarization‐activated cyclic nucleotide‐gated (HCN1) channels that are implicated in its hypnotic actions (Chen et al. 2009) and as therapeutic targets for depressive disorders (Shah 2012). Astrocytes express HCN1 channels (Rusnakova et al. 2013; Honsa et al. 2014) and changes in local cation concentrations mediated by HCN channels, activated by cAMP, play a role in modulating the fusion pore (Calejo et al. 2014). Also cAMP has been shown to stabilize an intermediate stage where the vesicle fusion pore transiently opens, thus increasing the probability of its rhythmic reopening (Calejo et al. 2013).

Ketamine‐evoked bursts, in which an initial downward step started an episode of alternating on‐ and off‐steps, likely represent a single vesicle fused with the plasma membrane that repeatedly failed to break its membranous connection. Such flickering of the ‘fission pore’ also termed ‘frustrated endocytosis’ has been observed before (Henkel et al. 2000; Thiel et al. 2009) and was hypothesized to result from processes involving dynamin and the action of protein kinase (PK) inhibitors that influence steps in endo‐/exocytosis via protein phosphorylation (Henkel et al. 2000). PKC‐mediated phosphorylation was shown to affect the fusion pore via multiple phosphorylation sites (Scepek et al. 1998) and ketamine is associated with mechanisms involving PKC inhibition (Tomioka et al. 2009) as well as PKC phosphorylation or AMP‐activated PK activation in relation to antidepressant activity (Reus et al. 2011; Xu et al. 2013).

Experimental evidence indicates that the kinetics and closure of the endocytotic fission pore are Ca2+‐ and dynamin‐dependent (Cabeza et al. 2010). Exocytosis and endocytosis are coupled processes (Ales et al. 1999; Wu et al. 2013) that have been shown to share the same proteins, and ‘rapid’ Ca2+‐dependent endocytosis is the prevalent mode of vesicular recapture and recycling that could be mediated by de‐/phosphorylated dynamin‐1 (Artalejo et al. 2002). Dynamin has been demonstrated to also play a role in the exocytotic fusion pore and influence transient kiss‐and‐run events (Jackson et al. 2015). It has been additionally suggested that a loose soluble NSF‐attachment protein receptor complex is likely to be reversible and to provide a possible substrate for kiss‐and‐run events (Smith et al. 2008). Nevertheless, the exact mechanism(s) underlying fast endocytosis remain unclear, even though fusion/retrieval mechanisms are of great interest because of their implications in synaptic plasticity and pathophysiology (Kavalali 2006; Smith et al. 2008).

Astrocytes are part of the tripartite synapse and actively take part in regulating synaptic transmission and plasticity by vesicle‐based communication (Santello et al. 2012; De Pitta et al. 2016; Zorec et al. 2016). Distinct gliosignal‐containing vesicles with various sizes have been reported in rat astrocytes (Gucek et al. 2016). In this study, vesicles with diameters ranging from 34 to 425 nm were observed, and the median diameter of 125 nm corresponds well to the mean diameter of 127 nm observed in small LysoTracker‐labeled acidic vesicles (Gucek et al. 2016). 75% of vesicles in this study had diameters < 165 nm, suggesting that predominantly amino‐acid/peptide‐containing vesicles were observed and to a lesser extent larger ATP‐containing vesicles (~ 200 nm) (Gucek et al. 2016). In addition to vesicles undergoing kiss‐and‐run exocytosis (reversible events), we obtained indications for the involvement of less well‐characterized endocytotic vesicles in ketamine‐evoked flickering activity. As astrocytes influence numerous synapses within the neural network (Covelo and Araque 2016) via reuptake and release of neuro‐/glio‐transmitters, they are also implicated in the treatment of major depression (Etievant et al. 2013). The ketamine‐induced bursts observed in this study demonstrate an effect on exo‐/endocytosis of astrocytic vesicles, thus possibly affecting gliotransmission of neuroactive substances and retention of surface proteins such as ion channels, membrane receptors, and transporters (Zorec et al. 2016).

In summary, this study has demonstrated that subanesthetic doses (Tassonyi et al. 2002) of ketamine (0.25–2.5 μM) exert potent effects on fusion pore activity by stabilizing the fusion pore in a narrow configuration favoring bursts and decreasing full fission events. Ketamine‐elicited flickering activity of a narrow fusion pore suggests a release unproductive state of the vesicle and a role in vesicle recycling. The observed effects of ketamine on the fusion pore occurred almost immediately, were long‐lived, and thus warrant further research to test the hypothesis that stabilization of vesicle fusion pores may reset neural‐glia networks and represent a key mechanism in the behavioral effects of ketamine.