- Academic Editor
†These authors contributed equally.
Background: Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, is widely used as a general anaesthetic. However, the mechanisms of analgesic/anaesthetic effects induced by ketamine are only partially understood. Previously, studies have demonstrated that various general anaesthetics affect the primary somatosensory cortex (S1), a potential target of general anaesthetics in the central nervous system. However, it is unknown if astrocyte activities affect ketamine’s effects on information transmission in S1 pyramidal neurons. Methods: The whole-cell patch-clamp technique was employed to study the role of astrocytes in ketamine-induced anaesthetic actions. The whole-cell patch-clamp method was used to record the spontaneous postsynaptic currents (SPSCs) of rat S1 pyramidal neurons. We used the glia-selective inhibitor of the aconitase enzyme fluorocitrate (FC), to test if astrocyte activities alter the effects of ketamine on S1 pyramidal neurons. Results: Ketamine lowered the SPSCs of rat S1 pyramidal neurons in a concentration-dependent manner at clinically relevant doses. The concentration-effect curve revealed that ketamine had an EC50 value of 462.1 M for suppressing SPSCs. In rat S1 pyramidal neurons, the glia-selective metabolic inhibitor fluorocitrate (FC), which inhibits the aconitase enzyme, lowered the amplitude and frequency of SPSCs. The inhibitory impact of ketamine on the amplitude and frequency of SPSCs was significantly amplified in the presence of FC. Conclusions: Astrocytes impact the effects of ketamine on pre- and postsynaptic components and play a role in synaptic transmission.
Ketamine, a racemic combination of (S)- and (R)-ketamine, has been used in therapeutic trials since 1970. Because of its short half-life and lack of clinically significant respiratory depression, ketamine has proven to be a popular anaesthetic [1]. Ketamine has analgesic [2], anti-inflammatory [3], and antidepressant properties [4] in addition to its well-known anaesthetic action in adults, children, and obstetric patients. Numerous placebo-controlled trials have shown that ketamine, when administered intravenously (IV) at subanesthetic dosages (0.5 mg/kg over 40 min), can produce rapid (within hours), transitory antidepressant effects [5, 6, 7]. In contrast to the majority of other anaesthetics with sedative or hypnotic effects, ketamine does not predominantly operate through gamma-aminobutyric acid (GABA) receptors [8]. The direct ketamine-induced inhibition of N-methyl-D-aspartate receptors (NMDARs) is thought to be responsible for ketamine’s anaesthetic and analgesic characteristics [9]. The sensation of being aware while being drawn away from sensory perceptions can be used to describe the dissociative effects of ketamine. Because NMDARs are crucial for excitatory neurotransmission, long-term potentiation (LTP), and memory formation, a high dose of ketamine induces a condition of deep dissociation accompanied by amnesia and loss of consciousness. Notably, the rise in abuse of ketamine as a drug from the 1970s to the present shows that its psychedelic properties are also desired by recreational users [2]. GABA, dopamine, serotonin, sigma, opioid, and cholinergic receptors, as well as voltage-gated sodium and hyperpolarization-activated cyclic nucleotide-gated channels, are all potential therapeutic targets of ketamine [10, 11]. However, the underlying neurological mechanisms of unconsciousness induced by ketamine remain unknown. Previous research has suggested that the thalamocortical system is linked to the loss of consciousness caused by general anaesthetics [12, 13, 14]. The primary sensory cortex (S1) is the principal area of the brain that receives face sensory sensation information, and may play a significant role in general anaesthetic-induced unconsciousness in the central nervous system [15, 16]. In previous studies, synaptic transmission modulation has been found to be the primary mechanism of action for general anaesthetics [17, 18].
When astrocytes were first described, they were thought to serve just as a
structural framework to support and fill in the spaces between neurons. However,
evidence indicates that astrocytes protect neuronal networks in ways that go well
beyond providing nutrients or acting as a structural support system [19]. They
also play a minor role in information representation and processing. Previous
studies have demonstrated that astrocytes play a role in the physiological
control of synaptic transmission and that synapses and astrocytes communicate
bidirectionally [11, 20, 21, 22]. By using in vivo two-photon Ca
However, it is unclear whether the activity of astrocytes plays a role in the effect of ketamine on synaptic transmission. To address this issue, we investigated how ketamine affected spontaneous postsynaptic currents (SPSCs) in rat S1 pyramidal neurons before and after the astrocytes were suppressed by fluorocitrate (FC), an inhibitor of glia-selective metabolism that inhibits the aconitase enzyme and has been demonstrated to be efficient in reducing astrocytic function both in vivo [35, 36], to determine the role of astrocytes in the analgesic/anaesthetic actions of ketamine.
Forty Sprague–Dawley rats were obtained from the third
military medical university’s animal centre (Chongqing, China). All experimental
procedures were approved by the Animal Care and Use Committees of Zunyi Medical
University in Guizhou, China, and carried out in accordance with the ARRIVE
guidelines and the Guide for the Care and Use of Laboratory Animals [37].
Experiments were performed following the “Guide for the Care and Use of
Laboratory Animals” in China (No. 14924, 2001). All rats were housed in an
environment with an ambient temperature of 23
The process of brain
slice preparation was performed as previously described [10, 38]. Male
Sprague-Dawley rats (10–20 days after birth)
were anaesthetized with 2% isoflurane and assessed by the toe pinch reflex to
ensure adequate depth of anaesthesia. Then rats were decapitated using super-cut
scissors. The scissors were used to cut away the skin around the skull cap and
create minor incisions at the caudal/ventral base of the skull on either side.
Shallow cuts were made starting at the caudal/dorsal aspect of the skull and
working up the dorsal midline in a rostral direction, being careful not to injure
the underlying brain. A final “T” cut was made at the level of the olfactory
bulbs, perpendicular to the midline, and the complete brain mass was rapidly
separated with a metal spatula. The brain was submerged in an ice-cold artificial
cerebrospinal fluid (ACSF) consisting of 126 mM NaCl, 3 mM KCl, 2 mM CaCl
Individual coronal slices were placed into a thermoregulated (31–32
°C) recording container and continuously perfused with bubbled ACSF
(0.52 mL/min) after incubation. S1 stereotaxic coordinates were used to choose
electrophysiological recording sites in coronal rat slices (Paul Halasz & Lewis
Tsalis 6th). S1 pyramidal neurons were chosen for electrophysiological recordings
using an infrared camera on a microscope (BX51WI, Olympus,
Japan). Electrical signals were acquired using a
HEKA EPC10 amplifier (HEKA Instruments,
Ludwigshafen, Germany) and PatchMaster software (v2x80, Heka Instruments,
Ludwigshafen, Germany) in whole-cell voltage clamp settings. Data was sampled at
20 kHz and filtered at 3 kHz. When filled with the pipette
solution containing 120 mM CsCl, 10 mM NaCl, 10 mM HEPES, 2 mM MgCl
D, L-2-amino-5-phosphonovaleric acid (AP5), 6,7-dinitroquinoxaline-2,3-dione (DNQX), tetrodotoxin (TTX), gabazine, and fluorocitrate (FC) were among the substances employed in the analyses. Chemicals were provided by the Sigma-Aldrich Chemical Company (Shanghai, China). Nhwa Pharma. Corp. (Xuzhou, Jiangsu, China) provided the ketamine. All compounds were diluted in ACSF and used at a known concentration in the bath. Ketamine was dissolved into ACSF and the concentrations of ketamine in were 200 µMol/L, 400 µMol/L, 600 µMol/L, 800 µMol/L, 1000µMol/L, respectively. After impaling, brain slices were given 5 minutes to equilibrate. Brain slices were treated with several drug solutions for 5 minutes prior to whole-cell electrophysiological recording. The whole-cell recording lasted 10 min. During the recording, all brain slices were continuously perfused with regular artificial cerebrospinal fluid or various medication solutions through a gravity-fed device.
The amplitude and frequency of all events were compared to the mean values
observed during the initial control period and during and after the drug
application. Cumulative probability plots of the incidence of various amplitudes
and intervals, recorded under different
conditions from the same neuron, were subjected to the Kolmogorov–Smirnov (K–S)
test. StatView software (SAS, Cary, NC, USA). GraphPad Prism 5.0 (GraphPad
Software, Inc., San Diego, CA, USA) was used to create the concentration-effect
curve. N represents the number of neurons recorded. All results were presented as
mean
The patch electrodes were inserted in the S1 region (Fig. 1a,b). S1 neurons had
a multipolar or triangular-shaped soma, a bright and smooth appearance, and no
apparent organelles under the microscope [6] (Fig. 1b). The SPSCs were then
recorded by voltage-clamp. In S1 pyramidal neurons, SPSCs were
detected before and during ketamine administrations at various doses (Fig. 1c).
In the presence of AP5 (N-methyl-D-aspartate (NMDA) glutamate
receptors antagonist, 100
µM),
DNQX (20 µM, the antagonist of AMPA and kainite glutamate receptors),
gabazine (antagonist of
GABA
S1 pyramidal neurons recording location and SPSCs. (a) A coronal brain slice with a thickness of 350 m and the S1 recording region. (b) S1 pyramidal neuron with whole-cell recording electrode, Scale bar, 5 µM. (c) Typical traces of SPSCs obtained in pyramidal neurons under control circumstances (ACSF), ketamine, AP5 (100 µM) + DNQX (20 µM) + gabazine (10 µM) + TTX (1 µM) application and after washout with ACSF. DNQX, 6,7-dinitroquinoxaline-2,3-dione; TTX, tetrodotoxin; SPSCs, spontaneous postsynaptic currents; ACSF, artificial cerebrospinal fluid.
In brain slices,
ketamine (200 µM, 400 µM, 600
µM, 800 µM, 1000 µM) was applied to S1 pyramidal neurons by a
gravity-fed system; and decreased the frequency (n = 12, p
Ketamine’s impact on SPSCs in S1 pyramidal neurons. (a)
Ketamine (200 M) reduced SPSC frequency. (b) Ketamine (200 µM) reduced the
amplitude of SPSCs. Summary results for mean SPSCs. (c) amplitude and (d)
frequency from pyramidal neurons in control (n = 12) and ketamine (n = 12)
conditions. (e) amplitude and (f) frequency (n = 12, each point)
concentration-effect curve for ketamine-induced alterations in SPSCs. Data are
displayed as mean
The inhibitory effects
of ketamine on the amplitude and frequency of SPSCs were related to its
concentration. A concentration-effect curve was drawn (Fig. 2e,f) to calculate
the EC
Fluorocitrate (FC) is a selective aconitase enzyme inhibitor [40, 41], which can effectively inhibit astrocytes’ function in vivo and in vitro [36]. FC solution was prepared as previously described and diluted to 5 µM by adding standard ACSF, which does not appear to alter neuronal function [36]. To completely inhibit the activity of astrocytes, ACSF containing FC was used until the electrophysiological recording was completed.
To inhibit astrocytes, 5 µM ACSF containing FC was perfused after 5
minutes ACSF equilibration. Before and after astrocyte inhibition, SPSCs were
measured in S1 pyramidal neurons (Fig. 3a).
The amplitude of SPSCs decreased from 46.5
Astrocytes impact synaptic transmission in rat S1. (a) Typical
traces of SPSCs recorded in S1 pyramidal neurons in control circumstances (ACSF)
and FC (5 M). Summary results for mean SPSCs (b) amplitude and (c) frequency from
pyramidal neurons in control (n = 12) and FC (n = 12) conditions. Cumulative
probability plots for (d) amplitude (p
Consistent with the method described above, 5 µM ACSF containing FC was
perfused after 5 minutes ACSF equilibration. Before and during astrocyte
inhibition, we examined the inhibitory effects of ketamine on SPSCs in rat S1
pyramidal neurons. Ketamine (460 M) decreased the amplitude and frequency of
SPSCs from 46.5
The effects of ketamine on SPSCs in S1 pyramidal neurons are
influenced by astrocyte activity. (a) Typical SPSC traces obtained in S1
pyramidal neurons under control circumstances (ACSF), ketamine (460 µM), FC
(5 µM), and FC (5 µM) + ketamine (460 µM) application. Mean (b)
amplitude and (c) frequency of SPSCs from pyramidal neurons in control conditions
(ACSF), ketamine (460 µM), FC (5 µM), and FC (5 µM) + ketamine
(460 µM) application. Mean inhibition rate of SPSCs (d) amplitude and (e)
frequency in the presence of ketamine (460 M) and FC (5 M) + ketamine (460 M).
All data are presented as mean
In previous studies, the bidirectional connection between astrocytes and neurons was found to be critical for information integration [42, 43]. In this study, ketamine was observed to reduce the amplitude and frequency of SPSCs in rat S1 pyramidal neurons. The activity of astrocytes could determine the effect of ketamine on SPSCs.
We observed that clinically relevant ketamine doses lowered the amplitude and
frequency of SPSCs in a concentration-dependent manner without altering the decay
time. Changes in the amplitude and frequency of SPSCs suggest that there are
changes in the function of postsynaptic receptors. Therefore, our study indicates
that ketamine’s role on SPSCs is due partly to regulating postsynaptic receptors’
function. Ketamine also inhibits several ion channels, such as voltage-gated
Na
SPSC frequency and amplitude decreased when astrocytes were suppressed. The current findings suggest that astrocytes play a role in the synaptic information processing of rat S1 by modifying the function of postsynaptic receptors. Our findings are supported by the theory of a “tripartite synapse” [33, 51], in which the astrocyte functions as a component of the synapse. The structure is made up of pre- and postsynaptic synapse parts as well as an astrocytic process. A previous study showed that the “tripartite synapse” is present in S1 and participates in sensory information processing [52]. Tripartite synapses are also found in the human brain. Our study helps to further understand astrocyte function in the human central nervous system.
We also examined the inhibitory effect of ketamine on amplitude and frequency and found that the inhibitory effect of ketamine on astrocytes was enhanced. These findings show that astrocyte activity can reduce ketamine’s inhibitory effects on postsynaptic receptor function in rat S1 pyramidal neurons. There is increasing evidence that astrocyte activities are required to enhance synaptic transmission [34, 53, 54]. Thrane et al. [55] also found that ketamine can inhibit astrocytes through modulating its calcium signals. Recent evidence suggests that cortical astrocytes also express functional NMDA receptors [56, 57]. These results, suggest that ketamine, an NMDA receptor antagonist, might inhibit astrocytes’ activities at least partially through the NMDA receptor.
Astrocytes also express other receptors, such as metabotropic glutamate
receptors (mGluRs),
Gap junction channels are also necessary for astrocytes’ intercellular communication. Gap junctions produce a functioning syncytium between this type of glia cell [61]. Unlike other intravenous anaesthetics, such as propofol and etomidate, ketamine did not affect the permeability of gap junctions between astrocytes [50]. These results strongly suggest that astrocytes play a complex role in the mechanisms of ketamine’s analgesic/anaesthetic action. The role of astrocytes in the pharmacological effects of intravenous general anaesthetics requires further study.
This study has a few limitations. Firstly, only neonatal rats’ brain slice was examined, whether adult rat astrocytes are involved in synaptic transmission and alter the effects of ketamine on synaptic transmission deserves further study. Secondly, we only observed that astrocytes were engaged in synaptic transmission and altered ketamine’s effects on synaptic transmission in vitro. It would be interesting to see if the same (or different) alterations might be observed if ketamine were administered in vivo.
Our findings demonstrate that ketamine inhibits synaptic activity via presynaptic and postsynaptic components. Furthermore, we found that astrocytes engage in synaptic transmission and alter ketamine’s effects on synaptic transmission. This work adds to the growing body of evidence indicating astrocytes play a key role in the mechanisms of ketamine’s analgesic/anaesthetic actions.
NMDA, N-methyl-D-aspartate; S1, primary somatosensory cortex; SPSCs, spontaneous
postsynaptic currents; FC, fluorocitrate; GABA, g-amynobutyric acid; ACSF,
artificial cerebrospinal fluid; D, AP5, L-2-amino-5-phosphonovaleric acid; DNQX,
6,7-dinitroquinoxaline-2,3-dione; TTX, tetrodotoxin; mGluRs, glutamate receptors;
AMPARs,
On reasonable request, the corresponding author will provide the datasets created and analysed during the current work.
JY and YZ contributed equally to this work. JY performed the experiment, analyzed the data, and wrote the manuscript. YZ experimented and modified the paper. HY and SC designed and experimented. YL performed the experiment. TY designed and supervised the experiments and modified the paper. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.
This study was approved by the Medical Ethics Committee of the Zunyi Medical University Animal Care and Use Committee (approval code: 2021126).
We would like to express our gratitude to Yu Zhang of the Guizhou Key Laboratory of Anesthesia and Organ Protection at Zunyi Medical University for his patch-clamp technical assistance.
This study was funded by National Natural Science Foundation of China (No. 81960660, 82160683), The Basic Research Program of Science and Technology Department of Guizhou Province (202042940112211125), The Growth Project of Young Scientific and technological talents in the Department of Education of Guizhou Province ([2018]240), Doctor Foundation of Affiliated Hospital of Zunyi Medical University ([2018]12). These funds were utilised to purchase the study’s animal and materials.
The authors declare no conflict of interest.
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