“…acetylcholine could potentially play a more prominent role in emergence from anesthesia compared to induction.”

Image: A. Johnson, Vivo Visuals.

Image: A. Johnson, Vivo Visuals.

Although the precise neural correlates of consciousness have not yet been identified, there is one neurochemical marker that appears to track with our capacity to experience something: acetylcholine concentration in the cerebral cortex. Cortical acetylcholine concentration is high during wakefulness, decreases during slow-wave sleep, and increases again during rapid eye movement sleep, when we can have the conscious experience of dreaming.1  Based on the neuropharmacology of the particular anesthetic drug we might choose, the state of general anesthesia shows parallel changes. For example, the effects of the γ-aminobutyric acid–mediated (GABAergic) anesthetics propofol and sevoflurane are similar to slow-wave sleep and include low acetylcholine concentration, slower electroencephalographic frequency, and a low probability of experience.2,3  By contrast, the effects of the non-GABAergic anesthetics ketamine and nitrous oxide are similar to rapid eye movement sleep and include high acetylcholine concentration, faster electroencephalographic frequency, and a higher probability of experience in the form of dreams or hallucinations.2,4  In both humans5,6  and rodent models,7,8  interventions that raise acetylcholine concentration in the brain are associated with a reversal of anesthetic traits or a reduction in anesthetic potency. Conversely, lesions of cholinergic neurons in the basal forebrain—the main source of acetylcholine for the cortex—reduce anesthetic requirements for commonly used drugs such as isoflurane and propofol.8,9 

In this issue of Anesthesiology, Leung et al.10  leverage genetic mice models to probe the role of acetylcholine in general anesthesia by testing the hypothesis that deficiency of forebrain acetylcholine would enhance anesthetic sensitivity to isoflurane and ketamine and decrease the spectral power in the gamma frequency (i.e., faster frequencies in the electroencephalogram). The authors used mice that were genetically modified to alter the expression of the vesicular acetylcholine transporter by either reducing it centrally and peripherally (heterozygous knockdown mice) or completely eliminating it from the basal forebrain (forebrain knockout mice). Vesicular acetylcholine transporter is a transmembrane protein that moves acetylcholine from the cytoplasm to the synaptic vesicles. Upon neuronal depolarization, these synaptic vesicles are released from nerve terminals into the synaptic cleft, initiating the cascade of chemical neurotransmission. Decreased expression of vesicular acetylcholine transporter would be predicted to reduce the amount of acetylcholine available for synaptic signaling. Indeed, the heterozygous knockdown mice were characterized by a generalized decrease in acetylcholine concentration (~45%) across the brain, whereas forebrain knockout mice were characterized by complete elimination of acetylcholine in the brain regions innervated by the basal forebrain.

Using these genetic models of acetylcholine deficiency, the authors first showed that, compared to the wild-type control mice, the forebrain knockout mice required significantly less isoflurane (~8.8% difference) and ketamine (~15% difference) to lose the righting reflex, a surrogate for loss of consciousness in rodents. Estimation of the half-maximal effective dose (ED50) as a function of acetylcholine deficiency revealed a dose-dependent pattern. The ED50 for isoflurane was highest in the wild-type mice (0.76%), followed by heterozygous knockdown mice (0.72%), and decreased further in forebrain knockout mice (0.69%). A similar pattern was observed for ketamine, where an ED50 gradient paralleled the graded acetylcholine deficiency, i.e., 160 mg/kg for wild-type mice versus 150 mg/kg for heterozygous knockdown mice versus 124 mg/kg for forebrain knockout mice. These data demonstrate that a deficiency of forebrain acetylcholine enhances anesthetic sensitivity, which is consistent with previous findings showing a direct causal role of the cholinergic system in reversing anesthesia7  and increasing spontaneous wakefulness.11  The authors also showed that hippocampal high-gamma (63 to 100 Hz) power in acetylcholine-deficient mice was attenuated during isoflurane and ketamine anesthesia as compared to that observed under anesthesia in wild-type mice. However, a similar effect was not observed for high-gamma power in frontal cortex. Of note, it has been shown that electroencephalographic features, cortical acetylcholine concentrations, and anesthetic effects do not always correlate well.3,12  Thus, these relationships are complex, and it is not entirely surprising that there could be differences between the hippocampus and the cortex.

One limitation of these mice models is the lack of site specificity for regions innervated by the target sites. For example, given that both the frontal cortex and the hippocampus are innervated by basal forebrain, the mice models used in this study do not allow dissection of the individual roles of either of these areas. Furthermore, analysis of recovery could have been informative and revealed asymmetries relative to induction. Such asymmetry has been noted for orexin, a neurotransmitter synthesized in neurons of the hypothalamus, which has been shown to play a preferential role in anesthetic emergence.13  Of relevance to the study by Leung et al.,10  activation of orexin nerve terminals in the basal forebrain, which would increase cortical acetylcholine, accelerated emergence from isoflurane anesthesia in mice.14  Thus, acetylcholine could potentially play a more prominent role in emergence from anesthesia compared to induction. This will be a key question to answer for clinicians, with the ultimate goal of better controlling the emergence process. Drugs within our armamentarium such as physostigmine, ketamine, and caffeine are all known to increase cortical acetylcholine concentrations and have been explored in preclinical models or human studies as tools to accelerate emergence.

Despite these limitations, the manipulation of cholinergic system via genetic downregulation of acetylcholine release, as was done in this study,10  offers a model that overcomes the potential confounds associated with drug infusions (solubility, specificity, concentration) and nonspecific effects in lesion studies. Another advantage of these mice models is that only the acetylcholine transmission is affected, without impacting the corelease of other neurotransmitters, which will occur after cellular lesions. An interesting application of this model could be to understand the effect of endogenous acetylcholine modulation on sleep–wake states. This will be particularly important because it has been shown that specific lesions of basal forebrain cholinergic neurons do not produce any significant changes in sleep–wake states.15 

Although significant advances have been made in our understanding of the relationship between cortical acetylcholine and states of consciousness, including from the current study, there is much that remains to be explored. First, these data make it clear that, despite robust correlation with different levels of consciousness, acetylcholine makes only partial contributions to arousal and anesthesia, consistent with recent data highlighting the complexity of neurochemical changes during the anesthetized state.16  There is a need for a more comprehensive cartography of neurochemical changes associated with general anesthesia. Second, the ultimate clinical relevance of acetylcholine in the perioperative period requires further study, with a particular focus on reversal of general anesthesia and perioperative neurocognitive disorders. The current study by Leung et al.10  provides an important foundation for these future scientific and clinical investigations.

Supported by grant No. R01GM111293 from the National Institutes of Health, Bethesda, Maryland.

The authors are not supported by, nor maintain any financial interest in, any commercial activity that may be associated with the topic of this article.

1.
Lydic
R
,
Baghdoyan
HA
:
Sleep, anesthesiology, and the neurobiology of arousal state control.
Anesthesiology
.
2005
;
103
:
1268
95
2.
Shichino
T
,
Murakawa
M
,
Adachi
T
,
Arai
T
,
Miyazaki
Y
,
Mori
K
:
Effects of inhalation anaesthetics on the release of acetylcholine in the rat cerebral cortex in vivo.
Br J Anaesth
.
1998
;
80
:
365
70
3.
Pal
D
,
Silverstein
BH
,
Lee
H
,
Mashour
GA
:
Neural correlates of wakefulness, sleep, and general anesthesia: An experimental study in rat.
Anesthesiology
.
2016
;
125
:
929
42
4.
Pal
D
,
Hambrecht-Wiedbusch
VS
,
Silverstein
BH
,
Mashour
GA
:
Electroencephalographic coherence and cortical acetylcholine during ketamine-induced unconsciousness.
Br J Anaesth
.
2015
;
114
:
979
89
5.
Meuret
P
,
Backman
SB
,
Bonhomme
V
,
Plourde
G
,
Fiset
P
:
Physostigmine reverses propofol-induced unconsciousness and attenuation of the auditory steady state response and bispectral index in human volunteers.
Anesthesiology
.
2000
;
93
:
708
17
6.
Plourde
G
,
Chartrand
D
,
Fiset
P
,
Font
S
,
Backman
SB
:
Antagonism of sevoflurane anaesthesia by physostigmine: Effects on the auditory steady-state response and bispectral index.
Br J Anaesth
.
2003
;
91
:
583
6
7.
Pal
D
,
Dean
JG
,
Liu
T
,
Li
D
,
Watson
CJ
,
Hudetz
AG
,
Mashour
GA
:
Differential role of prefrontal and parietal cortices in controlling level of consciousness.
Curr Biol
.
2018
;
28
:
2145
52.e5
8.
Luo
TY
,
Cai
S
,
Qin
ZX
,
Yang
SC
,
Shu
Y
,
Liu
CX
,
Zhang
Y
,
Zhang
L
,
Zhou
L
,
Yu
T
,
Yu
SY
:
Basal forebrain cholinergic activity modulates isoflurane and propofol anesthesia.
Front Neurosci
.
2020
;
14
:
559077
9.
Leung
LS
,
Luo
T
,
Ma
J
,
Herrick
I
:
Brain areas that influence general anesthesia.
Prog Neurobiol
.
2014
;
122
:
24
44
10.
Leung
LS
,
Chu
L
,
Prado
MAM
,
Prado
VF
:
Forebrain acetylcholine modulates isoflurane and ketamine anesthesia in adult mice.
Anesthesiology
.
2021
;
134
:
588
606
11.
Parkar
A
,
Fedrigon
DC
,
Alam
F
,
Vanini
G
,
Mashour
GA
,
Pal
D
:
Carbachol and nicotine in prefrontal cortex have differential effects on sleep–wake states.
Front Neurosci
.
2020
;
14
:
567849
12.
Pal
D
,
Li
D
,
Dean
JG
,
Brito
MA
,
Liu
T
,
Fryzel
AM
,
Hudetz
AG
,
Mashour
GA
:
Level of consciousness is dissociable from electroencephalographic measures of cortical connectivity, slow oscillations, and complexity.
J Neurosci
.
2020
;
40
:
605
18
13.
Kelz
MB
,
Sun
Y
,
Chen
J
,
Cheng Meng
Q
,
Moore
JT
,
Veasey
SC
,
Dixon
S
,
Thornton
M
,
Funato
H
,
Yanagisawa
M
:
An essential role for orexins in emergence from general anesthesia.
Proc Natl Acad Sci USA
.
2008
;
105
:
1309
14
14.
Wang
D
,
Guo
Y
,
Li
H
,
Li
J
,
Ran
M
,
Guo
J
,
Yin
L
,
Zhao
S
,
Yang
Q
,
Dong
H
:
Selective optogenetic activation of orexinergic terminals in the basal forebrain and locus coeruleus promotes emergence from isoflurane anaesthesia in rats.
Br J Anaesth
.
2021
;
126
:
279
92
15.
Blanco-Centurion
C
,
Xu
M
,
Murillo-Rodriguez
E
,
Gerashchenko
D
,
Shiromani
AM
,
Salin-Pascual
RJ
,
Hof
PR
,
Shiromani
PJ
:
Adenosine and sleep homeostasis in the basal forebrain.
J Neurosci
.
2006
;
26
:
8092
100
16.
Zhang
X
,
Baer
AG
,
Price
JM
,
Jones
PC
,
Garcia
BJ
,
Romero
J
,
Cliff
AM
,
Mi
W
,
Brown
JB
,
Jacobson
DA
,
Lydic
R
,
Baghdoyan
HA
:
Neurotransmitter networks in mouse prefrontal cortex are reconfigured by isoflurane anesthesia.
J Neurophysiol
.
2020
;
123
:
2285
96