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Location of the Mesopontine Neurons Responsible for Maintenance of Anesthetic Loss of Consciousness


Abstract and Figures

The transition from wakefulness to general anesthesia is widely attributed to suppressive actions of anesthetic molecules distributed by the systemic circulation to the cerebral cortex (for amnesia and loss of consciousness) and to the spinal cord (for atonia and antinocice-ption). An alternative hypothesis proposes that anesthetics act on one or more brainstem or diencephalic nuclei, with suppression of cortex and spinal cord mediated by dedicated axonal pathways. Previously, we documented induction of an anesthesia-like state in rats by microinjection of small amounts of GABAA-receptor agonists into an upper brainstem region named the mesopontine tegmental anesthesia area (MPTA). Correspondingly, lesioning this area rendered animals resistant to systemically delivered anesthetics. Here, using rats of both sexes, we applied a modified microinjection method that permitted localization of the anesthetic-sensitive neurons with much improved spatial resolution. Microinjected at the MPTA hotspot identified, exposure of 1900 or fewer neurons to muscimol was sufficient to sustain whole-body general anesthesia; microinjection as little as 0.5 mm off-target did not. The GABAergic anesthetics pentobarbital and propofol were also effective. The GABA-sensitive cell cluster is centered on a tegmental (reticular) field traversed by fibers of the superior cerebellar peduncle. It has no specific nuclear designation and has not previously been implicated in brain-state transitions.
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Location of the Mesopontine Neurons Responsible for
Maintenance of Anesthetic Loss of Consciousness
XAnne Minert, Shai-Lee Yatziv, and XMarshall Devor
Department of Cell and Developmental Biology, Institute of Life Sciences, and Center for Research on Pain, The Hebrew University of Jerusalem, Jerusalem
91904, Israel
The transition from wakefulness to general anesthesia is widely attributed to suppressive actions of anesthetic molecules distributed by
the systemic circulation to the cerebral cortex (for amnesia and loss of consciousness) and to the spinal cord (for atonia and antinocice-
ption). An alternative hypothesis proposes that anesthetics act on one or more brainstem or diencephalic nuclei, with suppression of
cortex and spinal cord mediated by dedicated axonal pathways. Previously, we documented induction of an anesthesia-like state in rats
by microinjection of small amounts of GABA
-receptor agonists into an upper brainstem region named the mesopontine tegmental
anesthesia area (MPTA). Correspondingly, lesioning this area rendered animals resistant to systemically delivered anesthetics. Here,
using rats of both sexes, we applied a modified microinjection method that permitted localization of the anesthetic-sensitive neurons with
much improved spatial resolution. Microinjected at the MPTA hotspot identified, exposure of 1900 or fewer neurons to muscimol was
sufficient to sustain whole-body general anesthesia; microinjection as little as 0.5 mm off-target did not. The GABAergic anesthetics
pentobarbital and propofol were also effective. The GABA-sensitive cell cluster is centered on a tegmental (reticular) field traversed by
fibers of the superior cerebellar peduncle. It has no specific nuclear designation and has not previously been implicated in brain-state
Key words: anesthesia; arousal; MPTA; reticular formation; syncope; wet blanket hypothesis
Transient loss of consciousness (LOC, “reversible coma”) and the
other components of the anesthetic state, immobility, analgesia
and amnesia, are classically attributed to generalized pharmaco-
logical suppression of CNS function. Once ascribed to altered
physical properties of lipid membranes, suppression is now
known to be mediated by ubiquitous inhibitory ligand-gated ion
channels, most prominently the GABA
-receptor (GABA
Campagna et al., 2003;Franks, 2008). A refinement of this “gen-
eralized suppression” or “wet blanket” hypothesis adds that each
component of anesthesia is realized in a distinct CNS structure.
Thus, circulating anesthetic molecules suppress the neocortex
and hippocampus to bring about LOC and amnesia and the
brainstem and spinal cord to cause atonia and analgesia (Lukatch
and MacIver, 1996;Antognini et al., 2003;Grasshoff et al., 2005;
Hentschke et al., 2005;Bonhomme et al., 2012;Raz et al., 2014).
An alternative hypothesis holds that anesthetic molecules act
at a focus from which they engage dedicated axonal pathways.
The axons, in turn, project to the far-flung effector structures,
from cortex to cord, inducing anesthesia secondarily. This “ded-
icated pathways” hypothesis originated with Moruzzi and Ma-
Received Feb. 26, 2017; revised Aug. 6, 2017; accepted Aug. 9, 2017.
and M.D. analyzed data; A.M. and M.D. wrote the paper.
This work was supported by the Israel Health Ministry, the Seymour and Cecile Alpert Chair in Pain Research, the
Willem Bean Legacy Fund, and the Hebrew University Center for Research on Pain. The funders played no role in the
planning or execution of the experiments or the drafting of this manuscript. We thank Yelena Fishman, Adi Gold-
enberg,TamirAvigdor, and Mark Baron for their contributions and Clif Saper and Jun Lu for helpful comments on the
study and for hosting the senior author at BIDMC where the initial experiments were performed. The bonus time
method as a means of improving spatial resolution was originally suggested by Clif Saper.
The authors declare no competing financial interests.
Correspondenceshouldbe addressed to: Prof. M. Devor, Department of Cell and Developmental Biology, Institute
of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel. E-mail:
Copyright © 2017 the authors 0270-6474/17/379320-12$15.00/0
Significance Statement
General anesthesia permits pain-free surgery. Furthermore, because anesthetic agents have the unique ability to reversibly switch
the brain from wakefulness to a state of unconsciousness, knowing how and where they work is a potential route to unraveling the
neural mechanisms that underlie awareness itself. Using a novel method, we have located a small, and apparently one of a kind,
cluster of neurons in the mesopontine tegmentum that are capable of effecting brain-state switching when exposed to GABA
receptor agonists. This action appears to be mediated by a network of dedicated axonal pathways that project directly and/or
indirectly to nearby arousal nuclei of the brainstem and to more distant targets in the forebrain and spinal cord.
9320 The Journal of Neuroscience, September 20, 2017 37(38):9320 –9331
goun, who suggested that anesthetics suppress the mesopontine
ascending reticular activating system (aRAS) which, in turn, leads
to cortical suppression and LOC (Bremer, 1936;Moruzzi and
Magoun, 1949;French et al., 1953;Magni et al., 1959). Contem-
porary models parse the aRAS into numerous brainstem and
diencephalic sleep-promoting and sleep-suppressing “arousal
nuclei” with associated ascending and descending effector path-
ways. Anesthesia is induced when agents substitute for an endog-
enous neurotransmitter(s) in one or more nodes in this switching
circuitry, “hijacking” network function. Proposals about where
exactly this might occur include the locus coeruleus, tuberomam-
millary nucleus, medial thalamus, lateral hypothalamus, and pre-
optic area (Correa-Sales et al., 1992;Alkire et al., 2008;Franks,
2008;Lu et al., 2008;Brown et al., 2010;Moore et al., 2012;
Zecharia et al., 2012;Scharf and Kelz, 2013;Anaclet et al., 2014;
Baker et al., 2014;Weber et al., 2015).
A significant advance toward defining the locus of anesthetic
action was the discovery that an anesthesia-like state is induced
rapidly and reversibly by microinjecting GABA
-R agonists into
a circumscribed, bilaterally symmetrical region in the upper
brainstem, the mesopontine tegmental anesthesia area (MPTA)
(Devor and Zalkind, 2001;Voss et al., 2005; see video at http:// This state closely resembles systemic
anesthesia by behavioral, electrographic, and functional criteria
and can be sustained by repeated microinjections (Sukhotinsky et
al., 2007;Abulafia et al., 2009;Namjoshi et al., 2009;Devor et al.,
2016;Sukhotinsky et al., 2016). Anesthesia comes on much too
rapidly to permit drug diffusion to distant brain regions (seconds
to a few minutes) and effective doses are far too small to survive
dilution during vascular redistribution. These observations are
therefore inconsistent with generalized CNS suppression, but
they are compatible with the dedicated pathways hypothesis. In
addition, we found that lesioning the MPTA renders animals
resistant to anesthetic induction by the systemic route (Minert
and Devor, 2016). The MPTA cluster may thus be singular. No
comparable anesthetic-sensitive structure has been found despite
systematic searching and, if another exists, engaging it is apparently
insufficient because anesthesia cannot be induced in MPTA-
lesioned animals at clinically relevant doses. The intrinsic prop-
erties of MPTA neurons and their connectivity makes them both
sufficient and necessary for GABAergic anesthetic induction
(Sukhotinsky et al., 2016).
A limitation of our prior studies, however, is limited spatial
resolution. MPTA microinjections through indwelling catheters
likely exposed additional structures, including adjacent arousal
nuclei, to the agonists. This is the reason that we chose to call the
anesthetic-sensitive region an “area” without stipulating a specific
nuclear designation. More precise localization of the relevant neu-
rons is an important prelude to characterizing them in more detail
and determining the mechanism whereby they switch brain and spi-
nal cord state between wakefulness and anesthesia (Devor et al.,
2016). With this aim in mind, we have implemented a modified drug
delivery protocol with enhanced spatial resolution.
Materials and Methods
Overview of the “bonus time” method
Briefly, rats were anesthetized using the short-acting agent propofol and
mounted in a stereotaxic instrument. Then, minute volumes of GABA
agonists were microinjected unilaterally into the MPTA via a fine glass
micropipette. We monitored the degree to which localized deposition of
drug delayed the time to emergence from anesthesia (“anesthesia time“)
beyond what would have been expected without drug microinjection.
Although maintenance of anesthesia is not identical to anesthetic induc-
tion, it is a reasonable surrogate in light of the fact that induction per se
has already been documented after microinjection into the MPTA area
during the awake state. Prolongation of anesthesia time is referred to here
as a positive “bonus” and the approach is called “the bonus time”
Animals and surgery
Experiments were performed in two series using similar, but nonidenti-
cal methods. The first series, performed in the Department of Neurology,
Beth Israel Deaconess Medical Center (BIDMC)/ Harvard University
(Boston, MA) used adult male Sprague Dawley rats (270–330 g). In the
subsequent series, performed at the Institute of Life Sciences, the Hebrew
University of Jerusalem Israel (HUJI), we used adult female Wistar-
derived Sabra strain rats (250–350 g). At both venues, animals were
maintained under specific pathogen-free conditions, 1–3 per cage, room
temperature 21–23°C, 12 h:12 h day:night cycle, with lights on at 07:00.
Food and water were available ad libitum. Experimental protocols were
approved by the Institutional Animal Care and Use Committees of
BIDMC and HUJI and, at both venues, they were conducted in accor-
dance with the United States Public Health Service’s Policy on Humane
Care and Use of Laboratory Animals.
At BIDMC, a preliminary surgical procedure was performed to pro-
vide intravenous access for the main experiment. Under chloral hydrate
anesthesia (350 mg/kg, i.p.), a sterile chronic indwelling catheter was
introduced into the vena cava via a femoral vein tributary and firmly
anchored to local fascia. The intravenous part of the catheter was made of
soft silicone tubing. This was glued to a polyethylene tube (Intramedic
PE50) that was tunneled under the skin and exteriorized at the back of the
neck. Rats were given an analgesic postoperatively (Flunixin, 1 mg/kg, s.c.)
and were allowed to recover for 4–14 d, at which time they underwent a
terminal experiment in which drugs were microinjected into the MPTA.
During the recovery time, catheters were flushed intermittently with
heparin solution to help keep them patent. In experiments performed at
HUJI, the previously implanted catheter for intravenous access was sub-
stituted by placing, on the day of the experiment itself, an indwelling
neoflon catheter (26 G; Becton Dickinson) into the tail vein under brief
isoflurane sedation.
At both venues, experiments began by anesthetizing the rat with a
bolus dose of propofol (13 mg/kg, i.v., at BIDMC, propofol 1%; Gensia
Sicor Pharma; at HUJI, Propofol-Lipuro 1%; B. Braun Medical). It was
then mounted in a stereotaxic apparatus with head level between bregma
and lambda and a small craniotomy was made over the MPTA target
using a dental burr. At BIDMC, frontoparietal screw electrodes were
placed bilaterally for recording EEG and a pair of needle electrodes were
placed in the biceps femoris muscle to register the hindlimb EMG. The
empirical endpoint used to mark the emergence from anesthesia was the
sudden appearance of repetitive “pacing” movements of one or both
hindlimbs. As soon as this was observed, animals were given a fixed
supplemental dose of 10 mg/kg intravenous propofol to reinitiate anes-
thesia. This dose was determined in preliminary titration experiments to
yield 15 min of anesthesia before pacing reappeared. Pacing behavior
served as a convenient and unambiguous marker of emergence. However,
we also established that its onset was always accompanied by rhythmic
bursts in the biceps femoris EMG (Fig. 1C), nocifensive response to
hindpaw, and/or earlobe pinch. Slightly later theta waves (4– 8 Hz) and
sometimes higher-frequency EEG components (15–30 Hz) appeared, add-
ing to and later replacing the prominent propofol-induced delta-band
peak (0.5–4.0 Hz). In addition, in calibration trials in which the animal
was released from the stereotaxic and the supplemental propofol post-
poned, we observed prompt return of righting. Having determined in
this way that pacing reliably marks emergence from anesthesia, in subse-
quent experiments the behavioral endpoint was used as the primary
marker of emergence. In particular, pinch stimuli were avoided until
after pacing began because they are likely to accelerate emergence in an
uncontrolled manner and introduce an element of experimenter bias.
After one or more supplemental intravenous injections of propofol at
the fixed bolus dose, we began MPTA microinjections. The duration of
anesthesia provided by the fixed bolus dose before commencement of
drug microinjections was remarkably stable from trial to trial (Fig. 2) and
served as a basis for determining whether drugs microinjected into the
Minert et al. Transient Loss of Consciousness J. Neurosci., September 20, 2017 37(38):9320 –9331 • 9321
MPTA significantly extended the expected du-
ration of anesthesia, providing a positive bonus
MPTA microinjection
Procedure. First, a micropipette pulled from
fine borosilicate glass (inner diameter 0.3 or 0.5
mm (catalog #B100-30-7.5HP or #BF100-50-
15, respectively; Sutter Instruments) with tip
broken to a diameter 20–30
m was passed
through the burr hole and dura and lowered to
the level of the MPTA. In some experiments,
we deliberately targeted nearby off-target loca-
tions. We began with relatively large microin-
jections (200 and 500 nL) delivered to initial
coordinates located within the boundaries of
the MPTA as defined using the indwelling can-
nula method (7.6 to 8.8 mm caudal to
bregma, 1.3 mm lateral to the midline, and 6.3–
7.3 mm below the dura). Our strategy was to
roam from there, progressively reducing the
volume of drug microinjected, seeking a site(s)
that provided a significant extension of anes-
thesia time. Once such a “hotspot” was found,
additional small volume microinjections were
placed rostrocaudal, mediolateral, and dorso-
ventral to effective site. Other trials targeted
specific candidate nuclei including the pedun-
culopontine tegmental nucleus (PPTg), the
parabrachial complex (PB), and the oral pon-
tine reticular nucleus (PnO). All microinjec-
tions in this study were unilateral, favoring the
left side, although to ensure bilateral symme-
try, trials were also made on the right side.
Before insertion, microinjection pipettes
were filled to several millimeters above the
taper with one of the following test agents:
muscimol (2 mMin saline; Sigma-Aldrich, Re-
hovot, Israel), pentobarbital (200 mg/ml; Pen-
tal, CTS, Kiryat Malachi, Israel), propofol
(Propofol-Lipuro 1%; B. Braun Medical, or
97%, 2,6-diisopropylphenol; Sigma-Aldrich),
Figure 2. Anesthesia time was independent of the number of antecedent propofol boluses given. A, In rats given 2 (n60), 3
(n50), 4 (n20), or 5 (n7) consecutive bolus doses of propofol mean anesthesia time was the same on the first bolus as on
the n
.B, Lines represent anesthesia times of 60 individual rats upon repeated bolus dosing (all at HUJI). To ease separation, lines
representing the seven rats given five consecutive doses are colored. Error bars in Aindicate SD.
Figure1. Microinjections of muscimol into the MPTA. A,Sagittal section showingthe dye spread associatedwith a microinjectionof 200 nLof muscimol into theMPTA. B, Coronalsection showing
the dye spread associated with on-target muscimol microinjections of 20 nL, first on the left, then 20 min later on the right (rat #340). These trials yielded bonus times of 10 and 21 min, respectively.
The boundaries of the MPTA as defined by Devor and Zalkind (2001) are overlaid on the right-sided microinjection (1000 1500
m, solid outline). The common core of the MPTA is shown within
the 500 750
m dashed frame. C, The sudden appearance of repetitive hindlimb “pacing” movements was accompanied by rhythmic bursts in the biceps femoris electromyogram. Both were
silenced by the standard intravenous bolus of propofol.
9322 J. Neurosci., September 20, 2017 37(38):9320 –9331 Minert et al. Transient Loss of Consciousness
lidocaine (5% in PBS, prepared fresh from powder; Sigma-Aldrich), te-
trodotoxin (TTX, 1 mMin artificial CSF; Alomone Laboratories), or
saline (0.9% NaCl). In most experiments, the solution microinjected
included pontamine sky blue dye (1 or 2%; Gurr/BDH Chemicals) to
mark the microinjection site.
For microinjection, the butt of the pipette was attached by a length
of polyethylene tubing to a valve apparatus that delivered pulses of
positive air pressure. The pressure, duration, and duty cycle of the
pulses were adjustable and were varied with tip diameter and solution
viscosity to provide controlled extrusion of the test agent into the
brain. Typically, we used pressure of 1 kg/cm
, 20 ms pulses, 1–10
pulses/s. Between pulses, the back pressure on the pipette fell rapidly
to zero. Calibrated volumes of test solutions, 500-10 nL, were micro-
injected by monitoring the change in the position of the meniscus
inside the pipette using an ocular rule under optical magnification
(0.5 mm bore: 200 nL/mm, 0.3 mm bore: 70 nL/mm). The volume per
millimeter of internal pipette bore was calibrated previously using a 1
l Hamilton syringe.
Micropipettes were lowered to the target position and microinjection
was initiated 1–3 min before the anticipated time of emergence of the
animal from effects of the most recent intravenous bolus dose of propo-
fol. Microinjection took 20 –60 s. The micropipette was left in place for
3–5 min after microinjection, but was withdrawn immediately if pacing
began. Time was measured from the completion of the microinjection
until hindlimb pacing began. If this equaled the anticipated emergence
time (i.e., the time anticipated for the beginning of pacing), we registered
a “bonus” of zero for that trial. If the time to pacing was greater than
anticipated, then the difference was registered as a positive bonus, indi-
cating prolongation of the duration of anesthesia. As soon as the bonus
time was noted, an additional bolus dose of intravenous propofol was
given. Animals were not allowed to awaken from anesthesia while re-
strained in the stereotaxic.
Evaluation of bonus time. In the first experiments, we established the
anticipated time of emergence in each rat individually by averaging sev-
eral cycles of fixed-dose intravenous propofol injections. The time from
bolus injection until the beginning of pacing, “anesthesia time,” proved
to be stable with no consistent change over repeated cycles of bolus
dosing within and across rats (Fig. 2). This justified a modification of the
procedure such that, for each rat, we used the running average of (pre-
microinjection) anesthesia times obtained in all previous experimental
trials. This value converged rapidly. Over 130 injection cycles (at HUJI),
it averaged 11.0 2.0 min (SD, coefficient of variation 0.18; 42 rats
till #359). This value remained stable over subsequent experiments
with no detectable drift. Curiously, in experiments performed at
BIDMC, anesthesia time after the identical bolus dose of propofol was
significantly longer [19.0 6.3 min, coefficient of variation 0.33).
The difference is p0.001, 95% confidence interval (CI) 7.1–9.5].
Although this could be due to a variety of venue-specific factors, a
likely factor is the use of male Sprague Dawley rats at BIDMC and
female Sabra rats at HUJI. Clinical studies report significantly longer
emergence times from propofol-induced anesthesia in men than in
women when measurements begin at an equivalent anesthetic depth
based on bispectral index values (Hoymork and Raeder, 2005;
Choong et al., 2013).
In some experiments, only a single microinjection was given. In others,
we gave more either at an adjacent location or when attempting to rep-
licate a successful microinjection using a smaller volume (maximum of
four microinjections in a single rat; Table 1). Follow-up microinjections
were also given when, during the roaming process, muscimol yielded a
short bonus time, when saline was delivered as a control, or when a novel
Table 1. Summary of muscimol microinjection trials showing venue, volume delivered, and bonus time obtained (in minutes, 91 trials in 53 rats)
BIDMC rat #
500 nL or 200 nL
bonus time
HUJI rat #
200 nL
bonus time
100 nL or 50 nL
bonus time
20 nL
bonus time
10 nL
bonus time
Significant NS Significant NS Significant NS Significant NS Significant NS
465(R) 321 1, 2
5 13 358 10
6 35 359 21(R)
7 57(R) 322 26 5 35
839 323 14
9 26 324 36 24
10 34 325 10
11 22 327 50 23(R)
12 50 329 33 3(R) 23
13 45 370 9 2(R)
14 10 371 1, 2(R)
15 5 372 5(R), 6
29 28 330 32, 34 2(R)
30 45 331 22, 46(R)
31 45 332 15, 18(R)
32 28 338 3, 4(R), 6, 6(R)
33 36 339 1, 4(R)
36 28 351 2(R) 3
37 28 340 10, 21(R)
341 18, 30(R)
342 8
500 nL or 200 nL
bonus time
15, 19, 22(R)
11(R), 20 18
HUJI rat # Significant NS 346 16(R), 19(R) 5
369 15 347 30 4(R)
314 21 4 348 18, 22(R)
316 15 3 349 3(R) 31
317 2, 2, 3 350 3(R) 1(R)
318 58, 8 6 352 4(R) 3
Note that some rats had up to four microinjection trials. Scores marked (R) were from right-sided microinjections; italics indicate use of the volume set in italics. Criterion for statistical significance is described in the Materials and Methods.
NS, Not significant.
Minert et al. Transient Loss of Consciousness J. Neurosci., September 20, 2017 37(38):9320 –9331 • 9323
drug was involved for which we wanted to verify that the microinjection
had in fact been on target by follow-up delivery of muscimol or pento-
barbital to the same locus.
Definition of the microinjection site. At the end of each experiment, as
soon as pacing commenced, a final intravenous injection of propofol was
given and the animal was rapidly killed by transcardial perfusion with
0.9% saline followed by 10% neutral 0.1 MPO
buffered formalin (pH
7.3; Sigma-Aldrich), both at 37°C. The brain was dissected out shortly
after perfusion and postfixed at 4°C for at least 24 h in the same fixative.
It was then transferred to 20% sucrose in PBS containing 0.02% sodium
azide (PBS-azide) at 4°C. At least 24 h later, tissue was cut on a freezing
microtome into 50- or 100-
m-thick coronal sections. We collected all
sections in the region of microinjection beginning from the level at which
the first pontamine dye spot was seen in the block face 200–300
before dye actually appeared in sections and continuing until after dye
was no longer present. Every third section through each dye spot was
immediately mounted onto a glass slide in serial order. Intervening sec-
tions were saved in PBS-azide (4°C). The number and relative location of
individual dye spots seen on the block face as cutting proceeded always
matched the microinjection protocol. The slides were air dried over-
night, cleared with xylene, and coverslipped using Entellan (Merck).
Tissue sections containing dye spots were optically projected onto the
corresponding coronal level in the rat brain atlas of Paxinos and Watson
(1998) and the magnification was varied until a best fit was obtained to
the dorsal contour of the colliculi and the ventral contour of the pons.
The maximal visible extent of the contour of the dye spot was then
transferred by hand onto the atlas section by an individual blinded to
microinjected drug, volume, and the experimental outcome. The maxi-
mal cross-section of each microinjection was measured planimetrically
based on a section in which the micropipette tract emerged from the dye
spot (Fig. 1A,B).
Estimation of drug spread. We estimated the extent of drug spread
beyond the sphere defined by the volume microinjected based on the
maximal dye-spread cross-section measured. The calculation assumed
that spread was approximately isotropic and formed a sphere around the
tip of the injection micropipette. This assumption was based on the fact
that dye spots were round in both frontal and sagittal sections (Fig.
1A,B). The radius (r) of the droplet formed by drug solutions microin-
jected, before spread in the tissue, was calculated from the volume in-
jected. For the smallest volumes used (e.g., 10 nL), rwas taken as 134
calculated by solving for rin the formula for the volume of a sphere V
. The radius of the sphere formed by dye spread after microinjec-
tion was based on the cross-sectional area of dye-spread measured, cal-
culated by solving for rin the formula for a circle, A
. The
corresponding volume of spread was then calculated using 4/3
Neuronal density. Estimating the number of MPTA neurons exposed
to microinjected anesthetics requires a measure of neuronal density.
Four naive rats were perfused as above and brains were cut in serial 50
m frozen sections and immunolabeled for NeuN, a selective nuclear
marker of viable neurons. Briefly, sections were incubated overnight in
polyclonal mouse anti-NeuN antibody (Millipore catalog #MAB377,
1:25,000, room temperature), followed by 1.5 h in secondary antibody
(biotinylated goat anti-mouse IgG; Vector Laboratories, 1:1000; at 37°C).
Bound antibody was visualized using the ABC reaction and diaminoben-
zidine (Vectastain Elite ABC kit; Vector Laboratories).
One section from the mesopontine border (level 8.0 or 8.3 mm
relative to bregma; Paxinos and Watson, 1998) was selected for counting.
The field of view was overlaid with a 1000 1500
m counting frame
representing the caudal MPTA (Fig. 1B) and all immunolabeled neurons
within this region of interest (ROI) were counted bilaterally at 250
magnification using the Neurolucida system (version 10.51; MBF Biosci-
ence) and their locations were plotted. Plotting began in an upper corner
of the counting frame and proceeded systematically in a zig-zag manner
one field of view at a time until the entire frame was covered. Cells on the
edge of the ROI had to have 50% of their area within the frame to be
included in the count. The two counts (ROIs in the left and right MPTA)
for each rat were averaged and these values were averaged over the four
rats used. The average cell count per counting frame divided by the area
of the counting frame provided neuronal density (neurons per square
millimeter). This was converted to neurons per unit volume taking into
consideration the section thickness of 50
m. Therefore, the crude den-
sity in units of neurons per cubic millimeter was 20the measured
number of neurons per square millimeter. This crude density measure is
an overestimate, however, because some neurons are split by the mi-
crotome blade and counted twice. To correct for this factor, we used
Abercrombie’s method (Koningsmark, 1970), which estimates the de-
gree of overcounting based on section thickness (50
m) and mean
neuronal diameter (11.8
m) based on a measured mean cell area of
(see Results). The overcount was rectified by multiplying by the
calculated correction factor 0.83.
Neuronal size and shape. Neurons within a smaller ROI that included a
region we call the common core of the MPTA (see Results) were evalu-
ated for soma size and shape. This ROI covered 25% of the area of the
caudal MPTA counting frame (500 750
m) and was separated from
its upper, medial, and lateral borders by 250
m(Fig. 1B, dashed out-
line). Using the same sections as for neuronal counting, the perimeter of
each neuron for which at least 50% of the soma area fell within the
smaller ROI was traced with an on-screen cursor. From these measure-
ments, Neurolucida calculated the somatic area and aspect ratio as fol-
lows: (min diameter) (max diameter). As aspect ratio approaches 1,
the soma becomes less flat and more symmetric (e.g., circular or square).
We also calculated the form factor as follows: [(4
area) (perimeter
)], a
parameter reflecting the tortuosity of the perimeter, and roundness as
follows: [(4/
area) (maximum diameter)
], a parameter reflecting
the compactness of the soma, with a circle having a roundness of 1.
Statistical evaluation
Expected duration of anesthesia after repeated fixed-dose bolus injec-
tions of propofol was based on the observed average anesthesia time
SD as described above. In light of the stability of anesthesia time after
bolus injections of propofol (Fig. 2), it was possible to evaluate for indi-
vidual microinjection trials whether anesthesia time was increased sig-
nificantly; that is, whether the bonus time obtained was significantly
greater than zero. In doing this, we applied a highly conservative crite-
rion. Specifically, bonus time in a particular trial needed to be at least 3
SDs 0 to be declared statistically significant (Z-score 3, 1-tailed p
0.001). Therefore, the minimally significant bonus time was 18.8 min
(BIDMC) and 6.1 min (HUJI). Differences between group means were
evaluated using 2-tailed Student’s ttests after confirming normality of
the underlying data distributions. Significance of linear regressions (R
was tested using Pearson tests. The 95% CIs were calculated using Med-
calc ( All
means are given SD except where use of SEM is noted.
Locating the MPTA
A total of 91 unilateral muscimol microinjections, made in 53
rats, were targeted to the MPTA area and adjacent areas of inter-
est (Table 1). We began with a few 500 nL microinjections to
match the ones given in our prior studies using unilateral micro-
injections via chronically implanted cannulae (Devor and Zal-
kind, 2001;Devor et al., 2016). Then, we gradually reduced the
volume to as little as 10 nL at each step, attempting to refine the
boundaries of the effective area (Fig. 1A,B). The main result was
identification of a small subregion within the originally de-
fined MPTA area at which very small microinjections reliably
extended anesthesia time, whereas closely adjacent microin-
jections did not.
Figure 3 shows the location of the injection centers of all 91
trials, where filled symbols indicate loci that yielded a significant
bonus time and open symbols no bonus. The scatter of effective
loci using volumes of 500–50 nL was strikingly larger than that
using smaller volumes (20–10 nL). This is because, given the
greater spread of drug associated with larger volumes, the center
9324 J. Neurosci., September 20, 2017 37(38):9320 –9331 Minert et al. Transient Loss of Consciousness
of effective microinjections could be farther from the effective cell
cluster (hotspot). The loci of unsuccessful microinjections sur-
rounded the central effective core in all three dimensions. The
spatial spread of the pontamine sky blue dye used to track the
microinjected muscimol was typically spherical with a tear-
shaped extension running vertically along the track of the mi-
cropipette used for injection (Figs. 1A,B,4). Plots of all 17 small
volume (10 or 20 nL) microinjections that were effective (i.e.,
which yielded a significant bonus) clustered in a bilaterally sym-
metrical region just ventral and lateral to the periaqueductal gray,
at the far lateral edge of the decussation of the superior cerebellar
peduncle (SCP) (Fig. 5A). The dye spread area of each of these
microinjections is indicated in light pink in the figure, along with
the core area common to all, which is dark red (left and right
sides). Dye spread associated with the 14 microinjections cen-
tered on this plane that failed to yield a significant bonus sur-
round this area (volumes 10 –200 nL; light blue diagonal lines). In
only two of these ineffective trials was there likely to have been
overlap with the common core area (rat #15, 200 nL centered
8.0 mm from bregma, and rat #322, 200 nL centered 8.3 mm
from bregma, both bonus times 5 min; Fig. 5A, left).
In contrast, in our original study in which relatively large
(500 nL) microinjections were made through previously im-
planted guide cannulae, we logged failure to induce anesthesia in
nearly half of trials (46%) in which targeting appeared to be ad-
equate. A number of technical causes for this were suggested
(Devor and Zalkind, 2001). Here, we logged only two such fail-
ures (5.4% of trials). We attribute the improvement to the better
control of drug delivery afforded by the bonus time method and
to the use of smaller, more focal microinjections. For example, it
has been reported that muscimol delivered to the PnO promotes
arousal (Xi et al., 2001;Watson et al., 2011). Large microinjec-
tions that exposed much of the PnO together with the MPTA
might have attenuated effects obtained here using more focal
MPTA microinjections.
Figure 5Bshows all 35 effective microinjections (23 rats, in-
cluding the 50 –500 nL microinjections). In 33 of the cases (94%),
the dye spread overlapped the common core area on the left
and, in the remaining two, it approached very closely and in
fact did include the mirror common core (right side). Herein-
after, the term “common core” will refer to the dark red region
on the right side in Figure 5A. A number of effective microin-
jections using larger volumes had their centers rostral to the
8.0 plane or caudal to the 9.16 plane (n10 and 8, re-
spectively; Fig. 3). Extrapolating based on dye spread radius
(Myers, 1966;Myers and Hoch, 1978) in each case, the mus-
cimol would likely have reached the common core. Overall,
bonus times for the n37 muscimol microinjections that
included the common core averaged 25.1 14.0 min, whereas
bonus times of the n12 surrounding microinjections that
did not include the common core averaged 3.6 2.4 min
(Table 1). The difference is statistically significant (p
0.0001; 95% CI 29.7 13.3).
Microinjections that fell within the PPTg, the PB, and the
PnO, but in which the dye spread did not include the common
core of the MPTA, failed to yield a significant bonus (PPTg: 4 rats,
4 trials, bonus 3.0 1.8; PB: 3 rats, 6 trials, bonus 4.2 3.1
min; PnO: 3 rats, 5 trials, bonus 3.2 1.0 min; Figs. 3,4). A few
microinjections, including these nuclei, did produce a significant
bonus, but, in each one, the marker dye and presumably the
muscimol, reached the MPTA common core (Fig. 3). Saline
microinjections centered on the common core never yielded a
significant bonus (10 trials in 5 rats, bonus 2.6 1.1 min,
horizontal dark blue lines; Fig. 5C).
The laterodorsal tegmental nucleus (LDTg) is another nearby
nucleus implicated in sleep–wake regulation. No microinjections
were restricted to the LDTg, but 13 fell in the MPTA without
impinging on the LDTg and all of these yielded significant bonus
scores. Together, these observations indicate that the functional
hotspot of the MPTA region is not the PPTg, PB, PnO, or LDTg.
In addition, except for the LTDg, for which data are not available,
we can rule out the others as able to support anesthesia when ex-
posed to muscimol.
Figure3. Small volume muscimol microinjections(10 –20 nL) locate the hotspot for anesthetic induction withinthe MPTA area. Symbols mark centers of the microinjections listedin Table 1, with
the volume microinjected indicated for each symbol. Filled symbols indicate trials that yielded a significant bonus time; open symbols indicate bonus times that were not significant. Numbers above
the coronal sections indicate the section’s location caudal to bregma (in millimeters; outlines from Paxinos and Watson, 1998). Microinjections that fell on planes 7.0 to 8.0 are gathered on the
most rostral section outline (7.8 mm), those on planes 8.0 to 9.16 are gathered on the middle section (8.8 mm), and those that fell on planes 9.3 to 10.8 are gathered on the caudal
section (9.8 mm). Many effective microinjections overlap, especially in the dark (red) clusters.
Minert et al. Transient Loss of Consciousness J. Neurosci., September 20, 2017 37(38):9320 –9331 • 9325
Exposure of MPTA neurons to
GABAergic anesthetic agents
The MPTA area was originally discovered
using systematic intracerebral microin-
jections of the barbiturate anesthetic pen-
tobarbital (Devor and Zalkind, 2001).
However, because muscimol is a direct
-R agonist, in contrast to pento-
barbital, which acts at an allosteric modu-
latory site on the receptor (Lo¨scher and
Rogawski, 2012), and because it has a
much higher affinity for the GABA
than pentobarbital (K
2–10 nM), mus-
cimol was used here as a tool to refine the
functional boundaries of the effective MPTA
cluster (Beaumont et al., 1978;Davies et al.,
1998;Johnston, 2014). Muscimol, how-
ever, does not cross the blood– brain bar-
rier and is hence ineffective as a general
anesthetic agent upon systemic adminis-
tration. We therefore investigated whether
neurons in the MPTA common core also
respond to pentobarbital (Fig. 6B). Micro-
injected into the common core area, a sig-
nificant bonus was obtained on each of the 7
trials using pentobarbital (n6 rats, mean
bonus 19.3 8.4 min). Thus, accurately
targeted pentobarbital sustained anesthesia
for 20 min at 1/4000
of the systemic
dose (15 mg i.v. vs 4
g microinjected).
In addition, we tested microinjected
propofol, a second GABAergic anesthetic
currently in wide clinical use. Propofol
1% was effective in 7 microinjection trials
(4 rats, mean bonus 12.3 3.1 min)
and probably also in 2 additional trials in a
fifth rat (total 9 trials in 5 rats, 10.9 3.9
min all at HUJI; Fig. 6B). Both trials in the
fifth rat yielded bonus times of 6.0 min,
very close to our 6.1 min, p0.001 criterion. The short bonus
times obtained despite use of a saturating concentration of
propofol was probably due to rapid washout by the circulation
and perhaps also to a particularly low affinity to the GABA
isoform(s) expressed by the relevant MPTA neurons. With this in
mind, we also tested 97% propofol, which would have washed out
more slowly. Indeed, this significantly increased bonus times
(22.7 14.2 min, 3 trials in 3 rats compared with 1% propofol,
9 trials in 5 rats, ttest, p0.035; Fig. 6B). Rapid washout
could explain why Voss et al. (2005) obtained only a partial
effect with propofol using the indwelling cannula method (an-
algesia without LOC). Other contributing factors might have
included suboptimal targeting or co-recruitment of nearby
pro-arousal neurons by the relatively large volumes they mi-
croinjected (1000 nL).
Critical mass of neurons and neuronal characteristics
How many MPTA neurons need to be exposed to muscimol to
extend anesthesia time significantly? An upper-limit estimate of
this number can be derived from the number of neurons exposed
by 10 nL microinjections, the smallest volume used in this study.
The radius of dye marks associated with such microinjections
(mean 395
m, based on 6 on-target microinjections) repre-
sents a volume of 0.26 mm
(260 nL). Because the sphere radius
of 10 nL 134
m, the linear spread was ⬃⫻3 in each dimension
(volume spread is 26). Average neuronal density within the
MPTA counting frame based on NeuN-labeled histological sec-
tions was 435 neurons/mm
(Fig. 7B). This converts to 7200
after correcting for double counting of split cells
using Abercrombie’s formula (Koningsmark, 1970). Muscimol
and the marker dye are both small, water-soluble molecules
(m.w. 114 vs 992). Assuming that the spread of muscimol and
the marker was approximately the same (0.26 mm
), up to
1900 neurons would have been exposed to the drug by a 10 nL
microinjection. The actual number is probably less (see
The spherical volume associated with the MPTA common
core is considerably smaller than the dye-spread volume from 10
nL microinjections. It has a radius of 0.22 mm and a spherical
volume of 0.04 mm
(40 vs 260 nL; Fig. 5B, right). This volume
contains only 300 neurons. It is unclear whether it would suf-
fice to expose the common core neurons alone to muscimol to
obtain a significant bonus and clinical anesthesia. Microinjec-
tions smaller than 10 nL that do not spread beyond the common
core will be required to answer this question. However, it appears
that inclusion of the common core neurons is important, if not
absolutely necessary. Specifically, there were three microinjection
trials (Fig. 5A, left, blue diagonal lines) in which muscimol reached
Figure 4. Ineffective microinjections of muscimol surround the cluster of effective microinjections in all three planes. The full
extent of dye spread is shown in red (dark fill) for the 17 small volume microinjections (10 or 20 nL) that yielded a significant bonus
time. Dye spread of all of the ineffective microinjections represented at these levels (7.6 to 9.8 mm, n28 in 18 rats),
including large and small volume trials (10 –500 nL), are shown in light blue fill.
9326 J. Neurosci., September 20, 2017 37(38):9320 –9331 Minert et al. Transient Loss of Consciousness
neurons in the effective zone (pink), but missed the common core
(Fig. 5A, left, red). These trials failed to induce a significant bonus.
Conversely, two microinjections that induced a significant bonus
(Fig. 5B, left) hit the pink area, but missed the common core.
Given the ambiguity as to whether common core neurons are
sufficient and/or essential, we examined the relation between the
volume of muscimol microinjected and the bonus time obtained.
For successful microinjection trials, bonus values varied con-
siderably, averaging in the range of 20–30 min. This was much
longer than the anesthesia time provided by our standard intra-
venous bolus dose of propofol (11 min). However, interestingly,
bonus values did not vary systematically with the volume micro-
injected and thus with the overall muscimol dose or the number
of MPTA area neurons exposed to the drug (Fig. 6B). This was so
even for individual rats that received mi-
croinjections of several volumes. For ex-
ample, in 3 rats that received 2 muscimol
microinjections at the same location, 200
nL (45.6 ng) and later 50 nL (11.4 ng),
bonus scores obtained were 26 and 35
min, 36 and 24 min, and 33 and 23 min,
respectively (Table 1). Likewise, bonus
time values did not differ despite numer-
ous differences at the two venues where
the experiments were performed (Fig.
6A). This suggests that as long as the neu-
rons in the common core (and perhaps a
limited area beyond) are exposed to mus-
cimol, there is little further effect of expos-
ing a much larger number of neighboring
neurons to the drug.
Neurons residing in the common core
area vary in size and shape. Although most
had a somatic area 220
(90%), there
was a significant population of much larger
size extending up to 1080
(Fig. 7A,B).
Interestingly, projection neurons, MPTA
neurons retrogradely labeled from a variety
of projection targets (Reiner et al., 2008),
are considerably larger than the average
(188 vs 109
). MPTA common core
neurons were also heterogeneous in aspect ratio, form factor, and
roundness (Fig. 7CE). In addition to their size, the larger cells
tended to be flatter (smaller aspect ratio and form factor) and had
a more tortuous circumference (lower roundness) than the
smaller cells. A considerable amount is known about the afferent
and efferent connectivity of neurons in the MPTA area and on
other cellular characteristics (Sukhotinsky et al., 2016), but fur-
ther analysis is needed with respect to the MPTA’s common core.
Silencing MPTA neurons by exposing them to
local anesthetics
The GABAergic agents tested generally suppress activity in neu-
rons that bear GABA
-Rs. In previous microinjection studies
Figure 5. Location of the MPTA common core. AC, Maximal cross-sectional area of effective (solid pink) and noneffective (hatched blue) microinjections are collected onto a single rostrocaudal
plane (8.8 mm). The track of the injection pipette in these trials actually lay over the range of 8.0 to 9.16 mm caudal to bregma. A, Dye spread for the 17 small effective muscimol
microinjections largely overlap (10 or 20 nL, 9 rats, 10 on the left and 7 on the right). The region in common to all (intersection), on the right and on the left, is shown in solid red (dark). The term
“common core” refers to this area on the right. Noneffective microinjections (10 –200 nL) surround the effective ones (14 microinjections in 12 rats). B,AsinA, but showing areas of both large and
small effective muscimol microinjections (24 left, 11 right in 23 rats). Noneffective trials are not shown. The solid red area on the right is common to all 11 microinjections on that side. The (smaller)
solid red area on the left is common to 22 of the 24 left-sided microinjections. Dye spread areas of the two microinjections not included fell closely nearby (black outlines, rats #322 and #15, 50 and
200 nL, respectively). C, Dye spread areas of 10 saline (control) microinjections (5 rats; 50 –200 nL; horizontal dark blue lines) overlapped the common core zone bilaterally (5 left, 5 right). None of
these yielded a significant bonus time.
Figure 6. A, Average of the significant bonus times was the same (p0.24) at the two experimental venues (left:
BIDMC, male Sprague Dawley rats; right: HUJI, female Wistar-derived Sabra rats). B, Anesthesia bonus time did not
correlate significantly with the volume (and thus dose) of muscimol microinjected (open circles; 39 trials in which there was
a significant bonus; R
0.086, p0.07). The regression line for this relation is shown. Also shown are bonus times
obtained for on-target microinjections of pentobarbital (7 trials in 6 rats, filled diamonds) and propofol (12 trials in 9 rats,
filled triangles). The regression of all bonus times on volume microinjected also failed to reach statistical significance
0.011, p0.43). All data shown were from experiments performed at HUJI. Horizontal dashed lines indicate the
degree of variability of anesthesia times obtained using the standard bolus dose of propofol in trials in which there was no
drug microinjection (1SD2.0 min; 3 SDs 6.1 min).
Minert et al. Transient Loss of Consciousness J. Neurosci., September 20, 2017 37(38):9320 –9331 • 9327
using the indwelling cannula method, we showed that delivery of
lidocaine, a pan-sodium channel blocker that is expected to si-
lence MPTA neurons nonselectively, does not induce anesthesia.
This unexpected result is important because it constrains the
mechanism by which the MPTA might influence the brain-state
switching network (Devor and Zalkind, 2001;Devor et al., 2016).
For this reason, we attempted to replicate this experiment using
the new bonus time method. Indeed, on-target microinjection of
200 nL of 5% lidocaine yielded a nonsignificant bonus in each of
9 trials (9 rats, all at BIDMC, bonus 2.7 5.8 min). Finally,
because lidocaine action is pH sensitive and we could not mea-
sure the in situ pH in the MPTA, we repeated this experiment
with 1 mMTTX. Again, no significant bonus was obtained (4
trials in 2 rats, all at HUJI, 3 50 nL, 1 100 nL, all on-target,
mean bonus time 1.0 1.8 min).
The original borders of the MPTA were set by framing the centers
of effective microinjections (Devor and Zalkind, 2001). Agent
spread was not taken into account. The bonus time method, with
its improved spatial resolution, permitted a significant upgrade
in our ability to define the actual location of the MPTA neurons
that mediate general anesthesia upon exposure to GABAergic
anesthetics. Relatively large (200 nL) muscimol microinjec-
tions at a distance from the MPTA common core yielded signif-
icant bonus times, but the effect was lost using smaller volumes.
A progressive search using ever smaller volumes yielded a dorsal
region in the caudal half of the (original) MPTA frame, where as
little as 10 nL of muscimol reliably prolonged anesthesia time.
Deviation by as little as 0.5 mm in any direction eliminated the
effect. In a recent complementary experiment, we showed that
bilateral MPTA lesions markedly reduced sensitivity to sys-
temic GABAergic anesthetics (Minert and Devor, 2016). Impor-
tantly, the territory common to all effective lesions was centered
on the common core hotspot for anesthetic induction identified
in the present study.
Size and location of the MPTA
From knowledge of neuronal density and the extent of agent
spread, we estimated that unilateral exposure of 1900 MPTA
neurons to muscimol is sufficient to maintain anesthesia for 10s
of minutes. This number is probably an overestimate. The
marker dye, although somewhat larger than muscimol, had 1–2 h
to diffuse, the typical time from microinjection to perfusion. The
drug itself, in contrast, had to act within 2–3 min to prevent
pacing and termination of the trial. This brief diffusion interval
would probably have permitted the drug to access 1900 neu-
rons. If exposure of common core neurons is sufficient, then the
minimal number could be as few as 300 neurons. Concerning
the overall size of the MPTA, we adopt the conservative position
that GABA-receptive MPTA neurons involved in brain-state
switching might be present wherever small-volume trials were
successful (Fig. 8, right, pink zone). We refer to this as the “max-
imal MPTA” (mMPTA). With an equivalent radius of 0.75 mm
(volume 1.77 mm
), it contains 11,700 neurons
Nuclear designation
In the authoritative rat brainstem atlases of Paxinos and Watson
(Paxinos and Watson, 1998;Paxinos et al., 1999), the MPTA
common core occupies a tegmental (reticular) field traversed by
fibers of the SCP that has no nuclear designation. Common core
neurons lie interstitial to the SCP. The larger mMPTA is likewise
undesignated; the region ventral to the SCP is simply labeled
“subpeduncular tegmentum.” The mMPTA merges ventrally
with the PnO and is laterally and caudally adjacent to the PPTg,
Figure 7. Sizes and shapes of neurons populating the MPTA common core are heterogeneous. A total of 1198 neurons were measured bilaterally in four naive rats. A, Histogram showing somatic
area.B, Photomicrograph showing a typical field ofNeuN-immunolabeled neurons, includesone neuron that isexceptionally larger (arrow).CE, Histograms showingthe distribution of threeshape
parameters in the population of neurons shown in A. These were as follows: aspect ratio (a measure of flatness and radial symmetry), form factor (a measure of the complexity of the somatic
perimeter), and roundness.
9328 J. Neurosci., September 20, 2017 37(38):9320 –9331 Minert et al. Transient Loss of Consciousness
caudally adjacent to the PB, and medially adjacent to the LDTg.
These nuclei are implicated in sleep and arousal (Fuller et al.,
2011;Watson et al., 2011;Roeder et al., 2016), but all were ruled
out as the location of the anesthesia-promoting MPTA neurons.
The mMPTA approximately overlaps the rodent homolog of the
“mesencephalic locomotor region” (Sherman et al., 2015), an
area in which Shik et al. (1969) documented (in cats) that electri-
cal stimulation triggers locomotion. This is also the region where
direct application of teflurane or cyclopropane were shown to
induce sedation and sleep (in cats; Folkman et al., 1968). It con-
tains neurons that send ascending and descending axons to
nearby arousal nuclei and to more distant forebrain, hindbrain,
and spinal cord structures associated with arousal, memory,
movement, and pain. These pathways are presumably the effec-
tors of the canonical components of anesthesia. A subset of neu-
rons in the MPTA are known to express GABA
-R subunits.
These are presumably the neurons that are sensitive to GABAer-
gic anesthetics (Sukhotinsky et al., 2003,2007,2016).
Indwelling cannula versus bonus time methods
Both the original indwelling cannula method and the bonus time
method permit focal deposition of GABA
-R agonists in the
brain. A major difference is that, using the former, anesthetic
induction begins from the awake state; in the latter, the animal is
already anesthetized (with propofol). Effective drugs prolong an-
esthesia time, postponing emergence, but they do not actually
cause induction. This difference is potentially significant because
emergence from anesthesia might be an active process, not simple
reversal of induction as the anesthetic agent dissipates (Sleigh et
al., 2001;Kelz et al., 2008). A variety of actions promote arousal
and emergence from anesthesia. Noxious peripheral input (e.g.
by tail pinch) is a prime example. Reanimation can also be evoked
by activation of central pain pathways (Schiff et al., 2007;
Brischoux et al., 2009;Anaclet et al., 2014;Roeder et al., 2016;
Taylor et al., 2016;Morales and Margolis, 2017). It is not clear
whether arousal by pain-provoking stimuli is mechanistically
linked to mesopontine sleep–wake and anesthesia circuitry or is
more general in nature. Either way, MPTA neurons appear to be
key players in induction as well as emergence as between the two
experimental methods both have been documented. Whether the
same individual MPTA neurons contribute to both remains un-
certain. Also uncertain is the degree to which our findings apply
to anesthesia using non-GABAergic agents and to what extent
they apply to clinical anesthesia in humans.
Finally, we confirmed our earlier observation that nonselec-
tive suppression of the MPTA using lidocaine (and now TTX) do
neither. Our explanation of this apparent paradox is in a pro-
posed model of anesthetic induction by MPTA neurons (Minert
and Devor, 2016;Devor et al., 2016).
Network actions
Observations, mostly based on noninvasive imaging in humans,
have highlighted large-scale network connectivity and top-down
processing as central factors in conscious awareness, with
thalamocortical interactions taking center stage (Brown et al.,
2010;Bonhomme et al., 2012;Vijayan et al., 2013;Hudetz and
Mashour, 2016;Tononi et al., 2016;Mashour and Hudetz, 2017).
Degradation and recovery of effective connectivity are striking
correlates of anesthetic induction and emergence. These obser-
vations, however, do not bear directly on the question of where in
the brain anesthetics act. The temporal resolution of the BOLD
signal is too low to discriminate generalized suppression by cir-
culating drugs from activation of dedicated axonal pathways. In
principle, both are capable of mediating the observed changes.
Magnetoencephalography might have the required temporal
-Rs are ubiquitous in the CNS, including in the cere-
bral cortex, and they are surely engaged by circulating agonists.
Our microinjection results, however, suggest that their engage-
ment is not necessary for anesthetic LOC. It is probably also not
sufficient because, after MPTA lesions with the thalamus and
cortex intact, clinically relevant drug doses no longer induce an-
esthesia (Minert and Devor, 2016). We cannot exclude the pos-
sibility that certain parameters thought to be bona fide neural
correlates-of-consciousness are not in fact causal, as they require
direct agonist binding to cortical receptors. These could include
anesthetic-induced reduction in cortical blood flow and metab-
olism, aspects of cortical connectivity, and some features of al-
tered EEG. We note, for example, that bolus doses of systemic
propofol induce burst suppression, but agonists microinjected
into the MPTA, even at very high concentrations, do not (Devor
et al., 2016). Assuming that in clinical (systemic) anesthesia
MPTA-driven changes are engaged in addition to generalized
suppression-driven changes, it will be important moving forward
to unravel which changes are due to which process. This is true
for both the cortical correlates of LOC and for the sensory and
motor changes that accompany anesthesia, typically attributed to
the brainstem and spinal cord. Such dissociation is straightfor-
ward in rats using MPTA microinjection (Abulafia et al., 2009),
but in humans, microinjection is clearly impracticable. There
Figure 8. Graphical illustration of the MPTA common core and mMPTA. Left, Noneffective
microinjectionsfrom the right side ofFigure 5Aare mirrortransposed andplotted together with
noneffective microinjections actually made on the left side in Figure 5A(light blue shading).
These contours surround the transposed MPTA common core (Fig. 5A, right). Note the non-
shaded halo surrounding the common core that is devoid of failed microinjections. Right, Dye
spread areas of noneffective microinjections of all volumes (light blue shading) have been
mirror transposed and combined with all of the effective small volume microinjections
(10 –20 nL; Fig. 5A, both sides, pink fill). The union of these pink contours is the mMPTA. The
successfulmicroinjections (pink) are surrounded on allsides by failedmicroinjections (blue).For
clarity, the two negative trials noted in the Results that included the common core, whereas
indicated on the right side in this illustration, are absent on the left.
Minert et al. Transient Loss of Consciousness J. Neurosci., September 20, 2017 37(38):9320 –9331 • 9329
might be a pharmacological approach, however, if MPTA neu-
rons express GABA
-R isoforms or other propitious receptors
that can be activated using target-selective anesthetics.
Our results add to the growing understanding of brain-state
switching in general and the neural mechanisms underlying an-
esthesia in particular. Most notably, they support the dedicated
pathways hypothesis of general anesthesia by suggesting that sys-
temic drug administration induces immobility, analgesia, amne-
sia, and LOC by a primary action in the brainstem, followed by
recruitment of dedicated ascending and descending axonal path-
ways that secondarily modulate function in far-flung effector lo-
cations in the CNS. We have located the relevant GABA-sensitive
neurons to a very small, focal population within the larger region
originally designated as MPTA. This nucleus appears to consti-
tute a key node in a network that effects brain-state switching.
The improved localization of the functionally important
MPTA cluster will facilitate a more detailed characterization of
the relevant neurons, including their cellular properties and
functional connectivity. Of particular importance is determining
whether the same neurons participate in all of the functional
endpoints of anesthesia or if separate subpopulations mediate
atonia, analgesia, amnesia, and LOC (Reiner et al., 2007). Heter-
ogeneity might make it possible to recruit specific neuronal sub-
populations selectively permitting, for example, surgical quality
pain control without sedation or selective promotion of sleep (or
alertness). The improved localization will also facilitate determi-
nation of whether the flip-flop circuit associated with GABAergic
anesthesia participates in other instances of transient LOC such
as fainting due to hypotension (syncope), blood gas imbalance,
fear, or natural sleep (Meiri et al., 2016). Finally, the identifica-
tion of a localized cluster of neurons that are intimately related to
brain-state switching may provide a viable experimental lead into
the circuitry that realizes conscious experience itself.
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Minert et al. Transient Loss of Consciousness J. Neurosci., September 20, 2017 37(38):9320 –9331 • 9331
... The bottom line of the initial microinjection survey, and several follow-up studies, was the discovery of a single, small bilaterally symmetrical site located in the rat brainstem at which microinjection of minute quantities of a variety of GABA A -R agonists including PB and propofol rapidly evokes a state of surgical general anesthesia lasting tens of minutes (Figure 1; Minert et al., 2017). Although in the survey saturating concentrations of PB and propofol were used, we later documented that microinjecting clinically relevant concentrations of PB or propofol, the concentrations measured in the CSF during systemic-induced anesthesia, are also proanesthetic (Baron et al., in preparation). ...
... Unilateral PB microinjections as small as 10 nL, calculated to expose no more than about 1,900 neurons to the drug, proved to be pro-anesthetic. They generate both atonic and analgesic effects mediated by the spinal cord, as well as broad synchronization of the EEG reflecting an action in the cerebral cortex (Namjoshi et al., 2009;Minert et al., 2017;Avigdor et al., 2021). These effects must be mediated by impulse propagation along axonal pathways. ...
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The canonical view of how general anesthetics induce loss-of-consciousness (LOC) permitting pain-free surgery posits that anesthetic molecules, distributed throughout the CNS, suppress neural activity globally to levels at which the cerebral cortex can no longer sustain conscious experience. We support an alternative view that LOC, in the context of GABAergic anesthesia at least, results from anesthetic exposure of a small number of neurons in a focal brainstem nucleus, the mesopontine tegmental anesthesia area (MPTA). The various sub-components of anesthesia, in turn, are effected in distant locations, driven by dedicated axonal pathways. This proposal is based on the observations that microinjection of infinitesimal amounts of GABAergic agents into the MPTA, and only there, rapidly induces LOC, and that lesioning the MPTA renders animals relatively insensitive to these agents delivered systemically. Recently, using chemogenetics, we identified a subpopulation of MPTA "effector-neurons" which, when excited (not inhibited), induce anesthesia. These neurons contribute to well-defined ascending and descending axonal pathways each of which accesses a target region associated with a key anesthetic endpoint: atonia, anti-nociception, amnesia and LOC (by electroencephalographic criteria). Interestingly, the effector-neurons do not themselves express GABA A-receptors. Rather, the target receptors reside on a separate sub-population of presumed inhibitory interneurons. These are thought to excite the effectors by disinhibition, thus triggering anesthetic LOC.
... It seems likely that anesthesia transiently silenced a group of neurons necessary for wakefulness, and that these are the same neurons that, when silenced, produce the behavioral effects described by Bremer and others. Interestingly, however, while other GABAergic agents produce loss of arousal, neither silencing this region with lidocaine or tetrodotoxin Minert et al., 2017;Avigdor et al., 2021), nor ablating it caused loss of arousal Lanir-Azaria et al., 2018). No specific cell populations have been identified as mediating the effect of pentobarbital in this region (Minert et al., 2017). ...
... Interestingly, however, while other GABAergic agents produce loss of arousal, neither silencing this region with lidocaine or tetrodotoxin Minert et al., 2017;Avigdor et al., 2021), nor ablating it caused loss of arousal Lanir-Azaria et al., 2018). No specific cell populations have been identified as mediating the effect of pentobarbital in this region (Minert et al., 2017). Fuller et al. (2011) proposed that the neurons necessary for wakefulness are located caudal to this area, in the parabrachial nucleus (PB). ...
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Wakefulness is necessary for consciousness, and impaired wakefulness is a symptom of many diseases. The neural circuits that maintain wakefulness remain incompletely understood, as do the mechanisms of impaired consciousness in many patients. In contrast to the influential concept of a diffuse “reticular activating system,” the past century of neuroscience research has identified a focal region of the upper brainstem that, when damaged, causes coma. This region contains diverse neuronal populations with different axonal projections, neurotransmitters, and genetic identities. Activating some of these populations promotes wakefulness, but it remains unclear which specific neurons are necessary for sustaining consciousness. In parallel, pharmacological evidence has indicated a role for special neurotransmitters, including hypocretin/orexin, histamine, norepinephrine, serotonin, dopamine, adenosine and acetylcholine. However, genetically targeted experiments have indicated that none of these neurotransmitters or the neurons producing them are individually necessary for maintaining wakefulness. In this review, we emphasize the need to determine the specific subset of brainstem neurons necessary for maintaining arousal. Accomplishing this will enable more precise mapping of wakefulness circuitry, which will be useful in developing therapies for patients with coma and other disorders of arousal.
... Thus, loss of ACh may be a major contributor to propofol-induced loss of consciousness. The fact that our model requires neuromodulatory changes to produce propofol oscillations and their coupling suggests that the effects of propofol on the brain stem may be critical for its oscillatory phenomena, which is supported by active experimental research on propofol and other anesthetics (18,56,(75)(76)(77)(78). Since the transition from C-state to I-state in our model is associated with lowering ACh (without changing GABAergic effects), we predict that a smaller dose of physostigmine may produce less I-state and more C-state and thus may lead to more EEG low-beta, higherfrequency alpha, increased alpha power, less peak-max, and lower slow-wave amplitude. ...
Propofol-mediated unconsciousness elicits strong alpha/low-beta and slow oscillations in the electroencephalogram (EEG) of patients. As anesthetic dose increases, the EEG signal changes in ways that give clues to the level of unconsciousness; the network mechanisms of these changes are only partially understood. Here, we construct a biophysical thalamocortical network involving brainstem influences that reproduces transitions in dynamics seen in the EEG involving the evolution of the power and frequency of alpha/low beta and slow rhythm, as well as their interactions.Our model suggests propofol engages thalamic spindle and cortical sleep mechanisms to elicit persistent alpha/low-beta and slow rhythms, respectively. The thalamocortical network fluctuates between two mutually exclusive states on the timescale of seconds. One state is characterized by continuous alpha/low-beta frequency spiking in thalamus (C-state), while in the other, thalamic alpha spiking is interrupted by periods of co-occurring thalamic and cortical silence (I-state). In the I-state, alpha co-localizes to the peak of the slow oscillation; in the C-state, there is a variable relationship between an alpha/beta rhythm and the slow oscillation. The C-state predominates near loss of consciousness; with increasing dose, the proportion of time spent in the I-state increases, recapitulating EEG phenomenology. Cortical synchrony drives the switch to the I-state by changing the nature of the thalamocortical feedback. Brainstem influence on the strength of thalamocortical feedback mediates the amount of cortical synchrony. Our model implicates loss of low-beta, cortical synchrony, and coordinated thalamocortical silent periods as contributing to the unconscious state.
... Minert et al. discovered a small nucleus in an upper brainstem region, the mesopontine tegmental anesthesia area (MPTA), the lesioning of which rendered resistance to systemically delivered anesthetics. Therefore, the MPTA appears to be a key node responsible for anesthetic induction and maintenance (Minert et al., 2017). On-target MPTA lesions have been shown to reduce sensitivity to propofol GA (Minert et al., 2020), suggesting its vital role in propofol induction. ...
The development of cutting-edge techniques to study specific brain regions and neural circuits that regulate sleep-wake brain states and general anesthesia (GA), has increased our understanding of these states that exhibit similar neurophysiologic traits. This review summarizes current knowledge focusing on cell subtypes and neural circuits that control wakefulness, rapid eye movement (REM) sleep, non-REM sleep, and GA. We also review novel insights into their interactions and raise unresolved questions and challenges in this field. Comparisons of the overlapping neural substrates of sleep-wake and GA regulation will help us to understand sleep-wake transitions and how anesthetics cause reversible loss of consciousness. Significance Statement General anesthesia (GA), sharing numerous neurophysiologic traits with the process of natural sleep, is administered to millions of surgical patients annually. In the past decade, studies exploring the neural mechanisms underlying sleep-wake and GA have advanced our understanding of their interactions and how anesthetics cause reversible loss of consciousness. Pharmacotherapies targeting the neural substrates associated with sleep-wake and GA regulation have significance for clinical practice in GA and sleep medicine.
... The ventral tegmental area (VTA) is an important component of the mesolimbic system, playing an important role in reward circuits, the formation of long-term memory, and the regulation of sleep-awake cycles through the regulation of dopaminergic pathways (Lisman and Grace, 2005;Zellner and Ranaldi, 2010). In practice, the area of mesopontine tegmental anesthesia has a strong relation with individual sensitivity to anesthetics (Minert et al., 2017), but the role of adjacent areas remains unclear. Anxiety may decrease the emergence time from sevoflurane anesthesia by affecting VTA DA neurons, as shown by a significant decrease in GCaMP6 m fluorescence values measured by Ca 2+ signals using fiberoptic photometry in the VTA DA neurons of anxiety mice compared to controls. ...
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Sevoflurane is presently one of the most used inhaled anesthetics worldwide. However, the mechanisms through which sevoflurane acts and the areas of the brain associated with changes in consciousness during anesthesia remain important and complex research questions. Sevoflurane is generally regarded as a volatile anesthetic that blindly targets neuronal (and sometimes astrocyte) GABAA receptors. This review focuses on the brain areas of sevoflurane action and their relation to changes in consciousness during anesthesia. We cover 20 years of history, from the bench to the bedside, and include perspectives on functional magnetic resonance, electroencephalogram, and pharmacological experiments. We review the interactions and neurotransmitters involved in brain circuits during sevoflurane anesthesia, improving the effectiveness and accuracy of sevoflurane’s future application and shedding light on the mechanisms behind human consciousness.
... Direct electrical stimulation at appropriate subcortical locations in humans and animals readily evokes pain experience and pain behavior, subcortical seizure activity can be painful, appropriate subcortical regions are activated in functional imaging and pathway lesions can relieve pain [31,56,95]. What is more, localized microinjection of minute quantities of opiates into the brainstem PAG yields selective whole-body analgesia and microinjecting GABAergic anesthetics into the brainstem MPTA (mesopontine tegmental anesthesia area) yields LOC including insensitivity to noxious stimuli [96][97][98][99]. Most telling of all, localized lesions in the dorsal mesopontine tegmentum readily induce LOC in animals and humans, and lesions limited to the MPTA render animals relatively insensitive to otherwise clinically effective doses of anaesthetics delivered systemically [90,91,[100][101][102]. ...
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It is nearly axiomatic that pain, among other examples of conscious experience, is an outcome of still-uncertain forms of neural processing that occur in the cerebral cortex, and specifically within thalamo-cortical networks. This belief rests largely on the dramatic relative expansion of the cortex in the course of primate evolution, in humans in particular, and on the fact that direct activation of sensory representations in the cortex evokes a corresponding conscious percept. Here we assemble evidence, drawn from a number of sources, suggesting that pain experience is unlike the other senses and may not, in fact, be an expression of cortical processing. These include the virtual inability to evoke pain by cortical stimulation, the rarity of painful auras in epileptic patients and outcomes of cortical lesions. And yet, pain perception is clearly a function of a conscious brain. Indeed, it is perhaps the most archetypical example of conscious experience. This draws us to conclude that conscious experience, at least as realized in the pain system, is seated subcortically, perhaps even in the “primitive” brainstem. Our conjecture is that the massive expansion of the cortex over the course of evolution was not driven by the adaptive value of implementing consciousness. Rather, the cortex evolved because of the adaptive value of providing an already existing subcortical generator of consciousness with a feed of critical information that requires the computationally intensive capability of the cerebral cortex.
Propofol infusion is processed through the wake-sleep cycle in neural connections, and the ionotropic purine type 2X7 receptor (P2X7R) is a nonspecific cation channel implicated in sleep regulation and synaptic plasticity through its regulation of electric activity in the brain. Here, we explored the potential roles of P2X7R of microglia in propofol-induced unconsciousness. Propofol induced loss of the righting reflex in male C57BL/6 wild-type mice and increased spectral power of the slow wave and delta wave of the medial prefrontal cortex (mPFC), all of which were reversed with P2X7R antagonist A-740003 and strengthened with P2X7R agonist Bz-ATP. Propofol increased the P2X7R expression level and P2X7R immunoreactivity with microglia in the mPFC, induced mild synaptic injury and increased GABA release in the mPFC, and these changes were less severe when treated with A-740003 and were more obvious when treated with Bz-ATP. Electrophysiological approaches showed that propofol induced a decreased frequency of sEPSCs and an increased frequency of sIPSCs, A-740003 decrease frequency of sEPSCs and sIPSCs and Bz-ATP increase frequency of sEPSCs and sIPSCs under propofol anesthesia. These findings indicated that P2X7R in microglia regulates synaptic plasticity and may contribute to propofol-mediated unconsciousness.
Pentobarbital-induced anesthesia is believed to be mediated by enhancement of the inhibitory action of γ-aminobutyric acid (GABA)ergic neurons in the central nervous system. However, it is unclear whether all components of anesthesia induced by pentobarbital, such as muscle relaxation, unconsciousness, and immobility in response to noxious stimuli, are mediated only through GABAergic neurons. Thus, we examined whether the indirect GABA and glycine receptor agonists gabaculine and sarcosine, respectively, the neuronal nicotinic acetylcholine receptor antagonist mecamylamine, or the N-methyl-d-aspartate receptor channel blocker MK-801 could enhance pentobarbital-induced components of anesthesia. Muscle relaxation, unconsciousness, and immobility were evaluated by grip strength, the righting reflex, and loss of movement in response to nociceptive tail clamping, respectively, in mice. Pentobarbital reduced grip strength, impaired the righting reflex, and induced immobility in a dose-dependent manner. The change in each behavior induced by pentobarbital was roughly consistent with that in electroencephalographic power. A low dose of gabaculine, which significantly increased endogenous GABA levels in the central nervous system but had no effect on behaviors alone, potentiated muscle relaxation, unconsciousness, and immobility induced by low pentobarbital doses. A low dose of MK-801 augmented only the masked muscle-relaxing effects of pentobarbital among these components. Sarcosine enhanced only pentobarbital-induced immobility. Conversely, mecamylamine had no effect on any behavior. These findings suggest that each component of anesthesia induced by pentobarbital is mediated through GABAergic neurons and that pentobarbital-induced muscle relaxation and immobility may partially be associated with N-methyl-d-aspartate receptor antagonism and glycinergic neuron activation, respectively.
Although general anesthesia is normally induced by systemic dosing, an anesthetic state can be induced in rodents by microinjecting minute quantities of GABAergic agents into the brainstem mesopontine tegmental anesthesia area (MPTA). Correspondingly, lesions to the MPTA render rats relatively insensitive to standard anesthetic doses delivered systemically. Using a chemogenetic approach we have identified and characterized a small subpopulation of neurons restricted to the MPTA which, when excited, render the animal anesthetic by sensorimotor (immobility) and electroencephalographic (EEG) criteria. These “effector-neurons” do not express GABAAδ-Rs, the likely target of GABAergic anesthetics. Rather, we report a distinct sub-population of nearby MPTA neurons which do. During anesthetic induction these likely excite the effector-neurons by disinhibition. Within the effector population ~ 70% appear to be glutamatergic, ~30% GABAergic and ~ 40% glycinergic. Most are projection neurons that send ascending or descending axons to distant targets associated with the individual functional components of general anesthesia: atonia, analgesia, amnesia, and loss-of-consciousness.
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We all experience pain at one time or another. Pain is an essential “alarm bell” that tells us that something is wrong, and a “teacher” that reminds us not to do that same thing again. Usually, pain is felt when a stimulus, such as a pinch or an injury, causes electrical pulses to run along one of the cables of nerve fibers in our body and into the brain where they generate an unpleasant sensory and emotional experience. Sometimes pain occurs without any actual stimulus, such as when nerve fibers have been damaged. An example is the phantom pain that amputees sometimes feel in their missing limb. Certain drugs can stop pain by blocking the electrical pulses before they reach the brain. Other drugs stop pain in a different way, by preventing the brain from reading the pain message carried by the electrical pulses. This article explains what happens in the body when we are hurt, how the brain causes this to be felt as pain and how certain drugs can stop pain.
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There has been controversy regarding the precise mechanisms of anesthetic-induced unconsciousness, with two salient approaches that have emerged within systems neuroscience. One prominent approach is the “bottom up” paradigm, which argues that anesthetics suppress consciousness by modulating sleep-wake nuclei and neural circuits in the brainstem and diencephalon that have evolved to control arousal states. Another approach is the “top-down” paradigm, which argues that anesthetics suppress consciousness by modulating the cortical and thalamocortical circuits involved in the integration of neural information. In this article, we synthesize these approaches by mapping bottom-up and top-down mechanisms of general anesthetics to two distinct but inter-related dimensions of consciousness: level and content. We show how this explains certain empirical observations regarding the diversity of anesthetic drug effects. We conclude with a more nuanced discussion of how levels and contents of consciousness interact to generate subjective experience and what this implies for the mechanisms of anesthetic-induced unconsciousness.
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Dopamine-releasing neurons of the ventral tegmental area (VTA) have central roles in reward-related and goal-directed behaviours. VTA dopamine-releasing neurons are heterogeneous in their afferent and efferent connectivity and, in some cases, release GABA or glutamate in addition to dopamine. Recent findings show that motivational signals arising from the VTA can also be carried by non-dopamine-releasing projection neurons, which have their own specific connectivity. Both dopamine-releasing and non-dopamine-releasing VTA neurons integrate afferent signals with local inhibitory or excitatory inputs to generate particular output firing patterns. Various individual inputs, outputs and local connections have been shown to be sufficient to generate reward- or aversion-related behaviour, indicative of the impressive contribution of this small population of neurons to behaviour.
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Significance Although dopamine is known to promote wakefulness, the specific dopamine circuits in the brain that regulate arousal are not clear. Here we report that selective optogenetic stimulation of ventral tegmental area (VTA) dopamine neurons in mice produces a powerful arousal response sufficient to restore conscious behaviors, including the righting reflex, during continuous, steady-state general anesthesia. Although previous studies found that VTA dopamine neurons do not appear to play a central role in regulating sleep–wake transitions, our findings demonstrate that selective stimulation of these neurons is sufficient to induce the transition from an unconscious, anesthetized state to an awake state. These results suggest that VTA DA neurons play a critical role in promoting wakefulness.
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The rostral ventromedial medulla (RVM) has a well-documented role in pain modulation and exerts antinociceptive and pronociceptive influences mediated by 2 distinct classes of neurons, OFF-cells and ON-cells. OFF-cells are defined by a sudden pause in firing in response to nociceptive inputs, whereas ON-cells are characterized by a "burst" of activity. Although these reflex-related changes in ON- and OFF-cell firing are critical to their pain-modulating function, the pathways mediating these responses have not been identified. The present experiments were designed to test the hypothesis that nociceptive input to the RVM is relayed through the parabrachial complex (PB). In electrophysiological studies, ON- and OFF-cells were recorded in the RVM of lightly anesthetized male rats before and after an infusion of lidocaine or muscimol into PB. The ON-cell burst and OFF-cell pause evoked by noxious heat or mechanical probing were substantially attenuated by inactivation of the lateral, but not medial, parabrachial area. Retrograde tracing studies showed that neurons projecting to the RVM were scattered throughout PB. Few of these neurons expressed calcitonin gene-related peptide, suggesting that the RVM projection from PB is distinct from that to the amygdala. These data show that a substantial component of "bottom-up" nociceptive drive to RVM pain-modulating neurons is relayed through the PB. While the PB is well known as an important relay for ascending nociceptive information, its functional connection with the RVM allows the spinoparabrachial pathway to access descending control systems as part of a recurrent circuit.
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In this Opinion article, we discuss how integrated information theory accounts for several aspects of the relationship between consciousness and the brain. Integrated information theory starts from the essential properties of phenomenal experience, from which it derives the requirements for the physical substrate of consciousness. It argues that the physical substrate of consciousness must be a maximum of intrinsic cause–effect power and provides a means to determine, in principle, the quality and quantity of experience. The theory leads to some counterintuitive predictions and can be used to develop new tools for assessing consciousness in non-communicative patients.
Only a short time after the neuron became identified as the essential unit of the nervous system, the first attempts were made to estimate the number of neurons in different parts of the nervous system. During the past century, a great number of methods have been used in making such estimates. Although the most widely used and accepted method is that of direct counting in the microscope, various other techniques, including photographic, projection, homogenate, automatic, and ocular methods have been designed. Brief descriptions of these techniques will be given in the following account.
We review evidence that the induction of anesthesia with GABAergic agents is mediated by a network of dedicated axonal pathways, which convey a suppressive signal to remote parts of the central nervous system. The putative signal originates in an anesthetic-sensitive locus in the brainstem that we refer to as the mesopontine tegmental anesthesia area (MPTA). This architecture stands in contrast to the classical notion that anesthetic molecules themselves directly mediate anesthetic induction after global distribution by the vascular circulation. The MPTA came to light in a systematic survey of the rat brain as a singular locus at which microinjection of minute quantities of GABAergic anesthetics is able to reversibly induce a state resembling surgical anesthesia. The rapid onset of anesthesia, the observed target specificity, and the fact that effective doses are far too small to survive dilution during vascular redistribution to distant areas in the central nervous system are all incompatible with the classical global suppression model. Lesioning the MPTA selectively reduces the animal's sensitivity to systemically administered anesthetics. Taken together, the microinjection data show that it is sufficient to deliver γ-aminobutyric acid A receptor (GABAA-R) agonists to the MPTA to induce an anesthesia-like state and the lesion data indicate that MPTA neurons are necessary for anesthetic induction by the systemic route at clinically relevant doses. Known connectivity of the MPTA provides a scaffold for defining the specific projection pathways that mediate each of the functional components of anesthesia. Because MPTA lesions do not induce coma, the MPTA is not a key arousal nucleus essential for maintaining the awake state. Rather, it appears be a "gatekeeper" of arousal function, a major element in a flip-flop switching mechanism that executes rapid and reversible transitions between the awake and the anesthetic state.
Transient loss of consciousness (TLOC), frequently triggered by perturbation in essential physiological parameters such as pCO2or O2, is considered a passive consequence of generalized degradation in high-level cerebral functioning.However, the fact that it is almost always accompanied by atonia and loss of spinal nocifensive reflexes suggests that it might actually be part of a "syndrome" mediated by neural circuitry, and ultimately be adaptive. Widespread suppression by molecules distributed in the vasculature is also the classical explanation of general anesthesia. Recent data, however, suggest that anesthesia is due, rather, to drug action at a specific brainstem locus, the mesopontine tegmental anesthesia area (MPTA), with the spectrum of anesthetic effects resulting from secondary recruitment of specific axonal pathways. If so, might the MPTA also be involved in TLOC induced by hypercapnia and hypoxia?We exposed rats to gas mixtures that provoke hypercapnia and hypoxia and asked whether cell-selective lesions of the MPTA affect TLOC. Entry into TLOC, monitored as time to loss of the righting reflex (LORR) was unaffected. However, resumption of the righting reflex (RORR), and of response to pinch stimuli (ROPR), was significantly delayed. The extent of both effects correlated with the extent of damage in the MPTA, but was unrelated to damage that extended beyond the borders of the MPTA. The results implicate neurons in a specific common-core region of the MPTA in TLOC induced by both forms of asphyxia. This is the same area responsible for general anesthesia induced by GABAergic anesthetic agents. This implies the involvement of a common set of brain nuclei and dedicated axonal pathways, rather than nonspecific global suppression, in the mechanism mediating all three instances of TLOC.
A quest for a systems-level neuroscientific basis of anesthetic-induced loss and return of consciousness has been in the forefront of research for the past 2 decades. Recent advances toward the discovery of underlying mechanisms have been achieved using experimental electrophysiology, multichannel electroencephalography, magnetoencephalography, and functional magnetic resonance imaging. By the careful dosing of various volatile and IV anesthetic agents to the level of behavioral unresponsiveness, both specific and common changes in functional and effective connectivity across large-scale brain networks have been discovered and interpreted in the context of how the synthesis of neural information might be affected during anesthesia. The results of most investigations to date converge toward the conclusion that a common neural correlate of anesthetic-induced unresponsiveness is a consistent depression or functional disconnection of lateral frontoparietal networks, which are thought to be critical for consciousness of the environment. A reduction in the repertoire of brain states may contribute to the anesthetic disruption of large-scale information integration leading to unconsciousness. In future investigations, a systematic delineation of connectivity changes with multiple anesthetics using the same experimental design, and the same analytical method will be desirable. The critical neural events that account for the transition between responsive and unresponsive states should be assessed at similar anesthetic doses just below and above the loss or return of responsiveness. There will also be a need to identify a robust, sensitive, and reliable measure of information transfer. Ultimately, finding a behavior-independent measure of subjective experience that can track covert cognition in unresponsive subjects and a delineation of causal factors versus correlated events will be essential to understand the neuronal basis of human consciousness and unconsciousness.
General anesthetic agents induce loss of consciousness coupled with suppression of movement, analgesia and amnesia. Although these diverse functions are mediated by neural structures located in wide-ranging parts of the neuraxis, anesthesia can be induced rapidly and reversibly by bilateral microinjection of minute quantities of GABAA -R agonists at a small, focal locus in the mesopontine tegmentum (MPTA). State switching under these circumstances is presumably executed by dedicated neural pathways and does not require widespread distribution of the anesthetic agent itself, the classical assumption regarding anesthetic induction. Here we asked whether these pathways serve each hemisphere independently, or whether there is bilateral redundancy such that the MPTA on each side is capable of anesthetizing the entire brain. Either of two GABAA -R ligands were microinjected unilaterally into the MPTA in awake rats, the barbiturate modulator pentobarbital and the direct receptor agonist muscimol. Both agents, microinjected on either side, induced clinical anesthesia including bilateral atonia, bilateral analgesia and bilateral changes in cortical activity. The latter was monitored using c-fos expression and electroencephalography. This action, however, was not simply a consequence of suppressing spike activity in MPTA neurons as unilateral (or bilateral) microinjection of the local anesthetic lidocaine at the same locus failed to induce anesthesia. We propose a model of the state-switching circuitry that accounts for the bilateral action of unilateral microinjection and also for the observation that inactivation with lidocaine is not equivalent to inhibition with GABAA -R agonists. This is a step in defining the overall switching circuitry that underlies anesthesia. This article is protected by copyright. All rights reserved.