Effects of anesthesia on functional activation of
cerebral blood flow and metabolism

Yasuaki Nakao, Yoshiaki Itoh, Tang-Yong Kuang, Michelle Cook, Jane Jehle, and Louis Sokoloff*

Laboratory of Cerebral Metabolism, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892-4030

Contributed by Louis Sokoloff, April 11, 2001

Functional brain mapping based on changes in local cerebral blood
flow (ICBF) or glucose utilization (ICMRgi,) induced by functional
activation is generally carried out in animals under anesthesia,
usually «-chloralose because of its lesser effects on cardiovascular,
respiratory, and reflex functions. Results of studies on the role of
nitric oxide (NO) in the mechanism of functional activation of ICBF
have differed in unanesthetized and anesthetized animals. NO
synthase inhibition markedly attenuates or eliminates the ICBF
responses in anesthetized animals but not in unanesthetized ani-
mals. The present study examines in conscious rats and rats
anesthetized with a-chloralose the effects of vibrissal stimulation
on ICMRgic and ICBF in the whisker-to-barrel cortex pathway and
on the effects of NO synthase inhibition with N©-nitro-t-arginine
methyl ester (L-NAME) on the magnitude of the responses. Anes-
thesia markedly reduced the ICBF and ICMRgic responses in the
ventral posteromedial thalamic nucleus and barrel cortex but not
in the spinal and principal trigeminal nuclei. -NAME did not alter
the ICBF responses in any of the structures of the pathway in the
unanesthetized rats and also not in the trigeminal nuclei of the
anesthetized rats. In the thalamus and sensory cortex of the
anesthetized rats, where the ICBF responses to stimulation had
already been drastically diminished by the anesthesia, L-NAME
treatment resulted in loss of statistically significant activation of
ICBF by vibrissal stimulation. These results indicate that NO does
not mediate functional activation of ICBF under physiological
conditions.

whisker-to-barrel cortex pathway | cerebral glucose utilization |
deoxy["4C]glucose | functional brain imaging | iodo[4C]antipyrine

N euronal functional activation is normally associated with
increases in local cerebral glucose utilization (ICMRgi.) and
blood flow (ICBF) in anatomic units of the activated neural
pathways. These associations are now widely exploited to map
regions of the brain involved in specific neural and cognitive
processes. The mechanisms underlying the functional activation
of ICMRgi, are reasonably well understood. Glucose utilization
is increased by functional activation in direct proportion to the
increases in spike frequency in the afferent inputs to the
activated areas, and the increases are localized in neuropil and
not perikarya (1, 2). The increases in ICMR, appear to result
mainly from activation of Na*,K*-ATPase activity (2, 3), needed
to restore ionic gradients in the neuronal elements degraded by
the spike activity. Neuropil contains not only axonal and den-
dritic processes but also astroglial processes, and glutamate
stimulates glucose utilization in astroglia, also due in part to
activation of Nat ,K*-ATPase activity by coupled uptake of Nat
ions with the glutamate released during functional activation (4,
5). Glucose utilization is also increased to support the ATP-
dependent conversion to glutamine of the glutamate taken up by
the astroglia (6).

The mechanisms mediating the increases in ICBF during
functional activation are less clear. Roy and Sherrington (7)
hypothesized that the cerebral blood flow (CBF) is intrinsically
regulated by products of energy metabolism to adjust it to the
altered metabolic demands associated with changes in functional
activity. Subsequent findings that CBF is elevated by a rise in

www.pnas.org/cgi/doi/10.1073/pnas.121179898

Pco:, a fall in pH, or a lowering of PO2, expected consequences
of increased energy metabolism, and reduced by their changes in
the opposite direction, to be expected with decreased metabo-
lism (8), offered support to this hypothesis. There are now,
however, many other endogenous substances known to affect
cerebral blood vessels—e.g., neurotransmitters, neuromodula-
tors, nitric oxide, adenosine, adenine nucleotides, K*, prosta-
glandins, vasoactive intestinal peptide, etc. Some of these have
been considered as possible mediators, but not one, either alone
or in combination with others, has yet been proved to account
fully or even to be essential for the enhancement of CBF by
neuronal activation. Nitric oxide is a current popular candidate.
This potent vasodilator is synthesized in brain by two constitutive
isoforms of nitric oxide synthase, one in the vascular endothe-
lium and the other in neurons, and an inducible one in glia. There
have been a number of reports that activation of local CBF by
sensory stimulation is abolished or, at least, markedly diminished
by chemically induced inhibition of nitric oxide synthase (9-17).
Studies in knockout mice lacking either the endothelial or
neuronal nitric oxide synthase genes showed no loss of functional
activation of CBF, but when the remaining isoforms were
inhibited with drugs, results were obtained that were interpreted
as evidence of an essential role for neuronal nitric oxide synthase
in the CBF response to functional activation (18, 19). All of these
studies implicating nitric oxide were done, however, in anesthe-
tized animals. Studies in unanesthetized animals with quantita-
tive determinations of CBF have generally failed to confirm that
nitric oxide is essential for the functional activation of CBF. In
two studies in unanesthetized rats (20, 21), nonselective inhibi-
tion of all nitric oxide synthase isoforms by N°-nitro-L-arginine
methyl ester (L-NAME) had no significant effects on percent
enhancements of ICBF by vibrissal stimulation in all four ana-
tomical structures in the whisker-to-barrel cortex sensory path-
way that were examined. In another study in unanesthetized rats,
inhibition of neuronal nitric oxide synthase with 7-nitroindazole
did reduce but did not abolish activation of ICBF by vibrissal
stimulation in the thalamus and barrel cortex, stations of the
pathway containing higher level synapses, but it had no effects
on activation of ICBF in the principal and spinal trigeminal
nuclei, the stations with the primary synapses (22).

An obvious difference between studies supporting and not
supporting a role for nitric oxide in the functional activation of
CBF is the use of anesthesia, which has been reported to alter the
magnitude of functional activation of energy metabolism (23).
Therefore, to examine systematically the influence of anesthesia
on functional activation of energy metabolism and CBF and on
the role of nitric oxide, we have examined with quantitative

Abbreviations: CBF, cerebral blood flow; ICBF, local cerebral blood flow; ICMRgic, local
cerebral glucose utilization; L-NAME, NS-nitro-t-arginine methyl ester; VPM, ventral
posteromedial.

*To whom reprint requests should be addressed at: Laboratory of Cerebral Metabolism,
National Institute of Mental Health, Building 36, Room 14-07, 36 Convent Drive MSC 4030,
Bethesda, MD 20892-4030. E-mail: louis@shiloh.nimh.nih.gov.

The publication costs of this article were defrayed in part by page charge payment. This

article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.

§1734 solely to indicate this fact.

PNAS | June 19,2001 | vol.98 | no. 13 | 7593-7598

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methods the effects of anesthesia on baseline and functionally
activated glucose utilization and CBF in the stations of the
whisker-to-barrel cortex pathway before and after inhibition of
nitric oxide synthase activity.

Materials and Methods

Chemicals. Chemicals were obtained from the following sources:
2-deoxy-d-[1-!*C]glucose (2-deoxy[!*C]glucose; specific activity,
53 mCi/mmol) and 4-iodo[N-methyl-'4Clantipyrine,
iodo[!4C]antipyrine; specific activity, 54 mCi/mmol) from Du-
Pont NEN (Boston, MA); (L-NAME) and a-chloralose were
obtained from Sigma.

Animal Preparation. All procedures performed on the animals
were in strict accordance with the National Institutes of Health
Guide for Care and Use of Laboratory Animals and approved by
the local Animal Care and Use Committee.

‘Normal adult male Sprague~Dawley rats (308-398 g) were
"purchased from Charles River Laboratories and maintained in a
climate-controlied room on a normal 12-h light/dark cycle with
food and. water available ad libitum. They were fasted but
allowed free access to water for 16 h before the experiment. The
rats were anesthetized with halothane (5% for induction, 0.8-
1.0% for miaintenarice) in 70% Nz0/30% Op. Rats to be studied
later under a-chloralose anesthesia were intubated with an
endotracheal catheter while under halothane anesthesia and
then mechanically ventilated with the same anesthetic mixture
until completion of the surgical preparation. Polyethylene cath-
eters (PE 50; Clay Adams) were inserted in both femoral arteries
and veins; one arterial catheter was used for recording mean
arterial blood pressure and the other for sampling of arterial
blood. Catheter length was fixed at 16 cm to simplify correction
for lag and dead space washout in the sampling system (24, 25).
One venous catheter was used for injection of tracer and
L-NAME or saline vehicle and the other for a-chloralose when
administered. Lidocaine ointment (5%) was applied to the
surgical wounds after closure. Rats to be studied in the conscious
state were fitted with a loose-fitting plaster cast that was applied
to the pelvic area and taped to a lead brick to prevent locomo-
tion; anesthetized rats did not require such restraint. Body
temperature was continuously monitored by a rectal probe and
maintained at 37°C throughout the surgical and subsequent
experimental procedures by a thermostatically controlled
heating lamp or heating pad (model 73A; Yellow Springs
Instruments).

Anesthetic Conditions During Experimental Procedures. |CBF and
ICMRgic were determined in conscious unanesthetized rats and
in rats under a-chloralose anesthesia. a-Chloralose anesthesia
was chosen because it is believed to be least depressant of neural
functions. It is often used in studies of functional activation, and
we administered it as described in one such previous study (26).
Immediately after termination of the halothane anesthesia, an
acute dose of a-chloralose (50 mg/kg), dissolved in physiological
saline, was injected i.v. and followed by a continuous i.v. infusion
at a rate of 40 mg/kg per h. The rats were ventilated with 30%
oxygen/70% nitrogen throughout the procedure. Unanesthe-
tized rats received equivalent injections and infusions of saline.
ICBF and ICMR,), were determined about 3 h after termination
of the halothane anesthesia in the unanesthetized group and
about 45 min after switching from halothane to a-chloralose in
the anesthetized group.

Inhibition of Nitric Oxide Synthase. Effects of nitric oxide synthase
inhibition on baseline and functionally activated [CBF and
ICMRg, were studied in unanesthetized and anesthetized rats.
About 30 min before determination of ICBF or ICMRg, L
NAME (30 mg/kg), dissolved in normal saline, was infused iv.

7594 | www.pnas.org/cgi/doi/10.1073/pnas. 121179898

over a 3-min period; control rats were infused with equivalent
amounts of saline.

Vibrissal Stimulation. Vibrissae were stimulated unilaterally by
stroking the whiskers on the left side of the face continuously
with a soft paint brush at a frequency of 2-3/s. It was initiated
simultaneously with the onset of determination of either ICBF
or ICMRgi, and continued throughout the 1-min period of ICBF
determination or the 45 min needed for ICMR,). determination.
The whiskers on the right side had previously been clipped flush
with the skin to avoid spurious stimulation on the control side.

Monitoring of Physiological Variables. Several physiological vari-
ables with influence on CBF and metabolism were measured
immediately after the surgery and periodically thereafter. Mean
arterial blood pressure was monitored with a blood pressure
analyzer (model 300; Digi-Med, Louisville, KY) that had been
calibrated with an air-damped mercury manometer. Arterial
blood carbon dioxide (PaCOz) and oxygen (PaOz) tensions and
pH were measured with a blood gas analyzer (model 288 Blood
Gas System; CIBA-Corning Diagnostics Corp., Medfield, MA).
Arterial plasma glucose levels were determined in a Glucose
Analyzer 2 (Beckman Instruments, Fullerton, CA).

Measurements of ICBF and ICMRgic. ICMRgi- was determined by the
quantitative autoradiographic 2-deoxy[!4C]glucose method (27).
ICBF was determined by the quantitative autoradiographic
iodo[!4C]antipyrine method as described by Sakurada et al. (28),
except that the iodo[!*C]antipyrine (40 wCi in 0.8 ml of physi-
ological saline) was infused continuously via the femoral venous
catheter by a computer-driven infusion pump (model 2400-003;
Harvard Apparatus) programmed to produce an approximately
linear rise in arterial tracer concentration throughout the 1-min
experimental period. Corrections for delay and dispersion in the
arterial sampling system were included in the computation of
ICBF (24, 25).

ICBF and ICMR,g), were determined bilaterally in four stations
of the whisker-to-barrel cortex pathway, i.e., spinal trigeminal
and principal sensory trigeminal nuclei, ventral posteromedial
(VPM) nucleus of the thalamus, and the barrel field of the
somatosensory cortex. Values for ICBF or ICMR, in the
homologous structures of the stimulated and unstimulated sides
were compared to assess the effects of stimulation. ICBF or
ICMRgic was also determined bilaterally in four structures
outside this sensory pathway (i.c., cerebellar white matter,
caudate putamen, primary motor cortex, and nucleus accum-
bens) to evaluate possible effects of vibrissal stimulation, L-
NAME, and anesthesia on structures not directly related to the
activation. Anatomical structures were identified in the autora-
diograms by comparison of the thionine-stained sections of brain
corresponding to the autoradiograms with the rat brain atlas of
Paxinos and Watson (29).

Statistical Analyses. Data are presented as means + SEM. Sta-
tistical significance of differences in ICBF or ICMRgi, between
stimulated and unstimulated sides was determined by paired
tests with Bonferroni correction. Significance of the effects of
a-chloralose and/or L-NAME on baseline values of ICBF and
ICMR, in the unstimulated control side was evaluated by
unpaired ¢ tests with Bonferroni correction. The Kruskal-Wallis
test (one-way ANOVA by ranks) was used to determine statis-
tical significance of the effects of anesthesia and/or L-NAME on
percent differences in ICBF or ICMR,;. between unstimulated
and stimulated sides followed by Dunn’s test if statistical signif-
icance (P < 0.05) was found.

Nakao et al.
Table 1. Physiological variables

 

 

 

Conscious a-Chloralose anesthesia
Physiological variables Saline control t-NAME Saline control L-NAME
Before measurement of CBF n=6 n=6 n=7 n=6
Mean arterial blood pressure, mmHg 118 +2 152 + 2*** 1425 141 + 3t
Pacoz, mmHg 34+1 32+1 3241 3341
Paoz, mmHg 89 + 3 9342 138 + 6*** 128 +9
Arterial pH 7.45 + 0.01 7.45 + 0.01 7.49 + 0.01*** 7.48 + 0.01
Plasma glucose, mM 7.6 + 0.3 6.9 + 0.2 65+ 0.3 5.7+0.3
Before measurement of cerebral glucose utilization n=6 n=6 n=5 =5
Mean arterial blood pressure, mmHg 12124 158 + 4*** 12144 140 + 6
Pacoz, mmHg 3621 3641 33 +1 34+1
Pao2, mmHg 90 +3 95 +2 128 + B*** 111+ 10
Arterial pH 7.44 + 0.01 7.44 + 0.01 7.48 + 0.01** 7.45 + 0.01
Plasma glucose, mM 7140.4 6.2 + 0.1 6.2 + 0.1 5.8+ 0.3

 

Values are means + SEM of number of animals in parentheses. *, P < 0.05; **, P< 0.01; ***, P< 0.001 compared to the conscious saline controls. t, P< 0.001

compared to the a-chloralose-anesthetized saline-treated groups.

Results

Physiological Variables. Rats anesthetized with a-chloralose were
hypersensitive to loud sounds, and hand clapping evoked myo-
clonic responses. Their arterial Pao2 and pH levels were higher
than those of the unanesthetized groups, undoubtedly because of
the mechanical ventilation (Table 1). L-NAME raised systemic
arterial blood pressure substantially in both conscious and
anesthetized groups, evidence that at least systemic endothelial
nitric oxide synthase activity was effectively inhibited (Table 1).
Other physiological variables were unaffected by L-NAME.

Effects of a-Chloralose Anesthesia and L-NAME on Baseline CBF and
Glucose Utilization. Unilateral vibrissal stimulation produced no
statistically significant side-to-side differences in ICMRgi, or
1CBF in any of the structures outside the whisker-to-barrel cortex

pathway (Table 2). Effects of a-chloralose anesthesia and L-
NAME on baseline ICMRg), and ICBF were ascertained from
their effects in these structures outside the pathway, as well as in
the structures of the whisker-to-barrel cortex pathway on the
unstimulated side of the brain.

a-Chloralose anesthesia markedly lowered baseline levels of
both ICMR, and ICBF in all of the structures outside the
whisker-to-barrel cortex pathway that were examined (Table 2).
These effects on ICBF are similar to those previously observed
with isoflurane anesthesia (30). Anesthesia also significantly
reduced baseline ICMR, in all of the stations of the pathway on
the unstimulated control side (Fig. 1) but reduced ICBF statis-
tically significantly only in the VPM thalamic nucleus and
sensory cortex and not in the two trigeminal nuclei (Fig. 2).

L-NAME administration reduced baseline ICBF in all of the

Table 2. Effects of a-chloralose anesthesia and t-NAME on CBF and glucose utilization in some representative cerebral structures

outside whisker-to-barrel cortex sensory pathway
CBF, ml/100 g/min

Cerebral glucose utilization, zmol/100 g/min

 

 

Structure and treatment n Left side Right side % L-R diff. n Left side Right side % L-R diff.
Cerebellar white matter
Conscious + saline 6 50+2 50+1 2+3 6 3342 34+2 3+2
Conscious + L-NAME 6 34 + 2** 35241 244 6 3141 31+1 0.342
Anesthetized + saline 7 24 + 2*** 24 +3 -12+4 5 18 + 1*** 81 —2+2
Anesthetized + t-NAME 6 18 +1 18+ 1 4+3 5 19+1 6+2
Caudate-putamen
Conscious + saline 6 164 + 13 165+ 11 0.542 6 120 +3 72144 1+1
Conscious + t-NAME 6 139 +8 138 +9 -1+2 6 129 +4 129 +4 -0.2+1
Anesthetized + saline 7 68 + 5*** 66 +6 -4+3 5 44 + 3*** 43 +3 -1+1
Anesthetized + t-NAME 6 47 +17 44+2 -6+2 5 49+1 47 +1 —3+2
Motor cortex
Conscious + saline 6 165 + 10 197 +17 19+5 6 103 +2 108 + 3 542
Conscious + t-NAME 6 139 +11 152 + 14 9+3 6 110+3 11543 4+1
Anesthetized + saline 7 58 + 4** 57+4 -14+1 5 37 + 3*** 37 +3 222
Anesthetized + L-NAME 6 42+ 17 42+ 1 + 5 4242 41+2 —2+2
Nucleus accumbens core
Conscious + saline 6 172 +11 171+ 14 -1+3 6 7242 7343 1+
Conscious + t-NAME 6 117 + 13* 112 +11 —-4+3 6 76244 74+3 —2+1
Anesthetized + saline 5 59 + 8** 59 +8 -1+2 5 32 + 3*** 32+3 -1+1
Anesthetized + L-NAME 6 34+2 36+1 524 5 3341 3341 -1+2

 

Values are means + SEM of number of animals indicated. Percent L-R diff. (left-right difference) is the mean of the individual side-to-side percent differences
obtained in each rat and not the percent difference between the means. Conscious saline-treated controls vs. anesthetized saline-treated or conscious
t-NAME-treated groups (only left side statistically analyzed), *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Bonferroni corrected). Anesthetized saline-treated vs.
anesthetized L-NAME-treated groups (only left side statistically analyzed), t, P < 0.05 (Bonferroni corrected).

Nakao et ai.

PNAS | June 19,2001 { vol.98 | no.13 | 7595

 
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Effects of a-chloralose anesthesia and .-NAME (30 mg/kg) on baseline rates of glucose utilization and on the increases in glucose utilization due to

unilateral vibrissal stimulation in four stations of the Whisker-to-barrel cortex pathway. Bar heights and error bars represent mean rates of local glucose
utilization + SEM obtained in the number of rats indicated. **, P < 0.01; ***, P< 0.001 for side-to-side differences between stimulated and unstimulated sides
(paired t test with Bonferroni correction). Bracketed P values are for comparison of the values of glucose utilization in the structures and sides indicated by the
brackets (unpaired t test with Bonferroni correction). t, P < 0.05; tt, P < 0.01, statistical significance of difference in percent increase in glucose utilization
attributable to stimulation in indicated condition from percent increases due to stimulation found in conscious saline-treated controls, determined by

Kruskal-Wallis test (one-way ANOVA by ranks) with Dunn’s test.

structures outside the whisker-to-barrel cortex pathway in both
conscious and anesthetized rats, although some of these reduc-
tions fell short of statistical significance after Bonferroni cor-
rection for multiple comparisons (Table 2). L-NAME also
tended to lower ICBF in the stations of the pathway on the
unstimulated side in both conscious and anesthetized rats, but
the reductions were statistically significant only in the spinal
trigeminal nucleus and barrel cortex of the anesthetized rats. As
previously observed (31, 32) NAME had no effects on baseline
ICMRagi (Table 2), except for a slight but statistically significant
increase in the sensory cortex of the conscious rats (Fig. 1).

Functional Activation of ICBF and ICMRgic. In conscious saline-
treated rats, whisker stroking on the left side of the face
markedly and statistically significantly increased both ICBF and
ICMR, in the left spinal and principal trigeminal nuclei and
right VPM thalamic nucleus and sensory cortex above their
levels in the contralateral homologous structures (Figs. 1 and 2).
Because both anesthesia and L-NAME altered the baseline levels
of ICMR,;, and ICBF in these structures on the unstimulated
side, the percent difference between the stimulated and un-
stimulated sides were used to assess their effects on the magni-
tude of functional activation.

a-Chloralose anesthesia did not significantly alter percent
increases in ICMR,), due to vibrissal stimulation in the spinal and
principal trigeminal nuclei from their values in unanesthetized
rats, but it markedly reduced them from 75 + 5% to 39 + 6%
(P < 0.01) in the VPM thalamic nucleus and from 54 + 3% to
28 + 4% (P < 0.05) in the barrel cortex (Fig. 1). In contrast,
anesthesia tended to enhance percent differences in ICBF be-

7596 | www.pnas.org/cgi/doi/10.1073/pnas.121179898

tween stimulated and unstimulated sides in both trigeminal
nuclei; the increase from 51 + 9% to 115 + 11% in the spinal
trigeminal nucleus reaching statistical significance (P < 0.01).
Anesthesia, however, markedly depressed the percent increases
due to stimulation from 30 + 6% to 12 + 1% (P < 0.05) in the
VPM thalamic nucleus and from 97 + 15 to 17 + 2% (P < 0.01)
in the barrel cortex (Fig. 2).

L-NAME tended to reduce the percent differences in ICMRgic
between stimulated and unstimulated sides in all structures of
the pathway in both conscious and anesthetized rats. None of
these reductions was statistically significant, however, and side-
to-side differences retained their statistical significance in all of
the structures (Fig. 1). Similarly, L-NAME had no statistically
significant effects on percent differences in ICBF between
stimulated and unstimulated sides in any of the stations of the
pathway in both the conscious and anesthetized rats (Fig. 2). It
did, however, tend to reduce slightly the effects of stimulation in
the VPM thalamic nucleus and barrel cortex, and in anesthetized
rats in which anesthesia had already markedly reduced both
baseline and stimulated ICBF, this was sufficient to eliminate the
statistical significance of the differences between stimulated and
unstimulated sides (Fig. 2).

Discussion

Functional brain imaging, as currently used, is based largely on
localizing changes in rates of CBF or glucose utilization, or
indices of such changes, that are induced by alterations in specific
neural functions. In humans it is usually applied in the conscious
state, but its use in animals is often limited to the anesthetized
state because of regulatory and/or physical constraints. General

Nakao et al.
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Effects of a-chloralose anesthesia and L-NAME (30 mg/kg) on baseline blood flow and increases in blood flow due to vibrissal stimulation in four stations

of the whisker-to-barrel cortex pathway. Bar heights and error bars represent mean rates of blood flow + SEM obtained in the number of rats indicated. *, P<
0.05; **, P< 0.01; ***, P< 0.001 for side-to-side differences between stimulated and unstimulated sides (paired ¢ test with Bonferroni correction). Bracketed
P values are for comparison of the values of blood flow in the structures and sides indicated by the brackets (unpaired t test with Bonferroni correction). t, P<
0.05; tt, P< 0.01, statistical significance of difference in percent increase in blood flow attributable to stimulation in indicated condition from percent increases
due to stimulation found in conscious saline-treated controls, determined by Kruskal-Wallis test (one-way ANOVA by ranks) with Dunn’s test.

anesthetics havé many effects on CBF and metabolism (33, 34),
but a-chloralose is commonly used in studies involving func-
tional activation because of its lesser effects on cardiovascular
and respiratory functions and reflex activities. Ueki et al. (35)
compared the effects of a-chloralose, halothane, pentobarbital,
and nitrous oxide on activation of ICMRg), evoked by forepaw
electrical stimulation in rats and found that the pattern of
metabolic activation in the primary somatosensory cortex under
a-chloralose anesthesia to be most similar to that in conscious
animals. Lindauer et al. (26) compared effects of «-chloralose,
halothane, and thiobarbiturate on ICBF responses to vibrissal
stimulation in the sensory cortex in rats; they concluded that
a-chloralose anesthesia is the one most suitable for studying
ICBF coupling to neuronal functional activation because the
ICBF responses were greater in a-chloralose-anesthetized
(+16.9%) than in thiobarbiturate-anesthetized (+10.6%) rats
and more stable than those in halothane-anesthetized rats. None
of these studies, however, included direct comparisons between
anesthetized and unanesthetized states.

a-Chloralose does, in fact, have many effects on neuronal
functions (36). These include not only analgesia and uncon-
sciousness, but also enhanced y-aminobutyric acid type A re-
ceptor activity similar to that produced by barbiturates (37) and
nonuniform localized reductions in ICMRgic (38). The results of
the present studies confirm that a-chloralose anesthesia lowers
ICMRg, throughout the brain (Table 2 and Fig. 1) and also show
that it tends to lower ICBF generally but not statistically signif-
icantly in all structures examined (Table 2 and Fig. 2). Further-
more, anesthesia altered not only baseline levels of ICMRgi, and
ICBF but also the magnitude of their responses to neuronal
functional activation that varied in the different stations of the
activated neural pathway (Figs. 1 and 2). Anesthesia had rela-

Nakao et al.

tively little effect on baseline and activated levels of ICMRg). and
ICBF in the spinal and principal trigeminal nuclei, but it mark-
edly diminished all of them in the VPM thalamic nucleus and
sensory cortex to the point where effects of functional activation,
particularly on ICBF, were barely, if at all, detectable (Figs. 1 and
2). It is of interest that the effects of anesthesia were mainly in
the VPM thalamic nucleus and sensory cortex, which contain
secondary and tertiary synapses of the pathway, but were small
or absent in the brainstem nuclei, the direct projection zones
from the peripheral receptors and sites of the primary synapses.
Inasmuch as evoked metabolic responses to functional activation
have been shown to be localized in neuropil and directly related
to spike frequency in the afferent inputs to the synapses (1, 2),
this finding suggests that the anesthesia inhibits mainly synaptic
functions and has little effect, if any, on the output of the
peripheral receptors. Such regional differences in the effects of
anesthesia on the functional activation of ICBF or CMR, could
lead to biases in identifying and localizing structures related to
specific neural functional activations.

a-Chloralose anesthesia is also widely used in studies of the
mechanisms of the ICBF responses to functional activation. The
role of nitric oxide has been intensively examined, and most of
these studies were carried out in anesthetized animals in which
ICBF was not measured quantitatively but assessed indirectly by
laser-Doppler flowmetry and only in the cerebral cortex. These
studies generally showed that nonspecific inhibition of nitric
oxide synthase activity with L-NAME or only of the neuronal
isoform with 7-nitroindazole markedly depresses or eliminates
\CBF responses to sensory stimulation in the sensory cortex
(9-17). In contrast, those studies that were carried out in
unanesthetized animals with quantitative measurements of |CBF
in several stations of the activated pathway have failed to show

PNAS | June 19,2001 | vol.98 | no. 13 | 7597

 
that inhibition of nitric oxide synthase activity by L-NAME
reduces the percent increases in ICBF evoked by vibrissal
stimulation in any of the stations of the pathway (20-22). In the
study by Gotoh et al. (22), inhibition of the neuronal nitric oxide
synthase with 7-nitroindazole had no effects in the spinal and
principal trigeminal nuclei but did diminish somewhat the in-
creases in ICBF evoked by vibrissal stimulation in the VPM
thalamic nucleus and barrel cortex. The restricted localization of
the effects of 7-nitroindazole to the thalamus and cortex, sites of
the higher level synapses in the pathway, suggests that the
diminished CBF responses might have been due more to the
effects of nitric oxide deprivation on neuronal functional acti-
vation than on the mechanisms of coupling of blood flow to
functional activation. Nitric oxide, produced by neurons, may be
involved in interneuronal signal transduction (39-41), which
might be disrupted by 7-nitroindazole, an inhibitor of neuronal
nitric oxide synthase, although it has been reported that
7-nitroindazole has no effects on baseline ICMRgi. in conscious
rats (42) and that it does not reduce the ICMRg;, responses to
whisker stimulation in rats under a-chloralose anesthesia (17).
The present studies show, however, that the anesthesia itself
markedly reduces these responses, at least in the VPM thalamic

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