Rapid erasure of long-term memory associations in the cortex by an inhibitor of PKM zeta.
ABSTRACT Little is known about the neuronal mechanisms that subserve long-term memory persistence in the brain. The components of the remodeled synaptic machinery, and how they sustain the new synaptic or cellwide configuration over time, are yet to be elucidated. In the rat cortex, long-term associative memories vanished rapidly after local application of an inhibitor of the protein kinase C isoform, protein kinase M zeta (PKMzeta). The effect was observed for at least several weeks after encoding and may be irreversible. In the neocortex, which is assumed to be the repository of multiple types of long-term memory, persistence of memory is thus dependent on ongoing activity of a protein kinase long after that memory is considered to have consolidated into a long-term stable form.
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ABSTRACT: Balanced control of neural progenitor maintenance and neuron production is crucial in establishing functional neural circuits during brain development, and abnormalities in this process are implicated in many neurological diseases. However, the regulatory mechanisms of neural progenitor homeostasis remain poorly understood. Here, we show that mammalian target of rapamycin (mTOR) is required for maintaining neural progenitor pools and plays a key role in mediating glycogen synthase kinase 3 (GSK3) signaling during brain development. First, we generated and characterized conditional mutant mice exhibiting deletion of mTOR in neural progenitors and neurons in the developing brain using Nestin-cre and Nex-cre lines, respectively. The elimination of mTOR resulted in abnormal cell cycle progression of neural progenitors in the developing brain and thereby disruption of progenitor self-renewal. Accordingly, production of intermediate progenitors and postmitotic neurons were markedly suppressed. Next, we discovered that GSK3, a master regulator of neural progenitors, interacts with mTOR and controls its activity in cortical progenitors. Finally, we found that inactivation of mTOR activity suppresses the abnormal proliferation of neural progenitors induced by GSK3 deletion. Our findings reveal that the interaction between mTOR and GSK3 signaling plays an essential role in dynamic homeostasis of neural progenitors during brain development.Development 10/2014; · 6.27 Impact Factor
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ABSTRACT: Patienten und Methode Im Zeitraum von September 1997 bis Dezember 2008 wurde im Rahmen des klinikinternen Polytrauma-CT-Algorithmus (CT: Computertomographie) bei 95 Patienten eine Darm- und Mesenterialverletzung (DMV) verdächtigt, von denen 55 operiert und 40 nicht operiert wurden. Die initialen CT-Untersuchungen wurden retrospektiv unter dem Aspekt der CT-Befunde einer DMV ausgewertet und die Treffgenauigkeit in Bezug auf die Unterscheidung von operationspflichtigen und konservativ therapierbaren Verletzungen bestimmt. Ergebnisse Freie Flüssigkeit ist das einzige CT-Zeichen mit einer hohen Sensitivität (96 %). Spezifische, jedoch selten nachweisbare CT-Zeichen für eine operationspflichtige DMV sind extraluminale Luft (98 %), mesenterialer Kontrastmittelaustritt (90 %), pathologisches Darmwandenhancement (100 %) und der Abbruch von Mesenterialgefäßen (97 %). Kein CT-Zeichen war zugleich sensitiv und spezifisch. Diskussion Patienten mit operationspflichtiger DMV haben signifikant mehr positive CT-Zeichen als konservativ therapierbare Patienten der Vergleichsgruppe. Eine operationspflichtige DMV kann verdächtigt werden, wenn in der initialen Untersuchung ein spezifisches oder mindestens 3 positive CT-Zeichen nachweisbar sind.Trauma und Berufskrankheit 09/2013; 15(3):193-202.
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ABSTRACT: Methamphetamine (MA) is a toxic, addictive drug shown to modulate learning and memory, yet the neural mechanisms are not fully understood. We investigated the effects of 2 weekly injections of MA (30 mg/kg) on working memory using the radial 8-arm maze (RAM) across 5 weeks in adolescent-age mice. MA-treated mice show a significant improvement in working memory performance 1 week following the first MA injection compared to saline-injected controls. Following 5 weeks of MA abstinence mice were re-trained on a reference and working memory version of the RAM to assess cognitive flexibility. MA-treated mice show significantly more working memory errors without effects on reference memory performance. The hippocampus and dorsal striatum were assessed for expression of glutamate receptors subunits, GluA2 and GluN2B; dopamine markers, dopamine 1 receptor (D1), dopamine transporter (DAT) and tyrosine hydroxylase (TH); and memory markers, protein kinase M zeta (PKMζ) and protein kinase C zeta (PKCζ). Within the hippocampus, PKMζ and GluA2 are both significantly reduced after MA supporting the poor memory performance. Additionally, a significant increase in GluN2B and decrease in D1 identifies dysregulated synaptic function. In the striatum, MA treatment increased cytosolic DAT and TH levels associated with dopamine hyperfunction. MA treatment significantly reduced GluN2B while increasing both PKMζ and PKCζ within the striatum. We discuss the potential role of PKMζ/PKCζ in modulating dopamine and glutamate receptors after MA treatment. These results identify potential underlying mechanisms for working memory deficits induced by MA.Frontiers in Behavioral Neuroscience 12/2014; 8:438. · 4.16 Impact Factor
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(K.S.), the W. M. Keck Foundation (K.S.), and the Searle
Supporting Online Material
Materials and Methods
Figs. S1 to S6
29 May 2007; accepted 22 June 2007
Published online 12 July 2007;
Include this information when citing this paper.
Rapid Erasure of Long-Term
Memory Associations in the Cortex
by an Inhibitor of PKMz
Reut Shema,1Todd Charlton Sacktor,2Yadin Dudai1*
Little is known about the neuronal mechanisms that subserve long-term memory persistence in the
brain. The components of the remodeled synaptic machinery, and how they sustain the new synaptic or
cellwide configuration over time, are yet to be elucidated. In the rat cortex, long-term associative
memories vanished rapidly after local application of an inhibitor of the protein kinase C isoform,
protein kinase M zeta (PKMz). The effect was observed for at least several weeks after encoding and
may be irreversible. In the neocortex, which is assumed to be the repository of multiple types of
long-term memory, persistence of memory is thus dependent on ongoing activity of a protein kinase
long after that memory is considered to have consolidated into a long-term stable form.
tion (LTP) in hippocampus and for sustaining
hippocampus-dependent spatial memory (1). Itis
to store multiple types of long-term memory in
the mammalian brain (2, 3). We set out to deter-
ersistent phosphorylation by the atypical
for maintenance of long-term potentia-
is critical for storage of long-term memory in
We trained rats on conditioned taste aversion
(CTA) (5) using saccharin as the conditioned
stimulus (CS), and 3 days later, microinfused the
selective PKMz pseudosubstrate inhibitor ZIP
(1, 6) bilaterally into the IC. Controls received
vehicle only. We tested one ZIP group 1 week
later and another 1 month later. ZIP in the IC
blocked CTA memory in both groups [one-way
analysis of variance (ANOVA), F(2,16) = 7.61,
P < 0.005] (Fig. 1A). Post hoc comparisons
unveiled no difference between the ZIP groups;
however, each was different from the control
(P < 0.05). The difference persisted in extinc-
tion[repeated-measures ANOVA, group effect,
F(2,16) = 6.17, P < 0.01, test effect, F(2,32) =
8.91, P < 0.001]. The ZIP groups did not differ
from each other, but each was different from
control (P < 0.05).
Although consolidation of memory in the IC
is considered to be over within hours, judged by
loss of vulnerability to amnesic agents (7), we
wondered whether the vulnerability to ZIP re-
flects a longer consolidation process (8). We ad-
ministered ZIP at various times 3 to 25 days after
training, followed by CTA testing. The PKMz
Fig. 1. Erasureoflong-termCTAmemorybyasingleapplicationofthePKMz
inhibitor ZIP into the IC. (A) ZIP was administered 3 days after training, and
memorywas tested 1 week or1 month later.Controls weretested at 1month.
Data are shown for three successive tests, 1 day apart. The dashed line
for the CS may develop over time in naïve or CTA-extinguished rats, but AI
usually does not decline below 20 to 30 even in naïve rats. For statistics, see
text. (B) ZIP was microinfused into the IC at the indicated times after training,
which was a single conditioning session (3 and 7 days groups) or two
successive conditioning sessions, a day apart (25 days group). Memory was
tested 2 hours (3 and 7 days groups) or 1 day (25 days group) later. (C) Rats
weretrained onCTA and tested once3dayslater,followed 1to4 min laterby
microinfusion of ZIP into the IC. Although spontaneous recovery was seen in
the no-test interval between days 4 and 12 in the control group, the ZIP-
reinstatement (LiCl, day 13).
1Department of Neurobiology, The Weizmann Institute of
Science, Rehovot 76100, Israel.2Departments of Physiol-
ogy, Pharmacology, and Neurology, The Robert F. Furchgott
Center for Neural and Behavioral Science, SUNY Downstate
Medical Center, 450 Clarkson Avenue, Brooklyn, NY 11203,
*To whom correspondence should be addressed. E-mail:
VOL 317 17 AUGUST 2007
[(Fig. 1B); P < 0.001 for the difference between
ZIP groups at 3 days and 7 days and the controls,
P < 0.005 for the difference between the 25 days
group and control]. There is no evidence, there-
fore, for closure of a consolidation window even
after several weeks. The effect of ZIP on long-
term memory is rapid [within 2 hours at most;
intensifying training [(Fig. 1B), at 25 days after
two successive CTA trainings to the same taste].
inhibitor is unique to the CS used, we replaced
saccharin with glycine. ZIP was administered 3
days after training. Scrambled inactive ZIP was
used as control (1). A test 1 day later showed an
and 98.2 ± 1.05 in the control (n = 8 each, P <
0.005). The effect is thus not unique to the CS
used and requires inhibition of PKMz activity.
In the experiments above, ZIP was admin-
istered before the first test. Because reactivation-
induced reconsolidation may reinforce the
memory trace (9–11), we wondered whether re-
trieval under conditions that promote reconsoli-
dation (8) might render the trace immune to
PKMz inhibition. We subjected rats to two CTA
week later [before ZIP, AI = 94.5 ± 2.76, n = 10;
retested a day later. The effect of ZIP was not
eliminated [after ZIP, 58.68 ± 7.9; after vehicle,
92.1 ± 3.8; F(1,16) = 12.27, P < 0.005].
To examine the possibility that the inhibitor
blocks memory performance only transiently, we
continued testing ZIP-treated rats over time, to
unveil spontaneous recovery, and in addition,
after about 2 weeks, reapplied the unconditioned
stimulus (UCS) to elicit potential reinstatement.
None of these manipulations yielded evidence
Repeated-measures ANOVA on days 4 and 12
ery might occur) shows significant group effect,
F(1,12) = 5.79, P < 0.05, and a trend toward
Post hoc comparisons reveal nonsignificant
difference between groups on test day 4 (P =
0.28), but significant difference on day 12 (P <
0.01). All in all this indicates spontaneous
recovery in the control but not in the ZIP group.
The lack of expected extinction in the control
between days 12 and 14 (paired t test, P = 0.84)
suggests a reinstatement effect. No evidence for
an effect from UCS reapplication was observed
in the ZIP group.
Can ZIP disrupt more than one association at
a time? Rats were trained on CTA to saccharin
(CS1), and 2 days later to glycine (CS2). These
tastants are perceived differently (12). One week
later, a test schedule was initiated in which the
rats (n = 8) were tested on CS1 and CS2, con-
secutively, 1 day apart over 6 days. Both CS1-
UCS and CS2-UCS associations were disrupted:
3.16 in the control (n = 7), 70.3 ± 7.09 in the ZIP
group [F(1,13) = 8.41, P < 0.05]. AI on the first
test for CS2 association was 97.6 ± 0.97 in the
control, 78.9 ± 5.8 in the ZIP group [F(1,13) =
8.61, P < 0.05]. No significant difference was
detected among groups in extinction rate, indi-
cating lack of recovery from the ZIP effect on
repetitive testing [repeated-measures ANOVA,
group × test interactions, F(2,26) < 1, not
We tested the effect of the PKMz inhibitor on
the ability to encode, as opposed to retain, CTA
memory in the IC. First, we microinfused ZIP
into the IC 2 hours before exposure to a glycine
CS in CTA training and tested 3 days later. We
found no effect of ZIP on acquisition of CTA
[ZIP group,85.4 ±5.0,n= 8;vehicle,87.4 ±5.9,
n = 7; one-way ANOVA, F(1,13) <1, P = 0.81].
Similar results were obtained using saccharin as
the CS [ZIP group, 79.6 ± 3.9, n = 6; vehicle,
80.0 ± 3.8, n = 10; one-way ANOVA, F(1,14)
<1, P = 0.95]. Second, rats that were trained on
CTAtosaccharinand thentreatedwith ZIP,were
subjected a week later to a new CTA training to
and the control rats in their ability to reacquire
CTA[ZIP group 93.2 ±2.3, n =9; vehicle 95.0 ±
2.9, n = 5, tested 3 days after retraining; one-way
ANOVA, F(1,12) <1, P = 0.62].
The IC subserves detection and consolidation
of taste familiarity (13–17). We used two par-
disrupts taste familiarity once formed. The first is
latent inhibition (LI) (17). Preexposure to the CS
in a LI protocol attenuates later CTA training to
the same CS; hence, CTA performance serves as
a familiarity detector (17). Introduction of ZIP
LI protocol had no effect on LI [(Fig. 2A); one-
0.005]. Post hoc comparisons showed no sig-
nificant difference between the ZIP-LI and the
vehicle-LI groups; however, both groups were
significantly different from the no-LI group (P <
0.01). Repeated-measures ANOVA showed sig-
nificant group effect, F(2,37) = 6.83, P < 0.005,
and significant test effect, F(2,74) = 15.94, P <
0.001. Again, post hoc comparisons showed no
significant difference between the two LI groups,
and each of these groups was significantly
different from the no-LI group (P < 0.01).
We also examined attenuation of neophobia
(18). Here, rats are presented with an unfamiliar
tastant that invokes fear-of-the-new and then
repeatedly presented with the same tastant. Over
time, the neophobia decreases, serving as a
measure of familiarity. PKMz inhibition had no
effect in this paradigm [(Fig. 2B), repeated-
measures ANOVA, significant attenuation of
neophobia in the repeating tests, F(8,112) = 21.01,
P < 0.001]; however, no significant difference was
seen between the groups, F(1,14) < 1, P= 0.85].
PKMz has been previously shown to main-
tain LTP andspatial memoryin the hippocampus
(1). The role of the hippocampus in CTA is still
unsettled (19). Hippocampal lesions do not
impair CTA and were even reported to enhance
it (20). Microinfusion of ZIP into the dorsal
hippocampus 3 days after CTA did not impair
CTA memory when tested a day after ZIP ad-
ministration (Fig. 3). If at all, there was a trend
toward memory enhancement [repeated-measures
ANOVA, F(1,16) = 2.98, P = 0.1]. Besides dem-
onstrating that PKMz in the hippocampus is not
essential for long-term CTA memory, these data
also indicate that the effect of ZIP on memory in
the IC is region-specific.
The effect of the PKMz inhibitor on long-
term CTA memory in IC is consistent with
Fig. 2. (A) In LI, familiarity with the tastant at-
tenuates the potency of that tastant to serve as CS
in subsequent CTA training (compare vehicle, LI
the IC after the exposure to the tastant in the LI
protocol (ZIP, LI training) has no effect on famil-
iarity. (B) Neophobia declines over repeated non-
reinforced exposures to the tastant (days 1 to 6).
Application of ZIP into the IC has no effect on
familiarity in this protocol either.
Fig. 3. PKMz inhibitor in the hippocampus does
not impair CTA memory. ZIP was microinfused
bilaterally into the hippocampus 3 days after CTA
training, and memory was tested starting a day
later. These data also demonstrate that the effect
of ZIP on CTA memory in the IC is region-specific.
17 AUGUST 2007VOL 317
reports that the IC is critical for consolidation,
storage, and extinction of CTA (7, 13, 21, 22).
CTA encoding is still incomplete and probably
includes subcortical structures (23), once the
association is formed, the IC is likely to store
elements of the associative hedonic or incentive
value of the CS (24). In contrast, whereas the IC
is documented to detect taste novelty that
facilitates encoding of CTA (4, 7, 13, 14, 21),
the present data are in line with the possibility
that taste familiarity per se is not stored in the IC.
So far, we have found no evidence that the
is reversible; hence, we heuristically propose that
the PKMz inhibitor might practically erase some
long-term memory associations. We are aware of
the difficulties in concluding that a memory trace
in performance that is attributed to that trace. This
on whether amnesia is a storage or a retrieval
deficit, yet does not preclude the assumption that
amnesia is a storage deficit (8, 9, 25).
Recent data on reactivation-dependent vul-
nerability of memory to amnesic agents (8, 9)
reemphasize the frailty of the engram—an attri-
bute long recognized by cognitive psychologists
neuroscientists.Ourdata reinforcethenotion that
memory traces are prone to swift interferences
long after their encoding. In contrast to these ear-
lierstudies, however,no reactivation is needed to
render the trace susceptible to ZIP. The possi-
bility that the trace reactivates implicitly is low
given that classical amnesic agents, e.g., macro-
molecular synthesis inhibitors, have no effect on
the long-term CTA trace that has not been
reactivated (7, 13).
The possibility cannot yet be excluded that
vulnerability of memory to PKMz inhibition in
cortex might wane. If so, then the temporal
window of “cellular consolidation,” i.e., the sta-
bilization process that is postulated to occur in
synapses and cell bodies after memory encoding
(8), lingers far longer than originally thought.
This conclusion is even more striking given that
excluding a “systems consolidation” process in
which the hippocampal trace invades neocortex
over days to weeks (8). An alternative possibility
is that PKMz permanently maintains long-term
memory and, thus, is a target for amnesic agents
as long as the memory persists. In this case,
defining consolidation on the basis of vulnerabil-
How does PKMz inhibition disrupt memory
is a guide, the effect of PKMz might be on the
microstructure of preexisting synapses, resulting
in a doubling of the number of functional post-
synaptic AMPA-type glutamate receptors (27).
even weeks after learning, are not indelible mod-
ifications of synaptic structure, but remain de-
are capable of rapid and dynamic alterations by
experimental manipulation or, perhaps, in the
course of incorporation of new experience into
associative knowledge schemas in cortex (28).
The idea that persistent enzymatic activity keeps
memory going has been raised on the basis of
theoretical considerations (29–31). The finding
that this takes place, via PKMz, not only in LTP
memory in neocortex, has, in addition to theo-
retical implications, potential clinical signifi-
References and Notes
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Supporting Online Material
Materials and Methods
26 April 2007; accepted 6 July 2007
Detection of Near-Atmospheric
Concentrations of CO2by an Olfactory
Subsystem in the Mouse
Ji Hu,1,2* Chun Zhong,1,2* Cheng Ding,1Qiuyi Chi,3Andreas Walz,4Peter Mombaerts,4
Hiroaki Matsunami,3Minmin Luo1†
Carbon dioxide (CO2) is an important environmental cue for many organisms but is odorless to humans.
It remains unclear whether the mammalian olfactory system can detect CO2at concentrations
around the average atmospheric level (0.038%). We demonstrated the expression of carbonic
anhydrase type II (CAII), an enzyme that catabolizes CO2, in a subset of mouse olfactory neurons that
express guanylyl cyclase D (GC-D+neurons) and project axons to necklace glomeruli in the olfactory
bulb. Exposure to CO2activated these GC-D+neurons, and exposure of a mouse to CO2activated
bulbar neurons associated with necklace glomeruli. Behavioral tests revealed CO2detection thresholds
of ~0.066%, and this sensitive CO2detection required CAII activity. We conclude that mice detect
CO2at near-atmospheric concentrations through the olfactory subsystem of GC-D+neurons.
animal respiration, plant photosynthesis, and the
decomposition of organic matter. CO2signals
regulate many insect innate behaviors, such as
seeking food and hosts, avoiding stressful envi-
ronments, and ovipositioning (3–6). CO2has no
discernable odor to humans, but at high concen-
O2 is an olfactory stimulus for many
invertebrates (1, 2). CO2levels fluctuate
locally with biological activities, such as
sensation in the nasopharynx (7). Carbonic an-
hydrase (CA), an enzyme that is implicated in
chemoreceptors (2, 8), is expressed in a subset
of olfactory sensory neurons (OSNs) in several
vertebrate species (8,9).Studies indicate that rats
can detect CO2at levels above 0.5% (10, 11). It
remains unknown whether mammals can detect
VOL 31717 AUGUST 2007