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Brief Communication

Inactivation of the anterior cingulate cortex blocks
expression of remote, but not recent, conditioned
taste aversion memory
Hoi Ki Ding,1,2 Cátia M. Teixeira,1,3 and Paul W. Frankland1,2,4,5
1

Program in Neurosciences and Mental Health, The Hospital for Sick Children, Toronto M5G 1X8, Canada; 2Departmentof Physiology, University of Toronto, Toronto M5S 1A8, Canada; 3Graduate Program in Areas of Basic and Applied Biology(GABBA), Universidade do Porto, 4050-465 Porto, Portugal; 4Institute of Medical Science, University of Toronto,Toronto M5S 1A8, Canada

Previous studies have shown that medial prefrontal cortical regions, such as the anterior cingulate cortex (ACC), playa key role in the expression of remote spatial and contextual memory. To evaluate whether this role is conserved in
hippocampal-independent tasks we trained mice in the conditioned taste aversion (CTA) paradigm. Lidocaine-induced
inactivation of the ACC blocked the expression of CTA tested one month (remote), but not one day (recent), after
conditioning with either a weak or strong unconditioned stimulus (US). These data suggest that the ACC may play aconserved role in remote memory, regardless of memory strength or content.

The hippocampus plays an essential role in the formation ofspatial, contextual, and trace conditioning memories (Morris etal. 2003; Eichenbaum 2004; Kesner and Hopkins 2006). However,as these memories mature they may become additionally (or exclusively) dependent on extra-hippocampal structures, including
the medial prefrontal cortex (mPFC) (Frankland and Bontempi
2005; Squire and Bayley 2007). For example, lesions of the mPFC
(including the ACC and prelimbic cortex) disrupt expression ofmonth-old (remote), but not day-old (recent), trace eye-blink conditioning memories (Takehara et al. 2003). Similarly, pharmacological inactivation of the ACC specifically disrupts expression of
remote, but not recent, contextual fear (Frankland et al. 2004),
five-arm discrimination (Maviel et al. 2004), and water maze
(Teixeira et al. 2006) memories. These studies are consistent with
the idea that while the hippocampus may play an essential rolein the expression of recent memory (e.g., by integrating information stored in distributed cortical modules representing various features of an experience), regions such as the ACC mightassume a similar integrative function at more remote time points(Frankland and Bontempi 2005). However, whether this role extends to memories that do not initially depend on the hippocampus is not known.
To address this issue we used a conditioned taste aversion(CTA) paradigm (Garcia et al. 1955). In this task mice learn toavoid a novel taste (e.g., saccharin-flavored water [SACC]) that ispaired with a malaise-inducing agent (such as lithium chloride[LiCl]). When later given a choice between SACC and water, micethat were previously treated with LiCl will avoid the SACC anddrink the water. In these and subsequent experiments, C57B6/
129 mice (F1 offspring from a cross between C57B6Tac and129svev, Taconic Farms) were water-restricted for the duration ofthe experiment. During the initial habituation phase, mice wereplaced in a cage and given access to two water bottles. Over thecourse of 5 d, mice learned to concentrate their daily water consumption in these 30 min sessions. On the training day, thewater bottles were replaced by a single bottle containing SACC

(0.1%) and the mice were allowed to drink for 30 min. Forty
minutes later mice were treated with LiCl (0.15 M, 2% body weight,i.p.; N = 10) or PBS (N = 10). In the choice test 1 d later, mice wereplaced back into the same cage and had access to bottles containing either water or SACC. LiCl-treated mice exhibited a preference for water over SACC (t(9) = 5.09, P < 0.01) (Fig. 1A). In
contrast, PBS-treated mice drank roughly equal amounts of SACC
and water in the choice test (t(9) = ⳮ0.36, P > 0.05) (Fig. 1B), similar to naïve mice (N = 20) given a free choice between SACC andwater (t(19) = ⳮ1.53, P > 0.05) (Fig. 1C). These data indicate thata single SACC-LiCl pairing is sufficient to establish CTA inC57B6/129 mice.
Hippocampal manipulations have no effect or only a mildeffect on CTA memory formation, suggesting that the hippocampus does not play an essential role in CTA (Yamamoto and Fujimoto 1991; Josselyn et al. 2004). To verify that CTA memory
expression does not depend on the dorsal hippocampus (dHPC)
using our protocol, cannulae were implanted bilaterally above
the dHPC using standard stereotaxic procedures (Teixeira et al.
2006). At least 1 wk following surgery, mice were habituated (asabove) and conditioned (0.1% SACC paired with 0.15 M LiCl).
CTA memory was assessed one day later in a choice test. Ten
minutes before this test, mice received an infusion of PBS (N = 8)
or lidocaine (N = 9; 0.5 µL, 4% w/v) into the dHPC. Lidocaine isa sodium channel blocker and therefore transiently suppresses
both excitatory and inhibitory neural activity (Sandkuhler et al.
1987). In the choice test, both PBS-treated and lidocaine-treatedmice consumed significantly more water compared with SACC
(PBS: t(7) = 7.13, P < 0.01; lidocaine: t(8) = 5.98, P < 0.01), suggesting that inactivation of the dHPC does not affect the expressionof CTA (Fig. 1D,E). In contrast, using identical procedures wepreviously showed that intra-dHPC lidocaine infusions block theexpression of water maze memories in C57B6/129 mice (Teixeiraet al. 2006).
Lidocaine-induced inactivation of the ACC blocks the expression of remote, but not recent, spatial (water maze [Teixeiraet al. 2006] or five-arm discrimination [Maviel et al. 2004]), andcontextual (Frankland et al. 2004) memory. Therefore, we next
tested whether the ACC plays a similar role in the expression ofremote CTA memory. Cannulae were implanted in the ACC

5Corresponding author.
E-mail paul.frankland@sickkids.ca; fax (416) 813-7177.
Article is online at http://www.learnmem.org/cgi/doi/10.1101/lm.905008.

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The anterior cingulate cortex and conditioned taste aversion

Figure 1. (A–C) CTA in normal mice. Mean consumption (ⳲSEM) of water (black) and SACC (white) in the choice test is shown for three groups ofmice: (A) mice where SACC was paired with 0.15 M LiCl during training; (B) mice where SACC was paired with PBS during training; and (C) naïve mice(habituated as above and then given a choice test). (D) Inactivation of the dHPC does not block expression of CTA. During training SACC was pairedwith 0.15 M LiCl. Mean consumption (ⳲSEM) of water (black) and SACC (white) in the choice test 1 d following training is shown for mice receiving
pre-test intra-dHPC infusions of either PBS or lidocaine. (E) Representative cannula placements (triangles) in the dHPC.

retention delay (no main effect of infusion [F (1,52) = 2.59,
P > 0.05] nor infusion ⳯ delay interaction: [F (1,52) = 0.75,
P > 0.05]) (Fig. 2C), indicating that ACC inactivation did nothave any nonspecific effects on drinking behavior. The effects of
ACC inactivation on remote CTA memory parallel those observed in contextual fear conditioning (Frankland et al. 2004)
and water maze (Teixeira et al. 2006).
Increasing the intensity of the US should increase thestrength of the CTA memory (Garcia et al. 1955). Therefore, inthe next series of experiments we evaluated whether lidocaineinduced inactivation of the ACC would also block the expressionof a remote CTA following training using a stronger protocol. As
before, mice were implanted with cannulae in the ACC, and, 1wk later, habituated and then conditioned. On the conditioning(Teixeira et al. 2006), and 1 wk later mice were habituated andthen conditioned (0.1% SACC paired with 0.15 M LiCl). CTA wasthen assessed in separate groups of mice either 1 d (recent test) or
30 d (remote test) later. Ten minutes prior to this test, mice received an infusion of PBS or lidocaine (0.5 µL; 4% w/v) into theACC. In the choice test, PBS-treated mice consumed significantly
more water than SACC at both the recent (t(15) = 2.61, P < 0.05,N = 16) and remote (t(13) = 2.42, P < 0.05, N = 14) time points,indicating that a single SACC-LiCl pairing is sufficient to produce
a CTA that lasts as long as 1 mo. In contrast, intra-ACC infusionof lidocaine blocked CTA expression in the remote test(t(11) = 0.16, P > 0.05, N = 12), but spared CTA in the recent test(t(13) = 2.94, P < 0.05, N = 14) (Fig. 2A,B). Importantly, lidocaine
infusions had no effect on overall liquid consumption at either

Figure 2. Inactivation of the ACC blocks expression of remote CTA memory following training with a weak or strong US. (A–C) Conditioning with weak
US (0.15 M LiCl). During training SACC was paired with 0.15 M LiCl, and mice were given a choice test either 1 d (recent) (A) or 30 d (remote) (B) later.
For each choice test, mean consumption (ⳲSEM) of water (black) and SACC (white) is shown. (C) The total fluid consumed (water + SACC) is shownfor PBS-treated (black) and lidocaine-treated (white) mice in the recent and remote choice tests. (D–F) Conditioning with strong US (0.3 M LiCl). During
training SACC was paired with 0.3 M LiCl, and mice were given a choice test either 1 d (recent) (D) or 30 d (remote) (E) later. For each choice test, mean
consumption (ⳲSEM) of water (black) and SACC (white) is shown. (F) The total fluid consumed (water + SACC) for PBS-treated (black) and lidocainetreated (white) mice in the recent and remote choice tests. (G) Representative cannula placements (triangles) in the ACC.
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The anterior cingulate cortex and conditioned taste aversion

PBS, 0.15 M or 0.3 M LiCl (all N = 7) and then immediately
placed in an open field (44 ⳯ 44 ⳯ 19 cm), and their activity wasmonitored for 24 min. Exploration declined as a function of time(F(3,54) = 86.55, P < 0.01), indicating that all groups of mice habituated to the open field (Fig. 3D). Most importantly, LiCl reduced overall levels of exploration in a dose-dependent manner(Fig. 3E) (F(1,18) = 10.17, P < 0.01; Newman-Keuls post-hoc analyses indicate that PBS total activity > 0.15 M total activity > 0.3
Mtotal activity, Ps < 0.05). The more pronounced reduction in exploration in the group of mice treated with 0.3 M LiCl, suggests
this higher dose of LiCl is more efficacious in inducing malaise inC57B6/129 mice.
Lesion, electrophysiological, and immediate early gene
mapping studies have helped to establish that CTA engages anetwork of regions including the nucleus of the solitary tract, theparabrachial nucleus of the pons, the medial thalamus, theamygdala, and the insular cortex (Houpt et al. 1994; Yamamotoet al. 1994; Lamprecht and Dudai 1995; Welzl et al. 2001; Bermudez-Rattoni 2004; Bernstein and Koh 2007). Whereas previous studies have primarily been concerned with identifying circuits involved in the encoding and initial recall of CTA memories, we have instead focused on CTA expression at remote time
points following training. Our primary observation is that inactivation of the ACC blocks the expression of remote (month-old),but not recent (day-old), CTA memory.
Similar to contextual and spatial memory, these data indicate that expression of a CTA memory at remote retention delays
necessarily involves the ACC. The time-dependent recruitmentof the ACC into circuits supporting this and other forms ofmemory might reflect one of two processes. First, it might reflecta role for the ACC in effortful recall in that older memories might
be harder to access and therefore likelier to engage the ACC(Rudy et al. 2005). Our experimental design addresses this issue
since we manipulated both memory age and strength. Whereas
an effortful recall account would predict that ACC inactivation
would affect weak (rather than strong)memory, regardless of age, we found theopposite pattern: ACC inactivation disrupted remote (rather than recent)memory, regardless of strength. While
we did not exhaustively vary memory
strength in these studies, the data nonetheless do not support an effortful recall
account.
A second (but not necessarily mutually exclusive) possibility is that the recruitment of the ACC reflects a timedependent process of memory reorganization (Frankland and Bontempi 2005).
That the ACC seems to play a similarrole in the expression of remote memory
in contextual and spatial tasks suggeststhat its role in remote memory does notdepend on (1) the content of learningand (2) whether or not the task is initially hippocampal-dependent. This last
observation is perhaps the most surprising since it implies that time-dependentsystems-wide reorganization might be a
general process that is conserved acrossFigure 3. (A) Water preference in recent and remote choice tests in PBS-treated mice trained withmemory systems.

day, SACC was paired with 0.3 M LiCl (2% body weight, i.p.).
Either 1 d (recent test) or 30 d (remote test) later, mice were givena choice test. Ten minutes prior to this test, mice received aninfusion of PBS or lidocaine (0.5 µL; 4% w/v) into the ACC. In thechoice test, PBS-treated mice consumed more water than SACC atboth the recent (t (12) = 3.85, P < 0.01, N = 13) and remote(t(15) = 3.33, P < 0.01, N = 16) time points. In contrast, intra-ACC
infusion of lidocaine blocked CTA expression in the remote(t(11) = 1.46, P > 0.05, N = 12), but not the recent (t(12) = 2.56,
P < 0.05, N = 13), test (Fig. 2D,E). While there was a trend for
reduced liquid consumption in the lidocaine-treated mice in the
remote group, this effect was not statistically reliable: an ANOVA
revealed no main effect of infusion (F(1,50) = 3.20, P > 0.05) norinfusion ⳯ delay interaction (F(1,50) = 1.08, P > 0.05) (Fig. 2F).
Representative cannula placements for these experiments areshown in Figure 2G. Similar to our first study, these results indicate that lidocaine-induced inactivation of the ACC blocks theexpression of remote CTA memory, even following training witha higher intensity US.
Comparison of the two experiments reveals that water preference was generally (but not significantly) stronger in control
mice trained with 0.3 M LiCl (F(1,55) = 1.80, P = 0.18) (Fig. 3A).
This lack of statistical reliability raises the possibility that increasing the intensity of the US did not necessarily induce a stronger
CTA memory. However, when the PBS-treated mice from the tworemote groups were retested 1 d later, extinction was only evident in the weakly-conditioned group. Whereas the mice conditioned with 0.3 M LiCl continued to express a preference forwater over SACC (t(15) = 2.83, P < 0.05) (Fig. 3B), mice conditioned with 0.15 M LiCl consumed similar quantities of waterand SACC (t(13) = 0.72, P > 0.05) (Fig. 3C). Resistance to extinction in the group of mice conditioned with 0.3 M LiCl is consistent with the interpretation that CTA memory was stronger inthis group. We further evaluated the efficacy of the LiCl treatment in an additional experiment. Mice were treated with either

either 0.15 M (gray circles) or 0.3 M (black circles) LiCl. (B–C) When retested 1 d later the mice trainedwith 0.3 M LiCl (B) continued to prefer water (black) over SACC (white), whereas the mice trained with0.15 M LiCl (C) consumed roughly equal amounts of both. (D–E) Effects of LiCl treatment of activity inopen field. (D) Distance traveled in open field is plotted in 6 min blocks for mice treated with PBS(black), 0.15 M (gray), or 0.3 M (white) LiCl. (E) Total distance traveled in 24 min test is shown for micetreated with PBS (black), 0.15 M (gray), or 0.3 M (white) LiCl.
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Acknowledgments
We thank Sheena Josselyn and AlonsoMartinez-Canabal for comments on anearlier version of this manuscript. This
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The anterior cingulate cortex and conditioned taste aversion

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work was supported by grants from CIHR, NARSAD, and the EJLBFoundation (PWF).

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Received January 13, 2008; accepted in revised form February 21, 2008.

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