ABSTRACT In isolated hippocampal slices, decaying long-term potentiation can be stabilized and converted to late long-term potentiation lasting many hours, by prior or subsequent strong

high-frequency tetanization of an independent input to a common population of neurons—a phenomenon known as ‘synaptic tagging and capture’. Here we show that the same phenomenon occurs in

the intact rat. Late long-term potentiation can be induced in CA1 during the inhibition of protein synthesis if an independent input is strongly tetanized beforehand. Conversely, declining

early long-term potentiation induced by weak tetanization can be converted into lasting late long-term potentiation by subsequent strong tetanization of a separate input. These findings

full access to this article via your institution. Download PDF SIMILAR CONTENT BEING VIEWED BY OTHERS BEYOND BOUNDARIES: EXTENDED TEMPORAL FLEXIBILITY IN SYNAPTIC TAGGING AND CAPTURE Article

Open access 04 April 2025 MEMORY CONSOLIDATION AND IMPROVEMENT BY SYNAPTIC TAGGING AND CAPTURE IN RECURRENT NEURAL NETWORKS Article Open access 03 March 2021 A REVISED VIEW OF THE ROLE OF

CAMKII IN LEARNING AND MEMORY Article 18 November 2024 INTRODUCTION Throughout the history of neuroscience, discoveries about brain function made in both humans and living animals have

triggered research into circuit-level, neuronal and molecular mechanisms. For example, the _in vivo_ discovery of long-term potentiation (LTP) of synaptic strength initiated an explosion of

interest in the cellular and molecular mechanisms of synaptic change1. Similarly, ocular dominance plasticity was observed almost six decades ago in the cortex of the living cat2, triggering

computational models3 and experimental studies _in vitro_ to reveal its physiological basis4. The converse is also true; new discoveries at the molecular and cellular level, often in

reduced preparations, have led to insights concerning the functioning of the intact nervous system. Examples include classical studies of neuronal plasticity in _Aplysia_5, the _in vitro_

discovery of the homeostatic scaling of synaptic weights6, and adult neurogenesis7. This interdisciplinary interplay between different levels of analysis is both an exciting feature of

contemporary neuroscience and a necessary step towards an integrated functional and mechanistic account of the operation of the brain. Although it is generally naïve to explain complex

processes such as vision or memory with reference only to molecular mechanisms, there may be instances in which cellular processes place such rigid constraints on systems-level properties

that the gap between levels can be bridged to realize a full understanding. For example, the encoding of memory traces in the mammalian brain requires rapid changes in synaptic efficacy in

response to glutamatergic activity, and engages similar cellular mechanisms to those that underlie LTP1. If such changes in synaptic strength at a set of synapses cannot be stabilized, it is

difficult to imagine how lasting memory traces could be formed8. The initial phase of early LTP is supported by the post-translational modification or trafficking of existing proteins,

whereas late LTP lasting at least 4–6 h requires new protein synthesis9. _In vitro_ studies indicate that the events causing the upregulation of protein synthesis, in the soma or in

dendrites, need not occur at exactly the same time as the trigger for LTP induction10,11,12,13,14,15,16,17,18,19,20,21. Two critical observations that underlie the ‘synaptic tagging and

capture’ (STC) framework are: (1) late LTP in hippocampal area CA1 can be blocked by protein-synthesis inhibitors such as anisomycin, but prior strong tetanization of an independent input to

an overlapping population of postsynaptic neurons stabilizes the decaying LTP10—in other words late LTP can be induced without new protein synthesis at the time of induction if the relevant

plasticity-related proteins have been synthesized beforehand; and (2) a strong tetanus can also ‘rescue’ decaying LTP induced by subsequent, or prior, weak tetanization of an independent

input10,11,13,14,15,16,17,18,19,21. This extension of the time window for associative interactions during the stabilization of synaptic changes (sometimes called ‘late associativity’),

likely has important implications for our understanding of the association of information across time and the formation of lasting memories. According to the STC hypothesis, glutamatergic

stimulation during memory encoding sets temporary ‘tags’ at activated synapses, in a post-translational manner, that then sequester plasticity-related proteins as they become available, thus

stabilizing synaptic changes22,23,24. However, the phenomenon of STC has neither been reported nor validated _in vivo_. Much information has been gathered over the past 10 years concerning

the molecular mechanisms underlying the STC process, extending to the level of individual dendritic spines20, but it is not yet known whether the level of available proteins places

significant restrictions on the persistence of synaptic changes _in vivo_. Spontaneous activity levels are reduced _in vitro_, neuromodulatory afferents are severed, and baseline levels of

plasticity-related proteins are low if sufficient time is allowed for metabolic stabilization25. In the living animal, free from these artificial constraints on protein synthesis, the

availability of relevant proteins may not limit the duration of synaptic changes; all synapses that are activated above a certain threshold—and tagged—might successfully capture the proteins

necessary for lasting potentiation. In other words, the STC phenomenon might be specific to _in vitro_ preparations, with no relevance in the intact animal. Despite the need for an

assessment of synaptic tagging and capture _in vivo_, the demand for two independent synaptic inputs to a common population of postsynaptic neurons presents a technical challenge26. In CA1

slices, two stimulating electrodes can be placed in the Schaffer-collateral pathway, one on either side of a recording electrode in the stratum radiatum, but this configuration does not

yield independent inputs in the intact animal. To overcome this difficulty, we chose to exploit the extensive long-range longitudinal connectivity of the Schaffer-collateral/commissural

system, in addition to the transverse connectivity that forms part of the ‘traditional’ trisynaptic circuit. The axons of CA3 are extensively collateralized, forming both associational

connections with other CA3 cells, as well as the Schaffer-collateral projection to CA1. Axons in both of these projections can extend for distances of several millimetres along the

septotemporal (dorso-ventral) axis of the hippocampus; the commissural projection comprises axons that cross the midline via the ventral hippocampal commissure and terminate in the

contralateral hippocampus27. Functional and anatomical studies indicate that interhippocampal CA3–CA1 connectivity is maximal between regions located at equivalent septotemporal levels, and

ipsilateral and contralateral projections converge on an overlapping population of CA1 neurons28. By placing stimulating electrodes bilaterally in CA3 at equivalent locations, independent

populations of afferents to CA1 can be activated29. Using this arrangement, we set out to examine the phenomenon of synaptic tagging and capture in the living animal. In experiment 1, we

present evidence that strong tetanization can rescue decaying early LTP induced by subsequent tetanization of a second input in the presence of the protein-synthesis inhibitor anisomycin

(‘strong before strong’10). In experiment 2, we show that early LTP induced by weak tetanization can be stabilized by the later delivery of a strong tetanus to a second input (‘weak before

strong’11). In experiment 3, we investigate the dopamine-dependence of late LTP induced by our strong-tetanus protocol. Overall, our results confirm that ‘synaptic tagging and capture’ can

occur in the intact animal, and is not merely an artefact of _in vitro_ hippocampal preparations. RESULTS INDEPENDENCE OF IPSILATERAL AND CONTRALATERAL PROJECTIONS Bilateral stimulation of

CA3 under urethane anaesthesia activated independent ipsilateral (s1i) and contralateral (s2c) populations of afferents converging on CA1 (Fig. 1). Owing to the challenging nature of the

experimental set-up, and the need for long baseline periods to ensure signal stability, the time between the induction of anaesthesia and tetanization was typically 5–6 h, comparable to the

incubation periods employed _in vitro_ to minimize background levels of plasticity-related proteins25. ‘STRONG-BEFORE-STRONG’ PROTOCOL Experiment 1 involved strong tetanization of pathway

s1i after artificial cerebrospinal fluid (aCSF) infusion (‘strong+aCSF’ group; _n_=6), which induced persistent LTP lasting at least 5 h, relative to a non-tetanized contralateral control

pathway (s2c) (Fig. 2a and d). Intraventricular infusion of anisomycin (ANI) before strong tetanization of s1i (‘strong+ANI’ group; _n_=7) spared post-tetanic potentiation (PTP), and the

early phase of LTP, but late LTP was completely blocked; potentiation lasted ~3 h relative to the control pathway (Fig. 2b and d). Strong tetanization of s2c before anisomycin infusion and

subsequent tetanization of s1i (‘strong-before-strong+ANI’ group) yielded late LTP not only in s2c, but also in s1i, despite the fact that LTP in s1i was induced during the inhibition of

protein synthesis (Fig. 2c and d). These results indicate that the persistence of LTP is dissociable from its initial strength, consistent with the STC hypothesis. An analysis of the

opposite, mirror-image situation—the rescue of LTP in the contralateral projection (s1c) by strong tetanization of the ipsilateral pathway (s2i) in the same animals—is presented in

Supplementary Figs S1 and S2, and Supplementary Table S1. An analysis of variance (ANOVA) of the percentage field excitatory postsynaptic potential (fEPSP) slope LTP 4–5 h after tetanization

(Fig. 2d), with group (‘strong+aCSF’, ‘strong+ANI’, and ‘strong before strong+ANI’) as a between-subjects factor, and pathway (s1i and s2c) as a within-subjects factor, revealed a group x

pathway interaction (F(2,19)=4.81; _P_<0.02; Fig. 2d). Following the overall ANOVA, a separate ANOVA of late LTP in s1i only revealed a main effect of group (F(2,19)=3.86; _P_<0.05;

Fig. 2d; left-hand panel). _Post-hoc_ comparisons (Fisher’s LSD) revealed that anisomycin resulted in significantly lower late LTP relative to that in aCSF-treated controls (_P_<0.02),

but prior tetanization of s2c resulted in a significant increase in late LTP in s1i following anisomycin infusion (_P_<0.05); the resulting level of LTP did not differ from that in the

aCSF-treated group (_P_>0.5); and was significantly above baseline (t(8)=3.94; _P_<0.005; one-sample _t_-test). Analysis of simple main effects based on the overall ANOVA revealed

significant differences between s1i and s2c in the ‘strong+aCSF’ group (_P_<0.005), indicating significant late LTP, but no difference was observed in the ‘strong+ANI’ group

(_P_>0.6)—LTP had decayed to baseline values in this case. A main effect of group was also observed in s2c alone (F(2,19)=12.4; _P_<0.001; Fig. 2d; right-hand panel). _Post-hoc_

comparisons (Fisher’s LSD) revealed that a strong tetanus to s2c in the ‘strong-before-strong+ANI’ group caused significant late LTP relative to the aCSF control pathway (_P_<0.001), and

the anisomycin control pathway (_P_<0.0005); these control pathways did not differ from each other (_P_>0.6). At the dose used, anisomycin infusion typically caused a small but rapid

increase in the fEPSP, followed by a gradual, but again slight, fall evident in the untetanized control pathway (Fig. 2b). However, control pathways did not differ significantly between

aCSF- and anisomycin-treated groups 5 h after tetanization (see above), indicating that the block of late LTP cannot be attributed to baseline effects. Group differences in PTP (the mean

slope during the 5-min period after the final tetanus train) and early LTP (30–60 min after the final tetanus train) did not reach significance (F<1 in both cases; ANOVA), indicating that

anisomycin predominantly impaired late LTP. Nonetheless, higher doses caused pronounced baseline changes (Fig. 3). To assess the stability of rescued LTP, independently of its magnitude18,

we carried out an ANOVA of the percentage fEPSP slope LTP 30–60 min and 4–5 h after tetanization in the s1i pathways of the ‘strong+ANI’ (Fig. 2b), and ‘strong-before-strong+ANI’ (Fig. 2c)

groups only. This analysis revealed a significant group x time interaction (F(1,14)=10.0; _P_<0.001), and a subsequent analysis of simple main effects revealed a significant decay of LTP

between time points in the ‘strong+ANI’ group (F(1,14)=27.7; _P_<0.001), but no significant decline in the ‘strong-before-strong+ANI’ group (F(1,14)=1.41; _P_>0.2), confirming the

stability of rescued late LTP. There were no group differences in stimulation intensity (F<1; ANOVA) or fEPSP slope over the 1-h baseline period (F(2,19)=1.03; _P_>0.3); fEPSP slopes

in s2c were significantly smaller than in s1i (F(1,19)=17.9; _P_<0.0005), but stimulation intensity did not differ between pathways (F<1). Overall, no significant paired-pulse

facilitation (PPF) was observed (t(21)=0.04; _P_>0.9; one-sample _t_-test; comparison with chance=100%), and no group differences in values were obtained (F<1). See Table 1 for

details. ‘WEAK-BEFORE-STRONG’ PROTOCOL Experiment 2 avoided the use of drugs. Strong tetanization of s1i (‘strong-only’ group; _n_=7) induced robust late LTP (Fig. 4a and d), whereas weak

tetanization (‘weak-only’ group; _n_=11) induced only a decaying early LTP lasting ~3 h relative to the untetanized control pathway (s2c) (Fig. 4b and d). However, strong tetanization of s2c

after weak tetanization of s1i (‘weak-before-strong’ group; _n_=11) not only induced late LTP in s2c, but also rescued late LTP in s1i (Fig. 4c and d). In other words, decaying potentiation

induced by weak tetanization can be converted into late LTP by strong tetanization delivered later to an independent input. An analysis of the opposite, mirror-image situation—the rescue of

LTP in the contralateral projection (s1c) by strong tetanization of the ipsilateral pathway (s2i)—is presented in Supplementary Figs S1 and S3 and Supplementary Table S1. An ANOVA of the

mean percentage fEPSP slope LTP 4–5 h after tetanization, with group (‘weak only’, ‘strong only’ and ‘weak before strong’) as a between-subjects factor, and pathway (s1i and s2c) as a

within-subjects factor, revealed a group x pathway interaction (F(2,26)=9.35; _P_<0.002; Fig. 4d). Following the overall ANOVA, a separate ANOVA of late LTP in s1i only revealed a main

effect of group (F(2,26)=11.9; _P_<0.0005; Fig. 4d; left-hand panel). _Post-hoc_ comparisons (Fisher’s LSD) revealed that a weak tetanus to s1i caused significantly less late LTP than a

strong tetanus to s1i (_P_<0.0005), but pathway s1i in the ‘weak-before-strong’ group showed significantly more late LTP than the same pathway in the weak-only group (_P_<0.05)—in

other words a rescue of LTP was observed. Although the level of late LTP in pathway s1i of the ‘weak-before-strong’ group remained below that observed in the ‘strong-only’ group

(_P_<0.001), it was stable and the rescued LTP remained significantly above baseline 4–5 h after tetanization (t(10)=3.59; _P_<0.005; one-sample _t_-test). Analysis of simple main

effects based on the overall ANOVA revealed significant differences between pathways s1i and s2c in the ‘strong-only’ group (_P_<0.002), indicating significant late LTP, but no difference

was observed in the weakly tetanized group (_P_>0.6)—LTP had decayed to baseline values in these animals. A separate analysis of LTP 5–6 h after tetanization gave similar results

(Supplementary Fig. S4). A main effect of group was also observed in s2c alone (F(2,26)=6.21; _P_<0.01; Fig. 4d, right-hand panel). _Post-hoc_ comparisons (Fisher’s LSD) revealed that a

strong tetanus to s2c in the ‘weak-before-strong’ group caused significant L-LTP relative to the ‘strong-only’ control pathway (_P_<0.02), and the ‘weak-only’ control pathway

(_P_<0.005); these control pathways did not differ from each other (_P_>0.6). Owing to the slightly smaller PTP induced by weak (versus strong) tetanization, there was a trend towards

a significant overall group difference in PTP in s1i (F(2,26)=3.20; 0.1>_P_>0.05; ANOVA). Similarly, an overall analysis of early LTP (30–60 min after tetanization) revealed a

significant group effect owing to the inclusion of the ‘strong-only’ group (F(2,26)=5.46; _P_<0.02). Nevertheless, early LTP did not differ between ‘weak-only’ and ‘weak-before-strong’

groups (_P_<0.05; Fisher’s LSD), indicating that late, but not early LTP was significantly facilitated by the strong ‘rescue’ tetanus. As in experiment 1, to assess the stability of

rescued LTP induced by a weak tetanus, we conducted an ANOVA of the percentage fEPSP slope LTP 30–60 min and 4–5 h after tetanization in the s1i pathways of the ‘weak-only’ (Fig. 4b), and

‘weak-before-strong’ (Fig. 4c) groups only. This analysis revealed a significant group x time interaction (F(1,20)=7.14; _P_<0.02), and a subsequent analysis of simple main effects

revealed a significant decay of LTP between time points in the ‘weak-only’ group (F(1,20)=30.4; _P_<0.001), but no significant decline in the ‘weak-before-strong’ group (F(1,20)=2.96;

_P_>0.1). There were no group differences in stimulation intensity or fEPSP slope over the 1-h baseline period (F<1 in both cases; ANOVA); fEPSP slopes in s2c were significantly

smaller than in s1i (F(1,26)=44.7; _P_<0.0005), but stimulation intensity did not differ between pathways (F<1). Overall, no significant PPF was observed (t(28)=0.29; _P_>0.7;

one-sample _t_-test; comparison to chance=100%), and no group differences in values were obtained (F<1). See Table 1. Owing to the slight baseline rise evident in Fig. 4c, data from

‘weak-only’ and ‘weak-before-strong’ groups were re-analysed after the exclusion of data from all animals in which baseline fEPSP slope values rose or fell by more than 10 percentage points

between the first and last 20-min periods of the 1-h baseline in either s1i or s2c (an analysis that was possible owing to the relatively large _n_ of 11 in these two groups originally). The

re-analysed data are shown in Fig. 4e and f (‘weak only’: _n_=8; ‘weak before strong’: _n_=7). As expected, baseline values are stable, and the results are numerically very similar to those

in Fig. 4b and c. An ANOVA of the mean percentage fEPSP slope LTP 4–5 h after tetanization—with pathway as a within-subjects factor and group as a between-subjects factor—revealed a

significant overall main effect of group (F(1,13)=12.8; _P_<0.005). Comparison of late LTP in pathway s1i of the ‘weak-only’ and ‘weak-before-strong’ groups revealed a significant

difference (F(1,13)=6.52; _P_<0.025; analysis of simple main effects); late LTP in the latter group remained significantly above chance (t(6)=2.78; _P_<0.05; one-sample _t_-test). As

expected, the strong rescue tetanus resulted in significant potentiation relative to the untetanized control in the ‘weak-only’ group (F(1,13)=7.92; _P_<0.02; analysis of simple main

effects). THE ROLE OF DOPAMINE In experiment 3, we assessed the impact of the dopamine antagonist SCH23390 on late LTP. Neither aCSF (Fig. 5a; _n_=7) nor SCH23390 (5 μg μl−1; Fig. 5b; _n_=7)

blocked potentiation after 4 h (the point at which recording ended in this experiment), although a modest fall in both tetanized and control pathways was evident in the SCH23390 group by

the end of the recording period. An ANOVA of LTP 3–4 h after tetanization revealed a difference between tetanized and non-tetanized pathways (F(1,12)=13.16; _P_<0.005), but no effect of

drug group (F(1,12)=0.63; _P_>0.4), and no group × pathway interaction (F(1,12)=0.03; _P_>0.8). Similarly, no group differences in PTP (0–5 min after tetanus) or early LTP (30–60 min

after tetanus) were observed in s1i (F<1 in both cases). Fig. 5c shows the normalized fEPSP slope in each tetanized pathway expressed as a percentage of the value recorded in the

corresponding control pathway, in order to control for baseline changes. When analysed in this way, the time course and level of potentiation were identical in SCH23390 and vehicle-treated

groups. Mean potentiation 3-4 h after tetanization is summarized in Fig. 5d. No significant group (F(1,12)=0.08; _P_>0.7) or pathway (F(1,12)=2.07; _P_>0.1) differences were found in

stimulation intensity; baseline fEPSP slope likewise did not differ between groups (F(1,12=0.18; _P_>0.6), and on this occasion no differences were found between pathways s1i and s2c

(F(1,12)=1.28; _P_>0.2); see Table 1. DISCUSSION The results of experiment 1 reveal that late LTP of the ipsilateral Schaffer-collateral input to CA1 (s1i) can be induced in the living

rat during the inhibition of protein synthesis—a treatment that otherwise limits the duration of LTP to around 3 h—provided that strong tetanization of an independent contralateral

commissural input (s2c) occurs beforehand (Fig. 2). This suggests that proteins synthesized following strong tetanization of one pathway can subsequently be captured by tags set at the

synapses of a separate input. Doses of anisomycin comparable to that used here have previously been reported to block hippocampal LTP, long-term memory formation, and tetanus-induced protein

synthesis30,31,32. A large number of proteins are upregulated following the induction of late LTP (for example, McNair _et al_.33), and there is mounting evidence for the importance of

local dendritic translation (for example, refs 34,35,36,37). Although we cannot rule out the possibility that anisomycin also blocks long-lasting changes in the intrinsic properties of

neurons induced by strong tetanization38, a cell-wide mechanism cannot explain the ‘weak-before-strong’ data of experiment 2 (see below), unless weak tetanization sets a synaptic tag as our

hypothesis indicates that it must. In our hands, higher doses of anisomycin caused dose-dependent changes in baseline fEPSPs even in the absence of tetanization (Fig. 3), consistent with the

multiple effects of anisomycin reported at doses higher than that used in the present study32,39,40,41, including the suppression of neural activity42. However, no significant baseline

changes were observed at the dose used in experiment 1. Experiment 2 reveals that decaying early LTP induced by weak tetanization of the ipsilateral CA3–CA1 projection (s1i) can be converted

into late LTP by strong tetanization delivered later to the contralateral pathway (s2c) (Fig. 4). The delivery of the strong ‘rescue’ tetanus after the weak tetanus excludes the possibility

of sensitization, or a reduction in the threshold for the induction of late LTP11; it is consistent with the setting of synaptic tags that subsequently capture proteins synthesized after

the strong tetanus, and implies that the availability of plasticity-related proteins can indeed determine the duration of synaptic changes _in vivo_. Taken together, our results confirm the

symmetrical nature of the synaptic tagging and capture process as characterized _in vitro_—strong tetanization can rescue decaying potentiation whether delivered before or after the latter’s

induction10,11. Although we have focussed on the rescue of LTP in the uncrossed CA3–CA1 projection (s1i) by strong tetanization of the crossed pathway (s2c), the ability of strong

ipsilateral CA3–CA1 tetanization to rescue decaying LTP in the contralateral projection was qualitatively similar, although statistically less robust (Supplementary Figs S2 and S3), perhaps

owing to the typically smaller and more variable levels of LTP seen in the crossed CA3–CA1 pathway43. In order to obtain independent synaptic inputs to a common pool of CA1 neurons, we

stimulated bilaterally in CA3; our electrode locations yielded large fEPSPs in CA1 stratum radiatum following both ipsilateral and contralateral stimulation. The independence of crossed and

uncrossed projections was confirmed by the absence of PPF following alternate stimulation of each pathway at an interval of 50 ms (Fig. 1d, middle bar); similarly, tetanization of one

pathway caused no change in the other input. Although commissural projections to CA1 typically exhibit a denser pattern of termination in stratum oriens, relative to ipsilateral inputs that

primarily target stratum radiatum, afferents originating from the intrahilar region are predominantly directed toward the contralateral stratum radiatum44. In view of this, the recording of

large contralateral CA3–CA1 fEPSPs in stratum radiatum is consistent with our placement of stimulating electrodes towards the proximal (dentate) end of CA3. Although our experiments were

conducted under urethane—an anaesthetic that suppresses fEPSPs, and necessitates the use of stronger tetanization protocols for LTP induction45—strong tetanization induced persistent LTP

that was blocked by anisomycin (Fig. 2b), suggesting that protein synthesis-dependent late LTP can still be induced under these circumstances. Late LTP typically lasted as long as stable

recordings could be maintained (that is, at least 5 h); in some instances, we observed stable LTP for over 10 h following strong tetanization. Having established that synaptic tagging and

capture can occur _in vivo_, it is necessary to identify physiologically plausible neural analogues of the strong tetanus—that is, triggers for the heterosynaptic stabilization of synaptic

potentiation induced by the activation of glutamate receptors. As the activation of dopaminergic afferents is a leading candidate for this role46,47, the absence of an effect of dopamine

receptor blockade on LTP induced by the strong tetanus used in the current series of experiments (Fig. 5) might seem surprising. The route of administration and dosage were chosen to match

exactly those used in a previous behavioural study in which SCH23390 caused a marked impairment of long-term spatial-memory formation48. Moreover, dopamine D1/D5 receptor blockade can block

CA1 late LTP _in vivo_49,50, and dopamine receptor activation can induce dendritic protein synthesis51. But despite the frequent focus on the dopaminergic innervation of CA1, there are

likely to be multiple determinants of protein availability. For example, β-adrenergic activation facilitates a form of LTP requiring dendritic translation but not transcription52, and

somatic action potentials induced by alvear stimulation are sufficient to convert early to late LTP in CA1 (ref. 13). In our experiments, it is likely that strong glutamatergic stimulation

was sufficient to induce late LTP without the additional requirement for dopaminergic activity; the activation of CaMKIV or metabotropic glutamate receptors might, for example, serve this

function18,53. In fact, for anatomical reasons, the finding that our strong tetanus induces dopamine-independent LTP is fully consistent with the results obtained. The hippocampus receives

dopaminergic inputs from mesolimbic structures such as the ventral tegmental area; afferents terminate in CA1 and to a lesser extent in CA3 (ref. 54). However, the axons of dopaminergic

neurons exhibit limited collateralization, and only around 10% of ventral tegmental area dopaminergic neurons project contralaterally54. For these reasons, it is unlikely that tetanization

of CA3 could result in a significant recruitment of dopaminergic fibres terminating contralaterally in CA1. In other words, the ability of a strong tetanus delivered to the crossed CA3–CA1

pathway to rescue decaying LTP in the ipsilateral projection is unlikely to be mediated by dopaminergic afferents. Nonetheless, dopamine is likely to have an important role in the

stabilization of learning-induced synaptic changes triggered by physiological patterns of stimulation48,55,56. In summary, we find that the synaptic tagging and capture phenomenon,

consistent with its hypothesized role in memory57,58,55, is not limited to _in vitro_ preparations. As well as providing a mechanism for the association of information over time, our use of

long-range commissural projections to provide the ‘rescue’ stimulus indicates that the information to be associated can originate from cells located in opposite hemispheres of the brain. In

other words, the ‘late associativity’ of LTP operates not only over an extended time-frame, but also over a distance of several millimetres in the living rat. Our findings provide a bridge

between investigations of behavioural tagging in freely moving animals and _in vitro_ studies of underlying mechanisms, and so validate further work aimed at uncovering the molecular basis

of memory persistence. METHODS SUBJECTS All procedures were conducted in accordance with the UK Animals (Scientific Procedures) Act (1986). Before the experiment, adult male Lister-hooded

rats (250–500 g; _n_=91) were given _ad libitum_ access to food and water and maintained on a 12-h light/12-h dark cycle. ANAESTHESIA Rats were anaesthetized with urethane (ethyl carbamate;

1.5 g kg−1; 0.3 mg ml−1, i.p.), injected with carprofen (Rimadyl small animal solution, 4 mg kg−1; s.c.), and placed in a stereotaxic frame with the skull horizontal. Body temperature was

monitored by a rectal probe and maintained at 36.2 °C using an isothermic heating blanket. Depth of anaesthesia was assessed throughout the experiment, and urethane top-ups of 0.2 ml were

administered as required. Breathing rate was monitored continuously using a light-dependent resistor to detect thoracic movements, and analysed online using in-house software. If breathing

fell below 70 breaths per min, rats received an injection of atropine (0.4 mg kg−1; s.c.) or doxapram (5 mg kg−1; i.p.). Subcutaneous injections of a glucose/saline mixture were administered

every 3 h to maintain hydration (1.5 ml of 0.9% saline+0.5 ml of 5% glucose). ELECTROPHYSIOLOGICAL RECORDING PTFE-insulated monopolar platinum/iridium recording electrodes (diameter=0.103 

mm) were lowered bilaterally into the stratum radiatum of area CA1 (3.8 mm posterior and 2.5 mm lateral to bregma; depth ~−2.5 mm from dura). Bipolar stimulating electrodes comprising two

twisted wires identical in composition to the recording electrodes were lowered bilaterally into CA3 (3.5 mm posterior and 3.0 mm lateral to bregma; depth ~−3.0 mm from dura) in order to

activate independent populations of synaptic contacts made by ipsilateral Schaffer-collateral (s1i) and contralateral commissural projections (s2c) converging on the same neuronal

populations sampled by each of the recording electrodes. Fig. 1a shows a photomicrograph of representative marking lesions made at both stimulating sites and the left-hand recording site in

an individual rat. A schematic illustration of the electrode locations and pathways stimulated is shown Fig. 1b; for simplicity, CA3–CA3 projections are omitted, and only a single pair of

ipsilateral and contralateral projections is shown; in practice CA3 stimulation simultaneously activates crossed and uncrossed afferents, and recording electrodes were placed bilaterally to

capitalize on this (Supplementary Fig. S1). Nonetheless; in our main analysis (Figs 2 and 4), we focus solely on the rescue of LTP in an ipsilateral CA3–CA1 projection (s1i) by strong

tetanization of the crossed CA3–CA1 pathway (s2c) converging on the same recording site. Note that the hemisphere (left or right) containing the ipsilateral projection of interest (s1i) was

varied over days in a quasi-random fashion. An analysis of the opposite, mirror-image situation—the rescue of LTP in the contralateral projection (s1c) by strong tetanization of the

ipsilateral pathway (s2i) in the same animals—is presented in the Supplementary Information (Supplementary Figs S2 and S3). Throughout the experiment, fEPSPs were amplified and sampled at 20

 kHz using a PC running custom-written LabView software developed by Patrick Spooner; fEPSP amplitude and initial slope (measured by linear regression between two fixed time points) were

monitored on-line. Stimulation was delivered under computer control via a Neurolog system (NL800A; Digitimer LTD, Herts., UK), and consisted of biphasic constant-current pulses. At the start

of each experiment, electrodes were lowered into the hippocampus, and depths were adjusted to maximize the amplitude of the negative-going dendritic fEPSPs elicited in CA1 by stimulation of

CA3; typical depth profiles are shown in Fig. 6 (see Supplementary Methods for further information). Stimulation intensity was adjusted to elicit a contralateral fEPSP of ~3 mV in amplitude

(200–500 μA). Representative fEPSPs evoked by stimulation of s1i and s2c are shown in Figs 1c and 6. Table 1 shows mean stimulation intensities and baseline fEPSP slopes for all

experiments, groups, and pathways. The independence of s1i and s2c was confirmed at the start of the experiment in each animal by the delivery of pairs of biphasic stimulation pulses (50 μs

per phase) to s2c followed by s1i at an interval of 50 ms (six pairs; 10-s intra-pair interval), followed by single test pulses delivered to s1i only (6 pulses). PPF was calculated by

expressing the mean fEPSP slope recorded in s1i after stimulation of s2c as a percentage of the value obtained in s1i without prior stimulation. Mean data from all experiments reported in

Figs 2 and 4 are shown in Fig. 1d (middle panel) and Table 1. Paired stimulation was delivered first to s2c, then to s1i, as stimulation in the opposite sequence typically causes a modest

paired-pulse depression of s2c, a phenomenon that has been reported previously, and attributed to the recruitment of feed-forward and feedback inhibition by ipsilateral stimulation59. After

electrode placement, and a check for the absence of PPF, baseline recording began; single biphasic test pulses (50-μs pulse width per phase) were delivered alternately to each stimulating

electrode at 2-min intervals. The decision regarding the experiment to be conducted on any given day was always made before the start of baseline recording, with experiments alternating

between groups across days. After a baseline period typically lasting several hours, and once stable fEPSPs had been observed for at least 1 h, tetanic stimulation began. Pulse width (per

phase) was increased to 100 μs during a high-frequency tetanus. Strong tetanization consisted of three trains of 50 pulses at 250 Hz, with a 5-min inter-train interval; a weak tetanus

comprised 1 train of 50 pulses at 100 Hz. Data obtained from a specific recording electrode were discounted if the fEPSP slope elicited by ipsilateral or bilateral stimulation fell to 60% of

the baseline value or below within 4–5 h of tetanization (or the corresponding time point for untetanized pathways). In all cases, fEPSP slope data were normalized to the mean of the 1-h

baseline period (assigned a value of 100%), and group means were calculated. DRUGS An anisomycin solution was prepared by dissolving the powdered drug (Sigma-Aldrich, UK) in equimolar HCl

and diluting with artificial cerebrospinal fluid (aCSF; in millimolar: 150 Na+, 3 K+, 1.4 Ca2+, 0.8 Mg 2+, 155 Cl−, 0.2 H2PO4−, 0.8 HPO42−, in pyrogen-free water at pH 7.2). The pH of the

anisomycin solution was adjusted to 7.2 by the addition of NaOH, and the final concentration was 5 mg ml−1. Powdered SCH23390 (Sigma-Aldrich, UK) was dissolved in sterile aCSF to yield a

concentration of 5 mg ml−1. Solutions were vortexed, gently sonicated and stored in small aliquots at −20 C before use. DRUG ADMINISTRATION AND TIME LINES In experiment 1, bilateral

intraventricular (i.c.v.) infusion cannulae (24-gauge stainless steel) were lowered into the left and right lateral ventricles at the same time as electrode placement (co-ordinates: 0.9 mm

posterior and 1.3 mm lateral to bregma; depth=−4.5 mm from the skull surface). Bilateral i.c.v. infusions (5 μl per ventricle over 10 min) were delivered via 5-μl SGE syringes mounted in a

syringe pump (World Precision Instruments, Stevenage, UK), and connected to the infusion cannulae with plastic tubing. Control groups received a bilateral i.c.v. infusion (5 μl per ventricle

over 10 min) of either anisomycin (5 μg μl−1) or aCSF starting 15 min before strong tetanization of s1i. In the ‘rescue’ group, a strong tetanus was delivered to s2c ending 15 min before

infusion of anisomycin and subsequent strong tetanization of s1i; recording continued for a further 5 h. In experiment 2 (Fig. 4), control groups received either strong or weak tetanization

of s1i in the absence of infusion; in the ‘rescue’ group, s1i received weak tetanization 30 min before strong tetanization of s2c; recording continued for a further 5 h. In experiment 3,

intra-hippocampal infusion cannulae were implanted under recoverable anaesthesia before electrophysiological recording. Rats were anaesthetized with isoflurane, injected with Rimadyl

(carprofen; 5 mg kg−1, s.c.), and placed in a stereotaxic frame with the skull horizontal. Guide cannulae (outer diameter=0.46 mm; Plastics One) were implanted bilaterally into the dorsal

hippocampus (co-ordinates from bregma: AP=−4.5 mm; Lat.=3.0 mm; DV from dura=2.5 mm). These were fixed in place using dental cement, and the headcap was secured to the skull using jewellers’

screws. To prevent blockage or infection, dummy cannulae (or stylets; outer diameter=0.2 mm, protruding 0.5 mm from the end of the guide cannulae) were inserted into the guides during the

recovery period. After ~1 week, the rats were anaesthetized with urethane for electrophysiological recording. After stable baseline recordings were obtained, injection needles were inserted

into the guide cannulae. These were connected via plastic tubing to SGE syringes mounted in a syringe driver, and protruded 0.5 mm from the ends of the guide cannulae (that is, the infusion

site was −3.0 mm from the dura). Intrahippocampal infusions of either aCSF or SCH23390 (5 μg μl−1; 1 μl per hippocampus) were delivered over 5 min, starting 15 min before strong tetanization

of s1i. Post-tetanus recording continued for 4 h in Experiment 3, rather than 5 h in Experiments 1 and 2. In order to control for infusion-induced baseline changes, after normalization with

respect to baseline values (see above), fEPSP slopes in s1i (tetanized pathway) were subsequently expressed as a percentage of the corresponding values in s2c (control pathway) for each

animal and time point. HISTOLOGY At the end of experiments 1–3, marking lesions were made by the delivery of biphasic-mA constant-current pulses (1 s per phase) to both stimulating and

recording electrodes. Rats were killed by cervical dislocation and brains were removed and stored in 10% formalin. Thirty-micrometre coronal sections through the hippocampus were then cut

using a cryostat: 1 in 3 sections was mounted on a slide and stained with cresyl violet. After examination under a light microscope, stimulation sites were marked on the appropriate coronal

section taken from the Paxinos and Watson atlas60. All electrodes were correctly positioned (Fig. 7 and Supplementary Fig. S5), and, in experiment 1, all infusion cannulae were located in

the lateral ventricles. ADDITIONAL INFORMATION HOW TO CITE THIS ARTICLE: Shires, K.L. _et al_. Synaptic tagging and capture in the living rat. _Nat. Commun._ 3:1246 doi: 10.1038/ncomms2250

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references ACKNOWLEDGEMENTS This work was supported by a Wellcome Trust Project Grant held by S.J.M. (WT083601MA). We thank Patrick Spooner for programming, electronics and computing

support, Jane Tulloch for assistance with histology, and the members of the CCNS for discussion and advice. AUTHOR INFORMATION Author notes * K.L. Shires Present address: Present address:

Brain Repair Group, Cardiff School of Biosciences, Biomedical Sciences Building, Museum Avenue, Cardiff, CF10 3AX, UK, AUTHORS AND AFFILIATIONS * Centre for Cognitive and Neural Systems

(CCNS), University of Edinburgh, 1 George Square, Edinburgh, EH8 9JZ, UK K.L. Shires, B.M. Da Silva, J.P. Hawthorne, R.G.M. Morris & S.J. Martin Authors * K.L. Shires View author

publications You can also search for this author inPubMed Google Scholar * B.M. Da Silva View author publications You can also search for this author inPubMed Google Scholar * J.P. Hawthorne

View author publications You can also search for this author inPubMed Google Scholar * R.G.M. Morris View author publications You can also search for this author inPubMed Google Scholar *

S.J. Martin View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS K.L.S., B.M.daS., R.G.M.M. and S.J.M. designed the study. K.L.S., B.M.daS.,

J.P.H. and S.J.M. conducted the experiments and analysed the data. K.L.S., B.M.daS., R.G.M.M. and S.J.M. wrote the manuscript. CORRESPONDING AUTHOR Correspondence to S.J. Martin. ETHICS

DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURES, TABLE AND METHODS Supplementary Figures S1-S5,

Supplementary Table S1 and Supplementary Methods (PDF 1880 kb) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Shires, K., Da Silva, B., Hawthorne, J.

_et al._ Synaptic tagging and capture in the living rat. _Nat Commun_ 3, 1246 (2012). https://doi.org/10.1038/ncomms2250 Download citation * Received: 05 March 2012 * Accepted: 01 November

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