BACKGROUND
synaptic plasticity plays an important role in many aspects of brain function such as learning, memory and development  <cit> .
synaptic activity-induced changes in synaptic efficacy can persist for hours, days or even months  <cit>  given gene-transcription and subsequent synthesis of new protein  <cit> .
recently, gene chip analysis has identified large numbers of genes regulated by synaptic plasticity  <cit> .
the expression and synaptic localisation of activity-induced mrnas which encode proteins such as arc/arg <dig>  have been shown to be crucial for the regulation of synaptic plasticity  <cit> .
although numerous gene candidates are emerging, detailed knowledge is still lacking about the synaptic mechanisms affected by the gene expression which transforms early-phase ltp  into late-phase ltp .
while altered pre- and post-synaptic function in e-ltp has been demonstrated with quantal analysis of patch clamp recordings from acute hippocampal brain slices  <cit> , little is known about the nature, time course and mechanism of these changes in synaptically induced l-ltp  <cit> .
this lack arises in part from the difficulty of repeating measurements over more than  <dig> hours from a subset of synapses on the same cell in an acute brain slice.
here we assess the persistence and transcription dependency of potentiated synaptic transmission using a between cell analysis of brief recordings at multiple time points after its induction.
we use a culture based system of synaptically activated long-term plasticity where gene profiling has identified candidates for transcription-dependent late-phase plasticity  <cit> .
removal of inhibitory synaptic transmission in dissociated hippocampal cultures with the gabaa-receptor antagonist bicuculline for  <dig> minutes induces synchronous action potential bursting  which persists for more than  <dig> hours.
this potentiated state in culture mimics that of nmda receptor-dependent l-ltp in slices, requiring extracellular signal-regulated kinase 1/ <dig>  signalling and both gene transcription and translation.
neurons in bicuculline treated cultures show an increased frequency of ampa receptor-mediated mepscs at a time point  <dig> minutes after washout  <cit> .
mepscs represent the postsynaptic response to spontaneous release of single neurotransmitter vesicles at functional synapses and thus provide an index of synaptic efficacy and connectivity at a quantal level.
mepscs are known to be increased in frequency and/or amplitude in ltp in hippocampal slices  <cit> ; however it is not known whether such changes persist during l-ltp.
we show that synaptic potentiation, manifested as an increased mepsc frequency in the hippocampal culture model of ltp, is preserved at transcription-relevant time points  and requires transcription.
this finding points to a common transcription-dependent mechanism in the maintenance of synaptic potentiation and recurrent network bursting which is established within the first two hours after ltp induction.
RESULTS
assessment of ap bursting and synaptic transmission
in cultured hippocampal networks, it has previously been shown that ampa receptor-mediated mepscs show an increased frequency but not amplitude at a time point  <dig> minutes after ltp induction with a  <dig> minute bicuculline treatment  <cit> .
to examine the longevity of this upregulated quantal synaptic transmission, we randomly selected one neuron per coverslip and made whole-cell patch clamp recordings of mepscs at time points up to  <dig> hours after treatment with either bicuculline  or vehicle  for  <dig> minutes.
prior to mepsc-recordings, activity was assessed in all neurons by both cell-attached and whole-cell current clamp  recordings .
neurons showed distinct activity patterns categorised as no activity, random activity or recurrent bursting.
bursts were composed of clusters of  <dig> to  <dig> spikes separated by relatively spike-free intervals of  <dig> to  <dig> seconds  .
cumulative probability histograms for inter event intervals  and amplitudes were generated from at least  <dig> mepscs recorded from each cell .
mepscs were sensitive to  <dig> μm dnqx  confirming their identity as ampa receptor-mediated mepscs .
responses were also recorded to bath applied ampa  in the presence of cyclothiazide  to prevent desensitisation .
this produced a steady state plateau within  <dig> s whose amplitude was used as an indication of the total number of functional ampa receptors per cell.
potentiation in synaptic transmission over a  <dig> hour period following the induction of ap bursting
synaptic potentiation has previously been demonstrated in hippocampal cultures at a single time point  <dig> minutes after a  <dig> minute induction period of ap bursting  <cit> .
at this time point, mepsc frequency was shown to be increased but little effect was seen on mepsc amplitude or whole-cell ampa responses.
although this study showed that ap bursting persists more than  <dig> hours after induction, it is not known whether mepsc frequency remains increased and amplitude and ampa responses remain unchanged at later time points.
we first examined mepsc results pooled from recordings at all time points within  <dig> hours after the  <dig> minute induction period.
spontaneous and in particular, bicuculline-induced ap bursting significantly reduced mepsc iei but did not significantly affect the mepsc amplitude or the whole-cell ampa current .
to better illustrate the time course of these effects, data was split into three groups:  <dig> min to  <dig> h,  <dig> to  <dig> h and  <dig> to  <dig> h. these groups span the period of transcription dependency of synaptic plasticity and ap bursting whereby the transcription inhibitor actinomycin d having little effect over the first  <dig> hours after induction, causes a slow return to baseline activity levels between  <dig> and  <dig> hours after induction  <cit> .
neither mepsc amplitude nor whole-cell ampa current was significantly affected by ap bursting at any time point .
however, when bursting and non-bursting cell groups were pooled, whole-cell ampa response was slightly elevated in bicuculline vs. vehicle treated groups at the time points  <dig> min to  <dig> h  and  <dig> h to  <dig> h .
no difference in ampa whole-cell response was apparent in pooled data for the  <dig> to  <dig> h group.
in contrast, iei of mepscs was significantly shorter in comparison to untreated and vehicle treated cultures at all three time points .
thus, a  <dig> minute induction of ap bursting induces long lasting changes in mepsc iei but not amplitude or whole-cell ampa responses as was previously reported at a single  <dig> minute post-induction time point  <cit> .
this indicates that, along with ap bursting, synaptic transmission is retained at an elevated level at transcription-dependent time points .
ap bursting does not persist in all cells and is sensitive to actinomycin d
the persistence of bicuculline-induced bursting activity in hippocampal cultures and its sensitivity to the transcription inhibitor, actinomycin d has previously been investigated using arrays of planar extracellular electrodes, each of which can detect action potentials in multiple axons and somata within approximately  <dig> μm of the electrode  <cit> .
the prevalence, persistence and transcription dependency of ap bursting in the post induction period has not been examined on a single cell level to date.
we first tested whether ap bursting-induced gene transcription is indeed inhibited by actinomycin d. basal expression of the immediate-early gene c-fos in hippocampal cultures was very low in control conditions  and even lower in the presence of actinomycin d .
expression of c-fos was strongly induced  <dig> hours but not  <dig> minutes after induction of ap bursting with a  <dig> minute bicuculline treatment .
this induction of c-fos expression at the  <dig> h time point was abolished by the transcription inhibitor actinomycin d confirming that ap bursting-induced transcription is effectively blocked by this treatment.
the effects of bicuculline and actinomycin d treatment on ap bursting were next assessed on a single cell level  <dig> minutes to  <dig> hours after washout of bicuculline or vehicle.
bicuculline increased the overall incidence of ap bursting from  <dig> out of  <dig> neurons  to  <dig> out of  <dig> neurons  .
the proportion of cells displaying burst activity in either vehicle or bicuculline treated groups was considerably reduced however after exposure to actinomycin d  vehicle treated and in  <dig> out of  <dig>  bicuculline treated neurons) .
the time course of these effects is shown in figure 3c.
bicuculline induced ap bursting in 100% of neurons regardless of any pre-treatment with actinomycin d. the proportion of neurons bursting  <dig> minutes after bicuculline washout was reduced to 67%, indicating that bursting activity is not maintained in all neurons but that this does not originate in a failure to participate in ap bursting during the induction period.
however, this proportion declined only slightly over the next  <dig> hours to a level of 52%.
field recordings from meas have previously demonstrated that ap bursting persists for more than  <dig> hours in 90% of cultures  <cit> .
this figure represents the persistence of bursting at a single electrode in a  <dig> electrode array rather than the number of individual neurons which maintain ap bursting.
actinomycin d however, caused a sharp reduction in the incidence of ap bursting in the first  <dig> hours after its induction and had virtually abolished all bursting at later time points.
this single-cell data supports previous evidence from multi-cellular recordings that bicuculline-induced ap bursting requires gene transcription for its maintenance and further reveals an inhibitory effect of actinomycin d on bursting of a spontaneous origin.
thus even spontaneous ap bursting requires ongoing gene transcription.
the rapid shutdown of ap bursting in actinomycin d showed similar kinetics to the actinomycin d-induced decay of epsc potentiation in acute slices  <cit>  although somewhat faster than that reported by arnold et al.
.
the slower decay of potentiation in the mea study is likely due to the quantification of spikes within bursts at a single electrode rather than sampling single cells across the network as we have done or assessing the amplitude of extracellularly recorded signals which reflects to some extent the number of responsive cells.
the long-lasting, activity-induced increase in mepscs is actinomycin d sensitive
given the persistence of mepsc upregulation at time points later than  <dig> hours after the induction of ap bursting and the dependence of ap bursting on gene transcription, we wished to directly test whether the long lasting upregulation of mepscs was transcription-dependent.
mepscs were recorded from actinomycin d treated coverslips after the assessment of ap bursting at time points  <dig> minutes to  <dig> hours after bicuculline washout.
in actinomycin d treated neurons as in control conditions , ap bursting did not significantly affect the mepsc amplitude or the whole-cell ampa current .
actinomycin d did not affect the reduction in mepsc iei caused by ap bursting when data in each treatment group was pooled from all time points .
however, when this data from actinomycin d treated cells was separated into time groups, bursting and the reduction in iei induced by ap bursting were only apparent at early time points .
at time points more than  <dig> hours after induction of ap bursting, actinomycin d had abolished both ap bursting and any differences in mepsc iei between vehicle and bicuculline treated groups.
thus, actinomycin d had abolished both ap bursting and the effects of ap bursting on mepsc iei at transcription-dependent time points  but not at early time points.
we also noted that actinomycin d reduced mepsc iei in vehicle treated cells though this was only significant in the  <dig> min to  <dig> h time period .
similar effects have been noted previously where the slope of field evoked epsps is increased during the first  <dig> hours of actinomycin d application  <cit> .
pharmacological blockade of bursting immediately after the induction-period returns mepsc frequency to baseline.
given the correlation between mepsc frequency and ap bursting and their parallel time course of inhibition by actinomycin d, we wished to establish whether mepsc upregulation per se requires transcription or is just maintained by ongoing bursting which itself is transcription-dependent.
we investigated whether a brief induction of ap bursting is sufficed to cause a long-lasting increase in synaptic transmission which persists even if ap bursting is blocked in the post induction period.
alternatively, if mepsc upregulation were a short term effect of ap bursting then ap blockade would cause mepsc frequency to rapidly decay back to baseline levels.
to test these hypotheses, burst-activity was blocked immediately after the  <dig> minute induction period by ttx application before measuring mepscs within  <dig> minutes,  <dig> minutes or 2- <dig> hours.
since bursting was completely absent in all cells exposed to ttx in the post-induction period, data was pooled irrespective of burst activity prior to mepsc measurement.
a clear ap bursting-induced reduction in mepsc iei was still apparent at all time points in pooled control data .
pre-incubation with actinomycin d and/or ttx application after bicuculline washout did not disrupt the effects of ap bursting on mepsc iei within the first  <dig> minutes of the post-induction period .
this result indicates that upregulation of synaptic transmission outlasts a brief blockade of ap bursting.
however, blockade of ap bursting with ttx after the  <dig> minute induction period caused a gradual return of mepsc iei to baseline levels at the  <dig> h time point .
this indicates that the activity-induced increase in mepscs requires either ongoing ap bursting or more than  <dig> minutes of ap bursting to persist until transcription-dependent, "late-phase" time points.
for direct comparison of the effects of actinomycin d, data from both bursting and non-bursting cells were pooled and re-plotted in figure 5b.
actinomycin d treatment in the absence of ttx caused a more rapid loss of iei potentiation at both the  <dig> min and  <dig> h time points.
surprisingly, the combination of both actinomycin d and ttx treatment in the post-induction period resulted in a less rapid decay of mepsc iei back to baseline levels at the  <dig> h time point.
it is not clear why ap-blockade should slow the effects of actinomycin d in depotentiating synaptic transmission.
one possibility is that the early phase of de-potentiation caused by actinomycin d is mediated by some activity-dependent depletion of the vesicular pool and may relate to the increased amplitude of evoked responses seen within  <dig> hours of actinomycin d application  <cit> .
in summary, either ongoing activity or transcription or both are necessary for the potentiation of mepsc frequency to persist beyond the  <dig> hour post-induction time point.
a  <dig> hour critical period of activity is required to stabilise the increase in mepscs at the  <dig> hour time point
we next tested the possibility that potentiated synaptic transmission requires continued ap bursting activity after the  <dig> minute induction period to become independent of ap bursting during the transcription-dependent late phase.
therefore, ttx was applied  <dig> hours after the  <dig> minute induction period leaving a time-window of  <dig> hours for the stabilisation of potentiated synaptic transmission.
ttx was then applied for  <dig> hours before recording mepscs at the time point of  <dig> hours after the induction of ap bursting .
in control cells which did not receive ttx treatment, mepsc iei was significantly reduced in the bicuculline treated group compared to the vehicle treated group .
this reduced mepsc iei was also present in the group treated with ttx for  <dig> hours .
the amplitude of mepscs was unaffected by this  <dig> hour ttx-treatment indicating that synaptic scaling had not occurred .
thus activity blockade for  <dig> hours does not affect the long-lasting, activity-induced increase in mepsc frequency if ap bursting is allowed to proceed for the first  <dig> hours after induction.
this shows that mepsc upregulation is not a short term consequence of ongoing ap bursting but is a long lasting consequence of activity-induced synaptic potentiation.
the reappearance of ap bursting is also sensitive to activity blockade during but not after a  <dig> hour critical period
we next asked whether ap bursting, like mepsc upregulation, requires a  <dig> hour post-induction period for long-term stabilisation of a potentiated state at transcription-dependent time points .
since these experiments only required the assessment of burst activity, the use of multi-electrode arrays  was favoured over single-cell patch clamp measurements.
meas permit non-invasive, repeated assessments of the activity state at up to  <dig> electrodes across the culture.
we used the same protocols shown in the patch clamp assessment of mepscs shown in figures 5a and 6a to assess whether ap bursting reappears after reversible blockade of action potentials either in the first  <dig> hours or in the period between  <dig> and  <dig> hours after the induction period .
a control group without bicuculline treatment was also included to assess the effects of a  <dig> hour ttx application on activity after washout.
quantification of bursting at the same electrodes at repeated time points is shown in figure 6d.
as has been reported previously  <cit> , bicuculline treatment transformed low levels of baseline network activity into synchronous ap bursting upon the application of bicuculline.
at the  <dig> hour time point synchronous ap bursting was maintained in the group which had not yet been treated with ttx and absent in the other two groups.
six hours after the induction period, prior to ttx-washout, no spike activity was present in any group.
after ttx washout, ap bursting returned in networks where ttx was applied  <dig> hours after burst-induction but not in networks incubated with ttx immediately after bicuculline or vehicle treatment.
thus ongoing bursting during, but not after the  <dig> hour post-induction period is necessary for long-term maintenance of the potentiated network state.
in summary, a  <dig> minute induction of ap bursting establishes a potentiated synaptic and network state evident from an increased mepsc frequency and ap bursting respectively.
the persistence of this potentiated state in the first  <dig> hours does not require transcription but does require ongoing action potential activity.
persistence beyond the  <dig> hour time point requires both activity and transcription to have occurred in the first  <dig> hours but does not require ongoing activity thereafter.
the direct link between transcription and a sustained increase in mepscs points to genes modulating quantal transmission in late-phase plasticity.
discussion
this study addresses for the first time the long-term persistence and transcription dependency of synaptic potentiation at the level of quantal synaptic transmission in single cells.
we found that mepsc frequency remains elevated for more than  <dig> hours after the induction of ap bursting in hippocampal cultures, an nmda receptor-dependent model for synaptic activity-induced, long-lasting plasticity.
this long-lasting potentiation of quantal transmission was transcription-dependent and required action potentials in the initial  <dig> hours after induction but was independent of ongoing activity thereafter.
thus, elevated quantal transmission is a direct consequence of gene transcription and is likely to be the mechanism which drives the network into a bursting state at transcription-dependent time points.
this data identifies a functional consequence of the expression of genes previously identified by gene chip analysis as essential for the expression of long-lasting synaptic plasticity in this model.
a new technique to investigate quantal transmission in late-phase plasticity
it has been known for  <dig> years that l-ltp is transcription-dependent and long lists of activity-induced genes have subsequently been published.
however, the potential role of such genes in late-phase plasticity is obscured by a lack of knowledge about the mechanism of potentiated synaptic transmission in l-ltp.
minimal stimulation recordings have been used to assess quantal transmission in e-ltp but their application to l-ltp is impeded by the need for single-cell recordings over an extended time course of more than  <dig> hours.
our technique instead uses between-cell analysis based on brief recordings  which first begin after synaptic potentiation is established thus minimising the washout of intracellular messengers, a problem inherent with traditional whole-cell patch clamp recordings of l-ltp.
mepsc recordings also assess quantal transmission but unlike minimal stimulation experiments sample all synaptic connections rather than a potentiated subset thereof.
thus mepsc analysis requires widespread synaptic potentiation which is presumably afforded by bicuculline which induced ap bursting in 100% of neurons in our hippocampal cultures.
nonetheless, large cell numbers are required to overcome high between-cell variability in mepsc parameters arising from differences in the synaptic integration of neurons into the network.
mepsc analysis is often complemented by paired-pulse protocols, which detect changes in presynaptic release probability or coefficient of variation analysis which can estimate quantal content.
however, such methods require recordings of evoked epscs which mostly evoke compound epscs in bursting cultures making them as yet technically unfeasible.
the site of late-phase synaptic plasticity
previous studies investigating quantal synaptic transmission at late time points have used activators or analogs of camp to induce chemical l-ltp in a large subset of synapses and have shown an increased action potential evoked presynaptic release  <cit>  and mepsc frequency but not amplitude  <cit> .
in line with these results, we found an increased frequency of mepscs, with no change in amplitude.
such changes are mostly attributed to a higher release probability of presynaptic vesicles  <cit> .
however, postsynaptic mechanisms can also increase mepsc frequency without affecting mepsc amplitude in a process termed synapse unsilencing  <cit> .
to address this possibility we measured responses to bath application of ampa to detect unsilencing mediated by de novo ampa receptor insertion.
note that this method would not detect unsilencing mediated by the lateral diffusion of already functional ampa receptors into the synaptic cleft.
previously, our group has shown a significant increase in ampa response and mepsc amplitude in within-cell but not between-cell analysis  <cit> .
here we observed a small increase in the whole-cell ampa response at early time points but not at time points relevant for l-ltp.
therefore ap bursting appears to induce some de novo ampa receptor insertion into the postsynaptic membrane which combined with the robust increase in mepscs observed would indicate either synapse formation or unsilencing.
however, the fraction of this ampa receptor pool is small and the variability of whole-cell ampa-responses in between-cell experiments may preclude the detection of such subtle changes.
thus the side of the synaptic cleft at which potentiation of mepsc transmission occurs cannot yet be clearly identified.
distinct mechanisms of early- and late-phase synaptic potentiation
synaptic transmission was upregulated immediately after a  <dig> minute induction period of ap bursting and persists for  <dig> hours yet the mechanisms maintaining the increased frequency of mepscs do not remain constant over this timeframe.
ongoing action potential activity was necessary to maintain synaptic potentiation during the first  <dig> hours after induction but not thereafter.
in contrast, gene transcription during the initial  <dig> hour critical period was necessary for the long-term but not the short-term maintenance of network potentiation.
this distinguishes early and late phases of synaptic potentiation in this plasticity model which parallel those of e-ltp and l-ltp in acute brain slice experiments.
the dependence of early-phase potentiation on ongoing activity suggests that early-phase potentiation is a short-term consequence of ongoing burst-activity through mechanisms such as post-tetanic potentiation.
in addition, our results reveal that activity, in the form of action potentials, is necessary during the initial  <dig> hours for the late-phase persistence of both ap bursting and increased mepsc frequency.
this suggests that activity plays a permissive role for the stabilisation of potentiated synaptic transmission.
for example, the targeting or expression of mrna transcribed during the initial  <dig> hour period may require ongoing activity  <cit> .
after the initial  <dig> hour period, the potentiation of mepscs becomes permanently stabilised as a direct result of gene transcription.
an increased mepsc frequency but not amplitude has previously been reported in hippocampal cultures  <dig> minutes after application of d1/d <dig> agonists  <cit> .
this effect was due to protein kinase a-mediated activation of dendritic translation leading to local glur <dig> expression and unsilencing of synapses.
blockade of nmda receptor-mediated mepscs is known to activate dendritic translation and glur <dig> expression within a  <dig> hour timeframe  <cit> .
the direct link between transcription and a sustained increase in mepscs points to the involvement of genes modulating quantal transmission in late-phase plasticity.
indeed it seems likely that mepsc upregulation underlies the maintenance of ap bursting in the late phase, showing a parallel time course in their expression and their sensitivity to activity or transcription blockade.
in line with this, previous work has shown that single mepscs can influence firing in electrically compact neurons  <cit>  and a burst of mepscs can drive action potentials in hippocampal pyramidal neurons <cit> .
the identification of genes underlying the potentiation of quantal synaptic transmission will help to unravel the molecular mechanisms responsible for the long-term maintenance of synaptic plasticity.
candidate genes for late-phase synaptic potentiation
the transcription-dependent mechanism which mediates mepsc upregulation and is triggered by ap bursting now remains to be discovered.
screening the population of genes that were upregulated during ap-bursting  <cit>  reveals several candidate genes whose protein products can regulate synaptic transmission.
three gene candidates stand out as being upregulated by ap bursting in an nmda receptor-dependant manner and are known to act presynaptically to upregulate vesicle fusion and neurotransmitter release.
these are syntaxin <dig>  homer <dig> and synaptotagminiv.
syntaxin  <dig> belongs to the family of snare-proteins involved in the regulation of vesicle-fusion.
however, its function to date has only been described at ribbon synapses in the retina  <cit> .
homer 1a and c have been suggested to be involved in the regulation of glutamate release although the underlying mechanism is not known  <cit> .
synaptotagminiv is a presynaptically expressed protein involved in the regulation of transmitter release but which has been shown to impede synaptic potentiation and short-term memory in the hippocampus  <cit> .
investigating these genes in the light of synaptic potentiation in hippocampal networks is therefore of great interest.
four postsynaptic genes which are also upregulated by ap bursting and have the potential to modulate quantal transmission are ephrin b <dig>  catenin, brain-derived-neurotrophic factor  and arc/arg <dig>   <cit> .
retrograde messengers such as nitric oxide , arachidonic acid and cell adhesion molecules can account for increased efficacy of transmitter release at presynaptic terminals  <cit> .
ephrin b <dig> is a member of the ephrin family of cell adhesion molecules which together with their receptors are considered to modulate synaptic transmission by bidirectional signalling  <cit> .
catenin, which binds to the cell adhesion molecule cadherin is involved in the control of vesicle clustering at presynaptic terminals  <cit>  indicating a role in the control of release probability.
also, bdnf can acutely modify synaptic efficacy in ltp  <cit>  most likely through a pre-synaptic mechanism  <cit>  such as enhancing neurotransmitter release  <cit> .
other ap bursting-induced genes such as a rc/arg <dig>  and ac <dig>  have also been identified in l-ltp studies.
arc/arg <dig>  is of special interest since it was shown that upregulation of this gene in the postsynaptic neuron is directly linked to the modulation of synaptic transmission  <cit>  by regulating ampa receptor internalisation  <cit> .
this may directly affect synaptic transmission and thus underlie synaptic potentiation.
such identified candidate genes can now be individually manipulated to test for a causal role in the increased mepsc frequency which occurs in late-phase, transcription-dependent plasticity induced by ap bursting in hippocampal cultures.
CONCLUSIONS
long-term memory is thought to involve gene transcription which alters synaptic efficacy for extended periods; though the link between genes and synaptic function specifically altered in late-phase plasticity remains unknown.
we show that the frequency of quantal synaptic events is increased for over  <dig> hours by synaptic activity known to induce gene transcription-dependent and nmda receptor-dependent hippocampal plasticity.
this identifies a specific synaptic function which is altered by one or more gene candidates already identified by gene chip analysis in the same cellular model.
this puts us one step closer to matching gene to function in late-phase plasticity.

 synaptic plasticity plays an important role in many aspects of brain function such as learning, memory and development. synaptic activity-induced changes in synaptic efficacy can persist for hours, days or even months given gene-transcription and subsequent synthesis of new protein. recently, gene chip analysis has identified large numbers of genes regulated by synaptic plasticity. the expression and synaptic localisation of activity-induced mrnas which encode proteins such as arc/arg have been shown to be crucial for the regulation of synaptic plasticity. although numerous gene candidates are emerging, detailed knowledge is still lacking about the synaptic mechanisms affected by the gene expression which transforms early-phase ltp into late-phase ltp. while altered pre- and post-synaptic function in e-ltp has been demonstrated with quantal analysis of patch clamp recordings from acute hippocampal brain slices, little is known about the nature, time course and mechanism of these changes in synaptically induced l-ltp. this lack arises in part from the difficulty of repeating measurements over more than hours from a subset of synapses on the same cell in an acute brain slice. here we assess the persistence and transcription dependency of potentiated synaptic transmission using a between cell analysis of brief recordings at multiple time points after its induction. we use a culture based system of synaptically activated long-term plasticity where gene profiling has identified candidates for transcription-dependent late-phase plasticity. removal of inhibitory synaptic transmission in dissociated hippocampal cultures with the gabaa-receptor antagonist bicuculline for minutes induces synchronous action potential bursting which persists for more than hours. this potentiated state in culture mimics that of nmda receptor-dependent l-ltp in slices, requiring extracellular signal-regulated kinase 1/ signalling and both gene transcription and translation. neurons in bicuculline treated cultures show an increased frequency of ampa receptor-mediated mepscs at a time point minutes after washout. mepscs represent the postsynaptic response to spontaneous release of single neurotransmitter vesicles at functional synapses and thus provide an index of synaptic efficacy and connectivity at a quantal level. mepscs are known to be increased in frequency and/or amplitude in ltp in hippocampal slices; however it is not known whether such changes persist during l-ltp. we show that synaptic potentiation, manifested as an increased mepsc frequency in the hippocampal culture model of ltp, is preserved at transcription-relevant time points and requires transcription. this finding points to a common transcription-dependent mechanism in the maintenance of synaptic potentiation and recurrent network bursting which is established within the first two hours after ltp induction.  assessment of ap bursting and synaptic transmission in cultured hippocampal networks, it has previously been shown that ampa receptor-mediated mepscs show an increased frequency but not amplitude at a time point minutes after ltp induction with a minute bicuculline treatment. to examine the longevity of this upregulated quantal synaptic transmission, we randomly selected one neuron per coverslip and made whole-cell patch clamp recordings of mepscs at time points up to hours after treatment with either bicuculline or vehicle for minutes. prior to mepsc-recordings, activity was assessed in all neurons by both cell-attached and whole-cell current clamp recordings. neurons showed distinct activity patterns categorised as no activity, random activity or recurrent bursting. bursts were composed of clusters of to spikes separated by relatively spike-free intervals of to seconds. cumulative probability histograms for inter event intervals and amplitudes were generated from at least mepscs recorded from each cell. mepscs were sensitive to μm dnqx confirming their identity as ampa receptor-mediated mepscs. responses were also recorded to bath applied ampa in the presence of cyclothiazide to prevent desensitisation. this produced a steady state plateau within s whose amplitude was used as an indication of the total number of functional ampa receptors per cell. potentiation in synaptic transmission over a hour period following the induction of ap bursting synaptic potentiation has previously been demonstrated in hippocampal cultures at a single time point minutes after a minute induction period of ap bursting. at this time point, mepsc frequency was shown to be increased but little effect was seen on mepsc amplitude or whole-cell ampa responses. although this study showed that ap bursting persists more than hours after induction, it is not known whether mepsc frequency remains increased and amplitude and ampa responses remain unchanged at later time points. we first examined mepsc results pooled from recordings at all time points within hours after the minute induction period. spontaneous and in particular, bicuculline-induced ap bursting significantly reduced mepsc iei but did not significantly affect the mepsc amplitude or the whole-cell ampa current. to better illustrate the time course of these effects, data was split into three groups: min to h, to h and to h. these groups span the period of transcription dependency of synaptic plasticity and ap bursting whereby the transcription inhibitor actinomycin d having little effect over the first hours after induction, causes a slow return to baseline activity levels between and hours after induction. neither mepsc amplitude nor whole-cell ampa current was significantly affected by ap bursting at any time point. however, when bursting and non-bursting cell groups were pooled, whole-cell ampa response was slightly elevated in bicuculline vs. vehicle treated groups at the time points min to h and h to h. no difference in ampa whole-cell response was apparent in pooled data for the to h group. in contrast, iei of mepscs was significantly shorter in comparison to untreated and vehicle treated cultures at all three time points. thus, a minute induction of ap bursting induces long lasting changes in mepsc iei but not amplitude or whole-cell ampa responses as was previously reported at a single minute post-induction time point. this indicates that, along with ap bursting, synaptic transmission is retained at an elevated level at transcription-dependent time points. ap bursting does not persist in all cells and is sensitive to actinomycin d the persistence of bicuculline-induced bursting activity in hippocampal cultures and its sensitivity to the transcription inhibitor, actinomycin d has previously been investigated using arrays of planar extracellular electrodes, each of which can detect action potentials in multiple axons and somata within approximately μm of the electrode. the prevalence, persistence and transcription dependency of ap bursting in the post induction period has not been examined on a single cell level to date. we first tested whether ap bursting-induced gene transcription is indeed inhibited by actinomycin d. basal expression of the immediate-early gene c-fos in hippocampal cultures was very low in control conditions and even lower in the presence of actinomycin d. expression of c-fos was strongly induced hours but not minutes after induction of ap bursting with a minute bicuculline treatment. this induction of c-fos expression at the h time point was abolished by the transcription inhibitor actinomycin d confirming that ap bursting-induced transcription is effectively blocked by this treatment. the effects of bicuculline and actinomycin d treatment on ap bursting were next assessed on a single cell level minutes to hours after washout of bicuculline or vehicle. bicuculline increased the overall incidence of ap bursting from out of neurons to out of neurons. the proportion of cells displaying burst activity in either vehicle or bicuculline treated groups was considerably reduced however after exposure to actinomycin d vehicle treated and in out of bicuculline treated neurons). the time course of these effects is shown in figure 3c. bicuculline induced ap bursting in 100% of neurons regardless of any pre-treatment with actinomycin d. the proportion of neurons bursting minutes after bicuculline washout was reduced to 67%, indicating that bursting activity is not maintained in all neurons but that this does not originate in a failure to participate in ap bursting during the induction period. however, this proportion declined only slightly over the next hours to a level of 52%. field recordings from meas have previously demonstrated that ap bursting persists for more than hours in 90% of cultures. this figure represents the persistence of bursting at a single electrode in a electrode array rather than the number of individual neurons which maintain ap bursting. actinomycin d however, caused a sharp reduction in the incidence of ap bursting in the first hours after its induction and had virtually abolished all bursting at later time points. this single-cell data supports previous evidence from multi-cellular recordings that bicuculline-induced ap bursting requires gene transcription for its maintenance and further reveals an inhibitory effect of actinomycin d on bursting of a spontaneous origin. thus even spontaneous ap bursting requires ongoing gene transcription. the rapid shutdown of ap bursting in actinomycin d showed similar kinetics to the actinomycin d-induced decay of epsc potentiation in acute slices although somewhat faster than that reported by arnold et al.. the slower decay of potentiation in the mea study is likely due to the quantification of spikes within bursts at a single electrode rather than sampling single cells across the network as we have done or assessing the amplitude of extracellularly recorded signals which reflects to some extent the number of responsive cells. the long-lasting, activity-induced increase in mepscs is actinomycin d sensitive given the persistence of mepsc upregulation at time points later than hours after the induction of ap bursting and the dependence of ap bursting on gene transcription, we wished to directly test whether the long lasting upregulation of mepscs was transcription-dependent. mepscs were recorded from actinomycin d treated coverslips after the assessment of ap bursting at time points minutes to hours after bicuculline washout. in actinomycin d treated neurons as in control conditions, ap bursting did not significantly affect the mepsc amplitude or the whole-cell ampa current. actinomycin d did not affect the reduction in mepsc iei caused by ap bursting when data in each treatment group was pooled from all time points. however, when this data from actinomycin d treated cells was separated into time groups, bursting and the reduction in iei induced by ap bursting were only apparent at early time points. at time points more than hours after induction of ap bursting, actinomycin d had abolished both ap bursting and any differences in mepsc iei between vehicle and bicuculline treated groups. thus, actinomycin d had abolished both ap bursting and the effects of ap bursting on mepsc iei at transcription-dependent time points but not at early time points. we also noted that actinomycin d reduced mepsc iei in vehicle treated cells though this was only significant in the min to h time period. similar effects have been noted previously where the slope of field evoked epsps is increased during the first hours of actinomycin d application. pharmacological blockade of bursting immediately after the induction-period returns mepsc frequency to baseline. given the correlation between mepsc frequency and ap bursting and their parallel time course of inhibition by actinomycin d, we wished to establish whether mepsc upregulation per se requires transcription or is just maintained by ongoing bursting which itself is transcription-dependent. we investigated whether a brief induction of ap bursting is sufficed to cause a long-lasting increase in synaptic transmission which persists even if ap bursting is blocked in the post induction period. alternatively, if mepsc upregulation were a short term effect of ap bursting then ap blockade would cause mepsc frequency to rapidly decay back to baseline levels. to test these hypotheses, burst-activity was blocked immediately after the minute induction period by ttx application before measuring mepscs within minutes, minutes or 2- hours. since bursting was completely absent in all cells exposed to ttx in the post-induction period, data was pooled irrespective of burst activity prior to mepsc measurement. a clear ap bursting-induced reduction in mepsc iei was still apparent at all time points in pooled control data. pre-incubation with actinomycin d and/or ttx application after bicuculline washout did not disrupt the effects of ap bursting on mepsc iei within the first minutes of the post-induction period. this result indicates that upregulation of synaptic transmission outlasts a brief blockade of ap bursting. however, blockade of ap bursting with ttx after the minute induction period caused a gradual return of mepsc iei to baseline levels at the h time point. this indicates that the activity-induced increase in mepscs requires either ongoing ap bursting or more than minutes of ap bursting to persist until transcription-dependent, "late-phase" time points. for direct comparison of the effects of actinomycin d, data from both bursting and non-bursting cells were pooled and re-plotted in figure 5b. actinomycin d treatment in the absence of ttx caused a more rapid loss of iei potentiation at both the min and h time points. surprisingly, the combination of both actinomycin d and ttx treatment in the post-induction period resulted in a less rapid decay of mepsc iei back to baseline levels at the h time point. it is not clear why ap-blockade should slow the effects of actinomycin d in depotentiating synaptic transmission. one possibility is that the early phase of de-potentiation caused by actinomycin d is mediated by some activity-dependent depletion of the vesicular pool and may relate to the increased amplitude of evoked responses seen within hours of actinomycin d application. in summary, either ongoing activity or transcription or both are necessary for the potentiation of mepsc frequency to persist beyond the hour post-induction time point. a hour critical period of activity is required to stabilise the increase in mepscs at the hour time point we next tested the possibility that potentiated synaptic transmission requires continued ap bursting activity after the minute induction period to become independent of ap bursting during the transcription-dependent late phase. therefore, ttx was applied hours after the minute induction period leaving a time-window of hours for the stabilisation of potentiated synaptic transmission. ttx was then applied for hours before recording mepscs at the time point of hours after the induction of ap bursting. in control cells which did not receive ttx treatment, mepsc iei was significantly reduced in the bicuculline treated group compared to the vehicle treated group. this reduced mepsc iei was also present in the group treated with ttx for hours. the amplitude of mepscs was unaffected by this hour ttx-treatment indicating that synaptic scaling had not occurred. thus activity blockade for hours does not affect the long-lasting, activity-induced increase in mepsc frequency if ap bursting is allowed to proceed for the first hours after induction. this shows that mepsc upregulation is not a short term consequence of ongoing ap bursting but is a long lasting consequence of activity-induced synaptic potentiation. the reappearance of ap bursting is also sensitive to activity blockade during but not after a hour critical period we next asked whether ap bursting, like mepsc upregulation, requires a hour post-induction period for long-term stabilisation of a potentiated state at transcription-dependent time points. since these experiments only required the assessment of burst activity, the use of multi-electrode arrays was favoured over single-cell patch clamp measurements. meas permit non-invasive, repeated assessments of the activity state at up to electrodes across the culture. we used the same protocols shown in the patch clamp assessment of mepscs shown in figures 5a and 6a to assess whether ap bursting reappears after reversible blockade of action potentials either in the first hours or in the period between and hours after the induction period. a control group without bicuculline treatment was also included to assess the effects of a hour ttx application on activity after washout. quantification of bursting at the same electrodes at repeated time points is shown in figure 6d. as has been reported previously, bicuculline treatment transformed low levels of baseline network activity into synchronous ap bursting upon the application of bicuculline. at the hour time point synchronous ap bursting was maintained in the group which had not yet been treated with ttx and absent in the other two groups. six hours after the induction period, prior to ttx-washout, no spike activity was present in any group. after ttx washout, ap bursting returned in networks where ttx was applied hours after burst-induction but not in networks incubated with ttx immediately after bicuculline or vehicle treatment. thus ongoing bursting during, but not after the hour post-induction period is necessary for long-term maintenance of the potentiated network state. in summary, a minute induction of ap bursting establishes a potentiated synaptic and network state evident from an increased mepsc frequency and ap bursting respectively. the persistence of this potentiated state in the first hours does not require transcription but does require ongoing action potential activity. persistence beyond the hour time point requires both activity and transcription to have occurred in the first hours but does not require ongoing activity thereafter. the direct link between transcription and a sustained increase in mepscs points to genes modulating quantal transmission in late-phase plasticity. discussion this study addresses for the first time the long-term persistence and transcription dependency of synaptic potentiation at the level of quantal synaptic transmission in single cells. we found that mepsc frequency remains elevated for more than hours after the induction of ap bursting in hippocampal cultures, an nmda receptor-dependent model for synaptic activity-induced, long-lasting plasticity. this long-lasting potentiation of quantal transmission was transcription-dependent and required action potentials in the initial hours after induction but was independent of ongoing activity thereafter. thus, elevated quantal transmission is a direct consequence of gene transcription and is likely to be the mechanism which drives the network into a bursting state at transcription-dependent time points. this data identifies a functional consequence of the expression of genes previously identified by gene chip analysis as essential for the expression of long-lasting synaptic plasticity in this model. a new technique to investigate quantal transmission in late-phase plasticity it has been known for years that l-ltp is transcription-dependent and long lists of activity-induced genes have subsequently been published. however, the potential role of such genes in late-phase plasticity is obscured by a lack of knowledge about the mechanism of potentiated synaptic transmission in l-ltp. minimal stimulation recordings have been used to assess quantal transmission in e-ltp but their application to l-ltp is impeded by the need for single-cell recordings over an extended time course of more than hours. our technique instead uses between-cell analysis based on brief recordings which first begin after synaptic potentiation is established thus minimising the washout of intracellular messengers, a problem inherent with traditional whole-cell patch clamp recordings of l-ltp. mepsc recordings also assess quantal transmission but unlike minimal stimulation experiments sample all synaptic connections rather than a potentiated subset thereof. thus mepsc analysis requires widespread synaptic potentiation which is presumably afforded by bicuculline which induced ap bursting in 100% of neurons in our hippocampal cultures. nonetheless, large cell numbers are required to overcome high between-cell variability in mepsc parameters arising from differences in the synaptic integration of neurons into the network. mepsc analysis is often complemented by paired-pulse protocols, which detect changes in presynaptic release probability or coefficient of variation analysis which can estimate quantal content. however, such methods require recordings of evoked epscs which mostly evoke compound epscs in bursting cultures making them as yet technically unfeasible. the site of late-phase synaptic plasticity previous studies investigating quantal synaptic transmission at late time points have used activators or analogs of camp to induce chemical l-ltp in a large subset of synapses and have shown an increased action potential evoked presynaptic release and mepsc frequency but not amplitude. in line with these results, we found an increased frequency of mepscs, with no change in amplitude. such changes are mostly attributed to a higher release probability of presynaptic vesicles. however, postsynaptic mechanisms can also increase mepsc frequency without affecting mepsc amplitude in a process termed synapse unsilencing. to address this possibility we measured responses to bath application of ampa to detect unsilencing mediated by de novo ampa receptor insertion. note that this method would not detect unsilencing mediated by the lateral diffusion of already functional ampa receptors into the synaptic cleft. previously, our group has shown a significant increase in ampa response and mepsc amplitude in within-cell but not between-cell analysis. here we observed a small increase in the whole-cell ampa response at early time points but not at time points relevant for l-ltp. therefore ap bursting appears to induce some de novo ampa receptor insertion into the postsynaptic membrane which combined with the robust increase in mepscs observed would indicate either synapse formation or unsilencing. however, the fraction of this ampa receptor pool is small and the variability of whole-cell ampa-responses in between-cell experiments may preclude the detection of such subtle changes. thus the side of the synaptic cleft at which potentiation of mepsc transmission occurs cannot yet be clearly identified. distinct mechanisms of early- and late-phase synaptic potentiation synaptic transmission was upregulated immediately after a minute induction period of ap bursting and persists for hours yet the mechanisms maintaining the increased frequency of mepscs do not remain constant over this timeframe. ongoing action potential activity was necessary to maintain synaptic potentiation during the first hours after induction but not thereafter. in contrast, gene transcription during the initial hour critical period was necessary for the long-term but not the short-term maintenance of network potentiation. this distinguishes early and late phases of synaptic potentiation in this plasticity model which parallel those of e-ltp and l-ltp in acute brain slice experiments. the dependence of early-phase potentiation on ongoing activity suggests that early-phase potentiation is a short-term consequence of ongoing burst-activity through mechanisms such as post-tetanic potentiation. in addition, our results reveal that activity, in the form of action potentials, is necessary during the initial hours for the late-phase persistence of both ap bursting and increased mepsc frequency. this suggests that activity plays a permissive role for the stabilisation of potentiated synaptic transmission. for example, the targeting or expression of mrna transcribed during the initial hour period may require ongoing activity. after the initial hour period, the potentiation of mepscs becomes permanently stabilised as a direct result of gene transcription. an increased mepsc frequency but not amplitude has previously been reported in hippocampal cultures minutes after application of d1/d agonists. this effect was due to protein kinase a-mediated activation of dendritic translation leading to local glur expression and unsilencing of synapses. blockade of nmda receptor-mediated mepscs is known to activate dendritic translation and glur expression within a hour timeframe. the direct link between transcription and a sustained increase in mepscs points to the involvement of genes modulating quantal transmission in late-phase plasticity. indeed it seems likely that mepsc upregulation underlies the maintenance of ap bursting in the late phase, showing a parallel time course in their expression and their sensitivity to activity or transcription blockade. in line with this, previous work has shown that single mepscs can influence firing in electrically compact neurons and a burst of mepscs can drive action potentials in hippocampal pyramidal neurons. the identification of genes underlying the potentiation of quantal synaptic transmission will help to unravel the molecular mechanisms responsible for the long-term maintenance of synaptic plasticity. candidate genes for late-phase synaptic potentiation the transcription-dependent mechanism which mediates mepsc upregulation and is triggered by ap bursting now remains to be discovered. screening the population of genes that were upregulated during ap-bursting reveals several candidate genes whose protein products can regulate synaptic transmission. three gene candidates stand out as being upregulated by ap bursting in an nmda receptor-dependant manner and are known to act presynaptically to upregulate vesicle fusion and neurotransmitter release. these are syntaxin homer and synaptotagminiv. syntaxin belongs to the family of snare-proteins involved in the regulation of vesicle-fusion. however, its function to date has only been described at ribbon synapses in the retina. homer 1a and c have been suggested to be involved in the regulation of glutamate release although the underlying mechanism is not known. synaptotagminiv is a presynaptically expressed protein involved in the regulation of transmitter release but which has been shown to impede synaptic potentiation and short-term memory in the hippocampus. investigating these genes in the light of synaptic potentiation in hippocampal networks is therefore of great interest. four postsynaptic genes which are also upregulated by ap bursting and have the potential to modulate quantal transmission are ephrin b catenin, brain-derived-neurotrophic factor and arc/arg. retrograde messengers such as nitric oxide, arachidonic acid and cell adhesion molecules can account for increased efficacy of transmitter release at presynaptic terminals. ephrin b is a member of the ephrin family of cell adhesion molecules which together with their receptors are considered to modulate synaptic transmission by bidirectional signalling. catenin, which binds to the cell adhesion molecule cadherin is involved in the control of vesicle clustering at presynaptic terminals indicating a role in the control of release probability. also, bdnf can acutely modify synaptic efficacy in ltp most likely through a pre-synaptic mechanism such as enhancing neurotransmitter release. other ap bursting-induced genes such as a rc/arg and ac have also been identified in l-ltp studies. arc/arg is of special interest since it was shown that upregulation of this gene in the postsynaptic neuron is directly linked to the modulation of synaptic transmission by regulating ampa receptor internalisation. this may directly affect synaptic transmission and thus underlie synaptic potentiation. such identified candidate genes can now be individually manipulated to test for a causal role in the increased mepsc frequency which occurs in late-phase, transcription-dependent plasticity induced by ap bursting in hippocampal cultures.  long-term memory is thought to involve gene transcription which alters synaptic efficacy for extended periods; though the link between genes and synaptic function specifically altered in late-phase plasticity remains unknown. we show that the frequency of quantal synaptic events is increased for over hours by synaptic activity known to induce gene transcription-dependent and nmda receptor-dependent hippocampal plasticity. this identifies a specific synaptic function which is altered by one or more gene candidates already identified by gene chip analysis in the same cellular model. this puts us one step closer to matching gene to function in late-phase plasticity. 
reversible blockade of action potentials during, but not after this hour transcription period suppressed the increase in mepsc frequency and the recovery of burst activity at a time point hours after induction. the persistence of recurrent action potential bursting in these cultures is transcription-, translation- and nmda receptor-dependent thus providing an accessible model for long-lasting plasticity. blockade of gabaa-receptors with bicuculline for minutes induced action potential bursting in all neurons and was maintained in 50-60% of neurons for more than hours. the transcription blocker actinomycin d abrogated, within hours of burst induction, both action potential bursting and the increase in mepscs. this long-lasting mepsc upregulation is transcription-dependent and requires ongoing action potential activity during the initial hour period but not thereafter. thus mepsc upregulation may underlie the long term, transcription-dependent persistence of action potential bursting. the magnitude and longevity of synaptic activity-induced changes in synaptic efficacy is quantified by measuring evoked responses whose potentiation requires gene transcription to persist for more than 2- hours. while miniature epscs are also increased in amplitude and/or frequency during long-term potentiation, it is not known how long such changes persist or whether gene transcription is required. these results indicate that increased mepsc frequency persists well beyond the hour transcription-independent phase of plasticity in this model. we use whole-cell patch clamp recordings from dissociated hippocampal cultures to characterise for the first time the persistence and transcription dependency of mepsc upregulation during synaptic potentiation. this provides mechanistic insight to link gene candidates already identified by gene chip analysis to long lasting plasticity in this in vitro model.
 the magnitude and longevity of synaptic activity-induced changes in synaptic efficacy is quantified by measuring evoked responses whose potentiation requires gene transcription to persist for more than 2- hours. while miniature epscs are also increased in amplitude and/or frequency during long-term potentiation, it is not known how long such changes persist or whether gene transcription is required.  we use whole-cell patch clamp recordings from dissociated hippocampal cultures to characterise for the first time the persistence and transcription dependency of mepsc upregulation during synaptic potentiation. the persistence of recurrent action potential bursting in these cultures is transcription-, translation- and nmda receptor-dependent thus providing an accessible model for long-lasting plasticity. blockade of gabaa-receptors with bicuculline for minutes induced action potential bursting in all neurons and was maintained in 50-60% of neurons for more than hours. throughout this period, the frequency but neither the amplitude of mepscs nor whole-cell ampa currents was markedly increased. the transcription blocker actinomycin d abrogated, within hours of burst induction, both action potential bursting and the increase in mepscs. reversible blockade of action potentials during, but not after this hour transcription period suppressed the increase in mepsc frequency and the recovery of burst activity at a time point hours after induction.  these results indicate that increased mepsc frequency persists well beyond the hour transcription-independent phase of plasticity in this model. this long-lasting mepsc upregulation is transcription-dependent and requires ongoing action potential activity during the initial hour period but not thereafter. thus mepsc upregulation may underlie the long term, transcription-dependent persistence of action potential bursting. this provides mechanistic insight to link gene candidates already identified by gene chip analysis to long lasting plasticity in this in vitro model. 