Cholinergic inputs induce timing-dependent hippocampal plasticity in septo-hippocampal co-cultures as in acute hippocampal slices
Previous studies have shown that the septo-hippocampal co-culture system provides an excellent model to study cholinergic modulation of hippocampal functions (Gahwiler and Brown, 1985
; Fischer et al., 1999
), even including complex network activities like theta oscillations (Fischer et al., 1999
). In addition a major advantage of using the co-culture system (instead of acute hippocampal slices) is that this allows for the expression of proteins (e.g. nAChR subunits or GECIs) to restricted hippocampal subregions either in wildtype or transgenic mice (). We have previously shown that septal cholinergic inputs, activated either by electrical stimulation or via an optogenetic approach, can induce three types of hippocampal Schaffer collateral (SC) to CA1 synaptic plasticity, depending on the timing of the cholinergic input activation relative to the SC inputs in acute hippocampal slices (Gu and Yakel, 2011
). In this study, we used GECIs to measure calcium activity changes at both pre- and postsynaptic sites to understand their respective contributions to the expression of synaptic plasticity (). Secondly, we wanted to express the α7 nAChR subunit to either pre- or postsynaptic sites (or both) in α7 nAChR knockout slices in order to dissect out the roles of pre- and postsynaptic α7 nAChRs in inducing the α7 nAChR-dependent LTP and STD. To do this, medial septum tissues and hippocampi were dissected out from brain slices and placed next to each other on transwell membranes and cultured (). The following day, viruses encoding either the GECIs or the α7 nAChR subunit were microinjected into the hippocampal CA1 or CA3 regions, and experiments were done 7 days after viral infection to allow protein expression and cholinergic innervation into the hippocampus ().
Monitoring neuronal synaptic activities with calcium imaging in septo-hippocampal slice co-cultures
First we verified that the three different forms of ACh release-induced synaptic plasticity that we had previously observed in acute hippocampal slices using an optogenetic approach was well preserved with the same timing-sensitive modulation in the septo-hippocampal co-culture system. We selectively expressed the light-activated cation channel channelrhodopsin-2 (ChR2) in cholinergic neurons by microinjecting a Cre-inducible adeno-associated virus (AAV) containing a double floxed inverted ChR2 (fused with mCherry for visualization) (Tsai et al., 2009
; Witten et al., 2010
)to the septal tissue from cholineacetyltransferase (ChAT)-Cre transgenic mice. Extensive cholinergic innervations to the hippocampus can be observed 7 days later (). Cholinergic inputs were then activated with 488 nm light exposure (20 ms) in the stratum oriens (SO) layer above the CA1 pyramidal neuron under patch-clamp recording. The SC pathway was activated via an electrode (). Pairing of cholinergic fiber activation (via light exposure) with SC pathway (electrical) stimulation at three time intervals were selected to mimic the corresponding pairings of SO and SC electrical stimulation that produced the three types of synaptic plasticity previously observed in acute hippocampal slices (Gu and Yakel, 2011
Optogenetically-activated cholinergic inputs induce similar timing-dependent hippocampal plasticity in septo-hippocampal co-cultures as in acute hippocampal slices
Consistent with the results from acute hippocampal slices, when the cholinergic input was activated 100 ms before SC stimulation, LTP was induced (). When cholinergic inputs were activated 10 ms before SC stimulation, STD was induced (). Finally when cholinergic input was activated 10 ms after SC stimulation, LTP was induced (). These results demonstrated that the complex cholinergic modulation of hippocampal plasticity was well preserved in the septo-hippocampal co-culture system as in the acute hippocampal slices (). Furthermore, these results reinforced our previous observation of the robust timing-dependent cholinergic control of hippocampal function by successfully duplicating it in another system. Here we focus on these first two forms of plasticity (i.e. the LTP and STD), both of which were previously shown to be dependent on activation of the α7 nAChR, and attempt to dissect out the individual contribution of pre- and postsynaptic α7 nAChRs in inducing this plasticity.
The α7 nAChR-dependent LTP involves prolonged calcium activity enhancement at both pre- and postsynaptic sites
Initially we examined whether the α7 nAChR-dependent LTP was due to enhanced presynaptic or postsynaptic activity. By expressing the genetically-encoded calcium indicator (GECI) GCaMP3 (Tian et al., 2009
) in restricted hippocampal subregions, we have been able to monitor the SC stimulation-induced intracellular calcium increases in either pre- or postsynaptic sites during and after the induction of LTP. To measure postsynaptic activity in the CA1 region, GCaMP3 was selectively expressed in hippocampal CA1 neurons and their dendrites, and the fluorescent signal was monitored in the CA1 stratum radiatum (SR) layer (). To measure presynaptic activity in the CA1 region instead, GCaMP3 was selectively expressed in CA3 neurons by viral microinjection in the CA3 region, and the fluorescent signal from the projecting axons was monitored in the CA1 SR layer (). To avoid activating ChR2 while imaging GCaMP3 with 488 nm light, in these cases the cholinergic neurons were electrically stimulated with an electrode placed in the septal tissue (rather than using light to activate ChR2); this induced the same timing-dependent plasticity when paired with SC stimulation (data not shown).
Calcium imaging was carried out in the CA1 SR region 5 sec before and after SC stimulation to measure the basal transient increase in intracellular calcium levels (i.e. GCaMP3 intensity increase) due to SC stimulation. When measuring the averaged fluorescence from the whole image, stimulation of the SC pathway usually induced a 5% increase over the background fluorescence, with the peak reached within 0.5 sec and returning to basal levels within 2 sec (). This sampling was repeated every 1 to 2 min until stable responses were achieved (usually after about 10 min), after which the pairing protocol was introduced to induce synaptic plasticity. The sampling was then carried out for another 30 min to monitor any changes in the SC stimulation-induced calcium responses, and then the peak amplitude of the responses at 10 and 30 min after the pairing protocol were compared with the basal SC responses.
The α7 nAChR-mediated LTP involves prolonged calcium activity enhancement in both pre- and postsynaptic sites in septo-hippocampal slice co-cultures
When monitoring the postsynaptic calcium activity in the CA1 SR region (i.e. when GCaMP3 was expressed in CA1 neurons and the dendrites were monitored), the SC stimulation-induced response amplitudes were significantly increased after pairing the cholinergic input 100 ms before SC pathway stimulation (43 ± 8% and 58 ± 6% increase at 10 and 30 min, respectively, n = 6; ). This increase lasted for at least 30 min, demonstrating that a protocol that induces LTP at CA1 synapses induces prolonged increases in postsynaptic calcium activity. When monitoring presynaptic calcium activity in the same region (i.e. when the GCaMP3 was expressed in the CA3 neurons and the projecting SC axons were monitored in the CA1 SR region), SC stimulation-induced response amplitudes were also increased after pairing cholinergic input 100 ms before SC pathway stimulation (54 ± 3% and 41 ± 7% increase at 10 and 30 min, respectively, n = 6; ), also demonstrating that the LTP-inducing protocol significantly increases presynaptic calcium activity for a prolonged period of time. This presynaptic data is significant as our previous work had suggested that there was only a transient increase in presynaptic activity when using the paired-pulse ratio (PPR) as an indicator (Gu and Yakel, 2011
Both pre- and postsynaptic α7 nAChRs are required to induce the α7 nAChR-dependent LTP
We previously showed that the α7 nAChR-dependent LTP was likely due to a postsynaptic effect since it appeared to require the activation of the NMDAR and prolongation of the NMDAR-mediated calcium transients in the spines, and GluR2-containing AMPAR synaptic insertion. To examine the contributions of pre- and/orpostsynaptic α7 receptors to plasticity in more detail, we used a strategy of restoring the expression of the α7 nAChR subunit to slices obtained from α7 nAChR knockout mice; in these mice, this form of LTP was absent. Initially we verified that in septo-hippocampal co-cultures from α7 nAChR knockout mice (the septal tissue providing cholinergic inputs was from wildtype mice), the pairing protocol that normally would induce the α7 nAChR-dependent LTP failed to induce any significant prolonged increase in calcium signals at either post- or presynaptic sites (−6 ± 4% and −3 ± 5% change at 30 min, respectively, n = 5; ).
Next, we then restored the α7 nAChR (Seguela et al., 1993
) to either postsynaptic (by expressing the α7 nAChR along with GCaMP3 into CA1 neurons) or presynaptic sites (by expressing the α7 receptor into CA3 neurons in α7 nAChR knockout slices). The α7 nAChR was also subcloned to the AAV vector under the synapsin promoter. The restricted localization of α7 nAChRs and GCaMP3 to either CA1 or CA3 was verified with the GCaMP3 expression pattern before imaging experiments. When the α7 receptor was restored in the postsynaptic CA1 neurons only, the SC stimulation-induced GCaMP3 responses were transiently
increased in the postsynaptic sites after the pairing (44 ± 5% and −1 ± 2% change at 10 and 30 min, respectively, n = 5; ). Similarly when the α7 nAChR was restored in the presynaptic CA3 neurons only, the SC stimulation- induced GCaMP3 responses were also transiently increased in the presynaptic sites after the pairing (50 ± 7% and 4 ± 4% increase at 10 and 30 min, respectively, n = 5; ). When we expressed the α7 nAChR in both CA1 and CA3 neurons in the same slice (i.e. both post- and presynaptically), this restored the LTP that was seen in wildtype slices (i.e. a prolonged enhancement in the SC stimulation-induced GCaMP3 responses both pre- and postsynaptically; 31 ± 6% and 70 ± 7% increase at 30 min, respectively, n = 5; ) These data demonstrate that both pre- and postsynaptic α7 nAChRs play a role in regulating synaptic activities, and that both pre- and postsynaptic α7 receptors must be present in order to induce the α7 nAChR-dependent LTP. Furthermore these findings suggest that coordination between the pre- and postsynaptic modulation is required to induce this form of LTP.
Expression of α7 nAChR in post- or presynaptic sites in α7 nAChR KO slice only induced transient activity enhancement in post- or presynaptic sites, respectively
Both pre- and postsynaptic α7 nAChRs are required to induce the α7 nAChR-dependent STD
We have previously shown that the STD induced by pairing cholinergic input stimulation 10 ms before SC pathway stimulation was also mediated by the α7 nAChR, although a presynaptic mechanism was previously suggested from the change in the paired-pulse ratio. Therefore we tested whether a presynaptic mechanism alone is sufficient to explain this STD. In wildtype co-cultured slices, we observed a transient decrease in the SC stimulation-induced GCaMP3 responses at both post- (−39 ± 5% and 8 ± 5% change at 10 and 30 min, respectively, n = 5; ) and presynaptic sites (−37 ± 6% and −3 ± 3% change at 10 and 30 min, respectively, n = 5; ). Furthermore, both responses were abolished in α7 nAChR knockout slices (). When we restored the α7 nAChR to either the presynaptic (5 ± 7% change at 10 min, n = 5; ) or the postsynaptic (6 ± 7% change at 10 min, n = 5; ) sites alone (inα7 nAChR knockout slices), we could not restore the STD (); restoring the α7 receptor to both pre- and postsynaptic sites in the same slice was required to restore the STD in the α7 knockout slices (). This suggests that a presynaptic mechanism alone is not sufficient to induce this form of STD, again further implicating some type of coordination between pre- and postsynaptic sites.
The α7 nAChR-mediated STD involves transient calcium activity depression in both post- and presynaptic sites
The expression of functional α7 nAChRs in pyramidal neurons was verified by directly measuring choline (10 mM)-induced α7 nAChR currents (Fayuk and Yakel, 2004
). The α7 nAChR currents were induced in all of the CA1 and CA3 pyramidal neurons we tested (8 to 12 neurons for either group), with peak amplitudes of ~ 50–100 pA which were completely blocked by the α7-selective antagonist MLA (10 nM); choline-induced responses were absent in α7 nAChR knockout slices (). Virus-introduced α7 nAChRs (fused and visualized with YFP in AAV virus under the synapsin promoter) restored α7 nAChR currents in both CA1 and CA3 neurons from α7 nAChR knockout slices, with peak amplitudes not significantly different than those from wildtype slices () in both CA1 and CA3 neurons. Expression of the reporter YFP alone (in AAV under the synapsin promoter) did not result in any α7 nAChR currents in knockout slices, and had no obvious effects in wildtype slices (data were combined with the respective knockout and wildtype groups as shown in ).
Postsynaptic functionalα7 nAChRs in CA1 pyramidal neuron were required to induce either LTP or STD
To further verify the direct involvement of postsynaptic α7 nAChRs in CA1 pyramidal neurons in this plasticity, we compared the plasticity in individual α7 nAChR-positive (fused with YFP) and neighboring α7 nAChR-negative CA1 pyramidal neurons from the knockout rescue (both CA1 and CA3 rescue) slices with whole cell patch clamp. Consistent with the GCaMP3 imaging data, both LTP and STD were induced in α7 nAChR-positive CA1 pyramidal neurons (). However, only STP was induced inα7 nAChR-negative CA1 pyramidal neurons with the LTP pairing protocol, and no plasticity was induced with the STD protocol (), which is consistent with the presynaptic-only rescue imaging data. These results strongly suggest the direct involvement of α7 nAChRs in postsynaptic CA1 pyramidal neurons in inducing either LTP or STD.
Differential pre- and postsynaptic mechanisms in the expression of both α7 nAChR- dependent LTP and STD revealed with dual-color GECI imaging
Although the α7 nAChR rescue experiments suggest that the induction of both the α7 nAChR-dependent LTP and STD requires coordination between pre- and postsynaptic activities, the similar time courses between the pre- and postsynaptic changes could suggest that both of these two forms of plasticity were expressed primarily via presynaptic mechanisms; the observed postsynaptic change during the plasticity expression may simply be the result of presynaptic modulation. However we did see some differences in the time course of pre- and postsynaptic changes during either LTP or STD (especially during LTP). For example the increase at 30 min (58%) was larger than that at 10 min (43%) at postsynaptic sites, but smaller (41% at 30 min versus 54% at 10 min) at presynaptic sites during the LTP (). Another important issue is that the pre- and postsynaptic activities observed above were not from the same slices since the recordings were done separately. To clarify this question, we expressed two differently colored GECIs (green GCaMP3 and red calcium indicator R-GECO1) (Tian et al., 2009
; Zhao et al., 2011
) in the same slices, one at presynaptic and the other at postsynaptic sites. In this way we could observe the pre- and postsynaptic activities in the same slice simultaneously during the expression of either LTP or STD. A high degree of correlation between the changes in the pre- and postsynaptic activities would suggest a major role for the presynaptic sites in the expression of either form of plasticity.
We expressed green GCaMP3 in CA3 neurons and red R-GECO1 in CA1 neurons in the same slices to observe green presynaptic and red postsynaptic activities simultaneously while monitoring in the CA1 SR region. With this dual-color imaging, we found that in many cases, the activity changes in the pre- and postsynaptic sites were not correlated for either LTP or STD. For the example shown in during the α7 nAChR-dependent LTP, both pre- and postsynaptic activities were increased at 10 min and 30 min after the pairing protocol. However, while the postsynaptic increase was similar to presynaptic increase at 10 min, it was significantly larger at 30 min (). The ratio of post- to presynaptic response was significantly larger at 30 min than that at 10 min (1.46 ± 0.09 at 30 min versus 0.82 ± 0.06 at 10 min, n = 5, p < 0.001, t-test), suggesting that at least the later stage postsynaptic increase was not mainly driven by an increased presynaptic release. Instead the data suggest that an independent postsynaptic mechanism is driving the expression of later stage LTP.
Differential pre- and postsynaptic changes inα7 nAChR-dependent LTP and STD revealed with dual-color GECI imaging.M
Similarly, differential modulation of pre- and postsynaptic activities was seen in the expression of STD. As shown in during the α7 nAChR-dependent STD, both pre- and postsynaptic activities were similarly decreased at 10 min, however they were significantly different at 30 min. The post- to presynaptic ratio were significantly changed at 30 min as compared with that at 10 min (−1.52 ± 0.32 at 30 min versus 1.2 ± 0.04 at 10 min, n =5, p < 0.001, t-test). These results again strongly suggest the existence of an independent postsynaptic mechanism during the expression of STD.
In summary, our data show that both pre- and postsynaptic mechanisms contributed to the expression of the α7 nAChR-dependent LTP and STD. More importantly, we have provided direct evidence that the α7 nAChR-dependent modulation of both pre- and postsynaptic sites is required to induce both of these forms of plasticity. Therefore we conclude that cholinergic inputs, through activating α7 nAChRs located at both synaptic sites, coordinate pre- and postsynaptic activities to induce synaptic plasticity.