fosGFP+ cells show high spontaneous firing activity in vivo
To determine whether fosGFP expression was correlated with elevated spontaneous firing activity in vivo
, targeted-juxtacellular recordings were carried out in fosGFP+ and fosGFP− cells pairs within layer 2/3 of primary somatosensory (barrel) cortex of anesthetized animals. Under basal, unstimulated conditions, the percentage of both fos-immunoreactive neurons in wildtype () and fosGFP+ neurons in transgenic animals () was similar across different neocortical areas, (~15% of layer 2/3 cells; Figure S1
Fos-expressing pyramidal neurons in layer 2/3 exhibit higher spontaneous firing rates in vivo
Two-photon imaging of GFP expression combined with local illumination of cell bodies using a red fluorescent dye (shadow-patching; (Kitamura et al., 2008
) enabled identification of fosGFP+ and fosGFP− neurons (). A great deal is known about neurons in this layer with respect to local network properties (Feldmeyer et al., 2006
; Wang et al., 2006
; Kapfer et al., 2007
; Adesnik and Scanziani, 2010
), their activity during perception and ability to drive behavior (Kerr et al., 2007
; Houweling and Brecht, 2008
; Huber et al., 2008
; Poulet and Petersen, 2008
; Gentet et al., 2010
) and their capacity for experience-dependent plasticity (Glazewski and Fox, 1996
; Allen et al., 2003
; Celikel et al., 2004
; Clem et al., 2008
); as such they offer a strong entry point for analyses of neocortical networks.
Targeted neurons were 185±46 µM from the pial surface (n=12 pairs), and were located 38.6±19 µM apart (n=7 pairs; not all pairs measured). As in previous studies, firing rates across simultaneously-recorded cell pairs varied substantially (range 0.017–1.43 Hz). However, expression of the immediate-early gene fosGFP was a strong predictor of a cell having a higher overall firing rate compared to neighboring, unlabeled cells (; firing rate for simultaneously recorded fosGFP− cells 0.099±0.2 Hz versus fosGFP+ cells 0.25±0.4 Hz, n=12, p=0.03). On average, fosGFP+ cells fired at ~4-fold higher rates compared to fosGFP− cells ().
Thus, fosGFP expression is predictive of neurons with elevated firing activity in vivo. Because of the well-characterized delay between induction of fos expression and intrinsic GFP fluorescence, these data suggest that highly active neuronal subsets may be stable for hours in vivo.
fosGFP+ neurons fire more ex vivo
To carry out a detailed mechanistic analysis of the cellular and synaptic basis of this increased firing within fosGFP+ neurons, we examined whether fosGFP expression was correlated with elevated spontaneous firing activity for layer 2/3 pyramidal neurons in acute brain slices, using paired-cell recordings.
Because fosGFP expression is not induced by slice preparation (Barth et al., 2004
), the stimulus responsible for induction of fosGFP expression was likely to have occurred at least several hours prior to tissue preparation. Ex vivo
, fosGFP+ cells maintained significantly higher rates of overall firing activity compared to neighboring fosGFP− cells (; firing rate for simultaneously recorded fosGFP− cells 0.050±0.08 Hz versus fosGFP+ cells, 0.12±0.14 Hz, n=13, p=0.01) . Elevated firing rates in fosGFP+ cells could be observed for many hours (3+) after slice preparation and did not decline over the recording session.
In wild-type animals, mean firing rates of individually recorded neurons were similar to those of fosGFP− neurons (Figure S1
; 0.103±0.023 Hz, n=30, p=0.9). However, a subset of wild-type neurons exhibited high firing rates comparable to those observed in fosGFP+ cells, suggesting that this subset is present in wild-type animals but can be uniquely visualized in fosGFP transgenic mice.
FosGFP+ neurons fire earlier and more often during network activity
In both our experiments and others’ (Steriade et al., 1993
; Sanchez-Vives and McCormick, 2000
; MacLean et al., 2005
), spontaneous firing in neocortical neurons ex vivo
tends to occur during epochs of depolarization, similar to what has been termed Upstates in vivo
. Although the precise trigger for these events is unknown, epochs are observed in both neurons that fire frequently and those that do not fire at all, where they appear as prolonged subthreshold events (). Are fosGFP+ neurons differentially recruited during these epochs of network activity?
IEG-expressing neurons fire at higher frequencies during network activity
FosGFP+ cells fired more spikes during a depolarizing epoch compared to fosGFP− cells, although epoch duration was identical (fosGFP− 2.7±0.17, n=134 epochs over 18 cells, versus fosGFP+ 2.7±0.14, n=149 epochs over 18 cells, p=0.8). FosGFP+ cells showed significantly more spikes per epoch (s/e) than simultaneously recorded, neighboring fosGFP− cells (; fosGFP− 3.61±0.47 s/e; fosGFP+ 9.8±1.1 s/e, p<0.01).
Epoch frequency (including subthreshold depolarizations) was not significantly increased in fosGFP+ cells (; fosGFP− cells 0.035±0.007 Hz; fosGFP+ cells 0.034±0.007 Hz, p=0.26), indicating that network activity can engage both cell populations.
Identification of high-firing neurons using expression of an alternate IEG
To verify that the elevated spontaneous firing activity observed in fosGFP+ neurons was not due to expression of the fosGFP transgene, a second strain of transgenic mice expressing GFP under the control of the Arc/Arg3.1 promoter was analyzed (GENSAT BAC transgenic resource, Rockefeller University; (Gong et al., 2003
)). Similar to fosGFP+ neurons, ArcGFP+ neurons tended to fire more than ArcGFP− neurons within a cell pair ( and data not shown; mean overall firing rate, ArcGFP− 0.23±0.21 Hz versus ArcGFP+ 0.32±0.14 Hz; n=9 pairs, p=0.07). Like fosGFP+/− cell pairs, the frequency of depolarizing epochs was identical, and ArcGFP+ neurons showed significantly more spikes/epoch than ArcGFP− cells (; ArcGFP− 6.4±0.7 s/e, n=83 epochs over 9 cells versus ArcGFP+ 8.1±0.6 s/e, n=89 epochs over 9 cells, p=0.003). On average, ArcGFP+ cells fired 2.5-fold more than ArcGFP−cells, a significant difference (p=0.04). Although values from ArcGFP+ neurons were more variable compared to fosGFP+ neurons, it is remarkable that the basic observations made in both transgenic mice are so similar. Thus, it is unlikely that the increased firing activity characterized in fosGFP+ neurons is due to expression of the fosGFP transgene.
Network activity rapidly engages fosGFP+ neurons
Simultaneous recordings of fosGFP+ and fosGFP− cells enabled a direct comparison of cell engagement during an epoch of network activity. We found that fosGFP+ neurons were recruited into a depolarizing epoch significantly earlier than fosGFP− neurons (; mean onset timing for fosGFP− was 67.3±27 ms after onset in fosGFP+ cells; n=31 epochs over 9 cell pairs; p<0.001). Thus, although spontaneous network activity engages both cell types, fosGFP+ cells are activated earlier and are more likely to fire during a depolarizing epoch.
FosGFP+ neurons show reduced intrinsic excitability
Why do fosGFP-expressing neurons display elevated spontaneous firing activity? One explanation is that these neurons show greater intrinsic excitability (i.e., depolarized resting membrane potential, action potential (AP) threshold, or input resistance). However, comparison between fosGFP+ and fosGFP− cells showed that these properties were identical between groups (Table S1
To evaluate intrinsic excitability, input-output curves were constructed, using constant current injection to elicit firing (Figure S2
). FosGFP+ cells required more current to generate a single spike (mean rheobase current fosGFP− 37.12±1.6 pA versus fosGFP+ 45.6±2.99 pA, n=16 for both; p=0.02) and exhibited fewer spikes at all levels of current injection compared to fosGFP− cells (Figure S2
). AP threshold for fosGFP+ and fosGFP− cells was similar, despite the finding that fosGFP+ cells required more current to spike. This suggests that other voltage-dependent, spike-delaying conductances are differentially regulated in fosGFP+ neurons. However, increased spontaneous activity in fosGFP+ neurons is unlikely to result from cell-intrinsic electrophysiological properties.
FosGFP+ cells receive increased excitatory and reduced inhibitory drive
Under our recording conditions, spontaneous activity in layer 2/3 neurons requires synaptic input since it is abolished in the presence of GABA- and glutamate receptor antagonists (Shruti et al., 2008
). To examine whether fosGFP+ neurons receive differential synaptic drive, the frequency and amplitude of post-synaptic currents (PSCs) during spontaneous network activity was analyzed.
Although the amplitude of spontaneous excitatory PSCs (sEPSCs) was similar between fosGFP+ and fosGFP− neurons, fosGFP+ neurons showed a significantly greater sEPSCs frequency (; sEPSC frequency fosGFP− 1.3±0.2 Hz versus fosGFP+ 1.7±0.2 Hz; n=13 cells for both; p=0.001), a finding further confirmed by paired-cell recordings ().
FosGFP+ neurons receive increased excitatory and decreased inhibitory synaptic drive during network activity
FosGFP+ neurons showed a significantly reduced amplitude of spontaneous inhibitory PSCs (sIPSCs; ; fosGFP− 24±3 pA versus fosGFP+ 19±2 pA; p=0.02), although frequency was not significantly different (; frequency fosGFP− 3.1±0.5 Hz versus fosGFP+ 2.6±0.5 Hz; p=0.3). Paired-cell recordings show a significant reduction in sIPSC frequency for fosGFP+ cells (). These data indicate that fosGFP+ neurons receive both more excitation and less inhibition during spontaneous network firing.
To determine whether this difference in excitatory and inhibitory input would be maintained in the absence of spiking, miniature EPSC and IPSCs (mEPSCs and mIPSCs) were assessed in the presence of the Na+ channel blocker tetrodotoxin (TTX). In TTX, no significant differences in mEPSC or mIPSC frequency or amplitude were detected (). This result is intriguing, since it indicates that differences in synaptic drive require network firing to be manifested. During unconstrained network activity, presynaptic excitatory neurons may fire more onto fosGFP+ neurons, inputs that may provide a basis for increased spontaneous activity of this cell subset.
Direct and indirect connectivity between fosGFP+ neurons
Are fosGFP+ neurons are non-randomly wired into the neocortical network, receiving presynaptic input from excitatory neurons that are themselves more active? We speculated that fosGFP+ neurons might show a high degree of either direct or indirect interconnectivity.
Connectivity between pairs of layer 2/3 pyramidal neurons was assessed by a second series of dual-cell recording experiments. In total, 214 paired recordings were performed representing all combinations of fosGFP+ and fosGFP− cells as well as cells from wild-type animals (). Using the criterion of a short latency EPSP (1–5 ms) with high (>50%) trial-to-trial reliability, six cells (all fosGFP+/fosGFP+ pairs) out of the group (162 pairs), exhibited a direct, unidirectional synaptic input (). In no other case were examples of direct synaptic connectivity observed. In connected cells, mean EPSP amplitude was 0.99+0.21 mV (n=6 cells; 10 responses per cell averaged), similar to previously described unitary EPSP amplitudes (Thomson et al., 2002
; Feldmeyer et al., 2006
FosGFP+ cells show greater interconnectivity within the cortical network
Distance between connected pairs was similar to that of all cells overall (68.8+16 µm; n=6 pairs; p=0.2 versus group mean) and showed a similar distance from the pia surface (222.6+30 µm; n=6; p=0.7 versus group mean). Thus, the location of the cell soma was not a critical variable in predicting cell connectivity.
Although the frequency of directly connected cells was low, we noted that current injection into the trigger cell often led to an increase in PSP frequency in the second cell (). These putative EPSPs (since recordings were carried out at the experimentally determined reversal potential for Cl−) were of variable latency (~5–50 ms) with respect to the presynaptic AP, suggesting that they were of polysynaptic origin. APs triggered in a fosGFP− cell led to a ~1.2-fold, not significant, increase in EPSP frequency in the “follower” cell compared to the prestimulus window (; fold-change in EPSP frequency from baseline: fosGFP− trigger to fosGFP− follower: 1.18±0.05, n=45 cells; fosGFP− trigger to fosGFP+ follower: 1.17±0.07, n=23 cells; fosGFP+ trigger to fosGFP− follower: 1.13±0.1, n=19, p>0.5).
However, depolarization of fosGFP+ cells often led to a significant increase in EPSP frequency in unconnected fosGFP+ cells. Stimulation of the trigger cell led to a significant, 1.5-fold increase in EPSP frequency compared to that in the prestimulus window (fold-change in EPSP frequency from baseline: fosGFP+ trigger to fosGFP+ follower: 1.53±0.1, n=45 cells; p=0.004; ). Because EPSPs were not evoked at a consistent time interval following each presynaptic AP but were distributed between 10–50 ms after each stimulus, it is likely that they were polysynaptic in origin.