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Previously, we have shown that these compound IPSPs were abolished by application of either the GABAA receptor antagonist, bicuculline, or the AMPA receptor antagonist, CNQX, suggesting glutamatergic input drives burst-firing PV+ interneurons to release GABA at multiple sites onto BLA principal neurons (Rainnie, 1999a). Each parvalbumin interneuron can innervate more than 150 BLA principal neurons (Muller et al., 2006), further suggesting that spontaneous, compound IPSPs are highly synchronized across a larger population of principal neurons than the pairs we show here.
Elsewhere in the brain, IPSPs have been shown to interact with subthreshold membrane potential oscillations (MPOs) to improve stimulus discrimination and action potential precision in neurons (Mainen and Sejnowski, 1995; Schaefer et al., 2006). We were therefore interested in the ability of synchronized, compound IPSPs to coordinate firing activity within networks of BLA principal neurons. As illustrated in Figure 6.2, compound IPSPs are capable of coordinating activity in the BLA, improving the temporal coherence of spontaneous action potentials between pairs of primate BLA principal neurons. When neurons were depolarized to threshold for action potential generation, action potentials occurring upon rebound from an IPSPinduced membrane hyperpolarization were highly coincident across cells (Figure 6.2A, shaded regions). To identify periods with consistent spike-timing across cells, we used a correlationbased metric with a sliding window (see Methods; Schreiber et al., 2003), where a value of 1 indicates identical spike-timing and a value of 0 indicates no timing correlation. Action potentials during a window directly following spontaneous, compound IPSPs had improved temporal coherence across pairs of neurons compared to those preceding IPSPs (Figure 6.2B).
Interestingly, upon rebound from compound IPSPs, a subpopulation of primate BLA principal neurons (3/11 cells) exhibited an increased and more consistent firing rate (from 3.6 to 7.4 Hz, coefficient of variation from 0.56 to 0.28) (Figure 6.2C). Moreover, clusters of action potentials showing a consistent firing rate and high coherence following compound IPSPs were also observed in 2/6 paired recordings (Figure 6.2D). In the course of these experiments it was noted that compound IPSPs could elicit a damped oscillation on rebound, suggesting the observed effects on action potential patterning may be due to an interaction with an intrinsic MPO (Figure
Together these observations strongly suggest that compound IPSPs coordinate the firing activity of principal neurons in both the rat and primate BLA, and their prevalence and synchrony further suggest that this coordination extends across large groups of principal neurons. To better assess the interactions of compound IPSPs with intrinsic properties of BLA principal neurons, subsequent experiments examined the effects of IPSPs on spike trains in the absence of synaptic noise. Moreover, given the scarcity of primate tissue, all experiments were performed in the rat.
6.4.2 Compound IPSPs enhance spike-timing precision in rat BLA principal neurons.
We first examined the effect of IPSPs on the precision of action potential timing in a neuron depolarized to action potential threshold with DC current injection. In order to better isolate the effects of intrinsic currents on spike timing, we blocked synaptic currents with a mixture of glutamate and GABA receptor antagonists (see Methods). As illustrated in Figure
6.3A, BLA principal neurons displayed a regular action potential firing pattern when held at -45 mV. When ten sweeps from the same neuron were aligned using an action potential as the trigger (Figure 6.3B), it was apparent that subtle variations in inter-spike interval accumulated over the course of the train, such that the timing of spikes at the end of the train was less consistent than at the beginning. Conversely, when two simulated IPSPs were injected during 10 sweeps captured randomly in time (Figure 6.3C), the phase of spiking was reset and spike times became much more consistent across sweeps.
Having established that artificial IPSPs can improve spike-timing precision in free-firing neurons, we next sought to quantify this effect. Specifically, we used transient (2.5 s) steps of injected current to elicit a spike train and determine the effect of IPSPs on spike-timing precision in individual principal neurons, and between pairs of principal neurons. Similar to when neurons are free-firing, the timing of the first few spikes in a train was extremely consistent across sweeps, but the timing of subsequent spikes became less consistent as the train progressed because small variations in the inter-spike interval accumulated (Figure 6.4A). Here we used the same correlation-based metric as described for Figure 6.2B, adapted to compare across five sweeps recorded in a single neuron (see 6.3 Methods). This analysis revealed that, at the onset of the train, spike-timing was extremely precise with an initial correlation value of 0.75 ± 0.13 (mean ± SD), which then diminished to 0.26 ± 0.20 within 300 ms (Figure 6.4D, Control, n = 11).
We next evaluated spike-timing precision in the presence of stimulus-evoked IPSPs (Figure 6.4B). Electrical stimulation of the dorsolateral BLA in the presence of glutamate receptor antagonists elicited a monosynaptic IPSP in principal neurons that had a similar amplitude and duration to the spontaneous compound IPSPs. We also examined the effects of artificial IPSPs, elicited with hyperpolarizing current injection, on spike-timing precision (Figure
6.4C). Activation of either evoked or artificial IPSPs during the action potential train resulted in a significant improvement in spike-timing precision compared to the control condition (Two-way ANOVA with repeated measures, effect of group: F2,800 = 136.3, p 0.0001). Both types of IPSPs significantly increased correlation values relative to the control condition for approximately 270 ms following each IPSP (effect of interaction: F78,800 = 4.72, p 0.0001, Bonferroni post-tests). Evoked IPSPs improved correlation values from a baseline of 0.19 ± 0.11 to a peak of 0.47 ± 0.11 (n = 11) immediately following the IPSPs (Figure 6.4D). As illustrated in Figure 4F, artificial IPSPs had a more pronounced effect on spike-timing precision than evoked IPSPs, with a peak correlation value of approximately 0.71 ± 0.21 (n = 11) following each IPSP.
Only at the peak points of the correlation, however, was there any significant difference in how the two IPSP manipulations affected spike-timing precision.
6.4.3 Compound IPSPs synchronize the firing activity of multiple BLA principal neurons.
We next quantified the ability of compound IPSPs to improve firing coherence across multiple BLA principal neurons, using a similar metric as above to measure the correlation of spike times in simultaneously recorded sweeps across the two neurons. In the absence of IPSPs, spike-timing across BLA neurons showed low coherence, such that the correlation-based metric reached an initial peak of only 0.27 ± 0.39 which then declined rapidly to 0.09 ± 0.07 (n = 6) within 300 ms (Figure 6.5C). The introduction of 2 evoked IPSPs was not able to significantly improve the coherence of spike times between neurons, likely due to the observed inconsistency in the amplitude and duration of the evoked IPSP waveform between neurons (data not shown).
Because artificial IPSPs have a highly consistent waveform across pairs of neurons and therefore mimic the consistency of spontaneous, compound IPSPs better than do evoked IPSPs, we also tested the effect of 2 artificial IPSPs on spike-timing. Artificial IPSPs significantly increased the coherence of spike times between pairs of neurons in the period immediately following the IPSPs (Two-way ANOVA with repeated measures, effect of interaction: F39,200 = 2.123, p 0.001, Bonferroni post-tests), with an improvement from a baseline of 0.09 ± 0.12 to a peak of approximately 0.42 ± 0.27 (n = 6) in the correlation-based metric (Figure 6.5B, D). These data strongly suggest that synchronized IPSPs enhance spike-timing precision of BLA principal neurons and can serve to entrain the firing activity of multiple neurons, despite inherent differences in their intrinsic electrophysiological properties (e.g., membrane input resistance, time constants of membrane charging, and firing frequency). Based on our prior observation that spontaneous, compound IPSPs not only entrain action potential firing, but also promote rhythmic firing and unmask a damped membrane potential oscillation, we hypothesized that the ability of compound IPSPs to coordinate firing would be facilitated by an interaction with intrinsic oscillatory properties of principal neurons. Therefore, we next characterized the interaction of compound IPSPs with intrinsic oscillatory properties of BLA principal neurons.
6.4.4 Compound IPSPs facilitate an intrinsic membrane potential oscillation in BLA principal neurons.
Most central nervous system neurons exhibit a preferred resonance frequency that provides them with the ability to filter synaptic input based on frequency (Hutcheon et al., 1996a, b; Hutcheon and Yarom, 2000). Pape and colleagues have reported that principal neurons in the lateral amygdala of the cat have an intrinsic resonance frequency in the range of 1-3.5 Hz (1998).
Here we extend these observations to show that BLA principal neurons of the rat also have an intrinsic resonance (Figure 6.6A1-D1, n = 8), with a preferred frequency at 4.2 ± 0.1 Hz (Figure
Many of the membrane currents that contribute to the resonant properties of neurons have also been implicated in mediating long-lasting, sub-threshold MPOs in the BLA as well as other brain regions (Hutcheon et al., 1994; Hutcheon et al., 1996a, b; Pape and Driesang, 1998; Pape et al., 1998). To determine whether compound IPSPs interact with an intrinsic MPO in principal neurons, we next examined the effect of IPSPs on membrane voltage in neurons depolarized to threshold in the presence of TTX (1 µM; n = 6). As illustrated in Figure 6.6A2, depolarizing current injection evoked a transient depolarizing voltage deflection at the onset of current injection but did not elicit an MPO in BLA principal neurons. Furthermore, injection of artificial IPSPs evoked a similar depolarizing voltage deflection on the rebound of each IPSP, but did not elicit an MPO (Figure 6.6A3). We hypothesized that the basal state of the neurons in the slice preparation might not be conducive to the expression of an MPO, and that modulation of intrinsic currents might be necessary to reveal the presence of an MPO.
6.4.5 The membrane potential oscillation is sensitive to modulation of its component currents.
Work by Pape and colleagues has shown that MPOs in the BLA can be enhanced by modulating a select population of voltage-activated currents including, but not limited to, the hyperpolarization-activated cation current (IH) and the low-threshold Ca2+ current (IT) (Pape et al., 2005). Significantly, an interaction between IH and IT is also thought to be a key element in the regulation of intrinsic resonance (Hutcheon et al., 1994; Hutcheon et al., 1996a, b). The IT current is often opposed by the transient K+ current, IA (Russier et al., 2003; Molineux et al., 2005; Hammack et al., 2007; Anderson et al., 2010), which has been shown to regulate firing activity in BLA principal neurons (Gean and Shinnick-Gallagher, 1989). Thus, we reasoned that blocking IA channels could effectively enhance IT and thus facilitate resonance behavior in BLA principal neurons and unmask an MPO. Bath application of the non-selective IA channel blocker, 4-aminopyridine (4-AP), at 100µM (Figure 6.6E) and 500µM (Figure 6.6B1, E) both significantly enhanced the amplitude of the peak resonance (One-way ANOVA, Tukey post-tests, F3,46 = 8.763, p 0.05). Application of 500 µM 4-AP also enhanced the expression of the transient depolarizing voltage deflection and unmasked a small, transient MPO at the onset of the depolarizing step (Figure 6.6B2). Furthermore, in the presence of 4-AP, the introduction of artificial IPSPs (Figure 6.6B3) enhanced the amplitude of the MPO, which had peak power at approximately 5 Hz (Figure 6.7A-C, n = 6).
Importantly, IT, IH, and IA channels are all substrates for phosphorylation by protein kinase-A (PKA), which decreases the conductance of IA channels and increases the conductance of IH and IT channels (Kamp and Hell, 2000; Kim et al., 2006a; Ramadan et al., 2009) (Ingram and Williams, 1996; Hoffman and Johnston, 1998; Gerhardstein et al., 1999; Vargas and Lucero, 2002). Thus, we next examined the effects of the PKA activator, forskolin, on the resonance properties of BLA principal neurons. As illustrated in Figure 6.6C1, bath application of forskolin (10 µM) in combination with 4-AP (500 µM) significantly increased the amplitude of the resonance peak compared to TTX controls (One-way ANOVA, Tukey post-test, F3,46 = 8.763, p 0.05). However, the peak power of the resonance in 4-AP and forskolin was not significantly different than that observed in the presence of 4-AP alone (Figure 6.6E). In the context of the depolarizing step, the addition of forskolin (10 µM) in combination with 4-AP (500 µM) enhanced both the amplitude and duration of the MPO in all neurons tested (Figure 6.6C2). The MPO resembled a damped oscillation (Pape and Driesang, 1998) and, as can be seen in Figure
6.6F, the power of the MPO was greatest at the onset of the depolarizing current injection and declined over time. In the majority of neurons the MPO was seen to terminate before the conclusion of the depolarizing current injection. The introduction of artificial IPSPs further enhanced the oscillation (Figure 6.6C3, G) without changing the preferred frequency (Figure
6.7E, compared to 6.7C and 6.7D). Application of forskolin (10 µM) alone also unmasked an MPO, similar to the effects of 500 µM 4-AP, with a peak frequency at 4.8 Hz in all neurons tested (n = 4) (Figure 6.7D). Hence, activation of the cAMP-signaling cascade alone can facilitate the expression of the MPO in BLA principal neurons.
In other brain regions, MPOs are partially dependent on the activation of IT channels, and as the transient depolarizing voltage deflections observed upon rebound from the IPSPs were reminiscent of low-threshold calcium spikes, we next determined whether blocking IT channels with 500 µM NiCl (Lee et al., 1999) would inhibit the combined response to forskolin and 4-AP.