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There was a more than two-fold reduction in Rin from P7 to P14, and a nearly three-fold reduction from P14 to P21. Specifically, Rin decreased significantly from a median value of 523.7 MΩ at P7 (n = 45) to 374.8 MΩ at P10 (n = 37), 238.4 MΩ at P14 (n = 54), 88.0 MΩ at P21 (n = 43), and 55.9 MΩ at P28 (n = 58; Wilcoxon rank sum post-hoc tests). By P28, Rin had achieved its mature value and was not significantly different (p 0.05) from neurons aged P35 (52.67 MΩ, n = 56). Membrane time-constant followed a similar developmental trajectory to Rin (Figure 2.2B, C), with a nearly four-fold reduction from P7 to P28. Overall there was a significant effect of age (Kruskal-Wallis, χ25 = 221.4), with a nearly two-fold reduction from P7 to P14 and another twofold decrease from P14 to P28. Whereas the change from P7 to P10 was not significant (p 0.05, Wilcoxon rank sum post-hoc tests; median = 59.1 ms at P7, n = 45; 50.5 ms at P10, n = 39), there was a significant (p 0.001) decrease from P10 to P14 (32.0 ms, n = 53), P14 to P21 (18.0 ms, n = 45), and P21 to P28 (15.1 ms, n = 56). Similar to Rin, τmemb reached its mature value by P28 and did not change significantly between P28 and P35 (15.1 ms, n = 54; p 0.05). Having observed significant age-dependent changes in the passive membrane properties of BLA principal neurons, we next examined concomitant changes in active properties, beginning with the voltage-gated current, Ih.

2.4.2 Postnatal Maturation of Intrinsic Currents Development of the Voltage-Gated Current, Ih One current which classically contributes to passive membrane properties like Rin and τmemb is the hyperpolarization-activated cation current, Ih. This current mediates a depolarizing voltage sag observed in response to hyperpolarization from rest that is readily apparent at all ages (Figure 2.1). When neurons were manually held at -60 mV and the amplitude of the transient hyperpolarizing current steps adjusted to elicit peak voltage deflections to -80 mV, the depolarizing sags observed in the voltage response had a similar amplitude at all time-points (Figure 2.3A); however, the rate of onset of the sag increased with age. Significantly, the voltage sag was abolished at all ages by bath application of 5 mM Cs+, suggesting it is likely mediated by activation of Ih. An example of the Cs+ blockade of the depolarizing sag at P28 is illustrated in Figure 2.3A, upper trace. Considering the differences in Rin across ages and the visible differences in the kinetics of the voltage sag, we next quantified the maturation of Ih in voltage clamp.

To quantify the amplitude and kinetics of Ih activation, the membrane potential was stepped from -60 to -100 mV, both before and after application of 5 mM Cs+, and the resulting currents were then subtracted to isolate Ih. The mean isolated Ih for each time-point is illustrated in Figure 2.3B. Analysis of the peak amplitude revealed a steady increase of Ih amplitude across the entire first postnatal month (Figure 2.3C), such that the mean Ih amplitude was 91.5 ± 28.1 pA at P7 (n = 10), 359.8 ± 82.3 pA at P14 (n = 14), 518.1 ± 239.2 pA at P21 (n = 11), and 641.9 ± 239.8 pA at P28 (n = 11). Notably, significant transitions occurred from P7 to P14 and from P14 to P28 (p 0.01, One-way ANOVA with Tukey’s post-hoc, F3,42 = 19.93). The activation kinetics of Ih were estimated by fitting its charging curve with a two-term exponential equation, except at P7, when the curve was sufficiently parameterized with a one-term exponential. The fast time constant decreased from 25.4 ± 6.3 ms at P14 (n = 19) to 19.6 ± 2.4 ms at P21 (n = 15), but remained steady between P21 and P28 (19.1 ± 2.7 ms, n = 13; Figure 2.3D, black squares).

The single time constant at P7 was 188.5 ± 61.1ms (n = 11), which was similar to the slow time constant of activation for the other ages (253.5 ± 65.2 at P14, 252.9 ± 127.7 at P21, and 361.7 ± 340 at P28; Figure 2.3D, black triangles). Development of Intrinsic Resonance Membrane properties like τmemb and active currents like Ih help shape intrinsic resonance, which acts as a band-pass filter to enhance responsiveness to synaptic input at particular frequencies. BLA principal neurons in the adult rat, guinea pig, and primate exhibit a preferred resonance frequency between 2.5 and 6 Hz (Pape and Driesang, 1998; Ryan et al., 2012).

Resonance properties of neurons likely contribute to the production of network oscillations (Lampl and Yarom, 1997; Desmaisons et al., 1999; Richardson et al., 2003; Tohidi and Nadim, 2009), and recent evidence suggests that network oscillations in the BLA at the frequency of principal neuron resonance are intimately related to fear memory formation and expression (Popa et al., 2010; Lesting et al., 2011). Considering the potential contribution of BLA principal neuron resonance to the generation of fear expression and learning, we next examined the ontogeny of resonance properties in these neurons.

By injecting a sinusoidal ZAP current of increasing frequency (see Methods) we were able to determine the peak resonance frequency, the input frequency that elicits the greatest membrane deflection, of BLA principal neurons across development. As illustrated in Figure

2.4A, the population responses to ZAP currents at P7, P14, P21, and P28 clearly showed a shift in the resonance frequency of principal neurons, with more mature neurons showing higher resonance frequencies. By taking the ratio of power spectra for output voltage and ZAP input current, we generated functions of impedance vs. input frequency (Figure 2.4B). Here, the peak resonance frequency increased sharply and significantly until P21 (p 0.001, One-Way ANOVA with Tukey’s post-tests, F3,91 = 74.31), with values (mean ± SD) of 0.97 ± 0.33 Hz at P7 (n = 21),

2.63 ± 1.48 Hz at P14 (n = 24), 5.47 ± 1.30 Hz at P21 (n = 21), and 5.69 ± 1.49 Hz at P28 (n = 29; Figure 2.4C).

To determine how developmental changes in Ih may contribute to the maturation of resonance, we blocked Ih using Cs+ and measured changes in the responses to ZAP currents across the first postnatal month (Figure 2.5A). At all ages, Cs+ application reduced the peak resonance frequency to below 1 Hz (0.60 ± 0.09 Hz at P7 (n = 8), 0.67 ± 0.22 Hz at P14 (n = 9),

0.85 ± 0.29 Hz at P21 (n = 8), and 0.94 ± 0.49 Hz at P28 (n = 11)). However, the effect of Cs+ blockade on resonance involves more than a change in peak frequency; therefore, to highlight differences in the contribution of Ih to resonance across ages, we quantified the effect of Cs+ on prominence, the proportion of total power between 1 and 10 Hz found in a given frequency band (1-2, 2-4, 4-6, 6-8, or 8-10 Hz)(Burton et al., 2008).

As shown in Figure 2.5B, the change in prominence due to Cs+ application varied by age as an inverted ‘U’ with Cs+ having relatively little impact at P7 and causing robust changes at P14, then having a progressively weaker effect at later time-points. There were significant main effects on change in prominence of frequency band (F4,272 = 518.1, p 0.0001, Two-way ANOVA with repeated measures) and age (F3,272 = 28.9, p 0.0001), as well as a significant interaction effect (F12,272 = 40.9, p 0.0001). Specifically, at P7 Cs+ increased prominence (mean ± SD) at 1-2 Hz by 32.9 ± 20.1% and reduced prominence in the higher bands by between 1 and 3% each (n = 18). Compared to at P7, Cs+ application at P14 caused a significantly greater increase in prominence in the 1-2 and 2-4 Hz bands (+157.3 ± 49.4% and +28.2 ± 12.6 % respectively; p 0.001, Bonferroni post-hoc tests), and a significantly greater reduction in the 4and 8-10 Hz bands (-31.8 ± 7.5%, -53.0 ± 9.0%, and -58.3 ± 7.6%, respectively; n = 19; p 0.01). The effect of Cs+ at P21 was significantly weaker than at P14 (p 0.001) in the 1-2, 4-6, and 6-8 Hz bands and significantly greater (p 0.01) in the 2-4 Hz band (+113.9 ± 43.1% at 1-2 Hz, +44.6 ± 16.3% at 2-4 Hz, -9.5 ± 6.7% at 4-6 Hz, -35.0 ± 9.3% at 6-8 Hz, and -46.5 ± 11.5% at 8-10 Hz; n = 19). The trend continued at P28, with Cs+ having a weaker effect than at P21.

Specifically, Cs+ caused a significantly smaller increase in prominence in the 1-2 Hz band (p 0.001; +68.9 ± 27.4%) but effects in the remaining bands were not significantly different than at P21 (+33.4 ± 13.5% at 2-4 Hz, -2.2 ± 4.3% at 4-6 Hz, -23.7 ± 8.9% at 6-8 Hz, and -34.8 ± 11.8% at 8-10 Hz; n = 16).

The effects of Cs+ on resonance were largely attributable to a direct effect on τmemb. In control conditions, peak resonance frequency was correlated with τmemb using a standard exponential equation (see Methods), yielding a R2 value of 0.76 with A1 = 12.42, τ1 = 17.24, and C = 0.28 (Figure 2.5C). Application of Cs+ increased τmemb at P14, P21, and P28 by an average of 67.0, 19.0, and 24.5 ms, respectively, causing a corresponding reduction in peak resonance frequency at each age (Figure 2.5D). Based on these observed changes to oscillatory properties of BLA principal neurons, we next characterized the maturation of spontaneous expression of membrane oscillations. Development of Spontaneous Membrane Potential Oscillations The oscillatory properties of adult BLA principal neurons manifest not only as resonance, but also as spontaneous membrane potential oscillations (MPO; Pape and Driesang, 1998; Ryan et al., 2012). These MPOs can influence spike-timing and interact with resonance to filter synaptic input based on frequency (Desmaisons et al., 1999; Izhikevich, 2002; Sancristobal et al., 2010). Furthermore, we have recently shown that phase-locked MPOs and coordinated spiking in adult BLA principal neurons are promoted by spontaneous, synchronous inhibitory postsynaptic potentials, highlighting a mechanism by which MPOs could contribute to network oscillations in the BLA (Ryan et al., 2012). We were therefore interested in the ontogeny of network oscillations in the BLA, and next examined the expression of MPOs in neurons at P7, 14, 21, and

28. Here, principal neurons were depolarized to action potential threshold with DC current injection. As illustrated in Figure 2.6A, neurons at P7 were more likely to fire action potentials in bursts, whereas P28 neurons had a more stable membrane potential and fired sporadically (Figure

2.6A). When depolarized to threshold, neurons also became more likely to exhibit spontaneous MPOs across the first postnatal month (Figure 2.6B, C). A blinded, qualitative analysis of current-clamp recordings near threshold revealed that only 5% of BLA principal neurons exhibited spontaneous MPOs at P7 (n = 20) and P10 (n = 19), but MPOs were present in 23% of neurons at P14 (n = 26), 64% at P21 (n = 25), and 60% at P28 (n = 48). Moreover, when we measured the frequency of spontaneous MPOs for neurons with a discernible peak in the power spectrum of their activity during a low-spiking period at threshold, we observed that it increased with development (3.8 ± 1.0 Hz at P14, n = 3; 3.7 ± 1.9 Hz at P21, n = 12; 4.4 ± 1.9 Hz at P28, n = 22). In other brain regions, MPOs are capable of organizing action potential timing (Llinas et al., 1991; Gutfreund et al., 1995; Desmaisons et al., 1999), and the same appears to be true in the BLA. An example of this is illustrated in Figure 2.6D, which shows a spike-triggered average from a P28 neuron.

2.4.3 Postnatal Maturation of Spiking Development of Spike Trains Having observed significant developmental changes in the sensitivity of BLA principal neurons to input as well as gross changes in action potential output (see Figures 2.1 and 2.6A), we next quantified the maturation of action potential trains across the first postnatal month. Here, spike trains were elicited with a transient, 1s, square-wave depolarizing current injection to action potential threshold. As illustrated in Figure 2.7 (inset), there was a gradual emergence across the first postnatal month of doublet and triplet firing at the onsets of the spike trains. Moreover, analysis of the instantaneous firing rates based on the first 6 inter-spike-intervals (ISIs) for principal neurons at P7, 14, 21, and 28 revealed that at P7 the firing rate was relatively consistent across the entire train, starting at 29.7 ± 13.3 Hz and stabilizing at 16.8 ± 8.1 Hz by the third interval (Figure 2.7A). By P14, doublets became more apparent with an initial firing rate of 62.2 ± 52.1 Hz that dropped to 17.3 ± 12.5 Hz by the second interval. At P21, the doublet became faster and a triplet emerged in some neurons, with firing rates of 138.8 ± 77.9 and 45.0 ± 44.5 Hz for the first and second intervals, respectively, which stabilized around 20 Hz for the remainder of the train. Firing at P28 was very similar to that at P21, with slightly faster rates for the first pair of spikes (166.9 ± 95.3 Hz). We quantified the emergence of doublets using the first ISI, which significantly decreased from P14 to 21 and from P21 to 28 (Figure 2.7B; p 0.001, KruskalWallis with Wilcoxon rank sum post-hoc tests, χ25 = 194.0). Every transition between neighboring pairs of time-points was significant as well (p 0.001).

We next measured the input-output relationship for action potential generation at P7, 14, 21, and 28 (n = 7, all groups), using 1 s, square-wave current injections applied from a resting membrane potential of -60 mV. As illustrated in Figure 2.7C, as neurons matured they required more current to generate the same output frequency. Interestingly, although the maximal firing frequency (mean ± SD) significantly increased (p 0.001, One-way ANOVA with Tukey’s posthoc tests, F3,23 = 25.96) from P7 (15.9 ± 3.3 Hz) to P14 (33.9 ± 7.3 Hz), the transitions (p 0.05) from P14 to P21 (34.7 ± 3.7 Hz) and from P21 to P28 (36.5 ± 4.2 Hz) were not significant. Development of the Action Potential Waveform We reasoned that the observed changes in spike trains were likely due, in part, to maturation of the waveform of individual action potentials. Consequently, to quantify changes in action potential waveform, neurons were probed with a depolarizing current ramp lasting 250 ms, whose amplitude was adjusted to elicit a single action potential. Figure 2.8A illustrates the mean action potential waveforms from each age group. As can be seen, action potential threshold exhibited a significant, negative shift of approximately 7 mV from P7 to P28 (Kruskal-Wallis, = 164.5, Figure 2.8B). The median threshold was -33.5 mV at P7 (n = 51), -34.7 mV at P10 (n = 35), -37.0 mV at P14 (n = 52), -40.9 mV at P21 (n = 43), -40.3 mV at P28 (n = 56), and -41.3 mV at P35 (n = 55). Statistically significant transitions occurred from P10 to P14 and P14 to P21 (p 0.001, Wilcoxon rank sum post-hoc tests). In addition, action potentials became much faster;

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