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«Distribution Agreement In presenting this thesis or dissertation as a partial fulfillment of the requirements for an advanced degree from Emory ...»

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2: Input resistance and membrane time constant decrease with age. (A, B) Box and whisker plots show input resistance (A) and membrane time-constant (B) of BLA principal neurons across the first postnatal month and in adulthood (n = 45 (P7), 37-39 (P10), 53-54 (P14), 43-45 (P21), 56-58 (P28), and 54-56 (P35)). Significance was assessed using a Wilcoxon ranksum test and pairwise comparisons were made for each age group with up to four neighboring time-points (see inset tables for results) using a Bonferroni correction for the resulting 9 comparisons (***, p 0.001; NS: not significant, p 0.05). (C) Maturation of membrane timeconstant is illustrated with the average, normalized membrane charging in response to a small, hyperpolarizing current step (approximately 5 mV deflection) for each developmental time point (n = 48 (P7), 28 (P10), 56 (P14), 45 (P21), and 58 (P28)).

Figure 2.3: Developmental increase in Ih amplitude and kinetics in BLA principal neurons Figure 2.

3: Developmental increase in Ih amplitude and kinetics in BLA principal neurons.

(A) Representative voltage responses of neurons to a hyperpolarizing, square step of magnitude adjusted to elicit a 20 mV peak deflection. Neurons were recorded in the presence of 1 μM TTX at each time point, and blockade of Ih by 5 mM cesium (Cs+) is depicted for a neuron at P28.

Neurons had baseline membrane potential adjusted to -60 mV with direct current. (B) Voltageclamp recordings following Cs+ application (5 mM) subtracted from those prior to Cs+ application depict the maturation of a Cs+-sensitive current. Neurons were held at -60 mV and stepped to mV, and all recordings were performed in the presence of 1 μM TTX (n = 10 (P7), 14 (P14), 11 (P21), 11 (P28)). (C) Peak amplitudes of subtraction currents from B are plotted as mean ± SEM for each time point. Significance was assessed using a one-way ANOVA with Bonferonni’s post-tests, and pairwise comparisons were made for each age group with up to three neighboring time-points (see inset table for results; ***, p 0.001; **, p 0.01; NS: not significant, p 0.05).

(D) Plotted as mean ± SEM for each age, the time constant of Ih activation was measured from a double exponential fit to the subtraction currents in B at all ages except P7, which was sufficiently fit with a single exponential.

Figure 2.4: Maturation of intrinsic resonance towards higher frequencies Figure 2.

4: Maturation of intrinsic resonance towards higher frequencies. (A) Membrane potential response, shown as mean (black line) and standard deviation (grey band), to a ZAP current (Iin, fixed amplitude and logarithmically increasing frequency, shown at bottom) is depicted for each age (n = 22 (P7), 24 (P14), 22 (P21), and 32 (P28)). Neurons were first hyperpolarized to -70 mV with direct current, and ZAP current amplitude was adjusted for each neuron to elicit a 20 mV depolarizing deflection. Instantaneous frequency of the injected current is highlighted with gray bars above each trace, and the mean, peak resonance frequency for each age is depicted amid the gray bars with a black triangle. (B) Relative impedance for input from 0.1-10 Hz, calculated by normalizing the power spectra of the voltage responses in A to the power spectra of injected current, were fit with polynomials and plotted as the mean (n consistent with A). (C) Peak resonance frequency is plotted as mean ± SEM, measured at the maximum of each neuron’s individual power spectrum (n = 21 at P7, 24 at P14, 21 at P21, and 29 at P28).

Statistical significance was assessed with a one-way ANOVA using Bonferroni’s post-tests to compare all data sets (** denotes p 0.001 versus all other groups).

Figure 2.5: Contribution of Ih to intrinsic resonance of BLA principal neurons changes with age Figure 2.

5: Contribution of Ih to intrinsic resonance of BLA principal neurons changes with age. (A) Mean membrane potential response to a ZAP current (Iin, fixed amplitude and logarithmically increasing frequency, shown at bottom) in 1 μM TTX alone (black line, taken from Figure 6) or with 5 mM Cs+ (red line) is depicted for each age (in Cs+, n = 19 at P7, 21 at P14, 20 at P21, and 17 at P28). Neurons were first hyperpolarized to -70 mV with direct current, and ZAP current amplitude was adjusted for each neuron and condition to elicit a 20 mV, maximal depolarizing deflection. Instantaneous frequency of the injected current is highlighted with gray bars between traces. (B) Effect of Cs+ application on prominence, calculated as ratio of the prominence before and after Cs+ application, plotted as mean & SEM at P7, P14, P21, and P28. (C, D) Peak resonance frequency is plotted against membrane time constant (τ) for neurons at each time point, recorded in TTX alone (C, D) or following application of 5 mM Cs+ (D). A black line depicts the results of an exponential regression (R2 = 0.76) of the data shown in C.

Figure 2.6: Spontaneous membrane potential oscillations emerge as BLA principal neurons develop Figure 2.

6: Spontaneous membrane potential oscillations emerge as BLA principal neurons develop. (A, B) Representative current-clamp recordings, shown at two scales, of neurons depolarized to action potential threshold with direct current, highlighting maturation of spiking pattern (A) and spontaneous membrane potential oscillations (B). (C) Pie charts depict the proportion of neurons expressing spontaneous membrane potential oscillations at each time-point (n = 20 (P7), 26 (P14), 25 (P21), and 48 (P28)). (D) Representative spike-triggered average from a 30 s recording of a P28 neuron held near threshold, displaying entrainment of spiking to a spontaneous membrane potential oscillation.





Figure 2.7: Maturation of spike trains in BLA principal neurons Figure 2.

7: Maturation of spike trains in BLA principal neurons. (A) Instantaneous action potential frequency is plotted for individual neurons (grey lines) and group mean (black lines) at each time point. Neurons were depolarized such that the mean inter-spike membrane potential was near spike threshold (see Methods for details). The start of a representative spike train is inset in each plot to highlight differences in initial spike rate (n = 39 (P7), 75 (P14), 97 (P21), and 103 (P28) trains). (B) First interspike-interval for the spike trains in A is depicted in a box and whisker plot, with the later time-points shown on both y-axes (n = 45 (P7), 37 (P10), 53 (P14), 43 (P21), 54 (P28), and 59 (P35)). Significance was assessed using a Wilcoxon rank-sum test and pairwise comparisons were made for each age group with up to four neighboring time-points (see inset table for results) using a Bonferroni correction for the resulting 9 comparisons (***, p 0.001; NS: not significant, p 0.05). (C) Input-output curves for neurons at each time point are depicted as mean (line) and standard deviation (grey band) of average firing frequency in response to a 1 s square current step from holding potential at -60 mV (n = 7 for all time points).

Figure 2.8: Action potentials of BLA principal neurons develop a more hyperpolarized threshold and become faster with age Figure 2.

8: Action potentials of BLA principal neurons develop a more hyperpolarized threshold and become faster with age. (A) Action potential waveform, depicted as mean (black line) and standard deviation (grey band), for neurons across postnatal development (n = 48 (P7), 34 (P10), 46 (P14), 40 (P21), 54 (P28)). (B-E) Box and whisker plots depict action potential threshold (B), half-width (C), 10-90% rise-time (D), and 90-10% decay time (E) for neurons at each time point (n = 49-51 (P7), n = 35-37 (P10), n = 52-55 (P14), n = 43-45 (P21), n = 56-57 (P28), and n = 55-57 (P35)). Significance was assessed using a Wilcoxon rank-sum test and pairwise comparisons were made for each age group with up to four neighboring time-points (see inset tables for results) using a Bonferroni correction for the resulting 9 comparisons (***, p 0.001; **, p 0.01; NS: not significant, p 0.05).

Figure 2.9: Action potential medium AHP matures and a fast AHP emerges with age Figure 2.

9: Action potential medium AHP matures and a fast AHP emerges with age. (A) Changes in afterhyperpolarization (AHP) waveform are illustrated by spike-triggered averages (from at least 8 action potentials) of one free-firing, representative neuron for each time point. (B,

D) Derived from spike-triggered averages, the voltage difference between a neuron’s action potential threshold and its medium (B) or fast (D) AHP peak (mean ± SEM) is plotted versus time elapsed from spike initiation to AHP peak (mean ± SEM) for each time point (n = 8 (P7), 15 (P14), 22 (P21), and 31 (P28)). There was a significant effect of age on mAHP amplitude (p 0.001, One-way ANOVA) and duration (p 0.01, Kruskal-Wallis). Only neurons with a discernible fast AHP were included in analysis for D, and the proportions of neurons expressing a fast AHP at each time point are depicted as pie charts (C).

Chapter 3: Morphology and Ion Channel Expression of Developing

–  –  –

Adapted from: Ryan SJ*, Ehrlich DE*, Hazra R, Guo JD, Rainnie DG (2013). Morphology and Ion Channel Expression of Developing Principal Neurons in the Rat Basolateral Amygdala. Under revision at J Comp Neurol.

3.1 Abstract The basolateral nucleus of the amygdala (BLA) assigns emotional valence to sensory stimuli, and many amygdala-dependent behaviors undergo marked development during postnatal life. We recently showed principal neurons in the rat BLA undergo dramatic changes to their electrophysiological properties during the first postnatal month, but no study to date has thoroughly characterized changes to morphology or gene expression that may underlie the functional development of this neuronal population. We addressed this knowledge gap with reconstructions of biocytin-filled principal neurons in the rat BLA and single-cell RT-PCR at postnatal days 7 (P7), 14, 21, 28, and 60. BLA principal neurons underwent a number of morphological changes, including a two-fold increase in soma volume from P7 to P21 followed by a comparable decrease by P60. Dendritic arbors expanded significantly during the first postnatal month and achieved a mature distribution around P28, in terms of total dendritic length and distance from soma. The number of primary dendrites and branch points were consistent with age, but branch points were found farther from the soma in older animals. Dendrites of BLA principal neurons at P7 had few spines, and spine density increased nearly five-fold by P21.

Corresponding with these morphological changes were shifts in the expression of transcripts for voltage-gated ion channels, with a number of subtypes present at P7 or P14 replaced by P28.

Together, these developmental transitions in BLA principal neuron morphology and gene expression help explain a number of concomitant electrophysiological changes and identify a critical period in amygdala development.

3.2 Introduction The amygdala is critical for the expression of normative and maladaptive emotional behaviors, but relatively few studies have characterized how BLA structure and function change with age. Many prior studies addressing maturation of the BLA have been performed in rats.

These studies have identified a window during the first postnatal month wherein the morphology and physiology of the nucleus undergo rapid and pronounced change. In particular, during this window the volume of the BLA increases while the density of neurons is reduced by half (Morys et al., 1998; Rubinow and Juraska, 2009; Chareyron et al., 2012). Neurons in the BLA grow during this period, with somas and dendritic arbors expanding (Escobar and Salas, 1993). We characterized the developmental changes to electrophysiological properties of BLA principal neurons, which comprise approximately 85% of BLA neurons and mediate virtually all output of the nucleus (Chapter 2; Ehrlich et al., 2012). Specifically, we showed that BLA principal neurons exhibit significant changes to their excitability and sensitivity to synaptic input across the first postnatal month, including a ten-fold reduction in input resistance and a hyperpolarization of action potential threshold greater than 5 mV. There are also concomitant changes to the waveform and patterning of action potential output, including the emergence of a fast afterhyperpolarization and spike doublets.

While previous studies have addressed gross morphological changes to the BLA and its component neurons throughout postnatal development, several important knowledge gaps remain.

The only study to date examining the morphology of individual neurons in the BLA was not specific to principal neurons and did not address features such as the quantity or branching of dendritic material or the expression of spines (Escobar and Salas, 1993). Developmental changes to these features should substantially alter neuronal function, as the surface area of BLA principal neurons and the types of ion channels inserted into their membranes directly impact neurophysiology, including firing patterns (Mainen and Sejnowski, 1996). We have addressed this knowledge gap by characterizing BLA principal neuron morphology throughout the first postnatal month and in adulthood. Additionally, we identified corresponding changes to the expression of subtypes for various voltage-gated ion channels. Specifically, we used whole cell patch clamp at postnatal days 7, 14, 21, 28, and 60 to fill neurons with biocytin for post-hoc morphological reconstruction and analysis. We also recovered principal neuron cytosol for analysis with single-cell RT-PCR, testing for the expression of transcripts for voltage-gated ion channels in the HCN, KIR, KV, and CaV families. Here, we describe a number of changes to the soma, dendritic arbor, dendritic spines, and ion channel expression in developing BLA principal neurons.

3.3 Methods 3.3.1 Ethical approval All experimental protocols strictly conform to National Institutes of Health guidelines for the Care and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee of Emory University.



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