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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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Using Sholl analysis, we identified specific portions of the dendritic arbor where significant growth and retraction occur during postnatal development (Figure 3.3D; Two-way ANOVA with Bonferroni post hoc tests; main effect of age: P 0.0001, F4,1155 = 85.67; main effect of distance from soma: P 0.0001, F34,1155 = 187.8; interaction effect: P 0.0001, F136,1155 = 6.586). In P7 neurons (n = 7), more than 99.7% of dendritic length is found within 200 µm of the soma. By P14, the proportion found within 200 µm drops to 89.8%, while 99.8% of dendritic length is found within 400 µm of the soma. From P7 to P14, there is also a significant expansion of dendrites in the region 40 to 180 µm from the soma (P 0.01). By P21, only 93.5% of dendritic material is found within 400 µm of the soma, with the remainder extending as far as 660 µm from the soma. From P14 to P21, significant growth of dendrites occurs in the region 140 to 220 µm from the soma (P 0.01). At P28, dendritic arbors occupy a similar space as those at P21, extending as far as 680 µm from the soma, with 96.5% of dendritic length found within 400 µm of the soma. The greater proportion of dendrites within 400 µm of the soma at P28 corresponds with a significant increase from P21 in dendrites found in the region 80 to 120 µm from the soma (P 0.001). The developmental expansion of dendrites in this window is reversed by P60, which exhibits a significant decrease from P28 in the region 60 to 120 µm from the soma (P 0.05). By P60, the reduction in dendritic length also occurs in the most distal parts of the arbor, with 99.4% of dendritic length being found within 400 µm of the soma.

We also examined the pattern of dendrite maturation by considering the growth of specific orders of dendritic branches (Figure 3.3C). When we normalized the aggregate dendritic length for individual orders of branches to the total for all branch orders of a neuron, we found the majority of dendritic length in second through sixth order dendrites.

The total length of dendrites varied significantly by branch order, but age did not significantly affect this distribution (Two-way ANOVA; main effect of branch order: P 0.0001, F8,268 = 52.96; main effect of age:

P 0.05, F4,268 = 0.28).

3.4.3 Maturation of Dendritic Branching We also investigated the maturation of branch points in the dendritic arbor, because the location of branch points determines the relationship between dendrite order and proximity to the soma (Figure 3.4). The number of primary dendrites was consistent throughout postnatal development (Figure 3.4A; P 0.05, Kruskal-Wallis, H(4) = 5.93; mean ± SD: 7 ± 2.4 at P7,

5.75 ± 1.5 at P14, 5.5 ± 1.1 at P21, 6.1 ± 1.5 at P28, and 7.75 ± 2.4 at P60; n = 8). We analyzed the total number of branch points in the dendritic arbor for neurons at each time-point, and found no significant effect of age (Figure 3.4B; P 0.05, Kruskal-Wallis, H(4) = 7.10; mean ± SD: 42 ± 18.0 at P7, 34.7 ± 13.3 at P14, 35.0 ± 8.5 at P21, 45.0 ± 8.8 at P28, and 34.5 ± 8.8 at P60; n = 7Furthermore, we quantified the proximity of branch points to the soma using a Sholl analysis with 40 µm thick rings. We found significant changes in the proximity of branch points to the soma with age (Figure 3.4C; Two-way ANOVA with Bonferroni post hoc tests; main effect of distance from soma: P 0.0001, F15,512 = 185.6; interaction of age and distance from soma: P 0.0001, F60,512 = 3.998). In P7 neurons, branch points were found very close to the soma, with

65.1 ± 9.4% occurring within 40 µm of the soma and 99.3 ± 0.7% occurring within 160 µm (mean ± SEM, n = 7). With age, branch points transitioned away from the soma: by P14, branch points were found more distally, extending as far as 320 µm from the soma, and with significantly fewer within 40 µm (45.3 ± 6.6%, P 0.001, n = 7). The proportion of branch points within 40 µm of the soma decreases further from P14 to P21 (40.0 ± 6.4%, n = 7), and decreases significantly from P14 to P28 (33.5 ± 5.5%, P 0.001, n = 8) and P60 (31.6 ± 3.4%, P 0.001, n = 8). Conversely, the proportion of branch points located more distally increases significantly with age. Specifically, in P7 neurons 4.4 ± 1.8% of branch points are located 80 to 120 µm from the soma, while this number increases to 10.1 ± 1.7% at P14 and significantly increases to 14.7 ± 3.5% at P21 (P 0.05), 17.8 ± 3.6% at P28 (P 0.001), and 18.8 ± 3.1% at P60 (P 0.001). A similar trend occurs for branch points located 120 to 160 µm from the soma (see Figure 3.4C).

3.4.4 Developmental Emergence of Dendritic Spines We next investigated the maturation of dendritic spines (Figure 3.5), which were much more apparent on the arbors of neurons at later time-points. Neurons at P7 frequently possessed smooth dendrites with few spines, while neurons at P21 and older had spine-laden dendrites (Figure 3.5A). To quantify the emergence of dendritic spines with age, we counted spines on 10 random segments of dendrite for neurons at each time-point (see Methods). We found that the density of dendritic spines changed significantly with age, increasing nearly six-fold across the time period studied, reaching adult levels at the end of the first postnatal month (Figure 3.5B; P 0.0001, One-way ANOVA, F4,25 = 59.41; n = 6 neurons per time-point). Specifically, neurons at P7 had a spine density of 0.21 ± 0.03 spines/µm (mean ± SEM) which increased significantly to

0.53 ± 0.05 spines/µm at P14 (P 0.001), 1.03 ± 0.12 at P21 (P 0.001), 1.18 ± 0.05 at P28 (P 0.001), and 1.29 ± 0.07 at P60 (P 0.001). Spine density also increased significantly from P14 to all later time points (P 0.001). The distribution of dendritic spine density vs. age was fit with a sigmoidal Boltzmann function (Eqn. 3.1), which estimated the inflection point at V1/2 = 15.97 days with a slope of α = 3.89 days. The lower asymptote for spine density was estimated to be A2 = 0.10 spines and the upper asymptote to be A1 = 1.26 spines. The goodness of fit was R2 = 0.868.

Using our measurements of mean spine density and the aggregate dendritic length from our reconstructions, we were able to estimate the total number of dendritic spines for each neuron (Figure 3.5C). These estimates suggest the total number of spines is more than fifteen times larger at P60 than at P7, as the number of spines increases significantly across postnatal development (P 0.0001, One-way ANOVA, F4,25 = 59.92; n = 6). Specifically, neurons at P7 had an estimated 533 ± 146 spines (mean ± SEM) which increased significantly to 2530 ± 392 spines at P14 (P 0.001), 6204 ± 512 at P21 (P 0.001), 7675 ± 704 at P28 (P 0.001), and 8357 ± 999 at P60 (P 0.001). Total spine number also increased significantly from P14 to all later time-points (P 0.01). As with spine density, the distribution of total dendritic spines vs.

age was fit with a sigmoidal Boltzmann function (Eqn. 3.1), which estimated the inflection point at V1/2 = 17.0 days with a slope of α = 3.80 days. The lower asymptote for spine density was constrained at A2 = 0 spines and the upper asymptote was estimated to be A1 = 8256.0 spines. The goodness of fit was R2 = 0.826.

3.4.5 Expression of Ion Channel Transcripts We next investigated the expression of ion channel mRNA transcripts, which are thought to endow principal neurons with their basic electrophysiological properties. Our previous study of the electrophysiological properties of developing BLA principal neurons provided several candidate ion channels for further study. Here, we investigated the presence of transcripts for multiple ion channel subtypes by isolating cytosolic mRNA from individual BLA principal neurons throughout the first postnatal month (Figure 3.6). Specifically, we assessed the presence of mRNA transcripts for subtypes of 4 different ion channels: namely, the hyperpolarizationactivated, cyclic nucleotide-gated (HCN) channel that mediates the H-current (IH); the inwardly rectifying potassium channel, KIR; the voltage-gated potassium channels mediating the A-current (IA); and the voltage-gated calcium channels mediating the T-current (IT). HCN Subtype Expression Four subtypes of HCN channel (HCN 1-4) mediate IH and are primarily differentiated by their differing gating kinetics (Pena et al., 2006). We previously reported a decrease in the activation time-constant for IH across the first postnatal month, leading us to hypothesize that HCN subtype expression may also change across development (Ehrlich et al., 2012). Consistent with our hypothesis, a developmental transition in the expression of mRNA for IH channel subtypes was observed in BLA principal neurons (Figure 3.6A). HCN2 and HCN4 were the only HCN subtypes present at P7, and at this age they were mutually exclusive, being expressed by 6/11 and 5/11 neurons, respectively. At P14 no HCN2 transcript expression was detected, however we did detect HCN3 and HCN4 transcript expression in 6/11 and 7/ 11 neurons, respectively. At P21, transcripts for all three HCN subtypes found previously were expressed;

HCN2 was detected in 4/12 neurons, HCN3 in 3/12, and HCN4 in 4/12. At P28, HCN1 expression emerged, being detected in 7/12 neurons, while HCN2 and HCN3 transcripts were detected in 5/12 and 9/12 neurons, respectively. HCN4 transcript expression was absent at P28. KIR Subunit Expression KIR channels are a family of inwardly-rectifying potassium channels. KIR2.1-2.4 are responsible for the “anomalous rectifying current” that is, like IH, activated at membrane potentials hyperpolarized to rest. We also found changes in the expression of mRNA transcripts for these channel subunits (Figure 3.6B). At P7, KIR2.1 was the only subunit expressed, and was found in all 11 neurons tested. At P14, KIR2.1 was only expressed in 4/11 neurons, while KIR2.3 expression emerged, being mutually exclusive with KIR2.1 expression, and found in the remaining 7/11. KIR2.1 and 2.3 were the only subunits expressed at P21, in 7/12 and 9/12 neurons, respectively. A similar pattern of expression was found at P28, with 7/12 neurons expressing KIR2.1, 1/12 expressing KIR2.2, and 8/12 expressing KIR2.3. There was no detectable expression of KIR2.4 transcripts during the first postnatal month. KV Subunit Expression We also investigated the developmental expression of several voltage-gated potassium (KV) channel subunits, specifically those channels mediating IA. As illustrated in Figure 3.6C, KV1.4 was expressed robustly and exclusively in immature neurons, being found in 10/11 neurons at P7, and 9/11 neurons at P14. However, at P21 and P28 KV1.4 mRNA transcripts were not detectable in any neuron tested. Contrastingly, KV3.4 transcripts were absent from neurons at P7 or P14, but were present in 10/12 and 12/12 neurons at P21 and P28, respectively. The pattern of expression for Kv4.1, 4.2, and 4.3 transcripts was consistent throughout development. KV4.1 transcripts were expressed by 8/11 neurons at P7, 7/11 at P14, 6/12 at P21, and 6/12 at P28.

KV4.2 was expressed by all 11 neurons at P7, 9/11 at P14, all 12 at P21, and 6/12 at P28. KV4.3 was expressed by 2/11 neurons at P7, 5/11 at P14, 5/12 at P21, and 4/12 at P28. Notably, expression of KV 4.1 and 4.3 transcripts were mutually exclusive in most neurons, aside from a single neuron at each of P14, P21, and P28 that expresses both subunits. CaV3 Subunit Expression Finally, we examined expression of the three alpha subunits of the heteromeric channels that conduct the transient calcium current, IT (Figure 3.6D). We found a clear shift in expression of CaV3 subunits from CaV3.1 to CaV3.2 with age. At P7 and P14, 10/11 neurons had detectable expression of CaV3.1, while that proportion decreased to 2/12 at P21 and 0/12 at P28. Conversely, no neurons had detectable expression of CaV3.2 transcripts at P7 or P14, whereas 7/12 neurons showed expression at P21 and 4/12 neurons at P28 expressed CaV3.2 transcripts. Transcripts for CaV3.3 were not detected at any time-point.

3.5 Discussion In this study, we presented a detailed analysis of the morphological properties of BLA principal neurons, conducted across the first two postnatal months in rats. During this window, BLA principal neurons exhibit a variety of structural changes with the most dramatic maturation occurring before P21. Significant morphological changes included: soma size developing as an inverted U with a peak at P28; a three-fold increase in aggregate dendritic length from P7 to P21;

growth of distal dendrites until P21 followed by retraction at P28; a shift of branch points more distally in the dendritic arbor through P60; and an increase in the density of dendritic spines, reaching maturity around P28. During the first postnatal month, we also observed transitions in the expression of specific subtypes of voltage-gated ion channels, generally away from channels that operate at more depolarized voltages and with slower kinetics. Taken together, these developmental changes to principal neuron morphology and gene expression help explain a wealth of electrophysiological changes occurring in the first postnatal month, including dramatic changes to passive membrane properties, action potential waveform and patterning, and intrinsic frequency preference (Ehrlich et al., 2012). The structural and functional maturation of amygdala neurons may underlie a variety of developmental changes to emotional behavior (King et al., 2013), which also occur during the first postnatal month and include conditioned avoidance (Sullivan et al., 2000), fear-potentiated startle (Hunt et al., 1994; Richardson et al., 2000), trace conditioning (Moye and Rudy, 1987), and extinction (Kim and Richardson, 2007). Our observations suggest that developmental changes in the mammalian amygdala extend from birth until adolescence, based on developmental milestones in the rat (Quinn, 2005).

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