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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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3.3.2 Animals Male rats born in-house to time-mated Sprague–Dawley female rats (embryonic day 5 on arrival from Charles River, Wilmington, MA, USA) were used in all experiments. Pups were housed with the dam prior to weaning on postnatal day (P)22 or P23 (considering P1 as day of birth). After weaning, rats were isolated by sex and housed three to four per cage with access to food and water ad libitum. Animals attributed to each developmental time-point (P7, P14, P21, P28, and P60) were used on that day or the following day (P7–8, P14–15, P21–22, P28–29, and P60-61, respectively).

3.3.3 Slice Physiology To perform neuronal reconstructions at each time-point, we performed whole-cell patch clamp to identify BLA principal neurons based on electrophysiological properties as described previously (Ehrlich et al., 2012) and to visualize neurons, biocytin (0.35%, Sigma-Aldrich, St Louis, MO, USA) was included in the patch recording solution. Acute brain slices containing the BLA were obtained as previously described (Rainnie, 1999b). Briefly, animals were decapitated under isoflurane anesthesia (Fisher Scientific, Hanoverpark, IL, USA) if older than 11 days, and the brains rapidly removed and immersed in ice cold, 95% oxygen–5% carbon dioxide-perfused ‘cutting solution' with the following composition (in mM): NaCl (130), NaHCO3 (30), KCl (3.50), KH2PO4 (1.10), MgCl2 (6.0), CaCl2 (1.0), glucose (10), ascorbate (0.4), thiourea (0.8), sodium pyruvate (2.0) and kynurenic acid (2.0). Coronal slices containing the BLA were cut at a thickness of 300–350 μm using a Leica VTS-1000 vibrating blade microtome (Leica Microsystems Inc., Bannockburn, IL, USA). Slices were kept in oxygenated cutting solution at 32°C for 1 h before transferring to regular artificial cerebrospinal fluid (ACSF) containing (in mM): NaCl (130), NaHCO3 (30), KCl (3.50), KH2PO4 (1.10), MgCl2 (1.30), CaCl2 (2.50), glucose (10), ascorbate (0.4), thiourea (0.8) and sodium pyruvate (2.0).

3.3.4 Patch clamp recording Individual slices were transferred to a recording chamber mounted on the fixed stage of a Leica DMLFS microscope (Leica Microsystems Inc., Bannockburn, IL, USA) and maintained fully submerged and continuously perfused with oxygenated 32°C ACSF at a flow rate of 1–2 mL min−1. The BLA was identified under 10x magnification. Individual BLA neurons were identified at 40x using differential interference contrast (DIC) optics and infrared (IR) illumination with an IR sensitive CCD camera (Orca ER, Hamamatsu, Tokyo Japan). Patch pipettes were pulled from borosilicate glass and had a resistance of 4–6 MΩ. Patch electrode solution had the following composition (in mM): potassium gluconate (130), KCl (2), HEPES (10), MgCl2 (3), K-ATP (2), Na-GTP (0.2), phosphocreatine (5), and 0.35% biocytin, titrated to pH 7.3 with KOH, and 290 mosmol L−1. Data acquisition was performed using either a MultiClamp 700A or an Axopatch 1D amplifier in conjunction with pCLAMP 10.2 software and a DigiData 1322A AD/DA interface (Molecular Devices, Sunnyvale, CA, USA). Whole-cell patch clamp recordings were obtained and low-pass filtered at 2 kHz and digitized at 10 kHz. The membrane potential was held at −60 mV for all neurons if not specified. Cells were excluded if they did not meet the following criteria: a stable resting membrane potential more negative than −55 mV; access resistance lower than 30 MΩ; stable access resistance throughout recording, changing less than 15%; and action potentials crossing 0 mV. The identity of BLA principal neurons was verified by injecting a series of 10 hyperpolarizing and depolarizing, 1 s long, square-wave current steps. They were scaled so that, for each cell, the peak voltage deflections were to approximately −80 mV and −40 mV.

Traces were analyzed using Clampfit 10.2 (Molecular Devices, Sunnyvale, CA, USA).

3.3.5 Histochemical Processing Patched neurons were labeled with biocytin (Sigma-Aldrich, St Louis, MO, USA) included in the patch pipette recording solution. After neurons were recorded for at least 15 minutes, slices were fixed in 10% buffered formalin (Fisher Scientific, Hanoverpark, IL, USA) for 12-72 hours, and then transferred to cryo-protectant for storage at -20 C. After three consecutive 10 minute washes in 0.05 M phosphate buffered saline (PBS), slices were permeabilized for 30 minutes in phosphate buffered saline (PBS) and 0.5% Triton X-100 (SigmaAldrich, St Louis, MO, USA). Slices were then treated with Alexa Fluor 488-conjugated Streptavidin (Invitrogen, Grand Island, New York, USA) diluted to 1:1000 in PBS with Triton Xovernight at room temperature. Slices were then washed 2 times for 1 hour each in 0.05 M PBS and washed for 10 minutes in 0.05 M phosphate buffer. Finally, the slices were mounted on glass slides, air dried for two to twelve hours, and cover-slipped with mowiol mounting medium (Sigma-Aldrich, St Louis, MO, USA).

3.3.6 Neuronal reconstruction and Data Analysis For morphological analysis, the dendritic arbor of each neuron was first reconstructed by hand using the Neurolucida neuron tracing software (MicroBrightField, Colchester, VT) from single z-stack images taken at 10x magnification with a 0.4 μm step size using a Leica DM5500B spinning disk confocal microscope (Leica Microsystems Inc., Bannockburn, IL, USA) and SimplePCI data acquisition software (Compix, Sewickley, PA). Slices were examined live at 63x to support tracing of fine or overlapping dendritic segments. Reconstructions of neuronal somas were performed using AutoNeuron workflow in Neurolucida from image stacks obtained with a 63x objective. Quantitative analysis of reconstructions was performed using Neurolucida Explorer (MicroBrightField). The volume and surface area of somas were estimated using the ‘Marker and Region Analysis’ subroutine to provide a 3-D contour summary. Dendritic length and branching were analyzed in Neurolucida Explorer using Sholl analyses with ring radius increments of 2 µm, and data were analyzed in Matlab (The MathWorks, Natick, MA, USA).

Corrections were not made for shrinkage due to tissue processing.

To estimate the average spine density for BLA principal neurons, we manually counted dendritic spines with Neurolucida on image stacks of dendrite segments taken at 100x magnification. For each neuron, 10 dendritic segments were analyzed and the counter was blinded to the originating neuron of each segment. The 10 segments for counting were chosen pseudo-randomly using a custom Matlab script (available upon request), which selected random locations throughout the dendritic arbor that were separated from each other by a minimum of 50 µm. Dendritic spines were counted on segments centered on each of these 10 locations, spanning 50 µm (or less if near an ending) along a random path through the dendritic arbor. Total spine number was estimated for each neuron individually using the product of average spine density and aggregate dendritic length, defined as the sum of the lengths of all dendritic segments.

3.3.7 Single Cell RT-PCR At the end of a subset of patch clamp recording sessions without biocytin present in the patch solution, the cell cytoplasm was aspirated into the patch recording pipette by applying gentle negative pressure under visual control. Pipettes contained ~5 μL of RNase-free patch solution. The contents of the patch pipette were expelled into a microcentrifuge tube containing 5 μL of the reverse transcription cocktail (Applied Biosystems, Foster City, CA, USA), and the reverse transcription product was amplified in triplicate and screened for 18S rRNA. Only those cell samples positive for 18S rRNA were subjected to amplification with primers. All cells were screened for the vesicular glutamate transporter 1 (VGLUT1) expression to confirm a glutamatergic phenotype, and all 46 neurons presented here were positive. The procedure used to determine mRNA transcript expression in single cells has been described in detail previously (Hazra et al., 2011). The sequences for the oligonucleotide primers are listed in Table 3.1. PCR products were visualized by staining with ethidium bromide and separated by electrophoresis in a 1% agarose gel.

3.3.8 Statistics To compensate for age-dependent changes in variance (assessed using Bartlett’s test with GraphPad, GraphPad Software Inc., La Jolla, CA, USA), data for soma volume, critical value of dendritic length, dendritic spine density, and total spine estimate were log-transformed before statistical analysis. Unless otherwise noted, data are presented as mean ± SEM. The values for aggregate dendritic length, critical value of dendritic length, dendritic spine density, and total dendritic spine estimate were fit with a Boltzman sigmoidal equation (Equation 3.1) in GraphPad. Most data sets were analyzed with a One-way ANOVA to determine effects of age (GraphPad). Data from Sholl analyses were analyzed with Two-way ANOVA to determine effects of age and distance from soma. Single-cell RT-PCR data were analyzed using logistic regression with a binomial distribution in R (The R Foundation for Statistical Computing, Vienna, Austria), with gene expression as the dependent variable (no expression defined as 0, expression defined as 1) and age as the independent variable.

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3.4 Results In total, 40 neurons from 15 male rats were filled with biocytin at postnatal day 7 (P7), P14, P21, P28, and P60 (8 per time-point). In addition, we characterized mRNA expression at each time-point using single cell reverse transcription – polymerase chain reaction (scRT-PCR) after recovering mRNA from BLA principal neurons at P7, P14, P21, and P28 (n = 11 to 12 neurons from 1 animal per time-point). Basic electrophysiological properties were measured and found to be consistent with previously reported values (Ehrlich et al., 2012). Brain slices containing filled, recorded neurons were stained and neurons visualized post-hoc to make neuronal reconstructions (see Methods). Representative neuronal reconstructions are depicted for each time-point in Figure 3.1, and illustrate a variety of developmental changes to BLA principal neuron morphology that are quantified in detail below. Principal neurons at all ages lacked a consistent orientation in the slice.

3.4.1 Soma size We first quantified somatic volume and surface area of BLA principal neurons throughout postnatal development (Figure 3.2). Somatic diameters ranged between 10 and 16 µm and somatic volume changed significantly during postnatal development (Figure 3.2A, P 0.0001, One-way ANOVA, F4,29 = 12.97), gradually increasing across the first postnatal month and then decreasing by adulthood. Mean somatic volume increased by 94% from P7 to P21, followed by a 39% reduction by P60. Somatic volume also exhibited an inverted-U relationship with age, increasing significantly from 793.9 ± 56.1 µm3 (mean ± SEM) at P7 (n = 7) to 1291.5 ±

60.7 µm3 at P14 (n = 7; Tukey’s post hoc test, P 0.01), 1541.5 ± 156.8 µm3 at P21 (n = 7, P 0.001), and 1507.1 ± 61.8 µm3 at P28 (n = 6, P 0.001). However, by P60, somatic volume measured 946.0 ± 103.7 µm3 (n = 7), constituting a significant decrease from P14 (P 0.05), P21 (P 0.01), and P28 (P 0.01).

A similar trajectory was exhibited by somatic surface area, which also changed significantly across postnatal development (Figure 3.2B, P 0.001, One-way ANOVA, F4,29 = 6.143). Somatic surface area increased from 376.5 ± 27.1 µm2 at P7 (n = 7) to 498.3 ± 25.8 µm2 at P14 (n = 7), and significantly increased from P7 to 536.0 ± 52.2 µm2 at P21 (n = 7; Tukey’s post hoc test, P 0.05) and 558.5 ± 20.0 µm2 at P28 (n = 6; P 0.01). By P60, the somatic surface area of 390.2 ± 29.9 µm2 was comparable to that at P7 and constituted a significant decrease from P21 (P 0.05), and P28 (P 0.05).

3.4.2 Growth and Retraction of Dendritic Arbor Extensive remodeling of dendritic architecture of BLA principal neurons also occurred across postnatal development (Figure 3.3). The aggregate length of dendrites for each neuron changed significantly with age (P 0.0001, One-way ANOVA, F4,34 = 23.27), increasing more than threefold across the first postnatal month (Figure 3.3A). Aggregate dendritic length increased significantly from 2.106 ± 0.168 mm at P7 (n = 7) to 4.863 ± 0.466 mm at P14 (n = 8;

Tukey’s post hoc test, P 0.001), 6.185 ± 0.358 mm at P21 (n = 8; P 0.001), 6.809 ± 0.402 mm at P28 (n = 8; P 0.001), and 6.378 ± 0.409 mm at P60 (n = 8; P 0.001). The aggregate dendritic length also increased significantly from P14 to P28 (P 0.01). The distribution of aggregate dendritic length vs. age was fit with a sigmoidal Boltzmann function (Eqn. 3.1), which estimated the inflection point at V1/2 = 10.92 days with a slope of α = 3.48 days. The lower asymptote of aggregate length was estimated to be A2 = 659.8 µm and the upper asymptote to be A1 = 6578 µm. The goodness of fit was R2 = 0.726.

The observed increase in aggregate dendritic length with age corresponded with an increased distance of that material to the soma (Figure 3.3B). Using a Sholl analysis with concentric rings of 4 µm thickness, we were able to determine the critical value for dendritic length, defined as the radius of the Sholl ring with the greatest amount of dendritic length. With age, dendrites became concentrated farther from the soma, with the critical value increasing significantly across the first postnatal month (P 0.0001, One-way ANOVA, F4,33 = 10.69).

Specifically, the critical value for dendritic length increased from 32.3 ± 2.9 µm at P7 (n = 7) to

52.0 ± 5.7 µm at P14 (n = 8) and increased significantly to 71.7 ± 10.1 µm at P21 (n = 7; Tukey’s post hoc test, P 0.01 vs P7), 91.0 ± 9.6 µm at P28 (n = 8; P 0.001), and 93.0 ± 11.8 µm at P60 (n = 8; P 0.001). The critical value also increased significantly from P14 to P28 and P60 (P 0.05). As for the aggregate dendritic length values, the distribution of critical value vs. age was fit with a sigmoidal Boltzmann function (Eqn. 3.1), which estimated the inflection point at V1/2 =

16.37 days with a slope of α = 4.91 days. The lower asymptote for critical value was estimated to be A2 = 23.70 µm and the upper asymptote to be A1 = 94.26 µm. The goodness of fit was R2 = 0.507.

The proximity of dendrites to the soma matures in a specific pattern, exemplified by the representative reconstructions in Figure 3.1. Across the first few postnatal weeks, dendrites extend farther from the soma. From P21 to P28 there is expansion of dendrites near the soma, and by P60 there is a reduction of dendrites in the most proximal and distal portions of the arbor.

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