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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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Despite the compelling evidence of early-life transitions in BLA function, no study to date has examined how changes in the physiological properties of individual BLA neurons contribute to these critical periods of development. This information is essential if we are to understand how the adult BLA becomes organized, how it comes to communicate with other brain regions, and how early-life perturbations could influence mature BLA function. We have begun to address this knowledge gap using whole-cell patch clamp recording to characterize the physiological development of BLA principal neurons during the first postnatal month. We show that these neurons undergo significant transitions in intrinsic properties which define their sensitivity to input and characteristic activity, including passive and oscillatory membrane properties, action potential waveform, and spike-train characteristics.

2.3 Methods 2.3.1 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.

2.3.2 Animals Rats born in-house to time-mated Sprague-Dawley female rats (embryonic day 14 on arrival, Charles River, Wilmington, MA) were used in all experiments. Pups were housed with the dam prior to weaning on postnatal day 22 (P22) or P23 (considering P1 as day of birth). After weaning, rats were isolated by sex and housed 3-4 per cage with access to food and water ad libitum. Animals attributed to each recording day (P7, P10, P14, P21, and P28) were recorded on that day or the following day (P7-8, P10-11, P14-15, P21-22, and P28-29, respectively).

2.3.3 Slice preparation 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, 95/5%

oxygen/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 vibratome (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), sodium pyruvate (2.0).

2.3.4 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. 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). A subset of neurons was filled using patch solution with added biocytin (0.3%) to confirm localization within the BLA. After some recordings, cytosol was recovered and screened using single-cell reversetranscriptase PCR, as described previously (Hazra et al., 2011), to confirm the presence of the glutamate transporter, VGluT, which was seen in 58/60 neurons tested across all ages. Patch pipettes were pulled from borosilicate glass and had a resistance of 4–6 MΩ. Patch electrode solution had the following composition (in mM): K-gluconate (130), KCl (2), HEPES (10), MgCl2 (3), K-ATP (2), Na-GTP (0.2), and phosphocreatine (5), titrated to pH 7.3 with KOH, and 290 mOsm. 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, Burlingame, CA, USA). Whole-cell patch clamp recordings were obtained and 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%; action potentials crossing 0 mV. Where indicated, Cs+ (5 mM, Sigma Aldrich, St. Louis, MO) was administered through bath application.

2.3.5 Data Analysis Data were analyzed by importing the raw voltage and current traces into Matlab (Mathworks, Natick, MA, USA) using scripts provided with sigTOOL (http://sigtool.sourceforge.net/, developed at King’s College, London) and processed with customized scripts (available upon request). To characterize neurons in current clamp, first, a series of ten hyperpolarizing and depolarizing, 1 second long, square-wave current steps were injected. They were scaled so that, for each cell, the peak voltage deflections were to approximately -80 mV and -40 mV (amplitude of negative current injections ranged from a minimum of -20 pA at P7 to a maximum of -1000 pA at P28, and positive current injections from +16 pA at P7 to +800 pA at P28). Second, linear ramps of depolarizing current were injected, lasting 250 ms and scaled to depolarize the neuron to -35 mV and elicit an action potential within the final 50 ms (peak current ranged from a minimum of +85 pA at P7 to a maximum of 555 pA at P28).

2.3.6 Membrane Properties and Intrinsic Currents Input resistance and time constant were calculated using the deflection (approx. 5 mV) in response to the smallest, hyperpolarizing current step (minimum of -4 pA at P7, maximum of pA at P28). Time constant was defined as the time necessary for the cell to reach 63.2% of its maximal deflection; input resistance was calculated as the ratio of peak voltage deflection to the current injected. To measure the hyperpolarization-activated, non-specific cation current, Ih, neurons were voltage clamped at a holding potential of -60 mV and stepped to -100 mV for 600 ms in the presence of 1 µM TTX. The waveform of the Ih current was generated by subtracting a current trace measured in the presence of bath-applied, 5mM Cs+, known to block Ih, from that measured in its absence. This subtraction current was used to measure amplitude and activation time constants of Ih. A two-term exponential fit (Equation 2.1, k = 1) was used to extract fast and slow time constants of Ih activation, except at P7, where two terms over-parameterized the fit and a one-term exponential (Equation 2.1, k = 2) was used instead.

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2.3.7 Action Potentials and Spike Trains Action potentials were detected using a heuristic to locate peaks in the second derivative of the membrane potential waveform. The time of the peak was assigned to be the time of spike initiation and the voltage assigned as action potential threshold, which correlated well with visual inspection of the data. Since the sampling rate used here was not fast compared to the frequency of the action potential waveform (10 kHz compared to ~1 kHz), linear interpolation between data points was used to enhance the temporal resolution of measurements of 10-90% rise time, 90decay time, and half-maximal width. These parameters and average action potential waveforms were calculated using spikes collected in the ramp protocol described above. Fast and medium afterhyperpolarizations (AHPs) were measured on spike-triggered averages from every spike captured (at least 8) during a 1 minute recording from neurons clamped manually at action potential threshold with DC current injection. Fast AHP peak was measured at a local minimum directly following spike re-polarization, if visibly distinct from the medium AHP and occurring within 15 ms of spike initiation. Medium AHP peak was measured at the minimum voltage following the spike, if it occurred within 150 ms of spike initiation (Storm, 1989).

Data on spike trains were collected from responses to the depolarizing, 1 second-long, square-wave current injections described above. Spike trains were included in the analysis if the mean of their inter-spike membrane potential fell within one standard deviation of the spike threshold measured for each age. Inter-spike intervals (ISIs) were calculated using the times for spike initiation in these traces, and the instantaneous firing frequency of the spike train was calculated as the reciprocal of these ISIs.

2.3.8 Resonance and Oscillations Resonance was assessed by injecting neurons with a ZAP current, a sinusoidal current of fixed amplitude that sweeps logarithmically from 0.1 to 12 Hz over 30 seconds. The amplitude of the current was adjusted to elicit a 20 mV maximal depolarization from a baseline potential of -70 mV. Impedance was calculated as a function of input frequency for each neuron by deriving a power spectrum for the voltage response to the ZAP current, using fast Fourier transforms in the Chronux toolbox for Matlab (Bokil et al., 2010), and normalizing it to the power spectrum of the injected current. In order to extract peak values from noisy power spectra and generate averages, the raw impedance traces were fit with a 6th order polynomial. Prominence was calculated using power spectra as the proportion of total power in the entire range considered (1-10 Hz) found in a given frequency band (1-2, 2-4, 4-6, 6-8, or 8-10 Hz). The change in prominence due to Cs+ application was calculated as a ratio of the power in a given frequency band in Cs+ and TTX to that in TTX alone. Correlation analysis of the relationship between membrane time constant (τmemb) and peak resonance frequency was performed using GraphPad Prism 4 (GraphPad Software Inc., La Jolla, CA, USA). The presence of membrane potential oscillations (MPOs) in recordings of neurons gradually depolarized to action potential threshold was assessed by an observer blinded to age group. Spike-triggered averages of these depolarized traces were generated using Matlab to observe phase relationships between action potentials and putative MPOs.

2.3.9 Statistics Data points greater than 2 standard deviations from the mean were deemed outliers and removed from statistical analysis. All data sets were tested for normality using the Shapiro-Wilk test (α = 0.05) and for homoscedasticity using Levene’s test (α = 0.0001), implemented in Matlab.

Data sets for mAHP and fAHP amplitude, maximal firing rate, peak resonance frequency, and Ih amplitude passed the Shapiro-Wilk and Levene’s tests, so one-way ANOVA with Tukey’s posthoc tests (α = 0.05) were used to assess significance. To account for age-dependent changes in variance, values of Rin, τmemb, and action potential rise- and decay-times were log-transformed before statistical analysis. Because the data sets for 6 of 7 basic electrophysiological parameters (Rin, τmemb, action potential half-width, rise-time, decay-time, and first inter-spike interval, but not action potential threshold) failed either the Shapiro-Wilk or Levene’s test, all of these data sets were analyzed using the Kruskal-Wallis test for overall effect of age on each parameter (α = 0.05). The Kruskal-Wallis test was also used for mAHP and fAHP times-to-peak. When a main effect was found, pair-wise comparisons were made for each age group with two nearest older and younger groups (i.e. P21 vs. P10, 14, 28, and 35) using Wilcoxon rank sum tests (Matlab) with a Bonferroni correction for the resulting 9 comparisons (α = 0.0056). Significant changes in prominence were assessed using a two-way, repeated measures ANOVA with Bonferroni posthoc tests, with frequency band as a within-subjects factor and age as a between-subjects factor.

Peak resonance frequency was exponentially correlated with τmemb using a least-squares method with Equation 2.1 (k = 1) in GraphPad for all data from P14, P21, and P28.

An analysis of the effects of sex on physiological maturation was performed using a twoway ANOVA with factors of sex and age for the following parameters using GraphPad: Rin, τmemb, action potential threshold, half-width, rise-time, decay-time, and first inter-spike interval.

Significance was assessed for a main effect of sex in each parameter (α = 0.05). This analysis included 16 male and female neurons from 5 male and 3 female rats at P14, 12 male and female neurons from 3 male and 2 female rats at P21, and 12 male and 8 female neurons from 4 male and 2 female rats at P28. No neurons were included from P7 or P10 due to difficulty assessing sex in the young rats. A posthoc power analysis for the effect of sex was conducted using G*Power (Faul et al., 2007) with α = 0.05.

2.4 Results Data were collected from a total of 499 BLA neurons from 93 rats on postnatal day 7 (P7), P10, P14, P21, and P28. Also included were data from 53 neurons from 26 older animals (P35) for comparison with the preadolescent data. To make gross comparisons of neural properties across development, we first examined the voltage response of patch clamped principal neurons to transient (1s) hyperpolarizing and depolarizing current injections at P7, P14, P21, and P28. As illustrated in Figure 2.1, BLA principal neurons at each developmental time-point had distinct voltage responses to DC current injection. The most obvious changes were to input resistance (Rin), membrane time-constant (τmemb), the depolarizing voltage sag upon membrane potential hyperpolarization likely caused by Ih, and the pattern of action potentials. Below we quantify these and many other physiological changes to BLA principal neurons across the first postnatal month.

2.4.1 Postnatal Maturation of Passive Membrane Properties We measured the passive electrical properties of principal neurons at all time-points when manually held at -60 mV with DC current injection. Here, Rin and τmemb were estimated from small, 5 mV hyperpolarizing voltage deflections elicited by transient current injection. We observed a significant reduction in Rin of nearly ten-fold across the first postnatal month (Figure

2.2A; p 0.001, Kruskal-Wallis, χ25 = 241.8), with the greatest changes occurring before P21.

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