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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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In studying the effects of PS, we focused on the GABA system in the basolateral nucleus of the amygdala (BLA) for several reasons. The BLA is a hub in emotional processing with widespread connectivity in the brain that support its roles mediating fearful and anxious behavior (Davis et al., 2003; LeDoux, 2007; Pape and Pare, 2010; Stuber et al., 2011), regulating sensory perception and memory (Pessoa and Adolphs, 2010; Chavez et al., 2013; Chen et al., 2013), and influencing reward systems (Ambroggi et al., 2008; Stuber et al., 2011). Activity of the BLA is strictly regulated by GABAergic interneurons, especially those expressing parvalbumin (Rainnie et al., 1991a; Ehrlich et al., 2009; Ryan et al., 2012), and ablation of BLA interneurons causes deficits in social behavior (Truitt et al., 2007). We have recently demonstrated that GABAergic transmission in the rat BLA undergoes pronounced maturation in the first three postnatal weeks (Ehrlich et al., 2013), suggesting it contributes to amygdala development and may be sensitive to early life perturbation. Furthermore, neurons in the BLA mature with a similar time course, exhibiting robust changes to intrinsic electrophysiological properties and excitability (Ehrlich et al., 2012). Early postnatal changes to GABAergic function in the BLA may therefore influence the development of neurons in the amygdala.

A recent study in a rodent model of Fragile X syndrome, which often presents autisticlike features, found that expression in the BLA of the GABAA receptor α1 subunit failed to emerge during the second and third postnatal weeks (Vislay et al., 2013). The only related study of PS found reduced expression of the GABAA receptor γ2 subunit as early as two weeks after birth, but no consistent effect on the α1 subunit (Laloux et al., 2012). Various forms of early postnatal stress reduce expression of the α1 subunit and increases the density of PV+ interneurons in the adult BLA (Caldji et al., 2003; Jacobson-Pick et al., 2008; Seidel et al., 2008), but the effects on developing GABA circuits, specifically when they coordinate critical period plasticity, are largely unknown. Considering these studies, PS is likely to influence GABAergic transmission in the developing BLA, which could broadly influence outcomes for emotional brain circuitry.

Here we describe the effects of PS on the developmental trajectory of BLA neuron electrophysiology, focusing on intrinsic excitability and GABAergic transmission. Male offspring of rat dams exposed to unpredictable shock stress from embryonic day (E)17-20 were sacrificed for patch clamp recordings in acute brain slices, and electrophysiological responses were measured throughout amygdala development (at postnatal days (P)10, 14, 17, 21, 28) and in adulthood (day 60). Expression in the BLA of mRNA for the GABAA receptor α1 subunit was also measured at each time point using quantitative RT-PCR. These studies were paired with behavioral investigations to validate our PS paradigm as a model of risk for ASD and SZ.

Offspring were tested for anxiety-like behavior on the elevated-plus maze in adulthood and isolation-induced ultrasonic vocalizations during infancy (Morgan et al., 1999; Shu et al., 2005) and sociability and social novelty preference were measured with the social choice test (Moy et al., 2004).

5.3 Methods 5.3.1 Ethical Approval All experimental protocols strictly conform to the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health, and were approved by the Emory University Institutional Animal Care and Use Committee.

5.3.2 Animals Male rats born in-house to time-mated, Sprague-Dawley rats (embryonic day (E)4 on arrival, Charles River, Wilmington, MA, USA) were used in all experiments. Pups were housed with the dam prior to weaning on P22 or P23, with the day of birth considered P1. After weaning, rats were isolated by sex and housed three to four per cage with ad libitum access to food and water. Throughout this chapter, animal ages are attributed to a single day for brevity, but labels describe developmental windows as follows: ‘P10’ for P10-11, ‘P14’ for P13-14, ‘P17’ for P17P21’ for P21-22, ‘P28’ for P27-30, and ‘P60’ for P60-70.

5.3.3 Prenatal Stress An unpredictable shock stress paradigm was applied as previously described (Hazra et al., 2012) to pregnant dams as follows. On E17-20, dams were placed in an operant conditioning chamber measuring 60 × 34 × 26 cm, with aluminum and polycarbonate walls (Lafayette Instruments, Lafayette, IN, USA). The floor of the chamber, composed of 0.4 cm-diameter stainless steel bars, conducted the electric shock. Dams in the PS group were allowed to habituate to the chamber for 5 min and then received two 8 min periods of shocks separated by an 8 min period without shocks (Figure 5.1). Each shock period consisted of 8 pseudo-randomly applied footshocks (0.5 s, 0.5 mA, inter-shock interval ranging from 30 to 90 s). Non-stressed control dams received the same handling procedures as the PS group and were placed in the shock chamber for the same duration without shocks applied. After stress exposure on E20, dams were returned to their home cage and left to give birth and rear pups normally.

5.3.4 Electrophysiology Tissue Preparation Offspring of stress-exposed and control dams were used for electrophysiological studies.

Slices were prepared from rats at various ages as previously described (Ehrlich et al., 2013).

Briefly, animals were decapitated, with those beyond P11 under isoflurane anesthesia (Fisher Scientific, Hanover Park, IL), and brains were rapidly removed and immersed in ice-cold 95%

oxygen-5% carbon dioxide-perfused “cutting solution” with the following composition (in mM):

130 NaCl, 30 NaHCO3, 3.50 KCl, 1.10 KH2PO4, 6.0 MgCl2, 1.0 CaCl2, 10 glucose, 0.4 ascorbate,

0.8 thiourea, 2.0 sodium pyruvate, and 2.0 kynurenic acid. Coronal slices containing the basolateral nucleus of the amygdala (BLA) were cut at a thickness of 300–350 μm with a Leica VTS-1000 vibrating blade microtome (Leica Microsystems, Bannockburn, IL). Slices were incubated for 1h in oxygenated cutting solution at 32°C before being transferred to regular artificial cerebrospinal fluid (ACSF) containing (in mM) 130 NaCl, 30 NaHCO3, 3.50 KCl, 1.10 KH2PO4, 1.30 MgCl2, 2.50 CaCl2, 10 glucose, 0.4 ascorbate, 0.8 thiourea, and 2.0 sodium pyruvate. Whole Cell Patch Clamp Individual slices were transferred to a recording chamber mounted on the fixed stage of a Leica DMLFS microscope (Leica Microsystems) 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, and individual BLA neurons were identified at 40x with differential interference contrast optics and infrared illumination with an infrared-sensitive CCD camera (Orca ER, Hamamatsu, Tokyo, Japan). Patch pipettes of 4–6 MΩ were pulled from borosilicate glass. Two patch electrode solutions were used, one based on potassium gluconate for current-clamp recordings and one based on cesium gluconate for voltage-clamp recordings.

The potassium gluconate patch solution had the following composition (in mM): 140 potassium gluconate, 2 KCl, 10 HEPES, 3 MgCl2, 2 K-ATP, 0.2 Na-GTP, and 5 phosphocreatine, was titrated to pH 7.3 with KOH, and was 290 mosM. The cesium gluconate patch solution had the following composition (in mM): 131 CsOH, 131 gluconate, 10 HEPES, 2 CaCl2, 10 glucose, 10 EGTA, 5 Mg-ATP, and 0.4 Na-GTP, was titrated to pH 7.3 with gluconate, and was 270 mosM.

Data acquisition was performed with a MultiClamp 700A amplifier in conjunction with pCLAMP 10.2 software and a DigiData 1322A AD/DA interface (Molecular Devices, Sunnyvale, CA). Whole cell patch-clamp recordings were obtained, low-pass filtered at 2 kHz, and digitized at 10 kHz. Cells were excluded if they did not meet the following criteria: a resting membrane potential more negative than −50 mV and drifting 5 mV over the course of the recording session; access resistance lower than 30 MΩ; stable access resistance throughout recording, changing 15%; and action potentials crossing 0 mV. Recordings were only included from BLA principal neurons, whose physiological profile we have recently characterized during development; they can be distinguished from BLA interneurons for electrophysiological recordings by a combination of their large somatic volume, low input resistance, slow action potentials, and relatively infrequent synaptic input (Ehrlich et al., 2012). In that study, we reported 58 of 60 putative principal neurons recorded in the immature BLA were found positive for mRNA for the vesicular glutamate transporter by single-cell RT-PCR. Intrinsic Properties Raw voltage and current traces were imported into Matlab (The MathWorks, Natick, MA, USA) using scripts provided with sigTOOL (http://sigtool.sourceforge.net/, developed at King's College London) and processed with customized scripts developed in the Rainnie Lab (first reported in Hazra et al., 2011). To determine input resistance and membrane time constant in current clamp, neurons were held at -60 mV with direct current injection and hyperpolarizing, 1 s-long, square-wave current steps were injected to elicit 5 mV voltage deflections. Input resistance was calculated as the ratio of peak voltage deflection to the current injected, averaged across 5 sweeps. The time constant was defined as the time necessary for the cell to reach 63.2% of its maximal deflection, averaged across 5 sweeps.

Action potential properties were measured from the responses to 5 linear ramps of depolarizing current, each lasting 250 ms and scaled to depolarize the neuron to approximately −35 mV and elicit an action potential within the final 50 ms. Action potential threshold was defined from a peak in the second derivative of the membrane potential waveform, which correlated well with visual inspection of the data. Linear interpolation between data points was used to enhance the temporal resolution of measurements of 10–90% rise time, 90–10% decay time and half-maximal width. Fast afterhyperpolarization (fAHP) peaks were measured from neurons free-firing near action potential threshold (at least 8 spikes) to remove the confound of current step artifacts, at a local minima following spike repolarization, if occurring within 15 ms of spike initiation and visibly distinct from the medium AHP.

Data on spike trains were collected from responses to depolarizing, 1 s-long, square-wave current injections with sweeps iterated at 0.1 Hz. Rheobase was measured at resting membrane potential with a series of current injections in increments of 5-20 pA depending on the age, and was defined as the magnitude of the injection of the first of two consecutive steps to elicit at least one action potential. F-I curves were generated from a series of sweeps that iterated beyond the magnitude required to elicit the maximum spike rate for each neuron, and data were excluded for current injections greater than the minimum current required to elicit the maximum spike rate. To facilitate plotting F-I curves, due to the diminishing number of cells with responses as current injection magnitude increased, data were not plotted at large current injection values for which less than half of neurons had responses at that time point. For neurons from animals at least 21 days old, neurons were excluded from plotting if they could not fire at least 30 spikes, based on previous findings (Ehrlich et al., 2012). This resulted in the exclusion of 5 of 58 neurons at P21, 28 and 60 combined (3 from control animals, 2 from PS animals). GABAergic Transmission To elicit GABAergic postsynaptic currents (PSCs), a bipolar stimulating electrode was placed in the dorsal end of the BLA, just medial to the external capsule, and the GABAA component of the response was pharmacologically isolated as previously described (Ehrlich et al., 2013). Stimulation at 0.2 Hz was applied after application of the following cocktail of synaptic blockers: the AMPA/kainate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX, 20 μM; Sigma-Aldrich), the NMDA receptor antagonist 3-(2-carboxypiperazin-4-yl)propyl-1phosphonic acid (RS-CPP, 10 μM; Tocris Bioscience, Bristol, UK), and the GABAB receptor antagonist (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl] (phenylmethyl)phosphinic acid hydrochloride (CGP52432, 2 μM; Tocris). Stimulation intensity was adjusted to elicit a half-maximal response. To verify that the isolated response was mediated purely by GABAA receptors, after some experiments the GABAA antagonist 6-imino-3-(4methoxyphenyl)-1(6H)-pyridazinebutanoic acid hydrobromide (SR95531, 5 μM; Tocris) was applied.

Due to the hyperpolarization of the GABAA reversal potential with age (Ehrlich et al., 2013), stimulation responses were recorded in voltage clamp at holding potentials of -50 and -70 mV, and the potential with the larger amplitude responses was used. Decay time constant was calculated in Clampfit from the average of 5 sweeps, from just after the peak to baseline, using a one-term exponential (Equation 1).

–  –  –

To measure short-term plasticity of GABAA PSCs, the response used to measure kinetics was elicited in trains of five pulses at 20 and 50 Hz, in five sweeps each at 0.1 Hz. Sweeps were averaged in Clampfit, and amplitudes were measured for each pulse from the 1 ms prior to the stimulation artifact to the absolute peak deflection. The ratio of the amplitudes of pulse 5 to pulse 1 was calculated for each neuron at both stimulation frequencies.

5.3.5 Quantitative RT-PCR BLA sections were harvested for PCR during the tissue preparation for electrophysiology. Bilateral BLA from a single coronal slice was microdissected and flash frozen.

Quantitative PCR was performed as previously described (Hazra et al., 2011). Total RNA was isolated by homogenizing each sample in Trizol (Invitrogen, Carlsbad, CA, USA), and isolated RNA was reverse transcribed using a cocktail of 5 µl of 10x RT buffer, 10 mM dNTP mix, 10x random hexanucleotide and Multiscribe RT 5U/µl and RNAase free water. The mixture was incubated in a thermal cycler at 25° C for 10 min and then at 37° C for 120 min, and the resulting cDNA samples were stored at -20° C. All reagents were obtained from Applied Biosystems (Foster City, CA, USA).

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