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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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Inhibitory synaptic transmission has been well characterized in the mature BLA (Rainnie et al., 1991a; Washburn and Moises, 1992; Martina et al., 2001), playing a fundamental role in determining the excitability of the region and thereby regulating emotional behavior (Quirk and Gehlert, 2003; Shekhar et al., 2003; Ehrlich et al., 2009). Synaptic inhibition in the BLA is produced by intrinsic and extrinsic afferents activating local-circuit interneurons; these inhibitory neurons then release the neurotransmitter γ-aminobutyric acid (GABA) onto principal neurons, which comprise 80–85% of the BLA neuronal population (McDonald, 1985, 1996). Blocking GABAA receptors in the BLA promotes fearful and anxious behaviors (for review, see Quirk and Gehlert, 2003), whereas enhancing GABA function attenuates these behaviors (Davis et al., 1994;

Sanders and Shekhar, 1995). GABAergic transmission in the BLA can also organize network activity; rhythmic inhibition of BLA principal neurons by burst-firing, parvalbumin-expressing interneurons can coordinate and synchronize firing of the principal neurons and may underlie network oscillations in the BLA related to fear (Chapter 6; Ryan et al., 2012).

In addition to regulating neuronal activity in the adult brain, GABAA receptors undergo functional maturation and serve to coordinate brain development. One well-established trigger for the onset of “critical periods,” or developmental windows of high plasticity, is activity at GABAA receptors (Hensch, 2005). This action depends on the subunit composition of GABAA receptors, which are comprised of five subunits and typically contain two α-, two β-, and one γ-subunit. There are six known α-subunits, and the relative expression of these subunits changes throughout development. The α-subunits influence the kinetics, localization, and drug sensitivity of GABAA receptors (Nusser et al., 1996; Hevers and Luddens, 2002). As the brain develops, expression shifts from high levels of the α2-subunit toward the α1-subunit, which confers faster kinetics (Hornung and Fritschy, 1996; Dunning et al., 1999; Davis et al., 2000;

Bosman et al., 2002; Mohler et al., 2004; Eyre et al., 2012).

GABAA receptor function in the brain changes in another fundamental way, switching from excitatory at birth to inhibitory in adulthood (for review, see Ben-Ari et al., 2012). This switch is believed to result from developmental changes to the concentration gradient of chloride, the ion mediating GABAA currents. At birth, there is greater expression of sodium-potassiumchloride cotransporter 1 (NKCC1), which accumulates intracellular chloride and renders GABAA receptors excitatory. In adulthood, potassium-chloride cotransporter 2 (KCC2) is expressed more highly, extruding chloride from the cell and rendering GABAA receptors inhibitory (Ben-Ari et al., 2012). Excitatory GABA early in development is thought to promote calcium influx and modulate neuronal growth and synapse formation (Ben-Ari et al., 1997).

Aside from the precedent for GABAergic maturation observed elsewhere in the brain, there are good reasons to expect that similar changes occur in the developing BLA. For example, we recently showed that the electrophysiological properties of BLA principal neurons mature rapidly from postnatal day (P)7 to P21, including a 10-fold reduction in input resistance and a 3fold reduction in membrane time constant, suggesting that the sensitivity to synaptic inhibition would also vary greatly (Ehrlich et al., 2012). There are also concurrent changes in the expression and connectivity of GABAergic interneurons in the BLA, including the emergence and maturation of parvalbumin-expressing interneurons between P14 and P30 (Berdel and Morys, 2000; Davila et al., 2008). Between P14 and P20, there is a significant increase in the density of GABAergic fibers and a decrease in the density of GABAergic cell bodies in the BLA (Brummelte et al., 2007). Further evidence comes from the development of behaviors related to BLA function. There is a switch from paradoxical approach to an aversively conditioned stimulus to the mature, avoidance behavior at P10, corresponding with a change in synaptic plasticity that is reversed by GABAA blockade (Sullivan et al., 2000; Thompson et al., 2008). In addition, rats exhibit suppression of fear learned at P18 but not P23, a phenomenon called infantile amnesia that is GABAA receptor dependent (Kim et al., 2006b). The mechanisms of fear extinction also change in this window, becoming amygdala- and GABA dependent between P17 and P24 (Kim and Richardson, 2008). There are several additional examples of rapid, developmental changes to the expression and underlying physiology of fear learning (Campbell and Ampuero, 1985; Moye and Rudy, 1987; Hunt et al., 1994; Tang et al., 2007).

Despite the variety of documented changes to the BLA circuit and emotional behavior across the first postnatal month, and the critical role that synaptic inhibition plays in the function of the adult amygdala, no study to date has examined the developmental profile of GABAergic transmission in the immature BLA. To address this knowledge gap, we have used a combination of patch-clamp electrophysiology and single-cell reverse transcription-polymerase chain reaction (RT-PCR) to characterize the properties of synaptic inhibition of BLA principal neurons across the first postnatal month. Here we outline significant changes in terms of the kinetics, reversal potential, and short-term synaptic plasticity (STP) of GABAA receptor activation as well as underlying changes in gene expression. In this study we characterized normative amygdala development to enable future studies to address the contribution of the amygdala to both healthy and maladaptive emotional development.

4.3 Methods 4.3.1 Ethical approval.

All experimental protocols strictly conform to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Emory University.

4.3.2 Animals.

Rats born in-house to time-mated female Sprague-Dawley rats (Charles River, Wilmington, MA) were used in all experiments. Pups were housed with the dam prior to weaning on P22 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 were killed for electrophysiological recordings at P7–10, P13–15, P20–22, P27–29, and P33–36, and for brevity these windows are described as single time points. To maximize the use of animals, experiments on GABAA reversal and for single-cell RT-PCR included data from the offspring of dams used as negative control subjects for other studies. In these cases, data were grouped because there was no observable difference in standard-raised animals and those born from dams receiving either manipulation.

4.3.3 Slice preparation.

Slices containing the BLA were obtained as previously described (Rainnie, 1999b).

Briefly, animals were decapitated under isoflurane anesthesia (Fisher Scientific, Hanover Park, IL) if older than P11, and the 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 BLA were cut at a thickness of 300–350 μm with a Leica VTS-1000 vibrating blade microtome (Leica Microsystems, Bannockburn, IL). Slices were kept in oxygenated cutting solution at 32°C for 1 h before transfer 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.

4.3.4 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 ×10 magnification. Individual BLA neurons were identified at ×40 with differential interference contrast optics and infrared illumination with an infrared-sensitive CCD camera (Orca ER, Hamamatsu, Tokyo, Japan). Patch pipettes were pulled from borosilicate glass and had a resistance of 4–6 MΩ. We used two patch electrode solutions, 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. To visualize the recording sites of some neurons, 0.35% biocytin (Sigma-Aldrich, St.

Louis, MO) was added to the patch solution and tissue was stained as previously described (Rainnie et al., 2006) and imaged at ×5 magnification on a Leica DM5500B microscope (Leica Microsystems) equipped with a CSU10B Spinning Disk (Yokagawa Electronic, Tokyo, Japan).

Data acquisition was performed with a MultiClamp 700A or Axopatch 1D 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 −55 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, which 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 low synaptic input (Rainnie et al., 1993).

Furthermore, we previously reported that 58 of 60 putative principal neurons recorded in the immature BLA were found positive for mRNA for the vesicular glutamate transporter by singlecell RT-PCR (Ehrlich et al., 2012).

4.3.5 Spontaneous inhibitory postsynaptic currents.

To quantify changes in the kinetics of GABAA inhibitory postsynaptic currents (IPSCs) in developing BLA principal neurons, spontaneous IPSCs were measured from 30-s-long recordings in voltage-clamp mode with a cesium gluconate-based patch solution. Outward synaptic currents were measured at −50 mV, unless discernible outward currents were observed at −60 mV. Neurons were only included in this analysis if their outward synaptic currents exhibited a reversal potential below −50 mV. Synaptic currents were selected by hand by a blinded experimenter from traces low-pass filtered at 500 Hz, and kinetics were analyzed off-line with

Mini Analysis 6.0.3 (Synaptosoft, Decatur, GA). Event detection parameters were as follows:

time before a peak for baseline (7.5 ms), period to search a decay time (50 ms), fraction of peak to find a decay time (0.368), and period to average a baseline (5 ms). Ten to ninety percent rise time and decay time constant were measured automatically based on the detected baseline and peak, using the time to reach 0.1 and 0.9 of the peak on the rising phase and 0.368 of the peak on the falling phase. An individual IPSC was excluded from analysis if its detected rise time was longer than its decay time, and a neuron was excluded if it had fewer than five IPSCs for analysis.

For illustration, IPSC waveforms were temporally aligned by rise time in MiniAnalysis and then smoothed with a sliding window of 2-ms width and averaged with MATLAB (The MathWorks, Natick, MA).

4.3.6 Stimulation-evoked postsynaptic potentials and currents.

To measure the duration of the network response and the reversal potential and STP of GABAA receptor-mediated events in BLA principal neurons, a bipolar stimulating electrode was placed in the dorsal end of the BLA, just medial to the external capsule (Figure 4.1). The initial, half-maximal response was recorded in current clamp when using potassium-based patch solution and voltage clamp when using cesium-based patch solution. Durations of the voltage-clamp responses were measured with Clampfit 10.2 (Molecular Devices) by an experimenter blind to postnatal age, from the stimulation artifact to the return to resting membrane potential, and the duration was averaged for the responses at −50, −60, and −70 mV.

To isolate from the initial response monosynaptic GABAA postsynaptic currents (PSCs) and potentials (PSPs), stimulation at 0.2 Hz was applied after application of a cocktail of synaptic blockers, including the AMPA/kainate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX, 20 μM; Sigma-Aldrich), the NMDA receptor antagonist 3-(2-carboxypiperazin-4yl)propyl-1-phosphonic 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). Placement of the stimulating electrode medial to the external capsule was often required to elicit a response in the presence of glutamatergic synaptic blockers. Stimulation intensity was adjusted to elicit a halfmaximal response after application of blockers. To verify that the isolated response was purely GABAA, some experiments culminated with the application of the GABAA antagonist 6-imino-3methoxyphenyl)-1(6H)-pyridazinebutanoic acid hydrobromide (SR95531, 5 μM; Tocris).

Reversal potential was estimated from GABAA PSPs evoked in neurons at least 15 min after patching with the potassium gluconate-based solution. Reversal potential was interpolated from stimulation responses in neurons adjusted with direct current injection to baseline recording potentials spanning the reversal, including three of the following: approximately −50, −60, −70, and −80 mV. The average response of five sweeps at each baseline potential was used for calculation with Clampfit.

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