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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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1: Schematic of recording and stimulation sites. A photomicrograph of a coronal slice of medial temporal lobe, depicting a representative, filled BLA principal neuron at P14 in the target recording site. The bipolar stimulating electrode was placed medial to the external capsule (EC) within the BLA. The lateral nucleus (LA) and central nucleus (CeA) of the amygdala are also labeled. The compass gives directions for dorsal (D), ventral (V), lateral (L), and medial (M).

Figure 4.2: Maturation of the stimulation response within the BLA Figure 4.

2: Maturation of the stimulation response within the BLA. (A,B) The average waveform of the response to five stimulations (0.1 Hz) of the dorsolateral BLA is shown for representative neurons at P7, P14, P21, and P28 from recordings in current clamp (A) and voltage clamp (B). Neurons in current clamp were adjusted before stimulation to a baseline membrane potential of approximately -50 or -70 mV, and the responses are plotted on a single voltage axis.

Highlighted on the plots are the GABAA (closed arrowheads) and GABAB (open arrowheads) components of the response. Neurons in voltage clamp were recorded from holding potentials of and -70 mV, and baselines were subtracted for comparison. The GABAB component was blocked in the voltage clamp recordings to highlight the GABAA response. (C) The duration of the stimulation response in voltage clamp is plotted for each time-point as mean ± SEM.

Significance was assessed with a one-way ANOVA (F3,60 = 5.807, p 0.001) and Tukey’s posttests (* p 0.05, ** p 0.01, n = 18 (P7), 17 (P14), 18 (P21), 11 (P28)).

Figure 4.3: Development of spontaneous IPSC kinetics across the first postnatal month Figure 4.

3: Development of spontaneous IPSC kinetics across the first postnatal month. (A) The waveforms of spontaneous IPSCs are illustrated for representative neurons at P7, P14, P21, and P28, depicted as the mean (black line) of all IPSCs recorded in a 30s window, as well as the first 40 IPSCs observed in these windows (grey lines). Scale bars represent 10 pA. Mean waveforms are superimposed (bottom) for comparison. (B-F) At each time-point, mean ± SEM is plotted for IPSC 10-90% rise-time (B), decay time-constant (C), half-width (D), and peak conductance (E), as well as the coefficient of variation of the peak conductance for each neuron (F). Significance was assessed using One-way ANOVAs and Tukey’s posttests (* p 0.05, ** p 0.01, *** p 0.001), identifying a significant main effect of age on IPSC rise-time (F3,36 = 7.80, p 0.001) and decay time-constant (F3,36 = 3.45, p 0.05), but not half-width (n = 6 (P7), 16 (P14), 11 (P21), 7 (P28)). No significant effect of age was detected for peak conductance (F3,23 = 0.87, p 0.05) or coefficient of variation (F3,22 = 1.28, p 0.05; n = 5-6).

Figure 4.4: Maturation of the response to exogenous GABAA agonist Figure 4.

4: Maturation of the response to exogenous GABAA agonist. (A) BLA principal neurons at P7, P14, P21, and P28 were patch clamped with a patch electrode (‘patch’) and a microelectrode containing 100 µM muscimol (‘pico’) was brought in close proximity to the soma.

Scale bar represents 10 µm. (B) Mean responses in voltage clamp at -50 mV to picospritzer application of muscimol in a representative neuron at each time-point. (C) Responses from panel B are normalized and superimposed to highlight decay kinetics. (D, E) The decay time-constant (D) and peak conductance (E) of the muscimol response are plotted as mean ± SEM for each time-point. Significance was assessed using One-way ANOVAs and Tukey’s posttests (* p 0.05, ** p 0.01), identifying a significant main effect of age on decay time-constant (F3,53 = 5.57, p 0.01; n = 12 (P7), 16 (P14), 13 (P21), 13 (P28)) and peak conductance (F3,43 = 5.67, p 0.01; n = 10 (P7), 14 (P14), 10 (P21), 10 (P28)).

Figure 4.5: Development of GABAA receptor subunit gene expression Figure 4.

5: Development of GABAA receptor subunit gene expression. (A) The expression of mRNA for seven GABAA receptor subunits (α1, α 2, α 3, α 5, β2, β3, and γ2) are depicted for ten BLA principal neurons at P14, P21, and P28. Each row depicts the expression of each gene for a single neuron, with positive signal represented by a filled box. Ten neurons at P7 were also analyzed but are not presented because they lacked detectable expression for all genes but 18S.

Only neurons with signal for the housekeeper gene 18S rRNA were included. Gel pictures are presented for representative neurons identified by open arrowheads. (B) Gel pictures depict the expression of particular receptor subunits for one to two representative neurons at each timepoint, reassembled from gels organized by gene instead of individual neuron. (C) A gel picture showing detectable mRNA expression for all GABAA receptor subunits tested in whole-BLA from a representative animal at P7 (n = 4).

Figure 4.6: Maturation of GABAA reversal potential and chloride pump expression Figure 4.

6: Maturation of GABAA reversal potential and chloride pump expression. (A,B) Reversal potential of evoked GABAA PSPs is plotted as mean ± SEM (A) for neurons at P7 (n = 8), P14 (n = 7), P21 (n = 5), and P28 (n = 5), and the average response at three different baseline membrane potentials is plotted for a representative neuron at each time-point (B). The reversal potentials shown in (B) are specific to the individual neurons depicted. Significance was assessed with a One-way ANOVA (F3,21 = 19.91) and pairwise comparisons were made with Tukey’s posttests (***, p 0.001 vs. P14, P21, and P28). The time of stimulation is depicted with an arrow. (C) The linear fits used to estimate GABAA reversal potential for each neuron are plotted in grey for neurons at each time-point, with the average line for each group plotted in black. (D) Expression of mRNA for the chloride pumps KCC2 and NKCC1, assessed in individual BLA principal neurons using single-cell RT-PCR, is plotted as the proportion of neurons with detectable expression at P7 (n = 15), P14 (n = 20), P21 (n = 23), and P28 (n = 8).

Figure 4.7: Development of short-term synaptic plasticity of IPSCs Figure 4.

7: Development of short-term synaptic plasticity of IPSCs. (A) Short-term plasticity of GABAA IPSCs in representative neurons at P7, P14, P21, P28, and P35 are illustrated as the average response to stimulation of the dorsolateral BLA with 5 pulses at 20 Hz. Neurotransmitter receptor antagonists were used to isolate the GABAA component of the response (see Methods).

Stimulation responses were aligned to and normalized by the first pulse, and stimulation artifacts were cropped for clarity. The area under the curve was filled to aid visual comparison across time-points. (B) As in A, average, normalized IPSCs in response to 20Hz stimulation are overlaid for comparison, taken from a representative neuron at P7, P21, and P28. (C, D) Short-term plasticity was quantified for 10 Hz and 20 Hz stimulation as a ratio of pulse amplitudes. The ratios of the second pulse (early STP, C) and fifth pulse (late STP, D) to the first pulse are plotted for each time-point as mean ± SEM. Significance was assessed with Two-way ANOVAs with repeated measures, and all pair-wise comparisons were made with Bonferroni posttests. Bars above each plot illustrate the significant pair-wise comparisons for stimulation at 20 Hz (* p 0.05, ** p 0.01, *** p 0.001). No pair-wise comparisons for 10 Hz stimulation were significant. N = 3 (P7), 5 (P14), 7 (P21), 6 (P28), and 7 (P35).

Figure 4.8: Maturation of spontaneous, rhythmic IPSCs Figure 4.

8: Maturation of spontaneous, rhythmic IPSCs. (A) Representative BLA principal neurons at P7 and P28 spontaneously exhibit rhythmic, compound IPSCs. Both neurons were recorded in voltage clamp with a holding potential of -60 mV. Insets highlight the waveform of individual events. (B) Pie charts depict the proportion of neurons spontaneously receiving rhythmic IPSCs at each time-point (n = 14 at P7, 28 at P14, 25 at P21, and 16 at P28). (C) Traces from a pair of simultaneously recorded BLA principal neurons illustrate that rhythmic IPSCs are synchronized across BLA principal neurons as early as P7.

Chapter 5: The Developmental Trajectory of Amygdala Neuron Excitability and GABAergic Transmission are

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5.1 Abstract The basolateral amygdala (BLA) plays a key role in emotional processing and is implicated in a variety of psychiatric disorders, many of which have early ages of onset and high incidence in juveniles. However, the cellular processes that contribute to the etiology of developmental psychiatric disorders are largely unknown. In Chapters 2 and 3, we showed principal neurons of the BLA undergo morphological and physiological transitions between postnatal days 7 (P7) and 21, including a 3x expansion of the dendritic arbor, 10x increase in dendritic spine density, and 10x decrease in input resistance. This developmental window coincides with maturation of GABAergic transmission, including a 15mV hyperpolarization of the GABAA reversal potential and a decrease in the duration of GABAA postsynaptic currents from P14 to P21, corresponding with the emergence of the fast GABAA receptor subunit, α1. We hypothesized that an early-life risk factor for anxiety disorders, prenatal stress (PS), alters the trajectory of BLA maturation. We tested this hypothesis by exposing pregnant dams to 30 min of daily unpredictable shock stress during gestational days 17-20. Male offspring of PS and control dams were sacrificed for whole-cell patch clamp studies at P10, 14, 17, 21, 28 & 60 to characterize the developmental trajectory of intrinsic membrane properties and synaptic transmission in BLA principal neurons. In adulthood, PS had an anxiolytic effect, as measured in the elevated plus-maze, and reduced sociability in the social choice test. Whole-cell patch clamp studies revealed no impact of PS on input resistance or membrane time-constant at any age.

However, PS reduced neuronal excitability in several ways. PS caused an increase in the rheobase that began to emerge at P28 (2-way ANOVA, p 0.01; N = 9-18); rheobase in PS neurons at P28 and P60 was 186 ± 21 and 323 ± 32 pA (mean ± SEM), respectively, compared to 170 ±19 and 176 ± 18 pA in controls. PS significantly increased action potential amplitude across all timepoints (2-way ANOVA, p 0.05; N = 10-19) including ~10mV increase at P17. In addition, neurons from PS animals exhibited slower GABAA receptor-mediated currents (2-way ANOVA, P 0.01) at P14 (decay time constant = 15.7 ± 1.9 ms, N = 20) compared to controls (9.8 ± 1.1 ms, N = 11). Finally, PS animals exhibited reduced expression in the BLA of the GABAA receptor α1 subunit, which plays a key role in coordinating developmental critical periods, as early as P17 and persisting into adulthood. We identified a number of effects of PS on the immature BLA that influence amygdala function during emotional development. These changes likely underlie the reduction in emotionality and sociability caused by PS, and may contribute to the etiology of psychiatric disorders, including autism and schizophrenia.

5.2 Introduction Autism spectrum disorders (ASDs) and schizophrenia (SZ) are neurodevelopmental disorders characterized by deficits in social interaction and emotional behavior, and these disorders are thought to involve pathophysiology in parts of the brain that mediate socioemotional processing, including the amygdala (Baron-Cohen et al., 2000; Sweeten et al., 2002; Aleman and Kahn, 2005; Schultz, 2005; Shayegan and Stahl, 2005; Bachevalier and Loveland, 2006; Amaral et al., 2008; Benes, 2010; Bellani et al., 2013; Tottenham et al., 2013). While the specific causes of ASDs and SZ are likely distinct and include both genetic and environmental factors (State and Levitt, 2011; Geoffroy et al., 2013; van Dongen and Boomsma, 2013), both disorders share the risk factor of prenatal stress (PS; Koenig et al., 2002; Beversdorf et al., 2005; Khashan et al., 2008; Kinney et al., 2008; Ronald et al., 2010). Stress early in life is known to alter amygdala maturation (Ono et al., 2008; Moriceau et al., 2009; Maheu et al., 2010; Tottenham et al., 2011), and early life deficits in amygdala dysfunction could mediate the contribution of PS to these neurodevelopmental disorders.

The contribution of PS to ASD and SZ etiology may be mediated by changes to the coordination of amygdala development by the neurotransmitter GABA. Neurodevelopmental disorders are thought to follow from early life deficits in inhibitory GABA systems in the brain, because GABAergic neurotransmission plays an organizing role in nervous system development (Ramamoorthi and Lin, 2011; Sgado et al., 2011; Chattopadhyaya and Cristo, 2012; King et al., 2013). In the immature brain, GABAergic transmission regulates cell proliferation, migration and differentiation, synapse maturation and stabilization, and circuit wiring (Owens and Kriegstein, 2002; Huang and Scheiffele, 2008; Le Magueresse and Monyer, 2013). Furthermore, activation of GABA receptors control the timing of developmental critical periods, windows of heightened plasticity and sensitivity to external stimuli (Hensch, 2005); specifically, activation of GABAA receptors containing the α1 subunit closes critical periods (Fagiolini et al., 2004), which coincides with the emergence of parvalbumin-expressing (PV+) interneurons that preferentially innervate these receptors (Nusser et al., 1996; Fritschy et al., 1998; Pawelzik et al., 1999; Klausberger et al., 2002; Hensch, 2005; Nowicka et al., 2009). Therefore, if PS alters the development of GABAergic transmission and the expression of GABAA receptor α1 subunit and PV+ interneurons in the amygdala, it may thereby influence critical periods to induce broad changes to emotional brain circuits. This mechanism has been broadly hypothesized for both SZ and ASD (Di Cristo, 2007; LeBlanc and Fagiolini, 2011; Volk and Lewis, 2013). Expression of the α1 subunit is reduced by 40% in cortex of human subjects with SZ (Glausier and Lewis, 2011), and genetic variation in GABAA receptor subunits has been associated with autism (Ma et al., 2005).

In addition, dysfunction of PV+ interneurons is proposed to underlie the deficits observed in SZ (Curley and Lewis, 2012).

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