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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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The changes we observed in HCN transcript expression are likely to impact the function of principal neurons in the maturing BLA. Ih is known to contribute to input resistance at rest (Surges et al., 2004), oscillatory properties of neurons including resonance (Hutcheon et al., 1996a; Hu et al., 2002; Marcelin et al., 2012), and synaptic integration in dendrites (Atkinson and Williams, 2009). Interestingly, the first postnatal month also includes a dramatic shift in the intrinsic resonance frequency of BLA principal neurons towards higher frequencies that could engage HCN channels with faster kinetics (Ehrlich et al., 2012). Resonance to higher frequencies can also be promoted by a reduction of input resistance, which is likely promoted by the expansion of the dendritic arbor and increase in soma volume across the first postnatal month.

HCN1 and 2 have been found in distal dendrites of adult pyramidal neurons (Notomi and Shigemoto, 2004), and HCN1 channels have been shown to impact synaptic transmission and plasticity in distal dendrites (Nolan et al., 2004). Interestingly, the expression of this subtype is lacking when dendrites are shorter early in development.

The KIR2 subfamily of inwardly rectifying potassium channels also pass current at voltages hyperpolarized to rest. Consistent with previous observation (Hibino et al., 2010), expression of these channel subunits, which form homo- and hetero-tetramers, was limited to KIR2.1 and 2.3 in BLA principal neurons. KIR2.3 mRNA expression was absent at P7, but otherwise KIR2.1 and 2.3 were expressed throughout postnatal development. In addition to their role regulating resting membrane potential and excitability of neurons (Isomoto et al., 1997;

Hibino et al., 2010), KIR2.1 and 2.3 are also found localized to the postsynaptic density in dendritic spines through an interaction with the scaffolding protein, PSD-95, where they regulate local resting membrane potential (Inanobe et al., 2002). This interaction seems likely to occur in the amygdala, considering the concomitant emergence of KV2.3 mRNA and dendritic spines.

Notably, KV2.3 becomes dissociated from PSD-95 following phosphorylation by protein kinase A (Cohen et al., 1996), which is activated in the BLA during stimulus-induced synaptic plasticity and may provide a mechanism for disinhibition of spines.

Similarly, the subunits for the tetrameric potassium channel mediating the A-type current also exhibited a distinct developmental trajectory. KV1.4 subunit mRNA was detected in BLA principal neurons at P7 and 14, but was lost by P21, whereas KV3.4 had an opposite profile of expression, only emerging at P21. Conversely, no significant change in mRNA expression was observed for KV4.1, 4.2, and 4.3 transcripts at any time-point. In the adult BLA, a previous in situ hybridization study reported stronger expression of mRNA transcripts for KV4.3 than 4.1 or 4.2 (Serodio and Rudy, 1998), but the study did not examine the expression profile across development. However, protein expression of KV4 subunits has been shown to increase in the BLA gradually with age until P30, then increase drastically by P60, suggesting development of these channels may continue beyond the window studied here (Vacher et al., 2006). Similarly, in pyramidal neurons of rat somatosensory cortex, protein expression of KV4.1, 4.2, and 4.3 increases across the first postnatal month as A-type potassium current increases in amplitude (Guan et al., 2011). While our results would predict a decrease in the contribution of KV1.4 to the A-current in BLA principal neurons with age, Guan and colleagues reported this subunit contributes proportionally to the A-current throughout the first postnatal month.

The loss of KV1.4 and emergence of KV3.4 during the first postnatal month should impact the electrical properties of these neurons, considering the functional differences of the two channels. Specifically, KV3.4 has more depolarized activation and inactivation curves than KV1.4 (Chandy et al., 2012b, a), suggesting more mature BLA principal neurons would be less likely to activate A-current at voltages below action potential threshold, which hyperpolarizes with age (Ehrlich et al., 2012). In addition, KV3.4 inactivates nearly five times faster than KV1.4 (Chandy et al., 2012b, a), which would potentially contribute to the faster action potential repolarization, larger spike after-hyperpolarization, and higher frequency action potential trains observed later in the first postnatal month (Ehrlich et al., 2012). Interestingly, KV1.4 is targeted to axons in central neurons, suggesting the loss of this subunit with age may preferentially affect axon function or synaptic transmission (Sheng et al., 1992).

Classically opposed to the A-current is the calcium-mediated T-current, a low-voltageactivated current that is conducted by the CaV3 family of ion channels (Pape et al., 1994;

Molineux et al., 2005; Hammack et al., 2007). Here we report a developmental shift in the CaV3 channel subunit composition, with mRNA transcripts for CaV3.1 expressed in almost all BLA principal neurons at P7 and P14. By P21, however, few neurons expressed mRNA transcripts for this subunit, and neurons began to express transcripts for CaV3.2. In situ hybridization studies in the adult rat brain show strong labeling for both transcripts in the BLA, as well as for CaV3.3 (Talley et al., 1999). Interestingly, the signal was strongest for the CaV3.1 subunit, suggesting other types of neurons in the BLA may prominently express this subunit. This discrepancy reinforces the need to assess levels of protein expression for the various subunits.





The T-current plays a variety of roles in neuronal function. Importantly, T-type calcium channels can be distributed throughout the dendritic arbor, where they contribute to dendritic calcium flux and back-propagation of action potentials (Errington et al., 2010). T-current and Hcurrent in adult BLA principal neurons are critically involved in the generation of membrane potential oscillations that emerge during the first postnatal month (Chapters 2 and 6; Ehrlich et al., 2012; Ryan et al., 2012), and the shift from CaV3.1 to 3.2 may play a role. As mentioned above, T-type and A-type channels are classically opposed, and CaV3.2 has been shown to form a complex with KV4.2 channels that allows the calcium influx from the T-current to modulate the activity of KV4.2 channels (Anderson et al., 2010).

Here we have shown how the morphology and ion channel expression of BLA principal neurons mature radically across the first two postnatal months. We also discussed these changes in the context of concurrent, substantial changes to neuronal physiology, animal behavior, and synaptic plasticity. Together, these findings clearly show that the first postnatal month of rodent development is a critical period in the development of brain regions that process emotion.

However, while morphology and intrinsic electrophysiology influence neuronal function, the functional output of the amygdala, and therefore its contribution to emotional behavior, relies on synaptic transmission. Various neurotransmitters regulate the activity of BLA principal neurons and influence the maturation of neural circuits. In Chapter 4, we describe a number of developmental changes to the function in the BLA of GABA, a neurotransmitter that regulates amygdala excitability and the expression of fear and anxiety, and, most importantly, influences the timing of neural circuit development. Due to its role coordinating processes in brain development, changes to GABAergic transmission in the developing BLA may act upstream of the maturation of morphology and intrinsic physiology. Therefore, understanding the properties of the GABA system in the developing BLA will be necessary for understanding how early life experience can shape emotional development.

Table 3.1: PCR Primers used for single cell RT-PCR

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Figure 3.1: Soma and dendrites of BLA principal neurons grow during the first postnatal month Figure 3.

1: Soma and dendrites of BLA principal neurons grow during the first postnatal month. Reconstructions of representative, biocytin-filled BLA principal neurons at postnatal days 7 (P7), 14, 21, 28, and 60.

Figure 3.2: Soma size increases across the first postnatal month, then decreases in adulthood Figure 3.

2: Soma size increases across the first postnatal month, then decreases in adulthood. (A, B) Soma volume (A) and surface area (B) of BLA principal neurons are depicted as mean ± SEM at postnatal days 7 (P7), 14, 21 and 28. At P7, P14, P21 and P60, n = 7; at P28, n = 6. * P 0.05; ** P 0.01. The results of Tukey’s tests following one-way ANOVA are depicted in grids (* P 0.05; ** P 0.01; *** P 0.001; NS: not significant).

Figure 3.3: Dendritic arbors expand with a specific pattern across postnatal development Figure 3.

3: Dendritic arbors expand with a specific pattern across postnatal development.

Sholl analysis was performed on reconstructed neurons with 20 µm steps between rings. (A, B) Aggregate length (A) and the critical value for length (B) of the dendritic arbor are plotted versus age as mean ± SEM, with a best-fit sigmioidal Boltzmann curve. The results of Tukey’s tests following two-way ANOVA are depicted in inset grids (* P 0.05; ** P 0.01; *** P 0.001;

NS: not significant; n = 8 per time-point). (C) Percentage of total dendritic length found in branches of a given order is plotted as mean ± SEM for each age (n = 8 neurons per age). (D) The profile of dendritic length derived from the Sholl analysis is plotted as mean (black line) and SEM (grey band) versus distance from the center of the soma, illustrating the expansion and pruning of the dendritic arbor with age (n = 8).

Figure 3.4: Dendritic branch points become more distant from the soma during postnatal development Figure 3.

4: Dendritic branch points become more distant from the soma during postnatal development. (A, B) Bar graphs illustrate the mean ± SD of the total number of primary dendrites (A) and branch points (B) for BLA principal neurons at each time-point (n = 8). (C) The proximity of branch points to the soma was derived from Sholl analyses on reconstructed neurons with 40 µm thick rings, and the proportion of total branch points found in each ring is plotted as mean ± SEM (n = 8). The results of Tukey’s tests following two-way ANOVA are depicted in inset grids for Sholl rings with significant effects of age, namely those centered at 20, 100, and 140 µm from the soma (* P 0.05; ** P 0.01; *** P 0.001; NS: not significant).

Figure 3.5: Dendritic spines emerge the first month of postnatal development in BLA principal neurons Figure 3.

5: Dendritic spines emerge the first month of postnatal development in BLA principal neurons. (A) Representative photomicrographs of segments of dendrites from filled BLA principal neurons at each time-point. (B,C) Dendritic spine density and the estimated total number of spines are plotted as mean ± SEM for neurons at each time-point (n = 6). The results of Tukey’s tests following one-way ANOVA are depicted in inset grids (* P 0.05; ** P 0.01;

*** P 0.001; NS: not significant).

Figure 3.6: Ion channel transcript expression in individual BLA principal neurons matures across the first postnatal month Figure 3.

6: Ion channel transcript expression in individual BLA principal neurons matures across the first postnatal month. Single-cell RT-PCR results for BLA principal neurons are depicted for 11 to 12 neurons at each time-point, with filled blocks signifying detectable expression for a given transcript. The results for all genes from a single neuron are depicted in a single row, with varying shades of grey for contrast. Representative gel images are included at each age to illustrate the expression profile of a single neuron, indicated with a grey arrow.

Chapter 4: Postnatal maturation of GABAergic transmission in the rat

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Adapted from: Ehrlich DE, Ryan SJ, Hazra R, Guo JD, Rainnie DG (2013). Postnatal maturation of GABAergic transmission in the rat basolateral amygdala. J Neurophysiol 110(4): 926-41.

4.1 Abstract Many psychiatric disorders, including anxiety and autism spectrum disorders, have early ages of onset and high incidence in juveniles. To better treat and prevent these disorders, it is important to first understand normal development of brain circuits that process emotion. Healthy and maladaptive emotional processing involve the basolateral amygdala (BLA), dysfunction of which has been implicated in numerous psychiatric disorders. Normal function of the adult BLA relies on a fine balance of glutamatergic excitation and GABAergic inhibition. Elsewhere in the brain GABAergic transmission changes throughout development, but little is known about the maturation of GABAergic transmission in the BLA. Here we used whole cell patch-clamp recording and single-cell RT-PCR to study GABAergic transmission in rat BLA principal neurons at postnatal day (P)7, P14, P21, P28, and P35. GABAA currents exhibited a significant twofold reduction in rise time and nearly 25% reduction in decay time constant between P7 and P28. This corresponded with a shift in expression of GABAA receptor subunit mRNA from the α2- to the α1-subunit. The reversal potential for GABAA receptors transitioned from depolarizing to hyperpolarizing with age, from around −55 mV at P7 to −70 mV by P21. There was a corresponding shift in expression of opposing chloride pumps that influence the reversal, from NKCC1 to KCC2. Finally, we observed short-term depression of GABAA postsynaptic currents in immature neurons that was significantly and gradually abolished by P28. These findings reveal that in the developing BLA GABAergic transmission is highly dynamic, reaching maturity at the end of the first postnatal month.

4.2 Introduction There is a critical need to characterize the developmental trajectory of the BLA and brain circuits that process emotion. Previously we have shown that the electrophysiological and morphological properties of neurons in the BLA change dramatically over the first postnatal month {Chapters 2 and 3`; \Ehrlich, 2012 #1297}. Here we focused on inhibitory synaptic transmission, which has been shown to be developmentally regulated (Ben-Ari et al., 2012; Kilb,

2012) and implicated in the pathophysiology of neurodevelopmental disorders (Chattopadhyaya and Cristo, 2012; King et al., 2013).



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