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3.5.1 Somatic Development The most basic metrics of morphological development we examined were somatic volume and surface area. Values for both measures increased dramatically from P7 to P28, with soma volume nearly doubling in this window. From P28 to P60, however, some volume and surface area decreased back to the values observed at P7. Our measurements of adult soma size are consistent with previous reports in rat amygdala (Chareyron et al., 2012) and somatosensory cortex (Romand et al., 2011). An early increase in soma size with a subsequent decrease has been observed in other brain regions in the rat (Vidal et al., 2004; Liao and Lee, 2012) and gerbil (Gleich and Strutz, 2002), while less expansive studies in rat motoneurons and cerebellar granule cells have corroborated the late decrease in soma size, suggesting a general principle of neuron development (Monteiro et al., 1998; Gleich and Strutz, 2002; Monteiro et al., 2005). In contrast, no significant effect of postnatal age was found on soma size in the macaque amygdala, although there was a peak during infancy (Chareyron et al., 2012). Nevertheless, we have shown that in rat BLA principal neurons the increase in soma size precedes the major period of dendritic outgrowth, while the soma shrinks once dendritic growth has ceased; this suggests the soma may enlarge specifically during a period of dendritic outgrowth, potentially to produce proteins or support the assembly of microtubules for dendritic growth (Baas and Lin, 2011). Interestingly, the soma is a key site for synaptic contacts from inhibitory basket cells, including parvalbuminexpressing interneurons, which increase in expression in the BLA until P30, when principal neurons reach a peak in soma surface area (Berdel and Morys, 2000).

3.5.2 Dendritic Morphology Our data suggests almost all growth of the dendritic arbor of BLA principal neurons occurs by P28, with a large proportion occurring between P7 and P21. While the aggregate length of dendritic material reaches the mature value around P21, Sholl analyses suggest the dendritic arbor is still remodeled beyond this time-point, with dendrites retracting from P21 to P28. We also used a Sholl analysis to compare the distribution of branch points throughout development.

Interestingly, the proportion of dendritic length for each branch order was comparable across ages, suggesting there is an optimal distribution of branch points in the dendritic arbor that is maintained through development. The total number of branch points was consistent at every age, as was the number of primary dendrites. Our Sholl analysis further revealed a shift of branch points more distally with age. While P7 neurons have nearly two-thirds of their dendritic branch points within 40 µm of the soma, for P60 the proportion is below one-third. On the other hand, at P60 nearly one-sixth of branch points are found between 120 and 160 µm from the soma, a much greater proportion than at any earlier time-point. The net effect of these changes is that the critical value is found gradually farther from the soma until P28. Maturation of the dendritic arbor is dependent on the excitatory actions of GABA early in development, and loss of excitatory GABA in development severely limits dendritic arborization and complexity (Cancedda et al., 2007). We have reported that GABAergic transmission onto BLA principal neurons is excitatory at P7 but switches to inhibitory by P14, suggesting the first two postnatal weeks represent a critical period for dendrite maturation (Ehrlich et al., 2013). Furthermore, activation of GABAB receptors early in development has been shown to promote dendrite outgrowth, and we have previously demonstrated large GABAB responses in P7 and P14 BLA principal neurons that diminish by P21, when dendrite expansion ends (Bony et al., 2013; Ehrlich et al., 2013).

The developmental trajectory of dendritic arbor morphology we report here is corroborated by previous studies in the BLA and developmental studies of pyramidal neurons in other brain regions. A previous study of BLA principal neurons in adult rats found a comparable spatial distribution of dendrites to our P60 time-point using a traditional Sholl analysis (Yajeya et al., 1997). A morphological analysis of developing layer V pyramidal neurons in somatosensory cortex by Romand and colleagues revealed a very similar growth pattern in the distribution of dendritic material, although their Sholl curves were notably broader, likely due to larger aggregate dendritic lengths (2011). Stereological studies in the rat have also shown an increase in the volume of the rat BLA between P7 and P21, consistent with an expansion of dendritic arbors of BLA neurons during this period (Chareyron et al., 2012). Furthermore, a Golgi-Cox study of developing BLA neurons, which reported the area encompassed by the dendritic arbor, found a similar expansion during the first few postnatal weeks, albeit with substantially smaller arbors (Escobar and Salas, 1993). Notably, previous Golgi-Cox studies of adult BLA neurons have estimated the aggregate dendritic length between 300 and 2000 µm (Tosevski et al., 2002;

Johnson et al., 2009; Pillai et al., 2012; Torres-Garcia et al., 2012), which differs greatly from our measurement of ~6,400 microns at P60. We argue that the Golgi-Cox technique provides underestimates of dendritic length, possibly by selectively sampling smaller neurons or staining only proximal dendritic segments. However, it is possible we are overestimating the dendritic length due to bias in the visual selection of neurons for patch clamp.

The growth of the dendritic arbor in both quantity and complexity has substantial implications for neuronal physiology, particularly passive electrical properties. The increase in neuronal surface area across the first postnatal month undoubtedly contributes to the concurrent, nearly ten-fold decrease in input resistance and three-fold decrease in membrane time constant we previously reported in this cell population (Ehrlich et al., 2012). Furthermore, multi-compartment modeling has revealed that expansion of the dendritic arbor can promote the expression of doublets of action potentials, driven by depolarization of the soma due to a dendritic spike (Mainen and Sejnowski, 1996). In our hands, the expansion of the dendritic arbor of BLA principal neurons during the first postnatal month reported here does in fact correspond with the emergence of doublets (Ehrlich et al., 2012). In addition, expansion of the dendritic arbor has the potential to effectively increase the diversity of presynaptic partners or sensory modalities of input for a BLA principal neuron, due to the topographical organization of sensory input to the BLA (McDonald, 1998). Interestingly, these inputs also undergo developmental change; tract tracing studies have demonstrated thalamic afferents are present in the BLA at P7 and remain relatively unchanged with age, while cortical afferents continue to mature throughout the first postnatal month (Bouwmeester et al., 2002b). It will be critical for future studies to address the sensitivity of this developmental trajectory to experience, considering the well documented effects of stressors on the dendritic arborization of principal neurons in the adult BLA (Roozendaal et al., 2009; Padival et al., 2013) and in the case of autism spectrum disorders and Fragile X syndrome (Kaufmann and Moser, 2000; Beckel-Mitchener and Greenough, 2004;

Puram et al., 2011).

3.5.3 Dendritic Spine Emergence As the dendritic arbor expands throughout the first postnatal month, BLA principal neurons come to express many more dendritic spines. We observed a progressive increase in the density of dendritic spines between P7 and P28, by which time spines are as dense as in adulthood (~1.2 spines/µm at P60). Comparable studies examining the development of dendritic spines in other brain regions have reported similar spine densities and developmental trajectories.

For example, the spine density of layer V neurons in somatosensory cortex stabilizes around P21 at ~0.6 spines/µm (Romand et al., 2011). Previous measurements of spine density in the BLA have yielded values slightly lower than ours, ~0.7 spines/µm in late-adolescence (Torres-Garcia et al., 2012). This discrepancy may be because this study utilized the Golgi-Cox staining method, which, as discussed above, may be biased towards proximal dendrites.

Interestingly, previous studies of synapse formation in the developing BLA, measured by synaptophysin staining, show the number of presynaptic terminals reaches a peak at P14 (Morys et al., 1998), while our data show that dendritic spines reach about half their mature density at this age. Although synaptophysin is not specific for afferents of principal neurons or those targeting dendritic spines, this mismatch in synaptophysin and spine development suggests during the first few postnatal weeks glutamatergic presynaptic terminals may form synapses with dendritic shafts or release transmitter without direct synaptic contact. The emergence of dendritic spines corresponds with the age when glutamate removal from synapses switches from primarily diffusion-based to uptake-dependent (Thomas et al., 2011). The early peak of synaptophysin expression may indicate an increase in glutamatergic transmission that could trigger the outgrowth of dendritic spines (Calabrese et al., 2006). In support of this notion, tract-tracing studies have shown that putative glutamatergic inputs to the BLA mature between P7 and P13 (Bouwmeester et al., 2002b) and stabilize by P25, before undergoing pruning in late adolescence (Cressman et al., 2010). Dopaminergic and noradrenergic inputs to the BLA, which largely target spine shafts and heads on distal dendrites (Muller et al., 2009; Muly et al., 2009; Zhang et al., 2013), become more dense between P14 and P20 (Brummelte and Teuchert-Noodt, 2006). Our own previous work demonstrates the presence of stimulation-evoked and spontaneous glutamatergic transmission onto BLA principal neurons as early as P7 (Ehrlich et al., 2013), when very few spines are present.

The emergence of dendritic spines in BLA principal neurons has numerous potential implications for neurotransmission in the amygdala. Glutamatergic afferents to the BLA are thought to provide representations for sensory stimuli that are critical to amygdala function, including noxious and neutral stimuli that undergo plasticity during associative fear learning (Rodrigues et al., 2004; Maren, 2005; Pape and Pare, 2010). Dendritic spines provide a means of compartmentalization of biochemical and electrical signals related to neurotransmission (Shepherd, 1996; Lee et al., 2012), meaning the lack of spines early in development should impact the specificity of synaptic plasticity. Coincidentally, during the same window when spines emerge and reach mature numbers, there is increased abundance in BLA synaptic terminals of zinc, which promotes long-term potentiation of glutamategic synapses in the BLA (Mizukawa et al., 1989; Li et al., 2011). Interestingly, juvenile mice exhibit generalization of conditioned fear, which could be related to poor specificity of synaptic plasticity (Ito et al., 2009). During infancy, rats also exhibit deficits to fear learning, and many forms of associative emotional learning emerge during the first few postnatal weeks (for review, see King et al., 2013). Perhaps most interesting is the observation that the amygdala is activated by odor-shock pairing after but not before P10, corresponding with the emergence of aversive conditioning and a change in amygdala synaptic plasticity (Sullivan et al., 2000; Thompson et al., 2008), precisely when dendritic spines begin to emerge.

3.5.4 Voltage-gated Ion Channel Expression In addition to the morphological changes we observed throughout development, we found a variety of changes in the expression of ion channel subtype mRNA transcripts in BLA principal neurons. These channels are distributed throughout the neuronal membrane and impact the electrophysiological function of BLA principal neurons. Using single-cell RT-PCR of patch clamped neurons at each time point, we found transitions in the expressed subtypes expressed of all the ion channels tested: HCN, KIR, KV, and CaV. The subtype changes, discussed individually below, correspond with and in many cases are corroborative of the maturation of membrane currents in these neurons. Further experiments will be required to determine whether quantitative changes are found for expression of mRNA for these ion channel subtypes and whether these translate to changes in protein and the function of ion channels using pharmacological manipulations. In addition, because we assessed gene expression on a binary scale and the technique has high potential for false negatives, we interpret these results as indications of trends in expression levels.

HCN channels mediate the H-current, a hyperpolarization-activated, nonselective cation current (Pena et al., 2006). We have shown a developmental shift in the expression of mRNA for HCN channel subtypes in BLA principal neurons from HCN4 to HCN1. Specifically, HCN4 mRNA was detected only at time-points before P28, while HCN1 mRNA emerged at P28. HCN4 has much slower kinetics and is classically expressed early in development, while HCN1 exhibits the fastest kinetics of the 4 subtypes and has stronger expression in the mature brain (Monteggia et al., 2000; Vasilyev and Barish, 2002; Surges et al., 2006; Bender and Baram, 2008;

Kanyshkova et al., 2009). The shift in expression from HCN4 to HCN1 in BLA principal neurons corresponds with the activation time-constant of IH in these neurons (Ehrlich et al., 2012). The transition in Ih kinetics does not occur sharply between P21 and P28, as the expression of HCN4 and 1 mRNA would suggest, possibly due to contributions of HCN2 and HCN3 or more gradual changes in protein expression compared to mRNA. Future studies should address quantitative changes in HCN subtype mRNA expression, which the present study failed to capture.

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