. 2 B, D; n 7, 4 animals, ANOVA analysis revealed significant difference in mIPSC frequency by age: F(2,19) 7.3, p 0.004; P13 15 vs young adult: t(19) 3.8, p 0.01, post hoc Bonferroni correction). These observations are consistent with the idea that orexigenic NAG neurons have a lower inhibitory tone during postnatal development. To explore this issue, we used an immunohistological approach to characterize the changes in the appearance of inhibitory synapses onto NAG neurons in the ARH. We filled ARH NPY-GFP neurons with biocytin (2 ) during recording, and performed postrecording immunohistochemistry using an antibody against VGAT. We analyzed the area and circularity of VGAT-labeled synaptic boutons from pups to adults. We found that there were no age-dependent differences in VGAT-labeled synaptic boutons across all ages tested (Fig. 1 A, B; n 6 ? optical sections, 9 animals; p 0.05). Given these results, we next investigated the number of VGAT appositions on the first 50 M of proximal processes. We determined that synaptic boutons containing VGAT in NAG neurons were relatively low at P13, with a rapid increase by P21 (Fig. 3 A, B,D; For P13 15, n 2? opticalsections, 6 animals, For P21 23, n 2? optical sections, 4 animals). However, there were no developmental changes that were significant between P13 15 and P21 23 ( p 0.05). In young adults, the number of VGAT appositions in proximal processes of NAG neurons was greater than any other age. However, these results were only significantly different between young adult and P13 15 mice (Fig. 3C,D; n 2? optical sections, 6 animals, ANOVA with post hoc Tukey’s revealed significant difference in the number of VGAT appositions by age: F(2,23) 5.5, p 0.011; young adult vs P13 15: q(23) 3.3, p 0.0001). Furthermore, there was an increase in the density of VGAT-labeled synaptic boutons at P21?P23 and in the young adult, but these changes were not significant (Table 1). The observed changes in our immunohistological assays for VGAT correlated well with those seen in IPSC frequency in these neurons. Development of afferent projections from the DMH to the ARH It is well established that NAG neurons receive abundant excitatory and inhibitory synaptic inputs in the adult, however, there8562 ?J. Neurosci., June 3, 2015 ?35(22):8558 ?SCR7 web Baquero et al. ?Synaptic Distribution in Arcuate Nucleus Neuronsis no current evidence about the development of afferent axonal projections into the ARH (Pinto et al., 2004; Zeltser et al., 2012). Recently, the DMH, which contains both GABAergic and glutamatergic neurons, has been postulated as an upstream regulator of NAG neuronal activity (Krashes et al., 2014). To investigate whether afferent axonal projections from DMH to the ARH are developed during the third week of postnatal development, we performed DiI implants in the DMH of NPYhrGFP mice at P15 and P21 (Fig. 3 E, H ). At P15, we observed that most of the labeled BMS-5 web fibers were located in a 5 periventricular pathway (Fig. 3F; n animals). While a few scattered labeled fibers have reached the outer edge of the ARH at this age, there were no fibers observed in the ventral medial region of the ARH where the NPY neurons are located (Fig. 3G). We did observe a few labeled fibers in both the central (VMHC) and dorsomedial (VMHdm) part of ventral medial hypothalamic nucleus (VMH; Fig. 3F ) at P15. By P21, there was a dramatic increase in the number of labeled fibers extending down into the ARH (Fig. 3 I, J; n 4 animals). The p.. 2 B, D; n 7, 4 animals, ANOVA analysis revealed significant difference in mIPSC frequency by age: F(2,19) 7.3, p 0.004; P13 15 vs young adult: t(19) 3.8, p 0.01, post hoc Bonferroni correction). These observations are consistent with the idea that orexigenic NAG neurons have a lower inhibitory tone during postnatal development. To explore this issue, we used an immunohistological approach to characterize the changes in the appearance of inhibitory synapses onto NAG neurons in the ARH. We filled ARH NPY-GFP neurons with biocytin (2 ) during recording, and performed postrecording immunohistochemistry using an antibody against VGAT. We analyzed the area and circularity of VGAT-labeled synaptic boutons from pups to adults. We found that there were no age-dependent differences in VGAT-labeled synaptic boutons across all ages tested (Fig. 1 A, B; n 6 ? optical sections, 9 animals; p 0.05). Given these results, we next investigated the number of VGAT appositions on the first 50 M of proximal processes. We determined that synaptic boutons containing VGAT in NAG neurons were relatively low at P13, with a rapid increase by P21 (Fig. 3 A, B,D; For P13 15, n 2? opticalsections, 6 animals, For P21 23, n 2? optical sections, 4 animals). However, there were no developmental changes that were significant between P13 15 and P21 23 ( p 0.05). In young adults, the number of VGAT appositions in proximal processes of NAG neurons was greater than any other age. However, these results were only significantly different between young adult and P13 15 mice (Fig. 3C,D; n 2? optical sections, 6 animals, ANOVA with post hoc Tukey’s revealed significant difference in the number of VGAT appositions by age: F(2,23) 5.5, p 0.011; young adult vs P13 15: q(23) 3.3, p 0.0001). Furthermore, there was an increase in the density of VGAT-labeled synaptic boutons at P21?P23 and in the young adult, but these changes were not significant (Table 1). The observed changes in our immunohistological assays for VGAT correlated well with those seen in IPSC frequency in these neurons. Development of afferent projections from the DMH to the ARH It is well established that NAG neurons receive abundant excitatory and inhibitory synaptic inputs in the adult, however, there8562 ?J. Neurosci., June 3, 2015 ?35(22):8558 ?Baquero et al. ?Synaptic Distribution in Arcuate Nucleus Neuronsis no current evidence about the development of afferent axonal projections into the ARH (Pinto et al., 2004; Zeltser et al., 2012). Recently, the DMH, which contains both GABAergic and glutamatergic neurons, has been postulated as an upstream regulator of NAG neuronal activity (Krashes et al., 2014). To investigate whether afferent axonal projections from DMH to the ARH are developed during the third week of postnatal development, we performed DiI implants in the DMH of NPYhrGFP mice at P15 and P21 (Fig. 3 E, H ). At P15, we observed that most of the labeled fibers were located in a 5 periventricular pathway (Fig. 3F; n animals). While a few scattered labeled fibers have reached the outer edge of the ARH at this age, there were no fibers observed in the ventral medial region of the ARH where the NPY neurons are located (Fig. 3G). We did observe a few labeled fibers in both the central (VMHC) and dorsomedial (VMHdm) part of ventral medial hypothalamic nucleus (VMH; Fig. 3F ) at P15. By P21, there was a dramatic increase in the number of labeled fibers extending down into the ARH (Fig. 3 I, J; n 4 animals). The p.
