Fig 2: Orexin augments voltage-dependent Ca2+ transients in nNOS+ LDT neurons, TpH+ DR neurons, and TH+ LC neurons in slices from OX-/-1 mice. Orexin-A augmented the voltage-dependent Ca2+ transient in identified neurons. Left column illustrates low-power fluorescent micrographs of the recorded slices immunostained with Alexa-488 (green) for nNOS in LDT (A), TpH in DR (B), and TH in the LC (C arrows). The second column illustrates a higher-power image of the recorded and fluorescently labeled neurons (Alexa-594) from each nucleus. The third column shows the same field with Alexa-488 visualized. The right column shows the images merged, revealing that each recorded neuron was immunopositive for nNOS, TpH, and TH, respectively.

Fig 3: Antibody staining in ET-like cell populations within the GL. a–d Example of ET-like cells (arrows) expressing HCN1 channels (green HCN1α) and CCK (red CCK). Some of the HCN1- and CCK-expressing cells (orange arrows) are also immunostained by antibodies directed against vilip1 (blue vilip1). e–l All TH-positive (green) ET-like cells (arrows) express hippo (red). Two representative examples are shown (e–h, i–l). TH-positive ET-like cells are either faintly stained (g) by NOS antibodies (blue) or devoid of NOS staining (k). Note the pronounced thick primary tuft in the ET-like cells. Antibodies used in e–l were: hippo, NOSgt, and TH. Further staining is shown in Electronic Supplementary Material, Fig. 5. All staining patterns are summarized in Table 2. Color codes of arrows as in Table 2. Bars 10 μm

Fig 4: Identification of amacrine cells expressing neuronal nitric oxide synthase loaded with molecular tracer via cut-loading.(a) Image created from z-stack slices spanning from the inner nuclear layer to the ganglion cell layer, flattened using the z-project function in Fiji. Open arrows, type-1 nNOS amacrine cells; solid arrows, displaced nNOS amacrine cells. (b, c) Vertical retinal sections labelled for nNOS (red) and DAPI (blue). (b) Open arrows highlight type-1 nNOS amacrine cells located in the inner nuclear layer (INL), (c) solid white arrows highlight displaced nNOS amacrine cells located in the ganglion cell layer (GCL). (d, e) Fluorescent intensity of NeurobiotinTM in cut-loaded type -1 and displaced nNOS amacrine cells analysed using (d) Method 2 (fitted with Eq 6); or (e) Method 3 (fitted with Eq 9). (f-g) Comparison of nNOS type-1 and nNOS displaced amacrine cells in their mean: (f) cell density, (g) cell-coupling coefficients kj, and (h) relative diffusion coefficients. Error bars are SEM. * p < 0.05.

Fig 5: Fluorescence markers for cell survival in LDT. (A) Monitoring of cell survival with DAPI (blue) and PI (red) in sections of the cultured slices with and without flow system at DIV 0, 7 and 14 (Scale bar = 70 µm). (B) Cell fractions showing neuronal viability in the cultured slices with and without flow system during the days of culture (*p = 0.020, ****p = 0.0001, two way ANOVA), indicating statistical differences in this fraction after the first 5 days. (C1) Immunohistochemical evaluation of cholinergic neurons with anti-bNOS antibody (green), and astrocytes with anti-GFAP antibody (red). The survival of these cells is evident during two weeks of culture, with a greater impact on those cultured with the flow system. (C2) Comparison of the number of cholinergic neurons (ACh; bNOS positive) (****p = 0.0001, ***p = 0.0018, two way ANOVA). (C3) Immunoreactivity measure of astrocytes, with significant differences in the corrected total cell fluorescence (CTCF) (****p = 0.0001, **p = 0.0025, two way ANOVA) on day 14 with and without the flow system. (D1) Evaluation of cell survival in hippocampal slices cultured for 14 days with and without flow system. The DAPI/PI fraction indicates a lower number of surviving cells with use of the conventional methodology. (D2) Bar graph of statistically significant difference between the two culture methodologies in hippocampal slices (***p = 0.0012, paired t-test).