MRC Brain Network Dynamics Unit, Department of Pharmacology, University of Oxford, Oxford OX1 3TH, UKDepartment of Biomedicine, Aarhus University, 8000 Aarhus C, DenmarkThe Danish Research Institute of Translational Neuroscience (DANDRITE), Nordic EMBL Partnership for Molecular Medicine, Aarhus University, 8000 Aarhus C, Denmark
Neurochemical profile and localization of nNOS+ type I neurons of the BLA. A, Confocal stack (z-stack, 27 μm) showing colocalization of SOM and NPY in BLA neurons with strong nNOS expression (nNOS+ type I, arrows) and no SOM and NPY immunoreactivity in neurons with weak nNOS expression (nNOS+ type II, arrowheads). B, Confocal stack (z-stack, 13 μm) showing colocalization of NK1 in BLA nNOS+ type I neurons (arrows) and no NK1 immunoreactivity in nNOS+ type II neurons (arrowheads). C, 93.2 ± 6.5% of nNOS+ type I neurons coexpressed SOM (n = 3 brains), 95.4 ± 5.4% coexpressed NPY (n = 6 brains), and 84.6 ± 3.1 coexpressed NK1 (n = 3 brains). D, Three coronal sections illustrating the distribution of BLA nNOS+ type I neurons at different rostrocaudal positions. nNOS+ type I cells plotted at each level were mapped by collapsing three neighboring 60-μm-thick sections. Data are presented as the mean ± SEM. BLV, Basolateral ventral amygdala; BMA, basomedial amygdala; CeA, central amygdala; CoA, cortical amygdala; CPu, caudate–putamen; Pir ctx, piriform cortex; PRh, perirhinal cortex.
Electrophysiological properties of pc-nNOS neurons. A, Colocalization of tdTomato and nNOS in BLA neurons from a Nos1-CreER;Ai9 mouse. B, C, Voltage responses to hyperpolarizing–depolarizing current pulses (calibration: −30/+15 pA, 5 pA steps, 400 ms; B) used to construct the I–V plot shown in C and to determine the value of Rin. D, Adapting instantaneous firing, obtained by injecting twice the rheobase current for 1 s. E, Spontaneous firing at resting Vm (Vm rest). F, Action potential evoked by a short depolarizing current (3 ms, +800 pA). G, Voltage sag and rebound depolarization generated by hyperpolarizing current injection (500 ms, −150 pA) and blocked by Ih blocker ZD7288 (30 μm).
Axonal and dendritic arborization of pc-nNOS neurons. A, Biocytin-labeled pc-nNOS neuron coexpressing tdTomato. B, Neurolucida reconstruction from a pc-nNOS cell (MB131202_1) with dendrites (black) running parallel with the intermediate capsule and axon (green) innervating both the BLA and the caudate–putamen (CPu). C, Neurolucida reconstruction from another pc-nNOS cell (MB151113_2) with dendrites (black) mostly running parallel with the external capsule and axon (green) innervating mostly the BLA, but also the dorsal endopiriform claustrum (DEn), the perirhinal cortex (PRh Ctx), and the amygdalostriatal transition area (AStria)/caudate–putamen (CPu). CeA, Central amygdala; ec, external capsule. Data are presented as the mean ± SEM.
VGAT expression and connectivity of pc-nNOS cells. A, Top, VGAT immunoreactivity of biocytin-filled axonal varicosities of a pc-nNOS neuron (arrows). Bottom, VGAT immunoreactivity in a biocytin-filled bouton from another pc-nNOS cell (arrow). B, Dual whole-cell recording (voltage clamp) showing a presynaptic pc-nNOS neuron functionally connected to a postsynaptic principal neuron (PN). Top, Schematic showing the dual whole-cell recording configuration. Middle, Action current evoked in the presynaptic pc-nNOS. Bottom, uIPSC recorded in the postsynaptic PN (holding potential, −40 mV; gray, overlap of 10 sweeps repeated every 10 s; black, average of the 10 sweeps). Inset, Stereotypical PN firing evoked by 500-ms-long, +100 pA current injection in the postsynaptic cell held at −65 mV. C, Rate of connectivity between a pc-nNOS cell and BLA cells. One of eleven nearby PN cells received a uIPSC, whereas no uIPSC could be recorded in five nearby pc-nNOS cells or three nearby nNOS-negative interneurons.
pc-nNOS neurons are activated during sleep. A, The 24 h profile of EEG SWA (EEG power between 0.5 and 4.0 Hz, displayed as the percentage of mean 24 h baseline; white bar, 12 h light period; dark bar, 12 h dark period) recorded in the frontal cortex and below the distribution of sleep–wake stages (W, wakefulness; N, NREM sleep; R, REM sleep) from a representative mouse. Note, as expected, that sleep predominates, and SWA shows a typical decline during the 12 h light period. B, EEG power spectral density during waking, NREM sleep, and REM sleep shown for the frontal EEG (n = 7). Note the state-dependent differences in cortical activity. C, Top, Representative profile of SWA during the 4 h SD and subsequent sleep opportunity/RS in one individual mouse. Bottom, The distribution of sleep–wake stages. Mice in the SD group were killed at the end of SD at ZT4 (n = 4), while the remaining mice in the SD+RS group (n = 4) were killed after the sleep opportunity. D, EEG spectral density in NREM sleep (displayed as a ratio of the mean 24 h baseline) during the sleep opportunity after SD (n = 4). Note the typical increase in SWA relative to the corresponding baseline interval after a period of prolonged waking. Thin lines represent the power density from single mice, whereas thick lines represent the mean power density from all four mice. E, Top panels, Confocal stack (z-stack, 29 μm) showing lack of c-Fos immunoreactivity in pc-nNOS cells after SD. A median filter was applied (x–y radius, 5 pixels). Arrowhead, A c-Fos+ cell immunonegative for nNOS. Bottom panels, Confocal stack (z-stack, 31 μm) showing c-Fos immunoreactivity in two pc-nNOS cells (arrows) following SD+RS. A median filter was applied (x–y radius, 5 pixels). Insets, Magnification of one the c-Fos+ pc-nNOS cells (z-stack, 5 μm; no filtering was applied). F, Quantification of c-Fos expression in pc-nNOS neurons. No pc-nNOS neuron expressed c-Fos following SD, whereas 31.4 ± 16.4% were c-Fos+ after subsequent RS (n = 4 per condition). G, Quantification of c-Fos expression in paracapsular Nissl-stained cells. Overall, c-Fos+ neurons were more abundant following SD (3.7 ± 1.1%) than during the subsequent RS (0.6 ± 0.5%, n = 4 per group) **p < 0.01; *p < 0.05. Data are presented as the mean ± SEM.
Dorsal raphe 5-HT neurons innervate pc-nNOS neurons. A, Cre-dependent expression of an anterograde tracer in the DRN of SERT-Cre mice B, Confocal stack (z-stack, 5.61 μm) showing selective expression of eYFP in 5-HT-immunopositive neurons in the dorsal raphe nuclei. MRN, Median raphe nuclei. C, nNOS immunoreactivity and innervation by dorsal raphe 5-HT neurons in the amygdaloid complex. The external paracapsular region displays prominent innervation. D, Confocal stack (z-stack, 11.1 μm) showing axonal varicosities from a dorsal raphe 5-HT neuron juxtaposed to a pc-nNOS neuron soma (arrow). Inset, Magnification of the somatic apposition (single optical section, 0.37 μm thickness). E, Confocal stack (z-stack, 5.49 μm) showing an axonal varicosity from a dorsal raphe 5-HT neuron juxtaposed to a pc-nNOS neuron dendrite (arrow). Inset, Magnification of the dendritic apposition (single optical section, 0.37 μm thickness).
5-HT inhibits pc-nNOS neurons. A, Representative cell-attached recording from a pc-nNOS neuron (voltage-clamp mode) inhibited by bath application of 5-HT (50 μm). In this cell, 5-HT did not trigger burst firing. B, Representative cell-attached recording from a pc-nNOS neuron (voltage-clamp mode) in which bath application of 5-HT elicited a reduction in both firing rate and burst firing. Insets, Magnified examples of tonic firing in control conditions and burst firing upon bath application of 5-HT (50 μm). C, Significant decrease in firing rate promoted by 5-HT (from 3.6 ± 1.6 Hz to 1.6 ± 0.4 Hz; p < 0.0001, paired t test; n = 18). D, Significant increase in firing irregularity (measured by the CV of the ISI: from 0.5 ± 0.06 to 2.9 ± 0.4; p < 0.0001, paired t test; n = 18) caused by 5-HT. E, 5-HT application enhances the burstiness of pc-nNOS neurons: the peak of the ISI histogram (in Log scale) shifts to the left (n = 18). F, 5-HT triggered spike bursts in only 7 of 18 pc-nNOS neurons. The remaining neurons displayed a reduction in firing rate only upon 5-HT application. G, In four cells displaying bursts upon 5-HT application, 5-HT was reapplied in the presence of synaptic blockers (10 μm NBQX, 50 μm D-APV, and 10 μm SR95531). In these conditions, 5-HT still triggered bursting (the peak of the Log ISI histogram shifted to the left), suggesting that synaptic inputs are not necessary for bursting activity. ****p < 0.0001. Data are presented as the mean ± SEM.
Direct hyperpolarization of pc-nNOS neurons by 5-HT via 5-HT1A receptors. A, Effect of 5-HT on voltage responses to depolarizing current injection of representative pc-nNOS neuron (20 pA, 300 ms) in control conditions (left) and in the presence of 5-HT1A antagonist WAY100635 (10 μm, right). B, Time course of the effect of 5-HT on the Vm of pc-nNOS neurons (n = 10). C, Time course of the effect of 5-HT on the Rin of pc-nNOS neurons (n = 10). D, 5-HT significantly hyperpolarizes pc-nNOS cells (from −59.3 ± 0.2 to −64 ± 0.7 mV; p = 0.001, one-way ANOVA with Bonferroni post hoc test; n = 10). E, 5-HT significantly reduced the Rin of pc-nNOS cells (by 11.3 ± 1.9%; p = 0.0006, one-way ANOVA with Bonferroni post hoc test; n = 10). F, G, 5-HT-evoked hyperpolarization and Rin reduction are significantly reduced by 10 μM WAY100635 (p = 0.0235 and p = 0.0064, respectively, paired t tests; n = 5). H, 5-HT significantly hyperpolarizes pc-nNOS cells, even in the presence of synaptic blockers (10 μm NBQX, 50 μm D-APV, and 10 μm SR95531; p = 0.0017, paired t test; n = 5), suggesting a direct effect. ***p < 0.001; **p < 0.01; *p < 0.05. Data are presented as the mean ± SEM.
Abbreviations: Rin: input resistance; CV ISI: coefficient of variation of the interspike interval (calculated on the instantaneous firing rate); fAHP: fast after-hyperpolarization; Vm rest: resting Vm.