Coreleased Orexin and Glutamate Evoke Nonredundant Spike Outputs and Computations in Histamine Neurons

Summary Stable wakefulness requires orexin/hypocretin neurons (OHNs) and OHR2 receptors. OHNs sense diverse environmental cues and control arousal accordingly. For unknown reasons, OHNs contain multiple excitatory transmitters, including OH peptides and glutamate. To analyze their cotransmission within computational frameworks for control, we optogenetically stimulated OHNs and examined resulting outputs (spike patterns) in a downstream arousal regulator, the histamine neurons (HANs). OHR2s were essential for sustained HAN outputs. OHR2-dependent HAN output increased linearly during constant OHN input, suggesting that the OHN→HANOHR2 module may function as an integral controller. OHN stimulation evoked OHR2-dependent slow postsynaptic currents, similar to midnanomolar OH concentrations. Conversely, glutamate-dependent output transiently communicated OHN input onset, peaking rapidly then decaying alongside OHN→HAN glutamate currents. Blocking glutamate-driven spiking did not affect OH-driven spiking and vice versa, suggesting isolation (low cross-modulation) of outputs. Therefore, in arousal regulators, cotransmitters may translate distinct features of OHN activity into parallel, nonredundant control signals for downstream effectors.


Genetic targeting and viral transduction
Animal procedures were in accordance with UK Home Office regulations. Expression of ChR2 in OHNs was carried out and functionally confirmed as illustrated in Fig. S1.
A borosilicate glass pipette tip (20-40 µm diameter) was stereotaxically lowered into the lateral hypothalamus. Three injections (each 50 nl, delivered at 75 nl/min) were made into LH in each hemisphere (bregma: -1.3 to -1.4 mm; midline ±0.9 mm; dorsal surface: -5.30, -5.15, and -5.00 mm). The pipette was gently withdrawn 6 min after final injection. Injections of identical AAV constructs into the lateral hypothalamus of WT mice did not generate ChR2-eYFP expression in the lateral hypothalamus (n = 3 mice), confirming that ChR2 expression was specific to Cre-containing cells. 2
To label HANs during whole-cell recordings, these were filled with 0.2 % biocytin (Tocris) that was added to the intracellular recording solution. Cells were kept in the whole-cell mode for a minimum of 20 min. After recovery for at least 20 min, the tissue was fixed in 4 % PBS overnight. Biocytin filled cells were labeled with rabbit anti adenosine deaminase (Chemicon; 1:250). Immunofluorescence was achieved using goat anti-rabbit Alexa 555 (Invitrogen; 1:1000) antibodies and streptavidin tagged with Cy2 (Invitrogen; 1:1000).
Images were taken using an Olympus BX61WI confocal microscope (Olympus FluoView v 2.1b software) in a dynamic range of 16 bit using a 25x water immersion objective (NA 1.05, Olympus). Cy2 was excited with an argon laser at 488 nm, and its fluorescence collected between 570 and 670 nm using a spectral detector (Olympus).
Alexa 555 was excited with a diode-pumped solid-state (DPSS) laser at 559 nm, and fluorescence emission collected at 570-670 nm using a spectral detector (Olympus).
When more than one fluorophore was detected, the sequential "between-lines" scanning mode of the microscope was used to achieve optimal separation of fluorescent signals.

Photostimulation and electrophysiology
Coronal slices containing the tuberomammillary hypothalamic nucleus ( Control experiments revealed that basal tone (without optical stimulation) of orexin/hypocretin or glutamate was too low to drive histamine neuron firing.
Specifically, blocking OH2Rs with TCS did not alter spontaneous histamine neuron firing, as analyzed by examining 10 s intervals preceding optical stimulation (control:

Analysis
Data were analysed with Minianalysis (Synaptosoft), Matlab (Mathworks) or Prism (Graphpad). Histamine neuron firing responses to optical orexin neuron stimulation were separated from baseline firing by subtracting the average baseline firing during 10 s prior to stimulation in each cell. In group data plots, results are presented as means ± sem of multiple experimental trials (1-2 trials were recorded per cell), except 5 where stated otherwise. Student's unpaired or paired t-test, or one or two-way ANOVA followed by Bonferroni or Newman-Keuls posthoc tests, were used for statistical hypothesis testing; the significance labels in the figures are *p < 0.05, **p < 0.01, ***p < 0.001, and p > 0.05 was taken as non-significant (ns).
6 Figure S1. Studying functional transmission in orexin  histamine microcircuit (Illustration of methodological background to experiments in main Figures 1-3).
A. Schematic of virally-delivered constructs used to transduce orexin-Cre neurons (OHNs) with ChR2 (top), after which OHN spikes routinely followed optical stimuli in brain slices (bottom, typical example of n = 10 cells).
B: Schematic of experimental strategy for probing postsynaptic responses of histamine neurons (HANs) to OHN stimulation (top), and typical confocal images (bottom) of a HAN filled with biocytin (BC) and confirmed by post-recording immunocytochemistry to contain HAN marker adenosine deaminase (ADA, arrowheads, n = 3/3 cells). ChR2-eYFP fibers (shown in red pseudo-color) make close contact with HAN (arrows in bottom images). Scale bar, 10 µm.