. 2015 Jul 1;87(1):164-78.
Epub 2015 Jun 18.
Wakefulness Is Governed by GABA and Histamine Cotransmission
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Wakefulness Is Governed by GABA and Histamine Cotransmission
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Histaminergic neurons in the tuberomammilary nucleus (TMN) of the hypothalamus form a widely projecting, wake-active network that sustains arousal. Yet most histaminergic neurons contain GABA. Selective siRNA knockdown of the vesicular GABA transporter (vgat, SLC32A1) in histaminergic neurons produced hyperactive mice with an exceptional amount of sustained wakefulness. Ablation of the vgat gene throughout the TMN further sharpened this phenotype. Optogenetic stimulation in the caudate-putamen and neocortex of "histaminergic" axonal projections from the TMN evoked tonic (extrasynaptic) GABAA receptor Cl(-) currents onto medium spiny neurons and pyramidal neurons. These currents were abolished following vgat gene removal from the TMN area. Thus wake-active histaminergic neurons generate a paracrine GABAergic signal that serves to provide a brake on overactivation from histamine, but could also increase the precision of neocortical processing. The long range of histamine-GABA axonal projections suggests that extrasynaptic inhibition will be coordinated over large neocortical and striatal areas.
Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
Pharmacogenetic Activation of Histaminergic Neurons Increased Motor Activity of
HDC-hM3Dq Mice (A) AAV-flex-hM3Dq-mCherry was bilaterally injected into the TMN region of HDC-Cre mice to give hM3Dq-mCherry expression selectively within histaminergic neurons (see inset image). (B) Activity of HDC-hM3Dq mice in an open field arena 30 min after saline (black trace, n = 4 mice) or CNO (red trace, n = 4 mice) injection. The distance moved was calculated every 5 min and average values (mean ± SEM) plotted over 60 min ( ∗p < 0.05; ∗∗p < 0.01). (C) Double-label immunocytochemisty of HDC- and hM3Dq-mCherry-positive neurons with HDC antisera and mCherry antibody; arrowheads indicate examples of double-labeled neurons. DAPI labeling was included to locate all cells. (D) Two examples of voltage traces recorded from HDC-hM3Dq mice during the application of CNO. The top trace shows an example of a silent neuron that was sufficiently depolarized to fire APs. The bottom trace shows a spontaneously active neuron with increased AP firing in the presence of CNO. On average there was a significant (paired t test, p < 0.05) ∼5 mV depolarization of TMN neurons (n = 11 cells, control, −46 ± 3 mV; CNO, −41 ± 2 mV). The results from each cell are shown on the scatterplot on the right.
The Majority of Histaminergic Neurons Express GABAergic Markers (A) Coronal section of the mouse posterior hypothalamus stained with HDC (green) and GAD67 (red) antisera. The diagram summarizes the staining from the whole TMN region: HDC-positive cells (green) and double-positive cells (orange). 3V, third ventricle; VTM, ventral tuberomammillary; DTM, diffuse tuberomammillary. (B)
HDC-Cre mice were crossed with Rosa26-loxP-stop-loxP-YFP mice to generate HDC-YFP mice. TMN sections from HDC-YFP mice were costained with EYFP and GAD67 antisera. Most YFP-positive (HDC neurons) in the TMN were also GAD67-positive. (C) vGAT-Cre mice were crossed with Rosa26-loxP-stop-loxP-tdTomato mice to generate vGAT-tdTomato mice. Sections from vGAT-tdTomato mice were stained with HDC antisera. Most HDC neurons were tdTomato positive.
Knocking Down and Knocking Out
vgat Expression from Histaminergic Neurons in the TMN (A) Three hairpin ( sh) oligonucleotides ( sholigo), targeting the vgat transcript, were designed. Each sholigo was separately cloned into the pPRIME-dsRed vector; a scramble shRNA was also cloned into pPRIME-dsRed. (B) To test the knockdown efficiency of the three shvgat oligonucleotides in vitro, CMV-dsRed-scramble or CMV-dsRed-shvgat (1, 2, or 3) were cotransfected with pCMV-vGAT-3′UTR (a cDNA containing the vgat open reading frame and 3′ untranslated region) into HEK293 cells. Thirty-six hours later, cells were fixed and stained with vGAT antisera. vGAT expression was best reduced with sholigo1 and sholigo3. (C) Three independent transfections were performed, and relative vGAT immunofluorescence in HEK cells was quantified. (D) AAV-flex-dsRed-shvgat (oligo3) was bilaterally injected into the TMN region of HDC-Cre mice. Cre recombination produced dsRed-shvgat expression in HDC-positive neurons, and the resulting mice were termed HDC-vgat KD mice. Arrowheads indicate examples of colabeled cells. (E) HDC-vgat KD mice were more active than HDC-scramble ( AAV-flex-dsRed-scramble-injected HDC-Cre) mice in an open field assay ( ∗p < 0.05; ∗∗p < 0.01). (F) AAV-Cre-2A-Venus was bilaterally injected into the TMN region of vgat mice to generate lox/lox TMN-Δvgat mice. Sections from virus-injected ( TMN-Δvgat) mice were costained with HDC and GFP (Venus) antisera. In the TMN region, GFP expression was in 77% ± 2% of HDC neurons. Arrowheads indicate examples of colabeled cells. (G) TMN-Δvgat mice ran further than TMN-GFP ( AAV- GFP-injected vgat ) mice in an open field assay ( lox/lox ∗∗p < 0.01; ∗∗∗p < 0.001). During each running episode TMN-Δvgat mice also ran faster, as evidenced by instantaneous speed measurements ( ∗∗p < 0.01; ∗∗∗p < 0.001).
GABA Release from Histaminergic Neurons Governs the Amount of Sleep (A) Continuous EMG, EEG, delta power, wake, NREM sleep, and REM sleep scoring data recorded for an
HDC-scramble and an HDC-vgat KD mouse for 24 hr. (B) The graphs illustrate the average 24 hr sleep scoring (percentage of wake, NREM, or REM sleep) for HDC-scramble (black trace) and HDC-vgat KD (red trace) mice; bars, SEM. (C) Comparison of the power spectra of wake obtained from EEG data recorded during the day and night for the HDC-scramble (black trace) and HDC-vgat KD (red trace) mice. (D) Continuous EMG, EEG, delta power, wake, NREM sleep, and REM sleep scoring data recorded from TMN-GFP and TMN-Δvgat mice. (E) The graphs illustrate the average 24 hr sleep scoring (percentage of wake, NREM, or REM sleep) for TMN-GFP (black trace) and TMN-Δvgat (red trace) mice; bars, SEM. (F) Comparison of the power spectra of wake obtained from EEG data recorded during the day and night for the TMN-GFP (black trace) and TMN-Δvgat (red trace) mice.
Selective ChR2-EYFP Expression in Histaminergic Neurons and Tracing Their Axons to Neocortex and Striatum (A)
AAV-flex-ChR2-EYFP was bilaterally injected into the TMN region of HDC-Cre mice. The blue circle indicates the area of the slice that was optically stimulated. (B) Cre recombination produced ChR2-EYFP expression within HDC-expressing neurons. Shown is a combined bright-field and primary fluorescence photograph, taken at low magnification, of a freshly cut coronal brain slice used for electrophysiological recording from TMN neurons. (C) ChR-EYFP expression in the TMN imaged with antisera to GFP (green) and HDC (red), with arrowheads indicating ChR2 expression in processes. (D) The duration of the LED power output (blue trace) measured from the objective lens, and the membrane voltage recorded (black trace) from the soma of a HDC-ChR2-EYFP neuron. Increasing the LED power depolarized the membrane to generate action potentials. Inset image: four neurons recorded from this slice with cofluorescence for ChR-EYFP (green) and postrecording neurobiotin fill (red). (E) The same cell as (D) firing action potentials with 5 Hz light stimulation. (F) Schematic of the fibers (axons) which, following AAV-flex-ChR2-EYFP injection into the TMN of HDC-Cre mice, transported ChR-EYFP from the HDC-ChR2-EYFP soma into the neocortex and caudate-putamen. (G and H) Low-power photographs of the ChR2-EYFP-positive fibers in the caudate-putamen (CPu) (G) and sensory and visual cortex (H). Blue, DAPI; I, layer I; II, layer II; III, layer III; IV, layer IV; V, layer V; VI, layer VI; WM, white matter. (I) Schematic of the ChR2-EYFP fiber distribution in the caudate-putamen (CPu) and neocortex.
Histaminergic Axons Produce Slow GABAergic Responses in Visual Cortex Pyramidal and Striatal Neurons (A) Double injection of
AAV-Cre-Venus and AAV-flex-ChR2-EYFP into the TMN of vgat mice to make light-sensitive histaminergic neurons that lack vGAT ( lox/lox HDC-ChR TMN-Δvgat mouse). (B) The top photomicrograph shows combined expression of the two AAV transgenes in the bilateral TMN area (coronal section). The lower picture shows double staining in the TMN with HDC and EGFP antisera. (C) Two layer IV pyramidal neurons filled with neurobiotin/Alexa 555 (red) postrecording. (D) Electrophysiological data from a HDC-ChR mouse. The gray symbols superimposed upon the current trace (black line) show the average holding current calculated for every 1 s epoch during the entire recording. An increase in the holding current begins during the 5 Hz optogenetic stimulation but takes minutes to reach its peak response following termination of the stimuli. (E) Scatterplot for all recordings made from the HDC-ChR mice. The lines indicate paired recordings made before and after the 5 Hz optogenetic stimulation. On average, G significantly increased by 1.1 ± 0.3 nS (paired t test, p < 0.01; n = 8) in the tonic HDC-ChR slices. (F) Scatterplot for all recordings made from the HDC-ChR mice in the presence of TTX and 4-AP. On average, G tonic significantly increased by 0.5 ± 0.1 nS (paired t test, p < 0.005; n = 9) in the HDC-ChR slices. (G) Electrophysiological data from a HDC-ChR TMN-Δvgat mouse. The gray symbols superimposed upon the current trace (black line) show the average holding current calculated every 1 s epoch for the entire recording. No change in the holding current occurred in response to the 5 Hz optogenetic stimulation, but there was an increase in the frequency of spontaneous synaptic activity (transient downward deflections). (H) Scatterplots for recordings made from the HDC-ChR TMN-Δvgat mice. The lines indicate the paired recordings made before and after the 5 Hz optogenetic stimulation. G was reduced by −0.2 ± 0.1 nS in the tonic TMN-Δvgat/HDC-ChR mice (paired t test, p = 0.225, n = 9). (I) Scatterplots for recordings made from the HDC-ChR mice in the presence of H1 (pyrilamine) and H2 (ranitidine) blockers. G increased by 0.6 ± 0.2 nS (paired t test, p < 0.05; n = 11). (J) Medium spiny neuron (red, neurobiotin fill postrecording) from the caudate-putamen of a tonic HDC-ChR mouse; several ChR-EYFP-positive axons (green) are in the vicinity of the cell. (K and L) Scatterplots for all recordings made from medium spiny neurons in the HDC-ChR mice in control conditions and with the GABA A receptor antagonist gabazine (10 μM). G increased by 1.9 ± 0.6 nS (paired t test, p < 0.05; n = 7) in control conditions and decreased by −0.8 ± 0.5 nS in the presence of gabazine (paired t test, p = 0.3; n = 6). tonic
Activated Neocortical “GABA-Histamine” Axons Increase Synaptic Drive and
G by Independent Mechanisms (A) Whole-cell voltage-clamp recording from a pyramidal cell in layer IV of visual cortex during 5 Hz LED stimulation of axon fibers from an tonic HDC-ChR mouse. The left-hand trace is from the start, and the right-hand trace is at the end of, the 5 Hz LED stimuli. The increased holding current between the first and last trace was used to calculate the increase in tonic conductance (ΔG ). (B) Cross-correlation analyses between the LED trigger and the occurrence of sPSCs. There was no peak in the histogram, consistent with a lack of LED-triggered PSCs. (C) Whole-cell voltage-clamp recording in a layer IV pyramidal cell in the presence of TTX and 4-AP during 5-Hz LED stimulation of axon fibers in the visual neocortex from an tonic HDC-ChR mouse. There was an increase in G but in this experiment little change in sPSC rate. (D) Scatterplot of the change in synaptic drive (Hz) onto layer IV visual cortex pyramidal cells for the four main experiments: stimulation of tonic HDC-ChR axons, stimulation of HDC-ChR axons in the presence of TTX/4-AP, stimulation of HDC-ChR axons in the presence of H1 and H2 receptor antagonists, and stimulation of TMN-Δvgat/HDC-ChR axons. In HDC-ChR mice the sPSC frequency significantly increased from 6.4 ± 1.7 Hz (n = 9) to 9.5 ± 2.2 Hz (paired t test, p < 0.05). In TMN-Δvgat/HDC mice, a similar increase in sPSC frequency was observed from 8.7 ± 2.8 Hz (n = 9) to 15.6 ± 3.3 Hz (paired t test, p < 0.05). With the H1 receptor antagonist pyrilamine (20 μM) and the H2 receptor antagonist ranitidine (5 μM), the ChR-stimulated increase in the rate of asynchronous sPSCs was 23.9 ± 6.0 Hz at the start and 21.9 ± 5.4 Hz at the end of stimulation (n = 11, paired t test, p = 0.3).
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Research Support, Non-U.S. Gov't
Gene Knockdown Techniques
Hypothalamic Area, Lateral / metabolism*
Neostriatum / metabolism*
Neural Inhibition / genetics
Neural Inhibition / physiology*
Pyramidal Cells / metabolism
Receptors, GABA-A / metabolism
Vesicular Inhibitory Amino Acid Transport Proteins / genetics
Wakefulness / physiology*
gamma-Aminobutyric Acid / metabolism*
Vesicular Inhibitory Amino Acid Transport Proteins
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