Parallel Arousal Pathways in the Lateral Hypothalamus

57 Pages·2017·2.15 MB·English

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Research Article: New Research | Integrative Systems
Parallel Arousal Pathways in the Lateral Hypothalamus
Jaime E. Heiss1, Akihiro Yamanaka2 and Thomas S. Kilduff1
1Biosciences Division, Center for Neuroscience, SRI International, Menlo ParkCA 94025, USA
2Department of Neuroscience II, Res. Inst. of Environmental Medicine, Nagoya University, Nagoya Japan
DOI: 10.1523/ENEURO.0228-18.2018
Received: 7 June 2018
Revised: 11 July 2018
Accepted: 12 July 2018
Published: 1 August 2018
Author Contributions: JH designed research, performed research, contributed unpublished reagents/ analytic
tools, analyzed data and wrote the paper. AY designed research and contributed unpublished reagents/ analytic
tools. TK designed research, contributed unpublished reagents/ analytic tools and wrote the paper.
Funding: http://doi.org/10.13039/100000065HHS | NIH | National Institute of Neurological Disorders and Stroke
(NINDS)
R01NS077408
R01NS098813
Funding: KAKENHI, MEXT, Japan
16H01271
15H01428
Conflict of Interest: Authors report no conflicts of interest.
Research was supported by R01NS077408 and R01NS098813 from the National Institutes of Health and by
KAKENHI grants (16H01271, 15H01428 to A.Y.) from Ministry of Education, Culture, Sports Science, MEXT,
Japan. The content is solely the responsibility of the authors and does not necessarily represent the official
views of the National Institutes of Health.
Corresponding author: Thomas S. Kilduff, 333 Ravenswood Ave, Menlo Park, CA 94025. E-mail:
[email protected]
Cite as: eNeuro 2018; 10.1523/ENEURO.0228-18.2018
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Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading
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Copyright © 2018 Heiss et al.
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Parallel Arousal Pathways in the Lateral Hypothalamus
1
2 Abbreviated Title: Parallel hypothalamic arousal mechanisms
3 Authors: Jaime E. Heiss1, Akihiro Yamanaka2, Thomas S. Kilduff1
4 1Center for Neuroscience, Biosciences Division, SRI International, Menlo Park, CA, 94025 USA
5 2Dept. of Neuroscience II, Res. Inst. of Environmental Medicine, Nagoya Univ., Nagoya, Japan
6 Author Contributions: JH designed research, performed research, contributed unpublished
7 reagents/ analytic tools, analyzed data and wrote the paper. AY designed research and
8 contributed unpublished reagents/ analytic tools. TK designed research, contributed
9 unpublished reagents/ analytic tools and wrote the paper
10 Corresponding author: Thomas S. Kilduff ([email protected]). 333 Ravenswood Ave,
11 Menlo Park, CA 94025
12 Figures: 8, Tables: 2
13 Pages: 46 Figures: 8 Tables: 2
14 Number of words in Abstract: 249, Significance: 91, Introduction: 615, Discussion: 1,511
15 Acknowledgements: We thank Dr. Michael Schwartz for comments on the manuscript, Dr.
16 Ling Jong for almorexant synthesis and Messrs. Webster Lincoln, Jeremiah Palmerston and
17 Gabriel Orellana, as well as Ms. Deepti Warrier and Ms. Jennifer Walters for technical
18 assistance.
19 Authors report no conflicts of interest.
20
21 Funding Sources: Research was supported by R01NS077408 and R01NS098813 from the
22 National Institutes of Health and by KAKENHI grants (16H01271, 15H01428 to A.Y.) from
23 Ministry of Education, Culture, Sports Science, MEXT, Japan. The content is solely the
24 responsibility of the authors and does not necessarily represent the official views of the
25 National Institutes of Health
26 Abstract
27 Until recently, hypocretin (Hcrt) neurons were the only known wake-promoting neuronal
28 population in the lateral hypothalamus (LH), but subpopulations of inhibitory neurons in this
29 area and glutamatergic neurons in the nearby supramammillary nucleus (SuM) have recently
30 been found that also promote wakefulness. We performed chemogenetic excitation of LH
31 neurons in mice and observed increased wakefulness that lasted more than 4h without unusual
32 behavior or EEG anomalies. The increased wakefulness was similar in the presence or absence
33 of the dual orexin receptor blocker almorexant (ALM). Analysis of hM3Dq transfection and c-
34 FOS expression in LH inhibitory neurons and in the SuM failed to confirm that the increased
35 wakefulness was due to these wake-promoting populations, although this possibility cannot be
36 completely excluded. To evaluate the relationship to the Hcrt system, we repeated the study in
37 Orexin-tTA mice in the presence or absence of dietary doxycycline (DOX), which enabled us to
38 manipulate the percentage of Hcrt neurons that expressed hM3Dq. In DOX-fed mice, 18% of
39 Hcrt neurons as well as many other LH neurons expressed hM3Dq; these mice showed a
40 profound increase in wake after hM3Dq activation even in the presence of ALM. In mice
41 switched to normal chow, 62% of Hcrt neurons expressed hM3Dq along with other LH cells;
42 chemogenetic activation produced even more sustained arousal which could be reduced to
43 previous levels by ALM treatment. Together, these results indicate an LH neuron population
44 that promotes wakefulness through an Hcrt-independent pathway that can act synergistically
45 with the Hcrt system to prolong arousal.
46
47
2
48
49 Visual Abstract
50 Activation of lateral hypothalamic (LH) neurons transfected with hM3Dq (denoted in blue)
51 induces profound Hcrt-independent wakefulness (% W) that can further increase after
52 activation of Hcrt signaling pathways, suggesting the existence of independent, parallel wake-
53 promoting mechanisms in LH. Arrows denote injection of either vehicle (VEH) or the dual Hcrt
54 receptor antagonist almorexant (ALM) at ZT4 and either CNO or saline at ZT5.
55
56 Significance
57 Despite nearly a century of evidence suggesting that the lateral hypothalamus contains neurons
58 essential to sustain wakefulness, the primary wake-promoting cells in this brain region studied
59 to date have been the hypocretin/orexin neurons. Here, we show that chemogenetic excitation
60 of lateral hypothalamus neurons can induce sustained wakefulness even in absence of
61 hypocretin signaling and that concurrent hypocretin neuron activation can prolong arousal.
62 These results indicate the existence of parallel arousal pathways in the lateral hypothalamus
63 and suggest a new therapeutic target for disorders of excessive sleepiness such as hypersomnia
64 and narcolepsy.
65
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66 Introduction
67 The lateral hypothalamus (LH) has been thought to play a critical role in sleep-wake regulation
68 since the early studies of Von Economo (Von Economo, 1930). Lesions and acute inhibition of
69 LH neurons are known to increase sleep time (Ranson, 1939; Nauta, 1946; de Ryck and
70 Teitelbaum, 1978; Shoham and Teitelbaum, 1982; Cerri et al., 2014) while excitation produces
71 arousal and increases in activity (Krolicki et al., 1985; Sinnamon et al., 1999; Alam and Mallick,
72 2008; Li et al., 2011). Several studies have identified wake-active neurons in the LH (Alam et al.,
73 2002; Koyama et al., 2003; Lee et al., 2005) but their neurochemical identity is unknown. More
74 recently, inhibitory LH neuronal populations have been identified that induce wakefulness upon
75 optogenetic or chemogenetic stimulation (Herrera et al., 2016; Venner et al., 2016). The wake-
76 promoting LH cell population that has been studied most extensively to date are the
77 hypocretin/orexin (Hcrt) neurons (de Lecea et al., 1998; Peyron et al., 1998; Kilduff and Peyron,
78 2000; Gerashchenko et al., 2001; Bonnavion and de Lecea, 2010). These cells project to several
79 wake-promoting areas of the brain including the basal forebrain (BF), the tuberomamillary
80 nucleus (TMN) and the locus coeruleus (LC) where they can release the Hcrt peptides,
81 glutamate, dynorphin and perhaps other neurotransmitters (Torrealba et al., 2003; Eriksson et
82 al., 2004; Schone et al., 2012; Schone et al., 2014). In this regard, the Hcrt neurons have been
83 proposed to act like a stabilizer of a flip-flop switch that controls behavioral state (Saper et al.,
84 2005).
85 Narcolepsy is a neurological disease characterized by the extensive loss of Hcrt neurons
86 without degeneration of neighboring cells (Kilduff and Peyron, 2000; Thannickal et al., 2000).
87 Narcoleptic patients are unable to sustain long periods of consolidated wakefulness and must
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88 nap frequently; some patients also experience attacks of cataplexy which greatly affects their
89 quality of life (Kornum et al., 2017). However, total sleep time in untreated narcoleptic patients
90 as well as in mouse models of narcolepsy does not differ from controls over the 24h period
91 (Dantz et al., 1994; Hara et al., 2001; Tabuchi et al., 2014), a phenotype that is distinct from the
92 lethargy observed after LH lesions or acute LH neuronal inhibition. Chemogenetic excitation of
93 Hcrt neurons in mice (Sasaki et al., 2011) produced a 30% increase in wake time during the first
94 hour of stimulation compared to controls but no significant difference was observed after the
95 second hour, which also differs from the effects of LH excitation cited above. Thus, Hcrt neuron
96 stimulation and ablation studies do not fully recapitulate the effects on wake and sleep
97 observed after manipulations of LH neuron activity, indicating that there are likely other wake-
98 promoting neuronal populations in the LH that may be involved in sleep/wake regulation.
99 To determine whether LH stimulation has wake-promoting effects distinct from those
100 exhibited by Hcrt neuron stimulation, we performed chemogenetic excitation of LH
101 neurons(cid:3)while conducting EEG and EMG recordings of the transfected mice. We then evaluated
102 the effect of time-of-day on LH-mediated wake promotion and whether Hcrt signaling was
103 necessary to evoke arousal during LH activation. Finally, we combined transfection of LH
104 neurons with a diet-dependent expression of hM3Dq in Hcrt neurons to test whether the Hcrt
105 and non-Hcrt arousal pathways are parallel or overlapping. Our results show that LH neuronal
106 stimulation evokes profound but physiological arousal independent of Hcrt signaling and that
107 concurrent activation of Hcrt and non-Hcrt LH neurons has synergistic effects on arousal that
108 are attenuated in presence of the dual orexin receptor antagonist almorexant (ALM). These
109 results indicate the existence of an LH arousal pathway that can be activated even when Hcrt
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110 signaling is pharmacologically blocked, suggesting a novel target for treatment of sleep/wake
111 disorders such as narcolepsy.
112
113 Materials and Methods
114 Animals. Adult Gad2-IRES-Cre;R26R-EYFP (Jax stock # 010802 and 006148; n = 6 males
115 and 4 females, 2-4 months old) and Orexin-tTA mice (C57BL/6-Tg(orexin/tTA)G5/Yamanaka; n =
116 21 males, 2-4 months old ), which express the tetracycline transactivator (tTA) exclusively in the
117 Hcrt neurons (Tabuchi et al., 2014), were singly-housed under a 12h/12h light/dark cycle with
118 ad libitum access to food and water in temperature (24 ± 2°C) and humidity (50 ± 20% relative
119 humidity) controlled rooms. 16 of the Orexin-tTA mice were reared with chow that included
120 doxycycline (DOX). An additional cohort of C57BL/6J WT mice (Jax stock # 000664; n =4 males
121 and 4 females, 3-6 months old) were housed under the same conditions and used to assess the
122 effects of clozapine-N-oxide (CNO) treatment on sleep/wake in naïve mice (see Table 1). All
123 studies were conducted in accordance with the Guide for the Care and Use of Laboratory
124 Animals and were approved by the Institutional Animal Care and Use Committee at SRI
125 International. Every effort was made to minimize animal discomfort throughout these studies.
126 Surgical Procedures. Under isoflurane anesthesia, Gad2-IRES-Cre;R26R-EYFP and
127 Orexin-tTA mice were injected bilaterally into the LH (-1.4 AP, 1 ML relative to bregma and -5
128 DV from pia) with 340 nl of an AAV encoding hM3Dq-mCherry (AAV-TetO-hM3Dq-mCherry
129 (Sr10); titer 2x1011; Nagoya University, Japan) using a Picospritzer II (Parker Instrumentation,
130 Cleveland, OH) connected to a BK Precision 4003A Pulse Generator (B&K Precision Corporation,
131 Yorba Linda, CA) which delivered 5-20 ms pulses of pressurized air every 2 s into a glass
6
132 micropipette for 5-10 min. After a recovery period of at least two weeks, injected mice as well
133 as naïve C57BL/6J mice underwent surgery for implantation of EEG and EMG leads by
134 cementing a reference EEG screw over the occipital bone, one in the left frontal bone (1 AP, 1
135 ML), and another over the parietal bone (-2 AP, 2 ML). EMG electrodes were two stainless
136 steel-coiled wires anchored to the neck musculature. A 5-pin connector was connected to the
137 EEG and EMG leads and cemented to the skull for conventional tethered EEG/EMG recordings.
138 Experimental Procedures. To allow acclimation to the recording apparatus, mice were
139 connected to a tether that was attached to a commutator (Pinnacle Technology Inc., Lawrence,
140 KS) for at least 5 d prior to experiments. The commutator allowed the mice to move freely in
141 their home cages. For acclimation to the dosing procedure, mice underwent 3 i.p. injections of
142 saline on different days prior to initiation of the dosing study. Experiments occurred no sooner
143 than 21 d after the EEG/EMG implantation surgery. EEG and EMG data were collected at 800
144 Hz and digitally band-passed at 0.5-300 Hz for Gad2-IRES-Cre;R26R-EYFP and WT mice, and at
145 500 Hz and digitally band-passed at 0.5-200 Hz for Orexin-tTA mice using a TDT RZ2 system
146 connected to a 96-channel PZ2 amplifier and a low impedance RA16LI-D headstage (Tucker-
147 Davis Technologies, Alachua, FL). To monitor activity, infrared analog video was collected using
148 CNB B1760N4.3 cameras (CNB Technology Inc., Seoul, Korea) and digitized at 30 fps using a Q-
149 See QT5616 system (Q-See, Anaheim, CA). All animals underwent an undisturbed 24 h baseline
150 recording in their home cages prior to dosing experiments.
151 Dosing procedures. Clozapine N-oxide (Cat No. 4936, Tocris Bioscience, Avonmouth,
152 UK), the ligand for the modified muscarinic receptor hM3Dq, was injected i.p. at 3 mg/Kg in
153 saline solution at a concentration of 0.5 mg/ml in all experiments. To block Hcrt signaling, the
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154 dual orexin receptor antagonist almorexant (ALM), synthesized at SRI International (Black et al.,
155 2013), was injected i.p. at a concentration of 200 mg/Kg in vehicle (VEH) consisting of 1.25%
156 hydroxypropyl methyl cellulose (SKU 09963), 0.1% dioctyl sodium sulfosuccinate (SKU 323586)
157 and 0.25% methyl cellulose (SKU 274429) in water (all from Sigma-Aldrich, St. Louis, MO) after
158 vortexing a solution of 30 mg/ml for 2 h. Dosing sessions were at least 4 d apart and at least 7 d
159 elapsed between CNO dosings.
160 In Gad2-IRES-Cre;R26R-EYFP mice, dosing occurred either at ZT12 and EEG/EMG was
161 recorded for the subsequent 12 h or at ZT4 and the EEG/EMG was recorded for 9 h starting at
162 ZT3. In Orexin-tTA mice, dosing studies were also performed at ZT4 and EEG/EMG was
163 recorded from ZT4 to ZT11. For mice belonging to both the DOX(+) and DOX(-) cohort (n = 10),
164 the DOX-containing chow was replaced by normal chow at least 3 weeks before the dosing
165 study was repeated under the DOX(-) condition.
166 Locomotor activity data collection and analysis. Synchronization of EEG/EMG data and
167 time-stamped video was achieved by synchronizing the Q-See system’s clock to the TDT
168 computer’s clock and manually entering the start and end time of all behavioral videos. The
169 position of the mice was detected automatically using Noldus EthoVision XT 11.5 software
170 (Noldus Information Technology, Wageningen, Netherlands). To calculate the speed of the
171 mice in Figs. 3F,G, after manually curating the automatic tracking of the mouse’s position, we
172 obtained a matrix with the X and Y coordinates of each mouse 30 times/sec as well as a vector
173 with the relative time of each data point. A median filter and then an average filter with a
174 moving window of 10 time points and step of 1 time point was applied to both sets of
175 coordinates and the Euclidean distance with respect to the origin of the coordinate system was
8
176 obtained for every time point. The distance traveled was calculated as the absolute value of
177 the difference between the calculated distance and the distance from the first data point.
178 Finally, the speed was calculated as the absolute difference between two consecutive points of
179 the distance travelled, divided by the difference in time between the two consecutive points.
180 All data were processed using custom scripts created in Matlab 2015b (Mathworks, Natick,
181 MA).
182 EEG/EMG data collection and analysis. Using the criteria described previously (Morairty
183 et al., 2013; Dittrich et al., 2015), EEG/EMG data were visually scored as wake (W), non-rapid
184 eye movement sleep (NREM) or rapid eye movement sleep (REM) in 4 s epochs. A bout of W,
185 NR or REM was defined as three consecutive 4 s epochs (i.e., 12 s) within the same state. For
186 quantitative EEG analysis, the power spectrum was calculated for each 4 s artifact-free epoch
187 using the squared amplitude coefficients of the fast Fourier transform and the mean power in
188 the delta ((cid:71)), 0.5-4 Hz; low theta (L(cid:84)(cid:12)(cid:15) 4-8 Hz; high theta (H(cid:84)(cid:12)(cid:15)(cid:3)8-10 Hz; low gamma (L(cid:74)(cid:12)(cid:15) 15-50
189 Hz; high gamma (H(cid:74)(cid:12)(cid:15) 60-80 Hz; and very high gamma (Vh(cid:74)(cid:12)(cid:15)(cid:3)90-200 Hz range. To avoid 60 Hz
190 artifact, the spectral power values at 60 ± 1 Hz and the harmonics at 120 and 180 Hz were
191 replaced by a linear interpolation from the values at 59 and 61 Hz, 119 and 121 Hz and 179 and
192 181 Hz. Normalized W power was obtained by dividing the mean power spectrum during W by
193 the mean power in W during the same time period of a baseline recording.
194 Experimental design and statistical analyses. Table 1 summarizes the 6 dosing
195 experiments that were conducted. Results obtained from male mice were similar to those of
196 female mice and thus data from both sexes were grouped. For statistical analysis, we
197 performed 2-way repeated measures ANOVA using Sigmaplot (Systat Software Inc., San Jose,
9