The lateral habenula regulates stress-related respiratory responses via the monoaminergic system

Fig. 1. Histologic examination. a Histologic examination of the stimulation site. The slice is taken from 3.7 mm caudally from the bregma, which corresponds to 3.6 mm caudally from the bregma on the Paxinos and Watson rat brain atlas [21]. The arrow indicates a lesioned site that indicates a stimulation site in the LHb. The area enclosed by the dotted line is the LHb. D3V: Dorsal 3rd ventricle, f: fornix, LHb: lateral habenula, MD: Mediodorsal thalamic nucleus, mt: mammillothalamic tract, PV: Palaventricular thalamic nucleus, VPM: Ventral posteromedial thalamic nucleus, 3V: 3rd ventricle. b The orange dots show the stimulation sites in the experiments used to observe the effects of LHb stimulation on respiratory movement (n = 5). The red, blue, and purple dots show the stimulation sites in the LHb with the administration of clozapine (n = 5), the vehicle (n = 5), and methysergide (n= 5). The green dots show the stimulation sites in the LHb with the administration of muscimol to the VTA (n = 6)

Fig. 2. LHb stimulus frequency-dependent changes in respiratory responses. a Respiratory responses elicited by LHb stimulation at each stimulus frequency (10 Hz, 30 Hz, 50 Hz, and 100 Hz). The black bars at the top show the period of LHb stimulation (10 s). LHb: lateral habenula. cpm: cycles per minute. b Analyzed respiratory responses during LHb stimulation at each stimulus frequency. Asterisks indicate p < 0.05 vs prestimulus (n = 5). cpm: cycles per minute, bpm: beats per minute

Fig. 3. Stimulus intensity-dependent changes in respiratory responses elicited by LHb stimulation. a Respiratory responses elicited by each stimulus intensity (50 µA, 100 µA, 300 µA, 500 µA, 700 µA, 1 mA). The black bars at the top show the period of LHb stimulation (10 s). LHb: lateral habenula. cpm: cycles per minute. b Analyzed respiratory responses during LHb stimulation at each stimulus intensity. Asterisks indicate p < 0.05 vs prestimulus (n = 5). cpm: cycles per minute, bpm: beats per minute

Fig. 4. Effects of administering vehicle on LHb-induced respiratory responses. a Respiratory responses before (top) and after (bottom) administration of clozapine. The black bars at the top show the period of LHb stimulation. LHb: lateral habenula. cpm: cycles per minute. b Changes in respiratory responses after administration of the vehicle compared with respiratory responses before the administration (n= 5)

Fig. 5. Effects of blockade of dopaminergic receptors on LHb-induced respiratory responses. a Respiratory responses before (top) and after (bottom) administration of clozapine. The black bars at the top show the period of LHb stimulation. LHb: lateral habenula. cpm: cycles per minute. b Changes in respiratory responses after administration of clozapine compared with respiratory responses before the administration. Asterisks indicate p < 0.05 vs prestimulus (n = 5)

Fig. 6. Effects of blockade of serotonergic receptors on LHb-induced respiratory responses. a Respiratory responses before (top) and after (bottom) administration of methysergide. The black bars at the top show the period of LHb stimulation. LHb: lateral habenula. cpm: cycles per minute. b Changes in respiratory responses after administration of methysergide compared with respiratory responses before the administration. Asterisks indicate p < 0.05 vs prestimulus (n= 5)

Fig. 7. Effects of inactivation of the VTA on LHb-induced respiratory responses. a Respiratory responses before (top) and after administration of muscimol to the ipsilateral (middle) and bilateral (bottom) VTA. b Changes in respiratory responses after ipsilateral and bilateral administration of muscimol compared with respiratory responses before the administration. Asterisks indicate p < 0.05 vs prestimulus (n= 6)