Acute Administration of the Nonpathogenic, Saprophytic Bacterium, Mycobacterium vaccae, Induces Activation of Serotonergic Neurons in the Dorsal Raphe Nucleus and Antidepressant-Like Behavior in Association with Mild Hypothermia

Abstract

Peripheral immune activation can have profound physiologic and behavioral effects. One mechanism through which immune activation may affect physiology and behavior is through actions on brainstem neuromodulatory systems, such as serotonergic systems. To test this hypothesis, in Experiment 1, adult male BALB/c mice were implanted with telemetric recording devices and then immunized with Mycobacterium vaccae NCTC 11659 (0.1 mg, s.c.; Days - 28, - 14; N = 36). On Day 1, mice received an acute challenge with M. vaccae (0.1 mg, s.c.) or borate-buffered saline vehicle. Core body temperature and locomotor activity recordings were conducted during a 36 h period beginning 24 h prior to challenge; 12 h following acute challenge, mice were either tested in a 6-min forced swim test, or served as home cage controls (n = 9 per group). In Experiment 2, the protocol was repeated, but with the aim of assessing c-Fos expression in brainstem serotonergic neurons, assessed 90 min following exposure to forced swim (N = 32; n = 8 per group). In Experiment 1, acute M. vaccae challenge in M. vaccae-immunized mice, relative to vehicle-challenged controls, decreased locomotor activity and core body temperature measured 3 h following challenge, as measured by continuous telemetric recordings, and decreased immobility in the forced swim test measured 12 h following challenge. In Experiment 2, acute M. vaccae challenge in M. vaccae-immunized mice decreased home cage locomotion, in alignment with findings in Experiment 1, as measured by video-based behavioral analysis, and, among mice exposed to the forced swim test, increased c-Fos expression in subsets of serotonergic neurons within the dorsal raphe nucleus (DR) measured 13.5 h following challenge. Together, these data are consistent with the hypothesis that acute peripheral immune activation with a heat-killed preparation of M. vaccae transiently induces mild hypothermia in association with suppression of locomotor activity, activates subsets of serotonergic neurons in the DR, and induces antidepressant-like behavioral responses.

Keywords: Actinobacteria; Antidepressant; Body temperature; Hypothermia; M. vaccae; Mycobacterium vaccae.

Fig. 1

Experimental timelines for a Experiment 1 and b Experiment 2. Day 1 is defined as the day of the challenge with M. vaccae or vehicle, s.c., whereby Day 1 is defined as the day immediately prior to the challenge. DR dorsal raphe nucleus, FST forced swim test, IHC immunohistochemistry, i.p intraperitoneal, LMA locomotor activity, s.c subcutaneous, Tb core body temperature

Fig. 2

Graphs illustrating effects of s.c. M. vaccae challenge on a core body temperature (Tb) and b locomotor activity (LMA) in Experiment 1. All mice were immunized (s.c) with heat-killed M. vaccae NCTC 11659 (0.1 mg/100 µl, s.c.) on Days − 28 and − 14 and were challenged (s.c.) with either M. vaccae (n = 17; 0.1 mg/100 µl s.c.) or borate-buffered saline (BBS) vehicle (n = 16) on Day 1. Core body temperature (Tb; °C) and locomotor activity (LMA; arbitrary units, a.u.) were monitored in the home cage environment; cages were placed on receivers 24 h before challenge injections for habituation. Telemetry data were sampled for a 5 s period at 128 Hz at 1 min intervals. The graphs display the mean ± S.E.M. for average values for each 30-min interval, illustrated at the start time of each averaged 30-min interval. Vertical dashed lines indicate the time at which the s.c. injections were given; horizontal black bars indicate the dark phase of the light/dark–light cycle. Statistical significance was assessed by linear mixed model analysis followed by Fisher’s least significant difference (LSD) tests at individual time points; *p < 0.05, relative to vehicle-treated controls at the same time point. a.u., arbitrary units; Tb, core body temperature

Fig. 3

Effects of s.c. M. vaccae challenge, in undisturbed M. vaccae-immunized mice, on forced swim test (FST) behavior in Experiment 1. All mice were immunized (s.c) with M. vaccae (0.1 mg/100 µl, s.c.) on Days − 28 and − 14 and were challenged (s.c.) with either M. vaccae (0.1 mg/100 µl, s.c.) or borate-buffered saline (BBS) vehicle on Day 1. Mice underwent a 6-min FST 12 h after challenge. Bars representing group means + SEM are shown for  % time spent engaged in each behavior during the final 4 min of the test. Statistical significance was assessed by Student’s t-tests. *p < 0.05, compared to vehicle-challenged mice. Sample sizes: vehicle, n = 9; M. vaccae, n = 9

Fig. 4

Graphs illustrating effects of s.c. M. vaccae challenge on a core body temperature (Tb) and b locomotor activity (LMA) in Experiment 2. All mice were preimmunized (s.c) with heat-killed M. vaccae NCTC 11659 (0.1 mg/100 µl, s.c.) on Days − 28 and − 14 and were challenged (s.c.) with either M. vaccae (0.1 mg/100 µl s.c.) or borate-buffered saline (BBS) vehicle on Day 1. Core body temperature (Tb; ºC) and locomotor activity (LMA; arbitrary units) were monitored in the home cage environment during three 30-min periods, (1) a baseline period from − 30 min to 0 min, immediately before acute challenge with M. vaccae or vehicle (HC1), (2) from 2 h to 2 h, 30 min following challenge (HC2), and (3) from 10 h to 10 h, 30 min following challenge (HC3). Statistical significance was assessed by linear mixed model analysis followed by Fisher’s least significant difference tests. Sample sizes vehicle, n = 15; M. vaccae, n = 16. a.u., arbitrary units; Tb, core body temperature

Fig. 5

Effects of s.c. M. vaccae challenge, in M. vaccae-immunized mice, on home cage behavior in Experiment 2. All mice were immunized (s.c) with M. vaccae (0.1 mg/100 µl s.c.) on Days − 28 and − 14 and were challenged (s.c.) with either M. vaccae (0.1 mg/100 µl s.c.) or borate-buffered saline (BBS) vehicle on Day 1. Behaviors were monitored in the home cage environment during three 30-min periods, (1) a baseline period from − 30 to 0 min, immediately before acute challenge with M. vaccae or vehicle (HC1), 2) from 2 h to 2 h, 30 min following challenge (HC2), and 3) from 10 h to 10 h, 30 min following challenge (HC3). Bars representing group means + SEM are shown for time spent engaged in each behavior. Statistical significance was assessed by linear mixed model analysis followed by Fisher’s least significant difference tests. *p < 0.05, compared to M. vaccae-immunized, vehicle-challenged mice. ⁺ p < 0.01, main effect of time. Sample sizes: vehicle, n = 15; M. vaccae, n = 16

Fig. 6

Effects of s.c. M. vaccae challenge, in M. vaccae-immunized mice, on c-Fos expression in serotonergic neurons in subregions of the dorsal raphe nucleus (DR) in Experiment 2. a Photomicrographs illustrating subregions of the DR. b Cell count data for c-Fos-immunoreactive/tryptophan hydroxylase (Tph)-immunoreactive neurons (closed bars) and the total number of serotonergic neurons sampled (i.e. c-Fos-immunoreactive/Tph-immunoreactive neurons and c-Fos-immunonegative/Tph-immunoreactive neurons; open bars) in selected subdivisions of the DR, measured 13.5 h following immunization with either vehicle or M. vaccae, and 1.5 h following exposure to home cage control conditions or a 6-min forced swim test. All mice were immunized (s.c.) with M. vaccae (0.1 mg/100 µl, s.c.) on Days − 28 and − 14 and were challenged (s.c.) with either M. vaccae (0.1 mg/100 µl, s.c.) or borate-buffered saline (BBS) vehicle on Day 1. Mean cell counts + S.E.M. are shown. Statistical significance was assessed by linear mixed model analysis followed, when appropriate, by Fisher’s least significant difference tests; *p < 0.05, compared to vehicle-challenged mice within the same forced swim test condition. Sample sizes: vehicle/home cage, n = 7; vehicle/forced swim test (FST), n = 8; M. vaccae/home cage, n = 8; M. vaccae/FST), n = 8. Abbreviations: Aq, cerebral aqueduct; CLi, caudal linear nucleus; DRC, dorsal raphe nucleus, caudal part; DRD, dorsal raphe nucleus, dorsal part; DRI, dorsal raphe nucleus, interfascicular part; DRV, dorsal raphe nucleus, ventral part; DRVL/VLPAG, dorsal raphe nucleus, ventrolateral part/ventrolateral periaqueductal gray; ir, immunoreactive; mlf, medial longitudinal fasciculus; M.v., M. vaccae; Tph, tryptophan hydroxylase; Veh, borate-buffered saline vehicle. Rostrocaudal coordinates are indicated in mm with reference to bregma. Scale bar = 200 µm

Fig. 7

Effects of s.c. M. vaccae challenge, in M. vaccae-immunized mice, on c-Fos expression in non-serotonergic cells in subregions of the dorsal raphe nucleus (DR) in Experiment 2. a Photomicrographs illustrating subregions of the DR. b Cell count data for c-Fos-immunoreactive/tryptophan hydroxylase (Tph)-immunonegative neurons in selected subdivisions of the DR, measured 13.5 h following immunization with either vehicle or M. vaccae, and 1.5 h following exposure to home cage control conditions or a 6-min forced swim test (FST). All mice were immunized (s.c.) with M. vaccae (0.1 mg/100 µl, s.c.) on Days − 28 and − 14 and were challenged (s.c.) with either M. vaccae (0.1 mg/100 µl, s.c.) or borate-buffered saline (BBS) vehicle on Day 1. Mean cell counts + S.E.M. are shown. Statistical significance was assessed by linear mixed model analysis followed, when appropriate, by Fisher’s least significant difference tests; +p < 0.05, compared to home cage controls within the same M. vaccae challenge condition. Sample sizes: vehicle/home cage, n = 7; vehicle/FST, n = 8; M. vaccae/home cage, n = 8; M. vaccae/FST, n = 8. Aq, cerebral aqueduct; CLi, caudal linear nucleus; DRC, dorsal raphe nucleus, caudal part; DRD, dorsal raphe nucleus, dorsal part; DRI, dorsal raphe nucleus, interfascicular part; DRV, dorsal raphe nucleus, ventral part; DRVL/VLPAG, dorsal raphe nucleus, ventrolateral part/ventrolateral periaqueductal gray (Note: serotonergic neurons in the DRVL and VLPAG appear to be functionally related (Johnson et al. 2004) and were counted as a single population); ir, immunoreactive; mlf, medial longitudinal fasciculus; M.v., M. vaccae; Tph, tryptophan hydroxylase; Veh, borate-buffered saline vehicle. Rostrocaudal coordinates are indicated in mm with reference to bregma. Scale bar = 200 µm