Mounting evidence supports the influence of the gut microbiome on the local enteric nervous system and its effects on brain chemistry and relevant behavior. Vagal afferents are involved in some of these effects. We previously showed that ingestion of the probiotic bacterium Lactobacillus rhamnosus (JB-1) caused extensive neurochemical changes in the brain and behavior that were abrogated by prior vagotomy. Because information can be transmitted to the brain via primary afferents encoded as neuronal spike trains, our goal was to record those induced by JB-1 in vagal afferents in the mesenteric nerve bundle and thus determine the nature of the signals sent to the brain. Male Swiss Webster mice jejunal segments were cannulated ex vivo, and serosal and luminal compartments were perfused separately. Bacteria were added intraluminally. We found no evidence for translocation of labeled bacteria across the epithelium during the experiment. We recorded extracellular multi- and single-unit neuronal activity with glass suction pipettes. Within minutes of application, JB-1 increased the constitutive single- and multiunit firing rate of the mesenteric nerve bundle, but Lactobacillus salivarius (a negative control) or media alone were ineffective. JB-1 significantly augmented multiunit discharge responses to an intraluminal distension pressure of 31 hPa. Prior subdiaphragmatic vagotomy abolished all of the JB-1-evoked effects. This detailed exploration of the neuronal spike firing that encodes behavioral signaling to the brain may be useful to identify effective psychoactive bacteria and thereby offer an alternative new perspective in the field of psychiatry and comorbid conditions.

it is known that the acquisition of a normal microbiome is necessary for the development of a complete immunological repertoire (8, 19), including that of the immune regulatory system (29). Even monoassociation of germ-free mice with, for example, the segmented filamentous bacterium can restore the immune system to normality, indicating the strain dependency of these events (35, 52). Also, the presence of a normal microbiome may be constitutively anti-inflammatory and antinociceptive (31, 51, 54). However, very little is known about the role of the gut microbiome in the development or function of the nervous system.

It has been recently reported that nonpathogenic intestinal microorganisms can signal to the brain as part of the so-called microbiome-gut-brain axis (9, 19, 22, 38). Changes in the microbiome have been noted in the behavior of healthy noninfected animals, the most striking of which have been demonstrated in recent experiments showing that certain aspects of behavior in different strains of adult mice were dependent on the content of their respective fecal microbiomes (1). Most of the information reaching the brain via primary sensory afferents, including the vagus fibers, has to be encoded in the language of neuronal spike trains that represent the nature and intensity of the stimulus (20). Therefore, knowing how the sensory spike trains are affected by commensals or probiotics might enable us to screen for and identify new potentially beneficial gut microorganisms, and eventually their active molecules by their effects on primary afferent firing.

In terms of nociception, treatment of mice with nonabsorbable antibiotics increased pseudoaffective responses to colorectal distension, and this could be prevented by ingestion of Lactobacillus paracasei (54). Similar responses in rats reflecting perception of visceral pain evoked by colorectal distension were almost completely inhibited by 9 days of gavage with L. rhamnosus (JB-1) (31). Spontaneous and distension-evoked action potential firing recorded from single dorsal root fibers was also decreased by prior ingestion of the same bacteria (31, 37). In addition, JB-1 ingestion quenched long-term dorsal root ganglion neuron hyperexcitability elicited by repeated colorectal distension in healthy animals. Ingestion of JB-1 within minutes of application to the epithelium (34, 55) facilitates firing of enteric intrinsic primary afferent neurons (34) and moderates peristaltic reflexes (55). These observations have taught us that changes in the gut microbiome can significantly influence the activity of the enteric nervous system and spinal pathways.

Moreover, we recently reported that feeding JB-1 for 28 days to conventionally housed mice decreased anxiety-related behavioral scores and induced widespread changes in the expression of brain γ-aminobutyric acid (GABA) receptors (7). An important finding in this study was that the intact vagus was essential since prior vagotomy abrogated these behavioral and central neurochemical effects.

It is not clear from current knowledge whether psychoactive bacteria are characterizable in terms of their potential vagal activation or even other evidence of neuroactivity. For example, JB-1 decreases constitutive and distension-evoked firing in dorsal root ganglia neurons (31), which is consistent with its antinociceptive properties, yet its effects on myenteric intrinsic primary afferent neurons is one of enhanced intrinsic excitability (34).

The present experiments were therefore designed to study how probiotic bacteria might affect the electrical discharges in the mesenteric nerve bundle, which contains vagus afferent fibers. We have recorded from afferent fibers, since they emerge from the intestine in an ex vivo intestinal jejunal segment mesenteric nerve recording preparation (32). This has allowed us to control bacterial concentrations in the lumen and measure spike firing onset latencies from the moment of bacterial application to the epithelium (55). Our hypothesis was that JB-1 might reduce the firing rate of vagal fibers, since this bacteria had a vagally dependent anxiolytic effect (7). We have shown that introduction of JB-1, and not another Lactobacillus strain, in the lumen of such a segment within minutes causes an increase in the constitutive firing rate, increases the neuronal response to gut distension, and that both of these effects are prevented by prior subdiaphragmatic vagotomy.

MATERIALS AND METHODS

All procedures adhered to Canadian Council on Animal Care guidelines and were approved by the Animal Research Ethics Board of McMaster University (permit no. 08-08-35).

Extracellular Recordings

Adult male Swiss Webster mice (20–30 g) were procured from Charles River Laboratories (Wilmington, MA). The mice were killed by cervical dislocation. All ensuing procedures were ex vivo.

Segments of excised distal jejunum (∼2.5 cm) with attached mesenteric tissue were removed from dead animals and immediately placed in a Sylgard-coated recording petri dish filled with Krebs buffer of the following composition (in mM): 118 NaCl, 4.8 KCl, 25 NaHCO 3 , 1.0 NaH 2 PO 4 , 1.2 MgSO 4 , 11.1 glucose, and 2.5 CaCl 2 bubbled with carbogen (95% O 2 -5% CO 2 ). The oral and anal portions of gut were cannulated with plastic tubing and gently emptied using an attached syringe filled with oxygenated Krebs buffer. The gut and mesenteric tissue were pinned out, the mesenteric nerve bundle was isolated by careful dissection under a stereomicroscope, and the preparation was transferred to an inverted microscope and then lumen gravity perfused at 0.5–1 ml/min with room temperature (∼22°C) oxygenated Krebs and/or additives using several Mariotte bottles (56). The serosal compartment was separately perfused with prewarmed (34°C) Krebs solution at 3–5 ml/min. The nerve bundle was gently sucked into a glass pipette attached to a patch-clamp electrode holder (CV-7B; Molecular Devices, Sunnyvale, CA), and extracellular nerve recordings were made using a Multi-Clamp 700B amplifier and Digidata 1440A signal converter (Molecular Devices). Electrical signals were bandpass-filtered at 0.1–2 kHz, sampled at 20 kHz, and stored on a personal computer running pClamp 10 software (Molecular Devices) for post hoc analysis. Constitutive multiunit electrical activity was always recorded from the mesenteric nerve bundle, even in the absence of active distension. Repeated distensions of the segment were made by infusing Krebs in the lumen of jejunal segments at a constant gravity pressure head of 14 or 31 hPa and closing the outflow tube for 1 min each time (up to a maximum of 5 times consecutively). Segments were allowed to rest with no imposed distension for 9 min between repeated distensions. We chose several single units in selected multiunit recordings and made templates with them using the spike identification module in Clampfit. The templates were then used to identify the individual single units by template matching successive spikes for shape, duration, and size.

Vagotomy

For some experiments, a subdiaphragmatic vagotomy was carried out as previously described (53). Animals were allowed to recover for 10–14 days before harvesting the jejunum and mesenteric tissue for electrophysiological experiments. Sham vagotomy was performed in three animals. Postoperatively, the body weight and general health of the mice were measured daily (5, 26). We found no evidence of significant differences in weight gain 1 wk postsurgery in either vagotomized or sham-treated animals [data not shown (43)]. All vagotomized mice were tested for completeness of the procedure by recording after each experiment the responses to serosal application of cholecystokinin (CCK). Vagotomy was deemed to have been effective when CCK did not increase mesenteric nerve firing rate (25).

Integrity of Jejunal Segments

To test the integrity of the jejunal segments with respect to translocation of bacteria across the epithelium during an experiment, we labeled JB-1 with 5-(6)-carboxyfluorescein succinimidyl ester (CFSE), placed these at a concentration of 109/ml Krebs in the lumen, and, after incubation for 75 min, fixed the tissue in paraformaldehyde and examined sections for their presence below the epithelium in confocal microscopy (dual-laser microscopy, LSM 510; Carl Zeiss).

JB-1 were washed two times in PBS and suspended in a final concentration of CFSE of 5 μM in PBS supplemented with 5% fetal calf serum and incubated for 25 min at 37°C. The tissues (n = 3) were fixed in 4% paraformaldehyde at 4°C overnight and then washed three times in PBS for 10 min each and sectioned at 10 and 30 μm; transverse sections were transferred to microscope slides and mounted. These were then reviewed in optical slices by the Z-stacking methodology.

Probiotics and Drugs

L. rhamnosus (JB-1), previously referred to as L. reuteri and subsequently identified by genomic analysis as an L. rhamnosus, were taken from in-house stock (34, 37, 54); for more information, please see the supplementary information in Bravo et al. (7). L. salivarius was a gift from the Alimentary Pharmabiotic Centre (University College Cork, Cork, Ireland) (44). Bacterial cell numbers were determined optically, and viability was checked by the ability to grow after plating on growth medium agar plates. All other methods were as reported previously (34, 37, 55). Briefly, live L. rhamnosus were grown from frozen (−80°C) 1-ml aliquots that consisted of 5 × 109 cells in Man-Rogosa Sharpe broth (Difco Laboratories, Sparks, MD). Bacteria from frozen stocks were thawed and centrifuged at 2,000 rpm for 15 min, and the pellet was suspended in an equal volume of Krebs buffer. The suspension was again centrifuged, and the bacteria was removed and resuspended in Krebs at the original concentration. Just before use, the bacteria were diluted to working concentrations with Krebs buffer. For some experiments the bacteria were diluted directly to working concentrations after thawing (in broth); the bacteria were always applied in the lumen of the jejunal segment. Cholecystokinin (25–33) sulfated (CCK; AnaSpec, Fremont, CA) was dissolved in dimethyl sulfoxide (DMSO) to make a 1 mM stock solution. Aliquots were diluted on the day of the experiment to a working concentration in Krebs buffer, with a final DMSO concentration ≤0.0001%.

Off-Line Data Analysis

Multi- and single-unit spontaneous firing frequencies of mesenteric afferent nerve bundles were measured using Clampfit 10.2 (Molecular Devices) and Origin 8.5 (Northampton, MA) software. Both methods (multiunit spike discharge and waveform analysis) of measurement are routinely used to determine changes in the excitability of the mesenteric nerve fibers induced by different treatments (6, 25, 27, 32, 45, 49). The timing of the multiunit spikes was determined using the peak detection module of Clampfit, and average frequency was calculated from spike intervals. Single-unit activity was isolated from the multiunit activity using the spike shape automatic template detection tool of Clampfit (computerized waveform analysis). After template detection, discrimination was always checked by visual inspection, and nonmatching spike events were discarded (<0.2%).

Statistics

Data are expressed as means ± SE with n referring to the total number of the jejunal segments recorded; the maximum number of segments recorded from the same animal was two. The Wilcoxon test was used for paired data comparisons, and Friedman test with Dunn's post hoc test for repeated measures ANOVA were performed using Prism software 5.0 (GraphPad Software, San Diego, CA). Because large variations in spontaneous activity may occur between one preparation and another in multiunit neural activity (49), all comparisons were paired with before and after treatment recordings made where each nerve bundle served as its own control. Differences were considered significant if P ≤ 0.05.

RESULTS

Stability of the Recordings and Integrity of the Tissue

We always recorded spontaneous neural activity for at least 5–10 min before any stimuli were applied (gut distension, bacteria, etc.). Application of 1 μM of the sodium channel blocker tetrodotoxin always eliminated this neural activity (data not shown). Complete experiments lasted ∼1 h, and during this time the preparation remained stable and healthy (see Fig. 1, A–C). The mucosa of distal jejunum can remain healthy up to ∼4 h in similar experiments involving Ussing chambers (28). Effects of bacteria began to be evident after 10–15 min of luminal application.

Fig. 1.Extracellular multiunit activity from mesenteric nerve bundle is stable during experiments. A: firing frequency during 60 min does not vary significantly in the absence of stimuli. Distension stimulus increases firing frequency only transiently. Each point illustrates the average of firing frequency ± SE of each 0.6 min of one nerve bundle recording. B: firing frequency of first and last 5-min recording not exposed to drugs or bacteriological treatment do not vary significantly (n = 7, P = 0.46, Wilcoxon test). C: average rundown in the same group as B is 3.3 ± 4%. D: gut distension with 32 cmH 2 O (31 hPa) applied with a gravity system during ∼1 min (each 10 min) induces a remarkable increase in basal firing frequency. After the first distension, subsequent distensions induce a smaller increase in firing frequency that remains constant for up to three further distensions (n = 4, P = 0.67, Friedman test with Dunn's post hoc). The second distension was used as a control in further experiments. E: labeled Lactobacillus rhamnosus (JB-1) with 5-(6)-carboxyfluorescein succinimidyl ester (CFSE) placed in the lumen at 109/ml Krebs during 75 min. The section reveals fluorescent bacteria in the lumen but not in the epithelial layer or below in the mucosa or deeper layers (n = 3).

Distensions consistently increased the average multi- and single-unit firing frequency (Fig. 1A); the response to the first distension was the largest with subsequent (up to 4 tried) responses being of equal intensity (Fig. 1D). Because of this, in further experiments bacteria were applied after the second distension, and we made up to three further measures of the gut response to distension.

We failed to find evidence of translocation of bacteria across the epithelium. Examination of the slides in confocal microscopy from several sections in each of three jejunal segments taken over the time course of the experiments revealed numbers of labeled bacteria in the lumen, but no fluorescent bacteria were seen in the epithelial layer or below it in the mucosa, submucosa, or myenteric plexus (Fig. 1E).

Effects of JB-1 on the Spontaneous Firing Frequency of Mesenteric Afferent Nerve Bundles

Application of 5 × 107 colony-forming units (cfu)/ml of JB-1 (in broth) had no effect on basal multiunit firing frequency (before and after JB-1: 20.7 ± 2.4 vs. 19.7 ± 2.1 Hz) (n = 10, P = 0.15, Fig. 2A). JB-1 at 1 × 108 cfu/ml also had no apparent effect on the nerve firing frequency (data not shown). When the JB-1 concentration was increased to 1 × 109 cfu/ml, we observed a transient increase in the basal firing frequency in both conditions (with Krebs and broth; no significant differences were found between these two groups) [from 14.2 ± 2.5 to 19.6 ± 3.3 Hz, n = 17/24 (70.8% of cases), P = 0.0002; pooled results, Fig. 2, B and F]. These effects were evoked within 10–15 min of the onset of intraluminal application, even when the bacteria were previously washed and diluted in Krebs solution, and we used this effective concentration (1 × 109 cfu/ml) in all subsequent experiments. Table 1 summarizes the degree of JB-1 actions (% of increase) on constitutive firing frequency and on the response to distension in all experimental groups. We saw no time-dependent rundown in mesenteric nerve discharge, and no differences when Krebs was substituted for diluted broth (25.1 ± 3.8 vs. 23.9 ± 3.4 Hz) (n = 12, P = 0.21, Fig. 2C). Because we previously showed that L. salivarius had no effect on gut contractility (55), we tested it in our system. This strain of Lactobacillus had no effect at 1 × 109 cells/ml on the basal firing frequency (11.1 ± 2.5 vs. 9.9 ± 2.0 Hz; n = 11, P = 0.36, Fig. 2D) so we used it as a further negative control.

Fig. 2.L. rhamnosus (JB-1) increases the spontaneous firing frequency in the gross fiber population of the mesenteric afferent nerve bundle. A: graph shows spontaneous firing frequency before and after 5 × 107 JB-1/ml (in broth; P = 0.15, n = 10). B: JB-1 [1 × 109 colony-forming units (cfu)/ml; ***P = 0.0002, n = 17/24 (70.8%), Wilcoxon test]. C: effects of broth alone (P = 0.21, n = 12). D: 1 × 109L. salivarius/ml (in Krebs, P = 0.36, n = 11). E: 1 × 109 JB-1/ml (in Krebs) after treatment with 3 μM of nicardipine [L-type Ca2+ channels blocker; **P = 0.002, n = 9/13 (69.2%), Wilcoxon test]. F: representative traces of multiunit extracellular recordings in control conditions (Krebs in lumen; left). Right, after addition of 1 × 109 JB-1/ml. Dotted rectangles show details with faster time base. Scales are the same for both traces.

Table 1. Degree of JB-1 actions on constitutive firing frequency and on the response to distension in all experimental groups Multiunit constitutive firing frequency 5 × 107 2 ± 4% (n = 10 of 10) 1 × 109 50 ± 12% (n = 17 of 24†) 1 × 109 + 3 μM nicardipine 26 ± 5% (n = 9 of 13†) 1 × 109 (vagotomized) 2 ± 6% (n = 11 of 11) 1 × 109 (sham-treated) 51 ± 10% (n = 5 of 5†) Single-unit constitutive firing frequency 1 × 109 59 ± 13% (n = 12 of 17†) 1 × 109 + 3 μM nicardipine 64 ± 13% (n = 12 of 18†) Multiunit response to distension* 1 × 109 108 ± 51% (n = 10 of 14†) 1 × 109 (vagotomized) 11 ± 13% (n = 5 of 5) Single-unit response to distension* 1 × 109 118 ± 29% (n = 11 of 17†)

To test if the JB-1 effect on the multiunit activity was mediated via muscle contraction, the smooth muscle was paralyzed using the L-type calcium channel blocker nicardipine (33). When 3 μM of nicardipine was added to the Krebs in the perfusion system, before applying intraluminal JB-1, the bacteria still increased the multiunit firing frequency [15.1 ± 7 vs. 19.3 ± 9 Hz, n = 9/13 (69.2%), P = 0.002, Fig. 2E].

The Firing Frequency Increase Induced by JB-1 Could be Detected Using Single-Unit Recording

The extracellular multiunit recording data were derived from the average discharge of multiple single fibers, and such averaging may have obscured specific treatment effects. Because the mesenteric nerve bundle contains both spinal and vagal fibers (Fig. 3B) (4), it is possible that subpopulations of fibers could respond differently to different treatments (25). To control for this we measured the effects of applying intraluminal bacteria on single-unit firing frequency (49). Therefore, we tested the effects of JB-1 on shape-identified (25, 49) single-unit activity on 1-min time segments taken before and at 10–15 min after adding bacteria (see materials and methods and Fig. 3A) (49). We compared the effects on single-unit firing frequency with results from several responsive multiunit data. Seventy percent of single units showed a clear increase in the spontaneous firing frequency when JB-1 (1 × 109 cfu/ml) was applied. When we averaged the results for all the analyzed single units, the increase was from 2 ± 0.4 to 2.8 ± 0.5 Hz (n = 17, P = 0.001), which is similar to the percentage of increase found in the multiunit analysis with this JB-1 concentration. Next, we included for analysis only fibers that had an increase in the firing frequency [increase from 2.3 ± 0.6 to 3.3 ± 0.7 Hz, n = 12/17 (70%), P = 0.001, Fig. 4, A and D] to show that the increase in multiunit firing frequency was not primarily due to an increase in the total number of active fibers but to an increase in the firing rate of individual previously active primary afferent fibers. Broth alone did not have an effect on firing frequency of extracted single fiber activity (1.9 ± 0.2 vs. 1.8 ± 0.2 Hz, n = 16, P = 0.07, Fig. 4B). With 3 μM of nicardipine previously added to the Krebs, JB-1 still increased the single-unit firing frequency (1.1 ± 0.1 vs. 1.4 ± 0.1 Hz, n = 18, P = 0.006); we again wanted to include in the analysis only the fibers that increased the firing frequency with JB-1; in this case the effect was from 1 ± 0.2 to 1.5 ± 0.2 Hz [n = 12/18 (66.6%), P = 0.001, Fig. 4C].

Fig. 3.Single-unit activity extracted from multiunit recordings. A: top, example of extracellular multiunit recording segment that contains the activity of 3 distinguishable single fibers comparable in shape, size, and duration. Individual action potentials of each single fiber are marked on top of the voltage trace with different grayscale tone and detected according to fit to a template from the same recording (Clampfit program analysis). Bottom, superimposed recordings from multiple events from examples of each single fiber. B: illustration of how the mesenteric nerve bundle is constituted by a mixed population of single vagal and spinal fibers that innervates the gut wall.

Fig. 4.Spontaneous firing frequency increase induced by JB-1 can be detected in single-unit activity. A: 1 × 109 JB-1 effects on basal single-fiber firing frequency [**P = 0.001, n = 12/17 (70.5%), Wilcoxon test]. B: effects of broth alone (P = 0.07, n = 16). C: 1 × 109 JB-1/ml (in Krebs) after treatment with 3 μM of nicardipine [**P = 0.001, n = 12/18 (66.6%), Wilcoxon test]. D: representative traces of multiunit recordings: Krebs in lumen (left) and after addition of 1 × 109 JB-1/ml (right); enlarged single-unit waveforms extracted from the traces shown above (bottom).

Effect of JB-1 on Distension-Evoked Increases in Firing Frequency

Distensions consistently increased average multiunit and single-unit firing frequencies (see materials and methods, Figs. 1A and 5A). The percentage firing frequency increase above resting spontaneous discharge during the second distension was 352 ± 115 vs. 305 ± 82% on the third distension and 345 ± 112% on the fourth distension (the lumen contained only Krebs in these experiments) (Fig. 1D).

Fig. 5.JB-1 augmented the increase of nerve bundle firing frequency induced by gut distension. A: multiunit activity; increased firing frequency during gut distension (for 1 min each 10 min) at 31 hPa or 32 cmH 2 O. Dotted rectangles show details with faster time base before (left) and after (right) gut distension. B: firing frequency increase induced by gut distension before and after addition of 1 × 109 JB-1/ml [**P = 0.001, n = 10/14 (71.4%), Wilcoxon test]. C: single-unit waveforms exemplify the firing frequency increase (%) of single fibers before and after the addition of 1 × 109 JB-1/ml. D: graph showing the results exemplified in C [**P = 0.001, n = 11/17 (64.7%), Wilcoxon test].

In another set of experiments, we examined if JB-1 could modify the frequency increase evoked by distension. Distension in the presence of JB-1 at 1 × 109 cfu/ml (added after the 2nd distension) induced a robust increment compared with the response evoked previously by gut distension when only Krebs was present [244.4 ± 34% on the 2nd distension vs. 406.8 ± 62% on the 4th distension; n = 10/14 (71.4%), P = 0.001, Fig. 5B]. Also, JB-1 at 1 × 108 cfu/ml or broth by itself (data not shown) did not have an effect on the nerve response to gut distension. To test if this bacteria increases the response to distension in all the population of responsive mesenteric nerve fibers or in a subset of them, we carried out single-unit extractions before and during distension from the experiments with and without (Krebs in the lumen) the effective dose of JB-1; we have found that 64.7% of the single fibers showed an increase in the response to distension; this percentage is similar to the percentage of single fibers that increased their constitutive firing frequency (see Fig. 2A) [314.9 ± 61% on the 2nd distension vs. 577.9 ± 77% on the 4th distension; n = 11/17 (64.7%), P = 0.001, Fig. 5, C and D].

Vagotomy Eliminated the JB-1 Effects on Spontaneous Nerve Activity and on the Response to Gut Distension

Mesenteric nerve response to CCK.

The mesenteric nerve contains vagal and spinal afferent fibers. Because CCK strongly stimulates vagal but not spinal afferents, it can be used as a control to test that vagal afferent fibers are functionally intact and to establish that surgical vagotomy quenched all afferent vagal traffic (25, 48). Hence, we tested the effects of adding CCK to the serosal perfusate.

CCK increased spontaneous multiunit activity, had a short onset latency (∼30 s), and was reversible and dose-dependent. The response to a range of CCK doses (1 pM-1 nM) was examined in individual jejunal segments (n = 15). The lowest dose that produced a just discernible effect was 1 pM, whereas 100 pM increased the basal firing frequency from 8.8 ± 3 to 15 ± 5 Hz (83.4 ± 14% of increase; n = 5, P = 0.03, Fig. 6, A and B, top). The percentage of increase in firing frequency vs. CCK concentration plot was fitted by a Hill equation of the form Y = bottom + (top − bottom)/[1 + 10(LogEC 50 − X)]. EC 50 for CCK firing frequency increase was 11 pM (Fig. 6A). We also tested if JB-1 is able to induce an increase in the actions of a low dose of CCK (10 pM) on intact mesenteric nerve bundles; there were no significant differences in the percentage of firing frequency increase induced by CCK before and after the administration of JB-1 in the luminal jejunum (data not shown).

Fig. 6.Increase in basal firing frequency induced by cholecystokinin (CCK) is lost after vagotomy. A: dose-response plot from 15 experiments was fitted with a logistic equation, EC 50 = 11 pM (intact animals). ○, CCK effects on intact animals; □, CCK effects after vagotomy. B: effect of 100 pM CCK in intact (P = 0.03, n = 5; top) or vagotomized (P = 0.1, n = 13; bottom) animals.

After vagotomy, 0/13 of individual jejunal segments tested responded with an increase in multiunit firing when 100 pM CCK was applied; firing frequencies were 28.4 ± 5 vs. 28.1 ± 5 Hz (n = 13, P = 0.1, Fig. 6, A and B, bottom). We conclude that the vagotomies were functionally effective.

JB-1 Effects on Neuronal Activity were Abolished After Vagotomy

We added 11 additional animals to our study to include the effects of JB-1 on the mesenteric nerve bundle after vagotomy. Six animals were vagotomized, and five were sham-treated.

We tested the JB-1 effects on the spontaneous firing frequency and on the response induced by gut distension after performing vagotomies. No effect on the spontaneous firing frequency of JB-1 was seen (28 ± 7 vs. 29.9 ± 7 Hz; n = 11, P = 0.06, Fig. 7, A and B). After vagotomy, JB-1 also failed to augment the response to gut distension [percentage of firing frequency increase: 308.2 ± 93 and 321.6 ± 75% before and after the addition of JB-1, respectively (n = 5, P = 0.4, Fig. 7C)]. The basal response to gut distension induced by 14 or 31 hPa did not differ from that seen in intact animals. We conclude that the excitatory actions of JB-1 on the mesenteric afferents were carried by vagal fibers. To demonstrate that spinal afferents were still intact after vagotomy we always confirmed that adding 1 μM capsaicin to the serosal compartment evoked a strong excitatory multiunit response (data not shown) (39). JB-1 effects on the mesenteric nerve bundles with sham-treated animals were similar to those found in intact animals; here, the effective dose of this bacteria increased the spontaneous firing frequency from 12.6 ± 3.3 to 18.7 ± 5 Hz (n = 5, P = 0.03, Fig. 7D).

Fig. 7.JB-1 effects on firing frequency lost after vagotomy. A: representative traces of multiunit spontaneous firing frequency before (left) and after (right) addition of 1 × 109 JB-1. Dotted rectangles show details with faster time base. B: graph showing the results exemplified in A (P = 0.06, n = 11). C: firing frequency increase induced by gut distension before and after the addition of 1 × 109 JB-1/ml (P = 0.4, n = 5). D: effects of 1 × 109 JB-1/ml on spontaneous firing frequency of sham-treated mice (*P = 0.03, n = 5/5, Wilcoxon test).

DISCUSSION

The intestinal microbiome influences gut-brain communication (1, 7, 9, 14, 19, 38, 47). This is further supported by observations that the enteric nervous system and brain differ between germ-free and conventional animals (1, 15, 25, 40, 41). However, the underlying mechanisms are unknown, and most studies focus on the impact of altered signaling on the brain (9, 13, 19, 38) and not on the intervening pathways from gut luminal microbiome to the brain. Our data are incompatible with the hypothesis we initially offered in the Introduction. Indeed, we present the first evidence that in a normal mouse jejunum a single probiotic (L. rhamnosus JB-1) provokes an increase of the constitutive mesenteric nerve multi- and single-unit firing rate within minutes of luminal application. In addition, JB-1 further augmented distension-evoked increases in multi- and single-unit firing of mesenteric vagal afferents. These effects were due to single fibers that increase their firing frequency rather than an increase in the number of active fibers recruited.

The vagus nerve is an important afferent signaling pathway between the gastrointestinal tract and the brain (24, 47). This pathway is bidirectional, and clear evidence for a vagal efferent anti-inflammatory pathway exists (50). There are several lines of evidence that suggest that the vagus is involved in both mood regulation and gut inflammation (13, 23). Because low-grade, even unrecognizable gut inflammation, induced by pathogenic bacteria appears to promote anxiety-like behavior (36), the introduction of bacteria such as probiotics, many of which themselves induce anti-inflammatory circuits (18, 29), cannot be assumed to be acting on the brain independently from their effects on inflammation. There is also evidence that changes in the gut microbiota and/or ingestion of probiotics can experimentally influence behavior in animals that have undergone some experimental manipulations (1, 3), but these occurred separately from influences of the autonomic nervous system. The anxiolytic effect of B. longum also inhibited the generation of anxiety-like behavior in a chemical colitis model, and this effect, however, was inhibited by prior vagotomy (2).

We have recently published that chronic feeding of conventionally housed healthy mice with L. rhamnosus (JB-1) induced anxiolytic changes in behavior and in the level of GABA Aα2 , GABA Aα1 , and GABA B1b receptor mRNA expression in several brain structures (hippocampus, prefrontal cortex, cingulate cortex, amygdala, and locus coeruleus). These effects were abrogated after vagotomy (7). These data establish the importance in this model of the intact afferent vagus for the brain GABA receptor mRNA changes and related anxiolytic effects (12), but they provide no information about how afferent signals are altered by the probiotic. Our present data show the nature of the change that one anxiolytic probiotic bacteria produces in afferent vagal firing. This contrasts with the effects of another Lactobacillus, L. salivarius, which is not neuroactive in the gut (55) and also did not alter vagal firing. The evidence suggests that neuroactive and psychoactive effects are bacterial strain specific (16). Also, we have found that if we apply JB-1 when the muscle is paralyzed by nicardipine, an L-type calcium channel blocker, the degree of effects on the spontaneous firing frequency is still present although lower than with JB-1 administered alone. Even so, we cannot discard the possibility that JB-1 may modify the constitutive firing frequency partially through changes in the peristaltic reflexes that we have previously reported (55). However, because the degree of changes in the constitutive firing frequency induced by JB-1 on single fibers is very similar with and without nicardipine, we conclude that our results with multiunit recordings were probably obscured by the number of nonresponsive single fibers.

Our experiments could not directly address the underlying mechanisms of cellular transduction and signaling between JB-1 and the vagal primary afferents within the mucosa. However, commensals produce a large range of molecules, including fatty acids, cell wall oligosaccharides, and neurotransmitters (17, 30, 42). Some of these may interact with mucosal epithelial cells that release paracrine mediators to act on neuronal processes (38); certain molecules such as hydronium ions could also act directly on vagal afferents. A cogent approach to the study of which bacterial molecule(s) mediate neural effects on the host awaits genomic and metabolomic analysis of the probiotic (10). JB-1 did not facilitate the vagal response to CCK, suggesting that these bacteria probably did not act via the CCK pathway. It is not clear which of these effects might explain the neuronal effects with onset latencies of 10–15 min that we report here.

It remains to be elucidated exactly how the increase in the constitutive vagal firing frequency and the augmentation of its response to gut distension may change brain neurochemistry and behavior. However, electrical stimulation of vagal fibers alters concentrations of serotonin, norepinephrine, GABA, and glutamate within the brain (46), and, although controversial, vagal stimulation is a Food and Drug Administration (FDA)-approved treatment for patients with depression who do not respond to classical drug therapy (11, 46) and is also FDA approved for the adjunctive treatment of epilepsy (21).

In summary, we have shown that, within minutes of introduction of probiotic bacteria in the jejunal lumen of mice, vagal afferents were activated and that this activation was recorded as an increase in the spontaneous frequency of both multi- and single-unit firing frequency. This procedure further augmented the distension-evoked increases of firing frequency that in turn were all abrogated by prior subdiaphragmatic vagotomy. Thus, the mechanisms whereby some probiotic bacteria influence brain and behavior may critically involve the facilitation of vagal firing. It is therefore plausible that certain bacteria might be useful and safe adjuncts to therapy in some conditions. Our results support the fact that use of this pathway is bacterial strain dependent as has been also shown for bacterial effects on behavior. Further detailed exploration of the neuronal spike trains that encode behavioral signaling to the brain may be useful to identify those psychoactive bacteria that are effective. It may also help uncover how they signal the brain and thus offer novel approaches to the development of new forms of treatment for some psychiatric and other comorbid disorders.

GRANTS

We authors gratefully acknowledge financial support for the conduct of this research to the National Science and Engineering Research Council (no. RPGIN371955-09 to W. Kunze) and the Giovanni and Concetta Guglietti Family Foundation. A. Perez-Burgos was supported by the Consejo Nacional de Ciencia y Tecnología postdoctoral fellowship and the Brain-Body Institute.

DISCLOSURES

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

AUTHOR CONTRIBUTIONS

Author contributions: A.P.-B., Y.-K.M., B.M., and K.-A.M.N. performed experiments; A.P.-B. and W.A.K. analyzed data; A.P.-B. and W.A.K. interpreted results of experiments; A.P.-B. prepared figures; A.P.-B. drafted manuscript; A.P.-B., J.B., and W.A.K. edited and revised manuscript; A.P.-B., B.W., Y.-K.M., B.M., K.-A.M.N., J.B., and W.A.K. approved final version of manuscript; B.W., J.B., and W.A.K. conception and design of research.