Human brain imaging studies have revealed the anterior cingulate cortex (ACC) as a key brain region for mediating visceral-pain-cognitive interactions. Recently, we characterized impairments of long-term potentiation and spike-field coherence in the basolateral amygdala (BLA)-ACC network in association with a decision-making deficit in rats with visceral hypersensitivity (VH). Now, by combining integrative neurobiological approaches, we show that ACC-reactive astrogliosis and activity-dependent impairment of lactate release occur in VH rats. Exogenous lactate supply rescues chronic-visceral-pain-caused impairments of ACC phase locking and decision making, which can be mimicked by optogenetic activation of ACC astrocytes. Large-scale electrophysiological recordings in free-moving animals during a decision-making task indicate that optogenetic astrocytic activation improves decision-making performance and engages ACC phase locking and BLA-to-ACC information flow. Collectively, these observations support the idea of an “astrocyte-neuron l -lactate shuttle” and suggest that targeting astrocytes may help with cognitive dysfunctions under chronic visceral pain.

In the current study, we reveal that administration of-lactate to the ACC rescues impairments of ACC LTP and spike field coherence (SFC) and mitigates a decision-making deficit in VH rats. Recently, we have developed an optogenetic approach to specifically manipulate astrocytic functions in vivo (). We postulate that, if astrocyte-neuronal lactate signaling plays a fundamental role in modulating neuronal electrophysiological functions, then optogenetic targeting of ACC astrocytes could have a major physiological effect on pro-cognitive functions under chronic visceral pain. To address these key questions, we employed integrative neurobiological approaches combining large-scale neural recordings and optogenetics in free-moving animals during the RGT. We show that disruptions of ACC phase-locking and BLA-to-ACC information flow closely track poor decision-making performance in VH rats. Optogenetic astrocytic activation released-lactate, enhanced phase locking and BLA-ACC information flow during the RGT, and repaired the VH-caused decision-making deficit via the lactate pathway. These findings provide insight into cognitive and pain treatment in the management of chronic visceral pain.

Neuronal oscillations are likely to be a fundamental mechanism for modulating, filtering, and redirecting information in the nervous system. Within individual brain areas, oscillations can synchronize neurons, creating coherent cell assemblies () and appropriate plasticity, depending on the precise timing of pre- and post-synaptic activity (). Our published data show that phase locking and synchronization within the ACC and between the ACC and BLA play a major role in the modulation of decision-making behavior in rats ().

Impairment of cognitive function by chemotherapy: association with the disruption of phase-locking and synchronization in anterior cingulate cortex.

Impairment of decision making and disruption of synchrony between basolateral amygdala and anterior cingulate cortex in the maternally separated rat.

Astrocytes, but not neurons, store glycogen (), which can be rapidly mobilized and directed to the glycolytic pathway to produce-lactate. Activity-dependent lactate release has been detected in the activated brain areas during sensory and cognitive stimulations both in humans and intact animals (). Here, by employing microdialysis, we show failure of lactate release under theta burst stimulation (TBS) of the basolateral amygdala (BLA) and in awake rats after a rat gambling task (RGT) under chronic visceral pain. Previous studies reported that an inhibitor of glycogen phosphorylase, 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), injected either before or immediately after training, blocked long-term memory retrieval and disrupted the maintenance of hippocampal LTP (). However, the precise roles and mechanisms of lactate on neuronal functions, particularly how astrocytic-neuronal lactate signaling affects large-scale neural oscillations in the brain circuitry, remain unclear.

There is a growing body of evidence indicating that glial cells, particularly astrocyte activation, can augment or suppress both excitatory and inhibitory synaptic transmission, which participate in some forms of spike timing-dependent cortical plasticity and working memory (). Astrocytes respond to all forms of CNS damage and disease by undergoing cellular, molecular, and functional changes commonly referred to as reactive astrogliosis (). However, the functions and effects of reactive astrocytes are unclear (), as are their roles in specific visceral-pain processes and cognitive disorders.

The other brain: from dementia to schizophrenia, how new discoveries about the brain are revolutionizing medicine and science.

Visceral hypersensitivity (VH) is a key factor in the pathophysiology of irritable bowel syndrome (IBS) (). In a series of previous publications, we have provided electrophysiological evidence that the anterior cingulate cortex (ACC) is a component of a functional circuit that plays a crucial role in the mediation of perception and processing of chronic visceral pain (). We further characterized impairments of long-term potentiation (LTP) in the ACC synapses of VH rats (), which implied that this pain-associated synaptic metaplasticity may lead to other emotional and cognitive disturbances. The clinical connection between visceral pain and increases in levels of anxiety, depression, and cognitive disorders has long been recognized (). Remarkably, these emotional and cognitive signs are less studied than the sensory components of visceral pain. Decision making is a basic cognitive process for adaptation that has attracted increasing attention in recent years. Previous studies demonstrated that decision making relies mainly on the integrating functions of specific prefrontal cortex (PFC) regions, including the ACC (). More recently, we have shown that VH causes impairment of decision making in rats (); however, the underlying brain neural network mechanisms are less clear.

Impairment of decision making associated with disruption of phase-locking in the anterior cingulate cortex in viscerally hypersensitive rats.

Impairment of cognitive function by chemotherapy: association with the disruption of phase-locking and synchronization in anterior cingulate cortex.

Electrophysiological data showed that, during the RGT, light stimulation increased the percentage of within-ACC phase-locked neurons to 52.56% ± 3.20% ( Figure 7 B; paired t test, n = 6 undecided and poor decision makers, t= 9.2, p < 0.001). In parallel, the percentage of ACC to BLA theta phase-locked neurons was increased to 47.67% ± 4.90% with light stimulation ( Figure 7 D; t= 4.66, p = 0.006). Additionally, two-way ANOVA revealed that the peak of cross-correlation coefficient was higher during the RGT with light stimulation ( Figure 7 E; F= 12.54, p = 0.001). The mean lags at peak also decreased to −16.89% ± 5.50% with light stimulation ( Figure 7 F; t= 2.63, p = 0.047), suggesting predominant directionality from the BLA to the ACC under light stimulation. In contrast, using another 6 good decision makers, we showed that light stimulation did not further increase the percentages of within-ACC ( Figures S7 A and S7B), ACC spike-to-BLA theta phase-locked neurons ( Figures S7 C and S7D), and BLA-to-ACC information flow ( Figures S7 E and S7F), suggesting a specific role of astrocytic activation in undecided and poor decision makers during the RGT. Hence, it appears that ACC phase locking and BLA-to-ACC information flow engage the pro-cognitive action of astrocytic activation.

Data are presented as mean ± SEM (p < 0.05,p < 0.01, andp < 0.001 by paired t test). See also Figures S6 and S7 and Table S1

(F) During re-RGT, the mean lags at the peak were reduced by light stimulation.

(A–D) Light stimulation increased ACC phase-locking in undecided and poor decision makers during re-RGT. (A and C) Averaged percentages of phase-locked neurons of within-ACC (A) and ACC spike to BLA theta (C) across each rat (n = 6) were increased under light stimulation. (B and D) Averaged percentages of phase-locked neurons during re-RGT show that light stimulation increased both within-ACC and ACC spike to BLA theta phase locking.

Then we investigated whether the improvement of optogenetic activation of astrocytes in decision-making performance involves increases in spike-field synchrony during the RGT; eight VH subjects with ChR2 expression (two undecided and six poor) were re-tested on the following day with light stimulation. RGT data revealed that, under light stimulation, six of eight rats were identified as good decision makers in the re-RGT and the other two as poor decision makers ( Table S1 ). In a separate study, seen VH rats with eYFP expression (five undecided and two poor) were also re-tested with the same stimulating pattern as sham treatment. During light stimulation, only one undecided rat changed its behavior to be identified as a good decision maker ( Table S1 ). The unchanged behavioral types of decision-making rats are consistent with previous findings showing that the preference of high risks and disadvantageous choices in poor decision makers are stable over time ().

First we examined whether light stimulation could mimic the effects of exogenous lactate on ACC SFC and phase locking when rats were in a quiet waking state in the home cage. We found that, in control rats (n = 4 rats), light activation of ACC astrocytes increased the mean SFC values in the theta band ( Figure S6 B; unpaired t test, n = 47 neurons, t= 2.06, p = 0.043) and the percentage of within-ACC phase-locked neurons ( Figure S6 C; paired t test, n = 4 rats, t= 5.74, p = 0.011). In parallel, in VH rats (n = 6), the mean SFC values in the theta band was increased by light stimulation ( Figure S6 E; one-way ANOVA followed by Bonferroni’s test, n = 113 neurons, t= 3.06, p = 0.014). Prior infusion of DAB (t= 4.52, p < 0.001) or 4-CIN (t= 3.62, p = 0.002) markedly reduced the effects of light stimulation. Moreover, astrocytic activation also increased the percentage of phase-locked neurons ( Figure S6 F; one-way ANOVA followed by Bonferroni’s test, n = 6 rats, t= 4.8, p = 0.029), and DAB (t= 5.53, p = 0.016) or 4-CIN (t= 8.87, p = 0.0018) prevented light actions, indicating the involvement of astrocyte-neuronal lactate signaling on spike-timing synchrony.

To investigate the directionality and strength of neural information flow in the BLA-ACC network, we performed information flow analysis by cross-correlation of instantaneous amplitudes of theta oscillations (). A BLA-ACC lag estimate revealed that, before the RGT, the peak of the cross-correlation is located at negative lags among good, undecided, and poor decision makers ( Figure 6 D), suggesting a predominant information flow directionality from the BLA to the ACC. Interestingly, during the RGT, good decision makers depicted a consistently predominant information flow from the BLA to the ACC ( Figure 6 F; mean lag = −16.07 ± 2.08; Wilcoxon signed-rank test, n = 7 rats, p = 0.016 from zero); however, poor decision makers showed no significant directionality in the BLA-ACC network (mean lag = −0.51 ± 5.58, n = 8 rats, p = 0.84). Also, during the RGT, the peak of the cross-correlation coefficient dramatically decreased in undecided and poor decision makers ( Figure 6 G, left; two-way ANOVA, F= 30.38, p < 0.001). The peak of the cross-correlation coefficient was positively correlated with the percentage of advantageous choices during the RGT in good but not undecided and poor ones ( Figure 6 G, right). These findings indicate that reduced BLA-to-ACC information flow contributes to undecided and poor decision-making performance.

Cross-correlation of instantaneous amplitudes of field potential oscillations: a straightforward method to estimate the directionality and lag between brain areas.

To further investigate possible mechanisms underlying the actions of astrocytic activation on improving decision making in the VH state, we performed multiple-channel optetrode recordings in the BLA and ACC during the RGT. The placements of electrodes in the BLA and ACC are shown in Figures S1 C–S1F. Seven rats were identified as good, two rats as undecided, and six rats as poor decision makers by their behavioral performance ( Table S1 ). We found that, during the RGT, the within-ACC phase-locked neurons were significantly lower in undecided and poor subjects compared with good decision makers ( Figure 6 B, left; two-way ANOVA, n = 8 undecided and poor and 7 good decision makers, F= 21.48, p < 0.001), although, before the RGT, in their home cages, there was no difference between the two sub-groups (t = 0.26, p > 0.99). The percentage of within-ACC phase-locked neurons was positively correlated with the percentage of advantageous choices made by good decision makers but not undecided and poor subjects ( Figure 6 B, right). Similarly, we found that, during the RGT, the ACC spike phase locking to BLA theta was also lower in undecided and poor decision makers compared with good subjects ( Figure 6 C left; two-way ANOVA, F= 34.29, p < 0.001). The percentage of ACC-BLA phase-locked neurons positively correlated with behavioral advantageous choices in good but not undecided and poor subjects ( Figure 6 C, right). These results suggest that the strength of ACC phase-locking in the theta range can closely track good decision performance; however, undecided and poor decision makers failed to increase phase locking during the RGT.

Data are presented as mean ± SEM. See also Figure S1 and Table S1

(G) Left: during the RGT, the undecided and poor decision makers showed a lower value of cross-correlation coefficients. Right: averaged peaks of cross-correlation coefficients for each rat show a positive correlation with the percentages of advantage choices during the RGT in good but not undecided and poor subjects.

(F) The mean lags at the peak in good and undecided and poor decision makers during the RGT. ∗ p < 0.05 by Wilcoxon signed-rank test.

(E) Distributions of cross-correlation coefficients at different lags during the RGT in the good and poor decision makers shown in (D). Dark stars show the peaks of the cross-correlation.

(B and C) Left: percentage of within-ACC (B) and ACC spikes to BLA theta (C) phase-locked neurons for each rat in undecided and poor decision makers (n = 8) compared with good performers (n = 7). Right: Spearman cross-correlation of the percentage of phase-locked neurons and the percentages of advantageous choices during the RGT for each rat. The corresponding correlation r and p values are indicated. The black and red lines indicate the regression lines of good and undecided and poor decision makers, respectively.

(A) Examples of an ACC single unit recorded from a good decision maker (top) phase-locked to local theta oscillation during the RGT, and a unit from a poor decision maker showing no phase locking (bottom).

To determine the effects of optogenetic stimulation of astrocytes in the ACC on visceral pain sensation, in a separate group of VH rats, the nociceptive response (the visceromotor response to graded pressures of colorectal distention [CRD]) were performed. Measurement of visceral sensitivity in animals is mainly based on brain stem reflexes (). Two-way ANOVA revealed that there was no difference in the factor of light effect ( Figure S5 ; two-way repeated-measures ANOVA, n = 5 rats, F= 0.86, p = 0.37), indicating that light activation of ACC astrocytes does not affect visceral pain sensation.

Next, we conducted RGT experiments in VH rats with ChR2-eYFP or eYFP expression in bilateral ACC regions ( Figure 5 A). Three types of decision makers were identified in eYFP (n = 24), ChR2 (n = 27), and ChR2+4-CIN (n = 19) rats ( Figure 5 B). There was no significant three-way interaction between treatments, types of decision makers, and time (F= 0.57, p = 0.93). The general activities in these three treated groups were similar ( Figure 5 C; one-way ANOVA, F= 2.02, p = 0.14). However, the proportion of three subgroups of decision makers in the three treated populations was significantly different ( Figure 5 D; Kruskal-Wallis test, p = 0.008). Post hoc analysis revealed a significant difference between the eYFP and ChR2 rats (p = 0.044). Blocking the uptake of lactate from the extracellular space to neurons by prior 4-CIN administration abolished the action of light stimulation (p = 0.003). The final reward obtained was also different in the three treated groups ( Figure 5 E; one-way ANOVA, F= 9.21, p < 0.001); it increased to 138.1 ± 8.9 in the ChR2 group (t= 3.67, p = 0.0014) compared with 90.9 ± 8.93 in the eYFP group and returned to 88.5 ± 11.1 in the ChR2+4-CIN group (t= 3.61, p = 0.0017). These results suggest that manipulation of intrinsic astrocytes in the ACC regions rescues the decision-making deficit of VH rats through lactate signaling.

(E) The food pellets obtained during the testing session in the three groups ( ∗∗ p < 0.01 by Bonferroni’s post hoc test).

(D) The proportions of different types of decision makers in the three groups ( ∗ p < 0.05 and ∗∗ p < 0.01 by Dunn-Bonferroni’s post hoc test).

(B) Identification of different types of decision makers in eYFP-expressing (n = 24, left), ChR2-expressing (n = 27, center), and ChR2-expressing (n = 19, right) rats with 4-CIN infusion.

To further determine whether-lactate in the rat ACC is derived from astrocytes and whether specifically manipulating intrinsic astrocytes can reinstate astrocytic physiological functions and, thereby, palliate decision-making impairments in VH rats, we employed an optogenetic approach. The expression of the virus in the ACC was identified ( Figure S3 A), and the specificity of virus expression in ACC astrocytes was confirmed ( Figures S3 B–S3G; n = 4 rats). To test whether optogenetic activation of astrocytes is sufficient to release lactate, in vivo microdialysis was performed in virus-infected VH rats under anesthesia ( Figure S4 A). We found that light stimulation induced an increase in-lactate concentration in ChR2-expressing rats but not in enhanced yellow fluorescent protein (eYFP)-expressing rats ( Figure S4 B; two-way ANOVA, n = 4 per group, F= 91.77, p < 0.001). Then we examined whether optogenetic activation-induced lactate release is repeatable in awake VH rats. We found that light stimulation induced a marked increase in lactate level in the ACC of conscious VH rats ( Figure S4 C; one-way ANOVA, n = 5, F= 5.34, p < 0.001) and that the increased levels over the 4 consecutive days had no significant change ( Figure S4 D; one-way ANOVA, F= 0.062, p = 0.98). These results indicate that daily optogenetic activation of ACC astrocytes induced a repeatable increase of local-lactate concentration in chronic VH rats.

How does ACC astrogliosis result in failure of-lactate release? Previous studies revealed that astrocytic lactate release is mediated by glutamate uptake into astrocytes through the excitatory amino acid transporter 2 (EAAT2) (). Accordingly, we tested the expression of EAAT2 and found that it was reduced to 84.3% ± 1.4% compared with control ( Figure 4 F, right; t= 5.20, p = 0.0013) in VH rats. These observations suggest that a decrease in glutamate uptake may reduce lactate release from astrocytes.

Role of glutamate transporters in the modulation of stress-induced lactate metabolism in the rat brain.

We then investigated whether the failure of lactate release was indeed due to the astrocytic dysfunctions that occur under chronic visceral pain. We first checked the expression of glial fibrillary acidic protein (GFAP) and S100 calcium-binding protein beta (S100β) in VH rats, both of which are sensitive and reliable markers of astrocytes that respond to CNS damage and disease (). Immunohistochemical staining of GFAP Figure 4 B) and S100βcells ( Figure 4 D) showed thicker processes and larger, more densely stained cell bodies in VH rats. Quantifying analyses revealed that the density of GFAPcells in the ACC was increased to 2,111 ± 234.6 in VH rats compared with 1,175 ± 112.7 cells/mmin controls ( Figure 4 C; unpaired t test, n = 3, t= 3.60, p = 0.023). The density of S100βcells in the ACC was also increased to 1,005.0 ± 39.5 in VH rats compared with 759.5 ± 9.9 cells/mmin controls ( Figure 4 E; n = 3, t= 7.32, p = 0.0019). In addition, western blot analyses of protein expression in the ACC showed increases of GFAP ( Figure 4 F, left; n = 4 control and 5 VH rats, t= 4.21, p = 0.004) and S100β expression ( Figure 4 F, center; t= 3.23, p = 0.012) in VH rats. These observations indicate development of reactive astrogliosis in the chronic VH rat model.

(F) Examples (top) and averaged western blot analysis (bottom) showing increasing expression of GFAP (left) and S100β (center) and decreasing expression of EAAT2 (right) in VH rats (n = 4 control and 5 VH rats).

(C and E) Increases in the number of GFAP + (C) and S100β + (E) cells in VH rats (n = 3 rats/group).

(B and D) Representative GFAP-stained sections (B) and S100β-stained sections (D) from a control and a VH rat ∼2.8 mm from the bregma. Arrows show more densely stained cell bodies in VH rats. Scale bars, 50 μm.

(A) Schematic showing the quantified area in the ACC (gray squares) for immunostained sections from 3.8 to 2.2 mm from the bregma. cc, corpus callosum; Cg1, cingulate cortex, area 1; Cg2, cingulate cortex, area 2; PrL, prelimbic cortex.

Upregulation of GFAP and S100β Is Associated with Downregulation of EAAT2 in VH Rats

Figure 4 Upregulation of GFAP and S100β Is Associated with Downregulation of EAAT2 in VH Rats

We reason that decreased release of lactate may be responsible for impaired decision-making behavior in the VH state. We found that the basal levels of extracellular-lactate in the ACC were similar in control and VH rats ( Figure 3 A; unpaired t test, n = 4 control and 5 VH rats, t= 0.83, p = 0.44). Activity-dependent lactate release has been detected previously (). If lactate signaling is involved in impairment of synaptic plasticity and decision making in VH rats, then the lactate concentrations might be different in VH rats under corresponding afferent electrical stimulation and during the behavioral task. We found that TBS to the BLA elicited a marked increase in lactate concentration in the ACC from the basal level to 115.2% ± 3.0% in control rats ( Figure 3 B; n = 4). Further, after the RGT, the lactate concentration was markedly increased in control rats ( Figure 3 C; n = 4, 140.8% ± 4.9% of baseline at 10 min). In contrast, in a separate study using VH rats, we showed that TBS failed to elicit obvious changes in ACC lactate concentration (n = 4; 101.3% ± 3.1%; two-way ANOVA, F= 8.45, p = 0.0049). The RGT only induced a minor increase in lactate concentration in the VH group (n = 5, 126.7% ± 1.4%, two-way ANOVA, F= 29.83, p < 0.001). These findings suggest that impairments of ACC lactate release may play a crucial role in deficits of synaptic plasticity and decision making in VH rats.

(C) The effect of the RGT on the ACC lactate level in control (n = 4) and VH rats (n = 5).

(B) The effect of TBS to the BLA on the ACC lactate level in control and VH rats (n = 4 rats/group).

We also observed that, in control rats (n = 7), blocking lactate production by DAB (1 nmol) () reduced the ACC SFC values ( Figure S2 B; one-way ANOVA, n = 137 neurons, F= 16.14, p < 0.001) and decreased the percentage of phase-locked neurons ( Figure S2 D; one-way ANOVA, n = 7 rats, F= 6.40, p = 0.008). Administration of lactate into the ACC recovered the effects of DAB (p < 0.01 for SFC and p < 0.05 for phase locking). Together, these results suggest that-lactate is necessary and sufficient for ACC spike-timing synchrony.

To further study the involvement of lactate signaling in the spike-timing synchrony of ACC single units, we recorded neuronal spikes and LFP simultaneously and analyzed SFC and phase locking between ACC spikes and LFP. We found that, in VH rats (n = 7), lactate infusion tremendously increased SFC values in the low-frequency range, and the monocarboxylate transporter 2 (MCT2) blocker α-cyano-4-hydroxycinnamate (4-CIN, 0.5 nmol) () prevented the action of lactate ( Figure 2 C; two-way ANOVA, n = 159 neurons, F= 52.53, p < 0.001). The averaged SFC values in theta band (4–10 Hz) were significantly different among the three treated groups ( Figure 2 D; one-way ANOVA, F= 8.73, p < 0.001). Lactate infusion increased theta band SFC from 1.01% ± 0.13% to 1.604% ± 0.23% (t= 2.62, p = 0.028), and prior 4-CIN infusion suppressed it to 0.67% ± 0.09% (t= 4.13, p < 0.001). Moreover, the percentage of phase-locked neurons increased from 27.35% ± 8.02% to 59.27% ± 9.89% following lactate infusion ( Figure 2 F; one-way ANOVA, n = 7 rats, t= 2.68, p = 0.046), and prior 4-CIN infusion reduced that to 19.65% ± 7.16% (t= 3.32, p = 0.011).

Inhibition of monocarboxylate transporter 2 in the retrotrapezoid nucleus in rats: a test of the astrocyte-neuron lactate-shuttle hypothesis.

We then asked whether lactate is sufficient to reinstate LTP induction in VH rats. We found that, in control rats (n = 5 rats), a robust LTP was induced by TBS to the BLA ( Figure 2 A). The local field potential (LFP) amplitude was increased up to 136.3% ± 5.7% 30 min after TBS compared with the baseline level ( Figure 2 B; paired t test, t= 5.17, p = 0.0067). In VH rats (n = 5), after ACSF infusion, no obvious change in LFP amplitude was observed after TBS (99.0% ± 4.1%; t= 0.40, p = 0.71). In contrast, in another group of VH rats (n = 5), after infusion of-lactate (10 nmol) () into the ACC, a 129.0% ± 6.1% potentiation was observed in response to the BLA stimulus (t= 4.75, p = 0.009). These observations suggest that the induction of LTP-like plasticity in BLA-ACC synapses was restored by-lactate infusion into the ACC.

Data are presented as mean ± SEM (p < 0.05 andp < 0.001 by Bonferroni’s post hoc test). See also Figure S2

(F) Averaged percentages of phase-locked neurons (n = 7 rats) showing that l -lactate enhanced ACC spikes phase-locking to local theta in VH rats, and the enhancement was prevented by 4-CIN.

(E) Example showing polar histograms of the spike-theta phase distribution of an ACC single unit recorded in one VH rat with ACSF, lactate, or 4-CIN + lactate administration. The length of the mean resultant vector (MRL, range 0–1 with 1.0 = exact phase synchronization of the neurons) and Rayleigh p values are depicted at the bottom. The red arrow in the center polar histogram represents the angle of the mean resultant vector.

(C) The SFC distribution of ACC single units as a function of frequencies in VH rats with ACSF, lactate, and 4-CIN + lactate administration. Lactate enhanced SFC values in the low-frequency range of VH rats, and the enhancement was prevented by prior 4-CIN infusion (n = 159 neurons/7 rats). Shadow areas represent SEM.

(B) Time course plot of normalized LFP amplitude showing that impairment of LTP in VH rats was recovered by lactate administration (n = 5 rats).

(A) Representative curves of evoked LFP responses in the ACC to BLA stimuli before TBS and 30 min after TBS in a control rat (top), a VH rat with ACSF infusion (center), and a VH rat with l -lactate infusion (bottom).

We first investigated whether exogenous lactate is able to facilitate decision-making performance in both physiological and chronic states of visceral pain. Chronic cannulation was performed in bilateral ACC regions ( Figure 1 A). The injection sites and estimation of drug diffusion are shown in Figures S1 A and S1B. In compliance with our previous findings (), three types of decision-making performers were identified in control (n = 26 rats), control + lactate (n = 22), VH (n = 26), and VH + lactate rats ( Figure 1 B; n = 24). There was no significant three-way interaction between treatments, types of decision makers, and time (F= 0.41, p > 0.99). We found that the general activities, as assessed by number of nose pokes per minute, during the last training session were similar among these four groups ( Figure 1 C; one-way ANOVA, F= 0.67, p = 0.57). However, the proportion of the three subgroups of decision makers among the four treated populations was significantly different ( Figure 1 D; Kruskal-Wallis test; p = 0.011). Post hoc analysis revealed that the proportions of the three subgroups of decision makers were significant different in both lactate-treated control (p = 0.024), and VH rats (p = 0.020) compared with those with artificial cerebrospinal fluid (ACSF) infusions. The final mean pellets rewarded during the RGT test were increased in lactate-treated control and VH rats ( Figure 1 E; one-way ANOVA, F= 8.77, p < 0.001). In control rats, the final mean pellets rewarded increased to 191.2 ± 12.5 in the lactate-treated group compared with 143.9 ± 13.6 in the ACSF group (t= 2.75, p = 0.043). Similarly, in VH rats, the final mean pellets rewarded increased to 167.0 ± 12.2 in the lactate-treated group compared with 107.2 ± 9.4 in the ACSF group (t= 3.56, p = 0.0035). These findings suggest a fundamental physiological role of-lactate signaling in improving decision-making behavior during the RGT not only in normal subjects but also, in particular, in rats with chronic visceral pain.

(E) The food pellets obtained during the testing session in the four groups. ∗ p < 0.05 and ∗∗ p < 0.01 by one-way ANOVA followed by Bonferroni’s post hoc test.

(D) The proportions of different types of decision makers in the four groups. ∗ p < 0.05 by Kruskal-Wallis test followed by Dunn-Bonferroni’s test for post hoc comparisons.

(C) The general activities show no significant difference among the four treated groups.

(B) Identification of good, undecided, and poor decision makers in control (n = 26, top left), control with lactate infusion (n = 22, top right), VH (n = 26, bottom left), and VH with lactate infusion rats (n = 24, bottom right).

(A) Top: schematic illustrating cannula implantation to the bilateral ACC. Bottom: examination timeline of RGT training and testing sessions with l -lactate (or ACSF) infusions.

Impairment of cognitive function by chemotherapy: association with the disruption of phase-locking and synchronization in anterior cingulate cortex.

Discussion

Volterra and Meldolesi, 2005 Volterra A.

Meldolesi J. Astrocytes, from brain glue to communication elements: the revolution continues. l -lactate in the ACC region was similar in VH rats compared with controls, TBS application in the BLA, which was used to elicit classical LTP, could reliably induce an increase in extracellular lactate level in the ACC of control rats but failed to induce this alteration in VH rats. Further, 140.8% ± 4.9% increases in l -lactate over basal levels were observed after RGT in normal rats. On the other hand, the activity-dependent lactate release was attenuated in visceral pain state ( Pellerin and Magistretti, 1994 Pellerin L.

Magistretti P.J. Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization. Uehara et al., 2007 Uehara T.

Sumiyoshi T.

Itoh H.

Kurachi M. Role of glutamate transporters in the modulation of stress-induced lactate metabolism in the rat brain. +-dependent “high-affinity” transporters, has a predominant role in clearing glutamate throughout the synaptic cleft. The Na+-coupled reuptake of glutamate by astrocytes following activation of the Na+-K+ ATPase triggers glycolysis process for lactate production ( Pellerin and Magistretti, 1994 Pellerin L.

Magistretti P.J. Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization. Gosselin et al., 2010 Gosselin R.D.

O’Connor R.M.

Tramullas M.

Julio-Pieper M.

Dinan T.G.

Cryan J.F. Riluzole normalizes early-life stress-induced visceral hypersensitivity in rats: role of spinal glutamate reuptake mechanisms. +-K+ ATPase and reduction of lactate release from astrocytes. The brain requires a continuous supply of oxygen and energy-yielding substrates involving glucose (). Indeed, recent investigations reported that lactate also plays a key role in the supply of energy to the brain, especially during acute neural activation. Performing in vivo microdialysis, we found that, although the basal concentration of-lactate in the ACC region was similar in VH rats compared with controls, TBS application in the BLA, which was used to elicit classical LTP, could reliably induce an increase in extracellular lactate level in the ACC of control rats but failed to induce this alteration in VH rats. Further, 140.8% ± 4.9% increases in-lactate over basal levels were observed after RGT in normal rats. On the other hand, the activity-dependent lactate release was attenuated in visceral pain state ( Figure 3 ). Currently, the molecular mechanisms underlying the causality between pathological astrocytes and reduced lactate release is not fully understood. Nevertheless, we realize that astrocyte lactate release is mediated by glutamate uptake (). EAAT2, a family of Na-dependent “high-affinity” transporters, has a predominant role in clearing glutamate throughout the synaptic cleft. The Na-coupled reuptake of glutamate by astrocytes following activation of the Na-KATPase triggers glycolysis process for lactate production (). In line with previous findings that show downregulation EAATs in stress-induced chronic VH rats (), the current study shows significant suppression of EAAT2 ( Figure 4 F), suggesting that decreases of glutamate uptake may not only trigger an excitatory-inhibitory imbalance but also cause inactivity of Na-KATPase and reduction of lactate release from astrocytes.

Uehara et al., 2007 Uehara T.

Sumiyoshi T.

Itoh H.

Kurachi M. Role of glutamate transporters in the modulation of stress-induced lactate metabolism in the rat brain. Gibbs and Hertz, 2008 Gibbs M.E.

Hertz L. Inhibition of astrocytic energy metabolism by D-lactate exposure impairs memory. Suzuki et al. (2011) Suzuki A.

Stern S.A.

Bozdagi O.

Huntley G.W.

Walker R.H.

Magistretti P.J.

Alberini C.M. Astrocyte-neuron lactate transport is required for long-term memory formation. l -lactate levels is derived from glycogen, and that the astrocyte-neuronal l -lactate shuttle is involved in hippocampal LTP and long term memory formation. The existing literature has well demonstrated that l -lactate potentiates NMDA receptor-mediated currents following an increase in intracellular calcium. In parallel, l -lactate increases the expression of plasticity-related genes, including Arc, c-Fos, Zif268, and BDNF, in cortical neurons ( Yang et al., 2014b Yang J.

Ruchti E.

Petit J.M.

Jourdain P.

Grenningloh G.

Allaman I.

Magistretti P.J. Lactate promotes plasticity gene expression by potentiating NMDA signaling in neurons. l -lactate surely plays a role as a signaling molecule for neuronal plasticity ( Mosienko et al., 2015 Mosienko V.

Teschemacher A.G.

Kasparov S. Is L-lactate a novel signaling molecule in the brain?. l -lactate into the ACC could recover TBS-induced LTP in the BLA-ACC network, suggesting that failure of lactate release in response to TBS plays a causal role in the impairment of LTP induction in the VH state. Moreover, exogenous l -lactate supply enhanced SFC and spike-to-theta phase locking within the ACC region in VH rats. It appears that lactate is necessary and sufficient for ACC spike-theta synchrony. The brain prefers lactate over glucose as an energy substrate when both are available, and lactate exerts a direct neuroprotective effect (). A study showed that bead discrimination memory in chickens requires glycogenolysis in astrocytes (). Another elegant study byhas demonstrated that learning leads to a significant increase in extracellular-lactate levels is derived from glycogen, and that the astrocyte-neuronal-lactate shuttle is involved in hippocampal LTP and long term memory formation. The existing literature has well demonstrated that-lactate potentiates NMDA receptor-mediated currents following an increase in intracellular calcium. In parallel,-lactate increases the expression of plasticity-related genes, including Arc, c-Fos, Zif268, and BDNF, in cortical neurons (). Therefore,-lactate surely plays a role as a signaling molecule for neuronal plasticity (). In compliance with these findings, in the present study, we found that infusion of-lactate into the ACC could recover TBS-induced LTP in the BLA-ACC network, suggesting that failure of lactate release in response to TBS plays a causal role in the impairment of LTP induction in the VH state. Moreover, exogenous-lactate supply enhanced SFC and spike-to-theta phase locking within the ACC region in VH rats. It appears that lactate is necessary and sufficient for ACC spike-theta synchrony.

In behavioral studies, daily lactate infusion into the ACC did not change rats’ general activities in the RGT; however, lactate rescued the decision-making deficit under chronic visceral pain. Although the effect of lactate does not appear to be specific to VH rats, these observations suggest an essential role of attenuated activity-dependent lactate release in the VH-caused decision-making deficit and imply a therapeutic target in the lactate signaling pathway to rescue the VH-caused decision-making deficit.

l -lactate increases lactate level in the hippocampus and produces an antidepressant-like effect ( Carrard et al., 2016 Carrard A.

Elsayed M.

Margineanu M.

Boury-Jamot B.

Fragnière L.

Meylan E.M.

Petit J.M.

Fiumelli H.

Magistretti P.J.

Martin J.L. Peripheral administration of lactate produces antidepressant-like effects. Newman et al., 2011 Newman L.A.

Korol D.L.

Gold P.E. Lactate produced by glycogenolysis in astrocytes regulates memory processing. Under normal physiological conditions, the role of lactate supply on emotional and cognitive functions has also been recognized. For instance, peripheral administration of-lactate increases lactate level in the hippocampus and produces an antidepressant-like effect (). Another study demonstrated that intrahippocampal infusion of lactate in normal rats enhances memory when rats performed a spatial working memory task (). Consistent with these observations, in this study, we found that daily lactate infusion into the ACC increased the percentage of good decision makers ( Figure 1 D) and the final food pellets rewarded ( Figure 1 E), indicating that exogenous lactate improves decision-making performance in control rats.

In this study, we found that, under physiological conditions, optogenetic activation of astrocytes facilitated ACC SFC ( Figures S6 A and S6B) and phase locking to local theta oscillations ( Figures S6 C) in control rats. These data suggest that the lactate signaling pathway plays an essential role in astrocyte-promoted neuronal spike-field synchrony. In a chronic visceral pain state, optogenetic activation of astrocytes also increased local SFC and phase-locking in VH rats, and both DAB and 4-CIN abolished the actions of light ( Figures S6 D–S6F). Moreover, we revealed that optogenetically activating astrocytes in the ACC did not alter acute visceral pain responses ( Figure S5 ); however, it rescued the decision-making deficit in VH rats ( Figure 5 ). 4-CIN blocked the effects of light stimulation on promoting VH-related decision-making deficit, suggesting that lactate plays a core role in rescuing poor decision-making performance under chronic visceral pain.

Rutishauser et al. (2010) Rutishauser U.

Ross I.B.

Mamelak A.N.

Schuman E.M. Human memory strength is predicted by theta-frequency phase-locking of single neurons. Buzsáki and Draguhn, 2004 Buzsáki G.

Draguhn A. Neuronal oscillations in cortical networks. Mu et al., 2015 Mu L.

Wang J.

Cao B.

Jelfs B.

Chan R.H.

Xu X.

Hasan M.

Zhang X.

Li Y. Impairment of cognitive function by chemotherapy: association with the disruption of phase-locking and synchronization in anterior cingulate cortex. Fields, 2010 Fields R.D. The other brain: from dementia to schizophrenia, how new discoveries about the brain are revolutionizing medicine and science. Fields, 2010 Fields R.D. The other brain: from dementia to schizophrenia, how new discoveries about the brain are revolutionizing medicine and science. have shown that memory formation in humans is predicted by close coordination of spikes phase-locking with theta band oscillations. A growing body of evidence suggests that cortical oscillations in the theta band (in particular, the synchrony between spike timing and the theta oscillating phase) facilitates neuronal communications, modifies synaptic weights between anatomically distant but functionally associated brain regions, and is related to behavioral outputs (). Moreover, this broad spatial integration is achieved across exceedingly wide temporal scales ranging well beyond the millisecond to seconds of electrical signaling in neurons to encompass, instead, hours, days, and months, which are well-suited to the temporal dynamics of glial communication and plasticity (). It is important to examine how glial cells in the brain may participate in these processes, especially those requiring broad temporal and spatial scales of integration (). Performing large-scale electrophysiological recordings in behaving animals during the RGT, we showed that light stimulation specifically facilitated both ACC spikes phase locking and BLA-to-ACC information flow in undecided and poor decision makers, which may play a causal role in pro-cognitive functions of astrocytic activation.