The anterior cingulate cortex controls the hyperactivity in subthalamic neurons in male mice with comorbid chronic pain and depression

Neurons in the subthalamic nucleus (STN) become hyperactive following nerve injury and promote pain-related responses in mice. Considering that the anterior cingulate cortex (ACC) is involved in pain and emotion processing and projects to the STN, we hypothesize that ACC neurons may contribute to hyperactivity in STN neurons in chronic pain. In the present study, we showed that ACC neurons enhanced activity in response to noxious stimuli and to alterations in emotional states and became hyperactive in chronic pain state established by spared nerve injury of the sciatic nerve (SNI) in mice. In naïve mice, STN neurons were activated by noxious stimuli, but not by alterations in emotional states. Pain responses in STN neurons were attenuated in both naïve and SNI mice when ACC neurons were inhibited. Furthermore, optogenetic activation of the ACC-STN pathway induced bilateral hyperalgesia and depression-like behaviors in naive mice; conversely, inhibition of this pathway is sufficient to attenuate hyperalgesia and depression-like behaviors in SNI mice and naïve mice subjected to stimulation of STN neurons. Finally, mitigation of pain-like and depression-like behaviors in SNI mice by inhibition of the ACC-STN projection was eliminated by activation of STN neurons. Our results demonstrate that hyperactivity in the ACC-STN pathway may be an important pathophysiology in comorbid chronic pain and depression. Thus, the ACC-STN pathway may be an intervention target for the treatment of the comorbid chronic pain and depression.


Introduction
Chronic pain affects approximately 15% of the population and is commonly associated with psychological maladaptations, including depression [1,2].The presence of depression prolongs and exacerbates pain [2,3].The interplay between pain and depression creates a vicious cycle that poses a significant challenge to effective treatment for comorbid chronic pain and depression.Fortunately, many neuroplasticity changes have been shared by chronic pain and depression [4][5][6][7][8][9][10][11].Identifying neural circuit pathways implicated in chronic pain and depression will enrich the repertoire of targets used to develop effective therapies for the comorbid chronic pain and depression.
The subthalamic nucleus (STN), an innate glutamatergic nucleus in the basal ganglia, has been extensively investigated in both patients with Parkinson's disease (PD) and rodent PD models.Evidence demonstrates that deep brain stimulation in the STN relieves pain in PD patients [12,13].Alterations in neuronal activity of the STN also correlate with depression in PD patients [14,15].Moreover, STN neurons readily respond to painful and emotional stimuli [16][17][18].Our group showed that activation of STN neurons is sufficient to trigger pain-like behavior and emotional dysfunction in naive mice; inhibition of the hyperactive STN neurons and some of their projections in chronic pain mouse models alleviates pain-and depressionlike behaviors [7,17,19,20].These results implicate the STN in the pathophysiology of comorbid chronic pain and depression.However, the mechanisms underlying dysfunctions of STN neurons and their related circuits remain poorly understood.
Cortical regions, including the anterior cingulate cortex (ACC), play important roles in the high-level integration of pain signals with negative emotions.Because ACC neurons carry value-related information such as rewards, fear, or pain [21][22][23][24], it plays a key role in regulating emotional states associated with pain [25].In humans, neuroimaging studies have consistently found that patients with chronic pain and depression exhibit hyperactivity in the ACC [26,27].In terms of neural circuits, the ACC is among the cortical inputs to the STN forming the hyperdirect pathway in the cortico-basal ganglia-thalamo-cortical loop [28][29][30][31].However, it remains unknown whether the ACC plays a role in the enhancement of STN neuron activity in chronic pain and whether the ACC and STN coordinate to control the manifestation of pain-and depression-like behaviors in chronic pain.
In the present study, we hypothesized that the ACC controls pain and emotional responses in STN neurons and the dysfunction of the ACC-STN pathway is an important pathophysiology contributing to the comorbid chronic pain and depression.We observed that ACC neurons were hyperactive in mice subjected to the spared nerve injury (SNI), a chronic pain model; inhibition of STN-projecting ACC neurons attenuated pain-like and aversion-like responses in STN neurons in SNI mice; optogenetic activation of the ACC-STN pathway is sufficient to trigger hyperalgesia and depression-like behaviors in naive mice; optogenetic inhibition of the ACC-STN pathway ameliorated hyperalgesia and depression-like behavior in SNI mice, which was annihilated by chemogenetic activation of STN neurons.Overall, this study demonstrated a significant role of the ACC-STN pathway in the pathophysiology of comorbid chronic pain and depression.

STN-projecting ACC neurons are hyperactive in neuropathic pain
To provide synaptic evidence for the projection from the ACC to the STN (ACC-STN projection), we injected AAV-CaMKII-ChR2-eYFP into the ACC of wild-type (WT) mice (Fig 1A).Four weeks later, we observed a great many of eYFP-labeled neurons in the ACC and eYFP-labeled axonal fibers in the STN (Fig 1A and 1B).Whole-cell patch-clamp recording from acute brain slices showed that eYFP-labeled ACC neurons displayed time-locked firing in response to blue light stimulation (473 nm, 20 Hz, 5 ms; Fig 1C ); in 64% (16 out of 25 neurons from 4 mice) of STN neurons among eYFP-labeled fibers, 5 blue light pulses (473 nm, 5 ms, 20 Hz) evoked excitatory postsynaptic currents (photo-EPSCs) (Fig 1D and 1E).These photo-EPSCs were not blocked by the mixture of tetrodotoxin (TTX; 1 μM), a voltage-dependent sodium channel blocker, and 300 μM 4-aminopyridine (4-AP), a blocker of voltage-gated potassium channels, but were abolished by subsequent application of CNQX (20 μM), an antagonist for glutamate receptors (Fig 1D and 1F).The amplitude and latency for lightevoked EPSCs were 91.57± 4.95 pA (n = 4 mice) (Fig 1E) and 5.5 ± 0.3 ms (n = 4 mice), respectively.These data demonstrate a valid protocol to manipulate the monosynaptic glutamatergic ACC-STN projection.
To visualize the location of STN-projecting ACC neurons (ACC-STN neurons), we injected a retrograde AAV vector (AAV retro-CaMKII-eYFP) into the STN of WT mice (Fig 1G).After 3 weeks' recovery, we observed numerous eYFP-labeled neurons along the anterior-posterior axis in the ACC, more specifically, in layer V (Fig 1H and 1I).The results show that ACC-STN neurons distribute throughout the ACC preferentially in layer V.
We then established SNI neuropathic pain mouse models and examined whether the excitability of ACC-STN neurons is modified in SNI mice.The mice subjected to SNI surgery on the right side exhibited long-lasting mechanical and thermal hypersensitivity since the third day after surgery and developed depression-like behaviors 5 weeks after surgery (S1A-S1F Fig) .Our histological data showed that c-Fos-positive neurons in the ACC were significantly increased 4 weeks after surgery in SNI mice (S1G and S1H Fig).These data support that ACC neurons are hyperactive in neuropathic pain, consistent with a previous study [32].
We next examined whether ACC-STN neurons were among the hyperactive ACC neurons in SNI mice.To answer this question, we injected AAV retro-hSyn-mCherry-Cre into the STN to label ACC-STN neurons, then we performed whole-cell patch-clamp recording from the labeled ACC-STN neurons 4 weeks after SNI or sham operation (Fig 1J and 1K).The excitability of ACC-STN neurons in SNI mice was enhanced significantly compared with that in sham mice (Fig 1L -1R).Specifically, ACC-STN neurons in SNI mice responded to depolarizing current injections with higher firing rates than those in sham mice (Fig 1L and 1M).In addition, ACC-STN neurons displayed higher membrane resistance, lower rheobase, depolarization threshold to evoke action potentials, though resting membrane potentials, the correlation between rheobase and membrane depolarization did not differ significantly between SNI and sham mice (Fig 1N -1R).We then performed patch-clamp recording from STN neurons in mice subjected to AAV-CaMKII-ChR2-eYFP injection into the ACC (Fig 1S and 1T).We observed bigger amplitude, but similar paired pulse ratio of photo-EPSCs in STN neurons in SNI mice, relative to sham mice (Fig 1U and 1V).
These data suggest that chronic pain not only enhances the excitability of ACC-STN neurons, but also synaptic strength of the ACC-STN projection.

ACC and STN neurons respond similarly to pain-like and aversive stimulation but differently to alterations in emotional states
The ACC receives highly diverse somatosensory inputs [21,33].Accumulating evidence suggests that the ACC and STN may have common roles in pain processing in both somatosensory and emotional aspects [5,7,17,19,34,35].However, comparison of the role of the ACC and STN in these aspects in the same study has been lacking, and it remains unknown whether and how the ACC-STN pathway regulates pain responses in STN neurons.We conducted several experiments to address these issues.
We first monitored the responses of ACC neurons to pain-like stimulation and to events causing changes in emotional states using in vivo fiber photometry.To fulfill this, we transfected AAV-CaMKII-GCaMP6s into excitatory neurons across all layers of the right ACC and implanted an optical fiber above the injection site (Fig 2A and 2B).Three weeks later, we recorded GCaMP6 signal from ACC neurons in freely moving mice upon mechanical and thermal stimuli (Fig 2C).The GCaMP6s signal exhibited a significant increase when the mice showed pain-like responses to a mechanical stimulus with a von Frey filament (with a fiber force of 2 g) or a thermal stimulation (with a 50˚C metal block) on both the ipsilateral and contralateral hind paws (Fig 2D -2I).But the GCaMP6s signal in the ACC remained unchanged when the mice were subjected to hind paw stimulation by a sub-threshold von Frey filament (a force of 0.16 g) and while the mice lifted their paws during free exploration in the test chamber (Fig 2D -2I).SNI mice exhibited a comparable enhancement in GCaMP6s signal when subjected to the von Frey filament stimulation corresponding to a supra-threshold mechanical stimulation (0.16 g) or a 48˚C thermal stimulation on the hind paws (Fig 2J -2L).Control mice expressing eYFP did not show any fluorescence change in response to von Frey filament or thermal stimulation (Figs 2G-2I and S2A-S2D).These results confirm that hyperactivity of ACC neurons is associated with pain-like hypersensitivity.
To investigate the effect of stimuli related to negative emotional states on GCaMP6s signal in the ACC, we conducted the following experiments.First, we applied 1 s air puff (an aversive stimulation) onto the faces of the mice (Fig 2M) and observed a significant increase in GCaMP6s signal in the ACC (Fig 2N -2P).When the mice explored in an elevated plus maze (EPM) (Fig 2Q ), we observed an increase in GCaMP6s signal upon their entrance into the open arms, but a subsequent decrease in the GCaMP6s signal upon their retreat into the closed arms (Fig 2R -2T).In separate trials, changes of GCaMP6s signal in the ACC were observed in SNI mice when they were subjected to air puff onto the face, and explored the open and closed arms in the EPM with similar amplitude as naïve mice, and such effects were not observed in In summary, the data from GCaMP6 mice suggest that ACC neurons in both naïve and SNI mice detect pain-like stimulation and alterations in emotional states.
Our previous studies showed that STN neurons can also be activated by pain-like stimulation and alternation in emotional states [17,20], we next asked whether ACC neurons contribute to pain-like and aversive responses in STN neurons.To solve this puzzle, we injected AAV-CaMKII-hM4Di-mCherry or AAV-CaMKII-mCherry into the ACC and AAV-CaM-KII-GCaMP6 into the STN and implanted an optical fiber in the STN (Figs 3A, 3B, and S3A).In this configuration, ACC neurons can be inhibited by clozapine-N-oxide (CNO), the ligand of hM4Di.After ACC neurons were inhibited by intraperitoneally administered CNO (3 mg/ kg), the increase in GCaMP6 signal in the STN evoked by von Frey filament, thermal stimulation and air puff were significantly attenuated (Fig 3C -3Q).In control mice that mCherry was virally transfected into ACC neurons, intraperitoneal administration of CNO did not alter GCaMP6s responses of STN neurons to mechanical and thermal stimulation (S3B-S3M Fig).Considering that ACC neurons respond to emotional stimulation [23,36], we investigated whether the activity of STN neurons changes in response to emotional stimulation, and these responses are modulated by ACC neurons in naïve and SNI mice.Similar to the response of ACC neurons to air puff, we found that transient air puff reliably elicited an increase in GCaMP6s signal in STN neurons of both naïve and SNI mice and chemogenetic inhibition of ACC neurons attenuated the responses (Figs 3O-3Q and S4Y-S4Z2 and S2 Table ).Air puffevoked GCaMP6s responses were not affected by CNO in control mice with mCherry-expressing ACC neurons (S3N-S3P Taken together, these data suggest that ACC neurons influence the processing of pain signals and emotion in the STN.In the following paragraphs, we determined the behavioral consequences of modulating the ACC-STN pathway.(D, E, G, H, J, K, N, O, and S

Activating ACC-STN neurons induces hyperalgesia and depression-like behaviors in naïve mice
ACC neurons are consistently suggested to participate in pain perception and neuropsychiatric disorders, including depression [34,37,38].We tried to determine whether ACC-STN neurons, a group of ACC neurons, exert similar effect on pain-like and depression-like behavior.We transfected ChR2 into ACC-STN neurons by injecting AAV retro-hSyn-mCherry-Cre into the STN and injecting AAV-EF1α-DIO-ChR2-eYFP or AAV-EF1α-DIO-eYFP into the ipsilateral ACC (Figs 4A-4C and S6A-S6D).Immunofluorescence staining showed that most mCherrylabeled neurons in the ACC expressed a marker protein of glutamatergic neurons, vesicular glutamate transporter 1 (VgluT1) (S6E and S6F Fig) .Using patch-clamp technique, we confirmed that blue light pulses evoked time-locked inward currents in ChR2-expressing ACC neurons, suggesting that this strategy allows for efficient manipulation of ACC-STN neurons (S6G and S6H Fig) .In behavioral tests, we observed that optogenetic activation of ACC-STN neurons reduced mechanical and thermal thresholds on both hind paws in naïve mice (Fig 4D -4G).Note that mechanical and thermal thresholds in control eYFP mice were not affected by blue light illumination of the ACC (Fig 4D -4G).The data from ChR2 mice support that stimulation of ACC-STN neurons induces pain-like behaviors in naïve mice.
We employed conditioned place preference/aversion (CPP/CPA) paradigm to determine whether repetitive activation of ACC-STN neurons leads to CPP/CPA.On day 1, the mice were allowed for freely roaming in 2 chambers in CPP/CPA box for 20 min in the absence of light stimulation.In days 2 to 4, blue light pulses (473 nm, 20 Hz, 5 ms, 4 mW) (15 min/day) were delivered into the ACC when the mice were placed in the chamber with meshed floor (Fig 4H).On day 5, ChR2 mice displayed significant CPA, while control eYFP mice did not show either CPP or CPA (Fig 4I and 4J).We also observed that blue light illumination of the ACC did not alter the traveling velocity in the blue light-paired chamber in either ChR2 or eYFP mice (Fig 4K).These data exclude the possibility that CPA was confounded by altered locomotor activity.
To determine whether hyperactivity in ACC-STN neurons is sufficient to induce depression-like behaviors in naïve mice, we performed 2 routine assays including the tail suspension test (TST) (increased immobility) and forced swim test (FST) (increased immobility).We observed that optogenetic activation (473 nm, 5 ms pulses, 20 Hz, 4 mW) of ACC-STN neurons in mice significantly prolonged immobility time in TST and FST (Fig 4L and 4M).These data suggest that ACC-STN neurons are among ACC neurons triggering depression-like behaviors.

Inhibition of ACC-STN neurons ameliorates hyperalgesia and depressionlike behavior in neuropathic pain
The above data demonstrate that ACC-STN neurons are implicated in pain-like behaviors and negative emotions (Fig 4A -4M).These data prompted us to investigate whether inhibition of Heat maps (C, F, I, and L), average traces (D, G, J, and M), and summary (E, H, K, and N) of GCaMP6s signal in the STN of mice receiving von Frey filament (2 g) or thermal stimulation on hind paws with intraperitoneal (i.p.) injection of saline or CNO (3 mg/kg).(E) t = 3.54, P = 0.0008.(H) t = 2.18, P = 0.039.(K) t = 2.91, P = 0.0062.(N) t = 2.29, P = 0.03.(O-Q) Heat maps (O), average traces (P), and summary (Q, t = 2.21, P = 0.029) of GCaMP6s signal in the STN of mice exposed to 1 s air puff onto the face after intraperitoneal injection of saline or CNO.CNO or the same volume of saline was applied 45 min prior to initiation of GCaMP6s signal recording.Dashed lines in ( C, D, F, G, I, J,  L, M, O, P) indicate the onset of the stimulation.* P < 0.05, ** P < 0.01; two-tailed unpaired t test for panels (E, H, K, N, and Q, n = 5 mice.).Scale bars: 100 μm.Source data can be found in the fourth worksheet of S1 Data.ACC, anterior cingulate cortex; CNO, clozapine-N-oxide; STN, subthalamic nucleus.https://doi.org/10.1371/journal.pbio.3002518.g0034V).Furthermore, in SNI mice with ACC-STN neurons expressing NpHR, yellow light illumination throughout the test session shortened immobility time in the TST and FST, compared with SNI mice with ACC-STN neurons expressing eYFP (Fig 4W and 4X).We further performed female urine sniffing test as a non-operant test for measuring anhedonia in depression-like behavior [39].SNI mice with NpHR or eYFP transfected into ACC-STN neurons were subjected to sniffing test with water and urine of female mice.Time of female urine sniffing was significantly increased in NpHR mice upon yellow light illumination compared with that in eYFP mice, but time of water sniffing was similar between NpHR and eYFP mice (Fig 4Y and 4Z).
These results demonstrate that reversing hyperactivity in ACC-STN neurons is sufficient to mitigate hyperalgesia and depression-like behaviors in chronic pain.eYFP, or AAV-CaMKII-eYFP into the right ACC and implanted optical fiber in the right STN (Figs 5A, 5H-5J, and S8A-S8D).As expected, we observed that blue light stimulation (20 Hz, 5 ms pulse, 473 nm, 4 mW) of the ACC-STN projection reduced mechanical and thermal thresholds on both hind paws in ChR2 mice, but not in eYFP mice (Fig 5B and 5C).We performed the CPP paradigm described in Fig 4H and unexpectedly found that optogenetic stimulation of the ACC-STN projection did not induce preference or aversion to the light-paired chamber (Fig 5D and 5E).Similarly, optogenetic stimulation of the ACC-STN projection did not affect depression-like behaviors assessed with the TST and FST (Fig 5F and 5G).Therefore, the ACC-STN projection only replicates the effect of the activation of ACC-STN neurons on pain modulation but not that on aversion-and depression-like behaviors.

The modulation of pain thresholds and depression-like behaviors by ACC-STN projection in naïve and SNI mice
We then examined the behavioral outcomes following optogenetic inhibition of the ACC-STN projection in naïve and SNI mice.We observed that optogenetic inhibition of the ACC-STN projection (continuous light, 589 nm laser) did not alter mechanical and thermal thresholds on either hind paw in naïve mice (S8E-S8H Fig), but optogenetic inhibition of the ACC-STN projection replicated the effects of inhibition of ACC-STN neurons on several aspects in SNI mice, such as, a significant increase of mechanical paw withdrawal threshold (PWT) and thermal paw withdrawal latency (PWL) (Fig 5K), the establishment of CPP (Fig 5L -5N), and a dramatic attenuation of depression-like behaviors measured with the TST, FST, and female urine sniffing test (Fig 5O -5Q).
Together, these experiments support that the ACC-STN projection mediates the role of ACC-STN neurons in pain modulation in naïve mice, but in modulation of both pain-like and depression-like behaviors in SNI mice.

The ACC-STN projection interplays with STN neurons to modulate pain thresholds and depression-like behavior
Our previous studies demonstrate that stimulation of STN neurons reduces pain threshold in naïve mice [19,20].We then tested whether inhibition of the ACC-STN projection is enough to affect the activity of STN neurons and mitigates hyperalgesia induced by stimulation of STN neurons.To address this question, we injected AAV-CaMKII-hM3Dq-mCherry or AAV-CaMKII-mCherry into the STN and AAV-CaMKII-NpHR-eYFP into the ACC, and implanted optical fiber into the STN (Figs 6A, 6B, S9A, and S9B).Thus, we were able to activate STN neurons by intraperitoneal administration of CNO and inhibit the ACC-STN projection by yellow light illumination of the STN.Our patch-clamp recording from STN neurons expressing hM3Dq-mCherry in brain slices showed that bath perfusion of CNO (3 μM) increased firing rate in these neurons (S9C and S9D Fig).
We measured mechanical and thermal thresholds before and after intraperitoneal injection of CNO with or without yellow light illumination of the STN (Fig 6C).We found that CNO injection significantly decreased mechanical PWT and thermal PWL on both hind paws in hM3Dq mice, but not in mCherry mice (Fig 6D and 6E); subsequent optogenetic inhibition of the ACC-STN projection elevated both mechanical PWT and thermal PWL in hM3Dq mice received CNO injection; optogenetic inhibition of the ACC-STN projection had no effect on either mechanical or thermal thresholds in mCherry mice (Fig 6D and 6E).Therefore, inhibition of the ACC-STN projection is sufficient to attenuate hyperalgesia following hyperactivity of STN neurons.
Considering the difference in temporal resolution between optogenetic inhibition/stimulation and CNO-based chemogenetic modulation, we made some modifications in the CPA paradigm.hM3Dq or mCherry mice freely roamed in the CPA chamber for 20 min on day 1.On day 2 through day 4, these mice were intraperitoneally injected with saline and placed to 1 chamber for 20 min, and 4 h later, the mice were intraperitoneally injected with CNO and placed to the other chamber for 20 min.On day 5, the mice were allowed roaming freely in 2 chambers, and the time in each chamber was recorded (Fig 6F).We found that CNO injection induced significant place aversion for CNO-paired side in hM3Dq mice but not in mCherry mice (Fig 6G -6I).In another set of experiment, during 3 days' conditioning, we applied yellow light illumination of the STN when mice stayed in CNO injection-paired chamber (Fig 6J ).We observed that CNO-induced CPA in hM3Dq mice was blocked by optogenetic inhibition of the ACC-STN projection (Fig 6K and 6L).Optogenetic inhibition of the ACC-STN projection did not alter the time spent in the CNO-paired chamber in mCherry mice (Fig 6K and  6L).We found that neither CNO injection nor CNO injection with yellow light illumination of the STN changed the traveling velocity in hM3Dq or mCherry mice (Fig 6I and 6M).Therefore, inhibition of the ACC-STN projection is an effective approach to mitigate aversion induced by hyperactivity of STN neurons.
We next explored whether analgesic effect of inhibition of the ACC-STN projection in SNI mice is related to regulation of STN neurons.First, we compared c-Fos-positive STN neurons in SNI mice with and without receiving optogenetic inhibition of the ACC-STN projection.We found that optogenetic inhibition of the ACC-STN projection significantly reduced the number of c-Fos-positive STN neurons in SNI mice (S10A-S10C Fig) .Second, we examined the effect of optogenetic inhibition of the ACC-STN projection on pain thresholds and CPP in SNI mice with and without chemogenetic activation of STN neurons.We observed that activation of STN neurons with CNO prevented the analgesic effects of inhibition of the ACC-STN projection in SNI mice (Fig 6O).In contrast, analgesic effect of optogenetic inhibition of the ACC-STN projection was not affected by CNO injection in mCherry mice in which STN neurons were not activated by CNO (Fig 6O).Activation of STN neurons also eliminated CPP and antidepressant effects of inhibition of the ACC-STN projection in SNI mice (Fig 6Q -6U).Therefore, the analgesic and antidepressant effect of the inhibition of the ACC-STN projection in SNI mice is relevant to its regulation of STN neurons.

Discussion
The comorbidity of chronic pain and psychiatric disorders, such as depression, has long been recognized clinically [40,41], and preclinical studies have reported depression-like behaviors in animal models of chronic pain [6][7][8]11].These findings suggest that common neural circuit bases may exist for these pathological states.In this study, we focused on the ACC-STN pathway.Using viral vector-assisted circuit tracing, in vivo Ca 2+ imaging, optogenetic and  (B, C, E, K, M, N, and Q  chemogenetic modulation, and electrophysiological and morphological assay in mice, we demonstrated that inhibition of the ACC-STN pathway had benefit to not only hyperalgesia but also depression-like behaviors in chronic pain mice.First, inhibition of ACC neurons attenuated responses of STN neurons to mechanical, thermal, and aversive stimulation.Second, optogenetic inhibition of ACC-STN neurons and the ACC-STN projection alleviated pain sensitivity, established CPP, and mitigated depression-like behaviors in SNI mice.Third, the analgesic and antidepressant effect of the inhibition of the ACC-STN projection in SNI mice was blocked by chemogenetic activation of STN neurons.These data suggest that the ACC-STN pathway plays an important role in the maintenance of hyperalgesia and negative emotion in chronic pain.Additionally, our findings support a notion that the hyperactivity of STN neurons is important for the manifestation of hyperalgesia and depression-like behavior in chronic pain; the ACC-STN pathway is one but not the only synaptic inputs contributing to the hyperactivity of STN neurons in chronic pain; only when normal activity of STN neurons is restored, are symptoms of chronic pain effectively mitigated. The basal ganglia, including the STN, relays nociceptive signals to brain nuclei implicated in pain processing and modulation [19,20,42].Our recent studies demonstrated that the STN and its projections are involved in both somatosensory and emotional aspects of chronic pain [7,19,20].The ACC has similar function as the STN in these aspects [25][26][27].Using optogenetic and patch-clamp techniques, we confirmed that ACC glutamatergic neurons monosynaptically innervated STN glutamatergic neurons.Although the ACC is a source of hyperdirect pathway to the STN, which plays an important role in motor control [31,43,44], it is unclear whether the ACC-STN pathway is involved in the modulation of pain threshold and emotional processing by the STN.We found that enhancing the activity of STN neurons by optogenetic stimulation of the ACC-STN projection led to hyperalgesia, but did not cause CPA and depression-like behavior in naïve mice; chemogenetic inhibition of ACC glutamatergic neurons suppressed activation of STN neurons induced by pain-like and aversion-like stimulation.Therefore, the hyperdirect pathway provides a bridge between the ACC and STN conferring regulatory effects on pain thresholds.These results expand the roles of the hyperdirect pathway.(D, E, H, I, L, M, O, R, and S); two-tailed unpaired t test for (T and U).Scale bars: 100 μm.Source data can be found in the seventh worksheet of S1 Data.ACC, anterior cingulate cortex; CNO, clozapine-N-oxide; CPP, conditioned place preference; FST, forced swim test; PWL, paw withdrawal latency; PWT, paw withdrawal threshold; SNI, spared nerve injury; STN, subthalamic nucleus.https://doi.org/10.1371/journal.pbio.3002518.g006 Photostimulation of ACC glutamatergic neurons establishes CPA and induces depressionlike behavior in naïve animals [5,34,35,37].Similar to these studies, we observed that optogenetic activation of ACC-STN neurons was sufficient to elicit aversion-and depression-like behavior in naïve mice.However, activation of the ACC-STN projection did not elicit aversion-and depression-like behaviors in naive mice.In contrast, chemogenetic activation of STN neurons results in aversion-and depression-like behavior in naïve mice.These inconsistencies may suggest the complexity of the ACC-STN pathway.Together with previous studies [5,10], this study shows that besides the STN, ACC-STN neurons project to the basolateral amygdala, bed nucleus of stria terminalis, nucleus accumbens, caudate putamen, claustrum, periaqueductal gray, thalamic nuclei, and superior colliculus, etc., it suggests that these projections may work together to regulate emotion.This may explain why the ACC-STN projection did not regulate emotions like ACC-STN neurons.The activation of ACC axonal terminals in the STN mobilizes a proportion of STN neurons which may be less than optogenetic stimulation of the STN does.This may explain why the effects of STN neuron stimulation differ from stimulation of the ACC-STN projection.These results suggest that in the ACC-STN pathway, the information in ACC-STN neurons may not completely transmit to the STN, and STN neurons are influenced by other brain regions in addition to the ACC.
Hyperactivity in STN neurons has been reported in several pain states [7,19,20].Although the STN receives projections from many brain regions implicated in various pain states [29,45], the contribution of these brain regions to the hyperactivity in STN neurons has not been profoundly studied.Our recent study reported that reduced synaptic inputs from GABAergic neurons in the substantia nigra pars reticulata (SNr) may contribute to the hyperactivity of STN neurons in mice with chronic pain [7].The present study shows that the excitability of ACC-STN neurons and synaptic strength of the ACC-STN projection in chronic pain mice were enhanced.We observed that optogenetic activation of either ACC-STN neurons or the ACC-STN projection induced hypersensitivity to mechanical and thermal stimuli and CPA in naïve mice.These data suggest that enhanced ACC-STN pathway may be sufficient to cause hyperalgesia and depression-like behavior in chronic pain.In contrast, we found that inhibition of this projection mitigated hyperalgesia and depression-like behavior in chronic pain, suggesting that the enhancement of the ACC-STN pathway may be necessary for these phenotypes in chronic pain.These evidence underscores a significant role of the ACC-STN pathway in facilitating and maintaining hyperalgesia and depression-like behavior in chronic pain.
We have demonstrated that the activity of STN neurons is elevated in mice with comorbid chronic pain and depression [7] and in mice exposed to acute stress [17].Parolari and colleagues [46] reported that repeated inhibition of a subpopulation of STN neurons expressing Gabrr3 gene suppressed grooming in a mouse model for obsessive-compulsive disorder (OCD).These findings collectively suggest that increased activity of STN neurons may be a common neuronal mechanism underlying emotional dysfunctions, such as depression, occurred in chronic pain, acute stress, and OCD.
While our study provides evidence showing the involvement of the ACC-STN pathway in chronic pain and depression, it is important to acknowledge that the experiments have some limitations.First, as sex differences have been observed in relation to chronic pain and emotional disorders [9,47,48], future studies should investigate whether sex acts as a biological variable in the effects of the ACC-STN pathway on hyperalgesia and depression-like behaviors in chronic pain.Second, in fiber photometry recording, we examined the responses of ACC and STN neurons to pain, aversive, and anxiety-like stimulation, while in neuromodulation experiments, we focused on pain threshold and depression-like behaviors.Anxiety and depression, 2 major psychiatric disorders with a comorbidity of about 50% [48], affect each other [48], but their neural circuit mechanisms may not be identical [49][50][51].Further investigations are needed to address the role of the ACC-STN pathway in representation and modulation of anxiety and depression.
In summary, our study demonstrates that the ACC-STN pathway plays a significant role in regulating pain and emotion with somewhat difference among its components, including ACC-STN neurons, the ACC-STN projection, and STN neurons.Notably, a significant interplay may exist between the ACC-STN projection and STN neurons to control the manifestations in comorbid chronic pain and depression.This study supports that the ACC-STN pathway may be a therapeutic target to treat comorbid chronic pain and depression.

Ethics statement
The care and use of animals and the experimental protocols (No. 202207S123) used in this study were approved by the Institutional Animal Care and Use Committee and the Office of Laboratory Animal Resources of Xuzhou Medical University under the Regulations for the Administration of Affairs Concerning Experimental Animals (1988) in China.

Animals
Wild-type C57BL/6J (WT) mice were purchased from the animal facility at Xuzhou Medical University.The mice were group-housed (no more than 5 per cage) in a temperature-and humidity-controlled housing facility on a 12-h light/dark cycle with ad libitum access to food and water.Male mice at least 8 weeks old were used in the experiments.Efforts were made to minimize animal suffering and to reduce the number of animals used.

Stereotaxic surgeries and viral injection
Mice were deeply anesthetized with isoflurane (3% for induction, 1.5% for maintenance) (RWD Life Science Co., Shenzhen, China), placed on a heating pad, and stabilized on a stereotaxic apparatus (RWD Life Science Co., Shenzhen, China).Small holes were drilled in the skull above brain regions of interest.Injections (120 nl of virus per site at 50 nl/min) were made using an automatic microinjection pump (KD Scientific, Holliston, Massachusetts, United States of America).
The viral vectors were injected into the following coordinates (relative to the Bregma): STN (AP, −1.85 mm; ML, 1.48 mm; DV, 4.75 mm) and ACC (AP, +0.85 mm; ML, 0.4 mm; DV, 1.85 mm).Optical fiber implants (200 μm in diameter, NA 0.37) (Inper, Hangzhou, China) were placed 200 μm above (for optogenetic manipulation) or in (for fiber photometry recording) the injection site and were fixed to the skull with dental cement.Mice with virus injections and optical fiber implants were allowed to recover for at least 3 weeks before electrophysiological recordings and morphological assays.Viral expression and the position of fiber implants in each mouse were confirmed histologically after the termination of the experiments.We only included mice with viral expression confined to the STN or ACC and optical fibers in the right places for either optogenetic modulation or fiber photometry recording.
Meloxicam (4 mg/kg) (Aladdin Biochemical Technology, Shanghai, China) was administered subcutaneously once per day for 3 days for postoperative pain relief.

Fiber photometry
A fiber photometry instrument (ThinkerTech, Nanjing, China) [6,18,20,52] was used to monitor GCaMP6 and eYFP signals in ACC and STN neurons.We adjusted the instrument by setting the excitation light to 50 μW and the gain to a level that gave a background signal of 3 units.The input optical cable was then connected to the optical implant in the mouse brain.To evaluate responses of STN and ACC neurons to mechanical, thermal, and emotional stimulation, we designated 3 s GCaMP6 and eYFP signal prior to the stimulation as the baseline value (F 0 ) and calculated the mean and standard deviation (SD) of F 0 .We used these parameters to transform GCaMP6 and eYFP signal (F) into the z-score ((F-Mean of F 0 )/SD of F 0 ) and measured the area under the curve (AUC, 12 s post stimulation) of the z-score plot to quantify the response of ACC neurons and STN neurons to sensory and emotional stimulation.

Optogenetic manipulation
For ChR2-mediated optogenetic stimulation, 473-nm laser pulses (5 ms, 20 Hz, 4 mW) were delivered.For NpHR-mediated optogenetic inhibition, a 3 mW 589-nm laser was kept on continuously for 1 to 2 min (constant, 3 mW).All optogenetic manipulations were performed unilaterally in the right hemisphere.Therefore, the contralateral and ipsilateral sides refer to the left and the right sides, respectively.

von Frey filament test
Individual mice were acclimatized for at least 1 h in a test compartment on a wide gauge wire mesh supported by an elevated platform.The von Frey filaments with fiber force between 0.01 and 2 g were used to measure mechanical PWT of both hind paws.The 50% threshold was determined with the up-down method [7,53].

Thermal nociception threshold
Individual mice were acclimatized for at least 1 h in a test compartment on a glass surface.Thermal PWLs in both hind paws were measured with a plantar anesthesia tester (Boerni, Tianjin, China) [7,54].

Spared nerve injury (SNI)
Chronic neuropathic pain model was established with spared nerve injury of the sciatic nerve according to a previously reported protocol [7,55].Mice were deeply anesthetized using isoflurane (3% for induction, 1.5% for maintenance) (RWD Life Science Co., Shenzhen, China).The fur in the surgical area, extending from the knee to the hip, was shaved, and the skin was sterilized with 75% alcohol.A longitudinal incision was made in the shaved region, allowing for blunt dissection of the biceps femoris muscle to expose the sciatic nerve and its branches (sural, common peroneal, and tibial nerves).Two nylon sutures 3 mm apart were tightly ligated around the common peroneal and tibial nerves, and the nerves between the sutures were subsequently cut and removed.The mice were then allowed to recover on a heating pad.
Mice that did not receive nerve ligation and severing were used as sham controls.Pain thresholds were assessed using von Frey filaments and a heating beam targeting the skin area innervated by the sural nerve.

Conditioned place preference (CPP) test
The CPP was performed in a custom-made two-chamber box (length × width × height: 40 × 20 × 30 cm 3 ): the right chamber had vertical black-and-white stripes on the walls and a smooth floor, whereas the left chamber had horizontal black-and-white stripes on the walls and a mesh floor.The CPP test was performed according to a previous study with some modifications [20].
Day 1 was the preconditioning test (precondition) day.Mice were given free access to the 2 chambers and the time that the mice spent in each chamber was recorded.On days 2 to 4, the mice were restricted to 1 chamber for 20 min and received optogenetic modulation.At least 4 h later, the mice were restricted to the other chamber for 20 min without optogenetic modulation.On day 5 (around 18 h after the last conditioning), the mice were allowed to freely explore the 2 chambers for 20 min and the time spent in each chamber was recorded.On the precondition and test days, the animal's movement was video-tracked and analyzed online or offline with an EthoVision XT video tracking software (Noldus Information Technology, Wageningen, the Netherlands) [56,57].We calculated the time spent in the light-paired side on the precondition and test days.Mice were not used if they spent more than 75% of the total time in 1 chamber on the pre-test day.

Measurement of depression-like behaviors
TST, FST, and female urine sniffing test have been used for assessing depression-like behaviors in rodents [6,7,39].A longer immobility time in the TST and FST refers to despair, while a shorter time spent sniffing female urine refers to anhedonia in male mice.

Tail suspension test (TST)
A mouse was suspended by taping its tail onto a horizontal bar 50 cm above the floor.The mouse was allowed to hang undisturbed for 6 min and its behavior was video-recorded.The total duration that the mouse remained immobile in the last 5 min was used to evaluate depression-like behavior.

Forced swim test (FST)
Mice were placed in a 1 liter glass beaker filled with 26˚C water.The movement of the mice was recorded for 5 min using a camera placed beside the beaker, and the immobility time was measured.

Female urine sniffing test
This is a sex-related reward-seeking behavior [39].Male mice were singly housed for 1 week before testing.In the test day, male mice in their home cage were first exposed for 3 min to a cotton tip dipped in tap water; 45 min later, male mice were exposed for 3 min to another cotton tip dipped in fresh urine collected from female mice in the estrus phase.The total times of water and female urine sniffing were measured.

Statistical analysis
GraphPad Prism 7.0 was used for statistical analyses.Clampfit 10.7 (Molecular Devices) was used for analysis of electrophysiological and GCaMP6 data.Figures were prepared with Adobe Illustrator CS6.All summarized data are expressed as mean ± SEM.Two-tailed paired or unpaired t tests were used for comparison of a parameter between 2 groups if data were normally distributed.One-way or two-way, ordinary or repeated measures ANOVAs followed by Tukey's post hoc analysis were used for multiple comparisons.If the equal-variance assumptions were not valid, statistical significance was evaluated with the Mann-Whitney test or ANOVA rank tests.The mean and SEM, n (the number of animals), statistical test, and t, F, and P values are reported in the figure legends.A value of P < 0.05 was considered statistically significant.The minimal number of mice used in each experiment was calculated in a priori power analysis (StatMate 2.0) and the power of each experiment was set to 0.8.The sample sizes in each experiments are larger than the minimal numbers.

Fig 1 .
Fig 1. STN-projecting ACC neurons are hyperactive in chronic pain.(A and B) Viral vector was injected in the ACC to label ACC neurons (A) and enable visualization of their AU : AbbreviationlistshavebeencompiledforthoseusedinFigs1to6:Pleaseverifythatallentriesarecorrect projections to the STN (B).(C) Whole-cell patch-clamp recordings were performed to record firing in ACC neurons evoked by 5 ms blue light stimulation (50 ms interval, 2 mW) (upper panel, schematic diagram; lower panel, a typical trace).(D-F) Blue light (5 ms, 10 Hz, 2 mW)-evoked excitatory postsynaptic currents (photo-EPSCs) in STN neurons were not blocked by perfusion of 1 μM TTX and 0.3 mM 4-AP, but were blocked by 20 μM CNQX.(D) Traces.(E) Percentage of STN neurons (25 neurons from 4 mice) responding to photostimulation of the ACC-STN projection.(F) Summary of amplitude of photo-EPSCs in different conditions (F (2, 9) = 125.5,P < 0.0001, n = 8 neurons recorded from 4 mice).(G and H) Schematic diagram (G), image (H), and summary (I) showing that virus injected into the STN retrogradely labeled layer V neurons in the ACC in coronal sections (n = 5 mice).(J) Schematic diagram for retrograde labeling of STN-projecting ACC neurons and whole-cell patch-clamp recording of STN-projecting ACC neurons 4 weeks after SNI or sham surgery.(K) Left: A representative image of mCherry-labeled STN-projecting ACC neurons in the layer V of the ACC from an SNI mouse 4 weeks after SNI surgery.Right: higher magnification fluorescent image (upper right) and IR-DIC image (lower right).Arrow head points to a patched mCherry-labeled ACC neuron.(L and M) Representative traces (L) and summary (M) of evoked firing recorded from mCherry-labeled ACC neurons 4 weeks after sham (black) or SNI (red) surgery (Group: F (1, 806) = 241.6,P < 0.0001; n = 37 neurons in SNI, n = 29 neurons in sham, from 4 mice in each group).(N-Q) Quantification of resting membrane potential (N, t = 0.28, P = 0.78, n = 15 neurons in each group), membrane resistance (O, Rm, t = 2.77, P = 0.0098, n = 15 neurons in each group), rheobase (P, t = 5.19, P < 0.0001, n = 15 neurons in each group), and depolarization threshold (Q, t = 2.86, P = 0.008, n = 15 neurons in each group) of STN-projecting ACC neurons from SNI and sham mice.(R) Scatter plot and linear relationship between depolarization voltage and rheobase in SNI and sham mice.SNI: r = 0.68, P = 0.0058; sham: r = 0.80, P = 0.0004; n = 15 neurons in each group.P = 0.51, SNI vs. sham mice.(S) Diagram showing AAV-CaMKII-ChR2-eYFP injection into the ACC and patch-clamp recording from STN neurons of SNI and sham mice.(T) Photo-EPSCs recorded from STN neurons in SNI (red) and sham (black) mice.(U) Amplitude of photo-EPSCs in STN neurons from SNI and sham mice (t = 2.45, P = 0.018, n = 32 neurons in SNI, n = 24 neurons in sham, from 4 mice in each group).(V) Paired-pulse ratio of photo-EPSCs in STN neurons from sham and SNI mice (t = 0.78, P = 0.44, n = 31 neurons in SNI, n = 19 neurons in sham, from 4 mice in each group).* P < 0.05, ** P < 0.01; one-way repeated measures ANOVA with Tukey's post hoc analysis for (E); two-way repeated measures ANOVA with Tukey's post hoc analysis for (M); two-tailed unpaired t test for (N-Q, U, V).A z-test on Fisher ztransformed correlation coefficients was used in (R).Scale bars: 100 μm.Source data can be found in the first worksheet of S1 Data.ACC, anterior cingulate cortex; EPSC, excitatory postsynaptic current; SNI, spared nerve injury; STN, subthalamic nucleus.https://doi.org/10.1371/journal.pbio.3002518.g001

Fig 2 .
Fig 2. ACC neurons respond to pain-like stimuli and alterations in emotional states.(A-C) Schematic diagram (A, C) and a representative image (B) of virus injection into the ACC for fiber photometry recording of GCaMP6s-or eYFP-labeled ACC neurons in response to von Frey filament and heat stimulation on the hind paws or during paw lifting in freely moving mice, and 2 g but not 0.16 g von Frey filament induced paw withdrawal behavior in naïve mice.(D-I) Heat maps (D, G), averaged normalized traces (E, H), and summary (F, I) of changes in GCaMP6s and eYFP signals in the ACC in response to von Frey filament stimulation (F, F (3, 86) = 8.57, P < 0.0001) or thermal stimulation (I, F (3, 118) = 16.23,P < 0.0001) on hind paws or in response to random paw lifting in naïve mice.n = 5 mice in each group.(J-L) Heat maps (J), averaged normalized Furthermore, SNI mice exhibited similar GCaMP6s responses of STN neurons to mechanical and thermal stimulation as naïve mice (S1 Table), which were attenuated by chemogenetic inhibition of ACC neurons (S4A-S4X Fig).These results indicate that ACC neurons regulate pain signals in STN neurons in both physiological and neuropathic pain states.
Fig).Unlike ACC neurons, STN neurons in naïve mice did not exhibit similar changes in GCaMP6s signal during exploration in the EPM (S5A-S5L Fig).Additionally, intraperitoneal administration of CNO to the mice expressing hM4Di or mCherry in the ACC had no discernible impact on GCaMP6s signal in the STN during EPM exploration (S5A-S5L Fig).In contrast, we observed significant increase or decrease in GCaMP6s signal in the STN of SNI mice upon their entry into the open arms and into the closed arms in the EPM (S2 Table); chemogenetic inhibition of ACC neurons by CNO did not attenuate the alterations of GCaMP6s signal when the mice entered into the open or closed arms (S5M-S5O Fig).These results indicate that STN neurons in neuropathic pain state respond to aversive stimulation and changes in emotional state.
traces (K), and summary (L) of changes in GCaMP6s signals in response to 0.16 g von Frey filament or 48˚C thermal stimulation on the hind paw on the injured side in SNI mice that spared nerve injury was performed on the side either contralateral or ipsilateral to GCaMP6s side.(L) F (4, 108) = 7.75, P < 0.0001.n = 5 mice in each group.(M-P) Schematic diagram (M), heat maps (N), averaged normalized traces (O), and summary (P, F (2, 110) = 38.77,P < 0.0001) of changes in GCaMP6s and eYFP signals in naïve or SNI mice in response to 1 s air puff onto the face.(Q-T) Schematic diagram (Q), heat maps (R), averaged normalized traces (S), and summary (T, F (4, 118) = 42.97,P < 0.0001) of changes in GCaMP6s and eYFP signal in naïve or SNI mice during exploration of the open or closed arms in the EPM.(Q) Includes color coding for (S) and (T).** P < 0.01; oneway ANOVA with Tukey's post hoc analysis for (F, I, L, P, and T).n = 5 mice in each group.Dashed lines in

Fig 3 .
Fig 3. Chemogenetic inhibition of ACC neurons attenuates the response of STN neurons to pain-like and aversive stimuli.(A) Experimental strategy for recording of GCaMP6s signal in STN neurons with or without chemogenetic inhibition of ACC neurons.(B) Example images of hM4Di and GCaMP6s expression in the injection sites.(C-N)

S1Fig.
Neurons in the anterior cingulate cortex are hyperactive in SNI mice.Related to Fig 1. (A) Schematic diagram of SNI surgery (upper panel) and time line of experiments for measurement of pain-like and depression-like behaviors.(B and C) Time course of mechanical (B) and thermal (C) thresholds after SNI surgery.(B) F (1, 12) = 212.9,P < 0.0001.(C) F (1, 12) = 475.9,P < 0.0001.n = 6 sham mice, n = 8 SNI mice.PWT: paw withdrawal threshold; PWL: paw withdrawal latency.(D and E) Immobility time in the TST (D, t = 2.54, P = 0.026, n = 6 in sham, n = 8 in SNI) and the FST (E, t = 3.38, P = 0.0055, n = 6 sham mice, n = 8 SNI mice).(F) Comparison of time spending sniffing water and female urine between sham and SNI mice.SNI mice showed reduced time spent sniffing female urine compared with sham mice (F (1, 28) = 24.15,P < 0.0001, n = 8 mice in each group).(G and H) Representative images (G) and quantification (H) of c-Fos-(+) neurons in the ACC 4 weeks after sham operation or SNI surgery on the right side (t = 8.9, P < 0.0001; n = 5 mice in each group).** P < 0.01; two-way repeated measures ANOVA with Tukey's post hoc analysis for (B and C); two-tailed t test for (D, E, and H); one-way ANOVA was used for (F).* P < 0.05; ** P < 0.01; ns, not significant.Scale bars: 200 μm.Source data can be found in the first worksheet of S2 Data.(TIFF) S2 Fig. eYFP signal in ACC neurons does not change with pain-like and emotion-like behavior.Related to Fig 2. (A) Schematic diagram showing virus (AAV-CaMKII-eYFP) injection and fiber implantation into the ACC for fiberphotometry.(B-D) Heat maps (B), average traces (C), and quantification (D, t = 0.67, P = 0.51) of eYFP signal in the ACC of naïve mice in response to von Frey filament (vF) and thermal stimulation of hind paws.(E-G) Heat maps (E), average traces (F), and quantification (G, t = 0.31, P = 0.67) of eYFP signal in the ACC of naïve or SNI mice expressing eYFP in the ACC in response to 1 s air puff on the face.(H-J) Heat maps (H), average traces (I), and quantification (J, F (2, 57) = 2.69, P = 0.076) of eYFP signal in the ACC of naïve or SNI mice expressing eYFP in the ACC during exploration of the open or closed arms in the elevated plus maze.Two-tailed t test for panels (D, G, n = 5 mice.);one-way ANOVA with Tukey's post hoc analysis for (J, n = 5 mice in control and ); two-tailed unpaired t test for (F, G, O, and P).Scale bars: 100 μm.Source data can be found in the sixth worksheet of S1 Data.ACC, anterior cingulate cortex; FST, forced swim test; PWL, paw withdrawal latency; PWT, paw withdrawal threshold; SNI, spared nerve injury; STN, subthalamic nucleus; TST, tail suspension test.https://doi.org/10.1371/journal.pbio.3002518.g005