Top-down descending modulation of dorsal spinal excitatory transmission from the insular cortex

Abstract

The insular cortex (IC), a critical hub for nociception, emotion, and cognition processing, has emerged as a key role in the descending modulation of spinal cord excitability. Although previous studies have suggested that IC may influence spinal nociceptive reflexes through direct or indirect top-down pathways, the specific effects of IC stimulation on spinal nociceptive transmission remain unclear. In this study, by combining in vivo whole-cell patch-clamp, behavioral and morphological approaches, we identified a direct projection from the IC to the contralateral dorsal spinal cord. To determine whether IC activation affect the spinal nociceptive reflex, we measured the spinal nociceptive tail-flick (TF) reflex during IC stimulation. We found that activating the IC by electric stimulation did not significantly alter the spinal TF reflex. Furthermore, in vivo whole-cell patch-clamp recordings from spinal dorsal horn neurons revealed that IC stimulation produced delayed inhibition of spontaneous excitatory transmission in some neurons, while exciting or having no significant effect on others. These results indicate that the top-down modulation from the IC to the spinal cord is not uniformly facilitatory, distinguishing it from the consistently facilitatory effects observed in the anterior cingulate cortex (ACC)–spinal cord projection.

Introduction

The anterior cingulate cortex (ACC) and insular cortex (IC) are two key cortical regions involved in pain perception.^1–3 While the ACC has been extensively studied, the role of the IC is less understood. The IC is a multimodal brain region integrating sensory, emotional, and cognitive processes in both humans and rodents.^4–8 Imaging and electrophysiological studies in humans have consistently highlighted its role in pain perception.^9–12 Electrical stimulation of the IC in humans evoked pain sensations. ¹³ There are also some reports that lesions in the IC can exert an antinociceptive effect in both human ¹⁴ and animal studies. ¹⁵ Additionally, lesions of the insula showed higher pain ratings without emotional response in humans ¹⁶ indicating a complex modulatory role. It is generally accepted that the posterior IC is involved in the sensory-discriminative aspects of pain, while the anterior IC mediates affective components.^5,8,17

Anatomic evidence has demonstrated that the IC receives afferent inputs from thalamic nuclei^4,18–20 and projects to various regions, including the caudate nucleus, thalamus, parabrachial nucleus, solitary tract nucleus, and spinal cord, have been found by using the anterograde tracing technique in primates.^21–24 The IC also forms reciprocal connections with the amygdala, limbic system, and cortical association areas. Some of these projections are involved in pain modulation. For example, activation of the posterior IC– mid-cingulate cortex pathway induces mechanical pain hypersensitivity in mice. ²⁵ A descending pathway from the caudal granular IC that passes through the primary somatosensory cortex evokes potentials in the spinal dorsal horn (SDH). ²⁶ In addition, increasing GABA in the rostral agranular IC produces lasting analgesia by enhancing the descending inhibition of spinal nociceptive neurons, while selectively activating GABA_B-receptor-bearing rostral agranular IC neurons produces hyperalgesia through projections to the amygdala, an area involved in pain and fear in rats. ²⁷ Brainstem structures, such as the periaqueductal gray (PAG) and rostroventral medial medulla (RVM), which receive IC projections,^28–30 are known to bidirectionally modulate SDH neuronal activity and nociceptive responses in rats.^31,32 This pathway laid another foundation of indirect descending modulation from the IC to the spinal cord.

There are also a few studies reported direct projections from the IC to the spinal cord. For instance, the IC projects to the spinal trigeminal nucleus of the medulla that facilitates nociception in rats. ³³ An early morphological study on rats identified a direct somatostatinergic projection from the IC to the spinal cord. ³⁴ Our previous study, using in vivo whole-cell patch recording and Ca²⁺ imaging on the SDH neurons, reported that electric stimulation of the ACC facilitates spinal nociceptive transmission in rats and mice,^35,36 and directly potentiated the spinal excitatory synaptic transmission and spinal neuronal Ca²⁺ responses. ³⁷ However, whether the IC sends projections to the SDH directly and modulates the spinal excitatory transmission remains unclear. Therefore, in the present study, we investigated the effects of unilateral IC stimulation on mechanical nociception. By using a virus tracking strategy, we labeled neurons in the IC that project to the contralateral SDH. We further recorded the spontaneous excitability in the SDH neurons before and after the IC stimulation.

Methods

Animals

Adult Sprague-Dawley rats (5–6 weeks) were used. Animals were randomly housed under a 12-h light-dark cycle (9 a.m.–9 p.m. light), with food and water ad libitum, at least one week before carrying out experiments. Experimental procedures involving animals were approved by the Experimental Animal Center of the Kunming Medical University (kmmu20241949).

In vivo patch recording

The methods used for the in vivo whole-cell patch-clamp recording from SDH neurons were similar to those described previously. ³⁶ Sixteen rats were deeply anesthetized with intraperitoneal urethane (1.2–1.5 g/kg). A thoraco-lumbar laminectomy exposed the spinal cord at the L3–L5 level, and the animal was secured in a stereotaxic frame. To access the SDH, the dorsal roots above the recording site were gently retracted bilaterally using a glass tool, revealing Lissauer’s tract. A small section of the pia-arachnoid membrane was excised with microforceps to create an entry point for the recording electrode. The spinal cord surface was continuously perfused with oxygenated (95% O₂, 5% CO₂) artificial cerebral spinal fluid solution (10–15 ml/min, 36.5 ± 0.5℃), containing (in mM): NaCl 117, KCl 3.6, CaCl₂ 2.5, MgCl₂ 1.2, NaH₂PO₄ 1.2, glucose 11, and NaHCO₃ 25.

Patch pipettes (8–12 MΩ) were filled with an intracellular solution for EPSC recordings (in mM): K-gluconate 135, KCl 5, CaCl₂ 0.5, MgCl₂ 2, EGTA 5, ATP-Mg 5, and HEPES-KOH 5 (pH 7.2). Electrodes were advanced at a 30–45° angle into the SDH (30–150 μm depth) using a micromanipulator. Neurons with a stable resting membrane potential (<−50 mV) and held at −70 mV (voltage-clamp) were selected for experiments. Signals were amplified (Axopatch 200B), digitized (Digidata 1440A), and recorded using pCLAMP 10 software.

IC electric stimulation

For IC electrical stimulation, a small cranial burr hole was made, and a concentric bipolar electrode (0.15 mm OD; PlasticsOne, USA) attached to a 30-gauge injector was positioned in the IC (coordinates: AP −1.08 mm, ML −6.0 mm, DV −7.5 mm). High-frequency stimuli (HFS) consisted of 100 μs rectangular pulses (100 μA, 100 Hz) delivered in 1-s trains every 10 s for 50 s. After obtaining stable recordings from an SDH neuron, IC stimulation was applied to assess its effects on spinal neuronal activity. For the spinal nociceptive tail-flick reflex experiments, the intensity range from a low of 20 µA to a maximum of 300 µA with a stable frequency at 100 Hz, is to find whether there is a dimorphic behavioral modulation. ³⁵ For the in vivo patch recording experiments, the 100 µA intensity and 100 Hz frequency are chosen as a supra-threshold, strong stimulus likely to reliably activate a significant number of axons and trigger any potential modulatory effects on the recorded neuron, according to our previous reports. ³⁷

Spinal nociceptive tail-flick (TF) reflex

Six rats were anesthetized with 2%–3% isoflurane. After surgery, rats were maintained at a lightly anesthetized state (about 0.75% isoflurane). The TF reflex was induced using radiant heat stimulation on the ventral side of the tail. Randomly chosen locations at distances of 3, 4, 5, 6, or 7 cm from the tail tip were heated at intervals of 2–3 min to elicit the response.^38–41 The time taken for the tail to withdraw from the heat source was recorded with a photocell timer, accurate to 0.1 s. To prevent tissue injury, a maximum exposure time of 10 s was set. The 2–3 min gap between consecutive heat applications ensured consistent reflex latencies throughout the study. To determine stimulation thresholds for facilitation or inhibition of behavioral nociceptive reflexes, electrical stimulation started 10 s before the onset and continued during noxious stimulation of peripheral tissue. This stimulation paradigm has been determined experimentally to require the lowest intensity of stimulation to inhibit the TF reflex. ⁴¹ At each site of stimulation in the IC, stimulation at a low intensity (20 µA) was tested first, and the intensity was increased stepwise thereafter to a maximum of 300 µA.

Virus tracing

Four rats were anesthetized by isofluorane (RWD, China, 3%) and were placed in a stereotactic rig. The skull was exposed, and a small burr hole was made above the right IC. The adeno-associated viral vector AAV2/9-hSyn-FLEX-EGFP-WPRE-pA (Taitool, Shanghai, China) at a titer of 5E+11 was delivered via a micro-syringe pump (23 nl/min, Nanoject III, DRUMMOND). The coordinates of the injection were as follows: AP −1.44, DV −7.3 and ML −6.0 mm. The L3-L5 was exposed by surgery, and the retrograde tracing AAV2/2Retro-hSyn-Cre-WPRE-pA (Taitool, Shanghai, China) at a titer of 1E+12 was delivered in the left SDH ( DV −0.32 and ML −0.86 mm). The skin was closed with sutures. Mobility, eating, drinking, and weight were monitored for the 5-day postoperative duration.

At 3–4 weeks after injections, rats were anesthetized and transcardially perfused with saline, followed by 4% PFA. Brains were removed and postfixed overnight. Serial coronal 30 µm sections were made with a freezing microtome (Leica CM1860, USA), and slices were plated on slides. Immunofluorescence images were collected with a laser scanning confocal microscope (Nikon AX R, Tokyo, Japan).

Statistical analysis

All data were reported as means ± SEM. OriginPro 2021, GraphPad Prism 9, and SPSS 22.0 software were used for figure plotting and data analysis. A two-tailed paired or unpaired t-test was used to examine statistical differences between the two groups. One-way ANOVA followed by Dunnett T3 post hoc test was used for comparison among multiple groups. The significance levels of the statistical tests were presented as *p < 0.05, **p < 0.01, ***p < 0.001; ns means no significance with p > 0.05.

Results

Electrical stimulation of the IC did not affect the tail-flick reflex of lightly anesthetized adult rats

Previous investigations found that focal electrical stimulation of the ACC facilitated spinal nociceptive TF reflex. ³⁵ To observe whether the IC has a similar effect, we anesthetized the rat with 0.75% isoflurane and implanted the electrode stereotaxically into the left IC. Then we measured the latency of the TF reflex (Figure 1(a) and (c)). Weak (20 μA), medium (110 μA), and strong (300 μA) HFS were applied in the IC sequentially in each rat. The TF latency was measured before, during, and after the HFS. Different from the reports in the ACC, we found that the HFS at any intensity failed to facilitate the spinal nociceptive reflex in all the stimulating sites among all six rats shown in Figure 1(c) (Figure 1(b)). These results suggest that the pattern of spinal nociceptive modulation by the IC is different from that of the ACC, which might be due to a different top-down modulation network.

Figure 1.

Spinal nociceptive tail-flick reflex before, during, and after the HFS applied in the IC at different intensities. (a) Schematic of electrode placement in the IC for electrical stimulation. (b) Summary data showing no significant changes in tail-flick latency during or after HFS at 20 μA, 110 μA, or 300 μA intensities compared to baseline (Baseline, white), stimulation (HFS, light gray), and post-stimulation (post-HFS, dark gray) periods. Data represent mean ± SEM. (c) Stimulating sites are shown in red dots. Each dot represents the stimulation site in one animal, n = 6 rats.

The IC sends direct projections to the contralateral SDH

To observe the possible top-down projection from the IC to the spinal cord directly, we injected the anterograde virus AAV2/9-hSyn-FLEX-EGFP-WPRE-pA in the right posterior IC, and injected the retrograde virus AAV2/2Retro-hSyn-Cre-WPRE-pA into the left cervical SDH (Figure 2(a) and (b)). We found GFP⁺ neurons in the posterior IC, and they were mainly distributed in the deep layers (Figure 2(c)). We did not observe GFP-labeled neurons in the ipsilateral IC. These results provide direct evidence that the posterior IC sends direct projections to the contralateral SDH.

Figure 2.

Viral tracing reveals direct projections from the posterior insular cortex (pIC) to the contralateral spinal dorsal horn (SDH). (a) Schematic of viral injection strategy: An anterograde AAV2/9-hSyn-FLEX-EGFP (green) was injected into the right pIC, while a retrograde AAV2/Retro-hSyn-Cre was delivered to the left cervical SDH. (b) Sagittal view of spinal cord injection site (dorsal side left) showing retrograde labeling (DAPI, blue). (c) Coronal sections of pIC with GFP+ neurons (green) in deep layers (DAPI, blue). Insets (white boxes) show magnified views (scale: 100 μm) of boxed regions in main panels (scale: 1000 μm). Anterior-posterior (AP) coordinates relative to Bregma are indicated (+0.01, −0.35, −1.23 mm).

IC electrical stimulation produced mixed effects on the sEPSCs of SDH neuron

To check whether activation of IC could affect the synaptic transmission of spinal cord neurons, we performed in vivo whole-cell patch-clamp recording of lumbar SDH neurons located in laminae I/II at a depth less than 150 μm in urethane (1.2–1.5 g/kg, i.p.)-anesthetized adult rats. We first recorded the spontaneous excitatory postsynaptic currents (sEPSCs) from 12 SDH neurons. After obtaining a stable recording of sEPSCs (under voltage-clamp conditions at a holding potential of −70 mV, we applied focal electrical stimulation (100 Hz at the intensity of 100 μA) to the contralateral posterior IC through pre-implanted electrodes. In six neurons of six rats, the frequency of the spinal sEPSCs decreased at 20 min after the HFS was applied (68.5% ± 10.1% of the baseline, p < 0.05), and the amplitude did not change (102.3% ± 9.3% of the baseline, p > 0.05; Figure 3). The frequency of the spinal sEPSCs in four neurons of four animals increased (167.3% ± 23.9% of the baseline, p < 0.05), and the amplitude remained stable (103.2% ± 10.0% of the baseline, p > 0.05; Figure 4). In two neurons of two animals, neither the frequency (94.4% ± 30.2% of the baseline, p > 0.05) nor the amplitude (103.2% ± 10.0% of the baseline, p > 0.05) of the spinal sEPSCs changed significantly (Figure 5), suggesting that contralateral IC activation modulates spinal excitatory sensory transmission in a mixed way.

Figure 3.

Contralateral IC stimulation depressed the sEPSCs in some SDH neurons. (a) A schematic diagram showing the experimental design for IC electronic stimulation and in vivo spinal cord recording. (b–c) One sample (b) and a histogram figure (c) showing IC stimulation-induced inhibition of the frequency and amplitude of the sEPSC. (d) Averaged normalized frequency and amplitude in a 5-min time epoch from the baseline (−5 to 0 min) and post-HFS (15–20 min) periods individually in six SDH neurons. *frequency: p = 0.019; amplitude: p = 0.60, paired t-test. (e) Mapping of the stimulation sites in the IC.

Figure 4.

Contralateral IC stimulation facilitated the sEPSCs in some SDH neurons: (a–b) One sample (a) and a histogram figure (b) showing IC stimulation-induced excitation of the frequency and amplitude of the sEPSC. (c) Averaged normalized frequency and amplitude in a 5-min time epoch from the baseline (−5 to 0 min) and post-HFS (15–20 min) periods individually in four SDH neurons. *frequency: p = 0.045; amplitude: p = 0.97, paired t-test. (d) Mapping of the stimulation sites in the IC.

Figure 5.

Contralateral IC stimulation had no significant effect on the sEPSCs of SDH neurons. (a–b) One sample (a) and a histogram figure (b) showing IC stimulation-induced neither a significant effect on the frequency nor the amplitude of the sEPSC. (c) Averaged normalized frequency and amplitude in a 5-min time epoch from the baseline (−5 to 0 min) and post-HFS (15–20 min) periods individually in two SDH neurons. Frequency: p = 0.27; amplitude: p = 0.13, paired t-test. (d) Mapping of the stimulation sites in the IC.

Considering the corticospinal projections might be bilateral, we also stimulated the ipsilateral IC and recorded the spontaneous activity of the SDH neurons. In four neurons of four rats, the frequency of the spinal sEPSCs decreased at 20 min after the HFS (74.2% ± 15.0% of the baseline, p < 0.05), with no change in amplitude (93.3% ± 7.5% of the baseline, p > 0.05; Figure 6). The frequency of the spinal sEPSCs in two neurons of two animals increased (119.1% ± 9.1% of the baseline, p < 0.05), and the amplitude did not change (101.6% ± 4.3% of the baseline, p > 0.05; Figure 7). This result indicates that ipsilateral IC activation can neither trigger consistent top-down facilitation of spinal excitatory sensory transmission. Thus, unlike the consistent descending facilitation observed with ACC electrical stimulation, IC activation modulates spinal excitatory transmission in a heterogeneous manner.

Figure 6.

Ipsilateral IC electrical stimulation depressed the sEPSCs in some SDH neurons. (a–b) One sample (a) and a histogram figure (b) showing IC stimulation-induced inhibition of the frequency and amplitude of the sEPSC. (c) Averaged normalized frequency and amplitude in a 5-min time epoch from the baseline (−5 to 0 min) and post-HFS (15–20 min) periods individually in four SDH neurons. *frequency: p = 0.041; amplitude: p = 0.95, unpaired t-test. (d) Mapping of the stimulation sites in the IC.

Figure 7.

Ipsilateral IC electrical stimulation enhanced the sEPSCs in some SDH neurons. (a–b) One sample (a) and a histogram figure (b) showing IC stimulation-induced excitation of the frequency and amplitude of the sEPSC. (c) Averaged normalized frequency and amplitude in a 5-min time epoch from the baseline (−5 to 0 min) and post-HFS (15–20 min) periods individually in two SDH neurons. *frequency: p = 0.016; amplitude: p = 0.35, paired t-test. (d) Mapping of the stimulation sites in the IC.

Discussion

The present study provides new insights into the role of the IC in descending modulation of spinal nociceptive transmission as well as behavioral reflex. Using a combination of viral tracing, electrophysiology, and behavioral assays, we confirmed a direct contralateral projection from the posterior IC to the SDH, primarily originating from deep cortical layers. Unlike the ACC, HFS of the IC did not reliably facilitate spinal nociceptive reflexes (tail-flick test). In vivo patch-clamp recordings revealed mixed effects of IC stimulation on spinal sEPSCs, including inhibition, facilitation, or no change, suggesting a lack of uniform descending facilitation. These findings contrast sharply with the well-documented pro-nociceptive role of the ACC, which consistently enhances spinal excitability via direct pathways.^35,37 We cannot rule out the possibility that electrical stimulation of IC may activate different direct and indirect descending pathways, and some of the effects produced may be mixed. Future studies using a selective activation of IC to the spinal pathway will be needed to investigate their impact on spinal TF reflex as well as spinal dorsal horn neuron responses to peripheral sensory stimulation.

IC Stimulation yields bidirectional modulation of the spinal excitatory transmission

For a long decade, the IC has been regarded as a hinge in pain transmission, especially chronic pain.^4,5,17 Both clinical and animal research found that activation of the IC caused analgesic effects in individuals under the condition of chronic pain.^42–45 The GABAergic, ²⁷ serotonergic, ⁴⁶ and dopaminergic ⁴⁷ systems in the IC can also achieve such an antinociceptive effect. However, there are also a few reports that claim the activation of the IC causes nociceptive hypersensitivity.^13,25 The bidirectional effects may arise from: (a) sensory-discriminative posterior IC versus affective-interoceptive anterior IC may engage distinct spinal microcircuits. (b) IC glutamatergic projections may excite GABAergic or glycinergic interneurons in the dorsal horn, leading to net inhibition in some neurons. (c) Electrical stimulation may simultaneously activate IC projections to brainstem nuclei (e.g., PAG/RVM) that bidirectionally modulate spinal transmission.

In the ACC, lesion or inhibition caused a robust analgesic effect under the condition of chronic pain.^1,3 Although there are also a few reports that indicate the ACC may have an analgesic effect due to its role in the Placebo effect and pleasure,^48,49 these cannot mask the difference between the IC and the ACC in pain modulation. Previous studies found that stimulation in the ACC facilitated the tail-flick reflex in 0.75% halothane-anesthetized rats. In our study, we applied a similar stimulation in the posterior IC of 0.75% isoflurane-anesthetized rats, but we did not observe the facilitation. The anesthetic depth can be ruled out from the effect on the detectability of descending modulation during tail-flick tests, because at concentrations surrounding the minimum alveolar concentration (MAC), halothane depresses dorsal horn neurons more than does isoflurane, and the MAC for isoflurane is larger than halothane.^50,51 One possible explanation for the differences between the ACC and the IC is that the ACC has robust direct spinal projections, whereas IC–spinal connections may be sparser or modulatory rather than driver-like. The ACC is a well-established facilitator of spinal nociception via direct glutamatergic projections to the SDH ³⁷ and indirect engagement of brainstem nuclei (periaqueductal gray, PAG; rostroventral medulla, RVM). ³¹ The IC heavily interacts with the amygdala and PAG,^27,28 which could gate its descending influence based on emotional or autonomic context.

Heterogeneity in IC–spinal pathway

By applying the in vivo whole-cell patch-clamp technique, we recorded different effects on the sEPSC of SDH neurons after IC stimulation. However, in the tail-flick reflex, we did not observe facilitatory or inhibitory effects on the latency after stimulating the IC at different sites. On the one side, the IC is anatomically and functionally segregated into the posterior part, which processes sensory-discriminative aspects of pain,^5,8 and the anterior part, which encodes affective and interoceptive components. ⁶ The posterior IC–spinal projections could be modulatory rather than driver-like, fine-tuning sensory input without overriding spinal circuits, ²⁶ while the anterior IC–amygdala/PAG projections ²⁷ might dominate indirect descending control, masking direct spinal effects. The study primarily examined posterior IC projections, which may explain the lack of strong facilitation/inhibition at a behavioral level. On the other side, the electrical HFS used in our experiment lacks specificity. This method activates all neurons, afferent and efferent fibers of the IC within the stimulation radius. Therefore, the mixed effects we observe on spinal excitability are likely the net result of simultaneously activating multiple, competing microcircuits within the IC that have opposing effects on descending modulation. For example, the HFS may co-activate the ACC-projecting neurons^25,52 to drive spinal facilitation directly or indirectly. The HFS can also activate efferents from the IC that project to the PAG or other brainstem nuclei that mediate descending inhibition. Future studies will increase the specificity of activation by combining optogenetic or chemogenetic approaches.

The IC–spinal descending projection can be observed by virus tracing

The descending projections from the IC have been mapped by a few studies using a variety of tracing strategies.^30,34,53 However, distinct results were obtained. Shimada et al. found a somatostatinergic projection by using a retrograde tracer biotin-horseradish peroxidase. However, they did not specify the injection sites in the cervical or lumbar spinal cord. ³⁴ Liang and Labrakakis ³⁰ used an anterograde virus in the posterior IC, and they found no projection in the spinal cord. Anterograde tracing may fail to detect sparse or weakly expressed projections due to low transduction efficiency in IC neurons with distant spinal targets, or dilution of signal across long axonal distances. ⁵⁴ Simone et al. reported a direct projection from the insula to the spinal cord in macaques by combining resting-state fMRI with data from tract-tracing injections. However, they injected the retrograde tracer in the lateral funiculus, a part of the motor system. ⁵³ In our study, we used anterograde and retrograde AAV encoding the Cre-FLEX system to label the IC–spinal pathway. Retrograde tracing could amplify detection by labeling cell bodies, even if spinal terminals are sparse. ⁵⁵ In addition, the present study found IC–SDH neurons primarily in deep layers (V/VI), consistent with corticofugal projections. ⁵⁶ Prior anterograde studies might have injected superficial layers, missing deeper projecting neurons.

For the neurochemical phenotype of the labeled neurons in the IC, the most probable phenotypes are glutamatergic. Over 80% of cortical pyramidal neurons in layers V/VI are glutamatergic. ⁵⁶ Both the facilitatory and inhibitory effects on spinal sEPSCs observed in the paper are most easily explained by the release of glutamate, which could directly excite SDH neurons or excite spinal inhibitory interneurons, leading to inhibition. According to Shimada et al.’s ³⁴ study, the IC-labeled neuron can also be somatostatinergic and cause inhibition on excitatory or inhibitory neurons within the SDH. In addition, although it is less common, it is possible. A small percentage of corticofugal projections are GABAergic. Activating these long-range inhibitory neurons could directly suppress activity in their target zones in the SDH.

Functional implications of the IC–spinal descending projection

The identification of a direct but sparse IC–SDH projection has important implications for pain management and neuropsychiatric disorders. It provides a direct circuit aside from the IC–other cortices/brainstem-spinal cord pathway, which benefits the understanding of the mechanisms of chronic pain as well as its treatment. For example, the bidirectional modulation of spinal nociception suggests it could be a target for non-opioid neuromodulation (e.g., transcranial magnetic stimulation, TMS) in chronic pain patients. ⁵⁷ Additionally, since the IC integrates pain affect, ⁸ its spinal projections could underlie comorbidities like pain-depression/anxiety syndromes. ² Modulating IC activity (e.g., via psychotherapy or drugs) might alleviate emotional suffering without blunting sensory pain.