Activation of the dorsal, but not the ventral, hippocampus relieves neuropathic pain in rodents

Abstract

Accumulating evidence suggests hippocampal impairment under the chronic pain phenotype. However, it is unknown whether neuropathic behaviors are related to dysfunction of the hippocampal circuitry. Here, we enhanced hippocampal activity by pharmacological, optogenetic, and chemogenetic techniques to determine hippocampal influence on neuropathic pain behaviors. We found that excitation of the dorsal (DH), but not the ventral (VH) hippocampus induces analgesia in 2 rodent models of neuropathic pain (SNI and SNL) and in rats and mice. Optogenetic and pharmacological manipulations of DH neurons demonstrated that DH-induced analgesia was mediated by N-Methyl-D-aspartate and μ-opioid receptors. In addition to analgesia, optogenetic stimulation of the DH in SNI mice also resulted in enhanced real-time conditioned place preference for the chamber where the DH was activated, a finding consistent with pain relief. Similar manipulations in the VH were ineffective. Using chemo-functional magnetic resonance imaging (fMRI), where awake resting-state fMRI was combined with viral vector-mediated chemogenetic activation (PSAM/PSEM89s) of DH neurons, we demonstrated changes of functional connectivity between the DH and thalamus and somatosensory regions that tracked the extent of relief from tactile allodynia. Moreover, we examined hippocampal functional connectivity in humans and observe differential reorganization of its anterior and posterior subdivisions between subacute and chronic back pain. Altogether, these results imply that downregulation of the DH circuitry during chronic neuropathic pain aggravates pain-related behaviors. Conversely, activation of the DH reverses pain-related behaviors through local excitatory and opioidergic mechanisms affecting DH functional connectivity. Thus, this study exhibits a novel causal role for the DH but not the VH in controlling neuropathic pain-related behaviors.

Figure 1.

Glutamate, but not indiplon, in the dorsal hippocampus reversed pain-like behaviors after SNI or SNL neuropathic injuries in rats. (A) Microinjection of glutamate (21.2 pmol in 1 µL volume) but not saline into the dorsal hippocampus (ipsilateral to SNI) reversed SNI-induced tactile allodynia. This effect lasted for 2 days and was replicated by a second injection of glutamate (n = 6 rats per group). (B) Coapplication of glutamate (5.3 pmol) with NMDA receptor antagonist APV in the dorsal hippocampus (5 nmol in 1 µL volume, 30 minutes before 5.3 pmol glutamate injection) diminished the effect of glutamate on SNI-induced tactile allodynia (n = 9 rats per group). Note the lower dose of glutamate resulted in shorter duration pain relief. (C) In Sham but not in SNI rats, activation of GABA_A receptors in the dorsal hippocampus by infusion of indiplon (positive allosteric modulator of GABA_A receptors, 10 pmol in 1 µL volume) decreased the tactile thresholds (n = 6 rats per group). (D) Representative coronal sections and histological verification for implanted cannulas. (E) In L5 SNL-induced neuropathic pain model, tactile allodynia on ipsilateral hind paws was reversed by glutamate microinjected into the dorsal hippocampus (5.3 pmol in 1 µL volume, n = 6 per group). Post hoc statistical significance of differences is indicated as follows: ***P < 0.0001 and ns P > 0.05 (compared with −1 hour before glutamate injection), #P < 0.05 (compared with the glutamate-treated SNI group at corresponding time from the start of testing). For detailed statistics, see Table S1, available at http://links.lww.com/PAIN/B350. SNI, spared nerve injury; SNL, spinal nerve ligation.

Figure 2.

Pharmacological modulation by glutamate or indiplon in the ventral hippocampus had no effect on the tactile thresholds in either SNI or Sham rats. (A) Ventral hippocampus microinjection of glutamate (21.2 pmol in 1 µL volume) or saline did not change the tactile thresholds in either Sham or SNI rats (n = 5 per group). (B) Representative coronal sections and histological verification for implanted cannulas. (C) Indiplon (10 pmol in 1 µL volume) in the ventral hippocampus had no effect on the tactile thresholds in either Sham or SNI rats (n = 5 per group). ns P > 0.05 (compared with −1 hour before glutamate or indiplon injections). For detailed statistics, see Table S1 for all statistical tests, available at http://links.lww.com/PAIN/B350. SNI, spared nerve injury.

Figure 3.

Continuous optogenetic activation of the dorsal, but not the ventral, hippocampus alleviated tactile allodynia in SNI mice. (A) Sample slices display expression of ChR2-GFP in virus injection sites: dorsal hippocampus (DH, left) or ventral hippocampus (VH, right). Intended targets, relative to the bregma, for implanting the optical device are shown in smaller images. (B) Slice recording shows depolarization and spiking evoked in GFP-positive dorsal hippocampal neurons with photostimulation. (C) On SNI day 28, continuous photostimulation of dorsal hippocampal neurons is sufficient to relieve tactile allodynia, at around 3 hours after dorsal hippocampus stimulation (20 Hz stimulation for 15 minutes, n = 7). No effect is observed in Sham mice (n = 9). (D) On SNI day 23, a similar but smaller effect was detected with ventral hippocampus stimulation (20 Hz stimulation for 15 minutes). Hippocampal stimulation is ineffective on Sham mice (n = 5 mice per group). Post hoc statistically significant differences are indicated as follows: *** P < 0.0001 and ns P > 0.05 (compared with −1 hour before stimulation). For detailed statistics, see Table S1, available at http://links.lww.com/PAIN/B350. SNI, spared nerve injury.

Figure 4.

Activation of the dorsal, but not the ventral hippocampus is sufficient to drive preferred behavior in SNI mice. (A) Schematic representation of the behavioral paradigm for the real-time conditioned place preference (rtCPP) test. (B) Representative heat maps of SNI and Sham mice activity during habituation (prestimulation) and last stimulation sessions (S8) of rtCPP (20 Hz, 15 mins/session, DH optical stimulation). (C) Preference in seconds, measured by time in the stimulation-paired chamber at a given session minus time in the stimulation-paired chamber at prestimulation habituation session during rtCPP optical stimulation (20 Hz) of the dorsal hippocampus in ChR2-expressing SNI (red, n = 5) and Sham (white, n = 10) mice. (D) Representative heat maps of SNI and Sham mice activity during habituation (prestimulation) and last stimulation sessions (S8) of rtCPP (20 Hz, 15 mins/session, VH optical stimulation). (E) In contrast with dorsal hippocampus rtCPP, ventral hippocampus optical stimulation showed no preference for the stimulation-paired chamber for SNI (blue, n = 5) and Sham (n = 5) mice. Post hoc statistically significant differences are indicated as *P < 0.05, **P < 0.05, and ns P > 0.05 (compared with habituation, h2). For detailed statistics, see Table S1, available at http://links.lww.com/PAIN/B350. SNI, spared nerve injury.

Figure 5.

Chemogenetically increasing excitability of dorsal hippocampal neurons diminishes neuropathic tactile allodynia in SNI rats, abolished by naloxone. (A) In AAV—SYN-PSAM-L141F-Y115F-5HT3HC-GFP–infected rats GFP fluorescence specific for the dorsal hippocampal neurons. (B–C) Slice recording showing the rapid depolarization and spiking evoked in GFP-positive dorsal hippocampal neurons by bath application of PSEM^89s (10 µM). (D) In PSAM-5HT3–infected SNI rats, intraperitoneal (i.p.) injection of PSEM^89s (30 mg/kg) rapidly and reversibly blunted tactile allodynia of the SNI ipsilateral paw, on day 5 after SNI, whereas the treatment with saline did not perturb tactile sensitivity of SNI (n = 4 per group). (E) Experimental timeline: Rats were first injected with AAV9-SYN-PSAM-L141F-Y115F-5HT3HC-GFP virus into the bilateral dorsal hippocampus (∼4 weeks before SNI/Sham surgeries). One day before SNI/Sham injury, tactile thresholds were assessed (baseline VF). Spared nerve injury/Sham surgeries were then performed unilaterally. Five days after surgery, tactile thresholds (−1 hour) were assessed, and afterwards, rats received PSEM^89s injections (i.p.) followed by another i.p. injection of either naloxone or saline 30 minutes after. Tactile thresholds were assessed 1 hour after the first injection. Two days later, rats that had received PSEM^89s and naloxone were injected with PSEM^89s and saline, and vice versa, and tactile thresholds were assessed again. (F) PSEM^89s injection in the virus expressed SNI rats (n = 16) showed a reduction of SNI-induced tactile allodynia. This effect was fully abolished by naloxone. By contrast, the tactile threshold of sham rats (n = 4) was unchanged by naloxone. Post hoc statistically significant differences are indicated as follows: #P < 0.01, ##P < 0.05, * < 0.05, and ns P > 0.05 (between before and after PSEM or saline ± naloxone or saline). For detailed statistics, see Table S1, available at http://links.lww.com/PAIN/B350. rtCPP, real-time conditioned place preference; SNI, spared nerve injury.

Figure 6.

Awake chemo-fMRI functional network reorganization with increasing excitability of the dorsal hippocampus (DH) in rats: Subdividing the brain into 96 clusters identifies whole-brain and dorsal hippocampus connectivity changes with increased DH excitability. (A) Experimental timeline: Rats (n = 7) were first injected with AAV9-SYN-PSAM-L141F-Y115F-5HT3HC-GFP virus into the bilateral dorsal hippocampus (∼9 weeks before SNI surgery). Two weeks later, they received head-posts (∼7 weeks) and then underwent a 2-week training period to enable awake resting-state fMRI; 1 to 2 days before SNI injury, baseline tactile thresholds were assessed for left and right hind paws (BL VF). Spared nerve injury surgery was then performed unilaterally. Four or 5 days after surgery, tactile thresholds (T0) were assessed, and the rats scanned for resting-state fMRI (rsfMRI); immediately afterwards, rats received either saline or PSEM^89s injections (i.p.), retested for tactile thresholds, and again underwent awake rsfMRI (2 consecutive scans). Two hours later, rats that had received PSEM^89s were injected with saline, and vice versa, and tactile thresholds and rsfMRI were assessed one more time. (B–C), Dorsal and lateral views of change in network connectivity with increased DH excitability, comparing PSEM^89s with the saline injection conditions. Change in connectivity across all nodes is shown in gray; increased and decreased connectivity for the 4 DH seeds (green) are shown in red and blue, respectively. (D) Histogram of the significant changes in the average correlation coefficient for PSEM^89s in contrast to saline: Contrasts were performed combining scan 1 and 2 data, using permutation testing, P < 0.05 (see Table S2 for statistical details, available at http://links.lww.com/PAIN/B350). A, anterior; D, dorsal; L, left; P, posterior; R, right; SNI, spared nerve injury; V, ventral.

Figure 7.

Awake chemo-fMRI identifies dorsal hippocampal–driven functional networks that reflect change in tactile allodynia as a function of increased dorsal hippocampal excitability in rats. (A) The map delineates brain regions where functional connectivity changes (ΔFC; between PSEM and saline conditions) were correlated with change in tactile allodynia (ΔThreshold; between PSEM and saline conditions), for stimulating the SNI hind paw (using only scan 1 data) (cluster corrected for multiple comparisons. Table S3, available at http://links.lww.com/PAIN/B350, summarizes identified regions and associated statistics). Standard atlas slices are also illustrated. (B) Group median (quartiles and minimum/maximum) tactile thresholds are shown, for the SNI hind paw (red) and healthy hind paw (gray). Tactile thresholds for the SNI hind paw diminished at 1 hour after PSEM^89s, but not after saline. Post hoc comparisons **P < 0.01 (between baseline and post-SNI, T0; also between T0 SNI and SNI + PSEM). (C) Average scatter plots across all 4 seeds for the primary somatosensory cortex (barrel field) between ΔFC and ΔThreshold are shown for discovery and replication groups for the SNI (red) and healthy hind paw (gray). We observe the SNI hind paw ΔThreshold is negatively correlated with the average ΔFC, but not for the non-SNI hind paw. Thus, we consider this brain regional FC a reliable outcome, indicating that high FC values in the region are related with high neuropathic pain (yellow circle at z = −2.8 mm in (A)). (D and E) Two additional brain regions identified in the discovery data could be replicated (yellow circles at z = −2.0 mm and z = 2.0 mm in (A)). In both regions, dorsolateral thalamus (D) and primary somatosensory cortex (limb region) (E), discovery and replication results show a positive relationship between ΔFC and ΔThreshold for the SNI hind paw, but not for the non-SNI hind paw. Thus, in these 2 regions, high FC values (in PSEM^89s) are associated with decreased neuropathic pain. Only across-seeds group averages are displayed. SNI, spared nerve injury.