Activation of Dopamine D2 Receptors Alleviates Neuronal Hyperexcitability in the Lateral Entorhinal Cortex via Inhibition of HCN Current in a Rat Model of Chronic Inflammatory Pain

Abstract

Functional changes in synaptic transmission from the lateral entorhinal cortex to the dentate gyrus (LEC-DG) are considered responsible for the chronification of pain. However, the underlying alterations in fan cells, which are the predominant neurons in the LEC that project to the DG, remain elusive. Here, we investigated possible mechanisms using a rat model of complete Freund's adjuvant (CFA)-induced inflammatory pain. We found a substantial increase in hyperpolarization-activated/cyclic nucleotide-gated currents (I_h), which led to the hyperexcitability of LEC fan cells of CFA slices. This phenomenon was attenuated in CFA slices by activating dopamine D2, but not D1, receptors. Chemogenetic activation of the ventral tegmental area -LEC projection had a D2 receptor-dependent analgesic effect. Intra-LEC microinjection of a D2 receptor agonist also suppressed CFA-induced behavioral hypersensitivity, and this effect was attenuated by pre-activation of the I_h. Our findings suggest that down-regulating the excitability of LEC fan cells through activation of the dopamine D2 receptor may be a strategy for treating chronic inflammatory pain.

Keywords: Dopamine D2 receptor; HCN current; Inflammatory pain; Lateral entorhinal cortex; Neuronal hyperexcitability.

Fig. 1

CFA evokes thermal hyperalgesia and mechanical allodynia in the injured hind paw. A Progression of thermal latency following intraplantar CFA or saline injection. B Progression of mechanical threshold following intraplantar CFA or saline injection. BL, baseline; saline right, the right hindpaw of rats received a saline injection. **P < 0.01.

Fig. 2

Firing property of LEC fan cells is changed after CFA injection. A Representative traces showing the firing response of LEC fan cells to a 1-min rheobase stimulus. B, C Action potential (AP) numbers and mean interspike intervals (ISI) of LEC fan cells elicited by a 1-min rheobase stimulus. D CFA injection induces a marked leftward shift of the ISI cumulative distribution curve. E Representative traces showing the firing responses of LEC fan cells to a depolarizing current stimulus (+ 300 pA, 500 ms). F Fast afterhyperpolarization potential (fAHP, upper panel) and instantaneous frequency (Inst., lower panel) of AP firing in response to 100, 200, or 300 pA current injection. Data are from the first 6–9 spike intervals; #, number of spike intervals. f_o, onset frequency; f_ss, steady-state frequency. *P < 0.05, **P < 0.01.

Fig. 3

I_h of LEC fan cells is up-regulated after CFA injection. A Representative traces of voltage sag induced by a current step that causes an approximately − 20 mV hyperpolarization at the steady state. V_bsl, baseline voltage; V_ss, steady-state voltage; V_min, minimum voltage. B Voltage sag ratios in the saline and CFA groups. C Representative traces of I_h induced by applying 1-s hyperpolarizing voltage steps from − 60 to − 120 mV (in 10-mV steps). D Current-voltage relationships of I_h in LEC fan cells from the saline and CFA groups. E I_h density at − 120 mV. F I_h is blocked after bath application of 10 µmol/L ZD7288 in LEC fan cells of the CFA group. G Onset frequency of the fan cells in F is significantly reduced after bath application of 10 µmol/L ZD7288. Left panel, representative traces showing that the onset frequency is measured between the 1st and 2nd AP elicited by a 200-pA depolarizing stimulus. H Representative immunohistochemistry images showing the expression pattern of HCN1 in the LEC (scale bar, 500 µm). Inserts, magnified images of HCN1-positive cells. I Relative IOD levels of HCN1 in the LEC are almost identical in the saline and CFA groups, as shown by immunohistochemistry. The IOD levels are expressed as a fold increase compared with the mean of the saline group in this and the following figures in this format. J Western blots and analysis of HCN1 expression levels in the saline and CFA groups. The relative protein levels are normalized as a ratio of β-actin levels and expressed as fold increase compared with the mean of the saline group in all Western blot figures. *P < 0.05, **P < 0.01.

Fig. 4

Effects of dopamine D1 and D2 receptor agonists on I_h and onset frequency of LEC fan cells in the CFA group. A, E Changes in I_h of LEC fan cells during bath application of 30 µmol/L SKF38393 or 10 µmol/L quinpirole. Right panel, amplified current amplitude at − 120 mV. C, G Changes in the firing of LEC fan cells during bath application of 30 µmol/L SKF38393 or 10 µmol/L quinpirole. Right panel, amplified onset frequency. Spikes are elicited by a 200-pA depolarizing stimulus (500 ms in duration). B I_h amplitude in pre-drug, SKF38393, and washout in fan cells at − 120 mV. ** vs pre. For this and the following figures in this format, the grey circles represent data from individual cells. D Onset frequency in pre-drug, SKF38393, and washout in fan cells. ** vs pre. F I_h amplitude in pre-drug, quinpirole, and washout in fan cells at − 120 mV. ** vs pre. H Onset frequency in pre-drug, quinpirole, and washout in fan cells. ** vs pre. **P < 0.01.

Fig. 5

D2R interacts with the HCN channel to modulate the onset frequency. A I_h of fan cells is almost completely blocked by 10 µmol/L ZD7288 and subsequent additional application of 10 µmol/L quinpirole fails to modulate it. C The onset frequency of fan cells decreases during bath application of 10 µmol/L ZD7288 and subsequent additional application of 10 µmol/L quinpirole has no effect. B, D I_h amplitude at −120 mV and onset frequency in pre-drug, ZD7288, and ZD7288 + quinpirole. ** vs pre. E I_h of fan cells is up-regulated by 1 mmol/L 8-Br-cAMP (cAMP) and subsequent additional application of 10 µmol/L quinpirole fails to decrease it. G The onset frequency of fan cells increases during bath application of 1 mmol/L 8-Br-cAMP and subsequent additional application of 10 µmol/L quinpirole has no effect. F, H I_h amplitude at −120 mV and onset frequency in pre-drug, 8-Br-cAMP, and 8-Br-cAMP + quinpirole. *, ** vs pre. *P < 0.05, **P < 0.01.

Fig. 6

AC mediates the effect of D2R on I_h. A I_h of fan cells is significantly down-regulated by 10 µmol/L SQ22536. B I_h of fan cells is up-regulated by 10 µmol/L forskolin (FSK) and subsequent additional application of 10 µmol/L quinpirole fails to decrease it. C I_h amplitude at −120 mV in pre-drug, SQ22536, and washout. ** vs pre. D I_h amplitude at −120 mV in pre-drug, forskolin, and forskolin + quinpirole. * vs pre. *P < 0.05, **P < 0.01.

Fig. 7

The expression level of D1R and D2R in LEC do not change after CFA injection. A Representative immunohistochemistry images showing the expression pattern of D1R in the LEC (scale bars, 500 µm; inserts, magnified images of D1R-positive cells). B Relative IOD levels of D1R in the LEC are almost identical in the saline and CFA groups, as shown by immunohistochemistry. C Representative immunohistochemistry images showing the expression pattern of D2R in the LEC (scale bars, 500 µm; inserts, magnified images of the D2R-positive cells.) D Relative IOD levels of D2R in the LEC are almost identical in the saline and CFA groups, as shown by immunohistochemistry. E Representative images showing the protein expression of D1R and D2R in the LEC in the saline and CFA groups. F Western blot analysis of D1R and D2R expression levels in the saline and CFA groups.

Fig. 8

Activation of VTA-LEC projection terminals or injection of quinpirole into the superficial layer of LEC alleviates behavioral hypersensitivity induced by CFA. Aa, Expression of AAV-hM3Dq-mCherry in the VTA 28 days after viral injection (scale bar, 500 µm). Ab, Expression of mCherry-positive fibers and terminals in the LEC (scale bar, 500 µm; insert, magnified image showing mCherry-positive fibers). Ac Histological verification of drug diffusion in the LEC (scale bar, 500 µm; L1, layer 1). B, C Effects of intra-LEC injection of vehicle or CNO (3 µmol/L) in AAV-hM3Dq-mCherry- or AAV-EGFP-infected CFA rats on paw withdrawal thermal latencies (B) and mechanical thresholds (C) for paws ipsilateral to the CFA injection. ** vs AAV-EGFP + vehicle. D, E Thermal latencies (D) and mechanical thresholds (E) of AAV-hM3Dq-mCherry-infected CFA rats with or without intra-LEC pre-injection of vehicle, SCH23390 (50 µmol/L), or sulpiride (50 µmol/L) before CNO application. * vs CNO. F Effect of intra-LEC injection of quinpirole, vehicle, or 8-Br-cAMP + quinpirole on thermal latency and mechanical threshold of the left and right hindpaws (CFA was injected into the left hindpaw; L, left; R, right). G Thermal latencies and mechanical thresholds of CFA-injured paws, normalized to the non-injured paws, in rats receiving quinpirole, vehicle, or 8-Br-cAMP + quinpirole injection. *P < 0.05, **P < 0.01; n.s., not significant.