Enhanced NMDA receptor NR1 phosphorylation and neuronal activity in the arcuate nucleus of hypothalamus following peripheral inflammation

Abstract

AbstractAim:To investigate the role of glutamate and N-methyl-D-aspartate (NMDA) receptors in central sensitization following peripheral inflammation in the arcuate nucleus (ARC) of the mediobasal hypothalamus.

Methods: Mediobasal hypothalamic slices were prepared from rats undergoing peripheral inflammation, which was induced by a unilateral injection of complete Freund's adjuvant (CFA) into hind paw. Neuronal activation levels in the ARC were monitored by recording extracellular unit discharges. The NMDA receptor NR1 subunit (NR1) was measured using Western blot analysis.

Results: Enhanced NR1 phosphorylation was observed in the ARC of CFA-inflamed rats. Compared with the control rats, the firing rate of spontaneous discharges in ARC neurons of inflamed rats was significantly higher, and it was significantly reduced both by an NMDA receptor antagonist (MK-801, 300 μmol/L) and by a non-NMDA receptor antagonist (CNQX, 30 μmol/L). Application of exogenous glutamate (200 μmol/L) or NMDA (25 μmol/L) resulted in increased neuronal discharges for ARC neurons, which was enhanced to a greater extent in inflamed rats than in control rats.

Conclusion: Glutamate receptor activation in the hypothalamic ARC plays a crucial role in central sensitization associated with peripheral inflammation.

Figure 1

Schematic drawing of a mediobasal hypothalamic slice that contains the ARC. Dashed lines indicate the boundaries of the nucleus, as indicated. DMN: dorsomedial nucleus; VMN: ventromedial nucleus; ME: medium eminence; 3V: third ventricle; f: fornix.

Figure 2

Enhanced pNR1 but not NR1 expression in the ARC from CFA-inflamed rats. (A) Immunoblots of phosphorylated NR1 (pNR1) and NR1 in the ARC of control and CFA-inflamed rats. β-actin was as control. (B) The relative density of pNR1 protein was significantly increased in the ARC of CFA-inflamed rats (n=6) compared to control rats (n=6; ^cP<0.01, unpaired t-test). (C) There was no significant difference in NR1 protein expression between control (n=6) and CFA-inflamed (n=6) rats.

Figure 3

The discharge patterns of arcuate neurons. Examples of electrical signals (left) and their inter-spike interval histograms (right) recorded from arcuate neurons in control and inflamed rats are shown in A and B, respectively. a, b, and c show examples of regular, irregular and burst firing, respectively.

Figure 4

Effects of exogenous glutamate and NMDA on neuronal discharges in the ARC. Relative increases in neuronal discharge frequency following glutamate (200 μmol/L, n=26 in control rats, n=23 in inflamed rats) or NMDA (25 μmol/L, n=29 in control rats, n=24 in inflamed rats) application are shown in A. The baseline discharge frequency before glutamate application in control and inflamed rats were 1.26±0.31 Hz and 1.58±0.22 Hz, respectively; before NMDA application they were 1.27±0.22 Hz and 1.69±0.34 Hz, respectively. The latency and duration of glutamate or NMDA-induced increases in neuronal discharges are shown in B and C, respectively. ^bP<0.05, ^cP<0.01 as compared with control rats.

Figure 5

MK-801 (NMDA receptor antagonist) and CNQX (non-NMDA receptor antagonist) inhibit spontaneous discharge of ARC neurons in inflamed rats. (A) A significant decrease in neuronal discharge frequency was observed following MK-801 application (300 μmol/L) in inflamed rats (n=11; P<0.01, unpaired t-test) but not in control rats (n=12). (B) A significant decrease in neuronal discharge frequency was observed following CNQX application (30 μmol/L) in inflamed rats (n=11; ^cP<0.01, unpaired t-test), but not in control rats (n=10).