Role of EGR1 in hippocampal synaptic enhancement induced by tetanic stimulation and amputation

Abstract

Hippocampal neurons fire spikes when an animal is at a particular location or performs certain behaviors in a particular place, providing a cellular basis for hippocampal involvement in spatial learning and memory. In a natural environment, spatial memory is often associated with potentially dangerous sensory experiences such as noxious or painful stimuli. The central sites for such pain-associated memory or plasticity have not been identified. Here we present evidence that excitatory glutamatergic synapses within the CA1 region of the hippocampus may play a role in storing pain-related information. Peripheral noxious stimulation induced excitatory postsynaptic potentials (EPSPs) in CA1 pyramidal cells in anesthetized animals. Tissue/nerve injury caused a rapid increase in the level of the immediate-early gene product Egr1 (also called NGFI-A, Krox24, or zif/268) in hippocampal CA1 neurons. In parallel, synaptic potentiation induced by a single tetanic stimulation (100 Hz for 1 s) was enhanced after the injury. This enhancement of synaptic potentiation was absent in mice lacking Egr1. Our data suggest that Egr1 may act as an important regulator of pain-related synaptic plasticity within the hippocampus.

Figure 1

Peripheral noxious stimuli induced EPSPs from CA1 pyramidal neurons. (A) Diagram of an in vivo intracellular recording in an anesthetized rat. (C) An example of intracellularly stained CA1 pyramidal neurons (C). Representative traces (B) show the evoked responses of a CA1 neuron to stimuli of different durations. Each is the average of four traces. (Arrow) The stimulus artifact. (D) Plot of EPSP amplitude versus intensity of peripheral stimulation with different stimulus durations (triangles: 1.0 ms; squares: 0.5 ms; circles: 0.1 ms). Each point is the mean ± SEM.

Figure 2

Amputation of a mouse distal tail segment increased hippocampal Egr1. Egr1 was isolated by immunoprecipitation from hippocampus and detected by Western blot in control mice (indicated by −) and mice 1 h after amputation (+). Increases in hippocampal Egr1 immunoreactivity at 45 min after amputation are compared with the hippocampus of normal mice (the bottom set of photos are high-magnification details of the indicated areas). Confocal images of double-labeled CA1 pyramidal neurons in the hippocampus of amputated mice for FITC-labeled Egr1 (green, top), Cy-3 labeled CaMKII (red, middle), and merged image (bottom) showing a strong nuclear Egr1 signal expression in many pyramidal neurons visualized by CaMKII immunofluorescence. (D) Pretreatment with MK-801 (1 mg/kg) almost completely blocked Egr1 activation. (E) Intraperitoneal morphine (10 mg/kg) and subcutaneous QX-314 (5%, 10 μl) significantly decreased amputation-induced Egr1 activation. (F) Amputation increased c-Fos but not Egr1 immunoreactivity in the spinal dorsal horn. Bars: (B, top and E) 400 μm; (B, bottom) 150 μm; (F) 100 μm.

Figure 3

Amputation affected hippocampal LTP but not LTD. A single tetanic stimulation (100 Hz, 1 s) produced short-term potentiation in normal mice (n = 6, open squares). But 45 min after amputation, tetanic stimulation caused enhanced synaptic potentiation lasting for at least 60 min (n = 6, filled squares). An example illustrates that synaptic potentiation in slices prepared from amputated mice persisted for at least 3 h. Synaptic potentiation is input specific. As in A, a single tetanic stimulation induced enhanced potentiation (n = 4, filled squares) but synaptic responses at the second, independent pathway remained unaffected (open squares). Summarized time course curve of the effect of amputation on synaptic potentiation induced by one train tetanic stimulation (filled squares; open circle indicates sham-animals). Keeping mice under general anesthesia during the 45 min between amputation and decapitation prevented synaptic potentiation caused by amputation (filled triangles). LTD was not affected by amputation (control, n = 7, open squares; amputated, n = 5, filled squares). Synaptic responses to 5 Hz stimulation (for 3 min) also revealed no difference (control: n = 5, 80.0 ± 14.5%, open squares; amputated: n = 4, 94.2 ± 20.5%, filled squares). Summary of frequency-dependent responses.

Figure 4

Hippocampal LTP and LTD in mice lacking Egr1. LTD was normal in mutant mice (wild-type, n = 8, open squares; mutant, n = 6, filled squares). Synaptic responses to 5 Hz stimulation was also normal (wild-type, n = 4, open squares; mutant, n = 5, filled squares). Synaptic potentiation induced by a single tetanic stimulation was similar (wild-type, n = 8, open squares; mutant, n = 5, filled squares). Amputation caused no synaptic enhancement of LTP in mutant mice (wild-type, n = 5, open squares; mutant, n = 6, filled squares). Summarized data of different treatments on the enhancement of LTP caused by amputation.

Figure 5

Egr1 contributes to NMDA receptor-dependent late-phase LTP. (A) Wild-type (n = 6, open squares) and mutant slices (n = 8, filled squares) showed no significant difference in paired-pulse facilitation of the field EPSP at different interpulse intervals. (B) Wild-type (n = 9, open squares) and mutant slices (n = 6, filled squares) showed no significant difference in NMDA receptor-mediated EPSPs. (C) The induction of late-phase LTP in wild-type mice was completely blocked by 100 μM AP-5 in bath solution (n = 4). (D) Late-phase LTP was significantly decreased in mutant mice (wild-type, n = 9, open squares; mutant, n = 5, filled squares).