Correspondence to: Min Zhuo, Department of Anesthesiology, Washington University School of Medicine, Campus Box 8054, 660 S. Euclid Avenue, St. Louis, MO 63110. Tel:(314) 747-0416 Fax:(314) 362-8571 E-mail:zhuom{at}morpheus.wustl.edu.
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Abstract |
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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.
Key Words: Egr1, NMDA, LTP, pain, hippocampus
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Introduction |
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The hippocampus and related structures are important for certain types of learning and memory in both rodents and humans (
Excitatory synaptic transmission within the CA1 region of the hippocampus is mediated by glutamate. Glutamatergic synapses exhibit divergent synaptic plasticity, including long-term potentiation (LTP)1 and long-term depression (LTD), depending on synaptic activity as well as postsynaptic membrane excitability (Bliss and Colingridge, 1993;
In this study, we tested the hypothesis that activity-dependent expression of Egr1 in hippocampal neurons may contribute to plastic changes in excitatory synaptic transmission. Electrophysiological, immunocytochemical, and genetic approaches were used to test this hypothesis. First, we recorded from CA1 pyramidal neurons in anesthetized adult rats and showed that some of these neurons responded to peripheral noxious stimulation. Second, using immunocytochemical staining, the IEG Egr1 was activated in hippocampal CA1 neurons after tissue injury in both rats and mice. Third, we demonstrated that in CA1 neurons, synaptic plasticity of excitatory glutamatergic transmission was altered after tissue injury. Finally, using mice lacking Egr1, we found that Egr1 was required for plastic changes in the hippocampus caused by tissue injury, in addition to contributing to long-lasting synaptic enhancement in the normal hippocampus.
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Materials and Methods |
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Animals and Treatment
Adult male rats (Sprague-Dawley rats, 220400 g; Harlan) and mice (wild-type and mutant Egr1 mice generated by Dr. J. Milbrandt) were used. As reported previously, we used two different amputation procedures under halothane anesthesia: in adult rats, the central digit of a rat hindpaw was removed (
In Vivo Electrophysiology
Intracellular recording and injection of dye were performed on adult rats under halothane anesthesia. A bipolar stimulating electrode was placed into one hindpaw. Recording electrodes had a tip resistance of 5070 M when filled with a solution of 4% neurobiotin (Vector) in 2 M potassium acetate. After placement of a microelectrode in the cortex above the hippocampus (AP 3.06.5 mm, ML 1.04.0 mm), the exposed surface of the brain was covered with soft paraffin wax. After impalement, neurons with stable membrane potentials of -60 mV or greater were selected for further study. After each successful recording, neurobiotin was iontophoresed into the cell by passing a positive current pulse (2 Hz, 300 ms, 0.51 nA) for 10 min. At the end of the experiment, the rat was deeply anesthetized and perfused transcardially with 0.01 M phosphate-buffered saline followed by 4% paraformaldehyde. The brain was removed and stored in fixative overnight. Coronal sections were cut at a 50-µm thickness using a vibratome and incubated in 0.1% horseradish peroxidaseconjugated avidin-D (Vector) in 0.01 M potassium phosphate-buffered saline (KPBS, pH 7.4) with 0.5% Triton X-100 at room temperature for 68 h. After detection of peroxidase activity with 3',3'-DAB, sections were examined in KBPS. Sections containing labeled neurons were mounted on gelatin-coated slides for light microscopy.
In Vitro Electrophysiology
Mice were anesthetized with halothane and the tail tip (2.5 cm) was removed (
In all experiments, slices recovered in the chamber for at least 2 h before recording. A bipolar tungsten stimulating electrode was placed in the stratum radiatum of the CA1 region, and extracellular field potentials were recorded using a glass microelectrode (312 M, filled with ACSF), also in the stratum radiatum. Test responses were elicited at 0.02 Hz. In some experiments, picrotoxin (100 µM) was included in bath solution to block inhibitory transmission. LTD was induced by low frequency stimulation (1 Hz for 15 min;
Immunocytochemistry
After different treatments, rats or mice were deeply anesthetized with halothane and perfused transcardially with 50100 ml saline followed by 150500 ml of cold 0.1 M phosphate buffer (PB) containing 4% paraformaldehyde. The brain block, including hippocampus and lower lumbar and sacral spinal cord were removed, post-fixed for 4 h, and then cryoprotected by storing in a 30% sucrose, 0.1 M PB solution for 2 d at 4°C. Coronal brain and spinal cord sections (30-µm thickness) were cut using a cryostat. Sections from sham-operated and experimental animals were simultaneously processed for immunostaining. Primary rabbit antibodies used included anti-Egr1 (A310, 1:5,000; antiserum (1:1,000, Oncogene). Secondary antibodies conjugated to fluorescent markers FITC (1:50, used with Egr1) and Cy-3 (1:600, used with CaMKII
; Jackson ImmunoResearch Laboratories) were used. Images of the CA1 areas of hippocampus sections at 0.7-µm intervals with 20x lens were obtained with Bio-Rad Laboratories MRC 1000 laser-scanning confocal fluorescent imaging system.
Immunoprecipitation of Egr1
For two mice under brief anesthesia with halothane, amputation of 2.5-cm long tail segments was performed. Two control mice received only the same brief anesthesia. After 1 h, mice were killed and hippocampi were rapidly dissected and extracted in 1.2 ml ice-cold lysis buffer (10 mM Tris, pH 7.5, 10 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 1 mM leupeptin, and 1 mM pepstatin). Samples were sonicated three times and extracts were centrifuged (10,000 g for 15 min) to remove insoluble materials. Supernatants were incubated with the Egr1-specific monoclonal antibody 6H10 (
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Results |
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Hippocampal Pyramidal Cells Respond to Peripheral Noxious Stimuli in Adult Rats In Vivo
Previous studies using extracellular field recording or spike-recording techniques revealed that neurons in the hippocampus show spike responses or mixed field potentials to peripheral noxious stimuli (
Intracellular recordings were performed from identified hippocampal CA1 pyramidal neurons in anesthetized adult rats (n = 30; Fig 1 A). All neurons were identified with neurobiotin staining as CA1 pyramidal cells (Fig 1 C). In >35% of recorded neurons (11/30), peripheral electrical stimulation of one hindpaw elicited EPSPs. The EPSPs were intensity-related and polysynaptic in nature (Fig 1B and Fig D). These results provide the first direct evidence that hippocampal CA1 pyramidal neurons receive sensory, including nociceptive, inputs from the periphery.
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Amputation Causes Rapid Expression of Egr1 in Hippocampus
Repetitive activation of excitatory glutamatergic synapses within the hippocampus activates various types of IEGs in postsynaptic neurons (
To use genetically manipulated mice, we tested whether similar changes could be observed in mice after tissue/nerve injury. Removal of the tip of a mouse tail induced an NMDA-dependent, long-lasting hyperalgesia in mice (
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Only minor changes were observed in CA3 or DG (Table 1). Egr1 expression is selective for noxious stimuli; in experiments using non-noxious mechanical brush (with paintbrush for 12 s, n = 3) or non-noxious heating (at 40°C for 12 s, n = 4), we did not see any significant increase of Egr1 expression in the hippocampus.
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To determine whether Egr1 was upregulated in neurons of the spinal dorsal horn or brainstem in response to injury, we examined its expression and that of c-Fos after amputation of the distal tail. While significant increases in c-Fos expression were observed in dorsal horn neurons, little or no change in Egr1 levels was observed in the spinal cord (Fig 2 F and Table 1) or rostral ventromedial medulla (RVM; data not shown).
Peripheral Sensory Inputs and Activation of NMDA Receptors
Heightened Egr1 expression caused by amputation was NMDA receptor dependent. Pretreatment with i.p. injected MK-801 (1 mg/kg, 30 min before amputation) attenuated amputation-induced increases in Egr1 expression in the hippocampus (Fig 2 D and Table 1). In mice receiving i.p. morphine (10 mg/kg; 30 min before amputation) and local anesthetic blockade with subcutaneously applied QX-314 (5%; 10 µl, 10 min before), the amputation induced significantly less Egr1 expression in the hippocampus (Fig 2 E and Table 1). These findings indicate that sensory inputs during tissue/nerve injury were critical for the induction of Egr1.
Plasticity within the Hippocampus after Amputation
Could changes in Egr1 expression also be accompanied by plastic changes in excitatory transmission in the CA1 region? Two major forms of synaptic plasticity have been reported in the hippocampus: LTP and LTD (
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To test if the conscious experience of pain during the 45 min between the tail amputation and decapitation contribute to the observed alterations in hippocampal synaptic potentiation, we kept mice anesthetized throughout the 45 min between amputation and slice preparation in some experiments. Interestingly, we found that no significant enhancement of synaptic potentiation (n = 4, 131.5 ± 3.7%; no significant difference from slices of sham-treated animals). In addition, we measured Egr1 activation in hippocampus from mice receiving continuous halothane anesthesia after amputation and found that activation of Egr1 was completely blocked in these mice (n = 2).
LTD induced by low frequency stimulation (1 Hz, 15 min) was not affected by amputation (sham: n = 7, 66.8 ± 13.4% of control at 30 min after stimulation; amputated: n = 5, 50.3 ± 11.8%, no significant difference between the two groups; Fig 3 E). To detect possible frequency-dependent changes, we applied the same number of pulses (n = 900) at two additional frequencies, 5 and 10 Hz. No significant difference was found between sham-operated and amputated mice (Fig 3F and Fig G). Basal synaptic responses were not significantly different between sham (n = 25 slices/10 mice) and amputated animals (45 min after the amputation; n = 30 slices/15 mice, data not shown). Furthermore, paired-pulse facilitation, an indication of possible presynaptic changes, was also not affected (sham: n = 9; amputated: n = 16, data not shown).
Activation of Egr1 in Hippocampal Slices
What is the molecular mechanism contributing to the enhancement of synaptic potentiation after amputation? We hypothesized that NMDA receptor-dependent Egr1 activation may play an important role in the synaptic enhancement caused by amputation. To test this, we first determined whether hippocampal slices could be used to detect possible changes in gene expression caused by amputation. Studies from different regions of the central nervous system have shown that some physiological changes can be detected using an in vitro brain slice technique (
Hippocampal Synaptic Potentiation and Depression in Mice Lacking Egr1
Second, we wanted to determine if Egr1 contributes to hippocampal LTD and LTP using mice lacking Egr1 (
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Egr1 Contributes to Late-Phase LTP
What could be the possible physiological functions of Egr1 in hippocampal plasticity? One possible function of Egr1 is to contribute to LTP. We performed several additional electrophysiological experiments in wild-type and mutant mice. First, we measured paired-pulse facilitation at different interpulse intervals. No significant difference was found between wild-type (n = 6 mice) and mutant mice (n = 8 mice; Fig 5 A). Basal synaptic responses to stimulation were also not significantly different between wild-type (n = 18 slices/11 mice) and mutant animals (n = 19 slices/12 mice). However, late-phase LTP induced by a four train tetanic stimulation (3940) was significantly decreased in mutant mice (wild-type: n = 9, 210.7 ± 21.3%; mutant: n = 5, 132.6 ± 29.0%, t(12) = 2.18, P < 0.05; Fig 5 D). Pharmacological experiments using 100 µM AP-5 demonstrated that the NMDA receptor is essential for induction and expression of late-phase LTP (n = 4, 92.2 ± 13.2%, Fig 5 C). To detect if NMDA receptor-mediated responses may be affected in mutant mice, we measured NMDA receptor-mediated EPSPs in the presence of the AMPA/kainate receptor antagonist CNQX (10 µM). We found no significant difference between wild-type (n = 9 slices/6 mice) and mutant animals (n = 6 slices/5 mice; Fig 5 B), indicating that NMDA receptor function is not significantly affected. Finally, we also measured late-phase LTP of mutant slices in the presence of picrotoxin (100 µM) and found a similar defect in late-phase LTP (n = 4, 138.9 ± 8.3%). These results suggest the observed defect in late-phase LTP in mutant mice is not due to changes in NMDA receptor functions or inhibitory tone.
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Discussion |
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In this study, we show that noxious somatosensory stimuli elicit both EPSPs and elevations in Egr1 expression in hippocampal CA1 neurons. The same maneuver, tail tip amputation, that led to the latter result also led to an enhancement of synaptic potentiation in the CA1 region. Moreover, this enhancement of plasticity was not present in mice lacking Egr1. Finally, late-phase LTP but not early-phase LTP or LTD was abolished in Egr1 knockout mice.
It may not be readily evident why tail tip amputation should induce changes in hippocampal physiology different from decapitation, which is performed during hippocampal slice preparation. We hypothesized that after an extremely painful stimulus, such as tail tip amputation, a mouse, awake and alert after recovering from the brief, light anesthesia, would react in many ways, in particular, that changes may occur in susceptibility to synaptic plasticity in the hippocampus. According to this model, decapitation would induce none of these effects, the animal never experiences that stimulation consciously. Supporting this hypothesis, we found that keeping mice under halothane anesthesia between tail amputation and decapitation blocked the enhancement of synaptic potentiation. Furthermore, experiments using slices harvested at different time points after amputation consistently demonstrated that a certain period of consciousness is required for synaptic enhancement to occur. Slices were allowed to recover in the recording chamber for at least 2 h (often more time elapsed before the beginning of a given recording). Since 45 min between amputation and slice preparation was sufficient time to achieve a maximal effect on hippocampal synaptic potentiation, more than sufficient time elapsed after slice preparation to allow any similar effect of that maneuver to take hold. Taken together, these observations lend strong support to our conclusion that the difference observed between sham-operated and amputated animals was specifically a result of the physiological responses of a conscious animal during the minutes following severe tissue/nerve injury.
Electrical and Biochemical Responses of Hippocampal Neurons to Somatosensory Stimuli
We demonstrate in this study that >35% of hippocampal CA1 pyramidal cells respond to peripheral noxious shocks, providing the first intracellular in vivo recordings to support previous observations implicating the hippocampus in pain-related physiological functions. Previous electrophysiological evidence using extracellular recording techniques failed to determine which neurons were the source of the signal recorded. In addition, peripheral stimulation generates many subthreshold depolarizations in CA1 neurons which would not generate spikes but may nonetheless influence the summated output.
Interestingly, these EPSPs showed intensity-related responses, that is, larger EPSPs were observed with higher-intensity peripheral electrical shocks. These findings suggest that these neurons may also encode the intensity of stimulation. Gentle touch, however, did not cause any significant responses. We should point out that not all CA1 neurons responded to peripheral noxious shocks, suggesting that only a subpopulation of CA1 neurons are responsive to noxious stimuli. Hippocampal neurons receive inputs from many areas of the CNS, including the rostroventral medulla (
Hippocampal neurons also respond to somatosensory stimuli by changes in gene expression. Mixed changes in the expression of c-Fos in the hippocampus after tissue injury has been reported previously, including increases after subcutaneous formalin injection (
Activation of NMDA receptors was important for the expression of Egr1 caused by tissue injury in vivo or bath application of glutamate to hippocampal slices in vitro. These results are consistent with previous studies in rats showing that Egr1 expression was induced by synaptic activity through NMDA receptors (see Introduction). In the spinal cord, we found that Egr1 was not significantly activated in dorsal horn sensory neurons. These results differ from a previous report using heat-induced tissue injury (
Egr1 Contributes to Hippocampal Synaptic Potentiation
Despite the well-documented, activity-dependent stimulation of Egr1 expression in hippocampal neurons, no report is available concerning a possible contribution of Egr1 to NMDA receptor-dependent synaptic plasticity within the hippocampus. In this study, using mice lacking Egr1 gene, we present the first evidence that Egr1 is important for late-phase LTP. This effect of Egr1 deletion is relatively selective. LTP induced by a single tetanic stimulation or LTD were normal in mice lacking Egr1. Furthermore, other basic electrophysiological properties of these synapses seem to be normal (e.g., paired-pulse facilitation, basal field EPSPs and NMDA receptor-mediated EPSPs). It is obviously important to identify, in future studies, further cellular target proteins downstream from Egr1 which may contribute to an enhancement of synaptic potentiation. Although Egr1 is also upregulated by seizure (
Synaptic Enhancement after Amputation
One typical question for gene-related pathways is whether they are activated under physiological/pathological conditions and, upon activation, how they may affect the properties of central synapses. This study provides a possible answer for Egr1 in the hippocampus. We found that activation of Egr1 within the hippocampus occurs after tissue injury. Parallel with the activation of Egr1 by tissue injury, changes in synaptic plasticity obtained from hippocampal slices from animals with tissue injury were observed. A single tetanic stimulation, which normally induced moderate synaptic potentiation, induced a larger and longer-lasting potentiation in the CA1 area of the hippocampus after amputation. Interestingly, we found that keeping amputated animals anesthetized throughout the 45 min between amputation and hippocampal slice preparation prevented synaptic enhancement. Consistent with electrophysiological observations, the activation of Egr1 by amputation was also blocked. Halothane is known to decrease central neuronal excitability by inhibiting excitatory glutamatergic transmission and enhancing inhibitory transmission in rats and mice (
The other forms of synaptic plasticity tested were not affected, such as paired-pulse facilitation and LTD. These changes are not likely due to general stress during or after the amputation. Hippocampal synaptic plasticity is differentially affected in animals under behavioral stress compared with animals after tail amputation. After behavioral stress, hippocampal LTP was inhibited (
We believe that the synaptic enhancement after amputation did not reflect a generally elevated neuronal excitability. First, in experiments using two-pathway stimulation in the same slice, we showed that synaptic enhancement was selectively observed in the pathway receiving a single tetanic stimulation. Synaptic responses were not affected at the second pathway. Second, we have showed that basal responses to stimulation as well as paired-pulse facilitation were not affected after amputation. Finally, if neuronal excitability were uniformly enhanced, we might be expected to see less LTP (due to an occlusive, or saturating, effect); instead, we observed the opposite effect (enhanced LTP).
We also further test the possible relationship between activated Egr1 and changes in synaptic plasticity using mice lacking Egr1. Interestingly, the enhancement of potentiation caused by a single tetanic stimulation was blocked in mice lacking Egr1. These results suggest that Egr1 or Egr1-related signaling pathways could serve as a temporary marker within neurons for peripheral tissue injury.
Physiological Significance
Although the evidence we have so far does not allow us to assign confidently any physiological role to Egr1 in vivo, we suggest that Egr1 might serve as an important molecule for nociception or pain-related plasticity within the hippocampus. Egr1 may not only serve as a signaling molecule downstream from the NMDA receptor, thereby contributing to late-phase LTP, but it may also associate sensory nociceptive, non-spatial information with spatial memory. The hippocampus and related structures are known to play a critical role in spatial as well as non-spatial memory formation (
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Footnotes |
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1 Abbreviations used in this paper: ACSF, artificial cerebrospinal fluid; DG, dentate gyrus; EPSPs, excitatory postsynaptic potentials; IEGs, immediate-early genes; LTD, long-term depression; LTP, long-term potentiation.
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Acknowledgements |
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We want to thank all members of the Zhuo's laboratory for helpful suggestions.
This work was supported in part by grants from the National Institutes of Health (NIDA 10833 and NINDS 38680 to M. Zhuo; National Cancer Institute to J. Milbrandt; and NINDS to Z.C. Xu).
Submitted: 8 December 1999
Revised: 8 May 2000
Accepted: 23 May 2000
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References |
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