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Journal of Physiology (2002), 539.3, pp. 735-741
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2001.013379
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ABSTRACT |
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To elucidate characteristic changes in the N-methyl-D-aspartate (NMDA) receptor on neurons following axotomy, subunit expressions and functional features of the NMDA receptor were examined in the dorsal motor nucleus of vagus (DMV) of rats receiving vagal axotomy at the neck. Western blotting analysis demonstrated that the expression of NR2A decreased 2-3 days after in vivo axotomy, while expression of NR1 and NR2B, NR2C and NR2D subunits did not change significantly. To examine the functional changes, patch clamp recordings in whole-cell mode were employed on the axotomized DMV neurons identified by retrograde labelling with fluorescent dye. The amplitude ratios of ifenprodil-sensitive components of NMDA response and D,L-2-amino-5-phosphovaleric acid (APV)-sensitive evoked postsynaptic current increased after axotomy. In addition, APV-sensitive postsynaptic currents exhibited a longer decay time in identified axotomized vagal motoneurons than in control neurons. No significant differences in the current density of the NMDA response and the peak amplitude of APV-sensitive synaptic currents were observed between axotomized and intact DMV neurons. In conclusion, a decrease in NR2A expression results in the appearance of functional characteristics of the NMDA receptor predominantly containing the NR2B subunit. This might lead to a long-term increase of the susceptibility of neurons to excitotoxicity.
(Resubmitted 9 October 2001; accepted after revision 27 November 2001)
Corresponding author J. Nabekura: Department of Cellular and System Physiology, Graduate School of Medical Sciences, Kyushu University, Japan. Email: nabekura{at}physiol2.med.kyushu-u.ac.jp
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INTRODUCTION |
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NMDA receptors play important roles in synaptic plasticity, neural development and neuronal degeneration. NMDA receptors are highly Ca2+ permeable and closely linked to neuronal calcium-dependent excitotoxicity (Choi, 1988). In rat hippocampus, elevated cytosolic Ca2+ concentration brought about by NMDA receptor activation stimulates proteases such as calpain I, which, in turn, degrades major neuronal structural proteins (Siman et al. 1989). Ca2+ overload also activates phospholipases capable of breaking down the cell membrane and liberating arachidonic acid, and endonucleases capable of breaking down genomic DNA (Choi, 1992). Excess intracellular Ca2+ can induce the formation of oxygen free radicals which initiate many destructive processes including lipid peroxidation (Braughler & Hall, 1992). Additionally, the resulting cell damage may further exacerbate excitotoxic injury by promoting glutamate release (Lustig et al.1992).
Excitotoxicity mediated by the NMDA receptor, associated with human disease states, has reportedly contributed to the pathogenesis of brain and spinal cord injury (Regan & Choi, 1991). Enhanced expression of NR1 mRNA in axotomized motoneurons with NMDA receptor blockade results in neuronal death (Sanner et al. 1994). Treatment with NMDA receptor antagonists or an increase in Mg2+ concentration in the cerebrospinal fluid provides protection from traumatic brain injury in vivo (Faden et al. 1989; Goldberg & Choi, 1993). In various states of neuronal injury, a number of studies revealed alterations in the NMDA receptor. Stretch injury rapidly reduces the sensitivity to extracellular Mg2+ in cultured neurons (Zhang et al. 1996). Axotomy induces a reduction in NR1, NR2B and NR2D mRNAs in spinal motoneurons (Piehl et al.1995), and reduces the voltage-dependent Mg2+ block of NMDA response in vagal motoneurons (Furukawa et al. 2000). In spite of increasing evidence for the involvement of the NMDA receptor in the neuronal destiny of injured neurons, the functional characteristics of the NMDA receptor in injured neurons has not been well elucidated. Furthermore, the correlation between molecular and functional changes of the NMDA receptor in injured neurons has not been reported.
In the present study we examined the alteration of NMDA receptor subunits and functional characteristics of vagal motoneurons following in vivo axotomy.
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METHODS |
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Axotomy
All experiments conformed to the Guiding Principles for the Care and use of Animals approved the Council of the Physiological Society of Japan. All efforts were made to minimize the number of animals used and their suffering. The method of vagal axotomy was described previously (Furukawa et al. 2000). Briefly, 17- to 19-day-old rats were deeply anaesthetized with diethyl ether, a vertical skin incision was made at the neck and the vagus nerve bundle on either side was exposed. Axotomy of the vagal motoneurons was performed with fine scissors at the left vagus nerve at the neck. Thereafter, a small piece of Di-I, or rhodamine-conjugated dextran (molecular weight, 10 000) was placed at the proximal crushed site of the nerve bundle (Furukawa et al. 2000). The skin incision was closed and the rats were returned to the cage after awaking from the anaesthetic.
Western blotting
Vagally axotomized rats (19-21 days old) were deeply anaesthetized with intraperitoneal pentobarbitone (100 mg kg-1) and killed by decapitation. The DMV was dissected and homogenized with 0.5 % SDS, 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 % Nonidet P-40, 2 mM EDTA, 0.1 % protease inhibitor cocktail (Sigma P-8340). The homogenate was fractionated on an SDS-polyacrylamide gel (7.5 %), electroblotted onto a Immobilon-P membrane (Millipore) and reacted with anti-NR1, anti-NR2A antibody (Chemicon International), anti-NR2B (Transduction), anti-NR2C (Chemicon International) or anti-NR2D (Santa Cruz Biotechnology Inc.). The reaction was visualized using a secondary antibody labelled with alkaline phosphatase (Promega) and quantified using a densitometer (GS-700, Bio-Rad). Data from five independent experiments were analysed statistically.
Dissociation of injured neurons and electrical recordings
Vagus neurons of the dorsal motor nucleus (DMV) were acutely dissociated from rats 2-3 days after axotomy. Following sodium pentobarbitone anaesthesia (50 mg kg-1 I.P.) rats were killed by decapitation and 400 µm thick coronal sections of the brainstem were made with a microslicer (DTK-1000, Dosaka). The motoneurons in the DMV receiving axotomy were easily identified as dye (1,1'-deoctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Di-I) or rhodamine) positive (Fig. 1). The dye positive neurons were mechanically dissociated (Furukawa et al. 2000). The neurons identified as dye positive under epifluoroscopic microscopy were prepared for electrophysiological experiments (Nabekura et al. 1995). Electrical measurements were performed with nystatin perforated patch whole-cell recordings (Nabekura et al. 1996). The resistance between the patch pipette filled with the internal solution and the reference electrode in the normal external solution was 3-5 M
. Ionic currents were measured with a patch clamp amplifier (EPC-7, List-Medical), low-pass filtered at 1 kHz (E-3201A, NF Electronic Instruments), monitored on both a digital storage oscilloscope (VC-6025, Hitachi) and a pen recorder (Linearcorder F), and then stored on a videotape after being digitized at 44 kHz (Nihon Koden, PCM 501 ESN). The membrane potential was held at -40 mV throughout the experiment. To examine the ifenprodil-sensitive component of NMDA-induced currents, ifenprodil (3 µM) was perfused for 10 min before NMDA application. All experiments using acutely dissociated neurons were performed in the standard solution at room temperature (24-26 °C). Whole-cell currents were normalized to cell size by dividing the current amplitude response to 0.3 mM NMDA by the cell capacitance (expressed in pF). NMDA at a concentration of 0.3 mM induces maximal responses both in the control and axotomized DMV neurons (Furukawa et al. 2000).
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Figure 1. Combined dim translucent and epifluorescent light image of injured DMV neurons stained with Di-I Unfixed coronal slice of the brainstem (400 µm thick) obtained from a rat which had been axotomized and which had received Di-I 2 days before. DMV motoneurons ipsilateral (ipsi.), but not contralateral (contra.), to axotomy were clearly identified as positively staining with Di-I. NTS, nucleus of tractus solitarius; XII, hypoglossal nucleus; CC, central canal; ST, solitari tract. Vertical bar, 200 µm. | ||
Synaptic currents
Brainstem slices (280 µM thick) were prepared from the rats with axonal injury. Slices were maintained at 32 °C in incubation solution. Dye-positive DMV neurons were identified using epifluorescent light in the slices (Fig. 1). Patch clamp recordings in whole-cell mode were employed on the identified neurons. To activate synaptic inputs to DMV neurons, constant current stimuli (0.1 Hz, 100 µs, 100-500 µA intensity) were delivered using a bipolar stimulating electrode positioned at the nucleus tractus solitarius close to the DMV. Recordings of NMDA receptor-mediated currents, the solutions contained bicuculline methiodide (10 µM), strychnine (10 µM) and CNQX (30 µM).
Solution
The standard external solution contained (mM): NaCl 150, KCl 5, MgCl2 1, CaCl2 2, Hepes 10 and glucose 10. The pH was adjusted to 7.4 with tris(hydroxymethyl)aminomethane (Tris-base). For recording the NMDA-induced currents in the acute dissociated neurons, nominally Mg2+-free solution was employed with 1 µM glycine and 10 µM strychnine. The composition of the patch pipette (internal) solution was (mM): CsMeSO4 100, CsCl 50, and Hepes 10 (pH 7.2 with Tris-base). Drugs used in the present experiments were ifenprodil, nystatin, CNQX and D,L-2-amino-5-phosphovaleric acid (APV) from Sigma and pronase from Calbiochem. CNQX was first dissolved in dimethyl sulphoxide (DMSO) at a concentration of 10 mM, respectively, for the preparation of a stock solution, and the final DMSO concentration in the experiments did not exceed 0.1 %. Rapid change of external solution was performed using the Y-tube method described previously (Nabekura et al. 1995).
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RESULTS |
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Change in NMDA receptor subunit expression after axonal injury
Changes in NR1, NR2A, NR2B, NR2C and NR2D subunit expression after the axonal injury were examined using Western blotting. Quantification of the Western blotting results was acheived by determining the relative optical densities (Fig. 2). The graph indicates that NR2A expression had significantly decreased 2 days after in vivo axotomy. In contrast, NR1, NR2B, NR2C and NR2D expression remained unchanged after injury.
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Figure 2. NMDA receptor subunit expression in the rats DMV receiving axonal injury Western blots from intact (Control) and axotomized (Axotomy) DMV tissue probed with polyclonal antibodies against NR1, NR2A, NR2B, NR2C and NR2D subunits. B, a significant decrease in immunoreactivity for NR2A was demonstrated, while immunoreactivity for NR1, NR2B, NR2C and NR2D was not significantly different between control and axotomized DMV. *P < 0.05, Student's unpaired t test. Data were analysed from five different experimental samples. Each sample contained the excised DMV from 7-10 rats. | ||
Change in NMDA receptor function after axonal injury
To investigate whether the decrease in NR2A expression affects NMDA receptor function after axonal injury, whole-cell patch recordings were employed on DMV neurons receiving in vivo axotomy 2-3 days prior to making preparations. Axotomized DMV neurons were easily identified from positive staining with dye.
First, the functional contribution of the NR2B-containing NMDA receptor to the NMDA response in axotomized DMV neurons was investigated because NR2A and NR2B expressions run antiparallel in development (Watanabe et al. 1992). At a holding potential (VH) of -40 mV in Mg2+-free extracellular solution, 3 
10-5 M NMDA induced an inward current in acutely dissociated DMV neurons stained with Di-I or rhodamine. The current density in the presence of 0.3 mM NMDA, which induced maximal currents in control and axotomized DMV neurons (Furukawa et al. 2000), was 12.2 ± 3.2 pA pf-1 (n = 8) in control and 10.7 ± 2.2 pA pf-1 (n = 7) in axotomized neurons, the difference was not significant (P > 0.1). Where ifenprodil (3 µM) was applied for 10 min before NMDA application, the NMDA response was reduced in amplitude by 35 ± 5 % (n = 10) in control DMV neurons obtained from sham operated rats, 49 ± 8 % (n = 11) in injured DMV neurons and 34 ± 7 % (n = 8) in DMV neurons contralateral to vagal axotomy (contralateral) (Fig. 3). A significant increase in the ratio of the ifenprodil-sensitive component of the NMDA response was demonstrated in injured DMV neurons compared with control and contralateral DMV neurons (P < 0.05, Student's unpaired t test in each comparison).
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Figure 3. Increase in ifenprodil-sensitive NMDA response in axotomized DMV neurons A, pretreatment with ifenprodil (3 µM) for 10 min suppressed the response to 30 µM NMDA at a VH of -40 mV in acutely dissociated control (upper traces) and axotomized (lower traces) DMV neurons. Axotomy was performed 2 days before recording. Axotomized neurons were identified by positive Di-I staining. B, the ratio of the ifenprodil-sensitive component of the NMDA response to total current was greater in injured DMV neurons (n = 11) than that in control DMV neurons (n = 10). Extracellular solution contained 10 µM bicuculline, 1 µM glycine, 10 µM strychnine, 10 µM CNQX and free Mg2+. P < 0.05, Student's unpaired t test. | ||
To examine whether this is also the case for the synaptic events, APV-sensitive postsynaptic currents evoked by electrical stimulation applied to the afferents in the slice preparation were recorded from the identified DMV in the presence of 10 µM CNQX, 10 µM bicuculline and 10 µM strychnine. No significant difference was observed in the absolute values of peak amplitudes of APV-sensitive postsynaptic currents induced by maximal afferent stimulation were 72 ± 22 pA (n = 5) in the control and 66 ± 18 pA (n = 7) in the injured neurons (P > 0.1, Student's unpaired t test). The ratio of the ifenprodil-sensitive components of to the total APV-sensitive synaptic current were 28 ± 7 % (n = 5) in control and 56 ± 12 % (n = 5) in the injured neurons (P < 0.05, Student's unpaired t test, Fig. 4A).
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Figure 4. Prolongation of decay times of NMDA receptor-mediated synaptic current after axonal injury A, the ifenprodil-sensitive component of the APV-sensitive evoked synaptic current in the slice preparation was also greater in injured than in control neurons. Axotomy was performed 2 days before recording. B, time constant of decay ( | ||
One of the characteristic differences in function among NMDA receptors composed of one of four NR2 subunits with NR1, is the closing time of the channel after washing out agonists (Vicini et al. 1998; Roberts & Ramoa, 1999). Slower kinetics of deactivation of the channel are demonstrated in the recombinant NMDA receptors composed of NR1 and NR2B, and NR1 and NR2D than NR1 and NR2A (Vicini et al. 1998). To examine whether the change in the subunit composition affects NMDA receptor-mediated synaptic events, the decay time of APV-sensitive postsynaptic currents evoked by afferent stimulation was measured in identified DMV neurons in the slice preparation (Fig. 1). The time constant of synaptic current decay after the peak was 28.1 ± 4.2 ms (n = 9) in the injured neurons and 15.9 ± 2.9 ms (n = 10) in control (Fig. 4). This result demonstrates a significantly slower decay of APV-sensitive synaptic current in the injured neurons (P < 0.05), which is compatible with the result of the decrease of NR2A expression after axotomy (Fig. 2). In addition, 3 µM ifenprodil significantly shortened the decay of APV-sensitive postsynaptic current in the injured neurons (19.1 ± 2.8 ms, n = 5), but affected the decay in control neurons to a lesser extent (13.2 ± 2.4 ms, n = 5, Fig. 4B).
These results suggest that the NMDA receptor in axotomized motoneurons acquired the functional characteristics of NMDA-receptor complexes containing predominantly the NR2B subunit.
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DISCUSSION |
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Alteration of NMDA receptor characteristics in injured neurons
Injury-induced functional changes have been well studied in peripheral neurons. Alterations in receptor and channel properties became evident 1-3 days after in vivo axonal injury, such as 5-HT3 receptors in peripheral sympathetic neurons (Rosenberg et al. 1997), ACh receptors in the ciliary ganglion (Levey & Jacob, 1996), and Na+ channels in spinal sensory neurons (Dib-Haii et al. 1996). However, little is known about the changes in NMDA receptor characteristics of the central neurons in various pathophysiological conditions. Ischaemia decreases the expression of NR2A and NR2B (Zhang et al. 1997), but not NR1 (Pellegrini-Giampietro et al. 1994) in the hippocampus. The alterations in NMDA receptor subunits after axotomy remain contraversial. Axotomy decreases expression of NR1, NR2B and NR2D in motoneurons of the rat spinal cord (Piehl et al. 1995). In contrast, axotomy does not affect NR1 mRNA in rat spinal motoneurons (Sanner et al. 1994). In cultured central neurons, mechanical injury to the soma induces a reduction in the Mg2+ block of the NMDA response (Zhang et al. 1996). In vivo axonal injury reduces voltage-dependent Mg2+ block of the NMDA response (Furukawa et al. 2000) and NR2A expression of vagal motoneurons (Fig. 2).
A number of reports demonstrate the functional differences among the various subunit compositions of the recombinant NMDA receptor, e.g. sensitivity to Mg2+ (Monyer et al. 1992) and channel kinetics (Vicini et al. 1998). Ifenprodil inhibited the current response of the NMDA receptor containing NR2B more potently than that of NR2A (Williams, 1993). Slower kinetics of deactivation of the channel and slower recovery from desensitization are demonstrated in the recombinant NMDA receptors composed of NR1 and NR2B, and NR1 and NR2D than NR1 and NR2A (Vicini et al. 1998). Thus, the alteration in expression of the NMDA receptor subunits possibly contributes to the functional change of the NMDA receptor in various pathophysiological conditions. In the adult brainstem, NR2A is predominantly expressed among the four NR2 subunits (Monyer et al. 1992). The estimated functional change in NMDA receptor function brought about by a decrease in the NR2A subunit in ratio is the prolongation of the decay time of deactivation (Vicini et al. 1998). The shortening of the decay time of the NMDA receptor-mediated synaptic current was demonstrated to be associated with the developmental increase in NR2A subunit expression in various neurons, such as those in the cortex and (Flint et al. 1997; Roberts & Ramoa, 1999) and cerebellum (Takahashi et al. 1996; Rumbaugh & Vicini, 1999; Cathala et al. 2000). These reports are compatible with the present results that a decrease in NR2A expression and increased contribution of the ifenprodil-sensitive component (NR2B) to the NMDA response (Fig. 3) resulted in a longer decay time of NMDA receptor-mediated synaptic events (Fig. 4).
The synaptic current time constant of decay in the DMV (Fig. 4) was somewhat faster than those recorded in other preparations (Takahashi et al.1996; Flint et al. 1997; Cathala et al. 2000). Although many factors affect the decay of synaptic currents, the diversity of subunits from which the NMDA receptor is composed, e.g. greater content of NR2A and splice variant of NR1 (Rumbaugh et al. 2000), might contribute to a faster decay in the DMV. In the present study, the expression of NR1 (Fig. 1) and NMDA-induced current density was not changed significantly by axotomy. Thus, the replacement of NR2A with an NR2B or NR2B insertion into the NMDA receptor complex might be responsible for the their characteristic change after axotomy without changing the density of functional NMDA receptor. Other possible alterations in NMDA receptor subunits, such as NR3A, which is expressed in the motoneurons (Abdrachmanova et al. 2000) and which affects NMDA receptor function (Das et al. 1998), remain for the future study.
Functional significance of alteration in subunit composition
In injured neurons, various immature characteristics are re-expressed. The reappearance of immature characteristics after in vivo axonal injury has been reported, e.g. in the ACh receptor in the ciliary ganglion (Levey & Jacob, 1996) and in type III Na+ channels in spinal sensory neurons (Waxman et al. 1994). In the NMDA receptor, reduced Mg2+ block of NMDA response, which is one of the immature characteristics (Nabekura et al. 1994), is re-expressed in injured motoneurons (Furukawa et al. 2000). During development, NR2A replaces NR2B and NR2D in various brain areas (Watanabe et al. 1992). In the present study, NR2A was expressed to a lesser extent in injured neurons (Fig. 2). Lesser expression of NR2A is a characteristic in the immature animal (Monyer et al. 1992). If this is so, injured neurons should reacquire a greater plasticity. However, application of NR2B antagonist has been shown to effectively reduce brain damage in the case of stroke (Di et al. 1997), and cortical c-fos mRNA induction following focal injury (Menniti et al. 2000). In addition, the slow kinetics of deactivation of the NMDA receptor containing NR2B (Fig. 4) might induce more Ca2+ influx, which is directly linked to excitotoxicity. In this sense neuronal injury would cause a long-term increase in the susceptibility of neurons to excitotoxicity.
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Acknowledgements
The authors would like to thank Dr Mike Andresen at Oregon Health Science University and Dr Rita Balice-Gordon at the University of Pennsylvania for critical reading of the manuscript. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan on Advanced Brain Research (No. 13210108) and Integrated brain Research (No. 1303506) to J. Nabekura.
Author's present address
M. Okada: Division of Pharmacology, Course for Molecular and Cellular Medicine, Graduate School of Medical and Dental Sciences, Niigata University, Japan.
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  F. Fumagalli, A. Frasca, G. Racagni, and M. A. Riva Dynamic Regulation of Glutamatergic Postsynaptic Activity in Rat Prefrontal Cortex by Repeated Administration of Antipsychotic Drugs Mol. Pharmacol., May 1, 2008; 73(5): 1484 - 1490. [Abstract] [Full Text] [PDF]
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 T. Y. Nakamura, A. Jeromin, G. Smith, H. Kurushima, H. Koga, Y. Nakabeppu, S. Wakabayashi, and J. Nabekura Novel role of neuronal Ca2+ sensor-1 as a survival factor up-regulated in injured neurons J. Cell Biol., March 27, 2006; 172(7): 1081 - 1091. [Abstract] [Full Text] [PDF]
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 J. Nabekura, T. Ueno, A. Okabe, A. Furuta, T. Iwaki, C. Shimizu-Okabe, A. Fukuda, and N. Akaike Reduction of KCC2 Expression and GABAA Receptor-Mediated Excitation after In Vivo Axonal Injury J. Neurosci., June 1, 2002; 22(11): 4412 - 4417. [Abstract] [Full Text] [PDF]
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) after the peak was larger in DMV neurons receiving axotomy (n = 10, third bar from the left) than in control DMV neurons (n = 9, first bar, *, P < 0.05, Student's unpaired t test). Ifenprodil (3 µM, ifen) significantly decreased