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Research Communication

Magnesium Deficiency Differentially Affects the Retina and Visual Cortex of Intact Rats

Departments of Clinical Neurophysiology and ^* Neuropathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

¹To whom correspondence should be addressed. E-mail: ygoto{at}neurophy.med.kyushu-u.ac.jp.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

 
To determine the influence of magnesium (Mg) on the visual system, electroretinograms (ERG) and visual evoked potentials (VEP) were recorded under dark-(DA) and light-adapted (LA) conditions in intact rats. Weanling rats were fed either a Mg-deficient (Mg-D) or a control diet for 17 d before the tests, and ERG, VEP and immunohistopathological analyses of retinae and cortices were made. In the Mg-D rats, ear congestion, hair loss and loss of body weight were observed, and serum Mg concentration was 25% of that in the control rats (P < 0.01). The amplitudes of the DA a-wave and the second positive peak of the oscillatory potentials (OP₂) of the ERG, and the negative component of the VEP (N1) in Mg-D rats were significantly greater than those of control rats. However, the amplitudes of the DA b-wave, LA 2 Hz b-wave, the 20 Hz flicker responses and the implicit times of all response components did not differ between the two groups. The immunohistopathologic results also were not altered in the Mg-D rats. We suggest that the functional abnormalities induced by Mg deficiency may depend not only on the hyperactivity of the N-methyl-D-aspartate (NMDA) receptor, but also on the behavior of the Ca²⁺ and Mg²⁺ ions in the intact eye.

KEY WORDS: • magnesium deficiency • electroretinograms • visual evoked potentials • NMDA receptors • rats •

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

 
Disturbances in Mg metabolism significantly influence the central and peripheral nervous systems in humans (1 2 3) and animals (4 5 6 7) . For example, we reported that young rats fed a low Mg diet become hyperactive within a few days; shortly thereafter, they become highly susceptible to audiogenic stimuli, with episodes of rapid running, tonic and/or clonic convulsions, and rearing (5) . Tonic convulsions and muscle cramps were reported in young people whose serum Mg concentration was significantly lower than normal (1 2 3) . These behavioral changes may be caused by an alteration of the excitability of the N-methyl-D-aspartate (NMDA)² receptors based on in vitro (8 9 10) and intact animal studies (6) .

Recent research in the pharmacology and physiology of Mg²⁺ has been based on the finding that Mg²⁺ blocks Ca²⁺ channels at physiologic concentrations, particularly those coupled with the NMDA receptor (11) . Because Mg²⁺ gates Ca²⁺ import (11) , reduction in the extracellular concentration of Mg²⁺ activates Ca²⁺ conductance, thereby increasing neuronal excitability, which leads to the behavioral changes. Severe visual loss and legal blindness, which may be caused by the induced hyperexcitability and toxicity of the NMDA receptors, have been observed in Mg-deficient (Mg-D) patients (12) . However, the physiologic changes and lesions in the visual pathway of Mg-D animals and patients remain poorly understood.

In rodent electroretinograms (ERG), the dark-adapted (DA) a-wave originates from the rod outer segments (13 14 15) . The DA and light-adapted (LA) b-waves are thought to be initiated by depolarization of the ON bipolar neurons in response to flash, which induces potassium efflux from the Müller cell end-feet; the oscillatory potentials (OP) are generated from the amacrine cells (16 17 18 19) . In addition, the LA responses to 20-Hz stimulation (flicker stimulation) originate from the cone and cone-driven bipolar cells (20) . In the visual evoked potential (VEP), a negative component (N1) with an implicit time near 70 ms has been recorded from the occipital area using dim flash stimuli (21 22 23) . This component may originate from the primary visual cortex because it cannot be recorded from other cortical areas. Thus, many diseases of the visual system cause changes in the ERG and VEP, and examinations of their properties are of diagnostic value. The purpose of this study was to determine how Mg-D affects the responses of the retina and visual cortex elicited by high intensity stimuli in intact animals.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

 
Weanling male Sprague-Dawley rats (n = 22; Kyudo Co. Japan), weighing 50–60 g, were used; 12 rats were used for basic electrophysiologic and histologic studies, and 10 to study the effect of loading the NMDA receptor with a competitive blocker, DL-2-amino-7-phosphonoheptanoic acid (APH). Each group was divided into those fed a Mg-D diet and those fed a Mg-supplemented (control) diet. Experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research (ARVO Membership Directory, 2000, xvii-xviii), and were approved by the animal ethics committee of Kyushu University.

A synthetic diet was fed to each group. The Mg-D diet contained vitamin-free casein (200 g/kg diet, ICU Biochemicals, Cleveland, OH), glucose (702 g/kg diet, Katayama Chemical, Osaka, Japan), corn oil (50 g/kg diet, Katayama Chemical), vitamin mixture (10 g/kg diet, AIN-76, oriental Yeast, Tokyo, Japan), choline chloride (3 g/kg diet, Katayama Chemical) and salt mixture (35 g/kg diet, AIN-76, oriental Yeast) without added Mg (24 ,25) . This diet contained < 0.0099 g Mg/kg diet by analysis. The control diet was made by adding 0.4 g Mg/kg diet as MgO (24) . Deionized water containing <10 µg Mg/L (undetectable Mg by atomic absorption) was consumed ad libitum from polyethylene bottles with rubber stoppers and stainless steel lick-spouts. Rats were fed diets for 17 d before testing. They were kept in a pathogen-free facility at a constant room temperature at 24°C with a 12-h light:dark cycle and had free access to food and water.

Each rat was kept in a dark room for at least one night and prepared under dim red illumination (20 ,26) . Rats were anesthetized with intraperitoneal injection of 15 µL/g body of a 9 g/L saline solution containing ketamine (1 g/L), xylazine (0.4 g/L) and urethane (40 g/L). The pupils were dilated with 2.5 mmol/L phenylephrine HCl and 1.5 mmol/L proparacaine HCl drops; the rats were placed on a heating pad throughout the experiment. A wire electrode, coated with 1 mmol/L methylcellulose, was placed over the cornea to record the ERG. A similar wire electrode placed in the mouth served as a reference electrode, and a needle electrode inserted into the tail was grounded. The impedance between the corneal and reference electrodes was < 3.0 kÙ. Responses were differentially amplified between 0.8 and 1200 Hz. In addition, the OP were recorded using an amplifier set for a bandpass frequency of 30–300 Hz.

White (xenon) stroboscopic flashes (1.30 log cd s/m²) were presented in a commercial Ganzfeld stimulator (VPA-10; Cadwell, Kennewick, WA), either in the dark or against a white adapting field (1.50 log cd/m²). The responses to five successive flashes at an interstimulus interval of 1 min were averaged for the DA responses. After the DA VEP recording, the rats were exposed to a white adapting field for at least 25 min and the LA ERG, elicited by a flash of 1.30 log cd s/m², were recorded. Responses to 50 successive flashes presented at a rate of 2 or 20 Hz were averaged.

VEP were recorded after the DA ERG recordings using a needle electrode placed under the scalp overlying the visual cortex. The reference electrode was a needle electrode placed under the scalp overlying the frontal sinus, and an electrode in the tail was used to ground the rat. The impedance between the scalp and reference electrodes was < 3.0 kÙ. Responses were differentially amplified between 0.8 and 1200 Hz. Flash stimuli (1.30 log cd s/m²) were presented in a Ganzfeld stimulator under DA conditions. Twenty consecutive responses were averaged at 1 Hz.

On completion of the experiment, blood samples were taken from the aorta of control and Mg-D rats for the determination of the serum concentrations of electrolytes.

After the blood samples were taken, the Mg-D rats were perfused through the aorta with PBS followed by 4 mmol/L paraformaldehyde in PBS. Immunohistochemistry was performed on 10-µm thick paraffin-embedded eye and brain sections by the indirect immunoperoxidase method. The sections were deparaffinized in xylene, hydrated in ethanol and incubated with 0.15 mmol/L hydrogen peroxide in absolute methanol for 30 min at room temperature to inhibit endogenous peroxidase. The sections were then autoclaved at 120°C for 10 min in 10 mmol/L citrate buffer (pH 6.0) to enhance the immunoreactivities of glutamate receptors. The sections were then incubated with the primary antibodies to NMDAR1, NMDAR2A/B or metabotropic glutamate receptor 1 (mGluR1) (Chemicon, Temecula, CA) diluted 1:100 in PBS containing 0.005 mol/L normal goat serum at 4°C overnight. After rinsing, the sections were incubated with a peroxidase-conjugated anti-rabbit immunoglobulin G antibody diluted 1:200 in PBS for 60 min at room temperature. The colored reaction product was developed with 3,3'-diaminobenzidine tetrahydrochloride.

Mg-D (n = 5) and control (n = 5) rats were injected with 0.072 g/kg APH (6 ,7) intraperitoneally in a dark room. Before and 1 h after the APH injection, VEP were recorded and analyzed as above. Because we did not have any evidence that APH would cross the blood-retinal barrier, ERG were not recorded. In addition, we did not inject APH intraviteally because it is difficult to evaluate the effect of APH without the injection artifacts (e.g., bleeding, increased intraocular pressure).

Control and Mg-D rats were compared by one-way ANOVA; differences were considered significant when P < 0.05.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

 
Systemic effects of Mg-D.

The Mg-D rats had congested ears, hair loss and a significantly lower body weight (66.8 ± 1.5 g) than the control rats (120.3 ± 1.8 g). The serum Mg concentration in Mg-D rats (0.76 ± 0.012 mmol/L, P < 0.01) was 25% that of the control rats (3.4 ± 0.10 mmol/L), whereas serum Ca, K and Na were not different (data not shown), consistent with previous reports (5 6 7) .

Functional abnormalities of retinae in Mg-D rats.

Representative ERG from a control and a Mg-D rats are shown in Figure 1 . The ERG were elicited by flash stimuli (1.30 log cd s/m²) delivered under DA conditions (Fig. 1 A and B) and under LA conditions with a steady background field of 1.50 log cd/m² (Fig. 1 C and D). The amplitudes of the DA a-wave and the second positive OP peak (OP₂) in Mg-D rats were significantly greater (P < 0.05) than those in control rats (Table 1 ). However, the amplitudes of DA b-waves, the LA 2-Hz and LA flicker responses in Mg-D rats were not different (Table 1) . The implicit times of DA a-wave, DA b-wave, OP₂ and LA b-wave did not differ between control and Mg-D rats (data not shown).

View larger version (24K): [in this window] [in a new window]   Figure 1. Electroretinograms (ERG) and visual evoked potentials (VEP) recorded from representative control (upper waveforms) and magnesium deficient (Mg-D, lower waveforms) rats on d 18. A stimulus intensity of 1.30 log cd s/m2 was presented to the dark-adapted (DA) eyes (A, B, E), or superimposed on a background field of 1.50 log cd/m2 at light-adapted (LA) 20 Hz (C) or 2 Hz (D). The amplitudes and implicit times of the a-wave (a), b-wave (b) and oscillatory potentials (OP) under DA conditions, and the b-wave at 2 and 20 Hz stimuli under LA conditions were recorded. The amplitude of the a-wave was measured from the prestimulus baseline to the trough of the cornea-negative peak; the b-wave was measured from the negative trough to the positive peak, and the implicit times of the a- and b-waves were measured from the onset of the stimulus to the a- and b-wave peaks. Only the second positive peak (OP2) was measured. The amplitude of the negative component of VEP (N1) (E) was measured from the positive (almost baseline) trough to the negative peak, and the implicit time of N1 was measured from the onset of the stimulus to the N1 peak. Note that the dark-adapted a-wave and OP2 amplitudes in ERG and N1 amplitude in VEP of Mg-D rats were greater than those of control rats.

Our DA ERG results contradict previous results (27) that the b-wave amplitude was enhanced and the a-wave was only slightly increased in the isolated skate retina. The increased DA a-wave may have been due mainly to the alterations of Ca²⁺ in rods or to a blockage of transmission between the rods and bipolar cells. The bipolar, horizontal and ganglion cells receive synaptic input from glutamate receptors with only the latter apparently being blocked by Mg²⁺. The extracellular Mg²⁺ changes should influence the b-wave potentials of the ERG. However, our results showed no changes in the b-wave amplitude. It is still unknown how extracellular Ca²⁺ and Mg²⁺ behave in an intact retina in the presence of extracellular space (subretinal space), intact blood supply, retinal pigment epithelium and synaptic transmission between photoreceptors and second-order neurons. However, Mg deficiency may alter the complex interactions between retinal cells to affect the DA a-wave but not the DA b-wave in the intact retina.

Functional abnormalities of visual cortex in Mg-D rats.

The control VEP was dominated by the N1 component with an implicit time near 36 ms (Fig. 1 E), which was faster than the mouse flash VEP reported previously (21 22 23) . This VEP reflects both rod- and cone-mediated activities; thus the implicit time may be faster. The amplitude of the N1 component of the Mg-D rats was greater (P < 0.05) than that of control rats (Table 1 , Fig. 1 E); however, the implicit times did not differ in control (36.4 ± 0.9 ms) and Mg-D rats (36.2 ± 0.8 ms). The cortex histopathology of the Mg-D rats showed no obvious differences from the control rats (data not shown). In addition, Figure 2 shows representative VEP from control and Mg-D rats before and 1 h after an injection of APH. After the APH injection in Mg-D rats, the amplitude of N1 decreased significantly (27.8 ± 1.7 µV, P < 0.05) from before injection. The implicit time of N1 was significantly prolonged in Mg-D rats (67.5 ± 1.1 ms, P < 0.01), but was almost identical in waveform to that of control rats (before, 28.1 ± 1.1 µV; after, 64.3 ± 1.5 ms). Changes in the VEP had recovered by the following day (data not shown).

View larger version (14K): [in this window] [in a new window]   Figure 2. Visual evoked potentials (VEP) recorded from a control (upper waveforms) and a magnesium-deficient (Mg-D; lower waveforms) rat before (A) and 1 h after injection (B) of DL-2-amino-7-phosphonoheptanoic acid (APH). Stimulus intensity of 1.30 log cd s/m2 at 1 Hz was presented to the dark-adapted condition. Note that decreases in the amplitude of the negative component of VEP (N1) and prolongation of the implicit time of N1 in control and Mg-D rats were observed at 1 h after APH injection, and the waveforms were similar.

On the other hand, the VEP waveforms were almost identical in Mg-D and control rats 1 h after APH injection (Fig. 2) . Thus, the amplitude difference of the N1 component between Mg-D and control rats may depend mainly on the activity of the NMDA receptor. Decreasing the concentration of Mg²⁺ in the cortex seems to be the physiologically activated neurons. NMDA receptors with a weaker Mg²⁺ block would allow Ca²⁺ to enter postsynaptic neurons more easily than those with the normally strong Mg²⁺ blockers (28) . Calcium ion entry in a reduced Mg²⁺ block may decrease the threshold for synaptic plasticity without losing the uniqueness of the NMDA receptors as "coincidence detectors," sensing simultaneous occurrence of postsynaptic depolarization and presynaptic activation (28) . This may be another reason that the amplitude of the N1 component in Mg-D rats increased in our experiment. However, the prolonged peak of N1 after APH injection cannot be explained.

In conclusion, our results suggest that the influence of Mg-D on the intact visual system may be different from previous studies that used isolated retinas. This difference may depend not only on the hyperactivity of the NMDA receptor, but also on the behavior of the Ca²⁺ and Mg²⁺ ions in the intact eye.

	ACKNOWLEDGMENTS

 
We thank M. Nakamura, Neal S. Peachey and Duco I. Hamasaki for their comments on this manuscript, and also S. Abe, Department of Clinical Nutrition, Graduate School of Health and Nutrition Sciences, Nakamura-Gakuen College, for preparation of the magnesium-deficient rats.

	FOOTNOTES

 
² Abbreviations used: APH, DL-2-amino-7-phosphonoheptanoic acid; control, Mg-supplemented; DA, dark-adapted; ERG, electroretinograms; LA, light-adapted; Mg-D, Mg-deficient; mGluR1, metabotropic glutamate receptor 1; N1, negative component of visual evoked potentials; NMDA, N-methyl-D-aspartate; OP, oscillatory potentials; OP₂, second positive peak of the oscillatory potentials; VEP, visual evoked potentials.

Manuscript received March 27, 2001. Revision accepted June 13, 2001.

	LITERATURE CITED

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