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. Author manuscript; available in PMC: 2009 Jun 2.
Published in final edited form as: Restor Neurol Neurosci. 2008;26(6):467–473.

Protective effects of gastrodia elata on aluminium-chloride-induced learning impairments and alterations of amino acid neurotransmitter release in adult rats

He Shuchang a,b, Niu Qiao b,*, Niu Piye b,c, He Mingwei d, Sun Xiaoshu a, Shao Feng a, Wang Sheng e, Mark Opler f
PMCID: PMC2689815  NIHMSID: NIHMS114836  PMID: 19096134

Abstract

Purpose

High brain levels of aluminum (Al) can be neurotoxic and cause learning and memory deficits. Gastrodia elata (GE) is a Chinese herb widely used for improving mental function in traditional Chinese medicine. We measured changes in Al-induced neurotransmitter alteration and performance on a learning and memory task to elucidate the mechanism of Al toxicity and to assess whether these alterations could be attenuated by GE.

Methods

Thirty-six adult, male rats were randomly divided into six groups. Four Al-exposed groups were given aluminum chloride at 5 mg/kg/day or 10 mg/kg/day (i.p.) for two months, with two of these groups (one for each dose of Al) receiving GE (0.4 g/kg, via oral intubation, with the GE powder mixed in the drinking water) while the other two groups received vehicle. A GE control group was given injections of saline plus GE and a saline control group was given injections of saline and with 3 injection days and one day off. A step-down test was used to measure learning and memory ability. Al concentrations in the neocortex were assayed with a graphite furnace atomic absorption spectrophotometer. Amino acid neurotransmitter levels in the neocortex were determined by high performance liquid chromatogram-fluorescence.

Results

Al-exposed rats showed impaired learning and memory ability as indicated by shorter step down latency and more retention errors. Cortical concentrations (mean ± SEM) of Al were: 56.22 ± 34.10 ng/g (wet weight) in the Saline control group; 172.87 ± 111.06 in the 5 mg/kg/dayAl group; 289.15 ± 102.55 in the 10mg Al group; 74.98 ± 19.00 in the GE control group; 232.55 ± 35.74 in 5 mg Al+GE group; and 291.35 ± 98.38 in 10 mg Al+GE group respectively. Al exposure produced a significant increase in cortical GABA levels. Gastrodia elata reduced learning and memory deficits without affecting brain Al levels.

Conclusions

Rats exposed to AlCl3 suffer from deficits in learning and memory, accompanied by increases in GABA levels in the neocortex. Gastrodia elata is effective in improving memory functions and normalizing GABA levels.

Keywords: Aluminum, learning and memory, amino acid neurotransmitter, gastrodia elata

1. Introduction

Gastrodia elata (GE) is a traditional Chinese herb that has been used for the treatment of rheumatism, hemiplegia, lumbago, headaches and vertigo. The major active components in GE are gastrodin (GA), p-hydroxybenzyl alcohol (HA), vanillyl alcohol (VA), 4-hydroxybenzaldehyde (HD), vanillin (VL), succinic acid (Yan, 2006; Zeng, 2006). β-sitosterol and sucrose have also been isolated from GE. The components of GE play various roles in treatment of diseases. Gastrodin exerts neuroprotective effects, making it useful for treatment of dizziness, epilepsy, stroke and dementia (Zeng, 2006; Linda, 2006). Gastrodin and alcohol HA facilitate memory consolidation and retrieval, but not acquisition, as shown in the passive avoidance task test in rats (Hsieh et al., 1997). The aqueous extract of GE improved D-galaxies-induced memory impairment in mice and performance on a step-down, passive-avoidance task in senescent mice. GE reduces oxygen free radicals (Zeng et al., 2006), protects against neuronal damage (Kim et al., 2006; Huang et al., 2007), and exhibits anxiolytic-like effects via the GABAergic nervous system (Jung et al., 2006). Based on these findings, we hypothesized that GE might exert protective effects on the learning and memory impairments caused by Al exposure in rodent models.

Aluminum (Al), the most abundant metal in the earth’s crust, is ubiquitous in the environment and its extensive industrial utilization has stimulated considerable interest in the possible environmental toxicity of this metal. However, little is known about possible effects of Al as a trace element in animals and humans in normal conditions. It has recently become clear that when Al is mobilized from soil by acid rain, it poses a hazard to all exposed organisms (Birchall et al., 1989). Al is also thought to be a causal agent in some cases of encephalopathy and osteomalacia observed in patients with chronic renal failure caused by long-term hemodialysis (Tahara, 2004). Al toxicity in humans has been implicated in many neurodegenerative diseases such as Alzheimer’s disease, amyotrophic lateral sclerosis, and parkinsonism-dementia (Roberts, 1986; Garruto et al., 1994; Solomon et al., 2001). The mechanism of Al-inducted neurotoxicity and identification of effective treatment for such impairments is, therefore, an important public and occupational health priority for industrial and developing nations.

Al contributes to a variety of cognitive impairments in mice, rabbits, and rat pups (Muller et al., 1990; Yokel, 1985, Bilkei-Gorzo, 1993; Mari, 2001). Studies on workers exposed to Al dust in industrial environments demonstrate similar effects (Rifat, et al., 1991; Bast-pettersen, et al., 1994; White et al., 1992, Akila et al., 1999). Many researchers have found elevated Al levels to be associated with a decline in visual memory, attention, concentration, frontal lobe function and lower vocabulary scores in hemodialysis patients (Bolla et al., 1992). Our own research has demonstrated that exposure to electrolytic aluminum fumes adversely affects neurobehavioral performance in workers, including motor coordination, mood, and autonomic nervous function (He et al., 2003), although other reports on occupational Al exposure and neurological impairments demonstrate mixed findings (Sim et al., 1997). Despite strong experimental and clinical evidence for Al neurotoxicity, the mechanism of Al effects on the nervous system is still not completely clear. To gain further insight into potental mechanisms of toxicity and to explore the possible treatment effects of GE we studied the effects of exposure to AlCl3 and GE on behavioral and neurochemical endpoints in rats.

2. Materials and methods

2.1. Animal model

Adult, male Sprague-Dawley rats, weighing 200–220 g, were kept with food and water ad libitum. Rats were group housed in stainless steel cages, maintained on a 12 h light/dark cycle (lights on at AM).

2.2. Aluminum administration

Rats were divided into six groups: the control group (n = 6) received daily i.p. 0.9% saline injections. Two groups of six rats each were exposed to aluminum chloride (nonahydrate) at 5 mg/kg/day (i.p.) or 10 mg/kg/day (i.p.). Prior to injections, the solution was titrated to a pH of 7.2. Four-day injection cycles were administered for two months, with 3 injection days and one day off. The saline+GE (GE control), 5 mg+GE and 10 mg+GE groups were administrated with GE in a dosage of 0.4 g/kg/day (GE powder mixed with drinking water was administered via oral intubation).

2.3. Step-down test

Following a two-month treatment period, the rats were tested on a step-down test in a passive-avoidance chamber (30 cm × 30 cm × 25 cm). The floor of the chamber consists of copper rods and a well-insulated 4 cm × 4 cm × 5 cm platform made of rubber in one corner of the chamber. The rats were placed in the chamber for a three-minute adaptation period and were then placed on the platform. Their latency to step down on the grid with all four paws was measured. Upon stepping down on the copper bars, the rats received an immediate mild electrical shock (stimulus current was 0.5–0.6 mA). To avoid the shock, rats demonstrate an instinctive reaction to jump back onto the platform. Rats were tested in this manner for 5 minutes. The number of times rats stepped down from the platform within 5 minutes were considered acquisition errors. The following day, this procedure was repeated, and the step-down latency was used as a measurement of memory retention. The number of times the rats stepped down onto the platform within the 5-minute interval were recorded as retention errors. All behavioral testing was conducted with the experimenters being blind to group/treatment status.

2.4. Aluminum concentration in rat neocortex analysis

At the end of testing, rats were sacrificed by an overdose (2 ml) of chloral hydrate and the whole brains were immediately removed. Approximately 0.1 g fresh tissue (neocortex of right hemisphere of the brain) was heated in a solution of 1.6 ml nitric acid and 0.4 ml perchlorate acid in saline for resolution. This solution was then heated at 120–150°C for four hours and then transferred into an Eppendorf tube containing de-ionized water to a final total volume of 2.0 ml. Al concentration was determined by graphite atomic absorption spectrometry (AA-670, JAPAN) according to parameters described previously (Colomina et al., 1999) using a wavelength of 309.2 nm, slit width of 1.3 nm, lamp current of 10 mA, and an injection volume of 10 μl.

2.5. Determination of amino acid content

Amino acid content was quantified by means of high performance liquid chromatogram, using fluorescence detection (HPLC-FD). For this procedure, 0.1 g brain tissue was placed in a cuvette with 5 ml of artificial cerebrospinal fluid (2-aminoethylliso-thiouronium bromide (AET), 28.1 mg, PLP_1.0 mmol/L in 2.0 ml, 0.1 ml Tritonx-100, dissolved in 0.1 mmol/L 100 ml phosphate buffer, pH 7.0) and homogenized. The cuvette was then centrifuged at 4000 revs/min for 10 min. Then, 100 μl of the supernatant was placed in another tube in which 20 μl 3-phenyserine(1 mmol/L) and 100 μl acetonitrile was added. It was then centrifuged at 15000 revs/min for fifteen minutes. Next, 20 μl of the extract was placed in a culture dish and a derivatization solution was added. After being incubated for 2 min, 10 μL was taken for analysis of glutamate (Glu) and GABA concentrations. Standard solutions of amino acid neurotransmitters included Glu, GABA and 3-phenyserine (sigma) 2.5 mmol/l, dissolved in methanol/water mixture (methanol: water = 50:50) and stored frozen at −20°C.

Na—phosphate buffer (Na2HPO4·12H2O 0.64 g, NaH2PO4·H2O 0.39 g, NaN3 0.10 g), was put added into 1000 ml of de-ionized water to pH 7.0, with acetonitrile 2%, methanol 2.5%, and Na-phosphate buffer added to make up 100%. Derivatization solution consisted of methanol-PITC-water triethylamine at the following ratios: 7:1:1:1.

For HPLC-FD, a 5 μm Hypesil ODS 4.6 mm × 150 was use with a 4.6 mm × 30 mm C18 protection column, maintained at a temperature of 35 °C. The test wavelength was 254 nm, 0.05 AUFS. Isocratic fluid phases were employed, beginning with 0.01 mol/L acetic acid at a pH = 6.1. The second fluid phase consisted of methanol. A flow rate of 1.2 mL/min was maintained. Peak measurement was made against an internal standard using 3-phenyserine.

2.6. Statistical analyses

SPSS for Windows Version 10.0 was used to perform one-factor analysis of variance (ANOVA). followed by Fisher’s Protected Least Significant Difference (PLSD) post hoc tests, when warranted. The probability level at which the null hypothesis was rejected was p< 0.05.

3. Results

3.1. Step-down test

Learning and memory deficits were observed in all Al-exposed groups. It was observed that the latency to step down was shorter for rats in the 5 mgAl group and 10 mg Al group (Table 1). The number of acquisition errors in the 10 mg Al group increased significantly, compared with that of the saline group (Table 1). The number of retention errors was significantly greater for the 10 mg Al group, relative to saline controls (Table 1). Both acquisition and retention errors were greater in the 10 mg Al group compared with those of the 5 mg Al group. GE-treated rats showed fewer learning and memory errors than their Al-treated cournterparts. The latency to step down in the 5 mg Al+GE group was longer than that of the 5 mgAl group (p < 0.05). In the 10 mg Al+GE group, the latency to step down was significantly longer than that of the 10 mgAl group and both acquisition and retention errors were decreased significantly, compared with the 10 mgAl group. Findings between saline controls and GE controls were similar.

Table 1.

Step-down test results

Groups Number of acquisition
errors		 Latency to step down
(in seconds)		 Number of retention
errors
Saline (n =6) 0.67 ± 0.52 172.50 ± 107.28 0.83 ± 0.41
5 mg Al (n =6) 1.67 ± 1.21 35.00 ± 36.46* 1.50 ± 0.55
10 mg Al (n =6) 3.00 ± 1.55* 37.33 ± 48.46* 2.67 ± 1.63*
Saline+GE (n =6) 0.50 ± 0.54 123.50 ± 88.60 0.83 ± 0.41
5 mg Al+GE (n =6) 0.83 ± 0.75 125.33 ± 39.83† 1.8 ± 0.98
10 mg Al+GE (n =6) 0.67 ± 0.51‡ 145.88 ± 40.62‡ 1.17 ± 0.41‡
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*

Compared to the saline group (P < 0.01).

†

Compared to the 5 mg Al group (P < 0.05).

‡

Compared to the 10 mg Al group (P < 0.01).

3.2. Cortical Al levels

Cortical Al concentrations were significantly higher in Al-exposed groups than those in the control groups (Table 2). Al levels were also higher in the 10 mg Al group vs. the 5 mg Al group (Table 2). There was no significant difference in cortical Al concentrations between saline+GE group and saline group,and among 10 mg Al+GE group, 5 mg Al+GE group, 10 mg Al group and 5 mg group, respectively.

Table 2.

Cortical Al levels

Group n Brain Al(ng/g)
Saline 6 76.22 ± 34.10
5 mg Al 6 242.87 ± 111.06*
10 mg Al 6 289.15 ± 102.55*
Saline + GE 6 74.98 ± 19.00
5 mg Al + GE 6 232.55 ± 35.74*
10 mg Al + GE 6 291.35 ± 98.38*
Open in a new tab
*

Compared to the saline group (P < 0.01).

3.3. Amino acid neurotransmitter concentrations

As indicated in Table 3, Glu levels in 10 mg Al group was significantly lower than the saline-injected control group. We observed no significant difference between the 10 mg Al and 5 mg Al group in GABA and Glu levels. GABA levels was significantly higher in the 10 mg Al group as compared to control groups. In contrast with the 10 mg Al group, the GABA levels decreased significantly in the 10 mg+GE group (Table 3).

Table 3.

Glu and GABA concentrations in brain tissue (μg/g)

Groups Glu GABA
Saline 310.12 ± 43.72 165.05 ± 54.23
5 mg Al 251.68 ± 18.78 227.85 ± 44.02
10 mg Al 213.0 ± 51.06* 254.62 ± 69.64*
Saline + GE 212.47 ± 60.28 182.65 ± 92.73
5 mg Al group + GE 226.50 ± 78.98 180.03 ± 80.94
10 mg Al group + GE 247.90 ± 71.58 153.18 ± 54.90#
Open in a new tab
*

Compared to the saline group (P < 0.01).

#

compared to the 10 mg Al group (P < 0.05).

4. Discussion

This study describes the neurotoxic effects of intraperitoneal administration of aluminum chloride to adult rats over a two-month period and the treatment effects of GE on rats exposed to AlCl3. Aluminum exposure exerted adverse effects on learning and memory which were manifested in increases in the number of acquisition and retention errors of rats that were exposed to AlCl3 for two months. This finding supports other research that has indicated that higher-levels of Al in the brain can impair long-term potentiation (LTP), which is thought to be the major physiological basis of learning and memory (Llansola et al., 1999). The present study also demonstrated ameliorative effects of GE on Al-induced learning and memory impairments and these GE treatments also reversed the effects of Al on cortical levels of glutamate and GABA.

Previously, others have reported that several active ingredients of GE can facilitate memory consolidation and retrieval, but not acquisition (Hsieh et al., 1997). It is hypothesized that GE exerts its neuroprotective effects in the CA3 area of hippocampus following lead (Pb) exposure (Hu et al., 2003). The effect of GE on learning and memory has also been investigated by Wu et al. in (1996) who found that the methanol extract of GE significantly prolonged the shortened step-through latency induced by scopolamine in the passive-avoidance task in rats. Additionally, the ethyl acetate and n-butanol fractions of the methanol extract of GE has been shown to prolong the shortened step-through latency induced by scopolamine (Wu et al., 1996). Gastrodin, isolated from the n-butanol fraction of the methanol extract, and p-hydroxybenzyl alcohol, isolated from the ethyl acetate fraction of the methanol extract, also significantly prolonged the shortened step-through latency induced by scopolamine on a passive avoidance task (Wu et al., 1996).

In our study, the Al concentration in the rat neocortex among Al-exposed animals was significantly higher than that of controls, corresponding to learning and memory deficits. GE administration did not affect Al levels in the brain, suggesting that it does not influence certain toxicokinetic parameters, such as absorption and distribution. Certain brain regions are of particular interest for Al toxicokinetics and for their resulting cognitive effects. McDermott reported that the hippocampus has the highest concentration of Al in the human brain, 6.5 μg/g (dry weight) followed by frontal lobe and temporal lobe, while the corpus callosum has the lowest levels (1.5 μg/g) (McDermott et al., 1979). Our findings are in keeping with the results reported by Florence et al. (1994), who also found that chronic oral administration of Al citrate to male Wistar rats leads to an Al overload in a relatively short period of time. Our findings are also in concert with others (Candy et al., 1992; Shimizu et al., 1994). Al in the human brain accumulates primarily in neocortex, which may explain the learning and memory impairments reported experimentally and clinically. While GE does not decrease the Al levels in the brain, it is apparently capable of reversing behavioral and biochemical effects induced by Al exposure.

GABA and glutamate are two key amino acid neurotransmitters in the CNS. Changes in levels of GABA in either direction may be expected to impair normal nervous system function. In this study, Al-exposed rats had a lower glutamate levels and higher GABA level. These biochemical changes are difficult to interpret in detail, at least on the basis of this single set of experiments. Provan et al. (1992) reported Al levels in the brain were found to disturb G proteins, Ca2+ channel and PKC. Chronic exposure to Al has been shown to reduce the basal activity of guanylate cyclase, and impairs the glutamate—nitric oxide-cyclic GMP pathway in rats in vivo and in cultured neurons (Hermenegildo et al., 1999). Our results indicate that the active components of GE may impact one or more of these pathways, requiring further study using both in vivo and in vitro approaches, as well as the biochemistry of Al-toxicity on neurotransmitter metabolism in the CNS (Rahman et al., 2003).

GE could reverse the inhibitory effects on neuro-transmitters caused by Al exposure as demonstrated by relative decreases in GABA levels in experimental animals in our study. One potential mechanism that might explain this effect has been reported by Choi et al. (2006) who found that citryl glycoside from GE plays an inhibitory role on GABA transaminase and, in turn, increases the levels of GABA. In a different study, which examined the effects of GE on GABA, a major constituent of Gastrodin decreased immunoreactivities of the GABA shunt enzymes in the hippocampus of seizure-sensitive gerbils (An et al., 2003).

Our study provides preliminary data on how Al exposure affects neurotransmitters and suggests a novel treatment regimen in the form of GE. Based on available literature and on our own results, the active components of GE may protect neural tissue from various forms of damage, including aging (Kao et al., 1994), toxic exposures (Hu et al., 2003; Hsieh, 2005) and ischemia (Zeng et al., 2006; Yu, 2005).

We conclude that GE exerts positive effects on cognition in our experimental model of Al-neurotoxicity and helps to normalize GABA level. This may be of considerable public health significance as Al production is a major industrial activity, contributing both to primary industrial exposures (Sim & Benke, 2007), as well as to secondary ambient exposures (Zhang et al., 2007). More studies are needed to verify the potential protective mechanisms and the active compounds of GE before the therapeutic efficacy of this form of treatment for Al toxicity can be fully assessed.

Acknowledgment

This work was supported by the grants from National Natural Science Foundation of China (No 30400135, and No 30671777). We thank Dr. Sunny Kalara and Prof. Zhou Zhuan for their assistance.

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