Differential changes of neuroactive amino acids in samples obtained from discrete rat brain regions after systemic administration of saxitoxin

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Differential changes of neuroactive amino acids in samples obtained from discrete rat brain regions after systemic administration of saxitoxin

Rosa Carmina Cervantes Cianca

^a,

*, Rafael Dura´n Barbosa

^a,1

, Lilian Rosana Ferreira Faro

^a,1

, Lucia Vidal Adan

^a,1

, Ana Gago-Martı´nez

^b,2

, Miguel Alfonso Pallares

^a,1

aDepartamento de Biologı´a Funcional y Ciencias de la Salud, Facultad de Biologı´a, Campus Universitario de Vigo, Universidad de Vigo, 36200-Vigo, Spain

bDepartamento de Quı´mica Analı´tica y Alimentaria, Facultad de Quimica, Campus Universitario de Vigo, Universidad de Vigo, 36200-Vigo, Spain

1. Introduction

The CNS is a target site for toxicity of different neurotoxins altering parameters related to synaptic function and eliciting behavioural and neuropathological effects. A neurotoxin acts specifically on nerve cells by interacting with membrane proteins (receptors, carriers, ion channels). Paralytic Shellfish Poisoning (PSP) are powerful neurotoxic compounds. PSP cannot be considered as a major public health problem. Nevertheless, it is of considerable concern because its toxicity, its effects and because if a fatal dose is consumed, there is no an antidote. Saxitoxin (STX) was the first PSP isolated and it is considered between the most toxic of this group. It is among the most potent hypotensive agent known (Lagos and Andrinolo, 2000) and its main toxicological activity is observed through the blockade of the sodium channels (Henderson et al., 1973; Strichartz, 1984; Guo et al., 1987; Hu and Kao, 1991). These neurotoxins can originate a reduction in the amplitude and speed of conduction of the action potentials by the peripheral and central

nerves, as well as a weakening of the skeletal muscle contraction (Lipkind, 1994). STX cause numbness, paralysis and death by respiratory arrest. In recent times, it has been discovered that STX also binds to calcium and potassium channels; it too inhibits the neuronal nitric oxide synthase, an enzyme that stimulate nitric oxide (NO) production. NO is implicated in the liberation of some neurotransmitters (Llewellyn, 2006).

Amino acids are important classes of neuroactive substances (Boyd et al., 2000) that can act like neurotransmitters or non- neurotransmitters, depending on their function (Shah et al., 2002).

There is abundant evidence to suggest that alterations of neuroactive amino acids (excitatory and inhibitory amino acids) play a signiﬁcant role in the pathogenesis of different diseases.

Nevertheless there is no great evidence about the relationship between neurotoxins effects on neuroactive amino acids. For that reason we have investigated the possible effects of STX on neuroactive amino acids in some discrete rat brain regions.

Considering that LD

50

(Lethal dose medium) of STX is 10–

12 m g kg

¹

in male rats (Herna´ndez-Orozco and Ga´rate, 2006), we just choose 10 and 5 m g kg

¹

STX dose for this research. On the other hand, in previous studies we have reported (Cervantes et al., 2007) that with these doses, STX crosses the blood–brain barrier. In addition we have evaluated Aswad’s method modiﬁcation (1984) for the measurement of neuroactive amino acid.

A R T I C L E I N F O

Article history:

Received 20 July 2008

Received in revised form 25 November 2008 Accepted 12 December 2008

Available online 25 December 2008

Keywords:

Saxitoxin PSP

Neurotransmitters amino acids Voltage-gated sodium channels Release

A B S T R A C T

Aspartic acid, glutamic acid,

g

-amino-n-butyric acid (GABA) and 2-aminoethanesulfonic acid are neuroactive amino acids. They are found in the central rat nervous system. Here, we have studied if a relationship exists between the presence of saxitoxin (STX) a paralytic poisoning shellﬁsh (PSP) and the neuroactive amino acids. Samples of striatum (S), hypothalamus (H), mid brain (MB), frontal cortex (FC), brain stem (BS), right hemisphere (RH) and left hemisphere (LH) of rat brain were collected and analyzed for neuroactive amino acids (AAnt) by Aswad method (1984). Experiments, consisting of intraperitoneal injection of SXT (5 and 10

m

g kg ¹body weight) to young male rats, evoked signiﬁcant changes in AAnt

above basal values. Aspartic and glutamic acid signiﬁcantly increased for RH and LH (after 30 min the increased was 116% and 210%,P0.001 over basal values, respectively). On the other hand, aspartic, glutamic, taurine and GABA signiﬁcantly decreased for S (after 30 min the decreased was 77.4%; 84%;

93.8% and 95.3%,P0.001 over basal values, respectively). These results suggest that STX alters AAnt. It is produced at least in part, because STX blocks voltage-gated sodium channels and this blockade could decrease AAntrelease by exocytotic dependent mechanism of depolarization.

* Corresponding author. Tel.: +34 986 81 19 96; fax: +34 986 81 25 56.

E-mail address:[email protected](R.C.C. Cianca).

1Tel.: +34 986 81 19 96; fax: +34 986 81 25 56.

2Tel/Fax: +34 986 81 22 84.

Contents lists available atScienceDirect

Neurochemistry International

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / n e u i n t

doi:10.1016/j.neuint.2008.12.014

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2. Materials and methods 2.1. Reagents

Sodium hydroxide (NaOH), perchloric acid (HClO4), and hydrochloride acid (HCl) were purchased from Panreac (Madrid, Spain). Boric acid,L-glutamic acid (Glu),L- aspartic acid (Asp),g-amino-butyric acid (GABA), 2-aminoethanesulfonic acid (Tau),L-alanine (Ala),L-arginine (Arg), Glycine (Gly), o-phthaldialdehyde (OPA), N- acetyl-L-cysteine (NAC), acetic acid and sodium acetate were obtained from Sigma–

Aldrich (St. Louis, MO, USA). Methanol and acetonitrile (ACN) LC grade were obtained from Panreac. Chromatographic-grade water was produced by a Milli-Q system (Millipore, MA, USA). Standard solution of STX was purchased from the Institute for Marine Bioscience, National Research Council, Certiﬁed Reference Material Program (NRC-CRM Halifax, Canada).

2.2. Preparation of amino acid standards

GABA and Tau, were prepared at a concentration of 0.1 M in distilled water; being the solution adjusted to pH 7.4 with 0.1 M NaOH. Glu and Asp are insoluble in their acidic form (D’aniello et al., 2000), but they are soluble as sodium salts, so that, they were prepared at a concentration of 1 M in 50 ml of distilled water, using magnetic stirring, and 2 M NaOH solution was added dropwise to dissolve the amino acid in order to obtain a ﬁnal pH 7.4. After that, amino acid solutions were brought to 100 ml with distilled water to obtain an amino acid ﬁnal solution of 0.5 M for each one (D’aniello et al., 2000). A stock solution was prepared for every amino acid tested: Asp:

66.55mg/mL; Glu: 73.55mg/mL; Tau 12.52mg/mL and GABA 10.32mg/mL. An amino acid standard mixture was prepared by two dilutions: 1:100 and ﬁnally 1:10.

2.3. Animals

Male adult Sprague–Dawley rats (weighing between 150 and 200 g) were used in all the experiments. Animals were housed under monitored conditions of temperature (2228C) and photoperiod (light:dark 14:10 h) with free access to food and water. The experiments were performed according to theGuidelines of the European Union Council (86/609/EU)for the use of laboratory animals.

2.4. Determination of neuroactive amino acids in rat brain regions

We choose two STX doses (Cervantes et al., 2007), 5mg kg ¹low dose and 10mg kg¹high dose. The low dose (approximately 0.5 ml suspension of STX) was directly injected intraperitoneally (i.p.) using a syringe, amounting to the equivalent of 5mg kg¹body weight (bw). The high dose (approximately 1 ml suspension of STX) was also injected directly (i.p.) amounting to the equivalent of 10mg kg¹bw. After STX administration, 30, 60 and 120 min (reaction time) for the ﬁrst dose and 30 min (reaction time) for the second dose, animals were sacriﬁced by cervical dislocation. As soon as rats were killed, brains were removed and dissected in the following rat brain regions: hypothalamus (H), striatum (S), brain stem (BS), midbrain (MB), frontal cortex (FC), left hemisphere (LH) and right hemisphere (RH).

Four groups of 5 rats were evaluated (one group for each reaction time and tested dose) and one group of five control rats were killed in parallel to treated groups post-injection of 1 mL of saline solution (STX standard was diluted in 0.003 M HCl for each evaluated dose, final pH was 2.6; about saline solution final pH was adjusted as the STX solution administered). Fig. 1 shows representative chromatograms about amino acid standard mixture, it also includes a representative region (brain stem) of control and treated rat.

2.5. Sample preparation

Tissue samples (Cervantes et al., 2007) were weighed and then homogenized by sonication in a solution of 0.1 M HClO (1:20 for H and S; for BS, MB and FC in a proportion of 1:10 and LH and RH in a proportion of 1:8). After that, samples were centrifuged at 16,000g(48C) during 15 min. Supernatants were ﬁltered through 0.22mm nylon ﬁlters. Samples for amino acid measured were conserved at 48C until analysis.

2.6. Derivatization reaction

Derivatization was carried out using OPA-NAC as described byAswad (1984) with slight modifications: 20mL of the sample obtained after filtration, was mixed with 15–20ml of 0.1 M NaOH (to bring the pH to9.0) and 60–63.5mL of 0.01 M sodium borate buffer, pH 8.0 to a final volume of 100ml. After that 5ml of OPA-NAC reagent was added allowing it to react for 2 min at room temperature. Finally 20ml of this solution was injected into the HPLC-FLD system.

2.7. Chromatographic conditions for neuroactive amino acid analysis

The neuroactive amino acid determination was achieved by using a high performance liquid chromatography with fluorescence detection (HPLC/FLD), method proposed byAswad (1984). Briefly, 20mL of derivatizated sample was injected into the HPLC system using a Rheodyne injection valve. The HPLC/FLD system was equipped with a Jasco PU-1580 HPLC pump, and a CMA 280 fixed

wavelengths ﬂuorescence detector (CMA/Microdialysis, Sweden). The separation of neuroactive amino acid was achieved using Kromasil 100 C18 reversed-phase columns (250 mm4.6 mm and 5mm particle size). Aswad’s method considered gradient elution conditions; nevertheless, the elution was carried out in isocratic conditions. Column was eluted with a mobile phase consisting in a 12% acetonitrile in 30 mM sodium acetate buffer. The ﬂow rate was maintained at 2 ml/min.

Neuroactive amino acids were detected at 330–365 nm and 440–530 nm excitation and emission wavelength respectively. Other parameters like linearity, detection and quantiﬁcation limits (LOQ & LOD), relative recovery and precision were evaluated. Data were analyzed by the Cromanec XP software.

2.8. Statistical evaluation

Statistical analyses were carried out using SPSS 14.0. Data of neuroactive amino acids levels in discrete rat brain regions are represented as meansS.E.M. (n= 5). 1- way ANOVA was used to assess the statistical signiﬁcance of effects. Tukeypost hoc analyses were used as a posterior Test. The signiﬁcance level was set at^*P0.05;

**P0.01 and^***P0.001.

3. Results

In previous studies (Cervantes et al., 2007) have obtained

evidences that STX systemic administration (5 and 10 m g/kg

¹

bw)

crosses the blood–brain barrier. Table 1 shows STX concentration

Fig. 1.(a) Shows representative chromatogram of the measurement of the mixture of amino acid standard solution: Asp, Glu, Tau, GABA and other AA that appeared in the discrete brain samples. (b) Shows representative chromatogram of one brain region:brain stem, after systemic administration of saline solution. (c) Shows representative chromatogram of one brain region: brain stem, after systemic administration of 5mg/kg STX dose and 2 h time reaction. The chromatographic conditions are presented in Section2.

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got in the different rat brain regions analyzed. That study was realized before neuroactive amino acids research.

3.1. Determination of neuroactive amino acids (Aswad, 1984)

The HPLC/FLD method, once optimized, was applied for neuroactive amino acid determination in the different rat brain regions analyzed. Aswad’s method considered gradient elution conditions, so that after different assays about acetronitile (AcN) concentration, we found the ideal isocratic conditions about neuroactive amino acids elution. With AcN 12% concentration, we could separate neuroactive amino acids from other ones. Linearity was not less than 0.99 for all neuroactive amino acids. The LOQ varied from 150 to 220 pM and LOD varied from 46 to 65 pM for Asp, Glu and Tau respectively. About GABA, the LOQ and LOD was 38 and 11.4 nM respectively. The relative recovery was

>99%

(n = 3). The intra-day precision was

<6.6% (in term of % coefﬁcient

of variation). This assay was carried out in isocratic conditions for identiﬁcation and quantiﬁcation of neuroactive amino acids levels in the different rat brain regions analyzed.

3.2. Aspartic amino acid levels with 5 m g kg

¹

STX dose in the different rat brain regions analyzed

STX administration produced different alterations in Asp levels after 30, 60 and 120 min (time reaction) on the different rat brain regions studied (Fig. 2).

At 30 min, Asp level decreased at S and MB (77.4%, P 0.001 and 52.5%,

0.01 respectively). On the other hand, levels were

increased at BS and LH (66.7%, P 0.01 and 62.4%, P 0.001 respectively).

At 60 min, Asp levels decreased at S (55.4%, P 0.001) and were increased at FC, LH and RH (197%, P 0.01; 104.9%, P 0.001 and 116%, P 0.001) respectively).

At 120 min, just S presented a decreasing (41.9%, p 0.001).

3.3. Glutamic amino acid levels with 5 m g kg

¹

STX dose in the different rat brain regions analyzed

Fig. 3 shows Glu levels after 30, 60 and 120 min of STX administration.

At 30 min, Glu levels were decreasing at S and H (86% and 33%, P 0.001 respectively). At LH and RH, Glu levels were increasing (50.3% and 34.4%, P 0.05 respectively).

At 60 min, Glu levels were decreasing for S and H (72.4% and 32.8, P 0.001 respectively). For MB, FC, LH and RH, Glu levels were increasing (78.7%, P 0.01; 269.9%, P 0.01; 210%, P 0.001 and 197%, P 0.001, respectively).

At 120 min, Glu level was increased for FC (110.4% P 0.01). For S, H, BS, FC, LH and RH, Glu levels were decreasing (66%, P 0.001;

57%, P 0.001; 53.5%, P 0.01; 46%, P 0.001 and 36% P 0.001, respectively).

3.4. Taurine amino acid levels with 5 m g kg

¹

STX administration produced different alterations in Tau levels after 30, 60 and 120 min (time reaction) on the different rat brain regions studied (Fig. 4).

Table 1

pg of STX for mg of tissue in the different rat brain regions studied after i.p. injection of 5 and 10mg kg¹STX bw.

STX dose (mg kg ¹) Reaction time (min) Rat brain regions analyzed (pg/mg of tissue)

H S BS MB FC LH RH

5 30 0.760.02 0.500.02 0.260.01 0.270.02 0.580.03 0.310.01 0.320.02

5 60 1.050.97 0.680.07 0.630.0 1.150.02 1.130.08 0.470.01 0.400.08

5 120 2.360.19 1.180.28 1.500.15 1.380.04 1.510.13 0.760.15 0.800.20

10 30 3.500.22 3.260.02 3.530.05 2.740.02 3.450.05 1.770.05 1.560.07

Time reaction was 30, 60 and 120 min for the first dose and 30 min for the second one. After time reaction animals were sacrificed by cervical dislocation. Tissue samples were weighed and then homogenized by sonication in a solution of 0.1 M HClO4, the pH being adjusted to 3–4 with NaOH 0.1 M, and then boiled to 608C for 5 min. After that, samples were centrifuged at 16,000g(48C) during 15 min. Supernatants were filtered through 0.22mm nylon filters. 300mL of the filtrate was applied to hydrogen peroxide oxidation as describe by Lawrence and Menard (1995) for STX determination by HPLC/FLD. The values represent the mean and S.E.M. obtained from 6 determinations (Cervantes et al., 2007).

Fig. 2.Aspartic amino acid concentration in the different rat brain regions analyzed at 30, 60 and 120 min after systemic administration of 5mg kg¹STX dose. It also is included basal levels got in control group. In all cases values are the meanS.E.M.

obtained of 5 young male rats. Signiﬁcant differences^**P0.01,^***P0.001 compared to control group. Hypothalamus (H), Striatum (S), Brain stem (BS), Frontal cortex (FC), Mid brain (MB), Left hemisphere (LH) Right hemisphere (RH).

Fig. 3.Glutamic amino acid concentration in the different rat brain regions analyzed at 30, 60 and 120 min after systemic administration of 5mg kg¹STX dose. It also is included basal levels got in control group. In all cases values are the meanS.E.M.

obtained of 5 young male rats. Signiﬁcant differences^*P0.05,^**P0.01,^***P0.001 compared to control group. Hypothalamus (H), Striatum (S), Brain stem (BS), Frontal cortex (FC), Mid brain (MB), Left hemisphere (LH), Right hemisphere (RH).

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At 30 min, Tau levels were decreasing at S, H, LH and RH (89.6%, P 0.001; 82.3%, P 0.001; 12%, P 0.05 and 17%, P 0.05, respectively). For BS, Tau value was increasing (47.6%, P 0.01).

At 60 min, Tau levels were decreasing for S, H, LH and RH (81.6%, P 0.001; 56.4%, P 0.001; 51.7%, P 0.001 and 48.7%, P 0.001, respectively)

With respect to 120 min, Tau levels were decreased for S, H, LH and RH (72.7%, P 0.001; 45.5%, P 0.001; 60.2%, P 0.001 and 56.8%, P 0.001, respectively).

3.5. GABA amino acid values with 5 m g kg

¹

Fig. 5 shows GABA levels after 30, 60 and 120 min of STX administration.

For 30 min, GABA levels were decreased for S, H, MB, FC, LH and RH (95.3, P 0.001; 89.3%, P 0.01; 37.7%, P 0.001; 50.4%, P 0.001; 31.6%, P 0.001 and 30.1%, P 0.01, respectively).

At 60 min, GABA levels were decreased for S, H, BS, MB, FC, LH and RH (95.7, P 0.001; 80.5%, P 0.001; 47.9%, P 0.001; 73.4%, P 0.001; 28%, P 0.05; 78.1%, P 0.001 and 77.9%, P 0.001 respectively).

With respect to 120 min, GABA values were decreased for S, H, BS, FC, LH and RH (93.3%, P 0.001; 63.9%, P 0.01; 51.4%, P 0.001; 54.5%, P 0.001; 50.1%, P 0.001 and 48%, P 0.001, respectively).

3.6. Aspartic amino acid levels with 10 m g kg

¹

STX administration provoked some alterations in Asp levels in the different rat brain regions studied (Table 2). The administration of STX signiﬁcantly increased Asp values in BS 73.6% (P 0.01); in FC 184.5% (P 0.001); in LH and RH was 54.6% (P 0.05) and 44%

(P 0.01), whereas no signiﬁcant changes were detected in S, H and MB.

3.7. Glutamic amino acid levels with 10 m g kg

¹

The i.p. administration of STX produced a differential effect on Glu levels in the rat brain regions analyzed (Table 2). So, Glu levels increased signiﬁcantly in BS (47%, P 0.05); in MB (89%, P 0.001); in FC (271%, P 0.001); in LH (109%, P 0.001) and RH (116%, P 0.001). However, Glu levels decreased signiﬁcantly in S (56%, P 0.001) and H (39.2%, P 0.001).

3.8. Taurine amino acid variations with 10 m g kg

¹

The administration of STX increased signiﬁcantly Tau values (Table 2) in BS (53%, P 0.001); in FC (141%, P 0.001). However, Tau content decreased signiﬁcantly in S (94%, P 0.001); in H (70%, P 0.001); in LH (17.5%, P 0.05) and in RH (17.1%, P 0.05) of treated rats.

3.9. GABA amino acid variations with 10 m g kg

¹

The administration of STX reduced signiﬁcantly GABA values (Table 2) in S (93.9%, P 0.001); in H (95%, P 0.001); in FC (49.7%, P 0.001); in LH (34%, P 0.001) and RH (29.1%, P 0.001).

4. Discussion

Different chromatographic assays for the measurements of amino acids have been proposed (Aswad, 1984; Murai et al., 1991;

Fekkes, 1996; Reason, 2003; Grant et al., 2006; Mazzeo et al., 2006).

Precolumn derivatization with OPA-NAC (Davis et al., 1978;

Lindroth and Mopper, 1979; Medina et al., 1990a,b) followed by reversed-phase HPLC separation is a sensitive procedure for amino acids analysis. In this research we employed Aswad method (1984) for neuroactive amino acid analysis. This method was based on the diastereomeric separation of Asp, Glu, Tau and GABA from other amino acids by gradient elution. Nevertheless, this method was modiﬁed and adapted to our experimental conditions. We principally modiﬁed gradient elution to isocratic elution (Tcherkas and Denisenko, 2001; Wu et al., 2005). On the other hand, we optimized mobile phase concentrations. The best separation and elution was obtained decreasing the original concentration of Aswad’s method from 70% AcN up to 12% AcN without changing sodium acetate concentration. Four neuroactive amino acids were evaluated in 25 min. Other amino acids were detected but they did not interfere. We considered that chromatographic parameters were in an acceptable range for this kind of analysis (Graser et al., 1984; Donzanti and Yamamoto, 1988; Venta, 2001; Reason, 2003;

Bartolomeo and Maisano, 2006; Hsieh et al., 2006; Kang et al., 2006).

Fig. 4.Taurine amino acid concentration in the different rat brain regions analyzed at 30, 60 and 120 min after systemic administration of 5mg kg ¹STX dose. It also is included basal levels got in control group. In all cases values are the meanS.E.M.

obtained of 5 young male rats. Signiﬁcant differences^*P0.05,^***P0.001 compared to control group. Hypothalamus (H), Striatum (S), Brain stem (BS), Frontal cortex (FC), Mid brain (MB), Left hemisphere (LH), Right hemisphere (RH).

Fig. 5.GABA amino acid concentration in the different rat brain regions analyzed at 30, 60 and 120 min after systemic administration of 5mg kg¹STX dose. It also is included basal levels got in control group. In all cases values are the meanS.E.M.

obtained of 5 young male rats. Signiﬁcant differences^**P0.01,^***P0.001 compared to control group. Hypothalamus (H), Striatum (S), Brain stem (BS), Frontal cortex (FC), Mid brain (MB), Left hemisphere (LH), Right hemisphere (RH).

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On the other hand, the evaluation about saxitoxin effects (5 and 10 m g kg

¹

, body weight i.p. administration) in the intracellular levels of Asp, Glu, Tau and GABA was shown that STX administration leads to signiﬁcant changes in the content of all four neuroactive amino acids in the rat brain regions analyzed (Kolluri and Lakshimi, 1989).

The effects of systemic administration of 5 m g kg

¹

STX dose produced signiﬁcant decreases in aspartic levels at 30, 60 and 120 min (time reaction), whereas were increased at BS, LH (at 30 min) and FC, LH, RH (at 60 min). On the other hand, 10 m g kg

¹

STX dose produced signiﬁcant increases in the same regions.

As for Glu levels, both doses produced signiﬁcant decreases in S and H (in the three time reaction for low dose). Glu levels were increased at 30 and 60 min for LH and RH but they were decreased at 120 min; with respect to FC, Glu levels were increased at 60 and 120 min; and just in MB and BS, Glu levels were increased at 60 min and decrease at 120 min respectively. With high dose increases for BS, MB, FC, LH and RH were detected. The increases presented in the content of Glu probably reﬂect hyperactivation of the glutamatergic transmission and may serve as a marker of neurotoxicity.

About the content of GABA levels, signiﬁcant decreases were detected in all the rat brain regions analyzed with both doses, with exception of BS (at 30 min) and MB (at 120 min) that did not present signiﬁcant changes.

Intraperitoneal administration of both saxitoxin doses increased the aspartic and glutamic levels in FC. But GABA levels were decreased. In this case, saxitoxin could be producing an excitatory-inhibitory effect on neuroactive amino acids of the frontal cortex. Kulagina et al. (2004) describe the rapid effects of brevetoxin-2 (PbTx-2) and STX on embryonic murine frontal cortex neuronal networks grown on substrate integrated micro- electrode arrays.

On the other hand, Asp and Glu increases could be a consequence of GABAergic neurotransmission. Flores et al.

(2004), mention that an inhibition of GABAergic neurotransmission produces an effect about excitatory activity. On the other hand, the increases could be correlated with an imbalance between excitation and inhibition, as well as alterations of ion channels.

GABA is not an amino acid related to proteins. Its presence is a good indicative that is used like a neurotransmitter (Purves et al., 2001). Signiﬁcant decreases in GABA levels may be produced by inhibition or blocked of the enzyme glutamate decarboxylase (GAD,

responsible of GABA synthesis. GAD is only present at nervous system of mammals (Purves et al., 2001; Flores et al., 2004).

With respect Tau levels, both doses produced signiﬁcant decreases in H, S, LH and RH, and signiﬁcant increases in BS (just at 30 min for low dose), but high dose also increase FC amino acid levels.

Neuroactive amino acids levels were always decreased with systemic administration of both STX doses in S and H (with exception of Asp levels that just were decreased for S). On other hand, both saxitoxin doses produced alterations of neuroactive amino acids (excitatory and inhibitory) in both hemispheres. These data suggest that saxitoxin may have selective actions on the excitatory-inhibitory amino acid systems in these regions.

The changes observed in the amino acids levels are quite variable and sometimes difﬁcult to explain. This is because the amino acids content in brain regions does not reﬂect only the neurotransmitter levels but also changes in the metabolic functions of amino acids or their participation in the synthesis of proteins (except GABA).

Moreover, the blockade of the STX on voltage dependent sodium channels is not its only one pharmacological action. So, it is well know that STX can bind to calcium and potassium channels or can inhibit the nitric oxide synthase (NOS), enzyme responsible for the synthesis of nitric oxide, which is implicated in the modulation of the neurotransmitters release. All these additional mechanism could explain, at least in part, the changes in the amino acid levels in some regions.

Other researches about effects of some biotoxins on neurotransmitter amino acid levels in some discrete rat brain regions have been reported. Dura´n et al. (1995) studied domoic acid i.p.

administration (0.2 mg/kg). They reported signiﬁcant changes in Asp, Glu, Tau, GABA, Gly and Ala levels in H, S, FC, MB and amygdale. Allen et al. (1986) after homocysteine administration also observed differential changes in GABA levels (decreases in hippocampus and substantia nigra), whereas Tau levels just decreased in hippocampus and Glu levels were decreased at hypothalamus, hippocampus, striatum and substantia nigra.

Klivenlli et al. (2005) studied mitochondrial effects (MPTP) on neurotransmitter amino acid levels in some discrete rat brain regions. They detected signiﬁcant decreases in S and FC and increases in cerebellum neurotransmitter amino acids levels.

The nature and magnitude of these toxic effects produced by different toxins show interregional variations on the content of the

Table 2

It shows amino acids concentration in control and treated rats^*.

Rat brain regions analyzed Group Asp (ng/mg) Glu (ng/mg) Tau (ng/mg) GABA (ng/mg)

S CG 347.935.6 1,493.3148.9 482.938.7 644.334.9

TG 281.519.6 660.153.7^*** 29.94.1^*** 39.32.5^***

H CG 127.55.0 577.341.5 232.610.9 432.936.8

TG 171.917.9 350.926.0^*** 69.03.2^*** 21.61.8^***

BS CG 322.112.7 1,041.965.3 183.19.1 245.124.0

TG 559.350.2^** 1,531.7138.5^* 279.725.0^*** 212.921.1

MB CG 522.247.4 1,163.345.2 289.725.3 523.128.5

TG 693.424.7 2,202.0208.2^*** 363.717.8 444.925.9

FC CG 89.35.7 152.98.9 140.510.8 118.69.0

TG 263.016.1^*** 568.325.4^*** 338.831.8^*** 59.65.9^***

LH CG 309.713.6 1,094.919.1 823.339.7 368.7419.6

TG 478.744.1^* 2,293.2166.9^*** 679.225.5^* 242.95.6^***

RH CG 307.56.2 1,096.222.0 895.232.3 374.314.8

TG 444.040.6^** 2,369.2208.5^*** 751.418.8^* 265.28.6^**

Treated rats were exposed to 10mg kg¹STX dose during 30 min. The determination of neuroactive amino acids, sample preparation and the chromatographic conditions are described in Section2. CG = Control Group. Amino acid basal values, TG = Treated Group. Amino acid values after STX administration,^aConsisted of an i.p. injection of saxitoxin solution using an appropriate volume to administer 10mg kg¹body weight. The rats were injected and killed after 30 min. Data were obtained using the speciﬁc HPLC-FLD method for determination of neuroactive amino acids (see Section2). The results represent the meanS.E.M. obtained from 5 young male rats.

*Signiﬁcant differencesP0.05 respect to control group.

**Signiﬁcant differencesP0.01 respect to control group.

***Signiﬁcant differencesP0.001 respect to control group.

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neurotransmitters amino acids in the different rat brain regions studied. These results are in accordance with our investigation.

In conclusion, the present study indicates that results can be achieved by the easy to use OPA-NAC derivatization for neuroactive amino acid analyses. It is a basic tool in the quantitative study of neurotransmitters amino acids. Intraperitoneal injections of two saxitoxin doses caused complex and differential changes in the neuroactive amino acids levels of the different rat brain analyzed. On the other hand, there is not great difference between the two saxitoxin doses evaluated with respect to neuroactive amino acids effects. Changes in the levels of neuroactive amino acids could serve as indicators of disease and its prognosis.

Acknowledgements

The authors of this research acknowledge the ﬁnancial support of Xunta de Galicia and the University of Vigo for the development of this work.

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