Application of Overexcitation Model Induced by Penicillin Sodium in the Study of Inhibitory Effect of Sedative-Hypnotic Drugs

ABSTRACT

Excessive release of glutamate can cause many nervous system disorders. It has been reported that when a high dose of penicillin sodium is administered into the rat's brain, epilepsy, accompanied with glutamate elevation, will result. In this experiment, a low dose of penicillin sodium (1000 kIU/l) was microinjected into the rat's lateral ventricle to set up an overexcitation model, in which the concentration of ipsilateral hippocampal glutamate was monitored in vivo. by microdialysis-HPLC method as an indicator of the rat's excitatory state. Influences of sedative-hypnotic drugs on this model were verified by coadministration of diazepam or phenobarbital with Na-PCN intracerebroventricularly. In models, hippocampal glutamate concentration was elevated to 307% compared with its baseline level (p < 0.05), and this increase of glutamate was inhibited completely when different doses of diazepam or phenobarbital were administered (p < 0.05). The sedative effect of jujuboside A and trifluoperazine were then studied with this model. Jujuboside A (JuA) 0.1 g/l also reduced the glutamate level significantly (p < 0.05). Calmodulin antagonist trifluoperazine showed similar inhibitory effect as JuA, which may indicate that the effect of JuA is correlated with its anticalmodulin action. This model can be used to investigate the inhibitory effect of central nervous system drugs.

Keywords:

• Glutamate
• hippocampus
• overexcitation
• penicillin sodium
• sedative-hypnotic drug

Introduction

Overexcitation of the nervous system can induce many diseases, such as mania, hypochondriasis, and epilepsy (Ribak, 1987). Even though all the anticonvulsants inhibit excessive neuronal activity, this acute effect appears to be produced by several mechanisms that fall into three major categories: (1) blockade of voltage-gated sodium channels, (2) indirect or direct enhancement of inhibitory gamma-aminobutyric acid (GABA) neurotransmission, or (3) inhibition of excitatory glutamatergic neurotransmission (Soderpalm, 2002).

Excessive release of glutamate (Glu) is closely related to epilepsy (Bradford, 1995). Penicillin is a GABA_A receptor blocker and is often used to induce epilepsy (Horn & Esseling, 1993; Uysal et al., 1996; Fiacro et al., 1999). It can also promote the release of Glu indirectly (Chen et al., 2001). Epileptiform activity and behavioral hyperactivity would develop immediately after penicillin sodium (Na-PCN) was injected into the motor cortex or the olfactory cortex (Horn et al., 1991; El-Yamany & Horn, 2002). In this paper, a low dose of Na-PCN (1000 kIU/l, 3 µl) was introduced into the rat's left lateral ventricle (LV) to set up an overexcitation model instead of an epilepsy model. The Glu level change in the ipsilateral hippocampus of rats was monitored in vivo. to reflect the excitatory degrees of rats. Influence of sedative drugs on this model was verified by coadministration of diazepam or phenobarbital with Na-PCN intracerebroventricularly.

Diazepam, as a commonly used hypnotic, can inhibit the excitation effect induced by Na-PCN significantly. It was reported that injection of diazepam at a dose of 2 mg/kg 20 min before Na-PCN application results in the reduction of epileptiform discharges latency in the epileptogenic focus and in a decrease in their frequency before seizures as compared to the control animals (Samsonova et al., 1979). Phenobarbital, as a barbiturate, has also long been used in the treatment of epilepsy. But studies also suggest that antiepileptic drugs, in particular phenobarbital, can cause behavior disturbances, mostly hyperactivity (Domizio et al., 1993; Alvarez, 1998). Whether this side effect is correlated with post-Glu level remains to be studied.

The inhibitory effect of jujuboside A was then studied on this overexcitation model. JuA is a main component of jujubogenin extracted from the seed of Ziziphus jujuba. Mill var spinosa. (Bunge) Hu ex H F Chou (Rhamnaceae), which is widely used in Chinese traditional medicine for the treatment of insomnia and anxiety (Shou et al., 2001). Because JuA is an inhibitor of calmodulin (CaM), the effect of trifluoperazine (TFP), a type of CaM antagonist, was also studied in the experiment.

Materials and Methods

Animals and chemicals

All facets of animal care and use in our experiment met the requirements of the Chinese Code of Practice for the Care and Use of Animals for Scientific Purposes. Experiments were performed on adult male Sprague-Dawley rats, obtained from Zhejiang Center of Laboratory Animals (grade II, certificate no. 2001001), weighing 240–300 g.

JuA (purity 99%) was provided by the National Institute for the Control of Pharmaceutical and Biological Products; o.-phthaldialdehyde, β.-mercaptoethanol, and trifluoperazine were obtained from Sigma; urethane, penicillin sodium, diazepam injection solution, and phenobarbital injection solution were obtained from China Medical Bioproduct Co.

Surgery

The rats were kept on a 12-h light/12-h dark schedule with light on at 7:00 AM. Surgery was performed under anesthesia by injection of urethane (1.25 g/kg, s.c.). Intracerebral guide cannulas (BAS MD-2251, USA) were implanted in the rats to secure microdialysis probes. Bregma coordinates for hippocampus were P = − 5.8, L = + 5.0, H = − 3.0; coordinates for LV were P = − 0.8, L = + 1.5, H = − 3.5. The placement of the probes in the lateral ventricle and hippocampus is shown in Figures 1A and 1B, respectively. Five days was allowed for recovery.

Figure 1 (A) Schematic coronal section of the rat brain (bregma = P − 0.8) illustrating the placement of the infusion probe in the lateral ventricle. (B) Schematic coronal section of the rat brain (bregma = P − 5.8) illustrating the placement of the microdialysis probe in hippocampus.

Microdialysis procedure

Microdialysis probes (BAS, MD-2204, 4-mm membrane) were inserted 120 min before starting the experiments in order to allow for equilibration. Each experiment began by perfusing the probe with artificial cerebrospinal fluid (ACSF = 126 mM NaCl, 27.5 mM NaHCO₃, 2.4 mM KCl, 5 mM KH₂PO₄, 5 mM Na₂HPO₄, 0.5 mM Na₂SO₄, 0.82 mM MgCl₂ · 6H₂O, 1.1 mM CaCl₂ · 2H₂O, 5 mM glucose, pH 7.4) at the flow rate of 1 µl/min. Samples were manually collected (20 µl of dialysate per sample).

Glu detection

The commonly used o.-phthaldialdehyde(OPA)–β.-mercaptoethanol precolumn derivatization, reversed-phase gradient elution, and fluorescence detection (RF) method was applied (Ye, 1988; Begley et al., 1994). The HPLC (Shimadzu-10AVP, Japan) employed two mobile phases: (A) buffer (0.1 M KH₂PO_4, adjusted to pH 6.60 by NaOH): methanol = 65:35, v/v; (B) buffer (0.1 M KH₂PO_4, adjusted to pH 6.60 by NaOH): methanol = 10:90, v/v. Solution B was filtered and degassed through an 0.2-µm nitrocellulose membrane under vacuum. Separation was achieved on a C18 column (Hypersil, BDS, 5 µm, 4.0▪ 200 mm). Twenty microliter dialysate samples and 10 µl OPA derivating fluid were allowed to react for 1 min at room temperature before being injected onto the column through a 20 µl sample loop and separated with a gradient from A:B (100:0) to A:B (60:40) within 12 min; then it was eluted with 100% B for 5 min to elute other components. The flow rate was 1 ml/min; EX: 357 nm; EM: 455 nm. Glu concentration in the samples was evaluated by an external standard calibration curve method.

Overexcitation model and its applications

Rats were randomly assigned to nine groups: control group (solvent:ACSF), Na-PCN model group (1000 kIU/l Na-PCN), low-dose diazepam group (1.25 g/l diazepam + 1000 kIU/l Na-PCN), high-dose diazepam group (2.5 g/l diazepam + 1000 kIU/l Na-PCN), low-dose phenobarbital group (5 g/l phenobarbital + 1000 kIU/l Na-PCN), high-dose phenobarbital group (10 g/l phenobarbital + 1000 kIU/l Na-PCN), low-dose JuA group (0.05 g/l JuA + 1000 kIU/l Na-PCN), high-dose JuA group (0.1 g/l JuA + 1000 kIU/l Na-PCN), and TFP group (50 uM TFP + 1000 kIU/l Na-PCN). In each group, five rats were studied.

Infusion probe (BAS MD-2252) was inserted into the LV along with the settlement of microdialysis probe. Perfusate for the first 120 min was not collected to allow equilibration between the brain tissue and ACSF before sampling. Subsequently, samples were collected at 20-min intervals, and the first two samples collected after equilibration served as Glu baseline levels. At the beginning of the second stage, through the infusion probe placed in the LV, each rat was injected with 3 µl relevant drugs according to its group. Dialysates in the first 20 min after microinjection of relevant drugs were analyzed to evaluate the effects of the various drugs.

Histology and statistical analysis

After completion of experiments, the rats were euthanized with urethane overdose and decapitated. The brains were cut into slices with the width of 50 µm each piece by freezing microtome (Microm, HM505E) and probe placements were verified for all data presented in this study. Data are presented as mean±SEM. Statistical significance was evaluated with Student's t.-test. Significance was accepted at the p < 0.05 level.

Results

Low-dose Na-PCN induces hippocampal Glu elevation

The recovery of Glu obtained is 105±9.8%. In the range of 0.625 to 40 µM, Glu concentration was linearly related with the peak area (r = 0.9988). The detection limit for Glu in the dialysate was approximately 1 pmol/sample. The mean concentration of baseline Glu was 6.69±2.7 µM. In the model group, hippocampal Glu level was greatly elevated to 307% compared with its baseline (p < 0.05 vs. control group: Table 1). Accompanied with the increase of hippocampal Glu, rats were found in an overexcitation state, beard erecting and muscle tensed.

Table 1.. The influence of diazepam and phenobarbital on Na-PCN–induced hippocampus Glu elevation models (n = 5).

Group	Glu level after drug administration/baseline Glu level
ACSF^a.	1.14±0.19
1000 kIU/l Na-PCN^b.	3.07±1.60^c.
1.25 g/l diazepam + 1000 kIU/l Na-PCN	0.73±0.48^d.
2.5 g/l diazepam + 1000 kIU/l Na-PCN	0.68±0.39^e.
5 g/l phenobarbital + 1000 kIU/l Na-PCN	0.62±0.52^d.
10 g/l phenobarbital + 1000 kIU/l Na-PCN	0.46±0.31^e.

^a.ACSF group as control of penicillin group.

^b.Na-PCN group as control of diazepam group, phenobarbital group and JuA group.

^c.p < 0.05 vs. a..

^d.p < 0.05 vs. b..

^e.p < 0.01 vs. b..

Diazepam and phenobarbital inhibited Glu increase induced by Na-PCN

When diazepam or phenobarbital was co-injected into the LV with Na-PCN, the increase of hippocampal Glu was reduced significantly. The Glu concentrations were 0.73±0.48 and 0.68±0.39 times baseline with 1.25 and 2.5 g/l diazepam, respectively (Table 1). The Glu concentrations were 0.62±0.52 and 0.46±0.31 times baseline with 5 and 10 g/l phenobarbital, respectively (Table 1). Diazepam and phenobarbital inhibited the excitatory effect of Na-PCN completely by reducing Glu level below its baseline. In these four groups, when drug was injected, rats got very excited for the first several minutes, indicating an overexcitation state; right after that, inhibition appeared and the rats became drowsy.

Influence of JuA and TFP on Glu elevation

When JuA was co-injected into lateral ventricle with Na-PCN (1000 kIU/l), the increase of Glu caused by Na-PCN was reduced by JuA in a dose - dependent manner. The Glu concentrations were 2.38±0.76 and 1.58±0.69 times baseline with 0.05 and 0.1 g/l JuA, respectively. There was a significant difference (p < 0.05) between model group and high-dose JuA group. TFP 50 µM completely inhibited the effect of Na-PCN: the Glu concentration was 1.01±0.32 times baseline (Figure 2).

Figure 2 Influence of JuA and TFP on the increase in extracellular level of Glu induced by 1000 kIU/l Na-PCN in rat hippocampus in vivo.. JuA and TFP were co-injected with Na-PCN. Values are means±SD, n = 5. *p < 0.05 vs. control; #p < 0.05 vs. Na-PCN model.

Discussion

Optimization of HPLC method and intracerebroventricular drug administration

Low pH of buffer can increase the hydrophobicity of amino acid derivants and thus achieve better separation effect, but it also increases the instability of derivants and reduces the fluorescent intensity (Begley et al., 1994). We tried the 0.1 M KH₂PO₄ buffer at many different pH and finally set the buffer pH at 6.6, which provided the best separation effect. As the rat's hippocampus tissue is located at the inner side of the lateral ventricle, intracerebroventricular drug administration not only imitates drug diffusion process in the cerebral spinal fluid but also helps to investigate the action mechanisms between drug and hippocampus.

Overexcitation model

Glu is a major excitatory neurotransmitter in the hippocampus. Elevated levels of extracellular Glu are responsible for neuronal damage and degeneration in many brain disorders (Coyle & Puttfarcken, 1993). Na-PCN is a GABA_A receptor blocker. It is often used to induce epilepsy, and it can promote the release of Glu in special brain regions, as confirmed by our data (Fig. 2). This elevation in hippocampal Glu can reflect the overexcitaion state induced by Na-PCN to some degree. In this study, when a low dose of Na-PCN (1000 kIU/l) was introduced into the rat's LV, Glu level was greatly elevated compared to its baseline in hippocampus; though the rats were in excited conditions, they did not demonstrate any epileptic symptoms. When a high dose of Na-PCN (32000 kIU/l) was tried in another rat, clearly epileptic symptoms appeared, such as continuously grinding teeth, convulsions, and body stiffness. It is known that benzodiazepine receptor and GABA_A receptor compose a benzodiazepine-GABA receptor-ionophore complex. A variety of centrally acting anxiolytic, depressant, and anticonvulsant drugs bind to one of the sites in the complex and modulate the binding of ligands at the other sites (Ticku, 1983). Diazepam, as a benzodiazepine, may reduce Glu indirectly by potentiating GABAergic neurotransmission at GABA_A receptors via a modulatory binding site. Phenobarbital, however, had only minimal GABA-mimetic inhibitory action at high doses. Modulation of synaptic events mediated by GABA and Glu might contribute to its anticonvulsant activity (MacDonald & Barker, 1979). More experiments should be done in order to study whether the side effect of phenobarbital is correlated with Glu level. This overexcitation model can be used to assist pharmacological research on the inhibtion effect of other sedative-hypnotic drugs.

Application in study of inhibitory effect of JuA and TFP

When co-injected with Na-PCN and JuA, the increase in extracellular levels of Glu was dose-dependently inhibited (Fig. 2). TFP is often used as a CaM inhibitor. Evidence demonstrated that CaM regulates synaptic protein phosphorylation and plays a role in regulating neurotransmitter release (DeLorenzo, 1982; Sandoval et al., 1985). It is therefore likely that TFP reduced the extracellular level of Glu by suppressing Glu release from presynaptic terminals through inhibiting the activity of CaM. We found that TFP and JuA had similar effects in this case, which may indicate the effect of JuA is correlated with its anticalmodulin action. To verify it, further study is needed. Furthermore, TFP is a widely used phenothiazine antipsychotic. Phenothiazines were reported to alter GABA_A receptor kinetics in hippocampal cells (Mozrzymas et al., 1999). Thus, it seems that TFP may impact Na-PCN binding to the benzodiazepine site at the GABA_A ionophore and then directly inhibit the effect of Na-PCN.

Acknowledgments

This study was supported by the National Science Foundation of China, grant 30170275, the Science and Technology Department of Zhejiang Province (no. 011106239), and the Key Laboratory for Biomedical Engineering of Ministry of Education of China.