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Functional neurotoxic effects in rats elicited by 3-nitropropionic acid in acute and subacute administration
Environmental Toxicology and Pharmacology 19 (2005) 811–817
Functional neurotoxic effects in rats elicited by 3-nitropropionic acid in acute and subacute administration Andrea Szab´o ∗ , Andr´as Papp, L´aszl´o Nagymajt´enyi Department of Public Health, University of Szeged, H-6723 Szeged, D´om t´er 10, Hungary
Abstract Changes possibly induced by 3-NP in electrophysiological functional characteristics of the central nervous system are, in contrast to biochemical and morphological alterations, less well known. In this study, the usability of a standard neurophysiological investigation system to detect functional changes caused by 3-NP administration in rats was studied. In subacute treatment, 10 or 15 mg/kg 3-NP was given i.p. on five consecutive days to groups of 10 rats and the effects were checked 4 weeks later. Acutely treated rats received 20 mg/kg i.p. after several control records. For recording, the animals’ left hemisphere was exposed in urethane anesthesia. Silver electrodes were placed on the cortical sensory foci and tungsten needles in the subcortical (caudatum, globus pallidus) recording sites. Spontaneous electrical activity, as well as somatosensory, visual and auditory evoked potentials, were recorded. Following subacute treatment, the slowest (theta) and fastest (beta2 and gamma) frequencies of the spontaneous activity were changed, differently in the cortical versus subcortical sites. In the sensory evoked potentials after subacute treatment, an increase of the latency was seen in all sensory areas. In the acutely treated animals, the amplitude of the somatosensory evoked potential decreased after giving 3-NP. With double stimuli, the relation of the two responses was treatment- and interval-dependent. Understanding the mechanism of these effects may widen the knowledge base for using 3-NP in disease models. © 2005 Elsevier B.V. All rights reserved. Keywords: 3-Nitropropionic acid; Cortical activity; Subcortical activity; Rat
1. Introduction The ‘natural’ origin of most of our food by far does not mean that they cannot contain toxic substances. The compound 3-nitropropionic acid (3-NP) is naturally found in Astragalus species (Leguminosae; Johnson et al., 2000), and occasionally causes severe or fatal poisoning in grazing farm animals (James et al., 1980). Human intoxication may result from infestation of foodstuffs (sugar cane, cereals, etc.) with moulds of the Anthirium and Aspergillus genus (Scallet et al., 2001; Patocka et al., 2000; Peraica and Domijan, 2001), producing 3-NP. Human exposure to 3-NP, even in low doses, causes acute encephalopathy followed by dystonia (Liu et al., 1992). Torsion spasms and jerky movements were observed in the victims, and CT images indicated lesions of the basal ganglia (He et al., 1995). ∗
Corresponding author. Tel.: +36 62 545 119; fax: +36 62 545 120. E-mail address: [email protected] (A. Szab´o).
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The morphological and functional effects of 3-NP intoxication have been replicated in animal experiments. With a dose similar to ours (5 × 20 mg/kg i.p.), Teunissen et al. (2001) obtained decrease of motor performance in rats. Tissue degeneration, involving primarily the striatum but also the hippocampus and thalamus, was also described, both in vitro (Behrens et al., 1995) and in vivo (following one dose of 10–30 mg/kg 3-NP; McCracken et al., 2001), and was interpreted as a morphological correlate of the motor disorders. Based on that, 3-NP has been introduced in modeling Huntington’s disease in animals (Brouillet et al., 1999). At the cellular level, 3-NP inhibits succinate dehydrogenase, a key enzyme of oxidative energy production (Coles et al., 1979). The effect develops fast and is not limited to the sites of morphological damage (Brouillet et al., 1998). In mouse hippocampus, e.g., population spike amplitude was diminished by repeated doses of 20 mg/kg 3-NP, apparently due to inhibition of oxidative phosphorylation (Hellweg et al., 2003). 3-NP was also found to act on synaptic transmission by influencing NMDA receptors. The resulting excess in
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glutamatergic excitation led to chemically induced long-term potentiation in in vitro slices (Calabresi et al., 2001) and to excitotoxicity both in vitro and in vivo (Pubill et al., 2001). Although, a general mitochondrial impairment in the brain and the disturbance of glutamatergic transmission is likely to be reflected also in e.g. the spontaneous and stimulus-evoked cortical activity, and the description of such effects would be required to a comprehensive description of the chronic disease models based on 3-NP, no data of this kind were found in the literature. The aim of this work was therefore to see to what extent the neurophysiological investigation system established in our laboratory is suitable to detect functional changes caused by 3-NP administration in rats.
2. Methods The effects of 3-NP were investigated in three different time schemes—it is known from Brouillet et al. (1999) that dosing schemes and time until recording can have a major influence on the effects of 3-NP. In the subacute experiments, adult male Wistar rats (of 450–500 g body weight, 10 animals per treatment group) received 10 (low dose) and 15 (high dose) mg/kg b.w. 3-NP i.p. for five consecutive days and were kept for further 4 weeks before recording. Control animals were untreated. For acute exposure, 10 animals (of the same strain and weight) received 20 mg/kg 3-NP i.p. and were kept for 24 h. To see immediate effects, further 10 rats were first prepared for recording and four of the recording sequences described below were taken with 20 min interval. Then, 20 mg/kg 3-NP i.p. was given and further 6–8 records taken. For comparison, the intraperitoneal LD50 of 3-NP in rats is 67 mg/kg (Pass et al., 1985). For recording, the animals were anaesthetized with urethane (1000 mg/kg i.p.) and were placed in a stereotaxic instrument. The left hemisphere was exposed and silver electrodes were placed on the dura over the primary somatosensory (SS), visual (VIS) and auditory (AUD) areas. One steel needle electrode each was inserted in the caudato-putamen (stereotaxic coordinates: AP 0, L 3, V 5; Paxinos and Watson, 1982) and the globus pallidus (AP 2, L 3, V 6). The recording sequence started with spontaneous electrical activity (electrocorticogram, ECoG), recorded from these sites simultaneously for 6 min from the record, the relative spectral power of the frequency bands (delta to gamma; Kandel and Schwartz, 1985) was determined. Then, stimulusevoked activity was recorded from the mentioned cortical areas. Somatosensory stimulation was done by a pair of needles inserted into the whiskery skin, delivering rectangular electric stimuli of 3–4 V and 0.2 ms. Visual stimulation was performed by flashes (60 lx) from a flash generator via an optical fiber directed into the contralateral eye of the rat. For acoustic stimulation, clicks (40 dB) were applied into the ear of the rat. Of each modality, one series of 50 stimuli at 1 s repetition time (1 Hz frequency) was applied to each rat, and the evoked activity was recorded. After averaging, amplitude,
latency and duration of the evoked responses was measured manually. In previous studies with other neurotoxicants (Papp et al., 2000a,b) it was found that changes in the measurable parameters of cortical responses evoked with multiple stimulation are sensitive to the toxic influence. In the present study, doublepulse somatosensory stimulation was used (repetition time, 1 s; inter-stimulus time, 60–300 ms). From the double-pulse records, the second:first ratio for amplitude and latency was calculated and interpreted as measure of dynamic interaction between the two excitation processes. It was tested whether this interaction is sensitive to the toxic influence. The results of subacute and acute (24 h) exposure were tested for significance with one-way ANOVA. For the results of immediate treatment (pre- and post-application data), repeated measure ANOVA was used. Post hoc analysis was done by LSD with p < 0.05 as limit. During the whole study, the principles of the Ethical Committee for the Protection of Animals in Research of the University were strictly followed.
3. Results 3.1. Effects on the spontaneous cortical activity In subacute exposure, the most conspicuous effect was the significant (SS: F2,27 = 16.59, p < 0.001; VIS: F2,27 = 5.48, p < 0.014; AUD: F2,27 = 9.62, p < 0.001) decrease in the theta activity seen in all cortical foci in the high-dose animals (Fig. 1A–C). In the other bands, the changes were not uniform: increase of the delta activity was significant only in the SS and VIS area, decrease of alpha in the VIS area, and increase of beta2 in the SS and AUD areas. In the basal ganglia (Fig. 1D, E) activity decreased in the slow, delta and theta bands (significant in the high-dose group and only in the caudatum, delta: F2,27 = 5.51, p < 0.015; theta: F2,27 = 2.94, p < 0.05). Increase of the gamma band activity was seen in the caudatum and the globus pallidus, but was significant only in the latter (F2,27 = 3.33, p < 0.05). In the low-dose rats, the changes were mostly below significance. In the rats recorded 24 h after 3-NP application (Fig. 2), the trend of the ECoG bands was partly similar to that obtained by subacute exposure. In the SS area (Fig. 2A), delta activity increased significantly (F1,18 = 4.17, p < 0.054). The increase of theta and decrease of faster bands was not significant. In the VIS focus (Fig. 2B) the changes were similar but only the decrease of alpha activity was significant (F1,18 = 4.49, p < 0.046). In the AUD cortex (Fig. 2C) delta activity increased significantly (F1,18 = 13.43, p < 0.001), and alpha, beta1 and beta2, decreased (alpha: F1,18 = 20.66, p < 0.001; beta1: F1,18 = 10.10, p < 0.004; beta2: F1,18 = 4.02, p < 0.051). The ECoG changes observed within ca. 2 h after i.p. application of 20 mg/kg 3-NP are shown in Fig. 3. None of the changes was significant but it was clearly visible that
A. Szab´o et al. / Environmental Toxicology and Pharmacology 19 (2005) 811–817
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Fig. 2. Effects of 3-NP on the spontaneous cortical activity (A, somatosensory; B, visual; C, auditory area) in acute exposure (20 mg/kg 3-NP i.p., recording in 24 h), displayed as in Fig. 1. * p < 0.05 vs. control in the same band.
the change at 100 min was to the same direction as 4 weeks or 24 h after 3-NP administration. The time course of the alterations was, however, such that the immediate change (10 min after administration) was opposite to that seen at 100 min. 3.2. Effects on cortical evoked potentials
Fig. 1. Effects of 3-NP on the spontaneous cortical (A, somatosensory; B, visual; C, auditory area) and subcortical (D, caudato-putamen; E, globus pallidus) activity in subacute exposure (control, untreated; low dose: 10, high dose: 15 mg/kg 3-NP i.p. on five consecutive days; recording in 4 weeks). Ordinate: relative power of the standard frequency bands in control and treated animals (means, n = 10). Inset: frequency bands. * p < 0.05 vs. control in the same band.
The general slow-down of the cortical activity following subacute treatment with 3-NP was paralleled by the increased latency of the sensory evoked potentials (Fig. 4). The change was significant in all recorded cortical areas and in both treated groups versus control (SS: F2,27 = 7.19, p < 0.004; VIS: F2,27 = 16.82, p < 0.001; AUD: F2,27 = 10.72, p < 0.001). The change seemed to be dose-dependent but high versus low dose differences were not significant. The alteration of the duration of the responses was slight. In acute (24 h) 3NP exposure (Fig. 5), there was no consequent effect on the
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Fig. 3. Immediate effect of 3-NP (20 mg/kg i.p.) on the spontaneous cortical activity (A, somatosensory; B, visual; C, auditory area). Abscissa: time elapsed after drug administration, con is the average of pre-administration records. Other features as in Fig. 1.
latency: small decrease in the SS and AUD response but significant increase (F1,18 = 4.44, p < 0.049) in the VIS response. The duration of the potentials was, on the contrary, significantly increased in the SS (F1,18 = 4.54, p < 0.046), and VIS (F1,18 = 4.66, p < 0.031) but not in the AUD area. 3.3. 3-NP effects on the dynamic interaction in the somatosensory system revealed by double-pulse stimulation Double-pulse somatosensory stimulation with different inter-stimulus intervals was used to investigate dynamic interaction in the sensory system. It was supposed that the relative refractory state left behind by the first cortical response would detectably alter the measured parameters of the second one. In rats recorded 24 h after 20 mg/kg 3-NP i.p., the second:first ratio of the SS cortical response was less than 0.5, that is, the second response was considerably suppressed by the first one
Fig. 4. Effects of 3-NP on the cortical evoked potentials (A, somatosensory; B, visual; C, auditory area) in subacute exposure (low dose: 10, high dose: 15 mg/kg 3-NP i.p. on five consecutive days; recording in 4 weeks). Abscissa: latency (lat.) and duration (dur.) of the potentials. Ordinate: mean + S.D. (n = 10) of the respective value. Inset: doses. ** p < 0.01 vs. control.
(Fig. 6A) but the effect was practically the same in untreated control rats. The second:first ratio of latency values was different in the treated and control rats, but not significantly and with no clear dependence on the inter-stimulus interval. In rats injected with 3-NP during recording (immediate effect), the second:first ratio of the amplitude changed in dependence of the time elapsed and of the inter-stimulus interval. As seen in Fig. 6B, the ratio in the control period (−60 to 0) fluctuated around 0.5. After i.p. administration of 20 mg/kg 3-NP, the ratio started to increase (that is, the second response was less impeded by the first one) and went well over 1. For 300 and 240 ms inter-stimulus interval, the increasing trend was present from ca. the 80th minute on and by the end of recording it was significant (treatment effect, 300 ms: F1,18 = 4.53, p < 0.038; 240 ms: F1,18 = 4.62, p < 0.027; treatment–time interaction also significant). The increase started only later for 180 ms interval, and for 120 ms, it was negligible. The latency ratio in the same rats was influenced by 3-NP only ca. 100 min after injection (Fig. 6C). The change was de-
A. Szab´o et al. / Environmental Toxicology and Pharmacology 19 (2005) 811–817
Fig. 5. Effects of 3-NP on the cortical evoked potentials (A, somatosensory; B, visual; C, auditory area) in acute exposure (20 mg/kg 3-NP i.p., recording in 24 h), displayed as in Fig. 4. * p < 0.05 vs. control.
pendent on the inter-stimulus interval but remained below significance.
4. Discussion The 3-NP induced several alterations in the spontaneous and evoked cortical electrical activity of treated rats. The principal way of action of 3-NP is to cause energy insufficiency in the neurons by impeding mitochondrial oxidation (Coles et al., 1979), resulting in a tissue hypoxia-like state. In human volunteers, breathing low-oxygen gas mixture caused an EEG shift to lower frequencies (van der Post et al., 2002). Also, in human cases of inherited or idiopathic mitochondrial dysfunction, like mitochondrial encephalomyopathy (ME), cortical functions were affected (Montirosso et al., 2002). The EEG abnormalities seen were interpreted as a sensitive indicator of brain mitochondrial disorder (Smith and Hard
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Fig. 6. (A) denote the effect of 3-NP in acutely treated rats (20 mg/kg i.p., recorded in 24 h) on the second:first ratio of amplitude (diamonds) and first peak latency (squares) of the somatosensory evoked potential elicited with double stimuli (see Section 2). Abscissa: inter-stimulus time. Ordinate: second:first ratio (mean, n = 10). Inset: control and treated. (B) and (C) denotes time course of the amplitude and first peak latency, respectively, of the somatosensory evoked potentials obtained by double stimuli, in rats before and immediately after i.p. application of 20 mg/kg 3-NP. Abscissa: time (3NP was given at 0 ms). Ordinate: second:first ratio (mean, n = 10) of the parameters. Inset in (B): inter-stimulus time for (B) and (C). * p < 0.05 vs. pre-administration period.
ing, 1993). The main EEG abnormality of ME patients was slowed activity (Sciacco et al., 2001), which was similar to the ECoG alteration (increased slow – delta and theta – activity in all cortical foci) obtained in our work by subchronic 3-NP administration. In earlier works of our laboratory, it was found that various environmental neurotoxicants (pesticide agents, heavy metals) induce a shift in the ECoG power spectrum (Institoris et al., 2002; Schulz et al., 1997). Regarding that the EEG (or ECoG) signals result from the spatio-temporal summation of a high number of individual neuronal activities (Kandel and Schwartz, 1985) the non-specific nature of the EEG alteration, stressed by Smith and Harding (1993) is understandable. The fact that the ECoG alteration was similar when recorded 4 weeks after the last application (subacute
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treatment) or 24 h after a single application (acute treatment) suggested that a rapidly developing mechanism of 3-NP action was involved—most probably energetic impairment as suggested also by the similarity of this effect to that seen in hypoxic persons or ME patients. Brain cholinesterase, another enzyme affected by 3-NP (Ludolph et al., 1991) has most probably no role in the ECoG changes as the frequency shift is opposite to that obtained by organophosphates (D´esi and Nagymajt´enyi, 1999) which are established cholinesterase inhibitors. Further it can be supposed that the ECoG changes, caused by energetic impairment or other mechanism, are not likely to run in parallel with the morphological and functional alterations (Brouillet et al., 1999) serving as the base of modelling Huntington’s disease by applying 3-NP. In the subacutely treated rats, the effect of 3-NP on cortical-evoked potentials was similar to what had been described from ME patients. In particular, the alteration of certain visual-evoked potential components in ME (Finsterer, 2001; Scaioli et al., 1998) and the increase of evoked potential latency in the treated rats was similar. The changes in the measured parameters of the cortical evoked responses in the treated rats were dissimilar in subacute versus acute 3-NP treatment, possibly indicating another mechanism of action besides (or instead of) mitochondrial impairment. A known effect of 3-NP, inhibition of glutamate uptake (Tavares et al., 2001) may have led first to imbalance between excitation and inhibition—possibly explaining the increased duration of cortical-evoked responses in acute 3-NP exposure (Fig. 5). The final effect is excitotoxicity (observed in vitro and in vivo; Pubill et al., 2001)—possibly reflected in the slowed ECoG activity and lengthened evoked potential latency seen in our subacutely treated rats (Fig. 4). The results obtained with double-pulse somatosensory stimulation reflect probably a kind of disinhibition. In human subjects with inherited mitochondrial disorder, pairedpulse somatosensory stimulation via the median nerve revealed strongly reduced intracortical inhibition (Liepert et al., 2001). Although this was a phenomenon observed in humans and the inter-stimulus intervals were not comparable to those used by us, the similarity of the effects, and the known connection of mitochondriopathy with increased cortical excitability (Rosing et al., 1985) and altered GABA levels (Erecinska and Nelson, 1994), suggest that double-pulse stimulation can be a sensitive and specific means to reveal, and even more to follow-up, the action of mitochondrial toxins, such as 3-NP, in the CNS. The overall effect of 3-NP on the activity of the brain is complex and seems to go beyond the endpoints utilized in modelling Huntington’s disease. Further studies, to reveal the mechanisms responsible for the functional alterations described in this paper, will possibly contribute to a better understanding of the disease model based on 3-NP, first of all concerning effects of different (biochemical, physiological, morphological) nature.
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Functional neurotoxic effects in rats elicited by 3-nitropropionic acid in acute and subacute administration Andrea Szab´o ∗ , Andr´as Papp, L´aszl´o Nagymajt´enyi Department of Public Health, University of Szeged, H-6723 Szeged, D´om t´er 10, Hungary
Abstract Changes possibly induced by 3-NP in electrophysiological functional characteristics of the central nervous system are, in contrast to biochemical and morphological alterations, less well known. In this study, the usability of a standard neurophysiological investigation system to detect functional changes caused by 3-NP administration in rats was studied. In subacute treatment, 10 or 15 mg/kg 3-NP was given i.p. on five consecutive days to groups of 10 rats and the effects were checked 4 weeks later. Acutely treated rats received 20 mg/kg i.p. after several control records. For recording, the animals’ left hemisphere was exposed in urethane anesthesia. Silver electrodes were placed on the cortical sensory foci and tungsten needles in the subcortical (caudatum, globus pallidus) recording sites. Spontaneous electrical activity, as well as somatosensory, visual and auditory evoked potentials, were recorded. Following subacute treatment, the slowest (theta) and fastest (beta2 and gamma) frequencies of the spontaneous activity were changed, differently in the cortical versus subcortical sites. In the sensory evoked potentials after subacute treatment, an increase of the latency was seen in all sensory areas. In the acutely treated animals, the amplitude of the somatosensory evoked potential decreased after giving 3-NP. With double stimuli, the relation of the two responses was treatment- and interval-dependent. Understanding the mechanism of these effects may widen the knowledge base for using 3-NP in disease models. © 2005 Elsevier B.V. All rights reserved. Keywords: 3-Nitropropionic acid; Cortical activity; Subcortical activity; Rat
1. Introduction The ‘natural’ origin of most of our food by far does not mean that they cannot contain toxic substances. The compound 3-nitropropionic acid (3-NP) is naturally found in Astragalus species (Leguminosae; Johnson et al., 2000), and occasionally causes severe or fatal poisoning in grazing farm animals (James et al., 1980). Human intoxication may result from infestation of foodstuffs (sugar cane, cereals, etc.) with moulds of the Anthirium and Aspergillus genus (Scallet et al., 2001; Patocka et al., 2000; Peraica and Domijan, 2001), producing 3-NP. Human exposure to 3-NP, even in low doses, causes acute encephalopathy followed by dystonia (Liu et al., 1992). Torsion spasms and jerky movements were observed in the victims, and CT images indicated lesions of the basal ganglia (He et al., 1995). ∗
Corresponding author. Tel.: +36 62 545 119; fax: +36 62 545 120. E-mail address: [email protected] (A. Szab´o).
1382-6689/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2004.12.062
The morphological and functional effects of 3-NP intoxication have been replicated in animal experiments. With a dose similar to ours (5 × 20 mg/kg i.p.), Teunissen et al. (2001) obtained decrease of motor performance in rats. Tissue degeneration, involving primarily the striatum but also the hippocampus and thalamus, was also described, both in vitro (Behrens et al., 1995) and in vivo (following one dose of 10–30 mg/kg 3-NP; McCracken et al., 2001), and was interpreted as a morphological correlate of the motor disorders. Based on that, 3-NP has been introduced in modeling Huntington’s disease in animals (Brouillet et al., 1999). At the cellular level, 3-NP inhibits succinate dehydrogenase, a key enzyme of oxidative energy production (Coles et al., 1979). The effect develops fast and is not limited to the sites of morphological damage (Brouillet et al., 1998). In mouse hippocampus, e.g., population spike amplitude was diminished by repeated doses of 20 mg/kg 3-NP, apparently due to inhibition of oxidative phosphorylation (Hellweg et al., 2003). 3-NP was also found to act on synaptic transmission by influencing NMDA receptors. The resulting excess in
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glutamatergic excitation led to chemically induced long-term potentiation in in vitro slices (Calabresi et al., 2001) and to excitotoxicity both in vitro and in vivo (Pubill et al., 2001). Although, a general mitochondrial impairment in the brain and the disturbance of glutamatergic transmission is likely to be reflected also in e.g. the spontaneous and stimulus-evoked cortical activity, and the description of such effects would be required to a comprehensive description of the chronic disease models based on 3-NP, no data of this kind were found in the literature. The aim of this work was therefore to see to what extent the neurophysiological investigation system established in our laboratory is suitable to detect functional changes caused by 3-NP administration in rats.
2. Methods The effects of 3-NP were investigated in three different time schemes—it is known from Brouillet et al. (1999) that dosing schemes and time until recording can have a major influence on the effects of 3-NP. In the subacute experiments, adult male Wistar rats (of 450–500 g body weight, 10 animals per treatment group) received 10 (low dose) and 15 (high dose) mg/kg b.w. 3-NP i.p. for five consecutive days and were kept for further 4 weeks before recording. Control animals were untreated. For acute exposure, 10 animals (of the same strain and weight) received 20 mg/kg 3-NP i.p. and were kept for 24 h. To see immediate effects, further 10 rats were first prepared for recording and four of the recording sequences described below were taken with 20 min interval. Then, 20 mg/kg 3-NP i.p. was given and further 6–8 records taken. For comparison, the intraperitoneal LD50 of 3-NP in rats is 67 mg/kg (Pass et al., 1985). For recording, the animals were anaesthetized with urethane (1000 mg/kg i.p.) and were placed in a stereotaxic instrument. The left hemisphere was exposed and silver electrodes were placed on the dura over the primary somatosensory (SS), visual (VIS) and auditory (AUD) areas. One steel needle electrode each was inserted in the caudato-putamen (stereotaxic coordinates: AP 0, L 3, V 5; Paxinos and Watson, 1982) and the globus pallidus (AP 2, L 3, V 6). The recording sequence started with spontaneous electrical activity (electrocorticogram, ECoG), recorded from these sites simultaneously for 6 min from the record, the relative spectral power of the frequency bands (delta to gamma; Kandel and Schwartz, 1985) was determined. Then, stimulusevoked activity was recorded from the mentioned cortical areas. Somatosensory stimulation was done by a pair of needles inserted into the whiskery skin, delivering rectangular electric stimuli of 3–4 V and 0.2 ms. Visual stimulation was performed by flashes (60 lx) from a flash generator via an optical fiber directed into the contralateral eye of the rat. For acoustic stimulation, clicks (40 dB) were applied into the ear of the rat. Of each modality, one series of 50 stimuli at 1 s repetition time (1 Hz frequency) was applied to each rat, and the evoked activity was recorded. After averaging, amplitude,
latency and duration of the evoked responses was measured manually. In previous studies with other neurotoxicants (Papp et al., 2000a,b) it was found that changes in the measurable parameters of cortical responses evoked with multiple stimulation are sensitive to the toxic influence. In the present study, doublepulse somatosensory stimulation was used (repetition time, 1 s; inter-stimulus time, 60–300 ms). From the double-pulse records, the second:first ratio for amplitude and latency was calculated and interpreted as measure of dynamic interaction between the two excitation processes. It was tested whether this interaction is sensitive to the toxic influence. The results of subacute and acute (24 h) exposure were tested for significance with one-way ANOVA. For the results of immediate treatment (pre- and post-application data), repeated measure ANOVA was used. Post hoc analysis was done by LSD with p < 0.05 as limit. During the whole study, the principles of the Ethical Committee for the Protection of Animals in Research of the University were strictly followed.
3. Results 3.1. Effects on the spontaneous cortical activity In subacute exposure, the most conspicuous effect was the significant (SS: F2,27 = 16.59, p < 0.001; VIS: F2,27 = 5.48, p < 0.014; AUD: F2,27 = 9.62, p < 0.001) decrease in the theta activity seen in all cortical foci in the high-dose animals (Fig. 1A–C). In the other bands, the changes were not uniform: increase of the delta activity was significant only in the SS and VIS area, decrease of alpha in the VIS area, and increase of beta2 in the SS and AUD areas. In the basal ganglia (Fig. 1D, E) activity decreased in the slow, delta and theta bands (significant in the high-dose group and only in the caudatum, delta: F2,27 = 5.51, p < 0.015; theta: F2,27 = 2.94, p < 0.05). Increase of the gamma band activity was seen in the caudatum and the globus pallidus, but was significant only in the latter (F2,27 = 3.33, p < 0.05). In the low-dose rats, the changes were mostly below significance. In the rats recorded 24 h after 3-NP application (Fig. 2), the trend of the ECoG bands was partly similar to that obtained by subacute exposure. In the SS area (Fig. 2A), delta activity increased significantly (F1,18 = 4.17, p < 0.054). The increase of theta and decrease of faster bands was not significant. In the VIS focus (Fig. 2B) the changes were similar but only the decrease of alpha activity was significant (F1,18 = 4.49, p < 0.046). In the AUD cortex (Fig. 2C) delta activity increased significantly (F1,18 = 13.43, p < 0.001), and alpha, beta1 and beta2, decreased (alpha: F1,18 = 20.66, p < 0.001; beta1: F1,18 = 10.10, p < 0.004; beta2: F1,18 = 4.02, p < 0.051). The ECoG changes observed within ca. 2 h after i.p. application of 20 mg/kg 3-NP are shown in Fig. 3. None of the changes was significant but it was clearly visible that
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Fig. 2. Effects of 3-NP on the spontaneous cortical activity (A, somatosensory; B, visual; C, auditory area) in acute exposure (20 mg/kg 3-NP i.p., recording in 24 h), displayed as in Fig. 1. * p < 0.05 vs. control in the same band.
the change at 100 min was to the same direction as 4 weeks or 24 h after 3-NP administration. The time course of the alterations was, however, such that the immediate change (10 min after administration) was opposite to that seen at 100 min. 3.2. Effects on cortical evoked potentials
Fig. 1. Effects of 3-NP on the spontaneous cortical (A, somatosensory; B, visual; C, auditory area) and subcortical (D, caudato-putamen; E, globus pallidus) activity in subacute exposure (control, untreated; low dose: 10, high dose: 15 mg/kg 3-NP i.p. on five consecutive days; recording in 4 weeks). Ordinate: relative power of the standard frequency bands in control and treated animals (means, n = 10). Inset: frequency bands. * p < 0.05 vs. control in the same band.
The general slow-down of the cortical activity following subacute treatment with 3-NP was paralleled by the increased latency of the sensory evoked potentials (Fig. 4). The change was significant in all recorded cortical areas and in both treated groups versus control (SS: F2,27 = 7.19, p < 0.004; VIS: F2,27 = 16.82, p < 0.001; AUD: F2,27 = 10.72, p < 0.001). The change seemed to be dose-dependent but high versus low dose differences were not significant. The alteration of the duration of the responses was slight. In acute (24 h) 3NP exposure (Fig. 5), there was no consequent effect on the
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Fig. 3. Immediate effect of 3-NP (20 mg/kg i.p.) on the spontaneous cortical activity (A, somatosensory; B, visual; C, auditory area). Abscissa: time elapsed after drug administration, con is the average of pre-administration records. Other features as in Fig. 1.
latency: small decrease in the SS and AUD response but significant increase (F1,18 = 4.44, p < 0.049) in the VIS response. The duration of the potentials was, on the contrary, significantly increased in the SS (F1,18 = 4.54, p < 0.046), and VIS (F1,18 = 4.66, p < 0.031) but not in the AUD area. 3.3. 3-NP effects on the dynamic interaction in the somatosensory system revealed by double-pulse stimulation Double-pulse somatosensory stimulation with different inter-stimulus intervals was used to investigate dynamic interaction in the sensory system. It was supposed that the relative refractory state left behind by the first cortical response would detectably alter the measured parameters of the second one. In rats recorded 24 h after 20 mg/kg 3-NP i.p., the second:first ratio of the SS cortical response was less than 0.5, that is, the second response was considerably suppressed by the first one
Fig. 4. Effects of 3-NP on the cortical evoked potentials (A, somatosensory; B, visual; C, auditory area) in subacute exposure (low dose: 10, high dose: 15 mg/kg 3-NP i.p. on five consecutive days; recording in 4 weeks). Abscissa: latency (lat.) and duration (dur.) of the potentials. Ordinate: mean + S.D. (n = 10) of the respective value. Inset: doses. ** p < 0.01 vs. control.
(Fig. 6A) but the effect was practically the same in untreated control rats. The second:first ratio of latency values was different in the treated and control rats, but not significantly and with no clear dependence on the inter-stimulus interval. In rats injected with 3-NP during recording (immediate effect), the second:first ratio of the amplitude changed in dependence of the time elapsed and of the inter-stimulus interval. As seen in Fig. 6B, the ratio in the control period (−60 to 0) fluctuated around 0.5. After i.p. administration of 20 mg/kg 3-NP, the ratio started to increase (that is, the second response was less impeded by the first one) and went well over 1. For 300 and 240 ms inter-stimulus interval, the increasing trend was present from ca. the 80th minute on and by the end of recording it was significant (treatment effect, 300 ms: F1,18 = 4.53, p < 0.038; 240 ms: F1,18 = 4.62, p < 0.027; treatment–time interaction also significant). The increase started only later for 180 ms interval, and for 120 ms, it was negligible. The latency ratio in the same rats was influenced by 3-NP only ca. 100 min after injection (Fig. 6C). The change was de-
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Fig. 5. Effects of 3-NP on the cortical evoked potentials (A, somatosensory; B, visual; C, auditory area) in acute exposure (20 mg/kg 3-NP i.p., recording in 24 h), displayed as in Fig. 4. * p < 0.05 vs. control.
pendent on the inter-stimulus interval but remained below significance.
4. Discussion The 3-NP induced several alterations in the spontaneous and evoked cortical electrical activity of treated rats. The principal way of action of 3-NP is to cause energy insufficiency in the neurons by impeding mitochondrial oxidation (Coles et al., 1979), resulting in a tissue hypoxia-like state. In human volunteers, breathing low-oxygen gas mixture caused an EEG shift to lower frequencies (van der Post et al., 2002). Also, in human cases of inherited or idiopathic mitochondrial dysfunction, like mitochondrial encephalomyopathy (ME), cortical functions were affected (Montirosso et al., 2002). The EEG abnormalities seen were interpreted as a sensitive indicator of brain mitochondrial disorder (Smith and Hard
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Fig. 6. (A) denote the effect of 3-NP in acutely treated rats (20 mg/kg i.p., recorded in 24 h) on the second:first ratio of amplitude (diamonds) and first peak latency (squares) of the somatosensory evoked potential elicited with double stimuli (see Section 2). Abscissa: inter-stimulus time. Ordinate: second:first ratio (mean, n = 10). Inset: control and treated. (B) and (C) denotes time course of the amplitude and first peak latency, respectively, of the somatosensory evoked potentials obtained by double stimuli, in rats before and immediately after i.p. application of 20 mg/kg 3-NP. Abscissa: time (3NP was given at 0 ms). Ordinate: second:first ratio (mean, n = 10) of the parameters. Inset in (B): inter-stimulus time for (B) and (C). * p < 0.05 vs. pre-administration period.
ing, 1993). The main EEG abnormality of ME patients was slowed activity (Sciacco et al., 2001), which was similar to the ECoG alteration (increased slow – delta and theta – activity in all cortical foci) obtained in our work by subchronic 3-NP administration. In earlier works of our laboratory, it was found that various environmental neurotoxicants (pesticide agents, heavy metals) induce a shift in the ECoG power spectrum (Institoris et al., 2002; Schulz et al., 1997). Regarding that the EEG (or ECoG) signals result from the spatio-temporal summation of a high number of individual neuronal activities (Kandel and Schwartz, 1985) the non-specific nature of the EEG alteration, stressed by Smith and Harding (1993) is understandable. The fact that the ECoG alteration was similar when recorded 4 weeks after the last application (subacute
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treatment) or 24 h after a single application (acute treatment) suggested that a rapidly developing mechanism of 3-NP action was involved—most probably energetic impairment as suggested also by the similarity of this effect to that seen in hypoxic persons or ME patients. Brain cholinesterase, another enzyme affected by 3-NP (Ludolph et al., 1991) has most probably no role in the ECoG changes as the frequency shift is opposite to that obtained by organophosphates (D´esi and Nagymajt´enyi, 1999) which are established cholinesterase inhibitors. Further it can be supposed that the ECoG changes, caused by energetic impairment or other mechanism, are not likely to run in parallel with the morphological and functional alterations (Brouillet et al., 1999) serving as the base of modelling Huntington’s disease by applying 3-NP. In the subacutely treated rats, the effect of 3-NP on cortical-evoked potentials was similar to what had been described from ME patients. In particular, the alteration of certain visual-evoked potential components in ME (Finsterer, 2001; Scaioli et al., 1998) and the increase of evoked potential latency in the treated rats was similar. The changes in the measured parameters of the cortical evoked responses in the treated rats were dissimilar in subacute versus acute 3-NP treatment, possibly indicating another mechanism of action besides (or instead of) mitochondrial impairment. A known effect of 3-NP, inhibition of glutamate uptake (Tavares et al., 2001) may have led first to imbalance between excitation and inhibition—possibly explaining the increased duration of cortical-evoked responses in acute 3-NP exposure (Fig. 5). The final effect is excitotoxicity (observed in vitro and in vivo; Pubill et al., 2001)—possibly reflected in the slowed ECoG activity and lengthened evoked potential latency seen in our subacutely treated rats (Fig. 4). The results obtained with double-pulse somatosensory stimulation reflect probably a kind of disinhibition. In human subjects with inherited mitochondrial disorder, pairedpulse somatosensory stimulation via the median nerve revealed strongly reduced intracortical inhibition (Liepert et al., 2001). Although this was a phenomenon observed in humans and the inter-stimulus intervals were not comparable to those used by us, the similarity of the effects, and the known connection of mitochondriopathy with increased cortical excitability (Rosing et al., 1985) and altered GABA levels (Erecinska and Nelson, 1994), suggest that double-pulse stimulation can be a sensitive and specific means to reveal, and even more to follow-up, the action of mitochondrial toxins, such as 3-NP, in the CNS. The overall effect of 3-NP on the activity of the brain is complex and seems to go beyond the endpoints utilized in modelling Huntington’s disease. Further studies, to reveal the mechanisms responsible for the functional alterations described in this paper, will possibly contribute to a better understanding of the disease model based on 3-NP, first of all concerning effects of different (biochemical, physiological, morphological) nature.
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