1
ACUTE ELECTROPHYSIOLOGICAL EFFECTS
OF A METAL AND A NON-METAL
MITOCHONDRIAL TOXIN
Szabolcs TAKÁCS *, Sarolta BANKÓ, Andras PAPP
Department of Public Health, University of Szeged Faculty of Medicine. H-6720 Szeged, Dóm tér 10.
* E-mail : takszabi@gmail.com
ABSTRACT
Mitochondrial toxins are related both to causation and modelling of central
nervous damage. Manganese, often present in occupational settings and as
environmental pollutant, inhibits complex 2 and 3 of the mitochondria and is known to
cause a Parkinson-like CNS damage. 3-nitropropionic acid (3NP), a neurotoxin of
plant and microfungal origin, inhibits succinate dehydrogenase leading to various
biochemical and morphological alterations in the brain. Experimental animals treated
by 3NP are used to model Huntington’s disease. In this work, adult male Wistar rats
were acutely prepared for recording spontaneous and stimulus-evoked activity from
the projection area of the whiskers and the tail. It was tested whether the
dependence of the evoked response on the parameters of stimulation was influenced
by the mitochondrial toxins and what correlation existed between the cortical and
peripheral responses. Mn intensified frequency-dependent fatigue. 3-NP caused
latency decrease with minimal amplitude change, and had less effect on frequency
dependence. The observed effects were apparently limited to the brain.
Key words: manganese, 3-nitropropionic acid, evoked activity,
nerve action potential, stimulation parameters
INTRODUCTION
Mitochondrial toxins diminish oxidative energy production by blocking the
mitochondrial enzyme complexes. This causes a decline in the cells’ reserve energy
that can result in acute symptoms and contribute to various diseases.
Manganese is essential for living organisms in small amounts but toxic when
overdosed. The presence of Mn in the general environment is partly due to human
activity (petrol additive, fungicide, dry cells etc). Inorganic Mn can deposit in the brain
(Aschner et al., 1999), causing functional (Shinotoh et al., 1997) and structural
(Yamada et al., 1986) damage. The toxin 3-nitropropionic acid (3NP) is naturally
present in leguminous plants, occasionally poisoning grazing livestock. Human
poisoning may come from consumption of foodstuffs infested with certain moulds
producing 3NP (Brouillet et al., 1999). A common effect of both Mn and 3NP is the
inhibition of the enzyme complexes responsible for energy production. Mn decreases
the activity of all complexes, particularly complex 2 and 3 (Zhang et al. 2003)
whereas 3NP irreversibly inhibits succinate dehydrogenase in complex 2 (Coles et
al., 1979).
„Metal Elements in Environment, Medicine and Biology”, Tome VIIII, pp. 1-6. Publishing House
“Eurobit” Timişoara, 2008
Proc. of 8th Int. Symp. of Roumanian Academy - Branch Cluj-Napoca and Branch Timişoara,
2008, December 5-6, Timişoara, Roumania
2
Although Mn and 3-NP both are mitochondrial blockers, there are many
differences in their intra- and extra-mitochondrial mode of action which leads to
completely dissimilar clinical pictures in chronic intoxications. The symptoms of
excess Mn intake are, on the whole, called manganism, a state that can be very
similar to Parkinson’s disease, while 3NP poisoning resembles Huntington’s disease
and is used in an animal model of that (Brouillet et al., 1999).
Since the function of the nervous system requires lots of energy, the
mitochondrial damage is probably reflected in the electrical activity of the brain.
Beside damage in energy production, the two substances studied have also more
direct effects in the nervous system, so their excessive presence should be
detectable by electrophysiology. To study this, we examined the changes of evoked
activity in acute experiments in rats.
MATERIALS AND METHODS
Adult male Wistar rats (ca. 300 g) were given urethane (1000 mg/kg b.w.) ip.
for anaesthesia. The left hemisphere was exposed by removing a part of the bony
skull. Following a short recovery period, silver recording electrodes were placed on
the surface of the cortex at the primary somatosensory projection area of the tail and
the whiskers for measuring electrical activity. Evoked potentials (EPs) were triggered
by 0.05 ms square electric stimuli that were delivered by pairs of needle electrodes
placed in the contralateral whisker pad and in the tail base. To record the peripheral
nerve responses another pair of needles were inserted in the tail 50 mm distally from
the base. See Lukács and Szabó, 2007, for the recording techniques.
First, in 8 rats for both substances, the dependence of the obtained cortical EP
on parameters of the whisker stimulation was tested. The just-supramaximal stimulus
strength (around 3 - 5 V) was determined first, then trains of 50 stimuli were given at
frequency of 1, 2, 5 and 10 Hz. This sequence was done 3 times with 30 min
intervals for control. Then the appropriate solution was given in an intraperitoneal
injection, and further 4 records were taken. 3NP was given in a dose of 20 mg/kg, or
Mn2+ (in form of MnCl2) in 50 mg/kg, both dissolved in distilled water. In another
experiment both whiskers and tail of 8 rats was stimulated at 1 Hz. Most conditions
were the same as in the previous set except that beside the corresponding cortical
EPs, the tail nerve response was recorded also. The records were averaged, and
their onset latency and peak-to-peak amplitude was measured.
To eliminate individual variation, the latency and amplitude values were
normalized to the mean of the control period (first 3 series). From these normalized
data, group mean was calculated and plotted against the frequency (1 to 10 Hz) of
stimulation, and any difference between the resulting curves before vs. after
administration of the toxicant was sought for. In the second experiment, cortical and
peripheral response parameters were plotted in the same graph to see their similar or
dissimilar time trend, and their correlation was tested.
RESULTS AND DISCUSSION
Before application of either Mn or 3NP, or in the untreated parallel controls,
the latency data normalized to control mean and plotted against frequency of
stimulation gave a horizontal line, showing that stimulation with 10 Hz vs. 1 Hz had
no effect on the latency (Fig. 1). After injection of Mn, the latency started to increase
and developed nonlinear frequency dependence with maximum at 2 Hz. Treatment
with 3NP induced latency decrease which increased in time (curve 4 to curve 7) and
diminished with increasing stimulation frequency.
3
The time trend of the latency and amplitude of EPs obtained by whisker or tail
base stimulation was investigated together and was compared to the trend of the
peripheral response (nerve action potential) evoked by tail stimulation.
Rel. Lat.
1.06
Rel. Lat.
1.025
1
1.04
2
3
4 1.02
1
1
2
3
4 0.975
5
6
7
5
6 0.95
7
1
0.98
0.925
1
2
5
10
Frequency, Hz
1
2
5
10
Frequency, Hz
Fig. 1. Frequency dependence of the EP latency before and after administration of Mn (left) and 3NP
(right). Curves 1 to 3 show values before, and 4 to 7 after, application. Group means of normalized
individual data (see Methods).
In the Mn-treated rats, the latency of the cortical EPs evoked by whisker or tail
stimulation had a clear decreasing trend (Fig. 2, top left), while the amplitude of the
EPs increased (Fig. 2, top right), but neither parameter of the tail nerve response had
a noteworthy change. The correlation diagrams of Fig. 2 show that the two cortical
responses changed together but independently of the peripheral response.
Rel. Change
Rel. Change
yP = 0.0025x + 0.9974
1.05
1
2.2
0.95
1.8
2
yT = 0.1326x + 0.7171 R = 0.0062
R2 = 0.5773
yW = 0.0483x + 0.955
R2 = 0.384
yW = -0.0105x + 1.0177
0.9
yP = -0.0089x + 1.0531
2.6
R2 = 0.2082
1.4
R2 = 0.9216
yT = -0.0104x + 1.0174
0.85
R2 = 0.7271
1
0.6
0.8
1
2
Whiskers Lat.
3
Tail Lat.
4
5
Periph. Lat.
6
7
Record No
1
2
Whiskers Ampl.
3
5
Periph. Ampl.
6
7
Record No
2.5
1.04
yTP = 0,1243x + 0,8771
yWT = 2,1456x - 0,9847
R2 = 0,2759
R2 = 0,8398
Whiskers-Tail
Whiskers-Periph.
1.9
yWP = -0,3137x + 1,2967
1
yWP = 0,0999x + 0,8994
Tail-Periph.
R2 = 0,6623
R2 = 0,0429
0.96
Whiskers-Tail
1.3
Whiskers-Periph.
yWT = 1,3816x - 0,3947
yTP = -0,1453x + 1,1471
R2 = 0,8437
Tail-Periph.
R2 = 0,5916
0.92
0.92
4
Tail Ampl.
0.7
0.96
1
1.04
0.7
1.3
1.9
2.5
Fig. 2. Top: Relative change (compared to control mean) of the latency (left) and amplitude (right) of
the cortical and peripheral evoked responses in Mn-treated rats. Record N° 1 to 3 was take before,
and 4 to 7 after, Mn application. Bottom: correlation diagrams of the same data sets. Inserts: equations
and correlation coefficients of the fitted straight lines.
4
In the rats treated with 3NP the effect was highly similar. Latency decrease and
amplitude increase of the cortical responses evolved in parallel, shown by the linear
2
1.04
yTP = -0,131x + 1,135
yWT = 1,2673x - 0,2074
R2 = 0,0879
1
R2 = 0,3201
1.5
yWP = -0,2827x + 1,2831
yTP = -0,097x + 1,1384
R2 = 0,328
0.96
Whiskers-Tail
Whiskers-Periph.
Tail-Periph.
yWT = 0,9995x + 0,0004
R2 = 0,8001
0.92
0.92
Whiskers-Tail
Whiskers-Periph.
Tail-Periph.
0.96
1
1.04
R2 = 0,0222
1
yWP = 0,141x + 0,8556
R2 = 0,0093
0.5
0.5
1
1.5
2
Fig. 3. Correlation diagrams of the latency (left) and amplitude (right) of cortical and peripheral
responses obtained in 3NP-treated rats. Displayed as in Fig. 2.
trend line standing at about 45 degrees inclination; but there was no change in the
tail nerve response, as shown by the nearly horizontal trend lines. (Fig. 3).
Mitochondrial inhibition is the obvious common point in the action of Mn and 3NP,
and the cortical activity changes in inherited mitochondrial encephalopathy are known
(Smith and Harding, 1993). The correlation diagrams suggest, however, that a cortex- or
at least CNS-specific mechanism played a role, most probably the disturbance of
glutamate turnover (Mn: Hazell and Norenberg, 1997; 3NP: Tavares at al., 2001).
Electrophysiological recording may be of use in the study of mitochondrial toxins.
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