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Basal levels of metabolic activity are elevated in Genetic Absence Epilepsy Rats from Strasbourg (GAERS): measurement of regional activity of cytochrome oxidase and lactate dehydrogenase by histochemistry
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Experimental Neurology 182 (2003) 346 –352
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Basal levels of metabolic activity are elevated in Genetic Absence Epilepsy Rats from Strasbourg (GAERS): measurement of regional activity of cytochrome oxidase and lactate dehydrogenase by histochemistry Franck Dufour, Estelle Koning, and Astrid Nehlig* INSERM U398, Universite´ Louis Pasteur, Strasbourg, France Received 28 May 2002; revised 24 October 2002; accepted 9 December 2002
Abstract The Genetic Absence Epilepsy Rats from Strasbourg (GAERS) are considered an isomorphic, predictive, and homologous model of human generalized absence epilepsy. It is characterized by the expression of spike-and-wave discharges in the thalamus and cortex. In this strain, basal regional rates of cerebral glucose utilization measured by the quantitative autoradiographic [14C]2-deoxyglucose technique display a widespread consistent increase compared to a selected strain of genetically nonepileptic rats (NE). In order to verify whether these high rates of glucose metabolism are paralleled by elevated activities of the enzymes of the glycolytic and tricarboxylic acid cycle pathways, we measured by histochemistry the regional activity of the two key enzymes of glucose metabolism, lactate dehydrogenase (LDH) for the anaerobic pathway and cytochrome oxidase (CO) for the aerobic pathway coupled to oxidative phosphorylation. CO and LDH activities were significantly higher in GAERS than in NE rats in 24 and 28 of the 30 brain regions studied, respectively. The differences in CO and LDH activity between both strains were widespread, affected all brain systems studied, and ranged from 12 to 63%. The data of the present study confirm the generalized increase in cerebral glucose metabolism in GAERS, occurring both at the glycolytic and at the oxidative step. However, they still do not allow us to understand why the ubiquitous mutation(s) generates spike-and-wave discharges only in the thalamocortical circuit. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Absence epilepsy; Seizures; Spike-and-wave discharges; Glucose metabolism; Cytochrome oxidase; Lactate dehydrogenase; Histochemistry
Introduction The Genetic Absence Epilepsy Rats from Strasbourg (GAERS) are considered an isomorphic, predictive, and homologous model of human generalized absence epilepsy (Danober et al., 1998). In a previous work, we showed that basal regional rates of cerebral glucose utilization measured by the quantitative autoradiographic [14C]2-deoxyglucose (2DG) technique (Sokoloff et al., 1977) display a widespread consistent increase in these animals compared to a selected strain of genetically nonepileptic rats (NE). How* Corresponding author. INSERM U 398, Faculte´ de Me´decine, 11 rue Humann, 67085 Strasbourg Cedex, France. Fax: ⫹33-390.24.32.48. E-mail address: [email protected] (A. Nehlig).
ever, these levels of brain energy metabolism were measured in a mixed state during which seizures did occur spontaneously and represented a mean duration of about 16 s per min (Nehlig et al., 1991, 1992). Further studies on GAERS showed that the induction of absence status epilepticus decreased the levels of energy metabolism and suppressed the strain difference between GAERS and NE. On the other hand, the inhibition of occurrence of spike-andwave discharges (SWDs) by ethosuximide maintained regional rates of cerebral energy metabolism at a level at least as high and rather higher than in untreated GAERS (Nehlig et al., 1992, 1993). We also found that rates of cortical blood flow assessed by laser-Doppler flowmetry were reduced during the occurrence of SWDs in GAERS (Nehlig et al., 1996). The results from these studies are in accordance
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F. Dufour et al. / Experimental Neurology 182 (2003) 346 –352
with human data obtained with the [18F]fluorodeoxyglucose technique in positron emission tomography (PET) studies in patients with typical childhood absence epilepsy. In these patients, cerebral metabolism is diffusely and massively increased when absence seizures occur during the PET scan (Engel et al., 1982, 1985, 1988). As in our model, in human patients, no metabolic activation is measured when seizures turn to status epilepticus (Theodore, 1988; Theodore et al., 1985). Likewise, the use of the Doppler technique in human patients showed a drop in rates of cerebral blood flow in the middle cerebral artery during the occurrence of SWDs (Bode, 1992; Nehlig et al., 1996; Sanada et al., 1988). Thus, it seems that in absence epilepsy, the seizures are not responsible for the high metabolic level that could rather be attributed to interictal mechanisms requiring enough energy to stop the seizures and prevent their occurrence and their spread to limbic and motor seizures (Nehlig et al., 1991, 1992, 1993). To try to gain further knowledge of the characteristics of cerebral glucose metabolism in GAERS compared with NE rats, in the present study we measured by histochemistry the regional activity of the two key enzymes of glucose metabolism, lactate dehydrogenase (LDH) as the final enzyme of the anaerobic pathway and cytochrome oxidase (CO) as the enzyme of the final step of oxidative phosphorylation. The purpose of the present study was twofold: first, to verify whether the elevated rates of glucose metabolism measured by [14C]2DG technique in GAERS compared to NE were paralleled with high activities of the enzymes of the glycolytic and tricarboxylic acid cycle pathways; and second, to identify whether the mutation(s) that affects all brain cells in this strain and underlies the occurrence of SWDs in GAERS but allows the expression of SWDs only in the thalamocortical loop translates into changes in the level of activity of CO and LDH in all brain regions, as noted with the [14C]2DG technique in this strain (Nehlig et al., 1991, 1992, 1993), or only in the thalamic and cortical regions that are able to express SWDs.
Methods Animals The experiments were performed on seven adult male GAERS and nine adult male NE rats, 6 months old, originating from our breeding colony. The animals were maintained at 22°C room temperature under a 12-h/12-h normal light/dark cycle (lights on at 7:00 AM) with food and water ad libitum. Under those conditions, animals exhibit spontaneous SWDs alternating with periods of normal background EEG activity. The mean duration of SWDs reached 20 –24 s/min. All animal experimentation was performed in accordance with the rules of the European Communities Council Directive of November 24, 1986 (86/609/EEC), and the French Department of Agriculture (License No. 67–97).
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Histochemical measurement of cytochrome oxidase and lactate dehydrogenase activities For the histochemical measurement of CO and LDH activities, rats were deeply anesthetized with pentobarbital and immediately sacrificed by decapitation. This procedure was used to prevent any possible effect of the anesthesia on enzyme activity. Brains were dissected out and frozen in methylbutane chilled to ⫺30°C. Coronal brain sections of 20-m thickness obtained in a cryostat at 11 preselected brain levels were mounted onto gelatin- coated glass slides. Sections were taken at the following anteroposterior levels: prefrontal cortex, caudate–putamen, globus pallidus, anterior thalamus, dorsal hippocampus, medial thalamus, ventral hippocampus, superior colliculus, inferior colliculus, cerebellar nuclei, cerebellar cortex. For the assessment of either CO or LDH activity, all sections of all rat brains (three slides for each animal) were incubated together on the same day and in the same incubation mixture in order to avoid any variation in incubation conditions for the final measurement of optical density readings representative of enzyme activities. For the measurement of CO (cytochrome c oxidase; ferrocytochrome c: oxygen reductase, EC 1.9.3.1) activity, diaminobenzidine histochemistry was carried out according to the method of Wong-Riley (Wong-Riley, 1979), modified by Darriett et al. (Darriet et al., 1986). The incubation was performed at 38°C in the dark for 2 h in 0.1 M phosphate buffer, pH 7.4, containing 0.25 mg/ml cytochrome c (grade III, Sigma, St. Louis, MO, USA) and 0.05% 3,3⬘-diaminobenzidine tetrachloride (Sigma). In this reaction, cytochrome c becomes enzymatically reduced by electron transfer from diaminobenzidine. By oxidation, diaminobenzidine polymerizes and precipitates as a brown reaction product. At the end of the incubation, sections were rinsed, dehydrated in successive baths of increasing concentration of ethanol followed by LMR (Labo Moderne, Paris, France), and coverslipped in Histolaque (Labo Moderne). For the demonstration of LDH (EC 1.1.1.27), the method of Jacobsen (Jacobsen, 1969), modified by Borowsky and Collins (Borowsky and Collins, 1989a) was used. The incubation was carried out for 5 min at 37°C in 50 mM Tris–HCl buffer, pH 7.0, containing 100 mM DL-lactate (Sigma), 4 mM NAD⫹ (grade II, Boehringer Mannheim, Germany), 4.9 mM (0.4%) nitroblue tetrazolium, 0.33 mM phenazine methosulfate, and 10% (w/v) polyvinyl alcohol. This reaction utilizes phenazine methosulfate as electron carrier. Lactate oxidation leads to the formation of NADH ⫹ H⫹ which reduces nitroblue tetrazolium and generates blue formazan. Incubation time is shortened as much as possible and the presence of polyvinyl alcohol restricts movement of the tetrazolium reaction product. After incubation, sections were rinsed in water and fixed in 10% neutral formalin. They were then washed with distilled water and coverslipped in aquovitrex (Carlo Erba, Milan, Italy).
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For evaluation of both enzyme activities, relative optical density measurements were performed on sections using an image processing system (Biocom 500, Les Ulis, France). This procedure was used since a direct correlation between optical density and spectrophotometric measurements has been shown for both CO and LDH activities (Borowsky and Collins, 1989a; Darriet et al., 1986). Optical density measurements were made bilaterally for each structure anatomically defined according to the stereotaxic atlas of Paxinos and Watson (Paxinos and Watson, 1986). All readings were corrected for background (e.g., glass, inclusion medium, and coverslip) and nonspecific diaminobenzidine (CO) and nitroblue tetrazolium (LDH) reaction by incubation of a set of sections without substrate and cofactors. They always included the whole structure or nucleus of interest and were performed by an observer who was blind to the strain of the animals. Statistical analysis CO and LDH activities were determined in 30 brain regions in GAERS and NE rats. Optical density measurements of enzyme activities in each strain of rats were compared by means of an analysis of variance consisting of a regions ⫻ strains analysis for repeated measures. This analysis of variance was followed by a Scheffe test when the ANOVA indicated a significant effect.
Results Cytochrome oxidase activity As shown in Table 1, CO activities were significantly higher in GAERS than in NE rats in 24 of the 30 brain regions studied. The differences in CO activity between both strains were widespread, affected all brain systems studied, and ranged from 19% in hypothalamus to 63% in posterior and ventrolateral thalamic nuclei. Enzyme activity levels were not significantly different in the caudate–putamen, anteroventral and anterodorsal thalamic nuclei, substantia nigra pars reticulata, subthalamic nucleus, and red nucleus from GAERS and NE rats. Lactate dehydrogenase activity As shown in Table 2, LDH activities were significantly higher in GAERS than in NE rats in all brain regions studied, except two areas, namely the occipital cortex and the mediodorsal thalamic nucleus. The differences in LDH activity between both strains were widespread, affected all brain systems studied, and ranged from 12% in the thalamic medial geniculate nucleus to 52% in perirhinal cortex.
Table 1 Activity of cytochrome oxidase in various cerebral regions of GAERS and NE rats NE rats (n ⫽ 9) Cerebral cortex Frontal Cingulate Parietal Temporal Entorhinal Perirhinal Occipital Forebrain Hippocampal CA1 area, pyramidal cell layer Amygdala Caudate–putamen Globus pallidus Hypothalamus Thalamus Anteroventral Anterodorsal Ventrolateral Mediodorsal Laterodorsal Ventromedian Ventroposterior Posterior nuclei Reticular Medial geniculate Lateral geniculate Midbrain–Brainstem Substantia nigra pars reticulata Subthalamic nucleus Red nucleus Superior colliculus, superficial layer Inferior colliculus Pontine reticular nucleus, oral part Cerebellar cortex, granule cell layer
GAERS rats (n ⫽ 7)
% of variation from NE
8.70 ⫾ 0.74 9.72 ⫾ 0.86 9.11 ⫾ 0.74 7.14 ⫾ 0.62 6.25 ⫾ 0.64 7.21 ⫾ 0.68 8.63 ⫾ 0.84
11.10 ⫾ 0.26** 11.87 ⫾ 0.17* 11.31 ⫾ 0.34* 9.80 ⫾ 0.28** 8.87 ⫾ 0.24** 10.24 ⫾ 0.37** 11.50 ⫾ 0.48**
⫹28 ⫹22 ⫹24 ⫹37 ⫹42 ⫹42 ⫹33
6.65 ⫾ 0.75
9.29 ⫾ 0.24**
⫹40
7.46 ⫾ 0.66 8.43 ⫾ 0.89 4.79 ⫾ 0.64 8.23 ⫾ 0.54
9.70 ⫾ 0.24** 9.82 ⫾ 0.19 6.86 ⫾ 0.23** 9.81 ⫾ 0.32*
⫹30 ⫹17 ⫹43 ⫹19
10.48 ⫾ 0.83 11.79 ⫾ 0.96 6.25 ⫾ 0.89 7.03 ⫾ 0.63 8.32 ⫾ 0.80 7.26 ⫾ 0.61 8.50 ⫾ 0.73 6.62 ⫾ 0.82 7.81 ⫾ 0.72 6.42 ⫾ 0.68 7.93 ⫾ 0.66
11.99 ⫾ 0.27 13.15 ⫾ 0.31 10.20 ⫾ 0.35** 9.15 ⫾ 0.21** 10.37 ⫾ 0.27* 9.28 ⫾ 0.31** 11.24 ⫾ 0.38** 10.81 ⫾ 0.37** 9.47 ⫾ 0.24* 8.09 ⫾ 0.20* 10.38 ⫾ 0.38**
⫹14 ⫹12 ⫹63 ⫹30 ⫹25 ⫹28 ⫹32 ⫹63 ⫹21 ⫹26 ⫹31
7.86 ⫾ 0.85
9.27 ⫾ 0.26
⫹18
11.78 ⫾ 0.80 8.81 ⫾ 0.78 8.60 ⫾ 0.68
13.88 ⫾ 0.33 8.09 ⫾ 0.56 11.17 ⫾ 0.45**
⫹18 ⫺8 ⫹30
10.96 ⫾ 0.95 5.95 ⫾ 0.92
13.30 ⫾ 0.36* 9.45 ⫾ 0.39**
⫹21 ⫹59
8.56 ⫾ 0.81
11.14 ⫾ 0.28**
⫹30
Note. Values, expressed as optical density ⫻100, are means ⫾ SD of the number of animals in parentheses. * P ⬍ 0.05, ** P ⬍ 0.01, statistically significant differences between GAERS and NE rats.
Discussion The data of the present study show that the elevated levels of cerebral glucose utilization previously measured in GAERS (Nehlig et al., 1991, 1992) are paralleled by elevated activities of the enzymes of anaerobic and oxidative cerebral glucose metabolism. Both rates of glucose utilization (Nehlig et al., 1991, 1992) and the activity of the enzymes of its aerobic and anaerobic degradation (the present study) are diffusely increased all over the brain. Thus, the ubiquitous mutation(s) that underlies the expression of SWDs in GAERS translates into a ubiquitous change
F. Dufour et al. / Experimental Neurology 182 (2003) 346 –352 Table 2 Activity of lactate dehydrogenase in various cerebral regions of GAERS and NE rats
Cerebral cortex Frontal Cingulate Parietal Temporal Entorhinal Perirhinal Occipital Forebrain Hippocampal CA1 area, pyramidal cell layer Amygdala Caudate–putamen Globus pallidus Hypothalamus Thalamus Anteroventral Anterodorsal Ventrolateral Mediodorsal Laterodorsal Ventromedian Ventroposterior Posterior nuclei Reticular Medial geniculate Lateral geniculate Midbrain–brainstem Substantia nigra pars reticulata Subthalamic nucleus Red nucleus Superior colliculus, superficial layer Inferior colliculus Pontine reticular nucleus, oral part Cerebellar cortex, granule cell layer
NE rats (n ⫽ 9)
GAERS rats (n ⫽ 7)
% of variation from NE
5.11 ⫾ 0.32 5.08 ⫾ 0.53 5.45 ⫾ 0.29 6.54 ⫾ 0.35 6.65 ⫾ 0.34 5.31 ⫾ 0.39 5.28 ⫾ 0.53
6.69 ⫾ 0.29** 6.38 ⫾ 0.38* 6.48 ⫾ 0.20** 8.69 ⫾ 0.31** 9.06 ⫾ 0.30** 8.08 ⫾ 0.34** 5.98 ⫾ 0.48
⫹31 ⫹25 ⫹19 ⫹37 ⫹36 ⫹52 ⫹13
8.33 ⫾ 0.41
9.89 ⫾ 0.24**
⫹19
7.83 ⫾ 0.50 4.98 ⫾ 0.34 4.29 ⫾ 0.33 4.73 ⫾ 0.21
9.20 ⫾ 0.21* 6.35 ⫾ 0.19** 6.32 ⫾ 0.29** 6.43 ⫾ 0.16**
⫹18 ⫹28 ⫹47 ⫹36
4.91 ⫾ 0.41 4.82 ⫾ 0.44 4.44 ⫾ 0.35 6.72 ⫾ 0.37 5.80 ⫾ 0.14 5.94 ⫾ 0.25 5.70 ⫾ 0.25 5.20 ⫾ 0.23 4.96 ⫾ 0.41 5.43 ⫾ 0.28 4.59 ⫾ 0.33
6.46 ⫾ 0.35** 6.71 ⫾ 0.62** 6.57 ⫾ 0.41** 7.16 ⫾ 0.44 7.12 ⫾ 0.17** 7.44 ⫾ 0.28** 7.12 ⫾ 0.28** 6.84 ⫾ 0.27** 6.46 ⫾ 0.40* 6.05 ⫾ 0.14* 6.15 ⫾ 0.34**
⫹32 ⫹39 ⫹48 ⫹7 ⫹23 ⫹25 ⫹25 ⫹32 ⫹30 ⫹12 ⫹34
4.37 ⫾ 0.27
6.08 ⫾ 0.21**
⫹39
4.69 ⫾ 0.20 4.87 ⫾ 0.31 5.03 ⫾ 0.33
6.52 ⫾ 0.27** 6.00 ⫾ 0.17** 6.72 ⫾ 0.37**
⫹39 ⫹23 ⫹34
4.66 ⫾ 0.33 4.55 ⫾ 0.36
5.79 ⫾ 0.47* 6.01 ⫾ 0.46**
⫹24 ⫹32
3.63 ⫾ 0.43
5.19 ⫾ 0.48**
⫹43
349
ments of CO activity by diaminobenzidine histochemistry, as performed in the present study and the activity of the enzyme measured spectrophotometrically, has been established (Darriet et al., 1986). The two latter measurements of enzyme activity are performed in vitro under optimal environmental conditions mainly of pH, substrate, and cofactor availability and thus reflect the total enzyme activity present in the tissue but not the actual activity under in situ conditions. However, these measurements of enzyme activity have been shown to closely reflect the long-term functional use of the pathways of aerobic glucose metabolism. A direct positive correlation has been established between capillary
Note. Values, expressed as optical density ⫻100, are means ⫾ SD of the number of animals in parentheses. * P ⬍ 0.05, ** P ⬍ 0.01, statistically significant differences between GAERS and NE rats.
in the characteristics of cerebral glucose metabolism in this strain. Moreover, as shown in Fig. 1, in most brain regions, whether they express SWDs (cortex, thalamus) or not, the increases in enzyme activities and rates of glucose utilization are quite similar. A few exceptions can be noted; for example, in mediodorsal and posterior thalamic nuclei mainly, increases in glucose metabolism rates are quite larger than increases in LDH and CO activity while the reverse is true in parietal cortex and substantia nigra pars reticulata. The functional significance of these differences remains to be clarified. A close correlation between optical density measure-
Fig. 1. Activity of cytochrome oxidase (CO) and lactate dehydrogenase (LDH) and rates of glucose utilization (GLU) in various brain areas of GAERS. Values represent percentages of increase over control levels in NE rats. Abbreviations used: FTAL, frontal cortex; ACING, anterior cingulate cortex; PAR, parietal cortex; TEMP, temporal cortex; ENT, entorhinal cortex; AV, anteroventral thalamus; MD, mediodorsal thalamus; LD, laterodorsal talamus; VM, ventromedian thalamus; PO, posterior thalamus; CA1, CA1 layer of the hippocampus; AMY, amygdala; CAU, caudate nucleus; SNPR, substantia nigra pars reticulata; CBCX, cerebellar cortex. *P ⬍ 0.05; #P ⬍ 0.01, statistically significant differences between GAERS and NE rats.
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density and CO activity or cerebral glucose utilization in the rat (Borowsky and Collins, 1989a; Klein et al., 1986). Conversely, the relationship between LDH activity and capillary density is a negative one, raising the possibility that brain regions with a high glycolytic capacity may require fewer capillaries (Borowsky and Collins, 1989a). In the present study, we found also discrepancies in the activity of the two enzymes. The highest levels of CO activity were mostly found in sensory and motor systems and lower levels were recorded in limbic and associative areas (Table 1), as previously shown (Bilger and Nehlig, 1991; Hevner et al., 1995). On the other hand, LDH activity was especially high in limbic and associative regions such as hippocampus, amygdala, and some thalamic nuclei and low in motor regions (Table 2). Thus, the data from the literature and the present study are in favor of the hypothesis that the different brain regions are composed of a mixture of oxidative and glycolytic zones reflecting cellular heterogeneity, as well as a heterogeneous pattern of innervation (Borowsky and Collins, 1989a). Energy utilization by the brain is tightly coupled to neuronal functional activity (for review see (Clarke and Sokoloff, 1999; DiRocco et al., 1989; Erecinska and Silver, 1989)). Mature neurons are completely dependent on glucose as their sole energy substrate and on oxidative phosphorylation for the final production of ATP. Moreover, the brain has almost no energy reserves in the form of glycogen or fat, and even a brief interruption of substrate supply will be detrimental to brain function and cellular integrity. These biochemical properties, together with the high metabolic demand of the brain linked to neuronal activity, are responsible for the tight coupling between neuronal activity and oxidative energy metabolism in the brain. There is considerable available experimental evidence that the rates of cerebral glucose utilization measured by the quantitative [14C]2DG technique are tightly coupled to cerebral functional activity (for review see (Clarke and Sokoloff, 1999; Sokoloff, 1982)). Likewise, a clear coupling between brain functional activity and CO activity has been demonstrated (for review see (Wong-Riley, 1989)). Thus, CO histochemistry, representative of the full oxidative degradation of glucose, has been extensively used to map the functional activity in the normal adult brain (Borowsky and Collins, 1989a; Darriet et al., 1986; Gonzalez-Pardo et al., 1996; Hevner et al., 1995), and during development and aging (Curti et al., 1990; Hovda and Villablanca, 1998; Tuor et al., 1994). Decreases in CO activity have been described after visual deprivation (Rosa et al., 1991; Wong-Riley and Carroll, 1984), vibrissae removal, or deafferentation (Borowsky and Collins, 1989b; Villablanca et al., 1999; Wong-Riley and Welt, 1980), while increases in CO activity were recorded during recovery from sensory deprivation (Dietrich et al., 1982). In summary, CO and [14C]2DG are both metabolic markers but they do not always produce identical results. There are major differences between the two techniques. The [14C]2DG method measures the uptake and
phosphorylation of [14C]2DG to [14C]2DG-6P over a given period of time, usually 45 min, and represents the sum of the glycolytic and oxidative pathways (Sokoloff, 1982; Sokoloff et al., 1977). In contrast, the levels of the enzymes of glucose metabolism, i.e., LDH and CO, are regulated according to glycolytic and oxidative metabolic needs, respectively, during the hours or days before tissue is obtained (Wong-Riley, 1989). Thus, the two methods reveal cerebral activity through different temporal and metabolic windows. In the present study, this is not a limitation since the data from the [14C]2DG study and measurements of enzyme activity were both measured under basal conditions. Moreover, we observed a very good correlation between the degree of activation of cerebral glucose utilization and the activity of the glycolytic and oxidative pathway at the regional level in GAERS compared to NE rats. These data confirm the presence of a generalized change in the characteristics of cerebral glucose metabolism in this strain that is triggered by the ubiquitous mutation(s) that underlies the expression of SWDs in GAERS. At this time, the progress on the genetic analysis of this strain points to mutations on different chromosomes but the exact mutations have not yet been identified (G. Rudolf, personal communication). CO and LDH could also be considered markers of synaptic activity. Thus, strong excitatory input (Kageyama and Wong-Riley, 1982; Mjaatvedt and Wong-Riley, 1982) and high action potential frequency (Kageyama and Wong-Riley, 1982; Villablanca et al., 1999) have been associated with high CO levels. Ionic currents, which are mainly involved in synaptic activity, are thought to be of major importance because approximately 40 – 60% of the ATP produced in brain is used for ion pumping by Na⫹-K⫹ATPase (Clarke and Sokoloff, 1999). Furthermore, Na⫹K⫹-ATPase and CO are regulated in parallel by neuronal functional activity (Hevner et al., 1992). It has also been shown that synaptic reorganization in the hippocampus following lesions of the entorhinal cortex leads to changes in the laminar distribution of LDH (Borowsky and Collins, 1989b) and further that CO and LDH activities measured by histochemistry are regionally and developmentally regulated (Bilger and Nehlig, 1991). Taken together, these studies show that levels of CO and LDH reflect the intensity or pattern of synaptic activity. However, there is still a discussion about the existence of a coupling between neuronal spiking, synaptic activity, and energy supply (Lauritzen, 2001). In fact, it has been reported that functional activity changes depend on the afferent input function (all aspects of presynaptic and postsynaptic processing) but are totally independent of the efferent function (the spike rate in a given region) (Lauritzen, 2001). The absence of direct correlation between spiking and increase in brain functional activity (Lauritzen, 2001) is further confirmed in the present study since there is no metabolic difference between the regions like the cortex and thalamus that express SWDs and areas like limbic regions and brainstem that do not. In conclusion, the data of the present study confirm the
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generalized increase in cerebral glucose metabolism in GAERS, both at the glycolytic and at the oxidative step. However, they still do not allow us to understand why this (these) ubiquitous mutation(s) generates SWDs only in the thalamocortical circuit. Although there are no specific constitutive changes in the GABA and glutamate receptors, some slight changes have been evidenced at the level of glutamate transporters (Dutuit et al., 2002; Ingram et al., 2000) and enzymes of glutamate metabolism (Dutuit et al., 2000), specifically in the thalamus and cortex. Thus, it seems that subtle changes in the balance of glutamate and GABA neurotransmission may underlie the generation of SWDs in these regions particularly dependent on these amino acid neurotransmissions (Dufour et al., 2001). We suggest that a metabolic dysregulation of energy metabolism may favor a defect in cerebral environment of GAERS, which could generate a specific balance favoring the expression of SWDs.
Acknowledgments This work was supported by grants from the Institut National de la Sante´ et de la Recherche Me´ dicale (INSERM U398) and from the Fondation pour la Recherche Me´ dicale (Paris, France).
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Theodore, W.H., 1988. Cerebral metabolism in absence seizures and related syndromes, in: Myslobodsky, M.S., Mirsky, A.F. (Eds.), Elements of Petit Mal Epilepsy. Lang, New York, pp. 131–157. Theodore, W.H., Brooks, R., Margolin, R., Patronas, M., Sato, S., Porter, R.J., Mansi, L., Bairamian, D., Dichiro, G., 1985. Positron emission tomography in generalized seizures. Neurology 35, 684 – 690. Tuor, U.I., Kurpita, G., Simone, C., 1994. Correlation of local changes in cerebral blood flow, capillary density, and cytochrome oxidase during development. J. Comp. Neurol. 342, 439 – 448. Villablanca, J.R., Schmanke, T.D., Hovda, D.A., 1999. Effects of a restricted unilateral neocortical lesion upon cerebral glucose and oxidative metabolisms in fetal and neonatal cats. Dev. Brain Res. 117, 1–13. Wong-Riley, M.T.T., 1979. Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry. Brain Res. 171, 11–28. Wong-Riley, M.T.T., 1989. Cytochrome oxidase: an endogenous metabolic marker for neuronal activity. Trends Neurosci. 12, 94 –101. Wong-Riley, M., Carroll, E.W., 1984. Effect of impulse blockade on cytochrome oxidase activity in monkey visual cortex. Nature 307, 262–264. Wong-Riley, M.T.T., Welt, C., 1980. Histochemical changes in cytochrome oxidase of cortical barrels after vibrissae removal in neonatal and adult mice. Proc. Natl. Acad. Sci. USA 77, 2333–2337.
Experimental Neurology 182 (2003) 346 –352
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Basal levels of metabolic activity are elevated in Genetic Absence Epilepsy Rats from Strasbourg (GAERS): measurement of regional activity of cytochrome oxidase and lactate dehydrogenase by histochemistry Franck Dufour, Estelle Koning, and Astrid Nehlig* INSERM U398, Universite´ Louis Pasteur, Strasbourg, France Received 28 May 2002; revised 24 October 2002; accepted 9 December 2002
Abstract The Genetic Absence Epilepsy Rats from Strasbourg (GAERS) are considered an isomorphic, predictive, and homologous model of human generalized absence epilepsy. It is characterized by the expression of spike-and-wave discharges in the thalamus and cortex. In this strain, basal regional rates of cerebral glucose utilization measured by the quantitative autoradiographic [14C]2-deoxyglucose technique display a widespread consistent increase compared to a selected strain of genetically nonepileptic rats (NE). In order to verify whether these high rates of glucose metabolism are paralleled by elevated activities of the enzymes of the glycolytic and tricarboxylic acid cycle pathways, we measured by histochemistry the regional activity of the two key enzymes of glucose metabolism, lactate dehydrogenase (LDH) for the anaerobic pathway and cytochrome oxidase (CO) for the aerobic pathway coupled to oxidative phosphorylation. CO and LDH activities were significantly higher in GAERS than in NE rats in 24 and 28 of the 30 brain regions studied, respectively. The differences in CO and LDH activity between both strains were widespread, affected all brain systems studied, and ranged from 12 to 63%. The data of the present study confirm the generalized increase in cerebral glucose metabolism in GAERS, occurring both at the glycolytic and at the oxidative step. However, they still do not allow us to understand why the ubiquitous mutation(s) generates spike-and-wave discharges only in the thalamocortical circuit. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Absence epilepsy; Seizures; Spike-and-wave discharges; Glucose metabolism; Cytochrome oxidase; Lactate dehydrogenase; Histochemistry
Introduction The Genetic Absence Epilepsy Rats from Strasbourg (GAERS) are considered an isomorphic, predictive, and homologous model of human generalized absence epilepsy (Danober et al., 1998). In a previous work, we showed that basal regional rates of cerebral glucose utilization measured by the quantitative autoradiographic [14C]2-deoxyglucose (2DG) technique (Sokoloff et al., 1977) display a widespread consistent increase in these animals compared to a selected strain of genetically nonepileptic rats (NE). How* Corresponding author. INSERM U 398, Faculte´ de Me´decine, 11 rue Humann, 67085 Strasbourg Cedex, France. Fax: ⫹33-390.24.32.48. E-mail address: [email protected] (A. Nehlig).
ever, these levels of brain energy metabolism were measured in a mixed state during which seizures did occur spontaneously and represented a mean duration of about 16 s per min (Nehlig et al., 1991, 1992). Further studies on GAERS showed that the induction of absence status epilepticus decreased the levels of energy metabolism and suppressed the strain difference between GAERS and NE. On the other hand, the inhibition of occurrence of spike-andwave discharges (SWDs) by ethosuximide maintained regional rates of cerebral energy metabolism at a level at least as high and rather higher than in untreated GAERS (Nehlig et al., 1992, 1993). We also found that rates of cortical blood flow assessed by laser-Doppler flowmetry were reduced during the occurrence of SWDs in GAERS (Nehlig et al., 1996). The results from these studies are in accordance
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F. Dufour et al. / Experimental Neurology 182 (2003) 346 –352
with human data obtained with the [18F]fluorodeoxyglucose technique in positron emission tomography (PET) studies in patients with typical childhood absence epilepsy. In these patients, cerebral metabolism is diffusely and massively increased when absence seizures occur during the PET scan (Engel et al., 1982, 1985, 1988). As in our model, in human patients, no metabolic activation is measured when seizures turn to status epilepticus (Theodore, 1988; Theodore et al., 1985). Likewise, the use of the Doppler technique in human patients showed a drop in rates of cerebral blood flow in the middle cerebral artery during the occurrence of SWDs (Bode, 1992; Nehlig et al., 1996; Sanada et al., 1988). Thus, it seems that in absence epilepsy, the seizures are not responsible for the high metabolic level that could rather be attributed to interictal mechanisms requiring enough energy to stop the seizures and prevent their occurrence and their spread to limbic and motor seizures (Nehlig et al., 1991, 1992, 1993). To try to gain further knowledge of the characteristics of cerebral glucose metabolism in GAERS compared with NE rats, in the present study we measured by histochemistry the regional activity of the two key enzymes of glucose metabolism, lactate dehydrogenase (LDH) as the final enzyme of the anaerobic pathway and cytochrome oxidase (CO) as the enzyme of the final step of oxidative phosphorylation. The purpose of the present study was twofold: first, to verify whether the elevated rates of glucose metabolism measured by [14C]2DG technique in GAERS compared to NE were paralleled with high activities of the enzymes of the glycolytic and tricarboxylic acid cycle pathways; and second, to identify whether the mutation(s) that affects all brain cells in this strain and underlies the occurrence of SWDs in GAERS but allows the expression of SWDs only in the thalamocortical loop translates into changes in the level of activity of CO and LDH in all brain regions, as noted with the [14C]2DG technique in this strain (Nehlig et al., 1991, 1992, 1993), or only in the thalamic and cortical regions that are able to express SWDs.
Methods Animals The experiments were performed on seven adult male GAERS and nine adult male NE rats, 6 months old, originating from our breeding colony. The animals were maintained at 22°C room temperature under a 12-h/12-h normal light/dark cycle (lights on at 7:00 AM) with food and water ad libitum. Under those conditions, animals exhibit spontaneous SWDs alternating with periods of normal background EEG activity. The mean duration of SWDs reached 20 –24 s/min. All animal experimentation was performed in accordance with the rules of the European Communities Council Directive of November 24, 1986 (86/609/EEC), and the French Department of Agriculture (License No. 67–97).
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Histochemical measurement of cytochrome oxidase and lactate dehydrogenase activities For the histochemical measurement of CO and LDH activities, rats were deeply anesthetized with pentobarbital and immediately sacrificed by decapitation. This procedure was used to prevent any possible effect of the anesthesia on enzyme activity. Brains were dissected out and frozen in methylbutane chilled to ⫺30°C. Coronal brain sections of 20-m thickness obtained in a cryostat at 11 preselected brain levels were mounted onto gelatin- coated glass slides. Sections were taken at the following anteroposterior levels: prefrontal cortex, caudate–putamen, globus pallidus, anterior thalamus, dorsal hippocampus, medial thalamus, ventral hippocampus, superior colliculus, inferior colliculus, cerebellar nuclei, cerebellar cortex. For the assessment of either CO or LDH activity, all sections of all rat brains (three slides for each animal) were incubated together on the same day and in the same incubation mixture in order to avoid any variation in incubation conditions for the final measurement of optical density readings representative of enzyme activities. For the measurement of CO (cytochrome c oxidase; ferrocytochrome c: oxygen reductase, EC 1.9.3.1) activity, diaminobenzidine histochemistry was carried out according to the method of Wong-Riley (Wong-Riley, 1979), modified by Darriett et al. (Darriet et al., 1986). The incubation was performed at 38°C in the dark for 2 h in 0.1 M phosphate buffer, pH 7.4, containing 0.25 mg/ml cytochrome c (grade III, Sigma, St. Louis, MO, USA) and 0.05% 3,3⬘-diaminobenzidine tetrachloride (Sigma). In this reaction, cytochrome c becomes enzymatically reduced by electron transfer from diaminobenzidine. By oxidation, diaminobenzidine polymerizes and precipitates as a brown reaction product. At the end of the incubation, sections were rinsed, dehydrated in successive baths of increasing concentration of ethanol followed by LMR (Labo Moderne, Paris, France), and coverslipped in Histolaque (Labo Moderne). For the demonstration of LDH (EC 1.1.1.27), the method of Jacobsen (Jacobsen, 1969), modified by Borowsky and Collins (Borowsky and Collins, 1989a) was used. The incubation was carried out for 5 min at 37°C in 50 mM Tris–HCl buffer, pH 7.0, containing 100 mM DL-lactate (Sigma), 4 mM NAD⫹ (grade II, Boehringer Mannheim, Germany), 4.9 mM (0.4%) nitroblue tetrazolium, 0.33 mM phenazine methosulfate, and 10% (w/v) polyvinyl alcohol. This reaction utilizes phenazine methosulfate as electron carrier. Lactate oxidation leads to the formation of NADH ⫹ H⫹ which reduces nitroblue tetrazolium and generates blue formazan. Incubation time is shortened as much as possible and the presence of polyvinyl alcohol restricts movement of the tetrazolium reaction product. After incubation, sections were rinsed in water and fixed in 10% neutral formalin. They were then washed with distilled water and coverslipped in aquovitrex (Carlo Erba, Milan, Italy).
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For evaluation of both enzyme activities, relative optical density measurements were performed on sections using an image processing system (Biocom 500, Les Ulis, France). This procedure was used since a direct correlation between optical density and spectrophotometric measurements has been shown for both CO and LDH activities (Borowsky and Collins, 1989a; Darriet et al., 1986). Optical density measurements were made bilaterally for each structure anatomically defined according to the stereotaxic atlas of Paxinos and Watson (Paxinos and Watson, 1986). All readings were corrected for background (e.g., glass, inclusion medium, and coverslip) and nonspecific diaminobenzidine (CO) and nitroblue tetrazolium (LDH) reaction by incubation of a set of sections without substrate and cofactors. They always included the whole structure or nucleus of interest and were performed by an observer who was blind to the strain of the animals. Statistical analysis CO and LDH activities were determined in 30 brain regions in GAERS and NE rats. Optical density measurements of enzyme activities in each strain of rats were compared by means of an analysis of variance consisting of a regions ⫻ strains analysis for repeated measures. This analysis of variance was followed by a Scheffe test when the ANOVA indicated a significant effect.
Results Cytochrome oxidase activity As shown in Table 1, CO activities were significantly higher in GAERS than in NE rats in 24 of the 30 brain regions studied. The differences in CO activity between both strains were widespread, affected all brain systems studied, and ranged from 19% in hypothalamus to 63% in posterior and ventrolateral thalamic nuclei. Enzyme activity levels were not significantly different in the caudate–putamen, anteroventral and anterodorsal thalamic nuclei, substantia nigra pars reticulata, subthalamic nucleus, and red nucleus from GAERS and NE rats. Lactate dehydrogenase activity As shown in Table 2, LDH activities were significantly higher in GAERS than in NE rats in all brain regions studied, except two areas, namely the occipital cortex and the mediodorsal thalamic nucleus. The differences in LDH activity between both strains were widespread, affected all brain systems studied, and ranged from 12% in the thalamic medial geniculate nucleus to 52% in perirhinal cortex.
Table 1 Activity of cytochrome oxidase in various cerebral regions of GAERS and NE rats NE rats (n ⫽ 9) Cerebral cortex Frontal Cingulate Parietal Temporal Entorhinal Perirhinal Occipital Forebrain Hippocampal CA1 area, pyramidal cell layer Amygdala Caudate–putamen Globus pallidus Hypothalamus Thalamus Anteroventral Anterodorsal Ventrolateral Mediodorsal Laterodorsal Ventromedian Ventroposterior Posterior nuclei Reticular Medial geniculate Lateral geniculate Midbrain–Brainstem Substantia nigra pars reticulata Subthalamic nucleus Red nucleus Superior colliculus, superficial layer Inferior colliculus Pontine reticular nucleus, oral part Cerebellar cortex, granule cell layer
GAERS rats (n ⫽ 7)
% of variation from NE
8.70 ⫾ 0.74 9.72 ⫾ 0.86 9.11 ⫾ 0.74 7.14 ⫾ 0.62 6.25 ⫾ 0.64 7.21 ⫾ 0.68 8.63 ⫾ 0.84
11.10 ⫾ 0.26** 11.87 ⫾ 0.17* 11.31 ⫾ 0.34* 9.80 ⫾ 0.28** 8.87 ⫾ 0.24** 10.24 ⫾ 0.37** 11.50 ⫾ 0.48**
⫹28 ⫹22 ⫹24 ⫹37 ⫹42 ⫹42 ⫹33
6.65 ⫾ 0.75
9.29 ⫾ 0.24**
⫹40
7.46 ⫾ 0.66 8.43 ⫾ 0.89 4.79 ⫾ 0.64 8.23 ⫾ 0.54
9.70 ⫾ 0.24** 9.82 ⫾ 0.19 6.86 ⫾ 0.23** 9.81 ⫾ 0.32*
⫹30 ⫹17 ⫹43 ⫹19
10.48 ⫾ 0.83 11.79 ⫾ 0.96 6.25 ⫾ 0.89 7.03 ⫾ 0.63 8.32 ⫾ 0.80 7.26 ⫾ 0.61 8.50 ⫾ 0.73 6.62 ⫾ 0.82 7.81 ⫾ 0.72 6.42 ⫾ 0.68 7.93 ⫾ 0.66
11.99 ⫾ 0.27 13.15 ⫾ 0.31 10.20 ⫾ 0.35** 9.15 ⫾ 0.21** 10.37 ⫾ 0.27* 9.28 ⫾ 0.31** 11.24 ⫾ 0.38** 10.81 ⫾ 0.37** 9.47 ⫾ 0.24* 8.09 ⫾ 0.20* 10.38 ⫾ 0.38**
⫹14 ⫹12 ⫹63 ⫹30 ⫹25 ⫹28 ⫹32 ⫹63 ⫹21 ⫹26 ⫹31
7.86 ⫾ 0.85
9.27 ⫾ 0.26
⫹18
11.78 ⫾ 0.80 8.81 ⫾ 0.78 8.60 ⫾ 0.68
13.88 ⫾ 0.33 8.09 ⫾ 0.56 11.17 ⫾ 0.45**
⫹18 ⫺8 ⫹30
10.96 ⫾ 0.95 5.95 ⫾ 0.92
13.30 ⫾ 0.36* 9.45 ⫾ 0.39**
⫹21 ⫹59
8.56 ⫾ 0.81
11.14 ⫾ 0.28**
⫹30
Note. Values, expressed as optical density ⫻100, are means ⫾ SD of the number of animals in parentheses. * P ⬍ 0.05, ** P ⬍ 0.01, statistically significant differences between GAERS and NE rats.
Discussion The data of the present study show that the elevated levels of cerebral glucose utilization previously measured in GAERS (Nehlig et al., 1991, 1992) are paralleled by elevated activities of the enzymes of anaerobic and oxidative cerebral glucose metabolism. Both rates of glucose utilization (Nehlig et al., 1991, 1992) and the activity of the enzymes of its aerobic and anaerobic degradation (the present study) are diffusely increased all over the brain. Thus, the ubiquitous mutation(s) that underlies the expression of SWDs in GAERS translates into a ubiquitous change
F. Dufour et al. / Experimental Neurology 182 (2003) 346 –352 Table 2 Activity of lactate dehydrogenase in various cerebral regions of GAERS and NE rats
Cerebral cortex Frontal Cingulate Parietal Temporal Entorhinal Perirhinal Occipital Forebrain Hippocampal CA1 area, pyramidal cell layer Amygdala Caudate–putamen Globus pallidus Hypothalamus Thalamus Anteroventral Anterodorsal Ventrolateral Mediodorsal Laterodorsal Ventromedian Ventroposterior Posterior nuclei Reticular Medial geniculate Lateral geniculate Midbrain–brainstem Substantia nigra pars reticulata Subthalamic nucleus Red nucleus Superior colliculus, superficial layer Inferior colliculus Pontine reticular nucleus, oral part Cerebellar cortex, granule cell layer
NE rats (n ⫽ 9)
GAERS rats (n ⫽ 7)
% of variation from NE
5.11 ⫾ 0.32 5.08 ⫾ 0.53 5.45 ⫾ 0.29 6.54 ⫾ 0.35 6.65 ⫾ 0.34 5.31 ⫾ 0.39 5.28 ⫾ 0.53
6.69 ⫾ 0.29** 6.38 ⫾ 0.38* 6.48 ⫾ 0.20** 8.69 ⫾ 0.31** 9.06 ⫾ 0.30** 8.08 ⫾ 0.34** 5.98 ⫾ 0.48
⫹31 ⫹25 ⫹19 ⫹37 ⫹36 ⫹52 ⫹13
8.33 ⫾ 0.41
9.89 ⫾ 0.24**
⫹19
7.83 ⫾ 0.50 4.98 ⫾ 0.34 4.29 ⫾ 0.33 4.73 ⫾ 0.21
9.20 ⫾ 0.21* 6.35 ⫾ 0.19** 6.32 ⫾ 0.29** 6.43 ⫾ 0.16**
⫹18 ⫹28 ⫹47 ⫹36
4.91 ⫾ 0.41 4.82 ⫾ 0.44 4.44 ⫾ 0.35 6.72 ⫾ 0.37 5.80 ⫾ 0.14 5.94 ⫾ 0.25 5.70 ⫾ 0.25 5.20 ⫾ 0.23 4.96 ⫾ 0.41 5.43 ⫾ 0.28 4.59 ⫾ 0.33
6.46 ⫾ 0.35** 6.71 ⫾ 0.62** 6.57 ⫾ 0.41** 7.16 ⫾ 0.44 7.12 ⫾ 0.17** 7.44 ⫾ 0.28** 7.12 ⫾ 0.28** 6.84 ⫾ 0.27** 6.46 ⫾ 0.40* 6.05 ⫾ 0.14* 6.15 ⫾ 0.34**
⫹32 ⫹39 ⫹48 ⫹7 ⫹23 ⫹25 ⫹25 ⫹32 ⫹30 ⫹12 ⫹34
4.37 ⫾ 0.27
6.08 ⫾ 0.21**
⫹39
4.69 ⫾ 0.20 4.87 ⫾ 0.31 5.03 ⫾ 0.33
6.52 ⫾ 0.27** 6.00 ⫾ 0.17** 6.72 ⫾ 0.37**
⫹39 ⫹23 ⫹34
4.66 ⫾ 0.33 4.55 ⫾ 0.36
5.79 ⫾ 0.47* 6.01 ⫾ 0.46**
⫹24 ⫹32
3.63 ⫾ 0.43
5.19 ⫾ 0.48**
⫹43
349
ments of CO activity by diaminobenzidine histochemistry, as performed in the present study and the activity of the enzyme measured spectrophotometrically, has been established (Darriet et al., 1986). The two latter measurements of enzyme activity are performed in vitro under optimal environmental conditions mainly of pH, substrate, and cofactor availability and thus reflect the total enzyme activity present in the tissue but not the actual activity under in situ conditions. However, these measurements of enzyme activity have been shown to closely reflect the long-term functional use of the pathways of aerobic glucose metabolism. A direct positive correlation has been established between capillary
Note. Values, expressed as optical density ⫻100, are means ⫾ SD of the number of animals in parentheses. * P ⬍ 0.05, ** P ⬍ 0.01, statistically significant differences between GAERS and NE rats.
in the characteristics of cerebral glucose metabolism in this strain. Moreover, as shown in Fig. 1, in most brain regions, whether they express SWDs (cortex, thalamus) or not, the increases in enzyme activities and rates of glucose utilization are quite similar. A few exceptions can be noted; for example, in mediodorsal and posterior thalamic nuclei mainly, increases in glucose metabolism rates are quite larger than increases in LDH and CO activity while the reverse is true in parietal cortex and substantia nigra pars reticulata. The functional significance of these differences remains to be clarified. A close correlation between optical density measure-
Fig. 1. Activity of cytochrome oxidase (CO) and lactate dehydrogenase (LDH) and rates of glucose utilization (GLU) in various brain areas of GAERS. Values represent percentages of increase over control levels in NE rats. Abbreviations used: FTAL, frontal cortex; ACING, anterior cingulate cortex; PAR, parietal cortex; TEMP, temporal cortex; ENT, entorhinal cortex; AV, anteroventral thalamus; MD, mediodorsal thalamus; LD, laterodorsal talamus; VM, ventromedian thalamus; PO, posterior thalamus; CA1, CA1 layer of the hippocampus; AMY, amygdala; CAU, caudate nucleus; SNPR, substantia nigra pars reticulata; CBCX, cerebellar cortex. *P ⬍ 0.05; #P ⬍ 0.01, statistically significant differences between GAERS and NE rats.
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density and CO activity or cerebral glucose utilization in the rat (Borowsky and Collins, 1989a; Klein et al., 1986). Conversely, the relationship between LDH activity and capillary density is a negative one, raising the possibility that brain regions with a high glycolytic capacity may require fewer capillaries (Borowsky and Collins, 1989a). In the present study, we found also discrepancies in the activity of the two enzymes. The highest levels of CO activity were mostly found in sensory and motor systems and lower levels were recorded in limbic and associative areas (Table 1), as previously shown (Bilger and Nehlig, 1991; Hevner et al., 1995). On the other hand, LDH activity was especially high in limbic and associative regions such as hippocampus, amygdala, and some thalamic nuclei and low in motor regions (Table 2). Thus, the data from the literature and the present study are in favor of the hypothesis that the different brain regions are composed of a mixture of oxidative and glycolytic zones reflecting cellular heterogeneity, as well as a heterogeneous pattern of innervation (Borowsky and Collins, 1989a). Energy utilization by the brain is tightly coupled to neuronal functional activity (for review see (Clarke and Sokoloff, 1999; DiRocco et al., 1989; Erecinska and Silver, 1989)). Mature neurons are completely dependent on glucose as their sole energy substrate and on oxidative phosphorylation for the final production of ATP. Moreover, the brain has almost no energy reserves in the form of glycogen or fat, and even a brief interruption of substrate supply will be detrimental to brain function and cellular integrity. These biochemical properties, together with the high metabolic demand of the brain linked to neuronal activity, are responsible for the tight coupling between neuronal activity and oxidative energy metabolism in the brain. There is considerable available experimental evidence that the rates of cerebral glucose utilization measured by the quantitative [14C]2DG technique are tightly coupled to cerebral functional activity (for review see (Clarke and Sokoloff, 1999; Sokoloff, 1982)). Likewise, a clear coupling between brain functional activity and CO activity has been demonstrated (for review see (Wong-Riley, 1989)). Thus, CO histochemistry, representative of the full oxidative degradation of glucose, has been extensively used to map the functional activity in the normal adult brain (Borowsky and Collins, 1989a; Darriet et al., 1986; Gonzalez-Pardo et al., 1996; Hevner et al., 1995), and during development and aging (Curti et al., 1990; Hovda and Villablanca, 1998; Tuor et al., 1994). Decreases in CO activity have been described after visual deprivation (Rosa et al., 1991; Wong-Riley and Carroll, 1984), vibrissae removal, or deafferentation (Borowsky and Collins, 1989b; Villablanca et al., 1999; Wong-Riley and Welt, 1980), while increases in CO activity were recorded during recovery from sensory deprivation (Dietrich et al., 1982). In summary, CO and [14C]2DG are both metabolic markers but they do not always produce identical results. There are major differences between the two techniques. The [14C]2DG method measures the uptake and
phosphorylation of [14C]2DG to [14C]2DG-6P over a given period of time, usually 45 min, and represents the sum of the glycolytic and oxidative pathways (Sokoloff, 1982; Sokoloff et al., 1977). In contrast, the levels of the enzymes of glucose metabolism, i.e., LDH and CO, are regulated according to glycolytic and oxidative metabolic needs, respectively, during the hours or days before tissue is obtained (Wong-Riley, 1989). Thus, the two methods reveal cerebral activity through different temporal and metabolic windows. In the present study, this is not a limitation since the data from the [14C]2DG study and measurements of enzyme activity were both measured under basal conditions. Moreover, we observed a very good correlation between the degree of activation of cerebral glucose utilization and the activity of the glycolytic and oxidative pathway at the regional level in GAERS compared to NE rats. These data confirm the presence of a generalized change in the characteristics of cerebral glucose metabolism in this strain that is triggered by the ubiquitous mutation(s) that underlies the expression of SWDs in GAERS. At this time, the progress on the genetic analysis of this strain points to mutations on different chromosomes but the exact mutations have not yet been identified (G. Rudolf, personal communication). CO and LDH could also be considered markers of synaptic activity. Thus, strong excitatory input (Kageyama and Wong-Riley, 1982; Mjaatvedt and Wong-Riley, 1982) and high action potential frequency (Kageyama and Wong-Riley, 1982; Villablanca et al., 1999) have been associated with high CO levels. Ionic currents, which are mainly involved in synaptic activity, are thought to be of major importance because approximately 40 – 60% of the ATP produced in brain is used for ion pumping by Na⫹-K⫹ATPase (Clarke and Sokoloff, 1999). Furthermore, Na⫹K⫹-ATPase and CO are regulated in parallel by neuronal functional activity (Hevner et al., 1992). It has also been shown that synaptic reorganization in the hippocampus following lesions of the entorhinal cortex leads to changes in the laminar distribution of LDH (Borowsky and Collins, 1989b) and further that CO and LDH activities measured by histochemistry are regionally and developmentally regulated (Bilger and Nehlig, 1991). Taken together, these studies show that levels of CO and LDH reflect the intensity or pattern of synaptic activity. However, there is still a discussion about the existence of a coupling between neuronal spiking, synaptic activity, and energy supply (Lauritzen, 2001). In fact, it has been reported that functional activity changes depend on the afferent input function (all aspects of presynaptic and postsynaptic processing) but are totally independent of the efferent function (the spike rate in a given region) (Lauritzen, 2001). The absence of direct correlation between spiking and increase in brain functional activity (Lauritzen, 2001) is further confirmed in the present study since there is no metabolic difference between the regions like the cortex and thalamus that express SWDs and areas like limbic regions and brainstem that do not. In conclusion, the data of the present study confirm the
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generalized increase in cerebral glucose metabolism in GAERS, both at the glycolytic and at the oxidative step. However, they still do not allow us to understand why this (these) ubiquitous mutation(s) generates SWDs only in the thalamocortical circuit. Although there are no specific constitutive changes in the GABA and glutamate receptors, some slight changes have been evidenced at the level of glutamate transporters (Dutuit et al., 2002; Ingram et al., 2000) and enzymes of glutamate metabolism (Dutuit et al., 2000), specifically in the thalamus and cortex. Thus, it seems that subtle changes in the balance of glutamate and GABA neurotransmission may underlie the generation of SWDs in these regions particularly dependent on these amino acid neurotransmissions (Dufour et al., 2001). We suggest that a metabolic dysregulation of energy metabolism may favor a defect in cerebral environment of GAERS, which could generate a specific balance favoring the expression of SWDs.
Acknowledgments This work was supported by grants from the Institut National de la Sante´ et de la Recherche Me´ dicale (INSERM U398) and from the Fondation pour la Recherche Me´ dicale (Paris, France).
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