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Neuraminidase activity in different regions of the seizing epileptic and non-epileptic brain
Brain Research 964 (2003) 211–217 www.elsevier.com / locate / brainres
Research report
Neuraminidase activity in different regions of the seizing epileptic and non-epileptic brain * ´ ´ Alfonso Boyzo, Jose´ Ayala, Rafael Gutierrez, Jorge Hernandez-R ´ , Biof ´ısica y Neurociencias, Centro de Investigacion ´ y de Estudios Avanzados del IPN, Apartado Postal 14 -740, Departamento de Fisiologıa Mexico D.F. 07000, Mexico Accepted 16 October 2002
Abstract The sialic acid in the brain is split from sialoglucoconjugates by sialidases (neuraminidases, EC 3.2.1.18), and is postulated to act as an inhibitor of cellular adhesion and to play a role in various membrane functions. Since epilepsy alters cellular interactions and connectivity, it is reasonable to propose that sialidases can be affected by this pathological state or, alternately, by seizures. Therefore, we studied the activity of total, soluble, and membranal sialidases in various brain regions in normal, kindled epileptic and non-epileptic seizing rats. The results showed that in kindled rats, the total activity of the sialidases significantly decreased in cerebral cortex (11.38%) and cerebellum (28.58%), whereas it increased in brainstem (35.51%), hypothalamus (2.88%) and hippocampus (9.37%). The activity of the membranous sialidases in kindled rats followed the same pattern as the total activity, whereas the activity of soluble sialidase was significantly lower than membranous activity. Interestingly, the activity of total and membranal sialidases in non-epileptic seizing rats paralleled that observed in kindled rats. We suggest that the seizure-induced decrease of sialidasic activity may not modify the number of sialic acid molecules bound to gangliosides in cell membranes, as compared to areas of increased activity, that may decrease them. These changes in sialidases’ activity may reflect functional disturbances of membrane polysialylated gangliosides related to the functional and anatomical plastic changes associated to seizures. Our data indicate that these changes are related to the presence of seizures rather than to an established epileptic state. 2002 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Epilepsy: human studies and animals models Keywords: Brain sialidase; Neuraminidase; Epilepsy; Sialic acid; Kindling; Seizure
1. Introduction More than 50% of the sialic acid in the central nervous system (SNC) is related to sialoglucoconjugated macromolecules—sialoglucosphingolipids, sialoglucoproteins, sialogangliosides and polysialylated carbohydrates [44]. Sialic acid is an anionic molecule bound to the membranous polysialogangliosides. It is also abundant in the extracellular domain (HNK-1) of the cellular adhesion molecules (CAMs) [2]—the neural CAM (N-CAM), the axonic-glial CAM (MAG) and the neuroglia CAM (NgCAM, L1)—and in other molecules like glycoprotein of cell adhesion between neurites D2 [46], the a subunit of *Corresponding author. Fax: 151-78-5747-3754. ´ E-mail address: [email protected] (J. Hernandez-R).
the insulin receptor and the sialoglycoprotein clusterin [17]. In relation to its sialic acid content, N-CAM is subjected to a developmentally regulated post-translational modification [37]. The sialic acid has a highly regulated expression pattern and it is closely related to axonal growth [18,48], to the recognition of target cells, and to the construction of neuromuscular junctions during embryonic development, when it is particularly abundant. During postnatal development and in the mature brain, the expression of sialic acid is more restricted and is mainly associated to regions where clear morphological or physiological plasticity occurs, such as the hippocampus, the olfactory bulb and some hypothalamic nuclei [28,61,53]. It has been postulated that sialic acid exposed to cell surfaces acts as a global inhibitor of cell adhesiveness and it affects cellular interactions [50,6,35,30]. When the sialic acid is
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03985-9
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removed by sialidases, the speed of aggregation among neuronal membranes increases significantly [19,50,1], suggesting that its activity can be involved in cellular interactions and, therefore, in plastic phenomena of neural networks [51,15]. Furthermore, the expression of sialic acid after lesions of the fimbria-fornix suggests that it is upregulated in regenerating axons during axonal elongation and downregulated upon target innervation [2]. Moreover, in areas of reactive gliosis it may contribute to a permissive environment for axonal regrowth [2]. Thus, sialic acid seems to be a factor involved in morphological plasticity as a permissive factor that allows neuronal and glial remodeling to occur when the proper inductive stimulus intervenes [60]. Sialidases (neuraminidase, EC 3.2.1.18) constitute a family of glycosidases that catalyze the hydrolysis of sialic acid [5,38] in sialoglucoconjugated substrates, including sialoglucoproteins, polysialylated carbohydrates and gangliosides that liberate a-glucosides [21,9]. In mammals they are present as soluble enzymes in the cytoplasm or bound to lisosomes and to plasmatic membrane, including synaptic, myelin and nuclear membranes [52,14,11]. The activity of the sialidases in the regulation of the polysialylation state of molecules as N-CAM, L1, D2, MAG, gangliosides and clusterin, provides an example of their morphogenic effects, whenever regulated adhesion and synaptic activity depend on the sialylation state [40,49,26]. In the case of the gangliosides, their complex structural transformations during development support their proposed functional role [43,18]. On the other hand, it has been proposed that gangliosides play a role in the epileptic phenomenon, since antiganglioside antibodies induce epileptiform activity [23]. During seizures, and especially in the presence of an epileptic state, a number of changes in ion channels expression and function, extracellular and intracellular ion buffering, syntapic transmission, neurotransmitter release modulation, genetic expression and anatomical reorganization occur [57,12,7,33,34,3,47,10,58,16]. Many of these may involve sialylated molecules [63,48,60,15]. Considering that the epileptic state produces changes in the levels of cerebral gangliosides in cellular adhesion molecules and their sialylation state [65,22,59,4,24], we decided to evaluate the total, soluble and membranous activities of sialidases after a single pentylenetetrazol (PTZ)-induced seizure and after several kindled seizures, in which a permanent epileptic state has been developed.
stimulated with a 1-s train of square pulses (0.1 ms pulse duration; 60 Hz; 500 mA) until five generalized convulsive seizures were evoked [45]. Acute seizures were induced with a single i.p. administration of pentylenetetrazol (PTZ; 70 mg / kg) [16,25]. All experimental procedures were approved by the Committee on Ethical Animal Research of our Institution and of the Ministry of Health. Five minutes after the seizure, the rats were sacrificed by cervical dislocation and the brains were removed and rapidly dissected at 4 8C. The following regions were dissected: cerebral cortex, cerebellum, mesencephalon, medulla and pons, hypothalamus, hippocampus and olfactory bulb. Each region was homogenized in 10 volumes of cold of Tris–acetate 0.05 M, pH 7.4, solution. The total enzymatic activity of the sialidases was measured in triplicate samples from each cerebral region. The soluble and membrane fractions were separated as previously described [62]. The basal enzymatic activity was obtained from the cerebral cortex of normal rats. Aliquots of 30 mg of proteins from supernatants or precipitates were incubated in buffer solution of Tris– acetate 0.1 M, pH 3.9 at 37 8C for 15 min, in the presence of methylumbelliferyl N-acetylneuraminic acid 1 mM (MUB-NacNeu) as the substrate, in a final volume of 200 ml. To stop the enzymatic reaction, 3 ml of buffer solution of glycine buffer 0.2 M, were added to yield a pH of 10.5. The product of the reaction was quantified in a spectrophotofluorometer (Perkin Elmer LS-50B) at an excitation and emission wavelengths of 365 and 445 nm, respectively. The specific activity of sialidases is expressed as liberated sialic acid in nmol per mg protein per hour [42,8]. Protein concentration was determined by the method of Lowry et al. [29]. The changes of the enzymatic activity among the groups are expressed as the mean6S.D. computed from triplicate determinations from individual experiments of each region. The groups analyzed were: (a) healthy control animals (C, n542), control soluble and membranal (Cs and Cm, n510); (b) rats implanted with an electrode but not stimulated (SH, sham; n538); (c) kindled rats with five generalized convulsive seizures (K, kindling; n557), kindling soluble and membranal (Ks and Km, n512); (d) PTZ-treated rats with a single generalized seizure (PTZ, n57). Statistical comparisons were done with ANOVA and multiple comparisons test of Newman– Keuls, using the program Graph Pad Prism (ver. 3.2).
3. Results 2. Materials and methods Male Wistar rats (230–250 g body weight) were anaesthetized with ketamine (60 mg / kg i.p.) and implanted with bipolar stainless steel electrodes (80 kV) into the left basolateral amygdala (AP 2.5; L 5; H 8.5) [39]. Starting 7–10 days after the operation, the animals were daily
The kindled animals presented the first generalized convulsive seizure after 1362 stimuli and were further stimulated until five seizures were consecutively evoked. The animals injected with PTZ presented a generalized convulsive seizure within the first minute after the injection, which lasted up to 2 min. The total activity of sialidases determined in kindled
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epileptic animals is shown in Fig. 1. A decrease in cerebral cortex (11.38%; P,0.001) and cerebellum (28.58%; P, 0.001) was evident, as compared to the controls. Since no difference between the determinations in pons, medulla and mesencephalon were evident they were pooled together and hereafter reported as brainstem. The total enzymatic activity increased in the brainstem by 31.51% (P,0.001), and in the hippocampus by 9.73% (P,0.001). Interestingly, no changes were detected in the hypothalamus by 2.88% (P.0.05), and the olfactory bulb. Membranal activity diminished in the cerebral cortex (16.94%; P,0.001) (Fig. 2) and in the cerebellum (19.81%; P,0.001), while an increase was evident in the brainstem (30.73%; P,0.01), and hippocampus (10.4%; P,0.001). Again, no changes were observed in the hypothalamus (3.87%; P .0.05) and the olfactory bulb. It is important to notice that the activity of membrane sialidase in kindled rats showed the same pattern of changes as the total activity (see Figs. 1–3), and in all cases such membrane activity was always higher than the soluble one. Soluble sialidase activity did not have significant changes in the cerebral cortex (Fig. 3). In the brainstem and the hypothalamus it showed a non significant increase while the increase observed in the hippocampus were of 12.25% (P,0.05). On the contrary, its activity in the cerebellum diminished by 21.49% (P,0.05).
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In the PTZ group, the enzymatic activities for the different regions are as follows: cerebral cortex total (11.38%), membranal (16.92%), and soluble (7.13%); cerebellum, total (28.58%), membranal (19.81%), and soluble (21.47%); brainstem total (38.14%), membranal (30.73%), and soluble (35.88%); hypothalamus total (2.88%), membranal (3.87%), and soluble (7.49%); hippocampus total (9.73%), membranal (10.29%), and soluble (10.64%). Interestingly, neither of these values differed from the corresponding activity of the kindled group (Figs. 1 and 2).
4. Discussion It is known that the kindled state and seizures themselves are able to produce plastic functional and anatomical changes in several brain regions. Many of these changes are consequence of the epileptic activity, whereas other changes can arise during its establishment. Some promote or support the maintenance of the epileptic activity, while others tend to limit its generation. The changes in neuraminidases activity that we observed in the different brain regions of kindled epileptic and non-epileptic seizing animals are likely to reflect changes in the amount of sialic acid bound to or unbound from the gangliosides or from other polysialylated compounds
Fig. 1. Total activity of sialidases of control (C), sham (SH), kindled (K) and PTZ-treated rats in the different structures analyzed. The activity of sialidases is expressed as liberated sialic acid (N-acetylneuraminic acid, NANA) in nmol / mg protein / h (mean6S.D) computed from triplicate determinations from individual experiments (control, n542; sham, n538; kindling, n557; PTZ, n57 rats). Significance: * P,0.001, ** P,0.05, *** P,0.01; Newman–Keuls test.
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Fig. 2. Membranal (M) sialidase enzymatic activity of control (C), kindled (K) and PTZ-treated rats. The activity of sialidases is expressed as liberated sialic acid (N-acetylneuraminic acid, NANA) in nmol / mg protein / h (mean6S.D.) computed from triplicate determinations from individual experiments (control, n510; kindling, n512; PTZ n57 rats). Significance: * P,0.001, ** P,0.05, *** P,0.01; Newman–Keuls test.
[56,20,41]. From our results we can conclude that the sialidase activity presents regional specificity, whereby the pattern of activity possibly represents functional differences among the cerebral cortex, cerebellum and subcortical structures. During brain development, the sialic acid
bound to membranes is involved in the formation of branching of the cortico-thalamic axons and contribute to the formation of the neocortical layers by inhibiting the formation of contacts in inappropriate areas [27]. It has been shown that when the cerebral cortex is treated with
Fig. 3. Soluble (S) sialidase enzymatic activity of control (C), kindled (K) and PTZ-treated rats. The activity of sialidases is expressed as liberated sialic acid (N-acetylneuraminic acid, NANA) in nmol / mg protein / h (mean6S.D.) computed from triplicate determinations from individual experiments (control, n510; kindling, n512; PTZ n57 rats). Significance: * P,0.001, ** P,0.05, *** P,0.01; Newman–Keuls test.
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endosialidase, the number of branching points is increased significantly, but the length of the buds decreases [64]. The decrease of the enzymatic activity observed in the subcortical areas may be related to a possible increment in the number of sialic acid molecules present in the cell membrane, resulting in functional changes. On the other hand, it has been suggested that the removal of sialic acid by an enhanced sialidase activity may cause an increment in the number of mossy or collateral fibers in the hippocampus [13,32]. It is interesting that in epileptic animals, an aberrant growth of the mossy fibers occurs similar to the appearance of ectopical fibers observed in NCAM mutant animals or in animals treated with specific endosialidase against sialic acid [55,31]. In patients with Alzheimer’s disease, the polysialylated form of NCAM is massively expressed in areas of the hippocampus where neurodegenerative processes have taken place, especially in the dentate gyrus [32]. In the same way, in patients with epilepsy of the temporary lobe, polysialylation is increased in the hippocampus and in the entorhinal cortex, which correlates with the density of buds of mossy fibers [32]. One can propose that these types of changes can be related to the sialidase alterations that we observe in the hippocampus and the cerebral cortex of kindled epileptic and non-epileptic seizing rats. The balance between sialylation and desialylation that depends on sialyltransferase / sialidase activities would determine the course of the morphological rearrangements that possibly emerge as an adaptive response to the convulsive state [65,37,36]. A significant increase of sialidasic activity may have resulted as a consequence of the epileptiform process in the subcortical regions, most likely with a corresponding decrease of sialic acid content in cellular membranes. Some gangliosides, as GM1, located in the external surface of the synaptic membranes have been considered as active receptive molecules during epileptic activity [23]. It is known that the levels of sialic acid bound to gangliosides are increased after epileptic activity in layers III–V of the cerebral cortex, particularly in layer IV [22]. On the other hand, Yu [65] reported that during the epilepsy there is an increment of sialic acid in the cerebral gangliosides GM1, GD1a, GD1b and GT1. In spite of these results, it is difficult to establish a causal relationship between the epileptic activity and the sialylation state. Although we could determine that the activity of sialidases is altered by the epileptic state, it was also important to determine if the convulsive seizures per se were able to produce the same changes. Interestingly, we found that after producing a single generalized convulsive seizure, the sialidase activity presented the same pattern as that observed in the kindled animals, which presented several seizures. This result can be indicative of the involvement of the sialidases in the modulation of subsequent seizures and possibly in the maintenance of a permanent epileptic state.
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It is noteworthy that the activity of the membranal component prevails over the soluble one. Therefore, the modifications in soluble enzymatic activity may indicate the existence of various cellular metabolic changes that need to be further investigated. The changes that we describe may thus be related to compensatory mechanisms put in play by the establishment of the kindled epileptic state, by seizures themselves or by the electrical stimulation that produces the kindling effect. They can represent a consequence of an increased cerebral excitability, and also they can be involved in its progression. Moreover, they can represent a compensatory response to deleterious processes brought up by the seizures, providing an opportunity for the establishment of reinnervation and remodeling of particular circuits [54]. We can conclude that the described changes of neuraminidase activity, mainly in the membranous fraction, may be associated to a functional alteration of related sialylated molecules like gangliosides, possibly involved in the mechanism of the convulsive phenomenon.
Acknowledgements ´ We thank Ignacio Vargas Martınez for excellent technical assistance.
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Research report
Neuraminidase activity in different regions of the seizing epileptic and non-epileptic brain * ´ ´ Alfonso Boyzo, Jose´ Ayala, Rafael Gutierrez, Jorge Hernandez-R ´ , Biof ´ısica y Neurociencias, Centro de Investigacion ´ y de Estudios Avanzados del IPN, Apartado Postal 14 -740, Departamento de Fisiologıa Mexico D.F. 07000, Mexico Accepted 16 October 2002
Abstract The sialic acid in the brain is split from sialoglucoconjugates by sialidases (neuraminidases, EC 3.2.1.18), and is postulated to act as an inhibitor of cellular adhesion and to play a role in various membrane functions. Since epilepsy alters cellular interactions and connectivity, it is reasonable to propose that sialidases can be affected by this pathological state or, alternately, by seizures. Therefore, we studied the activity of total, soluble, and membranal sialidases in various brain regions in normal, kindled epileptic and non-epileptic seizing rats. The results showed that in kindled rats, the total activity of the sialidases significantly decreased in cerebral cortex (11.38%) and cerebellum (28.58%), whereas it increased in brainstem (35.51%), hypothalamus (2.88%) and hippocampus (9.37%). The activity of the membranous sialidases in kindled rats followed the same pattern as the total activity, whereas the activity of soluble sialidase was significantly lower than membranous activity. Interestingly, the activity of total and membranal sialidases in non-epileptic seizing rats paralleled that observed in kindled rats. We suggest that the seizure-induced decrease of sialidasic activity may not modify the number of sialic acid molecules bound to gangliosides in cell membranes, as compared to areas of increased activity, that may decrease them. These changes in sialidases’ activity may reflect functional disturbances of membrane polysialylated gangliosides related to the functional and anatomical plastic changes associated to seizures. Our data indicate that these changes are related to the presence of seizures rather than to an established epileptic state. 2002 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Epilepsy: human studies and animals models Keywords: Brain sialidase; Neuraminidase; Epilepsy; Sialic acid; Kindling; Seizure
1. Introduction More than 50% of the sialic acid in the central nervous system (SNC) is related to sialoglucoconjugated macromolecules—sialoglucosphingolipids, sialoglucoproteins, sialogangliosides and polysialylated carbohydrates [44]. Sialic acid is an anionic molecule bound to the membranous polysialogangliosides. It is also abundant in the extracellular domain (HNK-1) of the cellular adhesion molecules (CAMs) [2]—the neural CAM (N-CAM), the axonic-glial CAM (MAG) and the neuroglia CAM (NgCAM, L1)—and in other molecules like glycoprotein of cell adhesion between neurites D2 [46], the a subunit of *Corresponding author. Fax: 151-78-5747-3754. ´ E-mail address: [email protected] (J. Hernandez-R).
the insulin receptor and the sialoglycoprotein clusterin [17]. In relation to its sialic acid content, N-CAM is subjected to a developmentally regulated post-translational modification [37]. The sialic acid has a highly regulated expression pattern and it is closely related to axonal growth [18,48], to the recognition of target cells, and to the construction of neuromuscular junctions during embryonic development, when it is particularly abundant. During postnatal development and in the mature brain, the expression of sialic acid is more restricted and is mainly associated to regions where clear morphological or physiological plasticity occurs, such as the hippocampus, the olfactory bulb and some hypothalamic nuclei [28,61,53]. It has been postulated that sialic acid exposed to cell surfaces acts as a global inhibitor of cell adhesiveness and it affects cellular interactions [50,6,35,30]. When the sialic acid is
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03985-9
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removed by sialidases, the speed of aggregation among neuronal membranes increases significantly [19,50,1], suggesting that its activity can be involved in cellular interactions and, therefore, in plastic phenomena of neural networks [51,15]. Furthermore, the expression of sialic acid after lesions of the fimbria-fornix suggests that it is upregulated in regenerating axons during axonal elongation and downregulated upon target innervation [2]. Moreover, in areas of reactive gliosis it may contribute to a permissive environment for axonal regrowth [2]. Thus, sialic acid seems to be a factor involved in morphological plasticity as a permissive factor that allows neuronal and glial remodeling to occur when the proper inductive stimulus intervenes [60]. Sialidases (neuraminidase, EC 3.2.1.18) constitute a family of glycosidases that catalyze the hydrolysis of sialic acid [5,38] in sialoglucoconjugated substrates, including sialoglucoproteins, polysialylated carbohydrates and gangliosides that liberate a-glucosides [21,9]. In mammals they are present as soluble enzymes in the cytoplasm or bound to lisosomes and to plasmatic membrane, including synaptic, myelin and nuclear membranes [52,14,11]. The activity of the sialidases in the regulation of the polysialylation state of molecules as N-CAM, L1, D2, MAG, gangliosides and clusterin, provides an example of their morphogenic effects, whenever regulated adhesion and synaptic activity depend on the sialylation state [40,49,26]. In the case of the gangliosides, their complex structural transformations during development support their proposed functional role [43,18]. On the other hand, it has been proposed that gangliosides play a role in the epileptic phenomenon, since antiganglioside antibodies induce epileptiform activity [23]. During seizures, and especially in the presence of an epileptic state, a number of changes in ion channels expression and function, extracellular and intracellular ion buffering, syntapic transmission, neurotransmitter release modulation, genetic expression and anatomical reorganization occur [57,12,7,33,34,3,47,10,58,16]. Many of these may involve sialylated molecules [63,48,60,15]. Considering that the epileptic state produces changes in the levels of cerebral gangliosides in cellular adhesion molecules and their sialylation state [65,22,59,4,24], we decided to evaluate the total, soluble and membranous activities of sialidases after a single pentylenetetrazol (PTZ)-induced seizure and after several kindled seizures, in which a permanent epileptic state has been developed.
stimulated with a 1-s train of square pulses (0.1 ms pulse duration; 60 Hz; 500 mA) until five generalized convulsive seizures were evoked [45]. Acute seizures were induced with a single i.p. administration of pentylenetetrazol (PTZ; 70 mg / kg) [16,25]. All experimental procedures were approved by the Committee on Ethical Animal Research of our Institution and of the Ministry of Health. Five minutes after the seizure, the rats were sacrificed by cervical dislocation and the brains were removed and rapidly dissected at 4 8C. The following regions were dissected: cerebral cortex, cerebellum, mesencephalon, medulla and pons, hypothalamus, hippocampus and olfactory bulb. Each region was homogenized in 10 volumes of cold of Tris–acetate 0.05 M, pH 7.4, solution. The total enzymatic activity of the sialidases was measured in triplicate samples from each cerebral region. The soluble and membrane fractions were separated as previously described [62]. The basal enzymatic activity was obtained from the cerebral cortex of normal rats. Aliquots of 30 mg of proteins from supernatants or precipitates were incubated in buffer solution of Tris– acetate 0.1 M, pH 3.9 at 37 8C for 15 min, in the presence of methylumbelliferyl N-acetylneuraminic acid 1 mM (MUB-NacNeu) as the substrate, in a final volume of 200 ml. To stop the enzymatic reaction, 3 ml of buffer solution of glycine buffer 0.2 M, were added to yield a pH of 10.5. The product of the reaction was quantified in a spectrophotofluorometer (Perkin Elmer LS-50B) at an excitation and emission wavelengths of 365 and 445 nm, respectively. The specific activity of sialidases is expressed as liberated sialic acid in nmol per mg protein per hour [42,8]. Protein concentration was determined by the method of Lowry et al. [29]. The changes of the enzymatic activity among the groups are expressed as the mean6S.D. computed from triplicate determinations from individual experiments of each region. The groups analyzed were: (a) healthy control animals (C, n542), control soluble and membranal (Cs and Cm, n510); (b) rats implanted with an electrode but not stimulated (SH, sham; n538); (c) kindled rats with five generalized convulsive seizures (K, kindling; n557), kindling soluble and membranal (Ks and Km, n512); (d) PTZ-treated rats with a single generalized seizure (PTZ, n57). Statistical comparisons were done with ANOVA and multiple comparisons test of Newman– Keuls, using the program Graph Pad Prism (ver. 3.2).
3. Results 2. Materials and methods Male Wistar rats (230–250 g body weight) were anaesthetized with ketamine (60 mg / kg i.p.) and implanted with bipolar stainless steel electrodes (80 kV) into the left basolateral amygdala (AP 2.5; L 5; H 8.5) [39]. Starting 7–10 days after the operation, the animals were daily
The kindled animals presented the first generalized convulsive seizure after 1362 stimuli and were further stimulated until five seizures were consecutively evoked. The animals injected with PTZ presented a generalized convulsive seizure within the first minute after the injection, which lasted up to 2 min. The total activity of sialidases determined in kindled
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epileptic animals is shown in Fig. 1. A decrease in cerebral cortex (11.38%; P,0.001) and cerebellum (28.58%; P, 0.001) was evident, as compared to the controls. Since no difference between the determinations in pons, medulla and mesencephalon were evident they were pooled together and hereafter reported as brainstem. The total enzymatic activity increased in the brainstem by 31.51% (P,0.001), and in the hippocampus by 9.73% (P,0.001). Interestingly, no changes were detected in the hypothalamus by 2.88% (P.0.05), and the olfactory bulb. Membranal activity diminished in the cerebral cortex (16.94%; P,0.001) (Fig. 2) and in the cerebellum (19.81%; P,0.001), while an increase was evident in the brainstem (30.73%; P,0.01), and hippocampus (10.4%; P,0.001). Again, no changes were observed in the hypothalamus (3.87%; P .0.05) and the olfactory bulb. It is important to notice that the activity of membrane sialidase in kindled rats showed the same pattern of changes as the total activity (see Figs. 1–3), and in all cases such membrane activity was always higher than the soluble one. Soluble sialidase activity did not have significant changes in the cerebral cortex (Fig. 3). In the brainstem and the hypothalamus it showed a non significant increase while the increase observed in the hippocampus were of 12.25% (P,0.05). On the contrary, its activity in the cerebellum diminished by 21.49% (P,0.05).
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In the PTZ group, the enzymatic activities for the different regions are as follows: cerebral cortex total (11.38%), membranal (16.92%), and soluble (7.13%); cerebellum, total (28.58%), membranal (19.81%), and soluble (21.47%); brainstem total (38.14%), membranal (30.73%), and soluble (35.88%); hypothalamus total (2.88%), membranal (3.87%), and soluble (7.49%); hippocampus total (9.73%), membranal (10.29%), and soluble (10.64%). Interestingly, neither of these values differed from the corresponding activity of the kindled group (Figs. 1 and 2).
4. Discussion It is known that the kindled state and seizures themselves are able to produce plastic functional and anatomical changes in several brain regions. Many of these changes are consequence of the epileptic activity, whereas other changes can arise during its establishment. Some promote or support the maintenance of the epileptic activity, while others tend to limit its generation. The changes in neuraminidases activity that we observed in the different brain regions of kindled epileptic and non-epileptic seizing animals are likely to reflect changes in the amount of sialic acid bound to or unbound from the gangliosides or from other polysialylated compounds
Fig. 1. Total activity of sialidases of control (C), sham (SH), kindled (K) and PTZ-treated rats in the different structures analyzed. The activity of sialidases is expressed as liberated sialic acid (N-acetylneuraminic acid, NANA) in nmol / mg protein / h (mean6S.D) computed from triplicate determinations from individual experiments (control, n542; sham, n538; kindling, n557; PTZ, n57 rats). Significance: * P,0.001, ** P,0.05, *** P,0.01; Newman–Keuls test.
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Fig. 2. Membranal (M) sialidase enzymatic activity of control (C), kindled (K) and PTZ-treated rats. The activity of sialidases is expressed as liberated sialic acid (N-acetylneuraminic acid, NANA) in nmol / mg protein / h (mean6S.D.) computed from triplicate determinations from individual experiments (control, n510; kindling, n512; PTZ n57 rats). Significance: * P,0.001, ** P,0.05, *** P,0.01; Newman–Keuls test.
[56,20,41]. From our results we can conclude that the sialidase activity presents regional specificity, whereby the pattern of activity possibly represents functional differences among the cerebral cortex, cerebellum and subcortical structures. During brain development, the sialic acid
bound to membranes is involved in the formation of branching of the cortico-thalamic axons and contribute to the formation of the neocortical layers by inhibiting the formation of contacts in inappropriate areas [27]. It has been shown that when the cerebral cortex is treated with
Fig. 3. Soluble (S) sialidase enzymatic activity of control (C), kindled (K) and PTZ-treated rats. The activity of sialidases is expressed as liberated sialic acid (N-acetylneuraminic acid, NANA) in nmol / mg protein / h (mean6S.D.) computed from triplicate determinations from individual experiments (control, n510; kindling, n512; PTZ n57 rats). Significance: * P,0.001, ** P,0.05, *** P,0.01; Newman–Keuls test.
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endosialidase, the number of branching points is increased significantly, but the length of the buds decreases [64]. The decrease of the enzymatic activity observed in the subcortical areas may be related to a possible increment in the number of sialic acid molecules present in the cell membrane, resulting in functional changes. On the other hand, it has been suggested that the removal of sialic acid by an enhanced sialidase activity may cause an increment in the number of mossy or collateral fibers in the hippocampus [13,32]. It is interesting that in epileptic animals, an aberrant growth of the mossy fibers occurs similar to the appearance of ectopical fibers observed in NCAM mutant animals or in animals treated with specific endosialidase against sialic acid [55,31]. In patients with Alzheimer’s disease, the polysialylated form of NCAM is massively expressed in areas of the hippocampus where neurodegenerative processes have taken place, especially in the dentate gyrus [32]. In the same way, in patients with epilepsy of the temporary lobe, polysialylation is increased in the hippocampus and in the entorhinal cortex, which correlates with the density of buds of mossy fibers [32]. One can propose that these types of changes can be related to the sialidase alterations that we observe in the hippocampus and the cerebral cortex of kindled epileptic and non-epileptic seizing rats. The balance between sialylation and desialylation that depends on sialyltransferase / sialidase activities would determine the course of the morphological rearrangements that possibly emerge as an adaptive response to the convulsive state [65,37,36]. A significant increase of sialidasic activity may have resulted as a consequence of the epileptiform process in the subcortical regions, most likely with a corresponding decrease of sialic acid content in cellular membranes. Some gangliosides, as GM1, located in the external surface of the synaptic membranes have been considered as active receptive molecules during epileptic activity [23]. It is known that the levels of sialic acid bound to gangliosides are increased after epileptic activity in layers III–V of the cerebral cortex, particularly in layer IV [22]. On the other hand, Yu [65] reported that during the epilepsy there is an increment of sialic acid in the cerebral gangliosides GM1, GD1a, GD1b and GT1. In spite of these results, it is difficult to establish a causal relationship between the epileptic activity and the sialylation state. Although we could determine that the activity of sialidases is altered by the epileptic state, it was also important to determine if the convulsive seizures per se were able to produce the same changes. Interestingly, we found that after producing a single generalized convulsive seizure, the sialidase activity presented the same pattern as that observed in the kindled animals, which presented several seizures. This result can be indicative of the involvement of the sialidases in the modulation of subsequent seizures and possibly in the maintenance of a permanent epileptic state.
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It is noteworthy that the activity of the membranal component prevails over the soluble one. Therefore, the modifications in soluble enzymatic activity may indicate the existence of various cellular metabolic changes that need to be further investigated. The changes that we describe may thus be related to compensatory mechanisms put in play by the establishment of the kindled epileptic state, by seizures themselves or by the electrical stimulation that produces the kindling effect. They can represent a consequence of an increased cerebral excitability, and also they can be involved in its progression. Moreover, they can represent a compensatory response to deleterious processes brought up by the seizures, providing an opportunity for the establishment of reinnervation and remodeling of particular circuits [54]. We can conclude that the described changes of neuraminidase activity, mainly in the membranous fraction, may be associated to a functional alteration of related sialylated molecules like gangliosides, possibly involved in the mechanism of the convulsive phenomenon.
Acknowledgements ´ We thank Ignacio Vargas Martınez for excellent technical assistance.
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