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Distribution of voltage-dependent calcium channel beta subunits in the hippocampus of patients with temporal lobe epilepsy
VDCC b subunits in temporal lobe epilepsy
Pergamon PII: S0306-4522(99)00162-1
Neuroscience Vol. 93, No. 2, pp. 449–456, 1999 449 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00
DISTRIBUTION OF VOLTAGE-DEPENDENT CALCIUM CHANNEL BETA SUBUNITS IN THE HIPPOCAMPUS OF PATIENTS WITH TEMPORAL LOBE EPILEPSY ¨ MCKE,‡ S. G. VOLSEN,§ O. D. WIESTLER,‡ C. E. ELGER* and H. BECK* A. A. LIE,*† I. BLU Departments of *Epileptology and ‡Neuropathology, University of Bonn Medical Center, 53105 Bonn, Germany §Lilly Research Centre Ltd., Eli Lilly and Company, Windlesham, Surrey, U.K.
Abstract—Voltage-dependent Ca 21 channels constitute a major class of plasma membrane channels through which a significant amount of extracellular Ca 21 enters neuronal cells. Their pore-forming a1 subunits are associated with cytoplasmic regulatory b subunits, which modify the distinct biophysical and pharmacological properties of the a1 subunits. Studies in animal models indicate altered expression of a1 and/or b subunits in epilepsy. We have focused on the regulatory b subunits and have analysed the immunoreactivity patterns of the b1 , b2 , b3 and b4 subunits in the hippocampus of patients with temporal lobe epilepsy (n 18) compared to control specimens (n 2). Temporal lobe epilepsy specimens were classified as Ammon’s horn sclerosis (n 9) or focal lesions without alteration of hippocampal cytoarchitecture (n 9). Immunoreactivity for the b subunits was observed in neuronal cell bodies, dendrites and neuropil. The b1, b2 and b3 subunits were found mainly in cell bodies while the b4 subunit was primarily localized to dendrites. Compared to the control specimens, epilepsy specimens of the Ammon’s horn sclerosis and of the lesion group showed a similar b subunit distribution, except for b1 and b2 staining in the Ammon’s horn sclerosis group: in the severely sclerotic hippocampal subfields of these specimens, b1 and b2 immunoreactivity was enhanced in some of the remaining neuronal cell bodies and, in addition, strongly marked dendrites. Thus, hippocampal neurons apparently express multiple classes of b subunits which segregate into particular subcellular domains. In addition, the enhancement of b1 and b2 immunoreactivity in neuronal cell bodies and the additional shift of the b1 and b2 subunits into the dendritic compartment in severely sclerotic hippocampal regions indicate specific changes in Ammon’s horn sclerosis. Altered expression of these b subunits may lead to increased currents carried by voltage-dependent calcium channels and to enhanced synaptic excitability. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: Ammon’s horn sclerosis, human hippocampus, immunohistochemistry.
The hippocampal formation has been implicated in the pathogenesis of human temporal lobe epilepsy (TLE). Its involvement in TLE is supported by intracranial electroencephalography recordings during presurgical examination and by the fact that removal of the amygdala and of the hippocampus can eliminate or considerably reduce seizures in many patients with TLE. Furthermore, the hippocampus constitutes an anatomical structure where specific neuropathological and functional changes are observed in TLE. Several studies have indicated changes in the dynamics of intracellular Ca 21 in epilepsy, i.e. altered intracellular Ca 21 buffering 1,14,18,21 in addition to altered Ca 21 entry through voltage-dependent Ca 21 channels 27 (VDCCs) and a-amino3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. 13 VDCCs represent a major class of plasma membrane channels through which a significant amount of extracellular Ca 21 can enter neuronal cells. It is generally accepted that neuronal VDCCs are formed by one of the pore-forming a1 subunits (a1A, a1B, a1C, a1D, a1E, a1G and a1H). For the a1A–E subunits, an association with cytoplasmic regulatory b subunits (b1, b2, b3 and b4) has been demonstrated, which modify the distinct biophysical and pharmacological properties of these a1 subunits. 16 In particular, b
subunits have been shown to increase the current amplitude through a1 subunits and to alter activation and inactivation kinetics. In addition to this diversity, an additional a2d regulatory subunit may associate with any a1 subunit, act to increase current amplitude and potentiate the effect of b subunit association. 12,22 The potential diversity generated by this multitude of subunits is further increased by alternative splice variants. 23 To understand the functional role of this diversity, it is important to analyse the distribution of the different VDCC subunits in different types of neurons and also in different subcellular compartments. A number of physiological studies have shown that VDCCs on dendrites and soma of hippocampal pyramidal cells are differentially distributed, with low-threshold currents, N- and P/Q-type currents concentrated in the dendrites, and an L-type current primarily located in the soma. 9 Such a differential subcellular distribution is also suggested by immunohistochemical studies of a1 and b subunits in different areas of the brain. 6,7,25,26,30 In the kindling model and in the kainate model of epilepsy, increased VDCC amplitudes have been observed in CA1 neurons and dentate granule cells compared to control animals. 2,10 In CA1 neurons, alterations in the expression of a1A, a1D and a1E subunits may underlie these changes. 11 However, the distribution of VDCC subunits in the hippocampal formation of patients with TLE has not been described to date. Thus, it remains unknown whether these subunits are differentially expressed in the human hippocampus. In the present study, we have focused on the regulatory b subunits and have analysed the immunoreactivity patterns of the b1,
†To whom correspondence should be addressed. Abbreviations: AHS, Ammon’s horn sclerosis; AMPA, a-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid; EEG, electroencephalography; PBS, phosphate-buffered saline; TLE, temporal lobe epilepsy; VDCC, voltage-dependent calcium channel. 449
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Neuropathological evaluation of hippocampal specimens of temporal lobe epilepsy patients
Table 1. Clinical data of epilepsy patients and controls
Number Sex Age (years) Age at seizure onset (years) Duration of epilepsy (years) Frequency of CPS/month
AHS
Lesion
Control
9 7F, 2M 33 ^ 8 15 ^ 5 18 ^ 7 42 ^ 58
9 4F, 5M 24 ^ 11 10 ^ 9 11 ^ 8 51 ^ 32
2 1F, 1M 44 ^ 2 – – –
Clinical data of patients included in the study. Values are expressed as mean ^ S.E.M. CPS, complex partial seizures; F, female; M, male.
b2, b3 and b4 subunits in the hippocampal formation of different patient groups with TLE compared to control specimens.
All specimens were independently examined by two neuropathologists and classified with respect to the presence of AHS (n 9) or focal lesions (n 9). AHS was characterized by extensive neuronal cell loss and concomitant astrogliosis in the CA1, CA3 and CA4 subfields of the Ammon’s horn with relative sparing of CA2 and the granule cell layer of the dentate gyrus. In the non-AHS patients, focal lesions were found in the subcortical white matter or temporomesial neocortex. These did not involve the hippocampus proper. The histopathological diagnoses included glioneuronal hamartias (n 2), porencephalic cysts (n 2), cavernous angioma (n 1), focal cortical dysplasia (n 2), and dysembryoplastic neuroepithelial tumors (n 2). The clinical data of patients with AHS and lesion-associated epilepsy were not significantly different (Table 1). Hematoxylin and Eosin stains, Nissl stains, combined Hematoxylin and Eosin–Luxol Fast Blue stains and glial fibrillary acidic protein immunohistochemistry were available for all specimens. Immunohistochemistry
EXPERIMENTAL PROCEDURES
Hippocampal specimens Eighteen surgical specimens obtained from patients with chronic pharmaco-resistant TLE were examined (Table 1). All patients suffered from complex partial seizures. The epileptogenic focus was localized to the temporal lobe in all patients by non-invasive or invasive diagnostic procedures, as described elsewhere. 3 Many of these patients have undergone several different therapeutic regimens with different combinations of antiepileptic drugs prior to epilepsy surgery. At the time of presurgical evaluation, a monotherapy with carbamazepine was carried out in 3/9 Ammon’s horn sclerosis (AHS) and 5/9 lesion-associated TLE patients (for definition of patient groups see below). A combination of anticonvulsants including carbamazepine, gabapentin, lamotrigin, phenobarbital, valproate and vigabatrin was administered in 6/9 AHS and 4/9 lesion-associated TLE patients. Surgical removal of the hippocampus was necessary to achieve seizure control. Informed and written consent was obtained from all patients for additional histopathological studies. All procedures were conducted in accordance with the Declaration of Helsinki and approved by the ethics committee of the University of Bonn Medical Center. The surgical specimens were obtained within 30 min after resection and coronally dissected along the septotemporal axis into 3–4-mm-thick tissue blocks. All tissue blocks of this study were chosen from the middle segment of the main hippocampal body, immersion-fixed in 4% buffered formalin at room temperature for 8– 48 h and embedded into paraffin. In addition, surgical control hippocampal specimens were obtained from two patients without a history of epileptic seizures. These two specimens were processed like the epilepsy surgery tissue. Both patients suffered from a tumor (glioblastoma multiforme, WHO Grade IV and anaplastic oligodendroglioma, WHO Grade III) located adjacent to the hippocampal formation. Histopathological examination excluded tumor cell invasion into the hippocampus proper or other neuropathological alterations within the hippocampal formation.
Polyclonal antibodies specific for VDCC b1, b2, b3, and b4 subunits were produced and characterized as described previously. 26 Paraffin sections were cut at 4 mm, mounted on 3-amino-propyltriethoxysilanecoated slides (DAKO, Glostrup, Denmark) and air-dried overnight at 428C. All hippocampal specimens included in this study were stained under identical conditions using a capillary gap procedure and were processed in a single batch for each antibody. After deparaffination, slides were incubated in 2% hydrogen peroxide (Merck, Darmstadt, Germany) diluted in methanol for 15 min, rehydrated and rinsed in phosphate-buffered saline (PBS). Sections were transferred into 0.01 M citrate buffer (Sigma, St Louis, U.S.A.), boiled twice for 5 min in a microwave oven according to the standard Dako microwave treatment protocol and transferred into PBS. Preincubation with 2% goat serum (Vector Laboratories, Burlingame, U.S.A.), 10% fetal calf serum (Seromed, Berlin, Germany), and 5% non-fat dry milk (Bio-Rad Laboratories, CA, U.S.A.) in PBS as a blocking reagent was performed for 3 h at 378C, followed by incubation with the respective primary antibodies overnight at 48C in a humid chamber (concentrations used: 1 mg/ml for b1 and b2; 0.5 mg/ml for b3 and b4). Binding of primary antibodies was detected by the avidin–biotin complex peroxidase method (ABC Elite, Vector Labs, Burlingame, CA, U.S.A.), using 3,3 0 -diaminobenzidine (ICN, Cleveland, Ohio, U.S.A.) as a chromogen. Control experiments included omission of primary antibodies and substitution of the respective primary antibody by equivalent dilutions of non-immune rabbit IgG serum (DAKO), using the same staining protocol. RESULTS
In general, immunoreactivity for the VDCC subunits b1, b2, b3 and b4 was observed in neuronal somata, dendrites and, to a lesser degree, in the neuropil. While the b1, b2 and b3 subunits showed a similar, predominantly somatic distribution
Fig. 1. Immunoreactivity for the VDCC b1, b2, b3 and b4 subunits in the subiculum of a lesion-associated TLE specimen. Neuronal staining for b1 (A), b2 (B) and b3 (C) is strong and mainly found in the cell bodies, while labelling for b4 (D) is primarily localized to dendrites. Scale bar 15 mm.
CA1–4
b1, b2 and b3 immunoreactivity (IR) is mainly observed in neuronal somata, except for AHS specimens which show enhanced dendritic labelling for b1 and b2. In contrast, b4-IR is predominantly localized to dendrites. C, control specimens; DG, dentate gyrus; GCL, granule cell layer; L, lesion specimens; ML, molecular layer. –/ 1 / 1 1 / 1 1 1 , no/weak/strong/very strong labelling; *only very few neurons are labelled within CA1–4; †in CA1–3 neurons generally show weak IR, while in CA4 most neurons exhibit stronger staining; ‡ dendrites are only sporadically stained.
1 1 1/1 1 1 1/1 1 † 1 1/1 1 1 2/1 1 1 1/1 1 1 1/1 1 1 1/1 1 1 2/1 2/1 2/1 1* 1‡ 2/1 2/1 2/1 1 1‡ 2/1 DG
GCL ML neuronal somata dendrites neuropil
1 2/1 1 1‡ 2/1
2/1 2/1 1/1 1 1/1 1 2/1
1 2/1 1/1 1 1‡ 2/1
1 2/1 1/1 1 1‡ 2/1
1 2/1 1 1/1 1 1 1/1 1 2/1
2/1 2/1 1* 1‡ 2/1
2/1 2/1 1* 1‡ 2/1
1 1 1/1 1 1 1/1 1 1 1/1 1 1 2/1
AHS b4 L C AHS b3 L C AHS b2 L C AHS b1 L C Region
Table 2. Immunoreactivity for b subunits in the hippocampus of temporal lobe epilepsy patients compared to control tissue
VDCC b subunits in temporal lobe epilepsy
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pattern, b4 immunoreactivity was mainly present in dendrites. As staining for b3 was generally weaker than b1 and b2 labelling, no photomicrographs are shown for the b3 subunit. The results are summarized in Table 2. The strong, specific immunoreactivity for the b1, b2, b3 and b4 subunits in the subiculum of all hippocampal specimens served as an internal positive control (Fig. 1A–D). Negative control experiments included omission of primary antibodies and substitution of the respective primary antibody by equivalent dilutions of non-immune rabbit IgG serum. These control reactions did not yield immunoreactivity (not shown). Immunoreactivity pattern in the dentate gyrus of temporal lobe epilepsy specimens compared to controls In control specimens, weak b1, b2 and b3 immunoreactivity was found in granule cell somata, while the molecular layer showed only faint labelling (Fig. 2A, B). No immunoreactive dendrites were observed. In contrast, strong staining for b4 was seen in dendrites, while granule cell somata exhibited less intense b4 immunoreactivity (Fig. 2C). Lesion-associated TLE (Fig. 2D, E, F) and AHS (Fig. 2G, H, I) specimens showed a similar immunoreactivity pattern as the controls. In some TLE specimens of both groups, b1 labelling of granule cell somata was extremely weak or absent. Immunoreactivity pattern in the subfields CA1 to CA4 of temporal lobe epilepsy specimens compared to controls In the hippocampal subfields CA1 to CA4, the general distribution of immunoreactivity was similar to that in the dentate gyrus. In the control specimens, b1, b2 and b3 labelling was mainly found in neuronal cell bodies throughout the regions CA1 to CA4 (Figs 3A, B, 4A, B), while b4 staining was prominent in dendrites (Figs 3C, 4C). All hippocampal subfields displayed weak b1 immunoreactivity in most cell bodies of polymorphic hilar neurons (Fig. 3A), pyramidal cells (Fig. 4A) and of a few interneurons within the stratum oriens, while dendrites were only sporadically labelled. The staining pattern for b2 was similar to the one for b1, except for a stronger labelling intensity of neuronal cell bodies (Figs 3B, 4B). However, in CA1 to CA4 only a minority of neurons were b3-immunoreactive. In contrast, all hippocampal subfields revealed b4 staining in most cell bodies of polymorphic hilar neurons (Fig. 3C), pyramidal cells (Fig. 4C) and of a few stratum oriens interneurons and markedly labelled dendrites. Compared to the controls, lesion-associated TLE specimens showed similar immunoreactivity patterns for all b subunits (Figs 3D, E, F, 4D, E, F). The AHS specimens exhibited a characteristic substantial loss of principal neurons, which was most extensive in the hippocampal subfields CA1, CA3 and CA4. In parallel, the number of immunoreactive neurons decreased. Otherwise, the staining patterns for the b3 and b4 subunits were similar to the controls (Figs 3I, 4I). Interestingly, in those regions with severe neuronal cell loss, some of the remaining neurons showed stronger labelling for b1 and b2 compared to controls (Figs 3G, H, 4G, H). In addition, strongly b1- and b2-immunoreactive dendrites were found in these sclerotic areas (Figs 3G, H, 4G, H). DISCUSSION
In this study, we have examined the immunohistochemical
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Fig. 2. Immunoreactivity for the VDCC b1, b2 and b4 subunits in the dentate gyrus of patients with TLE. In the control specimen, the b1 (A) and b2 (B) subunits show a predominantly somatic distribution, while b4 immunoreactivity (C) is mainly seen in the dendritic compartment. A similar distribution pattern can also be observed in lesion-associated TLE specimens (D, E, F) and in the hippocampus with severe AHS (G, H, I). Scale bar 5 mm.
distribution of the VDCC b1, b2 , b3 and b4 subunits in the hippocampus of two pathogenetically different groups of patients with TLE and in human control specimens. Immunoreactivity for VDCC b subunits was observed in neuronal cell bodies, dendrites and neuropil. The b1, b2 and b3 subunits were found mainly in cell bodies while the b4 subunit was primarily localized to dendrites. Our findings corresponded well to the data of Day et al. 7
Compared to the control specimens, human TLE specimens of the AHS and of the lesion group showed a similar immunohistochemical distribution pattern for the b subunits, except for b1 and b2 staining in AHS specimens. In the severely sclerotic hippocampal subfields of these specimens, b1 and b2 immunoreactivity was enhanced in some of the remaining neuronal cell bodies and, in addition, strongly marked dendrites.
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Fig. 3. Immunoreactivity for the VDCC b1, b2 and b4 subunits in the CA4 region of patients with TLE. In the control hippocampus, b1 (A) and b2 (B) staining is mainly observed in the neuronal cell bodies, whereas b4 immunoreactivity (C) is prominent in dendrites and less intense in the somata. A similar distribution pattern can also be found in lesion-associated TLE specimens (D, E, F). In the hippocampus with severe AHS (G, H, I), the number of immunoreactive neurons decreases due to substantial neuron loss. Some of the remaining neurons show stronger labelling for b1 (G) and b2 (H) compared to the controls. In addition, markedly b1- and b2-immunoreactive dendrites are detectable (G, H). The primarily dendritic localization of the b4 subunit (I) is similar to the control. Scale bar 8 mm.
Functional considerations Although a1 subunits contain the ion pore and determine many of the kinetic and pharmacological properties of VDCCs, auxiliary subunits can significantly modify the biophysical and pharmacological properties of these
channels. 12 With regard to the b subunits, the modulatory effect on currents carried by the a1A–E subunits appears qualitatively similar in most subunit combinations. For example, a stimulation of current amplitude has been observed for almost all a1 subunits. In addition, auxiliary b subunits systematically shift the current–voltage relationship towards more
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Fig. 4. Immunoreactivity for the VDCC b1, b2 and b4 subunits in the CA1 region of patients with TLE. In the control hippocampus, b1 (A) and b2 (B) staining is mainly seen in the neuronal cell bodies, while b4 immunoreactivity (C) is prominent in the dendritic compartment and less intense in the somata. This distribution pattern can also be observed in lesion-associated TLE specimens (D, E, F). In the hippocampus obtained from a TLE patient with severe AHS (G, H, I), the number of immunoreactive neurons decreases due to substantial neuron loss. Some of the remaining neurons show enhanced staining for b1 (G) and b2 (H) compared to the controls. In addition, strongly b1- and b2-immunoreactive dendrites are found (G, H). The primarily dendritic localization of the b4 subunit (I) is similar to the control. Scale bar 8 mm.
hyperpolarized potentials and modify the time-dependent activation and inactivation behaviour of VDCCs (for review see Refs 8 and 29). In contrast to the a1A–E subunits, T-type currents probably corresponding to a1G and a1H subunits are unchanged following depletion of b subunits. 15 In our study, we have shown a differential subcellular
localization of the b4 and the b1–3 subunits. It is not yet clear which a1 and b subunits assemble in the brain to form native channels. However, it is generally assumed that different a1/b subunit combinations exist in different subcellular compartments. b3 and b4 have been demonstrated as the major L-type channel b subunits in the mammalian
VDCC b subunits in temporal lobe epilepsy
hippocampus, 19 while the N-type channel appears to be primarily associated with the b1b, b3 and b4 subunits. 20 Interestingly, L-type channels have been shown to be predominantly located in neuronal cell bodies, while N-type and P/ Q-type channels are concentrated in dendrites. 9 The subcellular localization of the remaining types of VDCCs has been less well characterized. Nevertheless, increasing evidence indicates that a differential segregation of the b subunits is important for the subcellularly specific modification of pharmacological and kinetic properties of a1 subunits. An interesting change was observed for the b1 and b2 subunits in AHS, i.e. localization of these subunits in dendrites of surviving pyramidal cells in intensely sclerotic regions of the Ammon’s horn. While we cannot completely exclude a minor contribution of presynaptic b1 and b2 subunit labelling, the characteristic staining pattern strongly suggests up-regulation of b1 and b2 subunits in dendrites. It is tempting to speculate that this up-regulation may enhance currents carried by VDCCs in AHS. An enhancement of Ca 21 current amplitude occurs in the kindling and kainate models of epilepsy in the CA1 region and the dentate gyrus, respectively. 2,10,27 In addition to enhanced expression of a1 subunits, 11 alterations in modulatory b subunits may contribute to this phenomenon. The increase of Ca 21 current amplitude may have profound influence on the discharge properties of neurons. For instance, altered dendritic Ca 21 currents may affect the electrogenic properties of dendrites which control the propagation of synaptic potentials towards the soma of hippocampal neurons. 17 In addition, Ca 21-dependent bursting has been observed in some neuronal cells, and it is tempting to speculate that Ca 21 current up-regulation might also affect the propensity of hippocampal neurons to burst. Finally, voltage-dependent Ca 21 currents represent one of the main entry routes for Ca 21 into neurons. As intracellular Ca 21 overload seems to be able to trigger neuronal cell death, 5 there may be a link between altered expression of Ca 21 channel subunits and selective vulnerability of pyramidal cells in AHS.
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In addition to the modification of specific pharmacological and kinetic properties of a1 subunits, it has recently been recognized that b subunits are also involved in targeting the translocation of a1 subunits to the neuronal membrane. 4,24 Thus, the almost exclusive presence of b4 subunits in dendrites raises the possibility that these subunits may be important in targeting specific a1 subunits to the dendritic compartment. It is interesting to note that a b4 subunit-specific interaction site has been discovered in the carboxy-terminal region of the a1A subunit, suggesting that specific interactions between these two channel components do exist. 28 In this context, the enhanced b1 and b2 immunoreactivity in neuronal cell bodies together with the novel appearance of b1 and b2 subunits in the dendritic compartment within severely sclerotic hippocampal subfields of AHS specimens may also affect the distribution of a1 subunits.
CONCLUSIONS
We have shown that hippocampal neurons express multiple classes of VDCC b subunits which appear to segregate into particular subcellular domains. In addition, we report an enhancement of b1 and b2 immunoreactivity in neuronal cell bodies and an additional shift of the b1 and b2 subunits into the dendritic compartment in severely sclerotic hippocampal regions of AHS specimens. These alterations in the distribution of VDCC b subunits may lead to considerably altered function of voltage-dependent Ca 21 channels and contribute to hyperexcitability in TLE associated with AHS.
Acknowledgements—We are grateful to our neurosurgical colleagues Prof. Schramm, Prof. Zentner, and Dr van Roost who supplied biopsy specimens. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 400; DFG EL 122/7-1), the joint German-Israeli Research Program and a University of Bonn Center Grant (BONFOR). A. A. Lie is recipient of a postdoctoral fellowship from the Gertrud Reemtsma Stiftung of the Max-Planck-Gesellschaft.
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Pergamon PII: S0306-4522(99)00162-1
Neuroscience Vol. 93, No. 2, pp. 449–456, 1999 449 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00
DISTRIBUTION OF VOLTAGE-DEPENDENT CALCIUM CHANNEL BETA SUBUNITS IN THE HIPPOCAMPUS OF PATIENTS WITH TEMPORAL LOBE EPILEPSY ¨ MCKE,‡ S. G. VOLSEN,§ O. D. WIESTLER,‡ C. E. ELGER* and H. BECK* A. A. LIE,*† I. BLU Departments of *Epileptology and ‡Neuropathology, University of Bonn Medical Center, 53105 Bonn, Germany §Lilly Research Centre Ltd., Eli Lilly and Company, Windlesham, Surrey, U.K.
Abstract—Voltage-dependent Ca 21 channels constitute a major class of plasma membrane channels through which a significant amount of extracellular Ca 21 enters neuronal cells. Their pore-forming a1 subunits are associated with cytoplasmic regulatory b subunits, which modify the distinct biophysical and pharmacological properties of the a1 subunits. Studies in animal models indicate altered expression of a1 and/or b subunits in epilepsy. We have focused on the regulatory b subunits and have analysed the immunoreactivity patterns of the b1 , b2 , b3 and b4 subunits in the hippocampus of patients with temporal lobe epilepsy (n 18) compared to control specimens (n 2). Temporal lobe epilepsy specimens were classified as Ammon’s horn sclerosis (n 9) or focal lesions without alteration of hippocampal cytoarchitecture (n 9). Immunoreactivity for the b subunits was observed in neuronal cell bodies, dendrites and neuropil. The b1, b2 and b3 subunits were found mainly in cell bodies while the b4 subunit was primarily localized to dendrites. Compared to the control specimens, epilepsy specimens of the Ammon’s horn sclerosis and of the lesion group showed a similar b subunit distribution, except for b1 and b2 staining in the Ammon’s horn sclerosis group: in the severely sclerotic hippocampal subfields of these specimens, b1 and b2 immunoreactivity was enhanced in some of the remaining neuronal cell bodies and, in addition, strongly marked dendrites. Thus, hippocampal neurons apparently express multiple classes of b subunits which segregate into particular subcellular domains. In addition, the enhancement of b1 and b2 immunoreactivity in neuronal cell bodies and the additional shift of the b1 and b2 subunits into the dendritic compartment in severely sclerotic hippocampal regions indicate specific changes in Ammon’s horn sclerosis. Altered expression of these b subunits may lead to increased currents carried by voltage-dependent calcium channels and to enhanced synaptic excitability. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: Ammon’s horn sclerosis, human hippocampus, immunohistochemistry.
The hippocampal formation has been implicated in the pathogenesis of human temporal lobe epilepsy (TLE). Its involvement in TLE is supported by intracranial electroencephalography recordings during presurgical examination and by the fact that removal of the amygdala and of the hippocampus can eliminate or considerably reduce seizures in many patients with TLE. Furthermore, the hippocampus constitutes an anatomical structure where specific neuropathological and functional changes are observed in TLE. Several studies have indicated changes in the dynamics of intracellular Ca 21 in epilepsy, i.e. altered intracellular Ca 21 buffering 1,14,18,21 in addition to altered Ca 21 entry through voltage-dependent Ca 21 channels 27 (VDCCs) and a-amino3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. 13 VDCCs represent a major class of plasma membrane channels through which a significant amount of extracellular Ca 21 can enter neuronal cells. It is generally accepted that neuronal VDCCs are formed by one of the pore-forming a1 subunits (a1A, a1B, a1C, a1D, a1E, a1G and a1H). For the a1A–E subunits, an association with cytoplasmic regulatory b subunits (b1, b2, b3 and b4) has been demonstrated, which modify the distinct biophysical and pharmacological properties of these a1 subunits. 16 In particular, b
subunits have been shown to increase the current amplitude through a1 subunits and to alter activation and inactivation kinetics. In addition to this diversity, an additional a2d regulatory subunit may associate with any a1 subunit, act to increase current amplitude and potentiate the effect of b subunit association. 12,22 The potential diversity generated by this multitude of subunits is further increased by alternative splice variants. 23 To understand the functional role of this diversity, it is important to analyse the distribution of the different VDCC subunits in different types of neurons and also in different subcellular compartments. A number of physiological studies have shown that VDCCs on dendrites and soma of hippocampal pyramidal cells are differentially distributed, with low-threshold currents, N- and P/Q-type currents concentrated in the dendrites, and an L-type current primarily located in the soma. 9 Such a differential subcellular distribution is also suggested by immunohistochemical studies of a1 and b subunits in different areas of the brain. 6,7,25,26,30 In the kindling model and in the kainate model of epilepsy, increased VDCC amplitudes have been observed in CA1 neurons and dentate granule cells compared to control animals. 2,10 In CA1 neurons, alterations in the expression of a1A, a1D and a1E subunits may underlie these changes. 11 However, the distribution of VDCC subunits in the hippocampal formation of patients with TLE has not been described to date. Thus, it remains unknown whether these subunits are differentially expressed in the human hippocampus. In the present study, we have focused on the regulatory b subunits and have analysed the immunoreactivity patterns of the b1,
†To whom correspondence should be addressed. Abbreviations: AHS, Ammon’s horn sclerosis; AMPA, a-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid; EEG, electroencephalography; PBS, phosphate-buffered saline; TLE, temporal lobe epilepsy; VDCC, voltage-dependent calcium channel. 449
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Neuropathological evaluation of hippocampal specimens of temporal lobe epilepsy patients
Table 1. Clinical data of epilepsy patients and controls
Number Sex Age (years) Age at seizure onset (years) Duration of epilepsy (years) Frequency of CPS/month
AHS
Lesion
Control
9 7F, 2M 33 ^ 8 15 ^ 5 18 ^ 7 42 ^ 58
9 4F, 5M 24 ^ 11 10 ^ 9 11 ^ 8 51 ^ 32
2 1F, 1M 44 ^ 2 – – –
Clinical data of patients included in the study. Values are expressed as mean ^ S.E.M. CPS, complex partial seizures; F, female; M, male.
b2, b3 and b4 subunits in the hippocampal formation of different patient groups with TLE compared to control specimens.
All specimens were independently examined by two neuropathologists and classified with respect to the presence of AHS (n 9) or focal lesions (n 9). AHS was characterized by extensive neuronal cell loss and concomitant astrogliosis in the CA1, CA3 and CA4 subfields of the Ammon’s horn with relative sparing of CA2 and the granule cell layer of the dentate gyrus. In the non-AHS patients, focal lesions were found in the subcortical white matter or temporomesial neocortex. These did not involve the hippocampus proper. The histopathological diagnoses included glioneuronal hamartias (n 2), porencephalic cysts (n 2), cavernous angioma (n 1), focal cortical dysplasia (n 2), and dysembryoplastic neuroepithelial tumors (n 2). The clinical data of patients with AHS and lesion-associated epilepsy were not significantly different (Table 1). Hematoxylin and Eosin stains, Nissl stains, combined Hematoxylin and Eosin–Luxol Fast Blue stains and glial fibrillary acidic protein immunohistochemistry were available for all specimens. Immunohistochemistry
EXPERIMENTAL PROCEDURES
Hippocampal specimens Eighteen surgical specimens obtained from patients with chronic pharmaco-resistant TLE were examined (Table 1). All patients suffered from complex partial seizures. The epileptogenic focus was localized to the temporal lobe in all patients by non-invasive or invasive diagnostic procedures, as described elsewhere. 3 Many of these patients have undergone several different therapeutic regimens with different combinations of antiepileptic drugs prior to epilepsy surgery. At the time of presurgical evaluation, a monotherapy with carbamazepine was carried out in 3/9 Ammon’s horn sclerosis (AHS) and 5/9 lesion-associated TLE patients (for definition of patient groups see below). A combination of anticonvulsants including carbamazepine, gabapentin, lamotrigin, phenobarbital, valproate and vigabatrin was administered in 6/9 AHS and 4/9 lesion-associated TLE patients. Surgical removal of the hippocampus was necessary to achieve seizure control. Informed and written consent was obtained from all patients for additional histopathological studies. All procedures were conducted in accordance with the Declaration of Helsinki and approved by the ethics committee of the University of Bonn Medical Center. The surgical specimens were obtained within 30 min after resection and coronally dissected along the septotemporal axis into 3–4-mm-thick tissue blocks. All tissue blocks of this study were chosen from the middle segment of the main hippocampal body, immersion-fixed in 4% buffered formalin at room temperature for 8– 48 h and embedded into paraffin. In addition, surgical control hippocampal specimens were obtained from two patients without a history of epileptic seizures. These two specimens were processed like the epilepsy surgery tissue. Both patients suffered from a tumor (glioblastoma multiforme, WHO Grade IV and anaplastic oligodendroglioma, WHO Grade III) located adjacent to the hippocampal formation. Histopathological examination excluded tumor cell invasion into the hippocampus proper or other neuropathological alterations within the hippocampal formation.
Polyclonal antibodies specific for VDCC b1, b2, b3, and b4 subunits were produced and characterized as described previously. 26 Paraffin sections were cut at 4 mm, mounted on 3-amino-propyltriethoxysilanecoated slides (DAKO, Glostrup, Denmark) and air-dried overnight at 428C. All hippocampal specimens included in this study were stained under identical conditions using a capillary gap procedure and were processed in a single batch for each antibody. After deparaffination, slides were incubated in 2% hydrogen peroxide (Merck, Darmstadt, Germany) diluted in methanol for 15 min, rehydrated and rinsed in phosphate-buffered saline (PBS). Sections were transferred into 0.01 M citrate buffer (Sigma, St Louis, U.S.A.), boiled twice for 5 min in a microwave oven according to the standard Dako microwave treatment protocol and transferred into PBS. Preincubation with 2% goat serum (Vector Laboratories, Burlingame, U.S.A.), 10% fetal calf serum (Seromed, Berlin, Germany), and 5% non-fat dry milk (Bio-Rad Laboratories, CA, U.S.A.) in PBS as a blocking reagent was performed for 3 h at 378C, followed by incubation with the respective primary antibodies overnight at 48C in a humid chamber (concentrations used: 1 mg/ml for b1 and b2; 0.5 mg/ml for b3 and b4). Binding of primary antibodies was detected by the avidin–biotin complex peroxidase method (ABC Elite, Vector Labs, Burlingame, CA, U.S.A.), using 3,3 0 -diaminobenzidine (ICN, Cleveland, Ohio, U.S.A.) as a chromogen. Control experiments included omission of primary antibodies and substitution of the respective primary antibody by equivalent dilutions of non-immune rabbit IgG serum (DAKO), using the same staining protocol. RESULTS
In general, immunoreactivity for the VDCC subunits b1, b2, b3 and b4 was observed in neuronal somata, dendrites and, to a lesser degree, in the neuropil. While the b1, b2 and b3 subunits showed a similar, predominantly somatic distribution
Fig. 1. Immunoreactivity for the VDCC b1, b2, b3 and b4 subunits in the subiculum of a lesion-associated TLE specimen. Neuronal staining for b1 (A), b2 (B) and b3 (C) is strong and mainly found in the cell bodies, while labelling for b4 (D) is primarily localized to dendrites. Scale bar 15 mm.
CA1–4
b1, b2 and b3 immunoreactivity (IR) is mainly observed in neuronal somata, except for AHS specimens which show enhanced dendritic labelling for b1 and b2. In contrast, b4-IR is predominantly localized to dendrites. C, control specimens; DG, dentate gyrus; GCL, granule cell layer; L, lesion specimens; ML, molecular layer. –/ 1 / 1 1 / 1 1 1 , no/weak/strong/very strong labelling; *only very few neurons are labelled within CA1–4; †in CA1–3 neurons generally show weak IR, while in CA4 most neurons exhibit stronger staining; ‡ dendrites are only sporadically stained.
1 1 1/1 1 1 1/1 1 † 1 1/1 1 1 2/1 1 1 1/1 1 1 1/1 1 1 1/1 1 1 2/1 2/1 2/1 1* 1‡ 2/1 2/1 2/1 1 1‡ 2/1 DG
GCL ML neuronal somata dendrites neuropil
1 2/1 1 1‡ 2/1
2/1 2/1 1/1 1 1/1 1 2/1
1 2/1 1/1 1 1‡ 2/1
1 2/1 1/1 1 1‡ 2/1
1 2/1 1 1/1 1 1 1/1 1 2/1
2/1 2/1 1* 1‡ 2/1
2/1 2/1 1* 1‡ 2/1
1 1 1/1 1 1 1/1 1 1 1/1 1 1 2/1
AHS b4 L C AHS b3 L C AHS b2 L C AHS b1 L C Region
Table 2. Immunoreactivity for b subunits in the hippocampus of temporal lobe epilepsy patients compared to control tissue
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pattern, b4 immunoreactivity was mainly present in dendrites. As staining for b3 was generally weaker than b1 and b2 labelling, no photomicrographs are shown for the b3 subunit. The results are summarized in Table 2. The strong, specific immunoreactivity for the b1, b2, b3 and b4 subunits in the subiculum of all hippocampal specimens served as an internal positive control (Fig. 1A–D). Negative control experiments included omission of primary antibodies and substitution of the respective primary antibody by equivalent dilutions of non-immune rabbit IgG serum. These control reactions did not yield immunoreactivity (not shown). Immunoreactivity pattern in the dentate gyrus of temporal lobe epilepsy specimens compared to controls In control specimens, weak b1, b2 and b3 immunoreactivity was found in granule cell somata, while the molecular layer showed only faint labelling (Fig. 2A, B). No immunoreactive dendrites were observed. In contrast, strong staining for b4 was seen in dendrites, while granule cell somata exhibited less intense b4 immunoreactivity (Fig. 2C). Lesion-associated TLE (Fig. 2D, E, F) and AHS (Fig. 2G, H, I) specimens showed a similar immunoreactivity pattern as the controls. In some TLE specimens of both groups, b1 labelling of granule cell somata was extremely weak or absent. Immunoreactivity pattern in the subfields CA1 to CA4 of temporal lobe epilepsy specimens compared to controls In the hippocampal subfields CA1 to CA4, the general distribution of immunoreactivity was similar to that in the dentate gyrus. In the control specimens, b1, b2 and b3 labelling was mainly found in neuronal cell bodies throughout the regions CA1 to CA4 (Figs 3A, B, 4A, B), while b4 staining was prominent in dendrites (Figs 3C, 4C). All hippocampal subfields displayed weak b1 immunoreactivity in most cell bodies of polymorphic hilar neurons (Fig. 3A), pyramidal cells (Fig. 4A) and of a few interneurons within the stratum oriens, while dendrites were only sporadically labelled. The staining pattern for b2 was similar to the one for b1, except for a stronger labelling intensity of neuronal cell bodies (Figs 3B, 4B). However, in CA1 to CA4 only a minority of neurons were b3-immunoreactive. In contrast, all hippocampal subfields revealed b4 staining in most cell bodies of polymorphic hilar neurons (Fig. 3C), pyramidal cells (Fig. 4C) and of a few stratum oriens interneurons and markedly labelled dendrites. Compared to the controls, lesion-associated TLE specimens showed similar immunoreactivity patterns for all b subunits (Figs 3D, E, F, 4D, E, F). The AHS specimens exhibited a characteristic substantial loss of principal neurons, which was most extensive in the hippocampal subfields CA1, CA3 and CA4. In parallel, the number of immunoreactive neurons decreased. Otherwise, the staining patterns for the b3 and b4 subunits were similar to the controls (Figs 3I, 4I). Interestingly, in those regions with severe neuronal cell loss, some of the remaining neurons showed stronger labelling for b1 and b2 compared to controls (Figs 3G, H, 4G, H). In addition, strongly b1- and b2-immunoreactive dendrites were found in these sclerotic areas (Figs 3G, H, 4G, H). DISCUSSION
In this study, we have examined the immunohistochemical
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Fig. 2. Immunoreactivity for the VDCC b1, b2 and b4 subunits in the dentate gyrus of patients with TLE. In the control specimen, the b1 (A) and b2 (B) subunits show a predominantly somatic distribution, while b4 immunoreactivity (C) is mainly seen in the dendritic compartment. A similar distribution pattern can also be observed in lesion-associated TLE specimens (D, E, F) and in the hippocampus with severe AHS (G, H, I). Scale bar 5 mm.
distribution of the VDCC b1, b2 , b3 and b4 subunits in the hippocampus of two pathogenetically different groups of patients with TLE and in human control specimens. Immunoreactivity for VDCC b subunits was observed in neuronal cell bodies, dendrites and neuropil. The b1, b2 and b3 subunits were found mainly in cell bodies while the b4 subunit was primarily localized to dendrites. Our findings corresponded well to the data of Day et al. 7
Compared to the control specimens, human TLE specimens of the AHS and of the lesion group showed a similar immunohistochemical distribution pattern for the b subunits, except for b1 and b2 staining in AHS specimens. In the severely sclerotic hippocampal subfields of these specimens, b1 and b2 immunoreactivity was enhanced in some of the remaining neuronal cell bodies and, in addition, strongly marked dendrites.
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Fig. 3. Immunoreactivity for the VDCC b1, b2 and b4 subunits in the CA4 region of patients with TLE. In the control hippocampus, b1 (A) and b2 (B) staining is mainly observed in the neuronal cell bodies, whereas b4 immunoreactivity (C) is prominent in dendrites and less intense in the somata. A similar distribution pattern can also be found in lesion-associated TLE specimens (D, E, F). In the hippocampus with severe AHS (G, H, I), the number of immunoreactive neurons decreases due to substantial neuron loss. Some of the remaining neurons show stronger labelling for b1 (G) and b2 (H) compared to the controls. In addition, markedly b1- and b2-immunoreactive dendrites are detectable (G, H). The primarily dendritic localization of the b4 subunit (I) is similar to the control. Scale bar 8 mm.
Functional considerations Although a1 subunits contain the ion pore and determine many of the kinetic and pharmacological properties of VDCCs, auxiliary subunits can significantly modify the biophysical and pharmacological properties of these
channels. 12 With regard to the b subunits, the modulatory effect on currents carried by the a1A–E subunits appears qualitatively similar in most subunit combinations. For example, a stimulation of current amplitude has been observed for almost all a1 subunits. In addition, auxiliary b subunits systematically shift the current–voltage relationship towards more
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Fig. 4. Immunoreactivity for the VDCC b1, b2 and b4 subunits in the CA1 region of patients with TLE. In the control hippocampus, b1 (A) and b2 (B) staining is mainly seen in the neuronal cell bodies, while b4 immunoreactivity (C) is prominent in the dendritic compartment and less intense in the somata. This distribution pattern can also be observed in lesion-associated TLE specimens (D, E, F). In the hippocampus obtained from a TLE patient with severe AHS (G, H, I), the number of immunoreactive neurons decreases due to substantial neuron loss. Some of the remaining neurons show enhanced staining for b1 (G) and b2 (H) compared to the controls. In addition, strongly b1- and b2-immunoreactive dendrites are found (G, H). The primarily dendritic localization of the b4 subunit (I) is similar to the control. Scale bar 8 mm.
hyperpolarized potentials and modify the time-dependent activation and inactivation behaviour of VDCCs (for review see Refs 8 and 29). In contrast to the a1A–E subunits, T-type currents probably corresponding to a1G and a1H subunits are unchanged following depletion of b subunits. 15 In our study, we have shown a differential subcellular
localization of the b4 and the b1–3 subunits. It is not yet clear which a1 and b subunits assemble in the brain to form native channels. However, it is generally assumed that different a1/b subunit combinations exist in different subcellular compartments. b3 and b4 have been demonstrated as the major L-type channel b subunits in the mammalian
VDCC b subunits in temporal lobe epilepsy
hippocampus, 19 while the N-type channel appears to be primarily associated with the b1b, b3 and b4 subunits. 20 Interestingly, L-type channels have been shown to be predominantly located in neuronal cell bodies, while N-type and P/ Q-type channels are concentrated in dendrites. 9 The subcellular localization of the remaining types of VDCCs has been less well characterized. Nevertheless, increasing evidence indicates that a differential segregation of the b subunits is important for the subcellularly specific modification of pharmacological and kinetic properties of a1 subunits. An interesting change was observed for the b1 and b2 subunits in AHS, i.e. localization of these subunits in dendrites of surviving pyramidal cells in intensely sclerotic regions of the Ammon’s horn. While we cannot completely exclude a minor contribution of presynaptic b1 and b2 subunit labelling, the characteristic staining pattern strongly suggests up-regulation of b1 and b2 subunits in dendrites. It is tempting to speculate that this up-regulation may enhance currents carried by VDCCs in AHS. An enhancement of Ca 21 current amplitude occurs in the kindling and kainate models of epilepsy in the CA1 region and the dentate gyrus, respectively. 2,10,27 In addition to enhanced expression of a1 subunits, 11 alterations in modulatory b subunits may contribute to this phenomenon. The increase of Ca 21 current amplitude may have profound influence on the discharge properties of neurons. For instance, altered dendritic Ca 21 currents may affect the electrogenic properties of dendrites which control the propagation of synaptic potentials towards the soma of hippocampal neurons. 17 In addition, Ca 21-dependent bursting has been observed in some neuronal cells, and it is tempting to speculate that Ca 21 current up-regulation might also affect the propensity of hippocampal neurons to burst. Finally, voltage-dependent Ca 21 currents represent one of the main entry routes for Ca 21 into neurons. As intracellular Ca 21 overload seems to be able to trigger neuronal cell death, 5 there may be a link between altered expression of Ca 21 channel subunits and selective vulnerability of pyramidal cells in AHS.
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In addition to the modification of specific pharmacological and kinetic properties of a1 subunits, it has recently been recognized that b subunits are also involved in targeting the translocation of a1 subunits to the neuronal membrane. 4,24 Thus, the almost exclusive presence of b4 subunits in dendrites raises the possibility that these subunits may be important in targeting specific a1 subunits to the dendritic compartment. It is interesting to note that a b4 subunit-specific interaction site has been discovered in the carboxy-terminal region of the a1A subunit, suggesting that specific interactions between these two channel components do exist. 28 In this context, the enhanced b1 and b2 immunoreactivity in neuronal cell bodies together with the novel appearance of b1 and b2 subunits in the dendritic compartment within severely sclerotic hippocampal subfields of AHS specimens may also affect the distribution of a1 subunits.
CONCLUSIONS
We have shown that hippocampal neurons express multiple classes of VDCC b subunits which appear to segregate into particular subcellular domains. In addition, we report an enhancement of b1 and b2 immunoreactivity in neuronal cell bodies and an additional shift of the b1 and b2 subunits into the dendritic compartment in severely sclerotic hippocampal regions of AHS specimens. These alterations in the distribution of VDCC b subunits may lead to considerably altered function of voltage-dependent Ca 21 channels and contribute to hyperexcitability in TLE associated with AHS.
Acknowledgements—We are grateful to our neurosurgical colleagues Prof. Schramm, Prof. Zentner, and Dr van Roost who supplied biopsy specimens. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 400; DFG EL 122/7-1), the joint German-Israeli Research Program and a University of Bonn Center Grant (BONFOR). A. A. Lie is recipient of a postdoctoral fellowship from the Gertrud Reemtsma Stiftung of the Max-Planck-Gesellschaft.
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