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Reduced density of parvalbumin- and calbindin D28k-immunoreactive neurons in experimental cortical dysplasia
Epilepsy Research 37 (1999) 63 – 71 www.elsevier.com/locate/epilepsyres
Reduced density of parvalbumin- and calbindin D28k-immunoreactive neurons in experimental cortical dysplasia Steven N. Roper a,d,*, Stephan Eisenschenk b, Michael A. King c,d a
Department of Neurological Surgery, Box 100265, Uni6ersity of Florida, Gaines6ille, FL 32610 -0265, Florida, USA b Departments of Neurology, Uni6ersity of Florida, Gaines6ille, FL 32610 -0265, Florida, USA c Departments of Neuroscience, Uni6ersity of Florida, Gaines6ille, FL 32610 -0265, Florida, USA d Gaines6ille VA Medical Center, Gaines6ille, FL 32610 -0265, Florida, USA Received 13 November 1998; received in revised form 5 March 1999; accepted 21 April 1999
Abstract Cortical dysplasia (CD) is a congenital brain malformation in humans that is closely associated with intractable epilepsy. This study utilized an animal model of CD, in utero irradiation in rats, to determine if experimental dysplastic cortex demonstrates a reduction in the density of inhibitory interneurons. Fetal rats were exposed to external irradiation on gestational day 17 to produce diffuse CD and heterotopic grey matter. As adults, these rats were processed for immunohistochemistry using primary antibodies for parvalbumin (PA), calbindin D28k (CA), the 67 kD subunit of glutamic acid decarboxylase (GAD67), and cresyl violet staining. Quantitative methods were used to determine the density of immunoreactive neurons and cresyl violet-stained neurons in the neocortex at the rostro-caudal level of the anterior commissure. Compared to control values, the density of PA- and CA-immunoreactive neurons was reduced in dysplastic cortex. Density of glutamic acic decarboxylase-immunoreactive neurons was not different between control and dysplastic cortex. Overall neuronal density, measured in cresyl violet-stained sections, was not significantly different between control and dysplastic cortex. These data suggest a selective reduction in inhibitory interneurons in experimental CD cortex or an impaired ability for these neurons to produce PA and CA. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Cortical dysplasia; Development; Inhibition; Neocortex
1. Introduction
* Corresponding author. Tel.: +1-352-3924331; fax: +1352-3928413. E-mail address: [email protected] (S.N. Roper)
Cortical dysplasia (CD) describes a human congenital brain abnormality that results from a deviation from normal development during the formation of the cerebral cortex. It is characterized by abnormalities in neuronal location, orien-
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tation, and morphology (Mischel et al., 1995). It has a strong clinical association with intractable epilepsy (Taylor et al., 1971; Palmini et al., 1991; Raymond et al., 1995). Abnormalities in electroencephalographic activity over areas of focal CD (Palmini et al., 1995) and surgical studies that correlate seizure control with completeness of resection (Hirabayashi et al., 1993; Palmini et al., 1995) suggest a causal relationship between CD and epilepsy. In spite of this, relatively little is known about the neurochemical identity and physiologic properties of neurons in dysplastic cortex. This study was undertaken to determine if the density of inhibitory neurons was altered in an animal model of CD. Exposure of pregnant rats to external g-irradiation on the 17th day of gestation (E17) produces abnormalities of cortical development in the offspring when they are examined as adults. These include microcephaly, diffuse CD, subcortical heterotopic grey matter, heterotopic neurons in the hippocampus, and agenesis or hypoplasia of the corpus callosum (Riggs et al., 1956; Cowan and Geller, 1960; Roper et al., 1995). Previous studies have demonstrated an increased propensity for electrographic seizures in vivo in the presence of certain sedating agents (Roper et al., 1995), and enhanced epileptiform activity in dysplastic cortex (compared to control neocortex) in vitro when GABAA-mediated inhibition is blocked (Roper et al., 1997). Most theories of epileptogenesis are based upon an imbalance of excitatory influences and inhibitory control over a population of connected neurons. Abnormalities that selectively reduce the density of inhibitory neurons would make the affected cortex more susceptible to seizure activity. Cortical inhibitory neurons have neurochemical profiles that allow them to be labeled using immunohistochemical techniques. The calcium binding proteins, parvalbumin (PA) and calbindin D28k (CA), are present in two, largely non-overlapping populations of inhibitory neocortical neurons (Hendry et al., 1989). The 67 kD subunit of glutamic acid decarboxylase (GAD 67) is a general marker for GABAergic neurons (Kaufman et al., 1991). The current study attempted to determine if there is a selective reduction in inhibitory
neurons in dysplastic cortex by comparing the densities of PA-, CA-, and GAD 67-labeled and cresyl-violet stained neurons with control neocortex.
2. Methods Animals were prepared by obtaining pregnant Sprague–Dawley rats with a known date of insemination (Harlan Sprague–Dawley Inc. PO Box 29176, Indianapolis, IN 46229-0176, USA). The date of insemination was designated E0. On E17, the pregnant rat was exposed to 225 cGy external l-irradiation from a linear accelerator source. Offspring were delivered normally and weaned on post-natal day 21 (P21). Rats were sacrificed for histology as adults (\ P60). All animal care was in accordance with protocols approved by the appropriate institutional review boards. Immunohistochemical studies were performed on 11 adult experimental rats from five litters and 12 adult control rats from five litters. All animals were deeply sedated with sodium pentobarbital (100 mg/kg), underwent intracardiac perfusion with 4% paraformaldehyde, and were decapitated. The brains were dissected free and placed in 4% paraformaldehyde overnight. They were cryoprotected by floating in 30% sucrose sodium-phosphate buffer solution until osmotically equilibrated. Thirty-mm thick coronal sections were obtained through the forebrain using a freezing microtome. Sections at the rostrocaudal level of the anterior commissure were evaluated. For quantitative assessment, one section was evaluated from each animal for each of the three antibodies and for cresyl violet-stained sections. Immunohistochemistry was performed on adjacent sections using primary antibodies for PA, CA, and GAD 67. The primary antibody for PA was a monoclonal mouse antibody (Accurate Chemical and Scientific Corp. 300 Shames Drive, Westbury, NY 11590, USA) used at a dilution of 1:2500. The primary antibody for CA was monoclonal mouse antibody (Sigma Chemical Co., PO Box 14508, St Louis, MO 63178-9974, USA) used at a dilution of 1:300. The primary antibody for
S.N. Roper et al. / Epilepsy Research 37 (1999) 63–71
GAD 67 was a polyclonal rabbit antibody (Chemicon International Inc., 28835 Single Oak Drive, Temecula, CA 92590, USA) used at a dilution of 1:1000. Primary antibodies were visualized using biotinylated secondary antibodies, avidin-horseradish peroxidase, and diaminobenzidine (DAB). Color intensification using nickel/ DAB was performed for CA and GAD 67 immunostaining but not in PA-labeled sections. Cresyl violet staining was performed on 16 experimental (from six litters) and 15 control rats (from six litters) using standard techniques. Eleven of the animals in both groups were the same as those used for immunohistochemical studies. In these cases, the sections used for cresyl violet staining were adjacent to those used for immunohistochemistry. Cell counting was performed on immunohistochemical sections using a digital imaging system attached to the microscope (ImagePro II (Media Cybernetics), 8484 Georgia Avenue, Silver Spring, MD 20910, USA). For each section, a region of the dorsal cortex above the lateral ventricle (at the level of the anterior commissure) was photographed at low magnification (4× objective). Using this image, a rectangular box that was 600 mm wide and extended through the depth of the cortex was placed over this region (Fig. 1). Subcortical heterotopic neurons were not included in this study. This rectangular image was cut and saved separately. A blinded reviewer analyzed each of these rectangular images. An automated counting program was used to count the number of immunoreactive cells in each box, but the reviewer set the windows and threshold for what was counted and visually ascertained that nonneuronal structures were not counted and that all neurons were included. Dysplastic cortex was significantly thinner than control cortex. Therefore, final counts were mathematically normalized to an area 600×1000 mm. Densities of PA-, CA-, and GAD 67-immunoreactive neurons were averaged and compared between the two groups (control vs. experimental) using the non-paired, two-tailed Students t-test. Densities of cresyl-violet stained neurons were counted using a computerized image acquisition system that controlled the objective stage and
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focusing of the microscope. Microscope images of selected brain sections were digitized at 12.5 × using a video camera connected to a PCI/Pentium microcomputer configured with a Flashpoint 128 (Integral Technologies Inc., 9855 Crosspoint Blvd. c 126, Indianapolis, IN 46256-3336, USA) video capture card controlled by ImagePro Plus software (Media Cybernetics, 8484 Georgia Avenue, Silver Spring, MD 20910, USA). Cortical count regions, bounded medially by the shoulder of the interhemispheric fissure, laterally by an imaginary line extending dorsally from the most lateral extent of the lateral ventricle, dorsally by the pia, and ventrally, by the subcortical white matter, were outlined on the low magnification images. The calibrated areas of these count regions were measured. An optical fractionator technique was used to generate non-biased estimates of neuronal density in the count regions. Optical dissectors (West et al., 1991) were positioned in a systematic random manner in the tissue sections by software
Fig. 1. Captured video image of a 30-mm thick, coronal section from a control rat immunolabeled with an antibody for PA. The dorsal cortex overlying the lateral ventricle at the level of the anterior commissure was the region sampled for determining the density of PA-, CA-, and GAD 67-immunoreactive neurons. A rectangular counting frame is superimposed on the image. The horizontal border of the counting frame =600 mm. The vertical length of the rectangle was set for each section so that it extended from the layer I/II interface through layer VI. The neurons within the counting frame appear darker because they have been digitally marked as counted objects during image processing.
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controlling a motorized microscope stage (Prior Scientific, 80 Reservoir park Drive, Rockland, MA 02370, USA) and referring to the count area outline. Dissector spacing was 150 mm in the X and Y dimensions. At each location the number of neurons containing definite nucleoli was counted, using a 100 × objective, in optical cubes of tissue 10 mm deep (controlled by stage control software) in unbiased count frames 724 mm2 (total volume= 7240 mm3) printed on acetate video monitor overlays. As is typical and necessary for unbiased counts, neurons present at the bottom of the cubes, or touching the exclusion lines of the count frame, were not counted. Section thickness was measured at each dissector location to determine the depth sampling frequency (count cube depth/section thickness). Neuronal density was calculated as the total number of cells counted divided by the total volume of all the dissector cubes. Densities were compared using one-way analysis of variance (factor: irradiation) using SAS (SAS Institute, Cary NC, USA). 3. Results Cortical abnormalities were consistently seen in the irradiated animals and have been described in Fig. 2. Low-magnification photomicrographs of normal (A,C,E) and dysplastic (B,D,F) dorsal neocortex at the level of the anterior commissure. Sections labeled for PA show immunoreactive neurons and terminals throughout the depth of the cortex in both control (A) and dysplastic (B) cortex. Sections from irradiated rats (B) also demonstrate PA-immunoreactive neurons in subcortical heterotopic grey matter (arrow) and hippocampus (asterisk). Quantitative measurements demonstrate a reduced density of PA-immunoreactive neurons in dysplastic cortex. CA-labeled sections show intensely immunoreactive neurons scattered sparsely throughout the cortex in both control (C) and dyplastic (D) cortex. CA-immunoreactive neurons are also present in subcortical heterotopic grey matter (arrow) in irradiated rats. Quantitative measurements show a reduced density of CA-labeled neurons in dysplastic cortex. Cresyl-violet stained sections (E,F) show that dysplastic cortex is significantly thinner than control neocortex. In image F, the deep border of the dysplastic cortex is demarcated by the dotted line with subcortical heterotopic grey matter (asterisk) lying below it. Total neuronal density is not signficantly different between control (E) and dysplastic (F) cortex. In all images, the pial surface is at the top. Scale bar =400 mm.
Fig. 2.
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detail elsewhere (Riggs et al., 1956; Cowan and Geller, 1960; Roper et al., 1995; Roper, 1998). All experimental animals showed CD with thinning of the neocortex, subcortical heterotopic grey matter, heterotopic neurons in the hippocampus, and agenesis or hypoplasia of the corpus callosum. Dysplastic cortex showed absence of normal lamination, groups of neurons extending through layer I to the pial surface, and abnormal orientation of many neurons. PA-immunoreactive cells were scattered throughout the depth of the cortex in experimental and control animals (Fig. 2A,B). Inspection of the cells at high power demonstrated neurons with a non-pyramidal morphology (Fig. 3A,B). Punctate labeling of terminals was present throughout the cortex but most dense in layers II/III and V. These PA-immunoreactive terminals could be seen surrounding and defining the outline of some of the PA-negative cell bodies. The density of PAimmunoreactive cells was significantly reduced in dysplastic cortex (53.7 911.4, mean 9 SD) (cells per counting frame with a dimension of 600 × 1000 mm) as compared to control cortex (88.0 9 18.2) (PB 0.0001). CA-immunoreactive cells fell into two categories; consistent with previously published reports (van Brederode et al., 1991; Alcanatara et al., 1993). Very lightly labeled cells were found in high numbers in layers II/III. These cells were neuronal and possessed a pyramidal cell morphology. These cells were not counted in the current study. The second population consisted of cells that were strongly immunoreactive for CA (Fig. 2C,D). They were relatively sparse but occurred throughout the depth of the cortex. These cells demonstrated a morphology consistent with nonpyramidal neurons (Fig. 3C,D). No labeling of terminals was seen with the CA antibody. The density of CA-immmunoreactive cells was significantly reduced in dysplastic cortex (20.19 13.7) (cells per counting frame with a dimension of 600×1000 mm) as compared to control neocortex (39.5 919.3) (PB0.02). GAD 67-immunoreactive cells occurred throughout the depth of the cortex. Cell bodies were labeled and punctate labeling of terminals was also seen. The density of GAD 67-immunore-
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active cells was not different between the dysplastic cortex (55.99 17.9) (cells per counting frame with a dimension of 600× 1000 mm) and control neocortex (51.09 21.4) (P = 0.57) The density of total neurons was determined using cresyl violet-stained sections (Fig. 2E,F and Fig. 3E,F). As mentioned above, the thickness (pia to subcortical white matter) of the dysplastic cortex was consistently reduced as compared to control cortex (by about 50%) (Fig. 2E,F). The mean neuronal density in dysplastic cortex was 169× 103 9 52× 103 neurons/mm3 compared to a mean of 186×103 9 36× 103 neurons/mm3 in control neocortex. Although the calculated mean density value was slightly lower in dysplastic cortex (91% of control), this difference was not statistically significant (P= 0.2893).
4. Discussion This study is the first to show a quantitative, selective reduction in the density of PA- and CA-immunoreactive neurons in experimenta CD. PA and CA are calcium binding proteins that are found in inhibitory interneurons in the neocortex and throughout the nervous system. They identify different morphologic subtypes of cortical GABAergic interneurons with PA expressed in basket cells and chandelier cells and calbindin expressed in double bouquet cells (DeFelipe et al., 1989a,b; Hendry et al., 1989). Therefore, the current findings would suggest a selective reduction in inhibitory neurons in dysplastic cortex. This evidence is somewhat indirect in that it is impossible to know if some other type of inhibitory neuron that is not labeled by PA or CA is still present in sufficient numbers to maintain inhibitory control in the cortical circuitry or if the irradiation treatment somehow altered the ability of some inhibitory interneurons to produce PA and CA, making them undetectable with the current techniques. This study did not find a difference in the density of GAD 67-immunoreactive neurons in dysplastic and control neocortex. The GAD 67 antibody should, theoretically, provide a more
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Fig. 3. High-magnification photomicrographs of normal (A,C,E) and dysplastic (B,D,F) dorsal neocortex at the level of the anterior commissure. Sections labeled with an antibody for PA show that immunoreactive neurons are less numerous in the dysplastic cortex (B) as compared to control neocortex (A). Punctate labeling of PA-immunoreactive terminals is also demonstrated. CA-labeled neurons are seen in both control (C) and dysplastic cortex (D). The non-pyramidal morphology of a CA-immunoreactive neuron is demonstrated in image D (arrow). Cresyl violet-stained sections show that overall neuronal density is similar between control (E) and dysplastic (F) cortex. In all images, the pial surface is to the left. Scale bar =100 mm.
complete labeling of all GABAergic neurons. Therefore, one would predict that the GAD 67-labeled cells would include both the PA- and CA-labeled cells (and possibly others) and that the total counts of the GAD 67-immunoreactive cells would be greater than or equal to the sum of the PA- and CA-labeled cells. In practice, however,
we found that labeling with GAD 67 was variable and our cell densities with this antibody were less than those of PA-labeled cells alone. Esclapez et al. (1994) have shown that in situ hybridization for GAD 67 mRNA can identify GABAergic cells that are not labeled by immunohistochemical techniques, presumably as a result of low levels of
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the protein in the soma. Since our data showed that the GAD 67 antibody was only labeling a fraction of the cells that we had intended for it to identify, we felt that the GAD 67 results did not provide a meaningful estimate of inhibitory interneurons in this study. Although indirect, the current data strongly suggest a significant reduction (by 40 to 50% from control values) in the density of a subpopulation (PA- and CA-immunoreactive) of inhibitory neurons in experimental CD. Reductions of inhibitory neurons to this degree (in the absence of any other compensatory changes) should significantly increase excitability in the local circuitry of the affected cortex. The quantitative analysis of total neuronal density using cresyl violet-stained sections shows that the decrease in PA- and CA-immunoreative neurons is a selective one. These counts showed that the total neuronal density was not significantly lower in dysplastic cortex when compared to control neocortex. This may sound surprising because the loss of PA- and CA-labeled neurons should be reflected in the total neuronal density. Inhibitory interneurons comprise about 25% of neurons in the neocortex (Jones, 1993). Since we saw a 40 – 50% reduction in PA- and CA-labeled neurons this would produce, at most, a 10 – 12% reduction in total neuronal density. If PA- and CA-immunoreactive neurons do not account for all inhibitory interneurons in the neocortex, then the expected percentage reduction in total neuronal density would be even smaller. Our measurements showed a calculated mean density of neurons in dysplastic cortex that was 10% less than in control; however, this difference was not statistically significant. Based on these estimates, our data from cresyl violet-stained sections are not inconsistent with a significant reduction in PA- and CA-labeled neurons and show that there is not a major reduction in overall neuronal density. This demonstrates that the reduction in PA- and CAlabeled neurons is, to some degree, a selective one. It does not rule out the possibility that there are other subpopulations of neurons (not identified in this study) that may also be diminished by in utero irradiation.
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The current study used non-stereologic cell counting techniques to quantify densities of PAand CA-immunoreactive neurons in somatosensory cortex of control and irradiated rats. The study was designed to complement physiologic experiments that are being performed using in vitro brain slices from the same region. In this narrow context, the density of inhibitory interneurons in a specific region becomes more important than the total number of such neurons throughout the entire cortex. The current cell-counting techniques do not determine the absolute number of labeled neurons throughout the entire cortex; nor do they provide absolute densities of labeled neurons in the volume that was measured. They are useful only as a means of quantitative comparison between the two experimental groups. The tendency for two-dimensional cell counting techniques to overestimate object density is well established (West and Gundersen, 1990). This is a result of the fact that portions of an object may be included in the sections and counted as a whole and the degree of bias is affected by the size of the object relative to the thickness of the volume analyzed. Since dysplastic cortex did not show abnormalities in the volume of individual neurons (this is a characteristic of some types of human CD that is not maintained in this animal model), any counting biases should apply equally to both experimental groups. Therefore, the quantitative measurements used for the PA- and CA-labeled cells should be valid for the purpose of comparison between the two groups. The two-dimensional counting techniques were not applicable to the cresyl violet-stained sections because the density of the imaged cells was too great. Therefore, modified stereologic techniques were used to determine cell density in cresyl violet-stained sections. A possible difference in the absolute number of PA- and CA-labeled cells throughout the cortex of control and irradiated rats is another important question and experiments to address this issue using stereologic techniques are underway. Studies on human CD have noted areas of reduction in neurons that are immunoreactive for calcium binding proteins. Ferrer et al. (1992, 1994) described specimens from two patients with
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CD. In both cases, they found abnormally large neurons that were immunoreactive for PA and CA. They also described areas within these specimens where PA- and CA-immunoreactivity was dramatically reduced. Spreafico et al. (1998) examined surgical specimens from three patients with CD. In all three cases, they reported a qualitative reduction in the density of neurons that were immunoreactive for PA, CA, and calretinin. They also described dense accumulations of PAimmunoreactive terminals surrounding the perikarya of PA-negative giant pyramidal cells and balloon cells. Both of these authors interpreted their results as suggestive of a selective reduction of inhibitory neurons in human CD. The current study would complement these findings in an injury-based animal model of CD. Several clinical studies have demonstrated the highly epileptogenic behavior of neurons in areas of human CD. Palmini et al. (1995) reported frequent or continuous epileptiform activity in human dysplastic cortex recorded during surgery for intractable epilepsy in 67% of such lesions. This markedly abnormal physiology seems to be a distinctive feature of CD because it was seen in only 1% of mesial temporal lobe epilepsy cases that were used for comparison. Others have demonstrated interictal and ictal epileptiform activity from scalp EEG that localizes to regions of dysplastic cortex (Guerrini et al., 1992; Desbiens et al., 1993; Hirabayashi et al., 1993; Raymond et al., 1995). Studies from in vitro brain slices of human dysplastic cortex demonstrate prolonged epileptiform discharges in response to stimulation and application of the proconvulsant compound, 4-aminopyridine, that are not seen in neocortical slices from patients with mesial temporal lobe epilepsy (Mattia et al., 1995). Although none of these studies establish the mechanisms that underlie this pronounced epileptogenicity of dysplastic cortex, an impaired inhibitory system would certainly be a strong contributing factor. The current study provides additional support for impaired inhibition as a cause of increased seizure susceptibility in some types of CD. This study provides evidence for a selective reduction of inhibitory interneurons in an animal model of CD. This represents a possible mecha-
nism for the excitability and epileptogenicity that has been identified in human CD. It also complements and supports findings from immunohistochemical studies of human dysplastic cortex. In addition, it demonstrates that a definable injury that occurs at a specific point in cortical development (in the absence of any pre-existing genetic abnormality) can have a major effect on the representation of neuronal subtypes in the neocortex of the mature animal. Investigations into the physiologic impact of these alterations and their effect on seizure susceptibility are ongoing.
Acknowledgements The authors are grateful to Dr Frank J. Bova of the Department of Neurological Surgery at the University of Florida for his assistance with animal irradiation. This work was supported by a grant from the National Institutes of Health (1R29NS35651) to S.N. Roper.
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Palmini, A., Gambardella, A., Andermann, F., et al., 1995. Intrinsic epileptogenicity of human dysplastic cortex as suggested by corticography and surgical results. Ann. Neurol. 37, 476 – 487. Raymond, A.A., Fish, D.R., Sisodiya, S.M., Alsanjari, N., Stevens, J.M., Shorvon, S.D., 1995. Abnormalities of gyration, heterotopias, tuberous sclerosis, focal cortical dysplasia, microdysgenesis, dysembryoplastic neuroepithelial tumor and dysgenesis of the archicortex in epilepsy: clinical, EEG and neuroimaging features in 100 adult patients. Brain 118, 629 – 660. Riggs, H.E., McGrath, J.J., Schwartz, H.P., 1956. Malformation of the adult brain (albino rat) resulting from prenatal irradiation. J. Neuropathol. Exp. Neurol. 15, 432 – 447. Roper, S.N., Gilmore, R.L., Houser, C.R., 1995. Experimentally induced disorders of neuronal migration produce an increased propensity for electrographic seizures in rats. Epilepsy Res. 21, 205 – 219. Roper, S.N., King, M.A., Abraham, L.A., Boillot, M.A., 1997. Disinhibited in vitro neocortical slices containing experimentally induced cortical dysplasia demonstrate hyperexcitability. Epilepsy Res. 26, 443 – 449. Roper, S.N., 1998. In utero irradiation of rats as a model of human cerebrocortical dysgenesis: a review. Epilepsy Res. 32, 63 – 74. Spreafico, R., Battaglia, G., Arcelli, P., et al., 1998. Cortical dysplasia: an immunocytochemical study of three patients. Neurology 50, 27 – 36. Taylor, D.C., Falconer, M.A., Bruton, C.J., Corsellis, J.A.N., 1971. Focal dysplasia of the cerebral cortex in epilepsy. J. Neurol. Neurosurg. Psychiat. 34, 369 – 387. West, M.J., Slomianka, L., Gundersen, H.J.G., 1991. Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator. Anat. Rec. 231, 482 – 497. West, M.J., Gundersen, H.J.G., 1990. Unbiased stereological estimation of the number of neurons in the human hippocampus. J. Comp. Neurol. 296, 1 – 22.
Reduced density of parvalbumin- and calbindin D28k-immunoreactive neurons in experimental cortical dysplasia Steven N. Roper a,d,*, Stephan Eisenschenk b, Michael A. King c,d a
Department of Neurological Surgery, Box 100265, Uni6ersity of Florida, Gaines6ille, FL 32610 -0265, Florida, USA b Departments of Neurology, Uni6ersity of Florida, Gaines6ille, FL 32610 -0265, Florida, USA c Departments of Neuroscience, Uni6ersity of Florida, Gaines6ille, FL 32610 -0265, Florida, USA d Gaines6ille VA Medical Center, Gaines6ille, FL 32610 -0265, Florida, USA Received 13 November 1998; received in revised form 5 March 1999; accepted 21 April 1999
Abstract Cortical dysplasia (CD) is a congenital brain malformation in humans that is closely associated with intractable epilepsy. This study utilized an animal model of CD, in utero irradiation in rats, to determine if experimental dysplastic cortex demonstrates a reduction in the density of inhibitory interneurons. Fetal rats were exposed to external irradiation on gestational day 17 to produce diffuse CD and heterotopic grey matter. As adults, these rats were processed for immunohistochemistry using primary antibodies for parvalbumin (PA), calbindin D28k (CA), the 67 kD subunit of glutamic acid decarboxylase (GAD67), and cresyl violet staining. Quantitative methods were used to determine the density of immunoreactive neurons and cresyl violet-stained neurons in the neocortex at the rostro-caudal level of the anterior commissure. Compared to control values, the density of PA- and CA-immunoreactive neurons was reduced in dysplastic cortex. Density of glutamic acic decarboxylase-immunoreactive neurons was not different between control and dysplastic cortex. Overall neuronal density, measured in cresyl violet-stained sections, was not significantly different between control and dysplastic cortex. These data suggest a selective reduction in inhibitory interneurons in experimental CD cortex or an impaired ability for these neurons to produce PA and CA. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Cortical dysplasia; Development; Inhibition; Neocortex
1. Introduction
* Corresponding author. Tel.: +1-352-3924331; fax: +1352-3928413. E-mail address: [email protected] (S.N. Roper)
Cortical dysplasia (CD) describes a human congenital brain abnormality that results from a deviation from normal development during the formation of the cerebral cortex. It is characterized by abnormalities in neuronal location, orien-
0920-1211/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 0 - 1 2 1 1 ( 9 9 ) 0 0 0 3 5 - 2
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tation, and morphology (Mischel et al., 1995). It has a strong clinical association with intractable epilepsy (Taylor et al., 1971; Palmini et al., 1991; Raymond et al., 1995). Abnormalities in electroencephalographic activity over areas of focal CD (Palmini et al., 1995) and surgical studies that correlate seizure control with completeness of resection (Hirabayashi et al., 1993; Palmini et al., 1995) suggest a causal relationship between CD and epilepsy. In spite of this, relatively little is known about the neurochemical identity and physiologic properties of neurons in dysplastic cortex. This study was undertaken to determine if the density of inhibitory neurons was altered in an animal model of CD. Exposure of pregnant rats to external g-irradiation on the 17th day of gestation (E17) produces abnormalities of cortical development in the offspring when they are examined as adults. These include microcephaly, diffuse CD, subcortical heterotopic grey matter, heterotopic neurons in the hippocampus, and agenesis or hypoplasia of the corpus callosum (Riggs et al., 1956; Cowan and Geller, 1960; Roper et al., 1995). Previous studies have demonstrated an increased propensity for electrographic seizures in vivo in the presence of certain sedating agents (Roper et al., 1995), and enhanced epileptiform activity in dysplastic cortex (compared to control neocortex) in vitro when GABAA-mediated inhibition is blocked (Roper et al., 1997). Most theories of epileptogenesis are based upon an imbalance of excitatory influences and inhibitory control over a population of connected neurons. Abnormalities that selectively reduce the density of inhibitory neurons would make the affected cortex more susceptible to seizure activity. Cortical inhibitory neurons have neurochemical profiles that allow them to be labeled using immunohistochemical techniques. The calcium binding proteins, parvalbumin (PA) and calbindin D28k (CA), are present in two, largely non-overlapping populations of inhibitory neocortical neurons (Hendry et al., 1989). The 67 kD subunit of glutamic acid decarboxylase (GAD 67) is a general marker for GABAergic neurons (Kaufman et al., 1991). The current study attempted to determine if there is a selective reduction in inhibitory
neurons in dysplastic cortex by comparing the densities of PA-, CA-, and GAD 67-labeled and cresyl-violet stained neurons with control neocortex.
2. Methods Animals were prepared by obtaining pregnant Sprague–Dawley rats with a known date of insemination (Harlan Sprague–Dawley Inc. PO Box 29176, Indianapolis, IN 46229-0176, USA). The date of insemination was designated E0. On E17, the pregnant rat was exposed to 225 cGy external l-irradiation from a linear accelerator source. Offspring were delivered normally and weaned on post-natal day 21 (P21). Rats were sacrificed for histology as adults (\ P60). All animal care was in accordance with protocols approved by the appropriate institutional review boards. Immunohistochemical studies were performed on 11 adult experimental rats from five litters and 12 adult control rats from five litters. All animals were deeply sedated with sodium pentobarbital (100 mg/kg), underwent intracardiac perfusion with 4% paraformaldehyde, and were decapitated. The brains were dissected free and placed in 4% paraformaldehyde overnight. They were cryoprotected by floating in 30% sucrose sodium-phosphate buffer solution until osmotically equilibrated. Thirty-mm thick coronal sections were obtained through the forebrain using a freezing microtome. Sections at the rostrocaudal level of the anterior commissure were evaluated. For quantitative assessment, one section was evaluated from each animal for each of the three antibodies and for cresyl violet-stained sections. Immunohistochemistry was performed on adjacent sections using primary antibodies for PA, CA, and GAD 67. The primary antibody for PA was a monoclonal mouse antibody (Accurate Chemical and Scientific Corp. 300 Shames Drive, Westbury, NY 11590, USA) used at a dilution of 1:2500. The primary antibody for CA was monoclonal mouse antibody (Sigma Chemical Co., PO Box 14508, St Louis, MO 63178-9974, USA) used at a dilution of 1:300. The primary antibody for
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GAD 67 was a polyclonal rabbit antibody (Chemicon International Inc., 28835 Single Oak Drive, Temecula, CA 92590, USA) used at a dilution of 1:1000. Primary antibodies were visualized using biotinylated secondary antibodies, avidin-horseradish peroxidase, and diaminobenzidine (DAB). Color intensification using nickel/ DAB was performed for CA and GAD 67 immunostaining but not in PA-labeled sections. Cresyl violet staining was performed on 16 experimental (from six litters) and 15 control rats (from six litters) using standard techniques. Eleven of the animals in both groups were the same as those used for immunohistochemical studies. In these cases, the sections used for cresyl violet staining were adjacent to those used for immunohistochemistry. Cell counting was performed on immunohistochemical sections using a digital imaging system attached to the microscope (ImagePro II (Media Cybernetics), 8484 Georgia Avenue, Silver Spring, MD 20910, USA). For each section, a region of the dorsal cortex above the lateral ventricle (at the level of the anterior commissure) was photographed at low magnification (4× objective). Using this image, a rectangular box that was 600 mm wide and extended through the depth of the cortex was placed over this region (Fig. 1). Subcortical heterotopic neurons were not included in this study. This rectangular image was cut and saved separately. A blinded reviewer analyzed each of these rectangular images. An automated counting program was used to count the number of immunoreactive cells in each box, but the reviewer set the windows and threshold for what was counted and visually ascertained that nonneuronal structures were not counted and that all neurons were included. Dysplastic cortex was significantly thinner than control cortex. Therefore, final counts were mathematically normalized to an area 600×1000 mm. Densities of PA-, CA-, and GAD 67-immunoreactive neurons were averaged and compared between the two groups (control vs. experimental) using the non-paired, two-tailed Students t-test. Densities of cresyl-violet stained neurons were counted using a computerized image acquisition system that controlled the objective stage and
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focusing of the microscope. Microscope images of selected brain sections were digitized at 12.5 × using a video camera connected to a PCI/Pentium microcomputer configured with a Flashpoint 128 (Integral Technologies Inc., 9855 Crosspoint Blvd. c 126, Indianapolis, IN 46256-3336, USA) video capture card controlled by ImagePro Plus software (Media Cybernetics, 8484 Georgia Avenue, Silver Spring, MD 20910, USA). Cortical count regions, bounded medially by the shoulder of the interhemispheric fissure, laterally by an imaginary line extending dorsally from the most lateral extent of the lateral ventricle, dorsally by the pia, and ventrally, by the subcortical white matter, were outlined on the low magnification images. The calibrated areas of these count regions were measured. An optical fractionator technique was used to generate non-biased estimates of neuronal density in the count regions. Optical dissectors (West et al., 1991) were positioned in a systematic random manner in the tissue sections by software
Fig. 1. Captured video image of a 30-mm thick, coronal section from a control rat immunolabeled with an antibody for PA. The dorsal cortex overlying the lateral ventricle at the level of the anterior commissure was the region sampled for determining the density of PA-, CA-, and GAD 67-immunoreactive neurons. A rectangular counting frame is superimposed on the image. The horizontal border of the counting frame =600 mm. The vertical length of the rectangle was set for each section so that it extended from the layer I/II interface through layer VI. The neurons within the counting frame appear darker because they have been digitally marked as counted objects during image processing.
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controlling a motorized microscope stage (Prior Scientific, 80 Reservoir park Drive, Rockland, MA 02370, USA) and referring to the count area outline. Dissector spacing was 150 mm in the X and Y dimensions. At each location the number of neurons containing definite nucleoli was counted, using a 100 × objective, in optical cubes of tissue 10 mm deep (controlled by stage control software) in unbiased count frames 724 mm2 (total volume= 7240 mm3) printed on acetate video monitor overlays. As is typical and necessary for unbiased counts, neurons present at the bottom of the cubes, or touching the exclusion lines of the count frame, were not counted. Section thickness was measured at each dissector location to determine the depth sampling frequency (count cube depth/section thickness). Neuronal density was calculated as the total number of cells counted divided by the total volume of all the dissector cubes. Densities were compared using one-way analysis of variance (factor: irradiation) using SAS (SAS Institute, Cary NC, USA). 3. Results Cortical abnormalities were consistently seen in the irradiated animals and have been described in Fig. 2. Low-magnification photomicrographs of normal (A,C,E) and dysplastic (B,D,F) dorsal neocortex at the level of the anterior commissure. Sections labeled for PA show immunoreactive neurons and terminals throughout the depth of the cortex in both control (A) and dysplastic (B) cortex. Sections from irradiated rats (B) also demonstrate PA-immunoreactive neurons in subcortical heterotopic grey matter (arrow) and hippocampus (asterisk). Quantitative measurements demonstrate a reduced density of PA-immunoreactive neurons in dysplastic cortex. CA-labeled sections show intensely immunoreactive neurons scattered sparsely throughout the cortex in both control (C) and dyplastic (D) cortex. CA-immunoreactive neurons are also present in subcortical heterotopic grey matter (arrow) in irradiated rats. Quantitative measurements show a reduced density of CA-labeled neurons in dysplastic cortex. Cresyl-violet stained sections (E,F) show that dysplastic cortex is significantly thinner than control neocortex. In image F, the deep border of the dysplastic cortex is demarcated by the dotted line with subcortical heterotopic grey matter (asterisk) lying below it. Total neuronal density is not signficantly different between control (E) and dysplastic (F) cortex. In all images, the pial surface is at the top. Scale bar =400 mm.
Fig. 2.
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detail elsewhere (Riggs et al., 1956; Cowan and Geller, 1960; Roper et al., 1995; Roper, 1998). All experimental animals showed CD with thinning of the neocortex, subcortical heterotopic grey matter, heterotopic neurons in the hippocampus, and agenesis or hypoplasia of the corpus callosum. Dysplastic cortex showed absence of normal lamination, groups of neurons extending through layer I to the pial surface, and abnormal orientation of many neurons. PA-immunoreactive cells were scattered throughout the depth of the cortex in experimental and control animals (Fig. 2A,B). Inspection of the cells at high power demonstrated neurons with a non-pyramidal morphology (Fig. 3A,B). Punctate labeling of terminals was present throughout the cortex but most dense in layers II/III and V. These PA-immunoreactive terminals could be seen surrounding and defining the outline of some of the PA-negative cell bodies. The density of PAimmunoreactive cells was significantly reduced in dysplastic cortex (53.7 911.4, mean 9 SD) (cells per counting frame with a dimension of 600 × 1000 mm) as compared to control cortex (88.0 9 18.2) (PB 0.0001). CA-immunoreactive cells fell into two categories; consistent with previously published reports (van Brederode et al., 1991; Alcanatara et al., 1993). Very lightly labeled cells were found in high numbers in layers II/III. These cells were neuronal and possessed a pyramidal cell morphology. These cells were not counted in the current study. The second population consisted of cells that were strongly immunoreactive for CA (Fig. 2C,D). They were relatively sparse but occurred throughout the depth of the cortex. These cells demonstrated a morphology consistent with nonpyramidal neurons (Fig. 3C,D). No labeling of terminals was seen with the CA antibody. The density of CA-immmunoreactive cells was significantly reduced in dysplastic cortex (20.19 13.7) (cells per counting frame with a dimension of 600×1000 mm) as compared to control neocortex (39.5 919.3) (PB0.02). GAD 67-immunoreactive cells occurred throughout the depth of the cortex. Cell bodies were labeled and punctate labeling of terminals was also seen. The density of GAD 67-immunore-
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active cells was not different between the dysplastic cortex (55.99 17.9) (cells per counting frame with a dimension of 600× 1000 mm) and control neocortex (51.09 21.4) (P = 0.57) The density of total neurons was determined using cresyl violet-stained sections (Fig. 2E,F and Fig. 3E,F). As mentioned above, the thickness (pia to subcortical white matter) of the dysplastic cortex was consistently reduced as compared to control cortex (by about 50%) (Fig. 2E,F). The mean neuronal density in dysplastic cortex was 169× 103 9 52× 103 neurons/mm3 compared to a mean of 186×103 9 36× 103 neurons/mm3 in control neocortex. Although the calculated mean density value was slightly lower in dysplastic cortex (91% of control), this difference was not statistically significant (P= 0.2893).
4. Discussion This study is the first to show a quantitative, selective reduction in the density of PA- and CA-immunoreactive neurons in experimenta CD. PA and CA are calcium binding proteins that are found in inhibitory interneurons in the neocortex and throughout the nervous system. They identify different morphologic subtypes of cortical GABAergic interneurons with PA expressed in basket cells and chandelier cells and calbindin expressed in double bouquet cells (DeFelipe et al., 1989a,b; Hendry et al., 1989). Therefore, the current findings would suggest a selective reduction in inhibitory neurons in dysplastic cortex. This evidence is somewhat indirect in that it is impossible to know if some other type of inhibitory neuron that is not labeled by PA or CA is still present in sufficient numbers to maintain inhibitory control in the cortical circuitry or if the irradiation treatment somehow altered the ability of some inhibitory interneurons to produce PA and CA, making them undetectable with the current techniques. This study did not find a difference in the density of GAD 67-immunoreactive neurons in dysplastic and control neocortex. The GAD 67 antibody should, theoretically, provide a more
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Fig. 3. High-magnification photomicrographs of normal (A,C,E) and dysplastic (B,D,F) dorsal neocortex at the level of the anterior commissure. Sections labeled with an antibody for PA show that immunoreactive neurons are less numerous in the dysplastic cortex (B) as compared to control neocortex (A). Punctate labeling of PA-immunoreactive terminals is also demonstrated. CA-labeled neurons are seen in both control (C) and dysplastic cortex (D). The non-pyramidal morphology of a CA-immunoreactive neuron is demonstrated in image D (arrow). Cresyl violet-stained sections show that overall neuronal density is similar between control (E) and dysplastic (F) cortex. In all images, the pial surface is to the left. Scale bar =100 mm.
complete labeling of all GABAergic neurons. Therefore, one would predict that the GAD 67-labeled cells would include both the PA- and CA-labeled cells (and possibly others) and that the total counts of the GAD 67-immunoreactive cells would be greater than or equal to the sum of the PA- and CA-labeled cells. In practice, however,
we found that labeling with GAD 67 was variable and our cell densities with this antibody were less than those of PA-labeled cells alone. Esclapez et al. (1994) have shown that in situ hybridization for GAD 67 mRNA can identify GABAergic cells that are not labeled by immunohistochemical techniques, presumably as a result of low levels of
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the protein in the soma. Since our data showed that the GAD 67 antibody was only labeling a fraction of the cells that we had intended for it to identify, we felt that the GAD 67 results did not provide a meaningful estimate of inhibitory interneurons in this study. Although indirect, the current data strongly suggest a significant reduction (by 40 to 50% from control values) in the density of a subpopulation (PA- and CA-immunoreactive) of inhibitory neurons in experimental CD. Reductions of inhibitory neurons to this degree (in the absence of any other compensatory changes) should significantly increase excitability in the local circuitry of the affected cortex. The quantitative analysis of total neuronal density using cresyl violet-stained sections shows that the decrease in PA- and CA-immunoreative neurons is a selective one. These counts showed that the total neuronal density was not significantly lower in dysplastic cortex when compared to control neocortex. This may sound surprising because the loss of PA- and CA-labeled neurons should be reflected in the total neuronal density. Inhibitory interneurons comprise about 25% of neurons in the neocortex (Jones, 1993). Since we saw a 40 – 50% reduction in PA- and CA-labeled neurons this would produce, at most, a 10 – 12% reduction in total neuronal density. If PA- and CA-immunoreactive neurons do not account for all inhibitory interneurons in the neocortex, then the expected percentage reduction in total neuronal density would be even smaller. Our measurements showed a calculated mean density of neurons in dysplastic cortex that was 10% less than in control; however, this difference was not statistically significant. Based on these estimates, our data from cresyl violet-stained sections are not inconsistent with a significant reduction in PA- and CA-labeled neurons and show that there is not a major reduction in overall neuronal density. This demonstrates that the reduction in PA- and CAlabeled neurons is, to some degree, a selective one. It does not rule out the possibility that there are other subpopulations of neurons (not identified in this study) that may also be diminished by in utero irradiation.
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The current study used non-stereologic cell counting techniques to quantify densities of PAand CA-immunoreactive neurons in somatosensory cortex of control and irradiated rats. The study was designed to complement physiologic experiments that are being performed using in vitro brain slices from the same region. In this narrow context, the density of inhibitory interneurons in a specific region becomes more important than the total number of such neurons throughout the entire cortex. The current cell-counting techniques do not determine the absolute number of labeled neurons throughout the entire cortex; nor do they provide absolute densities of labeled neurons in the volume that was measured. They are useful only as a means of quantitative comparison between the two experimental groups. The tendency for two-dimensional cell counting techniques to overestimate object density is well established (West and Gundersen, 1990). This is a result of the fact that portions of an object may be included in the sections and counted as a whole and the degree of bias is affected by the size of the object relative to the thickness of the volume analyzed. Since dysplastic cortex did not show abnormalities in the volume of individual neurons (this is a characteristic of some types of human CD that is not maintained in this animal model), any counting biases should apply equally to both experimental groups. Therefore, the quantitative measurements used for the PA- and CA-labeled cells should be valid for the purpose of comparison between the two groups. The two-dimensional counting techniques were not applicable to the cresyl violet-stained sections because the density of the imaged cells was too great. Therefore, modified stereologic techniques were used to determine cell density in cresyl violet-stained sections. A possible difference in the absolute number of PA- and CA-labeled cells throughout the cortex of control and irradiated rats is another important question and experiments to address this issue using stereologic techniques are underway. Studies on human CD have noted areas of reduction in neurons that are immunoreactive for calcium binding proteins. Ferrer et al. (1992, 1994) described specimens from two patients with
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CD. In both cases, they found abnormally large neurons that were immunoreactive for PA and CA. They also described areas within these specimens where PA- and CA-immunoreactivity was dramatically reduced. Spreafico et al. (1998) examined surgical specimens from three patients with CD. In all three cases, they reported a qualitative reduction in the density of neurons that were immunoreactive for PA, CA, and calretinin. They also described dense accumulations of PAimmunoreactive terminals surrounding the perikarya of PA-negative giant pyramidal cells and balloon cells. Both of these authors interpreted their results as suggestive of a selective reduction of inhibitory neurons in human CD. The current study would complement these findings in an injury-based animal model of CD. Several clinical studies have demonstrated the highly epileptogenic behavior of neurons in areas of human CD. Palmini et al. (1995) reported frequent or continuous epileptiform activity in human dysplastic cortex recorded during surgery for intractable epilepsy in 67% of such lesions. This markedly abnormal physiology seems to be a distinctive feature of CD because it was seen in only 1% of mesial temporal lobe epilepsy cases that were used for comparison. Others have demonstrated interictal and ictal epileptiform activity from scalp EEG that localizes to regions of dysplastic cortex (Guerrini et al., 1992; Desbiens et al., 1993; Hirabayashi et al., 1993; Raymond et al., 1995). Studies from in vitro brain slices of human dysplastic cortex demonstrate prolonged epileptiform discharges in response to stimulation and application of the proconvulsant compound, 4-aminopyridine, that are not seen in neocortical slices from patients with mesial temporal lobe epilepsy (Mattia et al., 1995). Although none of these studies establish the mechanisms that underlie this pronounced epileptogenicity of dysplastic cortex, an impaired inhibitory system would certainly be a strong contributing factor. The current study provides additional support for impaired inhibition as a cause of increased seizure susceptibility in some types of CD. This study provides evidence for a selective reduction of inhibitory interneurons in an animal model of CD. This represents a possible mecha-
nism for the excitability and epileptogenicity that has been identified in human CD. It also complements and supports findings from immunohistochemical studies of human dysplastic cortex. In addition, it demonstrates that a definable injury that occurs at a specific point in cortical development (in the absence of any pre-existing genetic abnormality) can have a major effect on the representation of neuronal subtypes in the neocortex of the mature animal. Investigations into the physiologic impact of these alterations and their effect on seizure susceptibility are ongoing.
Acknowledgements The authors are grateful to Dr Frank J. Bova of the Department of Neurological Surgery at the University of Florida for his assistance with animal irradiation. This work was supported by a grant from the National Institutes of Health (1R29NS35651) to S.N. Roper.
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