Brain Research, 535 (1990) 195-204 Elsevier
195
BRES 16113
Granule cell dispersion in the dentate gyrus of humans with temporal lobe epilepsy Carolyn R. Houser Neurology and Research Services, Veterans Administration Medical Center, West Los Angeles, Wadsworth Division, and Department of Anatomy and Cell Biology and Brain Research Institute, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90024 (U.S.A.) (Accepted 19 June 1990)
Key words: Hippocampal formation; Febrile seizures; Development; Neuronal migration
The distribution of granule cells in the dentate gyrus of the hippocampal formation was studied in control autopsy and temporal lobe epilepsy (TLE) specimens. In control tissue, the granule cell somata were closely approximated and formed a narrow lamina with a distinct, regular border with the molecular layer. In 11 of 15 TLE specimens, the granule cell somata were dispersed and formed a wider than normal granule cell layer. The granule cell somata extended into the molecular layer to varying extents, creating an irregular boundary between the lamina. The dispersed granule cells were frequently aligned in columns, and many of these neurons displayed elongated bipolar forms. The extent of granule cell dispersion appeared to be related to the amount of cell loss in the polymorph layer of the dentate gyms. Granule cell dispersion was not consistently associated with granule cell loss although 5 of the 11 specimens with granule cell dispersion also showed moderate to marked granule cell loss. The most common features in the histories of the TLE cases with granule cell dispersion were severe febrile seizures or seizures associated with meningitis or encephalitis during the first 4 years of life. The dispersion of the granule cells suggests that there has been some alteration in the patterns of cell migration in a subpopulation of cases with severe TLE. The resultant ectopic positions of the granule cells could lead to changes in both the afferent and efferent connections of these neurons and, thus, contribute to the altered circuitry of the hippocampal formation in TLE.
INTRODUCTION There are several unique features in the morphological organization of dentate granule cells in some cases of h u m a n temporal lobe epilepsy (TLE). While there is substantial cell loss in several regions of the hippocampal formation in T L E , m a n y granule cells are preserved 14'33 and, thus, could participate in the generation of seizure activity. F u r t h e r m o r e , the mossy fibers of the remaining granule cells exhibit reorganization in both kainatetreated rats 16'35'41,52 and humans with T L E 8'17'26'5°. Present observations indicate that there are also alterations in the distribution of the somata of granule cells in the dentate gyrus of a subpopulation of patients with severe TLE. The granule cell layer of the normal h u m a n dentate gyrus consists of a tightly assembled band of neuronal somata. This layer has a distinct border with the adjacent molecular layer that contains only a few neuronal cell bodies, most of which are local circuit neurons rather than granule cells 3'12. By contrast, in some hippocampal specimens from patients with T L E , the border between the granule cell and molecular layers is not distinct, and
n u m e r o u s granule cell somata extend into the molecular layer. The goals of this study were first, to provide a detailed qualitative and quantitative description of this alteration in the granule cell layer, and second, to determine if the granule cell dispersion was consistently associated with either granule cell loss or loss of n e u r o n s in the polymorph layer of the dentate gyrus. A preliminary report of this alteration was included in a paper describing the reorganization of mossy fibers in h u m a n T L E 26. MATERIALS AND METHODS
Patients and control cases Surgical specimens from the hippocampal formation were obtained from 15 patients with medically intractable TLE. The patients, 6 females and 9 males, ranged in age from 18 to 46 years (mean = 29.4) at the time of surgery. The age of epilepsy onset was 8 months to 23 years (mean = 9.4), and the duration of epilepsy was 12-38 years (mean = 20.0). None of the patients had tumors or structural lesions as evidenced by computed tomography or magnetic resonance imaging, and the absence of such lesions was confirmed postsurgically by pathological examination of excised tissues. Control autopsy specimens from the hippocampal formation of 6 unfixed brains were obtained from the Human Neurological
Correspondence: C.R. Houser, BRI 73-364 CHS, UCLA - - Brain Research Institute, Los Angeles, CA 90024-1761, U.S.A.
196 Specimen Bank, VA Wadsworth Medical Center, Los Angeles, CA. The case~, 6 males, were 44-77 years of age (mean = 60.0) and had no known history of neurological disease. Autolysis times ranged from 9 to 28 h (mean = 16.9).
Tissue processing Specimens from the hippocampal formation of the surgical TLE and autopsy specimens were sectioned perpendicular to the long axis of the hippocampus into 3-5 mm thick blocks and immersed in 4% paraformaldehyde in 0.12 M phosphate buffer (pH 7.3) for 2-3 h. After rinsing in phosphate buffer and infiltration with a 20% sucrose solution, the specimens were frozen with dry ice, and 30/~m thick sections were cut on a cryostat. Sections were stored in serial order in 0.1 M Tris buffer (pH 7.3), Every 10th section was stained with 0.5% Cresyl violet for visualization of neuronal cell bodies.
width through the granule cell layer, density of granule cells, and density of neurons in the polymorph region. Pearson Product Moment Correlation Coefficients between pairs of variables in the experimental group were also calculated. The width of the granule cell layer was paired successively with the number of granule cells within a column through the granule cell layer, the density of granule cells and the density of polymorph neurons. Correlations were not determined between these variables in the control group because of the small number of cases. RESULTS
Distribution of granule cells Control specimens. The dentate granule cells of control
Data analysis
specimens were arranged in a compact layer with distinct
Cresyl violet-stained sections from control and TLE cases were examined to determine the general morphological characteristics of the tissue, including the distribution of granule cells of the dentate gyrus and the presence of obvious cell loss in the hippocampal fields. The mid-rostrocaudal level of the dentate gyrus was chosen for quantitative analysis because this region was included in most of the epilepsy specimens and because the dentate gyrus forms a characteristic C-shaped structure at this level. The inferior limb of the dentate gyrus was relatively straight in this region (Fig. 1A,B), thus providing the most comparable region for measurements in control and epilepsy specimens. Three types of quantitative analyses were conducted. First, the average width of the granule cell layer was determined, and these measurements provided an index of the amount of granule cell dispersion. To determine the average width of the layer, 20 consecutive measurements, at 50-gm intervals, were taken along the central 1000gm of the lower, relatively straight limb of the dentate gyrus. The perpendicular distance from the inner (hilar) edge of the granule cell layer to the outer border of the most distal granule cell somata was determined with the aid of an image analysis system (Analytical Imaging Concepts), using a ×20 objective and a final magnification of ×730 on the monitor. The mean and standard deviation of 20 measurements were calculated for each case. Second, the number and density of granule cells in two 100/~m wide columns through the granule cell layer of a 30 Mm thick section were determined for each specimen. Nuclei of granule cells were mapped with the aid of a drawing tube at a magnification of × 1250 (× 100 objective, × 10 eyepiece and x 1.25 tube factor), and the cells were subsequently counted. Cell counts were made in 2 nonadjacent columns that were 100 gm in width but had variable vertical lengths that were determined by the extent to which the granule cell somata invaded the molecular layer. Both the number of granule cells within a column and the density of neurons per unit volume were calculated. Finally, the relative density of neurons in the polymorph layer was determined. This layer extends approximately 600 Mm deep to the granule cell layer and consists of several morphologically and chemically distinct classes of neurons ~'3'4°. Cell counts in the polymorph layer were made in an area near the relatively straight inferior limb of the dentate gyrus. The area was 1000/~m in length and extended from 100/~m above the hilar border of the granule cell layer to 600/~m into the hilus, and, thus, included the polymorph region with moderate cell densities but excluded the cell-sparse zone that lies immediately adjacent to the granule cell layer. Neuronal profiles with a nucleus and nucleolus were mapped with the aid of a drawing tube within a rectangular grid (500 × 1000 /xm) at a magnification of ×200 ( x 16 objective, x 10 eyepiece and x 1.25 tube factor), and the cells were subsequently counted. Cell counts were made in 2 sections (30 /~m thick) from each specimen, and the density of neurons per unit volume was calculated. Differences between control and epilepsy groups were analyzed with Student's t-tests for the following variables: width of the granule cell layer, number of granule cells within a column of fixed
upper and lower borders (Fig. 1A). This highly organized pattern characterized the straight portions of the ventral, medial and lateral limbs of the granule cell layer. Generally only a few granule cell somata were observed within the molecular layer although a small n u m b e r of presumptive local circuit n e u r o n s were randomly distributed throughout this layer. The width of the granule cell layer showed some variation along the length of each limb and frequently increased at the angles and enfolded regions of the dentate gyrus (Fig. 1A). In normal specimens, even when the width of the granule cell layer increased, the granule cell somata generally remained in close apposition to one another, and a clear b o u n d a r y between the molecular and granule cell layers was maintained (Fig. 1A). However, at the angles and enfolded regions of the dentate gyrus, small groups of granule cells occasionally protruded into the molecular layer. Such variations in the pattern and width of the granule cell layer were most frequently observed in the r o s t r a l ' a n d caudal regions of the dentate gyrus due, in part, to bending of the hippocampal formation at these locations. As indicated previously, these regions were not included in the quantitative analysis. In control specimens, the average width of the ventral limb of the granule cell layer at mid-rostrocaudal levels was 99.5/~m + 19.1.
TLE specimens. Two basic patterns of granule cell distribution could be identified in the T L E specimens. In some specimens, the granule cells formed a narrow, compact layer that was indistinguishable from that of control specimens (Fig. 2A). In a second group of specimens, the granule cell layer was wider, and the outer border with the molecular layer was poorly defined (Fig. 2B). In these cases, the granule cell somata extended into the molecular layer by varying degrees, creating an irregular or undulating outer border of the granule cell layer (Figs. 1B and 2B). In some cases, the deep (hilar) border of the granule cell layer was also less sharply defined than normal (Fig. 1B), but variations along this border were not as marked as those at the border with the
197
Fig. 1. Cresyi violet-stained sections from the hippocampal formation of control (A) and epilepsy (B) specimens. A: in the control specimen, the granule cell layer (G) is relatively narrow, and the cell bodies are closely approximated. B: in the epilepsy specimen, the granule cell layer (G) is wider and appears disorganized due to dispersion of many of the granule cells. Marked cell loss is evident in the polymorph region (PM), CA1 and CA3 fields. Scale bars, 500/~m.
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Fig. 3. Cresyl violet-stained specimens from the dentate gyrus of epilepsy specimens. A: many granule cells appear to be aligned in vertical columns (arrows) that extend into the molecular layer. B: some dispersed granule cells display elongated cell bodies with vertically oriented processes (arrows), and, thus, resemble bipolar neurons commonly observed during early development. Scale bars: A, 50/~m; B, 25/~m.
molecular layer. In specimens with a wider granule cell layer, there appeared to be lower densities of granule cell somata within the layer, creating a general impression of cell dispersion. In 2 of the cases the granule cell layer appeared bilaminar, with a cell-sparse region near the center of the layer (Fig. 2C). The average width of the
inferior limb of the granule cell layer in all epilepsy specimens was 182.1/~m + 57.9. The majority of the dispersed cell bodies in the inner molecular layer were considered to be granule cells rather than local circuit neurons or glia because the cytoplasmic and nuclear staining with Cresyl violet
Fig. 2. Cresyl violet-stained sections of the dentate gyrus from 3 epilepsy specimens. A: granule cell somata form a highly organized lamina that has distinct borders with the molecular layer (M) and polymorph region (PM). This pattern is similar to that of control specimens (Fig. 1A). B: granule cell somata are dispersed, and many extend into the molecular layer (M). Thus, the outer border of the granule cell layer (G) is quite irregular. C: granule cells are again dispersed but form a bilaminar pattern (G) with a relatively neuron-free zone between the two layers. Scale bar for all panels, 100/~m.
200 closely resembled that of granule cells in deeper parts of the granule cell layer (Fig. 3B), and the cells stained positively with antisera to neuron-specific enolase and neurofilament protein. In some specimens, the dispersed granule cells were arranged in single-cell-thick columns (Fig. 3A), and often displayed a bipolar form, with processes extending vertically within the molecular layer (Fig. 3B). In 2 of the epilepsy specimens that showed granule cell dispersion, aggregations of small neurons were also present within the hilus. These neurons were considered to be granule cells on the basis of their size, shape and Cresyl violet staining patterns. Furthermore, immediately adjacent regions were densely labeled with a monoclonal antibody to the GABA-A receptor, suggesting that this region was occupied by dendrites of the presumptive granule cells, which normally exhibit high concentrations of GABA-A receptors on their dendrites in the molecular layer2s. Thus, many of the morphological and chemical features of the small neurons were similar to those of granule cells in the normal human dentate gyrus. The average width of the granule cell layer was statistically significantly greater in ihe epilepsy group than in the control group (P < 0.01, Table I). The widths of the granule cell layer in 11 specimens from the epilepsy
300' A
E
0 0 0
250.
group were greater than 2 S.D. above the mean control value, and there was no overlap between the values for these epilepsy specimens and those of the control group (Fig. 4). The S.D. of the width measurements in these epilepsy specimens was also large (Table II), reflecting the irregular dispersion of the granule cells rather than a simple widening of the granule cell layer. The widths of the granule cell layer in the remaining 4 specimens did not exceed the range of control values (Fig. 4).
Granule cell loss and relationship to cell dispersion Granule cell counts within a 100/~m wide column in the control and epilepsy groups were not significantly different (Table I). However, there was a general trend toward fewer granule cells in the epilepsy specimens. In 5 epilepsy specimens, the granule cell counts were more than 2 S.D. below the mean of the control values, and all of these specimens showed granule cell dispersion. However, in 8 epilepsy specimens, there was little or no granule cell loss, as indicated by cell counts that were less than 1 S.D. from the mean of the control group, and 5 of these specimens showed granule cell dispersion. The correlation between the degree of cell loss and the extent of granule cell dispersion was not significant (R = -0.294, df = 13). In contrast, the average densities of granule cells in control and epilepsy specimens were significantly different (P < 0.01, Table I). Nearly all cases with granule cell dispersion had decreased neuronal densities in the granule cell layer, and there was a significant negative correlation between the width of the granule cell layer and the density of granule cells (R = -0.829, df = 13, P < 0.01).
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TABLE I
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Comparison of mean values for control and epilepsy specimens (+_ S,E.M.)
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Specimens Fig. 4. Comparisons of the average width of the granule cell layer in control and epilepsy specimens. Each value represents the m e a n of 20 m e a s u r e m e n t s along a central 1000-/~m segment of the inferior limb of the dentate gyrus. Horizontal lines indicate the mean for each group.
Control (n = 6) Width of granule cell layer ~um) 99.5___ 7.8
Epilepsy (n = 15)
t-values P-values (dr= 19) ~
182.1 ___14.9
-3.37
195
BRES 16113
Granule cell dispersion in the dentate gyrus of humans with temporal lobe epilepsy Carolyn R. Houser Neurology and Research Services, Veterans Administration Medical Center, West Los Angeles, Wadsworth Division, and Department of Anatomy and Cell Biology and Brain Research Institute, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90024 (U.S.A.) (Accepted 19 June 1990)
Key words: Hippocampal formation; Febrile seizures; Development; Neuronal migration
The distribution of granule cells in the dentate gyrus of the hippocampal formation was studied in control autopsy and temporal lobe epilepsy (TLE) specimens. In control tissue, the granule cell somata were closely approximated and formed a narrow lamina with a distinct, regular border with the molecular layer. In 11 of 15 TLE specimens, the granule cell somata were dispersed and formed a wider than normal granule cell layer. The granule cell somata extended into the molecular layer to varying extents, creating an irregular boundary between the lamina. The dispersed granule cells were frequently aligned in columns, and many of these neurons displayed elongated bipolar forms. The extent of granule cell dispersion appeared to be related to the amount of cell loss in the polymorph layer of the dentate gyms. Granule cell dispersion was not consistently associated with granule cell loss although 5 of the 11 specimens with granule cell dispersion also showed moderate to marked granule cell loss. The most common features in the histories of the TLE cases with granule cell dispersion were severe febrile seizures or seizures associated with meningitis or encephalitis during the first 4 years of life. The dispersion of the granule cells suggests that there has been some alteration in the patterns of cell migration in a subpopulation of cases with severe TLE. The resultant ectopic positions of the granule cells could lead to changes in both the afferent and efferent connections of these neurons and, thus, contribute to the altered circuitry of the hippocampal formation in TLE.
INTRODUCTION There are several unique features in the morphological organization of dentate granule cells in some cases of h u m a n temporal lobe epilepsy (TLE). While there is substantial cell loss in several regions of the hippocampal formation in T L E , m a n y granule cells are preserved 14'33 and, thus, could participate in the generation of seizure activity. F u r t h e r m o r e , the mossy fibers of the remaining granule cells exhibit reorganization in both kainatetreated rats 16'35'41,52 and humans with T L E 8'17'26'5°. Present observations indicate that there are also alterations in the distribution of the somata of granule cells in the dentate gyrus of a subpopulation of patients with severe TLE. The granule cell layer of the normal h u m a n dentate gyrus consists of a tightly assembled band of neuronal somata. This layer has a distinct border with the adjacent molecular layer that contains only a few neuronal cell bodies, most of which are local circuit neurons rather than granule cells 3'12. By contrast, in some hippocampal specimens from patients with T L E , the border between the granule cell and molecular layers is not distinct, and
n u m e r o u s granule cell somata extend into the molecular layer. The goals of this study were first, to provide a detailed qualitative and quantitative description of this alteration in the granule cell layer, and second, to determine if the granule cell dispersion was consistently associated with either granule cell loss or loss of n e u r o n s in the polymorph layer of the dentate gyrus. A preliminary report of this alteration was included in a paper describing the reorganization of mossy fibers in h u m a n T L E 26. MATERIALS AND METHODS
Patients and control cases Surgical specimens from the hippocampal formation were obtained from 15 patients with medically intractable TLE. The patients, 6 females and 9 males, ranged in age from 18 to 46 years (mean = 29.4) at the time of surgery. The age of epilepsy onset was 8 months to 23 years (mean = 9.4), and the duration of epilepsy was 12-38 years (mean = 20.0). None of the patients had tumors or structural lesions as evidenced by computed tomography or magnetic resonance imaging, and the absence of such lesions was confirmed postsurgically by pathological examination of excised tissues. Control autopsy specimens from the hippocampal formation of 6 unfixed brains were obtained from the Human Neurological
Correspondence: C.R. Houser, BRI 73-364 CHS, UCLA - - Brain Research Institute, Los Angeles, CA 90024-1761, U.S.A.
196 Specimen Bank, VA Wadsworth Medical Center, Los Angeles, CA. The case~, 6 males, were 44-77 years of age (mean = 60.0) and had no known history of neurological disease. Autolysis times ranged from 9 to 28 h (mean = 16.9).
Tissue processing Specimens from the hippocampal formation of the surgical TLE and autopsy specimens were sectioned perpendicular to the long axis of the hippocampus into 3-5 mm thick blocks and immersed in 4% paraformaldehyde in 0.12 M phosphate buffer (pH 7.3) for 2-3 h. After rinsing in phosphate buffer and infiltration with a 20% sucrose solution, the specimens were frozen with dry ice, and 30/~m thick sections were cut on a cryostat. Sections were stored in serial order in 0.1 M Tris buffer (pH 7.3), Every 10th section was stained with 0.5% Cresyl violet for visualization of neuronal cell bodies.
width through the granule cell layer, density of granule cells, and density of neurons in the polymorph region. Pearson Product Moment Correlation Coefficients between pairs of variables in the experimental group were also calculated. The width of the granule cell layer was paired successively with the number of granule cells within a column through the granule cell layer, the density of granule cells and the density of polymorph neurons. Correlations were not determined between these variables in the control group because of the small number of cases. RESULTS
Distribution of granule cells Control specimens. The dentate granule cells of control
Data analysis
specimens were arranged in a compact layer with distinct
Cresyl violet-stained sections from control and TLE cases were examined to determine the general morphological characteristics of the tissue, including the distribution of granule cells of the dentate gyrus and the presence of obvious cell loss in the hippocampal fields. The mid-rostrocaudal level of the dentate gyrus was chosen for quantitative analysis because this region was included in most of the epilepsy specimens and because the dentate gyrus forms a characteristic C-shaped structure at this level. The inferior limb of the dentate gyrus was relatively straight in this region (Fig. 1A,B), thus providing the most comparable region for measurements in control and epilepsy specimens. Three types of quantitative analyses were conducted. First, the average width of the granule cell layer was determined, and these measurements provided an index of the amount of granule cell dispersion. To determine the average width of the layer, 20 consecutive measurements, at 50-gm intervals, were taken along the central 1000gm of the lower, relatively straight limb of the dentate gyrus. The perpendicular distance from the inner (hilar) edge of the granule cell layer to the outer border of the most distal granule cell somata was determined with the aid of an image analysis system (Analytical Imaging Concepts), using a ×20 objective and a final magnification of ×730 on the monitor. The mean and standard deviation of 20 measurements were calculated for each case. Second, the number and density of granule cells in two 100/~m wide columns through the granule cell layer of a 30 Mm thick section were determined for each specimen. Nuclei of granule cells were mapped with the aid of a drawing tube at a magnification of × 1250 (× 100 objective, × 10 eyepiece and x 1.25 tube factor), and the cells were subsequently counted. Cell counts were made in 2 nonadjacent columns that were 100 gm in width but had variable vertical lengths that were determined by the extent to which the granule cell somata invaded the molecular layer. Both the number of granule cells within a column and the density of neurons per unit volume were calculated. Finally, the relative density of neurons in the polymorph layer was determined. This layer extends approximately 600 Mm deep to the granule cell layer and consists of several morphologically and chemically distinct classes of neurons ~'3'4°. Cell counts in the polymorph layer were made in an area near the relatively straight inferior limb of the dentate gyrus. The area was 1000/~m in length and extended from 100/~m above the hilar border of the granule cell layer to 600/~m into the hilus, and, thus, included the polymorph region with moderate cell densities but excluded the cell-sparse zone that lies immediately adjacent to the granule cell layer. Neuronal profiles with a nucleus and nucleolus were mapped with the aid of a drawing tube within a rectangular grid (500 × 1000 /xm) at a magnification of ×200 ( x 16 objective, x 10 eyepiece and x 1.25 tube factor), and the cells were subsequently counted. Cell counts were made in 2 sections (30 /~m thick) from each specimen, and the density of neurons per unit volume was calculated. Differences between control and epilepsy groups were analyzed with Student's t-tests for the following variables: width of the granule cell layer, number of granule cells within a column of fixed
upper and lower borders (Fig. 1A). This highly organized pattern characterized the straight portions of the ventral, medial and lateral limbs of the granule cell layer. Generally only a few granule cell somata were observed within the molecular layer although a small n u m b e r of presumptive local circuit n e u r o n s were randomly distributed throughout this layer. The width of the granule cell layer showed some variation along the length of each limb and frequently increased at the angles and enfolded regions of the dentate gyrus (Fig. 1A). In normal specimens, even when the width of the granule cell layer increased, the granule cell somata generally remained in close apposition to one another, and a clear b o u n d a r y between the molecular and granule cell layers was maintained (Fig. 1A). However, at the angles and enfolded regions of the dentate gyrus, small groups of granule cells occasionally protruded into the molecular layer. Such variations in the pattern and width of the granule cell layer were most frequently observed in the r o s t r a l ' a n d caudal regions of the dentate gyrus due, in part, to bending of the hippocampal formation at these locations. As indicated previously, these regions were not included in the quantitative analysis. In control specimens, the average width of the ventral limb of the granule cell layer at mid-rostrocaudal levels was 99.5/~m + 19.1.
TLE specimens. Two basic patterns of granule cell distribution could be identified in the T L E specimens. In some specimens, the granule cells formed a narrow, compact layer that was indistinguishable from that of control specimens (Fig. 2A). In a second group of specimens, the granule cell layer was wider, and the outer border with the molecular layer was poorly defined (Fig. 2B). In these cases, the granule cell somata extended into the molecular layer by varying degrees, creating an irregular or undulating outer border of the granule cell layer (Figs. 1B and 2B). In some cases, the deep (hilar) border of the granule cell layer was also less sharply defined than normal (Fig. 1B), but variations along this border were not as marked as those at the border with the
197
Fig. 1. Cresyi violet-stained sections from the hippocampal formation of control (A) and epilepsy (B) specimens. A: in the control specimen, the granule cell layer (G) is relatively narrow, and the cell bodies are closely approximated. B: in the epilepsy specimen, the granule cell layer (G) is wider and appears disorganized due to dispersion of many of the granule cells. Marked cell loss is evident in the polymorph region (PM), CA1 and CA3 fields. Scale bars, 500/~m.
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Fig. 3. Cresyl violet-stained specimens from the dentate gyrus of epilepsy specimens. A: many granule cells appear to be aligned in vertical columns (arrows) that extend into the molecular layer. B: some dispersed granule cells display elongated cell bodies with vertically oriented processes (arrows), and, thus, resemble bipolar neurons commonly observed during early development. Scale bars: A, 50/~m; B, 25/~m.
molecular layer. In specimens with a wider granule cell layer, there appeared to be lower densities of granule cell somata within the layer, creating a general impression of cell dispersion. In 2 of the cases the granule cell layer appeared bilaminar, with a cell-sparse region near the center of the layer (Fig. 2C). The average width of the
inferior limb of the granule cell layer in all epilepsy specimens was 182.1/~m + 57.9. The majority of the dispersed cell bodies in the inner molecular layer were considered to be granule cells rather than local circuit neurons or glia because the cytoplasmic and nuclear staining with Cresyl violet
Fig. 2. Cresyl violet-stained sections of the dentate gyrus from 3 epilepsy specimens. A: granule cell somata form a highly organized lamina that has distinct borders with the molecular layer (M) and polymorph region (PM). This pattern is similar to that of control specimens (Fig. 1A). B: granule cell somata are dispersed, and many extend into the molecular layer (M). Thus, the outer border of the granule cell layer (G) is quite irregular. C: granule cells are again dispersed but form a bilaminar pattern (G) with a relatively neuron-free zone between the two layers. Scale bar for all panels, 100/~m.
200 closely resembled that of granule cells in deeper parts of the granule cell layer (Fig. 3B), and the cells stained positively with antisera to neuron-specific enolase and neurofilament protein. In some specimens, the dispersed granule cells were arranged in single-cell-thick columns (Fig. 3A), and often displayed a bipolar form, with processes extending vertically within the molecular layer (Fig. 3B). In 2 of the epilepsy specimens that showed granule cell dispersion, aggregations of small neurons were also present within the hilus. These neurons were considered to be granule cells on the basis of their size, shape and Cresyl violet staining patterns. Furthermore, immediately adjacent regions were densely labeled with a monoclonal antibody to the GABA-A receptor, suggesting that this region was occupied by dendrites of the presumptive granule cells, which normally exhibit high concentrations of GABA-A receptors on their dendrites in the molecular layer2s. Thus, many of the morphological and chemical features of the small neurons were similar to those of granule cells in the normal human dentate gyrus. The average width of the granule cell layer was statistically significantly greater in ihe epilepsy group than in the control group (P < 0.01, Table I). The widths of the granule cell layer in 11 specimens from the epilepsy
300' A
E
0 0 0
250.
group were greater than 2 S.D. above the mean control value, and there was no overlap between the values for these epilepsy specimens and those of the control group (Fig. 4). The S.D. of the width measurements in these epilepsy specimens was also large (Table II), reflecting the irregular dispersion of the granule cells rather than a simple widening of the granule cell layer. The widths of the granule cell layer in the remaining 4 specimens did not exceed the range of control values (Fig. 4).
Granule cell loss and relationship to cell dispersion Granule cell counts within a 100/~m wide column in the control and epilepsy groups were not significantly different (Table I). However, there was a general trend toward fewer granule cells in the epilepsy specimens. In 5 epilepsy specimens, the granule cell counts were more than 2 S.D. below the mean of the control values, and all of these specimens showed granule cell dispersion. However, in 8 epilepsy specimens, there was little or no granule cell loss, as indicated by cell counts that were less than 1 S.D. from the mean of the control group, and 5 of these specimens showed granule cell dispersion. The correlation between the degree of cell loss and the extent of granule cell dispersion was not significant (R = -0.294, df = 13). In contrast, the average densities of granule cells in control and epilepsy specimens were significantly different (P < 0.01, Table I). Nearly all cases with granule cell dispersion had decreased neuronal densities in the granule cell layer, and there was a significant negative correlation between the width of the granule cell layer and the density of granule cells (R = -0.829, df = 13, P < 0.01).
lIB .J ,w •
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TABLE I
0
Comparison of mean values for control and epilepsy specimens (+_ S,E.M.)
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Control
Epilepsy
Specimens Fig. 4. Comparisons of the average width of the granule cell layer in control and epilepsy specimens. Each value represents the m e a n of 20 m e a s u r e m e n t s along a central 1000-/~m segment of the inferior limb of the dentate gyrus. Horizontal lines indicate the mean for each group.
Control (n = 6) Width of granule cell layer ~um) 99.5___ 7.8
Epilepsy (n = 15)
t-values P-values (dr= 19) ~
182.1 ___14.9
-3.37
