THE JOURNAL OF COMPARATIVE NEUROLOGY 302:206-219 (1990)

Quantitative,Three=DimensionalAnalysis of Granule Cell Dendrites in the Rat Dentate Gyrus BRENDA J. CLAIBORNE, DAVID G. AMARAL, AND W. MAXWELL COWAN The Salk Institute for Biological Studies, San Diego, California 92138

ABSTRACT The three-dimensional organization of dentate granule cell dendritic trees has been quantitatively analyzed with the aid of a computerized microscope system. The dendrites were visualized by iontophoretic injection of horseradish peroxidase into individual granule cells in the in vitro hippocampal slice preparation. Selection criteria insured that the analyzed cells were completely stained and that only neurons with two or fewer cut dendrites in the distal portion of the molecular layer were analyzed. Twenty-nine of the 48 sampled granule cells had no cut dendrites. The granule cells had between one and four primary dendrites. Granule cell dendritic branches were covered with spines and most extended to the hippocampal fissure or pial surface. The mean total dendritic length was 3,221 pm with a range from 2,324 pm to 4,582 km. The dendrites formed an elliptical plexus with the transverse spread averaging 325 pm and the spread in the septotemporal axis averaging 176 pm. On individual neurons, the maximum branch order ranged from four to eight and the number of dendritic segments ranged from 22 to 40.Approximately 63% of the dendritic branch points occurred in a zone that included the granule cell layer and the inner one-third of the molecular layer. The dendritic tree was organized so that, on average, 30% of the length was in the granule cell layer and proximal third of the molecular layer, 30% was in the middle third, and 40% was in the distal third. Comparisons were made between the dendrites of granule cells in the suprapyramidal and infrapyramidal blades of the dentate gyrus. Suprapyramidal cells had a significantly greater total dendritic length than infrapyramidal cells, their transverse spread was higher, and they had a greater number of dendritic segments. When neurons in the suprapyramidal blade were further subdivided on the basis of somal position within the depth of the cell body layer, superficial neurons were found to have a greater number of primary dendrites, more elliptical trees, and larger transverse spreads of their dendrites. There were no significant differences in dendritic segment number or total dendritic length between superficial and deep cells. Key words: computer-aidedmorphometry, hippocampal slices, horseradish peroxidase, intracellular injections

The hippocampal formation comprises several relatively simple cortical areas including the dentate gyrus, hippocampus, subicular complex, and entorhinal cortex. Functionally, the hippocampal formation appears to be involved in certain forms of learning and memory (Milner, '70; ZolaMorgan et al., '86; Squire, '87). There has been considerable interest, therefore, in understanding the physiological and anatomical features of neurons in this region. Despite extensive morphological analyses carried out over the past century (Golgi, 1886; Cajal, '11; Lorente de NO, '341, relatively little quantitative data has been generated on neurons in any of the hippocampal fields. Here we report our use of intracellular labeling and three-dimensional reconstruction techniques to characterize the morphology

o 1990 WILEY-LISS, INC.

of one population of hippocampal neurons, the dentate granule cells. Although the dentate gyrus is the simplest of the cortical fields that comprise the hippocampal formation, it plays a pivotal role in transferring information from the entorhinal cortex (the recipient of most of the sensory input to the hippocampal formation-Insausti et al., '87) to the other hippocampal fields. It contains one major class of neuron, ~~~

Accepted August 29,1990. Address reprint requests to Dr. Brenda Claiborne, who is now a t Division of Life Sciences, University of Texas at San Antonio, San Antonio, TX 78285. Dr. W. Maxwell Cowan's present address is Howard Hughes Medical Institute, 6701 Rockledge Drive, Bethesda, MD 28017.

GRANULE CELL DENDRITES the granule cell, whose dendrites extend into the molecular layer and receive input from the perforant fibers from the entorhinal cortex (Hjorth-Simonsen and Jeune, '72; Steward, '76). The granule cell axons, the mossy fibers, first traverse the dentate hilar region, where they give off numerous fine collaterals (Claiborne et al., '86), and then project into stratum-lucidum of the CA3 region of the hippocampus where they make synaptic contacts onto the proximal dendrites of pyramidal cells. The granule cell has been described qualitatively in several studies using the Golgi technique (Golgi, 1886; Cajal, '11; Lorente de N6, '34; Seress and Pokorny, '81; Wenzel et al., '81). There have also been several quantitative Golgi studies of granule cells in which parameters such as total dendritic length, total number of branch points, and segment number and length have been measured (Lindsay and Scheibel, '81; Seress and Pokorny, '81; Desmond and Levy, '82; Duffy and Rakic, '83; Williams and Matthysse, '83; Green and Juraska, '85). As Desmond and Levy ('82) have pointed out, however, the hallmark of this body of work is the variability, from study to study, of the numbers obtained for the measurements. As one example, estimates for total dendritic length of the rat granule cell dendritic tree ranged from a low of approximately 1,200 pm (Green and Juraska, '85) to an intermediate figure of about 2,400 bm (Seress and Pokorny, '81) to a high of about 3,600 bm (Desmond and Levy, '82). While a number of explanations could be offered for the variability in these Golgi studies, we are inclined to agree with Desmond and Levy ('82) that the problems are largely technical. The Golgi procedures used in these studies have a number of inherent difficulties that make them problematic for quantitative analyses. (See Desmond and Levy, '82, for a detailed discussion of technical limitations of the Golgi technique.) Impregnated neurons, for example, often have cut dendrites at the surface of a section. Unless corrected by serial reconstruction, dendritic length measurements of such cells underestimate their total dendritic length. When problems such as these are coupled with the inherent capriciousness of the Golgi method, it is hardly surprising that reported dendritic parameters vary markedly from study to study. In the present study, the in vitro hippocampal slice preparation was used and individual granule cells were injected with horseradish peroxidase (HRP). To avoid the problems inherent in reconstructing neurons from adjacent sections, the 400 pm-thick slices were processed as whole mounts so that a n entire HRP-labeled granule cell was visible in one slice. Strict selection criteria were employed (neurons with more than two cut dendrites were not included, for example) and acceptable neurons were analyzed in three dimensions.

MATEXUALSANDMETHODS The methods used in this study have been described in detail elsewhere (Claiborne et al., '86) and for this reason need only be summarized here.

htracellularlabeling Sprague-Dawley rats (Zivic-Miller)of both sexes, ranging in age from 35 to 49 days, were used. The rats were anesthetized with either Nembutal (25 mgikg) or open ether and then decapitated. The brains were quickly re-

207

moved and immersed in ice-cold oxygenated saline for 15 seconds. After removing the hippocampus, 400 bm-thick transverse slices were cut in a plane perpendicular to the long (or septotemporal) axis of the dentate gyrus by using a McIlwain tissue chopper. The tissue slices were then placed on filter membranes (12 pm, Nuclepore Corp.) in a heated slice chamber. Only slices from the middle three-fifths of the hippocampus were used. The methods used for effectively maintaining the viability of the slices were similar to those described by others (see Dingledine, '84) and are given in detail in our previous report (Claiborne et al., '86). In some cases rats were deeply anesthetized and perfused with an artificial blood substitute (Oxypherol, Green Cross of Japan) before decapitation. This technique had the merit of eliminating red blood cells which, due to their endogenous peroxidase activity, are intensely stained in the HRP procedure. Micropipettes were pulled on a Brown-Flaming pipette puller. After filling with a solution of 2-3% HRP (Boehringer-Mannheim, Grade I) in KCliTris buffer (pH 7.6), they were beveled to final resistances between 80 and 120 M a . To maximize the number of labeled cells that had all of their dendrites within the slice, we attempted to impale only neurons that were located near the middle of the slice. HRP was injected by using positive current pulses 250 msec in duration and 3 nA in amplitude a t a rate of 2Isecond for 5 to 8 minutes.

"issuepromsing After allowing several hours for the diffusion of the tracer, the slices were fixed between two pieces of filter paper in a solution containing 1%paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) for approximately 12 hours at 4°C. They were then either immediately processed or stored for several days in a 30% sucrose solution in phosphate buffer at 4°C. For visualization of the HRP, the slices were processed with diaminobenzidine (DAB). To insure that processes in the middle of the slice were well stained, slices were first incubated for 1hour in a 1% solution of the detergent Triton X-100 in 0.1 M phosphate buffer, washed for 20 minutes in phosphate buffer, and then preincubated in 0.05% DAB for 1 hour. The slices were then processed for 30 minutes in the same concentration of DAB to which 0.15% H,O, was added, thoroughly washed, cleared in ascending concentrations of glycerol, and finally mounted with a coverslip in 100% glycerol. Glycerol was used as the clearing agent because our initial studies had shown that ethanol dehydration usually caused wrinkling and distortion of the filled dendrites (Claiborne et al., '86).

Seledion and analysisoffilled neulyllls Filled neurons were first photographed with a Leitz Dialux 20 microscope and then drawn with the aid of a drawing tube by using a 6 3 plano-apo ~ oil immersion objective with a 0.6 mm working distance. In order to obtain accurate measurements of dendritic parameters, strict criteria were adopted for the selection of the filled neurons before quantitative analysis. First, the staining of the dendritic tree had to be uniformly dark throughout the entire extent of the molecular layer. If there was any indication that the distal dendritic segments were incompletely stained, the cell was discarded from further analysis. Second, if a neuron had any cut dendrites in the proximal third of the molecular layer, it was not analyzed. Lastly, if a

208

cell had more then two severed dendrites in the distal two-thirds of the layer, it was rejected. The camera lucida drawing of the dendritic tree served as a check that all dendrites were entered into the computer. The computer-microscope system has previously been described by Capowski and Sedivec ('81). Neurons were interactively digitized by an operator with each data point consisting of three (X, Y, and Z) coordinates. The digitized neuron could be reconstructed on a display screen, electronically rotated in three dimensions, and printed on a HewlettPackard plotter. Analysis software allowed various numeric and graphical representations of the dendritic data. Statistical analyses were carried out by using Minitab or Statgraphics; comparisons of sample means were carried out with a two-tailed Student's t test. Differences were considered significant at P < 0.05.

RESULTS General appearanceof HRP-fiIledgranule cells From a larger population of labeled granule cells, 48 met the criteria for quantitative analysis. Thirty of the 48 cells were located in the suprapyramidal blade and 18 were in the infrapyramidal blade of the dentate gyrus. In both blades the cells were located at various depths within the granule cell layer. The general appearance of the dendritic arborization of the HRP-labeled cells was similar to that observed in rapid Golgi preparations (Desmond and Levy, '82). The primary dendrites of the granule cells always emerged from the superficial aspect of the soma (Fig. lA,B) and nearly all branched within the outer part of the granule cell layer or in the inner third of the molecular layer. Toward their termination, the dendrites tapered and, as they approached the hippocampal fissure or pial surface, often turned sharply and traveled for a short distance (1to 10 pm) parallel to the fissure or pia; none were observed to turn back toward the granule cell layer. It should be emphasized that virtually all of the dendrites extended to the hippocampal fissure or the pial surface and that they were uniformly and darkly stained throughout (Fig. lB,C). Only occasionally did an uncut dendrite terminate at some intermediate point within the molecular layer. As seen in Golgi-impregnated material, numerous spines of various shapes and sizes were observed along the full extent of the dendrites (Fig. 1C). In the present study, we made no attempt to count the number of spines or to plot their distribution.

Quantitative analysis As noted previously, neurons that demonstrated a cut dendrite in the proximal third of the molecular layer were rejected from analysis, and only cells with two or fewer cut dendrites in the distal portion of the molecular layer were subjected to quantitative analysis. Of the 48 cells that met the selection criteria, 29 had no cut branches, 12 had one, and seven had two. Of these latter seven, only two cells had a cut dendrite at each surface of the slice. Of the neurons that were rejected for quantitative analysis, only a few were rejected because they had cut dendrites at both surfaces. To determine whether or not including neurons with cut branches affected our measurements, we compared the dendritic parameters (those listed in Table 1) of the cells without any severed branches to those with cut branches. When neurons were grouped according to their position in either the suprapyramidal or infrapyramidal blade, there

B.J. CLAIBORNE ET AL. were no significant differences in any dendritic characteristics between neurons with two or less cut branches and those without any cut dendrites. By using the three-dimensional capabilities of the computer, the number of dendritic segments, maximum branch orders, individual segment lengths, total dendritic length, and dendritic spread were quantified for each cell (Fig. 2 ) . Dendritic spread was considered to be the distance between the outermost dendritic tips and was measured first in the transverse plane of the hippocampus (the plane of the slice), and then in the longitudinal plane of the hippocampus (see Fig. 2). This latter measurement was obtained by rotating the neuron on the computer monitor 90" from the plane of the slice and noting the distance between the outermost tips. Maximum branch order referred to the highest order of branching observed in a cell. The number of primary dendrites, defined as those dendrites exiting the soma, and the lengths and widths of granule cell somata were determined from the two-dimensional drawings. In addition to the above dendritic parameters, the percentage of branch points and the percentage of the total dendritic length within various laminae of the molecular layer were determined from the three-dimensional data. To determine the percentage of branch points in portions of the molecular layer, the layer was divided into equal thirds and the branch points contained within each zone were counted. Branch points within the granule cell layer were included with those in the deepest third of the layer. Calculations of dendritic length within thirds of the layer were done directly from the three-dimensional data and again, length within the granule cell layer was included with that in the deepest third. Tree shape was estimated mathematically by rotating individual trees on the computer monitor and measuring the dendritic spread at its widest point and at the point perpendicular to the widest spread (Fig. 2). The ratio of the widest spread to this perpendicular spread was then determined for each neuron: a ratio of one indicated a circular tree at the base of the dendritic cone, whereas a ratio less than one indicated an elliptical tree. From these rotations, it was also possible to determine the orientation of the trees in relation to the axes of the hippocampus.

Summaryof quantihtive analysis-All

cells

In this section we will summarize various parameters of dendritic morphology based on all 48 labeled cells; we will then compare smaller groups of cells including those located in the supra- or infrapyramidal blades, those located at different radial positions (from deep to superficial) within the granule cell layer, and adjacent cells filled by the same injection. Data for the entire population of granule cells is

Fig. 1. Photomicrographs of HRP-filled granule cells. A: Lowmagnification photomicrograph of the dentate gyrus in a hippocampal slice preparation. A filled granule cell is present in the infrapyramidal blade (open arrow); its cell body is in the granule cell layer (GL) and its dendrites extend into the overlying molecular layer (ML). PL; pyramidal cell layer. B: A higher-magnification photomicrograph of an HRPfilled granule cell located in the suprapyramidal blade. Only a portion of the dendritic tree is in focus. Stained red blood cells can be seen scattered throughout the slice. The axon (a mossy fiber) exits from the base of the soma (asterisk). C: A higher-magnification photomicrograph of a portion of the dendritic tree of a granule cell showing dendritic spines of several sizes and shapes (arrows). Scale bars: A = 100 pm; B = 50 Fm; C = 10 pm.

Figure 1

a1

a2

a3

b2

b3

Figure 2

GRANULE CELL DENDRITES

211

TABLE 1. Dendritic Parametersfor Entire Population of Granule Cells' Mean (n = 48) Primary dendrites

Range

1.9 f .2

(1-41

Dendritic segments

1 29 I

(22 - 401

Manmum branch order

5.7 I .1

(4-8)

Transverse spread (bm)

325 I 11

(186-518)

Longitudmal spread (pml

176 L 6

(57 - 286)

3,221 2 78

(2,324- 4,582)

Total dendrhc length (pm)

Tree shape2

.56 I .03

(.14-.98)

Somalwidth (kml

10.3 i- .3

( 6 - 151

Somal length (pm)

18.6 i- .5

(15-30)

'Valuesarerneans f S.E.M. 'See text for method of d e t e n n i m g tree shape

summarized in Tables 1 and 2. Table 1 shows the average values and the ranges for all dendritic parameters, and Table 2 gives the distribution of branch points and the percentages of total dendritic length within thirds of the molecular layer. From Table 1 it can be seen that the average number of primary dendrites was 1.9, with a range from one to four. Neurons had on average 29 dendritic segments and a maximum branch order of approximately six. The mean value for the transverse spread was 325 pm and the longitudinal spread was about half that, 176 pm. Total dendritic length averaged 3,221 pm and ranged from a high of 4,582 pm to a low of 2,324 pm. Most trees were elliptical, with a dendritic spread ratio of 0.56, although from the maximum ratio of 0.98 it can be seen that some neurons had a nearly circular dendritic tree. The average width of granule cell somata was 10.3 pm and the average length was 18.6 pm. Results from the rotations done to measure tree shape were also used to determine the orientation of the trees in relation to the axes of the hippocampus. For most cells, the plane in which the neuron had its widest spread was at, or close to, the transverse plane (i.e., the plane in which the slice was cut). Of the entire population of neurons, 48% had their widest spread exactly at 0" rotation, another 46% had their widest spread within 30" of the transverse plane, and only 6% had their widest spread more than 30" (35" to 60") from the plane of the slice. In view of the fact that the majority of the afferents to the dentate gyrus terminate in a laminar fashion, it was of interest to determine the percentages of branch points and total length that were located within different laminae of the molecular layer. The results of such an analysis are

Fig. 2. Computer-generated plots of reconstructions of two HRPfilled granule cells from the suprapyramidal blade. Each neuron is viewed from three different spatial angles. The neuron shown in the top panel (a)did not have any cut dendrites while the neuron illustrated in the lower panel (b)had two cut dendrites (marked by asterisks) in the distal part of the molecular layer. In a1 and bl, the cells are viewed in the transverse plane of the dentate gyrus (i.e., in the same orientation as they were entered into the computer system). The top panel is used to illustrate nomenclature for branch order; 1st-, 2nd-, and 3rd-order dendritic branches are indicated, as are the primary dendrite and a dendritic segment. For the views in a2 and b2, the cells were rotated 90" about their vertical axis, illustrating their dendritic spreads in the longitudinal plane of the dentate gyrus. In a3 and b3, the cells are viewed from above (i.e., from the pial surface).

shown in Table 2. For the entire population of cells, 63% of the branch points occurred in the granule cell layer and the inner third of the molecular layer, with 27% in the middle third and only 10%in the outer third. The dendritic length was divided such that 30% was in the granule cell layer and proximal third of the molecular layer, 30% in the middle third, and 40% in the distal third. Individual segment lengths were determined for each neuron and are illustrated for six cells in the schematic tree diagrams shown in Figure 3. The main characteristic of these data was the large variability of lengths for segments even at equivalent hierarchical orders. For example, for the suprapyramidal cell illustrated in the top left portion of Figure 3, the shortest segment was 11 pm and the longest was 306 pm. Some secondary dendritic segments were nearly 300 pm long and extended to the hippocampal fissure whereas other dendritic segments extended less than 20 pm before branching.

Subgroupings of granule cells Suprapyramidal us. infrapyramidal cells. Dendritic characteristics of the 30 granule cells located in the suprapyramidal blade were compared to those of the 18 neurons located in the infrapyramidal blade. Three parameters were significantly different between the two populations (Table 3). The suprapyramidal cells had more total dendritic length (3,478 pm) than did the infrapyramidal cells (2,793 pm). On average, the suprapyramidal cells had more dendritic segments (31)than did cells in the infrapyramidal blade (27) and their transverse spread (347 pm) was significantly greater than that of the infrapyramidal neurons (288 pm). These differences are illustrated in Figures 3 and 4. The greater total dendritic length of the suprapyramidal neurons was not correlated with a wider molecular layer. (The molecular layer of the suprapyramidal blade was not significantly wider than that of the infrapyramidal b l a d e 2 5 4 pm vs. 240 pm.) When the percentages of branch points and total dendritic length within the inner, middle, and outer thirds of the molecular layer were compared, no differences were found between the two populations (data not shown). Because the cells of the infrapyramidal blade are generated somewhat later than those of the suprapyramidal blade (Schlessinger et al., '75), it is conceivable that the shorter dendritic lengths of the infrapyramidal cells simply reflected their relative immaturity in the 35- to 50-day-old animals. To test this possibility, three additional suprapyramidal granule cells were labeled in two older animals (71-day-old)and compared with two granule cells injected in the infrapyramidal blades of two 111-day-oldrats. The suprapyramidal cells had total dendritic lengths of 3,376

TABLE 2. Percentagesof Branch Points and Total Dendritic Length in Thirds of the Molecular Layer' Thirds Proximd'

Percent branch points 63 2 2

Percent total dendritic length

(31-881

30 f 1 (11-55)

Middle

27 2 2 (6-501

30 f 1 (23-41)

Distal

lo?

40 ? 1 (20 - 52)

1 (0-28)

'Values are means 2 S.E.M. All 48 neurons were included. 'Branch points and dendritic length in the cell layer were included in the proximal third percentages.

.

fi

I

Inf rapyramida I

I

3 uprapyramida I

A

D

92831

11231 122

208 105

115 41

48 233

232

215

277 148-

81

137

I

130

?,fin

131

88-

174 72

41 134 115

101 nLC

268

131

160

124

285

B

12431 268

78

27

107

95

73

-

110 121 Ad

47 189

#

47

44

-5

161

247

245 242 L l l

223

c

F

30143

MD2931

171

171 €€

196

77

I

I

..

1,

149

v 9

7 1

102

125

174

I

232

104 ,?

107

I

125 140

58 114

56 '"loP

74

-_

56

47 139

212

181

Fig. 3. Computer-generated dendritic tree diagrams from six different granule cells, three of which were located in the suprapyramidal blade (A-C) and three in the infrapyramidal blade (D-F). The threedimensional lengths (in km) of each segment are given. Scale bar = 100 +m.

GRANULE CELL DENDRITES

213 TABLE 5. Dendritic Parameters of Infrapyramidal Granule Cells: A Comparison of SuperficialVs. Deep Neurons'

TABLE 3. Dendritic Parameters: A Comparison of Suprapyramidal Vs. Infrapyramidal Granule Cells' Suprapyramidal (n = 30)

Infrapyramidal (n = 18)

P

Primary dendrites

21t.2 (1-4)

16t.2 (1-4)

Dendritic segments

3121 124 - 39)

2721 (22 - 40)

Maximum branch order

5.8 i .2 14 - 81

5.5 2 .2 (4 - 7)

Transverse spread (pm)

347 i 14 (186-518)

288 i 16 (187-445)

Longitudrnalspread (pm)

182 i 8 (87 - 286)

166 i 6 (132 -217)

3,478 i 88 (2,500- 4,583)

2,793 2 74 (2,324- 3,450)

.53 2 .04 (.14- .98J

.59 i .04 (31- .98)

S o d width (pml

10.4 t .4 ( 8 - 15)

10.1 .4 (6- 13)

-

S o d length (pm1

19 3 2 .6 (15-30)

17.6 2 .6 (15-25)

-

Molecular layer width (pml

254 i 7 (182-341)

240 i 4 (189-274)

-

-

Superficial' (n = 12)

Deep (n = 6)

1.7 i .3 I141 28 t 1

1.3 i 2 (1-2)

Primary dendrites Dendritic segments

-

Transverse spread (pm! Longitudinal spread (bmJ

Total dendntic length 1pm) Tree shape'

*

Total dendritic length (pm) -

-

TABLE 4. Dendritic Parameters of Suprapyramidal Granule Cells: A Comparison of SuperficialVs. Deep Neurons'

Primary dendrites

Deep (n = 11) 1.5t 2

Dendritic segments

31 ? 1 (2639)

30 t 1 (24-37)

Maomum branch order

5.5 i .2 147)

6.4 2 .3 (5-8)

Transverse spread (pmJ

378 t 16 (255-518)

293 t 16 (18-69)

Longitudinal spread (pm)

171 t 11 187-2531

200 t 12 (14S2863

-

Totaldendriticlength (pml

3,484 t 130 (2,50&4,582)

3,468 t 92 (3,0624,127)

-

.46 ? .04 t.lP.78)

67 t .05 (.42-.98)

13) -

'Values are means S.E.M. Ranges are shown in parentheses. 2Superlicialneurons had somata in the top half ofthe granule cell layer, whereas deep neurons had somata in the bottom half. %see text for method used to determine tree shape. 'Significant at P < .05.

pm, 3,244 pm, and 3,747 pm (mean 3,455 pm) while the cells located in the infrapyramidal blade had total lengths of only 2,620 pm and 2,537 pm (mean 2,578 pm). Thus, the smaller total dendritic length observed in the analyzed sample of infrapyramidal granule cells is likely representative of the mature situation in the dentate gyrus. Most of the neurons (40 out of 48) analyzed in the present study were from animals between the ages of 36 and 46 days, making it unlikely that any age-related differences would be detectable in our data. We did, however, examine this possibility, and results indicated that none of the

-

TABLE 6. Dendritic Parameters: A Comparison of Adjacent Granule Cells Infrapyramidal

b

a'

b

a'

Primary dendrites

2

1

3

1

Dendritic segments

32

21

25

25

Maximum branch order

6

6

4

5

Transverse spread !pm)

420

389

332

252

Total length (pm)

P

-

'Values are means ? S.E.M. Ranges are shown in parentheses. *Superficid neurons had somata in the top half of the granule cell layer, whereas deep neurons had somata in the bottom half. 3See text for method used to determine tree shape. *Signiiicantat P < .05.

Longitudinal spread (pm)

2.4 2 .3 11-4)

Tree shape'

(23-27) 5.5 1.3 (5-71 244 i 26 (187-321) 157 % 8 (132-188) 2,629 % 86 12,324-2,857) .69 ? .09 (.42-.98J

5.5 t .3 (4-7) 311 z 17 (222-445) 170 t 8 (132-217) 2,875 t 95 (2,3663,450) .55 t .04 ( 3 - 741

Suprapyramidal

'Values are means i S.E.M. Ranges are in parentheses. 'See text for method of determining tree shape. *Signfieant a t P < .05.

Superficial' (n = 19)

Tree shape'

-

251 1

(2240)

Ma%num branch order

P -

Tree s h a d

87

121

184

196

3,155

3,102

2,835

3,187

.14

.31

.55

.73

'Neurons labeled "a" and "b" w e n adjaeent to one another The cell b d i e s of ail neurons were located in the superficialhalfof each cell layer. None ofthe dendrites were cut 'Seetext for methodofdetermimgtreeshape.

dendritic parameters correlated with age for any of the subclasses (see also Rihn and Claiborne, '90). Superficial us. deep cells. The granule cells in each blade were further divided into subpopulations based on the location of their somata in the granule cell layer. First, cells in the suprapyramidal blade with somata in the superficial half of the cell layer were compared to those with somata in the deep half. Significant differences were seen between the two groups in number of primary dendrites, maximum branch order, transverse spread, and tree shape (Table 4). The superficial neurons had, on average, more primary dendrites (2.4 vs. 1.51, larger transverse dendritic spreads (378 pm vs. 293 pm), and more elliptical trees (.46 vs. .67) than the deeper cells. In addition, superficial cells had a lower maximum branch order than did the deep cells (5.5 vs. 6.4). There were no significant differences between the two subgroups in total dendritic length, somata width or length (Table 4), or in the percentages of branch points within thirds of the layer (data not shown). There was, however, a significant difference ( P < 0.04) in the percentage of dendritic length in the distal or outer third of the layer; superficial cells had 42% of their length in the distal third of the layer, whereas deeper cells had 37%.Correspondingly, superficial cells had slightly (but not significantly) less of their dendritic length in the proximal (29%vs. 31%) and middle thirds (29%vs. 32%)of the molecular layer.

214

Fig. 4. Computer-generated plots of reconstructions of the dendritic trees from granule cells located at various positions around the transverse axis of the dentate gyms. Each neuron was from a different animal. The total dendritic length of each neuron is indicated. Note that

the total dendritic lengths of the neurons in the suprapyramidal blade are greater than those in the infrapyramidal blade. C&,:field CA, of hippocampus.

When superficial cells in the infrapyramidal blade were compared to deep neurons in the same blade (Table 5 ) , superficial cells again had significantly larger transverse spreads (311 pm vs. 244 gm). They also tended to have more primary dendrites and were more elliptical than deep cells though these differences did not reach significance. AGacent cells. In approximately 50% of the injected slices, two or more adjacent granule cells were filled (Fig.5 ) . In eight cases (three in the suprapyramidal blade and four in the infrapyramidal blade), where at least two of the filled cells were adequately stained and all other selection criteria were met, these adjacent cells were subjected to quantitative analysis and were included in the total population of granule neurons. While in some cases of multiple labeling the stained cell bodies were not directly apposed (Fig. 5B,C),

in all cases they were within a few cell diameters of each other. Because the analyses of the subclasses described above showed that neurons could be grouped on the basis of location, we were interested in determining the dendritic similarity of adjacent neurons. In Table 6, data for two pairs of adjacent cells are presented. Analysis of dendritic parameters showed that the adjacent cells were very similar. In fact, the differences between any two adjacent cells were much less than the range of values seen for all neurons in the appropriate subclass (compare to minima and maxima in Tables 4 and 5 ) . For example, three sets of adjacent neurons were located in the superficial half of the infrapyramidal blade. Results showed that of the three sets, the maximum difference in dendritic segment number was 13,

GRANULE CELL DENDRITES

215

Fig. 5. Micrographs showing multiple labeling of granule cells from a single HRP injection. In A and C, two neurons were filled, while in B and D, three neurons were labeled. Close examination of each of these preparations indicates that a dendrite from one filled cell runs directly over (or under) the filled soma of another cell; we assume that it is at such sites that the HRP labeling has spread from the injection and into

the adjoining neuron(s1. This is especially clear in panel C, where one of the dendrites from the labeled cell at the bottom can be seen to course over the filled soma of the top cell (open arrow). In many of the micrographs, an axon (asterisk) can be seen exiting from the granule cell body. Scale bar = 100 wm.

as compared to 18 for all neurons in this region (Table 5). Similarly, the maximum differences in transverse spread and total length between adjacent neurons were 115 km and 626 pm, respectively, as compared to 223 IJ-mand 1,086 pm for all cells in the superficial portion of the infrapyramidal blade (Table 5 ) .

obtain complete dendritic trees of 48 neurons for quantitative analysis in three dimensions. Stringent selection criteria were employed to insure that the reconstructed dendritic trees represented an accurate likeness of the in vivo neurons. When the neurons were divided into subclasses based on their location, significant differences were seen in various dendritic parameters. Cells lociited in the suprapyramidal blade, for example, had greater total dendritic length, more dendritic segments, and larger transverse spreads than neurons located in the infr-apyramidal blade. When the cells located in the suprapyramidal blade were further subdivided into cells with somata in the superficial

DISCUSSION By intracellularly injecting single dentate granule cells in the in vitro hippocampal slice and by processing the entire 400 pm slice as a whole-mount preparation, we were able to

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216 half of the granule cell layer vs. those with somata in the deep half, cells located superficially were found to have a significantly larger number of primary dendrites, greater transverse spreads, and more elliptical trees than did deeper neurons.

Technicalconsiderations While the qualitative analysis of Golgi preparations has provided much of the existing information on the cellular organization of the central nervous system, using this technique for quantitative studies of neuronal form is prone to several technical problems. The capriciousness of the stain, the occurrence of cut dendritic branches at section planes, the possibility of incomplete impregnation, and the obscuring of fine dendritic processes by overlapping processes from other stained cells can hamper quantitative analyses. Due in part to these problems, quantitative studies on neurons with even simple branching patterns such as the dentate granule cell result in high variability and a wide range of average values. From studies of this kind it is difficult to resolve whether the variability reflects the inherent biological variation of the cell population or an artifact of the histological and technical procedures employed for making the observations. While it is certainly possible to compensate for many of the problems associated with the Golgi technique (Desmond and Levy, ’82),we have chosen to develop a different methodology to quantitatively study neurons of the hippocampal formation. The primary advantage of the techniques used here was that complete dendritic trees were analyzed directly from thick slices, thereby eliminating the need to reconstruct neurons from serial sections. During the iontophoretic procedures, attempts were made to only label cells located in the middle of the 400 pm slice to maximize the probability that the labeled dendritic tree would be contained within the slice. A second advantage of this labeling procedure was that the filled dendrites were clearly visible against the clear background of the slice; there were generally no overlapping dendrites as is often the case with Golgiimpregnated material. Third, we selected neurons with few if any cut dendrites to reduce artifactual variability in our results. Twenty-nine of the 48 analyzed cells had no cut dendrites, 12 had one cut dendrite, and seven had two cut dendrites. In each case, the cut dendrites were in the outer portion of the molecular layer. As noted in the Results, when neurons with cut branches were compared to neurons without any severed branches from the same blade, there were no significant differences in dendritic parameters.

Qualitative analysis The granule cells labeled by the intracellular injection of HRP resembled those stained in rodents by the Golgi method (Fricke, ’75; Lindsay and Scheibel, ’81; Seress and Pokorny, ’81; Wenzel et al., ’81; Desmond and Levy, ’82; Williams and Matthysse, ’83;Green and Jurasaka, ’85)and by the intracellular injection of Lucifer Yellow (Cobbett and Cottrell, ’81) or carboxyfluorescein (Rao et al., ’86). As noted by others, a granule cell had from one to four primary dendrites exiting the soma. We could not easily divide these primary dendrites into the “major” and “minor” categories described by Lindsay and Scheibel (’81). Of particular interest was the finding that virtually all of the dendritic branches of the HRP-filled neurons branched or extended to the hippocampal fissure or the pia; i.e., they rarely ended within the molecular layer itself (see also Durand et al.,

’83). Our results contrast with the observations from HRP-labeled granule cells in the guinea pig in which small “branchlets” appeared to terminate within the molecular layer (Turner and Schwartzkroin, ’83).

Quantitative analysisof all granule cells Several previous studies have quantified dendritic parameters of Golgi-impregnated granule cells in rodents, but to date the most exhaustive quantitative study has been carried out by Desmond and Levy (’82).Their observations were based on rapid Golgi-stained hippocampi that had been “flattened” along the long axis. While the dendritic trees of individual granule cells were analyzed in two dimensions from camera lucida drawings, appropriate corrections were applied to control for projection errors and cut branches when these authors computed parameters such as dendritic lengths. In general, the findings from the present analysis are consonant with the observations of Desmond and Levy. In the present study, we determined that granule cells had a mean of 1.9 primary dendrites which agrees with Desmond and Levy’s average of 2.2. Our HRP-filled neurons had between 22 and 40 segments, with an average of 29, and the cells they analyzed had between 18 and 35. In other Golgi studies, Seress and Pokorny (’81) reported 33 segments but Green and Juraska (’85)reported observing only 20 segments. Perhaps the most interesting parameter that can be obtained in these types of studies is the total dendritic length. With this parameter and an indication of spine density, it is possible to get an approximate indication of the amount of excitatory input that the granule cell receives (see below). The granule cells analyzed in this study had an average total dendritic length of 3,221 pm with values ranging from a low of 2,324 pm to a high of 4,582 pm. A recent study of the maturation of granule cells based on HRP labeling has confirmed these total lengths (Rihn and Claiborne, ’90). Using Golgi-stained tissue, Desmond and Levy (’82) reported uncorrected values for total dendritic length ranging from 1,100 km to 3,000 pm. Presumably these lower numbers reflect the effects of tissue shrinkage during the Golgi impregnation technique, the prevalence of twodimensional analyses, and the difficulties in analyzing entire trees from the thinner Golgi sections. Nonetheless, after applying correction factors Desmond and Levy reported an average total length of 3,662 pm, which is close to the value determined from our preparations. It is of interest to note that while total dendritic length varied about two-fold over the population as a whole, there was much greater variability in the lengths of individual granule cell segments (see Fig. 3). Other investigators have also commented on the wide range in lengths of granule cell segments, particularly of terminal segments, in the rat (Lindsay and Scheibel, ’81;Desmond and Levy, ’82) and the mouse (Williams and Matthysse, ’83). We found that 30% of the total dendritic length of the granule cell tree was located in the cell layer and proximal third of the molecular layer, 30% was in the middle third, and 40%in the most distal third. These results suggest that slightly more dendritic surface area is available for endings of the lateral entorhinal afferents that terminate in the outer third of the molecular layer. When we determined the percentages ofbranch points in thirds of the layer, we found 63% in the cell layer and proximal third of the molecular layer, 27% in the middle third, and only 10% in the distal

GRANULE CELL DENDRITES one-third. Desmond and Levy ('82) found similar percentages (70%, 25%, and 6%) for granule cells in the rat, and Williams and Matthysse ('83)also noted that approximately 75% of the branches of mouse granule cells occurred in the granule cell layer and inner third of the molecular layer. These results suggest that most of the branching occurs in the proximal third of the molecular layer where the associational and commissural afferents are known to terminate (Swanson et al., '78). From the three-dimensional data, tree shape was determined both visually and mathematically. When the trees were rotated on the computer monitor so that the viewer was looking down on a tree from the top, it was clear that most trees were elliptical (Fig. 2). This visual impression was confirmed by the finding that the average dendritic spread ratio (or "tree shape") was 0.56. Other investigators have also noted the elliptical shape of the granule cell tree in rats (Fricke, '75; Cowan et al., '80; Desmond and Levy, '82) and in mice (Williams and Matthysse, '83). We were interested in determining how the elliptical shape of the granule cell related to the axes of the hippocampus. A surprisingly high percentage of cells, 48%, had their widest dendritic spread exactly in the plane of the slice, and the vast majority of cells (93%) had their widest spread within 30" of the plane of the slice. Because our slices were cut as close as possible to the transverse plane of the hippocampus, these results suggest that most granule cells were oriented with the widest extents of their dendritic tree parallel to the transverse plane of the hippocampus.

Subgroupingsof granule cells Of the various ways that the granule cells could be divided into groups for comparison, we decided to compare cells of the suprapyramidal blade to those of the infrapyramidal blade and to compare granule cells which had their cell bodies located at various depths in the cell layer. These choices were prompted primarily by considerations of the development of the dentate gyrus. It has been well established that cells of the suprapyramidal blade are generated earlier than those of the infrapyramidal blade and that cells deep in the granule cell layer are generated after those located more superficially (Schlessinger et al., '75).

Suprapyramidalvs.infrapyramidal neurons When granule cells were divided into groups based on their transverse position within the dentate gyrus, significant differences were seen in various dendritic parameters. Suprapyramidal neurons had, on average, more dendritic segments, larger transverse spreads, and greater total dendritic length than did neurons in the infrapyramidal blade. The mean total dendritic length for the suprapyramidal cells was 3,478 ym and for the infrapyramidal cells was 2,793 pm. Assuming that the synaptic density was equal in both blades, the functional implication of this finding is that the suprapyramidal granule cells would receive a significantly greater number of terminals. Desmond and Levy ('85) have shown, however, that the density of dendritic spines is actually greater on suprapyramidal cells (1.6 spines/ym) than on infrapyramidal cells (1.3 spinedym). With these numbers and the mean dendritic lengths, an estimate of the number of spines on an average granule cell would be 5,564 in the suprapyramidal blade and 3,630 in the infrapyramidal blade. Desmond and Levy ('82) also reported that suprapyramidal cells had larger transverse spreads and greater total

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length than did infrapyramidal granule cells, although their absolute values were smaller than those reported here. Their suprapyramidal cells had an average transverse spread of 283 pm (compared to 347 ym reported here) and infrapyramidal neurons had a spread of 190 pm (compared to 288 pm). When they reconstructed dendritic trees from serial sections, they found that suprapyramidal cells had an average total length of 3,107 pm (compared to 3,478 ym in the present study), whereas infrapyramidal neurons had an average dendritic length of 2,078 ym (compared to 2,793 pm observed here). In the present study, the longitudinal spread of dendritic trees did not differ between the supraand infrapyramidal granule cell populations. In contrast, Desmond and Levy ('82) reported that infrapyramidal cells had greater longitudinal spreads (236 pm) than did suprapyramidal cells (194 ym).

Superficialvs.deep neurons Neurons in each blade were further subdivided into two classes based on the position of the cell body within the granule cell layer. In the suprapyramidal blade, the superficially located cells had a greater number of primary dendrites, larger transverse spreads of their dendrites, more elliptical trees, and a lower maximum branch order than did the deeper cells. In the infrapyramidal blade, superficial neurons differed from the deeper cells in having a significantly larger transverse spread of their dendritic trees. Green and Juraska ('85) reported that superficially located granule cells from both blades had a greater number of primary dendrites and larger transverse spreads than did deep cells. They also found that superficially located neurons had 10% more dendritic length than did neurons with deep somata. In contrast to this finding, we did not observe a significant difference in total dendritic length between superficial and deep cells in either blade, although an analysis of dendritic length within various thirds of the layer showed that superficial cells in the suprapyramidal blade had more dendritic length in the outer third of the molecular layer than did deep cells. The locations of branch points in the layer did not differ significantly between the two subgroups.

Adjacent granule cells In many of our slice preparations, two or more granule cells were labeled by one intracellular injection. Other laboratories have reported similar multiple cell fillings following intracellular injection of either HRP (Grant et al., '80; Triller and Horn, '81; Durand et al., '83; Turner and Schwartzkroin, '83) or the smaller fluorescent dyes, Lucifer Yellow (see Dudek et al., '86 for review) or carboxyfluorescein (Rao et al., '86). In the hippocampus, such multiple labeling after single injections of Lucifer Yellow or carboxyfluorescein is thought to be due, in part, to the transfer of the dyes across gap junctions (Dudek et al., '86; Rao et al., '861, which are known to be present in this region (Schmalbruch and Jahnsen, '81; MacVicar and Dudek, '82). Since HRP is a relatively large molecule that does not appear to cross gap junctions, the appearance of multiple stained cells has been attributed either to disruption of adjoining neuronal membranes by the microelectrodes, to fixationinduced membrane damage (Bennett, '73), or to the pinocytotic transfer of HRP between adjacent cells iTriller and Horn, '81). Preliminary electron microscopic observations of intracellularly injected granule neurons tend to favor the first of

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these possibilities. Electron micrographs taken from thin sections through the somata of adjacent HRP-labeled neurons show large gaps, up to 4 pm in length, between the two cells with the membranes of the two neurons apparently fused on either side of the gap (Claiborne, unpublished observations). Presumably, under the conditions of our experiments, the micropipette may damage the membranes of adjoining cells and then, either during or shortly after the filling process, HRP may diffuse from the impaled cell into the adjacent neuron. As the resting potentials of the cells studied were maintained throughout the period of filling, it seems likely that the membranes of the adjoining cells must have fused almost immediately after damage and impalement. Rather than being considered a bothersome artifact of the intracellular injection procedure, the double-labeled cells provided the unusual opportunity to compare dendritic characteristics of neurons that have common environmental and ontogenetic attributes. Results indicated that, in general, adjacent neurons were more similar to each other than to other neurons in the same subclass. This was particularly obvious when total dendritic lengths were compared. For example, one cell of two adjacent cells had a total length of 3,155 pm, and the other had a total length of 3,102 p,m (Table 6), a difference of only 53 Fm. In the population of all neurons located in the same region of the dentate, the superficial half of the suprapyramidal blade, the range in total length was from 2,500 Frn to 4,582 p,m, a difference of 2,082 pm.

CONCLUSION The hippocampal formation has been the subject of numerous anatomical studies over the past century. This is due, in part, to the seductive simplicity of this region. But more recently the importance of the hippocampal formation to normal memory function has added impetus to attempts to understand the organization of its neuronal circuitry. As noted by Brown et al. ('81) nearly a decade ago, there is a particularly pressing need for realistic morphological data that can be incorporated along with the results of increasingly sophisticated electrophysiological analyses into computer simulations of hippocampal structure and function (Mainen et al., '90). The techniques and data described in this paper are a first step towards providing reliable, quantitative data concerning the neuronal morphology and connectivity of the rat hippocampal formation.

ACKNOWLEDGMENTS We would like to thank Steve Pfeiffer and Kay Elledge for technical assistance and Kris Trulock for help with the photography. This work was supported in part by NIH grant N S 16980 to D.G.A. and by a grant from the Sloan Foundation.

Bennett, M.V.L. (1973) Permeability and structure of electrotonic junctions and intracellular movements of tracers. In S.B. Kater and C. Nicholson (eds): Intracellular Staining in Neurobiology. New York SpringerVerlag, pp. 115-134. Brown, T.H., R.A. Fricke, and D.H. Perkel (1981) Passive electrical constants in three classes of hippocampal neurons. J. Neurophysiol. 46:812827.

Cajal, S. Ramdn y (1911) Histologie du Systeme Nerveux de 1'Homme et des Vertebres. Val. 11.Paris: Maloine. Capowski, J.J., and M.J. Sedivec (1981) Accurate computer reconstruction and graphics display of complex neurons utilizing state-of-the-art interactive techniques. Comput. Biomed. Res. 14:518-532. Claiborne, B.J., D.G. Amaral, and W.M. Cowan (1986) A light and electron microscopic analysis of the mossy fibers of the rat dentate gyrus. J. Comp. Neurol. 246t435-458. Cobbett, P., and G.A. Cottrell (1981) The structural integrity of neurons in the hippocampal slice preparation as revealed by intracellular injection of Lucifer Yellow. J. Neurocytol. 10:671-678. Cowan, W.M., B.B. Stanfield, and K. Kishi (1980) The development of the dentate gyms. In R.K. Hunt (ed): Current Topics in Developmental Biology. New York: Academic Press, pp. 103-157. Desmond, N.L., and W.B. Levy (1982) A quantitative anatomical study of the granule cell dendritic fields of the rat dentate gyrus using a novel probabilistic method. J. Comp. Neurol. 212:131-145. Desmond, N.L., and W.B. Levy (1985) Granule cell dendritic spine density in the rat hippocampus varies with spine shape and location. Neurosci. Lett. 54.219-224. Dingledine, R. (1984) Brain Slices. New York: Plenum Press. Dudek, F.E., R.W. Snow, and C.P. Taylor (1986) Role of electrical interactions in synchronization of epileptiform bursts. In A.V. Delgado-Escueta, A.A. Ward, D.M. Woodbury, and R.J. Porter (eds): Advances in Neurology. New York: Raven Press 44593-617. Duffy, C.J., and P. Rakic (1983) Differentiation of granule cell dendrites in the dentate gyrus of the rhesus monkey: A quantitative Golgi study. J. Comp. Neurol. 214:224-237. Durand, D., P.L. Carlen, N. Gurevich, A. Ho, and H. Kunov (1983) Electrotonic parameters of rat dentate granule cells measured using short current pulses and HRP staining. J. Neurophysiol. 50: 1080-1097. Fricke, R.A. (1975) Studies on the morphology and development of the hippocampus and dentate gyrus. Unpublished Ph.D. dissertation. St. Louis: Washington University. Golgi, C. (1886) Sulla fina anatomia degli organi centrali del sistema nervoso. Milano: U. Hoepli, p. 215. Grant, K., J.P. Gueritaud, G. Horcholle-Bossavit, and S. Tyc-Dumont (1980) Detailed morphology of two vestibular neurones following a single intracellular injection of HRP. Neurosci. Lett. 16223-228. Green, E.J., and J.M. Juraska (1985) The dendritic morphology of hippocampal dentate granule cells varies with their position in the granule cell layer: A quantitative Golgi study. Exp. Brain Res. 59:582-586. Hjorth-Simonsen, A., and B. Jeune (1972) Origin and termination of the hippocampal perforant path in the rat studied by silver impregnation. J. Comp. Neurol. 144.215-232. Insausti, R., D.G. Amaral, and W.M. Cowan (1987) The entorhinal cortex of the monkey: 11. Cortical afferents. J. Comp. Neurol. 264:356-395. Lindsay, R.D., and A.B. Scheibel (1981) Quantitative analysis of the dendritic branching pattern of granule cells from adult rat dentate gyrus. Exp. Neurol. 73286-297. Lorente de Nd, R. (1934) Studies on the structure of the cerebral cortex. 11. Continuation of the study of the ammonic system. J. Psychol. Neurol. (Leipzig) 46113-117. MacVicar, B.A., and F.E. Dudek (1982) Electrotonic coupling between granule cells of rat dentate gyrus: Physiological and anatomical evidence. J. Neurophysiol. 47:579-592. Mainen, Z.F., A.M. Zador, B.J. Claiborne, and T.H. Brown (1990) Hebbian synapses induce feature mosaics in hippocampal dendrites. Neurosci. Abstr. 16:492. Milner, B. (1970) Memory and the medial temporal regions of the brain. In K.H. Pribian and D.E. Broadbent (eds): Biology of Memory. New York: Academic Press, pp. 29-50. Rao, G., C.A. Barnes, and B.L. McNaughton (1986) Intracellular fluorescent staining with carboxyfluorescein: A rapid and reliable method for quantifying dye-coupling in mammalian central nervous system. J. Neurosci. Methods 16251-263. Rihn, L.L., and B.J. Claiborne (1990) Dendritic growth and regression in rat dentate granule cells during late postnatal development. Dev. Brain Res. 54t115-124. Schlessinger, A.R., W.M. Cowan, and D.I. Gottlieb (1975) An autoradiographic study of the time of origin and the pattern of granule cell migration in the dentate gyrus of the rat. J. Comp. Neurol. 159t149-176. Schmalbruch, H., and H. Jahnsen (1981) Gap junctions on CA3 pyramidal cells of guinea pig hippocampus shown by freeze-fracture. Brain Res. 21 7:175-178.

GRANULE CELL DENDRITES Seress, L., and J. Pokorny (1981) Structure of the granular layer of the rat dentate gyrus. A light microscopic and Golgi study. J. Anat. 133:181195. Squire, L.R. (1987) Memory and the Brain. New York: Oxford University Press. Steward, 0. (1976) Topographic organization of the projections from the entorhinal area to the hippocampal formation of the rat. J. Comp. Neurol. 167285-3 14. Swanson, L.W., J.M. Wyss, and W.M. Cowan (1978) An autoradiographic study of the organization of intrahippocampal association pathways in the rat. J. Comp. Neurol. 181:681-716. Triller, A,, and H. Horn (1981) Interneuronal transfer of horseradish peroxidase associated with exoiendocytotic activity on adjacent membranes. Exp. Brain Res. 43:233-236.

2 19 Turner, D.A., and P.A. Schwartzkroin (1983) Electrical characteristics of dendrites and dendritic spines in intracellularly stained CA3 and dentate hippocampal neurons. J. Neurosci. 3:2381-2394. Wenzel, J.,G. Stender, and D. Duwe (1981) Zur Entwicklungder Neuronenstruktur der Fascia dentata bei der Ratte. Neurohistologisch-morphometrische, ultrastrukturelle and experimentalle untersuchungen. J. Hirnforsch. 22629-683. Williams, R.S., and S. Matthysse (1983) Morphometric analysis of granule cell dendrites in the mouse dentate gyrus. J. Comp. Neurol. 215:154164. Zola-Morgan, S., L.R. Squire, and D.G. Amaral(1986) Human amnesia and the medial temuoral lobe: Enduring. memorv imuairment followinc a bilateral lesion iimited to field C A r o f the hippdcampus. J. Neurosci. 6:2950-2967.

Quantitative, three-dimensional analysis of granule cell dendrites in the rat dentate gyrus.

The three-dimensional organization of dentate granule cell dendritic trees has been quantitatively analyzed with the aid of a computerized microscope ...
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