JOURNAL OF COMPARATIVE NEUROLOGY 190:87 -114 (1980)

Development of the Hippocampal Region in the Rat I. Neurogenesis Examined With 3H-Thymidine Autoradiography

SHIRLEY A. BA YER Laboratory ofDevelopmental Neurobiology, Department ofBiological Sciences, Purdue University, West Lafayette, Indiana 47907

ABSTRACT Neurogenesis in the rat hippocampal region was examined with 3R-thymidine autoradiography. The rats in the prenatal groups were the offspring ofpregnant females given two injections oPR-thymidine on consecutive days in an overlapping series: embryonic (E) day E13+E14, E14+E15, ... , E21+E22. The rats in the postnatal (P) groups were injected in two nonoverlapping series: first, the day of birth (PO) and PI, P2+P3, ... , P18+P19; second, PO-P3, P4-P7, ... , P16-P19. On 60 days of age, the percentage oflabelled cells and the proportion of cells added during each day of formation were determined at several anatomical levels within each structure of the hippocampal region (entorhinal cortex, para­subiculum, presubiculum, subiculum, Ammon's horn, and the dentate gyrus) and the hippocampal rudiment (tenia tecta, indusium griseum). The neurons in each structure arise in overlapping, but still significantly different, waves: the hippo­campal rudiment between E16-E17; the entorhinal cortex between E15-E17; the para- and presubiculum between E16-E19; the subiculum between E16-E18; large cells in strata oriens, radiatum, lacunosum-moleculare of Ammon's horn between E 15- E 17; Ammon's horn pyramidal cells between E17-E 19; large cells in the dentate hilus and molecular layer between E15-E19. Dentate granule cells begin to originate on E17, and 10% of the population forms after P18.

There are three characteristic gradients of formation within structures. First, deep cells are generated before superficial cells. Second, cells closer to the rhinal fissure are formed before those lying farther away ("rhinal to dentate" gradient). Third, later forming cells are flanked by earlier forming superficial and deep cells ("sandwich gradient") in the entorhinal cortex (layer III cells originate after layers II and IV), Ammon's horn (pyramidal cells originate after large cells in strata oriens, radiatum, and lacunosum-moleculare), and the dentate gyrus (granule cells originate after large cells in the hilus and molecular layer). There is a "rhinal to dentate" gradient between structures. The entorhinal cortex starts first, next is the subiculum, then field CA3 of Ammon's horn, and finally, the dentate gyrus. Two structures are exceptions to this gradient. The para- and presubiculum form signi­ficantly later than the subiculum, and CAl forms significantly later than adjacent CA3 cells; this late neurogenesis may be related to prominent thalamic input to both structures.

Neurogenetic gradients between the cells providing laminated afferent input to the Ammonic pyramidal and dentate granule cells correlate with their order of termination: afferents from progressively later-originating cells terminate pro­gressively closer to the cell body. Topographic hippocampal projections along the dorsoventral axis correlate with formation patterns in target structures: dorsal hippocampal fibers project to zones occupied by earlier-forming cells in the lateral septal nucleus and pars posterior of the mammillary body; ventral hippocampal fibers project to zones occupied by later-forming cells in these structures.

0021-9967/80/1901-0087$04.70 © 1980 ALAN R. LISS, INC.

88 SA BAYER

To contribute to a better understanding of the origins of the intricate anatomical organi­zation of the limbic system, a detailed analysis of its development in the rat has been under­taken. The first reports of the series dealt with the septal region (Bayer, '79a, b). Limbic nuclei in the hypothalamus (Altman and Bayer, '78a, b, c) and thalamus (Altman and Bayer, '79a, b, c) have also been described. All ofthese studies show that the temporal order of neurogenesis is extremely regular and precise in each struc­ture. The present report and a companion paper (Bayer, '80) deal with the hippocampal region.

The anatomy of the hippocampal region has been studied in the rat since the time of Cajal's ('11) and Lorente de No's ('33, '34) observations using the Golgi method. The contributions of Blackstad ('56, '58), RaismanetaI. ('65, '66) and Hjorth-Simonsen ('72, '73) using lesion methods, combined with the work of Meibach and Siegel ('75, '77a, b), Swanson and Cowan ('75, '76, '77), and Steward ('76) using physio­logical transport methods, make the rat hippo­campus one of the best known neuroanatomical structures. These studies have shown that the hippocampal region is an important component of the limbic system, with strong links to the septum, limbic neocortex, and limbic nuclei in the diencephalon.

3H-thymidine autoradiography has been used for the past 15 years to date the time of neuron origin in the hippocampal region. Most of these studie$. were similar to Angevine's ('65) monograph on the mouse hippocampal region and used single injections oPH-thymidine dur­ing various days of embryonic (Hine and Das, '74; Schlessinger et aI., '78) and postnatal (Altman and Das, '65, '66; Altman, '66; Schles­singer et aI., '75) life. A modified 3H-thymidine autoradiographic procedure, the progressively delayed comprehensive labelling method (Bayer and Altman, '74), increased the accu­racy in timing the onset, proportion of daily acquisition, and cessation of neurogenesis in Ammon's horn and the dentate gyrus over blocks of four days between birth and postnatal day 16. The present comprehensive report ex­pands these observations to include a daily chronology of neuronal acquisition in all areas of the hippocampal region (entorhinal cortex, parasubiculum, presubiculum, subiculum, Ammon's horn, and the dentate gyrus) and the hippocampal rudiment (tenia tecta, indusium griseum) from embryonic days 15-21 and thereafter over blocks of two days up to post­natal day 18.

MATERIALS AND METHODS

Since neurogenesis in the rat hippocampal region extends well beyond the day ofbirth, one prenatal and two postnatal developmental series were used. All series contained groups of Purdue-Wistar rats given successive daily (be­tween 9 and 11 a.m.) subcutaneous injections of 3H-thymidine (Schwarz-Mann; specific activity 6.0 CmM; 5 p.Ci/gram body weight); multiple injections were given to insure comprehensive cell labelling. The prenatal developmental series contained nine groups, the offspring of pregnant females given two successive daily injections progressively delayed by one day be­tween groups (E13+E14, E14+E15, ... , E21 + E22). The day of sperm positivity was embryonic day one (E1); two or more pregnant females were injected for each group. One post­natal developmental series had 10 groups given two successive daily injections progressively delayed by two days between groups (PO+PI, P2+P3, ... , P18+P19). The other postnatal developmental series had five groups given four successive daily injections progressively delayed by four days between groups (PO- P3, P4-P7, ... , P16-P19). Birth normally occurs on the afternoon of E23 and is postnatal day 0 (PO).

All animals were perfused through the heart with 100b neutral formalin on P60. The brains were kept for 24 hours in Bouin's fixative, then transferred to 10% neutral formalin until they were embedded in paraffin. The brains offive to six animals from each prenatal group were blocked sagittally and one-half of the brain was cut sagittally (6 p.m, every 10th section was saved); the other half was cut horizontally (6 p.m, every 15th section was saved). The brains of two animals in each of the postnatal groups were cut sagittally (6 p.m, every 30th section was saved). The slides were dipped in Kodak NTB-3 emulsion exposed for 12 weeks, devel­oped in Kodak D-19, and post-stained with either hematoxylin and eosin (prenatal series) or cresyl-violet (postnatal series).

Anatomically matched sections were selected at four levels throughout either the dorsoventral extent or the mediolateral extent of the hippocampal region in the horizontal and sagittal planes, respectively. The proportion of labelled cells was determined microscopically at 312.5 x or 500 x with the aid of an ocular grid. All cells with reduced silver grains overly­ing the nucleus in densities above background levels «1 grain per 150 p.2) were considered labelled; obvious endothelial and glial cells

89 HIPPOCAt\1PAL NEUROGENESIS

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Fig. 1. Horizontal section 00 !-LID, paraffin) of the young adult rat (P90) hippocampal region, 1.4 mm dorsal to the temporal extreme of the dentate granular layer (gl), showing the structures quantified (hematoxylin and eosin; bar, 1 mm). Abbreviations: EC, entorhinal cortex; la, lateral; im, intermediate; m, medial; I-VI, layers; PA, parasubiculum; PR, presubiculum, d, cells deep to para- and presubiculum; SU, subiculum; a, zone near presubiculum; b, central zone; pro, prosubiculum; pI, pyramidal layer; ml, molecular layer; AH, Ammon's horn; CAl, pyramidal cell field adjacent to subiculum containing a and bc zones; CA3, pyramidal cell field adjacent to dentate hilus (h) containing ab and c zones; so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum; slm, stratum lacunosum moleculare; DG, dentate gyrus; h, hilus; gl, granular layer; ml, molecular layer; ec, ectal part facing CAl and subiculum; en, endal part; FI, fimbria.

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Fig. 2. The entorhinal cortex in an animal exposed to 3H-thymidine on E17 +E18 and killed on P60 (horizintal section, 6 J.l.m, paraffin; hematoxylin and eosin; bar 0.5 mm) assignment ofcell layers follows Krieg ('46a, b). The strong lateral to medial gradient of neurogenesis is indicated by the arrow.

were excluded. The determination of the pro­portion of cells arising (ceasing to divide) on a particular day utilized a modification of the progressively delayed comprehensive labelling procedure described by Bayer and Altman ('74). The method is based on the assumption that 3H-thymidine will only be incorporated by mitotic neuronal precursors, not by post­mitotic early neurons, Within a specific popula­tion, a maximal percentage (>95%) oflabelled neurons indicates that most of the precursors are still dividing at the time of the onset of the injections, and few neurons are originating. Specific neuronal populations in the hippocam­pal region are maximally labelled after two successive daily injections at some time during embryonic development. If the onset of the in­jections is progressively delayed by 24 hours, injection schedules will follow when the per­centage of labelled neurons within a specific population declines, reflecting the change of precursors into early neurons. The proportion of neurons originating each day is equal to the daily decline in the percentage of labelled

neurons. For instance, the neurons originating on day E 17 are determined as follows: E 17 = (% neurons labelled E17+E18) - (% neurons la­belled E18+ E19).

Throughout the quantitative analysis, it was noted that trends in cell labelling within ani­mals were very consistent. For example, in the entorhinal cortex, the percentage of labelled cells in layer II tended to be lower than the percentage of labelled layer III cells; however, variability between animals in an injection group were large enough to mask this trend. Accordingly, a statistical procedure was used, the sign test (Conover '71), to determine the con­sistency of sequential neuron production be­tween paired iocations within individual ani­mals. The comparisons are grouped into three categories: 1) X > Y, "-" comparison; 2) X < Y, "+" comparison; 3) X = Y, "0" comparison. The zero comparisons are discarded and, depending on the total number of remaining "+" and '~"

comparisons, either a binomial distribution or a normal approximation is used to calculate probabilities (p).

91 HIPPOCAMPAL NEUROGENESIS

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RESULTS The structures included in the hippocampal

region are shown in Figure 1. As one progresses from the rhinal fissure (RF) to the medial corti­cal edge, six distinct structures (separated by dashed lines) are found: the entorhinal cortex (EC, area 28), the parasubiculum (PA, area 49), the presubiculum (PR, area 27), the subiculum (SU), the Ammon's horn (AR), and the dentate gyrus (DG). The region is connected with sub­cortical telencephalic (septum) and dience­phalic (mammillary body, anterior thalamic nuclei, and others) centers by the prominent fiber tracts of the fimbria (FI) and fornix (not shown in Fig. 1). Rudiments of the hippocampal region (illustrated in Fig. 13) continue dorsally over the corpus callosum as the indusium griseum and forward to the medial part of the anterior olfactory nucleus as the tenia tecta. In the following discussion, the time of origin of neurons in each lamina of the six hippocampal region structures will be described in detail at four different levels. The data will be analyzed first to see what intrinsic gradients exist be­tween and within laminae in each structure, followed by a comparison of times of origin be­tween neurons in homologous laminae across different structures.

Entorhinal cortex

Figure 2 shows the entorhinal cortex in an animal exposed to 'JH-thymidine on E 17+E 18. Five distinct layers can be recognized. Layer I is an outer plexiform zone. Layer II contains the cell bodies of large stellate cells, grouped into islands laterally and separated from layer III by a cell-sparse zone; the separation between layers II and III in the medial entorhinal cortex is less obvious. Layer III contains the cell bodies of medium-sized pyramids. Layer IV is a cell­sparse zone (lamina dessicans) with a few scat­tered large pyramidal-type cells. Layers V-VI contain medium-sized and small cells in the lateral entorhinal cortex, a predominance of small cells in the medial entorhinal cortex; hor­izontally oriented, medium-sized cells are lo­cated next to the white matter. Only a few cells in layer III are labelled in the lateral entorhi­nal cortex, while many cells are labelled in the medial entorhinal cortex. This indicates a lat­eral to medial gradient of formation (arrow). Within the medial entorhinal cortex, no cells in layers V-VI, a few in layer IV, most oflayer III, and some layer II cells are labelled, indicating a modified deep to superficial gradient.

Quantification of the neurogenetic gradients observed in the entorhinal cortex is shown in

Figure 3. The percentage of labelled cells con­tained within strips (.39 mm) extending from the white matter to the pia were determined for each lamina, except I, at lateral (LA), medial (ME), and intermediate (1M) locations throughout levels one to four (drawings).! The line graphs show the decline in the percentage oflabelled cells for combined levels one through four2 within each lamina for the lateral (solid line), intermediate (dashed line), and medial (dotted line) entorhinal cortex. Neurons in lat­eral strips are first to originate (all levels and comparisons, p ,:; .0064); those in intermediate strips are next; those in medial strips are last (all levels and comparisons, p':; .0038). The bar graphs indicate the percentage ofcells originat­ing each day based on the data of the line graphs (see Materials and Methods) in lateral (black), intermediate (dotted), and medial (stippled) strips. The majority of neurons are formed (precursors cease to divide) on either one (for example, lateral entorhinal cortex, layers V-VI) or two (for example, medial en­torhinal cortex, layers V-VI) days. Most neurons in the lateral entorhinal cortex form on E15, a few on E16 (layer III). In the interme­diate entorhinal cortex, a few neurons form on E15 (layers V-VI), most on E16, and a few on E17 (layer Ill). Neurogenesis in the medial en­torhinal cortex occurs mainly on E16 and E17.

Neurons from each lamina originate at signi­ficantly different times within lateral, inter­mediate, or medial strips. The cells of layers V-VI are the first to form (all levels and com­parisons, p :os: .0007); the cells oflayers II and IV are generated next and simultaneously; the cells of layer III are last (all levels and compari­sons, p ,:; .0064). By examining the bar graphs for all lamina at either lateral, intermediate, or medial strips, the pattern emerges. For exam­ple, in the lateral entorhinal cortex (black bars), layers V-VI are mainly generated on E15; layers II and IV are generated largely on

J Level one is the most ventral section with definite islands of cells in layer II of the lateral entorhioal cortex; layerlV is indistinct laterally. layer II indistinct medially. Level two is the most ventral section to show a prominent layer IV extending throughout the lateral entorhi­nal cortex. Level three is the most ventral section to show 0. cell·sparse zone between layers n, TIl of the medial entorhinal cortex and the superficial cell layer of the parasubiculum. Level four is the section with a large angular bundle in the white matter which also contains two cell groups in the superficial cell layer of the parasubiculum.

2:There were no consistent significant differences within laminae across levels one through four for both the medial and lateral entorhi­nal cortex. There was a tendency for more dorsal intermediate strips to contain later-forming neurons. Since the placement of the intermedi­ate strip is shifted medially from levels one through four, the difference between levels was assumed to be a reflection of th~ strong lateral to medial gradient rather than a ventral to dorsal gr~dient.

92 SA BAYER

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FORMATION Fig. 3. Neurogenesis in the entorhinal cortex. Drawings show levels (L) quantified (bar, 1 mm) with regions

where cells were counted; Ll, most ventral, L4, most dorsal. Lateral (LA), intermediate (IM), and medial (ME) strips (.39 rom wide) were analyzed at levels indicated. In each strip, layer II (black), layer III (dotted), layer IV, (clear), and layers V-VI (stripes) were separately quantified. Line graphs, with mean and standard deviation, show decline in percentage of labelled cells in each layer after two successive daily "H-thymidine injections began on embryonic ages indicated. There were no significant differences between levels in any layer; data for all lateral strips (solid lines), all intermediate strips (dashed lines), and all medial strips (dotted lines) are combined. Bar graphs are percentages of cells originating in each layer during indicated embryonic days for lateral (black bars, based on data of solid lines), intermediate (dotted bars, based on data of dashed lines), and medial (stippled bars, based on data of dotted lines) strips. Bar graph data was computed like the following example from layers V-VI, lateral entorhinal cortex (lower set of graphs, solid line, black bars);

83% cells labelled 8% cells labelled 75% cells formed E15+E16 E16+E17 during E15

In each layer, laterally placed cells originate first, then intermediately placed cells, and finally medially placed cells. Layers V-VI form before layers IV and II; layer III is last.

93 IDPPOCAMPALNEUROGENESffi

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Fig. 4. Parasubiculum (PA), presubiculum (PR), and deep cells (d) from an animal exposed to 3H-thymidine on E19+E20 and killed on P60 (horiwntal section; 6 J.'.m, hematoxylin,eosin; bar, 0,25 mm). The "deep to superficial" and "rhinal to dentate" gradients of neurogenesis are indicated by the arrows.

E15, with a few left to form on E16; layer III is to determine the percentage of labelled cells. generated on both E15 and E16, with a few left The deep cells in levels one to three form simul­to form on E17. taneously and their data are combined (solid

line), while those in level four (dashed line)Parasubiculum and presubiculum form significantly earlier (p :s; ,0192). The bar

The structures (Fig. 4) wedged between the graphs show that deep cells from all levels orig­medial entorhinal cortex posterolaterally and inate mainly on E16 and E17, with more cells the subiculum anterolaterally are the para­ forming on E15 at level foW' than at the other subiculum (PA) and the presubiculum (PR). levels. The parasubiculum shows a different The outer lamina is occupied by medium­ dorsoventral gradient. The cells at level one sized cells in the parasubiculum and by small, (solid line) and at level four (dotted line) form densely packed cells in the presubiculum. The simultaneously, as do the cells oflevels two and deep cells are small to medium-sized, similar to three (data are combined in the dashed line). those in layers V-VI of the medial entorhinal The cells in levels two and tlu'ee originate sig­cortex. This animal was exposed to 3H-thymi­ nificantly later (all levels and comparisons, p :s; dine onE19+ E20. None of the deep neurons are .0352). The bar graphs show that more cells labelled; a few cells in the parasubiculum and arise on E16, fewer on E17, at levels one and many cells in the presubiculum are labelled. four; the reverse pattern is in levels two and This suggests both a deep to superficial cell three. The presubiculum is not present at level gradient of neurogenesis as well as a one, and neurons in the remaining levels form parasubicular to presubicular gradient in the simultaneously on E17-E19. There are strong outer lamina (arrows in Fig. 4). neurogenetic gradients between deep cells,

Neurogenesis in the para- and presubiculum para- and presubiculum which are similar to is quantified in Figure 5; drawings show the those found in the entorhinal cortex. First, deep levels studied (the same as those for the en­ cells originate before the superficial lamina torhinal cortex). All cells in the superficial (p ::::. .00001). In the superficial lamina, lamina ofthe para- and presubiculum, and deep parasubicular cells located nearer the rhinal cells within an area of 0.152 mm2 were counted fissure originate significantly earlier than the

94 S.A. BAYER

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DAY OFAGE AT INITIAL INJECTION FORMATION Fig. 5. Neurogenesis in presubiculum (dots), parasubiculum (stripes), and deep cells (solid black). Drawings show levels�

quantified (bar, 1 mm). Line graphs, with mean and standard deviation, show decline in percentage oflabelled cells after two� successive daily 3H-thymidine injections began on embryonic days indicated. Bar graphs are percentages of cells originating� during embryonic days based on line graphs specified by arrows. Deep cells (lower set ofgraphs) at L4 (dashed line, upper bar� graph) originate before those at LI-L3 (data are combined in solid line,lower bar graph). The parasubiculum (center set of� graphs) at L4 (short dashed line, upper bar graph) and Ll (solid line, lower bar graph) originate before those at L2-L3 (data� are combined in long-dashed line, center bar graph). The presubiculwn forms simultaneously at L2- L4 (data are combined in� top set of graphs).�

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95 HIPPOCAMPAL NEUROGENESIS

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hematoxylin-eosin; bar, 0,25 rom). Superficial and deep halves ofthe pyramidal layer (pI) were quantified separately in zone a (adjacent to the presubiculum, PRj, zone b (central), and the prosubiculum (pro; adjacent to Ammon's hom, AH). The "deep to superficial" and "rhinal to dentate" gradients of neurogenesis are indicated by the arrows.

presubicular cells located farther from the rhi­nal fissure (p ::.:; .00001).

Subiculum

The subiculum (Fig. 6) has a diffuse pyrami­dal layer (pI) lying beneath a wide molecular layer (mI). For purposes of quantification, the structure was di vided into three zones: a, b, and the prosubiculum (pro). Zone a lies adjacent to the presubiculum (PR), and the prosubiculum lies adjacent to field CAl of Ammon's horn (AH). The autoradiogram in Figure 6 is from an animal exposed to 3H-thymidine on E18+ E19. No cells are labelled in zone a; a few superficial cells are labelled in zone b, and several cells (mainly superficial) are labelled in the pro­subiculum. This suggests both a deep to super­ficial and also a zone a to prosubiculum gradi­ent of neurogenesis (arrows in Fig. 6).

Neurogenesis in the subiculum is quantified in Figure 7; drawings show the levels studied (the same as those for the entorhinal cortex). The percentage of labelled cells within strips (.39 mm) were determined separately for deep and superficial halves of the pyramidal layer for each zone indicated in Figure 6. All dorso­

ventral levels of the subiculum form simul­taneously; consequently, the line graphs show combined data. In all strips a strong gradient is for the deep cells (bottom graphs) to be gen­erated significantly earlier (p ::.:; .00001) than those lying superficially (top graphs). The bar graphs show that deep cells are generated mainly on E 15- E 17, while superficial cells originate mainly on E17 and E18. As for all structures thus far described, cells located closer to the rhinal fissure (zone a) originate earlier than those farther away (prosub­iculum). The deep cells in zone a (solid line) form significantly earlier (p ::.:; ,0192) than those forming later and simultaneously in zone b and the prosubiculum (dashed line), The su­perficial cells in zone a (solid line) originate first (p::.:; .0215), next are the superficial cells in zone b (dashed line), and last (p::.:; ,02) are the super­ficial prosubicular cells (dotted line).

Ammon's horn

Pyramidal cell formation The narrow stratum ofpyramidal cells (sp) in

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Fig, 7, Subiculum neurogenesis in rone a (solid black), rone b (clear), and the prosubiculum (stripes), Drawings show� levels quantified (bar, 1 mm), Line graphs, with mean and standard deviation, show decline in percentage of labelled cells� after two successive daily :lH-thymidine injections began on embryonic days indicated, Bar graphs are percentages of cells� originating during embryonic days based on line graphs specified by arrows, There were no significant differences between� levels in deep cells (data are combined in bottom set of graphs) and superficial cells (data are combined in top set of graphs),� Deep ceJls in rone a (solid line, lower bar graph) originate before those in rone b and the prosubiculum (data are combined in� dashed line, upper bar graph), Superficial ceJls in zone a (solid line, lower bar graph) originate first; next are superficial rone b� ceJls (dashed line, center bar graph); last are superficial cells in the prosubiculum (dotted line, upper bar graph),�

subiculum to the hilus of the dentate gyrus.3 'Adjacent to the prosubiculum is a field of tightly packed medium­�

The autoradiograms in Figure 8 are from an sized ceJls; Cajal ('II) named this regio superior; Lorente de No ('34)� called it CAl (fig. 1) and furtbersubdivided it into three zones. Zone a is� animal exposed to :JH-thymidine on E19+ E20. the region where scattered deep cells extend beneath the superficial�

A through D are from a ventral horizontal sec­ ceJls (see short dashed line in fig. I and fig, 8A); zones band c can only� be distinguished in fiber·stained preparations and are considered a� tion (similar to Fig. 1). A few superficial cells single subdivision in this study (fig, 8B), Beginning adjacent to CAl�

are labelled in CA1a (Fig. 8A), while the major­ (long dotted line in fig, I) is a field of large, less densely packed cells,� ity of cells are labelled in CA1b,c (Fig, 8B), Cajal ('ll) caJled this regio inferior; Lorente de No ('34) further divided�

it into fields CA2 and CA3, CA2 pyramidal ceJls occupy a narrow bandsuggesting a formation gradient between the adjacent to CAl which can be distinguished from CA3 pyramidal ceJls� two subdivisions. Only one cell is labelled in in Golgi preparations by the absence of characteristic thorns on the�

apical dendrite, In this study, fields CA2 and CA3 are combined. CA3� CA3a,b (Fig. 8C), while more cells are labelled has been subdivided into three zones (a,b, and c) on the basis of the�

in CA3c at the same level (Fig. 8D). More dor­ numberofSchalTer collaterals supplied by the cells, Zones a and b (fig,� 8c) cannot be dislingu ished in Nissl preparations, but zone c (fig, 80,E)�sally, CA3c (Fig. 8E) has a higher proportion of also lies partly wilhin the dentate hilus (its boundary is marked by the

labelled cells. This suggests gradients between short dashed line in fig, 1),

97 HIPPOCAMPAL NEUROGENESIS

AMMON'S HORN PYRAMIDAL CELLS A 8�

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Fig.8. Ammon's horn pyramidal cells from an animal exposed to 3H-thyrnidine on E19+E20 and killed on P60 (horizontal sections, 6 /Lm; hematoxylin-eosin; bar, 0.1 mm). A, field CAla near presubiculum, with scattered deep pyramidal cells; B, field CAlb,c, nearer CA3, with fewer deep pyramidal cells; C, field CA3a,b, with larger less densley packed pyramidal cells at L2; D, field CA3c, within dentate hilus at L2, showing distinct lamination similar to CA3a,b; E, field CA3c, within dentate hilus at lA, showing indistinct lamination. In field CAl, superficial pyramidal cells are labelled, deep cells are unlabelled. Arrows indicate unlabelled cells in stratum radiatum; asterisk indicates unlabelled cell in stratum oriens.

CA3a,b and CA3c and a ventral to dorsal gradi­�ent within CA3c.�

4Levels one and two are tbe same as those used for the entorhinalNeurogenesis of the pyramidal cells is quan­cortex. Level three is the most ventral section where the fimbri.a ex­

tified in Figure 9; drawings are of the levels tends in an unbroken path into the ventral hippocampal commissure studied.4 In each section quantified, the per­ and posterior septum. Level four is the most ventral section where the

enda) limbs of both anterior (septal) and posterior (temporal) portionscentage of labelled pyramidal cells were sepa­ of the granular layer join and lie in an unbroken line lateral to the rately determined after counts of all cells con- dorsal thalamus.

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Fig. 9. Neurogenesis in Ammon's horn pyramidal cells from fields separately analyzed; CAla (stripes), CAlb,c (clear), CA3a,b (solid black), CA3c (dots). Drawings show levels quantified (bar, I mm). Line graphs, with mean and standard deviation, show decline in percentage of labelled cells after two successive daily 'H-thymidine injections began on embryonic days indicated. Bar graphs are percentages of cells originating during embryonic days based on line graphs specified by arrows. There were no significant difTerences between levels in CAl (top set ofgraphs); CAla (data from all levels are combined in solid line,lower bar graph) originates before CAlb,c (data from all levels are combined in dashed line, upper bar graph). CA3a,b (center set of graphs) fonns significantly later at LI (solid line, lower bar graph) than at other levels (data are combined in dashed line, upper bar graph.) CA3c (bottom set ofgraphs) originates first at L2 (solid line, lower bar graph), next at L3 (dashed line, middle bar graph), finally at L4 (dotted line, upper bar graph).

99 HIPPOCAMPAL NEUROGENESIS

AMMON'S HORN: DEEP AND SUPERFICIAL LAYERS

.....

/Y{-~mr:·:·· ..~~~ @ % ill:----------====:=;;-J------l %

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E 15 16 17 18 19 20

AGE AT INITIAL INJECTION DAY OF FORMATION

Fig. 10. Neurogenesis in the stratum oriens (stripes) and strata radiatum, lacunosum-moleculare (dots) in Anunon's horn. Drawings show levels quantified (bar, 1 rrun). Line graphs, with mean and standard deviation, show decline in percentage oflabelled cells after two successive daily 'H-thymidine injections began on embryonic days indicated. Bar graphs are percentages of cells originating during embryonic days based on line graphs specified by arrows. There were no significant differences between levels and data were combined. Stratum oriens (solid line, lower bar graphs) originates simultaneously with strata radiatum and lacunosum-moleculare (dashed line, upper bar graph).

tained within the fields and subdivisions of level three (dashed line); finally, those at level Ammon's horn as shown in Figure 1. There are four (p oS .0367). At all levels, cells of CA3a,b no differences between levels in CAl (Fig. 9, top (nearer the rhinal fissure) originate before (p oS

set of graphs) and data from all levels are com­ .0003) those of CA3c (farther from the rhinal bined. The pyramidal cells in CAla (solid line) fissure). The deep to superficial gradient of are generated significantly before (all levels formation within the CA3 partofthe pyramidal and comparisons, p oS .0001) those of CAlb,c layer is not as sharp as that present in field (dashed line). Field CAl also conforms to the CAL The bar graphs in Figure 9 show that the previously noted pattern that cells nearer the majority of pyramidal cells in Ammon's horn rhinal fissure (CAla) originate before those originate between E 17- E 19. Both subdivisions farther away (CAlb,c). Throughout the CAl of CAl are generated significantly later (p oS

field, there is a very strong deep to superficial .0053) than those of CA3a,b. gradient of origin within the layer (see Fig. 8A,

Cell fonnation superficial and deep to theB). The CA3a,b cells (Fig. 9, middle set of pyramidal cellsgraphs) at level one (solid line) are slightly

smaller and form significantly later (all levels The stratum oriens, stratum radiatum, and and comparisons, (p oS .0148) than the larger stratum lacunosum-moleculare contain a scat­cells in levels two to four (data combined in the tered population of large and medium-sized dashed line). Within zone CA3c (Fig. 9, bottom cells (arrows and asterisk in Fig. 8). Neuro­set of graphs), there is a strong gradient along genesis was examined in these strata, and the ventrodorsal axis. The cells at level two the data are shown in Figure 10. Drawings in­(solid line) begin first (p oS .0053); next, those at dicate the levels quantified, the same as those

100 S.A. BAYER

- '. '."". ".

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Po, -.. Fig.l1. The dentate gyrus inan animal exposed to'H-thymidineon E17 +E18 and killed on P60 (horizontal section, 6 !Lm,

hematoxylin·eosin; bar, 0.25 mm). Hilar cells are included in the dentate gyrus (Cajal, '11; Blackstad, '56) rather than in field CA4 of Ammon's horn (Lorente de No, '34). Arrow indicates the "ectal to endal" gradient ofneurogenesis in the hilus.

for Ammon's horn. No distinction was made between CAl and CA3 or between strata ra­diatum and lacunosum-moleculare; all cells in each section were counted, and the percentage oflabelled cells was determined. Cells are gen­erated simultaneously throughout all layers early in development, with most forming be­tween E15-E17. These cells originate much earlier than the pyramidal cells (p s .00001) and have a similar neurogenetic period as the deep cells of other structures in the hippocam­pal region.

Dentate gyrus Hilus and molecular layer

The dentate hilus is composed of pyramidal, polymorph, and small cells diffusely scattered beneath, or embedded into the base (basket cells), of the granular layer. It can be distin­guished from the stratum radiatum and stratum oriens associated with CA3c of Am­mon's horn by a slight increase in cell density (see Fig. 1). Figure 11 is from an animal ex­posed to 3H-thymidine E17 and E18. Most cells underlying the ectal limb are unlabelled, while some are still labelled beneath the endal limb. This suggests an ectal to endal gradient (arrow)

which is quantified in Figure 12. The levels analyzed (Fig. 12, left) are the same as those used for Ammon's horn. The percentage of la­belled cells was based on total counts from either ectal or endal parts ofthe hilus. For both ectal (solid lines) and endal (dashed lines) parts, level one (bottom set of graphs) origi­nates significantly later (p s .0053) than levels two to four (data are combined in the center set ofgraphs). Up to E18, the cells in the ectal part originate significantly earlier than those in the endal part (p s .0007); fromE190n, the remain­ing cells form simultaneously throughout. Ap­proximately 5% (levels two to four) and 10% (level one) form after E21. Occasionally, a large pyramidal cell is labelled on E21, but the majority are ofthe small, granular type; these cells are frequent at level one and are still la­belled during the early postnatal period.

The medium-sized superficial granule cells in the dentate molecular layer were quantified (the same levels as for Ammon's horn, see draw­ings in Fig. 12). Due to the small number of cells (many times less than 20 per 6,.,. section), no distinction was made between ectal and endal limbs. There were no differences in neurogenesis across levels one to four, and the

101 HWPOCAMPALNEUROGENESffi

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AGE AT INITIAL INJECTION DAY OF FORMATION

Fig. 12. Neurogenesis in dentate molecular layer (stripes), ectal hilus (large dots), and endal hilus (small dots). Drawings of levels indicate regions where cells were counted (bar, 1 mm). Line graphs, with mean and standard deviation, show decline in percentage oflabelled cells after two successive daily jH-thymidine injections began on embryonic days indicated. Bar graphs are percentages of cells originating during embryonic days based on line graphs specified by arrows; lined bars indicate cells labelled on E21last day of ini tiation for embryonic injections. There were no differences between levels in the molecular layer (top set ofgraphs); data are combined in solid line, top bar graph. Levels 2-4 form simultaneously in the hilus (center set of graphs), with the ectal hilus (solid line, lower bar graph) originating before the endal hilus (dashed line, upper bar graph). At level 1, the hilus (bottom set of graphs) forms later than at other levels, but also with the ectal part (solid line, lower bar graph) forming earlier than the endal part (dashed line, upper bar graph).

102 S.A. BAYER

data were combined. The cells arise from E15 , through E19 with a peak on E17; they originate

.­... later than the hilar cells by having more

neurogenesis on E18 and E19. The cells in the hilus and molecular layer of the dentate gyrus arise much earlier than the granule cells (to be described in the next section), following the same pattern as the cells in the stratum oriens

, , and strata radiatum, lacunosum-moleculare of Ammon's horn bear in relation to the pyrami­

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'j­

.. dal cells. The hilus forms mainly on E15-E17, a time when the deep cells of all other hippo­campal region structures are forming. Dentate granular layer

Horizontal sections of the granular layer run nearly parallel to the septo-temporal (dorso­ventral) axis at dorsal levels and do not allow representative sampling ofboth ectal and endal limbs; consequently, the granular layer was analyzed in the sagittal plane which sections the hippocampus perpendicular to the septal (dorsal) the temporal (ventral) extremes. Fig­ure 13 shows the crest area (point of junction between ectal, ec, and endal, en, limbs) at the four levels quantified5 (see drawings in Fig. 14) in an animal exposed to :JH-thymidine on

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tal (Fig. 13A) and temporal (Fig. 13D) extremes than those in both the septal (Fig. 13B) and temporal parts (Fig. 13C). At both septal and

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f>The septal extreme was the most medial section to have a distinct ectal limb to the granular layer; the endallimb extends more medially and is prominent at this leveL The septal level was the first section to show the flDlbria separating from the septum, and lies approximately .8 mm lateral to the septal extreme. The temporal level was the most lateral section (lying approximately 2.6 mm from the septal extreme) to show a separation between the endal limbs of dorsal (the part quan­tified) and ventral cuts of the granular layer; the temporal extreme was

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the most medial section through the ventral granular layer which contained a definite endal limb; the ectal limb extends more medially and is prominent at this level.

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Fig. 13. Sagittal sections (6 fLm, paraffin) of dentate , .' .. granular layer crest, junction between ectal (ec) and endal

(en) limbs, in an animal given four successive daily injections of 3H-thymidine from birth (PO) to P3 and killed on P60 (hematoxylin-eosin; bar, 0.1 mm). A, septal extreme, show­ing thin ectal and thicker endallimbs; B, septal level, 0.8 mm lateral to A, showing thick granular layer with pronounced crest; C, temporal level, 2.6 mm lateral to A, showing thinner

.. granular layer and less pronounced crest; D, temporal ex­treme, showing indistinct crest with short endal limb and longer ectal limb. Unlabelled cells in A-D are most superfi­cial; beneath there are heavily labelled cells; lightly labelled cells are at base of granular layer. A and D have fewer labelled cells than Band C.

103

100

HIPPOCAMPAL NEUROGENESIS

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17 19 21 PO P2 P4 PG P8 PIa Pl2 Pl4 PIG PI8AGE AT INITIAL INJECTION +tt++++++ -+ + tON 1820 22 P1 P3 P5 P7 P9 PI1 P13 P15 P17

DAY OF FORMATION

Fig. 14. Dentate granular layer neurogenesis at four septal to temporal levels in sagittal plane; drawings (bar, 1 mm) indicate regions where cells were counted. Line graphs, with mean and standard deviation, show slow decline in percentage of labelled cells, after two successive daily 'H-thymidine injections began on days indicated; on PO, P4, PB, P12, P16 half the animals received four successive daily injections. Bar graphs are percentages of cells originating on two-day blocks during embryonic and postnatal life; lined bars indicate proportion of cells forming after PiB; arrow indicates time of birth. Ectal limbs (solid lines, solid black) originate significantly before endal limbs (dashed lines, stripes) at septal and temporal extremes; 5CflO of ectal limb cells at both extremes accumulate by birth; 50% of endallimb cells accumulate by Pi (temporal extreme) or P3 (septal extreme). Cytogenesis in ectal limb leads endallimb during embryonic life at both septal and temporal levels. Cells form simultaneously in both limbs from PO on; 5CflO of the cells accumulate at both levels and in both limbs around P5. Temporal level cytogenesis is ahead of the septal level by a maximum of 5%.

104 SA BAYER

temporal extremes, the ectal limb shows fewer labelled cells than does the endal limb. These gradients are analyzed in Figure 14. At each level, all granule cells were counted, and the percentage of labelled cells was separately de­termined for each limb.

Comparisons were made between ectal and endal limb development. Neurogenesis in the ectal limb is significantly earlier (p :s .00001) than that in the endallimb for both septal and temporal extremes. About half of the granule cells in the ectal limbs (solid lines) of the two extremes (see 50% level indicators in Fig. 14) have originated by birth, while granule cell formation in the endal limb lags behind by a few days. In both the septal and temporal levels (which sample granule cells from the bulk of the dentate gyrus) neurogenesis in the ectal limb precedes that in the endallimb only dur­ing the prenatal period (p :s .0001), when ap­proximately 15%-20% of the total population arises (see bar graphs, Fig. 14). During the postnatal period, between 80% and 85% of the granule cell population forms at the septal level. Neurogenesis is simultaneous (p :s .1003) throughout both limbs. At the temporal level, about 80% ofthe cells form postnatally; neuro­genesis in the endallimb is significantly earlier (p:s .0062) than that in the ectal limb. At both levels, during postnatal day 5, the endal limb accumulates 50% of its cells slightly before the ectal limb. The ectal to endal gradient ofneuro­genesis is transient during dentate gyrus de­velopment, being operative essentially during the prenatal period. The earlier forming granule cells (and hilar cells) are influenced by this gradient, but not the bulk of the granule cell population (80%-85%).

Comparisons were also made between levels, and a strong "edge to center" gradient is along the septo-temporal axis. The earliest forming (p :s .00001) regions of the granular layer lie at the two extremes; the granule cells lying be­tween, at the septal and temporal levels, origi­nate later. Cells form simultaneously in the ectal limbs of the two extremes; but temporal extreme endal limb cells are significantly ear­lier (p :s .00001) than those in the septal ex­

treme. Cells at the temporal level originate slightly (5%), but significantly (p :s .002), ear­lier than those in the septal level. The bar graphs in Figure 14 show that the granule cells accumulate very slowly; often less than 10% is formed over a two-day period. Near the end of the first week (postnatal day 6) approximately 70% have originated at the septal extreme, 54% at the septal level, 55% at the temporal level, and 78% at the temporal extreme. Throughout much of the granular layer, 5-10% of the cells form after P18 (lined bars in Fig. 14).

Hippocampal rudiment

A narrow strip of small densely packed cells extends over the corpus callosum as the in­dusium griseum (Fig. 15C, D). It curves around the genu to extend slightly posteriorly along the midline in the anterior septal region as the dorsal tenia tecta, or the septo-hippocampal nucleus (Fig. 15B). At the ventral anterior sep­tal region, a lamina of larger, less densely packed cells begins deep to the smaller cells. These continue forward as the ventral tenia tecta (Fig. 15A) to lie adjacent to the medial portion of the anterior olfactory nucleus. Neurogenesis (Fig. 14) occurs simultaneously within both the tenia tecta and the indusium griseum, respectively. Cells originate signifi­cantly (p :s .01) earlier in the indusium than in the tenia. The bar graphs show that most cells are formed on E 17 throughout the hippocampal rudiment.

DISCUSSION

The progressively delayed comprehensive labelling method employed here allowed the construction of a daily chronology of neuronal origin for each structure in the hippocampal region. This same method has also been used to obtain timetables ofneurogenesis in the septal region (Bayer, '79a), hypothalamus (Altman and Bayer, '78a), and thalamus (Altman and Bayer, '79a, b), each of which is anatomically linked to the hippocampal region. Within these structures, specific neuronal populations are generated during overlapping, but still signifi­cantly different, time periods which fit into a

Fig. 15. Neurogenesis in the hippocampal rudiment. A-D are horizontal sections from an animal exposed to "H·thymidine on E18+E19 and killed on P60; A is anteroventral to D; section orientation is given in D: A, anterior; P, posterior; M, medial; L, lateral; CC, corpus callosum (6 }Lm, paraffin; hematoxylin-eosin, bar, 0.1 rom). Ventral tenia tecta (A) has larger and more sparsely packed cells than the remainder (B-D). More cells are labelled in the tenia tecta (A,B) than in the indusium griseum (C,D). Line graphs, with mean and standard deviation, show decline in percentage oflabelled cells after two successive daily 3H-thymidine injections began on embryonic days indicated. Bar graphs are proportion of cells arising during embryonic life. There is a caudal to rostral gradient of new'ogenesis, with the indusium griseum originating slightly earlier than the tenia tecta.

105

80

HIPPOCAMPAL NEUROGENESIS

HIPPOCAMPAL RUDIMENT A­

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106 SA BAYER

larger pattern of regional gradients. The orien­tation of neurogenetic gradients varies in dif­ferent regions and probably reflects, among other things, the location of the respective neuroepithelia giving rise to components of the septum (Bayer, '7gb), hypothalamus (Altman and Bayer, '78b), thalamus (Altman and Bayer, '80), and hippocampal region (Bayer, '80) in the developing brain. The data of this report will first be discussed to see what common laminar and interstructural gradients charac­terize the hippocampal region.

Gradients within the hippocampal region

The wedge-shaped segments in Figure 16 represent the various components of the hippo­campal region. Each segment is transversely divided into a series of laminae representing each layer where cells were quantified. Shad­ings show the percentage of neurons which have accumulated by the morning of the em­bryonic day indicated. By following the draw­ings from E15 to E21 (two days before birth), the patterns of neurogenesis within and be­tween structures can be compared. There are three gradients ofneurogenesis commonly seen within the hippocampal region: 1) "deep to su­perficial," 2) "sandwich," and 3) "rhinal to den­tate."

Deep to superficial gradients

In all hippocampal structures there is a ten­dency for deep cells (those located nearer the white matter) to begin their formation before superficial cells (those located farther from the white matter). Cells in layers V-VI of the en­torhinal cortex are generated before those of layers II-IV (E16, Fig. 16). The deep cells of the parasubiculum and presubiculum precede those in the superficial lamina (E17-E18, Fig. 16); within the outer layer ofthe presubiculum, deep cells are generated earlier than superfi­cial cells (illustrated in Fig. 4). The subiculum has a strong "deep to superficial" intralaminar gradient in the pyramidal layer; this single layer is divided into a deep and superficial part (containing the letters a, b, c) in Figure 16. The cells in the stratum oriens are generated well before the pyramidal cells in Ammon's horn

(E16-E18, Fig. 16). The pyramidal cells are also generated in a "deep to superficial" intra­laminar pattern (illustrated in Fig. 8A- C). The hilar cells in the dentate gyrus form long before the granule cells (E16-E18, Fig. 16). Every developmental study in rodents has shown the "deep to superficial" or "inside out" gradient in the retrohippocampal cortex, and in Ammon's horn (Angevine, '65; Hine and Das, '74; Vaughn et a1., '77; Schlessinger et a1., '78).

Sandwich gradients

In many structures, later-forming cells are flanked both superficially and deeply by those originating earlier. Throughout the entorhinal cortex, layers II and IV precede the origin of cells in layer III (E16-E18, Fig. 16). The pyra­midal cells in Ammon's horn are flanked by much earlier-forming large and medium-sized cells in the strata oriens, radiatum, and lacunosum-moleculare (E16-E20, Fig. 16). Similarly, the granule cells in the dentate gyrus are "sandwiched" by early-forming large cells in the hilus and medium-sized cells in the molecular layer (E16-E21, Fig. 16). The sand­wich gradient has not been described in the entorhinal cortex (Angevine, '65; Schlessinger et a1., '78), possibly because such subtle differ­ences are difficult to notice with the single­injection technique. Hine and Das ('74) noted the sandwich pattern in Ammon's horn and the dentate gyrus. Such a gradient has also been found in the embryonic cat neocortex (Marin­Padilla, '72). These earlier-forming cells of the hippocampal region are important output elements. Layers II and IV in the entorhinal cortex project to the hippocampal rudiment (Haberly and Price, '78a), and layer II projects massively to CA3 and the dentate gyrus (Stew­ard and Scoville, '76). The large cells in the superficial and deep layers of Ammon's horn project to the septum (Chronister, '78); those in the hilus project to the contralateral dentate gyrus (Hjorth-Simonson and Laurberg, '77; Fricke and Cowan, '78; Swanson et a1., '78). Small cells in these areas form during the sec­ond postnatal week (Bayer and Altman, '74) and are probably interneurons.

Fig. 16. Diagrammatic summary of cytogenetic patterns in the hippocampal region. The wedge-shaped segments are divided into the layers quantified: entorhinal cortex (EC, layers II-VI); parasubiculum (PA), presubiculum (PR), and subiculum (SU}-superficial (s) and deep (d) layers; CA 1 and CA3-stratum oriens (SO), stratum pyramidale (SP), and strata radiatum and lacunosum-moleculare (SR); dentate gyrus (DG)-hilus (H), granular layer (GL), and molecular layer (ML). The shadings in each location indicate the percentage of neurons which have already originated (are not labelled) by the morning of the embryonic day indicated. By following the drawings from E15 to E21 (two days before birth), the pattern of neurogenesis within and between structures can be compared.

107 HIPPOCAMPAL NEUROGENESIS

~ E15

GRADIENTS OF� CYTOGENESIS IN THE� HIPPOCAMPAL REGION�

LEGEND

~~6 SHADINGS

NOT

I EJ 40-59%

...••. W 60-79°1 ':.':...:::' ~

:.< • 80-100% ..:. • STRUCTURE~ E 21 COMPLETE

Figure 16

10

108 S.A. BAYER

Rhinal to dentate gradients

Within each structure of the hippocampal re­gion cells lying nearer to the rhinal fissure begin to arise earlier than those lying nearer to the dentate gyrus. In each layer of the entorhi­nal cortex, the cells located in the lateral part are generated first, followed by those in the intermediate part, and finally by those in the medial part (E16-E17, Fig. 16). Neurogenesis in the superficial lamina ofthe parasubiculum precedes that of the presubiculum (El8-E19, Fig. 16). Zone a of the subiculum begins to form first, followed by zone b, then by the pro­subiculum (E17-E18, Fig. 16). Cytogenesis of the pyramidal cells in CA1a begins before that in CA1b,c (El8-E19, Fig. 16), similarly, CA3a,b originates earlier than CA3c (El8­E20, Fig. 16). Finally, the ectal limb of the dentate hilus forms before the endal limb (E16-E17, Fig. 16).

The "rhinal to dentate" gradient is also prom­inent interstructurally in the hippocampal re­gion. The entorhinal cortex is the first struc­tural complex to be completed by E18. By E19, the subiculum finishes before the CA3a,b pyramidal cells (p :5 .0003). Finally, the den­tate granular layer finishes postnatally. There are two notable exceptions in the "rhinal to dentate" gradient between structures. First, the superficial laminae of the parasubiculum and presubiculum are significantly later form­ing than the subiculum (p :5 .00001). Second, the pyramidal cells of CAl are significantly later forming than those in CA3a,b. The "rhi­nal to dentate" gradient between structures and the late formation of the parasubiculum, presubiculum, and CAl pyramidal cells have been observed by Angevine ('65) in the mouse, and by Schlessinger et al. ('78) in the rat.

Septo-temporal axis gradients

The septo-temporal or dorsoventral axis is the only dimension showing inconsistent gra­dients (not illustrated in Fig. 16) in the hip­pocampal region. The entorhinal cortex, presubiculum, subiculum, CAl pyramidal cells, stratum oriens, strata radiatum and lacunosum-moleculare, and the dentate mo­lecular layer have no cytogenetic differences along their lengths, in agreement with a previ­ous study (Schlessinger et al., '78). The parasubiculum and dentate granular layer (to be discussed below) have an "edge to center" gradient where cytogenesis in dorsal (septal) and ventral (temporal) levels precedes that at middle levels.

In the dentate hilus and in the CA3 pyrami­dal cells cytogenetic differences correlate with the cytoarchitectonic differences observed by Gaarskjaer ('78a) along the septo-temporal axis in the rat. Ventral levels ofthe hilus have many small, late-forming granule cells scattered both ectally and endally. At its most ventral level, CA3a,b has smaller, more densely packed and later-forming cells than throughout the rest of its extent in the hippocampus. The strong tem­poral to septal gradient in field CA3c can be related to stratification differences along the axis. Near their ventral border (level 2), CA3c cells are distinctly stratified (Fig. 8D) and can be easily delineated from the surrounding hilus; here, they lag behind the formation ofthe CA3a,b field by approximately 10%. At level three, the CA3c field is well-stratified near CA3a,b, but becomes progressively more scat­tered farther into the hilus; later-forming cells are always located in this area of spreading. At level four, CA3c is poorly laminated through­out much ofits extent (Fig. 8E), and cytogenesis here trails the CA3a,b field by approximately 40%. The poorly laminated part of field CA3c contributes commissural fibers to Ammon's ~orn, perhaps to the dentate molecular layer; mterestingly, commissural input to these structures also shows a temporal to septal den­sity gradient (Gottlieb and Cowan, '73; Hjorth-Simonsen and Laurberg, '77; Fricke and Cowan, '78; Swanson et al., '78). Thus, gradi­ents in neurogenesis, cytoarchetectonics, and anatomical terminations may be related.

Gradients of the dentate granule cells

The granule cells have a very long genera­tion time (compare the bar graphs of Fig. 14 with Fig. 3,5,7,9,10,12). This is typical ofpopu­lations formed by secondary germinal matri­ces: cells of the nucleus accumbens (Das and Altman, '70; Bayer, '79a, b), olfactory granule cells (Altman, '69b), and cerebellar granule cells (Altman, '69a). Early in hippocampal de­velopment, cells migrate from the neuroepithe­lium near the region offimbrial outgrowth into the dentate primordium and establish a new germinal zone (Bayer and Altman, '74; Schles­singer et al., '75; Bayer, '80), which remains active beyond postnatal day 18 (this study) and into the adult period (Altman and Das, '65; Bayer and Altman, '75; Kaplan and Hinds, '77). Immature granule cells migrate radially from the hilus and accumulate in the granular layer, stacked in order from oldest to youngest (Fig.

109 HIPPOCAMPAL NEUROGENESIS

13), confirming several previous observations (Angevine, '65; Altman and Das, '65, '66; Altman, '66; Bayer and Altman, '74). This "superficial to deep" gradient is found through­out the entire length of the granular layer dur­ing all developmental stages.

The granule cells also have a very strong "edge to center" gradient; neurogenesis in the extremes leads middle-level neurogenesis by between 20- 30%. Within the major part of the granular layer, the maximum temporal to sep­tal gradient does not exceed 5% (see Fig. 14). Schlessinger et aI. ('75) reported a strong tem­poral to septal gradient of granule cell devel­opment, but their conclusion was based on hori­zontal sections which did not include the septal extreme. Morphogenesis of the granular layer follows the pattern of neurogenesis. The layer first appears on E20 at the two extremes of the dentate gyrus; by E22, it is present throughout (Bayer, '80). Prenatally, the ectal limb is prominent, in accordance with the ectal to endal gradient operant during this time. The endallimb emerges perinatally; but by the end ofthe first postnatal week, it is as mature as the ectal limb (Bayer and Altman, '74; Bayer, '80), conforming to the simultaneous neurogenesis observed throughout the postnatal develop­ment of the granular layer.

The hippocampal rudiment

Cytogenesis in both the tenia tecta and in­dusium griseum occurs mainly on E17 (in agreement with Hine and Das, '74) in a caudal to rostral gradient. Both the tenia and the in­dusium get input from the entorhinal cortex (Haberly and Price, '78a). The ventral tenia tecta also gets prominent olfactory input (Price, '73; De Olmos et aI., '78; Haberly and Price, '78b) and may be a transition area between the olfactory system and the hippocampus.

Patterns of neurogenesis related to patterns of fiber termination

in the hippocampal region

Entorhinal afferents The entorhinal cortex sends a large ipsilat­

eral projection into the hippocampus which perforates the subiculum and runs in the stratum lacunosum-moleculare of Ammon's horn and in the superficial dentate molecular layer (Cajal, '11; Blackstad, '58). Entorhinal fibers terminate in CA3 and the dentate molecular layer (Nafstad, '67) in a strict super­ficial to deep pattern (Hjorth-Simonsen, '72;

Hjorth-Simonsen and Jeune, '72; Steward, '76). The lateral entorhinaI cortical fibers end in a superficial (distal) band; below them is a band of intermediate fibers; below them, a band of medial fibers (Fig. 17). The fibers originate in layers II (the majority) and III (Steward and Scoville, '76). The order of neurogenesis in the entorhinal cortex proceeds from lateral to me­dial (Fig. 16) and strictly correlates with the order of its termination on the distal dendrites of CA3 and the dentate granule cells: afferents with older cells of origin terminate distal to af­ferents with younger cells of origin.

There is a sparse topographic bilateral pro­jection from entorhinal layers II and III to the stratum lacunosum-moleculare of field CAl (Steward, '76; Steward and Scoville, '76). Inter­estingly, there is also a correlation between the pattern ofneurogenesis and the pattern offiber termination. Afferent fibers from older cells of origin (lateral entorhinal cortex) project to older CAl pyramidal cells (CAla); afferent fi­bers from younger cells of origin (medial en­torhinal cortex) project to younger CAl pyra­midal cells (CAlb,c).

Commissural and associational afferents

Lesions of the contralateral hippocampus or cutting the ventral hippocampal commissure gives pronounced degeneration in the stratum oriens and stratum radiatum of Ammon's horn and in the deep third of the dentate molecular layer (Blackstad, '56; Raisman et aI., '65; Laatsch and Cowan, '67). Entire field CA3 pro­jects to the commissural zones on both CAl and CA3 dendrites (Gottlieb and Cowan, '73, Swan­son et aI., '78); field CA3c and the large hilar cells project to the commissural zone in the dentate molecular layer (Gottlieb and Cowan, '73; Hjorth-Simonsen and Laurberg, '77; Fricke and Cowan, '78; Swanson et aI., '78). These same regions also project ipsilaterally, CA3a,b to CAl and CA3 via the Schaffer collateral sys­tem (Hjorth-Simonsen, '73; Swanson et aI., '78), CA3c and the large hilar cells to the dentate molecular layer (Zimmer, '71; Swanson et aI., '78). Thus, commissural and associational pathways have similar cells of origin and over­lapping zones oftermination on the dendrites of both the pyramidal and granule cells. The zone of entorhinal termination in CA3 and the den­tate gyrus, and the zone of nucleus reuniens termination in CAl are strictly isolated from the commissural-associational zones on the remainder of the dendrites (Fig. 17).

8.A. BAYER110

ORDER OF NEUROGENESIS OLD~~J _

1.� MEDIAL SEPTAL NUCLEUS

DIAGONAL BAND NUCLEUS E14- E16

ENTORHINAL CORTEX

LAYERS II ,m:

2� LATERAL E15 -E16

3. INTERMEDIATE E15- E17

*4 MEDIAL E16-E17

*5 THALAMUS

NUCLEUS REUNIENS E16-E17

6 CA3 (COMMISSURAL,

ASSOCIATIONAL) E17-E19

7 DENTATE

CELLS

GRANULE

PO-P14

CA1 DGYOUNGEST

Fig. 17. Order of neurogenesis in cells of origin of afferent fibers related to order of termination on dendrites in CAl, CA3, and the dentate gyrus. Cells supplying septal afferents (#1) begin to originate first and terminate diffusely in all dendritic areas; all other afferents represented terminate in specific laminar zones. Earlier originating cells from lateral entorhinal cortex (#2) project most distally to the cell body, later-{)riginating cells from intermediate (#3) and medial (#4) entorhinal cortex project progressively more proximally to the cell body in CA3 stratum lacunosum-moleculare layer (8LM) and superficial part ofdentate molecular layer (ML). Cells projecting from the nucleus reuniens (#5) arise simultaneously (*) with medial entorhinal cortex and terminate throughout stratum lacunosum-moleculare of CAL CA3 contains later-originating cells (#6) supplying the majority of commissural and associational afferents terminating in stratum radiatum (8R) and stratum oriens (80) of CA2, CA3, and the deep part of dentate molecular layer. Finally, the dentate granular layer has the latest-originating cells (#7) supplying associational afferents (mossy fibers) to the CA3 dendrite. Note that the apical dendrites of CAl and CA3 pyramidal cells and the dentate granule cell dendrite have laminar afferents stacked according to age of the cells of origin (earliest-forming cells -. most distal terminal zone; latest-forming cells -. most proximal terminal zone).

E17 is the last day of neurogenesis for cells sponds to the sequence of fiber growth into the projecting to restricted afferent zones in CAl dentate molecular layer; entorhinal fibers ar­from the nucleus reuniens and in CA3 and the rive before commissural-associational fibers dentate gyrus from the entorhinal cortex, while (Loy et al., '77; Fricke and Cowan, '77). E17 is the first day of neurogenesis for CA3 Finally, there is the prominent mossy fiber pyramidal cells (Fig. 17), the major source of associational pathway running from the den­the commissural-associational projection. tate granule cells to the CA3 pyramidal cells Hilar cells also project to the dentate molecular (Blackstad et al., '70; Gaarskjaer, '78b; Swan­layer; many form before E 17, but some of these son et al., '78). The granule cells are latest to large cells are also labelled up to E21 (Fig. 12), form in the hippocampal region and their fibers so a late neurogenesis cannot be ruled out. It is occupy a strictly isolated tenninal zone in CA3 interesting to note that the commissural-asso­ most proximal to the cell body (Fig. 17). ciational fibers tend to have later-forming cells

Septal afferentsoforigin and terminate more proximally to the cell body, continuing the sequence begun in the The medial septal and diagonal band nuclei entorhinal projection. There is some evidence project to all parts of the hippocampal region that the sequence of neurogenesis also corre- (Powell, '63; Raisman et al., '65; Raisman, '66;

111 HIPPOCAMPAL NEUROGENESIS

Lewis and Shute, '67; Mellgren and Srebro, '73; Segal and Landis, '74; Swanson and Cowan, '76; Meibach and Siegel, '77a), share the same dis­persed terminal field throughout the region (Swanson and Cowan, '76; Meibach and Siegel, '77a), and diffusely innervate (Mellgren and Srebro, '73) the dendrites of Ammon's horn pyramidal cells and dentate granule cells (Fig. 17). This pattern ofinnervation is similar to the scattered input from brain stem raphe and locus coeruleus nuclei (Fuxe, '65; B1ackstad et al., '67; Storm-Mathisen and Guldberg, '74; Conrad et al., '74; Moore and Halaris, '75). There is no anatomical information about gra­dients (such as density differences) between the projections of the medial septal and diagonal band nuclei. In contrast, neurogenesis proceeds in a sharp caudal to rostral gradient between the two nuclei. The posterior and intermediate parts of the medial septal nucleus form mainly on E14 and E15; the nucleus of the diagonal band forms mainly on E15-E16, with the en­tire complex finished by the morning of E18 (Bayer, '79a). Based on the present anatomical information, these defini te neurogenetic gradi­ents do not correlate with the diffuse nature of the septal projection to the hippocampal region. The midline septum is also an important output center to the basal telencephalon and to hypothalamic and thalamic nuclei (Swanson and Cowan, '76; Meibach and Siegel, '77a; and others) and the pattern of neurogenesis may correlate more positively with some of these connections.

Thalamic afferents

Thalamic afferents run in the cingulum bun­dle to the hippocampal region (White, '59). The anteromedial, anterodorsal, and anteroven­tral nuclei project sparsely to the entorhinal cortex and heavily to the superficial laminae of the parasubiculum and presubiculum (Domesick, '69, '72, '73). The nucleus reuniens projects lightly to the entorhinal cortex and massively to the stratum-Iacunosum-molecu­lare of CAl (Herkenham, '78; Fig. 17). It is interesting that all targets of heavy thalamic input form late (not finished until the morning of E20), and these structures (parasubiculum, presubiculum, and CAl) are the only excep­tions to the "rhinal to dentate" interstructural gradient. However, neurogenesis in t~e an­terior nuclear complex and nucleus reumens IS

finished earlier, about the same time as the medial entorhinal cortex (Altman and Bayer, '79a, b). The thalamic nuclei are farther from

the hippocampus than the entorhinal cortex. It follows that if thalamic and entorhinal fibers started to grow at about the same time, then the entorhinal fibers would arrive at their targets first. It is suggested that neurogenesis in thalamic targets is delayed to coincide with a possible later arrival of thalamic fibers. Exper­iments tracing developing fiber tracts are needed to test this hypothesis.

Patterns of fiber termination� related to patterns of�

neurogenesis in� target areas�

The lateral septal nucleus gets prominent input from the hippocampus via the precom­missural fornix (Nauta, '56; Raisman et al., '66; Raisman, '66); the pyramidal cells of Ammon's horn and the subiculum project topographically (Fig. 18) so that dorsal levels terminate pre­dominately in medial zones, intermediate levels terminate in dorsolateral zones, ventral levels terminate in ventrolateral zones (Siegel et al., '74; Swanson and Cowan, '77; Meibach and Siegel, '77b). The mammillary body also gets prominent hippocampal input via the post­commissural fornix (Nauta, '56; Guillery, '56; Raisman et al., '66); the subiculum (Swanson and Cowan, '75, '77) projects topographically so that its dorsal part terminates in a dorsal transverse zone in the pars posterior of the me­dial mammillary nucleus; intermediate parts terminate in a central transverse zone; ventral parts terminate in a ventral transverse zone (Meibach and Siegel, '75, '77b). These projec­tions are diagrammed in Figure 18.

Two important relationships are maintained in both target structures. First, the order of neurogenesis correlates with the order of termi­nation. The medial part of the lateral septal nucleus forms earlier than its lateral part (Bayer, '79a); the dorsolateral part has a ten­dency (significant in two-thirds of the cases)6 to originate slightly before the ventrolateral part. The dorsal part of the medial mammillary nu­cleus pars posterior forms before its ventral part (Altman and Bayer, '78a). Second, the dor­sal hippocampus (all fields in the lateral septal projection, the subiculum in the mammillary projection) always projects to zones occupied by earlier-forming cells, while progressively more ventral parts project to zones occupied by later­forming cells (Fig. 18). These relationships may be coincidental, but a hypothesis is offered.

'This slight gradient was not reported by Bayer ("79a) but it has a bearing on the hypothesis that is offered.

112 SA BAYER

NEUROGENESIS:

CLOSER

LATERAL SEPTAL NUCLEUS

NEUROGENESIS:

! Fig. 18. Gradients of neurogenesis in target areas correlated with topographical hippocampal projections along the

dorsoventral axis. The "dorsal" and "ventral" parts of the hippocampus are diagrammatic and may not exactly correspond to the anatomical boundaries of the topographic projections [see Swanson and Cowan ('77) and Meibach and Siegel ('77b) for further details]. In the lateral septal nucleus, the dorsal hippocampus (CAl, CA3, and subiculum) projects to a zone occupied by earlier forming medial cells; the ventral part projects to zones occupied by later-forming laterally placed cells (further dorsoventral organization within this projection is not shown). In the mammillary body, pars posterior, the dorsal subiculum projects to dorsal transverse zones occupied by earlier-forming cells; ventral subiculum, to ventral transverse zones occupied by later-forming cells. Note that the dorsal projection is always to locations occupied by older cells, ventral projection to locations occupied by younger cells. This may be related to differential fiber length; see text for discussion.

Since neurogenesis occurs simultaneously ACKNOWLEDGMENTS -­along the dorsoventral axis of both the hippo­ The author would like to thank Joseph campus and subiculum, their axons may start Altman for advice and encouragement, Sharon to grow simultaneously from both dorsal and Evander for the histology, Kathy Shuster forventral levels toward the targets. The dorsal preparation of the figures, and Mary Ward forhippocampus and subiculum are closer to the typing the manuscript. This research was sup­targets, and their fibers would arrive earlier ported by the National Science Foundation,and settle in zones where earlier-forming cells Grant BNS77-12622. are located; the same pattern would follow for axons from more ventral sources. Fiber tracing LITERATURE CITED methods have to be used in developing brains to

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113 HIPPOCAMPAL NEUROGENESIS

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