H.E. Scharfman (Ed.)

Progress in Brain Research, Vol. 163

ISSN 0079-6123

Copyright r 2007 Elsevier B.V. All rights reserved

CHAPTER 10

Morphological development and maturation ofgranule neuron dendrites in the rat dentate gyrus

Omid Rahimi1 and Brenda J. Claiborne2,�

1Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio,TX 78229, USA

2Department of Biology, University of Texas at San Antonio, One UTSA Circle, San Antonio, TX 78249, USA

Abstract: The first granule neurons in the dentate gyrus are born during late embryogenesis in the rodent,and the primary period of granule cell neurogenesis continues into the second postnatal week. On the day ofbirth in the rat, the oldest granule neurons are visible in the suprapyramidal blade and exhibit rudimentarydendrites extending into the molecular layer. Here we describe the morphological development of thedendritic trees between birth and day 14, and we then review the process of dendritic remodeling that occursafter the end of the second week. Data indicate that the first adult-like granule neurons are present on day7, and, furthermore, physiological recordings demonstrate that some granule neurons are functional at thistime. Taken together, these results suggest that the dentate gyrus may be incorporated into the hippo-campal circuit as early as the end of the first week. The dendritic trees of the granule neurons, however,continue to increase in size until day 14. After that time, the dendritic trees of the oldest granule neurons aresculpted and refined. Some dendrites elongate while others are lost, resulting in a conservation of totaldendritic length. We end this chapter with a review of the quantitative aspects of granule cell dendrites inthe adult rat and a discussion of the relationship between the morphology of a granule neuron and thelocation of its cell body within stratum granulosum and along the transverse axis of the dentate gyrus.

Keywords: dendritic trees; spines; filopodia; neonates; hippocampus

Introduction

Granule neurons are the principal cell type in thedentate gyrus, and their cell bodies are located instratum granulosum of the suprapyramidal and in-frapyramidal blades. Dendrites extend from theapical pole of the granule cell body into the over-lying molecular layer, and the axon, or mossy fiber,exits from the basal pole. The axon gives rise tocollateral branches in the hilar region and then

�Corresponding author. Tel.: +1 210 458 5487;

Fax: 1 210 458 5669; E-mail: brenda.claiborne@utsa.edu

DOI: 10.1016/S0079-6123(07)63010-6 167

forms synapses on pyramidal neurons in the CA3region of the hippocampus proper. The apicaldendrites of the granule neurons bifurcate as theytraverse the molecular layer, and the vast majorityof terminal branches reach the top of the layer in theadult. The dendritic trees of most granule neuronsare elliptical, and all dendrites of granule neurons inthe adult dentate gyrus are covered with spines.

The primary period of granule cell neurogenesisoccurs over a two- to three-week period in the ro-dent, beginning in late embryogenesis and contin-uing through the second postnatal week. In the rat,although a few granule neurons are born as early as

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embryonic day 14, over 80% are born after thebirth of the animal (which occurs at about embry-onic day 21) and neurogenesis peaks near the end ofthe first week of life (Bayer and Altman, 1974;Schlessinger et al., 1975). It is worth noting that thegranule neurons are the last cells to be generated inthe hippocampal formation, and it is well knownthat granule cell neurogenesis continues into adult-hood (Altman and Das, 1965; Kaplan and Hinds,1977). Here we focus on granule neurons that aregenerated in the neonatal rat. From recent evidencein the mouse, it appears that adult-generated gran-ule neurons progress through a similar set of stagesas they develop and mature (Zhao et al., 2006).

Any description of the development and matu-ration of the granule neurons must take into ac-count the temporal and spatial gradients ofgranule cell neurogenesis (Schlessinger et al.,1975; Cowan et al., 1980, 1981). The earliest borngranule neurons form stratum granulosum in theseptal portion of the dentate gyrus, and neuronsthat are generated later form the more temporalportions of the dentate gyrus. This gradient is re-ferred to as the septotemporal gradient. A secondgradient exists along the transverse axis of thedentate gyrus and is of considerable importancefor developmental and morphological studies. Asthe first granule neurons are born, they form thecell-body layer at the tip of the suprapyramidalblade. As additional neurons are generated, theyform the cell-body layer in the middle of the sup-rapyramidal blade and then the portions of stra-tum granulosum closest to the crest region. Thisgradient continues as more neurons are generatedsuch that the youngest neurons make up stratumgranulosum in the infrapyramidal blade. A thirdgradient exists within stratum granulosum. Theneurons that are born first move into their finalposition at the top of the cell-body layer near themolecular layer, and the younger neurons moveinto position beneath them such that they are lo-cated in the bottom portion of stratum granulo-sum near the hilar border. This developmentalpattern is in contrast to the ‘‘inside-out’’ patternfound in other areas of the mammalian cerebralcortex in which the later-generated neurons movethrough the earlier-generated cells to occupy po-sitions at the top of the cell-body layer.

Thus the oldest granule neurons are most likelyto be found at the top of stratum granulosum nearthe distal tip of the suprapyramidal blade at theseptal pole, whereas the youngest neurons are lo-cated predominantly in the infrapyramidal bladenear the temporal pole and in the deeper portionsof stratum granulosum along the entire extent ofthe transverse axis of the dentate gyrus. In the rat,the suprapyramidal blade begins to form in lateembryogenesis as the first granule neurons areborn — it is visible as a separate structure on theday of birth and consists of a cell-body layer and arelatively thin molecular layer (Cowan et al.,1980). The molecular layer increases greatly inwidth over the first several weeks. It is less than100 mm at day 4 and increases to just over 200 mmat day 14; it averages approximately 300 mm inwidth in young adult rats (Loy et al., 1977; Clai-borne et al., 1990; Rihn and Claiborne, 1990). Theinfrapyramidal blade is barely visible on the day ofbirth and grows more slowly than the suprapy-ramidal blade during the first week, increasingfrom approximately 45 mm in width on day 4 toapproximately 110 mm on day 10; it measures be-tween 205 and 240 mm in young adult rats (Loy etal., 1977; Claiborne et al., 1990).

Here we describe the development and matura-tion of the dendritic trees of the granule neurons inthe rat, and we review the quantitative data ondendritic morphology in the adult. We considerthe developmental period to encompass the timefrom the birth of the animal through day 14. Byday 14, the oldest granule neurons have assumedtheir adult form and size. From day 14 to 60,however, the neurons go through a period of mat-uration during which the dendritic tree is sculptedand refined and the density of spines continues toincrease. The dendritic trees of granule neuronsappear to be mature by day 60: unpublished datafrom our lab indicate that they do not undergo anyquantitative changes between 60 and 180 days.

Development of granule neuron dendrites

Because of the prolonged time-course of granulecell neurogenesis in neonatal rats, a wide range ofdendritic morphologies are observed on any one

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day during the first and second postnatal weeks(Fricke, 1975; Seress and Pokorny, 1981; Wenzelet al., 1981; Lubbers and Frotscher, 1988; Liuet al., 1996, 2000; Ye et al., 2000; Jones et al.,2003). It is possible, however, to identify distinctstages in the development of granule cell dendritictrees, either by comparing the various neuronalstructures present on a single day or by examiningthe progression of dendritic morphologies over thecourse of the neonatal period. As an example ofthe first approach, Lubbers and Frotscher (1988)identified several stages of developing granule neu-rons in a 5-day-old rat. Neurons in the earlieststages of development had only rudimentary den-dritic trees, each consisting of two or more pri-mary apical dendrites exiting from the cell body,along with one or more basal branches. The pri-mary apical dendrites gave rise to higher orderbranches that were relatively short and thin andthat exhibited varicosities and the occasionalgrowth cone. Spines were not present on theseimmature cells. In contrast, the most mature gran-ule neurons on day 5 had a more elaborate apicaltree with longer branches that exhibited numerousvaricosities as well as occasional growth cones andfilopodia. A few spines were present on the apicaldendrites.

Based on data from Lubbers and Frotscher(1988) and a number of other investigators, herewe describe a sequence of stages that characterizethe morphological development of granule neurondendrites in the young rat. A variety of techniqueshave been used to examine developing granule neu-rons during the first few weeks of life, includingGolgi impregnation (Fricke, 1975; Duffy andTeyler, 1978a, b; Wenzel et al., 1981; Lubbers andFrotscher, 1988; Zafirov et al., 1994), intracellularinjection (Liu et al., 1996, 2000; Ye et al., 2000), andretrograde labeling (Jones et al., 2003). Fricke(1975) characterized the dendritic structures ofGolgi-stained granule neurons in tissue from ratsat postnatal days 1, 2, 4, 8, 12, and 20, as well as intissue from adult rats over the age of 60 days. Otherlabs using Golgi-impregnated material have exam-ined a similar range of ages. Duffy and Teyler(1978a, b) analyzed granule neurons in sectionsfrom rats at 7, 14, 30, 60, and 210 days of age,Wenzel et al. (1981) determined morphologies at a

variety of ages between days 0 and 180, and Zafirovet al. (1994) described granule neurons at days 5,10, 15, and 20. Trommer and colleagues (Liu et al.,1996, 2000; Ye et al., 2000) injected granule neuronsin slices from rats between the ages of 5 and 32 dayswith biocytin, whereas Jones et al. (2003) used ret-rograde labeling with DiI to analyze the dendriticstructures of granule neurons in rats between theages of 2 and 9 days. The latter group focused onthe oldest granule neurons — those that were lo-cated near the tip of the suprapyramidal blade andin the top portion of stratum granulosum.

On the day of birth and on postnatal day 1 in therat, granule neurons are visible in the suprapyram-idal blade of Golgi-stained tissue and exhibit rudi-mentary trees. Some have only a few short, stubbybranches, whereas others have longer and morenumerous dendrites (Fricke, 1975; Wenzel et al.,1981). The dendrites are smooth and growth conesare not typically observed. Wenzel et al. (1981)considered granule neurons on the day of birth tobe in the first stage of development and labeledthem as primitive or early neuroblasts. As noted byFricke (1975), it is a bit surprising that all of thestained granule neurons at this age have such sparsedendritic trees — although most granule neuronsare likely to be only a few days old at this time, afew neurons are born as early as embryonic day 14and are already 7 days old on the day of birth.

On postnatal days 2 and 3, the oldest granuleneurons, in the suprapyramidal blade, have one ormore primary apical dendrites and varying num-bers of shorter, higher-order branches (Fig. 1Aand B; Jones et al., 2003). Granule cells located atthe very top of stratum granulosum have severalprimary dendrites whereas those located a bitdeeper in the layer are more likely to have only onethick primary dendrite emerging from the cellbody, as described previously for granule neuronsin adult rodents (Fricke, 1975; Desmond andLevy, 1982; Green and Juraska, 1985; Claiborne etal., 1990). In the developing rat, the primary dend-rites of both the superficial and the deep granulecells tend to branch at or near the top of stratumgranulosum, and the diameters of the dendriteschange abruptly at branch points. The majority ofbranches are still relatively smooth at this age,with only occasional varicosities or growth cones.

Fig. 1. Photomontage (A) and drawings (B and C) made from

serial micrographs of DiI-labeled granule neurons from a 3-

day-old (A, B) and a 6-day-old rat (C). Note the presence of

immature features on dendrites at this age. Solid-curved arrow,

growth cone; solid-straight arrow, varicosity; open-straight ar-

row, filopodia; long-filled arrow, abrupt diameter change; open

arrowheads, continuation of axon; a, axon; c, axon collateral.

The montage and the drawings are shown at the same magni-

fication. Scale bar ¼ 50mm. (Adapted with permission from

Jones et al., 2003.)

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A few dendrites, however, exhibit numerousgrowth cones, varicosities, and filopodia. Most in-vestigators report the absence of dendritic spinesat these early ages, although Wenzel et al. (1981)noted ‘‘spine-resembling’’ structures at day 0 and afew spines at day 2. Basal dendrites are commonon neurons at days 2 and 3. They tend to be

thinner than apical dendrites but exhibit the sameimmature features, including growth cones, vari-cosities, and filopodia. Basal dendrites are con-sidered to be an immature feature of granuleneurons in the rodent — they are not commonlyfound on adult granule neurons (Seress and Po-korny, 1981; Lubbers and Frotscher, 1988).

As development proceeds, the most maturegranule neurons exhibit more extensive dendritictrees with longer branches and a full complementof immature features (Fricke, 1975; Duffy andTeyler, 1978a, b; Wenzel et al., 1981; Lubbers andFrotscher, 1988; Jones et al., 2003). On day 4,dendrites have larger varicosities and an increasednumber of filopodia as compared to neurons inyounger animals (Jones et al., 2003). Diameterchanges are abrupt at branch points, and mostdendrites terminate before reaching the top of themolecular layer. Basal dendrites are present on themajority of cells. Although Jones et al. (2003) didnot report any spines on granule neuron dendritesat day 4, Fricke (1975) noted the occasional spineon Golgi-stained dendrites at this age.

On day 5, the oldest granule neurons are char-acterized by numerous apical branches with manyfilopodia and varicosities and several basalbranches (Fig. 2A; Wenzel et al., 1981; Lubbersand Frotscher, 1988; Zafirov et al., 1994; Jones etal., 2003). Abrupt diameter changes and growthcones are present but are less numerous than onday 4. Spines are scattered throughout the dendri-tic trees of most of the older neurons on day 5,although they are found in clusters on a few dend-rites on a small number of cells (Seress and Po-korny, 1981; Wenzel et al., 1981; Lubbers andFrotscher, 1988; Zafirov et al., 1994; Jones et al.,2003). Wenzel et al. (1981) characterized granuleneurons between 5 and 8 days of age as interme-diate neuroblasts and considered this period to bethe second stage of granule neuron development.

The most mature granule neurons observed onday 6 differ somewhat from those in the youngeranimals (Fig. 1C; Jones et al., 2003). Growth conesand varicosities are smaller and less numerous,whereas the number of filopodia is greater (Fig.2B). Abrupt diameter changes and basal dendritesare less frequent. Most dendrites on these neuronsreach the hippocampal fissure at the top of the

Fig. 2. Stacked series of individual images taken with a con-

focal laser-scanning microscope. Dendrites are from a 5-day-

old rat (A) and a 6-day-old rat (B). The dendrite shown in B is

from one of the most mature neurons seen on day 6. Although

many spines are present, immature features are still observed on

day 6. Open arrow, filopodia; filled arrow, spines. Scale

bar ¼ 10mm. (Adapted with permission from Jones et al.,

2003.)

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molecular layer, and spines are present in varyingdensities and on varying numbers of dendriticbranches.

Jones et al. (2003) observed the first adult-likegranule neurons on day 7 (Fig. 3). These neuronswere considered to be adult-like because they ex-hibited the three primary characteristics of adultgranule neurons: they were devoid of immaturefeatures except for occasional varicosities or filopo-dia, the vast majority of the dendrites reached thetop of the molecular layer, and all dendrites werecovered with spines. The cell bodies of the adult-likegranule neurons were located towards the top of thegranule cell layer in the suprapyramidal blade andwere most likely between 7 and 10 days old at thistime. Other investigators had suggested that adult-like granule neurons were present at about this time.

Fricke (1975) illustrated a granule neuron with nu-merous spines from an 8-day-old rat. Wenzel et al.(1981) noted that granule neurons in 10-day-old ratsresembled adult neurons, although they reportedthe presence of growth cones and varicosities andsuggested that such features were indicative of con-tinued dendritic growth. They considered granulecells at this age to be in the third stage of devel-opment and labeled them maturing or young neu-rons. Zafirov et al. (1994) reported that somegranule neurons display extensive dendritic treeswith spines and without immature features on day10. Similarly, Liu et al. (2000) described granuleneurons with at least some adult-like features onday 7 and illustrated an adult-like granule cell froma 12-day-old rat.

Thus it is now clear that a small proportion ofthe oldest granule neurons exhibit adult-like mor-phological features by the end of the first postnatalweek in the rat. These data, in combination withresults from other studies, suggest that the dentategyrus may be functional at this early age. For ex-ample, our in vivo studies demonstrated that long-term potentiation (LTP) and long-term depression(LTD) can be elicited at medial perforant pathsynapses onto the granule neurons at day 7(O’Boyle et al., 2004), confirming earlier in vitrostudies (Duffy and Teyler, 1978b; Trommer et al.,1995). It is also worth noting that the granule cellafferents are in their approximate adult locationsat this time, and that the dendritic trees of hilarinterneurons are well developed (Cowan et al.,1980; Seay-Lowe and Claiborne, 1992). Further-more, the axons (or mossy fibers) of the oldestgranule neurons reach region CA3 of the hippo-campus proper on the day of birth or soon there-after (Minkwitz, 1976; Stirling and Bliss, 1978;Jones et al., 2003), and granule cell stimulation caninduce LTD in CA3 pyramidal neurons by day 7(Battistin and Cherubini, 1994). Taken together,these data suggest that the traditional view of thedentate gyrus as a ‘‘late developing’’ structure maybe incorrect; at least some of the granule neuronsare capable of functioning within the hippocampalcircuit by the end of the first postnatal week, atabout the same time that adult-like properties arefirst observed in the pyramidal neurons of the hip-pocampus proper.

Fig. 3. Drawing made from serial micrographs of a DiI-labeled granule neuron from a 7-day-old rat. Note the absence of immature

features and the prevalence of spines. A varicosity is visible on the axon (short, filled arrow); open arrowhead, continuation of axon.

Scale bar ¼ 50mm. (Adapted with permission from Jones et al., 2003.)

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Although granule neurons with adult-like fea-tures are present by the end of the first week, it isimportant to note that granule neuron dendritescontinue to elongate during the second postnatalweek (Fricke, 1975; Duffy and Teyler, 1978a, b;Seress and Pokorny, 1981; Wenzel et al., 1981;Zafirov et al., 1994). Using one of the first com-puter-microscope systems designed for quantita-tive, three-dimensional morphological studies ofsingle neurons, Fricke (1975) analyzed the dendri-tic trees of developing Golgi-impregnated granuleneurons over the first postnatal month in the rat.He found that the total dendritic length (defined asthe sum of the lengths of all individual dendriticsegments) of a granule neuron increased betweenday 8 and day 12, from an average of approxi-mately 500 mm on day 8 to an average of approx-imately 1000 mm on day 12, with a wide variabilityat both ages. Duffy and Teyler (1978a, b) also re-ported that granule neurons increased dramati-cally in size between day 7 and day 14, and Zafirovet al. (1994) showed that total dendritic lengthsincreased from approximately 300 mm at day 5 to

approximately 465 mm on day 10, with no signifi-cant increase between days 10 and 15.

As granule neuron dendrites continue to elon-gate after day 7, spine densities and synaptic con-tacts also increase. As noted above, spines areobserved on granule neurons on days 4 and 5 inthe rat, and in some cases, spines have been re-ported as early as days 2 or 3. Jones et al. (2003)considered spines to be protrusions that were lessthan 2 mm in length (whereas longer protrusionswere considered to be filopodia) and noted thatdendritic spines on neonatal granule neuronsranged in shape from short, stubby protrusions,either with or without heads, to those with long,thin necks ending in definitive spine heads (Des-mond and Levy, 1985; Trommald and Hulleberg,1997). Counts of spines on DiI-labeled dendriteslocated in the middle of the molecular layer re-vealed an average of 0.40 spines/mm in 5-day-oldrats and 0.57 spines/mm in 6-day-old animals(Jones et al., 2003). Densities increased to 0.81spines/mm by day 7, although they were still farbelow the values reported for dendrites in the same

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region in the adult rat (1.66 spines/mm; Desmondand Levy, 1985). Zafirov et al. (1994) also dem-onstrated that spine densities increased after day 5.These densities, however, were slightly lower thanthose reported by Jones et al. (2003), perhaps be-cause they calculated spine densities across the en-tire dendritic tree. Based on measurements of thetotal dendritic length and the total number ofspines per cell, they found that spine densities in-creased from day 5 through day 15, with 0.11spines/mm on day 5, 0.47 spines/mm on day 10, and0.67 spines/mm on day 15. Duffy and Teyler(1978a, b) also reported that the number of spinesper granule neuron more than doubled betweenday 7 and day 14, increasing from approximately100 spines/cell on day 7 to approximately 255spines/cell on day 14. Data from the above studiesare substantiated by the detailed analysis of spinedensities on dendrites of various orders over thecourse of granule neuron development and matu-ration done by Wenzel et al. (1981). For example,they reported that densities on 4th order branchesincreased from 0.05 spines/mm on day 5 to 0.12spines/mm on day 8 and to 0.18 spines/mm onday 10.

Electron microscope studies demonstrate thatonly a few synapses are present in the molecularlayer of the suprapyramidal blade on postnatalday 1 (0.4 synapses/100 mm2; Cowan et al., 1980).There is, however, a dramatic increase in synapsesover the first 10 days (Cowan et al., 1980). On days4 and 5, there are approximately 2–3 synapses/100 mm2 and, by day 10, densities have increased to11.0 synapses/100 mm2 (Crain et al., 1973; Cowanet al., 1980). In the molecular layer of the infra-pyramidal blade, synapses are present on day 5(0.8 synapses/100 mm2) and synaptic density in-creases about sixfold by day 10 (6.1 synapses/100 mm2; Cowan et al., 1980). In addition to thisconsiderable increase in synapse densities, the mo-lecular layer also increases in volume approxi-mately fourfold between days 5 and 10, resulting inapproximately a 16-fold increase in absolutesynapse number. It is also worth noting that ahigh percentage of synapses are found on dendriticshafts during the neonatal period (Cowan et al.,1981). On day 5, about 50% of synapses are ontodendritic shafts whereas on day 10, approximately

40% are onto shafts. The percentage of shaftsynapses continues to decline into adulthood, andby day 41, only approximately 10% of synapsesare found on dendritic shafts with the remaindercontacting dendritic spines. In addition tosynapses in the molecular layer, synaptic contactsare found on granule cell bodies in the neonatalrat. A fair proportion is symmetric and stain forglutamate-decarboxylase (Lubbers and Frotscher,1988; Seress et al., 1989).

In summary, during the first week of life, granuleneuron dendrites undergo a sequence of morpho-logical changes. Immature features appear and thenregress, dendrites elongate, and spines and synapsesdevelop. By day 7, the oldest granule neurons ex-hibit adult-like characteristics immature featureshave regressed, the vast majority of dendrites reachthe top of the molecular layer, and all dendrites arecovered with spines. During the second postnatalweek, dendrites continue to elongate and spine andsynaptic densities increase dramatically. By day 14,a considerable number of granule neurons have at-tained adult-like characteristics. It also appears thatthe large en passant boutons of the mossy fibershave attained an adult-like shape and complexity byday 14, even as the number of boutons continue toincrease into young adulthood (Stirling and Bliss,1978; Amaral and Dent, 1981).

Maturation of granule neuron dendritic trees

While granule neurons in 14-day-old rats qualita-tively resemble adult cells, quantitative studies sug-gest that their dendrites continue to change as theanimal matures. Early work indicated that the den-dritic tree might increase in size after the secondweek. Fricke (1975) reported that the total dendriticlengths of Golgi-stained granule neurons increasedafter day 12, reaching adult values of a little over1500mm on day 20. Duffy and Teyler (1978a, b)also reported an increase in granule cell dendriticlength between days 14 and 30. In addition, theyfound a slight but not statistically significant de-crease between days 30 and 60 and another slightincrease at day 210 — the average length at 210days was approximately the same as that at day 30.It is not clear from the methods, however, whether

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the reported lengths reflect measurements of thesum of all dendritic branch lengths or the length ofthe longest dendrite. Duffy and Teyler (1978a, b)state that they measured ‘‘from mid-soma to themost distal dendritic process’’, and the reportedvalues are quite similar to the width of the molec-ular layer (Rihn and Claiborne, 1990). Thus, theselengths may reflect the distance from the soma tothe most distal dendritic tips. Liu et al. (2000), usingintracellular labeling techniques, also found thatgranule cells enlarged after day 14 although the in-creases in total dendritic length were not statisti-cally significant.

Whereas the studies described above suggestthat dendritic trees increase in size during the thirdand fourth weeks, Wenzel et al. (1981) reportedthat granule cell dendritic length and the degree ofbranching reached adult proportions at day 15. Toresolve this issue, Rihn and Claiborne (1990) usedintracellular labeling techniques and three-dimen-sional analyses to quantify the dendritic trees ofonly the oldest granule neurons in the suprapy-ramidal blade between days 14 and 60. The somataof these neurons were located in the top portion ofthe granule cell layer, between the tip and themiddle of the suprapyramidal blade. Neuronsfrom rats of five age groups were examined: thefirst group included rats between the ages of 14and 19 days and the oldest group included animalsbetween 50 and 60 days. The dendritic trees fromrats in each age group exhibited similar structures,having between 1 and 4 primary apical dendritesthat were covered with spines (Fig. 4). Severalqualitative differences, however, were apparentbetween neurons in the youngest rats and those inthe oldest animals. For example, neurons in theyoungest rats had thicker dendrites in the proximaland middle thirds of the molecular layer, and theyexhibited a higher number of short, terminal seg-ments in the distal third of the layer.

Analyses of these maturing granule neuronsdemonstrated that both dendritic growth and re-gression occurred between days 14 and 60, leadingto a conservation of total dendritic length duringthis period (Rihn and Claiborne, 1990). The mo-lecular layer of the suprapyramidal blade increasedby approximately 50% (from an average of 205 to305 mm) between days 14 and 60, and the vast

majority of dendrites reached the top of the mo-lecular layer in animals of all ages, suggesting thatindividual dendrites elongated as the animal ma-tured. The number of dendritic segments, however,decreased from an average of 36 segments in the14- to 19-day-old rats to an average of 28 segmentsin the 50- to 60-day-old animals, with the majorityof the decrease occurring by day 29. (Segmentswere defined as a length of dendrite between anorigin and a branch point, between two branchpoints, or between a branch point and a distaltermination point.) As a consequence of this con-current branch elongation and loss, the averagetotal dendritic length did not change significantlybetween days 14 and 60 (3086 and 3417 mm, re-spectively). A similar process of dendritic loss withno change in total dendritic length has been doc-umented for nonpyramidal neurons in the rat vis-ual cortex (Parnavelas and Uylings, 1980).

Thus, although granule cells have attained theiradult dendritic lengths by postnatal day 14, den-dritic remodeling occurs between days 14 and 60with the elongation of some branches and the lossof others. In addition, the number of spines andsynapses increase (Duffy and Teyler, 1978a, b;Cowan et al., 1981; Wenzel et al., 1981; Zafirov etal., 1994). Duffy and Teyler (1978a, b) showed aslight increase in the number of spines per cell be-tween days 14 and 30. Values remained stable be-tween days 30 and 60 and then increased againbetween days 60 and 210. Seress and Pokorny(1981) noted that spine densities increased betweendays 10 and 25, and Zafirov et al. (1994) reportedthat spine densities (calculated on the basis of totalspines per cell divided by the total dendritic length)increased from 0.67 spines/mm at day 15 to 1.16spines/mm at day 20. Wenzel et al. (1981) foundthat spine densities increased dramatically ondendrites in almost all branch orders betweendays 15 and 30. For example, densities increasedfrom approximately 0.2 to 0.59 spines/mm on 4thorder branches and from 0.14 to 0.56 spines/mm on6th order branches. Furthermore, they found thatdensities also continued to increase slightly afterday 30; when all branch orders were consideredtogether, densities increased from approximately0.5 to approximately 0.6 spines/mm between days30 and 180. Synaptic density in the molecular layer

Fig. 4. Camera lucida drawing of a granule neuron from a 53-day-old rat. Note the significant increase in size as compared to the

adult-like neuron from a 7-day-old rat shown in Fig. 3. Scale bar ¼ 50 mm.

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also increased with maturation (Cowan et al.,1980). In the molecular layer of the suprapyram-idal blade, densities increased from 11 synapses/100 mm2 on day 10 to 36 synapses/100 mm2 on day21. Only a slight increase was seen after day 21;densities were 37 synapses/100 mm2 on day 41.Similarly, the percentage of spine synapses in-creased considerably from day 10 to 21, but didnot exhibit much of a change between days 21 and41. Spine synapses comprise 47% of all synapseson day 10, 84% on day 21, and 88% on day 41.

In summary, the total dendritic lengths of theoldest granule neurons in the suprapyramidalblade appear to be established by day 14. Quan-titative changes in the dendritic tree do occur afterthis time, however; some dendrites elongate whileothers are lost, thus leading to a conservation oftotal length. Interestingly, there is one other reportof regression in the hippocampal formation during

maturation. The axons or mossy fibers of thegranule neurons exhibit filopodial-like extensionsthat elongate during the first two postnatal weeks,reach a peak in length on day 14, and then de-crease to adult lengths by day 28 (Amaral, 1979).The time course of their growth and retraction isremarkably similar to the time course of dendriticbranch growth and regression in the oldest granuleneurons — whether the two processes are gov-erned by the same or similar mechanisms is not yetknown. In this regard, preliminary data from ourlab indicate that dendritic branch loss may beaffected by incoming neuronal activity. Specifi-cally, we found that blockade of N-methyl-D-as-partate (NMDA) glutamate receptors betweendays 14 and 24 did not affect dendritic growth,but did result in a decrease in dendritic branch lossin the oldest granule neurons (Blake and Clai-borne, 1995). Because branches continued to grow

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and fewer branches were lost, the granule neuronsin the treated animals had greater total dendriticlengths than did those in the controls. It is worthnoting that the glutamate released from synapseson granule cell dendrites in the middle third of themolecular layer binds to NMDA receptors, andthat the afferents to this region arise from the ent-orhinal cortex. The entorhinal cortex, in turn, re-ceives inputs from the visual, auditory, andsomatosensory cortices, and a number of signifi-cant events occur in the maturation of these sen-sory systems soon after day 14 in the rat. Forexample, the eyes open approximately at day 15and adult sensitivities to sound are reached be-tween days 16 and 18 (Tilney, 1933; Crowley andHepp-Reymond, 1966). Therefore, it is not sur-prising that granule cell remodeling begins shortlyafter day 14 and may be governed by afferent in-puts from the entorhinal cortex.

Granule neuron dendrites in the adult rat

Quantitative data on the dendritic trees of adultgranule neurons are based on two- and three-di-mensional measurements of both Golgi-impreg-nated and intracellularly labeled cells. Fricke(1975) was the first to make use of a computer-microscope system to analyze Golgi-stained neu-rons in three dimensions, whereas Desmond andLevy (1982) developed a novel probabilisticmethod for quantifying dendritic trees of granuleneurons from Golgi-stained tissue in two dimen-sions. They corrected for cut dendrites at the edgeof sections by estimating their branching and ter-mination patterns based on the patterns observedfor intact dendrites. Corrected values were com-pared with data from tracings of six neurons (twofrom each region) that were followed through se-rial sections. Only granule neurons located in themiddle 80% of the longitudinal extent of the hip-pocampus and with a minimum of cut dendrites inthe proximal third of the molecular layer were in-cluded. Claiborne et al. (1990) and Rihn and Clai-borne (1990) applied the computational techniquesfirst used by Fricke (1975) to quantify the dendritictrees of intracellularly labeled granule neurons inrelatively thick transverse slices (400 mm) of the

hippocampal formation. They analyzed only thoseneurons that were completely stained, had no cutdendrites in the proximal portion of the molecularlayer, and had fewer than two-severed dendrites inthe distal two-thirds of the layer.

Here we review the quantitative parameters ofadult granule neurons reported by the above in-vestigators and by others employing similar tech-niques. We first discuss the available data on theentire population of granule neurons, and we thenreview the relationship between the morphology ofa granule neuron and the location of its cell bodywithin stratum granulosum and within the twoblades of the dentate gyrus.

The cell body of an adult granule neuron in therat is approximately 10 mm wide and approxi-mately 19 mm long (Claiborne et al., 1990). Be-tween 1 and 5 primary apical dendrites exit fromthe apical pole of the cell body and bifurcate rel-atively close to the soma (Fig. 5). Fricke (1975)noted that most branching occurs within 100 mm ofthe soma and within the vicinity of the boundarybetween stratum granulosum and stratum mole-culare. Desmond and Levy (1982) found that themajority of primary segments branch within thegranule cell layer and first ninth of the molecularlayer. Furthermore, they reported that nearly allfirst-order branch points occur within 50 mm of thecell body and within the first 30 mm of the molec-ular layer. Primary dendrites bifurcate into higherorder segments, and up to eighth-order brancheshave been reported, with an average maximumbranch order of 5.7 (Claiborne et al., 1990). Gran-ule neurons have a total of approximately 30 den-dritic segments, with reported numbers rangingfrom 22 to 40 (Fricke, 1975; Seress and Pokorny,1981; Desmond and Levy, 1982; Green andJuraska, 1985; Claiborne et al., 1990; Rihn andClaiborne, 1990).

Based on Golgi impregnations, Fricke (1975)reported an average total dendritic length of1602 mm (range from 773 to 2445 mm) for adultgranule neurons, whereas Seress and Pokorny(1981) reported an average of 2405 mm, and Greenand Juraska (1985) found total dendritic lengthsranging from 1161 to 1279 mm. After correcting forcut dendrites, Desmond and Levy (1982) calcu-lated an average total dendritic length of 3662 mm

Fig. 5. Computer-generated plots of three-dimensional reconstructions of the dendritic trees of granule cells located at various

positions along the transverse axis of the dentate gyrus. Each neuron is from a different animal, and the total dendritic length of each

dendritic tree is indicated. Note that the total dendritic lengths of the trees in the suprapyramidal blade are greater than those of the

trees in the infrapyramidal blade. CA3, field CA3 of the hippocampus proper; GL, granule cell layer. Scale bar ¼ 100mm. (Adapted

with permission from Claiborne et al., 1990.)

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for Golgi-stained neurons. Claiborne and col-leagues reported similar averages of 3221 and3417 mm, respectively, for intracellularly labeledgranule neurons analyzed in two different studies(Fig. 5; Claiborne et al., 1990; Rihn and Claiborne,1990). Desmond and Levy (1982) reported thatapproximately 12% of the total length was foundwithin stratum granulosum, approximately 25%within the proximal third of the molecular layer,and the remaining portion was restricted to thedistal two-thirds. Similarly, data from Claiborne etal. (1990) indicated that 30% of the total lengthwas found in the granule cell layer and proximalthird of the molecular layer, 30% in the middlethird and 40% in the distal third. These resultssuggest that more dendritic length is available forsynapses from the entorhinal afferents that

terminate in the distal two-thirds of the layer thanfor commissural and associational afferents thatmake contacts in the inner third of the layer.

The majority of synapses onto the granule neu-ron dendrites occur on spines in the adult. As dis-cussed above, a number of groups have shown thatspine and synaptic densities increase throughoutgranule neuron development and maturation, andtheir reported values for densities in maturingand young adult rats are reviewed above (Duffyand Teyler, 1978a, b; Cowan et al., 1981; Seress andPokorny, 1981; Wenzel et al., 1981; Zafirov et al.,1994). Other investigators have reported spine den-sities only for granule neurons in young adult rats.Fricke (1975) noted that spine densities were low ondendrites of adult granule neurons within the first20mm of the soma and within 50mm of dendritic

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terminations. He found a maximum of approxi-mately 1.3 spines/mm on dendrites in the remainingportion of the dendritic tree. Desmond and Levy(1985) reported that spines were infrequent ondendrites within stratum granulosum and that therewere three major types of spines on adult granulecell dendrites in the molecular layer: stubby, mush-room-shaped, and thin. They calculated spine den-sities for each type in the proximal, middle, anddistal thirds of the molecular layer of the twoblades. In both blades and in all three portions ofthe layer, densities were highest for the thin spinesand were lowest for mushroom-shaped protrusions.When all spine types were included and correctionsmade for obscured spines, densities averaged 1.6spines/mm on dendrites of neurons in the suprapy-ramidal blade and 1.3 spines/mm on those in theinfrapyramidal blade. These values are similar tothose reported by Fricke (1975) but are muchgreater than those reported by Wenzel et al. (1981;see above) perhaps because different criteria wereused for the inclusion of spines (Desmond andLevy, 1985).

In summary, the dendritic trees of granule neu-rons in young adult rats are composed of approx-imately 30 spine-covered segments and have totaldendritic lengths of approximately 3400 mm. It isnot surprising that these total lengths are muchless than those of the relatively large pyramidalneurons in the hippocampus proper. Reports ofthe total dendritic lengths of pyramidal neurons inregion CA1 vary from an average of 10,800 to17,400 mm (Ishizuka et al., 1995; Mainen et al.,1996; Pyapali and Turner, 1996; Pyapali et al.,1998; Megias et al., 2001), whereas the totallengths for pyramidal neurons in region CA3range from a low of 7000 mm for pyramidal neu-rons in region CA3c to a high of 19,800 mm forthose in region CA3a (Ishizuka et al., 1995; Turneret al., 1995; Gonzales et al., 2001). It is also notsurprising that the granule neurons are the mostelectrotonically compact of the three major classesof hippocampal neurons (Carnevale et al., 1997).

Given the gradients of granule cell neurogenesis,it is of interest that several parameters of granuleneuron dendrites are correlated with the locationof the parent cell body in the granule cell layer —both along the transverse axis of the dentate gyrus

and within the depth of the granule cell layer. Datademonstrate that the dendritic trees of neurons inthe suprapyramidal blade are larger than those inthe infrapyramidal blade (Fig. 5). Fricke (1975)reported that suprapyramidal neurons havegreater total dendritic lengths than do those inthe infrapyramidal blade (1674 mm vs. 1482 mm),and Desmond and Levy (1982) confirmed this re-sult for Golgi-stained granule neurons that weretraced through multiple sections (3107 mm for sup-rapyramidal neurons vs. 2078 mm for infrapyram-idal blade cells). Similarly, Claiborne et al. (1990)showed that suprapyramidal granule neurons havegreater total dendritic lengths (3478 mm vs.2793 mm), more dendritic segments (31 vs. 27),and wider dendritic spreads in the transverse planeof the hippocampal formation (347 mm vs. 288 mm)than do the infrapyramidal blade neurons. Theseresults demonstrate that suprapyramidal granuleneurons have more dendritic length available forafferent contacts than do neurons in the oppositeblade. In addition, Desmond and Levy (1985) re-ported that spine density is greater on dendrites ofsuprapyramidal neurons (1.6 spines/mm vs. 1.3spines/mm), and this result, combined with the in-creased length of the suprapyramidal neurons,suggests that neurons in this blade receive manymore synaptic contacts than do neurons in the in-frapyramidal blade.

Overall, these data indicate that the dendritictrees of granule neurons in the suprapyramidalblade are likely to be larger than those in the in-frapyramidal blade and to receive more afferentcontacts. It is of considerable interest that the axontrajectories of the granule neurons also vary withgranule cell location. Axons of granule neuronslocated toward the tip of the suprapyramidal bladetraverse stratum radiatum of region CA3 beforeentering stratum lucidum, avoiding both the hilarregion and the proximal part of the pyramidal celllayer, whereas axons of neurons in the crest andthe infrapyramidal blade travel through the hilarregion and contact pyramidal neurons in the prox-imal portion of field CA3 (Claiborne et al., 1986).It is likely that such morphological distinctions inthe granule neurons, in combination with physio-logical differences (Scharfman et al., 2002; Chawlaet al., 2005), may lead to functional differences

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between the two blades. This idea is best exempli-fied by recent results showing that induction of theactivity-regulated, immediate early gene Arc fol-lowing behavioral stimulation occurs in the sup-rapyramidal blade, but not in the infrapyramidalblade (Chawla et al., 2005).

Not only do some morphological parametersvary according to the location of the cell bodyalong the transverse axis of the dentate gyrus, sev-eral dendritic parameters also differ according tothe depth of the parent cell body in the granule celllayer. Neurons with somata located at the top ofthe layer tend to have multiple primary brancheswhereas those with cell bodies located deeper inthe layer tend to have only one primary dendrite(Fricke, 1975; Seress and Pokorny, 1981; Des-mond and Levy, 1982; Green and Juraska, 1985;Claiborne et al., 1990). Granule neurons in thesuperficial half of the granule cell layer also exhibitnearly twice the density of axosomatic synapses asdo cells in the bottom half of the layer (Lee et al.,1982). In addition, Green and Juraska (1985)showed that Golgi-stained granule neurons withsomata in the superficial third of the granule celllayer had greater maximum widths in the trans-verse plane of the dentate gyrus, more branches inorders 1 through 3, and fewer branches in orders 4through 6. These neurons also had greater totaldendritic lengths (1279 vs. 1161) than did deeperneurons; 80% of the neurons in their sample werelocated in the suprapyramidal blade. In contrast,Claiborne et al. (1990) did not find a statisticallysignificant difference between the average totallengths of neurons with somata in the superficialhalf of the granule cell layer and of those withsomata in the bottom half of the layer in eitherblade. When only neurons in the suprapyramidalblade were analyzed, however, they found thatsuperficial granule neurons had more primarydendrites (2.4 vs. 1.5) and larger transverse spreads(378 mm vs. 293 mm) than deeper neurons. In ad-dition, 42% of the total length of superficial cellswas in the distal third of the layer as compared to37% of the length of the deeper cells. For neuronsin the infrapyramidal blade, the only significantdifference was that superficial cells had largertransverse spreads than did deeper neurons(311 mm vs. 244 mm).

Thus, a number of structural differences existbetween the dendritic trees of neurons located inthe superficial portion of stratum granulosum andthose located in the deeper aspects of the layer. Atleast one of the differences may have functionalconsequences. Carnevale et al. (1997) demon-strated that the single primary dendrites of deeperneurons attenuated voltage signals spreading fromthe soma out to the dendrites, thereby reducing theeffect of somatic events, including action poten-tials, on molecular layer synapses. Given thatadult-generated granule neurons migrate into thelower portion of the granule cell layer, it will be ofinterest to determine whether other functionaldifferences correlate with the depth of granule cellbodies in stratum granulosum in adult animals.

Summary

Granule neurons in the dentate gyrus of the ratundergo periods of development and maturationbefore reaching their final adult form and size. Inthe rat, the primary period of neurogenesis beginsduring late embryogenesis and continues over thefirst two weeks of life. The oldest granule neuronsoccupy the suprapyramidal blade whereas latergenerated cells form the infrapyramidal blade. Onthe day of birth, granule neurons in the suprapy-ramidal blade exhibit a few sparse dendrites. Ru-dimentary trees appear over the next few days andby day 4, dendritic branching is quite extensive.Immature features are abundant at this time andinclude abrupt diameter changes, varicosities, filo-podia, growth cones, and basilar dendrites. On days5 and 6, elaborate dendritic trees are present, thereis a reduction in the frequency of immature fea-tures, and some spines are visible on the most ma-ture cells. On day 7, the first few adult-like trees arepresent on granule neurons in the suprapyramidalblade. Their dendrites reach the top of the molec-ular layer and no longer exhibit immature featuresexcept for an occasional filopodium or varicosity.All of the branches are covered with spines. Phys-iological recordings demonstrate that some granuleneurons are functional at this time, suggesting thatthe dentate gyrus may be incorporated into thehippocampal circuit by the end of the first week.

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After day 7, the dendritic trees increase in size, and,by day 14, numerous adult-like granule cells arepresent. The oldest granule neurons then undergo aprocess of refinement. Many dendrites continue toelongate during this period, and other branches arelost; the simultaneous growth and regression resultsin a conservation of total dendritic length after day14. Spine densities, however, continue to increase.Adult granule neurons in the rat have approxi-mately 30 dendritic segments and total dendriticlengths of approximately 3400mm. A variety ofmorphological features are correlated with the lo-cation of the granule cell body, both along thetransverse axis of the dentate gyrus and within thedepth of the granule cell layer. Recent evidencesuggests that there are functional distinctions be-tween the two blades of the dentate gyrus; it will beof considerable interest to determine whether differ-ences in granule cell morphologies underlie thesedistinctions.

Acknowledgment

This work was supported by NIH grant #GM08194to BJC.

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