Ultrastructural features and synaptic connections of hilar ectopic granule cells in the rat dentate gyrus are different from those of granule cells in the granule cell layer

Ultrastructural features and synaptic connections of hilar ectopic granule cells in the rat dentate gyrus are different from those of granule cells in the granule cell layer. Abstract Several investigators have shown the existence of dentate granule cells in ectopic locations within the hilus and molecular layer using both Golgi and retrograde tracing studies but the ultrastructural features and synaptic connections of ectopic granule cells were not previously examined. In the present study, the biocytin retrograde tracing technique was used to label ectopic granule cells following injections into stratum lucidum of CA3b of hippocampal slices obtained from epileptic rats. Electron microscopy was used to study hilar ectopic granule cells that were located 20–40 m m from the granule cell layer (GCL). They had ultrastructural features similar to those of granule cells in the GCL but showed differences, including nuclei that often displayed infoldings and thicker apical dendrites. At their origin, these dendrites were 6 m m in diameter and they tapered down to 2 m m at the border with the GCL. Both biocytin-labeled and unlabeled axon terminals formed exclusively asymmetric synapses with the somata and proximal dendrites of hilar ectopic granule cells. The mean number of axosomatic synapses for these cells was three times that for granule cells in the GCL. Together, these data indicate that hilar ectopic granule cells are postsynaptic to mossy ﬁbers and have less inhibitory input on their somata and proximal dendrites than granule cells in the GCL. This ﬁnding is consistent with recent physiological results showing that hilar ectopic granule cells from epileptic rats are more hyperexcitable than granule cells in the GCL. (cid:211)


Introduction
described morphological aspects of the ectopic granule-like cells in the albino rat hippocampal formation The dentate gyrus of the hippocampal formation has and classified them into two groups. Using Golgi preparathree major parts, the hilus, granule cell layer (GCL) and tions, they showed that ectopic granule-like cells were molecular layer [1, 12,23,32]. In rats, most granule cells distributed throughout the hilar region. The cell body of have their cell bodies in the GCL. Their dendrites typically the monopolar subtype was small (1239 mm), oval in extend into the molecular layer and their axons enter the shape, and had a few occasional somatic spines. One or hilus where they give rise to collaterals and ultimately two primary thick dendrites with spines arose from the cell terminate in stratum lucidum of CA3 [7,8].
body. The bipolar granule cell subtype was more in-Several investigators indicated that granule cell bodies frequent. Its cell body was oval or spherical (1339 mm). also exist in the hilus [1, 14,26,45]. Marti [14] used a retrograde fluorescent tracing technique to pine-treated rats that did not develop SE (n54) were not study hilar ectopic granule cells. They determined that 5% used for this study. of the neurons in the hilus projected to CA3 and that these cells resembled ectopic granule cells. They also showed 2.2. Granule cell labeling with biocytin that the hilar ectopic granule cells had 'dendrites oriented toward' the GCL.
Rats were anesthetized and decapitated 24.9-61.7 weeks More recent studies showed that hilar ectopic granule after convulsant administration. Brain slices (400 mm cells were increased in epileptic rats. Parent et al. [29] thick) obtained from the caudal one-third of the hippocamfound an increase of dentate granule cell neurogenesis pal formation, transverse to the long axis, were cut with a following severe seizures, and described newly generated Vibratome (Technical Products International). They were granule cells in an ectopic location, the hilus. Scharfman et then transferred to a static interphase chamber (Fine al. [41] observed a large increase in the number of Science Tools) containing oxygenated artificial cerebrospicalbindin immunoreactive neurons in the hilus after nal fluid (ACSF) at 32.58C. After a 45-min incubation pilocarpine-induced status epilepticus. Such cells were period to allow for the slice to reach equilibrium in the morphologically similar to granule cells and were sug-chamber, a 4% solution of biocytin was iontophoretically gested to contribute to the hyperexcitability in this model ejected into stratum lucidum of CA3b of the hippocampus of epilepsy. Electrophysiology of these ectopic granule to label the mossy fibers of dentate granule cells (see Ref. cells in the hilus from epileptic rats indicated normal [27]). A glass micropipette with a filament (WPI, 1.5 mm electrophysiological characteristics but they also dis-external diameter, 5-10 mm tip diameter) was filled with a charged synchronously with spontaneous epileptiform freshly prepared solution of 4% biocytin in 0.05 M Trisbursts of CA3 pyramidal cells [41]. This abnormal bursting HCl buffer, pH 7.3. The tip of the glass micropipette was property of hilar ectopic granule cells suggested that they about 200 mm deep to the surface. Biocytin was ejected receive mossy fiber input as do the CA3 pyramidal cells.
into the extracellular space with intermittent positive Previous morphological studies of hilar ectopic granule current pulses (600 nA, 7 s on, 7 s off) for 10-20 min. cells were limited to light microscopic observations. The Three hours following biocytin injection, slices were present study attempts to provide additional morphological immersed in a fixative solution containing 2% paraformaldata of these cells at the electron microscopic level.
dehyde and 2% glutaraldehyde in 0.1 M phosphate buffer Specifically, two issues are addressed. First, do hilar (PB), pH 7.4, and stored overnight at 48C. ectopic granule cells have similar features as the typical To visualize the biocytin-labeled granule cells, the fixed granule cells in the GCL? Second, what are the synaptic slices were re-sectioned at a thickness of 50 mm. The connections of hilar ectopic granule cells? Following sections were collected in 0.1 M PB and treated with biocytin injections into CA3, several retrogradely labeled hydrogen peroxide to suppress endogenous peroxidase granule cells were consistently found in the hilus of activity. Then, the sections were washed in 0.1 M PB for epileptic and control rats [39]. The goal of the present 30 min and incubated in avidin-biotin horseradish perstudy is to examine the electron microscopic features of oxidase solution (Vectastain Elite ABC Kit, Vector Labs) these biocytin-labeled, hilar ectopic granule cells and to for 3-5 h at room temperature and overnight at 48C. determine their synaptic connections.
Finally, they were washed several times in 0.1 M PB and incubated in 0.025% diaminobenzidine (DAB) and 0.01% nickel ammonium sulfate for 15-20 min.

Material and methods
After the reaction product was formed, the sections were thoroughly washed in PB. Sections were placed in 1% 2.1. Induction of status epilepticus osmium tetroxide for 20-60 min and then processed for embedding in plastic following dehydration through a Adult male Sprague-Dawley rats (165-265 g; Zivic-series of ethanols and immersion in propylene oxide. A Miller Labs., Allison Park, PA) were used in these studies.
flat-embedding procedure was used to facilitate the exami-The Institutional Animal Care and Use Committees at nation of each section with a light microscope (Leitz). Duke University and the University of California at Irvine After identifying ectopic granule cells in the hilus, the approved animal protocols in advance of the experiments. section was then trimmed using a single-edged razor blade Ten rats were injected with pilocarpine hydrochloride and a dissecting microscope (Nikon). Ultrathin sections (300-325 mg / kg, i.p.) preceded 30 min earlier by containing the dentate gyrus and biocytin-labeled hilar methylscopolamine (1 mg / kg, i.p.). Six of these animals ectopic granule cells were cut with an ultramicrotome developed status epilepticus (SE), a continuous limbic (Reichert-Jung) and collected on formvar-coated slot grids. motor seizure of stage 2 or higher [31]. SE was terminated Prior to placing the grids in the microscope, the sections after 2.5-4.5 h with a single injection of sodium phenobar-were stained with uranyl acetate and lead citrate to bital (50 mg / kg, i.p.). After cessation of SE, animals were enhance the contrast. Ultrathin sections containing maintained as described by Okazaki et al. [27]. Pilocar-biocytin-labeled, hilar ectopic granule cells were examined with a Philips CM-10 transmission electron microscope. cells [4,21,33]. These typical cell bodies have a diameter The hilus, GCL and molecular layer were identified using of approximately 10 mm and are either round or ovalknown morphological features [12]. Then, labeled hilar shaped. The oval-shaped cell bodies were commonly found ectopic granule cells and their synapses were identified and near the hilar border and had their long axis perpendicular photographed. Axosomatic synapses were counted for both to the GCL (Fig. 2). Most of the cell body of typical types of granule cells (hilar ectopic and labeled ones in the granule cells is occupied by the nucleus that displays GCL). The frequency of synapses for labeled cell bodies several chromatin aggregates and has a relatively smooth and proximal dendrites was expressed as the number per surface. The perikaryon is limited to a thin shell around the unit length of membrane for a single thin section.
nucleus and this part of biocytin-labeled granule cells contained electron dense reaction product. A few cisternae of the granular endoplasmic reticulum and free ribosomes 3. Results are found in the perikaryon but they are not aggregated together to form Nissl bodies, as they are in the large For this analysis 12 biocytin-labeled hilar ectopic pyramidal neurons of CA3. In addition, numerous mitogranule cells were examined from four of the six epileptic chondria, free ribosomes, lysosomes and Golgi apparatus rats. The light microscopic examination of biocytin-labeled are present (Fig. 2). Dendrites were observed to arise from preparations from rats with SE showed brown reaction the apical pole of granule cells (Fig. 2). They were product in many cell bodies in the GCL and in adjacent typically 2-3 mm in diameter at their origin. A thin axon processes, such as the dendrites in the molecular layer and initial segment was found to emanate from the basal pole. mossy fibers in the hilus and area CA3 (Fig. 1A). In addition, these preparations contained biocytin-labeled, 3.2. Ultrastructural features of hilar ectopic granule hilar ectopic granule cells that were identified by their cells location in the hilus and by the presence of retrograde labeling for biocytin throughout their somata and pro- The hilar ectopic granule cells have ultrastructural cesses. They were found either close to the GCL (Fig. 1B) features that are similar to those of granule cells in the or in the deep hilus ( Fig. 1A) and bordering the pyramidal GCL with some morphological differences. The size of the cell layer in area CA3c (see Ref. [41]). The cells that were cell body of these cells was in the same range as that for chosen for electron microscopic examination were located typical granule cells. The somata were round or oval 20-40 mm from the GCL (Fig. 1B) because this site shaped and located in the hilus with their long axis allowed for a direct comparison with the granule cells in perpendicular to the GCL (Fig. 2). The perikaryon of hilar the GCL. The hilar ectopic granule cells were usually ectopic granule cells was similar to that for the granule bipolar with thick dendrites extending from each end of the cells in the GCL and contained the same cytoplasmic cell body. The dendrites of these cells showed numerous organelles (Figs. 3A and 4). On the other hand, several spines and they often penetrated the GCL and arborized features were different than those for typical granule cells. within this layer but could not be followed into the The nuclei of hilar ectopic granule cells often displayed molecular layer. The long axis of the hilar ectopic granule infoldings or were notched as opposed to being round or cells examined in this study was perpendicular to the GCL oval (Figs. 3A and 4A). Also, the proximal portions of ( Fig. 1B) hilar ectopic granule cell dendrites were larger in diameter Electron microscopic preparations of these same speci-than those of typical granule cells in the GCL (Fig. 3B,C). mens showed many biocytin-labeled and unlabeled granule At their origin, these thicker dendrites were 4-6 mm in cells in the GCL and a few biocytin-labeled ectopic diameter, and they tapered down to about 2 mm at the granule cells in the hilus where they were surrounded by hilar / GCL border. Similar to typical granule cells, these neuropil (Fig. 2). The labeled granule cells had electron ectopic granule cells had numerous spines on their dendense reaction product in their perikaryon and dendritic drites (Figs. 3C and 5). The axon initial segments of cytoplasm (Fig. 2). In general, the somata of hilar ectopic granule cells were not observed in the electron microscopic granule cells had similar nucleoplasmic and cytoplasmic preparations because a complete serial section analysis was features as granule cells in the GCL. A brief description is not made of these cells. given below of biocytin-labeled granule cells in the GCL followed by a description of the features of hilar ectopic 3.3. Synaptic connections of hilar ectopic granule cells granule cells.
We examined five labeled somata of hilar ectopic 3.1. Ultrastructural features of typical granule cells granule cells for axosomatic synapses. In this analysis, asymmetric (presumed excitatory) and symmetric (pre-Biocytin-labeled granule cell bodies are common in the sumed inhibitory) synapses were distinguished using estab-GCL (Fig. 2) and have ultrastructural features that are lished criteria for hippocampal synapses [12,21,38,42]. similar to those previously described for dentate granule Unlabeled axon terminals formed exclusively asymmetric synapses with the somata of hilar ectopic granule cells core vesicles and mitochondria that were located away (Fig. 4B,C). Many of the unlabeled axon terminals were from the active synaptic sites (Fig. 4). No biocytin-labeled large (1-2 mm), contained clear round synaptic vesicles axon terminals formed axosomatic synapses with hilar spread throughout the terminal, and had occasional dense-ectopic granule cells. A total of 32 axosomatic synapses were found for 149 mm length of somal membrane for the examined in the GCL. Unlabeled axon terminals with a five examined cells. Thus, the mean number of axosomatic similar morphology as that described above for axosomatic synapses was 6.4 per soma per thin section. In contrast, synapses also formed asymmetric synapses with the denlabeled granule cells (n55) in the GCL had significantly drites of all the examined hilar ectopic granule cells ( also formed asymmetric synapses with dendrites and Labeled dendrites of hilar ectopic granule cells were spines of hilar ectopic granule cells (Fig. 5A). These axon also examined for synaptic connections. The thin sections terminals had the same morphology and size as those in facilitated an analysis of only the proximal portions of the previous studies that identified them as mossy fibers dendrites that were found in the hilus. Dendrites were not [27,39]. The frequency of axodendritic synapses was significantly greater than that of axosomatic synapses (one have mainly asymmetric synapses on their somata and per 3.08 mm for axodendritic versus one per 4.65 mm for proximal dendrites, this finding suggests that they are more axosomatic; P,0.05, Student's t-test).
hyperexcitable than granule cells in the GCL.

Technical considerations 4. Discussion
The injection of biocytin is an effective method for This study has shown the electron microscopic features labeling both dendritic and axonal processes of neurons of retrogradely labeled hilar cells following biocytin [22,27]. In the present study, biocytin was ejected into injections into CA3b and indicates that they are ectopic stratum lucidum of CA3b of the hippocampus to target the granule cells. Furthermore, these hilar ectopic granule cells mossy fiber projection system of dentate granule cells. In have three times more total synapses on their somata than so doing, many granule cells in the GCL are retrogradely granule cells in the GCL. Because ectopic granule cells labeled [27,39]. Biocytin was chosen for our study because  it selectively labels the neurons with axons projecting to characterized at the electron microscopic level [36]. The the injection site, is a non-fluorescent tracer that can be fusiform cells of the dentate gyrus are located in a portion visualized with the electron microscope, and does not leak of the hilus within 100 mm of the GCL and have ovoid out of membranes at synapses. The light microscopic somata and bipolar dendrites with a long axis that is results from this study and a previous one from this parallel to the border between the hilus and GCL [36]. laboratory [39] demonstrated hilar ectopic granule cells, These neurons are distinguished by their dendrites: either and were consistent with the previous results of Gaarskjaer spiny or sparsely spiny varieties. The spiny type of and Laurberg [14] who used a fluorescent retrograde fusiform neuron has a round or ovoid nucleus that lacks labeling method. In these studies, retrograde labeling was infoldings and intranuclear rods, and occupies more than mainly limited to granule cells, including those in the GCL half of the somal area. The spiny fusiform cell has and ectopic ones in both the hilus and molecular layer.
numerous spines along its dendrites. This cell type also displays somal spines that occasionally have mitochondria 4.2. Identification of biocytin-labeled hilar cells within their heads. In contrast, the sparsely spiny fusiform cell displays only a few spines. This cell has a highly Are the biocytin-labeled cells in the hilus ectopic infolded, often eccentrically located nucleus. A prominent granule cells or another type of hilar neuron as described nucleolus is also observed as well as intranuclear rods. by Amaral [1]? Several reasons indicate that the biocytin-Once again, these features are not observed for any of the labeled cells in the hilus are displaced granule cells and biocytin-labeled cells located in the hilus in the present they will be discussed below.
study. Therefore, the biocytin-labeled hilar cells in the First, biocytin is not transferred through the membrane present study are not fusiform cells. These data taken of labeled neurons, so biocytin-labeled cells in the hilus together with the other features described above allow us must have axonal projections to stratum lucidum in the to conclude that the biocytin-labeled cells are most likely ipsilateral CA3 region of Ammon's horn. Neurons with hilar ectopic granule cells and not one of the other major this projection are thought to be exclusively granule cells cell types observed in the hilus of the dentate gyrus. [1, 14,23,32]. Second, in addition to this observation which identifies the biocytin-labeled cells in the hilus as granule 4.3.

Connections of hilar ectopic granule cells cells, ultrastructural features of these ectopic cells show several morphological features that are characteristic of
In addition to the analysis of the cytological features of granule cells. These include: a soma with a diameter of ectopic granule cells, the present study analyzed their about 10 mm that is mainly occupied by a nucleus, a thin synaptic connections. Axon terminals presynaptic to the perikaryon with several organelles but no Nissl bodies, and somata and proximal dendrites of labeled hilar ectopic a nucleus with several heterochromatic regions. In addi-granule cells formed exclusively asymmetric synapses. The tion, the dendrites are orientated orthogonal to the GCL fact that some of these axon terminals were labeled (Fig.  and have many spines. These ultrastructural features are 5A) indicates that mossy fibers are forming synapses with the same as those described previously for granule cells in hilar ectopic granule cells. This finding suggests that the GCL [4, 21,33]. Third, in the following paragraphs, the granule cells target hilar ectopic granule cells and may known ultrastructural features of a limited number of hilar provide the basis for increased excitatory input to them. neurons are presented to show that the biocytin-labeled The absence of symmetric synapses on the somata and hilar cells in the present study can be excluded as one of proximal dendrites of hilar ectopic granule cells indicates these hilar cell types.
that the inhibitory basket cell input to a portion of these The most common cell type in the hilus, and certainly granule cells is missing. Pyramidal basket cells have an the most impressive in size and structure, is the mossy cell axonal plexus in the GCL that is responsible for the [1,37]. The mossy cell's ultrastructural features include a formation of symmetric synapses with the somata and large soma (20-30 mm diameter) with a round or oval, proximal dendrites of granule cells in the GCL euchromatic nucleus that lacks infoldings and intranuclear [12,15,35,44]. The pyramidal basket cell axon arises from rods [5,13,25,37,49]. The perikaryon of mossy cells con-the apical dendrite and ramifies in the inner molecular tains organelles typical of comparably sized pyramidal cell layer before dropping vertical axon collaterals into the bodies, including Nissl bodies. The mossy cell bodies also GCL [32]. Therefore, it is unlikely that the axons of display numerous somal spines. Most of these are complex pyramidal basket cells will synapse with the cell bodies of in shape and may branch into three or four smaller spines. hilar ectopic granule cells because they lie outside the The dendrites arise from the cell bodies of mossy cells as GCL. The location of the soma and lack of symmetric large thick tapering structures and have numerous complex axosomatic synapses seen in the present study suggests spines or thorny excrescences on their proximal portions.
that hilar ectopic granule cells lack inhibitory input on These features were not observed for any of the biocytin-their somata and proximal dendrites. It should also be labeled cells located in the hilus in the present study.
noted that granule cells in the GCL have a lower mean Two types of fusiform cells in the hilus also were number of axosomatic synapses than hilar ectopic granule cells. The data for axosomatic synapses per granule cell in nections among granule cells in the GCL [34,40] and few the GCL in the present study (mean52.0) was similar to hilar ectopic granule cells in the normal brain [14,39]. In that shown previously by Seress and Ribak [46] where 205 pilocarpine-treated epileptic rats, however, the numbers of granule cells in the GCL had a mean of 2.2 synapses per both increase dramatically [41]. Hilar ectopic granule cells soma per thin section.
become integrated into the circuitry of the dentate gyrus, such that they receive excitatory innervation from other 4.4. Functional significance of morphological findings granule cells (present study) and send axon collaterals into the hilus and area CA3 [41]. Furthermore, hilar ectopic A unique ultrastructural feature of the hilar ectopic granule cells in the epileptic brain differ from granule cells granule cells is a notched or ruffled nucleus. Typical in the GCL in that they exhibit spontaneous bursts that are granule cells in the GCL do not display this morphology time-locked to spontaneous population bursts of CA3 [4, 21,33]. What is the meaning of this type of nuclear pyramidal cells [41]. Their propensity for burst discharge morphology? Previous studies have shown that infolded may be related to the paucity of inhibitory synapses on the nuclei are characteristic of high levels of neuronal activity somata and proximal dendrites of hilar ectopic granule [33,43]. This notching or ruffling of the nucleus increases cells. Even in the epileptic brain, hilar ectopic granule cells the surface area of the nuclear membrane, and allows for account for only a small percentage of the total granule increased transfer of RNA in these cells. Thus, notched cell population. However, Traub et al. [48] showed that the nuclei in hilar ectopic granule cells indicate a high level of synchronous discharge of only a few CA3 pyramidal cells activity for these cells, which may include hyperexcit-in the normal brain could trigger a population burst ability and a role in hippocampal seizures [41].
because these neurons are synaptically interconnected. By The granule cells of the adult hippocampal dentate gyrus analogy, bursting of the relatively few hilar ectopic granule are now considered to be a dynamic population of neurons cells may recruit the normal granule cell population into that undergo neurogenesis throughout the adult mammalian epileptiform activity via their reciprocal synaptic conlife [2,6,11,17-20]. Parent et al. [29] and Bengzon et al.
nections. In this way, hilar ectopic granule cells could be [3] showed increased numbers of mitotically active cells critical for the synaptically driven reverberating excitation below the GCL differentiating into neurons with prepara-characteristic of the dentate gyrus in the epileptic, but not tions that labeled bromodeoxy-uridine (BrdU) sites of in the normal, brain [10,16,30,47]. incorporation into DNA and the early postmitotic marker, TOAD-64. They hypothesized that differentiation of a population of newly born granule cells, rather than re-Acknowledgements modeling of mature granule cells, is the basis for the network reorganization seen in some forms of human The authors gratefully acknowledge Marieta B. Leonor temporal lobe epilepsy. More recently, Scharfman et al.
and Debra A. Evenson for technical assistance and Donald [41] showed increased numbers of calbindin immuno-L. Pick and Alan M. Wong for comments on the manureactive granule-like cells in the deep hilus after pilocarscript. This work was supported by NIH grants NS 38331 pine-induced status epilepticus and suggested that newly to C.E. Ribak and NS 17771 to J. V. Nadler. born or previously existing granule cells migrate to this location. They also indicated that these ectopic cells contribute to the hyperexcitability present in this model References and may participate in the generation of spontaneous seizure activity because they display synchronous bursting  for understanding the mechanism of seizure propagation in from GABA interneurons [12,28] and lack of a synaptic