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. 2000 Aug 15;20(16):6144-58.
doi: 10.1523/JNEUROSCI.20-16-06144.2000.

Granule-like neurons at the hilar/CA3 border after status epilepticus and their synchrony with area CA3 pyramidal cells: functional implications of seizure-induced neurogenesis

H E Scharfman  1 , J H Goodman, A L Sollas
Affiliations

Granule-like neurons at the hilar/CA3 border after status epilepticus and their synchrony with area CA3 pyramidal cells: functional implications of seizure-induced neurogenesis

H E Scharfman et al. J Neurosci. .

Abstract

A group of neurons with the characteristics of dentate gyrus granule cells was found at the hilar/CA3 border several weeks after pilocarpine- or kainic acid-induced status epilepticus. Intracellular recordings from pilocarpine-treated rats showed that these "granule-like" neurons were similar to normal granule cells (i. e., those in the granule cell layer) in membrane properties, firing behavior, morphology, and their mossy fiber axon. However, in contrast to normal granule cells, they were synchronized with spontaneous, rhythmic bursts of area CA3 pyramidal cells that survived status epilepticus. Saline-treated controls lacked the population of granule-like cells at the hilar/CA3 border and CA3 bursts. In rats that were injected after status epilepticus with bromodeoxyuridine (BrdU) to label newly born cells, and also labeled for calbindin D(28K) (because it normally stains granule cells), many double-labeled neurons were located at the hilar/CA3 border. Many BrdU-labeled cells at the hilar/CA3 border also were double-labeled with a neuronal marker (NeuN). Taken together with the recent evidence that granule cells that are born after seizures can migrate into the hilus, the results suggest that some newly born granule cells migrate as far as the CA3 cell layer, where they become integrated abnormally into the CA3 network, yet they retain granule cell intrinsic properties. The results provide insight into the physiological properties of newly born granule cells in the adult brain and suggest that relatively rigid developmental programs set the membrane properties of newly born cells, but substantial plasticity is present to influence their place in pre-existing circuitry.

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Figures

Fig. 1.
Fig. 1.
Neuronal loss and mossy fiber sprouting in a kainic acid-treated rat and a saline-injected control.A, Immunocytochemistry using an antibody to a neuronal nuclear protein (NeuN) demonstrates neuronal distribution in a saline-treated control rat. B, Neuropeptide Y immunoreactivity in an adjacent section to the one shown inA demonstrates numerous immunoreactive cells in the hilar region (arrowheads) and also fibers in the molecular layer. In this and all other figures: G,granule cell layer; H, hilus; P,pyramidal cell layer. C, NeuN staining in a section from a kainic acid-treated rat that was perfused 2.75 months after status. There is cell loss in superficial layers of the entorhinal cortex (arrows), but substantial preservation of neurons elsewhere. D, Neuropeptide Y staining in an adjacent section to the one shown in C illustrates immunoreactivity in mossy fiber axons of dentate gyrus granule cells, which are unstained in the saline-treated control (B). This example is representative of other rats with spontaneous seizures, where mossy fiber sprouting in the inner molecular layer (arrows) was evident. Scale bar, 500 μm.
Fig. 2.
Fig. 2.
CaBP-immunoreactive cells at the hilar/CA3 border in a kainic acid-treated rat. A, B, CaBP immunocytochemistry illustrates a population of cells at the hilar/CA3 border that were absent in saline-treated controls (D). B is a higher magnification of A.Arrows in A,B point to CaBP-immunoreactive cells. Same animal as shown in Figure 1. C, In an adjacent section to the one shown in A and B, NeuN staining demonstrate numerous hilar neurons. Scale bar: A, D, 100 μm; B, 50 μm; C, 200 μm.
Fig. 3.
Fig. 3.
CaBP-immunoreactive neurons were not stained using an antibody to GABA. A, CaBP-stained cells in a pilocarpine-treated rat that was examined 10 months after status epilepticus. The asterisk marks a blood vessel that is also marked in B. B, An adjacent section to the one shown in A, stained with an antibody to GABA. Numerous immunoreactive cells are located in and around the granule cell layer (arrows), but not in the hilar region.C, CaBP immunoreactivity in a section from the septal pole of the same animal. Fewer immunoreactive cells and processes were evident compared to the section from the midhippocampal region (A), an example of variation in CaBP immunoreactivity along the septotemporal axis of the hippocampus. D, Higher magnification of the section in part A showing the CaBP-labeled cells (arrows). Scale bar: A, C, 250 μm;B, 100 μm; D, 50 μm.
Fig. 4.
Fig. 4.
Markers of GABA neurons and glia in the dentate gyrus in a pilocarpine-treated rat with chronic seizures.A, Cresyl stain at low power shows relative preservation of neurons in the dentate gyrus of a pilocarpine-treated rat, although other areas of the hippocampus are shrunken. This animal was examined 4 months after status epilepticus and had recurrent motor seizures. InA–H, CA3 is to the right, and CA1 is up. B, C, CaBP immunoreactivity in two different sections from the same animal. B was an adjacent section to the one inA, and C was a section from the extreme septal pole of the hippocampus. Immunoreactive neurons in the hilus are marked by arrows. D, Neuropeptide Y immunoreactivity of a section adjacent to B shows mossy fiber sprouting in the inner molecular layer (double arrows) and a few immunoreactive cells in the granule cell layer (arrow), but not the hilus. E, F,Sequential sections adjacent to D that show immunoreactivity to parvalbumin (E), calretinin (F), somatostatin (G), and GFAP (H). Immunoreactive cells are marked by arrows. Scale bar: A, 400 μm;B–H, 100 μm.
Fig. 5.
Fig. 5.
Intracellularly labeled “granule-like” cells in a pilocarpine-treated rat with chronic seizures. A,An intracellularly labeled granule-like cell recorded near the end of the pyramidal cell layer (arrowhead) and granule cells recorded in the granule cell layer (arrow). Orientation: CA3 is to the right, and CA1 is down. B, A different granule-like cell from another slice taken from an animal that had 32 observed seizures in 2.25 months after status. 1, A drawing of the dendrites; 2, orientation of the cell;3, axon; 4, 5, photomicrographs of mossy fiber boutons along the axon. Dotted lines in 2 and3 outline the cell layers. Arrowheads in3–5 point to mossy fiber boutons. Arrowin 3 points to the soma of the cell. Large arrows in 3 indicate areas where axon collaterals were found in the inner molecular layer. The segments shown in 4 and 5 correspond to the areas marked with those numbers in 3. F,Hippocampal fissure. Scale bar: A, 100 μm;B1, 50 μm; B2, 250 μm;B3, 200 μm; and B4,5, 25 μm.
Fig. 6.
Fig. 6.
APs of neurons recorded at the hilar/CA3 border in pilocarpine-treated rats with chronic seizures. A, An AP evoked at threshold by intracellularly injected current (150 msec pulse). The start and end of the pulse are marked by small arrows. The recording was from a granule cell in the granule cell layer in a pilocarpine-treated rat that was examined 4.5 months after status. Inset, The AP is shown with a different scale to illustrate the relatively fast rise and slow decay of granule cell APs compared to interneurons (C). Resting potential, −70 mV. B, An AP from a granule-like cell at the hilar/CA3 border in a different pilocarpine-treated rat that was killed 1 month after status. Note the similarity of the AHP inA and B. The three phases of the AHP are indicated by numerals at the peak of each phase. Resting potential, −73 mV. C, An AP from a hilar/CA3 neuron with the morphology and physiological characteristics of a GABA neuron (“interneuron”). Same slice as used for part A. The AHP differs from granule cell AHPs in both amplitude and kinetics. Resting potential, −65 mV. D, A triplet of APs evoked at threshold in a CA3c pyramidal cell at the hilar border in a pilocarpine-treated rat, 9 months after status. Intrinsic bursts of APs are characteristic of these cells (Wong and Prince, 1981; Scharfman, 1993; Smith et al., 1995). Resting potential, −60 mV.
Fig. 7.
Fig. 7.
Firing behavior of neurons at the hilar/CA3 border. Responses to current injection were recorded from the same cells shown in Figure 6. The amount of injected current was manipulated so that each neuron fired four APs within 150 msec. A, B, Firing behavior of a granule cell in the granule cell layer (A) and a granule-like cell at the hilar/CA3 border (B) were similar in that trains of APs occurred with strong spike frequency adaptation. A,Resting potential, −71 mV. B, Same cell as Figure6B. C, Firing behavior of an interneuron demonstrated weak spike frequency adaptation. Same cell as Figure 6C. D, A CA3c pyramidal cell had an intrinsic burst of APs in response to injected current. Same cell as Figure 6D. The arrowheads inC and D point to spontaneous synaptic potentials, depolarizing in C and hyperpolarizing inD. E, Comparison of the mean (± SEM) interspike intervals of the train of four APs are compared for the three cell types firing in trains (granule cells, granule-like cells, and interneurons). F, An f–I plot for the same group of cells as in part E. The mean value of the first interspike interval is plotted as a function of injected current.
Fig. 8.
Fig. 8.
Simultaneous bursts recorded from granule-like cells intracellularly and the CA3 pyramidal cell layer extracellularly.A, Recordings of spontaneous activity from a granule-like cell (top; same cell as shown in Fig.5B) and a simultaneous recording from the CA3b pyramidal cell layer (bottom) show spontaneous bursts that were periodic and synchronized. Resting potential, −74 mV. B, C, A sample record of one burst with different time bases illustrates the onset of APs in the granule-like cell, and the onset of population spikes in the CA3b field were tightly synchronized.
Fig. 9.
Fig. 9.
Simultaneous bursts recorded simultaneously in a CA3c pyramidal cell and CA3b of the pyramidal cell layer.A, Recordings in the same slice as used for Figure 8, showing simultaneous bursts in a CA3c pyramidal cell (top; resting potential, −58 mV) and the CA3b pyramidal cell layer. B, C, Expanded traces of a burst recorded simultaneously in the pyramidal cell (top) and pyramidal cell layer (bottom) show that the bursts begin at similar times.
Fig. 10.
Fig. 10.
Newly born CaBP-stained neurons at the hilar/CA3 border in pilocarpine-treated rats with spontaneous seizures. In a pilocarpine-treated rat 2.5 months after status, CaBP-stained hilar/CA3 neurons were often double-labeled with an antibody to BrdU. BrdU was injected 4–11 and 26–30 d after status epilepticus (see Materials and Methods). A, The hilar/CA3 border had numerous double-stained cells (i.e., CaBP-positive/BrdU-positive). Numerous BrdU-labeled cells were also located in the subgranular zone.Arrowhead in A points to the same CaBP-positive branched process that is marked by anarrowhead in B. Scale bar (inE), 100 μm. B–E, The hilar/CA3 border near the arrowhead in A is shown at higher power. Double-stained cells are marked by arrows. Several focal planes are shown to illustrate that the CaBP-positive cytoplasm and BrdU-positive nucleus go in and out of focus together. This indicates true double-labeling, as opposed to juxtaposed cells, such as a CaBP-positive, BrdU-negative cell lying under a BrdU-positive, CaBP-negative cell. Scale bar, 50 μm.
Fig. 11.
Fig. 11.
NeuN-stained neurons at the hilar/CA3 border in pilocarpine-treated rats with spontaneous seizures. A,The hilar/CA3 border stained for NeuN and BrdU. Same animal as Figure10. Numerous double-labeled cells (NeuN-positive/BrdU-positive) are located in this area (arrows), as well as the base of the granule cell layer. Asterisks in Aand B mark CA3c pyramidal cells. Scale bar (inE), 100 μm. B–E, A focal series through the hilar/CA3 border, showing numerous NeuN-labeled cells also are labeled by BrdU (arrows). Different animal fromA. Nine seizures were observed in this rat in the 2.25 months between status and perfusion-fixation. Scale bar, 50 μm.
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