Mossy fibers are the primary source of afferent input to ectopic granule cells that are born after pilocarpine-induced seizures - PubMed (original) (raw)

Comparative Study

Mossy fibers are the primary source of afferent input to ectopic granule cells that are born after pilocarpine-induced seizures

Joseph P Pierce et al. Exp Neurol. 2005 Dec.

Abstract

Granule cell (GC) neurogenesis increases following seizures, and some newborn GCs develop in abnormal locations within the hilus. These ectopic GCs (EGCs) display robust spontaneous and evoked excitatory activity. However, the pattern of afferent input they receive has not been fully defined. This study used electron microscopic immunolabeling to quantitatively evaluate mossy fiber (MF) input to EGCs since MFs densely innervate the hilus normally and undergo sprouting in many animal models of epilepsy. EGC dendrites were examined in tissue from epileptic rats that had initially been treated with pilocarpine to induce status epilepticus and subsequently had spontaneous seizures. MF terminals were labeled with a zinc transporter-3 antibody, and calbindin immunoreactivity was used to label hilar EGCs and GC layer GCs. The pattern of input provided by sprouted MF terminals to EGC dendrites was then compared to the pattern of MF input to GC dendrites in the inner molecular layer (IML), where most sprouted fibers are thought to project. Analysis of EGC dendrites demonstrated that MF terminals represented their predominant source of afferent input: they comprised 63% of all terminals and, on average, occupied 40% and 29% of the dendritic surface in the dorsal and ventral dentate gyrus, respectively, forming frequent synapses. These measures of connectivity were significantly greater than comparable values for MF innervation of GC dendrites located in the IML of the same tissue sections. Thus, EGCs develop a pattern of synaptic connections that could help explain their previously identified predisposition to discharge in epileptiform bursts and suggest that they play an important role in the generation of seizure activity in the dentate gyrus.

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Figures

Fig. 1

CaBP immunoperoxidase-labeled cell bodies and dendrites within the hilus of a pilocarpine-treated animal, clustered near CA3c. Panel (B) is an enlargement of the region marked by the box in panel (A). ML, molecular layer; GCL, granule cell layer. Scale bar = 50 μm.

Fig. 2

Immunoperoxidase labeling for ZnT-3 in a pilocarpine-treated animal visualized both the normal MF pathway and a dense region of sprouted MFs in the IML. ML, molecular layer; GCL, granule cell layer. Scale bar = 50 μm.

Fig. 3

CaBP immunogold-labeled GC dendrites and ZnT-3 immunoperoxidase-labeled MF terminals in the IML of a pilocarpine-treated animal. LD, labeled dendrites; LT, representative labeled terminals; UT, representative unlabeled terminals; SP, spines. Arrows indicate representative immunogold particles. Scale bar = 0.5 μm.

Fig. 4

CaBP immunogold-labeled GC dendrites and ZnT-3 immunoperoxidase-labeled MF terminals in the hilus of a pilocarpine-treated animal. Labeled as in Fig. 3. Scale bar = 0.5 μm.

Fig. 5

Synapses (large arrows) formed by ZnT-3-labeled (LT) and unlabeled (UT) terminals in pilocarpine-treated animals and saline-injected controls. (A and B) In the hilus of saline-injected controls, asymmetric synapses with thick postsynaptic densities were formed by both small (A) and large (B) ZnT-3-labeled MF terminals (in panel B, the MF terminal partially envelops two spines (SP)). (C) An unlabeled hilar terminal from a pilocarpine-treated animal that formed a symmetric synapse in which the postsynaptic density is virtually absent (note however the presence of filamentous material within the synaptic cleft, providing evidence that an active zone was present). (D, E, and F) In the hilus (D, E) and IML (F) of pilocarpine-treated animals, ZnT-3-labeled MF terminals formed asymmetric synapses with both dendritic shafts (D) and spines (E, F) that often had less pronounced postsynaptic densities (IML MF synapses tended to have relatively thicker densities (F)). Small arrows indicate dense core vesicles in MF terminals. Scale bar = 0.1 μm.

Fig. 6

Although labeled MF terminals could often be observed in apposition to labeled GC dendrites in the IML, they most frequently formed synapses with small spines, some of which were also immunogold-labeled for CaBP. Labeled as in Fig. 3. Scale bar = 0.5 μm.

Fig. 7

MF terminals were at times observed clustered along the shafts of GC dendrites in the IML. Labeled as in Fig. 3. Scale bar = 0.5 μm.

Fig. 8

The most prominent characteristic of CaBP-labeled dendrites (A, B) in the hilus was the extent to which they were contacted by MF terminals. Labeled as in Fig. 3. Scale bar = 0.5 μm.

Fig. 9

MF terminals within the hilus could be observed forming synapses with spines as they emerged from the dendritic shaft. Labeled as in Fig. 3. Arrows indicate representative immunogold particles. Scale bar = 0.5 μm.

Fig. 10

Hilar MF terminals frequently formed axodendritic synapses with both large (A) and small (B) diameter EGC dendrites (small arrows indicate synaptic specializations formed by MF terminals). Labeled as in Fig. 3. Scale bar = 0.5 μm.

Fig. 11

Labeled GC dendrites in the IML were often seen in direct apposition with each other. Labeled as in Fig. 3. Scale bar = 0.5 μm.

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Mossy fibers are the primary source of afferent input to ectopic granule cells that are born after pilocarpine-induced seizures - PubMed (original) (raw)