Selective death of hippocampal CA3 pyramidal cells with mossy fiber afferents after CRH-induced status epilepticus in infant rats

Abstract

Previous studies of CRH-induced status epilepticus in infant rats demonstrated neuronal loss in several limbic structures, including the CA3 region of the hippocampus. The goal of the present study was to identify the neurons affected by CRH-induced seizures and determine whether they formed synapses with afferent axon terminals. Clusters of neurons in the CA3 region of the hippocampus were osmiophilic when viewed in thick sections. Semi-thin 2-microns sections of the pyramidal cell layer contained dark, shrunken neurons with apical and basal dendrites among normal appearing pyramidal cells. Electron microscopy revealed degenerating pyramidal cells with intact cell membranes and electron dense nuclei and cytoplasm. The shrunken dendrites of these cells had spines and were postsynaptic to large immature-appearing mossy fibers. Thus, CA3 pyramidal neurons that are linked via mossy fibers to the tri-synaptic excitatory hippocampal circuit die subsequent to CRH-induced status epilepticus. The shrunken appearance and selective loss of these neurons are incompatible with necrosis as the mechanism of degeneration.

Fig. 1

Photomicrographs of a semi-thin 2-µm section of the hippocampus obtained from a 12-day-old rat following CRH-induced SE. A shows the CA3b region with several dark, shrunken neurons (large arrows) in the pyramidal cell layer (P). No degenerating neurons were observed in strata oriens (O) and lucidum (L). B shows an enlargement of the degenerating pyramidal cells (arrows) indicated in A. Note the normal looking adjacent pyramidal cells and the basal dendrites (arrowheads) that arise from some of the dark neurons. Magnifications: A = ×400, B = ×800.

Fig. 2

Electron micrographs of an electron dense, degenerating cell body of a neuron in the pyramidal cell layer of CA3b. This thin section was obtained from the same tissue block as Fig. 1. A shows the nucleus (N) of this neuron, its large apical dendrite oriented toward stratum lucidum, and two normal looking pyramidal cells (P). A portion of another degenerating neuron appears in the upper right-hand corner. B shows an enlargement of the nucleus in A with its prominent dark nucleolus (Nu) and dense heterochromatin. Granular endoplasmic reticulum (er) is evident in the electron dense cytoplasm of this neuron. Symmetric axosomatic synapses (large arrows) are found on adjacent pyramidal cells and on the degenerating cell body (small arrow). A = ×5000; B = 18,500.

Fig. 3

Electron micrographs of dendrites in stratum lucidum of CA3b that arise from degenerating neurons in the pyramidal cell layer. A shows two longitudinally-sectioned, degenerating dendrites (d and arrow) found in the neuropil that also contains many normal cross-sectioned dendrites and large axon terminals. B shows a stubby spine from the dendrite at the top of A. Two axon terminals with round synaptic vesicles form synapses (probably asymmetric, arrows) with the electron dense dendrite (left) and a stubby spine (right). C shows a pedunculate spine (large arrow) and an axodendritic synapse (small arrow) formed by a large axon terminal with round synaptic vesicles and a few dense core vesicles, similar in appearance to a developing mossy fiber axon terminal. A = ×10,000; B = ×37,000; C = ×28,000.