Deafferentation causes apoptosis in cortical sensory neurons in the adult rat

Abstract

The present study provides an experimental model of the apoptotic death of pyramidal neurons in rat olfactory cortex after total bulbectomy. Terminal transferase (TdT)-mediated deoxyuridine triphosphate (d-UTP)-biotin nick end labeling (TUNEL), DNA electrophoresis, and neuronal ultrastructure were used to provide evidence of apoptosis; neurons in olfactory cortex were counted by stereology. Maximal TUNEL staining occurred in the piriform cortex between 18 and 26 hr postbulbectomy. Within the survival times used in the present study (up to 48 hr postlesion), cell death was observed exclusively in the piriform cortex; there was no evidence of cell death in any other areas connected with the olfactory bulb. Neurons undergoing apoptosis were pyramidal cells receiving inputs from, but not projecting to, the olfactory bulb. The apical dendrites of these neurons were contacted by large numbers of degenerating axonal terminals. Gel electrophoresis of DNA purified from lesioned olfactory cortex showed a ladder pattern of fragmentation. Inflammatory cells or phagocytes were absent in the environment of degenerating neurons in the early stages of the apoptotic process. The present model suggests that deafferentation injury in sensory systems can cause apoptosis. In addition, olfactory bulbectomy can be used for investigating molecular mechanisms that underlie apoptosis in mature mammalian cortical neurons and for evaluating strategies to prevent the degeneration of cortical neurons.

Fig. 1.

Simplified anatomy of the lesion implemented in the present study and its consequences (left) as compared with the normal circuitry (right) between the olfactory bulb (OB) and piriform (olfactory) cortex (PO). Connections are projected against the ventral surface of the rat forebrain. Although the lesion disrupts many connections of the olfactory bulb, anterior olfactory nucleus, and piriform cortex, the critical disconnection leading to apoptosis of cortical neurons is the removal of the mitral cell input to pyramidal cells in layer IIa. The majority of these neurons projects to interneurons and other pyramids within the piriform cortex.

Fig. 2.

Specificity (A, B) and types (C) of TUNEL staining in olfactory cortical neurons postbulbectomy. A, B, Two magnifications of the basal forebrain region (at the level of the decussation of the anterior commissure ipsilateral to the lesion) showing conspicuous TUNEL labeling in olfactory cortex (arrows) against a clear background. C, Apoptotic olfactory neurons show distinct nuclear TUNEL patterns corresponding to chromatin fragmentation in round pieces (1, most common), uniform chromatin condensation (2, common), and chromatin margination against the nuclear membrane (3, occasional). Scale bars: A, 1 mm; B, 0.5 mm;C, 20 μm.

Fig. 3.

Primary olfactory cortex (PO) is the only brain area connected with the olfactory bulb that undergoes apoptosis after bulbectomy. Sections shown in this figure represent two forebrain planes in which olfactory cortex is continuous with other structures interconnected with the bulb [i.e., with the anterior olfactory nucleus (AON; A, B) and the olfactory tubercle (Tu; C, D)]. Sections have been processed for TUNEL and counterstained with cresyl violet.B and D represent magnifications of the areas of transition in A and C that are indicated with arrowheads. A, B, Apoptotic profiles (B, arrows) are contained strictly within the boundaries of PO; none is seen beyond thePO–AON border. A blood vessel (V) is indicated for the purpose of orientation. C, D, Visualization of apoptotic cells (D, arrows) stops abruptly at the border between PO and Tu. Other labeled structures are the endopiriform nucleus (En), fundus striati (FSt), substantia innominata (SI), and magnocellular preoptic nucleus (MPN). Scale bars: A, C, 250 μm;B, D, 60 μm.

Fig. 4.

Apoptotic profiles in the rat olfactory cortex 22 hr postbulbectomy as depicted with TUNEL (A, B), an aniline dye (cresyl violet; C, D), and a combination of the two (E, F). A, B, TUNEL-stained nuclei cluster in layer IIa.B, A magnification of the framed area inA. Magnifications: A, 20×;B, 40×. C, D, Apoptotic profiles in layer IIa depicted with cresyl violet. Magnifications:C, 40×; D, 100×. E, F, TUNEL staining followed by cresyl violet counterstain shows similarities and differences of profiles visualized by the two methods. Compare E and F with single-labeledA–D. Note fragmented TUNEL nuclei (single arrow), nuclei diffusely stained with TUNEL (two arrows), and TUNEL-stained nuclei with marginated chromatin (three arrows). Also note apoptotic profiles stained with cresyl violet (thin arrow) and with both TUNEL and cresyl violet (star). In general, cresyl violet preferentially reveals fragmented nuclei and stains small, intensely basophilic particles frequently seen outside the neurons and representing apoptotic bodies (the end products of apoptosis). These bodies are visualized almost exclusively with cresyl violet. Scale bars: A, 80 μm; B, C, 40 μm;D–F, 20 μm.

Fig. 5.

Morphological features of degenerating olfactory cortical neurons and the surrounding neuropil 22 hr postbulbectomy. A, A′, Four apoptotic profiles (arrows) are featured in this field. All apoptotic profiles show intense cytoplasmic blebbing. A few myelinated axons are also present (x). A′, A magnification of the rightmost apoptotic neuron and the associated neuropil in A. This dying neuron, like other apoptotic cells in A, is surrounded by newly formed astrocytic processes (a), which contain occasional mitochondria but are devoid of typical filament bundles. Dendrites (d), featured by regularly spaced, densely packed microtubules and the abundance of mitochondria, also are labeled to facilitate orientation in the illustrated field. B, This high-power micrograph reveals the close association (in this case, literal engulfment) of apoptotic neurons with astrocytic processes (a). Compare the morphology of astrocytic processes with that of longitudinally and transversely sectioned dendrites (d). An axonal terminal (t) and a myelinated axon (x) are shown also. Magnifications:A, 3000×; A′, 12,000×;B, 6000×.

Fig. 6.

Disposition of phagocytic glial cells toward apoptotic neurons of the olfactory cortex shown in preparations stained for the myelomonocytic epitope ED1. A, B, Cells in the vicinity of degenerating cortical neurons (A, arrowheads) do not express the phagocytic epitope ED1. However, adjacent vascular pericytes are ED1-positive (A,arrows), as are cells in pial vessels in the ventral aspect of the brain underneath the lesioned olfactory cortex (B, arrows). Scale bars:A, 40 μm; B, 20 μm.

Fig. 7.

Molecular characterization of the type of cell death in olfactory cortex of bulbectomized rats, based on the “ladder” pattern of DNA fragmentation by gel electrophoresis. Segments of DNA columns showing laddering are delineated (arrows). Note that at later time points (24hvs 22h) ladder bands assume smaller molecular sizes. Examples of oligonucleosomal bands are 360 Da (bottom arrow, 22h), 720 Da (top arrow,22h), and 540 Da (middle arrow,24h). Lane C depicts DNA from control, unlesioned olfactory cortex. Lane M contains molecular weight markers.

Fig. 8.

Quantitation of TUNEL-stained cells at different times postbulbectomy (A) and at different anteroposterior planes (B). A, This time course illustrates that cell death begins at 14 hr and reaches a peak 22–26 hr postlesion. By 48 hr, cell death has almost ceased. Numbers represent TUNEL profiles throughout the extent of the olfactory cortex. B, Distribution of cell death from the anterior part of the olfactory cortex (closest to the lesion) to the most posterior part at three time points postlesion. Distance (in mm) is measured from the beginning of the olfactory cortex (corresponding to 0.0). A minimal number of cells undergo death 14 hr postlesion. The marked increase in cell death at 18 hr postlesion is especially evident in the anterior olfactory cortex. As the apoptotic process evolves (26 hr), the intensity of cell death migrates caudally.

Fig. 9.

Neuronal loss in the olfactory cortex 48 hr postbulb-ectomy was measured by stereology. The total number of neurons in layer II of olfactory cortex 48 hr postlesion (n = 5) is 18% less than in unlesioned animals (n = 5). This difference represents an average loss of 53,400 neurons (p = 0.02).

Fig. 10.

The design (left) and results (right) of the retrograde tracing experiment. FB injections into the olfactory bulb (OB) and piriform cortex (PO) were implemented to define projection targets of dying cortical neurons. Axotomized neurons projecting to the olfactory bulb (top neuron in thediagram) remain intact after the lesion. Nonaxotomized neurons projecting within piriform cortex (bottom neuronin A, corresponding to neurons in A–C) undergo apoptosis. A, Portion of the olfactory cortex of a bulbectomized rat retrogradely labeled with FB injected into the olfactory bulb before the lesion and counterstained with propidium iodide. The animal survived for 24 hr. FB labels neurons in layer IIb and does not colocalize in apoptotic cells (arrowsindicate an apoptotic neuron). B, C, Two apoptotic profiles (arrows) labeled postinjection of FB into the posterior piriform cortex. Fluorescent labeling shows that they are projection (pyramidal) neurons. The fact that these neurons cannot be labeled postinjection into olfactory bulb but become apoptotic postbulbectomy indicates that they are trans-synaptically affected by the lesion. Arrowheads in B show the axon of an apoptotic profile undergoing Wallerian degeneration. Scale bars, 10 μm.

Fig. 11.

Degenerating neuronal profiles in olfactory cortex after bulbectomy belong to cells receiving inputs from the olfactory bulb, as evidenced by ultrastructural observations on layer I of the olfactory cortex 12–15 hr postlesion. A, A field taken from layer I of control olfactory cortex featuring normal asymmetrical synaptic contacts between two terminals (t₁ andt₂) and two synaptic spines (s₁ ands₂). Two adjacent normal dendrites are indicated also (d).B, A normal asymmetrical contact between an axonal terminal (t) and a dendritic spine.s, Spine; d, dendrite; sa, spinal apparatus. C, D, A field from the lesioned olfactory cortex showing a normal asymmetrical axonospinous synapse (t, terminal; s, spine; d, dendrite) beside a densely degenerating terminal (right, arrow). D, A further magnification of C. The uniformly round vesicles in terminals depicted in C and D (as well as in B) are consistent with excitatory neurotransmission. Transversely sectioned microtubules are in evidence in the receiving dendrite in D. E, F, A field from a lesioned olfactory cortex showing in detail a densely degenerating axonal terminal (t) contacting the spine (s) of a dendrite (d) in an asymmetrical manner. There is little residual structure in the receiving spine and dendrite, both of which exhibit abnormal vesicular elements (F, arrows). Magnifications: A, 26,500×; B, 58,500×; C, 41,000×;D, 66,000×; E, 64,500×;F, 86,500×.