Postnatal Changes of HRP-Labeled Corticospinal Neurons in Congenital Hydrocephalic Rats (HTX).

Postnatal Changes of HRP-Labeled Corticospinal Neurons in Congenital Hydrocephalic Rats (HTX)

Summary. In the present study we examined the influence of hydrocephalic changes on the growth of corticospinal neurons and the formation of their neuronal connection with the spinal cord in the congenital hydrocephalic rat (HTX) during the postnatal period, using the horseradish peroxidase (HRP)­ labeling method.

We injected 0.1-2.0J..Ll of 20%-50% HRP solution into the cervical cords of HTX rats which had either high or slight dilatation of the lateral ventricles, at postnatal days 1, 7, 14, and 21, and observed the HRP-labeled corticospinal neurons in the cerebral cortex and the structure of the brain stem each day.

At postnatal day 1, HRP-labeled neurons were found in layer V, which was in the dorsal and lateral areas of the cerebral cortex in both types of hydrocephalic rats. From this finding these neurons could be identified as corticospinal neurons and their axons seemed to reach into the spinal cord. By postnatal day 21, HRP-labeled corticospinal neurons were found in the area from the dorsal to the lateral part of the cerebral cortex in the hydrocephalic rats with slight dilatation of the lateral ventricles. However, in the hydro­ cephalic rats with high dilatation of the lateral ventricles, HRP-labeled cortico­ spinal neurons gradually decreased and a few neurons were found in the lateral part of the cerebral cortex. At postnatal day 21, also, degenerative changes appeared in the corticospinal tract through the pyramis of the medulla oblongata.

These findings indicate that the neuronal connection between the cerebral cortex and the spinal cord, made by the corticospinal axons, gradually dis­ appears when cortical neurons, including corticospinal neurons are destroyed, such destruction being caused by the progresive dilatation of the lateral ven­ tricles and subsequently by the secondary degeneration of the corticospinal axons.

Kohn et al. (1981) reported that, in the congenital hydrocephalic rat (HTX), dilatation of the lateral ventricles appeared at birth and that the thickness of the cerebral cortex gradually decreased, depending on the advancement of lateral ventricular dilatation. These changes seem to influence the differentia­ tion and growth of neurons and neuroglial cells in the cerebral cortex.

Some of the descending projection neurons in the cerebral cortex are corticospinal neurons, which connect the cerebral cortex and the spinal cord. In rats the corticospinal axons are already distributed in the spinal cord at postnatal day 1 (Joosten et al. 1987; Ohtani and Shirai 1988). Moreover, the synaptic formation of the corticospinal neurons advances on their axon terminals in the spinal cord and on their somas through postnatal day 14 (Ohtani et al. 1987; Miyabayashi and Shirai 1988).

In the present study the postnatal development of the corticospinal neurons in the congenital hydrocephalic rat (HTX) was investigated, using the HRP­ Iabeling method, in order to define the effect of hydrocephalic changes on the formation of neuronal connections by cerebral cortical neurons .

Keywords. Congenital hydrocephalic rat - HRP-Labeling method Corticospinal neurons- Cerebral cortex- Postnatal development.

Materials and Methods

HTX rats with slight and high dilatations of the lateral ventricles at postnatal days 1, 7, 14, and 21 were used. We anesthetized all rats with Nembutal, and immediately injected 0.1-2.0 J.d of 20%-50% solution of horseradish perox­ idase (HRP) into their cervical cords. One or two days after HRP injection, we perfused them with a fixative consisting of a 1% solution of paraformaldehyde

Fig. 1. HRP-injection site in the cervical cord of HTX rat at postnatal day 14. Most area of the spinal cord is stained dark brown with HRP. X 24

Fig. 2. A A frontal section of the cerebrum with slight dilatation of the lateral ventricles at postnatal day 1. HRP-labeled corticospinal neurons( ...) are found in layer V of the cerebral cortex. x 24. B A high magnification of the dorsal cerebral cortex in Fig. 2.

The corticospinal neurons (...) are labeled with HRP. x 380

and a 1.25% solution of glutaraldehyde and immersed their brains and spinal cords into a 20% solution of sucrose. We made serial sections of their brains and spinal cords in thicknesses of 60 or 100Jlm, reacted these using the tetra­ methylbenzidine (TMB) method (Mesulam 1978), and observed them with a light microscope.

Results

Injection Site of HRP in the Cervical Cord

HRP was injected into almost the whole area of the cervical cord at postnatal days 1, 7, 14, and 21 (Fig. 1).

Distribution of HRP-Labeled corticospinal Neurons in the Cerebral Cortex At postnatal day 1, the cerebral cortex consisted of the molecular layer, the cortical plate, including many small cells, and layer V, containing large cells with slight and high dilatations of the lateral ventricles. HRP-Iabeled corticospinal neurons were found in layer V of the dorsal cortex with both slight and high dilatations of the lateral ventricles (Figs. 2a and 3a). In higher magnification the somas and proximal dendrites of the corticospinal neurons were found to be labeled with HRP (Figs. 2b and 3b).

At postnatal day 7, the dorsal area of the cerebral cortex with the high dilatation of the lateral ventricles appeared to have a thinner cerebral wall than the area with the slight dilatation; HRP-labeled neurons were localized in layer V of both cortices.

Fig. 3. A A frontal section of the cerebrum with high dilatation of the lateral ventricles at postnatal day 1. HRP-labeled corticospinal neurons ( ) are found in layer V of the dorsal cerebral cortex. x 24. 8 A high magnification of the dorsal cerebral cortex in Fig. 3A. The corticospinal neurons ( ) are labeled with HRP. x 380

At postnatal day 14, the cerebral cortex with the high dilatation (Fig. 4a) became thinner than that with the slight dilatation (Fig. Sa). The thinnest area appeared in the dorsal area of the cortex with the high dilatation. Many rows of HRP-labeled neurons were found in layer V of the cortex with the slight dilatation (Fig. Sa); however, several rows of these neurons were found in the same layer of the dorso-lateral part of the cortex with the high dilatation (Fig. 4a). The HRP-labeled neurons formed pyramidal cells in the cortex with the slight dilatation and formed such cells in the dorso-lateral part of the cortex with the high dilatation [Figs. Sb and 4b). The same neurons became smaller and flattened in the dorsal part of the cortex with the high dilatation (Fig. 4c).

At postnatal day 21, HRP-labeled neurons were stratified in layer V of the cortex with the slight dilatation, and the growth of their somas increased (Fig. 6a). Only a few HRP-labeled neurons were found in the dorsal part and a few HRP-labeled small neurons were found in the lateral part (Fig. 6b).

Degenerative Changes Appearing in the Pyramis of the Medulla Oblongata of HTX Rats with High Dilatation of the Lateral Ventricles At postnatal day 21, many macrophages appeared in the pyramis of the lower medulla oblongata and, simultaneously, degeneration of the corticospinal axons was found in this area (Fig. 7).

Discussion

In the normal rat, the corticospinal tract distributed in the posterior horn and the intermediate zone made immature synapses with the developing neurons in these zones at postnatal day 1, and formed a compound synaptic organization, similar to a glomerulus, through postnatal day 14 (Miyabayashi and Shirai 1988).

Also, retrograde labeling studies with HRP have shown that corticospinal neurons were localized in layer V of the cerebral cortex and that their axons reached the cervical cord. By postnatal day 14, their distribution formed an adult pattern and synaptogensis on their somas advanced (Ohtani et al. 1987; Ohtani and Shirai 1988).

In this study we examined the effect of hydrocephalic changes on the postnatal development of corticospinal neurons in the cerebral cortex of the

Fig. 5. A A frontal section of the cerebrum with slight dilatation of the lateral ventricles at postnatal day 14. Several rows of HRP-labeled corticospinal neurons (..,.) are found in layer V of the dorsal cerebral cortex. X 24. B A high magnification of the dorsal cerebral cortex in Fig. 5A. The somas and the proximal dendrites of the corticospinal neurons (..,.) are labeled with HRP. x 380

congenital hydrocephalic rat (HTX), compared with corticospinal neurons in the normal rat. At postnatal day 1, HRP-labeled neurons were found in layer V of the cerebral cortex. Therefore, this pattern was the same as that found in the normal rat. Also, it was confirmed that axons of the corticospinal neurons reached the spinal cord not only in the hydrocephalic rat with slight dilatation of the lateral ventricles, but also in the hydrocephalic rat with high dilatation of these ventricles. These findings seemed to show that the outgrowth of axons from the corticospinal neuronal somas to the spinal cord advanced without

Fig. 6. A HRP-Iabeled corticospinal neurons( ) are found in the dorsal cerebral cortex with slight dilatation of the lateral ventricles at postnatal day 21. x 380. B A few HRP­ labeled corticospinal neurons ( ) are found in the lateral cerebral cortex with high dilatation of the lateral ventricles at postnatal day 21. x 380 being influenced by the progress of lateral ventricular dilatation caused by the birth.

However, as Kohn et al. (1981) have described, the dilatation of the lateral ventricles became remarkable, and subsequently the thickness of the cerebrum reduced during postnatal development. In this process HRP-labeled cor­ ticospinal neurons gradually decreased in number. In particular, a few HRP­ labeled neurons were scattered in layer V of the lateral part of the cerebral cortex at postnatal day 21. At the same time, axons of the corticospinal neurons appeared to undergo degenerative changes through the pyramis of the

Fig. 7. A transverse section of the lower medulla oblongata with high dilatation of the lateral ventricles in the same hydrocyphalic rat as that shown in Fig. 6B at postnatal day 21. The degenerative changes of the corticospinal tract appear in the pyramis of the ventral part. X 24 medulla oblongata. This degeneration in the corticospinal axons seemed to be caused by changes in the corticospinal neurons, depending on the advancement of lateral ventricular dilatation and immediately the anterograde degeneration of the corticospinal axons. The neuronal connection between the cerebral cortex and the spinal cord, through the corticospinal axons, was gradually lost by because of the disappearance of the corticospinal neurons and the degenera­ tion of their axons.

References

Joosten EA, Gribnau AAM, Dederen JWC (1987) An anterograde tracer study of the developing corticospinal tract in the rat: three components. Dev Brain Res 36: 121-130

Kohn DF, Chinookoswong N, Chou SM (1981) A new model of congenital hydro­ cephalus in the rat. Acta Neuropathol (Berl) 54: 211-218

Mesulam M-M (1978) Tetramethylbenzidine for horseradish peroxidase neurochemistry: a noncarcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents. J Histochem Cytochem 26 : 106-117

Miyabayashi T, Shirai T (1988) Synaptic formations of the corticospinal tract in the rat spinal cord . Okajimas Folia Anat Jpn 65:117-140

Ohtani R, Shirai T (1988) The development of corticospinal tract (CST) neurons. Proceedings of the 6th M Singer Symposium pp 473-483

Ohtani R, Shirai T, Kato H (1987) The postnatal distributional changes and synaptogenesis of corticospinal tract neurons in the cerebral cortex of the rat with HRP-labeling method (abstract). Soc Neurosci 13: 1430