Stimulation ofSnell dwarf mouse neuronal growth by GH and T4

@Copyright 1984 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0734-600X/84/0600-0123 $03.20

Stimulation of Snell Dwarf Mouse Neuronal Growth by GH and T4

TETSUYA NOGUCHI, ~' TETSURO SUGISAKI, AND YASUZO TSUKADAt

Department of Physiology, Toho University School of Medicine, Ohmori-nishi, Tokyo-143 and ?Department of Physiology, Keio

University School of lVledicine, Shinanornachi, Tokyo-160, Japan

Received December 27, 1983; Accepted May 22, 1984

ABSTRACT

An investigation of the Snell dwarf motor cortex, area 6 of Caviness, was carried out by means of a modified Golgi silver impregnation method. The pyramidal neurons located in layer V were found to have small perikarya, short primary dendrites with sparse branchings. Following administration of bovine growth hormone (bGH) and thyroxine (T4) during the first 20 d of postnatal life, this retarded neuronal growth was restored to normal: neuronal perikarya were enlarged, and the dentrites were thicker with denser branchings. These results were confirmed in the sections stained for neuron specific enolase (NSE).

These findings demonstrate that exogenous GH and T4, will (a) enhance neuronal growth in the Snell dwarf cerebrum, and (b) restore neuronal arborization to normal.

Index Entries: Neuronal growth, stimulation by GH and T4; growth, stimulation of neuronal by GH and T4; Snell dwarf mouse neuronal growth, stimulation by GH and T4; mouse neuronal growth, stimulation by GH and T4; growth hormone, stimulation of Snell dwarf mouse neurons by; thyroxine, stimulation of Snell dwarf mouse neuronal growth by; Golgi-Cox method; neuron-specific enolase.

*Author to whom all correspondence and reprint requests should be addressed.

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124 NoguchL Sugisaki, and Tsukada

INTRODUCTION

Pituitary-deficient Snell dwarf mice (dw/dw) (Snell, 1929), in comparison with normal controls (+/?), have lower brain weight, decreased DNA content and hypomyelination in the cerebrum, and a strikingly reduced level of spontaneous locomotion activity and indistinct diurnal periodic- ity (Noguchi et al., 1982b). More recently, we have observed that the pyramidal neurons of the Snell dwarf motor cortex have small perikarya, short primary dendrites with sparse branchings, and a low spine density on the dendrites (Noguchi et al., 1983).

There is a growing body of clinical (Laron and Galatzer, 1980) and experimental (Noguchi et al., 1980; 1982a; 1982b; Roger et al., 1974; Sarlieve, 1983) evidence that growth hormone (GH) potentiates cerebral development. In previous papers (Noguchi et al., 1982b; 1982c; 1982d), we reported that the first series of deficiencies found in the Snell dwarf cerebrum results from reduced oligodendroglial proliferation caused by lack of circulating GH, and that acceleration of retarded myelinogenesis, possibly through the enhancement of glial cell division, depends essen- tially upon GH level with the synergistic effects of thyroid hormone (T4). In addition, we have found that the administration of bovine GH (bGH) with T4 during the first 20 d of postnatal life, the period of maximal glial cell proliferation, greatly ameliorated the first series of deficiencies, as well as distinctly improving myelinogenesis in the corpus callosum (Sugisaki et al., submitted).

It is well established that the neuronal body, as well as its processes, also keeps growing during this early postnatal period (Fazekas et al., 1951; Schapiro, 1968). Accordingly, the hormones, GH and T4, might have effects on neuronal growth.

In an extension of this earlier work, we now report our findings on the effects of bGH and T 4 o n the abnormal neuronal arborization of the Snell dwarf motor cortex. To study these neuronal changes, we used the Ramon-Moliner modification of the Golgi-Cox silver impregnation method (Ramon-Moliner, 1975), and immunohistochemical staining for neuron specific enolase (NSE), a neuron marker.

MATERIALS AND METHODS

Reagents Bovine growth hormone (bGH, Somatotropin, code no. 77-001, lot

no. 12) was purchased from Miles Laboratories, USA. L-Thyroxine (T4) was purchased from Sigma Chemical Co., USA. Rabbit antineuron- specific enolase antiserum (code no. 61010, lot no. L-201) was from IBL Co., Takasaki, Japan. Sheep anti-rabbit IgG and rabbit antiperoxidase antisera were from Miles-Yeda Ltd., Israel. Other chemicals were of ana- lytical grade.

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GH and L in Neuronal Growth 125

Animals

The Snell dwarf mice were produced by mating known heterozygous pairs, originally obtained from the Jackson Laboratory, Maine, USA. The mice were kept in an air-conditioned room at 22-25~ with a controlled light cycle; light was preset to come on at 0600 and to go off at 1800. A standard mouse diet (Oriental Yeast Co., Tokyo) and water were given ad libitum.

Newborn mice from a heterozygous mating were used as experimental animals. Hormones were administered once daily at 15:00 by subcutaneous injection, in order to coincide with the diurnal changes of circulating GH level found in the control group. The volume injected was adjusted to 0.1 mL per mouse by dilution with saline. The normal control mice (designated as +/?) were used without regard to their actual geno- type, because examination of the organs of heterozygous (dw/+) or homozygous (+/+) normal animals at various ages revealed no statistical differences in value for total organ weight, content of DNA, RNA, or protein (Winick and Grant, 1968), or 2',3'-cyclic nucleotide 3'-phos- phohydrolase activity in the brain (Noguchi et al., 1982d).

The dwarf (dw/dw) mice were divided into two groups, according to treatment: (a) untreated (n = 5), physiological saline only during the first 20 d of postnatal life, and (b) treated (n = 3), bGH, 10 p,g/g body weight plus T4, 0.25 ~g/mouse during the first 20 d of postnatal life. In addition, we used the untreated normal mice (+/?) as the controls, because histological examination of the brains of normal mice receiving physiological saline or bGH plus T 4 treatment revealed no differences from those of the untreated normal mice.

The experimental animals were killed by exsanguination on the 40th day of age, and the brain and hypophysis were removed. To identify the treated homozygous dwarf (dw/dw), the hypophyses were examined immunohistochemically with rabbit anti-bGH antiserum according to our previous paper (Noguchi et al., 1982c).

Histological Examinations

Golgi-Cox Method, as Modified by Ramon-Moliner The whole brains of 40-d-old controls (+/?), untreated dwarfs, and

treated dwarf mice were put into the impregnating fluid for 30 d in the dark at room temperature. The samples then were transferred to the al- kaline solution for 24 h. After washing in several changes of distilled water, the samples were dehydrated with alcohol and embedded in 10% celloidin. The tissues were sectioned in a parasagittal plane at a thickness of 100 ~zm, dehydrated in alcohol-chloroform (3:1), cleared in iodoben- zene, and cover-slipped in mounting medium.

The cell size of the pyramidal neurons, located in layer V, was measured with an ocular micrometer and expressed as a major or minor axis of the cell.



lmmunohistochemistry Using Anti-NSE Antiserum The paraffin-embedded samples, previously fixed in Zamboni's so-

lution (Zamboni and De Martino, 1967) for 3 d, were sectioned in a parasasdttal plane, at a thickness of 4 ~m. The sections were deparaffinized and equilibrated in 0.0IM phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) for 30 min at room temperature. Antiserum against NSE (diluted 1:500) was added and the sections incubated for 12 h at 4~ The sections then were washed in three changes of PBS containing 1% BSA every 5 rain. Sheep anti-rabbit immunoglobulin (diluted 1:20) was added next, the sections incubated for 30 min, followed by three more washings with PBS containing 1% BSA every 5 min. A soluble antigen-antibody complex of horseradish peroxidase-rabbit antihorseradish peroxidase (diluted 1:60) was added, and the sections were incubated for 30 rain. Finally, the sections were incubated with 0.05% hydrogen peroxide in 0.05M Tris-HC1 buffer (pH 7.6), and the development of the brown insoluble reaction product was observed carefully under the microscope.

RESULTS

Golgi Study of Cortical Neurons

Brains were stained by the Golgi-Cox method, as modified by Ramon-Moliner (1975). Serial sagittal sections of the cerebral cortex were cut at 100 ~m. Our cytoarchitectonic description was based upon the six- layered conventional description of Brodmann (1905). The area of the brain we chose in which to monitor neurons morphologically was the motor cortex, area 6 of Caviness (1975), because the cell and fiber pattern of this area is fairly homogeneous (Caviness, 1975; Krieg, 1946; Vas Ferriera, 1951).

Examination of area 6 of Caviness in a normal cerebral cortex shows a mature appearance of the cortex with complete stratification and a well- developed dendritic arborization of the cortical neurons. The pyramidal neuron soma located in layer V give rise to lateral basal dendrites and, at the vertex, apical dentrites that rise towards the pia and divide into subpial terminal dendritic arches. Oblique dentrites arise from the apical dendrite along its course and near layer I (Fig. 1A). By contrast, in the cerebrum of the untreated dwarf, there is a diffuse defect in the dendritic arborization of the cortical neurons located in layer V. This is visible on inspection of the motor cortex under low magnification, which shows small neuronal perikarya and the poor development of the dendritic arborization, characterized by shorter, thinner and sparser branchings (Fig. 1B). The administration of hormones during the first 20 d of postnatal life, however, produced histologically detectable changes in the dwarfs (Fig. 1C). The perikarya of the pyramidal neurons located in layer V were

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GH and T4 in Neuronal Growth 127

Fig. 1. Cytoarchitectonic comparison of motor cortices of normal, untreated dwarf and treated dwarf mice. Brains were stained by the Golgi-Cox method as modified by Ramon-Moliner (1975). Serial sagittal sections of the cerebral cortex were cut at 100 p~m. Our cytoarchitectonic description was based upon the six-layered conventional description of Brodmann (1905). (A) Motor cortex of the 40-d-old control.



Fig. lB. Motor cortex of the 40-d-old untreated dwarf. The proportionate depths of layers and the cell number in each layer are almost identical to those of the control and the treated dwarf. Note small perikarya (arrow), the short apical and basal dendrite, and the sparsity of their branchings.

Neurochemical Pathology VoL 2, 1984


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Fig. 1C. Motor cortex of the 40-d-old dwarf treated with bGH, 10 ~g/g body weight plus T4, 0.25 ~g/mouse during the first 20 d of postnatal life. The perikarya of the pyramidal neurons located in layer V (arrow) are larger, and the dendritic arborization in both the apical and basal dendrites are found to be thicker and had denser branchings. Bar = 80 ~m

Meurochemical Pathology Vol. 2, 1984

130 Noguchi, Sugisata, and Tsukada

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Fig. 2. Comparison of pyramidal neurons in layer V of normal, untreated dwarf and treated dwarf mice. (A) Pyramidal neurons in layer V of the normal cortex. (B) Pyramidal neurons in layer V of the untreated dwarf cortex. Note the thinner dendrites. (C) Pyramidal neurons in layer V of the treated dwarf cortex. The thickness of the dendrites appears to be equivalent to those of the control pyramidal neurons. Arrows point to the same spots as shown in each plate of Fig. 1. Bar = 60 p,m.

Neurochemical Pathology It'ol. 2, 1984


larger, and the dendritic arborization in both the apical and basal dendrites were found to be thicker and had denser branchings.

The neurons of the motor cortex of the untreated dwarf mice (Fig. 2B) generally appear to be structurally less developed than those in the motor cortex of the control mice (Fig. 2A). The neurons of the treated dwarf mice (Fig. 2C) appear to be more abundantly developed than those of the untreated dwarf mice. Furthermore, when we measured the cell size of the pyramidal neurons located in layer V by random sampling with an ocular micrometer, it was found to be significantly reduced in the untreated dwarfs (p < 0.01), but in the control range in the treated dwarfs (Table 1). In addition, the thickness of both the apical and basal dendrites of the neurons in the treated dwarf motor cortex (Fig. 2C) appears to be equivalent to those of the control pyramidal neurons (Fig. 2A).

Observations of the Motor Cortex Stained for NSE

Examination of sections of the cerebrum from normal mice demon- strated the presence of the brown reaction product within the cytoplasm of numerous neurons and their processes. The peroxidase-labeled mate- rial extended from the perikarya into the axons and dendrites of the neurons, especially those neurons located in layer II-III and V. The stratification of neurons and the width of the several layers appears identical in the normal controls and the untreated and treated dwarf mice (Fig. 3). However, in layer V of the motor cortex, the neurons of the untreated dwarf mice were reduced in size and showed the weakly stained reaction products in the cytoplasm in comparison to those of the control mice and the treated dwarfs. In addition, we noted the darkly stained reaction products in the dendrites of the pyramidal neurons only in the controls and treated dwarfs, not in the untreated dwarfs (Fig. 4).

DISCUSSION

Thyroidectomy or the administration of antithyroid drugs to mammals during the postnatal period results either in a delay (Hamburgh,

TABLE 1 Pyramidal Cell Size in Layer V of Motor Cortex a

N Major axis, p~m Minor axis, ~m

Control 50 21.7 -+ 1.7 16.4 -+ 2.0 Dwarf untreated 50 15.4 + 2.0 b 11.0 + 1.P Dwarf treated 50 21.7 - 3.5 NS C 16.7 - 2.0 NS c

~ are expressed as mean + SD. bDifferences statistically significant at p ~ 0.01, by Student ' s t-test. cNS, not significant.

Heurochemical Pathology Vol. 2, 1984

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134 Noguchi, Sugisaki, and Tsukada

1963; Legrand, 1963; Legrand et al., 1961), or in the retardation of growth and maturation of the cerebral cortex (Eayrs, 1955; 1960; Gomez et al., 1966). Neonatal thyroidectomy of the rats results in a marked reduction in the growth of the brain after 14 d of age. This is caused by a decrease in myelination (Balazs et al., 1969; DeRaveglia et al., 1972; Walravens and Chase, 1969) and a reduction in the volume of neurons (Mitskevich and Moskovkin, 1971), but not to a reduction in the number of cells (Geel and Timiras, 1967; Marin-Padilla, 1970; Pasquini et al., 1967). The development of dendrites is retarded and the degree of dendritic branching is reduced in the cerebral cortex of hypothyroid mammals (Eayrs and Holmes, 1964). The number of axodendritic synapses in the cerebral cortex also seems to be reduced in rats after neonatal thyroidectomy (Balazs et al., 1968; Cragg, 1970; Nicholson and Altman, 1972a; 1972b).

On the other hand, GH deficiency impairs cell migration and differentiation, with a subsequent failure in myelination (Pelton et al., 1974). Histological and chemical studies of the adult cerebral somatosensory cortex in this model have disclosed findings similar to those found in cre- tin animals (Pelton, 1977).

Since the early work of Goldberg and Chaikoff (1949), numerous studies have reported the interrelationship between GH and T4 (Daughaday et al., 1968; Eayrs and Holmes, 1964; Iwatsubo et al., 1967; Schooley et al., 1966). In both cretinism and hypothyroidism, pituitary eosinophils are degranulated, and plasma GH is diminished (Contopoulos et al., 1958; Daughaday et al., 1968; Herlant, 1964; Schooley et al., 1966; Solomon and Greep, 1950). Therefore, both the above findings may act via a similar mechanism, a thyroid-pituitary linkage. The Snell dwarf mouse thus appears to be ideally suited for studying the effect of hormones on neuronal growth, because their anterior putuitary gland cannot synthesize detectable amounts of GH (Slabaugh et al., 1981) and only very low amounts of thyroid-stimulating hormone (Sinha et al., 1975).

It is well established that the neuronal body as well as its processes also keeps growing during this early postnatal period (Fazekas et al., 1951; Schapiro, 1968). Accordingly, the hormones, GH and T4, might have effects on neuronal growth.

To delineate the morphological changes in cortical neurons produced by treatment with the hormones, we firstly used Ramon-Moliner's method, a tungstate modification of the Golgi-Cox method. This method is especially useful for the study of entire dendritic trees, because it gives reliable, progressive, controlled impregnation of neuronal processes, and stains dendrites and dendritic spines darkly and clearly.

We found that the administration of the hormones during the first 20 d of postnatal life produced histologically detectable changes in the mutant. The perikarya of the pyramidal neurons located in layer V were larger, and the dendrites were found to be thicker and to have denser branchings.



In order to confirm the results obtained by the Golgi-Cox method, we immunohistochemically stained sections of the cerebrum from the controls, untreated dwarf and treated dwarf mice for NSE, neuron specific enolase, which is located throughout the cytoplasm (soma, axon, and dendrites) of nerve cells. It was substantiated that the stratification of neurons and the widths of the various layers are absolutely identical with one another among the three types of mice. However, the pyramidal neurons located in layer V of the untreated dwarf mouse appear to be weakly stained and reduced in size in comparison with those of the controls or the treated dwarfs. In addition, pyramidal neurons of both the control and treated dwarf mice show darkly stained reaction products in the dendrites as well as the cytoplasm, while the neurons of the untreated dwarf mice fail to show a reaction product in the dendrites. These findings are in agreement with observations on sections stained by the Golgi-Cox method. The cell size of the treated dwarf mouse measured by means of an ocular micrometer is equal to that of the controls, and the thickness of both the apical and basal dendrites of neurons in the treated dwarf cortex appears to be equivalent to that of the controls, sug- gesting that the dendrites of the dwarf neuron are functionally immature (Cicero et al., 1972). Indirect evidence supporting this concept is that dwarfs treated with bGH and T 4 during the first 20 d of postnatal life showed a remarkably higher level of spontaneous locomotion activity than that of the untreated dwarf mice and maintained a clear diurnal pe- riodicity, identical to the control mice (Sugisaki et al., submitted to Int. J. Devl. Neurosci. ).

Accordingly, it has been shown in the present study that exogenous GH and T4, in addition to restoring arrested glial proliferation (Noguchi et al., 1982b; 1982c), will enhance the retarded neuronal growth of the Snell dwarf cerebrum and will restore abnormal neuronal arborization to normal, resulting in successful myelination (Noguchi et al., 1982b).

ACKNOWLEDGEMENTS

We are grateful to Dr. Walter Stahl for his help in preparing the man- uscript. This research was partially supported by Toho University School of Medicine, by the Professor Kato Memorial Research Fund for Physiol- ogy and Medicine, and by Grant No. 83-13 from National Center for Nervous, Mental and Muscular Disorders (NCNMMD) of the Ministry of Welfare, Japan.

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Stimulation ofSnell dwarf mouse neuronal growth by GH and T4