Development of evoked potentials in specific brain systems after neonatal administration of estradiol

EXPERIMENTAL

34, 129-139 (1972)

NEUROLOGY

Development Systems

of after

Evoked

Neonatal

Potentials Administration

JOHN J. CURRY AND PAOLA Departmelzt

of Physiology-Anatomy, Rerkcleg, California Received

in Specific

Set tcmber

of

S. TIWRAS

lJt&wsity 94720

Brain

Estradiol

1

of Califorrzia.

29,1971

The effects of neonatal administration of estradiol on the development of the transcallosal response (TCR) and the thalamically evoked relayed pyramidal response (RPR) were studied in rats by analyzing evoked potentials at several ages. Estradiol-treated animals generally showed a lower threshold, shorter peak latency, and greater peak amplitude of the TCR at the younger ages studied, indicating accelerated development at these ages. The maximal effect on peak amplitude occurred at a different age than that of threshold and peak latency. The RPR was only slightly affected. The data also suggest that the effects of estradiol may be mediated by increased neuronal excitability, precocious myelination, and precocious development of synaptic connections. These findings extend those of other investigators by indicating that hormones act selectively upon specific systems as well as generally upon the whole brain. Introduction

Studies during the past several years have indicated that in the rat 6-to g-days of age is a critical period during which extracliol exerts profound influences on the development of the central nervous system (CNS) , and of the brain in particular. Administration of estradiol to rats during this age period results in precocious maturation of the brain as measured by the time of appearance of the adult responseto supramaximal electroshock stimuli (5, 6) and accelerated myelination in the hypothalamus and sensory-motor cortex (2). Estradiol-treated rats also have a higher level of cerebrosides, an important constituent of the myelin sheath, in the spinal cord and cerebrum than do corresponding control animals ( 1) . 1 This work, supported in part by USPHS GM-09267, is part of a thesis submitted by the senior author in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Physiology. It was performed during his tenure of a USPHS predoctoral fellowship. His present address is: Department of Physiology, Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118. The authors wish to thank Mr. C. .Johnson for technical assistance and Dr. C. S. Nicoll and Dr. F. A. Beach for their helpful comments and suggestions. 129 @ 1972

by Academic

Press,

Inc.

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However, little work has been reported on the specificity of estradiol during development and several questions remain unresolved. It was the purpose of the present investigation to determine whether the effects of estradiol constitute a generalized phenomenon or whether specific systems are influenced selectively and, if so, to what extent. The measurement of evoked potentials is not only a means of exploring the specificity of estradiol in discrete brain systems, but may also provide information concerning its mechanisms of action on the developing CNS by distinguishing effects on myelination from effects on the development of synaptic processes. It would also be of interest to learn whether estradiol has long-term effects on the development of specific brain systems, as has been demonstrated for the development of the whole brain (5, 6). Other studies have demonstrated that estradiol administered during critical periods of brain development results in permanent behavior changes (3) ; accordingly, evoked potentials were studied at selected periods beyond early devlopment. The evoked transcallosal response (TCR) and the thalamically evoked relayed pyramidal response (RPR) were selected for study. The role of the corpus callosum in the spread of seizure discharge has been established ( 13), and the development of this activity has been shown to be affected by neonatal administration of estradiol (6). The RPR represents a combination of afferent and efferent pathways constituting a “higher CNS refles” which may be affected by estradiol as are the lower reflex centers (14). Inasmuch as the pyramidal system is also a motor pathway, it may be influenced via the general effect of estradiol on motor activity (18). The TCR recorded from the cortical surface in adult rats following stimulation of the contralateral corpus callosum consists primarily of a surface-negative wave preceded by a small surface positive wave, sometimes obscured by the stimulus artifact. At 10 days old a similar response can be obtained; however, the threshold is much higher, the amplitude much smaller, and the peak latency and wave duration much longer. During development these parameters progressively approach those of the adult (4, 10). The RPR has not been previously described in rats. Preliminary experiments (unpublished) in this laboratory showed that it consists of a surface-positive monophasic wave, often followed by a long-duration surfacenegative wave of much lower amplitude similar to that described for cats (9). No response was obtained using anesthetic doses of barbiturates, but urethane anesthesia was suitable for the study of the RPR. These studies also revealed that the RPR could not be reliably obtained in animals younger than 12 days of age. At 13 days of age threshold was high and the response consisted of a broad surface-positive wave of very low ampli-

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tude. During development, threshold, crease and amplitude increases. Materials

peak latency, and wave duration

and

de-

Methods

Forty litters, each consisting of 6 female Long-Evans rats were used. Offspring were kept with mothers until they were killed or until weaned when 21 days of age. Food and water were given a.d libituwz to mothers and young throughout the experimental period. Food consisted of a special powdered diet for lactating mothers and a standard pellet diet for the young after weaning. Animal quarters were maintained at 23 C and illuminated by artificial light automatically controlled to give 12 hr of light and 12 hr of darkness per day. All animals were handled uniformly and as little as possible. Estradiol-treated rats received one injection of estradiol dipropionate (100 pg/lOO g body wt, SC) dissolved in Mazola oil daily from day 6 through day 9. Control animals were injected with equivalent amounts of Mazola oil alone. Each group was then divided into smaller groups in which either the TCR was studied at 10, 15, 21, 30, and 60 days of age, or the RPR at 13, 15, 21, and 30 days of age. Animals were killed immediately afterward by perfusion. Surgical and Stereotaxic Techniques. Animals in which the TCR was studied were anesthetized with sodium pentobarbital (30 mg/kg body w-t, ip). Part of the skull and dura mater were removed and the exposed parietal cortex covered with mineral oil. The corpus callosum was stimulated with a pair of stainless steel wire electrodes 200~ in diameter, insulated except at their tips and placed side by side with a tip separation of 1 mm. The electrodes were inserted stereotaxically (S), 1.2 mm lateral to the midline and 6 mm anterior to the interaural zero plane in adult rats or two-thirds of the way anterior between the Bregma and Lambda sutures in immature rats. Specially constructed ear bars were used in the lo-day-old animals. In all experiments a monopolar silver ball electrode, approsimately 0.5 mm in diameter, was placed on the surface of the contralateral homologous cortex. A stainless-steel screw in the skull over the frontal sinus, or a loose flap of skin (in younger animals) served as the reference electrode. Animals in which the RPR was studied were anesthetized with 100 mg/kg body weight urethane intraperitoneally.” Parts of the skull and dura mater were removed from the left side and the exposed parietal cortex covered with mineral oil. Parts of the skull and dura were also removed 2Unpublished observations from this laboartory have shown the TCR measured in the present study are not altered when anesthetic rather than sodium pentobarbital in adult animals.

that the parameters of urethane is used as an

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from over the cerebellum just posterior to the transverse sinus near the midline. A stainless steel monopolar recording electrode 250~ in diameter and insulated except at the tip was inserted into the bulbar pyramidal tract through the cerebellum, just posterior to the transverse sinus and 0.3-0.5 mm lateral to the midline. The ventrolateral nucleus of the thalamus was stimulated with a bipolar electrode composed of two stainless steel wires, 250~ each in diameter, insulated except at the tips and mounted side by side with a vertical tip separation of 0.5 mm. Electrodes were placed stereotaxically using coordinates obtained from a series of mapping experiments performed on animals at each of the ages studied here. Final positioning of the stimulating electrodes was accomplished using the evoked response as a guide. Electrophysiological Techniques. Square wave pulses 0.05 msec (TCR) or 0.1 msec (RPR) in duration were delivered at one pulse per second from a Tektronix Type 161 pulse generator and a Tektronix Type 162 waveform generator through an isolation transformer. To compare responses in different animals, the threshold stimulus for the evoked response was determined by reading the voltage drop across a 100 ohm resistor on an oscilloscope, and twice threshold strength stimuli were used for analysis of the response. Evoked responses were amplified with a Grass model P5 preamplifier and displayed on a Tektronix Type 502 dual beam oscilloscope. The sum of 100 evoked responses was obtained on a Mnemotron 400 B computer of average transients (CAT) and written on a strip chart. The following four parameters of each response were measured at all ages (Figs. 1 and 3) : threshold; peak amplitude of the major wave; peak latency; and wave duration at one-half peak amplitude. Means and standard errors were calculated for each group and the t test applied with a p of 0.05 being considered statistically significant. The position of the electrodes in the corpus callosum, thalamus, and pyramidal tract were verified at necropsy by macroscopic and low-magnification microscopic examination for Prussian blue-iron deposits at the site of the electrodes obtained by perfusing the animal with solutions of 0.9% NaCl, 10% formalin and 4% potassium ferrocyanide. Results

The characteristic changes in the TCR with age for estradiol-treated and control rats are illustrated in Fig. 1. Figure 2 shows the mean values for peak latency, threshold, peak amplitude, and wave duration for both groups of animals at all ages tested. In control animals, the threshold for the TCR fell rapidly between 10 and 30 days of age after which it showed little change through 60 days of

ESTRADIOL

Age

Age

FIG. 1. Characteristic changes with age in the evoked transcallosal response in control animals and rats treated with estradiol (lOOpg/lOO g body wt) on days 6 to 9 of age. Each tracing is the sum of 100 evoked responses. The 60 day control tracing also shows the parameters of the response measured. a, peak latency; c, peak amplitude ; b, wave duration at half peak amplitude.

age (Fig. 2B). In treated animals, the threshold was lower at all ages, although statistical significance was obtained only at 10 days of age. The difference between the two groups became progressively smaller until 30 days of age. (Fig. 2A). The peak amplitude in control animals showed a consistent increase from 10 to 60 days of age with the greatest rate of increase between 15 and 21 days (Fig. 2C). In treated animals, it was greater than in controls at all ages except 60 days, and the greatest, statistically significant difference in amplitude between control and treated animals occurred at 15 days of age. The duration of the negative wave at half peak amplitude in both control and treated animals increased between 10 and 15 days of age, decreased rapidly until 30 days, and gradually thereafter (Fig. 2D). In treated animals the wave duration was greater than in controls at all ages except at 21 days of age, although the differences were never statistically significant. Relayed Pyramidal Response. The RPR in adult and developing rats was similar to that reported for cats (9). The characteristic changes with

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age in the RPR for both estradiol-treated and control animals are illustrated in Fig. 3. The mean values for peak latency, threshold, peak amplitude and wave duration are shown in Fig. 4. In all parameters tested, changes in RPR closely paralleled those of the TCR in control animals. In terms of threshold, very little difference appears between control and estradiol-treated rats with the exception of day 15 when the threshold was higher in control animals (Fig. -1B). The peak latency in control animals decreased slightly between 13 and 15 days of age and more markedly between 15 and 21 days, and then leveled off through 30 days of age. (Fig. 1A). The peak latency in treated animals was significantly less than in controls at 15 days only. The peak amplitude in control animals increased continually throughout

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FIG. 2. Mean values for peak latency, threshold, peak amplitude and wave duration of the evoked transcallosal response in control animals and rats treated with estradiol (lOO~g/lOO g body wt) on days 6 to 9 of age. Vertical lines indicate standard errors of the means. Asterisks indicate statistical significance (by t test) of difference between control and experimental animals.

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the experimental period (Fig. 4C). Peak amplitudes for estradiol-treated animals were (generally) greater than in controls at all ages but because of widespread individual variation, these differences were not statistically significant. Changes in wave duration were similar in both groups at all ages, and consisted of an initial increase from day 13 to 15, and a gradual decrease through 30 days of age (Fig. -lD). Discussion

The results suggest that estradiol administered during infancy induces accelerated maturation of the TCR, particularly during the younger ages studied. At later ages, differences between estradiol-treated and control animals gradually disappear. The RPR is only slightly affected, showing marginal signs of accelerated maturation only at 15 and 20 days. The selective effects of estradiol on the development of the TCR in the present study further attest to the differential manner in which this hormone reportedlv affects the development of the whole brain and of specific brain systems (5, 11). The specificity of estradiol on the callosal system may he interpreted in several ways. The cortical cells primarily responsible for the TCR may have a greater ability to concentrate estradiol from the blood than do the neurons involved with pyramidal function, although many of the same neurons are undoubtedly involved in both systems. Differential maturation of the blood-brain barrier may also be responsible for the selective effects of the hormone. Woolley, Holinka, and Timiras (17) demonstrated that in the infant and adult rat, the uptake of tritiated estradiol varies in different brain regions and progressively decreases with age. Similarly, it was shown (16) that the time required for rat spinal cord to respond to a single injection of extradiol with an increase in excitability lengthens as the animal matures. Unpublished studies from this laboratory have shown that the TCR can be elicited in rats of 10 days of age whereas the RPR cannot be reliably obtained until 12 days of age. Consequently, estrogen administered from 6 to 9 days of age in this species may preferentially affect the earlier developing TCR. In vitro studies provide additional examples that the effects of estradiol vary with respect to the degree of maturation of each system at the time of hormonal administration (16). It is evident that the “critical age” in the development of one system may not be the same as that for another, which explains why estradiol administered “too early” or “too late” appears to be less effective in influencing whole brain maturation (6 ). These hormonal effects may be independent from or additive to the effects of differential maturation of the blood-brain barrier. Previous reports (5) showed that prolonged alterations in brain excita-

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bility, lasting up to 1.20 days, occur following neonatal administration of estradiol. In view of the numerous structures involved in convulsive responses, complex interneuronal factors may contribute to the prolonged effects of estradiol on CNS excitability. As was demonstrated by Vernadakis (14), the spinal cord of rats is functionally mature at birth, and estradiol administered neonatally is capable of increasing the excitability of these lower CNS structures (15). The prolonged effects of estradiol observed in previous studies may be related to the excitatory effects of the hormone on the mature spinal cord, whereas the effects of neonatally administered estradiol on the relatively immature systems studied here appear to be transient. The electrophysiological findings in the present work appear in agreement with those of others that estradiol administered during critical periods of brain development influences the rate of myelination ( 1, 2)) synaptic activity ( 16) and ionic distribution of the brain (12). For example, in the TCR, a shortening of peak latency, such as that seen at 10 days in estradiol-treated rats, was ascribed (4) to an increase in axonal conduction velocity, implicating precocious myelination, whereas the increase in peak amplitude at 15 days reflects the progressive establishment of synaptic connections. Lowering of the threshold in lo-day-old treated animals reflects changes in either the electrical excitability of the neurons involved or in development of synaptic processes, or both. Inasmuch as the effects of estradiol administration on threshold of the TCR in lo-day-old animals did not coincide with an alteration in peak amplitude, the changes in threshold in this case are more likely to represent alterations in neuronal excitability consequent to disturbances in ionic balance rather than precocious development of synaptic connections. Based on the specific findings that estradiol differentially affects peak latency and peak amplitude in the TCR depending upon age, that these effects are independent from those on threshold, and that only peak latency is affected in the RPR, we suggest that estradiol exerts a threefold effect on functional development of specific brain systems, acting independently on myelination, the development of synaptic processes and neuronal excitability. The present findings substantiate and extend to the developmental process, the observations of Kawakami and Sawyer (7) who suggested that sex hormones may have a dual effect on brain function, acting selectively on specific brain areas as well as on overall brain function. Neonatal administration of estradiol may affect the maturation of several brain areas and thus be responsible for long term effects involving those aspects of brain physiology exclusively concerned with reproduction. Many areas of inquiry are suggested by these findings and warrant further investigation.

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