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Audiogenic seizures and the pineal gland
734
BIOL
PSYCHIATRY
1988:23:73+740
Audiogenic
Seizures and the Pineal Gland
Belarmino Alves de Azevedo and Pedro Fontana Jr.
The efigcts qf sound stimulation (electric bell, 110 dB) on the pineal glands qf adult ,femule ruts tijere studied. TMV types qf animals were selected: audiogenic (Adg) und nonuudiogenic (Nadg). Unlike the Nadg rats, Adg ruts exhibited tonic-clonic .seizures in response to stimulation. In Adg rats, after a single seizure. all pinealocyte nuclei were pyknotic and the charucteristic lob&r organizution qf the pineal I~USmarkedly disrupted, indicating intense glandular stress; however, neither .serotonin levels nor its biosynthesis Itjere signijicantly altered. These results suggest a phv.si(~~~atholoXic,ulrelationship behlven uudiogenic .seixre.s and the pineal glund.
Introduction Lerner et al. ( 1960) exhaustively studied the melatonin hormone produced by the pineal gland, and other authors have analyzed the effects of light on glandular metabolism (Wurtman et al. 1964; Axelrod et al. 1965a, b; Snyder et al. I965 a. b, 1967; Quay 197 I ; Klein and Weller 1972: Cardinali and Wurtman 1975). Other published works include electrophysiological evidence for the action of light (Taylor and Wilson 1970) and for the convulsive response of parathyroidectomized rats to pineal gland removal (Reiter and Morgan 1972; Reiter 198 1). A psychiatric review has been published (Mullen and Silman 1977)) and Wetterberg ( 1986) has suggested that a depression response, which he characterized as the “low melatonin syndrome,” was associated with pineal hypofunction. In seasonal affective disorders, Rosenthal et al. (1983. 1984) review the theoretical reasons for suspecting that bright light may be biologically active in humans. as many photoperiodic effects on animals are mediated via secretion of the melatonin hormone. Contrary to the earlier view (Tapp and Huxley 1972) that the pineal gland remains active throughout life time and that its enzymatic levels do not change with advancing age and degree of calcification, other authors (Reiter et al. 1980; Igushi et al. 1982) have found that there is a marked drop in pineal gland biosynthetic activity in aging rodents. as well as in humans. Serotonin, the precursor of the melatonin hormone (Namboodiri et al. 19X3). exhibits a circannual rhythm (Philo and Reiter 1980). with a maximum level in winter and minimum in the summer (Texas, U.S.A.). This same circannual rhythm has likewise been observed in the fertility of cows. The important role of serotonin has also been demonstrated in
From
the O\waldo
Addrer:,
reprint
hwituto Received
Crw
Institute.
requests
Oswaldo December
to: Dr.
Crux. I’).
Mangumhw Belarmino
F~ocmr.
1984. revised
Av. July
Rto de Janr~ro. Alves
Brasd. IX.
de Arevedo. 436%Rio lYX7.
Brard Setor de Ps~colog~a e Paqulatnn
de Jane~ro.
RJ (CEP
21040).
Brawl
do Hc~\pltal
Esandro
Chaga,.
Audiogenic
BIOL PSYCHIATRY 1988;23:734-740
Seizures
735
schizophreniform syndromes experimentally induced by amphetamine (Angrist and Gershon 1970; Rastogi et al. 1981) and in depression (Van Praag 1981). By means of electrocorticograms on rats, Tacke et al. (1984) established the validity of audiogenic seizures as animal models for generalized human epilepsy. As far as is known, studies of the effects of sound and of adverse emotional responses on the pineal gland have been carried out only by Miline (1957). The purpose of the present work was to examine changes in parenchymatous cells and serotonin levels of the pineal gland in female rats exposed to sound stimulation.
Methods The albino rats used in our experiments weighed 130-160 g and ranged in age from 7 to 10 months. Exposure to light was effected by means of an incandescent lamp that was lighted at 7:00 PM and turned off at 7:00 AM by automatic control in order to maintain a well-defined circadian cycle. For stimulation, a powerful 12-inch diameter electric bell was used, with a sound intensity level of 110 dB, as determined by a Briiel and Kjaers type 2202 sound level meter and a microphone placed inside the 45 X 45 X 45 cm stimulation box (Azevedo and Fontana 1980). After individual stimulation for 3 min, the rats were separated into two groups: one with audiogenic (Adg) rats that responded with tonic-clonic seizures, the other composed of nonaudiogenic (Nadg) rats, that is, rats showing no signs of seizures when exposed to sound. Each Adg or Nadg group consisted of six rats. Immediately after stimulation, the animals were killed by cervical dislocation, followed by in situ perfusion through the carotid artery with 20 ml of a 10% formol solution at a rate of 0.2 pJsec. The animals were then decapitated, their pineal glands removed for histological examination by means of the hemotoxylin-eosin technique, and then compared with the control group (six rats placed individually in the stimulation box for 3 min, but not exposed to sound stimulation). These rats were of the same weight and age and had the same exposure to light as those in the experimental groups.
Determination
of Serotonin
The experiment was performed between 12:30 and 1:30 PM. The animals were decapitated and immediately pinealectomized-this operation being executed within a period of approximately 5 min. The pineal thus obtained was placed on cold filter paper that had been soaked in an NaCl 0.9% solution, and then dissected, with any attached fragments of dura mater being carefully removed and the conarii nervi and stalk severed. The time spent in dissection and weighing of the pineal had to be limited to 2 min to avoid errors in analysis of the gland’s humidity. The weights of the pineal glands of all animals ranged from 0.8 to 1.2 mg. For the serotonin determination, we employed a slightly modified method of Snyder et al. (1967). The pineal gland obtained was immediately weighed and homogenized in 0.5 ml of lo-‘N HCl. To the homogenate were added 0.5 ml of 0.5 M borate buffer (pH lO.O), NaCl to saturation, and 4 ml of i-butanol. This was then agitated for 10 min and centrifuged for 10 min at 3000 rpm. After centrifugation, 3 ml of the organic phase, containing serotonin in I-butanol, was added to 6 ml of heptane and 1 ml of 0.1 M phosphate buffer (pH 7.0). This total volume was agitated for 5 min and centrifuged for the same period of time at 3000 ‘pm. After this second centrifugation, all the supematant
736
BIOL
B. Alves de Azevedo and P. Fontana
PSYCHIATRY
198%X3:734-740
was carefulIy removed, and 0.1 ml of 0.1 M ninhydrin was added to the aqueous phase containing the serotonin. This was then heated and maintained at 60°C for 60 min. Immediately thereafter, the solution was allowed to cool at room temperature for 20 min and the fluorescence was read by means of a Farrand spectrophotofluorometer, with an activation wavelength of 385 p and extinction of 490~. Recovery of the serotonin that was present in the homogenate ranged from 74% to 81%. One control, obtained by substitution of the serotonin for the same volume of deionized water, was used with each sample. The use of ninhydrin with the serotonin, after heating. resulted in an approximate eightfold increase of serotonin fluorescence (Synder et al. 1965b). This method permits serotonin determinations in biological materials, such as the pineal gland, adrenal, and heart, where the serotonin pool is below the dosage boundaries of other methods. The I-butanol and n-heptane were purified: the same volume of reagent was mixed with 0. I M NaOH, stirred, and decanted. The reagent volume that was recovered was mixed with the same volume of 0.1 N HCl and then stirred and decanted. The fluorescence of each reagent was measured every week, and any reagent that displayed a fluorescence higher than 0.06 nA (nanoamperes - an arbitrary unit for measuring fluorescence of the spectrophotofluorometer) was eliminated. The serotonin (5-hydroxyt~ptamine creatinine sulphate complex) and 5-hydroxy-DL-t~ptophan used were manufactured by the Sigma Chemical Company, and the ninhydrin by E. Merck, Darmstadt.
Results With reference to the duration of the audiogenic seizure phases, such behavior was registered and compared to the phases found by Smith ( 1941 f. The values established in our experiments could be compared with Smith’s registers (a, b. c, d, e, f, g, h) as far as expectation goes (Table 1). A morphometric analysis of the pineal gland is shown below. The cellular density was determined by estimating the number of cells per 1000 p2 in a microscopic range of 7850 F’. always in the central regions of the pineal glands. Average counts of 183.7, 87. I, and 191.5 ceils and 0, 21.2, and 191.5 pyknotic nuciei were found in the control, Nadg. and Adg groups, respectively. The intermediate forms were not taken into account in this work. It was observed that in the pineal of Adg rats all the cellular nuclei were pyknotic (dense, low volume, hyperchromatic nuclei) (Table 2). had a greater cellular density due to glandular atrophy (possible due to water loss), and the characteristic lobuiar Table 1. Record of Temporal Variations in the Various Phases of the Adg Crises Observed in 33 Rats” Phases In our experience Latency Course Tonic Chronic ‘These rats were stimulated box
In Smith’s (1941) (a,b.c,d.e,f.g,hf afb fl e+t’+g+h
i~dividualiy
Duration” (set) 44.3 12.7 21.2 5S.h
2 13.5 t 3.1 + 103 + 13.9
for 3 mitt with an electric bell placed 30 cm from the bottom of a wcwden acourtical
Audiogenic Seizures
BIOL
737
PSYCHIATRY
1988;23:734-740
Table 2. Distributions of Cells, with Normal and Pyknotic Nuclear Densities of Pineal Glands of Female Rats Stimulated bv a Bell
Groups Control (n = 6) Nadg (n = 6) And (n = 6) Counting loo0
Normal nuclear densities (mean 2 1
Cellular densities (mean 2 1 SD)
23.4 f 0.5
234 2 0.5
0”
24.4 ? 0.2”
groups: “10.001,
and b<0.05).
Magnification
2.7 -t 0.2“ 24.4 2 0.2”
fields of 7850 pz in the central region of the pineal gland. Densities:
k’. The number of glands used in each group was six. The statistical
of the control
0
8.4 t 0.5”
11.1 2 0.7”
was done in microscopic
SD)
Pyknotic nuclear densities (mean 2 1 SD)
significance
number of elements/
level (p) is with reference to the values
1200 immersion.
organization was markedly disrupted. Comparison of the glandular parenchymas of Adg rats with those of Nadg rats suggests a greater glandular stress of the former and a physiopathological relationship between an Adg convulsion and the observed alterations. By determining the serotonin levels in the pineal gland, as in the histological study, three groups of animals were established: control, Nadg, and Adg. Comparisons of the levels of this biogenic amine among these groups were not statistically significant (Table 3), although a slight decrease did occur in the concentration of serotonin (5-hydroxytryptamine, 5-HT) in the Adg animals as compared with the control group. There was surprising, principally when one considers the alterations observed in the parenchymatous cells, resulting in a more stressed reaction of these epileptic rats. These results suggested that the problem in the metabolism of serotonin should be taken into account. Consequently, a new experiment using its precursor, 5hydroxytryptophane (5HTP) at 25 mg/kg i.p., was performed, which gave no evidence of any protective effect against audiogenic seizures (21.73% and 19.20%, respectively, for the animals treated with 0.9% NaCl and with 5-HTP). I In the incorporation kinetics of 5-HTP by the pineal gland, the maximum 5-HT levels occurred 30 min after 5-HTP injection, showing an increase of approximately 100%. The enzyme that is responsible for this transformation is 5-hydroxytryptophane-descarboxilase (Snyder and Axelrod 1964b; Snyder et al, 1967). Similarly, 1 hr after the precursor injection, the 5-HT level in the pineal was high (82%), and this interval was selected for the following experiments. The serotonin concentrations in Adg and Nadg rats, measured by the transformation of 5-HTP, also did not reveal any significant alterations between Table 3. Effects of a Single Sound Stimulus (3 min) on the Serotonin (5-HT) Levels and the 5-HT Syntheses of Controls and of Exuerimental Nadg and Adp Pineal Glands of Female Rats Serotonin (5-HT) ngimg of pineal gland
As a precursor
Groups
Without 5-HTP i.p. injection (mean t 1 SD)
With 5-HTP i.p. injection (mean t- 1 SD)
Control Nadg Adg
65 2 5 63 2 7 60 2 8
105 * 4 102 2 7 98 ? 10
of the 5.HT
rats were used. weighing groups was not statistically
between
syntheses,
the 5-HTP
was used m a single i.p. dose (25 mg/kg body weight).
143 and 155 g. The pineal glands weighed
significant.
from 0.8 to 1.2 mg. The difference
A total of 83 between the
738
BIOL PSYCHIATRY 1988:23-736740
t3.
their levels, as is shown in Table 3. Thus, no variations under these conditions were found.
Alves de Azevedo and P. Fontana
in the biosynthesis
of serotonin
Discussion The genetic, biochemical, and neurophysiological implications of the audiogenic seizure have been studied by many authors and reviewed by Kesner ( 1966) and Maxson and Cowen (1976), but very few studies of the neural and somatic alterations caused by it are found. For example, Hurders and Sanders (1953) observed the action of such seizures on the parenchyma cells of the adrenal glands, with the occurrence of the hypertrophy of the adrenal cortex and an increase in the weight of the adrenals. There was also a marked eosinophilia in the rats after audiogenic seizure. It is relevant to note the inverse physiological relationship between the pineal and the adrenal glands mediated by the melatonin hormone (De Fronzo and Roth 1972), as well as the pineal influence on the hypophysis, the thyroid, and the gonads (Baschieri et al, 1963: De Fronzo and Roth 1972). Seyfried et al. (1981) suggest that the thyroid hormone (thyroxine) may influence and may indeed be an important requirement for the development of audiogenic seizure susceptibility. At present we have very little information on the correlation between the pineal gland and epilepsy. Nir et al. (1969) reported that pinealeactomized rats demonstrate increased EEG convulsive activity with intermittent outbursts of slow waves. In another experiment, Reiter and Morgan (1972) observed that a vigorous seizure occurred when parathyroidectomized rats were subjected to pinealectomies. A promising result was obtained by Anton-Tay (1974) with the use of oral melatonin as an anticonvulsive in humans, indicating the necessity for more rigorously controlled clinical studies. Rudeen et al. ( 1980) suggested an antiepileptic effect of melatonin in the pinealectomized Mongolian gerbil, although the mechanism through which melatonin reduces seizure activity is unknown. Albertson et al ( 198 1) reported a substantial anticonvulsive action of parenteral melatonin in two rat models of epilepsy (electrically kindled amygdaloid seizures or seizures induced with pentylene-tetrazol injections). In our experiments, we observed some alterations in the parenchymatous cells of the pineal, but even in metabolic conditions, using 5hydroxytryptophane, the serotonin level variations were not statistically significant, although the Adg seizure causes a decrease in the encephalic serotonin level (Schlesinger et al. 1969; Schreiber and Schlesinger 1971). A lesion of the inferior colliculi resulted in 100% inhibition of Adg seizures (Chocholova 1966). indicating that this structure has an important function in the epileptic discharge in Adg stimulation. The inferior colliculus nucleus, deep superior colliculus. and the adjacent tegmentum are involved in the primary pathway of audiogenic seizure activity, whereas the unrestrainedly erratic attacks appear to be mediated at the inferior colhculus level; progression to clonic and tonic convulsions may require more rostra1 levels (Willot and Lu 1980). The structural alterations at the pineal gland observed in our experiments may possibly be inversely related to the variations in the supraoptic nuclei and paraventricular nuclei reported by Popovichenco ( 1971) under similar conditions, confirming the histophysiological antagonism described by Miline (1957). It is interesting to note that in our experiments, the pineal gland showed some alterations in response to the epileptic tonic seizure. It is thus another functional adaptive response to the extremely stressful stimulus
Audiogenic
BIOL PSYCHIATRY 1988;23:734740
Seizures
739
of intense levels of sound, similar to its responses to variance of light and dark, and to strong emotion (Miline 1957). We therefore agree with Miline’s concept that the pineal gland is an endocrine organ responsible for important adaptative functions, particularly those involving an animal’s response to stress.
References Albertson TE, Peterson SL, Stark LG (1981): The anticonvulsivant kindled seizures in rats. Neuropharmacology 20:61. Angrist BZ, Gershon S (1970): The phenomenology of experimentally chosis. Preliminary observations. Biol Psychiatry 2:95.
properties
of melatonin
induced amphetamine
Anton-Tay F (1974): Melatonin: Effects on brain function. Adv Biochem Psychopharmacol Axelrod J, Quay WB, Baker PC (1965a): Enzymatic synthesis of skin-lightening in amphibians. Nature 208:386.
on psy-
I 1:315.
agent, melatonin
Axelrod J, Wurtman RG, Snyder SH (1965a): Control of hydroxyindole-O-methyl activity in the rat pineal gland by environmental lighting. J Biol Chem 240:949.
transferase
Azevedo BA, Fontana P Jr (1980): Alteracbes celulares na gl&ndula pineal de ratas albinas. Efeito da estimulacao Sonora dikia. Mem Inst Oswald0 Cruz 75~33. Baschieri L, De Luca F, Cramarossa L, De Martin0 C, Olirerio A, Negri M (1963): Modifications of thyroid activity by melatonin. Experientia 19:15. Cardinali DP, Wurtman RJ (1975): In Altschule MD (ed), Frontiers bridge, MA: MIT Press, p 12. Chocholova
L (1966): Reflex and memory mechanism
Comparative
and Cellular
Pathophysiology
of Pineal Physiology
in seizure pathology. In Servitz Z (ed), Amsterdam: Excerpta Medica, p 313.
of Epilepsy,
De Fronzo RA, Roth WD (1972): Evidence for the existence of pineal-adrenal axis. Acta Endocrinol 70:35. Hurders WP, Sanders AF (1953): Audiogenic
Cam-
and a pineal-thyroid
seizure and the adrenal cortex. Science 117:324.
Iguchi M, Kato K, Ibayashi H (1982): Age-dependent reduction in serum melatonin concentrations in healthy human subjects. J Clin Endocrinal Metab 55:27. Kesner
RP (1966): Subcortical
Comparative
and Cellular
mechanisms
underlying
Pathophysiology
of Epilepsy,
Klein DC, Weller JL ( 1972): Rapid light-induced activity. Science 177:532.
audiogenic seizures. In Servit Z (ed), Amsterdam: Excerpta Medica, p 313.
decrease in pineal serotonin N-acetyltransferase
Lemer AB, Case JD, Takahashi Y (1960): Isolation of melatonin and 5-methoxyindole-3-acetic acid from bovine pineal glands. J Biol Chem 235:1992. Maxson SC, Cowen JS (1976): Electroencefalographic of inbred mice. Physiol Behav 16:623.
correlates of the audiogenic seizure response
Miline R (1957): In Congres National des sciences Medicales, dans le syndroma d’adaptation: Mullen PE, Silman RE ( 1977): The pineal and psychiatry: Namboodiri, Science
Bucarest, 421. La part de l’epiphyse
A review. Psycho1 Med 7:407.
MAA, Sugden D, Klein DC (1983): 5-Hydroxytryptophan 4611:659.
Nir I, Behroozik K, Assael M (1969): Changes pinealectomy. Neuroendocrinology 4:122.
elevates serum melatonin.
in the electric activity of the brain following
Philo R, Reiter RJ (1980): A circannual rhythm in bovine pineal serotonin. Popovichenco convulsive
NV ( 197 1): Changes in the hypothalamic paroxysms. Arkh Patol 33:31.
hypophyseal
Experientia
neurosecretory
36:664.
system during
Quay WB (1963): Circadian rhythm in rat pineal serotonin and its modification by estrous cycle and photoperiod.
Gen Comp Endocrinol
3:473.
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BIOL PSYCHIATRY 1988;23:73&740
Quay WB (1971): Dissimilar functional cerebral meningeai interruptions on phase shifts of circadian activity rhythms. Physiol Behuv 75.57. Rastogi RB, Singhal RL, Lapierre YD (1981): Effects of short- and long-term neuroleptic treatment on brain serotonin synthesis and turnover: Focus on the serotonin hypothesis of schizophrenia. Life Sci 291735. Reiter RJ (198 1): The mammalian pineal gland: Structure and function. Am J Anat 162:2X7. Reiter RJ, Morgan WW (1972): Attempts to characterize the convulsive ectomized rats to pineal gland removal. Physiol Behav 9:203.
response of parathyroid-
Reiter RJ, Richardson BA, Johnson LY, Ferguson BN, Dinh DT (1980): Pineal melatonin rhythm: Reduction in aging Syrian hamster. Science 210: 1372. Rosenthal NE, Lewy AJ, Wehr TA, Kern HE, Goodwin FK (1983): Seasonal cycling in a bipolar patient. Psychiatq Res 8:25. Rosenthal NE, Sack DA, Gillin JC, Lewy AJ, Goodwin FK, Davenport Y, Mueller PS, Newsome DA, Wehr TA (1984): Seasonal affective disorder. A description of the syndrome and preliminary findings with light therapy. Arch Gen Psychiatry 41:72. Rudeen PK, Philo RC, Symmer SK (1980): Antiepileptic effects of melatonin in the pinealectomized Mongolian gerbil. Epirepsiu 2 I : 149. Scheiber RA, Schlesinger K (1971): Circadian rhythms and seizure susceptibility: hydroxytryptamine and norepinephrine in brain. Physiol Behav 6:635.
Relation to S-
Schlesinger K, Stauner KL, Morgan W. (1969): Modification of audiogenic and pentylenetetrazol seizures with gamma aminobutyric. acid, norepinephrine and serotonin. P.sychophormaco/aX?:~ I5:226. Seyfried TN, Glasscr GH, Yu RK (1981): Thyroid hormone can restore the audiogenic susceptibility of hypothyroid DBA/2J mice. Exp Neurol 7 I :220.
scizurr
Sitaram N, Gillin JC, Bunney WE (1978): Circadian variations in the time of “switch” of a patient with 48-hour manic-depressive cycles. Biol Psychiatry 13:567. Smith KU (1941): Quantitative J Comp Psycho1 32: 3 I 1.
analysis of the pattern of activity in audio-epileptic
seizures in rats.
Snyder SH, Axelrod J (1964a): Influence of light and sympathetic nervous system on %hydroxytryptophan descarboxilase (5-HTPD) activity in the pineal gland. Fed Proc 23:206. Snyder SH, Axelrod J (1964b); A sensitive assay for 5hydroxytryptophan Phurmacol I3:805
decarboxilasc.
Snyder SH, Axelrod J, Zweig M (1965a): A sensitive and specific fluorescence scrotonin. Biochem Phurmucol 14:83 I
Biochem
assay for tissue
Snyder SH, Zweig M. Axelrod J. Fischer JE (1965b): Control of the circadian rhythm in serotonm content of the rat pineal gland. Proc Nurl Acad Sci USA 53:301. Snyder SH, Axelrod J, Zweig MJ (1967): Circadian rhythm in the serotonin content of the rat pineal gland regulating factors. J Pharmacol Exp Ther 158:206. Tackc U, Tuomisto L, Danner R (1984): Cortical spike wave discharge during audiogemc vulsions in rats. Neurology 85:233. Tapp E. Huxley M (1972): The histological to old age. J Pathol 108: 137.
appearance of the human pmcal gland from pubert)
Taylor AN, Wilson RW ( 1970): Electrophysiological gland in the rat. E.xperientir? 26:267. Van Praag HM (1981 ): Management lh:291.
con-
of depression
cvidencc for the action of Ilght on the pineal with serotonin
precursor:,.
Bid
Pswhicm
Wetterberg L (1986): The pineal hormone melatonin as a rnarkcr for a subgroup of depression. .Medicqraphia 8:4. Willot JF. Lu SM ( 1980): Midbrain pathways of audiogemc sclzures in DBAi7 mlcc. I?.I[J. Newd 70288.
BIOL
PSYCHIATRY
1988:23:73+740
Audiogenic
Seizures and the Pineal Gland
Belarmino Alves de Azevedo and Pedro Fontana Jr.
The efigcts qf sound stimulation (electric bell, 110 dB) on the pineal glands qf adult ,femule ruts tijere studied. TMV types qf animals were selected: audiogenic (Adg) und nonuudiogenic (Nadg). Unlike the Nadg rats, Adg ruts exhibited tonic-clonic .seizures in response to stimulation. In Adg rats, after a single seizure. all pinealocyte nuclei were pyknotic and the charucteristic lob&r organizution qf the pineal I~USmarkedly disrupted, indicating intense glandular stress; however, neither .serotonin levels nor its biosynthesis Itjere signijicantly altered. These results suggest a phv.si(~~~atholoXic,ulrelationship behlven uudiogenic .seixre.s and the pineal glund.
Introduction Lerner et al. ( 1960) exhaustively studied the melatonin hormone produced by the pineal gland, and other authors have analyzed the effects of light on glandular metabolism (Wurtman et al. 1964; Axelrod et al. 1965a, b; Snyder et al. I965 a. b, 1967; Quay 197 I ; Klein and Weller 1972: Cardinali and Wurtman 1975). Other published works include electrophysiological evidence for the action of light (Taylor and Wilson 1970) and for the convulsive response of parathyroidectomized rats to pineal gland removal (Reiter and Morgan 1972; Reiter 198 1). A psychiatric review has been published (Mullen and Silman 1977)) and Wetterberg ( 1986) has suggested that a depression response, which he characterized as the “low melatonin syndrome,” was associated with pineal hypofunction. In seasonal affective disorders, Rosenthal et al. (1983. 1984) review the theoretical reasons for suspecting that bright light may be biologically active in humans. as many photoperiodic effects on animals are mediated via secretion of the melatonin hormone. Contrary to the earlier view (Tapp and Huxley 1972) that the pineal gland remains active throughout life time and that its enzymatic levels do not change with advancing age and degree of calcification, other authors (Reiter et al. 1980; Igushi et al. 1982) have found that there is a marked drop in pineal gland biosynthetic activity in aging rodents. as well as in humans. Serotonin, the precursor of the melatonin hormone (Namboodiri et al. 19X3). exhibits a circannual rhythm (Philo and Reiter 1980). with a maximum level in winter and minimum in the summer (Texas, U.S.A.). This same circannual rhythm has likewise been observed in the fertility of cows. The important role of serotonin has also been demonstrated in
From
the O\waldo
Addrer:,
reprint
hwituto Received
Crw
Institute.
requests
Oswaldo December
to: Dr.
Crux. I’).
Mangumhw Belarmino
F~ocmr.
1984. revised
Av. July
Rto de Janr~ro. Alves
Brasd. IX.
de Arevedo. 436%Rio lYX7.
Brard Setor de Ps~colog~a e Paqulatnn
de Jane~ro.
RJ (CEP
21040).
Brawl
do Hc~\pltal
Esandro
Chaga,.
Audiogenic
BIOL PSYCHIATRY 1988;23:734-740
Seizures
735
schizophreniform syndromes experimentally induced by amphetamine (Angrist and Gershon 1970; Rastogi et al. 1981) and in depression (Van Praag 1981). By means of electrocorticograms on rats, Tacke et al. (1984) established the validity of audiogenic seizures as animal models for generalized human epilepsy. As far as is known, studies of the effects of sound and of adverse emotional responses on the pineal gland have been carried out only by Miline (1957). The purpose of the present work was to examine changes in parenchymatous cells and serotonin levels of the pineal gland in female rats exposed to sound stimulation.
Methods The albino rats used in our experiments weighed 130-160 g and ranged in age from 7 to 10 months. Exposure to light was effected by means of an incandescent lamp that was lighted at 7:00 PM and turned off at 7:00 AM by automatic control in order to maintain a well-defined circadian cycle. For stimulation, a powerful 12-inch diameter electric bell was used, with a sound intensity level of 110 dB, as determined by a Briiel and Kjaers type 2202 sound level meter and a microphone placed inside the 45 X 45 X 45 cm stimulation box (Azevedo and Fontana 1980). After individual stimulation for 3 min, the rats were separated into two groups: one with audiogenic (Adg) rats that responded with tonic-clonic seizures, the other composed of nonaudiogenic (Nadg) rats, that is, rats showing no signs of seizures when exposed to sound. Each Adg or Nadg group consisted of six rats. Immediately after stimulation, the animals were killed by cervical dislocation, followed by in situ perfusion through the carotid artery with 20 ml of a 10% formol solution at a rate of 0.2 pJsec. The animals were then decapitated, their pineal glands removed for histological examination by means of the hemotoxylin-eosin technique, and then compared with the control group (six rats placed individually in the stimulation box for 3 min, but not exposed to sound stimulation). These rats were of the same weight and age and had the same exposure to light as those in the experimental groups.
Determination
of Serotonin
The experiment was performed between 12:30 and 1:30 PM. The animals were decapitated and immediately pinealectomized-this operation being executed within a period of approximately 5 min. The pineal thus obtained was placed on cold filter paper that had been soaked in an NaCl 0.9% solution, and then dissected, with any attached fragments of dura mater being carefully removed and the conarii nervi and stalk severed. The time spent in dissection and weighing of the pineal had to be limited to 2 min to avoid errors in analysis of the gland’s humidity. The weights of the pineal glands of all animals ranged from 0.8 to 1.2 mg. For the serotonin determination, we employed a slightly modified method of Snyder et al. (1967). The pineal gland obtained was immediately weighed and homogenized in 0.5 ml of lo-‘N HCl. To the homogenate were added 0.5 ml of 0.5 M borate buffer (pH lO.O), NaCl to saturation, and 4 ml of i-butanol. This was then agitated for 10 min and centrifuged for 10 min at 3000 rpm. After centrifugation, 3 ml of the organic phase, containing serotonin in I-butanol, was added to 6 ml of heptane and 1 ml of 0.1 M phosphate buffer (pH 7.0). This total volume was agitated for 5 min and centrifuged for the same period of time at 3000 ‘pm. After this second centrifugation, all the supematant
736
BIOL
B. Alves de Azevedo and P. Fontana
PSYCHIATRY
198%X3:734-740
was carefulIy removed, and 0.1 ml of 0.1 M ninhydrin was added to the aqueous phase containing the serotonin. This was then heated and maintained at 60°C for 60 min. Immediately thereafter, the solution was allowed to cool at room temperature for 20 min and the fluorescence was read by means of a Farrand spectrophotofluorometer, with an activation wavelength of 385 p and extinction of 490~. Recovery of the serotonin that was present in the homogenate ranged from 74% to 81%. One control, obtained by substitution of the serotonin for the same volume of deionized water, was used with each sample. The use of ninhydrin with the serotonin, after heating. resulted in an approximate eightfold increase of serotonin fluorescence (Synder et al. 1965b). This method permits serotonin determinations in biological materials, such as the pineal gland, adrenal, and heart, where the serotonin pool is below the dosage boundaries of other methods. The I-butanol and n-heptane were purified: the same volume of reagent was mixed with 0. I M NaOH, stirred, and decanted. The reagent volume that was recovered was mixed with the same volume of 0.1 N HCl and then stirred and decanted. The fluorescence of each reagent was measured every week, and any reagent that displayed a fluorescence higher than 0.06 nA (nanoamperes - an arbitrary unit for measuring fluorescence of the spectrophotofluorometer) was eliminated. The serotonin (5-hydroxyt~ptamine creatinine sulphate complex) and 5-hydroxy-DL-t~ptophan used were manufactured by the Sigma Chemical Company, and the ninhydrin by E. Merck, Darmstadt.
Results With reference to the duration of the audiogenic seizure phases, such behavior was registered and compared to the phases found by Smith ( 1941 f. The values established in our experiments could be compared with Smith’s registers (a, b. c, d, e, f, g, h) as far as expectation goes (Table 1). A morphometric analysis of the pineal gland is shown below. The cellular density was determined by estimating the number of cells per 1000 p2 in a microscopic range of 7850 F’. always in the central regions of the pineal glands. Average counts of 183.7, 87. I, and 191.5 ceils and 0, 21.2, and 191.5 pyknotic nuciei were found in the control, Nadg. and Adg groups, respectively. The intermediate forms were not taken into account in this work. It was observed that in the pineal of Adg rats all the cellular nuclei were pyknotic (dense, low volume, hyperchromatic nuclei) (Table 2). had a greater cellular density due to glandular atrophy (possible due to water loss), and the characteristic lobuiar Table 1. Record of Temporal Variations in the Various Phases of the Adg Crises Observed in 33 Rats” Phases In our experience Latency Course Tonic Chronic ‘These rats were stimulated box
In Smith’s (1941) (a,b.c,d.e,f.g,hf afb fl e+t’+g+h
i~dividualiy
Duration” (set) 44.3 12.7 21.2 5S.h
2 13.5 t 3.1 + 103 + 13.9
for 3 mitt with an electric bell placed 30 cm from the bottom of a wcwden acourtical
Audiogenic Seizures
BIOL
737
PSYCHIATRY
1988;23:734-740
Table 2. Distributions of Cells, with Normal and Pyknotic Nuclear Densities of Pineal Glands of Female Rats Stimulated bv a Bell
Groups Control (n = 6) Nadg (n = 6) And (n = 6) Counting loo0
Normal nuclear densities (mean 2 1
Cellular densities (mean 2 1 SD)
23.4 f 0.5
234 2 0.5
0”
24.4 ? 0.2”
groups: “10.001,
and b<0.05).
Magnification
2.7 -t 0.2“ 24.4 2 0.2”
fields of 7850 pz in the central region of the pineal gland. Densities:
k’. The number of glands used in each group was six. The statistical
of the control
0
8.4 t 0.5”
11.1 2 0.7”
was done in microscopic
SD)
Pyknotic nuclear densities (mean 2 1 SD)
significance
number of elements/
level (p) is with reference to the values
1200 immersion.
organization was markedly disrupted. Comparison of the glandular parenchymas of Adg rats with those of Nadg rats suggests a greater glandular stress of the former and a physiopathological relationship between an Adg convulsion and the observed alterations. By determining the serotonin levels in the pineal gland, as in the histological study, three groups of animals were established: control, Nadg, and Adg. Comparisons of the levels of this biogenic amine among these groups were not statistically significant (Table 3), although a slight decrease did occur in the concentration of serotonin (5-hydroxytryptamine, 5-HT) in the Adg animals as compared with the control group. There was surprising, principally when one considers the alterations observed in the parenchymatous cells, resulting in a more stressed reaction of these epileptic rats. These results suggested that the problem in the metabolism of serotonin should be taken into account. Consequently, a new experiment using its precursor, 5hydroxytryptophane (5HTP) at 25 mg/kg i.p., was performed, which gave no evidence of any protective effect against audiogenic seizures (21.73% and 19.20%, respectively, for the animals treated with 0.9% NaCl and with 5-HTP). I In the incorporation kinetics of 5-HTP by the pineal gland, the maximum 5-HT levels occurred 30 min after 5-HTP injection, showing an increase of approximately 100%. The enzyme that is responsible for this transformation is 5-hydroxytryptophane-descarboxilase (Snyder and Axelrod 1964b; Snyder et al, 1967). Similarly, 1 hr after the precursor injection, the 5-HT level in the pineal was high (82%), and this interval was selected for the following experiments. The serotonin concentrations in Adg and Nadg rats, measured by the transformation of 5-HTP, also did not reveal any significant alterations between Table 3. Effects of a Single Sound Stimulus (3 min) on the Serotonin (5-HT) Levels and the 5-HT Syntheses of Controls and of Exuerimental Nadg and Adp Pineal Glands of Female Rats Serotonin (5-HT) ngimg of pineal gland
As a precursor
Groups
Without 5-HTP i.p. injection (mean t 1 SD)
With 5-HTP i.p. injection (mean t- 1 SD)
Control Nadg Adg
65 2 5 63 2 7 60 2 8
105 * 4 102 2 7 98 ? 10
of the 5.HT
rats were used. weighing groups was not statistically
between
syntheses,
the 5-HTP
was used m a single i.p. dose (25 mg/kg body weight).
143 and 155 g. The pineal glands weighed
significant.
from 0.8 to 1.2 mg. The difference
A total of 83 between the
738
BIOL PSYCHIATRY 1988:23-736740
t3.
their levels, as is shown in Table 3. Thus, no variations under these conditions were found.
Alves de Azevedo and P. Fontana
in the biosynthesis
of serotonin
Discussion The genetic, biochemical, and neurophysiological implications of the audiogenic seizure have been studied by many authors and reviewed by Kesner ( 1966) and Maxson and Cowen (1976), but very few studies of the neural and somatic alterations caused by it are found. For example, Hurders and Sanders (1953) observed the action of such seizures on the parenchyma cells of the adrenal glands, with the occurrence of the hypertrophy of the adrenal cortex and an increase in the weight of the adrenals. There was also a marked eosinophilia in the rats after audiogenic seizure. It is relevant to note the inverse physiological relationship between the pineal and the adrenal glands mediated by the melatonin hormone (De Fronzo and Roth 1972), as well as the pineal influence on the hypophysis, the thyroid, and the gonads (Baschieri et al, 1963: De Fronzo and Roth 1972). Seyfried et al. (1981) suggest that the thyroid hormone (thyroxine) may influence and may indeed be an important requirement for the development of audiogenic seizure susceptibility. At present we have very little information on the correlation between the pineal gland and epilepsy. Nir et al. (1969) reported that pinealeactomized rats demonstrate increased EEG convulsive activity with intermittent outbursts of slow waves. In another experiment, Reiter and Morgan (1972) observed that a vigorous seizure occurred when parathyroidectomized rats were subjected to pinealectomies. A promising result was obtained by Anton-Tay (1974) with the use of oral melatonin as an anticonvulsive in humans, indicating the necessity for more rigorously controlled clinical studies. Rudeen et al. ( 1980) suggested an antiepileptic effect of melatonin in the pinealectomized Mongolian gerbil, although the mechanism through which melatonin reduces seizure activity is unknown. Albertson et al ( 198 1) reported a substantial anticonvulsive action of parenteral melatonin in two rat models of epilepsy (electrically kindled amygdaloid seizures or seizures induced with pentylene-tetrazol injections). In our experiments, we observed some alterations in the parenchymatous cells of the pineal, but even in metabolic conditions, using 5hydroxytryptophane, the serotonin level variations were not statistically significant, although the Adg seizure causes a decrease in the encephalic serotonin level (Schlesinger et al. 1969; Schreiber and Schlesinger 1971). A lesion of the inferior colliculi resulted in 100% inhibition of Adg seizures (Chocholova 1966). indicating that this structure has an important function in the epileptic discharge in Adg stimulation. The inferior colliculus nucleus, deep superior colliculus. and the adjacent tegmentum are involved in the primary pathway of audiogenic seizure activity, whereas the unrestrainedly erratic attacks appear to be mediated at the inferior colhculus level; progression to clonic and tonic convulsions may require more rostra1 levels (Willot and Lu 1980). The structural alterations at the pineal gland observed in our experiments may possibly be inversely related to the variations in the supraoptic nuclei and paraventricular nuclei reported by Popovichenco ( 1971) under similar conditions, confirming the histophysiological antagonism described by Miline (1957). It is interesting to note that in our experiments, the pineal gland showed some alterations in response to the epileptic tonic seizure. It is thus another functional adaptive response to the extremely stressful stimulus
Audiogenic
BIOL PSYCHIATRY 1988;23:734740
Seizures
739
of intense levels of sound, similar to its responses to variance of light and dark, and to strong emotion (Miline 1957). We therefore agree with Miline’s concept that the pineal gland is an endocrine organ responsible for important adaptative functions, particularly those involving an animal’s response to stress.
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