Sperm Release Evoked by Electrical Stimulation of the Fish Brain: A Functional-Anatomical Study LEO S. DEMSKI,' DIANA H. BAUER AND JERRY W. GERALD2 Departments of Anatomy and Biology, T h e University of N e w Mexico School of Medicine, Albuquerque, N e w Mexico 871 31
ABSTRACT Acute brain stimulation experiments were carried out in anesthetized male green sunfish, Lepomis cyanellus. Semen discharge was evoked consistently by low level electrical stimulation (100 pA or less) in the following areas: the preoptic region, dorsal hypothalamus, thalamus, midbrain tegmentum and the basolateral midbrain and medulla. Areas which were stimulated repeatedly at 100 p A and were always negative for sperm release included: the telencephalon with the exception of the preoptic region, the optic tectum, the cerebellum, the inferior lobe of the hypothalamus, the nucleus rotundus and the dorsal medulla. Removal of most of the optic tectum and cerebellum failed to block responses evoked from the preoptic area; however, they were usually eliminated by transecting the rostra1 spinal cord. Electrical stimulation of an isQlated 4 mm segment of spinal cord located at the third vertebral level resulted in sperm release, indicating that adequate mechanisms for semen discharge are present within the upper spinal cord. The results of this study suggest that a sperm release system in the green sunfish extends from the preoptic area to the spinal cord passing through the hypothalamus, midbrain tegmentum and basal midbrain and medulla.
The preoptic area is involved in the control of reproductive behavior in many vertebrates including fishes (Demski and Knigge, '71 ; Peter, '73), amphibians (Aronsonnand Noble, '45; Schmidt, '68, '69), birds (Akerman, '66; Badield, '69, '71) and mammals (MacLean, '66; Roberts et al., '67; Robinson and Mishkin, '68; Pfaff et al., '73). Potential neural pathways involving this region in the control of penile erection in the squirrel monkey (MacLean, '66) and lordosis in the rat (Pfaff et al., '73) have been described; however, relatively little is known of the actual mechanisms by which preoptic control of these or other reproductive activities is mediated. We have approached this problem by studying analogous systems in teleosts, anticipating that their relatively small brain, in many cases stereotyped and elaborate behavior and in some species the ability to change sex will eventually aid in the identification and understanding of these mechanisms. This report describes a pathway by which the preoptic area may influence spinal mechariisms controlling sperm release. METHODS
Sperm release responses were evoked by J. EXP. ZOOL., 191: 2 1 S 2 3 2 .
electrical stimulation of the brain in 26 ripe male green sunfish, Lepomis cyanellus, that ranged in standard length from 11.3 to 25.5 cm. All animals were obtained from a local pond and maintained in laboratory aquaria under a 16 hour of light per day photoperiod. Fish were stimulated while anesthetized by a 0.3% urethane solution that was circulated through a hollow mouth holder and passed over their gills. Animals were held partially submerged in the anesthetic solution by a stainless steel apparatus previously described (Demski and Knigge, '71 ; Demski and Gerald, '72). Two observers recorded evoked semen discharges which were conspicuous as steady white streams discharged into the colorless anesthetic solution. Evoked responses usually began within 1 second of the onset of stimulation and terminated immediately following the stimulation. In some cases semen was examined microscopically and motile spermatozoa confirmed. Anal fin erection was often 1 Present address: Department of Anatomy, Louisiana State University Medical Center, 1190 Florida Avenue, New Orleans, Louisiana 70119. Guest investigator from the Department of Physiology and Behavioral Biology, San Francisco State University, San Francisco, California 94132.
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LEO S. DEMSKI, DIANA H. BAUER A N D JERRY W. GERALD
evoked and this response served as a good indicator that an electrode was being moved into an area from which sperm release could also be elicited. Monopolar electrodes were constructed from tapered 00 stainless steel insect pins insulated with Epoxylite except at the tip. The stimulation, provided by a Nuclear Chicago 7150 constant current stimulator, consisted of 50 Hz, 2 msec biphasic square wave pulse pairs of opposite polarity with currents equal to or less than 100 pA. The indifferent electrode was a bare wire placed in the solution bathing the fish. The stimulus current was measured on an oscilloscope as the voltage drop across a resistor in series with the animal. Most electrode tracks (167) were run in a dorsoventral direction; however, a few (14) were directed in an oblique lateromedial path. Using a micromanipulator, electrodes were lowered slowly into the brain with the stimulation set at 100 pA. The majority of our localized stimulation points (represented by the solid symbols in fig. 1) were obtained using the following procedure. When a positive response was observed, the threshold was determined. The electrode was then lowered in 0.1 mm increments and the threshold for sperm release was recorded at each point. After passing the lowest threshold site by at least 0.2 mm the electrode was returned to this site. If the threshold remained within 10 pA of the original determination, the area was marked with iron ions by passing a 20 pA anodal direct current through the electrode for a period of 10-20 seconds. In some cases the top and bottom of an electrode track were also marked in order to permit identification and plotting of either the entire track or various segments of it (dashed lines in fig. 1). Some responses were elicited at 10 FA, the lowest setting of the stimulator; when more than one 10 FA response was consecutively evoked, the midpoint of these-sites along the track was marked as the most sensitive region. Additional positive stimulation sites (represented as open symbols in fig. 1) were determined as indicated above, with the exception that the threshold was either nat redetermined prior to marking or had varied more than 10 pA from the original determination. A few points (also plotted as open symbols in fig. 1) were identified by “maximizing” the response, e.g., by mov-
ing the electrode up and down and marking the area from which the strongest and shortest latency responses could be evoked. The distribution of points identified using the three techniques listed above seemed to overlap and for this reason we have plotted them together on the same sections (fig. 1). It is our opinion, however, that the first method probably yielded the most reliable and reproducible results and for this reason we concluded the study using this procedure alone. Many negative tracks (current at 100 FA) were run, a few of which were marked by deposition of iron along the entire track. Histologically identified negative tracks are indicated as dotted lines in figure 1; each of these was followed by positive responses along subsequent electrode tracks in the same animal. Following testing, fish were decapitated and their brains fixed in 10% formalin, embedded in paraffin, serially sectioned in the frontal plane at 10 p and stained, using the Prussian blue method (Akert and Welker, ’61) for localization of iron ions deposited at the stimulation sites and neutral red (Humason, ’67) for identification of nuclear regions. Stimulation sites are plotted on a series of representative frontal sections of the brain of the green sunfish (fig. 1). The anatomical terminology used in this study was adopted from Ariens Kappers et al. (’36) and Demski and Knigge (‘71). In some experiments various portions of the brain were ablated using suction; in others the spinal cord was transected with a sharp instrument. RESULTS
Stim u 1ation mapping experiments Sperm release was evoked from a total of 68 histologically identified sites. These are plotted in figure 1 and include all points indicated by specific symbols as well as two marked sites in figure 1F (arrows at the ends of the solid line) located along an electrode track in the preoptic area (see further description below). Unless otherwise indicated, sites plotted in figure 1 were located along dorsoventrdy directed tracks. Positive responses were elicited from the following regions (table 1): preoptic area and adjacent sites in the area ventralis telencephali, the forebrain bundles and the optic chiasma (fig. 1C-F); dorsal hypothalamus in the region of the nucleus prerotun-
SPERM RELEASE EVOKED FROM THE FISH BRAIN TABLE I
Regional distribution of stimulation sites f r o m which s p e n n release was moked Threshold (PA) Region stimulated
0-25
Preoptic area and adjacentregions Dorsal hypothalamus-region of the nucleus prerotundus pars lateralis and the forebrain bundles Thalamic-rostra1 tegmental area Lateral tegmentum ofthemidbrain Medial tegmentum of the midbrain Basolateral midbrain and medulla
26-50
51-75
76-100
6
8
4
1
3
2
1
0
2
1
1
0
11
2
0
5
0
0
1
3
7
6
2
2
dus pars lateralis and the forebrain bundles (fig. 1G-I); the thalamus and rostral projection of the midbrain tegmentum (fig. 1HJ); the lateral tegmentum of the midbrain slightly dorsal to the nucleus prerotundus pars lateralis at rostral levels, and the nucleus rotundus at more caudal levels (fig. 11-L); the medial tegmentum of the midbrain (a) just dorsal to the nucleus prerotundus pars medialis (fig. 1J) and (b) medial to the medial longitudinal fasciculus (fig. 1M); and the basolateral region of the brainstem throughout the caudal midbrain (fig. lL,M) and medulla (fig. IN-S). The lowest threshold responses (10 PA) were evoked from the preoptic area, the lateral tegmentum and the basolateral medulla. Areas from which stimulation resulted in responses with only slightly higher thresholds (1 1-25 FA) included the dorsal hypothalamus and the region where the thalamus and rostral tegmentum merge. Relatively high threshold (55-100 PA) stimulation in the medial tegmentum probably resulted in sperm release by current spread and consequent activation of the low threshold area in the adjacent lateral tegmentum. In order to best pinpoint the most sensitive regions for evoking sperm release, we placed electrode tracks at nearly right angles to the dorsoventrally directed tracks initially run in these regions. Figure 1E illustrates two angled tracks which indicate that the lowest threshold area at the pre-
217
optic level was in the preoptic area rather than in the more laterally placed forebrain bundles. This conclusion is further supported by the finding that stimulation evoked sperm release at a very low current level (10 PA) only along the portion of an electrode track that was immediately adjacent to the preoptic area (region between arrows along the solid line in fig. l F ) , and the observation that the lowest threshold for responses evoked from pointscompletely within the forebrain bundles was 30 FA (site plotted in fig. 1D). Other positive sites in areas rostral (area ventralis telencephali) and ventral (optic chiasma) to the preoptic area also had thresholds generally higher than those obtained from nearby points in the preoptic region (fig. lC,E,F). This result is consistent with the idea that activation of the preoptic area, either directly or by current spread from adjacent areas, is responsible for all responses evoked at this rostrocaudal level of the brain. Angled tracks were also run in the thalamic-rostra1 tegmental transition area from which sperm release was elicited and these results were consistent with those from the dorsoventrally placed electrodes (fig. lH,J). In the lateral tegmentum, the most sensitive area appears to be slightly dorsal to the nucleus rotundus since the lowest threshold sites along two tracks run at nearly right angles are almost superimposed at this point (fig. 1J). Due to confined cranial space it was very difficult to run angled tracks in medulla and caudal midbrain. We were able, however, to run one track at the level of the ganglion isthmi and the results suggest a lateral position for the lowest threshold area at this level (fig. 1M). Observation of the relative thresholds of the positive sites in the basal medulla strongly suggests that the lowest threshold areas are located approximately midway between the midline and lateral aspect of the brain. In a few cases there was a bimodal distribution of low threshold areas along an electrode track. These occurred in areas where electrode tracks passed through or adjacent to two regions, each of which had previously been identified as areas from which relatively low threshold responses could be evoked. Two of these tracks are illustrated in figure 11. The more medial track had low threshold sites in the dorsal hypothalamus just ventral to the nucleus
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LEO S. DEMSKI, DIANA H. BAUER AND JERRY W. GERALD
prerotundus pars lateralis and in the rostral part of the lateral tegmentum slightly dorsal to the nucleus prerotundus pars lateralis. The lateral track on this section had two low threshold sites relatively close together. It can be suggested that the dorsal point is in the thalamic-tegmental transition area while the more ventral site is in the lateral tegmental zone since these areas overlap at this level. The third track with two separate loci for low threshold responses is shown in figure 1L. The dorsal point was in the caudal part of the lateral tegmentum while the more ventral site is in the basolateral area from which many positive responses were elicited. Many negative tracks were run in the telencephalon exclusive of the preoptic region and adjacent areas. A few of these were marked and are plotted as dotted lines in figure lB,C,F. Other major portions of the brain that were always negative for sperm release included the optic lobes (tectum), the inferior lobes, the nucleus rotundus, the body and valvula of the cerebellum and areas in the dorsal medulla. Several of these negative tracks are indicated in figure 10,P,R. Stimulation-lesion studies In order to further characterize the pathways involved in sperm release, we have studied the effects of lesions of the nervous system on responses evoked by preoptic stimulation. Removal of the body and valvula of the cerebellum and at least a major portion of the optic lobes (tectum) did not block evoked sperm release. Initial experiments in four animals suggested that transection of the spinal cord above the fourth vertebral level most often terminated semen discharge. Variations in these experiments may have been due to the incomplete transection of the spinal cord. In a subsequent experiment, transection of the spinal cord at the level of the junction of the third and fourth vertebrae did not stop sperm release evoked by preoptic stimulation. In order to be certain of complete transection, a portion of spinal cord extending several millimeters caudal to the cut was removed by suction, and a second transection was made at the level of the junction of the second and third vertebrae. This completely eliminated the response. A small portion of
spinal cord extending rostral to the second cut was then removed by suction. The remaining isolated piece of spinal cord (4 mm in length, situated 8 mm posterior to the obex at the level of the third vertebrae) was electrically stimulated and sperm discharges were elicited. This result indicates that adequate mechanisms for sperm release are present within a relatively small area of the rostral spinal cord. DISCUSSION
Without combined stimulation, lesion and recording experiments, the locus of points from which low threshold sperm discharges were evoked can only be tentatively considered to represent a single functional system. With this in mind, the present results suggest that a sperm release pathway extends from the preoptic area through the dorsal hypothalamus into the lateral tegmentum of the midbrain and possibly also part of the thalamus and then runs in a ventrolateral position into the upper spinal cord where the primary motor system for sperm release is located (fig. 2). Directionality within this pathway has not been determined. However, it appears that stimulation anywhere within the system is activating descending fibers which control the sperm release mechanism in the spinal cord, since similar low thresholds and short latencies for evoked semen discharges were found all along the pathway. The mechanism by which the spinal cord mediates sperm release in sunfish is not known. It probably involves regulation of a urogenital sphincter similar to the one described in the teleost Uranoscopus scaber in which the striated muscle of the sphincter is controlled by somatic spinal nerves (Young, '31). The present finding that preoptic stimulation in sunfish can result in sperm release is in accord with observations that electrical activation of this area can trigger various reproductive responses including: nestbuilding and courtship in sunfish (Demski and Knigge, '71); sexual calling (Schmidt, '68) and spermiation (Stutinsky and Befort, '64) in frogs; responsp associated with courtship in pigeons (Akerman, '66); and penile erection, seminal emission and mounting in various mammals (MacLean and Ploog, '62; Vaughan and Fisher,
SPERM RELEASE EVOKED FROM THE FISH BRAIN
'62; Robinson and Mishkin, '66, '68; Roberts et al., '67; Malsbury, '71). Our observations in fish which suggest that a sperm release pathway extends throughout the hypothalamus and midbrain tegmentum are consistent with the following results in other species: (a) electrical stimulation of the posterior lateral hypothalamus in the rat can elicit seminal discharge and mounting (Herberg, '63; Caggiula and Hoebel, '66); (b) the caudal tegmentum is necessary for normal spawning in frogs (Aronson and Noble, '45); and (c) tegmental regions are involved in suggested pathways for lordosis in the rat (Pfaff et al., '73) and penile erection, seminal emission and genital scratching in the squirrel monkey (MacLean et al., '63a,b). The basolateral location of the medullary portion of the fish sperm release system corresponds at least topographically to the general location of some of the points from which penile erection, genital scratching and seminal discharge have been evoked in the squirrel monkey (MacLean et al., '63a,b). Thus, these results can be interpreted to suggest that certain reproductive responses may be mediated by similar brainstem mechanisms in widespread vertebrate groups. In the rat, portions of the preoptic area, hypothalamus and midbrain tegmentum contain neurons that concentrate and are probably influenced by gonadal hormones (Pfaff et al., '73; Sar and Stumpf, '73). Similar concentrating cells have been demonstrated in the preoptic area, hypothalamus and midbrain in birds (Zigmond et al., '72, '73; Meyer, '73) and the African frog Xenop u s laevis (Kelley et al., '73; Morrell and Kelley, '74), while the preoptic area in the teleost Fundulus heteroclitus may contain neurons which mediate spawning reflexes in response to systemic injections of posterior pituitary extract (Peter, '73). If similar sensitive regions exist within the proposed sperm release pathway, it follows that elevated levels of certain reproductive hormones may act by decreasing the threshold for activation of the system by the various external stimuli present during courtship and spawning and thus result in appropriately timed seminal discharges. The present results suggest additional studies. For example, electrical stimulation of the proposed sperm release pathway in
219
free-swimming animals may be used to determine if it is involved solely in the control of semen discharge or rather in a variety of other reproductive activities as well. It will also be of interest to know if this pathway is specific to sunfish and whether or not i t is also controlling sexual activity in females. In this regard, preliminary studies in goldfish (unpublished observations) have demonstrated that stimulation near the preop tic area can evoke sperm release in ripe males and egg discharge in ovulated females. Additional mapping experiments will be necessary in order to determine any possible sexual and species differences in the proposed system. ACKNOWLEDGMENTS
The authors wish to thank Drs. A. J. Ladman, E. C. Palmer, A. Ratner and L. C. Saland for their comments on this manuscript. Supported in part by general research funds of The University of New Mexico School of Medicine. LITERATURE CITED Akerman, B. 1966 Behavioural effects of electrical stimulation in the forebrain of the pigeon. I. Reproductive behaviour. Behaviour, 26:323-338 Akert, K., and W. I. Welker 1961 Problems and methods of anatomical localization. In: Electrical Stimulation of the Brain. D. E. Sheer, ed. University of Texas Press, Austin, pp. 251-260. Ariens Kappers, C. U., G. C . Huber and E. C. Crosby 1936 The Comparative Anatomy of the Nervous System of Vertebrates including Man. Reprinted by Hafner, New York, 1965. Aronson, L. R., and G. K. Noble 1945 The sexual behavior of the anura. 2.Neural mechanisms controlling mating in the male leopard frog, R a n a p i p i e n s . Bull. Amer. Mus. Nat. Hist., 86: 83-140. Barfield, R. J. 1969 Activation of copulatory behavior by androgen implanted into the preoptic area of the male fowl. Horm. Behav., I : 37-52. 1971 Activation of sexual and aggressive behavior by androgen implanted into the male ring dove brain. Endocrinol., 89: 1470-1476. Caggiula, A. R., and B. G. Hoebel 1966 "Copulation-reward site" in the posterior hypothalamus. Science, 153: 12861285. Demski, L. S., and J. W. Gerald 1972 Sound production evoked by electrical stimulation of the brain in toadfish (Opsanus b e t a ) . Anim. B e hav., 20: 507-513. Demski, L. S., and K. M. Knigge 1971 The telencephalon and hypothalamus of the bluegill ( L e p o m i s m a c r o c h i r u s ) : Evoked feeding, aggressive and reproductive behavior with representative frontal sections. J. Comp. Neur., 143: 1-16. Herberg, L. J. 1963 Seminal ejaculation follow-
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ing positively reinforcing electrical stimulation of the rat hypothalamus. J. Comp. Physiol. Psychol., 56: 679-4385. Humason, G. L. 1967 Animal Tissue Techniques. Freeman, San Francisco. Kelley, D. B., D. W. Pfaf€ and J. I. Morrell 1973 Radioactivity in the brain of male South African clawed frogs (Xenopus laenis) following injection of H3-testosterone. An autoradiographic study. Amer. Zool., 13: 1287. MacLean, P. D. 1966 Studies on the cerebral representation of certain basic sexual functions. In: Brain and Behavior. Vol. 111. Brain and Gonadal Function. R. A. Gorski and R. E. Whalen, eds. University of California Press, Los Angeles, pp.
35-79. MacLean, P. D., R. H. Denniston and S . Dua 1963a Further studies on cerebral representation of penile erection: Caudal thalamus, midbrain and pons. J. Neurophysiol., 26: 273-293. 1963b Cerebral representation for scratching and seminal discharge. Arch. Neurol., 9:
485497. MacLean, P. D., and D. W. Ploog 1962 Cerebral representation of penile erection. J. Neurophysiol., 25:29-55. Malsbury, C. W. 1971 Facilitation of male rat copulatory behavior by electrical stimulation of the medial preoptic area. Physiol. Behav., 7:
797-805. Meyer, C. C. 1973 Testosterone concentration in the male chick brain: An autoradiographic survey. Science, 180: 1381-1383. Morrell, J. I., and D. B. Kelley 1974 3H-estradiol uptake in the brain of the female South African clawed frog (Xenopus laevis): A n autoradiographic study. Anat. Rec., 178: 422. Peter, R. E. 1973 Neuroendocrinology of teleosts. Amer. Zool., 13: 743-755.
Pfaff, D., C. Lewis, C. Diakow and M. Keiner 1973 Neurophysiological analysis of mating responses as hormonesensitive reflexes. In: Progress in Physiological Psychology. Vol. 5. E. Stellar and J. M. Sprague, eds. Academic Press, New York, pp. 253-297. Roberts, W. W., M. L. Steinberg and L. W. Means 1967 Hypothalamic mechanisms for sexual, aggressive and other motivational behaviors in the opossum, Didelphis virginiana. J. Comp. Physiol. Psychol., 64: 1-15. Robinson, B. W., and M. Mishkin 1966 Ejaculation evoked by stimulation of the preoptic area in monkey. Physiol. Behav., 1 : 269-272. 1968 Penile erection evoked from forebrain structures in Macaca mulatta. Arch. Neurol., 19: 184-198. Sar, M., and W. E. Stumpf 1973 Autoradiographic localization of radioactivity in the rat brain after the injection of 1,2-3H-testosterone. Endocrinol., 92: 251-256. Schmidt, R. S. 1968 Preoptic activation of frog mating behavior. Behaviour, 30: 239-257. Stutinsky, F., and J. J. Befort 1964 Effets des stimulations klectriques du diencephale de Rana esculenta male. Gen. Comp. Endocrinol., 4: 370-
379. Vaughan, E., and A. E. Fisher 1962 Male sexual behavior induced by intracranial electrical stimulation. Science, 137: 758-760. Young, J. 2 . 1931 On the autonomic nervous system of the teleostean fish, Uranoscopus scaber. Quart. J. Micr. Sci., 74: 491-535. Zigmond, R. E.,F. Nottebohm and D. W. Pfaff 1972 Distribution of androgen-concentrating cells in the brain of the chaffinch. Proc. IV Internat. Congr. Endrocrinol. (Abstract 340). 1973 Androgen-concentratingcells in the midbrain of a songbird. Science, 179: 1005-1007.
Abbreviations
AC, anterior commissure ADTC, area dorsalis telencephali pars centralis ADTD, area dorsalis telencephali pars dorsalis ADTL, area dorsalis telencephali pars lateralis ADTM, area dorsalis telencephali pars medialis ALA, acoustico-lateral area of medulla ALL, acoustico-lateral lemniscus AVT, area ventralis telencephali EG, eminentia granularis of cerebellum FB, forebrain bundles GI, ganglion isthmi GL, granule cell layer of cerebellum HAB, habenula ML, molecular layer of cerebellum MLF, medial longitudinal fasciculus MV, ventricle of the midbrain NDLI, nucleus diffusus lobi inferioris NDTL, nucleus diffusus tori lateralis NPRL, nucleus prerotundus pars lateralis
NR, nucleus rotundus NR3, oculomotor nerve N3, nucleus of oculomotor nerve OB, olfactory bulb OL, optic lobe OT, optic tract P, pituitary PC, posterior commissure PO, preoptic area POC, postoptic commissures SV, saccus vasculosus TC, transverse commissure TEG, tegmentum of the midbrain TH, thalamus TL, torus longi tudin alis TN, tuberal nuclei TS, torus semicircularis VAL, valvula of the cerebellum V3, third ventricle V4, fourth ventricle
PLATES 1-8 EXPLANATION OF FIGURES
1 A-T
Distribution of Prussian blue marked stimulation sites from which sperm release was evoked at or less than 100 pA. Points are plotted on representative frontal sections of the brain of the green sunfish. Solid symbols represent marked sites that were within 10 p A of the lowest threshold obtained along the electrode track (METHODS). Open symbols indicaie marked sites with thresholds not necessarily within 10 FA of the lowest threshold obtained along the track or sites in which the response was “maxirhized” (METHODS). Symbols: circles, stimulation sites with thresholds of 0-25 FA; squares, stimulation sites with thresholds of 2650 PA; triangles (pointed up), stimulation sites with thresholds of 51-75 PA; triangles (pointed down), stimulation sites with thresholds of 76100 FA; dotted lines, Prussian blue marked electrode tracks that were negative for sperm release at 100 p A ; dashed lines, reconstructed electrode tracks; solid line between arrows (fig. l F ) , part of an electrode track showing the locus of points from which sperm release was evoked at 10 p A .
22 1
SPERM RELEASE EVOKED FROM THE FISH BRAIN Leo S. Dernski, Diana H . Bauer and Jerry W. Gerald
222
PLATES 1 and 2
PO
F 223
SPERM RELEASE EVOKED FROM THE FISH BRAIN Leo S. Demski, D i a n a H. Bauer and Jerry W. Gerald
G
224
PLATES 3 and 4
I
225
SPERM RELEASE EVOKED FROM THE FISH BRAIN Leo S. Demski, D i a n a H. Bauer and Jerry W. Gerald
226
PLATES 5 and 6
N 227
SPERM RELEASE EVOKED FROM THE FISH BRAIN Leo S. Demski, D i a n a H. Bauer and Jerry W. Gerald
0
b w T
P 228
PLATES 7 and 8
0
t c w
2
Schematic representation of the locus of points (hatched area) from which low threshold sperm discharges were evoked by electrical stimulation of the brain in anesthetized green sunfish. The diagram is a composite representing several sagittal planes. The area indicated is thought to represent a n integrated system that extends from the preoptic area to the spinal cord, passing through the lateral tegmentum slightly dorsal to the nucleus rotundus (N. Rot.) and the basolateral midbrain and medulla ( D I S C U S S I O N ) .
EXPLANATION O F FIGURE
PLATE 9
BULB
SPERM RELEASE EVOKED FROM THE FISH BRAIN Leo S . Demski, Diana H. Bauer a n d Jerry W. Gerald PLATE 9
ABSTRACT Acute brain stimulation experiments were carried out in anesthetized male green sunfish, Lepomis cyanellus. Semen discharge was evoked consistently by low level electrical stimulation (100 pA or less) in the following areas: the preoptic region, dorsal hypothalamus, thalamus, midbrain tegmentum and the basolateral midbrain and medulla. Areas which were stimulated repeatedly at 100 p A and were always negative for sperm release included: the telencephalon with the exception of the preoptic region, the optic tectum, the cerebellum, the inferior lobe of the hypothalamus, the nucleus rotundus and the dorsal medulla. Removal of most of the optic tectum and cerebellum failed to block responses evoked from the preoptic area; however, they were usually eliminated by transecting the rostra1 spinal cord. Electrical stimulation of an isQlated 4 mm segment of spinal cord located at the third vertebral level resulted in sperm release, indicating that adequate mechanisms for semen discharge are present within the upper spinal cord. The results of this study suggest that a sperm release system in the green sunfish extends from the preoptic area to the spinal cord passing through the hypothalamus, midbrain tegmentum and basal midbrain and medulla.
The preoptic area is involved in the control of reproductive behavior in many vertebrates including fishes (Demski and Knigge, '71 ; Peter, '73), amphibians (Aronsonnand Noble, '45; Schmidt, '68, '69), birds (Akerman, '66; Badield, '69, '71) and mammals (MacLean, '66; Roberts et al., '67; Robinson and Mishkin, '68; Pfaff et al., '73). Potential neural pathways involving this region in the control of penile erection in the squirrel monkey (MacLean, '66) and lordosis in the rat (Pfaff et al., '73) have been described; however, relatively little is known of the actual mechanisms by which preoptic control of these or other reproductive activities is mediated. We have approached this problem by studying analogous systems in teleosts, anticipating that their relatively small brain, in many cases stereotyped and elaborate behavior and in some species the ability to change sex will eventually aid in the identification and understanding of these mechanisms. This report describes a pathway by which the preoptic area may influence spinal mechariisms controlling sperm release. METHODS
Sperm release responses were evoked by J. EXP. ZOOL., 191: 2 1 S 2 3 2 .
electrical stimulation of the brain in 26 ripe male green sunfish, Lepomis cyanellus, that ranged in standard length from 11.3 to 25.5 cm. All animals were obtained from a local pond and maintained in laboratory aquaria under a 16 hour of light per day photoperiod. Fish were stimulated while anesthetized by a 0.3% urethane solution that was circulated through a hollow mouth holder and passed over their gills. Animals were held partially submerged in the anesthetic solution by a stainless steel apparatus previously described (Demski and Knigge, '71 ; Demski and Gerald, '72). Two observers recorded evoked semen discharges which were conspicuous as steady white streams discharged into the colorless anesthetic solution. Evoked responses usually began within 1 second of the onset of stimulation and terminated immediately following the stimulation. In some cases semen was examined microscopically and motile spermatozoa confirmed. Anal fin erection was often 1 Present address: Department of Anatomy, Louisiana State University Medical Center, 1190 Florida Avenue, New Orleans, Louisiana 70119. Guest investigator from the Department of Physiology and Behavioral Biology, San Francisco State University, San Francisco, California 94132.
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LEO S. DEMSKI, DIANA H. BAUER A N D JERRY W. GERALD
evoked and this response served as a good indicator that an electrode was being moved into an area from which sperm release could also be elicited. Monopolar electrodes were constructed from tapered 00 stainless steel insect pins insulated with Epoxylite except at the tip. The stimulation, provided by a Nuclear Chicago 7150 constant current stimulator, consisted of 50 Hz, 2 msec biphasic square wave pulse pairs of opposite polarity with currents equal to or less than 100 pA. The indifferent electrode was a bare wire placed in the solution bathing the fish. The stimulus current was measured on an oscilloscope as the voltage drop across a resistor in series with the animal. Most electrode tracks (167) were run in a dorsoventral direction; however, a few (14) were directed in an oblique lateromedial path. Using a micromanipulator, electrodes were lowered slowly into the brain with the stimulation set at 100 pA. The majority of our localized stimulation points (represented by the solid symbols in fig. 1) were obtained using the following procedure. When a positive response was observed, the threshold was determined. The electrode was then lowered in 0.1 mm increments and the threshold for sperm release was recorded at each point. After passing the lowest threshold site by at least 0.2 mm the electrode was returned to this site. If the threshold remained within 10 pA of the original determination, the area was marked with iron ions by passing a 20 pA anodal direct current through the electrode for a period of 10-20 seconds. In some cases the top and bottom of an electrode track were also marked in order to permit identification and plotting of either the entire track or various segments of it (dashed lines in fig. 1). Some responses were elicited at 10 FA, the lowest setting of the stimulator; when more than one 10 FA response was consecutively evoked, the midpoint of these-sites along the track was marked as the most sensitive region. Additional positive stimulation sites (represented as open symbols in fig. 1) were determined as indicated above, with the exception that the threshold was either nat redetermined prior to marking or had varied more than 10 pA from the original determination. A few points (also plotted as open symbols in fig. 1) were identified by “maximizing” the response, e.g., by mov-
ing the electrode up and down and marking the area from which the strongest and shortest latency responses could be evoked. The distribution of points identified using the three techniques listed above seemed to overlap and for this reason we have plotted them together on the same sections (fig. 1). It is our opinion, however, that the first method probably yielded the most reliable and reproducible results and for this reason we concluded the study using this procedure alone. Many negative tracks (current at 100 FA) were run, a few of which were marked by deposition of iron along the entire track. Histologically identified negative tracks are indicated as dotted lines in figure 1; each of these was followed by positive responses along subsequent electrode tracks in the same animal. Following testing, fish were decapitated and their brains fixed in 10% formalin, embedded in paraffin, serially sectioned in the frontal plane at 10 p and stained, using the Prussian blue method (Akert and Welker, ’61) for localization of iron ions deposited at the stimulation sites and neutral red (Humason, ’67) for identification of nuclear regions. Stimulation sites are plotted on a series of representative frontal sections of the brain of the green sunfish (fig. 1). The anatomical terminology used in this study was adopted from Ariens Kappers et al. (’36) and Demski and Knigge (‘71). In some experiments various portions of the brain were ablated using suction; in others the spinal cord was transected with a sharp instrument. RESULTS
Stim u 1ation mapping experiments Sperm release was evoked from a total of 68 histologically identified sites. These are plotted in figure 1 and include all points indicated by specific symbols as well as two marked sites in figure 1F (arrows at the ends of the solid line) located along an electrode track in the preoptic area (see further description below). Unless otherwise indicated, sites plotted in figure 1 were located along dorsoventrdy directed tracks. Positive responses were elicited from the following regions (table 1): preoptic area and adjacent sites in the area ventralis telencephali, the forebrain bundles and the optic chiasma (fig. 1C-F); dorsal hypothalamus in the region of the nucleus prerotun-
SPERM RELEASE EVOKED FROM THE FISH BRAIN TABLE I
Regional distribution of stimulation sites f r o m which s p e n n release was moked Threshold (PA) Region stimulated
0-25
Preoptic area and adjacentregions Dorsal hypothalamus-region of the nucleus prerotundus pars lateralis and the forebrain bundles Thalamic-rostra1 tegmental area Lateral tegmentum ofthemidbrain Medial tegmentum of the midbrain Basolateral midbrain and medulla
26-50
51-75
76-100
6
8
4
1
3
2
1
0
2
1
1
0
11
2
0
5
0
0
1
3
7
6
2
2
dus pars lateralis and the forebrain bundles (fig. 1G-I); the thalamus and rostral projection of the midbrain tegmentum (fig. 1HJ); the lateral tegmentum of the midbrain slightly dorsal to the nucleus prerotundus pars lateralis at rostral levels, and the nucleus rotundus at more caudal levels (fig. 11-L); the medial tegmentum of the midbrain (a) just dorsal to the nucleus prerotundus pars medialis (fig. 1J) and (b) medial to the medial longitudinal fasciculus (fig. 1M); and the basolateral region of the brainstem throughout the caudal midbrain (fig. lL,M) and medulla (fig. IN-S). The lowest threshold responses (10 PA) were evoked from the preoptic area, the lateral tegmentum and the basolateral medulla. Areas from which stimulation resulted in responses with only slightly higher thresholds (1 1-25 FA) included the dorsal hypothalamus and the region where the thalamus and rostral tegmentum merge. Relatively high threshold (55-100 PA) stimulation in the medial tegmentum probably resulted in sperm release by current spread and consequent activation of the low threshold area in the adjacent lateral tegmentum. In order to best pinpoint the most sensitive regions for evoking sperm release, we placed electrode tracks at nearly right angles to the dorsoventrally directed tracks initially run in these regions. Figure 1E illustrates two angled tracks which indicate that the lowest threshold area at the pre-
217
optic level was in the preoptic area rather than in the more laterally placed forebrain bundles. This conclusion is further supported by the finding that stimulation evoked sperm release at a very low current level (10 PA) only along the portion of an electrode track that was immediately adjacent to the preoptic area (region between arrows along the solid line in fig. l F ) , and the observation that the lowest threshold for responses evoked from pointscompletely within the forebrain bundles was 30 FA (site plotted in fig. 1D). Other positive sites in areas rostral (area ventralis telencephali) and ventral (optic chiasma) to the preoptic area also had thresholds generally higher than those obtained from nearby points in the preoptic region (fig. lC,E,F). This result is consistent with the idea that activation of the preoptic area, either directly or by current spread from adjacent areas, is responsible for all responses evoked at this rostrocaudal level of the brain. Angled tracks were also run in the thalamic-rostra1 tegmental transition area from which sperm release was elicited and these results were consistent with those from the dorsoventrally placed electrodes (fig. lH,J). In the lateral tegmentum, the most sensitive area appears to be slightly dorsal to the nucleus rotundus since the lowest threshold sites along two tracks run at nearly right angles are almost superimposed at this point (fig. 1J). Due to confined cranial space it was very difficult to run angled tracks in medulla and caudal midbrain. We were able, however, to run one track at the level of the ganglion isthmi and the results suggest a lateral position for the lowest threshold area at this level (fig. 1M). Observation of the relative thresholds of the positive sites in the basal medulla strongly suggests that the lowest threshold areas are located approximately midway between the midline and lateral aspect of the brain. In a few cases there was a bimodal distribution of low threshold areas along an electrode track. These occurred in areas where electrode tracks passed through or adjacent to two regions, each of which had previously been identified as areas from which relatively low threshold responses could be evoked. Two of these tracks are illustrated in figure 11. The more medial track had low threshold sites in the dorsal hypothalamus just ventral to the nucleus
218
LEO S. DEMSKI, DIANA H. BAUER AND JERRY W. GERALD
prerotundus pars lateralis and in the rostral part of the lateral tegmentum slightly dorsal to the nucleus prerotundus pars lateralis. The lateral track on this section had two low threshold sites relatively close together. It can be suggested that the dorsal point is in the thalamic-tegmental transition area while the more ventral site is in the lateral tegmental zone since these areas overlap at this level. The third track with two separate loci for low threshold responses is shown in figure 1L. The dorsal point was in the caudal part of the lateral tegmentum while the more ventral site is in the basolateral area from which many positive responses were elicited. Many negative tracks were run in the telencephalon exclusive of the preoptic region and adjacent areas. A few of these were marked and are plotted as dotted lines in figure lB,C,F. Other major portions of the brain that were always negative for sperm release included the optic lobes (tectum), the inferior lobes, the nucleus rotundus, the body and valvula of the cerebellum and areas in the dorsal medulla. Several of these negative tracks are indicated in figure 10,P,R. Stimulation-lesion studies In order to further characterize the pathways involved in sperm release, we have studied the effects of lesions of the nervous system on responses evoked by preoptic stimulation. Removal of the body and valvula of the cerebellum and at least a major portion of the optic lobes (tectum) did not block evoked sperm release. Initial experiments in four animals suggested that transection of the spinal cord above the fourth vertebral level most often terminated semen discharge. Variations in these experiments may have been due to the incomplete transection of the spinal cord. In a subsequent experiment, transection of the spinal cord at the level of the junction of the third and fourth vertebrae did not stop sperm release evoked by preoptic stimulation. In order to be certain of complete transection, a portion of spinal cord extending several millimeters caudal to the cut was removed by suction, and a second transection was made at the level of the junction of the second and third vertebrae. This completely eliminated the response. A small portion of
spinal cord extending rostral to the second cut was then removed by suction. The remaining isolated piece of spinal cord (4 mm in length, situated 8 mm posterior to the obex at the level of the third vertebrae) was electrically stimulated and sperm discharges were elicited. This result indicates that adequate mechanisms for sperm release are present within a relatively small area of the rostral spinal cord. DISCUSSION
Without combined stimulation, lesion and recording experiments, the locus of points from which low threshold sperm discharges were evoked can only be tentatively considered to represent a single functional system. With this in mind, the present results suggest that a sperm release pathway extends from the preoptic area through the dorsal hypothalamus into the lateral tegmentum of the midbrain and possibly also part of the thalamus and then runs in a ventrolateral position into the upper spinal cord where the primary motor system for sperm release is located (fig. 2). Directionality within this pathway has not been determined. However, it appears that stimulation anywhere within the system is activating descending fibers which control the sperm release mechanism in the spinal cord, since similar low thresholds and short latencies for evoked semen discharges were found all along the pathway. The mechanism by which the spinal cord mediates sperm release in sunfish is not known. It probably involves regulation of a urogenital sphincter similar to the one described in the teleost Uranoscopus scaber in which the striated muscle of the sphincter is controlled by somatic spinal nerves (Young, '31). The present finding that preoptic stimulation in sunfish can result in sperm release is in accord with observations that electrical activation of this area can trigger various reproductive responses including: nestbuilding and courtship in sunfish (Demski and Knigge, '71); sexual calling (Schmidt, '68) and spermiation (Stutinsky and Befort, '64) in frogs; responsp associated with courtship in pigeons (Akerman, '66); and penile erection, seminal emission and mounting in various mammals (MacLean and Ploog, '62; Vaughan and Fisher,
SPERM RELEASE EVOKED FROM THE FISH BRAIN
'62; Robinson and Mishkin, '66, '68; Roberts et al., '67; Malsbury, '71). Our observations in fish which suggest that a sperm release pathway extends throughout the hypothalamus and midbrain tegmentum are consistent with the following results in other species: (a) electrical stimulation of the posterior lateral hypothalamus in the rat can elicit seminal discharge and mounting (Herberg, '63; Caggiula and Hoebel, '66); (b) the caudal tegmentum is necessary for normal spawning in frogs (Aronson and Noble, '45); and (c) tegmental regions are involved in suggested pathways for lordosis in the rat (Pfaff et al., '73) and penile erection, seminal emission and genital scratching in the squirrel monkey (MacLean et al., '63a,b). The basolateral location of the medullary portion of the fish sperm release system corresponds at least topographically to the general location of some of the points from which penile erection, genital scratching and seminal discharge have been evoked in the squirrel monkey (MacLean et al., '63a,b). Thus, these results can be interpreted to suggest that certain reproductive responses may be mediated by similar brainstem mechanisms in widespread vertebrate groups. In the rat, portions of the preoptic area, hypothalamus and midbrain tegmentum contain neurons that concentrate and are probably influenced by gonadal hormones (Pfaff et al., '73; Sar and Stumpf, '73). Similar concentrating cells have been demonstrated in the preoptic area, hypothalamus and midbrain in birds (Zigmond et al., '72, '73; Meyer, '73) and the African frog Xenop u s laevis (Kelley et al., '73; Morrell and Kelley, '74), while the preoptic area in the teleost Fundulus heteroclitus may contain neurons which mediate spawning reflexes in response to systemic injections of posterior pituitary extract (Peter, '73). If similar sensitive regions exist within the proposed sperm release pathway, it follows that elevated levels of certain reproductive hormones may act by decreasing the threshold for activation of the system by the various external stimuli present during courtship and spawning and thus result in appropriately timed seminal discharges. The present results suggest additional studies. For example, electrical stimulation of the proposed sperm release pathway in
219
free-swimming animals may be used to determine if it is involved solely in the control of semen discharge or rather in a variety of other reproductive activities as well. It will also be of interest to know if this pathway is specific to sunfish and whether or not i t is also controlling sexual activity in females. In this regard, preliminary studies in goldfish (unpublished observations) have demonstrated that stimulation near the preop tic area can evoke sperm release in ripe males and egg discharge in ovulated females. Additional mapping experiments will be necessary in order to determine any possible sexual and species differences in the proposed system. ACKNOWLEDGMENTS
The authors wish to thank Drs. A. J. Ladman, E. C. Palmer, A. Ratner and L. C. Saland for their comments on this manuscript. Supported in part by general research funds of The University of New Mexico School of Medicine. LITERATURE CITED Akerman, B. 1966 Behavioural effects of electrical stimulation in the forebrain of the pigeon. I. Reproductive behaviour. Behaviour, 26:323-338 Akert, K., and W. I. Welker 1961 Problems and methods of anatomical localization. In: Electrical Stimulation of the Brain. D. E. Sheer, ed. University of Texas Press, Austin, pp. 251-260. Ariens Kappers, C. U., G. C . Huber and E. C. Crosby 1936 The Comparative Anatomy of the Nervous System of Vertebrates including Man. Reprinted by Hafner, New York, 1965. Aronson, L. R., and G. K. Noble 1945 The sexual behavior of the anura. 2.Neural mechanisms controlling mating in the male leopard frog, R a n a p i p i e n s . Bull. Amer. Mus. Nat. Hist., 86: 83-140. Barfield, R. J. 1969 Activation of copulatory behavior by androgen implanted into the preoptic area of the male fowl. Horm. Behav., I : 37-52. 1971 Activation of sexual and aggressive behavior by androgen implanted into the male ring dove brain. Endocrinol., 89: 1470-1476. Caggiula, A. R., and B. G. Hoebel 1966 "Copulation-reward site" in the posterior hypothalamus. Science, 153: 12861285. Demski, L. S., and J. W. Gerald 1972 Sound production evoked by electrical stimulation of the brain in toadfish (Opsanus b e t a ) . Anim. B e hav., 20: 507-513. Demski, L. S., and K. M. Knigge 1971 The telencephalon and hypothalamus of the bluegill ( L e p o m i s m a c r o c h i r u s ) : Evoked feeding, aggressive and reproductive behavior with representative frontal sections. J. Comp. Neur., 143: 1-16. Herberg, L. J. 1963 Seminal ejaculation follow-
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ing positively reinforcing electrical stimulation of the rat hypothalamus. J. Comp. Physiol. Psychol., 56: 679-4385. Humason, G. L. 1967 Animal Tissue Techniques. Freeman, San Francisco. Kelley, D. B., D. W. Pfaf€ and J. I. Morrell 1973 Radioactivity in the brain of male South African clawed frogs (Xenopus laenis) following injection of H3-testosterone. An autoradiographic study. Amer. Zool., 13: 1287. MacLean, P. D. 1966 Studies on the cerebral representation of certain basic sexual functions. In: Brain and Behavior. Vol. 111. Brain and Gonadal Function. R. A. Gorski and R. E. Whalen, eds. University of California Press, Los Angeles, pp.
35-79. MacLean, P. D., R. H. Denniston and S . Dua 1963a Further studies on cerebral representation of penile erection: Caudal thalamus, midbrain and pons. J. Neurophysiol., 26: 273-293. 1963b Cerebral representation for scratching and seminal discharge. Arch. Neurol., 9:
485497. MacLean, P. D., and D. W. Ploog 1962 Cerebral representation of penile erection. J. Neurophysiol., 25:29-55. Malsbury, C. W. 1971 Facilitation of male rat copulatory behavior by electrical stimulation of the medial preoptic area. Physiol. Behav., 7:
797-805. Meyer, C. C. 1973 Testosterone concentration in the male chick brain: An autoradiographic survey. Science, 180: 1381-1383. Morrell, J. I., and D. B. Kelley 1974 3H-estradiol uptake in the brain of the female South African clawed frog (Xenopus laevis): A n autoradiographic study. Anat. Rec., 178: 422. Peter, R. E. 1973 Neuroendocrinology of teleosts. Amer. Zool., 13: 743-755.
Pfaff, D., C. Lewis, C. Diakow and M. Keiner 1973 Neurophysiological analysis of mating responses as hormonesensitive reflexes. In: Progress in Physiological Psychology. Vol. 5. E. Stellar and J. M. Sprague, eds. Academic Press, New York, pp. 253-297. Roberts, W. W., M. L. Steinberg and L. W. Means 1967 Hypothalamic mechanisms for sexual, aggressive and other motivational behaviors in the opossum, Didelphis virginiana. J. Comp. Physiol. Psychol., 64: 1-15. Robinson, B. W., and M. Mishkin 1966 Ejaculation evoked by stimulation of the preoptic area in monkey. Physiol. Behav., 1 : 269-272. 1968 Penile erection evoked from forebrain structures in Macaca mulatta. Arch. Neurol., 19: 184-198. Sar, M., and W. E. Stumpf 1973 Autoradiographic localization of radioactivity in the rat brain after the injection of 1,2-3H-testosterone. Endocrinol., 92: 251-256. Schmidt, R. S. 1968 Preoptic activation of frog mating behavior. Behaviour, 30: 239-257. Stutinsky, F., and J. J. Befort 1964 Effets des stimulations klectriques du diencephale de Rana esculenta male. Gen. Comp. Endocrinol., 4: 370-
379. Vaughan, E., and A. E. Fisher 1962 Male sexual behavior induced by intracranial electrical stimulation. Science, 137: 758-760. Young, J. 2 . 1931 On the autonomic nervous system of the teleostean fish, Uranoscopus scaber. Quart. J. Micr. Sci., 74: 491-535. Zigmond, R. E.,F. Nottebohm and D. W. Pfaff 1972 Distribution of androgen-concentrating cells in the brain of the chaffinch. Proc. IV Internat. Congr. Endrocrinol. (Abstract 340). 1973 Androgen-concentratingcells in the midbrain of a songbird. Science, 179: 1005-1007.
Abbreviations
AC, anterior commissure ADTC, area dorsalis telencephali pars centralis ADTD, area dorsalis telencephali pars dorsalis ADTL, area dorsalis telencephali pars lateralis ADTM, area dorsalis telencephali pars medialis ALA, acoustico-lateral area of medulla ALL, acoustico-lateral lemniscus AVT, area ventralis telencephali EG, eminentia granularis of cerebellum FB, forebrain bundles GI, ganglion isthmi GL, granule cell layer of cerebellum HAB, habenula ML, molecular layer of cerebellum MLF, medial longitudinal fasciculus MV, ventricle of the midbrain NDLI, nucleus diffusus lobi inferioris NDTL, nucleus diffusus tori lateralis NPRL, nucleus prerotundus pars lateralis
NR, nucleus rotundus NR3, oculomotor nerve N3, nucleus of oculomotor nerve OB, olfactory bulb OL, optic lobe OT, optic tract P, pituitary PC, posterior commissure PO, preoptic area POC, postoptic commissures SV, saccus vasculosus TC, transverse commissure TEG, tegmentum of the midbrain TH, thalamus TL, torus longi tudin alis TN, tuberal nuclei TS, torus semicircularis VAL, valvula of the cerebellum V3, third ventricle V4, fourth ventricle
PLATES 1-8 EXPLANATION OF FIGURES
1 A-T
Distribution of Prussian blue marked stimulation sites from which sperm release was evoked at or less than 100 pA. Points are plotted on representative frontal sections of the brain of the green sunfish. Solid symbols represent marked sites that were within 10 p A of the lowest threshold obtained along the electrode track (METHODS). Open symbols indicaie marked sites with thresholds not necessarily within 10 FA of the lowest threshold obtained along the track or sites in which the response was “maxirhized” (METHODS). Symbols: circles, stimulation sites with thresholds of 0-25 FA; squares, stimulation sites with thresholds of 2650 PA; triangles (pointed up), stimulation sites with thresholds of 51-75 PA; triangles (pointed down), stimulation sites with thresholds of 76100 FA; dotted lines, Prussian blue marked electrode tracks that were negative for sperm release at 100 p A ; dashed lines, reconstructed electrode tracks; solid line between arrows (fig. l F ) , part of an electrode track showing the locus of points from which sperm release was evoked at 10 p A .
22 1
SPERM RELEASE EVOKED FROM THE FISH BRAIN Leo S. Dernski, Diana H . Bauer and Jerry W. Gerald
222
PLATES 1 and 2
PO
F 223
SPERM RELEASE EVOKED FROM THE FISH BRAIN Leo S. Demski, D i a n a H. Bauer and Jerry W. Gerald
G
224
PLATES 3 and 4
I
225
SPERM RELEASE EVOKED FROM THE FISH BRAIN Leo S. Demski, D i a n a H. Bauer and Jerry W. Gerald
226
PLATES 5 and 6
N 227
SPERM RELEASE EVOKED FROM THE FISH BRAIN Leo S. Demski, D i a n a H. Bauer and Jerry W. Gerald
0
b w T
P 228
PLATES 7 and 8
0
t c w
2
Schematic representation of the locus of points (hatched area) from which low threshold sperm discharges were evoked by electrical stimulation of the brain in anesthetized green sunfish. The diagram is a composite representing several sagittal planes. The area indicated is thought to represent a n integrated system that extends from the preoptic area to the spinal cord, passing through the lateral tegmentum slightly dorsal to the nucleus rotundus (N. Rot.) and the basolateral midbrain and medulla ( D I S C U S S I O N ) .
EXPLANATION O F FIGURE
PLATE 9
BULB
SPERM RELEASE EVOKED FROM THE FISH BRAIN Leo S . Demski, Diana H. Bauer a n d Jerry W. Gerald PLATE 9
