Microfluorimetric quantitation of catecholamine fluorescence in rat median eminence. II. Turnover changes in hormonal states.

Catecholamine nerve terminals in the rat median eminence have been studied using the fluorescence histochemical technique of Falck and Hillarp in combination with quantitative microfluorimetry. The catecholamine fluorescence intensities recorded from various parts of the median eminence were all found to be within the linear part of the dopamine or noradrenaline concentration-fluorescence relationship as studied in an agar-albumin model system. The catecholamine fluorescence was also found to disappear with time in an exponential manner following tyrosine hydroxylase inhibition produced by alpha-methyl-p-tyrosine methylester (H44/68). Similar results were obtained when measuring the dopamine decline by mass fragmentography in the median eminence after H44/68 treatment. These results and analysis of fluorescence frequency histograms strongly indicate that the catecholamine fluorescence values recorded are proportional to the catecholamine concentration. It is concluded that the microfluorimetric technique used is a reliable method for catecholamine quantitation in discrete nerve terminal areas of the median eminence. The main advantages of the technique are that a high sensitivity and quantitative data on the transmitter content can be obtained in strict relation to the neuroanatomy. Measurement of the catecholamine fluorescence disappearance after H44/68 was used to evaluate catecholamine turnover during various endocrine states. The results showed that two dopamine systems with different transmitter turnover may be distinguished. Tuberinfundibular dopamine neurons projecting to the lateral palisade zone were thus shown to have a slower turnover than those projecting medially to the capillary loops. No definite changes in catecholamine turnover were observed after adrenalectomy and castration in the male, although there was a tendency toward increased noradrenaline turnover in both states. During pregnancy an increase in noradrenaline as well as dopamine turnover was noted. The present results therefore give further evidence for the view that catecholamine nerve terminals in the median eminence may participate in the regulation of gonadotrophin secretion.     

Dopamine D1 and D2 Receptor Immunoreactivities in the Arcuate-Median Eminence Complex and their Link to the Tubero-Infundibular Dopamine Neurons

Dopamine D1 and D2 receptor immunohistochemistry and Golgi techniques were used to study the structure of the adult rat arcuate-median eminence complex, and determine the distribution of the dopamine D1 and D2 receptor immunoreactivities therein, particularly in relation to the tubero-infundibular dopamine neurons. Punctate dopamine D1 and D2 receptor immunoreactivities, likely located on nerve terminals, were enriched in the lateral palisade zone built up of nerve terminals, while the densities were low to modest in the medial palisade zone. A codistribution of dopamine D1 receptor or dopamine D2 receptor immunoreactive puncta with tyrosine hydroxylase immunoreactive nerve terminals was demonstrated in the external layer. Dopamine D1 receptor but not dopamine D2 receptor immnunoreactivites nerve cell bodies were found in the ventromedial part of the arcuate nucleus and in the lateral part of the internal layer of the median eminence forming a continuous cell mass presumably representing neuropeptide Y immunoreactive nerve cell bodies. The major arcuate dopamine/ tyrosine hydroxylase nerve cell group was found in the dorsomedial part. A large number of tyrosine hydroxylase immunoreactive nerve cell bodies in this region demonstrated punctate dopamine D1 receptor immunoreactivity but only a few presented dopamine D2 receptor immunoreactivity which were mainly found in a substantial number of tyrosine hydroxylase cell bodies of the ventral periventricular hypothalamic nucleus, also belonging to the tuberoinfundibular dopamine neurons. Structural evidence for projections of the arcuate nerve cells into the median eminence was also obtained. Distal axons formed horizontal axons in the internal layer issuing a variable number of collaterals classified into single or multiple strands located in the external layer increasing our understanding of the dopamine nerve terminal networks in this region. Dopamine D1 and D2 receptors may therefore directly and differentially modulate the activity and/or Dopamine synthesis of substantial numbers of tubero-infundibular dopamine neurons at the somatic and terminal level. The immunohistochemical work also gives support to the view that dopamine D1 receptors and/or dopamine D2 receptors in the lateral palisade zone by mediating dopamine volume transmission may contribute to the inhibition of luteinizing hormone releasing hormone release from nerve terminals in this region.Key words: Dopamine D1 and D2 receptors, tubero-infundibular dopamine neurons, dopamine receptor colocalization, arcuate-median eminence complex, volume transmission, luteinizing hormone releasing hormone     

Quantitative radioautographic light and electron microscopic analysis of the localization of monoamines in the median eminence of the rat

Summary By means of light and electron microscopic radioautography a quantitative study of the regional distribution of catecholamines in the median eminence of the rat was carried out. One hour after intraventricular injection of ³H-dopamine the highest radioautographic reaction was recorded in the external zone, especially in the lateral palisade zone where many neurosecretory terminals are separated from the basal lamina of the portal pericapillary space by a glial cuff. This area showed the highest percentage (52%) of labelled catecholamine containing neurosecretory terminals as well as the maximal silver grain density per one terminal. In the medial palisade zone where direct neurovascular contacts with the capillary loops prevail, only 27 per cent of neurosecretory terminals were found to harbour tritiated dopamine. On the average 35 per cent of neurosecretory terminals in the median eminence of the rat contain catecholamines (both dopamine and noradrenaline). Pretreatment of animals with reserpine strongly reduced the binding of the label. Per cent of labelled neurosecretory terminals as well as grain density over terminals were decreased in both the medial and lateral palisade zones, although to a lesser degree in the latter. The site of origin of catecholamine fibers as well as the mode of catecholamine action at the level of the median eminence are discussed.This work was presented at the joint session of the Society of Anatomists, Histologists and Embryologists, the Society of Physiologists, and the Society of Neuroendocrinologists dedicated to the memory of Professor Wolfgang Bargmann, held in Leningrad, January 17, 1979It is a pleasure to express our deep gratitude to Prof. P.P. Rumyantzev and Dr. N.V. Seina of the Institute of Cytology of the USSR Academy of Sciences, Leningrad, for their valuable advice on electron microscopic radioautography     

Quantitative radioautographic light and electron microscopic analysis of the localization of monoamines in the median eminence of the rat

Summary Serotonin containing structures in the median eminence of the rat have been studied by quantitative light and electron microscopic radioautography following intraventricular infusion of tritiated 5-hydroxytryptophan. One hour after injection of the tracer the highest density of silver grains was recorded in the ependymal and external zones, especially in the lateral palisade zone. The proportion of labelled neurosecretory terminals was also larger in the lateral palisade zone (29%) as compared with the medial palisade zone (13%), although the mean number of developed silver grains per one terminal was higher in the latter. On the average, 16% of neurosecretory terminals sequestered radiolabelled 5-hydroxytryptophan in the external zone of the rat median eminence. It is suggested that serotonin, like catecholamines, is discharged from neurosecretory terminals localized in the external zone and via the portal circulation affects the function of the anterior pituitary. The sites of origin of serotoninergic structures of the median eminence as well as the possible role of monoamine (catecholamine and indolamine) neurohormones in a dual peptidergic and monoaminergic control of anterior pituitary functions are discussed.This work was presented at the joint session of the Society of Anatomists, Histologists and Embryologists, the Society of Physiologists, and the Society of Neuroendocrinologists dedicated to the memory of Professor Wolfgang Bargmann, held in Leningrad, January 17, 1979     

Neuronal pathways to the margin of the hypothalamic median eminence and to the pituitary stalk of the rat. I. A golgi and degeneration study.

The course and termination of nerve fibres approaching the median eminence from lateral direction were studied in Golgi specimens and by the axon-degeneration technique. Varicose nerve fibres could be traced from an area corresponding to the medial and superficial portion of the medial forebrain bundle. They run immediately underneath the free ventral surface of the hypothalamus. Parasagittal knife-cuts placed at various distances (0.5 to 1.4 mm) from the midline resulted in a large number of degenerated axon fragments along the margin of the median eminence, on both sides of the tuberoinfundibular sulcus. Scattered degenerated fragments were found in the lateral part of the palisade zone as well as in the pituitary stalk. No degeneration could be seen in the abo9ve mentioned areas if the cut was as far as 1.8 mm from the midline. Degenerated axon fragments appeared as soon as 5 hours following the lesion indicating that the time course of ultrastructural degenerative alterations is remarkably fast in this fibre system.     

Immunocytochemical localization of growth hormone releasing factor (GHRF)-containing structures in the rat brain using anti-rat GHRF serum

The distribution of growth hormone releasing factor (GHRF) immunoreactive structures in the rat hypothalmus was studied after colchicine treatment with PAP immunocytochemistry in vibratome sections using an antiserum directed to rat hypothalamic GHRF. The majority of the GHRF-immunoreactive cell bodies were found in the arcuate nucleus, the medial perifornical region, and the ventral premammillary nuclei of the hypothalamus. Scattered cells were seen in the lateral basal hypothalamus, the medial and lateral portions of the ventromedial nucleus, and the dorsomedial and paraventricular nuclei. Immunoreactive fibers were observed in all the regions mentioned above. GHRF terminals were located in the central region of the median eminence. In addition, GHRF-immunoreactive neuronal processes were seen in the ventral region of the dorsomedial nucleus, the medial preoptic and suprachiasmatic regions, dorsal portion of the suprachiasmatic nucleus, bed nucleus of the stria terminals and the hypothalamic portion of the stria terminals. The localization of GHRF-immunoreactive terminals in the median eminence reinforces the view that GHRF plays a physiological role in the regulation of pituitary function. In addition, the localization of GHRF-immunoreactive structures in areas not usually considered to project to the median eminence suggest that GHRF may act as a neuromodulator or neurotransmitter.     

The distribution of monoamines in the hypothalamus of the Japanese quail Coturnix coturnix japonica

Summary The distribution of monoamines in the hypothalamus of the Japanese quail (Coturnix coturnix japonica) has been studied using a histochemical fluorescence technique. In the posterior hypothalamus catecholamine-containing nerve fibres are localised in the nucleus tuberis and nucleus hypothalamicus posterior medialis and are linked by fluorescent tracts running in the stratum cellulare internum. Further tracts may be traced from the nucleus tuberis around the base of the third ventricle to the sub-ependymal layer of the median eminence, where they then appear to pass through the hypothalamo-hypophysial neurosecretory tract to terminate in the palisade zone on the portal vascular bed. The innervation of the palisade layer by catecholamines is sparse. The fluorescent terminals are spread evenly throughout both the anterior and posterior divisions of the median eminence. There is no monoamine innervation of the pars nervosa. The paraventricular organ has both 5-hydroxytryptamine- and catecholamine-containing cell bodies and axons may be traced into the region of the nucleus hypothalamicus posterior medialis. In the anterior hypothalamus the neurosecretory paraventricular nucleus contains many catecholamine nerve fibres and terminals. These are linked by fibre tracts to the nucleus basalis and to the nucleus hypothalamicus posterior medialis. The supraoptic nucleus is less well innervated although a dense accumulation of fibres lies in the preoptic recess. The latter is thought to give rise to long axons which pass in association with the neurosecretory tract to end in the nucleus tuberis.Supported by a Grant (AG 24/36) from The Agricultural Research Council. We are indebted to Dr. G. A. Clayton, Institute of Animal Genetics, University of Edinburgh, for supplying the birds.     

Monoamine fluorescence in the median eminence of the Japanese quail,Coturnix coturnix japonica,following medial basal hypothalamic deafferentation

Summary Monoamine fluorescence was examined in the ventral hypothalamus of the Japanese quail, Coturnix coturnix japonica after medial basal hypothalamic deafferentation. In sham-operated control birds, numerous yellow-green fluorescent fibers were observed in the median eminence and the nucleus tuberis. In the area of the paraventricular organ, a number of fluorescent fibers and cell bodies were observed. In birds with deafferented hypothalami, fluorescence disappeared both in the median eminence and the nucleus tuberis. In the area of the paraventricular organ, which was within the area of deafferentation, fluorescence of neuronal perikarya did not change, but fluorescent fibers decreased markedly in number. Disappearance of monoamine fluorescence in the median eminence and the nucleus tuberis is discussed in relation to the tanycyte absorptive function and gonadal development.Supported by Grants from the Ministry of Education to Professors T. Bando and H. Kobayashi, and a Grant from the Ford Foundation to Prof. H. Kobayashi.     

Relationship between neuropeptide Y and luteinizing-hormone-releasing hormone immunoreactivities in the hypothalamus and preoptic region

The purpose of this study was to examine the anatomical relationships of perikarya and fibers containing neuropeptide Y (NPY) and luteinizing-hormone-releasing hormone (LHRH) in the hypothalamus and preoptic region of female rats. In view of our previous report of stimulatory effects of estrogen on LHRH and NPY levels in the median eminence, animals were bilaterally ovariectomized and subsequently implanted subcutaneously with capsules containing estradiol benzoate in oil or vehicle. Following intracerebroventricular injection of colchicine, rats were perfused with fixative and their brains sectioned and processed for immunohistochemical visualization of NPY and LHRH in the same section and in consecutive sections. Estrogen treatment had no discernible effect on the distribution or relationship of these peptides. NPY-immunoreactive fibers were intimately associated with LHRH-labeled primary dendrites and perikarya in the medial preoptic region and horizontal limb of the diagonal band of Broca. Fibers containing NPY or LHRH overlapped extensively in the lateral palisade region of the median eminence and also in the subependymal and internal zones. The external zone of the median eminence displayed relatively less overlap of these peptide systems. LHRH-immunoreactive axons coursed among NPY-labeled perikarya in the arcuate nucleus and appeared to contact these cells. These results suggest that NPY-containing axons may influence LHRH-positive neurons at the cell body and also at the site of axon termination in the median eminence. LHRH-containing axons appear to contact NPY-immunoreactive perikarya in the arcuate nucleus and may interact with terminals in the median eminence. This arrangement may provide a mechanism for communication between NPY and LHRH neurons and for the neuroendocrine coordination of hypothalamic NPY and LHRH secretion before ovulation.     

Fluorescence microscopy of neurons containing primary catecholamines in the ventral hypothalamus of the white-crowned sparrow,Zonotrichia leucophrys gambelii

Summary The ventral hypothalamus of White-crowned Sparrows, Zonotrichia leucophrys gambelii, was examined for primary catecholamines with the fluorescence technique of Falck-Hillarp and Owman. Photorefractory and photosensitive birds, photoperiodically stimulated and non-stimulated, were used.Four groups of catecholaminergic fibers were demonstrated: (1) afferent fibers of extra-hypothalamic origin to the infundibular nucleus where there are extensive synaptic contacts with non-fluorescent perikarya; (2) fibers apparently extending ventrally from an area with fluorescent perikarya in the vicinity of the paraventricular organ to the infundibular nucleus via the stratum cellulare internum and lateral pathways; (3) afferent fibers to the subependymal layer of the median eminence; and (4) afferent fibers to the zona externa of the median eminence. Fluorescent fibers that pass transversely through the median eminence may be derived from any of the last three categories. It appears that the fibers of the zona externa (4) are not derived to any appreciable extent from those of the subependymal layer (3). Because fluorescent perikarya could not be demonstrated in the infundibular nucleus no conclusion can be reached with respect to this nucleus as an origin for fibers (3) and (4).Diurnal cycles in the number of fluorescent terminals observable and in the intensity of the fluorescence in the palisade layer in photosensitive birds subjected to different photoperiodic regimes, in castrates, and in photorefractory birds are described tentatively on the basis of subjective assessments. Some possible implications of the differences are discussed.This communication is based extensively on a thesis, Primary catecholamine fibers in the ventral hypothalamus of the White-crowned Sparrow, Zonotrichia leucophrys gambelii, submitted in partial fulfillment of the requirements for the degree of Master of Science, University of Washington, August 1968, by the senior author. The research on which it is based was supported extensively by Grant No. NB 06187 from the National Institutes of Health to Professor Donald S. Farner. We are grateful to Ciba Pharmaceutical Products, Inc. for a supply of Serpasil.     

Electrical stimulation of the median eminence in rats,changes in catecholamine content and in plasma prolactin and growth hormone concentrations

Effects of field stimulation of the medial basal hypothalamus (MBH) and coaxial stimulation of the median eminence was studied on the catecholamine and DOPAC levels of the median eminence and on the prolactin and growth hormone release. The field stimulation induced an increased prolactin and growth hormone secretion without altering the catecholamine and DOPAC level. The direct electrical stimulation of the median eminence reduced the noradrenaline and dopamine content without significant changes in DOPAC concentration and in hormone secretions. It is concluded that (1) variations of DOPAC content are inadequate indicators of neuronal activity in the median eminence; (2) the multiple interaction of the stimulated neurons in the median eminence may mask the expected biochemical and hormonal responses to electrical stimulation.     

The innervation of the salivary gland of the moth, Manduca sexta: evidence that dopamine is the transmitter.

1. Using the Falck-Hillarp histochemical technique for monoamines, evidence was found for the presence of a catecholamine in the salivary gland nerves of the moth, Manduca sexta. 2. The innervation was studied with the electron microscope. Only the fluid-secreting region of the gland is innervated and the nerve endings are characteristic of monoamine-containing terminals. 3. Using a sensitive enzymatic-isotopic assay for catecholamines, it was found that whole salivary glands contain 0.33 mug/g dopamine but no noradrenaline. 4. It seems likely that dopamine mediates fluid-secretion in the salivary gland of Manduca as it does a number of other arthropods.     

The location of LH-RH neurons in the rat hypothalamus and their pathways to the median eminence

Summary The location of the perikarya of LH-RH neurons in the rat hypothalamus and their pathways to the median eminence were studied by immunohistochemistry and radioimmunoassay after placing stereotaxic electrolytic lesions in several parts of the hypothalamus. The principal location of the cell somata was found to be in the ventral part of the medial preoptic area; their pathways were classified into a main baso-lateral pathway and an accessory descending pathway branching off from the former. The main pathway was found to cross in the vicinity of the corresponding neuronal perikarya. The central median eminence and the dorsal and ventral walls of the tubero-infundibular sulcus of the caudal part of the median eminence are innervated mainly by the baso-lateral pathway. On the other hand, the rostral and most caudal portions of the median eminence are innervated principally by the descending pathway and have a subsidiary dual innervation. The projection of LH-RH neurons to the OVLT is believed to originate from perikarya adjacent to this circumventricular organ.This work was supported in part by a grant (No. 248093, 321426) from the Ministry of Education, Science and Culture, Japan     

Distribution of LH-RH nerve endings in the median eminence of proestrus female rats: fluorescence and peroxidase anti-peroxidase (PAP) immunohistochemistry.

The morphological distribution of the nerve terminals containing LH-RH in the hypothalamus especially in the median eminence of the proestrus female rat was demonstrated by immunohistochemistry, using FITC and peroxidase antibody. The terminals containing LH-RH were classified into four groups on the topographic relationship. LH-RH nerve processes terminated mainly in the infurdibular radix within an elliptical zone surrounding the bases of the infundibular recessus. The heaviest concentration of LH-RH terminals immunohistochemically demonstrated lay on each side of the region extending from the dorsal part of tuberoinfundibular sulcus to the lateral part of the external layer of the superior labium of the infundibulum. We were unable to detect any neuronal soma with the immunoreactivity to LH-RH in the hypothalamic gray matter. The distributional patterns of LH-RH, GH-RIH and monoamine in the median eminence as well as their relationships were briefly discussed.     

A fluorescence microscopic study of the distribution of monoamines in the hypothalamus of the cat

Three distinct groups of monoamine (MA)-containing nerve cell bodies have been visualized in the hypothalamus and preoptic area of the cat by means of the Falck-Hillarp fluorescence histochemical technique. First, numerous small-sized catecholamine (CA) type neurons were disclosed within the ventral half of the periventricular area in the supraoptic and middle hypothalamic regions. The round to oval neurons of this medio-ventral group were more especially abundant around the base of the third ventricle, within the arcuate and supraopticus diffusus nuclei. Numerous medium-sized CA perikarya identified as the dorsal group, were also mapped out in the dorsal and posterior hypothalamic areas. Finally, a small population of both CA and serotonin (5-hydroxytryptamine, 5-HT)-containing neurons was disclosed within the lateral area of the middle and mammillary hypothalamic regions. These multipolar or elongated neurons which compose the lateral group were lying either along the ventrolateral surface of the hypothalamus or around the ventrolateral aspect of the fornix. In addition to these three MA cell groups, a few cells displaying a fluorescence of the CA type were also visualized in the so-called “dorsal chiasmatic nucleus” after α-methyl-dopa treatment. High density of CA axon terminals were found, on the other hand, in the external layer of the median eminence, in the dorsomedial, paraventricular, supraoptic and suprachiasmatic nuclei, and also within nucleus interstitialis of stria terminalis. In the present study, however, it was not possible to identify with certainty any concentration of 5-HT axon terminals in the cat hypothalamus. Therefore, except for the lateral cell group which could be peculiar to the cat, the topographical distribution of MA nerve cell bodies and axon terminals in the hypothalamus of the cat appears similar to the morphological organization of the MA neuronal elements in the hypothalamus of the rat.     

Hypothalamic and extrahypothalamic distribution of somatostatin-immunoreactive elements in the rat brain

Summary Using a highly sensitive antibody to somatostatin, its hypothalamic and extrahypothalamic distribution in the rat was re-examined by light microscopic immunohistochemistry (PAP-method). The scattered somatostatin-producing perikarya occur in multiple layers within the subependymal neuropil surrounding the third ventricle. They supply with short-distance projections the following hypothalamic nuclei: 1) preoptic nuclei (especially their suprachiasmatic and medial components), 2) the peripheral zones of the suprachiasmatic nuclei, 3) the ventromedial and 4) arcuate nuclei, and 5) the ventral premammillary nuclei. Furthermore, the following long-distance projections have been observed: In a rostral direction (A1) rostral of the anterior commissure to the lamina terminalis, (A2) to the OVLT, (A3) to the olfactory tubercle, and (A4) rostrally and caudally by-passing the anterior commissure to the dorsal part of the stria terminalis.More caudally, at the retrochiasmatic level an ascending dorso-lateral projection joins the ventral amygdalo-hypothalamic pathway in a reciprocal manner (B1). In addition, a descending ventrolateral tract projects to the optic tract bending dorsal to it in different directions: (C1) medial to the median eminence, (C2) lateral to the corticomedial amygdala, and (C3) caudal for additional support of the arcuate and ventral premammillary nuclei.The principal tract of somatostatin-containing fibers descends in the subependymal neuropil to the median eminence (D).The results are discussed with reference to a possible participation of the somatostatin fiber system in the afferent branch of the circuit connecting the hypothalamus with the amygdala via the stria terminalis.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr. 569/2) and Stiftung Volkswagenwerk.     

Immunocytochemical study of the GABAergic innervation of the mouse pituitary by use of antibodies against gamma-aminobutyric acid (GABA)

Summary The GABAergic innervation of the mouse pituitary, including the median eminence, was studied at light microscopic and ultrastructural levels by use of a pre-embedding immunocytochemical technique with antibodies directed against GABA. In the median eminence, a high density of GABA-immunoreactive fibers was found in the external layer where the GABAergic varicosities were frequently observed surrounding the blood vessels of the primary capillary plexus. In the internal and subependymal layers, only few fibers were immunoreactive. The intense labeling of the external layer was observed in the entire rostro-caudal extent of the median eminence. In the pituitary proper, a dense network of GABA-immunoreactive fibers was revealed throughout the neural and intermediate lobes, entering via the hypophyseal stalk. The anterior and tuberal lobes were devoid of any immunoreactivity. The GABA-immunoreactive terminals were characterized in the median eminence, and in the intermediate and posterior lobes at the electron-microscopic level. They contained small clear vesicles, occasionally associated with dense-core vesicles or neurosecretory granules. In the intermediate lobe they were seen to be in contact with the glandular cells. In the posterior lobe and in the median eminence, GABA-immunoreactive terminals were frequently located in the vicinity of blood vessels. These results further support the concept of a role of GABA in the regulation of hypophyseal functions, via the portal blood for the anterior lobe, directly on the cells in the intermediate lobe, and via axo-axonic mechanisms in the median eminence and posterior lobe.     

促生长素抑制素样免疫反应在大白鼠弓状核和正中隆起的超微结构分布

本文用免疫电镜方法证明:促生长素抑制素样免疫反应神经末梢分布于弓状棱并与未标记的树突形成轴树突触。在正中隆起的纤维层和栅状层内均可见上述免疫反应末梢,大多数紧贴门脉毛细血管基底膜周围甚至穿入基底膜内。免疫反应末梢尚可与未标记的末梢形成轴轴突触样结构。     

Fine structure of the palisade zone of the rat median eminence as revealed by freeze-fracturing

Summary Nerve terminals in the palisade zone of the rat median eminence were investigated with freeze-fracture electron microscopy. Fracture face P of the specific terminals showed two populations of intramembranous particles (IMP): a large and a small variety. The large IMP-s often formed small irregular clusters. In nerve terminals the total number of both populations of IMP-s was considerably less than that found on P membrane faces of ependymal feet. On fracture face E of the nerve terminals, the number of IMP-s was about a quarter of that seen on fracture face P.On both fracture faces of most terminals a few small round impressions (or elevations respectively) were found which may be interpreted as broken necks of either exo- or endocytotic vesicles. Neither gap nor tight junctions occurred at lateral membranes of the specific axon terminals. Similarly, no membrane specializations were observed with freeze-fracturing on membrane areas adjacent to the basement membrane. The findings are discussed in relation to a possible exocytosis mechanism of the hypothalamic releasing and inhibiting hormones.