Cell Tiss. Res. 166, 461-473 (1976)
Cell and Tissue Research 9 by Springer-Verlag 1976
Localization of Dopamine in the Endocrine Hypothalamus of the Rat* David E. Scott**, John R. Sladek, Jr., Karl M. Knigge, Gerda Krobisch-Dudley, Daniel L. Kent, and Celia D. Sladek Department of Anatomy, University of Rochester, School of Medicine and Dentistry, Rochester, New York, USA
Summary. Microspectrofluorometry, fluorescence histochemistry and light and electron microscopic autoradiography have established the presence of sub-populations of neurons in the arcuate-periventricular region of the rat hypothalamus that sequester both radiolabeled dopamine and demonstrate formaldehyde-induced fluorescence. These characteristics are consistent with a catecholaminergic function. Selective sequestration of 3H-dopamine at the light and ultrastructural level is discussed in the context of an ultrashort loop autoregulatory mechanism for this neuronal population.
Key words: Arcuate-periventricular region - (rat) - Microspectrofluorometry - Electron microscopic autoradiography - Radiolabeled dopamine.
Introduction The anatomical organization of the endocrine hypothalamus has, in the past, embraced traditional concepts that subsume releasing hormone synthesis, sequestration and release from discrete cellular (neuronal) compartments of the basal medial hypothalamus. Although elegant in its original conception (Green and Harris, 1947) the hypothesis of parvicellular neurosecretory neuronal control as the lone neuroendocrine mechanism that effects change in adenohypophyseal function is not consistent nor does it adequately confront the array of new data that has been amassed over the last 20 years. These new data from this and other laboratories employing a broad range of morphologic, physiologic and biochemical approaches, utilizing in vitro as well as in vivo models, has made it quite evident that the endocrine hypothalamus including the arcuate-median eminence complex is a region whose structural organization Send offprint requests to : Prof. David E. Scott, Department of Anatomy, University of Rochester,
School of Medicine and Dentistry, Elmwood Avenue, Rochester, New York 14642, USA. * Supported by USPHS Program Project Grants NS-11642, RR-05403, and ** USPHS Career Development Awardee K04 GM 70001.
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is vastly more complex than was originally thought. (For a recent review, see Knigge and Silverman, 1974.) This region of the diencephalon alluded to as the so-called "hypophysiotropic" region of the brain (Halfisz et al., 1962) includes chiefly the arcuate nucleus (nucleus infundibularis) and adjacent parvicellular components. Small numbers of apparent neurosecretory neuronal elements are also scattered throughout the extent of the hypendymal and fibrous zones of the mammalian median eminence (Zambrano, 1968); however, their numbers are insignificant in contrast to those that constitute the major arcuate and periventricular nuclear pools. The relatively inflexible unitarian model of parvicellular neuronal control does not provide for the possibility that other brain hormones active in the control of adenohypophyseal function may be synthesized, sequestered or released from cellular compartments in other regions of the vertebrate brain. Recent research has evolved the ideation that other highly specialized circumventricular organs (H6fer, 1958) may also play some role in neuroendocrine function. These regions that constitute the circumventricular brain include the median eminence, the neural lobe, organum vasculosum lamina terminalis, subfomical organ, subcommissural organ, the pineal gland, and the area postrema. These seven ancient regions of the vertebrate brain share a number of unique features with respect to their neuronal, glial, and vascular organization, and may serve to integrate blood, brain and cerebrospinal fluid. Accordingly, they have been analogized as the "Seven Windows of the Brain" (Knigge, 1975). In light of these new data, the unitarian paradigm of the parvicellular neuronal control of adenohypophyseal function must be reexamined. This becomes especially relevant due to the impetus given the hypothesis put forth by Oksche et al. (1974) of functional compartmentalization within the hypothalamus of a number of avian species. The present investigation is a correlative fluorescence light microscopic and electron microscopic autoradiographic analysis of the regional localization and cellular compartmentalization of dopamine in the endocrine hypothalamus of the rat. Material and Methods Male albino rats (Wistar strain) were anesthetized with sodium pentobarbital (5 mg/kg) and placed in a Kopf stereotactic device; 0.5 jal of tritiated dopamine (20 c/raM) was slowly infused (1.5 jal/ minute) into the lateral cerebral ventricle through a stereotactically implanted 28 gauge cannula. Animals were then killed by decapitation 5, l0 and 20 minutes following the infusion, and brains were prepared for light and electron microscopic autoradiography following the techniques of Scott et al. (1973). Control rats received non-radiolabeled vehicle and were also killed at the same time intervals. Additional male, albino rats were prepared for fluorescence microscopy in the following manner. One group was killed by decapitation. Each brain was rapidly removed and a block of tissue containing the anterior hypothalamus and median eminence was obtained by placing cuts anterior to the optic chiasm, lateral to the median eminence, and undercutting to a depth slightly greater than 1 mm, the block was incubated with 4 jaM L-dopa in Krebs Ringer bicarbonate for 20 minutes. A similar block of brain tissue was removed from each of a group of control rats. Each block was rapidly frozen in liquid freon-22 prechilled to - 1 0 0 ~ in liquid nitrogen and freeze-dried for one week in a Sladek-Kontes freeze dryer. Blocks were then exposed to gaseous p-formaldehyde for two hours at 80~ and embedded in paraffin according to the method of Bj6rklund and Falck (1968). Further details of the procedure as performed in this laboratory are described elsewhere (Hoffman and Sladek, 1973). Serial sections were cut at 10 jam and examined in a Leitz
Dopamine in the Endocrine Hypothalamus
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M P V I I microspectrofluorometer specially equipped with Schoeffel monochromators (emission: GM 100, excitation: GM 251A), M460 photometers, photomultipliers, and ratio computing circuitry. Spectra were recorded on an X-Y plotter; such measurements were made following the principles outlined by Bj6rklund etal. (1972). Routine examination was accomplished using a narrow band excitation (S-405) filter and a K460 barrier filter, which allowed the visualization of the true blue hue of the catecholamine fluorophor.
Observations
Fluorescence Microscopy. Intense, blue fluorescence was noted in the contact zone of the median eminence while a duller, but detectable blue fluorescence was seen in neuronal perikarya of the dorsal region of the arcuate nucleus and the ventral periventricular region of control, non-incubated rats (Fig. 1A, B). This fluorescence hue is characteristic of the catecholamines, norepinephrine and dopamine. Brain tissue incubated in L-dopa revealed two additional compartments of catecholamine fluorescence. Tanycytes of the median eminence possessed a prominent fluorescence which visually appeared as intense as known dopaminergic perikarya of the arcuate-periventricular complex (Fig. 5 A). Spectral analysis of fluorescent tanycytes identified the presence of catecholamine with an emission maximum at 480 nm (Fig. 6). This observation corresponds well with model spectra established in this laboratory for the dopamine and norepinephrine fluorophores. Corrected excitation spectra revealed a maximum activation at 410 nm, which also corresponds to catecholamine activity. In addition, an emission peak was seen as a shoulder at 516-520 nm on the slope of the catecholamines peak, suggestive of an indoleamine fluorophor, probably serotonin. A second pool of catecholamine fluorescence was seen in the apical rim of ependyma lining the third ventricle in the arcuate region. Here, intense catecholamine fluorescence was seen in a restricted ("arcuate") portion of the ventricular wall overlying the entire rostro-caudal extent of the median eminence (Fig. 5A, B). Microspectrofluorometric analysis identified this fluorescence as due to a catecholamine fluorophor. Ependymal cells lining other portions of the third ventricle failed to display either visually or spectrally detectable catecholamines. With light microscopic autoradiography, cells of the dorsomedial arcuate and periventricular regions of the endocrine hypothalamus consistently sequestered radiolabeled dopamine at levels that significantly exceeded background (Fig. 2A, B). Double blind analysis of variance of grain counts, using a microscopic reticle at a magnification of 1,000 diameters, yielded uptake values over arcuate-periventricular neurons that exceeded background by a factor of 40. Significant labeling with tritiated dopamine was restricted to the dorso-medial periventricular hypothalamus and was not observed in other regions of the diencephalon, or in cellular constituents of other circumventricular organs. At the electron microscopic level, grains in the form of distinct emission tracts were confined chiefly to the cytoplasmic matrix of dorsal arcuate and periventricular neurons (Fig. 3A). A significant proportion of these grains were associated with rough endoplasmic reticulum. Significant uptake was also observed over
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Fig. 1. (A) Ventromedial basal hypothalamic region of a female rat during estrus. Intense dopaminergic perikarya are seen within the arcuate nucleus and adjacent periventricular nucleus (arrows). Intense contact zone fluorescence is also quite apparent (F). V third ventricular lumen. • (B) High power insert illustrates dopaminergic perikarya of the dorsal arcuate region (arrows). The ventricular lumen is indicated (V). • 780
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Fig. 2. (A) Coronal semi-thin (1/a) section of arcuate-median eminence region of rat killed 10 minutes following intraventricular infusion of 3H-dopamine. Neurons in dorsal arcuate-periventricular region (arrows) appear heavily labeled. EC ependymal cisterns; M E median eminence; P T pars tuberalis; Vthird ventricle, x 100. (B) Insert high magnification light microscopic autoradiogram of alternate section from arcuate-periventricular hypothalamus of rat killed 20 minutes following intraventricular infusion of 3H-dopamine. Here selected neurons (arrows) adjacent to the ventricular wall in the same location as those depicted in Fig. 1A are virtually obscured by overlying silver grains. • 640
Fig. 3. (A) E M A R of neuron (Ar) in arcuate-periventricular region that corresponds sterically to those observed in Fig. 2 B. Note numerous emission tracks (arrows) confined chiefly to the cytoplasmic component of this cell. G Golgi cisterns; ER endoplasmic reticulum; M mitochondria; P polysomes, x 19,500. (B) E M A R of axon terminal (A) in palisade zone of median eminence. Here 3H-dopamine or its metabolite seen as emission tracks (arrows) is sequestered quite selectively in a terminal which in addition harbors both dense and clear microvesicles. This phenomenon is also commonly observed in the dorsal arcuate-periventricular region and is regarded as the ultrastructural correlate o f a monoamine reuptake mechanism, x 18,800
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Fig. 4. Neurosecretory neuron (N) in dorsal arcuate-periventricular region of control rat that received vehicle only. Here the photographic reduction of emulsion is negative as it is for most other areas of control brain as well. Note, however, the ultrastructural correlate of heightened synthetic activity with distinct Golgi cisterns (G), numerous dense core vesicles (DCV), endoplasmic reticulum (ER), and prominent nuclear clefting. M mitochondria; N nucleus; S synapse. • 18,000
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axon terminals in the dorsal arcuate nucleus and in all zones of the median eminence (Fig. 3 B). These axon terminals characteristically harbored numerous lucent and dense microvesicles which measured from 20-50 nm. Ultra-thin sections from control animals that received only vehicle demonstrated arcuate neurons with little or no uptake (Fig. 4). What few grains were observed in these areas of control rats were interpreted to be tissue background. In addition to the highly selective and restricted axonal and neuronal sequestration of radiolabeled dopamine or a metabolite, specialized ependymal cells (tanycytes) were
Fig. 5. (A) Distinct catecholamine fluorescence is indicated within apparent tanycytes (T) and arcuate ependyma (arrows) of median eminence incubated in L-dopa. V third ventricular lumen. • 215. (B) The ependymal surface is seen to contain an intense catecholaminergic fluorescence (arrows) apical to ependymal nuclei (N). The third ventricle is indicated (1I). x 780
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also observed to sequester exogenous radiolabeled dopamine. However, the degree of uptake by tanycytes was less than that recorded for neuronal perikarya in dorsal arcuate-periventricular neurons of this region of the diencephalon. For a review of ependymal uptake and transport in vitro and in vivo, see Silverman and Knigge (1972b), Silverman etal. (1972a) and Scott etal. (1974b).
Discussion
The occurrence of catecholamine fluorescence in or upon ependyma of the arcuate region of the third ventricle may represent catecholamine uptake by axon terminals which project into the ventricular lumen. This could support the suggestion that neuroendocrine cell processes or perikarya project to the ventricular-brain interface. Such a phenomenon is well documented in certain non-mammalian vertebrates as a result of investigations employing Golgi method, histofluorescence and TEM (McKenna and Rosenbluth, 1974; Vigh and VighTeichman, 1974). Additionally, tern and sem data generated by this laboratory pro-
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Fig. 6. This graph illustrates corrected emission spectra for norepinephrine in a model protein droplet, a tanycyte incubated with L-dopa, and a catecholamine-containing varicosity of the endocrine hypothalamus. All three emission spectra indicate the presence of a catecholamine with an emission peak at 480 nm. Wave lengths are expressed in nanometers along the X axis while relative intensity is expressed as a ratio along the Y axis. The emission curve for the tanycyte displays a secondary peak on the catecholamine shoulder at around 516 nm which may be indicative of the indoleamine serotonin
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vide evidence for the presence of axon terminals and/or neuronal perikarya in the third ventricle of certain mammals (Scott and Knigge, 1970; Scott et al., 1973, 1974, 1975) which corresponds positionally with current observations in the rat. That dopaminergic perikarya of the hypothalamus did not exhibit an enhanced fluorescence intensity following incubation with L-dopa is unexpected, but not without explanation. It is probable that the thickness of the tissue block (1-2 ram) prevented penetration of the L-dopa into cellular compartments of the hypothalamus. The ventricular space may have provided an easy access for this catecholamine precursor to reach putative nerve terminals of CSF contacting neurons lining the ventricle, wherein a rapid uptake could have occurred. Similarly, tanycytes of the median eminence would have access to the same pool of L-dopa and if functionally active in uptake mechanisms as previously demonstrated (Knigge et al., 1975), could have sequestered enough of the substance to afford fluorescence microscopic visualization. The ability of tanycytes to sequester monoamines from incubation media was reported earlier by this laboratory (Knigge et al., 1975), but at that time, the capability of microspectrofluorometric verification was not available. The present addition of this qualitative analysis lends support to the possible role of these specialized cells in the mediation of neuroendocrine interactions. Recent data (Knigge et al., 1975; Sladek et al., 1975) indicate an additional capability of tanycytes to synthesize the indoleamine, serotonin, as revealed by histofluorescence-microspectrofluorometric techniques. Serotonin fluorescence was spectrally, but not visually, detectable within tanycytes of L-dopa incubates. Very likely the catecholamine fluorescence of the tanycytes produced a masking effect of the weakly fluorescent serotonin fluorophor. This phenomenon serves to indicate the ultrasensitivity of the microspectrofluorometer as a tool for the detection and identification of monoamine histofluorescence. The elegant investigations of Holzwarth et al. (1976) and Paull and Lechan (1974) have amply demonstrated that acute and chronic monosodium induced chemical lesions in mice are effective in destroying as much as 80% of the pool of arcuate neurons. Despite the extensive nature of the lesion in this so-called "hypophysiotrophic" zone of the diencephalon (Halasz, 1962), no signs of anterograde degeneration have ever been observed in any region of the adjacent median eminence by these authors. This area, a neuroendocrine transducer, has been commonly regarded as the functional terminus for the majority of arcuate axons. Thus other possible patterns of projection for these neurons must be examined. A significant number of axon terminals do insinuate into the ventricular lumen in this region of the third ventricle (Scott et al., 1974, 1975, 1976), and it has been postulated that the cerebral ventricular lumen may be a likely terminus for a number of arcuate axons (Scott et al., 1974, 1975). However, the overall number of axons that penetrate the ventricular lumen is small, suggesting other or additional patterns of projection for the majority of arcuate neurons. Conceivably, those arcuate neurons which are spared by monosodium glutamate could represent the total neuronal input to the median eminence. This seems rather unlikely, requiring a high degree of collateralization. Furthermore, it is likely that a number of other regions of the diencephalon and brainstem
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(areas as yet undefined) may contribute catechol or indoleaminergic axonal input not only to the median eminence but to a large extent of the cerebral ventricular system as well (Chan-Palay, 1975; Aghajanian and Gallager, 1975; Richards et al., 1973). These suggestions coupled with correlative fluorescence and autoradiographic data of the present investigation necessitate a re-examination of the role and functional composition of the endocrine hypothalamus with special emphasis directed toward the arcuate-periventricular complex. Furthermore, other cellular compartments not necessarily neuronal, such as tanycytes (Horstmann, 1954), which constitute a significantly large input to the portal bed must be seriously considered in the overall control of adenohypophyseal metabolism. The notion that the arcuate nucleus, at least in avian (specifically passerine) species, may possess different functional capacities is an attractive one consistent with the data of this and earlier investigations (Oksche et al., 1974). The evidence of the present investigation would appear to confirm the concept that subpopulations in the dorsal arcuate-periventricular hypothalamus are catecholaminergic. Selective and statistically significant uptake of exogenous 3Hdopamine coupled with strong fluorescence for endogenous catecholamines in neurons in the same area of the hypothalamus is also confirmatory as the structural correlate of a monoaminergic reuptake mechanism. These data also argue that these catecholaminergic neurons are not actively involved in the synthesis or sequestration of releasing factors. On the contrary, earlier investigations using radiolabeled thyrotropic releasing factors have demonstrated very different patterns of selective uptake and localization for this hormone (Knigge etal., 1974; Scott etal., 1974; Joseph etal., 1973). Recent immunocytochemical investigations using antisera against LRF demonstrate pools of LRF-producing neurons which are also anatomically distinct from the catecholaminergic neurons of the present investigation (Hoffman, 1975). The rationale for believing that cellular compartments (in this case neurons of the dorsal arcuate-periventricular region) which selectively sequester 3Hdopamine may actually synthesize and store it, is based on the monoamine reuptake or recapture mechanism (Aghajanian and Bloom, 1966). Conceptually, this may not be limited to catecholamines but may embrace a broad range of physiologically active molecules (Scott et al., 1974). In this paradigm, a functional subdivision or subpool of neurons which belong to a heterogenous population comprising a larger nuclear group, such as the arcuate, may be sensitive or receptive to the very same hormone that they or adjacent neurons of the same subpool synthesize, and thus may actively recapture that hormone. Such a mechanism may serve a useful function as an ultra-short, autoregulatory feedback loop which may, in a finely tuned fashion, control the on-going synthesis, storage and possibly release of biologically active molecules from that specific pool of neurons. Thus, the present investigation has demonstrated a small subpopulation of neurons in the arcuate-periventricular complex that both sequester radiolabeled dopamine and show formaldehyde-induced fluorescence characteristics consistent with a catecholaminergic function. These cells are anatomically distinct and physically separate from known populations of cells in the arcuate
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region that may be involved in the production of releasing factors. In addition, radiolabeled dopamine and catecholamine fluorescence were detected in tanycytes of the median eminence. From these and other data it becomes increasingly clear that the arcuate-periventricular region of the endocrine hypothalamus is far more complex and diverse in its anatomical and functional organization. Extensive re-examination of this region of the mammalian diencephalon is necessary, combining a variety of correlative morphological, physiological and biochemical approaches.
References Aghajanian, G.K., Bloom, F.E. : Electron microscopic autoradiography of the rat hypothalamus after injection of 3H-norepinephrine. Science 153, 308-310 (1966) Aghajanian, G.K., Gallager, D.W. : Raphe origin of serotonergic nerves terminating in the cerebral ventricles. Brain Res. 88, 221-231 (1975) Bj6rklund, A., Falck, B. : An Improvement of the histochemical fluorescence method for monoamines. Observations on varying extractability of fluorophors in different nerve fibers. J. Histochem. Cytochem. 16, 717-720 (1968) Bj6rklund, A., Falck, B., Owman, Ch. : Fluorescence microscopic and microspectrofluorometric techniques for the cellular localization of biogenic amines. In (J. Rail and I. Kopin, eds.) Methods in investigative and diagnostic endocrinology. I. The thyroid and biogenic amines. Amsterdam 1972 Chan-Palay, V.: Serotonin axons of the supra- and subependymal plexuses and subarachnoid systems of the rat and monkey. Proc. neurosci. 166 (1975) Green, J.D. Harris, G.W.: The neurovascular link between the neurohypophysis and adenohypophysis. J. Endocr. 5, 136-146 (1947) Halasz, B., Pupp, L., Uhlarik, S.: Hypophysiotropic area in the hypothalamus. J. Endocr. 25, 147-159 (1962) Hofer, H. : Zur Morphologie der circumventrikularen Organe des Zwischenhirnes der S/iugetiere. Verh. Dtsch. Zool. Ges. Frankfurt, 202-251 (1958) Hoffman, D.L., Sladek, J.R., Jr.: The distribution of catecholamines within the inferior olivary complex of the gerbil and rabbit. J. comp. Neurol. 151, 101-112 (1973) Hoffman, G. : Personal communication (1975) Holzwarth-McBride, M.A., Hurst, E.M., Knigge, K.M.: Monosodium glutamate induced lesions of arcuate nucleus. I. Endocrine deficiency and ultrastructure of median eminence. Anat. Rec., (in press) (1976) Horstmann, E. : Die Faserglia des Selachiergehirns. Z. Zellforsch. 39, 588-617 (1954). Joseph, S.A., Scott, D.E., Vaala, S.S., Knigge, K.M., Krobisch-Dudley, G.: Localization and content of thyrotropin releasing factor (TRF) in median eminence of the hypothalamus. Acta endocr. (Kbh.) 74, 215-225 (1973) Knigge, K.M. : Opening Remarks. In (K.M. Knigge, D.E. Scott, H. Kobayashi and S. Ishii, eds.) Brain-endocrine interaction. The ventricular system in neuroendocrine mechanisms. Basel: Karger 1975 Knigge, K.M., Joseph, S.A., Schock, D., Silverman, A.J., Ching, M.C.H., Scott, D.E., Zeman, D., Krobisch-Dudley, G.: Role of ventricular system in neuroendocrine processes. Synthesis and distribution of thyrotropin releasing factor (TRF) in the hypothalamus and third ventricle. Canad. J. Neurol. Sci. 1, 74-84 (1974) Knigge, K.M., Schock, D., Sladek, J.R., Jr.: Monoamines of median eminence. In (K.M. Knigge, D.E. Scott, H. Kobayashi, and S. Ishii, eds.). Brain-endocrine interaction II. The ventricular system in neuroendocrine mechanisms, p. 252-294. Basel: Karger 1975 Knigge, K.M., Silverman, A.J. : Anatomy of the endocrine hypothalamus. In: Handbook of physiology. Part I, p. 1-32. Endocrinology IV. (1974) McKenna, O.C., Rosenbluth, J. : Cytological evidence for catecholamine containing sensory cells bordering the ventricle of the toad hypothalamus. J. comp. Neurol. 154, 133-148 (1974)
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Oksche, A., Oehmke, H.J., Hartwig, H.G. : A concept of neuroendocrine cell complexes. In: Neurosecretion. The final neuroendocrine pathway, p. 155-156. Berlin-Heidelberg-New York: Springer 1974 Paull, W.K., Lechan, R. : The median eminence of mice with an MSG induced arcuate lesion. Anat. Rec. 178, 346 (1974) Richards, J.G., Lorenz, H.P., Tranzer, J.P. : Indole-alkylamine nerve terminals in cerebral ventricles: Identification by electron microscopy and fluorescence histochemistry. Brain Res. 57, 277-288 (1973) Scott, D.E., Knigge, K.M. : Ultrastructural changes in the median eminence of the rat following deafferentation of the basal hypothalamus. Z. Zellforsch. 105, 1-32 (1970) Scott, D.E., Kozlowksi, G.P., Krobisch-Dudley, G.: A comparative ultrastructural analysis of the third cerebral ventricle of the North American mink, Mustela vison. Anat. Rec. 175, 155-168 (1973) Scott, D.E., Kozlowski, G.P., Sheridan, M.N. : Scanning electron microscopy in the ultrastructural analysis of the mammalian cerebral ventricular system. Int. Rev. Cytol. 37, 349-388 (1974) Scott, D.E., Krobisch-Dudley, G., Knigge, K.M. : The ventricular system in neuroendocrine mechanisms. II. In vivo monoamine transport by ependyma of the median eminence. Cell Tiss. Res. 154, 1-16 (1974) Scott, D.E., Krobisch-Dudley, G., Paull, W.K., Kozlowski, G.P. Ribas, J.: The primate median eminence. I. Correlative scanning-transmission electron microscopy. Cell Tiss. Res. 162, 61-73 (1975) Scott, D.E., Krobisch-Dudley, G., Weindl, A., Joynt, R.J. : An autoradiographic analysis of hypothalamic magnocellular neurons. Z. Zellforsch. 138, 421-437 (1973) Scott, D.E., Sladek, J.R., Jr., Kozlowski, G.P., McNeill, T.H., Paull, W.K., Krobisch-Dudley, G. : The median eminence as a neuroendocrine transducer. In (A. Kumar, ed.) Neuroendocrine regulation of fertility. Basel: Karger 1976 (in press) Silverman, A.J., Knigge, K.M., Peck, W.A. : Transport capacity of the median eminence I. Amino acid transport. Neuroendocrinology 9, 123-132 (1972a) Silverman, A.J., Knigge, K.M. : Transport capacity of the median eminence II. Thyroxine transport. Neuroendocrinology 10, 71-82 (1972b) Sladek, J.R., Jr., Sladek, C.D., Knigge, K.M. : Organ culture and histofluorescence of indoleamines in median eminence. Proc. Soc. Neurosci. 1:461 (1975) Vigh-Teichmann, I., Vigh, B.: The infundibular cerebrospinal fluid contacting neurons. In: Advances in anatomy, embryology and cell biology, p. 1-91. Berlin-Heidelberg-New York: Springer 1974 Zambrano, D. : On the presence of neurons with granulated vesicles in the median eminence of rat and dog. Neuroendocrinology 3, 141-155 (1968)
Received October 10, 1975
Cell and Tissue Research 9 by Springer-Verlag 1976
Localization of Dopamine in the Endocrine Hypothalamus of the Rat* David E. Scott**, John R. Sladek, Jr., Karl M. Knigge, Gerda Krobisch-Dudley, Daniel L. Kent, and Celia D. Sladek Department of Anatomy, University of Rochester, School of Medicine and Dentistry, Rochester, New York, USA
Summary. Microspectrofluorometry, fluorescence histochemistry and light and electron microscopic autoradiography have established the presence of sub-populations of neurons in the arcuate-periventricular region of the rat hypothalamus that sequester both radiolabeled dopamine and demonstrate formaldehyde-induced fluorescence. These characteristics are consistent with a catecholaminergic function. Selective sequestration of 3H-dopamine at the light and ultrastructural level is discussed in the context of an ultrashort loop autoregulatory mechanism for this neuronal population.
Key words: Arcuate-periventricular region - (rat) - Microspectrofluorometry - Electron microscopic autoradiography - Radiolabeled dopamine.
Introduction The anatomical organization of the endocrine hypothalamus has, in the past, embraced traditional concepts that subsume releasing hormone synthesis, sequestration and release from discrete cellular (neuronal) compartments of the basal medial hypothalamus. Although elegant in its original conception (Green and Harris, 1947) the hypothesis of parvicellular neurosecretory neuronal control as the lone neuroendocrine mechanism that effects change in adenohypophyseal function is not consistent nor does it adequately confront the array of new data that has been amassed over the last 20 years. These new data from this and other laboratories employing a broad range of morphologic, physiologic and biochemical approaches, utilizing in vitro as well as in vivo models, has made it quite evident that the endocrine hypothalamus including the arcuate-median eminence complex is a region whose structural organization Send offprint requests to : Prof. David E. Scott, Department of Anatomy, University of Rochester,
School of Medicine and Dentistry, Elmwood Avenue, Rochester, New York 14642, USA. * Supported by USPHS Program Project Grants NS-11642, RR-05403, and ** USPHS Career Development Awardee K04 GM 70001.
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is vastly more complex than was originally thought. (For a recent review, see Knigge and Silverman, 1974.) This region of the diencephalon alluded to as the so-called "hypophysiotropic" region of the brain (Halfisz et al., 1962) includes chiefly the arcuate nucleus (nucleus infundibularis) and adjacent parvicellular components. Small numbers of apparent neurosecretory neuronal elements are also scattered throughout the extent of the hypendymal and fibrous zones of the mammalian median eminence (Zambrano, 1968); however, their numbers are insignificant in contrast to those that constitute the major arcuate and periventricular nuclear pools. The relatively inflexible unitarian model of parvicellular neuronal control does not provide for the possibility that other brain hormones active in the control of adenohypophyseal function may be synthesized, sequestered or released from cellular compartments in other regions of the vertebrate brain. Recent research has evolved the ideation that other highly specialized circumventricular organs (H6fer, 1958) may also play some role in neuroendocrine function. These regions that constitute the circumventricular brain include the median eminence, the neural lobe, organum vasculosum lamina terminalis, subfomical organ, subcommissural organ, the pineal gland, and the area postrema. These seven ancient regions of the vertebrate brain share a number of unique features with respect to their neuronal, glial, and vascular organization, and may serve to integrate blood, brain and cerebrospinal fluid. Accordingly, they have been analogized as the "Seven Windows of the Brain" (Knigge, 1975). In light of these new data, the unitarian paradigm of the parvicellular neuronal control of adenohypophyseal function must be reexamined. This becomes especially relevant due to the impetus given the hypothesis put forth by Oksche et al. (1974) of functional compartmentalization within the hypothalamus of a number of avian species. The present investigation is a correlative fluorescence light microscopic and electron microscopic autoradiographic analysis of the regional localization and cellular compartmentalization of dopamine in the endocrine hypothalamus of the rat. Material and Methods Male albino rats (Wistar strain) were anesthetized with sodium pentobarbital (5 mg/kg) and placed in a Kopf stereotactic device; 0.5 jal of tritiated dopamine (20 c/raM) was slowly infused (1.5 jal/ minute) into the lateral cerebral ventricle through a stereotactically implanted 28 gauge cannula. Animals were then killed by decapitation 5, l0 and 20 minutes following the infusion, and brains were prepared for light and electron microscopic autoradiography following the techniques of Scott et al. (1973). Control rats received non-radiolabeled vehicle and were also killed at the same time intervals. Additional male, albino rats were prepared for fluorescence microscopy in the following manner. One group was killed by decapitation. Each brain was rapidly removed and a block of tissue containing the anterior hypothalamus and median eminence was obtained by placing cuts anterior to the optic chiasm, lateral to the median eminence, and undercutting to a depth slightly greater than 1 mm, the block was incubated with 4 jaM L-dopa in Krebs Ringer bicarbonate for 20 minutes. A similar block of brain tissue was removed from each of a group of control rats. Each block was rapidly frozen in liquid freon-22 prechilled to - 1 0 0 ~ in liquid nitrogen and freeze-dried for one week in a Sladek-Kontes freeze dryer. Blocks were then exposed to gaseous p-formaldehyde for two hours at 80~ and embedded in paraffin according to the method of Bj6rklund and Falck (1968). Further details of the procedure as performed in this laboratory are described elsewhere (Hoffman and Sladek, 1973). Serial sections were cut at 10 jam and examined in a Leitz
Dopamine in the Endocrine Hypothalamus
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M P V I I microspectrofluorometer specially equipped with Schoeffel monochromators (emission: GM 100, excitation: GM 251A), M460 photometers, photomultipliers, and ratio computing circuitry. Spectra were recorded on an X-Y plotter; such measurements were made following the principles outlined by Bj6rklund etal. (1972). Routine examination was accomplished using a narrow band excitation (S-405) filter and a K460 barrier filter, which allowed the visualization of the true blue hue of the catecholamine fluorophor.
Observations
Fluorescence Microscopy. Intense, blue fluorescence was noted in the contact zone of the median eminence while a duller, but detectable blue fluorescence was seen in neuronal perikarya of the dorsal region of the arcuate nucleus and the ventral periventricular region of control, non-incubated rats (Fig. 1A, B). This fluorescence hue is characteristic of the catecholamines, norepinephrine and dopamine. Brain tissue incubated in L-dopa revealed two additional compartments of catecholamine fluorescence. Tanycytes of the median eminence possessed a prominent fluorescence which visually appeared as intense as known dopaminergic perikarya of the arcuate-periventricular complex (Fig. 5 A). Spectral analysis of fluorescent tanycytes identified the presence of catecholamine with an emission maximum at 480 nm (Fig. 6). This observation corresponds well with model spectra established in this laboratory for the dopamine and norepinephrine fluorophores. Corrected excitation spectra revealed a maximum activation at 410 nm, which also corresponds to catecholamine activity. In addition, an emission peak was seen as a shoulder at 516-520 nm on the slope of the catecholamines peak, suggestive of an indoleamine fluorophor, probably serotonin. A second pool of catecholamine fluorescence was seen in the apical rim of ependyma lining the third ventricle in the arcuate region. Here, intense catecholamine fluorescence was seen in a restricted ("arcuate") portion of the ventricular wall overlying the entire rostro-caudal extent of the median eminence (Fig. 5A, B). Microspectrofluorometric analysis identified this fluorescence as due to a catecholamine fluorophor. Ependymal cells lining other portions of the third ventricle failed to display either visually or spectrally detectable catecholamines. With light microscopic autoradiography, cells of the dorsomedial arcuate and periventricular regions of the endocrine hypothalamus consistently sequestered radiolabeled dopamine at levels that significantly exceeded background (Fig. 2A, B). Double blind analysis of variance of grain counts, using a microscopic reticle at a magnification of 1,000 diameters, yielded uptake values over arcuate-periventricular neurons that exceeded background by a factor of 40. Significant labeling with tritiated dopamine was restricted to the dorso-medial periventricular hypothalamus and was not observed in other regions of the diencephalon, or in cellular constituents of other circumventricular organs. At the electron microscopic level, grains in the form of distinct emission tracts were confined chiefly to the cytoplasmic matrix of dorsal arcuate and periventricular neurons (Fig. 3A). A significant proportion of these grains were associated with rough endoplasmic reticulum. Significant uptake was also observed over
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Fig. 1. (A) Ventromedial basal hypothalamic region of a female rat during estrus. Intense dopaminergic perikarya are seen within the arcuate nucleus and adjacent periventricular nucleus (arrows). Intense contact zone fluorescence is also quite apparent (F). V third ventricular lumen. • (B) High power insert illustrates dopaminergic perikarya of the dorsal arcuate region (arrows). The ventricular lumen is indicated (V). • 780
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Fig. 2. (A) Coronal semi-thin (1/a) section of arcuate-median eminence region of rat killed 10 minutes following intraventricular infusion of 3H-dopamine. Neurons in dorsal arcuate-periventricular region (arrows) appear heavily labeled. EC ependymal cisterns; M E median eminence; P T pars tuberalis; Vthird ventricle, x 100. (B) Insert high magnification light microscopic autoradiogram of alternate section from arcuate-periventricular hypothalamus of rat killed 20 minutes following intraventricular infusion of 3H-dopamine. Here selected neurons (arrows) adjacent to the ventricular wall in the same location as those depicted in Fig. 1A are virtually obscured by overlying silver grains. • 640
Fig. 3. (A) E M A R of neuron (Ar) in arcuate-periventricular region that corresponds sterically to those observed in Fig. 2 B. Note numerous emission tracks (arrows) confined chiefly to the cytoplasmic component of this cell. G Golgi cisterns; ER endoplasmic reticulum; M mitochondria; P polysomes, x 19,500. (B) E M A R of axon terminal (A) in palisade zone of median eminence. Here 3H-dopamine or its metabolite seen as emission tracks (arrows) is sequestered quite selectively in a terminal which in addition harbors both dense and clear microvesicles. This phenomenon is also commonly observed in the dorsal arcuate-periventricular region and is regarded as the ultrastructural correlate o f a monoamine reuptake mechanism, x 18,800
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Fig. 4. Neurosecretory neuron (N) in dorsal arcuate-periventricular region of control rat that received vehicle only. Here the photographic reduction of emulsion is negative as it is for most other areas of control brain as well. Note, however, the ultrastructural correlate of heightened synthetic activity with distinct Golgi cisterns (G), numerous dense core vesicles (DCV), endoplasmic reticulum (ER), and prominent nuclear clefting. M mitochondria; N nucleus; S synapse. • 18,000
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axon terminals in the dorsal arcuate nucleus and in all zones of the median eminence (Fig. 3 B). These axon terminals characteristically harbored numerous lucent and dense microvesicles which measured from 20-50 nm. Ultra-thin sections from control animals that received only vehicle demonstrated arcuate neurons with little or no uptake (Fig. 4). What few grains were observed in these areas of control rats were interpreted to be tissue background. In addition to the highly selective and restricted axonal and neuronal sequestration of radiolabeled dopamine or a metabolite, specialized ependymal cells (tanycytes) were
Fig. 5. (A) Distinct catecholamine fluorescence is indicated within apparent tanycytes (T) and arcuate ependyma (arrows) of median eminence incubated in L-dopa. V third ventricular lumen. • 215. (B) The ependymal surface is seen to contain an intense catecholaminergic fluorescence (arrows) apical to ependymal nuclei (N). The third ventricle is indicated (1I). x 780
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also observed to sequester exogenous radiolabeled dopamine. However, the degree of uptake by tanycytes was less than that recorded for neuronal perikarya in dorsal arcuate-periventricular neurons of this region of the diencephalon. For a review of ependymal uptake and transport in vitro and in vivo, see Silverman and Knigge (1972b), Silverman etal. (1972a) and Scott etal. (1974b).
Discussion
The occurrence of catecholamine fluorescence in or upon ependyma of the arcuate region of the third ventricle may represent catecholamine uptake by axon terminals which project into the ventricular lumen. This could support the suggestion that neuroendocrine cell processes or perikarya project to the ventricular-brain interface. Such a phenomenon is well documented in certain non-mammalian vertebrates as a result of investigations employing Golgi method, histofluorescence and TEM (McKenna and Rosenbluth, 1974; Vigh and VighTeichman, 1974). Additionally, tern and sem data generated by this laboratory pro-
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Fig. 6. This graph illustrates corrected emission spectra for norepinephrine in a model protein droplet, a tanycyte incubated with L-dopa, and a catecholamine-containing varicosity of the endocrine hypothalamus. All three emission spectra indicate the presence of a catecholamine with an emission peak at 480 nm. Wave lengths are expressed in nanometers along the X axis while relative intensity is expressed as a ratio along the Y axis. The emission curve for the tanycyte displays a secondary peak on the catecholamine shoulder at around 516 nm which may be indicative of the indoleamine serotonin
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vide evidence for the presence of axon terminals and/or neuronal perikarya in the third ventricle of certain mammals (Scott and Knigge, 1970; Scott et al., 1973, 1974, 1975) which corresponds positionally with current observations in the rat. That dopaminergic perikarya of the hypothalamus did not exhibit an enhanced fluorescence intensity following incubation with L-dopa is unexpected, but not without explanation. It is probable that the thickness of the tissue block (1-2 ram) prevented penetration of the L-dopa into cellular compartments of the hypothalamus. The ventricular space may have provided an easy access for this catecholamine precursor to reach putative nerve terminals of CSF contacting neurons lining the ventricle, wherein a rapid uptake could have occurred. Similarly, tanycytes of the median eminence would have access to the same pool of L-dopa and if functionally active in uptake mechanisms as previously demonstrated (Knigge et al., 1975), could have sequestered enough of the substance to afford fluorescence microscopic visualization. The ability of tanycytes to sequester monoamines from incubation media was reported earlier by this laboratory (Knigge et al., 1975), but at that time, the capability of microspectrofluorometric verification was not available. The present addition of this qualitative analysis lends support to the possible role of these specialized cells in the mediation of neuroendocrine interactions. Recent data (Knigge et al., 1975; Sladek et al., 1975) indicate an additional capability of tanycytes to synthesize the indoleamine, serotonin, as revealed by histofluorescence-microspectrofluorometric techniques. Serotonin fluorescence was spectrally, but not visually, detectable within tanycytes of L-dopa incubates. Very likely the catecholamine fluorescence of the tanycytes produced a masking effect of the weakly fluorescent serotonin fluorophor. This phenomenon serves to indicate the ultrasensitivity of the microspectrofluorometer as a tool for the detection and identification of monoamine histofluorescence. The elegant investigations of Holzwarth et al. (1976) and Paull and Lechan (1974) have amply demonstrated that acute and chronic monosodium induced chemical lesions in mice are effective in destroying as much as 80% of the pool of arcuate neurons. Despite the extensive nature of the lesion in this so-called "hypophysiotrophic" zone of the diencephalon (Halasz, 1962), no signs of anterograde degeneration have ever been observed in any region of the adjacent median eminence by these authors. This area, a neuroendocrine transducer, has been commonly regarded as the functional terminus for the majority of arcuate axons. Thus other possible patterns of projection for these neurons must be examined. A significant number of axon terminals do insinuate into the ventricular lumen in this region of the third ventricle (Scott et al., 1974, 1975, 1976), and it has been postulated that the cerebral ventricular lumen may be a likely terminus for a number of arcuate axons (Scott et al., 1974, 1975). However, the overall number of axons that penetrate the ventricular lumen is small, suggesting other or additional patterns of projection for the majority of arcuate neurons. Conceivably, those arcuate neurons which are spared by monosodium glutamate could represent the total neuronal input to the median eminence. This seems rather unlikely, requiring a high degree of collateralization. Furthermore, it is likely that a number of other regions of the diencephalon and brainstem
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(areas as yet undefined) may contribute catechol or indoleaminergic axonal input not only to the median eminence but to a large extent of the cerebral ventricular system as well (Chan-Palay, 1975; Aghajanian and Gallager, 1975; Richards et al., 1973). These suggestions coupled with correlative fluorescence and autoradiographic data of the present investigation necessitate a re-examination of the role and functional composition of the endocrine hypothalamus with special emphasis directed toward the arcuate-periventricular complex. Furthermore, other cellular compartments not necessarily neuronal, such as tanycytes (Horstmann, 1954), which constitute a significantly large input to the portal bed must be seriously considered in the overall control of adenohypophyseal metabolism. The notion that the arcuate nucleus, at least in avian (specifically passerine) species, may possess different functional capacities is an attractive one consistent with the data of this and earlier investigations (Oksche et al., 1974). The evidence of the present investigation would appear to confirm the concept that subpopulations in the dorsal arcuate-periventricular hypothalamus are catecholaminergic. Selective and statistically significant uptake of exogenous 3Hdopamine coupled with strong fluorescence for endogenous catecholamines in neurons in the same area of the hypothalamus is also confirmatory as the structural correlate of a monoaminergic reuptake mechanism. These data also argue that these catecholaminergic neurons are not actively involved in the synthesis or sequestration of releasing factors. On the contrary, earlier investigations using radiolabeled thyrotropic releasing factors have demonstrated very different patterns of selective uptake and localization for this hormone (Knigge etal., 1974; Scott etal., 1974; Joseph etal., 1973). Recent immunocytochemical investigations using antisera against LRF demonstrate pools of LRF-producing neurons which are also anatomically distinct from the catecholaminergic neurons of the present investigation (Hoffman, 1975). The rationale for believing that cellular compartments (in this case neurons of the dorsal arcuate-periventricular region) which selectively sequester 3Hdopamine may actually synthesize and store it, is based on the monoamine reuptake or recapture mechanism (Aghajanian and Bloom, 1966). Conceptually, this may not be limited to catecholamines but may embrace a broad range of physiologically active molecules (Scott et al., 1974). In this paradigm, a functional subdivision or subpool of neurons which belong to a heterogenous population comprising a larger nuclear group, such as the arcuate, may be sensitive or receptive to the very same hormone that they or adjacent neurons of the same subpool synthesize, and thus may actively recapture that hormone. Such a mechanism may serve a useful function as an ultra-short, autoregulatory feedback loop which may, in a finely tuned fashion, control the on-going synthesis, storage and possibly release of biologically active molecules from that specific pool of neurons. Thus, the present investigation has demonstrated a small subpopulation of neurons in the arcuate-periventricular complex that both sequester radiolabeled dopamine and show formaldehyde-induced fluorescence characteristics consistent with a catecholaminergic function. These cells are anatomically distinct and physically separate from known populations of cells in the arcuate
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region that may be involved in the production of releasing factors. In addition, radiolabeled dopamine and catecholamine fluorescence were detected in tanycytes of the median eminence. From these and other data it becomes increasingly clear that the arcuate-periventricular region of the endocrine hypothalamus is far more complex and diverse in its anatomical and functional organization. Extensive re-examination of this region of the mammalian diencephalon is necessary, combining a variety of correlative morphological, physiological and biochemical approaches.
References Aghajanian, G.K., Bloom, F.E. : Electron microscopic autoradiography of the rat hypothalamus after injection of 3H-norepinephrine. Science 153, 308-310 (1966) Aghajanian, G.K., Gallager, D.W. : Raphe origin of serotonergic nerves terminating in the cerebral ventricles. Brain Res. 88, 221-231 (1975) Bj6rklund, A., Falck, B. : An Improvement of the histochemical fluorescence method for monoamines. Observations on varying extractability of fluorophors in different nerve fibers. J. Histochem. Cytochem. 16, 717-720 (1968) Bj6rklund, A., Falck, B., Owman, Ch. : Fluorescence microscopic and microspectrofluorometric techniques for the cellular localization of biogenic amines. In (J. Rail and I. Kopin, eds.) Methods in investigative and diagnostic endocrinology. I. The thyroid and biogenic amines. Amsterdam 1972 Chan-Palay, V.: Serotonin axons of the supra- and subependymal plexuses and subarachnoid systems of the rat and monkey. Proc. neurosci. 166 (1975) Green, J.D. Harris, G.W.: The neurovascular link between the neurohypophysis and adenohypophysis. J. Endocr. 5, 136-146 (1947) Halasz, B., Pupp, L., Uhlarik, S.: Hypophysiotropic area in the hypothalamus. J. Endocr. 25, 147-159 (1962) Hofer, H. : Zur Morphologie der circumventrikularen Organe des Zwischenhirnes der S/iugetiere. Verh. Dtsch. Zool. Ges. Frankfurt, 202-251 (1958) Hoffman, D.L., Sladek, J.R., Jr.: The distribution of catecholamines within the inferior olivary complex of the gerbil and rabbit. J. comp. Neurol. 151, 101-112 (1973) Hoffman, G. : Personal communication (1975) Holzwarth-McBride, M.A., Hurst, E.M., Knigge, K.M.: Monosodium glutamate induced lesions of arcuate nucleus. I. Endocrine deficiency and ultrastructure of median eminence. Anat. Rec., (in press) (1976) Horstmann, E. : Die Faserglia des Selachiergehirns. Z. Zellforsch. 39, 588-617 (1954). Joseph, S.A., Scott, D.E., Vaala, S.S., Knigge, K.M., Krobisch-Dudley, G.: Localization and content of thyrotropin releasing factor (TRF) in median eminence of the hypothalamus. Acta endocr. (Kbh.) 74, 215-225 (1973) Knigge, K.M. : Opening Remarks. In (K.M. Knigge, D.E. Scott, H. Kobayashi and S. Ishii, eds.) Brain-endocrine interaction. The ventricular system in neuroendocrine mechanisms. Basel: Karger 1975 Knigge, K.M., Joseph, S.A., Schock, D., Silverman, A.J., Ching, M.C.H., Scott, D.E., Zeman, D., Krobisch-Dudley, G.: Role of ventricular system in neuroendocrine processes. Synthesis and distribution of thyrotropin releasing factor (TRF) in the hypothalamus and third ventricle. Canad. J. Neurol. Sci. 1, 74-84 (1974) Knigge, K.M., Schock, D., Sladek, J.R., Jr.: Monoamines of median eminence. In (K.M. Knigge, D.E. Scott, H. Kobayashi, and S. Ishii, eds.). Brain-endocrine interaction II. The ventricular system in neuroendocrine mechanisms, p. 252-294. Basel: Karger 1975 Knigge, K.M., Silverman, A.J. : Anatomy of the endocrine hypothalamus. In: Handbook of physiology. Part I, p. 1-32. Endocrinology IV. (1974) McKenna, O.C., Rosenbluth, J. : Cytological evidence for catecholamine containing sensory cells bordering the ventricle of the toad hypothalamus. J. comp. Neurol. 154, 133-148 (1974)
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Oksche, A., Oehmke, H.J., Hartwig, H.G. : A concept of neuroendocrine cell complexes. In: Neurosecretion. The final neuroendocrine pathway, p. 155-156. Berlin-Heidelberg-New York: Springer 1974 Paull, W.K., Lechan, R. : The median eminence of mice with an MSG induced arcuate lesion. Anat. Rec. 178, 346 (1974) Richards, J.G., Lorenz, H.P., Tranzer, J.P. : Indole-alkylamine nerve terminals in cerebral ventricles: Identification by electron microscopy and fluorescence histochemistry. Brain Res. 57, 277-288 (1973) Scott, D.E., Knigge, K.M. : Ultrastructural changes in the median eminence of the rat following deafferentation of the basal hypothalamus. Z. Zellforsch. 105, 1-32 (1970) Scott, D.E., Kozlowksi, G.P., Krobisch-Dudley, G.: A comparative ultrastructural analysis of the third cerebral ventricle of the North American mink, Mustela vison. Anat. Rec. 175, 155-168 (1973) Scott, D.E., Kozlowski, G.P., Sheridan, M.N. : Scanning electron microscopy in the ultrastructural analysis of the mammalian cerebral ventricular system. Int. Rev. Cytol. 37, 349-388 (1974) Scott, D.E., Krobisch-Dudley, G., Knigge, K.M. : The ventricular system in neuroendocrine mechanisms. II. In vivo monoamine transport by ependyma of the median eminence. Cell Tiss. Res. 154, 1-16 (1974) Scott, D.E., Krobisch-Dudley, G., Paull, W.K., Kozlowski, G.P. Ribas, J.: The primate median eminence. I. Correlative scanning-transmission electron microscopy. Cell Tiss. Res. 162, 61-73 (1975) Scott, D.E., Krobisch-Dudley, G., Weindl, A., Joynt, R.J. : An autoradiographic analysis of hypothalamic magnocellular neurons. Z. Zellforsch. 138, 421-437 (1973) Scott, D.E., Sladek, J.R., Jr., Kozlowski, G.P., McNeill, T.H., Paull, W.K., Krobisch-Dudley, G. : The median eminence as a neuroendocrine transducer. In (A. Kumar, ed.) Neuroendocrine regulation of fertility. Basel: Karger 1976 (in press) Silverman, A.J., Knigge, K.M., Peck, W.A. : Transport capacity of the median eminence I. Amino acid transport. Neuroendocrinology 9, 123-132 (1972a) Silverman, A.J., Knigge, K.M. : Transport capacity of the median eminence II. Thyroxine transport. Neuroendocrinology 10, 71-82 (1972b) Sladek, J.R., Jr., Sladek, C.D., Knigge, K.M. : Organ culture and histofluorescence of indoleamines in median eminence. Proc. Soc. Neurosci. 1:461 (1975) Vigh-Teichmann, I., Vigh, B.: The infundibular cerebrospinal fluid contacting neurons. In: Advances in anatomy, embryology and cell biology, p. 1-91. Berlin-Heidelberg-New York: Springer 1974 Zambrano, D. : On the presence of neurons with granulated vesicles in the median eminence of rat and dog. Neuroendocrinology 3, 141-155 (1968)
Received October 10, 1975
