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Triple Colocalization of Tyrosine Hydroxylase, Calretinin, and Calbindin D-28k in the Periventricu lar-Hypophyseal Dopaminergic Neuronal System
D. M. Jacobowitz, K. R. Isaacs, and V. Cimini Laboratory of Clinical Sciences National Institute of Mental Health Bethesda, Maryland 20892
Triple Colocalization of Tyrosine Hydroxylase, Calretinin, and Calbindin D-28k in the Periventricular-Hypophyseal Dopaminergic Neuronal System Earlier studies have revealed that catecholamine inhibition of amelanocyte-stimulating hormone (a-MSH)release is mediated by doparninergic (DA) nerves in the intermediate lobe of the pituitary (ILP). Other studies have shown that the dopaminergic neurons terminating in the ILP originate in the periventricular nucleus of the hypothalamus ( 1). A recent observation revealed that tyrosine hydroxylase (TH) colocalized with calretinin (CR), a calciumbinding protein, in the rat ILP (2). In addition, T H also colocalized with calbindin-D28k (CB), another calcium-binding protein (CaBP) (unpublished results). This suggested to us that a triple colocalization of TH, CR, and CB existed in the fibers of the ILP. The purpose of this study was to determine, using an immunohistochernical colocalization procedure, whether a triple colocalization of TH, CR, and CB existed in the DA fibers of the ILP. Furthermore, having demonstrated that such a triple colocalization exists, we set out to map the dopaminergic cells in the rat hypothalamus in a search for the origin of these pituitary fibers. Four rats were fixed by perfusion with 200 ml of 10% neutral buffered formalin, and the brains and pituitaries were postfixed for 1-2 hr at 4°C. Sections were cut in a cryostat and were routinely processed for indirect immunofluorescence. For triple immunofluorescent labeling, goat anti-CR sera (1:2500), rabbit anti-CB sera (1: SOOO), and monoclonal antityrosine hydroxylase sera (1:2000) were diluted in phosphate-buffered saline (PBS) containing 1% normal donkey serum and 0.3 Triton X-100. The primary antibodies were visualized using donkey anti-goat Texas Red (1: loo), donkey anti-rabbit fluorescein isothiocyanate (FITC, 1 : loo), and donkey anti-mouse AMCA (1: SO). Double fluorescent irnmunostaining used rabbit anti-CR sera (1:2500) or rabbit anti-CB sera with monoclonal antibodies to TH with 1% normal goat serum and 0.3% Triton X-100 in PBS. Goat anti-rabbit FITC (1: 300) and goat anti-mouse Texas Red (1: 100)were used to reveal primary antibodies. Antibodies to dopamine-beta-hydroxylase (DBH) (1: 1000) were used to confirm that the fibers in the ILP were not noradrenergic. Specificity controls consisted of preadsorbing the CR antibodies with 0.1 p M recombinant CR using preimmune serum or no primary antibodies. Advances in Pharmacology, Volume 42
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Rat brain sections were assigned to 1 of 13 rostra1 to caudal levels according to a standard atlas. DA nuclei A l l to A15v were identified by their THimmunoreactive (TH-ir) cells and location in the brains in relation to standard landmarks. All TH-ir cells were classified as either TH-only, TH+CR, TH+CB, or TH+CR+CB. The percentage of DA cells in each category was calculated for each animal at each level. In immunocytochemical preparations of rat pituitary glands, varicose dopaminergic nerve fibers formed a plexus enveloping the endocrine cells of the intermediate lobe (Fig. l),and the lack of anti-DBH staining confirmed the DA nature of these fibers. Triple colocalization of TH, CR, and CB revealed that all three neurochemicals were present in these fibers in what appeared to be a 1: 1 relationship between the CaBPs (CR, CB) and the TH-containing fibers. No immunoreactive cell bodies were observed in the ILP. Pituitary stalk section resulted in the disappearance of the triple colocalized fibers in the intermediate lobe. We have identified four subpopulations of TH-ir cells: (1)TH-ir only, (2)TH+CR-ir cells, ( 3 ) TH+CB-ir cells, and (4) TH+CR+CB-ir cells. Triple colocalized cells (TH+CR+CB) were found in three subsets of DA perikarya
FIGURE I Triple colocalization of CR, TH, and CB in the intermediate lobe of the rat pituitary (A-C). Note the 1 : 1 correlation between the three antigens; all TH-ir fibers also include CR and CB. Magnification bar equals 25 pm. In the A14 nuclei (D-F), many of the TH-ir cells are also CR- and CB-ir (dosed arrows), although some TH- and CB-ir cells can be identified (open arrows). Magnification bar equals 55 pm.
Triple Colocalization of TH, Calretinin. and Calbindin D-28k
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in the diencephalon of the brain (i.e., the A l l , A14, and A15v cells) (see Fig. 1).No triple colocalized cells were noted in the A12 (arcuate nucleus) and A13 cell groups. In the A1 1 and A14 cell groups, the triple colocalized cells represent 3.1% and 3.3% of the total TH-ir population, respectively. In the A14 cell group, the TH+CR cells represent 24.3% of the total TH-ir population and the TH+CB cells represent 1.9%. The A15v group had a 12.8% colocalization rate in the TH+CR subpopulation, but only a 1 % colocalization rate for the TH+CB cells. Cells in this nucleus were triple colocalized only 1%of the time. This study revealed that the DA (TH-ir)fibers that innervate the ILP gland also contain two CaBPs: CR and CB. CR is a predominantly neuronal protein that shares 58% amino acid homology with CB. It has been suggested that CR and CB may act as intracellular calcium buffers within neurons, thereby serving a neuroprotective role. The fact that TH-ir nerves in the ILP and that A l l and A14 cell bodies in the hypothalamus are DA rather than noradrenergic has been amply documented. Smelik (3) first showed a DA innervation of the ILP, although he believed that the cell bodies were located in the arcuate nucleus (A12 cells). Bjorklund and Falck (4) reported that dopamine was the predominant catecholamine in the ILP, although in the cat, significant amounts of norepinephrine were also found. Anatomical, neurochemical, and neuroendocrinological evidence (1)indicates that dopamine terminals in the ILP originate from A14 cells in the hypothalamic periventricular nucleus (5).We have observed that a triple colocalization of TH, CR, and CB was contained in about 3.3% of the A14 DA cell bodies, and we suggest that these are the cells that innervate the ILP. Because of the triple colocalization within approximately 3.1% of the A l l cells, this system may also project to the ILP. In addition, a few cells (about YO) of the A15v system (6) may also project to the ILP. The DA nature of the A15 cells has not been established. The sites of innervation of the TH+CR and TH+CB cells remain to be studied. Rogers (7)studied TH-containing cells that colocalized with either CR or CB in the rat brain. He also reported that double colocalization (TH+CR and TH+CB) was present in the A l l , A14, and A15v cell groups. Numerical differences between these two studies probably represent sampling differences. Rogers did not study triple colocalizations. Many TH-ir cells show great heterogeneity in CaBP content. The presence of CaBP in a variety of neuronal subpopulations has repeatedly been shown to express neurochemical heterogeneity, the significance of which, however, continues to elude us. The functional significance of the CaBPs may very well be linked to our understanding of this property of heterogeneity within subsets of brain neurons, ganglia, and even nonneuronal CaBP-containing cells (testis, ovary, pituitary). In this regard, Rogers (7)suggested that the observed heterogeneity may be a reflection of neuronal activity. Much work has supported the conclusion that catecholamines inhibit the release of a-MSH from the ILP. Furthermore, the inhibition of a-MSH release has been shown to be mediated by dopamine. Blockade of DA neurotransmission in vivo has been reported to stimulate a-MSH release, whereas DA receptor stimulation seems to inhibit a-MSH release in vitvo, as well as in vivo. Prior studies have shown that isolated melanotrophs prepared from the intermediate lobe responded briskly, with an increased output of a-MSH when
40
D. M.Jacobowia et 01.
challenged with high K', and that this response depended on extracellular calcium and was inhibited by a calcium channel blocker ( 8 ) . Their results indicated that the activation of the voltage-dependent calcium channels allowed calcium to enter the cell in amounts sufficient to provide a strong stimulus to secretion. These results also suggested that the spontaneous secretory activity of these cells could be explained by the existence of some channels that were in the open state under basal conditions. The significance of the presence of CR and CB in the hypothalamic-ILP DA system is unknown. It has been proposed that the presence of CaBPs in neurons in general may provide protection from calcium-induced neurotoxicity. The release of dopamine, which inhibits a-MSH secretion, would result in an increase in calcium in the extracellular milieu. The presence of two CaBPs, CR and CB, would provide extra buffering capacity to prevent neurotoxicity. This, however, remains a working hypothesis, and we do not begin to fathom the true function of these CaBPs.
References 1. Goudreau, J. L., Lindley, S. E., Lookingland, K. J., and Moore, K. E. (1992). Evidence that periventricular dopamine neurons innervate the intermediate lobe of the rat pituitary. Neuroendocrinology 56, 100-105. 2. Cimini, V., Isaacs, K. R., and Jacobowitz, D. M. (1997). Calretinin in the rat pituitary: Colocalization with thyroid stimulating hormone. Neuroendocrinology 65, 179-188. 3. Smelik, P. G. (1966). A dopaminergic innervation of the intermediate lobe of the pituitary? Actu Physiol. Phurmacol. Nkerl. 14, 1. 4. Bjorklund, A,, and Falck, B. (1969).Pituitary monoamines of the cat with special reference to the presence of an unidentified monoaniine-like substance in the adenohypophysis. 2. Zellforsch. 93, 254-264. 5. Bjorklund, A., and Lindvall, 0. (1984). Dopamine-containing systems of the CNS. In Handbook of Chemical Neuroanatomy, Vol. 2. (A. Bjorklund and T. Hokfelt, eds.) pp. 55-122. Elsevier, New York. 6. Hokfelt, T., Martensson, R., Bjorklund, A., Kleinau, S., and Goldstein, M. (1984).Distributional maps of tyrosine-hydroxylase-immunoreactive neurons in the rat brain. In Handbook of Chemical Neuroanatomy, Vol2. (A. Bjorklund and T. Hokfelt, eds.)pp.277-379. Elsevier, New York. 7. Rogers, J. H. (1992).Immunohistochemicalmarkers in rat brain: Colocalization of calretinin and calbindin-D28k with tyrosine hydroxylase. Bruin Res. 587, 203-210. 8. Tomiko, S. A., Taraskevich, P. S., and Douglas, W. W. (1981).Potassium-induced secretion of melanocyte-stimulating hormone from isolated pars intermedia cells signals participation of voltage-dependent calcium channels in stimulus-secretion coupling. Neuroscience 6, 2259-2267.
Triple Colocalization of Tyrosine Hydroxylase, Calretinin, and Calbindin D-28k in the Periventricular-Hypophyseal Dopaminergic Neuronal System Earlier studies have revealed that catecholamine inhibition of amelanocyte-stimulating hormone (a-MSH)release is mediated by doparninergic (DA) nerves in the intermediate lobe of the pituitary (ILP). Other studies have shown that the dopaminergic neurons terminating in the ILP originate in the periventricular nucleus of the hypothalamus ( 1). A recent observation revealed that tyrosine hydroxylase (TH) colocalized with calretinin (CR), a calciumbinding protein, in the rat ILP (2). In addition, T H also colocalized with calbindin-D28k (CB), another calcium-binding protein (CaBP) (unpublished results). This suggested to us that a triple colocalization of TH, CR, and CB existed in the fibers of the ILP. The purpose of this study was to determine, using an immunohistochernical colocalization procedure, whether a triple colocalization of TH, CR, and CB existed in the DA fibers of the ILP. Furthermore, having demonstrated that such a triple colocalization exists, we set out to map the dopaminergic cells in the rat hypothalamus in a search for the origin of these pituitary fibers. Four rats were fixed by perfusion with 200 ml of 10% neutral buffered formalin, and the brains and pituitaries were postfixed for 1-2 hr at 4°C. Sections were cut in a cryostat and were routinely processed for indirect immunofluorescence. For triple immunofluorescent labeling, goat anti-CR sera (1:2500), rabbit anti-CB sera (1: SOOO), and monoclonal antityrosine hydroxylase sera (1:2000) were diluted in phosphate-buffered saline (PBS) containing 1% normal donkey serum and 0.3 Triton X-100. The primary antibodies were visualized using donkey anti-goat Texas Red (1: loo), donkey anti-rabbit fluorescein isothiocyanate (FITC, 1 : loo), and donkey anti-mouse AMCA (1: SO). Double fluorescent irnmunostaining used rabbit anti-CR sera (1:2500) or rabbit anti-CB sera with monoclonal antibodies to TH with 1% normal goat serum and 0.3% Triton X-100 in PBS. Goat anti-rabbit FITC (1: 300) and goat anti-mouse Texas Red (1: 100)were used to reveal primary antibodies. Antibodies to dopamine-beta-hydroxylase (DBH) (1: 1000) were used to confirm that the fibers in the ILP were not noradrenergic. Specificity controls consisted of preadsorbing the CR antibodies with 0.1 p M recombinant CR using preimmune serum or no primary antibodies. Advances in Pharmacology, Volume 42
37
38
D. M. Jacobowia et a/.
Rat brain sections were assigned to 1 of 13 rostra1 to caudal levels according to a standard atlas. DA nuclei A l l to A15v were identified by their THimmunoreactive (TH-ir) cells and location in the brains in relation to standard landmarks. All TH-ir cells were classified as either TH-only, TH+CR, TH+CB, or TH+CR+CB. The percentage of DA cells in each category was calculated for each animal at each level. In immunocytochemical preparations of rat pituitary glands, varicose dopaminergic nerve fibers formed a plexus enveloping the endocrine cells of the intermediate lobe (Fig. l),and the lack of anti-DBH staining confirmed the DA nature of these fibers. Triple colocalization of TH, CR, and CB revealed that all three neurochemicals were present in these fibers in what appeared to be a 1: 1 relationship between the CaBPs (CR, CB) and the TH-containing fibers. No immunoreactive cell bodies were observed in the ILP. Pituitary stalk section resulted in the disappearance of the triple colocalized fibers in the intermediate lobe. We have identified four subpopulations of TH-ir cells: (1)TH-ir only, (2)TH+CR-ir cells, ( 3 ) TH+CB-ir cells, and (4) TH+CR+CB-ir cells. Triple colocalized cells (TH+CR+CB) were found in three subsets of DA perikarya
FIGURE I Triple colocalization of CR, TH, and CB in the intermediate lobe of the rat pituitary (A-C). Note the 1 : 1 correlation between the three antigens; all TH-ir fibers also include CR and CB. Magnification bar equals 25 pm. In the A14 nuclei (D-F), many of the TH-ir cells are also CR- and CB-ir (dosed arrows), although some TH- and CB-ir cells can be identified (open arrows). Magnification bar equals 55 pm.
Triple Colocalization of TH, Calretinin. and Calbindin D-28k
39
in the diencephalon of the brain (i.e., the A l l , A14, and A15v cells) (see Fig. 1).No triple colocalized cells were noted in the A12 (arcuate nucleus) and A13 cell groups. In the A1 1 and A14 cell groups, the triple colocalized cells represent 3.1% and 3.3% of the total TH-ir population, respectively. In the A14 cell group, the TH+CR cells represent 24.3% of the total TH-ir population and the TH+CB cells represent 1.9%. The A15v group had a 12.8% colocalization rate in the TH+CR subpopulation, but only a 1 % colocalization rate for the TH+CB cells. Cells in this nucleus were triple colocalized only 1%of the time. This study revealed that the DA (TH-ir)fibers that innervate the ILP gland also contain two CaBPs: CR and CB. CR is a predominantly neuronal protein that shares 58% amino acid homology with CB. It has been suggested that CR and CB may act as intracellular calcium buffers within neurons, thereby serving a neuroprotective role. The fact that TH-ir nerves in the ILP and that A l l and A14 cell bodies in the hypothalamus are DA rather than noradrenergic has been amply documented. Smelik (3) first showed a DA innervation of the ILP, although he believed that the cell bodies were located in the arcuate nucleus (A12 cells). Bjorklund and Falck (4) reported that dopamine was the predominant catecholamine in the ILP, although in the cat, significant amounts of norepinephrine were also found. Anatomical, neurochemical, and neuroendocrinological evidence (1)indicates that dopamine terminals in the ILP originate from A14 cells in the hypothalamic periventricular nucleus (5).We have observed that a triple colocalization of TH, CR, and CB was contained in about 3.3% of the A14 DA cell bodies, and we suggest that these are the cells that innervate the ILP. Because of the triple colocalization within approximately 3.1% of the A l l cells, this system may also project to the ILP. In addition, a few cells (about YO) of the A15v system (6) may also project to the ILP. The DA nature of the A15 cells has not been established. The sites of innervation of the TH+CR and TH+CB cells remain to be studied. Rogers (7)studied TH-containing cells that colocalized with either CR or CB in the rat brain. He also reported that double colocalization (TH+CR and TH+CB) was present in the A l l , A14, and A15v cell groups. Numerical differences between these two studies probably represent sampling differences. Rogers did not study triple colocalizations. Many TH-ir cells show great heterogeneity in CaBP content. The presence of CaBP in a variety of neuronal subpopulations has repeatedly been shown to express neurochemical heterogeneity, the significance of which, however, continues to elude us. The functional significance of the CaBPs may very well be linked to our understanding of this property of heterogeneity within subsets of brain neurons, ganglia, and even nonneuronal CaBP-containing cells (testis, ovary, pituitary). In this regard, Rogers (7)suggested that the observed heterogeneity may be a reflection of neuronal activity. Much work has supported the conclusion that catecholamines inhibit the release of a-MSH from the ILP. Furthermore, the inhibition of a-MSH release has been shown to be mediated by dopamine. Blockade of DA neurotransmission in vivo has been reported to stimulate a-MSH release, whereas DA receptor stimulation seems to inhibit a-MSH release in vitvo, as well as in vivo. Prior studies have shown that isolated melanotrophs prepared from the intermediate lobe responded briskly, with an increased output of a-MSH when
40
D. M.Jacobowia et 01.
challenged with high K', and that this response depended on extracellular calcium and was inhibited by a calcium channel blocker ( 8 ) . Their results indicated that the activation of the voltage-dependent calcium channels allowed calcium to enter the cell in amounts sufficient to provide a strong stimulus to secretion. These results also suggested that the spontaneous secretory activity of these cells could be explained by the existence of some channels that were in the open state under basal conditions. The significance of the presence of CR and CB in the hypothalamic-ILP DA system is unknown. It has been proposed that the presence of CaBPs in neurons in general may provide protection from calcium-induced neurotoxicity. The release of dopamine, which inhibits a-MSH secretion, would result in an increase in calcium in the extracellular milieu. The presence of two CaBPs, CR and CB, would provide extra buffering capacity to prevent neurotoxicity. This, however, remains a working hypothesis, and we do not begin to fathom the true function of these CaBPs.
References 1. Goudreau, J. L., Lindley, S. E., Lookingland, K. J., and Moore, K. E. (1992). Evidence that periventricular dopamine neurons innervate the intermediate lobe of the rat pituitary. Neuroendocrinology 56, 100-105. 2. Cimini, V., Isaacs, K. R., and Jacobowitz, D. M. (1997). Calretinin in the rat pituitary: Colocalization with thyroid stimulating hormone. Neuroendocrinology 65, 179-188. 3. Smelik, P. G. (1966). A dopaminergic innervation of the intermediate lobe of the pituitary? Actu Physiol. Phurmacol. Nkerl. 14, 1. 4. Bjorklund, A,, and Falck, B. (1969).Pituitary monoamines of the cat with special reference to the presence of an unidentified monoaniine-like substance in the adenohypophysis. 2. Zellforsch. 93, 254-264. 5. Bjorklund, A., and Lindvall, 0. (1984). Dopamine-containing systems of the CNS. In Handbook of Chemical Neuroanatomy, Vol. 2. (A. Bjorklund and T. Hokfelt, eds.) pp. 55-122. Elsevier, New York. 6. Hokfelt, T., Martensson, R., Bjorklund, A., Kleinau, S., and Goldstein, M. (1984).Distributional maps of tyrosine-hydroxylase-immunoreactive neurons in the rat brain. In Handbook of Chemical Neuroanatomy, Vol2. (A. Bjorklund and T. Hokfelt, eds.)pp.277-379. Elsevier, New York. 7. Rogers, J. H. (1992).Immunohistochemicalmarkers in rat brain: Colocalization of calretinin and calbindin-D28k with tyrosine hydroxylase. Bruin Res. 587, 203-210. 8. Tomiko, S. A., Taraskevich, P. S., and Douglas, W. W. (1981).Potassium-induced secretion of melanocyte-stimulating hormone from isolated pars intermedia cells signals participation of voltage-dependent calcium channels in stimulus-secretion coupling. Neuroscience 6, 2259-2267.