- Email: [email protected]
Electron microscope immunocytochemical localization of thyrotropin-releasing hormone (TRH) prohormone in the rat hypothalamus
Ncwopepndes (1988) 11, 107-110 Q Longman Group UK Ltd 1988
Electron Microscope lmmunocytochemical Localization of Thyrotropin-Releasing Hormone (TRH) Prohormone in the Rat Hypothalamus N. LIAO, M. BULANT,
P. NICHOLAS,
H. VAUDRY
and G. PELLETIER
MRC Group in Molecular Endocrinology, Lava/ University Medical Center, CHUL, Quebec, Canada Gl V 4G.2; and (M. B., P. N. and H. V.), Laboratoire d’Endocrinologie Molhxlaire, UA CNRS 650, Unit& Allike B I’INSERM, Universitb de Rouen, France. [N.L. and G.P.]
Abstract-Using an antiserum to a synthetic peptide corresponding to the sequence Phe-17% Ser-199 of the precursor of thyrotropin-releasing hormone (pro-TRH), we have studied by immunoelectron microscopy the ultrastructural localization of pro-TRH in the rat hypothalamus. In the paraventricular nucleus neuronal cell bodies, dendrites and axons were labelled. The dense core vesicles were strongly immunoreactive. lmmunostained cell bodies, dendrites and endings were frequently observed in contact with unidentified neuronal elements. In the median eminence, numerous endings containing positive dense core vesicles were detected. These results suggest that pro-TRH and/or fragment(s) of pro-tRH could play a neuromodulator/neurotransmitter role in the hypothalamus and could also be released into the pituitary portal plexus.
Introduction The precursor of thyrotropin-releasing hormone (pro-TRH) has been characterized in the rat brain (4). Very recently, using an antiserum to a synthetic peptide corresponding to sequence 178-199 of pro-TRH (Ps.5 in Fig l), we have studied by immunocytochemistry the distribution of immunoreactive pro-TRH in the rat brain (5). The distribution of immunostaining found in different brain areas was similar to that reported for TRH. In the hypothalamus, immunostained neuronal cell bodies were observed in large amounts in the pars parvocellularis of the paraventricular nucleus Date received 15 December 1987 Date accepted 17 December 1987
and positive endings were found in high concentrations in the external zone of the median eminence. These results suggest that pro-TRH and/or fragments of pro-TRH migrate with TRH itself along the axons. In order to gain more information about the processing of pro-TRH and the possible role of pro-TRH and/or peptides derived from pro-TRH, we conducted an immunoelectron microscopic localization of the cryptic fragment Ps5 of proTRH. Materials and Methods Animals
Five adult female rats weighing 250-300g were used in these experiments. They received an 107
108
Fig 1 Structure of rat pre-pro-TRH sequence.
NEUROPEPTIDES
and amino acid sequence of peptide spacer 5 (Ps5) deduced from the corresponding cDNA
injection of 50 kg of colchicine into the left lateral ventricle 48 hours before fixation. Immunocytochemistry
All the animals were perfused with 200ml of 4% paraformaldehyde in 0.1 M phosphate buffer. Consecutive coronal sections (40-50pm) of the hypothalamus were obtained with the Vibratome (Oxford). The pre-embedding staining was performed on the Vibratome sections as described by Pickel et al. (6) using the peroxidase-antiperoxidase technique (8). Areas of the paraventricular nucleus containing positive cell bodies were dehydrated, flat-embedded and cut for electron microscopic observations. Since there are a large number of fibers staining for pro-TRH in the median eminence. Postembedding staining was performed in this area using the protein-A-gold technique (7). In this case, the tissue was fixed by perfusion of 2.5% glutaraldehyde and subsequently post-fixed in 1%
osmium tetroxide, dehydrated in ethanol and embedded in Araldite. The antiserum (No. 360) to the cryptic fragment Ps5 of pro-TRH (Fig 1) was used at a dilution ranging from 1:500 (for pre-embedding staining) to 1:3000 (for post-embedding staining). This antiserum recognized an epitope located in the Phe 178-Ser186 region of the molecule (5). Results Light microscopic examination of thick Vibratome sections confirmed our previous results on the distribution of pro-TRH in the rat brain (5). In the hypothalamus, several positive cell bodies were observed in the pars parvocellularis of the paraventricular nucleus. At the electron microscopic level immunostaining was detected in cell bodies, dendrites and axons. In the cell bodies, the staining was localized primarily in dense core vesicles (70-100nm in diameter) but was also found throughout the cytoplasm (Fig 2). In the positive dendrites, the
Immunostaining of a cell body with the immunoperoxidase technique. Several dense core vesicles are strongly stained (arrows). Two unidentified endings are in synaptic contact (arrowheads) with the cell body. N: nucleus. X 20000.
Fig 2
LOCALIZATION
OF (TRH) PROHORMONE
109
IN THE RAT HYPOTHALAMUS
reaction product was rather diffuse without any labeling of specific organelles (Fig 3). Stained dense core vesicles were occasionally observed. In the axon profiles and endings, the staining was mostly found in dense core vesicles (70-100nm in diameter) and also more diffusely throughout the axoplasm (Fig 4). In the paraventricular nucleus, most of the afferents to the pro-TRH neurons appeared to occur via axo-dendritic synapses. The endings making contacts with the positive dendrites were generally filled with agranular vesicles and few dense core vesicles and formed assymetric syn-
apses (Fig 3). Labeled cell bodies were ally seen being contacted by endings Immunoreactive endings were also making synaptic contacts with unlabeled (Fig 4).
occasion(Fig 2). observed dendrites
In the median eminence, the post-embedding staining technique revealed that several endings contained immunoreactive materials. In the positive endings, gold particles were associated with dense core vesicles which have the same diameter as those observed in the paraventricular nucleus (Fig 5).
Fig 3 Immunostaining of a dendrite (D) in the paraventricular nucleus. The staining is rather diffuse. between the dendrite and an unlabeled ending can be observed. X 28000.
A
synaptic contact (arrow)
Fig 4 Immunostaining of an ending making synaptic contact with a dendrite (D). A dense core vesicle (4) is strongly labelled. Diffuse staining can also be observed. X 29000.
110
NEUROPEPTIDES
Fig 5 Immunolabeling of a ending in the external zone of the median eminence with the protein-A gold technique. The gold particles are mostly associated with dense core vesicles (arrows). X 11000.
Discussion The results obtained in the present study clearly indicate that pro-TRH and/or a cryptic fragment of pro-TRH is localized in dense core vesicles in parvocellular neurons of the hypothalamic paraventricular nucleus. They confirm a previous light microscopic study performed with the same antiserum which indicated that immunoreactive material was present in parvocellular neurons of the paraventricular nucleus and endings in the median eminence (5). It has already been reported that immunoreactive TRH was present in dense core vesicles in both cell bodies and processes including endings in the median eminence (3). It might be then suggested that not only TRH but also pro-TRH and/or fragment(s) of the prohormone are first stored in dense core vesicles in cell bodies and migrated along the axons to reach the endings. TRH and other peptide derived from pro-TRH are probably simultaneously released during exocytosis of dense core vesicles. These results also strongly suggest that pro-TRH and/or fragments of the precursor could act as a neuromodulator or neurotransmitter. In the median eminence, it is likely that not only TRH but also cryptic fragment(s) of pro-TRH, such as Ps5, can reach the anterior pituitary via the portal system. Studies are in progress in this laboratory to determine the role of Ps5 and other cryptic fragments of pro-TRH on the anterior pituitary secretion. The axo-dendritic and occasional axo-somatic contacts on pro-TRH neurons certainly indicate that these neurons are modulated at the level of
the paraventricular nucleus. Since the endings which are contacting pro-TRH neurons contain granular vesicles, they probably belong to the category of monoaminergic endings which invariabIy contain large dense core vesicles (1, 2). The identity of the endings contacting pro-TRH neurons remains to be clarified.
References 1. Beaudet, A. and Descarries. L. (1978). The monoamine innervation of rat cerebral cortex: synaptic and nonsynaptic axon terminals. Neuroscience 4:.8.51-860. _ . 3_. Descarries. L.. Beaudet. A. and Walkins. K. C. (1975). Serotin nerve terminals in adult rat neocortex. Brain Res. 100: 563-588. 3. Johansson, O., Hokfelt, T.. Jeffcoate, S. L.. White. N. and Spindel, E. (1983). Light and electron microscopic immunohistochemical studies on TRH in the central nervous system of the rat. In: Griffiths, E. C. and Bennett. G. W.. (eds), Raven Press, New York, pp. 19-31. 4. Lechan, R. M., Wu, P., Jackson, I. M. D., Wolf, H., Cooperman, S., Mandel, G. and Goodman. R. H. (1986). Thyrotropin-releasing hormone precursor: characterization in rat brain. Science 231: 159-162. 5. Laio, N., Bulant, M., Nicholas, P.. Vaudry, H. and Pelletier. G. (1987). Immunocytochemical distribution of neurons containing a peptide derived from thyrotropinreleasing hormone precursor in the rat brain. Neurosci. Lett., in press. 6. Pickel, M., Joh, T. H. and Reis, P. J. (1975). Ultrastructural localization of tyrosine hydroxylase in noradrenergic neurons of brain. Proc. Natl. Acad. Sci. USA 72: 658663. I. Roth, J., Bendayan. M. and Orci, L. (1978). Ultrastructural localization of intracellular antigens by the use of protein-A-gold complex. J. Histochem. Cytochem. 26: 1074-1081. 8. Sternberger, L. A. (1974). Immunocytochemistry, Prentice-Hall Inc., Englewoods Cliffs, N.J., 246 pp.
Electron Microscope lmmunocytochemical Localization of Thyrotropin-Releasing Hormone (TRH) Prohormone in the Rat Hypothalamus N. LIAO, M. BULANT,
P. NICHOLAS,
H. VAUDRY
and G. PELLETIER
MRC Group in Molecular Endocrinology, Lava/ University Medical Center, CHUL, Quebec, Canada Gl V 4G.2; and (M. B., P. N. and H. V.), Laboratoire d’Endocrinologie Molhxlaire, UA CNRS 650, Unit& Allike B I’INSERM, Universitb de Rouen, France. [N.L. and G.P.]
Abstract-Using an antiserum to a synthetic peptide corresponding to the sequence Phe-17% Ser-199 of the precursor of thyrotropin-releasing hormone (pro-TRH), we have studied by immunoelectron microscopy the ultrastructural localization of pro-TRH in the rat hypothalamus. In the paraventricular nucleus neuronal cell bodies, dendrites and axons were labelled. The dense core vesicles were strongly immunoreactive. lmmunostained cell bodies, dendrites and endings were frequently observed in contact with unidentified neuronal elements. In the median eminence, numerous endings containing positive dense core vesicles were detected. These results suggest that pro-TRH and/or fragment(s) of pro-tRH could play a neuromodulator/neurotransmitter role in the hypothalamus and could also be released into the pituitary portal plexus.
Introduction The precursor of thyrotropin-releasing hormone (pro-TRH) has been characterized in the rat brain (4). Very recently, using an antiserum to a synthetic peptide corresponding to sequence 178-199 of pro-TRH (Ps.5 in Fig l), we have studied by immunocytochemistry the distribution of immunoreactive pro-TRH in the rat brain (5). The distribution of immunostaining found in different brain areas was similar to that reported for TRH. In the hypothalamus, immunostained neuronal cell bodies were observed in large amounts in the pars parvocellularis of the paraventricular nucleus Date received 15 December 1987 Date accepted 17 December 1987
and positive endings were found in high concentrations in the external zone of the median eminence. These results suggest that pro-TRH and/or fragments of pro-TRH migrate with TRH itself along the axons. In order to gain more information about the processing of pro-TRH and the possible role of pro-TRH and/or peptides derived from pro-TRH, we conducted an immunoelectron microscopic localization of the cryptic fragment Ps5 of proTRH. Materials and Methods Animals
Five adult female rats weighing 250-300g were used in these experiments. They received an 107
108
Fig 1 Structure of rat pre-pro-TRH sequence.
NEUROPEPTIDES
and amino acid sequence of peptide spacer 5 (Ps5) deduced from the corresponding cDNA
injection of 50 kg of colchicine into the left lateral ventricle 48 hours before fixation. Immunocytochemistry
All the animals were perfused with 200ml of 4% paraformaldehyde in 0.1 M phosphate buffer. Consecutive coronal sections (40-50pm) of the hypothalamus were obtained with the Vibratome (Oxford). The pre-embedding staining was performed on the Vibratome sections as described by Pickel et al. (6) using the peroxidase-antiperoxidase technique (8). Areas of the paraventricular nucleus containing positive cell bodies were dehydrated, flat-embedded and cut for electron microscopic observations. Since there are a large number of fibers staining for pro-TRH in the median eminence. Postembedding staining was performed in this area using the protein-A-gold technique (7). In this case, the tissue was fixed by perfusion of 2.5% glutaraldehyde and subsequently post-fixed in 1%
osmium tetroxide, dehydrated in ethanol and embedded in Araldite. The antiserum (No. 360) to the cryptic fragment Ps5 of pro-TRH (Fig 1) was used at a dilution ranging from 1:500 (for pre-embedding staining) to 1:3000 (for post-embedding staining). This antiserum recognized an epitope located in the Phe 178-Ser186 region of the molecule (5). Results Light microscopic examination of thick Vibratome sections confirmed our previous results on the distribution of pro-TRH in the rat brain (5). In the hypothalamus, several positive cell bodies were observed in the pars parvocellularis of the paraventricular nucleus. At the electron microscopic level immunostaining was detected in cell bodies, dendrites and axons. In the cell bodies, the staining was localized primarily in dense core vesicles (70-100nm in diameter) but was also found throughout the cytoplasm (Fig 2). In the positive dendrites, the
Immunostaining of a cell body with the immunoperoxidase technique. Several dense core vesicles are strongly stained (arrows). Two unidentified endings are in synaptic contact (arrowheads) with the cell body. N: nucleus. X 20000.
Fig 2
LOCALIZATION
OF (TRH) PROHORMONE
109
IN THE RAT HYPOTHALAMUS
reaction product was rather diffuse without any labeling of specific organelles (Fig 3). Stained dense core vesicles were occasionally observed. In the axon profiles and endings, the staining was mostly found in dense core vesicles (70-100nm in diameter) and also more diffusely throughout the axoplasm (Fig 4). In the paraventricular nucleus, most of the afferents to the pro-TRH neurons appeared to occur via axo-dendritic synapses. The endings making contacts with the positive dendrites were generally filled with agranular vesicles and few dense core vesicles and formed assymetric syn-
apses (Fig 3). Labeled cell bodies were ally seen being contacted by endings Immunoreactive endings were also making synaptic contacts with unlabeled (Fig 4).
occasion(Fig 2). observed dendrites
In the median eminence, the post-embedding staining technique revealed that several endings contained immunoreactive materials. In the positive endings, gold particles were associated with dense core vesicles which have the same diameter as those observed in the paraventricular nucleus (Fig 5).
Fig 3 Immunostaining of a dendrite (D) in the paraventricular nucleus. The staining is rather diffuse. between the dendrite and an unlabeled ending can be observed. X 28000.
A
synaptic contact (arrow)
Fig 4 Immunostaining of an ending making synaptic contact with a dendrite (D). A dense core vesicle (4) is strongly labelled. Diffuse staining can also be observed. X 29000.
110
NEUROPEPTIDES
Fig 5 Immunolabeling of a ending in the external zone of the median eminence with the protein-A gold technique. The gold particles are mostly associated with dense core vesicles (arrows). X 11000.
Discussion The results obtained in the present study clearly indicate that pro-TRH and/or a cryptic fragment of pro-TRH is localized in dense core vesicles in parvocellular neurons of the hypothalamic paraventricular nucleus. They confirm a previous light microscopic study performed with the same antiserum which indicated that immunoreactive material was present in parvocellular neurons of the paraventricular nucleus and endings in the median eminence (5). It has already been reported that immunoreactive TRH was present in dense core vesicles in both cell bodies and processes including endings in the median eminence (3). It might be then suggested that not only TRH but also pro-TRH and/or fragment(s) of the prohormone are first stored in dense core vesicles in cell bodies and migrated along the axons to reach the endings. TRH and other peptide derived from pro-TRH are probably simultaneously released during exocytosis of dense core vesicles. These results also strongly suggest that pro-TRH and/or fragments of the precursor could act as a neuromodulator or neurotransmitter. In the median eminence, it is likely that not only TRH but also cryptic fragment(s) of pro-TRH, such as Ps5, can reach the anterior pituitary via the portal system. Studies are in progress in this laboratory to determine the role of Ps5 and other cryptic fragments of pro-TRH on the anterior pituitary secretion. The axo-dendritic and occasional axo-somatic contacts on pro-TRH neurons certainly indicate that these neurons are modulated at the level of
the paraventricular nucleus. Since the endings which are contacting pro-TRH neurons contain granular vesicles, they probably belong to the category of monoaminergic endings which invariabIy contain large dense core vesicles (1, 2). The identity of the endings contacting pro-TRH neurons remains to be clarified.
References 1. Beaudet, A. and Descarries. L. (1978). The monoamine innervation of rat cerebral cortex: synaptic and nonsynaptic axon terminals. Neuroscience 4:.8.51-860. _ . 3_. Descarries. L.. Beaudet. A. and Walkins. K. C. (1975). Serotin nerve terminals in adult rat neocortex. Brain Res. 100: 563-588. 3. Johansson, O., Hokfelt, T.. Jeffcoate, S. L.. White. N. and Spindel, E. (1983). Light and electron microscopic immunohistochemical studies on TRH in the central nervous system of the rat. In: Griffiths, E. C. and Bennett. G. W.. (eds), Raven Press, New York, pp. 19-31. 4. Lechan, R. M., Wu, P., Jackson, I. M. D., Wolf, H., Cooperman, S., Mandel, G. and Goodman. R. H. (1986). Thyrotropin-releasing hormone precursor: characterization in rat brain. Science 231: 159-162. 5. Laio, N., Bulant, M., Nicholas, P.. Vaudry, H. and Pelletier. G. (1987). Immunocytochemical distribution of neurons containing a peptide derived from thyrotropinreleasing hormone precursor in the rat brain. Neurosci. Lett., in press. 6. Pickel, M., Joh, T. H. and Reis, P. J. (1975). Ultrastructural localization of tyrosine hydroxylase in noradrenergic neurons of brain. Proc. Natl. Acad. Sci. USA 72: 658663. I. Roth, J., Bendayan. M. and Orci, L. (1978). Ultrastructural localization of intracellular antigens by the use of protein-A-gold complex. J. Histochem. Cytochem. 26: 1074-1081. 8. Sternberger, L. A. (1974). Immunocytochemistry, Prentice-Hall Inc., Englewoods Cliffs, N.J., 246 pp.