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Centre National de la Recherche Scientifique, Unité Propre de Recherche 9055, Biologie des Neurones Endocrines, Centre de Pharmacologie-Endocrinologie, F-34094 Montpellier Cedex 5, France
Address all correspondence and requests for reprints to: Dr. F. Moos, Centre National de la Recherche Scientifique, Unité Propre de Recherche 9055, Centre de Pharmacologie-Endocrinologie, 141 rue de la Cardonille, F-34094 Montpellier Cedex 5, France. E-mail: moos{at}ccipe.montp.inserm.fr
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Abstract |
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The RT-PCR on supraoptic RNA extracts revealed that mainly V1a, but also V1b, subtypes of VP receptors are expressed from birth to adulthood. No V2 receptor messenger RNA (mRNA) was detected. Furthermore, the single-cell RT-nested PCR indicated that the V1a receptor mRNA is present in vasopressinergic magnocellular neurons.
In light of these results, in situ hybridization was performed to visualize the V1a and V1b receptor mRNAs in supraoptic and paraventricular nuclei. Simultaneously, we coupled this approach to: 1) in situ hybridization detection of oxytocin or VP mRNAs; or 2) immunocytochemistry to detect the neuropeptides. This provided a way of identifying the neurons expressing perceptible amounts of V1a or V1b receptor mRNAs as vasopressinergic neurons.
Here, we suggest that the autocontrol exerted specifically by VP on vasopressinergic neurons is mediated through, at least, V1a and V1b subtype receptors.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the central nervous system, VP has been demonstrated to modulate electrical activities of magnocellular VP neurons (19, 20, 21, 22) caused by a somato-dendritic release of VP in the extracellular space of the paraventricular and supraoptic nuclei (23, 24, 25). Microspectrofluorimetric studies on dissociated supraoptic neurons confirmed the presence of specific receptors on the cell body of VP neurons (26, 27, 28). Although the action of VP seems to be predominantly mediated by V1-type VP receptors (26, 27, 28, 29), there is also some electrophysiological (20) and pharmacological (30, 31) evidence that V2-type receptors are involved.
Receptor binding studies, using [3H-]- or [125I-]-labeled agonists or antagonists, indicate that the V1a subtype is predominant in the brain (6, 32, 33, 34, 35, 36). Binding sites or V1a receptor messenger RNA (mRNA) have been localized in various area of the central nervous system (33, 34, 37, 38). However, autoradiographic studies have failed to reveal VP-binding sites on the magnocellular neurons of supraoptic and paraventricular nuclei (6, 32, 34, 36). In situ hybridization has provided a way of detecting the V1a receptor mRNA only in the parvocellular part of the paraventricular nucleus and in areas just dorsal to the supraoptic nucleus (5). V1b receptor transcripts have been detected in several extrapituitary regions, including the hypothalamus (14); and V1b receptor-immunoreactive cell bodies have been observed in the paraventricular and supraoptic nuclei (39). Furthermore, neuroanatomical data revealed that VP binding sites in the hypo-thalamic magnocellular nuclei are localized on VP-containing neurons (40).
Functional evidence of VP receptors in hypothalamic magnocellular nuclei, therefore, has been established. However, the nature of the receptor expressed remains unclear. Therefore, we have identified and visualized the VP receptors expressed by the magnocellular neurons of supraoptic and paraventricular nuclei of the hypothalamus. Using the very sensitive RT-nested PCR technique, we showed that V1a predominantly and V1b (but not V2) receptor transcripts are detectable in dissected supraoptic nuclei of rats from birth to adulthood. RT-PCR, performed at the single-cell level, then showed the presence of V1a receptor mRNA in both VP and oxytocin (OT) neurons. Finally, using simultaneous in situ hybridization and immunocytochemistry or double in situ hybridization, we were able to show, for the first time, that V1a and V1b receptor genes are transcribed in perceptible amounts essentially in the vasopressinergic magnocellular neurons of supraoptic and paraventricular nuclei.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Wistar rats were used in accordance with the European laws for the care and use of experimental animals.
RT-PCR. Wistar rats (0-, 6-, 10-, 14-, 21-, 28-day-old, and adult) were decapitated, and the supraoptic nuclei were rapidly microdissected, frozen in liquid nitrogen, pooled by 10 animals (half male, half female), and stored at -80 C until processed.
Single-cell RT-PCR. Magnocellular neurons, freshly dissociated from supraoptic nuclei of 1-month-old female rats, were prepared using a combined enzymatic and mechanical cell dissociation procedure (41). The cytoplasm from individual cells was collected by suction using a glass micropipette, blown in an Eppendorf microtube, immediately frozen in liquid nitrogen, and kept at -80 C until processed.
In situ hybridization. One-month-old male Wistar rats were decapitated, and their brains were rapidly removed, frozen at -50 C in isopentane, and stored at -80 C until processed. Sections of 12 µm were cut at -20 C with a Leica Corp. CM3000 cryostat, thaw-mounted on polylysine-treated slides, and processed for hybridization.
Primers for RT-PCR and probes for in situ hybridization
The primers for RT and nested PCR, and the probes for in
situ hybridization (Table 1
), were
chosen on the rat V1a, V1b, and V2
VP receptor mRNA sequences using CPrimer 1.09 (G. Bristol and R.
D. Andersen, University of California, Los Angeles, CA) and Amplify 1.2
(W. Engels, University of Wisconsin, Madison, WI) softwares. The
specificity of the sequences chosen for each subtype of VP receptor, as
well as the absence of cross-reaction with the OT receptor mRNA, were
carefully verified. The BLAST software (National Center for
Biotechnology Information, Bethesda, MD), set at its maximal
sensitivity, was also used to check that the primers and probes did not
match significantly any other known RNA or DNA sequence. To improve the
sensitivity of detection by in situ hybridization, two
probes were selected for each receptor subtype. For the PCR study, an
intron lies between the forward and reverse primer sets chosen for the
three types of receptors studied (formally for the V2
receptor whose rat genomic DNA sequence is known; by analogy with the
human and sheep genomic sequences for the V1a receptor; by
analogy with the V1a, V2, and OT receptors for
the V1b receptor whose genomic sequence has not yet been
released in the gene databases, for any species). Because of the use of
the very sensitive nested-PCR procedure, this ensures rejection (on the
basis of the size) of PCR bands amplified on any contaminating genomic
DNA that could have resisted to the DNA digestion before the RT
step. Controls, by omitting the RT step, were also performed.
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).
The DNA amplification was then performed using PTC-100/60 or PTC-150/16
thermal controllers (MJ Research, Inc., Watertown, MA),
Taq DNA Polymerase (Life Technologies)
according to the manufacturer’s instructions, and the primer sets
described in Table 1. The first PCR was made using the F1 forward
primer and the R1 reverse primer previously used for RT. The nested PCR
was performed using the F2 forward primer and the R2 reverse primer.
Settings are provided in Table 1.
The PCR products were electrophoresed on a 2% agarose gel in TAE
buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8)
containing 0.5 µg/ml ethidium bromide, observed under UV light, and
photographed. The lengths of the PCR products were as expected (Table 1). Specificity of the DNA bands obtained after amplification was also
verified by digesting the PCR products with appropriate restriction
enzymes; the lengths of the main fragments were as expected (Table 1).
Single-cell PCR
The RT was performed directly on the cytoplasm (without RNA
extraction) using simultaneously three R1 reverse primers: one for
copying both VP and OT transcripts, one for the V1a, and
one for the V1b VP receptor transcripts (Table 1). A
first-nested PCR provided a way of identifying the magnocellular neuron
as a VP or OT neuron. A second-nested PCR, consuming the rest of the RT
product, was performed to detect either the V1a or
V1b receptor transcript. Four VP and four OT neurons were
studied for each receptor subtype.
Combined in situ hybridization and immunocytochemistry
The receptor probes were 3' end-labeled with biotin
16-deoxyuridine 5-triphosphate using terminal transferase
(Boehringer Mannheim). The sections were air-dried and
fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for
20 min at room temperature. After washing with PBS (130 mM
NaCl, 7 mM Na2HPO4, 3
mM NaH2PO4), the sections were
dehydrated in increasing concentrations of ethanol (70% and 95%) and
air dried. The sections were then flooded with the two specific probes
dissolved at 10 nM final concentration in hybridization
buffer (50% deionized formamide, 0.08 M Tris buffer (pH
7.5), 0.6 M NaCl, 4 mM EDTA, 0.05%
Na2H2P2O7, 0.05%
Na4P2O7, 0.2% N-lauroylsarcosine).
The hybridization was performed in a humid chamber overnight at 42 C.
After incubation, the slides were washed, at 45 C, in decreasing
concentrations of saline-sodium citrate (2x, 0.5x and 0.1x for 30
min each; 1x saline-sodium citrate is 150 mM NaCl, 15
mM sodium citrate, pH 7).
When in situ hybridization was combined with immunocytochemistry, the unrevealed hybridized sections were incubated overnight with the polyclonal rabbit anti-VP antibody (diluted to 1/400 in 1x PBS, 2% BSA, 0.1% N-lauroylsarcosine) or with the mouse anti-OT neurophysin PS38 monoclonal antibody (diluted to 1/200 in 1x PBS, 2% BSA, 0.1% N-lauroylsarcosine). The sections were then incubated for 1 h, simultaneously with Cy3-streptavidin (Sigma Chemical Co.) to reveal the oligonucleotidic probes, and either with oregon green-conjugated antirabbit antibody or oregon green-conjugated antimouse antibody (diluted to 1/200 in 1x PBS, 2% BSA) to reveal the peptide.
When detections of receptor and peptide mRNAs were combined (double in situ hybridization), the probes for OT or VP mRNA were 3' end-labeled with digoxigenin 11-deoxyuridine 5-triphosphate. After posthybridization washings, the sections were treated for 15 min with 1% H2O2 in 1x PBS to inactivate endogenous peroxidase. The hybridized biotinylated probes (detecting the receptor mRNAs) were revealed by 30 min-incubation with horseradish peroxidase-conjugated streptavidin [1/100 diluted in 0.1 M Tris buffer, 0.15 M NaCl, 0.5% blocking reagent (NEN Life Science Products)]. The peroxidase detection was performed by addition of Cy3-tyramide stock solution [(1/50 in 1x amplification diluent (NEN Life Science Products)] for 8 min at room temperature. The hybridized digoxigenin-labeled probes (detecting the peptide mRNAs) were incubated for 1 h with a sheep antidigoxigenin IgG (Boehringer Mannheim; 1/500 in 0.1 M Tris buffer, 0.15 M NaCl, 0.5% blocking reagent) and then for 1 h with a donkey Cy2-conjugated antisheep F(ab')2-fragment IgG (Jackson, West Grove, PA; 1/100 in 0.1 M Tris buffer, 0.15 M NaCl, 0.5% blocking reagent).
To detect combined V1a and V1b receptor mRNAs, the probes for V1a receptor mRNA (labeled with biotin) and those for V1b receptor mRNA (labeled with digoxigenin) were hybridized simultaneously (42). The V1a receptor mRNA was then revealed using the tyramide signal amplification system (Cy3-tyramide). After inactivation of resting peroxidase activity with 0.01 N HCl for 10 min at room temperature, V1b receptor mRNA was revealed by incubation for 1 h with sheep horseradish-peroxidase-conjugated antidigoxigenin Fab fragments (Boehringer; 1/100 in 0.1 M Tris buffer, 0.15 M NaCl, 0.5% blocking reagent) followed by amplification with fluorescein-tyramide (42).
The sections were then washed with 0.1 M Tris buffer, 0.15 M NaCl, 0.05% Tween 20, mounted in Mowiol, and observed with a Leica Corp. TCS 4D confocal microscope. Images were processed using Adobe Photoshop 3.0 (San Jose, CA).
The controls were: 1) use of unlabeled probes or omission of probes; 2) competition between an excess of unlabeled probes and labeled probes in the hybridization reaction; and 3) omission of different molecules involved in biotin or digoxigenin detection. All the controls returned the hybridization signals to background levels. For the V1a and V1b receptor mRNAs double hybridization, the complete inactivation of peroxidase rest activity between the two tyramide deposit reactions was essential because omission of this step could have lead to repeated deposit of the second tyramide in the vicinity of the mRNA target detected first. The 0.01 N HCl inactivation procedure (42), which appeared to affect neither the first fluorescent in situ hybridization signal nor the cell morphology, was checked as efficient, because a second round of amplification with fluorescein isothiocyanate-tyramide, after this treatment, did not add the fluorescein isothiocyanate-signal in the vicinity of the previous Cy3-signal.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Then, PCR
performed separately with the three F1 and R1 primer sets designed to
detect VP receptors (Table 1) revealed that the V1a subtype
is expressed from birth to adulthood in this brain area (Fig. 1A). The V1b and
V2 receptor transcripts were undetectable at all the ages
studied.
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). This nested amplification provided a way of detecting
V1b receptor transcripts at all the ages studied (Fig. 1B).
Conversely, nested PCR failed to detect V2 receptor
transcripts in the RNA extracts from supraoptic nuclei at all the ages
studied, even under very high amplification conditions (as a control,
it was verified that the primer set can detect V2
transcripts in kidney extracts). Consequently, the V1a
subtype is the major VP receptor detected in the supraoptic nucleus,
whereas V1b is a minor isoform.
Both VP and OT magnocellular neurons express the V1a
receptor
The cytoplasm from the single freshly dissociated neurons was used
as substrate for simultaneous search for transcripts of VP, OT, and
either V1a or V1b receptor genes. The RT-nested
PCR revealed that the V1a receptor mRNA is present in the
neurons that produce the prepro-VP mRNA, as well as in those that
produce the prepro-OT mRNA (Fig. 2). At
the single-cell level, the mRNA for V1b receptor remained
undetectable (not shown).
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) were hybridized simultaneously and revealed
using the tyramide signal amplification system. They provided a way of
visualizing V1a and V1b receptor mRNAs in
magnocellular neurons of both supraoptic (Fig. 3) and paraventricular nuclei (not
shown). The signal for both VP receptor mRNAs was detected all over the
cell body, generally with patches of intense fluorescence distributed
in the cell body (Fig. 3; G, J, M, P, and S–U). The punctate labeling
may arise from the combination of: observation on a confocal microscope
(about 1-µm depth), low level of mRNAs nonuniformly distributed,
and finally, use of an efficient amplification procedure.
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, A–C) and not to OT
(Fig. 3, D–F). The colocalization of V1a receptor mRNA and
VP was attested by the fact that all neurons were yellow-colored when
the two images of in situ hybridization (Fig. 3A:
red fluorescence for V1a receptor mRNA) and
immunocytochemistry (Fig. 3B: green fluorescence for VP)
were superimposed (Fig. 3C). In contrast, the yellow color never
appeared when the in situ hybridization image of
V1a (Fig. 3D) and the immunocytochemistry image of OT (Fig. 3E) were superimposed (Fig. 3F).
To confirm the identification of the neurons expressing VP receptors, a
double in situ hybridization was performed. One
oligonucleotidic digoxigenin-labeled probe, specific for either OT or
VP mRNA (P, Table 1), was hybridized simultaneously with the two
biotinylated probes designed to detect each VP receptor mRNA subtype
(P1 and P2, Table 1). The double labeling showed that the magnocellular
neurons exhibiting either V1a or V1b receptor
mRNAs also express mRNA for VP (Fig. 3, G–I and M-O). In the absence
of VP signal, a few cells displayed a V1a or
V1b signal (Fig. 3, G–I for V1a,
arrowheads; M–O for V1b). Conversely, with few
exceptions, the OT mRNA did not colocalize with V1a or
V1b receptor mRNA (Fig. 3, J–L and P–R): only 3–4 cells
coexpressing OT mRNA and V1a or V1b receptor
mRNAs were observed during the course of our investigation (not shown).
In fact, V1a and V1b receptor mRNAs were not
easily detectable in the dorsal and anterior parts of the supraoptic
nucleus, the region where OT neurons are mainly concentrated. In more
median areas of the supraoptic nucleus, some neurons exhibiting OT mRNA
were closely mixed with many other neurons exhibiting V1a
or V1b receptor mRNAs (Fig. 3, J–L and P–R). The rare
cells coexpressing OT mRNA and V1a or V1b
receptor mRNA were observed in these regions.
The double in situ hybridization, to detect V1a
and V1b receptor mRNAs revealed sequentially by the
tyramide signal amplification system, showed that the V1a
and V1b receptor mRNAs are colocalized in the same neurons,
with, however, varying signal intensity within the cell (which ruled
out the possibility of a cross-reaction between the two sequential
amplifications) (Fig. 3, S–U).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using in situ hybridization with an antisense riboprobe, Ostrowski et al. (4, 5) found no V1 receptor transcripts in the supraoptic nucleus and the magnocellular part of paraventricular nucleus. However, V1b receptor protein was detected in this area by immunocytochemistry (39). To improve the detection sensitivity of in situ hybridization, we used simultaneously two oligonucleotidic probes for each receptor mRNA, a procedure recently introduced by Trembleau and Bloom (43). We also used biotin labeling of the probes and detection of the hybrids, by the very efficient tyramide signal amplification, to reveal the hybridized molecules. The result of these methodological choices was the visualization (for the first time, to our knowledge) of V1a and V1b receptor transcripts in the magnocellular neurons of supraoptic and paraventricular nuclei.
Furthermore, using either double in situ hybridization or a combination of in situ hybridization with immunocytochemistry, we clearly show that the great majority of the magnocellular neurons displaying the V1a and V1b receptor signals are those that synthesize VP. A few exceptions may probably concern the magnocellular neurons that contain both VP and OT (27, 44, 45, 46). They may also comprise the very small proportion of magnocellular neurons (up to 0.1%) where a crossing-over occurs between the close VP and OT genes, leading to hybrid VP and OT mRNAs (47, 48).
The present results surprisingly showed that V1a receptor mRNA was detectable by single-cell RT-PCR in the magnocellular OT neurons. Nevertheless, they also showed that the amount of this mRNA is probably very low, because it is undetectable using highly sensitive in situ hybridization. However, the physiological relevance of the V1a receptor mRNA in oxytocinergic neurons remains to be determined, especially because these neurons do not respond to VP or V1 agonists, neither in vivo nor as freshly dissociated cells (22, 27). The presence of V1a receptor mRNA suggests that VP may act directly on OT neurons via this receptor, rather than at high concentrations via the OT receptor. Another possibility is that OT neurons possess the intrinsic potentiality to respond directly to VP under particular physiological demands. This dual potentiality could be expressed, for example, during parturition or lactation, when the percent of cells expressing both VP and OT mRNAs increases markedly (49, 50).
Taken together, our experimental data show that the magnocellular VP neurons of the supraoptic and paraventricular nuclei express simultaneously the V1a and V1b VP receptors. VP modulates the electrical activity of VP neurons by exerting either inhibitory or excitatory effects (19, 20, 21, 31). This modulation leads the population of VP neurons to discharge with an optimized specific phasic pattern (22), the most efficient for systemic hormone release. The complexity of the autocontrol exerted by VP on VP neurons may arise, in part, from the interplay between V1a and V1b receptors.
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Acknowledgments |
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Received April 23, 1998.
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