In Situ Hybridization Analysis of
Arginine Vasopressin Gene
Transcription Using Intron-Specific
Probes
James P. Herman, Martin K.-H. Schafer, Stanley J. Watson, and
Thomas G. Sherman
Mental Health Research Institute (J.P.H., M.K.-H.S., S.J.W.)
University of Michigan
Ann Arbor, Michigan 48109-0720
Department of Behavioral Neuroscience (T.G.S.)
University of Pittsburgh
Pittsburgh, Pennsylvania 15260
In situ hybridization histochemistry with a probe
directed against an intron sequence of the rat arginine vasopressin (AVP) gene was used to demonstrate localization and regulation of AVP heteronuclear RNA in discrete brain regions. Hybridization
with an AVP intron I (AVPinl) probe revealed specific
hybridization confined to cell nuclei of paraventricular nucleus, supraoptic nucleus (SON), and suprachiasmatic nucleus neurons of the rat hypothalamus.
Grain counts revealed that the signal generated by
the AVPinl probe represented 1.9% of that derived
from an AVP exon C probe (AVPexC) in the SON.
Interestingly, in the suprachiasmatic nucleus the
proportion of AVPinl to AVP exon C ratio was much
higher (12%), suggesting either increased transcription of the AVP gene or changes in posttranscriptional RNA processing. Regulatory experiments revealed that 2.6-fold increases in AVPinl signal could
be visualized in the SON as little as 30 min after an
acute salt load, a period during which no significant
change in cytoplasmic AVP mRNA could be observed. In response to chronic salt loading, both
AVP heteronuclear RNA and AVP mRNA were upregulated. These data compared favorably with transcription rate values determined by nuclear run-on
assay, suggesting that intronic in situ hybridization
affords a relatively reliable method for assessment
of rapid changes in gene transcription in individual
central nervous system neurons. (Molecular Endocrinology 5: 1447-1456, 1991)
merous advances in neuronal peptide mRNA localization and regulation in brain. Among the best characterized model systems for such study is the vasopressinergic magnocellular hypothalamo-neurohypophysial
system, due primarily to the well characterized functional properties of this neuronal population (fluid and
electrolyte balance) and to the abundance of both arginine vasopressin (AVP) (1) and its mRNA (2, 3) within
individual vasopressin neurons. The cell bodies of the
hypothalamo-neurohypophysial system are known to
reside in the hypothalamic supraoptic nucleus (SON)
and magnocellular divisions of the paraventricular nucleus (PVN) and to project to the posterior pituitary,
where their secretory products gain access to the general circulatory system. The effects of appropriate physiological stimulation on peptide secretion (4-7) and on
mRNA levels (2, 3, 8-14) within this neural circuit are
well characterized, rendering this neuronal system ideal
for study of the effects of stimulatory events on numerous parameters associated with cellular function.
In situ hybridization histochemical detection of cellular
mRNA levels allows assessment of localization and
regulation of gene expression in individual neurons.
Semiquantitative in situ analysis is particularly well
suited to studies of mRNA regulation associated with
long term changes in cellular biosynthetic adaptation,
such as with chronic physiological challenge (2,15-18)
and lesion-associated regulatory changes (19). However, the application of this technique to the study of
short term gene regulation is problematic, in that numerous events may intervene between actual gene
transcription and detection of mRNA changes. First,
changes in mRNA levels occur relative to a sizeable
preexisting pool of mRNA in the cell body, rendering
detection of stimulus-induced changes dependent on
the signal-to-noise ratio offered by the technique employed. Second, a change in cellular mRNA content is
the end result of a chain of events associated with gene
activation, including gene transcription, splicing of in-
INTRODUCTION
Application of in situ hybridization methodology to the
study of neuropeptide gene expression has led to nu0888-8809/91 /1447-1456$03.00/0
Molecular Endocrinology
Copyright © 1991 by The Endocrine Society
1447
MOL ENDO-1991
1448
tronic sequences, polyadenylation, etc., and therefore
detection of mRNA changes can occur only after the
time required to generate new mRNA (synthesis of new
transcripts, posttranscriptional RNA processing, and
translocation of mRNA) has elapsed. Third, cytoplasmic
mRNA is subject to regulation at the level of degradation [e.g. poly(A) tail length (9, 20-23)], rendering the
meaning of stimulus-induced changes in mRNA content
difficult to interpret. Thus, acute changes in mRNA
levels represent only a loose indication of the temporal
relationship between a given stimulus and gene transcription.
In an effort to more reliably assess rapid stimulusinduced changes in gene expression in individual central
nervous system neurons, we have employed cRNA
probes complementary to intronic sequences of the
AVP gene in our regulatory studies. This method affords
the ability to assess AVP gene transcription under basal
and stimulated conditions. Previous studies have demonstrated the ability to use intronic in situ hybridization
to assess POMC gene expression in the pituitary and
brain (using cRNA probes) (24, 25) and AVP gene
expression in hypothalamic neurons (using oligonucleotide probes) (16). In the present studies, we have
employed the intronic in situ hybridization methodology
to assess hypothalamic AVP gene expression, focusing
on rapid changes associated with appropriate stimulation of magnocellular AVP neurons and comparison of
the effects of acute stimuli with those associated with
chronic activation of AVP neurons. Stimulus-induced
changes in AVP gene expression are compared with
mRNA levels at equivalent time points to allow comparison of heteronuclear RNA (hnRNA) vs. mRNA measures as (relative) indices of transcriptional activation.
The in situ hybridization results are qualitatively validated as relative measures of AVP gene transcription
by comparison with nuclear transcription run-on studies.
RESULTS
Design of the Rat Intron I and Exon C Probes
The derivation of the intron and exon probes used in
the present study are detailed in Fig. 1. A 735-basepair
(bp) PvuW fragment localized entirely within intron I of
the rat vasopressin gene (AVPinl) was subcloned into
the HincW site of pGEM3 in an inverse orientation such
that transcription by SP6 RNA polymerase yields a
cRNA. Southern analysis of rat genomic DNA using a
32
P-labeled AVPinl fragment obtained a qualitatively
identical pattern to that obtained using an AVP genespecific exon C probe: an 8.2-kilobase (kb) EcoRI band,
a 3.8-kb H/ndlll band, a 9.4-kb SamHI band, and a 1.6kb Pst\ band (26); the AVP exon C probe identified a
different Pst\ band at 1.8 kb due to a Pst\ site within
exon B (data not shown). The Southern analysis confirmed the specificity of the AVPinl probe for AVP
genomic transcripts, discounting the possibility of its
Vol5No. 10
recognizing repetitive sequences. The AVP exon C
probe was a 225-bp sequence encoding exon C and a
portion of the 3' untranslated region of the rat AVP
mRNA (AVPexC), derived from a rat cDNA clone (11).
The AVPexC fragment does not share significant homology with rat prooxytocin mRNA (11, 26).
Localization of AVP Intronic RNA within
Hypothalamic Nuclei
Localization of AVPinl sequences to magnocellular AVP
neurons is shown in Figs. 2 and 3. Note the dense,
punctuate appearance of the AVPinl signal in Fig. 2A,
in contrast to the exon C signal observed in Fig. 2D,
consistent with a restriction of the AVPinl signal to the
cell nucleus. Sense-strand controls and sections preincubated with RNase A are devoid of positive signal
(Fig. 2, B and C), demonstrating specificity of the in situ
hybridization probe employed. Figure 3 presents a
higher-power view of single SON cells expressing
AVPinl and AVPexC. Comparison of the cellular distribution of the two signals emphasizes the nuclear localization of the intronic signal, as can be appreciated by
the dense, punctate distribution of grains. The exon C
label shows a considerably larger distribution of grains,
consistent with the predominant localization of mRNA
to the cytoplasmic compartment. The indicated cells in
Fig. 3B show examples of cells with characteristic
diminution of grain density over the cell nuclei (as levels
of mRNA in the cytoplasm greatly exceed the amount
of AVP mRNA and hnRNA typically found in the cell
nucleus).
Examination of hypothalamic sections revealed the
presence of AVPinl signal in all known AVP-containing
nuclei in the hypothalamus (Fig. 4). AVPinl signal could
be clearly localized in the posterior magnocellular PVN
(Fig. 4A), ventral SON (Fig. 4B), and the parvocellular
suprachiasmatic nucleus (SCN; Fig. 4C), all regions that
displayed dense hybridization with probes directed
against AVPexC in adjacent sections and that are
known to contain AVP peptide. AVPinl-positive nuclei
were never localized to structures not expressing AVP
mRNA (as demonstrated by hybridization with the
AVPexC probe). Examination of thionin-counterstained
tissue (Fig. 4D) clearly demonstrates the restriction of
AVPinl signal to cell nuclei.
A comparison of intron/exon ratios for determined
brain regions is shown in Table 1. In both the SON and
PVN, the amount of AVPinl signal reflects 1.9% of the
signal generated by AVPexC-positive mRNA, as determined from grain counts corrected for exposure time
and specific activity. In contrast, within the SCN, the
AVPinl:AVPexC ratio was substantially higher than that
of the SON or PVN (-12%). Note that while the relative
abundance of AVP mRNA was substantially greater in
SON vs. SCN neurons, detectable hnRNA levels were
similar across these two nuclei. This observation can
be appreciated in Fig. 5, which illustrates relative intensities of AVPinl and AVPexC signals in sections containing both the SON and SCN. While SON and SCN
1449
Detection and Regulation of AVP hnRNA
I Signal I AVP
RAT NEUROPHYSIN II
CPP
intron I
intron II
Intron I Probe (AVPinI)
Exon C Probe (AVPexC)
Fig. 1. Schematic of the Rat AVP Gene
The regions selected for construction of intron (AVPinI) and exon (AVPexC) probes. The preproAVP mRNA is depicted on top;
the gene structure is illustrated on the bottom, with dashes corresponding to the positions from which introns I and II are spliced
to generate the observed mRNA. Note that the exC probe is complementary to a region of the rat AVP gene which has no
significant sequence identity to the closely related oxytocin gene.
Fig. 2. Localization of AVP hnRNA in the Hypothalamic SON
A, Signal generated using an intron I probe (In1). Note the dense packing of grains in this region, commensurate with nuclear
localization of the In1 signal. B, Section treated with a sense-strand In1 probe. C, Section treated with RNase A before incubation
with the In1 probe. Note a lack of specific signal in both control sections. D, Signal generated using an AVPexC probe (ExC).
Distribution of grains is considerably more widespread than in A, in keeping with the dense cytoplasmic localization of AVP mRNA.
Magnification, x150.
signal intensity appears quite comparable using the
AVPinI probe, it is clear that the SON signal overwhelms
that of the SCN in sections hybridized with the AVPexC
cRNA.
Osmotic Regulation of AVP Intronic RNA
AVPinI signal intensity was reliably increased in the
SON of rats receiving an acute salt load (2 M NaCI, ip)
(one-way analysis of variance, significant effect of treatment, F(9) = 5.56, P < 0.05). Increases of 2.6-fold in
AVPinI autoradiographic signal were clearly visualized
30 min after an acute salt load (Fig. 6A) and remained
elevated 2 h later (time zero is significantly different
from 30 min and 120 min after acute saline treatment,
P < 0.05, Newman-Keuls test). In contrast, the AVPexC
signal was unchanged either 30 min or 2 h after the
Vol5No. 10
MOL ENDO-1991
1450
ExC
Fig. 3. Comparison of the Grain Distributions Generated using AVP Intron and Exon Probes
Note the densely packed, punctuate distribution of grains resulting from hybridization with the AVPinI (In1) probe (A) in the SON,
as opposed to the more widespread scatter of grains in the AVPexC (ExC) case (B). The denser localization of the In1 signal is
consistent with its confinement to the cell nucleus (see also Fig. 4D). The arrows in the ExC illustration delineate cells which show
an apparent decrease in density in the neighborhood of the cell nucleus, in accordance with the high abundance of cytopiasmic
AVP mRNA relative to nuclear AVP mRNA and hnRNA. Magnification, x550.
onset of an acute stimulus to AVP release, suggesting
a considerable lag time between transcriptional initiation
and changes in mRNA pools. A 4-day paradigm of 2%
saline for drinking water, however, resulted in elevations in both AVPinI (3.0-fold) (unpaired t test, F(7) =
4.37, P < 0.05) and AVPexC (1.8-fold) (unpaired t test,
F(6) = 6.85, P < 0.05) signal (Fig. 6B). Chronic stimulation of AVP gene transcription can thus be visualized
at both the hnRNA and mRNA levels.
Determination of AVP Gene Transcription by
Nuclear Run-On Assay
In a separate experiment, nuclear run-on assays were
performed as an alternative measure of transcriptional
activation. We investigated the transcriptional activity
of the AVP gene in pooled nuclei isolated from punch
dissections of the PVN and SON animals receiving 0,
24, or 72 h of 2% saline for drinking water (2, 3). In
Detection and Regulation of AVP hnRNA
1451
SCN
PVN
SCN
D
Fig. 4. Localization of AVPinl Signal in Selected Hypothalamic Nuclei
Positive signal is seen in the posterior magnocellular PVN (A), ventral SON (B), and SCN (C), all regions known to contain AVP
mRNA and peptide. Panel D illustrates the confinement of grains to the nucleus of AVP cells of the SCN in counterstained material
(thionin). Arrows depict examples of cells demonstrating nuclear localization. Magnification, A, B, and C, X110X; D, x375.
Table 1. AVP hnRNA/mRNA: Relative Abundance
Region
AVPexC probe
(grains per cell)
AVPinl probe
(grains per cell)
AVPinl probe
(norm grains per cell)8
AVPinl/exC ratio
(% ExC signal)
SON
SCN
698.8 ± 82.0
90.4 ±17.7
119.7 ±9.9
93.4 ± 7.8
13.5 ±1.2
10.6 ±0.9
1.9
11.8
8
Normalized grains per cell.
Grain counts are corrected for exposure time and specific activity of the respective probes and are normalized to AVPexC
counts.
vitro labeled RNAs recovered from nuclear run-on analysis were hybridized against a combination of the 735bp AVPinl and AVPinll/exC fragments or against control
pGEM3 DNA for background. The results are shown in
Fig. 7. From an average of 7.24 x 106 ± 7.03 x 105
dpm 32P incorporated into trichloroacetic acid-precipitable RNA synthesized by nuclei isolated from control,
24-h, and 72-h salt-loaded rats, an average of 104.5 ±
28.7 dpm, or 51.7 ± 14.2 ppm when corrected for
hybridization efficiency, specifically hybridized to the
immobilized bound AVP gene fragments. Hybridization
efficiency was estimated at 27.9 ± 4.2% by the addition
of 13,237 dpm 3H-labeled AVP mRNA added to the
hybridization reactions. Nonspecific hybridization to linearized pGEM4 was 37.1 ±11.1 dpm.
After 24 h of salt loading, the rate of AVP gene
transcription was increased 3.2-fold (Fig. 7). A similar
rate of transcription was observed after 72 h of salt
loading.
DISCUSSION
These studies indicate that intron probes can be used
to detect and quantitate gene-specific hnRNA within
Vol5No. 10
MOL ENDO-1991
1452
son
sen
Fig. 5. Differential Abundance of AVPinl vs. AVPexC signal in the SON vs. the SCN
Dramatic differences in the relative abundance of AVPexC vs. AVPinl in the SCN and SON nuclei can be appreciated by
comparison of panels A [exon C (ExC) probe] and B [intron I (Inl) probe]. Note that using the ExC probe, hybridization density over
the discrete anatomical structure and density of hybridization per cell is dramatically greater in the SON than the SCN. In contrast,
using the AVPinl probe, both the overall density of hybridization and the density of hybridization per cell are quite similar for the
two nuclei. Magnification, x60.
the central nervous system. Localization of AVPinl signal can be accurately localized to nuclei of AVP-producing cells, in the appropriate subdivisions of anatomical
structures known to contain AVP peptide and mRNA.
Sense-strand and RNase preincubated controls do not
generate a positive signal, demonstrating hybridization
specificity. In addition, counter stained sections demonstrate confinement of intronic hybridization signal to
cell nuclei. The inability of the AVPinl probe to detect
cytoplasmic species verifies that the nucleic acid sequence probed resides entirely within intronic se-
quences of the AVP gene, and that the probe is hybridizing only to AVP hnRNA.
Grain counts indicate that, in spite of the dense
localization over cell nuclei, the relative abundance of
AVPinl signal in magnocellular neurons is about 2% of
that for cytoplasmic AVPexC signal, when signals are
corrected for exposure time (to liquid emulsion) and the
specific activity of the respective cRNA probes. The
greatly reduced signal strength for the AVPinl signal is
consistent with the short-lived nature of primary gene
transcripts in cell nuclei (27-29) and agrees well with
intron/exon ratios calculated for the POMC gene in
1453
Detection and Regulation of AVP hnRNA
A. Acute Salt Loading
300-|
200-
• 0'
n 30'
T
f
*
i
150-
120'
2
&
100-
Hi
50-
0-
i
Control
AVPinI
AVPexC
B. Chronic Salt Loading
T
AVPinI
Control
2% Saline
24 hr
72 hr
Fig. 7. Osmotic Regulation of AVP Transcription Rate as
Determined by Nuclear Run-On
Pairs of male rats were given 2% NaCI for drinking water
for 0, 24, or 72 h. Nuclei was prepared from pooled punch
dissections of PVN and SON from each group and prepared
for transcription labeling and immobilized substrate hybridizations as described in Materials and Methods. An average of
7.24 x 106 ± 7.03 x 105 dpm 32P incorporated into trichloroacetic acid-precipitable RNA (n ~ 9). Hybridization efficiency
was estimated at 27.9 ± 4.2% by the addition of 13,237 dpm
3
H-labeled AVP mRNA added to the hybridization reactions.
Nonspecific hybridization to linearized pGEM3 was 37.1 ±
11.1 dpm. Each bar represents the average ± SEM for three
independent experiments. *, Statistical reliability (P < 0.05).
AVPexC
Fig. 6. Osmotic Regulation of AVPinI Expression
Effects of acute (A) and chronic (B) salt loading on the in
situ hybridization signal intensity generated by AVPinI and
AVPexC probes. All data are presented as percentage of
control. A, Acute treatment with 2 ml 2 M NaCI results in a
rapid induction of AVP hnRNA expression as early as 30 min
post stimulation. In contrast, AVP mRNA levels do not exceed
baseline as much as 2 h after salt loading. *, Statistically
reliable differences from control; P < 0.05, Newman-Keuls
test. B, Chronic salt loading (2% saline to drink for 4 days)
elicits increases in both AVP hnRNA and AVP mRNA levels,
indicating a persistent increase in AVP gene expression after
the prolonged impetus for AVP secretion. *, Statistically reliable
differences from control; P < 0.05, unpaired t test.
anterior pituitary corticotropes and arcuate nucleus
neurons (using nuclease protection assays) (24, 25),
and with an earlier report of Young et. al. (16) using
oligonucleotide AVP intron probes. However, it should
be noted that the low abundance of the AVP intron
signal relative to that generated by the AVPexC probe
does not allow for careful matching of hybridization
conditions, i.e. equivalent exposure times and probe
specific activities. In this regard, the calculation of the
relative signal intensities reported in Table 1 does not
take differential penetration of the two probes (the
AVPinI probe was substantially longer than the AVPexC
cRNA) and any nonlinear relationship between grains
and either exposure time or probe specific activity into
account, and the estimated ratios presented are best
thought of as approximations.
Interestingly, the ratio of AVPinl/AVPexC is greatly
increased in the SCN relative to magnocellular nuclei,
with the AVPinI signal some 12% of that generated by
the exon C probe. The absolute number of grains per
cell nucleus in the SCN is similar to that seen in magnocellular neurons, indicating that the discrepancy in
inl/exC ratio is generated by the smaller cytoplasmic
pool of AVP mRNA in neurons of this nucleus. The
unequal intron/exon ratios in these functionally distinct
neuronal populations suggests two interesting possibilities. First, SCN AVP neurons may be undergoing active
transcription of the AVP gene at the time of death
(morning). This is supported somewhat by studies indicating increased mRNA levels in the morning (30) and
may be connected with the role of the SCN in mediating
circadian rhythmicity. Second, the transcriptional activity estimated by the AVPinI grain counts may reflect a
basal AVP transcription rate similar in both magnocellular neurons (SON and PVN) and parvocellular SCN
cells. The difference between the two may be the
smaller mRNA pool maintained in the considerably
smaller SCN cells, dictated by differential mRNA stability.
The acute salt administration paradigm provides evidence for rapid gene regulation within individual AVP
neurons in vivo, as detected by the intronic in situ
hybridization method. At both 30-min and 2-h time
points after an acute salt load, periods during which
AVP release is rising and falling, respectively (6),
changes in nuclear AVPinI signal can be clearly appreciated. The rapid induction of AVP hnRNA levels (within
30 min after stimulation) is consistent with a stimulus-
Vol5No. 10
MOL ENDO-1991
1454
secretion-transcription mechanism in which membrane
receptors responsible for depolarization-induced secretory activity are also responsible for generating the
intracellular signal transduction cascades necessary for
transcriptional activation. In contrast, changes in mRNA
levels cannot be seen at either time point, consistent
with previous studies showing a lag time of several
hours between osmotic stimulation and changes in
mRNA pools (2). Taken together, these data indicate
that acute increases in cytoplasmic mRNA after an
osmotic stimulus considerably lag behind the actual
stimulatory event. The observed delay between transcriptional activation and changes in cytoplasmic mRNA
levels are probably the product of the time required to
generate and translocate mature mRNA, the amount of
new mRNA necessary to generate a detectable difference in cytoplasmic mRNA pools, and possibly differential mRNA stability related to factors influencing
mRNA half-life and poly(A)-adenylation (9, 20, 23).
In the case of chronic salt loading, both AVPinl and
AVPexC signals are elevated in the SON. These observations are consistent with an ongoing, prolonged activation of AVP mRNA and peptide synthesis in AVP
neurons, which have been extensively characterized (2,
3, 8-14). Here both the intronic and exonic detection
methods reliably show activation in AVP cells. With
prolonged stimulation, it is apparent that neurons maintain increased AVP gene transcriptional activity, accumulating a greater pool of AVP mRNA that, in effect,
increases their biosynthetic capacity in response to the
increased secretory demand. That this increased intron
signal indeed reflects transcriptional activation is substantiated by the results of comparable nuclear run-on
assay experiments, presented in Fig. 7, demonstrating
changes of similar magnitude after 1-3 days of salt
loading. The general agreement across techniques also
has been demonstrated for POMC expression in pituitary (comparing RNase protection using intron-exon
probes and transcription run-on; 24), and is not, therefore, a fortuitous finding unique to magnocellular AVP
expression.
These studies demonstrate that intronic in situ hybridization histochemistry can be used to estimate
steady state levels of gene-specific hnRNA in individual
neurons, which due to its short half-life can be used to
estimate actual rates of transcription. However, it is
important to realize that the temporal relationships between transcription and splicing events impact critically
on the accuracy of this method in estimating gene
transcription rates. For instance, it is known that introns
are spliced out at different rates, which are intron and
gene specific; therefore, partially spliced transcripts
(processing intermediates) may be present after activation of any multiple-intron gene (c.f. Ref. 32). If the
sequence probed is part of such a processing intermediate, some pool of this species will accumulate and
thus provide an overestimate of absolute transcription
rate. Thus, when processing intermediates persist
within the nucleus, both the primary transcript and a
transcript which is partially spliced (i.e. in the case of
the AVP gene, has one of the two introns removed) will
be detected by the intronic in situ hybridization method.
In addition, while it is generally considered that primary
transcripts or processing intermediates are short-lived,
the exact period of existence of this species for a given
gene is undetermined. It is presently unclear whether
introns themselves and/or the gene transcripts have
measurable and persistent pools within the cell nucleus.
Therefore, given the large number of unknown variables
involved in determination of gene transcription rate,
intronic in situ hybridization analysis and, for that matter, transcription run-on assays are best thought of as
relative measures of transcription rate, suitable for comparison of one or more experimental conditions.
In summary, intronic in situ hybridization holds considerable promise as a method for assessing gene
activation in anatomically defined neurons. This technique offers values comparable to those determined by
nuclear run-on assay and offers the advantages of
cellular localization, vital when one wishes to resolve
transcriptional activation within complex and heterogeneous regions of the nervous system.
MATERIALS AND METHODS
Acute and Chronic Osmotic Stimulation of Rats
Male Sprague-Dawley rats (Charles River, Wilmington, MA),
weighed between 250-300 g at the time of experimentation.
Animals were housed in a constant temperature and humidity
vivarium quarters on a 12 h light-12 h dark cycle.
For acute salt-loading studies, rats (n = 4 per group) were
injected with 2 ml 2 M NaCI and killed by rapid decapitation 0,
30, or 120 min after injection. Chronic salt loading was accomplished by supplying 2% saline as the sole source of drinking
water. Salt-loaded (n = 4) and control (n = 5) rats were killed
by rapid decapitation 4 days after initiation of the salt-loading
protocol. After decapitation, brains were rapidly removed and
frozen in isopentane cooled to - 5 0 C on dry ice. Brains were
stored at - 7 0 C until processing. For estimates of mRNA/
hnRNA ratios, two normal rats were rapidly decapitated and
tissue harvested as above.
In Situ Hybridization Histochemistry
Frozen rat brains were sectioned on a Bright-Hacker microtome (Fairfield, NJ). Frozen 10-Mm sections were taken
through the region of the hypothalamus. All sections were
thaw-mounted onto poly-L-lysine (Sigma, St. Louis, MO)
coated slides and stored at - 7 0 C until processing.
In situ hybridization was performed on sections sampled
through the regions of the SON, PVN, and SCN. Sections
were removed from the - 7 0 C freezer, fixed for 30 min in 4%
paraformaldehyde, rinsed three times in 2x SSC (1 x SSC =
0.15 M NaCI, 0.015 M Na citrate, pH 7.0), and deproteinated
with 0.1 Mg/ml proteinase K for 15 min at 37 C. After deproteination, slides were washed for 1 min in distilled H2O, 1 min
in 0.1 M triethanolamine, and 10 min in 0.1 M triethanolamine
containing 0.25% acetic anhydride. The latter acetylation steps
reduce electrostatic binding of cRNA probe to tissue sections.
Deproteinated, acetylated sections were rinsed in distilled H2O
and dehydrated through graded alcohols.
Antisense 35S-labeled cRNA probes for proAVP exon C
(H/ndlll-linearized pGEM4-AVPexCc) and proAVP intron I
(EcoRI-linearized pGEM3-AVPglnlc) were produced using the
SP6 transcription system. Plasmids containing subcloned
Detection and Regulation of AVP hnRNA
cDNAs or intron fragments were linearized with the appropriate
5'-overhang-producing restriction enzyme to yield probes of
desired length and G:C composition. The labeling reaction
mixture contained 1 fig linearized plasmid, 1 x SP6 transcription buffer (Bethesda Research Labs, Gaithersburg, MD), 250
AtCi a-[35S]UTP (>1000 Ci/mmol, dried; Amersham, Arlington
Heights, IL), 150 nM ATP, 150 nM CTP, 150 »M GTP, 12.5 mM
dithiothreitol, 3.0 U/^l RNAsin (Promega, Madison, Wl), and
0.5 U/MI SP6 RNA polymerase (Promega). The reaction was
incubated for 90 min at 37 C, and the labeled probe separated
from free nucleotide over a Sephadex G50-50 column equilibrated in 0.1 M Tris-HCI, pH 7.5,12.5 mM EDTA, 0.15 M NaCI,
0.2% sodium dodecyl sulfate, and 10 mM dithiothreitol. The
AVPexC probe was a 225-bp cRNA coding for the C-terminal
region of the proAVP molecule, which bears no significant
homology with prooxytocin. The AVPinl probe was a 735-bp
cRNA derived from a PvuW fragment from rat genomic clone
AVPgH3. 35S-Labeled a-UTP was added to the transcription
reaction in amounts calculated to yield specific activities estimated at 5.12 x 10" Ci/mmol for the AVPexC probe and 1.63
x 105 Ci/mmol for the AVPinl probe.
35
S-Labeled cRNAs were diluted in hybridization buffer (75%
formamide, 10% dextran sulfate, 3x SSC, 50 mM sodium
phosphate buffer, pH 7.4, 1x Denhardt's, 0.1 mg/ml yeast
tRNA, and 0.1 mg/ml sheared salmon sperm DNA) to yield
1,000,000 dpm/30 fi\. Aliquots of 30 n\ were applied to each
section, the sections were coverslipped, and the coverslips
sealed with rubber cement. Slides were incubated at 55 C in
sealed plastic boxes containing moistened foam. After an
overnight hybridization, the coverslips were removed, the
slides rinsed in 2x SSC, and immersed in fresh 2x SSC for
20 min. The tissue was treated with RNase A (200 M9/m') at
37 C for 30 min to degrade any remaining single-stranded
cRNA. Sections were then washed successively in 2x, 1x,
and 0.2x SSC for 10 min each, followed by a 60-min wash in
0.2x SSC at 65 C. Sections were dehydrated through alcohols, exposed to Kodak XAR x-ray film (Eastman Kodak Co.,
Rochester, NY), and emulsion-dipped in Kodak NTB2 nuclear
emulsion. Emulsion-dipped sections and standards were exposed for 10 days (AVPexC) or 21 days (AVPinl); batch
development was based on the signal strength and signal-tonoise ratio of test slides developed at regular intervals.
Semiquantitative Analysis of In Situ Hybridization
Autoradiographs
Semiquantitative analysis of in situ hybridization autoradiographs were conducted using Macintosh-based Image software (courtesy of Dr. Wayne Rasband, NIH, Bethesda, MD).
Sections from experimental and control animals were matched
for rostrocaudal level. For determination of changes in AVP
mRNA and hnRNA after acute or chronic salt loading, sections
from salt-loaded and control rats were exposed to x-ray film
for periods of time yielding appropriate signal intensities (i.e.
within the linear range of x-ray film; AVPexC, 24 h; AVPinl, 72
h). The SON, SCN, and PVN regions were digitized and
subjected to densitometric analysis, yielding measures of integrated optical density (area of nucleus x average optical
density). Data were expressed as percentage of the appropriate control group.
Grain counting was conducted on 10 SON and 10 SCN
sections from each of two normal animals. To limit effects of
intersection variability, grain counts were determined over
SON and SCN neurons within the same tissue section. The
number of grains per cell was determined using Loats Associates (Westminster, MD) In Situ/Gram grain-counting software. All sections were hybridized with AVPexC or AVPinl
probes from the same labeling reactions; the two probes were
synthesized in parallel using identical reaction conditions and
transcription times. The grain counts derived from sections
hybridized with the AVPexC and AVPinl probes are presented
in Table 1 and represent the number of grains per cell minus
the number of grains generated over an equivalent (neighbor-
1455
ing) area free of specific hybridization. For accurate comparison across the two probes, the AVPinl data was normalized
with respect to the AVPexC counts. The normalization procedure involved correcting the raw counts by multiplication of
the inl data by a constant expressing the differences in probe
specific activity [41 (exC) vs. 130 (inl) uracil residues per probe]
and exposure time [10 (exC) vs. 21 (inl) days].
Nuclear Run-On Transcription Assay
After salt loading, rats were decapitated and their brains
removed. Using a rat brain block (Zivic-Miller, Allison Park,
PA), 1.0-mm coronal sections were wet dissected through the
hypothalamus using paired razor blades. The PVN and SON
regions were bilaterally punch dissected using a 1.0-mm diameter cannula. Nuclei were isolated from pooled PVN and
SON punches from two animals. The isolation of nuclei, in vitro
transcription, and the purification of labeled hnRNA was performed as described by Blum (31). The DNA substrates were
vacuo-blotted onto Nytran membranes (Schleicher & Schuell,
Keene, NH) using a Hybri-Slot manifold (Bethesda Research
Labs). For AVP transcript determinations, each immobilized
slot contained 0.5 ng of a 735-bp PvuW intron I fragment (from
pGEM3-sdAVPglnl) and 0.5 »g of a 635-bp Pst\-Hind\\\ fragment spanning AVPinll and exC (from pGEM3-sdAVPg3'HPc).
Background hybridization was determined by blotting 1.0 ^g
EcoRI-linearized pGEM3.
Hybridizations were conducted in a final vol of 100 /xl (31)
in a sealed bag for 72 h at 50 C. The membranes were washed
to a stringency of 0.1 x SSC containing 0.1% sodium dodecyl
sulfate at 50 C. Individual bands were counted in 5.0 ml
EcoLite(+) scintillation fluid (ICN Biomedicals, Irvine, CA).
Experimental Animals
All animal housing and protocols were performed according to
the guidelines set in the NIH Guide for the Care and Use of
Laboratory Animals. All procedures were approved by the
University Committee on Use and Care of Animals at the
University of Michigan.
Acknowledgments
The authors would like to thank Dr. Robert Thompson for
helpful advice and insight and Sharon Burke and James Stewart for invaluable technical assistance.
Received June 7, 1991. Revision received July 19, 1991.
Accepted July 24,1991.
Address requests for reprints to: Dr. James P. Herman,
Mental Health Research Institute, 205 Zina Pitcher Place,
University of Michigan, Ann Arbor, Michigan 48109-0720.
This research was supported by NIH Grants NS-08267 (to
J.P.H.), BNS-9021307 (to T.G.S.), DA-02265 (to S.J.W.), and
MH-42251 (to S.J.W.).
REFERENCES
1. Sachs H 1963 Studies on the intracellular distribution of
vasopressin. J Neurochem 10:289-297
2. Sherman TG, McKelvy JF, Watson SJ 1986 Vasopressin
mRNA regulation in individual hypothalamic nuclei: a
Northern and in situ hybridization analysis. J Neurosci
6:1685-1694
3. Sherman TG, Day R, Civelli O, Douglass J, Herbert E, Akil
H, Watson SJ 1988 Regulation of hypothalamic magnocellular neuropeptides and their mRNAs in the Brattleboro
Vol5No. 10
MOL ENDO-1991
1456
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
rat: coordinate responses to further osmotic challenge. J
Neurosci 8:3785-3796
McKinley MJ, Denton DA, Weisinger RS 1978 Sensors
for antidiuresis and thirst: osmoreceptors or CSF sodium
detectors? Brain Res 141:89-103
Vernay EB 1947 The antidiuretic hormone and the factors
which determine its release. Proc R Soc Lond [Biol]
135:25-106
Strieker EM, Verbalis JG 1986 Interaction of osmotic and
volume stimuli in regulation of neurohypophyseal secretion in rats. Am J Physiol 250:R267-R275
Strieker EM, McCann MJ, Flanagan LM, Verbalis JG 1988
Neurohypophyseal secretion and gastric function: biological correlates of nausea. In: Takagi H, Oomura Y, Ito M,
Otsuka M (eds) Biowaming Systems in the Brain. University of Tokyo Press, Tokyo, pp 295-307
Burbach JPH, De Hoop MJ, Schmale H, Richter D, De
Kloet ER, Ten Haaf JA, De Wied D 1984 Differential
responses to osmotic stress of vasopressin-neurophysin
mRNA in hypothalamic nuclei. Neuroendocrinology
39:582-584
Carter DA, Murphy D 1991 Rapid changes in poly(A) tail
length of vasopressin and oxytocin mRNAs form a common early component of neurohypophyseal peptide gene
activation following physiological stimulation. Neuroendocrinology 53:1-6
Majzoub JA, Rich A, van Boom J, Habener JF 1983
Vasopressin and oxytocin mRNA regulation in the rat
assessed by hybridization with synthetic oligonucleotides.
J Biol Chem 258:14061-14064
Sherman TG, Watson SJ 1988 Differential expression of
vasopressin alleles in the Brattleboro heterozygote. J
Neurosci 8:3797-3811
Sherman TG, Robinson AG, Watson SJ 1988 Down regulation of vasopressin and oxytocin mRNAs: decay profile
differences between hyponatremia and rehydration. Proceedings of the Society for Neuroscience 14:16 (Abstract)
Zingg HH, Lefebvre D, Almazan G 1986 Regulation of
vasopressin gene expression in rat hypothalamic neurons.
J Biol Chem 261:12956-12959
Zingg HH, Lefebvre DL 1988 Oxytocin and vasopressin
gene expression during gestation and lactation. Mol Brain
Res 4:1-6
Sherman TG, Akil H, Watson SJ 1985 Vasopressin mRNA
expression: a Northern and in situ hybridization analysis.
In: Schrier RW (ed) Vasopressin. Raven Press, New York,
pp 475-483
Young III WS, Mezey E, Siegel RE 1986 Vasopressin and
oxytocin mRNAs in adrenalectomized and Brattleboro
rats: analysis by quantitative in situ hybridization histochemistry. Brain Res 387:231-241
Watson SJ, Sherman TG, Schafer MKH, Herman JP, Akil
H 1988 Regulation of mRNA in peptidergic systems:
quantitative and in situ studies. In: McKerns KW, Cretien
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
M (eds) Molecular Biology of Brain and Endocrine Peptidergic Systems. Plenum Press, New York, pp 225-241
Watson SJ, Sherman TG, Burke S, Lewis ME, Akil H 1985
In situ hybridization: localization of mRNA in endocrine
and nervous tissue. Society for Neuroscience Short
Course, Syllabus 1:73-93
Herman JP, Wiegand SJ, Watson SJ 1990 Regulation of
basal corticotropin-releasing hormone and arginine vasopressin mRNA expression in the paraventricular nucleus:
effects of selective hypothalamic deafferentations. Endocrinology 127:2408-2417
Carrazana EJ, Pasieka KB, Majzoub JA 1988 The vasopressin mRNA poly(A) tract is unusually long and increases during stimulation of vasopressin gene expression in vivo. Mol Cell Biol 8:2267-2274
Cockrane AW, Deeley RG 1988 Estrogen-dependent activation of the avian very low density apolipoprotein II and
vitellogenin genes: transient alterations in mRNA polyadenylation and stability early during induction. J Mol Biol
203:555-567
Paek I, Axel R 1987 Glucocorticoids enhance stability of
human growth hormone mRNA. Mol Cell Biochem
7:1496-1507
Zingg HH, Lefebvre DL, Almazan G 1988 Regulation of
poly(A) tail size of vasopressin mRNA. J Biol Chem
263:11041-11043
Fremeau Jr RT, Lundblad JR, Pritchett DB, Wilcox JN,
Roberts JL1986 Regulation of pro-opiomelanocortin gene
transcription in individual cell nuclei. Science 234:12651269
Fremeau Jr RT, Autelitano DJ, Blum M, Wilcox J, Roberts
JL 1989 Intervening sequence-specific in situ hybridization: detection of proopiomelanocortin gene primary transcript in individual neurons. Mol Brain Res 6:197-201
Ivell R, Richter D 1984 Structure and comparison of the
oxytocin and vasopressin genes from rat. Proc Natl Acad
Sci USA 81:2006-2010
Darnell Jr JE 1983 The processing of RNA. Sci Am
249(4):90-100
Lewin B 1980 Heterogeneous nuclear RNA. In: Lewin B
(ed) Gene Expression 2. John Wiley & Sons, New York,
pp 728-864
Perry RP, Bard E, Hames BD, Kelly DE, Schibler U 1976
The relationship between hnRNA and mRNA. Prog Nucleic Acid Res Mol Biol 19:275-292
Uhl GR, Reppert SM 1986 Suprachiasmatic nucleus vasopressin messenger RNA: circadian variation in normal
and Brattleboro rats. Science 232:390-393
Blum M 1989 Regulation of neuroendocrine peptide gene
expression. Methods Enzymol 168:618-633
Levin N, Blum M, Roberts JL 1989 Modulation of basal
and corticotropin-releasing factor-stimulated proopiomelanocortin gene expression by vasopressin in rat anterior
pituitary. Endocrinology 125:2957-2966