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Laboratory of Neurochemistry, Basic Neuroscience Program, National Institute of Neurological Disorders and Stroke (E.G., K.K., H.C., H.G.), In Situ Facility, BNP, NINDS (E.M.), and the Section on Neural Gene Expression, National Institute of Mental Health (W.S.Y.), Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Harold Gainer, Ph.D., Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 36, Room 4D20, Bethesda, Maryland 20892. E-mail: hgatnih{at}codon.nih.gov
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1A-D, 2, and ß1–4 are expressed
in virtually all MNCs. However, the 1E subunit gene is not expressed
at detectable levels in these cells. The expression of all of the
ß-subunit genes in each MNC may account for the variations in
physiological and pharmacological properties of the high
voltage-activated channels found in these neurons. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The magnocellular neurosecretory cells (MNCs) in the hypothalamo-neurohypophysial system (HNS) are among the most intensively studied peptidergic neurons in the central nervous system (5, 10, 11, 12). The MNCs are located primarily in bilateral supraoptic (SON) and paraventricular nuclei (PVN) in the hypothalamus and project their axons to the neurohypophysis, where they secrete the neuropeptides oxytocin (OT) and vasopressin (VP) into the bloodstream as neurohormones. The MNCs have been characterized as having only two distinct phenotypes, the OT and VP magnocellular neurons, in which the expression of the OT and VP genes have been reported to be mutually exclusive (13). Recently, the presence of a third MNC phenotype expressing both OT and VP has been reported (14, 15). The OT and VP MNCs also express other neuropeptides [e.g. galanin, cholecystokinin (CCK), CRH, dynorphin, enkephalin, etc.], which can vary depending on specific functional conditions (1, 5, 11, 16).
The MNCs have served as excellent models for the study of calcium-dependent secretion of neuropeptides from axonal terminals as well as from perikaryonal/dendritic domains (11, 17, 18, 19, 20, 21, 22, 23). A central issue in this field has been the elucidation of the calcium channel subtypes that subserve the neuropeptide secretion in these different topographic domains in the MNCs (18). Extensive physiological studies of the diverse calcium channel currents in the MNC somata and axonal terminals have shown that although candidates for all the known subtypes of high voltage-activated (HVA) calcium current can be found in the MNCs, they are distributed heterogeneously in the cell and have different physiological and pharmacological properties depending on their cellular location (18, 22, 23). Not only is there a differential distribution of HVA currents (e.g. somata do not contain Q-type currents, whereas axon terminals do not contain R-type and P-type currents), but the activation thresholds and/or inactivation kinetics of the pharmacologically identified HVA currents (e.g. L- and N-channel subtypes) appear to differ in the two cellular regions.
Given this diversity in the physiological and pharmacological
properties of the MNCs, we attempted to determine which of the - and
ß-subunits of the calcium channel complex that are believed to
underlie these distinct calcium currents (24, 25, 26, 27) are present in the
MNCs. We were particularly concerned with investigating the
representation of the various ß-subunits, as they are known to
profoundly modulate the physiological and pharmacological properties of
the calcium current carried through -subunit subtypes, but
themselves cannot be identified by any known pharmacological agents
(28, 29, 30, 31, 32, 33, 34). Correlating the calcium channel subtypes with the specific
MNC phenotypes was an important objective of these studies, and hence,
we used a single cell RT-PCR approach to assay for the expression of
more than 15 specific genes in each cell.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, inset) and transferred into
4 µl lysis buffer. One to 2 µl of medium were also transferred in
this process.
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PCR amplification
Amplification of VP and OT was performed as follows. A 50-µl
hot start PCR reaction was assembled with a 25-µl lower reaction mix
containing 1 x PCR buffer F (PCR optimization kit,
Invitrogen, San Diego, CA), 125 µM of each
dNTP, and 2.5 pM of each primer (Table 1). A wax bead was added to the reaction
mix, melted at 80 C for 5 min, then allowed to harden at 4 C, forming a
barrier on top of the lower reaction mixture. The upper reaction mix
contained 1 x buffer F, 0.5 µl single cell cDNA template, and
2.5 U Taq polymerase (Perkin-Elmer Corp.,
Norwalk, CT). Amplification was performed as follows: 40 cycles at 94 C
for 45 sec, 62 C for 45 sec, and 72 C for 2 min. After cycling, the
reaction was extended at 72 C for 7 min. Three microliters from the
50-µl reactions were run on a 1.5% agarose gel containing ethidium
bromide and digitally photographed with a Stratagene Eagle
Eye camera (La Jolla, CA).
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and 2), and 2.5 U
Taq Polymerase Gold (Perkin-Elmer Corp.).
Amplification was performed on a PCR Thermal Cycler 9600 as follows: 1)
hold at 94 C for 10 min; 2) 40 cycles at 94 C for 30 sec, 58 C for 30
sec, and 72 C for 2 min; 3) hold at 72 C for 20 min; and then 4) hold
at 4 C. Five microliters from the 20-µl reactions were run on 1.5%
agarose gels containing ethidium bromide and digitally photographed
with a Stratagene Eagle Eye camera.
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Two different ISHH methods were used in this study. Detailed protocols can be found at the website http://intramural.nimh.nih.gov/lcmr/snge/. In the first method, oligonucleotides complimentary to either OT-neurophysin (OT-NP) or VP-neurophysin (VP-NP) messenger RNA (mRNA) were used as 35S-labeled ISHH probes as previously described (35, 36). These probes had specific activities ranging from 12,000-15,000 Ci/mmol. After the ISHH washes, fluorescent IHC was performed using monoclonal antibodies to either OT-NP (PS-36) or VP-NP (PS-41) (37). The slides were incubated in primary antibody for 1 h at room temperature at a 1:100 dilution in 1 x PBS (pH 7.2) with 0.6% Triton X-100 to improve penetration. After two rinses, a rhodamine-conjugated antimouse IgG was applied (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at a 1:500 dilution for 1 h at room temperature. After two rinses, the sections were coated in Kodak NTB3 nuclear track emulsion (Eastman Kodak Co., Rochester, NY) and developed 3 weeks later. The sections were then coverslipped and viewed under a Leitz Dialux20 microscope (Rockleigh, NJ).
In the second method, antisense riboprobes were used for ISHH as described by Le Moine and Young (38). The OT probe was generated from a 476-bp SstI rat genomic fragment containing exon I inserted into the pGEM-3z vector (Promega Corp., Madison, WI). The VP probe was generated from a 229-bp DraI/PstI cDNA fragment targeting the 3'-end of the rat VP mRNA, also in pGEM-3z. These probes were used for both single and double labeling experiments. The specific activity of these riboprobes ranged from 64,000-133,000 Ci/mmol. In double labeling experiments, 106 cpm 35S-labeled probe were mixed with 4 µl digoxigenin-labeled probe/50 µl hybridization solution. After overnight hybridization at 55 C, the sections were washed, processed for the digoxigenin-labeled probe, and apposed to a Kodak AR film overnight. Next, the slides were coated with Ilford K.5D nuclear emulsion and developed 3 months later.
Calcium channel subunit expression analysis
Digitally photographed images of each gel (see Fig. 4A for an
example) are analyzed with an image analysis software program developed
at NIH (NIH Image version 1.60). Fluorescent intensities were measured
as optic density averaged over a fixed area. Optical densities were
taken for the 394-, 512/504-, 1,084-, 1,608-, and 2,168-bp bands of the
1-kb ladder DNA marker (Life Technologies, Inc.).
Fluorescent intensity for each band was expressed as the measured
optical density minus the average of two background measurements.
Fluorescent intensity was then plotted against the known mass of each
molecular marker band, creating a standard curve for each gel (Fig. 4B). The masses of the amplified calcium channel subunit cDNAs were
then determined from this standard curve. The average mass of each
calcium channel subunit cDNA was obtained by adding the mass derived
from one to four separate experiments and then dividing by the total
template volume analyzed (in microliters). The values shown in Table 3 represent the average mass of calcium
channel subunit cDNA divided by the mass of the endogenous
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), were individually collected into a glass micropipette and
immediately transferred into a tube containing lysis buffer. The
phenotype of each cell was determined by RT-PCR using primers specific
for OT and VP mRNAs, and GAPDH primers were used to measure mRNA of
this housekeeping gene.
Ten magnocellular neurons from a 90-g normal female (nonlactating) rat
were analyzed. Four cells (no. 1–4 in Fig. 1A) predominantly express
VP mRNA (40%) and six cells (no. 5–10 in Fig. 1A) predominantly
expressed OT mRNA (60%). As this RT-PCR assay is sensitive (can detect
about 30 copies of OT and VP mRNA; our unpublished
observations), but not quantitative (the maximum bands in Fig. 1A are
clearly saturated), the absolute levels of OT and VP are unknown.
However, these data clearly show that most of the time there was
detectable OT mRNA expression in VP cells (i.e. cells
expressing predominantly VP mRNA) and VP mRNA expression in OT cells
(expressing predominately OT mRNA). The OT and VP cells exhibiting this
level of coexistence does not represent the population of magnocellular
neurons that has previously been reported in IHC and ISHH experiments
to express both OT and VP. That OT- and VP-coexpressing cell population
in normal rats has been described as representing only about 1–3%
(14, 15) of the total magnocellular neuronal population. The
probability of detecting this population of neurons in a sample size of
10 (as in Fig. 1A) would be negligibly small. In contrast, we found
that the coexpression illustrated in Fig. 1A, occurs in the majority of
the magnocellular neuronal population in normal rats.
To identify the OT- and VP-coexpressing population that has been
described previously by IHC and ISHH, we also studied MNCs from a
lactating female SON, where this coexpressing population is reported to
be increased to about 18% of the total population (15). The RT-PCR
data from 16 individual magnocellular neurons from a lactating rat are
shown in Fig. 1B. Seven cells (no. 1, 3, 8, 9, 10, 12, and 14 in Fig. 1B) predominately express VP mRNA (44%), 6 cells (no. 6, 7, 11, 13,
15, and 16 in Fig. 1B) predominately express OT mRNA (37%), and 3
cells (no. 2, 4, and 5) appear to express VP and OT mRNAs at
indistinguishable levels (19%). Thus, this single cell RT-PCR approach
detects three phenotypes, previously identified by IHC and ISHH,
i.e. OT and VP cells that predominantly express one peptide
mRNA species (and small amounts of the opposite mRNAs) as well as an
OT/VP-coexpressing phenotype that appears to express similar levels of
the two peptide mRNA species. In the lactating rats, as in normal
female rats, lower levels of OT mRNA were typically detected in VP
cells and lower levels of VP mRNA were also detected in OT cells (Fig. 1B).
ISHH evidence for the expression of low levels of the alternative
nonapeptide mRNA in the OT and VP phenotypes
The high sensitivity of the RT-PCR procedure revealed for the
first time that most of the magnocellular OT and VP phenotypes also
expressed lower levels of VP and OT mRNAs, respectively (Fig. 1, A
and B). In another series of studies, which used competitive,
quantitative RT-PCR procedures on the single cell mRNAs, we determined
that the difference between the major and minor peptide mRNA species in
the OT and VP magnocellular phenotypes in normal rat SONs was about 2
orders of magnitude (39). In contrast in the OT/VP phenotype the
difference was about 2-fold. Clearly, the latter mRNA coexistence would
be in the detection sensitivity of conventional ISHH using
oligonucleotide probes (14, 15), whereas the predominant (>70%)
coexpression at lower levels was not.
We therefore reasoned that if the RT-PCR data were correct, then by
increasing the sensitivity of the ISHH procedure we should be able to
detect this more general form of coexpression by ISHH. Figure 2 provides evidence in favor of this
view. The expression of OT and VP mRNA in the SON was visualized by two
different double labeling techniques. The first method combines
conventional ISHH using radiolabeled oligonucleotides and IHC with
OT-NP or VP-NP antibodies. The second method combines ISHH using a
highly sensitive radiolabeled antisense RNA probe with a less sensitive
digoxigenin-labeled RNA probe for double label ISHH.
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) were located
mostly in the dorsal portion of the SON, whereas the
VP-NP-immunoreactive cells (green in Fig. 2A) occupied the
ventral region of the SON. Similarly, cells that were labeled by the
VP-NP oligonucleotide were located in the ventral portion of the
nucleus (red in Fig. 2B), whereas OT-NP-immunoreactive cells
occupied the dorsal region of the SON (green in Fig. 2B).
The absence of yellow cells shows that these findings are consistent
with previous conclusions stating that the two populations are distinct
and separate (13).
However, when the more sensitive ribonucleotide-based ISHH
technique was employed, most of the magnocellular neurons in the SON
contained detectable OT mRNA (Fig. 2C), and virtually all of the cells
contained detectable VP mRNA (Fig. 2D). The results of the double label
ISHH, using high sensitivity probes for minor mRNA species, also
confirmed the widespread coexpression of these two peptide mRNA species
in the magnocellular neurons. Many VP cells coexpressed OT mRNA (Fig. 2, E and G), and most OT cells coexpressed VP mRNA (Fig. 2, F and H).
This was difficult to visualize in the brightfield images (Fig. 2, E
and F). However, examination of these cells (rectangles in
Fig. 2, E and F) by epifluorescent light using a polarizing filter
allowed visualization of the grains even on top of the digoxigenin
stain. We also found that the VP neurons in the suprachiasmatic nucleus
did not show coexpression of OT mRNA, even with the higher specific
activity probe and long exposure times (not shown). Thus, this direct
demonstration by ISHH of coexpression of OT and VP mRNA in nearly all
magnocellular neurons of the SON serves as a validation of our results
obtained using RT-PCR phenotype analysis of dissociated, isolated
single cells.
Other coexpressed peptide mRNAs in the magnocellular
neurons
In addition to the major neuropeptide genes, OT and VP,
prominently expressed in the magnocellular neurons, many other peptide
genes are also known to be expressed in these neurons at varying levels
depending on specific experimental conditions (1, 5, 16, 40). From
these studies it is generally agreed that in the SONs of normal rats,
most VP neurons typically contain detectable levels of dynorphin and
galanin, whereas most OT neurons do not. In contrast, a significant
number of OT neurons express CCK and CRH peptides, whereas under normal
conditions very few, if any, VP neurons do. In addition, calbindin, a
calcium-binding cytosolic protein, is more highly represented in OT
neurons than VP neurons in the SON (41, 42).
Figure 3 illustrates RT-PCR analyses of
13 cells from lactating and nonlactating rats that strongly express VP
or OT, and which were examined for their coexpression of the above
peptide mRNAs. The presence of dynorphin, galanin, CRH, CCK, and
calbindin mRNAs in individual OT and VP cells was determined by
gene-specific RT-PCR (Table 1) using 1 µl of 50 µl total single
cell lysate for each reaction. The PCR-amplified product was run on a
1% agarose gel containing ethidium bromide. Results of gene-specific
PCR amplification are shown for identified OT cells (Fig. 3A) and VP
cells (Fig. 3B); the cell numbering is the same as that shown in Fig. 1.
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). However, heterogeneity in this
property was clearly evident, in that one VP neuron (cell 2 in Fig. 3B)
appeared to contain no dynorphin mRNA, and one OT neuron appeared to
exhibit a robust expression of dynorphin mRNA (cell 11 in Fig. 3A). A
similar pattern was observed for galanin mRNA, which is believed to be
predominately expressed in VP neurons vs. OT neurons. This
appears generally to be confirmed by the RT-PCR data in Fig. 3, as five
of six VP neurons exhibited detectable galanin mRNA, whereas in only
two of seven OT neurons could galanin mRNA be detected. Again, there
was heterogeneity, and the most robust galanin expression occurred in
an OT neuron (cell 7 from normal females in Fig. 3A). A similar pattern
was obtained for those peptide mRNAs that are believed to be
predominantly represented in OT cells. Calbindin mRNA was expressed at
easily detectable levels in five of seven OT cells and generally less
intensely in four of six VP cells. CCK mRNA was detected only in the
two OT cells from the lactating rat, and CRH mRNA was detected in one
of the two OT cells from the lactating rat. Thus, although this RT-PCR
analysis of these other coexisting peptide mRNAs confirms the
conclusions already present in the literature based on IHC and ISHH
data, the individuality of each cell’s peptide expression
profile (Fig. 3) is dramatically made evident by this single cell
RT-PCR analysis.
Calcium channel subunit expression profiles in individual,
identified magnocellular neurons
Given the above validation of the single cell RT-PCR approach to
determine peptide mRNA expression profiles and cellular identities in
the magnocellular neuronal population, we then extended this approach
to study the HVA calcium channel subunit mRNAs in these cells. The
expression of various and ß HVA calcium channel subunit mRNAs in
individual, phenotypically identified, magnocellular neurons was
determined using a single cell RT-PCR protocol (see Materials and
Methods). Specific primer pairs for the various HVA neuronal
calcium channel subunits (see Table 2) were used for RT-PCR analysis of
16 individual magnocellular neurons that were isolated from a lactating
rat and had previously been identified as VP-, OT-, or VP/OT-expressing
cells by RT-PCR. All of the primer pairs shown in Table 2 were
pretested using whole rat hippocampal cDNA and were found to be highly
effective in generating robust and appropriately sized PCR
products.
In these experiments, 1–4 µl single cell cDNA template were
typically used for PCR analysis. The PCR products were visualized by
agarose gel electrophoresis in the presence of ethidium bromide as
described previously for the phenotypic and peptide coexpression
analyses. In all cases, we observed a single band migrating at the
expected mol wt as predicted from the primer positions. A
representative example of the raw data obtained from a single cell
using several of the calcium channel subtype-specific primers is shown
in Fig. 4A. For the cell illustrated in
Fig. 4A, the most robust PCR products were from 1A-, 1D-, and
ß2-subunit mRNAs, with lower, but clearly detectable, PCR products
for ß3 and ß4. The 1B-subunit mRNA was not detected (Fig. 4A),
but 1C mRNA was quite abundant in this particular cell (not
illustrated). Estimates of the quantity of PCR products shown in Fig. 4A were performed by comparing their fluorescence intensities to the
mass values of known standards run in the same electrophoretic run, as
shown in Fig. 4B. By converting each cell’s PCR products to such
nanogram DNA values, it was possible to compare these values between
different cells.
Table 3 summarizes the calcium channel mRNA expression profiles found
in the OT, VP, and OT/VP neuronal phenotypes. The data are shown as PCR
product mass for each subunit (nanograms of cDNA; see Fig. 4), divided
by the PCR product mass of the endogenous GAPDH in each cell, and as an
average (mean ± SEM) value for the given phenotype.
As can be seen from these average values in Table 3, virtually all of
the and ß calcium channel subunit mRNAs were found in both the OT
and VP neuronal phenotypes. Only the PCR product from the 1E subunit
mRNA was consistently absent from all of the phenotypes. This was not
due to the inadequacy of the primers used, because amplification of
1E mRNA was highly efficient when this primer pair was used with
cDNAs obtained from rat brain stem, cerebellum, or hippocampus (not
shown). Although all of the calcium channel mRNAs (except for 1E)
were represented in all of the magnocellular phenotypes, there were
significant qualitative and quantitative variations between the
individual cell expression patterns, even within a given phenotype
(e.g. OT vs. VP). There do seem to be some
differential patterns between the phenotypes that are suggested by
comparing the average values in Table 3. The L-channel-associated
subunits, 1C and 1D, were more prominent in the OT vs.
the VP phenotype, and 1C was absent from the OT/VP phenotype and
present at very low levels in the VP phenotype. This absence of 1C
in the OT/VP cells was compensated for by abundant 1D expression in
the OT/VP phenotype. As expected, the 2-subunit mRNA, which was
present in all calcium channel subtypes, was abundantly expressed in
all the phenotypes.
The apparent differences in Table 3 between the ß-subunit mRNAs were
not found to be statistically significant (i.e. P
> 0.05). Only the difference in 1C between the OT and OT/VP
phenotypes was significant (P = 0.04). In summary, the
data in Table 3 show that virtually all of the calcium channel subtype
mRNAs are expressed in each individual magnocellular neuron, that the
levels of expression of any given subunit varies from cell to cell, and
that only subtle, if any, differences in expression exist between the
phenotypes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our studies at the single cell level confirm the existence of an OT/VP-coexisting MNC population in the SON especially during lactation. However, our data also reveal that in control rats, virtually all of the MNCs of the OT phenotype express some VP, and those of the VP phenotype contain OT mRNA. If this coexpression of OT and VP is so widespread in the MNCs, then why was it not previously detected by IHC and ISHH methods? We believe the reason is that in the OT and VP phenotypes the difference in expression level between the major and minor peptides is so great that morphologists are generally reluctant to use conditions that can detect the minor peptide, because these would lead to massive overdevelopment of the major phenotype and, therefore, would obscure the primary cell of interest. Consequently, we tested this idea by performing double label IHC-ISHH in the conventional manner (showing the absence of coexistence) as well as under conditions that could reveal the lower levels of mRNA and the widespread expression predicted by RT-PCR. The results of these ISHH experiments confirm the conclusion of widespread coexpression and suggest that in the OT and VP phenotypes the major peptide mRNA level is much higher than that of the minor peptide. In fact, recent quantitative measurements in our laboratory of the absolute levels of OT and VP mRNAs in single MNCs indicate that the major peptide mRNA is present at 2 orders of magnitude greater levels than the minor peptide mRNA (39).
It remains to be determined if the minor coexpressed peptide mRNA has a physiological role or if its expression leads to detectable biosynthesis of the peptide in the cell. In any case, these results bear on our understanding of how these genes are regulated. A model based on the premise that these cells exclusively express either OT or VP mRNA would predict that there must be a strong genetic switch regulating the expression of these two genes. In contrast, our data show that the minor expressed peptide gene is activated to a significant basal level with the predominant peptide gene being superactivated above this level. It is also interesting to note that expression of the OT and VP genes is quite variable from cell to cell, suggesting that the expression of OT and VP mRNA is determined dynamically. This cell to cell variability is even more pronounced when considering other coexpressed peptides.
Expression of other peptide mRNAs in MNCs
It is well known that MNCs express peptides other than OT and VP
(1, 5, 11, 16, 44, 45, 46), and that these copeptides are selectively
expressed between the two major phenotypes. IHC and ISHH studies have
shown that CRH and CCK are coexpressed in OT cells, and galanin and
dynorphin are coexpressed in VP cells.
We used the above information to produce additional validation of our RT-PCR method’s ability to assess lower levels of gene expression (these copeptides are generally expressed at 1–2 orders of magnitude lower levels than the major OT and VP mRNAs in the MNCs). In general, our results confirm expectations from the literature. CRH and CCK tend to primarily colocalize to a subset of OT cells, and galanin and dynorphin tend to colocalize to VP cells. However, by analyzing the colocalization of all of these genes in individual cells by gene profiling, we find that considerable variation occurs. For example, we find galanin mRNA predominantly in VP cells, as indicated in the literature; however, the highest level of galanin mRNA is in a single OT cell.
Expression of calcium channel subunit mRNAs in the
MNCs
Substantial physiological and pharmacological evidence implicates
multiple voltage-gated calcium channel subtypes in the secretion of OT
and VP (18, 22, 23). In the central nervous system, the various HVA
calcium channel currents are associated with 1 channel subunit
proteins encoded by five distinct subunit genes (1A, 1B, 1C,
1D, and 1E) (24, 25, 26, 27). IHC and ISHH studies of the distribution of
1-subunit mRNA and 1-subunit immunoreactivity in the central
nervous system suggest that most or all of the identified calcium
channel types are expressed in the hypothalamus (17). However, none of
these studies was at cellular resolution in the HNS, and differences in
expression between OT and VP cells were not considered. In addition to
the five distinct 1-subunits discussed above, there are also four
distinct ß-subunits (ß1, ß2, ß3, and ß4) to be considered.
The ß-subunits are of particular interest here, because they are
known to modulate the physiological and pharmacological properties of
the 1-subunits, but they themselves are not selectively sensitive to
any known pharmacological agents (28, 29, 30, 31, 32, 34). In addition, there are
very few, if any, good antibodies available that can distinguish among
the ß-subunits in IHC procedures.
The calcium channel subtypes in the somatic and axonal terminal domains
of the MNCs have been intensively studied, and the roles of these
subtypes in secretion of OT and VP from axonal terminals in the
neurohypophysis have also been evaluated by pharmacological agents (18, 23). Significant differences in the activation thresholds and
inactivation times of the specific calcium channel currents in the MNCs
were found depending on cellular locations where these parameters were
measured (18). For example, N-type channels in axon terminals rapidly
inactivated (
= 100 msec), whereas in somas they inactivated
very slowly ( = 1800 msec), and although pharmacologically
identified L-channels in somas had relatively low activation thresholds
(about -50 mV), in axonal terminals the L-type channels were activated
between -20 to -30 mV. In addition to these variations, there are
unusual pharmacological sensitivities that have been found for some of
the calcium currents (18). Consequently, we performed a gene expression
profile analysis of calcium channel subunit mRNAs.
The data, summarized in Table 3, show that all of the HVA calcium
channel subunit genes, with the exception of 1E, are expressed in
all of the MNC phenotypes, i.e. the OT, VP, and OT/VP
neurons. The absence of 1E mRNA is of some interest, as R-type
channels have been reported as being present in MNC somata.
The data in Table 3 are expressed as nanograms of gene-specific PCR
product cDNA units normalized to the endogenous GAPDH PCR product.
These values can only be compared for a given PCR product
(e.g. 1A, etc.) between different cells (or
phenotypes), but not between PCR products in a given cell
(e.g. 1A vs. 1B, etc.), as the
efficiencies of the primer pairs for each specific molecule are unknown
and could differ significantly. Determination of absolute mRNA content
per cell would require another type of PCR, i.e.
quantitative competitive PCR, which was not performed here. Given this
restriction it is still possible to compare subunit representations
between the MNC phenotypes by focusing on the mean values in Table 3.
In this regard, it is reassuring that the housekeeping gene, GAPDH, had
relatively consistent nanograms of cDNA per cell, independent of
phenotype. Similarly, the 2 calcium channel subunit, which is
present in all subtypes of calcium channels, was not significantly
different between different cells and pheno- types.
Comparing the mean values of the data and the different phenotypes in
Table 3, it can be seen that the 1A- and 1B-subunit mRNAs are
similarly represented in the OT and VP cells. Similarly, the
1C-subunit mRNA levels are not significantly different between the
OT cells and the VP cells. Only in the OT cell (but not the VP cell)
was the 1C-subunit mRNA significantly greater than that in the OT/VP
cells (P = 0.04). The 1D-subunit also appears to be
expressed in all three phenotypes, but possibly at a lower level in the
VP phenotype. Especially notable is that all the ß-subunits were
expressed in the MNCs, but not at statistically significantly different
levels between the MNC phenotypes.
The data in Table 3 also suggest a significant cell to cell variation
in the representation of various subunit mRNAs. All of the cells have
substantial levels of GAPDH and 2 mRNA; however, most of the other
subunit mRNAs are variably expressed from cell to cell. Similar cell to
cell variations were reported in a RT-PCR analysis of calcium channel
1-subunit mRNAs in single motor neurons of the rat facial nucleus
(see Table 2 in Ref. 8). The significance of this cell to cell
variation is unclear, and its clarification will require further RT-PCR
and combined physiological experimentation in the same single
neurons.
In summary, the single cell RT-PCR experiments described in this paper
have shown for the first time that virtually all of the MNCs exhibit OT
and VP coexistence, and that all three identifiable MNC phenotypes
express all 1-subtype calcium channel subunit mRNAs, except for
1E. Most important is the demonstration of a widespread expression
of all four ß-subunits in the MNCs. It is therefore possible that by
varying the combinations of 1A-D and ß1–4 assemblies in the MNCs
and by targeting these different complexes to either soma/dendritic or
axon terminal domains, the variations in physiological and
pharmacological properties of calcium channels found in these cells can
be generated.
Received April 28, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1E calcium channel. J Membr
Biol 160:127–140[CrossRef][Medline]
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 S. S. Gouraud, K. Heesom, S. T. Yao, J. Qiu, J. F. R. Paton, and D. Murphy Dehydration-Induced Proteome Changes in the Rat Hypothalamo-Neurohypophyseal System Endocrinology, July 1, 2007; 148(7): 3041 - 3052. [Abstract] [Full Text] [PDF]
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 W. Zhang, B. Star, W. R. A. K. J. S. Rajapaksha, and T. E. Fisher Dehydration increases L-type Ca2+ current in rat supraoptic neurons J. Physiol., April 1, 2007; 580(1): 181 - 193. [Abstract] [Full Text] [PDF]
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S. Di, R. Malcher-Lopes, V. L. Marcheselli, N. G. Bazan, and J. G. Tasker Rapid Glucocorticoid-Mediated Endocannabinoid Release and Opposing Regulation of Glutamate and {gamma}-Aminobutyric Acid Inputs to Hypothalamic Magnocellular Neurons Endocrinology, October 1, 2005; 146(10): 4292 - 4301. [Abstract] [Full Text] [PDF]
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 G. Gomez-Lira, M. Lamas, H. Romo-Parra, and R. Gutierrez Programmed and Induced Phenotype of the Hippocampal Granule Cells J. Neurosci., July 27, 2005; 25(30): 6939 - 6946. [Abstract] [Full Text] [PDF]
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Y. Ueta, H. Fujihara, R. Serino, G. Dayanithi, H. Ozawa, K.-i. Matsuda, M. Kawata, J. Yamada, S. Ueno, A. Fukuda, et al. Transgenic Expression of Enhanced Green Fluorescent Protein Enables Direct Visualization for Physiological Studies of Vasopressin Neurons and Isolated Nerve Terminals of the Rat Endocrinology, January 1, 2005; 146(1): 406 - 413. [Abstract] [Full Text] [PDF]
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 M. Toledo-Rodriguez, B. Blumenfeld, C. Wu, J. Luo, B. Attali, P. Goodman, and H. Markram Correlation Maps Allow Neuronal Electrical Properties to be Predicted from Single-cell Gene Expression Profiles in Rat Neocortex Cereb Cortex, December 1, 2004; 14(12): 1310 - 1327. [Abstract] [Full Text] [PDF]
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N. Mutsuga, T. Shahar, J. G. Verbalis, M. J. Brownstein, C. C. Xiang, R. F. Bonner, and H. Gainer Selective Gene Expression in Magnocellular Neurons in Rat Supraoptic Nucleus J. Neurosci., August 11, 2004; 24(32): 7174 - 7185. [Abstract] [Full Text] [PDF]
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N. Sudo, Y. Chida, Y. Aiba, J. Sonoda, N. Oyama, X.-N. Yu, C. Kubo, and Y. Koga Postnatal microbial colonization programs the hypothalamic-pituitary-adrenal system for stress response in mice J. Physiol., July 1, 2004; 558(1): 263 - 275. [Abstract] [Full Text] [PDF]
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S. E Wells, D. M Flavell, G. W Bisset, P. A Houston, H. Christian, K. M Fairhall, and I. C A F Robinson Transgenesis and neuroendocrine physiology: a transgenic rat model expressing growth hormone in vasopressin neurones J. Physiol., August 15, 2003; 551(1): 323 - 336. [Abstract] [Full Text] [PDF]
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C. J. Habermann, B. J. O'Brien, H. Wassle, and D. A. Protti AII Amacrine Cells Express L-Type Calcium Channels at Their Output Synapses J. Neurosci., July 30, 2003; 23(17): 6904 - 6913. [Abstract] [Full Text] [PDF]
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S. Di, R. Malcher-Lopes, K. Cs. Halmos, and J. G. Tasker Nongenomic Glucocorticoid Inhibition via Endocannabinoid Release in the Hypothalamus: A Fast Feedback Mechanism J. Neurosci., June 15, 2003; 23(12): 4850 - 4857. [Abstract] [Full Text] [PDF]
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 A. C. Scallet, M. Wofford, J. C. Meredith, W. T. Allaben, and S. A. Ferguson Dietary Exposure to Genistein Increases Vasopressin but Does Not Alter {beta}-Endorphin in the Rat Hypothalamus Toxicol. Sci., April 1, 2003; 72(2): 296 - 300. [Abstract] [Full Text] [PDF]
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J. Han, C. Gnatenco, C. D Sladek, and D. Kim Background and tandem-pore potassium channels in magnocellular neurosecretory cells of the rat supraoptic nucleus J. Physiol., February 1, 2003; 546(3): 625 - 639. [Abstract] [Full Text] [PDF]
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M. Yamashita, E. Glasgow, B.-J. Zhang, K. Kusano, and H. Gainer Identification of Cell-Specific Messenger Ribonucleic Acids in Oxytocinergic and Vasopressinergic Magnocellular Neurons in Rat Supraoptic Nucleus by Single-Cell Differential Hybridization Endocrinology, November 1, 2002; 143(11): 4464 - 4476. [Abstract] [Full Text] [PDF]
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B.-J. Zhang, K. Kusano, P. Zerfas, A. Iacangelo, W. S. Young III, and H. Gainer Targeting of Green Fluorescent Protein to Secretory Granules in Oxytocin Magnocellular Neurons and Its Secretion from Neurohypophysial Nerve Terminals in Transgenic Mice Endocrinology, March 1, 2002; 143(3): 1036 - 1046. [Abstract] [Full Text] [PDF]
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 J. P. H. Burbach, S. M. Luckman, D. Murphy, and H. Gainer Gene Regulation in the Magnocellular Hypothalamo-Neurohypophysial System Physiol Rev, July 1, 2001; 81(3): 1197 - 1267. [Abstract] [Full Text] [PDF]
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 E. Glasgow, T. Murase, B. Zhang, J. G. Verbalis, and H. Gainer Gene expression in the rat supraoptic nucleus induced by chronic hyperosmolality versus hyposmolality Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2000; 279(4): R1239 - R1250. [Abstract] [Full Text] [PDF]
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