L .M. Donahue et al. Brain Research 887 2000 335 –343
337
mm serial coronal sections. Sections were de-paraffinized, expression of the RH-I SkM2 cardiac sodium channel
dehydrated through a series of ethanol washes, washed in gene in P0 and adult rat brain Fig. 1. When qualitatively
PBS, subjected to Proteinase K 20 mg ml digestion, and normalized to levels of cytoplasmic actin mRNA expres-
pre-hybridized for 2–4 h at room temperature [32,38]. sion Fig. 1B, RH-I SkM2 mRNA was detected at
Sections were hybridized at 508C for 16–20 h with 2 ng of roughly equal levels in both P0 and adult rat brain in three
35
either the S-labeled RH-1 SkM2 sense or antisense RNA
separate experiments representative example shown in probe from the plasmid SK- 15-2-1 [33]; specific activity
Fig. 1A. As expected, RH-I SkM2 mRNA was detected of the probes ranged from 12 to 23 mCi mg. The sense
in the rat heart RNA positive control, but not in the probe was generated by linearizing the SK- 15-2-1 plas-
negative control, yeast RNA. mid with SalI followed by transcription with T3 poly-
merase. The antisense probe was generated by linearizing the plasmid with SacI followed by transcription with T7
polymerase. After hybridization, the sections were washed for 30 min in 50 formamide, 13 SSC 0.15 M NaCl
0.015 M Na citrate, 10 mM DTT at 508C, followed by a 30-min wash in 0.53 SSC at room temperature. The
sections were then treated for 30 min with 20 mg ml RNase A at room temperature, washed for 2 h in 0.13
SSC at 758C, and dehydrated through a series of ethanol washes. Sections were dipped in NTB2 emulsion Eastman
Kodak Company, Rochester, NY, exposed for 3 weeks at 48C, developed, and counterstained with Harris modified
hematoxylin [37,44]. The sections were examined under both brightfield and darkfield optics on an Olympus BX-60
microscope. Identification of rat P0 brain regions was based on Paxinos et al. [41].
2.4. RT-PCR amplification The GeneAmp RNA PCR Core Kit Perkin Elmer
Cetus, Norwalk, CT was used to reverse transcribe and amplify 500 ng of DNase-treated human heart and brain
total RNAs, as well as 200–500 ng of DNase-treated HeLa
1
poly A RNA. Here, 100 pmol of each primer were used
in 50 ml reactions. The primers used were from the domain I-II linker region of hH1 SCN5A [55] and were used to
amplify a 410 base pair bp fragment. The forward primer was 59-CAGGACTTCTATGAAGCCACG-39, which ex-
tends from nucleotides 1698 to 1718, and the reverse primer
was 59-AAGCCATCTACACACGGAGC-39,
which extends from nucleotides 2089 to 2108 [55]. Ampli- fication conditions were 958C for 5 min, 30 cycles of 948C
for 1 min, 628C for 1.5 min, and 728C for 1 min, followed by an incubation at 728C for 5 min. The PCR products
were separated by electrophoresis on 2 agarose gels.
Fig. 1. RNase protection assays of RH-I SkM2 and cytoplasmic actin. Lane designations: M, markers; P, probe; Y, yeast; Hrt, adult rat heart; Ad,
adult; P0, postnatal day 0; Wb, whole brain. A RH-I SkM2 RPA. Fifty
3. Results
mg of total RNA from P0 and adult rat brain were compared. Five mg of total RNA from adult rat heart was used as a positive control. Five mg of
total RNA from yeast was used as a negative control. The predicted size
3.1. The RH-I SkM2 rat cardiac sodium channel mRNA
of the protected fragment was 358 nt. B Cytoplasmic actin RPA. Five
is expressed in P 0 and adult rat brain
mg of each of the RNAs were compared. The predicted size of the protected fragment was 126 nt. In both A and B, the asterisk marks
RNase protection assays were performed to examine
the expected size of the protected fragment.
338 L
3.2. Expression of the RH-I SkM2 rat cardiac sodium tions with the sense probe did not reveal any detectable
channel mRNA is localized to limbic regions of the P expression in brain, as expected Figs. 2C, 2F, 3C, 4D.
rat brain In the forebrain of the P0 rat brain, intense RH-I SkM2
gene expression was localized to the developing septal In situ hybridization was performed to examine the
region and the vertical and horizontal limbs of the diagonal anatomic localization of RH-I SkM2 cardiac sodium chan-
band of Broca Fig. 2B. Within this section, signal nel mRNA in the P0 rat brain Figs. 2–4. Both sense and
continued ventrally in a diffuse, intense pattern into the antisense RNA probes were used. In situ hybridizations
developing olfactory tubercule and the piriform cortex with the antisense probe revealed that the expression of
Fig. 2B. Strong, localized gene expression of RH-I mRNA for the RH-I SkM2 cardiac sodium channel gene
SkM2 was observed in the region of the developing medial was localized to three major regions in the P0 rat brain, all
basal amygdala Fig. 2E. Furthermore, expression of RH- of which are part of the limbic system. In situ hybridiza-
I SkM2 was also observed in the developing neocortex at
Fig. 2. In situ hybridization of the RH-I SkM2 rat cardiac sodium channel in the cerebral hemisphere of P0 rats. A–C In situ hybridization of RH-I SkM2 in the developing septal region and the vertical and horizontal limbs of the diagonal band of Broca. A Drawing of the regions shown in B
and C. B Darkfield photomicrograph of a coronal section demonstrating RH-I SkM2 expression in the septal and diagonal band regions. Arrowheads indicate the midline of the brain. C Darkfield photomicrograph of the adjacent serial section to that seen in B hybridized with the sense RH-I SkM2
probe. Bar shown is 375 mm. B and C at higher magnification than A. D–F In situ hybridization of RH-I SkM2 in the developing medial basal amygdala. D Drawing of the region shown in E and F. E Dark-field photomicrographs of a coronal section demonstrating RH-I SkM2 expression in
the amygdala of one hemisphere. F Darkfield photomicrograph of the adjacent serial section to that seen in E hybridized with the sense RH-I SkM2 probe. E and F at higher magnification than D. Bar shown is 150 mm. Abbreviations: S, septal region; DB, diagonal band of Broca; Cx, cortex; Cpu,
caudate putamen; Pir, piriform cortex; Tu, olfactory tubercule; cc, corpus callosum; LV, lateral ventricle; Amg, amygdaloid region; Hi, hippocampus; Th, thalamus; Hy, hypothalamus; 3V, 3rd ventricle.
L .M. Donahue et al. Brain Research 887 2000 335 –343
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Fig. 3. In situ hybridization of the RH-I SkM2 rat cardiac sodium channel in the diencephalon of P0 rats. A–C In situ hybridization of RH-I SkM2 in developing hypothalamic and thalamic nuclei. A Drawing of the regions shown in B and C. B Darkfield photomicrograph of a coronal section
demonstrating RH-I SkM2 expression in the VMH, DM, PH regions and the medial habenular-pretectal complex. Bar shown is 745 mm. Arrowhead indicates the 3rd ventricle of the brain. The drawing A depicts an anterodorsal to ventrocaudal view, while the sections in the darkfield photomicrographs
B, C are oriented ventrocaudal to posterodorsal. C Darkfield photomicrograph of the adjacent serial section to that seen in B hybridized with the sense RH-I SkM2 probe. Abbreviations: Cx, cortex; Th, thalamus; VMH and V, ventral medial hypothalamic area; DM and D, dorsal medial hypothalamic area;
PH and P, posterior hypothalamic area; asterisk , medial habenular–pretectal complex; 3V, 3rd ventricle; LV, lateral ventricle.
low, but detectable levels data not shown. In this case, performed with and without reverse transcriptase to ensure
the signal appeared to be localized over clusters of that the 410 bp hH1 SCN5A product did not arise from
immature neurons. residual genomic DNA contamination of the RNA sam-
In the diencephalon of the P0 rat brain, relatively ples. In reactions performed with the enzyme, expression
moderate levels of RH-I SkM2 expression were localized of the hH1 SCN5A human cardiac sodium channel mRNA
to developing hypothalamic and thalamic nuclei Fig. 3. was detected in both human fetal and adult brain RNA, as
These included three hypothalamic groups corresponding well as in the positive control sample of adult human heart
most closely to the ventral medial, dorsal medial and RNA. Cloning and sequencing of the PCR products
posterior hypothalamic nuclei, as well as the medial confirmed that they were hH1 SCN5A data not shown.
habenular–pretectal complex Fig. 3B. Signal was located No expression of hH1 SCN5A mRNA was detected in
mostly over neurons data not shown. samples lacking reverse transcriptase or in the negative
1
In the brainstem, intense RH-I SkM2 signal was local- control sample of HeLa poly A
RNA. ized to the ventrolateral medulla in a well-circumscribed
area that most closely approximated the lateral paragigan- tocellular nuclei Fig. 4B. Expression was observed in
cells with morphology typical of small, immature neurons
4. Discussion
