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Endocrinology Vol. 143, No. 3 1097-1107
Copyright © 2002 by The Endocrine Society

NEUROENDOCRINOLOGY

Distribution, Neuronal Colocalization, and 17ß-E2 Modulation of Small Conductance Calcium-Activated K+ Channel (SK3) mRNA in the Guinea Pig Brain

Martha A. Bosch, Martin J. Kelly and Oline K. Rønnekleiv

Department of Physiology/Pharmacology, Oregon Health Sciences University, Portland, Oregon 97201; Division of Neuroscience, Oregon Regional Primate Research Center, Beaverton, Oregon 97206

Address all correspondence and requests for reprints to: Dr. Oline K. Rønnekleiv, Department of Physiology/Pharmacology, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97201. E-mail: . ronnekle{at}ohsu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Molecular cloning has revealed the existence of three distinct small conductance (SK1–3) Ca2+-activated K+ channels. Because SK channels underlie the afterhyperpolarization (AHP) that is critical for sculpturing phasic firing in hypothalamic neurons, we investigated the distribution of these channels in the female guinea pig. Both SK1 and SK3 cDNA fragments were cloned using PCR, and ribonuclease protection assay as well as in situ hybridization analysis illustrated that the SK3 channel was the predominant subtype expressed in the guinea pig hypothalamus. Combined in situ hybridization and fluorescence immunocytochemistry revealed that SK3 mRNA was expressed in GnRH, dopamine, and vasopressin neurons, and all of these neurons exhibited an AHP current. Moreover, SK3 mRNA was found in other brain areas, including the septum, bed nucleus, amygdala, thalamus, midbrain, and hippocampus. Using quantitative ribonuclease protection assay, the rank order of SK3 mRNA expression was septum >= midbrain > rostral thalamus >= rostral basal hypothalamus >= caudal thalamus >= preoptic area >> caudal basal hypothalamus >= hippocampus. Moreover, 17ß-E2 treatment, which reduces plasma LH during the negative feedback phase, significantly increased SK3 mRNA levels in the rostral basal hypothalamus (P < 0.05; n = 6). Therefore, these findings suggest that estrogen increases the mRNA expression of SK3 channels, which may represent a mechanism by which estrogen regulates hypothalamic neuronal excitability during negative feedback.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CALCIUM-ACTIVATED potassium channels play a fundamental role in all excitable cells. Calcium entering the cell during the action potential activates K+ channels that cause membrane hyperpolarization, which inhibits cell firing and limits the frequency of repetitive action potentials (spike frequency adaptation). One class of these Ca2+-activated K+ channels, the small conductance Ca2+-activated K+ channels (SK), is responsible for the medium and slow afterhyperpolarization (AHP) following action potential firing in a number of central nervous system (CNS) neurons, including cortical and hippocampal pyramidal neurons, hypothalamic neurosecretory neurons, midbrain dopamine neurons, and motor neurons of the vagal nuclei (1, 2). Recently, three SK channel genes (SK1–3) have been cloned (3). Expression of SK1 mRNA in Xenopus oocytes yielded a Ca 2+-activated K+ current that is relatively insensitive to the bee venom apamin, which is similar to the AHP current in the hippocampal CA1 pyramidal neurons (4). Moreover, in situ hybridization for SK1 mRNA revealed that it is expressed in the CA3 and CA1 hippocampal cell layers in the rodent (3, 5, 6). On the other hand, the SK2 and SK3 channels are both apamin-sensitive channels (3), and their mRNA distributions exhibit overlapping, but also distinct, localizations. For example, both SK2 and SK3 have been described in the hippocampus and in other brain areas such as the thalamus and olfactory regions of the rodent (3, 5). However, based on in situ hybridization analysis, SK3 mRNA appears to be the main subtype expressed in various nuclei of the hypothalamus and in the midbrain substantia nigra pars compacta (3, 6).

Monoamine neurotransmitters inhibit the AHP in CNS neurons, thereby increasing the frequency of action potentials in response to a depolarizing stimulation (7, 8, 9, 10, 11, 12). Recently, we found that activation of both {alpha}1- and ß-adrenergic receptors inhibits the AHP current in hypothalamic neurons and dramatically alters their firing characteristics (13). Moreover, estrogen significantly potentiates the {alpha}1-adrenergic inhibition of the AHP current in preoptic neurons (13).

It is well known that estrogen has multiple actions in the hypothalamus that lead to inhibition and subsequent activation of GnRH neurosecretion (14). At the cellular level, estrogen applied acutely has been found to hyperpolarize neurons directly through activation of inwardly rectifying K+ channels (15, 16, 17). Estrogen also may alter G protein coupling to K+ channels both acutely and after longer-term treatment, resulting in alterations of potassium channel functions (18, 19). However, although previous studies have illustrated that estrogen can activate potassium channels, essentially nothing is known about the effects of estrogen on the mRNA expression of potassium channels in the CNS. We hypothesized that after longer-term estrogen exposure, synthesis of new channels would occur to maintain persistent inhibition of neuronal firing during negative feedback. We chose to look at the expression of SK3 channels, because these channels are the predominant subtype in the hypothalamus (3), and it has been shown that an apamin-sensitive channel is critical for modulating the firing frequency in hypothalamic neurosecretory neurons (20, 21, 22, 23). To investigate the regulation of SK expression, we used a sensitive ribonuclease protection assay to quantify SK3 mRNA in microdissected brain areas and correlated the quantitative measurements with cellular distribution determinations using in situ hybridization and immunocytochemistry. In support of our hypothesis, 17ß-E2 benzoate (EB) up-regulated SK3 mRNA levels in the rostral basal hypothalamus (rBH) at the time of negative, but not positive, feedback of the steroid on the hypothalamus.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal treatment and tissue preparation
All procedures performed using animals were in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by our local committee on animal care and use. Female Topeka guinea pigs (470–660 g) were maintained under a 14-h light, 10-h darkness lighting schedule, with lights on at 0630 h and off at 2030 h. The animals (60–80 d old) were ovariectomized under ketamine/xylazine anesthesia, then after 7 d were injected with 25 µg EB or oil at 0830 or 2030 h and killed 24 or 42 h thereafter, during negative or positive feedback, respectively, according to our established model (19). Trunk blood was collected for measurements of plasma 17ß-E2 by nonequilibrium RIA (24). For this steroid assay, water blanks, percent recovery, and intraassay coefficient of variation were 4.4 pg, 91.4%, and 5.5%, respectively.

Each brain was sliced into 2- to 3-mm coronal blocks using a chilled brain slicer (EM Corp., Chestnut Hill, MA). The preoptic area (POA) block was 3 mm and extended from the rostral border of the organum vasculosum lamina terminalis to immediately caudal to the retrochiasmatic area. The lateral and dorsal borders were through the lateral POA and anterior commissure, respectively (see Figs. 4–18 in Ref. 25). The rBH block was 2 mm and extended from the retrochiasmatic area to the caudal arcuate nucleus. The lateral and dorsal borders were immediately lateral to the fornix and at the top of the third ventricle, respectively (see Figs. 18–26 in Ref. 25). The caudal basal hypothalamus (cBH) was also 2 mm and included the remainder of the hypothalamus, including the mammillary bodies and the posterior hypothalamus (see Figs. 26–34 in Ref. 25). From some of these coronal blocks, the septum (Sept) and rostral and caudal thalamus (rThal and cThal) were also dissected and used to quantify SK mRNA distribution. In addition, the ventral midbrain, including the substantia nigra and ventral tegmental area, and the hippocampus, including dorsal and ventral parts, were dissected from a 2-mm coronal block through the midbrain. For ribonuclease protection assay (RPA) the tissues were quickly frozen, and total RNA was extracted using TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD). For in situ hybridization, the tissue blocks (2–3 mm) were fixed in 4% paraformaldehyde for 6 h, soaked overnight in 20% sucrose solution, frozen, and sectioned at 15 µm.

Electrophysiology
For electrophysiological studies only ovariectomized guinea pigs were used. On the day of experimentation, the animal was decapitated, the brain was removed from the skull, and the hypothalamus was dissected. The resultant hypothalamic block was mounted on a plastic cutting platform that was then secured in a vibratome well (Leica VT 10005 vibratome, Leica Instruments, Nussloch, Germany) filled with ice-cold, oxygenated (95% O2/5% CO2), artificial cerebrospinal fluid (aCSF; NaCl, 124 mM; KCl, 5 mM; NaHCO3, 26 mM; NaH2PO4, 2.6 mM; dextrose, 10 mM; HEPES, 10 mM; MgSO4, 2 mM; CaCl2, 1 mM). Four coronal slices (350 µM) through the rBH were then cut. The slices were transferred to a multiwell auxiliary chamber containing oxygenated aCSF and kept there until electrophysiological recording.

Sharp electrode recordings in current clamp were performed as previously described (26). Briefly, slices were maintained in a chamber perfused with warmed (35 C), oxygenated aCSF perfused via a peristaltic pump at a rate of 1.5 ml/min. Microelectrodes (100–225 M{Omega}) were assembled from borosilicate glass pipettes (Sutter Instrument Co., Novato, CA; OD, 1.2 mm) pulled on a P-97 Flaming Brown puller (Sutter Instrument Co.), and filled with a 3% biocytin solution in 1.75 M KCl and 0.025 M Tris (pH 7.4). Cells were approached "blindly." After successful impalement of an arcuate neuron, spontaneous action potentials were collected using Axoscope software (Axon Instruments, Foster City, CA; sampling frequency, 50 kHz), and stored electronically for subsequent determination of the firing rate. Spike frequency adaptation and the appearance of the AHP was assessed by evoking action potentials with a 10- to 100-pA, 1-sec depolarizing current pulse. The AHP observed on the off-step of the depolarizing current pulse in both the absence and presence of 1 µM tetanus toxin was also documented.

Double labeling of GnRH neurons
After recording, the arcuate slice was fixed in 4% paraformaldehyde for 2 h, soaked overnight in 20% sucrose solution, frozen, and stored at -80 C until further processing. For immunocytochemical staining of the recorded neurons, 12-µm sections were cut through the slices, and the sections were then reacted with streptavidin-fluorescein isothiocyanate to identify the recorded cells (27). After localization of the biocytin-filled neurons, the appropriate slides were processed with the EL-14 GnRH antiserum at a 1:2500 dilution using fluorescence immunocytochemistry (27).

Cloning of guinea pig SK1 and SK3
Guinea pig-specific SK1 and SK3 cDNA fragments were cloned using RT-PCR. Oligonucleotide primers were designed that were 100% homologous to the respective human SK sequences (5'-primers; SK1, 1178–1197 bp; SK3, 2174–2192 bp; 3'-primers; SK1, 1593–1612 bp; SK3, 2486–2503 bp of the human sequences) (28, 29). Primer synthesis by Life Technologies, Inc., included at the 5'-end of both primer sets a 12-bp extension of deoxy-UMP residues used with the PCR cloning kit, CloneAmp pAMP10 System (Life Technologies, Inc.). cDNA synthesis and PCR were performed using the GeneAmp kit (Perkin-Elmer Corp., Foster City, CA). The guinea pig SK1 fragment was amplified from 200 ng total RNA extracted from the hippocampus, and the SK3 fragment was amplified from 600 ng total RNA extracted from the substantia nigra area of the guinea pig brain. In both instances, oligo deoxythymidine was used as a primer for the cDNA first strand synthesis. RT was carried out for 15 min at 42 C. PCR was carried out for 35 cycles of denaturation (92 C, 45 sec), annealing (51-52 C, 45 sec), and extension (72C, 45 sec), with a final 5-min extension. The result was a single 437-bp product for SK1 and a 330-bp product for SK3. The PCR products were gel purified and subcloned into the pAMP10 vector (Life Technologies, Inc.) using the CloneAmp System (Life Technologies, Inc.) and sequenced.

In situ hybridization
In situ hybridization was performed on tissues obtained from intact or ovariectomized oil- and estrogen-treated guinea pigs using the SK1 and SK3 riboprobes with minor modifications of a previously described method (19). Briefly, slides were postfixed in 4% paraformaldehyde, rinsed with Sorensen’s phosphate buffer (0.03 M; pH 7.2), and treated with proteinase K (1.0 µg/ml, 2 min, 37 C). All sections were then treated (3 min) with 0.1 M triethanolamine, followed by 0.25% acetic anhydride in 0.1 M triethanolamine (10 min). Thereafter, the sections were rinsed in 2x SSC and prehybridized with hybridization buffer (50% formamide, 10% dextran sulfate, 1x Denhardt’s solution, 2x SSC, 125 µg/ml yeast transfer RNA, and 100 mM dithiothreitol) for 60 min at 58 C. The 35S-labeled sense and antisense riboprobes were heat-denatured, chilled on ice, diluted with hybridization buffer, and used at a final saturating concentration of 2 x 104 dpm/µl. Subsequently, the sections were covered with glass coverslips, sealed, and hybridized for at least 18 h at 58 C. After hybridization, the slides were rinsed in 2x SSC buffer, reacted with RNase (20 µg/ml; 30 min; 37 C), and sequentially rinsed in 2.0, 1.0, and 0.5x SSC (~58 C). Slides were finally washed (30 min, 65 C) in 0.1x SSC containing 1.0 mM dithiothreitol. The sections were dehydrated and together with autoradiographic 14C-labeled microscales (Amersham Pharmacia Biotech, Piscataway, NJ) were exposed to Hyperfilm-ßmax x-ray film (Amersham Pharmacia Biotech) for 6 d at 4 C. Slides were then dipped in Kodak NTB-2 emulsion (Eastman Kodak Co., Rochester, NY) and exposed for up to 20 d at 4 C.

Immunocytochemistry and in situ hybridization
To identify which cell types expressed the SK3 channel, we performed immunocytochemistry for tyrosine hydroxylase (TH; Ink-Star, Stillwater, MN), GnRH (EL-14) (30), or vasopressin (31) and in situ hybridization sequentially on the same tissue sections, as previously described with minor modifications (19). Briefly, fluorescent immunocytochemistry was performed first using diethylpolycarbonate-treated Milli-Q H2O and molecular grade reagents. All solutions, including biotinylated IgG and streptavidin-Cy3, were prepared in the presence of RNasin (60 U/ml) and sodium heparin (1.25 mg/ml). In situ hybridization for SK3 was performed as described above, except that the final wash was performed at 58-60 C.

SK3 mRNA images alone and in combination with immunofluorescent images were evaluated and photographed under darkfield and fluorescent illumination using a Nikon E800 microscope (Melville, NY). Darkfield and fluorescent views of photomicrographs were illustrated from film negatives or color slides using a film/slide scanner (Polaroid Sprint Scan 35 Plus, Cambridge, MA) and Adobe Photoshop 5.5 software.

RPA
The antisense SK1 and SK3 riboprobes were labeled by in vitro transcription with [32P]rUTP, purified using the Fullengther Preparative Gel Apparatus (Biokey American Instrument, Aloha, OR), and used in the RPA as previously described (19). Briefly, the SK probes were incubated with 5 µg total RNA or 62–2000 fg sense standard RNA overnight at 45 C. Hybridization was terminated by RNase digestion; the protected fragments were loaded onto an acrylamide gel and exposed to film for visualization. Quantification was performed using a phosphor imager (Bio-Rad Laboratories, Inc., Hercules, CA). Each SK3 band was normalized with its corresponding cyclophilin band. A Kolmogorov-Smirnov test was used to ascertain that the RPA data exhibited a Gaussian distribution (32). Total RNA from oil- and estrogen-treated animals were always assayed together, and comparison between the two groups was performed using two-tailed paired t test. To determine differences in the distribution of SK1 and SK3 mRNA within each brain region, a one-way ANOVA was performed, followed by a Tukey-Kramer multiple comparison test. Differences were considered statistically significant at P < 0.05. For the RPA, the intraassay coefficient of variation was approximately 5%, and the level of mRNA detection was approximately 25 fg/µg. The specificity of the different probes in the RPA is illustrated by the following: 1) each antisense probe protects a band of the predicted size when hybridized with guinea pig tissue RNA, but not with rat RNA; 2) the corresponding sense probes do not protect any bands; and 3) when each antisense probe is hybridized with increasing concentrations of the respective synthesized sense RNA, a linear regression line (standard curve) is obtained.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
AHP current in GnRH neurons
As we have previously demonstrated in a number of hypothalamic neurons, including dopamine, {gamma}-aminobutyric acid (GABA), and vasopressin neurons (13, 22, 33), spike frequency adaptation with the accompanying AHP was found in all of the GnRH neurons that were tested with a depolarizing pulse protocol (n = 8; Fig. 1Go). The mean AHP magnitude (5.3 ± 0.9 mV) was similar to what we have reported for dopamine (A12) neurosecretory neurons (6.7 ± 0.7 mV) (33). Figure 1Go shows an example of the AHP in modulating repetitive firing (spike frequency adaptation) in a GnRH neuron recorded under current clamp conditions. A 30-pA depolarizing current pulse, 1 sec in duration, elicited an initial burst of action potentials, with each action potential exhibiting an AHP during the repolarization phase. Over time the interspike interval between action potentials increased, thereby decreasing the firing rate during the latter phase of the pulse. The full magnitude of the AHP was observed on the off-step of the current pulse. Although we did not test the bee venom apamin on GnRH neurons, we have found that this selective blocker of SK2 and SK3 channels inhibits the AHP current in hypothalamic vasopressin and GABAergic neurons (13, 22).



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  		Figure 1. Sharp electrode recording of a GnRH neuron illustrating the role of the AHP current in slowing action potential firing (denoted by the increased shading of the bar). The action potentials, which are not fully replicated by the chart record, are induced by a depolarizing 30-pA current of 1-sec duration. The full magnitude of the AHP (8 mV, arrow) is observed at the off-step of the current pulse. This cell was subsequently identified as a GnRH neuron using double labeling techniques. The resting membrane potential (denoted by the dotted line) was -53 mV.

 
Cloning of guinea pig SK1 and SK3 PCR fragments
To study the expression of SK1 mRNA and SK3 mRNA in the guinea pig brain, PCR cDNA fragments corresponding to guinea pig SK1 and SK3 potassium channel sequences were amplified and cloned. The guinea pig SK1 PCR product, 437 bp in length, included part of the putative fifth transmembrane domain, spanning the pore region and the entire sixth transmembrane domain. The guinea pig SK3 PCR product, 330 bp in length, consisted of sequences representing the putative 3'-intracellular region and extending into the 3'-nontranslated region. The guinea pig SK1 and SK3 riboprobes were only 43% identical, but showed high sequence identity with corresponding sequences of the human and rat SK1 and SK3 genes, respectively (Table 1Go).


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  		Table 1. DNA sequence identity with corresponding human and rat sequences

 
Distribution of SK1 and SK3 mRNA using in situ hybridization
To ascertain the distribution of SK1 and SK3 mRNA in the guinea pig brain, we performed in situ hybridization with the respective radiolabeled riboprobes in hypothalamic and other brain regions. SK1 and SK3 mRNA distribution was essentially identical in the hippocampus (Fig. 2Go). Both transcripts were highly expressed primarily in the granular cell layers and only lightly in the molecular cell layers of the dentate gyrus. Similarly, both SK subunits were highly expressed in the CA1–CA3 pyramidal cell layers (Fig. 2Go). In contrast, SK1 and SK3 mRNA were differentially expressed in brain regions such as the Sept, POA, supraoptic (SON) and suprachiasmatic (SCH) nuclei, and basal hypothalamus. In these regions, SK1 mRNA was absent (SCH) or barely above background (Sept, SON, basal hypothalamus), whereas SK3 mRNA was more robustly expressed. A comparison of SK1 and SK3 mRNA distribution in the anterior hypothalamus at the level of the SCH is illustrated in Fig. 3Go. SK3 mRNA exhibited a wide and distinct distribution pattern in brain regions such as the Sept, bed nucleus of the stria terminalis (BST), thalamus, hypothalamus, and amygdala (Figs. 4–6GoGoGo). SK3 mRNA expression was particularly dense in the lateral Sept, BST, and medial nucleus of the amygdala (see Figs. 4AGo and 6BGo). Within the thalamus, SK3 mRNA was found primarily in medial nuclei, including the reuniens and paraventricular nuclei, as illustrated in Fig. 4Go. In the hypothalamus, SK3 mRNA was robustly expressed in the anteroventral periventricular nucleus, median and the medial preoptic nuclei, the SCH, the SON, and the arcuate nucleus (Figs. 4–6GoGoGo). SK3 mRNA was also found in relatively high abundance in the paraventricular (Fig. 5Go) and ventromedial nuclei of the hypothalamus, but was more lightly expressed in dorsal and caudal hypothalamic regions (data not shown). In contrast, hybridization with the SK3 sense probe did not label any brain regions, with the exception of a light hybridization signal in the CA1–CA3 pyramidal cell layers (data not shown).



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  		Figure 2. Darkfield photomicrographs of coronal sections through the dorsal hippocampus illustrating small conductance calcium-activated potassium channel (SK1) mRNA (A) and SK3 mRNA expression (B). In the guinea pig hippocampus, SK1 and SK3 mRNA (white grains) are about equally expressed in the dentate gyrus granular cell layers (DGsg) and polymorph layer (DGpo) and in the CA1-CA3 pyramidal cell layers. Bar, 200 µm.

 


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  		Figure 3. Darkfield photomicrographs of coronal sections through the anterior hypothalamus at the level of the SCH, which illustrate the distribution of SK1 (A) and SK3 (B) mRNA. SK1 mRNA is not found in the SCH, but is expressed lightly in the medial preoptic nucleus (MPN). In contrast, SK3 mRNA is quite robustly expressed in the SCH as well as the MPN. Bar, 100 µm.

 


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  		Figure 4. Darkfield photomicrographs of coronal sections through the septal area (A) and the POA from rostral (B) to caudal (C), which illustrate the distribution and density of small conductance calcium-activated potassium channel (SK3) mRNA. SK3 mRNA expression is quite dense in the lateral Sept (LS) and BST. Also note that the expression of SK3 mRNA is particularly dense in the anteroventral periventricular nucleus (AVPV) and the medial preoptic nucleus (MPN). SK3 mRNA is also expressed in the median preoptic nucleus (MEPO), SCH, and SON. OC, Optic chiasm; 3V, third ventricle. Bar, 100 µm (A and B) and 200 µm (C).

 


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  		Figure 5. Darkfield photomicrographs of coronal sections through the anterior hypothalamus (A) and rThal (B) illustrating SK3 mRNA expression. PVH, Paraventricular nucleus of the hypothalamus; PVT, paraventricular nucleus of the thalamus; RE, reunion nucleus of the thalamus. Bar, 100 µm.

 


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  		Figure 6. Darkfield photomicrograph of coronal sections through the arcuate nucleus of the hypothalamus (Arc; A) and the amygdala (B). Note the dense concentration of SK3 mRNA in the dorsal arcuate nucleus and the medial nucleus of the amygdala (MEA). ME, Median eminence. Bar, 50 µm (A) and 100 µm (B).

 
SK3 mRNA expression in identified hypothalamic neurons
We combined in situ hybridization and fluorescence immunocytochemistry to ascertain the expression of SK3 mRNA in identified hypothalamic neurons. SK3 mRNA was colocalized with TH in the majority (>60%) of A12 dopamine neurons of the arcuate nucleus, A14 dopamine neurons of the anterior hypothalamic periventricular area, and A13 dopamine neurons of the zona incerta. In addition, SK3 mRNA was highly expressed in the majority (>75%) of A9/10 dopamine neurons in the midbrain. Examples of SK3 mRNA and TH colocalization in various dopaminergic cell groups are illustrated in Fig. 7Go, A–C. SK3 mRNA was also found in about 60% of GnRH neurons within the nucleus of the diagonal band, POA, and anterior hypothalamus (Fig. 7Go). In addition, SK3 mRNA was expressed in most (>75%) vasopressin-positive cells as well as in adjacent unidentified, but presumably oxytocin, neurons in the SON (Fig. 7Go). Representative examples of SK3 mRNA and GnRH or vasopressin colocalization are illustrated in Fig. 7Go, D–F.



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  		Figure 7. Colocalization of SK3 mRNA and TH in dopamine neurons (Aa–Cc). SK3 mRNA was also expressed in neurons containing vasopressin (Dd) and GnRH (Ee–Ff). Aa–Cc, Photomicrograph of same tissue section, on which immunocytochemistry for TH (A–C) was performed, followed by in situ hybridization for SK3 mRNA (a–c), illustrating the distribution of TH-containing neurons that also express SK3 mRNA in the periventricular area (A14;(Aa), the arcuate (A12; Bb), and the midbrain (A9; Cc). Dd–Ff, Photomicrograph of same tissue section, in which immunocytochemistry for vasopressin in the SON (D) or GnRH in the rostral POA (E) and anterior hypothalamus (F) was performed, followed by in situ hybridization for SK3 mRNA (d–f), illustrating the colocalization of the respective peptide with SK3 mRNA. The arrows in A–F and a–f point to the same cells. The inset in each figure illustrates examples of immunoreactive neurons with SK3 mRNA overlay. Bar, 25 µm (A–C) and 35 µm (D–F).

 
Quantification of SK1 and SK3 mRNA using RPA
SK3 mRNA was measured in discrete brain regions using a sensitive RPA, and various concentrations of sense RNA (31–2000 fg) were used as a standard to quantify mRNA expression (Figs. 8–10GoGoGo). Linear regression analysis of the SK3 mRNA sense standard curve revealed r = 0.996–0.998 (Figs. 9Go and 10Go). The antisense [32P]rUTP-labeled riboprobe protected a single RNA band of 330 bp in all tissues (Figs. 9Go and 10Go). Quantitative distribution of SK3 mRNA (mean ± SEM; femtograms per µg total RNA) in the brain from ovariectomized guinea pigs is illustrated in Fig. 8Go, and the rank order of expression was Sept >= midbrain > rThal >= rBH >= cThal >= POA >> cBH >= hippocampus. Statistical analysis of densitometry measurements revealed that there was a significantly higher expression of SK3 mRNA in the Sept and midbrain compared with the other brain regions (P < 0.05–0.001, by ANOVA/Tukey-Kramer test). The caudal BH and the hippocampus exhibited the lowest expression of SK3 mRNA (P < 0.05 to 001) compared with the other brain areas (Fig. 8Go). The concentration of SK3 mRNA was similar in the POA, rBH, rThal, and cThal. As we measured a detectable signal in the hippocampus with the SK3 sense riboprobe using in situ hybridization, we tested this probe in the sensitive RPA assay and did not find an expression of SK3 sense RNA in any brain region including the hippocampus.



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  		Figure 8. Distribution and quantitative analysis (ANOVA/Tukey-Kramer) of SK3 mRNA (A) and SK1 mRNA (B) in brain tissue obtained from oil-treated female guinea pigs (n = 6 and 4, respectively). A, The highest concentration of SK3 mRNA was found in the septum (Sept), and the lowest was found in the hippocampus (Hi). , P < 0.05, Sept vs. rBH and rThal; P < 0.01, Sept vs. POA and cThal; P < 0.001, Sept vs. cBH and Hi. , P < 0.05, cBH vs. cThal and POA; P < 0.01, cBH vs. rBH; P < 0.001, cBH vs. Sept, rThal, and MB. , P < 0.001, Hi vs. Sept, POA, rThal, cThal, and MB. B, The highest level of SK1 mRNA was found in the Sept, and the lowest was found in the rBH. , P < 0.05, cThal vs. POA, rBH, cBH, and rThal. , P < 0.01, Sept vs. POA, rBH, cBH, and rThal. Hi, Hippocampus; MB, midbrain.

 


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  		Figure 9. Quantification of GP SK3 mRNA 24 h after EB treatment. A, Representative film image of RPAs of total RNA (5 µg/lane, POA, rBH, cBH, MB; 25 µg/lane, hippocampus) from oil- and EB (25 µg)-treated female guinea pigs illustrating the levels of SK3 mRNA detected in the different brain regions from individual animals. UP, Undigested probe; DP, digested probe. Sixty-two to 2000 fg sense RNA were used to construct a standard curve. B, Linear regression analysis of the SK3 RNA sense standard curve using a phosphorimager revealed r = 0.994. MB, Midbrain; Hi, hippocampus. C, Distribution and quantitative analysis of SK3 mRNA in brain tissue obtained from oil (n = 6)- and EB (n = 6)-treated GP 24 h after the injection. Each SK3 band was normalized to its corresponding cyclophilin band and quantified from each sense mRNA standard curve. , P < 0.05, oil vs. EB in the rBH.

 


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  		Figure 10. Quantification of GP SK3 mRNA 42 h after EB treatment. A, Representative film image of RPAs of total RNA (5 µg/lane, POA, rBH, cBH, and MB; 25 µg/lane, hippocampus) from oil- and EB (25 µg)-treated female guinea pigs illustrating the levels of SK3 mRNA detected in the different brain regions from individual animals. UP, Undigested probe; DP, digested probe. Sixty-two to 2000 fg sense RNA were used to construct a standard curve. B, Linear regression analysis of the SK3 RNA sense standard curve using a phosphorimager revealed r = 0.998. MB, Midbrain; Hi, hippocampus. C, Distribution and quantitative analysis of SK3 mRNA in brain tissue obtained from oil (n = 4)- and EB (n = 4)-treated GP 24 h after the injection. Each SK3 band was normalized to its corresponding cyclophilin band and quantified from each sense mRNA standard curve. There was no effect of EB at 42 h.

 
We also measured SK1 mRNA in the various diencephalic regions and in the hippocampus. SK1 mRNA levels were quite low in all regions, with values ranging from a high of 68.6 ± 2.7 fg/µg in the Sept to a low of 29.2 ± 3.5 fg/µg in the rBH (Fig. 8Go). The rank order of expression for SK1 mRNA was Sept >= cThal >= hippocampus > rThal >= POA >= cBH >= rBH. Quantitative analysis using ANOVA revealed that SK1 mRNA levels in all regions, with the exception of the hippocampus, were significantly lower than SK3 mRNA levels (P < 0.01–0.001).

Estrogen treatment increased SK3 mRNA expression in the guinea pig hypothalamus
To determine whether potassium channel mRNA expression is modulated by estrogen, we measured the concentration of SK3 mRNA in the POA, basal hypothalamus, as well as the hippocampus and midbrain, two other estrogen-sensitive structures, in ovariectomized animals that received oil (n = 6) or EB (n = 6) injections 24 or 42 h earlier. As expected, animals injected sc with EB had significantly elevated plasma estrogen concentrations (117.3 ± 26.4 and 75 ± 11.8 pg/ml at 24 and 42 h, respectively) compared with ovariectomized oil-treated animals (2.0 ± 1.9 pg/ml). This estrogen regimen rapidly inhibited LH secretion and maintained the low LH levels (negative feedback) until an LH surge (positive feedback) was induced at approximately 42 h (19). Furthermore, the estrogen treatment caused a significant increase (P < 0.05) in SK3 mRNA expression in the rBH at 24 h compared with oil-treated animals, but not in other brain regions (Fig. 9Go). In contrast, SK3 mRNA levels were not different between oil- and EB-treated animals at 42 h (Fig. 10Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major findings of the present study reveal that the mRNA for the SK3 channel, which conducts an apamin-sensitive AHP current, is widely distributed in the CNS, including the hypothalamus of the female guinea pig. Moreover, SK3 mRNA is expressed in a number of hypothalamic neurosecretory neurons, such as dopamine, GnRH, and vasopressin neurons, and in all of these neurons we have identified spike frequency adaptation with an accompanying pronounced AHP. Quantitative analysis using RPA revealed that the expression of SK3 mRNA in the rBH is significantly increased in estrogen-treated female guinea pigs during a phase that coincides with the estrogen-induced inhibition of LH secretion.

In situ hybridization with SK3 riboprobe revealed that this transcript is expressed in various brain regions of the guinea pig, including hypothalamus, Sept, hippocampus, and midbrain, as described previously in rat and human brain (3, 5, 6, 34, 35). Presently, we characterized the cellular distribution as well as demonstrated for the first time that SK3 mRNA expression is modulated by estrogen. For example, SK3 mRNA was found in high quantities in the medial nucleus of the amygdala and in the BST. These are regions of the brain that are important for conveying sensory olfactory information to the hypothalamus (36). In the hypothalamus, SK3 mRNA was expressed more robustly in the medial nuclei. These hypothalamic nuclei contain releasing hormones important for neuroendocrine functions. In comparison, there was considerably less expression of SK3 mRNA in lateral hypothalamic areas, with the exception of the SON, which contains vasopressin and oxytocin neurosecretory neurons. Therefore, the SK3 mRNA appears to be highly expressed in hypothalamic areas in which there are neurosecretory neurons. The rationale for looking at the expression of the SK mRNA stems from the fact that this Ca2+-activated K+ channel is critical for modulating action potential firing in hypothalamic neurosecretory neurons, i.e. spike frequency adaptation (21, 22, 33, 37). Indeed, our colocalization studies revealed that SK3 mRNA is expressed in hypothalamic dopamine neurons, GnRH neurons, and supraoptic vasopressin neurons. Previously, we have identified an apamin-sensitive AHP in vasopressin, oxytocin, and GABAergic neurons in the female guinea pig (13, 22), and presently we have found the same characteristic signature current in GnRH neurons. This would suggest that all of these neuronal groups express SK3 mRNA, which translates to form functional channels in these neurons.

Currently, we found that in all dopamine neuronal groups investigated, including the A9, A10, A12, A13, and A14 groups, about 60–75% of the dopamine neurons expressed SK3 mRNA. In previous studies in rats, SK3 mRNA had been found in the substantia nigra pars compacta neurons (3, 6), suggesting that the SK3 channel is responsible for the AHP in midbrain dopamine neurons (38). In addition, Loose et al. (33) found that arcuate dopamine (A12) neurons exhibit a similar AHP after induction of repetitive firing (33). Recently, we characterized the AHP in the guinea pig preoptic neurons and found that the majority of preoptic neurons (~60%), including the A14 dopamine neurons, display an apamin-sensitive AHP (39). Moreover, the mean AHP amplitude is larger in burst-firing preoptic neurons than in nonbursting preoptic neurons, suggesting that the AHP may play an important role in regulating the interburst interval in burst-firing preoptic neurons (39). Based on previous in situ hybridization studies, SK3 is the dominant channel subunit expressed in the rodent hypothalamus (3, 6), which agrees with our findings in the guinea pig. However, we observed that SK1 mRNA is also present in the guinea pig hypothalamus, albeit at much lower concentrations than SK3 mRNA. Moreover, this would agree with our recent electrophysiological observations, that an apamin-insensitive component of the AHP is present in medial preoptic neurons (13). However, further studies are needed to elucidate the specific roles of SK1 vs. SK3 (and SK2) in hypothalamic neurons.

The activation of SK channels causes membrane hyperpolarization, which dramatically inhibits cell firing (2). It is, therefore, interesting that we observed that estrogen exposure increases the expression of SK3 mRNA in the basal hypothalamus during a time of negative feedback. Estrogen applied acutely to hypothalamic and amygdala neurons causes hyperpolarization by opening K+ channels (15, 16, 17). Longer-term (24-h) estrogen exposure, such as what was used in the current study, has a complex action on hypothalamic neurons that includes regulation of nuclear transcription (for reviews, see Refs. 14 and 18). It is therefore possible that after 24 h of estrogen exposure, synthesis of new channels would occur to maintain persistent inhibition of neuronal firing. Such estrogen exposure inhibits plasma LH levels presumably in part through a hypothalamic site of action that would result in inhibition of hypothalamic GnRH neurons. The mechanism of estrogen action to maintain prolonged inhibition of the GnRH/LH neurosecretory axis may involve direct action on potassium channels, including SK channels, in GnRH neurons or modification of GABA and perhaps dopamine input to GnRH neurons (13, 14, 17, 19). However, there have been few physiological studies concerning the modulation of the apamin-sensitive AHP currents. In this respect, most studies have focused on the transmitter-mediated inhibition of the apamin-insensitive slow AHP in hippocampal CA1 neurons. These include demonstration that the slow AHP is attenuated by activation of ß-adrenergic, serotonergic, histamine, dopamine, metabotropic glutamate, and muscarinic receptors (12, 40). Recently, using whole cell voltage clamp recording we found that activation of both {alpha}1- and ß-adrenergic receptors inhibits AHP current and that E2 increases the potency of the {alpha}1-adrenergic inhibition of AHP current in hypothalamic GABAergic neurons (13). The synergy between the effects of E2 to increase adrenergic input to some hypothalamic neurons and the effects of E2 to increase channel expression in other hypothalamic neurons remains to be determined.

Although this is the first report of estrogen-mediated up-regulation of K+ channel mRNA in the CNS, estrogen has been found to induce a small K+ channel (min-K) mRNA in the rat uterus (41, 42). In addition, there are a number of reports on the activation or inhibition of K+ channel activity by estrogen in nonneuronal cells via nongenomic mechanisms (for review, see Ref. 43). For example, estrogen can activate the maxi-K+ channel in smooth muscle cells through its interaction with the ß-subunit (44), which may be important in terms of the cardio-protective actions of estrogen in women. In contrast, estrogen inhibits the KATP channel in pancreatic ß-cells (45, 46). Therefore, there are multiple examples of modulation of K+ channel activity by estrogen that may have widespread clinical relevance.

In conclusion, we have ascertained the distribution of SK3 mRNA in the female guinea pig brain with both qualitative and quantitative analysis. Most importantly, we have colocalized SK3 mRNA in neurosecretory neurons. In addition, we have found that estrogen up-regulates SK3 transcripts in the rBH, an area that is critical for the negative feedback actions of estrogen on the hypothalamic-pituitary axis during the ovulatory cycle (47). Future studies will identify the changes in SK3 mRNA in individual neurons using single cell RT-PCR in conjunction with measuring the ramifications of these changes using whole cell recording of the AHP current.


   Acknowledgments
 
We thank Mr. Barry R. Naylor and Mr. Jason T. Deignan for their expert technical assistance, and Dr. Edward Wagner for his insightful comments during the preparation of the manuscript.


   Footnotes
 
This work was supported by NIH Grants NS-35944, NS-38809, and DA-00192 (Research Scientist Development Award to M.J.K.).

Abbreviations: aCSF, Artificial cerebrospinal fluid; AHP, afterhyperpolarization; BST, bed nucleus of the stria terminalis; cBH, caudal basal hypothalamus; CNS, central nervous system; cThal, caudal thalamus; EB, 17ß-E2 benzoate; GABA, {gamma}-aminobutyric acid; POA, preoptic area; rBH, rostral basal hypothalamus; rThal, rostral thalamus; RPA, ribonuclease protection assay; SCH, suprachiasmatic nucleus; Sept, septum; SK, small conductance; SON, supraoptic nucleus; TH, tyrosine hydroxylase.

Received April 6, 2001.

Accepted for publication November 28, 2001.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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