Heterogeneous Expression of the Potassium-Chloride Cotransporter ...

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Endocrinology 144(7):3031–3036 Copyright © 2003 by The Endocrine Society doi: 10.1210/en.2002-220995

Heterogeneous Expression of the Potassium-Chloride Cotransporter KCC2 in Gonadotropin-Releasing Hormone Neurons of the Adult Mouse SARAH M. LEUPEN, STUART A. TOBET, W. F. CROWLEY, JR.,

AND

KAI KAILA

Reproductive Endocrine Unit, Massachusetts General Hospital/Harvard Medical School (S.M.L., W.F.C.), and Department of Biochemistry, University of Massachusetts Medical School (S.A.T.), Boston, Massachusetts 02114; and Department of Biosciences, University of Helsinki (K.K.), SF-00014 Helsinki, Finland In mature central neurons, chloride extrusion mediated by the K-Cl cotransporter KCC2 appears to be largely responsible for the Clⴚ driving force that allows ␥-aminobutyric acidA (GABAA) receptor activation to trigger a hyperpolarization. In its absence, GABA’s effect is typically depolarizing and often excitatory. We examined the colocalization of KCC2 and GnRH in adult male and female mice using a combined in situ hybridization-immunofluorescence procedure. We found that KCC2 was localized to approximately

G

ABA (␥-AMINOBUTYRIC ACID) is the most common inhibitory transmitter in the adult central nervous system. Hyperpolarizing GABAA-mediated inhibition is caused by the inward flow of Cl⫺ through ion-specific channels opened by receptor activation, which suppresses excitatory postsynaptic potentials and attenuates voltage-gated Ca2⫹ currents (reviewed in Ref. 1). However, GABA’s effect on ion flow and membrane polarization is depolarizing and sometimes even excitatory in the developing brain (2, 3) and in some adult neuronal populations (4 – 6). The developmental switch in GABA’s effect from depolarizing to hyperpolarizing is accomplished through the expression of the neuron-specific potassium-chloride cotransporter, KCC2 (7). This protein is an electroneutral transporter that carries one potassium and one chloride ion together across the neuronal membrane. Under physiological conditions, KCC2 normally acts as an efflux pathway maintaining intracellular Cl⫺ lower than predicted by an electrochemical equilibrium (7–9). When GABAA receptor activation triggers the opening of chloride channels, chloride flows into the cell carrying a hyperpolarizing current. KCC2 is widely and strongly expressed in the adult central nervous system (7, 8, 10 –13), allowing GABA (or glycine) to function as a hyperpolarizing inhibitory neurotransmitter throughout the adult brain (but see Ref. 14). GABAergic neurons acting through GABAA receptors comprise a major direct input to the GnRH neuronal population (15–18), with a particular role in mediation of steroid feedback to the GnRH population (reviewed in Refs. 19 and 20). However, little is known about mechanisms of GABAmediated signaling in adult GnRH neurons. Although GABA Abbreviations: GABA, ␥-Aminobutyric acid; GFP, green fluorescent protein; PBT, PBS with Tween; SSC, standard saline citrate.

34% of GnRH neurons. This proportion was similar in females and males. However, females exhibited a marked rostrocaudal gradient of colocalization that was not seen in males. By contrast, KCC2 was localized to nearly all vasopressin neurons of the supraoptic nucleus. These results indicate that a substantial fraction of GnRH neurons may be depolarized and excited by GABAA receptor activation throughout life, supporting the existence of functionally heterogeneous subpopulations. (Endocrinology 144: 3031–3036, 2003)

is considered to be the primary inhibitory input to the GnRH population (21, 22), many stimulatory effects have been documented (23–27); for instance, muscimol, a GABA agonist, stimulates GnRH release in the median eminence (23, 24), and GABA stimulates GnRH gene expression (26) and secretion (27). The evidence that GABA has a solely hyperpolarizing action on GnRH neurons (28) is controversial (29). Because of the obvious importance of GABA in GnRH neuron function, we investigated the colocalization of GnRH and KCC2 in adult male and female mice. Materials and Methods In situ hybridization Digoxygenin-labeled sense and antisense riboprobes were generated using a digoxygenin RNA labeling procedure. pBluescript plasmids containing the KCC2 gene insert [245 bp; construct provided by Dr. Roderic L. Smith (30)] were linearized with EcoRI and transcribed with T7 RNA polymerase (Roche, Indianapolis, IN; antisense) or XhoI and T3 RNA polymerase (sense). Adult male and female mice with green fluorescent protein (GFP) genetically targeted to GnRH neurons (31) (founders provided by Dr. Suzanne Moenter; n ⫽ 8, three males and five females) were anesthetized with ketamine and xylazine and intraaortically perfused with 4% paraformaldehyde. Mice were 4 – 8 months old, and the estrous cycle stage of females was random (i.e. not known). All animal experimentation was conducted in accordance with accepted standards of humane animal care and approved by institutional committees. Brains were postfixed for 24 h in the same solution and sliced under ribonuclease-free conditions to 80 ␮m thickness. Sections were treated with 6% H2O2 in PBS with Tween (PBT), followed by washing in PBT and treatment with proteinase K (10 ␮g/ml in PBS with Tween) and glycine (2 mg/ml in the same buffer). Sections were again washed in PBT, then postfixed in 4% paraformaldehyde. After prehybridization, sections were hybridized overnight at 56 C in hybridization buffer [50% formamide, 5⫻ standard saline citrate (SSC), 50 ␮g/ml yeast tRNA (Sigma-Aldrich Corp., St. Louis, MO), and 50 ␮g/ml heparin]. After stringent washes (wash solution 1: 50% formamide/5⫻ SSC; wash solution 2: 50% formamide/2⫻ SSC) sections were washed in Tris-buffered saline with Tween, then incubated overnight in an alkaline-phos-

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Leupen et al. • GnRH-KCC2 Colocalization

FIG. 1. Colocalization of KCC2 and GnRH or vasopressin. A, Neuron coexpressing GnRH and KCC2. Upper left, GnRH; upper center, KCC2; lower left, DAPI; lower center, KCC2 and DAPI; right, KCC2 and GnRH. B, As above, for a GnRH neuron not coexpressing KCC2. C, As above, for three vasopressin neurons in the supraoptic nucleus, all coexpressing KCC2.

phatase-labeled antibody to digoxygenin (Roche) in Tris-buffered saline with Tween (1:2000). Finally, sections were incubated in color detection mix [10% polyvinyl alcohol, 100 mm Tris, 100 mm NaCl, 5 mm MgCl2, 1% levamisole (Vector Laboratories, Inc., Burlingame, CA), and 2% nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate stock solution (Roche)] until color developed.

Immunohistochemistry Because GFP fluorescence was compromised by the in situ hybridization procedure, we proceeded to identify GnRH neurons by immunohistochemistry. Immediately after in situ hybridization, sections were washed in PBT and incubated for 72 h in one of two anti-GnRH polyclonal antisera [LR1 at 1:10,000, or the Affinity BioReagents, Inc. (Golden, CO) anti-GnRH polyclonal PA1–121 at 1:400] or a polyclonal vasopressin antiserum (Chemicon, Temecula, CA; catalogue no. 1565) at 1:1500 in

PBS with 0.3% Triton. After washing, sections were incubated in goat antirabbit immunoglobulin G (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), washed again, and incubated in Cy3-conjugated streptavidin (Jackson ImmunoResearch Laboratories, Inc.), both at 1:500 dilution in the same buffer. Tissue was mounted and coverslipped using Vectashield H-1000 with DAPI (Vector Laboratories, Inc.) for nuclear localization. Slides were viewed using a Axioplan microscope (Carl Zeiss, New York, NY) and digital images (1080 ⫻ 1520 pixels) captured using an RT color SPOT digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI).

Analysis After the sequential in situ hybridization-immunohistochemical procedure, each GnRH (or vasopressin)-immunopositive neuron was coded, and its position was recorded on an anatomical map appropriate


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to its rostrocaudal position within the GnRH population. Then, for each neuron, a triad of digital photographs was taken without adjusting the focus or stage position: a brightfield image to capture the hybridization deposition, and two fluorescent images, with the appropriate TRITC and UV filters to capture Cy3 fluorescence and DAPI fluorescence, respectively. Subsequently, an investigator blind to anatomical position combined the coded images in Adobe Photoshop and scored each GnRH (or vasopressin) neuron as colocalized with KCC2, not colocalized, or ambiguous. Ambiguous neurons, comprising about 3% of the total, were excluded from the analysis. A neuron was considered to be KCC2 positive if the associated KCC2 deposition was 1) deposited in a distinct pattern ringing the cell body, and 2) sufficiently dense as to clearly indicate signal over and above deposition noise. DAPI was used 1) to reveal cases in which an apparent double label was two separate neurons, and 2) to reveal the total population of neurons on a slice and thus aid in associating KCC2 deposits with specific neurons. GnRH neurons overlain with more than one cell nucleus as revealed by DAPI imaging were not considered double labeled even if this appeared to be the case from GnRH and KCC2 images. After this scoring procedure, scores were transferred to the anatomical maps for pattern analysis. For comparison of males and females and analysis of anatomical trends, neurons were collapsed into position bins, with each bin representing a 480-␮m coronal brain slice. For reference, the organum vasculosum of the lamina terminalis marks the Bin I-II boundary, and the anterior commissure juncture marks the Bin II-III boundary. Potential differences were assessed by ANOVA, followed by post hoc t tests for pairwise comparison (JMP 4.0 Statistical Package, SAS Institute, Cary, NC). Males and females were analyzed separately because the variance among the males was 2-fold greater than that among the females.

Results

On the average, there were 151 ⫾ 32 GnRH neurons counted per brain (counted in alternate sections only) after detection by immunohistochemistry. This represents a loss of about 20% detection vs. nonperformance of the in situ hybridization procedure before immunostaining; hence, the degree of double labeling can only provide a broad estimate of the actual degree of colocalization. Only a subset of GnRH neurons (34.4 ⫾ 1.6%) was colocalized with KCC2 in the adult mouse (Fig. 1). There was no difference in the number of GnRH neurons per brain (males, 148 ⫾ 49; females, 154 ⫾ 19) or total degree of colocalization (males, 35.0 ⫾ 3.6%; females, 34.0 ⫾ 1.3%) between the sexes. Conversely, colocalization with KCC2 was detected in 98.3% of vasopressin neurons of the supraoptic nucleus (n ⫽ 91; Fig. 1C). The likelihood of colocalization of GnRH and KCC2 exhibited a rostrocaudal gradient in female brains (by ANOVA, P ⫽ 0.012; Fig. 2, top panel). However, there was no evidence for this gradient in males (by ANOVA, P ⫽ 0.916; Fig. 2, lower panel). Because the caudal portions of the GnRH population are also more ventral, colocalized neurons in the female were more likely to be relatively ventral within the overall population. For an appreciation of the overall pattern, the location of each neuron, grouped such that each slice represents a rostrocaudal bin for a representative male and female brain (according to rostrocaudal colocalization pattern, best match to bin means), is shown in Fig. 3. The total number of neurons in each bin for all animals was as follows: bin I, 272; bin II, 572; bin III, 181; and bin IV, 125. Discussion

The results of the current study suggest that GABAergic stimulation of GnRH neurons through GABAA receptors is likely to cause depolarization in some GnRH neurons and

FIG. 2. Colocalization of GnRH and KCC2 by rostrocaudal levels in females (upper panel) and males (lower panel) as a percentage of the total number of GnRH neurons in each bin. Each bin represents a 480-␮m coronal slice through the GnRH population, with bin I containing the most rostral neurons. In females, there was a significant difference in percent colocalization across bins (by ANOVA, P ⬍ 0.02).

hyperpolarization in others. Virtually all GnRH neurons have been shown to contain GABAA receptors (16, 32), yet only a subset of GnRH neurons coexpresses the chloride transporter KCC2. By contrast, nearly all vasopressin neurons coexpressed KCC2, suggesting that the result in GnRH neurons was not due to insufficient sensitivity of the procedure. A previous failure to detect KCC2 message (10) in the SON may have been due to differences in probe sequences between the studies or insufficient sensitivity of procedures.

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FIG. 3. Anatomical representation of each neuron processed for KCC2 ISH-GnRH ICC in the most representative female (left) and male (right) brain by best match of individual to group means. Each slice map represents one anatomical bin. Gray, GnRH neurons not colocalized with KCC2; black, GnRH neurons colocalized with KCC2.

Due to the inefficiency of double-labeling procedures and because of the conservative nature of our scoring method, 35% could represent an overestimate (if undetected GnRH neurons are predominantly not double labeled) or an underestimate (if undetected GnRH neurons are predominantly double labeled) of actual colocalization. Nonetheless, it is clear that both KCC2-expressing and nonexpressing populations exist. Further, we found a rostrocaudal gradient of colocalization in the female, but not the male, GnRH population. The electrophysiological response of GnRH neurons in vivo to GABAA receptor activation is not yet known. Two groups have recently addressed this issue directly in slice preparations in vitro but have reported conflicting results; one group (29) observed depolarization across an array of conditions, whereas another (28) found hyperpolarizing

GABA actions in 9 of 10 neurons from the adult mice examined. It is possible that the differential results derived from the different transgenic mice that were used, GnRHGFP (29) vs. GnRH-lacZ (28). The mice in the current study were derived from the same background as the former (29). Clearly, further studies are needed to resolve the ways in which different GnRH neurons respond to GABAA receptor activation. Differential KCC2 colocalization in the GnRH neuronal population, whether qualitative (on/off) or quantitative (various levels of KCC2 expression), affords a possible mechanism of functional heterogeneity to the GnRH population, allowing for dramatically different GnRH secretion responses to GABAA receptor activation. A great deal of previous work has indicated or suggested heterogeneity in the GnRH population. Recent examples include the demonstra-


tion that individual GnRH neurons exhibit heterogeneous electrophysiological properties (33). Additionally, Nunemaker et al. (20) found that neurons located within the midventral preoptic area were responsive to blockade of ionotropic GABA transmission, whereas GnRH neurons located outside this area were not. Our data support these researchers’ conclusions that within the GnRH population there exists a possible correlation between anatomical location and use of GABA as mediators of estradiol effects. In other work, GnRH subpopulations have been correlated to metabolic activity (34), estrous cycle stage (35, 36), or hormone replacement (37). Visual inspection of individual KCC2-GnRH colocalization patterns (e.g. Fig. 3) suggests the tendency for KCC2-colocalized GnRH neurons to be centralized within the population, which is consistent with the previously described concept of differing roles for central and peripheral GnRH subpopulations (34). Furthermore, differential expression of a GABA response-switching mechanism, such as KCC2, is a possible explanation for the numerous reports of either or both stimulatory and inhibitory GABA effects on GnRH gene expression and secretion. There are few clear examples of sex differences in GnRH neurons beyond the physiological differences in GnRH release. GnRH neurons may be more widespread in males than females (reviewed in Ref. 38), and there may be subtle differences in neuronal shape during development (39). The rostrocaudal gradient of colocalization seen in females, but not in males, may reflect the different requirements of the two sexes for contrasting responses to GABA input. The preovulatory switch in sign of gonadal steroid feedback from negative to positive is female specific, and the expression or nonexpression of KCC2 in the relevant GnRH neurons could potentially account for this. Further work is clearly required to elucidate possible differences in spatial heterogeneity of GnRH neurons in males and females. The existence of KCC2-defined subpopulations has recently been documented in the retina (5), where both depolarizing and hyperpolarizing responses to GABA were reported. The researchers observed that these two responses occur distinctly from KCC2-expressing and nonexpressing populations, with different roles in signaling. Additionally, a related protein, the Na-K-Cl transporter, which transports Cl⫺ into the cell under normal physiological conditions (the reverse of KCC2’s action), was found to be mutually exclusive with KCC2 expression in the retina. Hence, these two transporters were suggested to explain the difference in the GABA reversal potential in these two populations (5). However, because GABAA receptors show a significant permeability to bicarbonate (14, 40), it will be important to assess the expression in GnRH neurons of a number of anion transporters (1) that are relevant for GABAergic signaling. Finally, it is critical that the electrophysiological responses of GnRH neurons to GABAA receptor activation in vivo be determined. Acknowledgments We thank Sue Moenter for providing founder mice and continuing discussion; Aline Davis, Heather Walker, and Marianne Seney for technical assistance; and Mark Palmert for helpful discussions. Received October 25, 2002. Accepted January 29, 2003.

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Address all correspondence and requests for reprints to: Dr. Sarah M. Leupen, Reproductive Endocrine Unit, BHX-519, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114. E-mail: leupen@ world.oberlin.edu. This work was supported by NIH Grants HD-08630-02 (to S.M.L.), MH-57748 (to S.A.T.), and HD-33441 (to G. A. Schwarting and S.A.T.) and the Academy of Finland (to K.K.). Present address for S.A.T.: Colorado State University, Department of Biomedical Sciences, 1680 Campus Delivery, Fort Collins, Colorado 80523. E-mail: [email protected].

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