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Chloride Accumulators NKCC1 and AE2 in Mouse GnRH Neurons: Implications for GABAA Mediated Excitation.

Taylor-Burds C, Cheng P, Wray S - PLoS ONE (2015)

Bottom Line: A developmental "switch" in chloride transporters occurs in most neurons resulting in GABAA mediated hyperpolarization in the adult.In the remaining GnRH neurons, HCO3- mediated mechanisms accounted for the remaining calcium responses to muscimol.Collectively these data reveal mechanisms responsible for maintaining depolarizing GABAA mediated transmission in GnRH neurons.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Developmental Neurobiology Section, NINDS/NIH, Bethesda, Maryland, United States of America.

ABSTRACT
A developmental "switch" in chloride transporters occurs in most neurons resulting in GABAA mediated hyperpolarization in the adult. However, several neuronal cell subtypes maintain primarily depolarizing responses to GABAA receptor activation. Among this group are gonadotropin-releasing hormone-1 (GnRH) neurons, which control puberty and reproduction. NKCC1 is the primary chloride accumulator in neurons, expressed at high levels early in development and contributes to depolarization after GABAA receptor activation. In contrast, KCC2 is the primary chloride extruder in neurons, expressed at high levels in the adult and contributes to hyperpolarization after GABAA receptor activation. Anion exchangers (AEs) are also potential modulators of responses to GABAA activation since they accumulate chloride and extrude bicarbonate. To evaluate the mechanism(s) underlying GABAA mediated depolarization, GnRH neurons were analyzed for 1) expression of chloride transporters and AEs in embryonic, pre-pubertal, and adult mice 2) responses to GABAA receptor activation in NKCC1-/- mice and 3) function of AEs in these responses. At all ages, GnRH neurons were immunopositive for NKCC1 and AE2 but not KCC2 or AE3. Using explants, calcium imaging and gramicidin perforated patch clamp techniques we found that GnRH neurons from NKCC1-/- mice retained relatively normal responses to the GABAA agonist muscimol. However, acute pharmacological inhibition of NKCC1 with bumetanide eliminated the depolarization/calcium response to muscimol in 40% of GnRH neurons from WT mice. In the remaining GnRH neurons, HCO3- mediated mechanisms accounted for the remaining calcium responses to muscimol. Collectively these data reveal mechanisms responsible for maintaining depolarizing GABAA mediated transmission in GnRH neurons.

No MeSH data available.


Related in: MedlinePlus

GnRH neurons retain depolarization to MUS in the absence of NKCC1.A) As in vivo, GnRH cells (green) maintained in explants express NKCC1 (red), and AE2 (red). GnRH neurons were negative for KCC2, although a few non-GnRH neurons expressed KCC2 (lower panel, GnRH neuron fiber near KCC2 positive cell). B1) Example of GnRH neurons used for calcium imaging: bright field (BF), after loading with the calcium indicator (CaGreen), and after fixing and staining (GnRH). (B2) Examples of calcium imaging traces from NKCC1+/+ and NKCC1-/- GnRH neurons showed spontaneous activity in both genotypes (“Spontaneous Ca2+ transients”, B3). All GnRH neurons in both NKCC1+/+ (+/+) and NKCC1-/- (-/-) explants had calcium responses to MUS (“Ca2+ responses to MUS”, B3). Error bars = S.E.M. O.D. = optical density. Scale bars, A and B1 = 10 μM.
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pone.0131076.g003: GnRH neurons retain depolarization to MUS in the absence of NKCC1.A) As in vivo, GnRH cells (green) maintained in explants express NKCC1 (red), and AE2 (red). GnRH neurons were negative for KCC2, although a few non-GnRH neurons expressed KCC2 (lower panel, GnRH neuron fiber near KCC2 positive cell). B1) Example of GnRH neurons used for calcium imaging: bright field (BF), after loading with the calcium indicator (CaGreen), and after fixing and staining (GnRH). (B2) Examples of calcium imaging traces from NKCC1+/+ and NKCC1-/- GnRH neurons showed spontaneous activity in both genotypes (“Spontaneous Ca2+ transients”, B3). All GnRH neurons in both NKCC1+/+ (+/+) and NKCC1-/- (-/-) explants had calcium responses to MUS (“Ca2+ responses to MUS”, B3). Error bars = S.E.M. O.D. = optical density. Scale bars, A and B1 = 10 μM.

Mentions: To determine if NKCC1 and/or AE2 was critical for the depolarizing response to GABAA activation, experiments were performed using intact primary GnRH neurons maintained in explants. The properties and receptors of GnRH cells in this model system mimic GnRH cells in vivo or slice preparations, and large numbers of GnRH cells are accessible for analysis [31]. Immunocytochemical analysis revealed that GnRH cells in 1–3 week old explants from WT mice, like GnRH neurons in vivo, expressed NKCC1 but not KCC2 (Fig 3A; N>4 for each group: 7–10, 14–17, and 21–24 div). A few GnRH negative/KCC2 positive cells were detected in most explants, however unlike GnRH cells, they did not migrate into the periphery. Their scarcity, and location in the tissue mass precluded them from analysis in imaging and recording experiments (Fig 3A). PCR on single cell cDNAs from GnRH neurons confirmed that transcripts for AE2 (S1 Fig) and AE3 (data not shown) were present in the majority of GnRH cells (7 div: AE2: 6/7, AE3: 5/7; and 28div: AE2: 4/7 cells, AE3: 4/8). However, protein expression of AE3 was not detected, while, similar to that found in vivo, ICC confirmed AE2 in some, but not all, GnRH neurons in explants (Fig 3A).


Chloride Accumulators NKCC1 and AE2 in Mouse GnRH Neurons: Implications for GABAA Mediated Excitation.

Taylor-Burds C, Cheng P, Wray S - PLoS ONE (2015)

GnRH neurons retain depolarization to MUS in the absence of NKCC1.A) As in vivo, GnRH cells (green) maintained in explants express NKCC1 (red), and AE2 (red). GnRH neurons were negative for KCC2, although a few non-GnRH neurons expressed KCC2 (lower panel, GnRH neuron fiber near KCC2 positive cell). B1) Example of GnRH neurons used for calcium imaging: bright field (BF), after loading with the calcium indicator (CaGreen), and after fixing and staining (GnRH). (B2) Examples of calcium imaging traces from NKCC1+/+ and NKCC1-/- GnRH neurons showed spontaneous activity in both genotypes (“Spontaneous Ca2+ transients”, B3). All GnRH neurons in both NKCC1+/+ (+/+) and NKCC1-/- (-/-) explants had calcium responses to MUS (“Ca2+ responses to MUS”, B3). Error bars = S.E.M. O.D. = optical density. Scale bars, A and B1 = 10 μM.
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Related In: Results  -  Collection

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pone.0131076.g003: GnRH neurons retain depolarization to MUS in the absence of NKCC1.A) As in vivo, GnRH cells (green) maintained in explants express NKCC1 (red), and AE2 (red). GnRH neurons were negative for KCC2, although a few non-GnRH neurons expressed KCC2 (lower panel, GnRH neuron fiber near KCC2 positive cell). B1) Example of GnRH neurons used for calcium imaging: bright field (BF), after loading with the calcium indicator (CaGreen), and after fixing and staining (GnRH). (B2) Examples of calcium imaging traces from NKCC1+/+ and NKCC1-/- GnRH neurons showed spontaneous activity in both genotypes (“Spontaneous Ca2+ transients”, B3). All GnRH neurons in both NKCC1+/+ (+/+) and NKCC1-/- (-/-) explants had calcium responses to MUS (“Ca2+ responses to MUS”, B3). Error bars = S.E.M. O.D. = optical density. Scale bars, A and B1 = 10 μM.
Mentions: To determine if NKCC1 and/or AE2 was critical for the depolarizing response to GABAA activation, experiments were performed using intact primary GnRH neurons maintained in explants. The properties and receptors of GnRH cells in this model system mimic GnRH cells in vivo or slice preparations, and large numbers of GnRH cells are accessible for analysis [31]. Immunocytochemical analysis revealed that GnRH cells in 1–3 week old explants from WT mice, like GnRH neurons in vivo, expressed NKCC1 but not KCC2 (Fig 3A; N>4 for each group: 7–10, 14–17, and 21–24 div). A few GnRH negative/KCC2 positive cells were detected in most explants, however unlike GnRH cells, they did not migrate into the periphery. Their scarcity, and location in the tissue mass precluded them from analysis in imaging and recording experiments (Fig 3A). PCR on single cell cDNAs from GnRH neurons confirmed that transcripts for AE2 (S1 Fig) and AE3 (data not shown) were present in the majority of GnRH cells (7 div: AE2: 6/7, AE3: 5/7; and 28div: AE2: 4/7 cells, AE3: 4/8). However, protein expression of AE3 was not detected, while, similar to that found in vivo, ICC confirmed AE2 in some, but not all, GnRH neurons in explants (Fig 3A).

Bottom Line: A developmental "switch" in chloride transporters occurs in most neurons resulting in GABAA mediated hyperpolarization in the adult.In the remaining GnRH neurons, HCO3- mediated mechanisms accounted for the remaining calcium responses to muscimol.Collectively these data reveal mechanisms responsible for maintaining depolarizing GABAA mediated transmission in GnRH neurons.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Developmental Neurobiology Section, NINDS/NIH, Bethesda, Maryland, United States of America.

ABSTRACT
A developmental "switch" in chloride transporters occurs in most neurons resulting in GABAA mediated hyperpolarization in the adult. However, several neuronal cell subtypes maintain primarily depolarizing responses to GABAA receptor activation. Among this group are gonadotropin-releasing hormone-1 (GnRH) neurons, which control puberty and reproduction. NKCC1 is the primary chloride accumulator in neurons, expressed at high levels early in development and contributes to depolarization after GABAA receptor activation. In contrast, KCC2 is the primary chloride extruder in neurons, expressed at high levels in the adult and contributes to hyperpolarization after GABAA receptor activation. Anion exchangers (AEs) are also potential modulators of responses to GABAA activation since they accumulate chloride and extrude bicarbonate. To evaluate the mechanism(s) underlying GABAA mediated depolarization, GnRH neurons were analyzed for 1) expression of chloride transporters and AEs in embryonic, pre-pubertal, and adult mice 2) responses to GABAA receptor activation in NKCC1-/- mice and 3) function of AEs in these responses. At all ages, GnRH neurons were immunopositive for NKCC1 and AE2 but not KCC2 or AE3. Using explants, calcium imaging and gramicidin perforated patch clamp techniques we found that GnRH neurons from NKCC1-/- mice retained relatively normal responses to the GABAA agonist muscimol. However, acute pharmacological inhibition of NKCC1 with bumetanide eliminated the depolarization/calcium response to muscimol in 40% of GnRH neurons from WT mice. In the remaining GnRH neurons, HCO3- mediated mechanisms accounted for the remaining calcium responses to muscimol. Collectively these data reveal mechanisms responsible for maintaining depolarizing GABAA mediated transmission in GnRH neurons.

No MeSH data available.


Related in: MedlinePlus