SCI 2020 - Resources and Networking from the SCI 2020 Conference

A resource and networking site to build from the NIH hosted conference "SCI 2020: Launching a Decade for Disruption in Spinal Cord Injury Research

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

<div><p>A developmental “switch” in chloride transporters occurs in most neurons resulting in GABA_A mediated hyperpolarization in the adult. However, several neuronal cell subtypes maintain primarily depolarizing responses to GABA_A 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 GABA_A receptor activation. In contrast, KCC2 is the primary chloride extruder in neurons, expressed at high levels in the adult and contributes to hyperpolarization after GABA_A receptor activation. Anion exchangers (AEs) are also potential modulators of responses to GABA_A activation since they accumulate chloride and extrude bicarbonate. To evaluate the mechanism(s) underlying GABA_A mediated depolarization, GnRH neurons were analyzed for 1) expression of chloride transporters and AEs in embryonic, pre-pubertal, and adult mice 2) responses to GABA_A 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 GABA_A 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 GABA_A mediated transmission in GnRH neurons.</p></div

GnRH neurons retain depolarization to MUS in the absence of NKCC1.

<p>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.</p

GnRH neurons are positive for NKCC1 and negative for KCC2 throughout development.

<p>A1-2) Robust NKCC1 staining (brown) was detected at E14.5 in the nasal area (N) including cells in the olfactory epithelium and cells and fibers (OA, black arrows) at the nasal forebrain junction (J), with many fibers seen entering the developing olfactory bulb (OB). A2 is higher magnification of boxed area in A1. A3) GnRH neurons (green, arrows) in nasal areas were NKCC1 positive (red), showing light labeling over their cell soma (white arrows). Labeling of GnRH processes was obscured by dense labeling of olfactory axon bundles (black arrow). B) NKCC1 (red) is co-localized in GnRH neurons (green) in pre-pubertal (B1) and adult (B2) mice. NKCC1 labeling was more robust in GnRH neurons and surrounding preoptic tissue at pre-pubertal ages compared to adult. However, in the adult, some GnRH neurons remained clearly positive for NKCC1 in the cell soma and proximal processes (B2). C) NKCC1 (red) was present in the median eminence (ME), but did not co-localized with GnRH fibers (green). D) Chromagen double-labeling amplification of NKCC1 signal with NiDAB (blue-black) in sections from WT (D1) and NKCC1^-/- (D2) mice demonstrates ubiquitous NKCC1 staining throughout the pre-optic area (POA) and staining of GnRH neurons (red), both cell soma and processes in WT mice (D1). E1-2) KCC2 staining (brown) was robust in the developing OB, ventral forebrain and areas around the cephalic flexure (CF). E2) Higher magnification of boxed area in E1 showing no KCC2 labeling in nasal areas (N) or at the nasal forebrain junction (J). E3) GnRH cells (green, arrows) at forebrain junction (J) are negative for KCC2 (red), while cells in the ventral forebrain (B) robustly stain for KCC2. F) GnRH cells (green) are KCC2 (red) negative in both pre-pubertal (F1) and adult (F2) mice, although robust KCC2 labeling is found throughout the brain. G) Although present in the median eminence (ME), KCC2 (red) did not co-localize with GnRH fibers (green). F) Double labeling was performed for GnRH neurons with anti-GFP and/or anti-cGnRH. Arrows indicate GnRH cells and large arrow indicate cells magnified on the right. Scale bars: A1 and B2 = 200 μM, E, F, and G = 100 μM; A3, B3, C and D = 10 μM. B = brain; T = tongue. N ≥ 3 at all stages.</p

GABA_A activation elicits similar response in GnRH neurons from NKCC1^-/- explants but is reduced by BUME.

<p>A) Example of a gramicidin perforated patch clamp recording used to measure response to MUS. Trace from NKCC1^-/- GnRH cell shows MUS depolarization is present before and after TTX. B) Peak amplitudes of MUS responses in GnRH neurons are similar in NKCC1^+/+ (+/+, black dots) and NKCC1^-/- (-/-, red dots) (B1) In contrast, BUME treatment significantly reduces the response to MUS in GnRH neurons in WT mice (B2). C) Reversal potential for GABA in GnRH neurons was evaluated by acute application of MUS (100 μM, arrowhead). All recordings were performed with CNQX/AP5 (10 μM each) and TTX (1 μM). Examples of recordings from GnRH neurons in untreated (WT) and bumetanide treated (+BUME) groups, and the voltage protocol used (C1). Reversal potentials were more negative when NKCC1 was inhibited by BUME (20 μM, red dots) than in control (WT, black dots) cells (*p<0.05, error bars = SEM) (C2).</p

Anion exchanger 2 (AE2) is expressed in GnRH neurons throughout development.

<p>GnRH cells (green) in prenatal (A, B) and pre-pubertal mice (C1-2) expressed AE2 (red). C3-4) Chromagen intensified double-labeling for AE2 (NiDAB, black) and GFP (ABC-AP, purple) confirmed that GnRH neurons express AE2 in pre-pubertal (C3) and adult (C4) mice. D) No AE2 (red) expression was detected on GnRH fibers (green) in the median eminence, whereas astrocytes (GFAP, green) in neighboring regions were AE2 positive (E). Scale bars: B, C and E = 10 μM; D = 100 μM.</p

Behavioral comparisons of the tastes of L-alanine and monosodium glutamate in rats

Recent research has implicated T1R1/T1R3 as the primary taste receptor in mammals for detecting L-amino acids, including L-monosodium glutamate (MSG) and L-alanine. Previous behavioral studies with rodents found only minimal evidence that these two substances share perceptual qualities, but those studies did not control for the taste of sodium associated with MSG. This study used several behavioral methods to compare the perceptual qualities of MSG and L-alanine in rats, using amiloride (a sodium channel blocker) to reduce the sodium component of MSG taste. Detection thresholds of L-alanine in rats ranged between 0.4 and 2.5 mM, with or without amiloride added, which are similar to threshold estimates for MSG. Conditioned taste aversion (CTA) found that rats showed strong cross-generalization of CTA between MSG and L-alanine when mixed with amiloride, indicating the two substances have similar perceptual qualities. Discrimination methods showed that rats easily discriminated between L-alanine and MSG unless the cue function of sodium was reduced. The discrimination became significantly more difficult at concentrations \u3c100 mM when amiloride was added to all stimuli and became even more difficult when NaCl was also added to L-alanine solutions to match the sodium concentrations of MSG. These results indicate that, perceptually, MSG and L-alanine have quite similar taste qualities and support the hypothesis that these two L-amino acids activate a common taste receptor. The differences in perceptual qualities also suggest separate afferent processing of one or both substances may also be involved. © Oxford University Press 2004; all rights reserved

Monosodium glutamate and sweet taste: Discrimination between the tastes of sweet stimuli and glutamate in rats

Generalization of a conditioned taste aversion (CTA) is based on similarities in taste qualities shared by the aversive substance and another taste substance. CTA experiments with rats have found that an aversion to a variety of sweet stimuli will cross-generalize with monosodium glutamate (MSG) when amiloride, a sodium channel blocker, is added to all solutions to reduce the taste of sodium. These findings suggest that the glutamate anion elicits a sweet taste sensation in rats. CTA experiments, however, generally do not indicate whether two substances have different taste qualities. In this study, discrimination methods in which rats focused on perceptual differences were used to determine if they could distinguish between the tastes of MSG and four sweet substances. As expected, rats readily discriminated between two natural sugars (sucrose, glucose) and two artificial sweeteners (saccharin, SC45647). Rats also easily discriminated between MSG and glucose, saccharin and, to a lesser extent, SC45647 when the taste of the sodium ion of MSG was reduced by the addition of amiloride to all solutions, or the addition of amiloride to all solutions and NaCl to each sweet stimulus to match the concentration of Na + in the MSG solutions. In contrast, reducing the cue function of the Na+ ion significantly decreased their ability to discriminate between sucrose and MSG. These results suggest that the sweet qualities of glutamate taste is not as dominate a component of glutamate taste as CTA experiments suggest and these qualities are most closely related to the taste qualities of sucrose. The findings of this study, in conjunction with other research, suggest that sweet and umami afferent signaling may converge through a taste receptor with a high affinity for glutamate and sucrose or a downstream transduction mechanism. These data also suggest that rats do not necessarily perceive the tastes of these sweet stimuli as similar and that these sweet stimuli are detected by multiple sweet receptors. © Oxford University Press 2004; all rights reserved

FAIR SCI Ahead: The Evolution of the Open Data Commons for Pre-Clinical Spinal Cord Injury Research

Over the last 5 years, multiple stakeholders in the field of spinal cord injury (SCI) research have initiated efforts to promote publications standards and enable sharing of experimental data. In 2016, the National Institutes of Health/National Institute of Neurological Disorders and Stroke hosted representatives from the SCI community to streamline these efforts and discuss the future of data sharing in the field according to the FAIR (Findable, Accessible, Interoperable and Reusable) data stewardship principles. As a next step, a multi-stakeholder group hosted a 2017 symposium in Washington, DC entitled “FAIR SCI Ahead: the Evolution of the Open Data Commons for Spinal Cord Injury research.” The goal of this meeting was to receive feedback from the community regarding infrastructure, policies, and organization of a community-governed Open Data Commons (ODC) for pre-clinical SCI research. Here, we summarize the policy outcomes of this meeting and report on progress implementing these policies in the form of a digital ecosystem: the Open Data Commons for Spinal Cord Injury ( ODC-SCI.org ). ODC-SCI enables data management, harmonization, and controlled sharing of data in a manner consistent with the well-established norms of scholarly publication. Specifically, ODC-SCI is organized around virtual “laboratories” with the ability to share data within each of three distinct data-sharing spaces: within the laboratory, across verified laboratories, or publicly under a creative commons license (CC-BY 4.0) with a digital object identifier that enables data citation. The ODC-SCI implements FAIR data sharing and enables pooled data-driven discovery while crediting the generators of valuable SCI data