First Photoswitchable Neurotransmitter Transporter Inhibitor: Light-Induced Control of γ‑Aminobutyric Acid Transporter 1 (GAT1) Activity in Mouse Brain

Inhibition of mGAT1, the most abundant GABA transporter in the brain, enhances GABA signaling and alleviates symptoms of CNS disorders such as epilepsy assumed to be associated with low GABA levels. We have now developed a potent and subtype selective photoswitchable inhibitor of this transporter, which for the first time extends the photoswitch concept for the light-induced control of ligand affinity to active membrane transporters. The new inhibitor exhibited reduced activity upon irradiation with light, as demonstrated in GABA uptake assays and electrophysiological experiments with brain slices, and might be used as a tool compound for deepening the understanding of mGAT1 function in brain

Development of New Photoswitchable Azobenzene Based γ‑Aminobutyric Acid (GABA) Uptake Inhibitors with Distinctly Enhanced Potency upon Photoactivation

A series of nipecotic acid derivatives with new azo benzene based photoswitchable N-substituents was synthesized and characterized in their (<i>E</i>)- and (<i>Z</i>)-form for their functional inhibitory activity at γ-aminobutyric acid transporters subtype 1 (GAT1), the most common γ-aminobutyric acid (GABA) transporter subtype in the central nervous system (CNS). This led to the identification of the first photoswitchable ligands exhibiting a moderate uptake inhibition of GABA in their (<i>E</i>)- but distinctive higher inhibitory potency in their (<i>Z</i>)-form resulting from photoirradiation. For the most efficient photoactivatable nipecotic acid derivative displaying an <i>N</i>-but-3-yn-1-yl linker with a terminal diphenyldiazene unit, an inhibitory potency of 4.65 ± 0.05 (pIC₅₀) was found for its (<i>E</i>)-form. which increased by almost two log units up to 6.38 ± 0.04 when irradiated. The effect of this photoswitchable mGAT1 inhibitor has also been evaluated and confirmed in patch-clamp recordings in acute hippocampal slices from mice

Development of New Photoswitchable Azobenzene Based γ‑Aminobutyric Acid (GABA) Uptake Inhibitors with Distinctly Enhanced Potency upon Photoactivation

A series of nipecotic acid derivatives with new azo benzene based photoswitchable N-substituents was synthesized and characterized in their (<i>E</i>)- and (<i>Z</i>)-form for their functional inhibitory activity at γ-aminobutyric acid transporters subtype 1 (GAT1), the most common γ-aminobutyric acid (GABA) transporter subtype in the central nervous system (CNS). This led to the identification of the first photoswitchable ligands exhibiting a moderate uptake inhibition of GABA in their (<i>E</i>)- but distinctive higher inhibitory potency in their (<i>Z</i>)-form resulting from photoirradiation. For the most efficient photoactivatable nipecotic acid derivative displaying an <i>N</i>-but-3-yn-1-yl linker with a terminal diphenyldiazene unit, an inhibitory potency of 4.65 ± 0.05 (pIC₅₀) was found for its (<i>E</i>)-form. which increased by almost two log units up to 6.38 ± 0.04 when irradiated. The effect of this photoswitchable mGAT1 inhibitor has also been evaluated and confirmed in patch-clamp recordings in acute hippocampal slices from mice

Development of New Photoswitchable Azobenzene Based γ‑Aminobutyric Acid (GABA) Uptake Inhibitors with Distinctly Enhanced Potency upon Photoactivation

A series of nipecotic acid derivatives with new azo benzene based photoswitchable N-substituents was synthesized and characterized in their (<i>E</i>)- and (<i>Z</i>)-form for their functional inhibitory activity at γ-aminobutyric acid transporters subtype 1 (GAT1), the most common γ-aminobutyric acid (GABA) transporter subtype in the central nervous system (CNS). This led to the identification of the first photoswitchable ligands exhibiting a moderate uptake inhibition of GABA in their (<i>E</i>)- but distinctive higher inhibitory potency in their (<i>Z</i>)-form resulting from photoirradiation. For the most efficient photoactivatable nipecotic acid derivative displaying an <i>N</i>-but-3-yn-1-yl linker with a terminal diphenyldiazene unit, an inhibitory potency of 4.65 ± 0.05 (pIC₅₀) was found for its (<i>E</i>)-form. which increased by almost two log units up to 6.38 ± 0.04 when irradiated. The effect of this photoswitchable mGAT1 inhibitor has also been evaluated and confirmed in patch-clamp recordings in acute hippocampal slices from mice

Identification of a Role for the Ventral Hippocampus in Neuropeptide S-Elicited Anxiolysis

<div><p>Neuropeptide S (NPS) increasingly emerges as a potential novel treatment option for anxiety diseases like panic and posttraumatic stress disorder. However, the neural underpinnings of its anxiolytic action are still not clearly understood. Recently, we reported that neurons of the ventral hippocampus (VH) take up intranasally administered fluorophore-conjugated NPS and, moreover, that application of NPS to mouse brain slices affects neurotransmission and plasticity at hippocampal CA3-CA1 synapses. Although these previous findings define the VH as a novel NPS target structure, they leave open whether this brain region is directly involved in NPS-mediated anxiolysis and how NPS impacts on neuronal activity propagation in the VH. Here, we fill this knowledge gap by demonstrating, first, that microinjections of NPS into the ventral CA1 region are sufficient to reduce anxiety-like behavior of C57BL/6N mice and, second, that NPS, via the NPS receptor, rapidly weakens evoked neuronal activity flow from the dentate gyrus to area CA1 <i>in vitro</i>. Additionally, we show that intranasally applied NPS alters neurotransmission and plasticity at CA3-CA1 synapses in the same way as NPS administered to hippocampal slices. Thus, our study provides, for the first time, strong experimental evidence for a direct involvement of the VH in NPS-induced anxiolysis and furthermore presents a novel mechanism of NPS action.</p> </div

HAB (black circles) and NAB (open circles) mice exhibit differences in electrophysiological properties in the vHPC.

<p>(<i>A</i>) HAB mice show weaker basal neurotransmission since the input-output curve is shifted towards smaller fEPSP amplitudes compared to NAB animals. (<i>B</i>) PPF is stronger in HAB compared to NAB animals at interstimulus-intervals of 25, 50, 100, and 200 ms, suggesting a lower probability of neurotransmitter release. (<i>C</i>) LTP at ventral CA3-CA1 synapses is more pronounced in slices from HAB mice. For clarity, stimulus artifacts in recording traces (<i>A</i>) were truncated in part. <i>n</i> numbers are indicated in brackets as follows (slices / animals).</p

NPS rapidly weakens evoked neuronal activity flow from the DG to area CA1 in hippocampal slices prepared from C57BL/6N mice.

<p>A, (Upper panel) Illustration of the position of the stimulation electrode (Stim) and the three ROIs used for the calculation of neuronal population activity within the dentate hilus, the CA3 subfield, and area CA1. (Lower panels) Representative filmstrips depicting the propagation of VSDI signals from the DG to the CA1 region before (‘Baseline’) and after bath application of 1 µM NPS (‘NPS’). Warmer colors represent stronger neuronal activity. Time specifications are given relative to the electrical stimulation pulse. B, Time course of the experiments depicted for the CA1 output subfield of the VH. NPS (1 µM) decreased FDS peak amplitudes (84±5% of baseline, n = 7 slices from 6 mice). This effect was completely abolished by pretreatment (15 min) of slices with the specific NPSR antagonist (R)-SHA 68 (10 µM) (101±3% of baseline, n = 5 slices from 4 mice). Data were normalized to the mean FDS peak amplitude of the last two acquisitions during baseline recording. C, Quantification of NPS effects on FDS peak amplitudes in the dentate hilus, the CA3 region, and area CA1. NPS decreased FDS peak amplitudes in the dentate hilus to 86±2% (p<0.001, t = 8.357, df = 6), in CA3 to 93±3% (p = 0.037, t = 2.664, df = 6), and in CA1 to 84±5% (p = 0.019, t = 3.165, df = 6) of baseline values. These effects were completely abolished by the NPSR antagonist (R)-SHA 68 (10 µM). Statistical evaluation was performed by comparing the mean FDS peak amplitudes of the last two acquisitions during baseline recording with the mean FDS peak amplitudes of the last two acquisitions during application of NPS.</p

Cy3-NPS is locally restricted to the site of injection into area CA1 of the VH.

<p>A, Injection site on an anatomical plate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060219#pone.0060219-Franklin1" target="_blank">[24]</a>. Overlay of DAPI (nuclear staining, blue) and Cy3-NPS (red) signals. Arrow indicates the injection site in the brain section. B, Anatomical plate showing the lateral (LA) and basolateral (BLA) amygdala, and overview of the amygdala in a brain section after Cy3-NPS injection (inset: Cy3 channel only). n = 4. Scale bars, 200 and 20 µm.</p

Intranasally applied NPS (open diamonds and open circles) causes enhanced basal neurotransmission (<i>A</i>) and reduced PPF (<i>B</i>) and LTP (<i>C</i>) at ventral CA3-CA1 synapses in HAB and NAB mice compared to Veh-treated (black diamonds and black circles) animals.

<p><i>n</i> numbers are indicated in brackets as follows (slices / animals). ★ Comparison NAB Veh <i>vs</i>. NAB NPS. $ Comparison HAB Veh <i>vs</i>. HAB NPS.</p

EPM parameters of the HAB (<i>n</i> = 15) and NAB (<i>n</i> = 12) mice used for the electrophysiological experiments shown in Fig. 2.

<p>EPM testing time was 5 min.</p