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The concept of the tripartite synapse describes the close interaction of pre- and postsynaptic elements and the surrounding astrocyte processes. For glutamatergic synapses, it is established that the presence of astrocytic processes and their structural arrangements varies considerably between and within brain regions and between synapses of the same neuron. In contrast, less is known about the organization of astrocytic processes at GABAergic synapses although bi-directional signaling is known to exist at these synapses too. Therefore, we established super-resolution expansion microscopy of GABAergic synapses and nearby astrocytic processes in the stratum radiatum of the mouse hippocampal CA1 region. By visualizing the presynaptic vesicular GABA transporter and the postsynaptic clustering protein gephyrin, we documented the subsynaptic heterogeneity of GABAergic synaptic contacts. We then compared the volume distribution of astrocytic processes near GABAergic synapses between individual synapses and with glutamatergic synapses. We made two novel observations. First, astrocytic processes were more abundant at the GABAergic synapses with large postsynaptic gephyrin clusters. Second, astrocytic processes were less abundant in the vicinity of GABAergic synapses compared to glutamatergic, suggesting that the latter may be selectively approached by astrocytes. Because of the GABA transporter distribution, we also speculate that this specific arrangement enables more efficient re-uptake of GABA into presynaptic terminals.

While the bladder vasculature is considered as a target of PDE5 inhibitors to improve bladder storage dysfunctions, its characteristics are largely unknown. Thus, the functional and morphological properties of arteries/arterioles of the bladder focusing on the NO-mediated signal transmission were explored.Diameter changes in rat bladder arteries/arterioles were measured using a video-tracking system. Intercellular CaIn rat suburothelial arterioles and vesical arteries, tadalafil (100 nM) attenuated nerve-evoked sympathetic vasoconstrictions. In both vascular segments, tadalafil-induced inhibition of sympathetic vasoconstriction was prevented by N ω-propyl-l-arginine hydrochloride (l-NPA, 1 μM), an nNOS inhibitor or N ω-nitro-l-arginine (l-NA, 100 μM). Both vascular segments were densely innervated with nNOS-positive nitrergic nerves in close apposition to tyrosine hydroxylase-immunoreactive sympathetic nerves. In pericyte-covered pre-capillary arterioles of the mouse bladder where sympathetic nerves were absent, nerve stimulation evoked transient reductions in pericyte CaBoth nitrergic nerve and nerve-evoked endothelial NO release appear to be involved in vasodilatory signal transmission in bladder vasculature. The NO-mediated signal transmission is a potential target for PDE5 inhibitor therapy in bladder dysfunctions.

Photoswitchable bioprobes enable bidirectional control of cell physiology with different wavelengths of light. Many current photoswitches use cytotoxic UV light and are limited by the need for constant illumination owing to thermal relaxation in the dark. Now a photoswitchable tetrafluoroazobenzene(4FAB)-based ion channel antagonist has been developed that can be efficiently isomerized in two separate optical channels with visible light. Importantly, the metastable cis configuration showed very high stability in the dark over the course of days at room temperature. In neurons, the 4FAB antagonist reversibly blocks voltage-gated ion channels with violet and green light. Furthermore, photoswitching could also be achieved with two-photon excitation yielding high spatial resolution. 4FAB probes have the potential to enable long-term biological studies where both ON and OFF states can be maintained in the absence of irradiation.

Summary: Synaptically released glutamate is largely cleared by glutamate transporters localized on perisynaptic astrocyte processes. Therefore, the substantial variability of astrocyte coverage of individual hippocampal synapses implies that the efficacy of local glutamate uptake and thus the spatial fidelity of synaptic transmission is synapse dependent. By visualization of sub-diffraction-limit perisynaptic astrocytic processes and adjacent postsynaptic spines, we show that, relative to their size, small spines display a stronger coverage by astroglial transporters than bigger neighboring spines. Similarly, glutamate transients evoked by synaptic stimulation are more sensitive to pharmacological inhibition of glutamate uptake at smaller spines, whose high-affinity N-methyl-D-aspartate receptors (NMDARs) are better shielded from remotely released glutamate. At small spines, glutamate-induced and NMDAR-dependent Ca2+ entry is also more strongly increased by uptake inhibition. These findings indicate that spine size inversely correlates with the efficacy of local glutamate uptake and thereby likely determines the probability of synaptic crosstalk.

Caged compounds enable fast, light-induced, and spatially-defined application of bioactive molecules to cells. Covalent attachment of a caging chromophore to a crucial functionality of a biomolecule renders it inert, while short pulses of light release the caged molecule in its active form. Caged neurotransmitters have been widely used to study diverse neurobiological processes such as receptor distribution, synaptogenesis, transport, and long-term potentiation. Since the neurotransmitters glutamate and gamma-aminobutyric acid (GABA) are the most important, they have been studied extensively using uncaging. However, to be able to probe their interactions on a physiologically relevant timescale, fast and independent application of both neurotransmitters in an arbitrary order is desired. This can be achieved by combining two caging chromophores absorbing non-overlapping and thus orthogonal wavelengths of light, which enables the precise application of two caged molecules to the same preparation in any order, a technique called two-color uncaging. In this chapter, we describe the principles of orthogonal two-color uncaging with one- and two-photon excitation with an emphasis on caged glutamate and GABA. We then give a guide to its practical application and highlight some key studies utilizing this technique.

KEY POINTS: A new caged nicotinic acetylcholine receptor (nAChR) agonist was developed, ABT594, which is photolysed by one‐ and two‐photon excitation. The caged compound is photolysed with a quantum yield of 0.20. One‐photon uncaging of ABT594 elicited large currents and Ca(2+) transients at the soma and dendrites of medial habenula (MHb) neurons of mouse brain slices. Unexpectedly, uncaging of ABT594 also revealed highly Ca(2+)‐permeable nAChRs on axons of MHb neurons. ABSTRACT: Photochemical release of neurotransmitters has been instrumental in the study of their underlying receptors, with acetylcholine being the exception due to its inaccessibility to photochemical protection. We caged a nicotinic acetylcholine receptor (nAChR) agonist, ABT594, via its secondary amine functionality. Effective photolysis could be carried out using either one‐ or two‐photon excitation. Brief flashes (0.5–3.0 ms) of 410 nm light evoked large currents and Ca(2+) transients on cell bodies and dendrites of medial habenula (MHb) neurons. Unexpectedly, photorelease of ABT594 also revealed nAChR‐mediated Ca(2+) signals along the axons of MHb neurons.

A subset of hippocampal GABAergic neurons, which are cholecystokinin-positive, highly express cannabinoid type 1 (CB1) receptors. Activation of these receptors inhibits GABA release and thereby limits inhibitory control. While genetic deletion of CB1 receptors from GABAergic neurons led to behavioural alterations and neuroinflammatory reactions, it remained unclear whether these changes in the knockout animals were a direct consequence of the enhanced transmitter release or reflected developmental deficits. The hippocampus is vital for the generation of spatial, declarative and working memory. Here, we addressed the question how CB1 receptors in GABAergic neurons influence hippocampal function. Patch clamp and field potential recordings in mice devoid of CB1 receptors in GABAergic neurons revealed an enhanced frequency and faster kinetics of spontaneous inhibitory postsynaptic currents in CA1 pyramidal neurons while tonic inhibition, paired-pulse facilitation and long-term potentiation in the hippocampus were not affected. Evaluation of cognitive functions demonstrated impaired acquisition of spatial memory and deficits in novel object recognition and partner recognition in the knockout mice, while working memory and spatial memory remained intact. The density of GABAergic neurons was also similar in knockout mice and their littermates, which argues against global deficits in hippocampal development. Together, these results suggest that CB1 receptors in GABAergic neurons influence specific aspects of neuronal excitability and hippocampal learning.