In vivo labeling and in vitro characterisation of central complex neurons involved in the control of sound production.

Injection of muscarine into the central complex of the grasshopper brain can stimulate species-specific sound production through activation of the phospholipase C-initiated transduction pathway. We introduce a strategy, to label central complex interneurons that are directly stimulated by the injected muscarine and to study their physiology in dissociated primary cell culture. Fluorescent dextranes, co-injected to brain sites where muscarine stimulates sound production, are incorporated from the extracellular space by 3-14 central complex neurons. Most labeled neurons are columnar neurons that express muscarinic acetylcholine receptors. An average of 3-4 dextrane-labeled central complex neurons per brain can be recognised by their fluorescence in dissociated cell cultures. Their function as potential direct targets of previous in vivo pharmacological stimulation of the intact brain was supported by expression of muscarinic receptors in cytomembranes of isolated neuronal cell bodies and muscarine-stimulated calcium responses in vitro. Pharmacological inhibition of phospholipase C function and removal of extracellular calcium indicated that release from inositolphosphate-regulated internal stores mediates the increase of cytosolic calcium concentrations. The experimental procedures described in this study can be applied to any preparation in which focal drug application elicits, terminates or modulates behavior in order to label and physiologically analyse those interneurons within the circuit that serve as direct targets of the injected drug.

Synaptic SAP97 isoforms regulate AMPA receptor dynamics and access to presynaptic glutamate.

The synaptic insertion of GluR1-containing AMPA-type glutamate receptors (AMPARs) is critical for synaptic plasticity. However, mechanisms responsible for GluR1 insertion and retention at the synapse are unclear. The synapse-associated protein SAP97 directly binds GluR1 and participates in its forward trafficking from the Golgi network to the plasma membrane. Whether SAP97 also plays a role in scaffolding GluR1 at the postsynaptic membrane is controversial, attributable to its expression as a collection of alternatively spliced isoforms with ill-defined spatial and temporal distributions. In the present study, we have used live imaging and electrophysiology to demonstrate that two postsynaptic, N-terminal isoforms of SAP97 directly modulate the levels, dynamics, and function of synaptic GluR1-containing AMPARs. Specifically, the unique N-terminal domains confer distinct subsynaptic localizations onto SAP97, targeting the palmitoylated alpha-isoform to the postsynaptic density (PSD) and the L27 domain-containing beta-isoform primarily to non-PSD, perisynaptic regions. Consequently, alpha- and betaSAP97 differentially influence the subsynaptic localization and dynamics of AMPARs by creating binding sites for GluR1-containing receptors within their respective subdomains. These results indicate that N-terminal splicing of SAP97 can control synaptic strength by regulating the distribution of AMPARs and, hence, their responsiveness to presynaptically released glutamate.

Astrocytic calcium signals induced by neuromodulators via functional metabotropic receptors in the ventral respiratory group of neonatal mice.

A controlled, periodic exchange of air between lungs and atmosphere requires a neuronal rhythm generated by a network of neurons in the ventral respiratory group (VRG) of the brainstem. Glial cells, e.g. astrocytes, have been shown to be supportive in stabilizing this neuronal activity in the central nervous system during development. In addition, a variety of neuromodulators including serotonin (5-HT), Substance P (SP), and thyrotropin-releasing hormone (TRH) stimulate respiratory neurons directly. If astrocytes in the VRG, like their neuronal neighbors, are also directly stimulated by neuromodulators, they might indirectly affect the respiratory neurons and consequently the respiratory rhythm. In the present study, we provide support for this concept by demonstrating expression of NK1-R, TRH-R, and 5-HT(2)-R in astrocytes of the VRG with immunohistochemistry. Additionally, we showed that the external application of the neuromodulators 5-HT, SP, and TRH activate calcium transients in VRG astrocytes. Consequently, we postulate that in the VRG of the neonatal mouse, neuromodulation by SP, TRH, and serotonin also involves astrocytic calcium signaling.

Possible roles of the weakly inward rectifying k+ channel Kir4.1 (KCNJ10) in the pre-Bötzinger complex.

Rhythmic activity of respiratory neurons is dependent on the clearance of neurotransmitter by astrocytes. Astrocytes should also be involved in the permanent and rapid clearance of extracellular ions. We analyzed the expression of the weakly inwardly rectifying K+ channel Kir4.1 (KCNJ10) in the respiratory network and studied the possible functions for neuronal activity in the pre-Bötzinger complex.

Calcium influx mediated by the inwardly rectifying K+ channel Kir4.1 (KCNJ10) at low external K+ concentration.

COS-1 cells with heterologeous expression of the Kir4.1 (KCNJ10) channel subunit, possess functional Kir4.1 channels and become capable to generating cytosolic Ca2+ transients, upon lowering of the extracellular K+ concentration to 2 mM or below. These Ca2+ transients are blocked by external Ba2+ (100 microM). Acute brain stem slices from wild-type mice (second post-natal week), which were loaded with the fluorescent Ca2+ indicator Oregon Green BAPTA-1-AM, were exposed to 0.2 mM K+. Under these conditions astrocytes, but not neurons, responded with cytosolic Ca2+ elevations in wild-type mice. This astrocyte-specific response has previously been used to identify astroglial cells type [R. Dallwig, H. Vitten, J.W. Deitmer, A novel barium-sensitive calcium influx into rat astrocytes at low external potassium. Cell Calcium 28 (2000) 247-259]. In Kir4.1 knock-out (Kir4.1-/-) mice, the number of responding cells was dramatically reduced and the Ca2+ transients in responding cells were significantly smaller than in wild-type mice. Our results indicate that Kir4.1 channels are the molecular substrate for the observed Ca2+ influx in astrocytes under conditions of low external K+-concentration.

Glycine transporter 1 expression in the ventral respiratory group is restricted to protoplasmic astrocytes.

The lack of the glycine transporter 1 (GlyT1) leads to early postnatal death due to failure of respiratory network activity. Here we demonstrate a segregated expression of GlyT1 on different astroglial cell populations of the ventral respiratory group. In TgN(hGFAP-EGFP) mice a combined immunohistochemical and electrophysiological approach was used to define the cellular expression of GlyT1 in the respiratory network. EGFP-labeled cells with outwardly rectifying current-voltage relationship did not express glycine transporter 1, while GlyT1 was abundantly expressed in mature protoplasmic astrocytes, which are electrophysiologically characterized by a large potassium conductance, a more negative membrane potential and the expression of glutamate transporters. Taken together, the vital capacity for the clearance of extracellular glycine is restricted to a subpopulation of astroglial cells.