GABA_B receptors modulate Ca²⁺ but not G protein‐gated inwardly rectifying K⁺ channels in cerebrospinal‐fluid contacting neurones of mouse brainstem

Abstract

Key points Medullo‐spinal CSF contacting neurones (CSF‐cNs) located around the central canal are conserved in all vertebrates and suggested to be a novel sensory system intrinsic to the CNS. CSF‐cNs receive GABAergic inhibitory synaptic inputs involving ionotropic GABAA receptors, but the contribution of metabotropic GABAB receptors (GABAB‐Rs) has not yet been studied. Here, we indicate that CSF‐cNs express functional GABAB‐Rs that inhibit postsynaptic calcium channels but fail to activate inhibitory potassium channel of the Kir3‐type. We further show that GABAB‐Rs localise presynaptically on GABAergic and glutamatergic synaptic inputs contacting CSF‐cNs, where they inhibit the release of GABA and glutamate. Our data are the first to address the function of GABAB‐Rs in CSF‐cNs and show that on the presynaptic side they exert a classical synaptic modulation whereas at the postsynaptic level they have an atypical action by modulating calcium signalling without inducing potassium‐dependent inhibition. Abstract Medullo‐spinal neurones that contact the cerebrospinal fluid (CSF‐cNs) are a population of evolutionary conserved cells located around the central canal. CSF‐cN activity has been shown to be regulated by inhibitory synaptic inputs involving ionotropic GABAA receptors, but the contribution of the G‐protein coupled GABAB receptors has not yet been studied. Here, we used a combination of immunofluorescence, electrophysiology and calcium imaging to investigate the expression and function of GABAB‐Rs in CSF‐cNs of the mouse brainstem. We found that CSF‐cNs express GABAB‐Rs, but their selective activation failed to induce G protein‐coupled inwardly rectifying potassium (GIRK) currents. Instead, CSF‐cNs express primarily N‐type voltage‐gated calcium (CaV2.2) channels, and GABAB‐Rs recruit Gβγ subunits to inhibit CaV channel activity induced by membrane voltage steps or under physiological conditions by action potentials. Moreover, using electrical stimulation, we indicate that GABAergic inhibitory (IPSCs) and excitatory glutamatergic (EPSCs) synaptic currents can be evoked in CSF‐cNs showing that mammalian CSF‐cNs are also under excitatory control by glutamatergic synaptic inputs. We further demonstrate that baclofen reversibly reduced the amplitudes of both IPSCs and EPSCs evoked in CSF‐cNs through a presynaptic mechanism of regulation. In summary, these results are the first to demonstrate the existence of functional postsynaptic GABAB‐Rs in medullar CSF‐cNs, as well as presynaptic GABAB auto‐ and heteroreceptors regulating the release of GABA and glutamate. Remarkably, postsynaptic GABAB‐Rs associate with CaV but not GIRK channels, indicating that GABAB‐Rs function as a calcium signalling modulator without GIRK‐dependent inhibition in CSF‐cNs.

Graded spikes differentially signal neurotransmitter input in cerebrospinal fluid contacting neurons of the mouse spinal cord

“…How might the different spike types we describe contribute to transmitter release? Typically, the HVA P/Q and N-type Ca 2+ channels are coupled to transmitter release in neural cells and N-type channels are present in CSFcNs (Jurčić et al, 2019). However, numerous examples of T-type Ca 2+ channel activity governing transmitter release exist, including in: neuroendocrine cells in the pituitary (Tomić et al, 1999), adrenal glands (Mlinar et al, 1993), retinal bipolar cells (Pan et al, 2001) and olfactory bulb neurons (Johnston and Delaney, 2010; Fekete et al, 2014).…”

“…Such dual modes of release have been described in chick auditory hair cells where T-type Ca 2+ currents regulate rapid vesicle release and L-type Ca 2+ channels regulate sustained neurotransmitter release (Levic and Dulon, 2012). Furthermore, the N-type Ca 2+ channel in medullar CSFcNs can be modulated by GABA B receptors (Jurčić et al, 2019), which provides further means for CSFcNs to modulate their spike properties dependent on their synaptic input.…”

GABA excitatory actions in cerebrospinal-fluid contacting neurones of adult mouse spinal cord

“…Voltage-gated K + channels were activated with voltage ramps (-140 to +200 mV for 100 ms) applied every 1 s, in control and during pressure application of GABA (1 mM, 10 s duration). K + currents were recorded to prevent their inhibitory effect on voltage-gated calcium channels (Jurčić et al, 2019). In experiments with calcium-free aCSF (0-Ca 2+ ), slices were perfused with external solution containing (in mM): NaCl 115, NaHCO3 26, NaH2PO4 1.25, KCl 3, MgSO4 4, glucose 15, ascorbic acid 2, Napyruvate 2, myo-Inositol 3, EGTA 0.2.…”

“…PKD2L1 are non-selective cationic channels and act as a potential generator in CSF-cNs for action potentials firing, suggesting a key role of PKD2L1 in tuning their excitability (Orts-Del'Immagine et al, 2016). CSF-cNs excitability is also adjusted by GABAergic synaptic inputs involving ionotropic GABAA-Rs as well as G proteincoupled metabotropic GABAB receptors (GABAB-Rs) (Orts-Del'immagine et al, 2012;Jurčić et al, 2019). Despite these apparent hallmarks of functionally mature neurones, CSF-cNs were also shown to express molecular markers of immaturity even into adulthood, such as the migrating and neuronal shaping proteins doublecortin (DCX) and PSA-NCAM (polysialylated neuronal cell adhesion molecule) as well as the homeobox protein Nkx6.1 (Stoeckel et al, 2003b;Shechter et al, 2007;Orts-Del'immagine et al, 2014).…”

Respiratory rhythm generation and pattern formation: oscillators and network mechanisms

“…ated by ligand binding through distal intracellular signal transduction effects (Fritschy et al, 1999). The GABAB receptor demonstrates wide expressivity throughout the cerebrum (Connors, 1992;Kuriyama et al, 1993;Malcangio and Bowery, 1995;Sil'kis, 1996), brainstem (Burman et al, 2003;Jurčić et al, 2019;Pfeiffer and Zhang, 2007), cerebellum (Kuriyama et al, 1993;Malcangio and Bowery, 1995), and spinal cord (Lev-Tov et al, 1990;Malcangio and Bowery, 1995), as well as cells of a plethora of peripheral effect organs (Castelli et al, 1999), including pan-creatic beta islet cells (Braun et al, 2004;Crivello et al, 2013), adrenocortical cells (Häusler et al, 1993;Metzeler et al, 2004), cardiomyocytes (Lorente et al, 2000), chondrocytes (Tamayama et al, 2005), and osteoblasts (Fujimori et al, 2002). Studies have variably elucidated the functional significance of these peripheral GABAB receptors in generating and modulating the breathing rhythm and neural circuitry underlying the genesis of various rhythmic and patterned behavioral outputs.…”