A single polycystic kidney disease 2-like 1 channel opening acts as a spike generator in cerebrospinal fluid-contacting neurons of adult mouse brainstem

Highlights

• In vertebrates CSF contacting neurons (CSF-cNs) are found around the central canal.

• They selectively express a spontaneously active unitary current carried by PKD2L1.

• Extracellular acidification activates ASICs while alkalinization enhances PKD2L1 activity.

• PKD2L1 is capable at the level of a single channel of triggering action potential.

• Increased PKD2L1 activity directly enhances CSF contacting neurons excitability.

Abstract

Cerebrospinal fluid contacting neurons (CSF-cNs) are found around the central canal of all vertebrates. They present a typical morphology, with a single dendrite that projects into the cavity and ends in the CSF with a protuberance. These anatomical features have led to the suggestion that CSF-cNs might have sensory functions, either by sensing CSF movement or composition, but the physiological mechanisms for any such role are unknown. This hypothesis was recently supported by the demonstration that in several vertebrate species medullo-spinal CSF-cNs selectively express Polycystic Kidney Disease 2-Like 1 proteins (PKD2L1). PKD2L1 are members of the ‘transient receptor potential (TRP)’ superfamily, form non-selective cationic channels of high conductance, are regulated by various stimuli including protons and are therefore suggested to act as sensory receptors.

Using patch-clamp whole-cell recordings of CSF-cNs in brainstem slices obtained from wild type and mutant PKD2L1 mice, we demonstrate that spontaneously active unitary currents in CSF-cNs are due to PKD2L1 channels that are capable, with a single opening, of triggering action potentials. Thus PKD2L1 might contribute to the setting of CSF-cN spiking activity. We also reveal that CSF-cNs have the capacity of discriminating between alkalinization and acidification following activation of specific conductances (PKD2L1 vs. ASIC) generating specific responses. Altogether, this study reinforces the idea that CSF-cNs represent sensory neurons intrinsic to the central nervous system and suggests a role for PKD2L1 channels as spike generators.

Introduction

Medullo-spinal cerebrospinal fluid contacting neurons (CSF-cNs) are present around the ependymal layer along the central canal from the filum terminale to the brainstem of all vertebrates examined so far (Vigh et al., 2004). CSF-cNs are GABAergic neurons and have a characteristic morphology with a unique dendrite that ends in the CSF with a ciliated protuberance (Vigh et al., 1983, Shimosegawa et al., 1986, Bruni and Reddy, 1987, Dale et al., 1987, Stoeckel et al., 2003, Orts-Del'Immagine et al., 2014). Although, little information is available regarding their function(s), CSF-cNs were suggested to have sensory functions by sensing either CSF movement within the central canal or variations in its composition, notably its pH (Huang et al., 2006). To support this hypothesis, recent data demonstrated that the Polycystic Kidney Disease 2-Like1 protein (PKD2L1), a channel with putative sensory functions (Nauli et al., 2003, Huang et al., 2006, Shimizu et al., 2009), represented a selective marker for medullo-spinal CSF-cNs in several vertebrate species (Djenoune et al., 2014, Orts-Del'Immagine et al., 2014).

PKD2L1 (or TRPP3 and initially named Polycystin-L) is a member of the polycystin family of TRP proteins (Clapham et al., 2010). Mutations of pkd1 and pkd2 genes coding for, polycystin-1 and polycystin-2, respectively, account for almost all cases of autosomal dominant polycystic kidney disease (ADPKD), the most common form of inherited polycystic kidney disease (Harris and Torres, 2014). pkd2l1 and pkd2l2 are two homologs of the pkd2 gene identified so far (Veldhuisen et al., 1999, Wu et al., 1998) but are unlikely to be directly linked to ADPKD (Basora et al., 2002, Nomura et al., 1998). Although the functional role of PKD2L1 is still unclear, it was suggested that PKD2L1 might be involved in sensory physiology. Indeed, PKD2L1 has a wide expression pattern especially in several brain nuclei and sensory organs such as retina, taste bud and inner ear (Basora et al., 2002, Huang et al., 2006, Ishimaru et al., 2006, LopezJimenez et al., 2006, Cuajungco et al., 2007, Li et al., 2007, Takumida and Anniko, 2010). Second, in expression systems, PKD2L1 forms a non-selective cationic channel of high conductance and was shown to be regulated by several stimuli such as: voltage, calcium (Chen et al., 1999, Liu et al., 2002), protons (Shimizu et al., 2011), extracellular osmolarity (Shimizu et al., 2009) and temperature (Higuchi et al., 2014). Finally, the levels of channel insertion in the plasma membrane as well as its functional properties were shown to depend on its association with other proteins in particular of the polycystin 1 type (Inada et al., 2008, Ishii et al., 2009, DeCaen et al., 2013, Delling et al., 2013).

In murine medullar CSF-cNs, we recently reported the presence of a spontaneously active unitary current bearing all the functional properties of PKD2L1 currents and we suggested that its activation could modulate CSF-cN excitability (Orts-Del'immagine et al., 2012). Nevertheless, the nature of the channels expressed in CSF-cNs could not be definitively demonstrated because of the lack of a selective blocker for PKD2L1 channels.

Here, using patch-clamp recording techniques on brainstem slices prepared from PKD2L1 mice lacking the channel and their wild type littermates (Horio et al., 2011), we demonstrate that functional PKD2L1 channels are indeed expressed in CSF-cNs. They are capable, at a single channel level, to generate a depolarization large enough to trigger action potentials and would act as spike generator. They play a role in the setting of basal excitability and in sensing extracellular variations in pH. Finally, and because of the lack of any excitatory synaptic entries, PKD2L1 appears to represent an important excitatory input to these peculiar neurons.