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A recent discovery: the cerebrospinal fluid-contacting neurons form an interoceptive system in the spinal cord

The cerebrospinal fluid (CSF) is a physiological fluid circulating throughout the different brain ventricles, the subarachnoid space and the central canal of the spinal cord, and is secreted by the choroid plexuses in brain ventricles (Figure 8). Classically, CSF has been known to hydro-mechanically protect the brain, as well as to maintain brain homeostasis. The CSF contains molecules crucial for central nervous system development (Gato et al., 2005; Lehtinen et al., 2011), and plays a role in metabolites clearance during sleep in mice (Xie et al., 2013).
CSF also contains a large variety of active neuromodulators: neurotransmitters like acetylcholine (Welch, Markham and Jenden, 1976), dopamine, noradrenaline and serotonin (Strittmatter et al., 1997); neuropeptides like somatostatin, neuropeptide Y (Nilsson et al., 2001), vasopressin and oxytocin (Martin et al., 2014); but also neurosteroids (Kim, Creekmore and Vezina, 2003; Meletti et al., 2017), purines (Schmidt et al., 2015), and endocannabinoids (Romigi et al., 2010). Besides its passive role, CSF could also have an active role in signaling different neurons of the central nervous system. Then, we can ask about the potential targets for CSF active molecules, which cells detect and respond to these molecules?

Definition of the cerebrospinal fluid-contacting neurons

Kolmer (Kolmer, 1921, 1931) and Agduhr (Agduhr, 1922) described a century ago, in over a hundred vertebrates, neurons in contact with the cerebrospinal fluid that they called cerebrospinal fluid-contacting neurons (CSF-cNs, also known as Kolmer-Agduhr “KA” cells) (Figure 9A). Based on their location at the interface between the central nervous system and the CSF (Figure 9), CSF-cNs could sense the active molecules of the CSF. In their description, Kolmer and Agduhr hypothesized that CSF-cNs may be sensory cells forming a sensory organ detecting changes in the CSF. Since their discovery, CSF-cNs have been extensively studied to investigate this question, with the help of recent technological progress in genetics or optical tools notably.
CSF-cNs are located in the spinal cord, and different structures of the brain, like the subcomissural organ (SCO), the hypophysis, the retina, the paraventricular organ or the pineal gland for examples (Vigh and Vigh-Teichmann, 1981, 1998; Vígh et al., 2004). Depending on the location, CSF-cNs display different morphologies and express different markers, suggesting that they might carry different functions. For example, the rat CSF-cNs of the brain mesencephalon have been found to express the TRPM8 receptor channel involved in cold sensation (Du et al., 2009), and so could be involved in cold detection. The CSF-cNs, described as forming a novel interoceptive sensory system, are the ones located in the spinal cord and will be called spinal CSF-cNs.
Spinal CSF-cN somata ultrastructure. The spinal CSF-cN soma is located in the intra or the sub-ependymal layers of the area around and including the central canal, called the central gelatinosa (Vigh and Vigh-Teichmann, 1973, 1998; Nagatsu et al., 1988). Spinal CSF-cN soma can exhibit a round or ovoid shape (Barber, Vaughn and Roberts, 1982; Jaeger et al., 1983; Shimosegawa et al., 1986; Dale et al., 1987; Stoeckel et al., 2003), as well as a triangular and fusiform shape (Barber, Vaughn and Roberts, 1982; Jaeger et al., 1983; Shimosegawa et al., 1986; Stoeckel et al., 2003). Compared to their neighboring cells in the central gelatinosa, spinal CSF-cNs are less-electron dense and possess a round to oval nucleus (Schueren and DeSantis, 1985; Alibardi, 1990; Marichal et al., 2009; Alfaro- Cervello et al., 2012). The soma of spinal CSF-cNs is commonly small with a diameter of around 10μm (Barber, Vaughn and Roberts, 1982; Nagatsu et al., 1988; Stoeckel et al., 2003; Orts-Del’Immagine et al., 2014), however larger spinal CSF-cN soma was described in rat with diameters of 13 to 28μm (Shimosegawa et al., 1986). In rat, fusiform spinal CSF-cNs with a diameter of 22μm were also described (Barber, Vaughn and Roberts, 1982). In the cytoplasm of spinal CSF-cNs, the presence of a rough endoplasmic reticulum (RER) (Barber, Vaughn and Roberts, 1982; Schueren and DeSantis, 1985; Alfaro-Cervello et al., 2012), as well as free ribosomes (Barber, Vaughn and Roberts, 1982; Alibardi, 1990; Marichal et al., 2009; Alfaro-Cervello et al., 2012) suggests a high level of protein synthesis. In the central canal, rat spinal CSF-cNs form apical zonulae adhaerens to link to ependymal cells (Stoeckel et al., 2003).
Spinal CSF-cN dendritic extension. Electron microscopy revealed that spinal CSF-cNs bear a dendritic apical extension protruding into the CSF in the central canal. This apical extension expresses the dendritic marker microtubule-associated protein2 (MAP2) (Djenoune et al., 2014; Orts-Del’Immagine et al., 2014). Spinal CSF-cNs apical extension slightly differs depending on the species, exhibiting or not multiple microvilli at the dendritic extension (Vigh and Vigh-Teichmann, 1973), as well as a cilium for some species like Xenopus (Dale et al., 1987), mice (Alfaro-Cervello et al., 2012) and zebrafish for example (Djenoune et al., 2017). Ultrastructure analysis in zebrafish showed that this brush of actin-based microvilli at the apical extension extends in the central canal and encircle a single cilium with a “9 + 2” microtubule organization, typical of a kinocilium. Further analysis in vitro and in vivo in zebrafish showed that this kinocilium is motile (Hubbard et al., 2016; Sternberg et al., 2018).
Spinal CSF-cN axonal projections. Spinal CSF-cNs project a single axon from the basal part of the cell body. In some species like lamprey or Xenopus, spinal CSF-cNs project axons into the ventrolateral margin of the spinal cord (Vigh, Vigh-Teichmann and Aros, 1977; Megías, Álvarez-Otero and Pombal, 2003), forming neurohormonal nerve endings attached by hemidesmosomes to the basal lamina of the spinal cord, allowing possible direct contact with the facing subarachnoid space CSF. In lamprey, spinal CSF-cNs also project into the lateral margin of the spinal cord where they can connect with intraspinal stretch receptors called the Edge cells (Christenson et al., 1991; Jalalvand et al., 2014), that can play a role of intraspinal mechanoreceptor (Grillner, Williams and Lagerbäck, 1984). In zebrafish, spinal CSF-cNs ventrally project ipsilateral ascending axons (Djenoune et al., 2014, 2017).
Molecular markers to label spinal CSF-cNs. Spinal CSF-cNs are gamma-aminobutyric acid (GABA)-ergic neurons, as it has been shown in multiple species like the rat (Barber, Vaughn and Roberts, 1982; Stoeckel et al., 2003), African clawed frog (Dale et al., 1987; Binor and Heathcote, 2001), lamprey (Christenson et al., 1991; Jalalvand, Robertson, Tostivint, et al., 2016), eel and trout (Roberts et al., 1995), dogfish (Sueiro et al., 2004), turtle (Reali et al., 2011) zebrafish (Bernhardt et al., 1992; Higashijima, Mandel and Fetcho, 2004; Higashijima, Schaefer and Fetcho, 2004; Wyart et al., 2009; Yang, Rastegar and Strähle, 2010; Djenoune et al., 2014), mouse and primate (Djenoune et al., 2014) (Figure 10). GABA expression is consistent in all spinal CSF-cNs and, coupled with their peculiar morphology, constituted for a long time the best way to identify the spinal CSF-cNs. However, to facilitate further investigation of spinal CSF-cNs, specific genetic markers were needed to specifically label all spinal CSF-cNs in the spinal cord. The marker of choice appeared to be the polycystic kidney disease 2-like 1 (PKD2L1) channel, a non-selective cationic channel that was found expressed in spinal CSF-cNs (Huang et al., 2006). Later on, PKD2L1 was also found expressed in spinal CSF-cNs in other species such as non-human primates (macaques, Djenoune et al., 2014) or zebrafish (Djenoune et al., 2014; Sternberg et al., 2018; Orts-Del’Immagine et al., 2020), and Pkd2l1 is co-expressed with Pkd1l2 protein (England et al., 2017) (Figure 10). Spinal CSF-cNs also express molecular markers of “immature” neurons, such as polysialylated neural cell adhesion molecule (PSA-NCAM), HuC/D, NKX6.1 and doublecortin (DCX) (Stoeckel et al., 2003; Russo et al., 2004; Marichal et al., 2009; Reali et al., 2011; Kútna et al., 2014; Orts-Del’Immagine et al., 2014). The specific marker PKD2L1 rapidly became the best way to label spinal CSF-cNs, allowing the generation of transgenic lines targeting spinal CSF-cNs that became essential for dissecting the functional roles of CSF-cNs, as well as their developmental origin.

Spinal CSF-cNs are mechanosensory cells

The spinal CSF-cNs extend a motile kinocilium into the CSF suggesting a mechanosensory function, as hypothesized by Kolmer a century ago. A first evidence of mechanosensory function in mice was whole-cell recordings showing that hypo-osmotic shocks increase the single-channel activity of the spinal CSF-cNs (Orts-Del’Immagine et al., 2012). In lamprey, spinal CSF-cNs respond to direct fluid pressure application (Jalalvand, Robertson, Wallén, et al., 2016). This mechanical detection is dependent on the ASIC3 channel, as APETx2 (an ASIC3 channel inhibitor) inhibits mechanosensory response to fluid pressure application (Jalalvand, Robertson, Wallén, et al., 2016). These data suggest a potential mechanical activation of spinal CSF-cNs by fluid pressure, maybe to detect changes in the CSF flow.
In zebrafish, spinal CSF-cNs were found to detect both passive and active spinal bending (Böhm et al., 2016; Hubbard et al., 2016). Spinal CSF-cNs are activated on the concave side of the tail bending, meaning that the cells respond to the mechanical compression applied with the bending, not the stretch (Figure 14A). Dorsal spinal CSF-cNs respond to lateral tail bending (Böhm et al., 2016), while ventral ones respond to longitudinal bending (Hubbard et al., 2016) (Figure 14A). These results are concordant with the evidence that spinal CSF-cNs in vitro respond to mechanical stimulation with a glass probe directly on the plasma membrane of the cell (Sternberg et al., 2018). Detection of mechanical cues in zebrafish is dependent on the PKD2L1 channel (Böhm et al., 2016; Sternberg et al., 2018), as PKD2L1 KO abolishes spinal CSF-cN responses to both lateral tail bending (Böhm et al., 2016) and glass probe mechanical stimulation (Sternberg et al., 2018). As spinal CSF-cNs respond to fluid pressure application (Jalalvand, Robertson, Wallén, et al., 2016), and that embryonic activity of spinal CSF-cNs in vivo correlates with the integrity of CSF flow (Sternberg et al., 2018), it was hypothesized that lateral tail bending induces a change in CSF flow that is detected by spinal CSF-cNs leading to their activation through PKD2L1 or ASIC3 ion channels. Recently, a study showed that spinal CSF-cNs mechanosensory functions rely on the Reissner fiber (RF) (Orts-Del’Immagine et al., 2020). The Reissner fiber is an agglomeration of the SCO-spondin glycoprotein, extending from the SCO in the third brain ventricle (where SCO-spondin is secreted) to the end of the spinal cord in the central canal.


The role of spinal CSF-cNs in the modulation of locomotion and posture in response to mechanical stimuli

In response to spinal mechanical stimuli, spinal CSF-cNs modulate locomotion and posture through the release of neurotransmitters such as GABA or somatostatin (Figure 15). The first evidence for spinal CSF-cN modulation of locomotion was the discovery that optogenetic activation of these neurons induces slow swim bouts in zebrafish (Wyart et al., 2009). Later on, it was discovered by combining pkd2l1 transgenic line, electrophysiology and optogenetics that dorsolateral spinal CSF-cNs form GABAergic synapses onto V0-v interneurons in zebrafish, an essential component of the slow locomotion (Fidelin et al., 2015; Djenoune et al., 2017) (Figure 15B). Through these connections, spinal CSF-cNs modulate slow locomotion in a state-dependent manner through the release of GABA (Fidelin et al., 2015). At rest, optogenetic activation of spinal CSF-cNs triggered delayed slow locomotion, such a delay let think that the locomotor response followed a long period of inhibition leading to a rebound swimming activity (Fidelin et al., 2015). During ongoing fictive locomotion, spinal CSF-cNs activation silenced locomotor activity through V0-v interneurons (Fidelin et al., 2015). In addition to GABA, a recent study showed that somatostatin 1.1 zebrafish KO mutant showed longer spontaneous swims, meaning that Somatostatin1.1 is also involved in slow locomotion inhibition (Quan et al., 2020).
Zebrafish spinal CSF-cNs also project GABAergic synapses onto fast motor neurons and excitatory sensory interneurons of the escape circuit, namely the caudal primary (CaP) motoneurons and the commissural primary ascending (CoPa) interneurons respectively (Figure 15A) (Hubbard et al., 2016; Djenoune et al., 2017). Activation of ventral spinal CSF-cNs was shown to directly silence the CaP motoneurons that innervate fast skeletal muscles (Hubbard et al., 2016). Silencing of neurotransmission in spinal CSF-cNs using botulinum toxin-induced postural defects during fast locomotion (Hubbard et al., 2016).
Recently, a study showed that rostralmost spinal CSF-cNs synapse onto occipital motor neurons in the caudal hindbrain, responsible for the control of head position (Wu et al., 2021) (Figure 15). Spinal rostralmost CSF-cNs also form axo-axonic synapses onto reticulospinal neurons located in the brain stem, that send descending information to the spinal cord, particularly to central pattern generators to control voluntary movements (Wu et al., 2021). Ablation of the hindbrain-projecting rostralmost spinal CSF-cNs induced defect of postural control, with more rolling events during acousto-vestibular (A-V) induced escapes (Wu et al., 2021). This result suggests that rostralmost spinal CSF-cNs send information to occipital motor neurons controlling head position, as well as to spinal motor neurons to maintain the stability of the fish during the initiation of the escape.

Are spinal CSF-cNs chemosensory cells forming a novel chemosensory system in the CNS?

The first evidence for a chemosensory role in spinal CSF-cNs came from the observation that pH stimulation of mice spinal CSF-cNs triggered action potentials (Huang et al., 2006). Further analysis in mice showed that spinal CSF-cNs detect both acidic and alkaline pH, through ASICs and PKD2L1 channels, respectively (Orts-Del’Immagine et al., 2012, 2016). Because their apical extensions directly bath into the CSF, we can hypothesize that spinal CSF-cNs can sense pH variations of the CSF itself. Recent evidence in rodents showed that spinal CSF-cNs can also respond to the neuromodulators acetylcholine (Corns et al., 2015; Johnson et al., 2020) and adenosine triphosphate (ATP) (Stoeckel et al., 2003; Johnson et al., 2020), but there is no evidence yet for a physiological relevance.
In other species, lamprey spinal CSF-cNs responded to pH acidification and alkalinization through ASICs and PKD2L1 channels (Jalalvand, Robertson, Tostivint, et al., 2016; Jalalvand, Robertson, Wallén, et al., 2016), confirming what was established in mice. Spinal CSF-cNs also detect acidic pH when protons are directly uncaged in the central canal of zebrafish (unpublished data, Böhm thesis, 2017).
In conclusion, spinal CSF-cNs can sense pH, potentially to detect CSF pH variations. Despite some evidence for detection by spinal CSF-cNs of neuromodulators, the chemosensory role of these neurons remains poorly understood. More investigations are required to decipher the role of spinal CSF-cNs in chemical detection from the CSF.

Table of contents :

Main Abbreviations
1/ Classical view: sensory information solely comes from the periphery to the spinal cord15
1.1) Dorsal root ganglia neurons
1.2) Cutaneous mechanoreceptors of touch
1.3) Cutaneous low threshold thermoreceptors
1.4) Proprioceptive DRG neurons (proprioceptors)
1.5) Nociceptive DRG neurons (nociceptors)
2/ A recent discovery: the cerebrospinal fluid-contacting neurons form an interoceptive system in the spinal cord
2.1) Definition of the cerebrospinal fluid-contacting neurons
2.2) Spinal CSF-cNs are mechanosensory cells
2.3) The role of spinal CSF-cNs in the modulation of locomotion and posture in response to mechanical stimuli
3/ Chemosensory systems in fish
3.1) The olfactory system
3.2) The gustatory system
3.3) The solitary chemosensory cells system
3.4) Are spinal CSF-cNs chemosensory cells forming a novel chemosensory system in the CNS?
Aim of the thesis
Chapter I: Development of a novel protocol for primary cell cultures of spinal CSF-cNs
1) Optimization of a new primary cell culture protocol of spinal CSF-cNs
1.1) Preparations prior to cell culture
1.2) Initial feeder layer of wild-type zebrafish cells
1.3) Second layer of fluorescently labeled spinal CSF-cNs
2) Electrophysiological properties of spinal CSF-cNs in vitro
2.1) In vitro spinal CSF-cNs exhibit characteristic high membrane resistance
2.2) Spinal CSF-cNs showed phasic and tonic firing in vitro
2.3) Spinal CSF-cNs conserved their channel opening properties in vitro
3) Molecular characterization of spinal CSF-cNs in vitro
Conclusion and perspectives
Chapter II: Transcriptome analysis of spinal CSF-cNs reveals numerous receptors to chemical cues
1) Transcriptome analysis of spinal CSF-cNs
1.1) Receptors for neurotransmitters and neuromodulators
1.2) Hormones receptors
1.3) Peptides receptors
1.4) Taste receptors
1.5) Immune-related receptors
2) Stimulation of spinal CSF-cNs with receptor agonists in vitro
Conclusion and perspectives
Chapter III: Sensory neurons detect pneumococci and promote survival in central nervous system infection
Sensory neurons detect pneumococci and promote survival in central nervous system infection
Discussion and perspectives
1) Spinal CSF-cNs as sensors for neuromodulators in the CSF
2) Spinal CSF-cNs are interoceptive chemosensory neurons involved in innate immunity
2.1) Spinal CSF-cNs detect bacterial metabolites during bacterial infection
2.2) Spinal CSF-cNs increase host survival during bacterial infection
2.3) What other immune-related metabolites are detected by spinal CSF-cNs?
3) Spinal CSF-cNs chemosensory functions in the regulation of locomotion
4) Spinal CSF-cNs contribute to morphogenesis
5) Can spinal CSF-cNs detect or respond to sex hormones?
Conclusion and Perspectives


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