Vesicular glutamate transporter isoforms: The essential players in the somatosensory systems

Accepted Manuscript Title: Vesicular glutamate transporter isoforms: The essential players in the somatosensory systems Authors: Fu-Xing Zhang, Shun-Nan Ge, Yu-Lin Dong, Juan Shi, Yu-Peng Feng, Yang Li, Yun-Qing Li, Jin-Lian Li PII: DOI: Reference:

S0301-0082(18)30047-9 https://doi.org/10.1016/j.pneurobio.2018.09.006 PRONEU 1581

To appear in:

Progress in Neurobiology

Received date: Revised date: Accepted date:

20-3-2018 28-8-2018 23-9-2018

Please cite this article as: Zhang F-Xing, Ge S-Nan, Dong Y-Lin, Shi J, Feng YPeng, Li Y, Li Y-Qing, Li J-Lian, Vesicular glutamate transporter isoforms: The essential players in the somatosensory systems, Progress in Neurobiology (2018), https://doi.org/10.1016/j.pneurobio.2018.09.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Vesicular glutamate transporter isoforms: the essential players in the somatosensory systems Fu-Xing Zhang a1, Shun-Nan Ge a, b1, Yu-Lin Dong a, Juan Shi a, Yu-Peng Feng a, Yang Lib, Yun-Qing Li a, c*, Jin-Lian Li a* a

Department of Anatomy and K.K. Leung Brain Research Centre, Preclinical School of Medicine, The

Department of Neurosurgery, Tangdu Hospital, The Fourth Military Medical University, Xi’an 710038, P.R. China

c

SC R

b

IP T

Fourth Military Medical University, Xi’an 710032, P.R. China

Collaborative Innovation Center for Brain Science, Fudan University, Shanghai 200032, P.R. China

Telephone: 86-29-84773087

N

Fax: 86-29-83283229

U

*Correspondence to: Dr. Jin-Lian Li, E-mail: [email protected]

Telephone: 86-29-84772706

M

Fax: 86-29-83283229

A

*Correspondence to: Dr. Yun-Qing Li, E-mail: [email protected]

PT

ED

1 These authors contributed equally to the present work.

A

CC E

Highlights  VGLUT mRNAs and proteins expression defining different subpopulations of neurons and/or subdivisions is elaborated in detail, targeting relay at each level of somatosensory pathways including dorsal root and trigeminal ganglia, superficial layers of spinal cord, trigeminal sensory nuclear complex, sensory thalamus and cortex.  Patterns of VGLUTs distribution, in terms of cellular mRNA and VGLUT protein-containing axonal endings, are generalized along the somatosensory pathways.  Physiological role(s) of VGLUT1 and VGLUT2 are reviewed by referring to data from global or conditioned knock-out mice.  Important issues impacting on insights into VGLUTs’ action in sensory transmission are tentatively proposed for consideration.

1

Abstract In nervous system, glutamate transmission is crucial for centripetal conveyance and cortical perception of sensory signals of different modalities, which necessitates vesicular glutamate transporters 1-3 (VGLUT1-3), the three homologous membrane-bound protein isoforms, to load glutamate into the presysnaptic vesicles. These VGLUTs, especially VGLUT1 and VGLUT2, selectively label and define functionally distinct neuronal subpopulations at each relay level of the neural hierarchies comprising spinal and trigeminal sensory systems. In this review, by scrutinizing each structure of the organism’s fundamental

IP T

hierarchies including dorsal root/trigeminal ganglia, spinal dorsal horn/trigeminal sensory nuclear complex, somatosensory thalamic nuclei and primary somatosensory cortex, we summarize and characterize in detail

to their

SC R

within each relay the neuronal clusters expressing distinct VGLUT protein/ transcript isoforms, with respect regional distribution features (complementary distribution in some structures), axonal

terminations/peripheral innervations and physiological functions. Equally important, the distribution pattern

U

and characteristics of VGLUT1/VGLUT2 axon terminals within these structures are also epitomized. Finally,

N

the correlation of a particular VGLUT isoform and its physiological role, disclosed thus far largely via studying the peripheral receptors, is generalized by referring to reports on global and conditioned

A

VGLUT-knockout mice. Also, researches on VGLUTs relating to future direction is tentatively proposed,

M

such as unveiling the elusive differences between distinct VGLUTs in mechanism and/or pharmacokinetics

CC E

PT

ED

at ionic/molecular level, and developing VGLUT-based pain killers.

Abbreviations:

A

AMPA: -amino-3-hydroxy-5-methyl-4-isoxazole proprionate acid ATP: adenosine triphosphate Bhlhb5: class B basic helix-loop-helix protein 5 BNPI: brain-specific Na+-dependent inorganic phosphate transporter I CCI: chronic constriction injury 2

CFA: complete Freund's adjuvant CGRP: calcitonin gene related peptide ChR2: channelrhodopsin-2 C-LTMR: C-type low-threshold mechanosensory receptor CNQX: 6-cyano-7-nitroquinoxaline-2,3-dione CNS: central nervous system CTb: cholera toxin B subunit

DNPI: differentiation-associated Na+-dependent inorganic phosphate transporter DRG: dorsal root ganglion

SC R

ECu: external cuneate nuclei EGFP: enhanced green fluorescent protein EPSPs: excitatory postsynaptic potentials

U

FNB: follicular (neural) network B

N

GRP: gastrin-releasing peptide

A

Ht-Pa: human tissue plasminogen

M

IB4: isolectin B4 IR: immunoreactivity

LTP: long-term potentiation

PT

MDH: medullary dorsal horn

ED

ISH: in situ hybridization

mEPSC: miniature excitatory postsynaptic currents

CC E

ML: medial lemniscus

IP T

DCN: dorsal column nuclei

MrgprA3: mas-related G-protein coupled receptor A3 MRGPRD: Mas-related G protein-coupled receptor D

A

Nav1.8: voltage-gated sodium channel 1.8 NF: neurofilament NGF: nerve growth factor NMDA: N-methyl-D-aspartic acid NPPB: natriuretic polypeptide b NPY: neuropeptide Y 3

NSE: neuron-specific enolase P2X3: purinergic receptor P2X, ligand-gated ion channel, 3 PAG: phosphate-activated glutaminase PGP 9.5: protein gene product 9.5 PKC: protein kinase C  Po: posterior nucleus of thalamus SAI: adapting type I

SI: primary somatosensory areas SII: secondary somatosensory areas

SC R

SNARE: soluble N-ethylmaleimide sensitive factor attachment receptor SNI: spared nerve injury SP: substance P

U

SSEA4: stage-specificembryonic antigen-4

TH: tyrosine hydroxylase

M

TRPM8: transient receptor potential melastatin 8

A

N

TG: trigeminal ganglion

TRPV1: transient receptor potential vanilloid type 1

ED

TSNC: trigeminal sensory nuclear complex VB: ventrobasal complex of thalamus

PT

Vc: subnucleus caudalis

IP T

SDH: spinal dorsal horn

VGLUT: vesicular glutamate transporter

CC E

Vi: subnucleus interpolaris

VIP: vasoactive intestinal peptide Vme: mesencephalic trigeminal nucleus

A

Vmo: trigeminal motor nucleus Vo: subnucleus oralis VP: ventral posterior nucleus of thalamus Vp: principal sensory nucleus Vpdm: dorsomedial part of Vp Vpvl: ventrolateral part of Vp 4

VPL: ventroposterolateral nucleus of thalamus

A

CC E

PT

ED

M

A

N

U

SC R

IP T

VPM: ventral posteromedial nucleus of thalamus

5

1. Introduction Neural transmission of different sensory modalities via either exteroceptors or interoceptors is crucial for survival of living organisms in ever-changing environments (Lechner and Siemens, 2011). Mediated through an array of orderly interconnected neurons into neural pathways (Willis, 2007), somatic sensory signals are first transferred by primary afferents encoding mechanical, thermal, noxious or pruritic stimuli

IP T

(Andrew and Craig, 2001; Dong and Dong, 2018; Ross et al., 2014) whose somata aggregate peripherally into the dorsal root ganglion (DRG) (Huang et al., 2007; McCarter et al., 1999) or trigeminal ganglion (TG) (Gazerani et al., 2010; Laursen et al., 2014). The TG and DRG supply the periphery and spinal cord or

SC R

sensorimotor trigeminal nuclei with nerve endings of neuronal bifurcated axons, respectively (Jacquin et al., 1993; Jacquin et al., 1988; Renehan et al., 1986; Willis, 2007; Wilson and Kitchener, 1996). The spinal cord and sensory trigeminal nuclei then forward the signals via the somatosensory thalamus to the cortices for

U

perception or processing (Minnery and Simons, 2003; Nash et al., 2010; Nathan et al., 2001).

N

Glutamate transmission in trigeminal and spinal sensory pathways has long since been an orthodox in

A

neuroscience (Miller et al., 2011; Miyata, 2007). Abundant glutamatergic neurons in spinal dorsal horn

M

(SDH), trigeminal nuclei, thalamic sensory nuclei and somatosensory cortex can be identified using antibodies against glutamate or phosphate-activated glutaminase (PAG), a glutamate producing enzyme

ED

(Diaz-Ruiz et al., 2016; Kaneko and Mizuno, 1988; Kaneko et al., 1987; Magnusson et al., 1987; Magnusson et al., 1986; Yezierski et al., 1993). Also, in vivo and in vitro electrophysiological studies record

PT

glutamate transmission inherent to somatic pathways. From 1990s to early 2000s, three isoforms of vesicular glutamate transporter (VGLUT) 1-3 were

CC E

identified in the central nervous system (CNS) (Aihara et al., 2000; Bai et al., 2001; Bellocchio et al., 1998; Bellocchio et al., 2000; Fremeau et al., 2002; Gras et al., 2002; Hayashi et al., 2001; Herzog et al., 2001; Hisano, 2003; Liguz-Lecznar and Skangiel-Kramska, 2007a; Ni et al., 1994; Otis, 2001; Schafer et al., 2002;

A

Shigeri et al., 2004; Takamori, 2006; Takamori et al., 2000, 2001; Varoqui et al., 2002). These are vesicular membrane-bound proteins trafficking glutamate into presynaptic vesicles priming for exocytosis during synaptic activity (Fillenz, 1995; Ozkan and Ueda, 1998; Palmada and Centelles, 1998). Unlike glutamate per se which can alternatively exist in GABAergic neurons, serving as metabolic precursor of GABA, VGLUT1 and VGLUT2 more specifically label glutamatergic neurons (Fremeau et al., 2002; Fremeau et al., 2004b; Gras et al., 2002; Hisano, 2003; Liguz-Lecznar and Skangiel-Kramska, 2007a; Otis, 2001; Schafer et al., 6

2002; Shigeri et al., 2004; Takamori, 2006). Thus, they are considered the most reliable markers for glutamatergic neurons. Accordingly, extensive studies on the trinity of VGLUTs, from the rudimental work of in situ protein/mRNA mapping to functional, pharmacological and bioenergetics elaboration, have surged since their discovery, attempting to unveil VGLUTs’ role(s) in the nervous system under either normal or pathophysiological conditions such as depression, anxiety and schizophrenia (Elizalde et al., 2010; Garcia-Garcia et al., 2009; Tordera et al., 2007; Uezato et al., 2009; Zink et al., 2010). Up to date, ample data available collectively represent an impressing panorama of a mutual exclusive

IP T

expression of VGLUT1 and VGLUT2 in the brain as a whole or in some brain regions/structures, as well as a separation of VGLUTs in the periphery with each isoform selectively expressed by nerve endings of a specific modality (Fremeau et al., 2004b; Kaneko and Fujiyama, 2002). In this article, the profile and

SC R

cellular compartmental localization of VGLUT mRNA/protein isoforms, together with their physiological role(s), in relation to different neuronal populations within each relay structures along spinal/trigeminal

U

sensory systems were, among others, comprehensively reviewed.

N

2. Expression of VGLUTs in the brain

A

2.1 The complementary distribution of VGLUTs in CNS

M

Numerous studies contribute to detailing the global or regional mRNA and/or protein expression of

ED

VGLUT isoforms in CNS. Unlike VGLUT3 which mostly labels neurons initially identified as non-glutamatergic, i.e., serotonergic, cholinergic or GABAergic (El Mestikawy et al., 2011; Fremeau et al., 2002; Gras et al., 2002; Schafer et al., 2002), VGLUT1 and VGLUT2 are mostly expressed in glutamatergic

PT

neurons (El Mestikawy et al., 2011; Liguz-Lecznar and Skangiel-Kramska, 2007a) and they show a complementary distribution pattern in the brain or a brain region/structure (Fremeau et al., 2004a; Fremeau

CC E

et al., 2004b; Kaneko and Fujiyama, 2002). A negligible exception is that the VGLUT1 or VGLUT2 phenotype also appears in some cholinergic, GABAergic, noradrenalinergic, adrenalinergic or dopamineric neurons within a few discrete brain structures (El Mestikawy et al., 2011). Generally, VGLUT1 mRNA

A

signal occurs in telencephalon and the granular layer of the cerebellum, precerebellar nuclei and medial habenular nucleus (Bai et al., 2001; Ni et al., 1996; Ni et al., 1994; Ni et al., 1995), whereas VGLUT2 mRNA mostly concentrates in diencephalon and lower brainstem (Fremeau et al., 2001; Herzog et al., 2001; Hisano et al., 2000). This non-overlapping expression mode also applies to individual brain regions where the neuronal co-expression of VGLUT1 and VGLUT2 is rare (Hisano et al., 2002; Sakata-Haga et al., 2001). The omnipresence of this VGLUT1 and VGLUT2 complementary distribution pattern in the brain was 7

appreciated and confirmed technically mostly by multiple immunofluorescence of axon terminals or by in situ hybridization for somatic mRNAs. 2.2 VGLUTs in somatic sensory transmission systems VGLUT1/VGLUT2 mRNAs and/or proteins are broadly expressed by neurons constituting the spinal and trigeminal sensory pathways. An overview is summarized below and in Table 1.

IP T

3. VGLUTs in spinal sensory pathway 3.1 Dorsal root ganglion and the axonal terminals

SC R

3.1.1 The peripheral nerve endings

Each isoform of the three VGLUTs was immunohistochemically detected in peripheral nerve endings, involving sensory receptors transducing either nociceptive, thermal or mechanical stimuli. This intimates

U

glutamate release from peripheral primary afferent terminals (Miller et al., 2011).

N

Mice hypodermis and glabrous skin, including dermis and epidermis, contain VGLUT1- and

A

VGLUT2-immunoreactive endings (Brumovsky et al., 2007). Some VGLUT2-immunostained nerve bundles

M

terminate either as free nerve endings or extensions contacting Merkel cells, and both transporters also appear in follicular (neural) network B (FNB) at the base of hair follicles, as were immunolabeled in hairy

ED

skin (Brumovsky et al., 2007; Haeberle, et al., 2004; Woo et al., 2012). Thus, it is obvious that skin VGLUT1 and VGLUT2 are implicated in transmitting different modalities (Fig. 1A). Physiologically, the peripheral VGLUT2 is inferably related to nociceptive sensation, since VGLUT2 is colocalized with

PT

calcitonin gene related peptide (CGRP), a pain-related molecule, and protein gene product 9.5 (PGP9.5,

CC E

pan-neuronal marker) (Brumovsky et al., 2007). The detection of highly VGLUT2- but not VGLUT1-positive tenocytes in tendinosis, a condition presenting with pain, also adds to the evidence (Scott et al., 2008).

In addition, VGLUT1 is remarkably expressed in muscle spindle afferent endings, the annulospiral

A

endings coiling intrafusal fibers around the equatorial and juxta-equatorial regions, as detected in triceps surae muscle (Fig.1C), indicating that VGLUT1 is the VGLUT solo engaged in proprioception at skeletal muscles (Wu et al., 2004). However, correlation of VGLUT1 and mechanoreception other than proprioception is also implicated by the presence of VGLUT1 in large-diameter fibers innervating external genital tract of female guinea pigs and mice, which were simultaneously labeled by neuron-specific enolase 8

(NSE) but not pain-related molecule CGRP (Vilimas et al., 2011). Colocalization of VGLUT1/VGLUT2 in the Merkel cell-neurite complex or VGLUT2-immunoreactivity (IR) in Merkel cells of rat hair follicle (Brumovsky et al., 2007; Haeberle et al., 2004; Woo et al., 2012), the sensor of gentle touch, also represent the indication for this role of these VGLUT isoforms. Although less abundant in skin, VGLUT3 was also reported to be: (1) persistently expressed by longitudinal lanceolate endings of hair follicles—unmyelinated, tyrosine hydroxylase-positive (TH+) and low-threshold C-mechanoreceptors (C-LTMR), or by TH- and unmyelinated, epidermal free nerve endings; transiently

expressed

at

prenatal/neonatal

stages

during

development

by

myelinated,

IP T

(2)

A-mechanoreceptors forming Merkel cell-neurite complex (Lou et al., 2013; Lou et al., 2015). The engagement of VGLUT3 in these receptors implicates VGLUT3’s role in pain and mechanical sensation

SC R

(Lou et al., 2013; Lou et al., 2015).

It is worthy to note that nerve endings of the interoreceptor (visceral receptor) also express VGLUT1 and VGLUT2; the sensory modalities transmitted by these VGLUTs fibers, however, remain unclear

U

(Brouns et al., 2006; Pintelon et al., 2007).

N

A collection of proteins including synapsin, synaptophysin, neuronal calcium sensor-1 (a Ca2+-binding

A

protein), bassoon and VGLUTs has been well established as constituting a presynaptic machinery for

M

synaptic vesicle exocytosis (Averill et al., 2004; Miller et al., 2011; Scarfone et al., 1988; Zhang et al., 2014). As regards the functions of these proteins in peripheral nerve endings, earlier studies presumably reported a

ED

priori their involvement in transmitter release, based mainly on the morphologically located synaptic-like microvesicles and vesicle membrane-associated proteins in peripheral nerve endings, as is exactly for the

PT

case of vestibular nerve calyx (Scarfone et al., 1988; Zhang et al., 2011). In fact, on condition that the nerve endings are subjected to natural/electrical stimulation, or

CC E

chemical/inflammatory insults, peripheral nerve release of glutamate, as well as neuroactive substances such as adenosine triphosphate (ATP) and CGRP, has been well documented (Averbeck and Reeh, 2001; deGroot et al., 2000; Miller et al., 2011). Chemicals such as endothelin-1 and glutamate injected into skin increased

A

(Cairns et al., 2007; Khodorova et al., 2009; Omote et al., 1998), or cyclooxygenase-2 inhibitor and hyaluronic acid intraarticularly into osteoarthritis-like knee decreased glutamate level (Jean et al., 2006; 2007; Miller et al., 2011). The released glutamate, putatively from the peripheral nerve endings, may contribute to sensitized nociception or pain pathology (Miller et al., 2011). By using styryl dye FM1-43 to label synaptic vesicles in lanceolate nerve endings and muscle spindle afferents, nerve activity- and Ca2+-dependent endocytosis and exocytosis, as assessed by dynamic alteration in fluorescent intensity, were 9

observed (Banks et al., 2013; Bewick et al., 2005). These findings lend convincing evidence of glutamate release directly from mechanoreceptive and proprioceptive peripheral afferent endings. Also, together with the localization to muscle spindle afferents of synapsin, synaptophysin, bassoon and VGLUT1, these findings strongly imply that the synaptic proteins of peripheral endings are implicated in glutamate release (Wu et al., 2004；Zhang et al., 2014). Thus, it suffices to reason that the different VGLUT isoforms in the peripheral nerve endings are involved in peripheral glutamate release, which can act back serving an autogenic modulation of the excitability of the sensory endings (Banks et al., 2013; Bewick et al., 2005).

IP T

3.1.2 The DRG

Both mRNA and protein for either VGLUT1, VGLUT2 or VGLUT3 were detected in DRG, with

SC R

VGLUT3-positive cells constituting a small subset (Fig. 1A). Initially, null transcripts of VGLUT2 and VGLUT3 in DRG was reported through in situ hybridization (ISH) (Oliveira et al., 2003), but this was otherwise rectified by subsequent studies. Perikaryal VGLUT1 and VGLUT2 proteins/mRNAs in almost all

U

L4-L5 and L4-S2 DRG of rats and mice (Landry et al., 2004; Malet et al., 2013; McCarthy et al., 2016), and

N

in most DRG neurons of pigeon and mouse were characterized (Atoji and Islam, 2009; Woo et al., 2012).

A

Further evidence for DRG VGLUT2 also comes from VGLUT2-immunostaining in DRG stem cell (Singh et

M

al., 2009). By contrast, VGLUT3 cells in DRG (~10%) were identified by enhanced green fluorescent protein (EGFP) controlled under VGLUT3 regulatory genes in transgenic mice (Seal et al., 2009) and was

ED

confirmed by presence of soma VGLUT3 mRNA in ~17% or 18.9% of the DRG neurons (Lou et al., 2013; Malet et al., 2013). Noteworthily, the three VGLUTs label distinct DRG cell types. Generally, VGLUT1 (protein or mRNA) occurs mostly in medium- and large-sized DRG neurons, while VGLUT2 and VGLUT3

PT

favor small- to medium-sized ones (Brumovsky et al., 2007; Malet et al., 2013; Seal et al., 2009). However,

CC E

a “moderate” number of or “most” DRG neurons co-express VGLUT1 and VGLUT2 (Brumovsky et al., 2007; Landry et al., 2004). To sum up, it is obvious that the profile of VGLUTs expression in DRG echoes well with the trinity of VGLUT isoforms present in different types of peripheral nerve endings. Recent studies, using RNA sequencing technique, categorized DRG neurons into distinct sets of sensory

A

neuron types and branched subtypes, characterized by different hierarchy of typing genes (Flegel et al., 2015; Li et al., 2016; Lopes et al., 2017; Manteniotis et al., 2013; Usoskin et al., 2015). These transcriptomics-based classifications, in most cases, didn’t include VGLUT transporter genes (Li et al., 2016; Usoskin et al., 2015). In Usoskin’s typing system, the NF1-NF5 groups, sharing parvalbumin and neurofilament genes (Pvalb and Nefh, respectively), correspond to A mechanoreceptors and proprioceptors, 10

and are most probably express VGLUT1, since they fit with the conventional large-sized DRG neurons (Usoskin et al., 2015). Groups PEP1-PEP2 (sharing CGRP) and NP1-NP3 (sharing MRGPRD and P2X3; NP: non-peptidergic nociceptors, MRGPRD: Mas-related G protein-coupled receptor D, P2X3: purinergic receptor P2X, ligand-gated ion channel, 3) correspond to nociceptors and pruriceptors, are the medium to small in size, and most likely express VGLUT2 and/or VGLUT3 (Usoskin et al., 2015). TH group is C-LTMR, the only cell type documented by the author to contain Vglut3 gene (VGLUT3) (Usoskin et al., 2015). In comparison, another lab (Li et al., 2016) reported the following DRG types: C1-C6 (including 10

IP T

subtypes) and C7-C10 (including 4 subtypes and another five subtypes) corresponding to small- and large-sized DRG neurons (Li et al., 2016), respectively. As such, C1-C6 and C7-C10 are predicted to express mainly VGLUT1 and VGLUT2/3, respectively.

SC R

VGLUT isoforms in DRG are differently regulated in response to neural insults such as axotomy. A relative consistent finding is the moderate to significant down-regulation of VGLUT1 and a moderate decrease of VGLUT3 in DRG ipsilateral to nerve injury (Brumovsky et al., 2007; Malet et al., 2013;

U

McCarthy et al., 2016; Wang et al., 2016). In contrast, nerve injuries induce DRG VGLUT2 change towards

N

different directions (Brumovsky et al., 2007; McCarthy et al., 2016; Wang et al., 2016) that are, presumably,

A

contingent on the severity of and the distance of DRG from the lesion site and most importantly, on the cell

M

type affected. Following nerve injuries (transaction, spared nerve injury or compression), an overall decrease or no alteration of VGLUT2 in ipsilateral DRG was reported respectively (Brumovsky et al., 2007; Malet et

ED

al., 2013; McCarthy et al., 2016; Wang et al., 2016). However, considering the DRG cell type involved, these studies showed an up-regulation of VGLUT2 in small neurons, down-regulation in medium-sized

PT

neurons and no change in large neurons ipsilateral to the injury (Brumovsky et al., 2007; McCarthy et al., 2016).

CC E

In mice L4/L5 DRG about 10% of neurons express VGLUT3 (Seal et al., 2009) and about 12% to 37% vs 65% were VGLUT1- and VGLUT2-IR, respectively (Brumovsky et al., 2007; Malet et al., 2013; Morris et al., 2005). Interestingly, a larger percentage of these neurons, about 45% or 69%, contained VGLUT1 or

A

VGLUT2 mRNAs, respectively (Brumovsky et al., 2007; Landry et al., 2004; Malet et al., 2013; Morris et al., 2005). The discrepancy between the ratios of VGLUT neurons estimated at mRNA and protein levels may implicate either a failure or low level of VGLUT synthesis, or immediate transport of the somatic protein off the cell body post-mRNA translation with some neurons. Anterograde transport of VGLUTs targeting axon endings can be readily evidenced by the canonical experimental paradigms of neurorectomy or nerve crush (e.g., sciatic nerve) which resulted in complete loss of skin VGLUT1-/VGLUT2-IR, or 11

axonal distention astride the constricted site that retained more abundant VGLUT1/VGLUT2 proximally than distally (Brumovsky et al., 2007). VGLUT1’s preference for medium-, especially large-sized DRG neurons indicates its functional implication in low-threshold mechanoreception. Large DRG neurons serve mechanoreception (Woolf and King, 1987) and they send VGLUT1-IR, myelinated primary afferents to terminate in spinal cord (Todd et al., 2003). Liu et al. (2010a) reported that spinal premotor interneurons mediating motor reflex responded, with evoked postsynaptic potentials (PSPs), to electrical stimulation of group I/II muscle spindle afferents

IP T

which terminate centrally with VGLUT1 synapses onto the recorded interneurons. This study directly certifies the VGLUT1’s involvement in mechanoreceptive sensation.

Lines of evidence support a close correlation of VGLUT2 and superficial senses, in particular, pain and

SC R

itch sensation. First, VGLUT2 are expressed preferentially in small- to medium-sized DRG neurons (Brumovsky et al., 2007; Malet et al., 2013), a majority of which belong to the subpopulations functionally sensitive to nociceptive stimulus (including the polymodal nociceptors) (Li et al., 2016; Usoskin et al., 2015;

U

Woolf and King, 1987).

N

Second, VGLUT2 was frequently detected to be expressed by DRG neurons containing pain-related

A

molecules/markers. Around 30% and 40% of the VGLUT2-IR L4/L5 DRG neurons co-expressed CGRP and

M

bound isolectin B4 (IB4), the markers for small-sized, pain sensitive DRG neurons, respectively; contrasting strikingly with the VGLUT1 containing large neurons that are incompatible with CGRP/IB4 (Brumovsky et

ED

al., 2007). In agreement with this, bladder-and colon/rectum-innervating DRG neurons in mouse were mostly found to be VGLUT2 and CGRP positive (Brumovsky et al., 2011; Brumovsky et al., 2013).

PT

Additionally, the temperature- and capsaicin-sensitive cation channel, transient receptor potential vanilloid type 1 (TRPV1), was found to be co-expressed in DRG, and nearly all the TRPV1-positive DRG neurons

CC E

co-express VGLUT2 (Hwang et al., 2004; Liu et al., 2010b). Further consolidating the involvement of VGLUT2 in nociceptive signaling is a recent study which, through double fluorescence labeling on lumbar DRG of mice (postnatal day 30), demonstrated quantitatively that VGLUT2, rather than VGLUT1, were

A

extensively expressed in sensory nociceptors labeled with IB4, CGRP and TRPV1. 100% of IB4- and CGRP-, and 94.2% TRPV1-postive DRG neurons were simultaneously labeled by VGLUT2; while 2.66%, 25.5% and 0.45% of these neurons by VGLUT1, respectively (Liu et al., 2010b). Third, when VGLUT2 was specifically deleted in DRG by using Cre line, where the promoter-driven Cre is under the control of tyrosine hydroxylase (Th), human tissue plasminogen (Ht-Pa), voltage-gated sodium channel 1.8 (Nav1.8), or transient receptor potential vanilloid 1 (Trpv1), the animals showed 12

attenuated responses to a range of various types of pain stimulation, accompanied by increased itch scratching (Liu et al., 2010b; Lagerstrom et al., 2010). These findings directly support the VGLUT2’s role in pain sensation (See also section “7.3”). Finally, apart from VGLUT2’s relevance to pain, VGLUT2 may likewise be implicated in itch. There exist distinct groups of itch-specific primary afferents, i.e., pruriceptors that respond to itch-evoking stimuli such as pruritogens and particular mechanical stimulus and express VGLUT2. These itch receptors include chloroquine -responsive mas-related G-protein coupled receptor (Mrgpr) A3 (MrgprA3)-expressing,

IP T

gastrin-releasing peptide (GRP)-expressing and histamine-sensitive pruriceptors (Akiyama et al., 2014; Dong and Dong, 2018; Liu et al., 2009; Sun and Chen, 2007 ); and all neurons in the first two groups express VGLUT2, and 80% and 77% of the DRG neurons responsive to chloroquine and histamine, are (Akiyama

et

al.,

2014;

Liu

et

al.,

2010b).

No

VGLUT1

SC R

VGLUT2-IR

was

found

in

MrgprA3-/GRP-expressing neurons (Liu et al., 2010b). Based on these data, we are tempted to infer that glutamate may act as pruriceptor-released transmitter. However, studies reported that itch behavior is

U

increased, not abolished, following removal of VGLUT2 from Nav1.8-/TRPV1-expressing DRG neurons

N

that include MrgprA3/GRP active neurons (Liu et al., 2010b; Lagerstrom et al., 2010), suggesting that

F/F

; Nav1.8

Cre

mice in this experiment is, mostly but not completely, removed in

M

since VGLUT2 in Vglut2

A

glutamate may be dispensable for itch sensation. Cautions should be taken in explaining these observations,

all TRPV1-expressing DRG neurons. In these mice, 3.88%, 0.88% and 12.3% of the TRPV1-, IB4- and

ED

CGRP-expressing DRG neurons still express VGLUT2, respectively (Liu et al., 2010b). More importantly, in these mice, although no VGLUT1 is expressed in Nav1.8

Cre

DRG neurons, but 2.08% and 2.16% of the

PT

MrgprA3- and GRP-active neurons, respectively, express VGLUT2 (Liu et al., 2010b). Furthermore, chloroquine-evoked scratching and spinal neuronal firing was partially reduced by spinal application of the

CC E

6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), the antagonists of -amino-3-hydroxy-5-methyl-4-isoxazole proprionate acid (AMPA)/kainate receptor antagonist (Akiyama et al., 2014). Therefore, the exact role of glutamate released from pruriceptors containging VGLUT2 in itch sensation and/or modulation requires to

A

be further investigated (See also section “7.3”). In adult, DRG VGLUT3 is predominantly expressed in small-sized neurons that include the

unmyelinated, C-LTMR. However, at perinatal stage during development, a small portion of medium to large-diameter DRG neurons transiently express VGLUT3, these neurons co-express neurofilament 200 (NF200) and thus represent myelinated A-mechanoreceptors (Lou et al., 2013; Seal et al., 2009). These morphological features, together with all the skin receptor types containing VGLUT3 (see above), clearly 13

suggest that DRG VGLUT3 is functionally either pain- or mechanoreception-related (Lou et al., 2013; Seal et al., 2009). In case of VGLUT3-containing unmyelinated and TH+ C-LTMR, Seal and colleagues reported its specific implication in mechanical hypersensitivity and mechanical pain under conditions of nerve injury or inflammation (Seal et al., 2009). However, further interrogation of the functional identity of this receptor is required, since a recent study showing that mechanical pain was largely unaffected in a mutant mice, Runx1F/F; Vglut3Cre/+, where DRG VGLUT3 was selectively, and most peripheral VGLUT3 C-LTMRs were, lost (Lou et al., 2013).

IP T

3.1.3 The central terminals of primary afferent neurons

Primary afferents project somatotopically to spinal cord by way of the dorsal root, where their central

SC R

terminals carrying different sensory information are partially separated by modality and area of innervation (Grant, et al., 2004). As such, spinal cord lamina I receives afferents mainly of cutaneous A and unmyelinated C fibers, together with projections from deep somatic structures, e.g., small diameter

U

myelinated and unmyelinated fibers (group III and IV fibers conveying nociceptive information from

N

articular structures); Lamina II receives unmyelinated C fibers evoking nociception or itch, in addition to A

A

fibers subserving high threshold mechanoreceptors that project to its outer subdivision (IIo) and cutaneous

M

mechanoreceptive A and A fibers that terminate in its inner subdivision (Iii) (Grant, et al., 2004). A, A and fine C fibers projects to lamina III-VI (Brumovsky, 2013; Grant, et al., 2004).

ED

VGLUT1- and VGLUT2-IR terminals are complementarily expressed in laminae of the SDH showing no overlapping, with laminae I/II showing high density of VGLUT2-IR terminals and sparse VGLUT1-IR fibers (laminae I and IIo); and laminae III/IV robust VGLUT1 but weak VGLUT2 (Alvarez et al., 2004;

PT

Brumovsky et al., 2007; Landry et al., 2004; Oliveira et al., 2003; Persson et al., 2006; Rethelyi et al., 2008;

CC E

Wiedey et al., 2008; Wu et al., 2012) (Fig.1A,central part). Generally, the VGLUT1-IR puncta are larger than the VGLUT2- and VGLUT3-IR ones (Landry et al., 2004). This pattern well characterizes the partially separated projections of primary sensory afferents to SDH: VGLUT1-IR afferents conveying cutaneous/muscle mechanosensory signals to deep laminae while VGLUT2-IR inputs transducing

A

nociceptive stimuli to superficial layers (Malet and Brumovsky, 2015; Shrestha et al., 2012). In contrast to VGLUT1 and VGLUT2 which show similar distribution patterns across laminae in spinal cord between rat and mouse (Bromovsky, et al., 2007; Landry, et al., 2004), VGLUT3-IR terminals occupy an area SDH of rat that is distinctinct from that in mice. Seal and colleagues immunostained in mouse a distinct narrow bands of VGLUT3-IR within lamina I and protein kinase C (PKC) layer of lamina II in the neuropil of 14

SDH (Seal et al., 2009), while Landry et al. detected abundant VGLUT3-IR occupying broad area of the whole grey matter of rat spinal cord (Landry, et al., 2004). Both the species and antibodies used by the authors may account for the difference. SDH VGLUT1- and VGLUT2-IR axon terminals have both peripheral and central origins (Brumovsky, 2013). The peripheral origin can be directly confirmed by axotomy. Significant depletion of VGLUT1-IR in laminae III/ IV and medial part of lamina V occurred following dorsal rhizotomy (Alvarez et al., 2004; Brumovsky et al., 2007), so did slight reduction of SDH VGLUT2-IR (Alvarez et al., 2004). Additionally, it

IP T

has been shown that Group I and II muscle afferents innervating sartorius send VGLUT1-IR terminals to synapse upon spinal premotor interneurons (Liu et al., 2010a). Moreover, SDH lamina III is enriched with terminals colabeled by VGLUT1 and stage-specific embryonic antigen-4 (SSEA4), a marker of cutaneous

SC R

mechanoreceptors (Alvarez et al., 2004). More directly, myelinated mechanoreceptive (e.g., tactile and proprioceptive) primary afferents, as revealed by transganglionic labeling of sciatic nerve with cholera toxin B subunit (CTb), provide laminae III-VI with large number of VGLUT1-IR or VGLUT1/VGLUT2

U

co-labeled axon terminals (Hughes et al., 2004; Todd et al., 2003). This finding confirms VGLUT1’s

N

peripheral origin and, incidentally, reveals VGLUT1’s relevance to proprioception which is also supported

A

by in vivo studies documenting spinal motoneurons’ excitatory response to stimulation of stretch responsive

M

IA afferents that form VGLUT1-only varicosities closely apposed to these neurons (Alvarez et al., 2011; Rotterman et al., 2014).

ED

Peripheral source of SDH VGLUT2 terminals was once considered dubious (Schneider and Walker, 2007) due to lack of significant SDH VGLUT2-IR alteration post dorsal nerve rhizotomy (Alvarez et al.,

PT

2004). Nevertheless, the fact might probably be that the peripheral-originated spinal VGLUT2 terminals are obscured by the overwhelming quantity of central-originated ones. Actually, some SDH (especially lamina I)

CC E

VGLUT2 and VGLUT1-IR terminals co-express CGRP and/or bound IB4, a marker for primary afferent, unmyelinated fibers (Landry et al., 2004). Similarly, some substance P-/somatostatin-expressing terminals in SDH, most of which are unmyelinated primary afferents, show low levels of VGLUT2-IR but not

A

VGLUT1-IR (Todd et al., 2003). Moreover, most non-peptidergic, not IB4 binding, small-caliber glutamatergic nociceptive primary afferents project VGLUT2-IR terminals centrally to lamina I of spinal cord (Clarke et al., 2011). In addition, SDH possesses many terminals colabeled by VGLUT2 and TRPV1, a thermal and capsaicin sensitive sensor on nociceptive primary afferents (Hwang et al., 2004; Zhou et al., 2009). More importantly, transganglionically identified myelinated primary afferents were found to send VGLUT2-IR terminals to laminae I of SDH (Todd et al., 2003) and accumulation of VGLUT2 in dorsal 15

roots on the DRG side after dorsal root crush indicates VGLUT2’s flow towards the central (Brumovsky et al., 2007). All these data indicate that the primary nociceptive spinal afferents employ VGLUT2 for glutamate transmission and contribute to the SDH VGLUT2 repository (Fig.1A). Dual origins of SDH VGLUT3-IR terminals from peripheral and central were similarly confirmed (Brumovsky, 2013). Dorsal rhizotomy yielded almost depletion of VGLUT3 signals in superficial laminae of SDH (Seal et al., 2009) and in VGLUT3fl/fl; Lbx1Cre mice where VGLUT3 are genetically knocked out in spinal neurons but retains in DRG neurons, heavy VGLUT3-IR were observed as a discrete band in spinal

IP T

cord lamina II, representing the projections from C-LTMR afferents (Peirs et al., 2015). Moreover, RosaTomato/+; VGLUT3Cre/+ also confirms the peripheral contribution of spinal VGLUT3 from VGLUT3 containing A type-mechanoreceptors that were fluorescently labeled (Lou et al., 2013). In contrast, the

SC R

presence of VGLUT3-positive neurons in laminae III-V implies the central origin of VGLUT3 (Landry et al., 2004; Malet et al., 2013; Peirs et al., 2015; Seal et al., 2009).

The VGLUT-IR profiles manifested by DRG neurons and their SDH central terminals disagree. Most

U

of the neuronal somata in DRG co-express VGLUT1 and VGLUT2 (e.g., mRNAs) (Brumovsky et al., 2007;

N

Landry et al., 2004), while this is rarely seen in SDH where appears a clear separation between VGLUT1

A

and VGLUT2 in spinal projections (Landry et al., 2004; Persson et al., 2006). Also, co-localization of

M

VGLUT2 and CGRP or SP, or VGLUT2 neurons binding IB4 abound in DRG cells, much less is the case for SDH terminals, however (Brumovsky et al., 2007). Finally, despite the enriched VGLUT2 somata in DRG,

ED

dorsal rhizotomy yields no significant VGLUT2-IR loss in SDH (Alvarez et al., 2004; Brumovsky et al., 2007). The most parsimonious explanation for this central-periphery mismatch is that neurons distinctly

PT

regulate protein translation. Some perikaryon VGLUT transcripts may possibly be penuriously translated into proteins or the somatic proteins are not/insufficiently transported to central terminals such that they

CC E

elude immunohistochemical detection. Alternatively, DRG neurons co-expressing VGLUT1 and VGLUT2 may utilize targeting mechanisms specific for each transporter to be sorted and separately targeted towards neuronal compartments (for instance, peripheral endings) other than SDH terminals (Landry, et al., 2004;

A

Malet et al., 2013).

3.2 Neurons in spinal cord and dorsal column nuclei (DCN) and their projections Somatosensory information from DRG is transmitted to higher brain regions through ascending spinal pathways, including spinothalamic tract and dorsal column pathways (Tracey, 2004). Most spinothalamic pathways signaling nociceptive, thermal and innocuous stimuli terminate in structures like the 16

ventroposterolateral nucleus of thalamus (VPL), posterior thalamic group (Po) and intralaminar nuclei (Tracey, 2004), while dorsal columns, consisting of ascending collaterals of myelinated primary afferents, send discriminative tactile and proprioceptive signals to DCN (Tracey, 2004) which in turn project contralaterally to VPL and the Po through medial lemniscus in the brainstem (Kemplay and Webster, 1989; Mantle-St John and Tracey, 1987). Afferents to somatosensory thalamus involved in both spinothalamic and dorsal column pathways utilize glutamate as neurotransmitter.

IP T

3.2.1 Spinal neurons and the efferent fibers thereof By in situ hybridization (ISH), somatic mRNAs for all the three VGLUT isoforms were identified. In rat spinal cord, VGLUT1-3 mRNAs are distributed throughout laminae I-X, although VGLUT3 is very weakly

SC R

expressed, especially in laminae I-IV, by scattered neurons as compared to the much higher levels of VGLUT1 and VGLUT2 mRNAs (Landry et al., 2004) (Fig.1, SDH). Among these VGLUT mRNA positive neurons, co-expression of VGLUT1 and VGLUT2 exists with different neuronal proportion across the

U

laminae, being 14-17% in laminae I-II (Landry et al., 2004). Commissural interneurons in lumbar spinal

N

cord of newborn mice contain VGLUT2 mRNA (Restrepo et al., 2009) and several studies identified

A

VGLUT2 terminals of SDH interneurons, also confirming the presence of VGLUT2 interneurons in SDH

M

(Bannatyne et al., 2006; Brooke et al., 2006; Maxwell et al., 2007). Recently, RNA-sequencing studies documented the SDH neuron types (Häring, et al., 2018; Sathyamurthy, et al., 2018), and SDH neurons are

ED

either glutamatergic, expressing Slc17a6 (VGLUT2) or GABAergic, expressing Slc32a1 (vesicular GABA transporter) (Häring et al., 2018). The glutamatergic type, i.e., VGLUT2-type neurons was further classified into 15 molecular subtypes, each demonstrating a particular spatial distribution pattern within SDH (Häring

PT

et al., 2018). Within these excitatory neurons, five subtypes, “Glut8-Glut12”, showed more neurons located

CC E

in lamina I, and subtypes Glut1, Glut10, Glut13 and Glut14 had more neurons in laminae III-V (Häring et al., 2018), suggesting that these subgroups may contain the VGLUT2-expressing spinothalamic projection neurons

relaying

pain/itch

signals,

consistent

with

the

immunohistochemically

identified

VGLUT2-expressing, second-order neurons along the pain/itch pathway.

A

It seems that distribution pattern of VGLUTs mRNAs differs across species. Using radioactivity in situ

hybridization on lumbar segments of mouse, only isolated neurons positive for VGLUT1 mRNA in laminae III-V and a small number of VGLUT3-postive neurons in III-IV were observed, while numerous VGLUT2 mRNA-positive ones were distributed throughout whole grey matter except lateral aspects of ventral horn (Malet, et al., 2013). The rarity of spinal cord VGLUT3 mRNA-containing neurons is consistent with the 17

presence of very few EGFP-positive spinal neurons in VGLUT3 EGFP BAC transgenic mice (Seal et al., 2009). This mRNA expression profiles contrasts those of proteins; more neurons in lamninae I to VI of rat were detected to be immunoreactive to all three VGLUT isoforms (Landry, et al., 2004). It is understandable that one species differ from another in the quantity of neurons of a particular VGLUT phenotype and in the topography of this neuron type within SDH; however, the technical sensitivity for screening a VGLUT mRNA by in situ hybridization employing different cRNA probes and the signal detecting paradigms used by different research groups may constitute additional factors contributing to the differences between

IP T

species. Spinal VGLUT2 neurons serve as local interneurons, as well as long-range projecting neurons. Complete abolishment of VGLUT2-IR, but not VGLUT1-IR, in the ipsilateral ventral posterior nucleus of

SC R

the thalamus (VP) following surgical disruption of unilateral spinothalamic tract strongly suggested that the spinothalamic projection neurons contain exclusively VGLUT2 mRNA (Graziano et al., 2008) (Fig.1B). In addition, Persson et al. and Gebre et al. independently confirmed that axon terminals of spinocervical and

U

spinocerebellar tracts were only VGLUT2 immunopositive (Gebre et al., 2012; Persson et al., 2006).

N

Similarly, SDH VGLUT1 or VGLUT3 neurons serve as interneurons (Brumovsky, 2013; Peirs et al.,

A

2015); however, little is known about whether the superficially located neurons with VGLUT1 or VGLUT3

M

target and transmit sensory signals to higher structures. A recent study, using VGLUT3Cre; lsl-tdTomato mice, reported transiently expressing VGLUT3 spinal neurons mostly located in lamina III, to a lesser extent in

ED

lamina II and sporadically in lamina I (Peirs et al., 2015). All these VGLUT3 neurons co-express VGLUT2 mRNA but not the inhibitory marker gad67 and act as a key component of multiple microcircuits

PT

gate-controlling the mechanical allodynia and mechanical hypersensitivity (Peirs et al., 2015). In contrast, some spinal VGLUT1 neurons in the dorsomedial part in the intermediate zone of thoracic spinal cord,

CC E

resembling those in dorsal nucleus of Clarke, are supposed to be neurons sending signals to anterior and posterior zones of cerebellum (Brumovsky, 2013). 3.2.2 DCN and the associated efferent fibers

A

DCN comprises gracile, cuneate and external cuneate nuclei (ECu) and receives low-threshold,

mechanoreceptive primary afferents encoding pressure/distortion. All constituent nuclei of DCN express intense VGLUT2 mRNA signal (Hioki et al., 2003; Hisano et al., 2002; Pang et al., 2009). The cuneate-originated VGLUT2-IR projections to cochlear nuclei coincide well with the preponderance of VGLUT2 mRNA in DCN (Zeng et al., 2011) (Fig.1D). Strong VGLUT1 mRNA signals in ECu contrast the 18

weak (Hioki et al., 2003; Hisano et al., 2002; Pang et al., 2009) or no VGLUT1 mRNA (Graziano, et al., 2008) in gracile and cuneate nuclei (Fig.1D). In DCN, VGLUT2-expressing neurons send afferents with long-range projections to contralateral thalamus, as portions of medial lemniscus (ML) (Graziano et al., 2008) (Fig.1D). This is evidenced by significant decrease of VGLUT2-IR in VP, without detectable changes of VGLUT1-IR in somatosensory thalamus, following deafferentation of VP via ML lesion (Graziano et al., 2008). In contrast, DCN-cerebellum projections expressed VGLUT1 (Gebre et al., 2012). The segregation of DCN VGLUT1

IP T

and VGLUT2 with respect to their projection targets strongly suggests that these transporters are selectively assigned to neurons sending mechanical signals for fulfilling different physiological roles.

SC R

4. VGLUTs in trigeminal sensory pathway

U

4.1 Trigeminal ganglion and its terminals

N

4.1.1 Peripheral terminals

The trigeminal sensory pathways convey neural signals from head and orofacial regions. All three

A

isoforms of VGLUTs were observed in Merkel cells, corpuscular nerve endings and intraepithelial free nerve

M

endings of rodent hard palate (Hitchcock, et al., 2004; Nunzi et al., 2004). However, VGLUT3 seems limited only to these structures (Nunzi et al., 2004). VGLUT1, not VGLUT2/3, is present in non-taste epithelium

ED

and taste papillae of the tongue (Braud et al., 2010); and the Ruffini endings and mechanoreceptors

(Fig.1E).

PT

innervating periodontal ligament of incisors display VGLUT1/2-but not VGLUT3-IR (Honma et al., 2012)

Both VGLUT1 and VGLUT2 are probably involved in nociception in the dental pulp. Nerve endings in

CC E

human dental pulp exhibited VGLUT1-IR and the majority co-expressed CGRP, while a few axons expressed VGLUT2 (Paik et al., 2012; Zerari-Mailly et al., 2012). In addition, axon terminals supplying dental pulp co-expressed VGLUT2 and the transient receptor potential melastatin 8 (TRPM8), a sensor

A

sensitive to noxious cold, suggests implication of VGLUT2 in cold nociception (Kim et al., 2015). Moreover, dental pulp VGLUT2-IR axons increased in response to complete Freund's adjuvant (CFA) stimulation (Yang et al., 2014). Additionally, VGLUT isoforms may participate in mechanoreception, as are characterized by Merkel cell-neurite complex (see above). For comprehensive understanding, here we incorporate the data on tactile VGLUTs from both spinal and trigeminal fields. Structurally, in Merkel cell-neurite complex, the paired 19

elements may possibly form reciprocal synapses, possibly with each element being presynaptic to the other (Mihara et al., 1979). It has been documented that both the Merkel cell and nerve endings (the A-type mechanoreceptive fibers) each were observed to express VGLUT2 and/or VGLUT3 (Brumovsky et al., 2007; Haeberle et al., 2004; Hitchcock et al., 2004; Lou et al., 2013; Nunzi et al., 2004; Woo et al., 2012). Also, VGLUT1 occurs at the region of Merkel cell-neurite complex (Hitchcock, et al., 2004), and it occurs in Merkel cells in palate (Nunzi et al., 2004). VGLUTs-associated glutamate released from Merkel cell may be the mediator signaling tactile

IP T

information to activate sensory afferents. The responses of slowly adapting type I (SAI) units (A-mechanoreceptors) to vibrissa movement was attenuated following blockade of inotropic glutamate receptors by kynurenate support the identity of Merkel cell-released glutamate as transmitter (Fagan et al.,

SC R

2001; Maksimovic et al., 2013). Structures and molecules observed in Merkel cell that characterize presynaptic features are strongly reminiscent of Merkel cell as presynaptic element, being equipped with machinery capable of release glutamate, among others. These include active–zone scaffolding proteins,

U

SNARE complex genes, synaptotagmins 1 and 7, large-cored vesicles, and the VGLUT2 and/or VGLUT3

N

(Haeberle et al., 2004; Lou et al., 2013; Mihara et al., 1979). In addition, by using optogenetic approach on

A

heterozygote CckCre/+;ChR2loxP/+ mice, where the photosensitive Channelrhodopsin-2 (ChR2) is specifically

M

expressed in the touch-dome Merkel cells but not in SAI afferents, recent study reported that SAI fired in response to light activation of Merkel cells, suggesting Merkel cell activates SAI endings (Maksimovic, et

ED

al., 2014). Collectively, these data indicate that Merkel cell may use glutamate as transmitter to activate SAI afferents. However, this is more or less questioned by the observations that NMDA subunit (NR2A/B),

PT

component to functional NMDA receptors, were located in Merkel cell proper instead of sensory afferents (Cahusac et al., 2005) and that both AMPA/kainate receptor antagonists and classical competitive NMDA

CC E

receptor antagonists did not depress the SAI responses to mechanical stimuli (Cahusac et al., 2005). More importantly, a recent study using patch-clamp to record SAI responses on whisker pad preparation showed SAI responded, among eleven likely transmitter candidates including glutamate, only to 5-HT, mechanically

A

by activating 5-HT3 receptor and 5-HT2A and 2B receptors (Chang et al., 2016). Still, Merkel cell glutamate as transmitter cannot be completely ruled out and further studies are needed (Chang et al., 2016). In addition, the VGLUTs-associated glutamate from Merkel endings may signal back on itself. In this case, Merkel cells assumes a role of neuroendocrine (paracrine and autocrine) cells, releasing glutamate and co-releasing a plethora of transmitters/regulators such as ATP, VIP (vasoactive intestinal peptide), SP and CGRP (Alvarez et al., 1988; Hartschuh et al., 1983; Maksimovic et al., 2013; Nakamura and Strittmatter, 20

1996; Tachibana et al., 2003; 2005), to modulate the activities of Merkel cell per se, as well as sensory afferents. In fact, Merkel cells express metabotropic glutamate receptors such as mGluR5 and NR2A/B subunit, and other receptors like P2Y receptor, etc. (Cahusac et al., 2005; Tachibana et al., 2003). As for Merkel nerve endings, these sensory afferents may likewise serve as mechanoreceptor, functioning as rapidly adapting fibers to transduce dynamic stimuli while Merkel cells transduce static phase stimui, as postulated by “two receptor-site model” (Maksimovic et al., 2014; Nakatani et al., 2015). In this case, VGLUTs-packaged glutamate released from Merkel cells may, like mentioned above, possibly be the

IP T

transmitter acting on the sensory afferents. Since these afferents contain VGLUT2 and VGLUT3 (Haeberle et al., 2004; Hitchcock et al., 2004; Lou et al., 2013; Nunzi et al., 2004), they may, like other peripheral nerve endings also release glutamate

(See section 3.1.1), for regulating the activities of Merkel cell and/or

SC R

nerve endings themselves. 4.1.2 Trigeminal ganglion (TG)

U

TG resembles DRG in neuronal VGLUTs’ profile, with nearly all somata expressing VGLUT1 and/or

N

VGLUT2 and contains more VGLUT2- than VGLUT1-positive neurons. VGLUT3 is also expressed by

A

some TG neurons (Ren, et al., 2018; Seal et al., 2009) and about 11% of the small TG neurons, of which

M

more than 90% are unmyelinated, express VGLUT3 (Seal et al., 2009). Over 80% of VGLUT-expressing TG neurons co-express VGLUT1 and VGLUT2 (Li et al., 2003b) (Fig.1E). Morphometrical analysis showed

ED

that VGLUT2 preferred small TG cells (averaged diameter ≤20 mm) (Li et al., 2003b), suggesting the involvement of VGLUT2 in orofacial nociception which is also supported by the co-existence of the VGLUT2 and pain-related molecular such as TRPM8 in TG (Kim et al., 2015). Actually, the proportion of

PT

VGLUT2-ir neurons in TG following pulpal inflammation was elevated (Yang et al., 2014). However, active

CC E

nociceptive TG neurons may also engage VGLUT1, since VGLUT1 was induced by lipopolysaccharides and co-expressed with ionotropic purinergic receptor family (P2X) member (s) in small-sized TG neurons (Chen et al., 2014). The VGLUT3 TG neurons share with their DRG counterparts the similar biochemical properties: no expression of SP and CGRP and very few binding with IB4 (Seal et al., 2009). Thus, these

A

neurons may functionally subserve mechanical hypersensitivity in orofacial region, similar to the case that was evidenced for unmyelinated, low-threshold mechanoreceptors with VGLUT3 in DRG. 4.1.3 The central processes of TG neurons in the trigeminal sensory nuclear complex (TSNC) The central processes of TG cells mainly terminate in the trigeminal sensory nuclear complex (TSNC), the structure involving the principal sensory nucleus (Vp) and the spinal trigeminal nucleus which is 21

rostrocaudally organized into three subnuclei: subnucleus oralis (Vo), subnucleus interpolaris (Vi) and subnucleus caudalis (Vc) or the medullary dorsal horn (MDH) (Waite, 2004).The superficial layers of Vc receive primary afferents from small-sized TG neurons with unmyelinated or thinly myelinated fibers, while the rest of TSNC receives myelinated axon terminals from medium to large TG cells (Waite, 2004). Vc (MDH) possesses a laminar arrangement of VGLUTs comparable to that in SDH. The superficial layers of MDH, in particular the marginal layer (lamina I) and outer part of substantia gelatinosa (lamina II) demonstrate intense VGLUT2-IR and weak VGLUT1-IR; while deeper layer, or the magnocellular part of

IP T

MDH, showed intense VGLUT1-IR and weak to moderate VGLUT2-IR (Li et al., 2003a) (Fig.1E). IB4-bound terminals projecting to the superficial layer of the MDH were frequently labeled with VGLUT2 but rarely with VGLUT1 (Li et al., 2003a), suggesting VGLUT2’s preference to the orofacial primary

SC R

unmyelinated afferents (Fig.1E).

In contrast, other components of TSNC exhibited even distribution of VGLUT1- and VGLUT2-IR (Pang et al., 2009; Xiang et al., 2012) (Fig.1. Vp, Vo and Vi). Unilateral trigeminal nerve rhizotomy nearly

U

abolished VGLUT1-IR in ipsilateral Vp, Vo, Vi and the rostral part of Vc (Pang et al., 2009), suggesting that

N

the myelinated primary afferents of the cranial nerves mainly use VGLUT1 for glutamate transmission, as in

A

the case of SDH. By using CTb to label corneal afferents, it was observed that the rostro-caudal extension of

M

Vi-Vc complex received distinct types of VGLUT afferents, with more rostrally-terminated terminals containing VGLUT1 than VGLUT2 and vice versa for the caudal projections; however, both VGLUTs

ED

preferentially label the rostral termini (Hegarty et al., 2010). This projection pattern may imply a functional difference between VGLUT1 and VGLUT2 trigeminal primary afferents. As far as we know, no report on

PT

VGLUT3 related to central terminals has been documented.

CC E

4.2 Mesencephalic trigeminal nucleus The trigeminal primary afferents transducing mechanoreceptive stimuli at periodontal ligments and orofacial muscles (e.g., masticatory muscle and supratarsal Müller muscle) have their somata ectopically dispersed in the pontine and mesencephalon, forming the trigeminal mesencephalic nucleus (Vme), (Byers

A

et al., 1986; Holstege et al., 1995; Matsuo et al., 2015; Rokx and van Willigen, 1988). VGLUT1 is the solo functionally engaged in muscle proprioception. Only VGLUT1-IR was detected in

intrafusal fibers of masseteric spindles (Lund et al., 2010; Pang et al., 2006) (Fig.1G), and centrally, in trigeminal motor nucleus (Vmo)

jaw-closing subdivision, a critical motor neuron pool receiving Vme

innervations involved in jaw jerk reflex (Faunes et al., 2016; Pang et al., 2009). Additionally, only VGLUT1 22

mRNA occurs in Vme neurons (Pang et al., 2006) (Fig.1, Vme), and their central terminals, traced to Vmo by transganglionic tracer CTb injected into masseter, selectively contain VGLUT1 (Li et al., 2012; Pang et al., 2006). Confirmation of the VGLUT1’s involvement in jaw-muscle spindle afferent feedback also comes from the axotomy of muscle spindle afferents via transaction of motor root of trigeminal nerve which yielded VGLUT1-IR elimination but no VGLUT2-IR alteration in the Vmo and dorsal division of the Vp, the important Vme projection targets (Dong et al., 2007; Pang et al., 2009). Lastly, indirect evidence for Vme’s VGLUT1 phenotype also exists. The ventromedial division of Vmo, i.e., the jaw-opening subdivision

IP T

known to be devoid of Vme innervation, lacks VGLUT1-IR (Pang et al., 2009), and VGLUT2-IR terminals, enriched in entire Vmo, obviously originates from neurons in surrounding structures such as Vp, the spinal trigeminal nucleus, the supratrigeminal region (supV) and the pontomedullary reticular formation (Pang et

SC R

al., 2009). Consistent with our findings, ultrastructural observation confirms the selective distribution of VGLUT1 and VGLUT2 in Vmo by showing that VGLUT1-labeled boutons were much more numerous in jaw-closing than in jaw-opening part, and VGLUT2 boutons were relatively evenly distributed in Vmo (Park

U

et al., 2018).

N

Interestingly, in neonates younger than 11 days VGLUT1 protein is expressed by Vme neurons,

A

presenting in both somata and axon endings, but dissipated progressively from the cell bodies afterwards

M

over development (Pang et al., 2006). Perikaryal VGLUT1 mRNA, however, persists through all developmental stages (Pang et al., 2006). It is likely that during later developmental stages Vme neurons

ED

transport centrifugally via an unknown cellular mechanism the synthesized VGLUT1 protein to nerve endings, priming for the physiologically maturing muscle spindle afferents to guarantee proprioceptive

PT

feedback during coordinated jaw movements while chewing.

CC E

4.3 TSNC and its efferents

Perikaryal VGLUT1 and VGLUT2 mRNAs in TSNC were complementarily distributed (Ge et al., 2014; Ge et al., 2010; Hisano et al., 2002; Pang et al., 2009; Xiang et al., 2012). VGLUT2 mRNA positive neurons preponderate in TSNC, evenly distributed across TSNC nuclei (including Vc, Vi, Vo and Vp). VGLUT1

A

mRNA-expressing neurons of TSNC are heterogeneously distributed: hardly any in the superficial layer of Vc, moderately and evenly across dorso-ventral divisions of Vi, less densely in dorsal division of Vo and abundantly throughout Vp (Fig.1). Co-expression of VGLUT1 and VGLUT2 mRNA signals is hardly seen in Vc and Vi, sporadically or singly encountered in the dorsal part of Vo (Ge et al., 2014; Ge et al., 2010), thus contrasting to a high 23

percentage (~64%) of co-expression in Vp where the remainder is either VGLUT1 or VGLUT2 mRNA positive (Ge et al., 2010). Notably, the VGLUT1/VGLUT2 co-expressing neurons in the ventrolateral part (Vpvl) and ventral part of dorsomedial part of Vp (Vpdm) are small-sized; while the larger neurons in the Vpdm are those expressing VGLUT1 mRNA and rarely colabeled with VGLUT2 mRNA (Ge et al., 2010) (Fig.1). TSNC transmits craniofacial sensory signals to the thalamus, mainly to the ventral posteromedial (VPM) and posterior (Po) nuclei, before reaching somatosensory cortex for perception. The trigemino-thalamic

IP T

projection neurons mostly express VGLUT2 mRNA (Ge et al., 2014; Graziano et al., 2008), contributing to the thalamic VGLUT2 terminal pool (Fig.1B). This is corroborated by the pronounced decrease of thalamic VGLUT2-IR but not VGLUT1-IR following lesion of unilateral trigeminal lemniscus (Graziano et al., 2008).

SC R

Accordingly, the two thalamic VGLUT isoforms in VPM and Po originate from distinct populations of neurons, with VGLUT1-IR arising from cortex, as proposed by Graziano et al. from the presence of corticothalamical projections with VGLUT1-IR and the unilateral SI lesion-evoked complete loss of

U

VGLUT1 in ipsilateral Vp (Graziano et al., 2008). However, our findings that Vp neurons projecting to the

N

VPM or Po of the thalamus co-expressed VGLUT1/VGLUT2 (Ge et al., 2014) (Fig.1F) also support the

A

trigeminal origin of thalamic VGLUT1-IR. Such view agrees with that of Nakamura et al (2005). The

M

aforementioned invisibility of VGLUT1 decrease following interruption of trigeminal lemiscus may be due to relatively less contribution of Vp to the VGLUT1 reservoir consisting overwhelmingly of corticothalamic

ED

VGLUT1-IR terminals.

Physiologically, the VGLUT1/VGLUT2 double-labeled Vpdm neurons may possibly, or at least, be

PT

responsible for sending muscle spindle afferent signals to VPM. First, these neurons project to VPM (Ge et al., 2014). Second, VPM-projecting neurons in Vpdm receive spindle afferent signals, since these neurons

CC E

are closely apposed by dense collateral swellings of muscle spindle afferents, as revealed via intracellular labeling of physiologically identified Vme neurons (Luo et al., 1991; 1995). As for VGLUT1-single labeled TSNC neurons, they constitute an overwhelming part of trigemino-cerebellar pathways (Ge et al., 2014)

A

(Fig.1H), suggesting VGLUT1’s role in conveying trigeminal signals to cerebellum for the purpose of coordinating head movements and balance. The functional considerations of the VGLUT2 neurons in TSNC include the following. First, based on the foregoing hodology data on TSNC-thalamic projections, TSNC VGLUT2-positive neurons are implicated in relaying orofacial senses to higher brain region. Second, Neurons of VGLUT2 phenotype in TSNC, inferably receptive to trigeminal stimulation (Waite, 2004), project to and synapse upon Vm neurons 24

with VGLUT2 terminals (Pang et al., 2009). Therefore, these neurons can, serving as premotor neurons, feedback orofacial signal to Vm for facilitating the jaw-reflex. Finally, diverse projections from Vc to brain structures such as parabrachial, cochlear nuclei and premotor nuclei in reticular formation and supratrigeminal nucleus, exclusively express VGLUT2 (Dong et al., 2012; Li et al., 2017; Ye et al., 2009; Zhou et al., 2007), indicating TSNC VGLUT2’s role in transmitting orofacial signals such as nociceptive and tactile signals for coordination of jaw reflex and/or further processing or signal integration. Similarly, VGLUT1 single-labeled TSNC neurons, albeit much less densely and heterogeneously

IP T

distributed, may also function in jaw-reflex.

5. Somatosensory thalamus

SC R

The ventral posterior complex of thalamus that includes VPL, the VPM and the Po relays somatic sensory signals to cerebral cortices. Among these, VPL and Po are recipients of spinal-relayed signals from body and limbs, whereas VPM and Po receive from craniofacial afferents via the relay of TSNC (Dong and

U

Dong, 2018; Tracey, 2004).

N

All these three somatosensory thalamic nuclei of rats (Barroso-Chinea et al., 2007) and mice (Graziano

A

et al., 2008) contain somatic VGLUT1 and VGLUT2 mRNA signals, with VGLUT1 mRNA signal being

M

low to moderately expressed and VGLUT2 mRNA being more significant (Barroso-Chinea et al., 2007). Among these nuclei, neurons in Po display the lowest level of VGLUT1 mRNA (Barroso-Chinea et al.,

ED

2007). Using dual fluorescent ISH, the sensory relay neurons in VPL and VPM were mostly observed to coexpress VGLUT1 and VGLUT2 mRNAs (Barroso-Chinea et al., 2008; Barroso-Chinea et al., 2007). This

PT

co-localization of VGLUTs at mRNA level is consistent with thalamocortical projections in lamina IV of SI which display co-immunostaining for VGLUT1 and VGLUT2 (Nakamura et al., 2005). As a generality for

CC E

the whole thalamus, the VGLUT1 and VGLUT2 mRNAs demonstrate a complementary distribution, since the complete lack of VGLUT1 mRNA in nonspecific thalamic nuclei (midline and intralaminar nuclei) and its low/no expression in principle relay nuclei and association thalamic nuclei contrast the high level of

A

VGLUT2 mRNA in these thalamic structures (Barroso-Chinea et al., 2007; Graziano et al., 2008). However, the “complementary” mode seems not applicable to VGLUT1 and VGLUT2 mRNAs in VPL and VPM due to extremely high incidence of neuronal co-expression in these nuclei (Barroso-Chinea et al., 2007). At the protein level, VPL, VPM and Po of mice display large number of VGLUT1- and VGLUT2-IR puncta, and in VPM but not VPL, co-localization of VGLUT1 and VGLUT2 exists (Graziano et al., 2008; Nakamura et al., 2005; Varoqui et al., 2002). The abundance and intensity of the two transporters are 25

dynamically regulated over development, possibly independently of each other as revealed from postnatal day 0 (P0) to P22, showing a diminution of the co-localization incidence or the down-regulation of VGLUT1 after P22 or in adult (Nakamura et al., 2005). These single-labeled VGLUT1 or VGLUT2 and co-labeled VGLUT1/VGLUT2 puncta in VPM and Po reflects neuron populations with distinct VGLUT phenotypes in TSNC (Ge et al., 2014) and cortex (Graziano et al., 2008) projecting to somatosensory thalamus. Indeed, cortical VGLUT1 mRNA exists in SI, in agreement with the VGLUT1-IR puncta in VP originating from SI cortex (Graziano et al., 2008); and we observed VGLUT1- and VGLUT2-mRNA

IP T

single-labeled neurons in TSNC and co-labeled neurons in Vp that send axons to VP (Ge et al., 2014).

6. Somatosensory cortex

SC R

The primary somatosensory areas (SI) receive its thalamic inputs dominantly from ventrobasal complex of thalamus (VB, including VPL and VPM) (Dong and Dong, 2018; Saporta and Kruger, 1977) to terminate mainly in layer IV of the “granular zones”, and also accept afferents from Po to layers I and Va of the

U

“dysgranular” and “perigranular” zones (Fig. I). The secondary somatosensory areas (SII) receive axonal

N

terminations from VB and Po as well (Carvell and Simons, 1987; Pierret et al., 2000), with those from Po

A

distributed in layers I and IV (Herkenham, 1980).

M

Both VGLUT1- and VGLUT2-IR puncta in SI are distributed across cortical laminae of rats and mice (Graziano et al., 2008; Nakamura et al., 2005; Varoqui et al., 2002). In SI, VGLUT2-IR is more enriched in

ED

superficial neuropil of layer I, deep layer III, layer IV and in upper layer VI; whereas VGLUT1-IR, although present across layers, is much weaker in layer IV (Fujiyama et al., 2001; Graziano et al., 2008). Notably, in

PT

layer IV the barrel core regions, characterized by VGLUT2-IR and VGLUT1-IR, are pronounced thanks to the much weaker VGLUT2-IR and more intense VGLUT1-IR surrounding them (Fujiyama et al., 2001;

CC E

Nakamura et al., 2005). Colocalization of VGLUT1 and VGLUT2 exists in layer IV, the incidence, however, vary between developmental stages (Graziano et al., 2008; Nakamura et al., 2005) (Fig. I). As is the case in other brain regions, the cortical VGLUTs are ultrastructurally localized to the synaptic vesicular membrane

A

of asymmetrical synapses (Conti et al., 2005; Fujiyama et al., 2001). The immunostained VGLUT puncta in SI have different origins. First, most VGLUT2-IR terminals

may arise from thalamic nuclei including VPM/VPL and ventral anterior, anteroventral, lateroposterior nuclei (Fujiyama et al., 2001; Hur and Zaborszky, 2005) (Fig. I), and some may be of cortical origin (Graziano et al., 2008).

Still, a little portion of the corticopetal VGLUT2-IR projections originate from

discrete neurons in claustrum, hypothalamus, amygdala, ventral tegmental area, globus pallidus, internal 26

capsule, and substantia innominata (Hur and Zaborszky, 2005). The marked decrease of cortical VGLUT2-IR induced by lesion of thalamic nuclei (VPM) with kainic acid (Fujiyama et al., 2001) and the layer IV VGLUT2-IR represented by biotinylated dextran-amine labeled thalamocortical axon terminals (Nahmani and Erisir, 2005) support VGLUT’s thalamic origin. Specifically, those relay neurons in VP with colocalized VGLUT1 and VGLUT2 can supply lamina IV of SI with thalamocotical projections showing colocalization of VGLUT1- and VGLUT2-IR, and intralaminar nuclei may send VGLUT2 projections to layer I of SI (Graziano et al., 2008). Second, almost no change in cortical VGLUT1-IR following lesion of

IP T

thalamic relay neurons (Fujiyama et al., 2001) imply that the cortical VGLUT1 may be of cortical origin (Nakamura et al., 2005).

In rodents, cortical VGLUT1 and VGLUT2 transcripts are complementarily distributed: strong

SC R

VGLUT1 mRNA signals in layers II-VI and weak VGLUT2 mRNA expression in layers IV and VI (Bai et al., 2001; Herzog et al., 2001; Hisano et al., 2000; Liguz-Lecznar and Skangiel-Kramska, 2007b; Ni et al., 1994; Ni et al., 1995). This topographic feature was elaborated by Graziano et al., demonstrating heavy

U

VGLUT1mRNA signal in SI except layers I and IV where the signal is weak, and diverse intensity of

N

VGLUT2 mRNA: none in layers I/II, high level in layer III and scattered in layers IV to VI (Graziano et al.,

A

2008). In human brain abundant VGLUT1 mRNA is present in layers V–VI and VGLUT2 mRNA restricted

M

to layer V (Vigneault et al., 2015), suggesting obvious species difference in cortical VGLUTs expression.

ED

7. Functional relevance of VGLUTs in the sensory systems

PT

7.1 Putative roles of VGLUT1/2 in the somatosensory pathways The profile of VGLUT phenotypes manifested by spinothalamic and trigeminal sensory systems are

CC E

recapitulated in Fig.1. In these pathways the data on the elemental neurons with a certain VGLUT isoform, considered under the context of fiber connections of these neurons with function-defined structure(s), and complemented by the in vivo physiological/behavioral tests, render it possible to resolve, at least partially,

A

the physiological role(s) of the VGLUT isoform in the specified cell type. In periphery, large-sized DRG neurons express abundant VGLUT1, while small-sized ones frequently display robust VGLUT2 expression. Similarly, in the trigeminal system, the proprioceptive afferents (Vme neurons) only express VGLUT1, while the primary afferents projecting to the putative nociceptive zone (superficial layer) of Vc contain intense VGLUT2-IR. Thus, it is reasonable to presume that the peripheral VGLUT1 and VGLUT2 are more implicated in proprioception and nociception, respectively. Centrally, the two VGLUT isoforms are also 27

segregated in neuronal populations, probably used for coding of distinct sensory signals. In trigeminal system, the clear-cut VGLUT1-labeled trigeminocerebellar and VGLUT2-labeled trigeminothalamic projection pathways may possibly send trigeminal signals of different modalities. Similarly, the trigeminothalamic projections such as Vp-VPM and Vp-Po projections, which engage co-expressed VGLUT1/VGLUT2 and VGLUT2, respectively, may transfer signals that are differently coded.

7.2 The bioenergetic and physiological difference between VGLUTs

IP T

Since the VGLUT isoforms each serve as vehicle for glutamate transport into synaptic vesicles, it seems a redundancy to recruit distinct VGLUTs for glutamate transmission by brain neurons ensemble. Differences, however, exist indeed between them in structure (e.g., composition of amino acids), understanding these differences, which were observed mostly in functional structures

SC R

bioenergetics and the

other than somatic sensory systems, will undoubtedly advance our insight into the VGLUTs’ role(s) in somatic sensory systems.

U

Initially characterized as plasma membrane transporters proposed to co-transport inorganic phosphate

N

(Pi) and glutamate, brain-specific Na+-dependent inorganic phosphate transporter I (BNPI) and

A

differentiation-associated Na+-dependent inorganic phosphate transporter (DNPI) (Bellocchio et al., 2000;

M

Takamori et al., 2001), were later verified to be the currently known VGLUT1 and VGLUT2. Structurally, these VGLUTs shared 82% homology in amino acid sequence; VGLUT1 features two polyproline motifs in

ED

cytoplasmic C terminus, however (Aihara et al., 2000).

Also, the driving forces for transport of glutamate into vesicles by VGLUT1 and VGLUT2 differ.

PT

VGLUT1 rely on electrical component (membrane potential, △) of the electrochemical proton gradient as the predominant driving force, generated by a Mg2+-activated vacuolar H+-ATPase on the vesicular

CC E

membrane; while VGLUT2 can be driven by both △ and pH gradient (△pH) (Bai et al., 2001, El Mestikawy et al., 2011). In addition, VGLUT1 and VGLUT2 differentially regulate basal synaptic function and synaptic strength. VGLUT1 synapses were observed to have a lower transmitter release probability than

A

VGLUT2 synapses (Fremeau et al., 2001; Fremeau et al., 2004b; Varoqui et al., 2002). The neuronal underpinning for this VGLUT-dependent difference in release probability is still elusive, but these transporters probably were observed to have different compartments as their docking sites in the presynaptic element. In PC12 cells heterologously transfected with VGLUT1 or VGLUT2, VGLUT2 shows a more diffuse cytoplasmic location than VGLUT1, while VGLUT1 has a peripheral somatic distribution (Fremeau et al., 2001). 28

VGLUT1 and VGLUT2 differentially regulate synaptic plasticity. Gene knockout study demonstrated that VGLUT1 and VGLUT2 synapses exhibited different forms of short-term plasticity (Fremeau et al., 2004a); and VGLUT1 but not VGLUT2, was more frequently expressed in synapse exhibiting long-term potentiation (LTP) (Varoqui et al., 2002). In vitro study showed that deletion of VGLUT1 impaired hippocampal LTP, while heterozygous VGLUT2 mice did not (Balschun et al., 2010). This phenomenon may possibly be explained by the structured polyproline motifs constituting VGLUT1 protein but not VGLUT2 and VGLUT3, which enables long-lasting active transmitter endocytosis/exocytosis recycling (Fei

IP T

et al., 2008; Voglmaier et al., 2006). On the other hand, different VGLUT isoforms may possibly impact on the quantal size of glutamate released by being assembled into and thus forming constitutively different presynaptic machineries for loading of glutamate into vesicles (Wilson et al., 2005; Wojcik et al., 2004).

SC R

Understandably, variation in the elements of this machinery causes glutamate release. Thalamic cultures and striatal neurons showing VGLUT2 expression level on vescicles have been documented to impact quantal size (Moechars et al., 2006). However, discrepancy was reported, i.e., no control of quantal size by VGLUTs,

U

as was demonstrated by comparing the miniature EPSC (mEPSC) amplitudes recorded on neurons from

N

VGLUT1 knockout mice and their wild-type littermates (Fremeau et al., 2004a). Obviously, more studies are

A

needed to elaborate on VGLUTs’ role and the nuances between different VGLUT isoforms in transmitter

M

release control.

ED

7.3 Behavioral relevance of VGLUT isoforms in sensory transmission As far as we know, no behavior studies using VGLUT1-deleted mice, including homozygotes

PT

(VGLUT1-/-) and heterozygotes (VGLUT1+/-), were designed particularly for addressing VGLUT1’s role in somatosensory system. Reportedly, global VGLUT1 knockout mice (VGLUT1-/-) developed hunched

CC E

posture from the third postnatal week onwards, and the sensorimotor gating deficits occurred in VGLUT1+/mice (Fremeau et al., 2004a; Inta et al., 2012). The neural substrates for developing such behavioral abnormalities are not known, but presumably involve proprioceptive signals made aberrant by genetically

A

nullifying VGLUT1. These behavioral data available yield no further and definite insights into VGLUT1’s role in these sensorimotor disorders, since the genetically maneuvered VGLUT1 deficiency is not regionand/or cell type-specific. VGLUT2 is involved in pain regulation, which is suggested by the VGLUT2 up-regulation (DRG and spinal cord) under the neuropathic pain state (Wang et al., 2016). However, in vivo studies on VGLUT2-pain relationship are hindered largely by the lack of specific VGLUT inhibitors (Izumi et al., 2015; Wang et al., 29

2016; Wang et al., 2015). In recent years, thanks to the genetic strategy using Cre/LoxP system which rendered possible the conditioned knockout of either VGLUT isoforms associated with specific circuits or cell types, we have advanced our insights into the function of VGLUTs (Fremeau et al., 2004a; Wallen-Mackenzie et al., 2010; Wojcik et al., 2004). Global knockout of VGLUT2 also reveals a correlation of VGLUT2 and pain; however, the degree to which VGLUT2 is related to pain seems depend on the type and/or nature of pain involved. VGLUT2 heterozygotes (VGLUT2 +/-) showed no significant change in acute, formalin- or carrageenan-induced pain but showed an alteration in development and/or maintenance

IP T

of neuropathic pain (Table 2). Reportedly, spared nerve injury (SNI)-modeled neuropathic pain was completely impaired in VGLUT2 +/- animals when compared with their wild type, scoring no (male) or weak (female) mechanical allodynia and deficient cold allodynia as tested by von Frey filament poking at

SC R

and acetone spraying onto plantar surface, respectively (Moechars et al., 2006). Similar reduced cold allodynia was also observed in VGLUT2 +/-, but not VGLUT1 +/- mice with pain modeled with chronic constriction injury (CCI) (Leo et al., 2009). These data suggest an essential role of VGLUT2 in acquisition

U

of neuropathic pain.

N

Details parsing the role of VGLUT2 in pain transmission owe considerably to conditional VGLUT

A

knockout targeting biochemically distinct DRG cell types, including those positive for TRPV1, Nav1.8,

M

Ht-Pa and Th (Lagerstrom et al., 2010; Lagerstrom et al., 2011; Liu et al., 2010b; Rogoz et al., 2014a; Rogoz et al., 2014b; Rogoz et al., 2012; Rogoz et al., 2015) (Table 2). Mice lacking VGLUT2 in Nav1.8-

ED

expressing DRG neurons (VGLUT2f/f; Nav1.8-Cre) behaved normally when modeled for neuropathic pain (Lagerstrom et al., 2011), but those with null VGLUT2 in DRG neurons expressing TRPV1, Ht-Pa and Th

PT

showed a reduced mechanical and thermal/cold allodynia (Liu et al., 2010b; Rogoz et al., 2012; Rogoz et al., 2015; Scherrer et al., 2010) (Table 2). These findings incidentally suggest that Nav1.8-expressing DRG

CC E

neurons may possibly be not responsible for neuropathic pain development. Mutants such as VGLUT2f/f; Per-Cre, VGLUT2f/f; ThIRES-Cre, VGLUT2f/f; Ht-Pa-Cre and VGLUT2f/f; Trpv1-Cre, all with conditioned VGLUT2 knockout in distinct DRG cell types showed decreased response

A

to heat nociception (Lagerstrom et al., 2010; Rogoz et al., 2014b; Rogoz et al., 2012; Scherrer et al., 2010), implicating VGLUT2’s role in thermal detection. In contrast, mice with null VGLUT2 in Nav1.8 expressing DRG neurons (VGLUT2f/f; Nav1.8Cre) manifested inconsistent responses to nociceptive heat stimulation, rating as no alteration or decreased response as compared with their wild types. However, most probably, VGLUT2-Nav1.8 expressing DRG neurons transduce mechanical pain stimulation, as was observed via Randall–Selitto

test

(Lagerstrom

et

al.,

2011; 30

Liu

et

al.,

2010b),

and

are

engaged

in

inflammatory/thermal-induced pain, as was revealed on pain model established by subcutaneous injection of complete Freund’s adjuvant (CFA) or nerve growth factor (NGF) (Lagerstrom et al., 2011; Liu et al., 2010b). It is clear that VGLUT2 is engaged in multiple aspects/types of pain which are transduced and coded inherently through nociceptors of different types. For instance, the type of TRPV1-DRG neurons, containing VGLUT2, is possibly more responsible for neuropathic pain generation; whereas the DRG neuron subtypes expressing, Th and Ht-Pa, which contain VGLUT2 as well, are more involved in inflammatory pain and thermal-induced acute pain, as exhibited on animal by an alleviated pain if provoked by inflammatory

IP T

algesic agents such as formalin; carrageenan; CFA and NGF (Lagerstrom et al., 2011; Lagerstrom et al., 2010; Liu et al., 2010b; Rogoz et al., 2012; Scherrer et al., 2010).

Similar to pain, itch signal from periphery to somatosensory cortex (SI) is transmitted via a “labeled

SC R

line” (Andrew and Craig, 2001; Dong and Dong, 2018; Duan et al., 2018; Handwerker, 2010; Lagerstrom et al., 2010; Ross et al., 2014). Then, what is the physiological role of VGLUTs in itch sensation? First, there exist in spinal lamina I both GRP receptor-expressing neurons and the spinothalamic tract

U

projection neurons that are indispensible for itch sensation (Sun et al., 2009; Ross et al., 2014). Since these

N

neurons along the itch transmitting pathway are excitatory (Andrew and Craig, 2001; Mu et al., 2017; Ross

A

et al., 2014), they are most likely to be VGLUT2 active, as is revealed by single cell RNA sequencing study

M

showing that all spinal excitatory contain Slc17a6, a gene encoding VGLUT2 (Häring et al., 2018). Thus, the itch-specific spinal neurons may possibly engage VGLUT2 in glutamate transmission of itch.

ED

Second, as mentioned above in the section “3.1.2”, pruriceptors contain VGLUT2. In addition, multiple types of pain deficits and increased itch manifested in VGLUT2 knockout mice clearly verify the critical

PT

functions of VGLUT2 in regulating itch (Liu et al., 2010b; Lagerstrom et al., 2010). However, up to date, the transmitter identity of glutamate released from the pruriceptors is still controversial.

CC E

Intradermal chloroquine-induced scratching itch was not altered following VGLUT2 knockout in MrgprA3-/GRP-positive pruriceptors (Liu et al., 2009; Liu et al., 2010b). These results speak against the VGLUT2-associated glutamate as neurotransmitter from primary afferent itch receptors (Liu et al., 2010b;

A

Ma, 2014). In contrast, other experimental observations tell otherwise. The chloroquine-evoked scratching and spinal neuronal firing could be attenuated by spinal application AMPA/kainate receptor antagonist CNQX, and could be abolished by co-application of CNQX plus neurokinin-1 and/or GRP receptor antagonists (Akiyama et al., 2014). These data suggest that glutamate, together with GRP, SP and natriuretic polypeptide b (NPPB), may constitute a cohort of neurotransmitters from pruriceptors (Akiyama et al., 2014; Ma, 2014). The idea of pruriceptor-released glutamate as neurotransmitter is also supported by the 31

observation that spinal GRP-responsive neurons receive exclusively C-type inputs, and the evoked responses of these neurons are actually mediated by glutamate, but not GRP (Koga et. al., 2011). Therefore, further studies are still required to address the exact role of VGLUT2-associated release of glutamate. Third, itch signal may be gated by the spinal circuits by using VGLUT2 associated glutamate. Itch is processed, extracted and gated by highly complicated spinal circuits. The selectivity (or population coding) theory hypothesizes that, putting it in a simplest way, both the itch- and pain-specific neurons connect, directly and/or indirectly, to pain and itch projection neurons in the spinal cord, respectively (Akiyama et

IP T

al., 2014; Duan et al., 2018; Handwerker, 2010; Lagerström et al., 2010; Liu et al., 2010b; McMahon and Koltzenburg, 1992). The pain-specific inputs, in addition, can inhibit itch projection neurons by activating interneurons such as those expressing transcription factor Bhlhb5, or the glycinergic ones and neuropeptide

SC R

Y (NPY) lineage neurons, effecting a gate control (Duan, et al., 2018; Ross, et al., 2010; 2014). In this scenario, itch is perceived only when itch-specific population is selectively activated. During this process, pruriceptor-released glutamate (mediated by VGLUT2) may functionally facilitate itch transmission, by

U

acting on the “interneuron-itch projection neurons” gating circuits to counteract the “tonic inhibition” made

N

possibly by pain signals (Ma, 2014). When both nociceptors and pruriceptors are activated, the pain-specific

A

inputs will release VGLUT2 mediated glutamate which, in turn, excite the gating interneurons and finally

M

suppress itch projection neurons. This model well explains our frequently used practice of relieving itch via scratching in our daily life and the deficits in pain and increased scratching manifested in mice

ED

VGLUT2-nulled mice (Liu et al., 2010b; Lagerstrom et al., 2010; Rogoz et al., 2014b). In summary, VGLUT2 plays a essential role in pain and itch perception (Malet and Brumovsky, 2015;

PT

Seal, 2016). As for VGLUT1, despite no direct evidence in vivo of VGLUT1’s involvement in the

CC E

somatosensory transmission, morphological and in vitro data strongly indicate its role in mechanoreception.

8. Future work

Morphological characterization of VGLUTs at relay of each order in the sensory pathways, however

A

comprehensive and elaborate, yield valuable but only limited insight into VGLUTs’ role in sensation. A full understanding of VGLUTs’ function in transmission and processing of neural signals from different modalities necessitates an all-around knowledge on these transporters, ranging from molecular, pharmacological, physiological to behavioral. The following are some points considered , among others, necessary to be addressed. First, the detailed molecular mechanisms and pharmacokinetics of, and the difference thereof between, 32

VGLUT isoforms in glutamate loading still remain incompletely clarified, thus hindering a full understanding of VLGUTs’ roles in spinal/trigeminal sensory systems. Future work unraveling the nuanced properties of VGLUTs will unequivocally necessitate exquisite cellular/ reconstituted proteosome working models and the currently cutting-edge research tools. Glutamate loading by VGLUTs depends on the electrochemical proton gradient generated by v-type proton-pump ATPase (Forgac, 2000; Wolosker, et al., 1996). Various studies (Guillaud, et al., 2017; Rossano, et al., 2017), including reconstitution of VGLUT, or together with vacuolar-type ATPase or purified bacterial

IP T

FoF1-ATPase into liposomes (Eriksen, et al., 2016; Juge, et al., 2006, 2010; Omote, et al., 2011; Preobraschenski, et al., 2014; Schenck, et al., 2009; Takamori, 2016a,b), demonstrated that inorganic ions like anion Cl (both the extravescicular and intraluminal), K+ and H+ can influence glutamate uptake by

SC R

VGLUT. These ions act on VGLUTs, for example, either through allosteric action, as proposed for the role of Cl which facilitates (low concentration, e.g., 4 mM) and inhibits (high concentration) glutamate uptake, respectively; or via binding to VGLUT, eliciting ion transportation into and out of vesicles to modulate

U

vesicular membrane potential (△) (Preobraschenski et al., 2014; Guillaud, et al., 2017; Rossano, et al.,

N

2017). Based on these findings, several models for VGLUTs’ working mechanism have been proposed,

A

including the intriguing one by Preobraschenski et al. (2014) which implicates VGLUT as an antiporter for

M

K+/H+ and Cl as an allosteric effector. Nevertheless, molecular mechanisms on ion-VGLUT interaction are still short of full understanding. For instance, in the process of transporting glutamate, whether can VGLUTs

ED

concomitantly function as ion channels for Cl permeation? No detectable Cl transportation was observed in reconstituted VGLUT-liposomes (Juge et al., 2010; Takamori, 2016a-b). This is in striking contrast to the

PT

large VGLUT2-associated Cl conductance recorded on oocytes (Eriksen et al., 2016). In addition, plasma membrane VGLUT2 showed a property of Na+dependent inorganic phosphate (Pi) uptake independent of

CC E

glutamate transportation, as confirmed via reconstituted proteoliposomes with VGLUT2 and FoF1-ATPase (Juge et al., 2006). Then, whether VGLUTs also function as Pi transporter in synaptic vesicles under physiological state? This is also a question that remains to answer relating to VGLUT’s functional property

A

at the molecular level.

Second, an intriguing, important, nevertheless pending issue is whether the DRG/TG neurons enriched

with VGLUT1-3 proteins have a property of releasing glutamate from neuronal somata, i.e., non-synaptic release. Although not touched in the foregoing sections, insight into this aspect is as equally important for disclosing VGLUTs’ somatosensory role(s) as VGLUTs’ profiling and their functional analysis at ionic/molecular/behavioral levels. It has been reported that stimulation DRG/TG neurons induced somatic 33

release of neuroactive substances such as CGRP and ATP via cytoplasmic vesicles (Thalakoti, et al., 2007; Ulrich-Lai, et al., 2001; Zhang, et al., 2007). More importantly, when heterogeneously expressed in Xenopus oocytes, the plasma membrane-bound VGLUT2 mediated glutamate release in a Ca2+-independent manner (Mackenzie, et al., 2008). Third, co-expression of VGLUT1 and VGLUT2 exists in DRG neurons, their bifurcated axon terminals, however, usually possess one single VGLUT isoform. Therefore, efforts should be made to clarify the cellular mechanisms underpinning sorting and transporting VGLUT1 and VGLUT2 from perikarya to

IP T

peripheral/central axon terminals. An ensuing question is: why one particular transporter is targeted for periphereal rather than central axon terminal, or vice versa? What are the nuances between these isoforms, located in distinct terminals in terms of their physiological roles, biochemical and/or pharmacokinetic

SC R

properties?

In addition, phylogenetic analysis indicates that animals subordinate to gnathostomes in the hierarchy of taxonomic ranks possesses one gene, referred to as Slc17a6/7/8 by Sreedharan et al. (2010), which encodes a

U

member of glutamate vesicular transporters; whereas other animals contain three orthologues, i.e., Slc17a6,

N

Slc17a7 and Slc17a8 encoding VGLUT2, VGLUT1 and VGLUT3, respectively. In the case of

A

somatosensory pathways in mammals, many neurons in DRG and trigeminal Vp express VGLUT1 and

M

VGLUT2 simultaneously. Then, what is the physiological rationale, in perspective of evolution, for animals to evolve from one common ancestor into three gene isoforms performing the seemingly same function?

ED

Fourth, in relay nucleus (e. g., TSNC), the physiological role(s) of neuronal subtypes with distinct VGLUT phenotypes should be clarified in terms of the sensory modalities they receive and relay. As have

PT

been reported, the signal-accepting/transmitting neurons positioned in any of the relay structures along the sensory pathways are assorted by VGLUT phenotype into over two chemically distinct groups. When

CC E

signals encoding a particular stimulation reach the relay structure, which type of the VGLUT-labeled neurons are responsive? Clearly, clarifying the modality-cell type relationship will undoubtedly deepen our understanding of the VGLUTs’ functions and may provide therapeutic clues for treatment of diseases such as

A

pain by targeting VGLUT(s). In vivo work using intracellular recording on the relay neuron responsive to peripheral stimulation (e.g., nociceptive stimulation), paired with labeling work post mortem to identify the neuron’s VGLUT isoform, may contribute much to this aspect. Finally, future endeavor is required to develop new drugs targeting VGLUT(s) to relieve pain, since intervention of glutamate transmission clearly affects pain sensation (see above and Weng et al., 2006). VGLUT2 abounds along somatosensory pathways. Under neuropathic conditions,VGLUT2 expression may 34

be up-regulated in some pain-related relay neurons (Wang et al., 2015). Thus, suppression of glutamate transmission may promise an analgesic stratagem. VGLUT inhibitors such as glutamate analogues and diazon-/monoazo-dyes are available (Ueda, 2016); these compounds, however, are either less potent, membrane-impermeable, or potentially toxic. Therefore, new drugs, developed either through screening or synthesis, without such drawbacks are anticipated. Notably, by removal of diazo group and replacement of sulphonate group with carboxyl group, two derivatives (BYA 1 and BYA 2) from the tissue toxic Brilliant Yellow were successfully synthesized, reportedly devoid of cell toxicity, and retain the property of high

IP T

specificity to VGLUT (Kehrl et al., 2017). Also, they are membrane-permeable and can depress glutamate transmission (Kehrl et al., 2017), thus putting forward a step towards developing more specific and effective VGLUTs inhibitors.

SC R

Additionally, the cutting-edge of genetic techniques enabling specific control of the transcription and/or translation of a target gene offers an alternative hope for pain control. For instance, by using siRNA to suppress or vector (e.g., virus)-carried gene(s) to over-express a certain VGLUT subtype in a specific cell

N

U

type, it may be possible to relieve debilitating, intractable pain occurring in cases such as cancer.

A

9. Concluding remarks

M

A large pool of data has represented for us an expressional profile of VGLUT1 and VGLUT2 along somatosensory systems, although further detailing VGLUTs at mRNA and/or protein levels is still required

ED

to perfect the VGLUTs topography of the spinal and trigeminal pathways. In addition, the tantalizing and important issues, especially those about VGLUTs’ functions, still stand as big tasks that merit cooperative

CC E

PT

effort from neuroscientists to fulfill.

Acknowledgements

A

This work is supported by National Natural Science Foundation of China (Nos. 81571074, 81171279, 81870866, 81071105, 31871216)

References Aihara, Y., Mashima, H., Onda, H., Hisano, S., Kasuya, H., Hori, T., Yamada, S., Tomura, H., Yamada, Y., Inoue, I., Kojima, I., Takeda, J., 2000. Molecular cloning of a novel brain-type Na(+)-dependent inorganic phosphate cotransporter. J. Neurochem. 74, 2622-2625. Akiyama, T., Tominaga, M., Takamori, K., Carstens, M.I., Carstens, E., 2014. Roles of glutamate, substance P, and 35

gastrin-releasing peptide as spinal neurotransmitters of histaminergic and nonhistaminergic itch. Pain. 155, 80-92. Alvarez, F.J., Cervantes, C., Blasco, I., Villalba, R., Martinez-Murillo, R., Polak, J.M., Rodrigo, J., 1988. Presence of calcitonin gene-related peptide (CGRP) and substance P (SP) immunoreactivity in intraepidermal freenerve endings of cat skin. Brain Res. 442, 391-395. Alvarez, F.J., Villalba, R.M., Zerda, R., Schneider, S.P., 2004. Vesicular glutamate transporters in the spinal cord, with special reference to sensory primary afferent synapses. J. Comp. Neurol. 472, 257-280. Alvarez, F.J., Titus-Mitchell, H.E., Bullinger, K.L., Kraszpulski, M., Nardelli, P., Cope, T.C., 2011. Permanent central synaptic disconnection of proprioceptors after nerve injury and regeneration. I. Loss of VGLUT1/IA synapses on motoneurons. J. Neurophysiol. 106, 2450-2470. Andrew, D., Craig, A.D., 2001. Spinothalamic lamina I neurons selectively sensitive to histamine: a central neural pathway for itch. Nat. Neurosci. 4, 72-77. (Columba livia). Anat. Histol. Embryol. 38, 475-478.

IP T

Atoji, Y., Islam, M.R., 2009. Localization of vesicular glutamate transporter 2 mRNA in the dorsal root ganglion of the pigeon Averbeck, B., Reeh, P.W., 2001. Interactions of inflammatory mediators stimulating release of calcitonin gene-related peptide, substance P and prostaglandin E(2) from isolated rat skin. Neuropharmacology. 40, 416-423.

SC R

Averill, S., Robson, L.G., Jeromin, A., Priestley, J.V., 2004. Neuronal calcium sensor-1 is expressed by dorsal root ganglion cells, is axonally transported to central and peripheral terminals, and is concentrated at nodes. Neuroscience. 123, 419-427. Bai, L., Xu, H., Collins, J.F., Ghishan, F.K., 2001. Molecular and functional analysis of a novel neuronal vesicular glutamate transporter. J. Biol. Chem. 276, 36764-36769.

U

Balschun, D., Moechars, D., Callaerts-Vegh, Z., Vermaercke, B., Van Acker, N., Andries, L., D'Hooge, R., 2010. Vesicular glutamate transporter VGLUT1 has a role in hippocampal long-term potentiation and spatial reversal learning. Cereb.

N

Cortex. 20, 684-693.

Banks, R.W., Cahusac, P.M., Graca, A., Kain, N., Shenton, F., Singh, P., Njå, A., Simon, A., Watson, S., Slater, C.R., Bewick, G.S., mammalian hair follicles. J. Physiol. 591, 2523-2540.

A

2013. Glutamatergic modulation of synaptic-like vesicle recycling in mechanosensory lanceolate nerve terminals of

M

Bannatyne, B.A., Edgley, S.A., Hammar, I., Jankowska, E., Maxwell, D.J., 2006. Differential projections of excitatory and inhibitory dorsal horn interneurons relaying information from group II muscle afferents in the cat spinal cord. J. Neurosci.

ED

26, 2871-2880.

Barroso-Chinea, P., Castle, M., Aymerich, M.S., Lanciego, J.L., 2008. Expression of vesicular glutamate transporters 1 and 2 in the cells of origin of the rat thalamostriatal pathway. J. Chem. Neuroanat. 35, 101-107. Barroso-Chinea, P., Castle, M., Aymerich, M.S., Perez-Manso, M., Erro, E., Tunon, T., Lanciego, J.L., 2007. Expression of the

PT

mRNAs encoding for the vesicular glutamate transporters 1 and 2 in the rat thalamus. J. Comp. Neurol. 501, 703-715. Bellocchio, E.E., Hu, H., Pohorille, A., Chan, J., Pickel, V.M., Edwards, R.H., 1998. The localization of the brain-specific inorganic phosphate transporter suggests a specific presynaptic role in glutamatergic transmission. J. Neurosci. 18,

CC E

8648-8659.

Bellocchio, E.E., Reimer, R.J., Fremeau, R.T., Jr., Edwards, R.H., 2000. Uptake of glutamate into synaptic vesicles by an inorganic phosphate transporter. Science 289, 957-960. Bewick, G.S., Reid, B., Richardson, C., Banks, R.W., 2005. Autogenic modulation of mechanoreceptor excitability by glutamate

A

release from synaptic-like vesicles: evidence from the rat muscle spindle primary sensory ending. J. Physiol. 562, 381-394.

Braud, A., Boucher, Y., Zerari-Mailly, F., 2010. Vesicular glutamate transporters localization in the rat lingual papillae. Neuroreport 21, 64-67. Brooke, R.E., Atkinson, L., Edwards, I., Parson, S.H., Deuchars, J., 2006. Immunohistochemical localisation of the voltage gated potassium ion channel subunit Kv3.3 in the rat medulla oblongata and thoracic spinal cord. Brain Res. 1070, 101-115. Brouns, I., De Proost, I., Pintelon, I., Timmermans, J.P., Adriaensen, D., 2006. Sensory receptors in the airways: neurochemical coding of smooth muscle-associated airway receptors and pulmonary neuroepithelial body innervation. Auton. Neurosci. 126-127, 307-319. 36

Brumovsky, P., Watanabe, M., Hokfelt, T., 2007. Expression of the vesicular glutamate transporters-1 and -2 in adult mouse dorsal root ganglia and spinal cord and their regulation by nerve injury. Neuroscience 147, 469-490. Brumovsky, P.R., 2013. VGLUTs in peripheral neurons and the spinal cord: Time for a review. ISRN Neurol. 2013, 829753. Brumovsky, P.R., Robinson, D.R., La, J.H., Seroogy, K.B., Lundgren, K.H., Albers, K.M., Kiyatkin, M.E., Seal, R.P., Edwards, R.H., Watanabe, M., Hokfelt, T., Gebhart, G.F., 2011. Expression of vesicular glutamate transporters type 1 and 2 in sensory and autonomic neurons innervating the mouse colorectum. J. Comp. Neurol. 519, 3346-3366. Brumovsky, P.R., Seal, R.P., Lundgren, K.H., Seroogy, K.B., Watanabe, M., Gebhart, G.F., 2013. Expression of vesicular glutamate transporters in sensory and autonomic neurons innervating the mouse bladder. J. Urol. 189, 2342-2349. Byers, M.R., O'Connor, T.A., Martin, R.F., Dong, W.K., 1986. Mesencephalic trigeminal sensory neurons of cat: axon pathways and structure of mechanoreceptive endings in periodontal ligament. J. Comp. Neurol. 250, 181-191. in slowly adapting type I mechanoreceptor responses? Neuroscience. 133, 763–773.

IP T

Cahusac, P.M., Senok, S.S., Hitchcock, I.S., Genever, P.G., Baumann, K.I., 2005. Are unconventional NMDA receptors involved Cairns, B.E., Dong, X., Mann, M.K., Svensson, P., Sessle, B.J., Arendt-Nielsen, L., McErlane, K.M., 2007. Systemic administration of monosodium glutamate elevates intramuscular glutamate levels and sensitizes rat masseter muscle afferent fibers. Pain. 132, 33-41.

SC R

Carvell, G.E., Simons, D.J., 1987. Thalamic and corticocortical connections of the second somatic sensory area of the mouse. J. Comp. Neurol. 265, 409-427.

Chang, W., Kanda, H., Ikeda, R., Ling, J., DeBerry, J.J., Gu, J.G., 2016. Merkel disc is a serotonergic synapse in the epidermis for transmitting tactile signals in mammals. Proc. Natl. Acad. Sci. U S A. 113, E5491-5500. receptors in rat trigeminal ganglion. Neuroreport 25, 991-997.

U

Chen, Y., Zhang, L., Yang, J., Chen, Z., 2014. LPS-induced dental pulp inflammation increases expression of ionotropic purinergic

N

Clarke, J.N., Anderson, R.L., Haberberger, R.V., Gibbins, I.L., 2011. Non-peptidergic small diameter primary afferents expressing VGluT2 project to lamina I of mouse spinal dorsal horn. Mol. Pain 7, 95.

A

Conti, F., Candiracci, C., Fattorini, G., 2005. Heterogeneity of axon terminals expressing VGLUT1 in the cerebral neocortex. Arch. Ital. Biol. 143, 127-132. stimulation. Neuroreport. 11, 497-502.

M

deGroot, J., Zhou, S., Carlton, S.M., 2000. Peripheral glutamate release in the hindpaw following low and high intensity sciatic

ED

Diaz-Ruiz, A., Montes, S., Salgado-Ceballos, H., Maldonado, V., Rivera-Espinosa, L., Rios, C., 2016. Enzyme activities involved in the glutamate-glutamine cycle are altered to reduce glutamate after spinal cord injury in rats. Neuroreport 27, 1317-1322.

Dong, X,, Dong, X., 2018. Peripheral and central mechanisms of itch. Neuron. 98, 482-494.

PT

Dong, Y.L., Wang, W., Li, H., Li, Z.H., Zhang, F.X., Zhang, T., Lu, Y.C., Li, J.L., Wu, S.X., Li, Y.Q., 2012. Neurochemical properties of the synapses in the pathways of orofacial nociceptive reflexes. PLoS One 7, e34435. Dong, Y.L., Zhang, F.X., Pang, Y.W., Li, J.L., 2007. VGluT1- and GAD-immunoreactive terminals in synaptic contact with

CC E

PAG-immunopositive neurons in principal sensory trigeminal nucleus of rat. Acta. Pharmacol. Sin. 28, 180-184. Duan, B., Cheng, L., Ma, Q., 2018. Spinal circuits transmitting mechanical pain and itch. Neurosci. Bull. 34, 186-193. El Mestikawy, S., Wallen-Mackenzie, A., Fortin, G.M., Descarries, L., Trudeau, L.E., 2011. From glutamate co-release to vesicular synergy: vesicular glutamate transporters. Nat. Rev. Neurosci. 12, 204-216.

A

Elizalde, N., Pastor, P.M., Garcia-Garcia, A.L., Serres, F., Venzala, E., Huarte, J., Ramirez, M.J., Del Rio, J., Sharp, T., Tordera, R.M., 2010. Regulation of markers of synaptic function in mouse models of depression: chronic mild stress and decreased expression of VGLUT1. J. Neurochem. 114, 1302-1314.

Eriksen, J., Chang, R., McGregor, M., Silm, K., Suzuki, T., Edwards, R.H., 2016. Protons regulate vesicular glutamate transporters through an allosteric mechanism. Neuron 90, 768-780. Fagan, B.M., Cahusac, P.M., 2001. Evidence for glutamate receptor mediated transmission at mechanoreceptors in the skin. Neuroreport. 12,:341-347. Faunes, M., Onate-Ponce, A., Fernandez-Collemann, S., Henny, P., 2016. Excitatory and inhibitory innervation of the mouse orofacial motor nuclei: A stereological study. J. Comp. Neurol. 524,738-758. 37

Fei, H., Grygoruk, A., Brooks, E.S., Chen, A., Krantz, D.E., 2008. Trafficking of vesicular neurotransmitter transporters. Traffic 9, 1425-1436. Fillenz, M., 1995. Physiological release of excitatory amino acids. Behav. Brain Res. 71, 51-67. Flegel, C., Schöbel, N., Altmüller, J., Becker, C., Tannapfel, A., Hatt, H., Gisselmann, G., 2015. RNA-Seq analysis of human trigeminal and dorsal root ganglia with a focus on chemoreceptors. PLoS One. 10, e0128951. Forgac, M., 2000. Structure, mechanism and regulation of the clathrin-coated vesicle and yeast vacuolar H(+)-ATPases. J. Exp. Biol. 203, 71-80. Fremeau, R.T., Jr., Burman, J., Qureshi, T., Tran, C.H., Proctor, J., Johnson, J., Zhang, H., Sulzer, D., Copenhagen, D.R., Storm-Mathisen, J., Reimer, R.J., Chaudhry, F.A., Edwards, R.H., 2002. The identification of vesicular glutamate transporter 3 suggests novel modes of signaling by glutamate. Proc. Natl. Acad. Sci. U S A 99, 14488-14493. Fremeau, R.T., Jr., Kam, K., Qureshi, T., Johnson, J., Copenhagen, D.R., Storm-Mathisen, J., Chaudhry, F.A., Nicoll, R.A.,

IP T

Edwards, R.H., 2004a. Vesicular glutamate transporters 1 and 2 target to functionally distinct synaptic release sites. Science 304, 1815-1819.

Fremeau, R.T., Jr., Troyer, M.D., Pahner, I., Nygaard, G.O., Tran, C.H., Reimer, R.J., Bellocchio, E.E., Fortin, D., Storm-Mathisen, J., Edwards, R.H., 2001. The expression of vesicular glutamate transporters defines two classes of excitatory synapse.

SC R

Neuron 31, 247-260.

Fremeau, R.T., Jr., Voglmaier, S., Seal, R.P., Edwards, R.H., 2004b. VGLUTs define subsets of excitatory neurons and suggest novel roles for glutamate. Trends. Neurosci. 27, 98-103.

Fujiyama, F., Furuta, T., Kaneko, T., 2001. Immunocytochemical localization of candidates for vesicular glutamate transporters in

U

the rat cerebral cortex. J. Comp. Neurol. 435, 379-387.

Garcia-Garcia, A.L., Elizalde, N., Matrov, D., Harro, J., Wojcik, S.M., Venzala, E., Ramirez, M.J., Del Rio, J., Tordera, R.M.,

N

2009. Increased vulnerability to depressive-like behavior of mice with decreased expression of VGLUT1. Biol. Psychiatry 66, 275-282.

A

Gazerani, P., Dong, X., Wang, M., Kumar, U., Cairns, B.E., 2010. Sensitization of rat facial cutaneous mechanoreceptors by activation of peripheral N-methyl-d-aspartate receptors. Brain Res. 1319, 70-82.

M

Ge, S.N., Li, Z.H., Tang, J., Ma, Y., Hioki, H., Zhang, T., Lu, Y.C., Zhang, F.X., Mizuno, N., Kaneko, T., Liu, Y.Y., Lung, M.S., Gao, G.D., Li, J.L., 2014. Differential expression of VGLUT1 or VGLUT2 in the trigeminothalamic or

ED

trigeminocerebellar projection neurons in the rat. Brain Struct. Funct. 219, 211-229. Ge, S.N., Ma, Y.F., Hioki, H., Wei, Y.Y., Kaneko, T., Mizuno, N., Gao, G.D., Li, J.L., 2010. Coexpression of VGLUT1 and VGLUT2 in trigeminothalamic projection neurons in the principal sensory trigeminal nucleus of the rat. J. Comp. Neurol. 518, 3149-3168.

PT

Gebre, S.A., Reeber, S.L., Sillitoe, R.V., 2012. Parasagittal compartmentation of cerebellar mossy fibers as revealed by the patterned expression of vesicular glutamate transporters VGLUT1 and VGLUT2. Brain Struct. Funct. 217, 165-180. Grant, G., Robertson, B. 2004. CHAPTER 4 - Primary Afferent Projections to the Spinal Cord A2 - Paxinos, George. In: The Rat

CC E

Nervous System (THIRD EDITION). pp. 111-119. Academic Press: Burlington. Gras, C., Herzog, E., Bellenchi, G.C., Bernard, V., Ravassard, P., Pohl, M., Gasnier, B., Giros, B., El Mestikawy, S., 2002. A third vesicular glutamate transporter expressed by cholinergic and serotoninergic neurons. J. Neurosci. 22, 5442-5451. Graziano, A., Liu, X.B., Murray, K.D., Jones, E.G., 2008. Vesicular glutamate transporters define two sets of glutamatergic

A

afferents to the somatosensory thalamus and two thalamocortical projections in the mouse. J. Comp. Neurol. 507, 1258-1276.

Guillaud, L., Dimitrov, D., Takahashi, T., 2017. Presynaptic morphology and vesicular composition determine vesicle dynamics in mouse central synapses. Elife 6. Haeberle, H., Fujiwara, M., Chuang, J., Medina, M.M., Panditrao, M.V., Bechstedt, S., Howard, J., Lumpkin, E.A., 2004. Molecular profiling reveals synaptic release machinery in Merkel cells. Proc. Natl. Acad. Sci. U S A 101, 14503-14508. Handwerker, H. O., 2010. Microneurography of pruritus. Neurosci. Lett. 470, 193-196. Häring, M., Zeisel, A., Hochgerner, H., Rinwa, P., Jakobsson, J.E.T., Lönnerberg, P., La Manno, G., Sharma, N., Borgius, L., Kiehn, O., Lagerström, M.C., Linnarsson, S., Ernfors, P., 2018. Neuronal atlas of the dorsal 38

horn defines its architecture

and links sensory input to transcriptional cell types. Nat. Neurosci. 21, 869-880. Hartschuh, W., Weihe, E., Yanaihara, N., Reinecke, M., 1983. Immunohistochemical localization of vasoactive intestinal polypeptide (VIP) in Merkel cells of various mammals: evidence for a neuromodulator function of the Merkel cell. J. Invest. Dermatol. 81, 361-364. Hayashi, M., Otsuka, M., Morimoto, R., Hirota, S., Yatsushiro, S., Takeda, J., Yamamoto, A., Moriyama, Y., 2001. Differentiation-associated Na+-dependent inorganic phosphate cotransporter (DNPI) is a vesicular glutamate transporter in endocrine glutamatergic systems. J. Biol. Chem. 276, 43400-43406. Hegarty, D.M., Tonsfeldt, K., Hermes, S.M., Helfand, H., Aicher, S.A., 2010. Differential localization of vesicular glutamate transporters and peptides in corneal afferents to trigeminal nucleus caudalis. J. Comp. Neurol. 518, 3557-3569. Herkenham, M., 1980. Laminar organization of thalamic projections to the rat neocortex. Science 207, 532-535. Herzog, E., Bellenchi, G.C., Gras, C., Bernard, V., Ravassard, P., Bedet, C., Gasnier, B., Giros, B., El Mestikawy, S., 2001. The RC181.

IP T

existence of a second vesicular glutamate transporter specifies subpopulations of glutamatergic neurons. J. Neurosci. 21, Hioki, H., Fujiyama, F., Taki, K., Tomioka, R., Furuta, T., Tamamaki, N., Kaneko, T., 2003. Differential distribution of vesicular glutamate transporters in the rat cerebellar cortex. Neuroscience 117, 1-6.

SC R

Hisano, S., 2003. Vesicular glutamate transporters in the brain. Anat. Sci. Int. 78, 191-204.

Hisano, S., Hoshi, K., Ikeda, Y., Maruyama, D., Kanemoto, M., Ichijo, H., Kojima, I., Takeda, J., Nogami, H., 2000. Regional expression of a gene encoding a neuron-specific Na(+)-dependent inorganic phosphate cotransporter (DNPI) in the rat forebrain. Brain Res. Mol. Brain Res. 83, 34-43.

U

Hisano, S., Sawada, K., Kawano, M., Kanemoto, M., Xiong, G., Mogi, K., Sakata-Haga, H., Takeda, J., Fukui, Y., Nogami, H., 2002. Expression of inorganic phosphate/vesicular glutamate transporters (BNPI/VGLUT1 and DNPI/VGLUT2) in the

N

cerebellum and precerebellar nuclei of the rat. Brain Res. Mol. Brain Res. 107, 23-31. nerve terminal in rats. Neurosci. Lett. 362, 196-199.

A

Hitchcock, I.S., Genever, P.G., Cahusac, P.M., 2004. Essential components for a glutamatergic synapse between Merkel cell and Holstege, G., Blok, B.F.M., ter Horst, G.J., 1995. CHAPTER 27 - Brain stem systems involved in the blink reflex, feeding

M

mechanisms and micturition - Paxinos. George. In: The Rat Nervous System (SECOND EDITION). pp. 257-275. Academic Press: Armsterdam.

ED

Honma, S., Kato, A., Shi, L., Yatani, H., Wakisaka, S., 2012. Vesicular glutamate transporter immunoreactivity in the periodontal ligament of the rat incisor. Anat. Rec. (Hoboken) 295, 160-166. Huang, C.W., Tzeng, J.N., Chen, Y.J., Tsai, W.F., Chen, C.C., Sun, W.H., 2007. Nociceptors of dorsal root ganglion express proton-sensing G-protein-coupled receptors. Mol. Cell. Neurosci. 36, 195-210.

PT

Hughes, D.I., Polgar, E., Shehab, S.A., Todd, A.J., 2004. Peripheral axotomy induces depletion of the vesicular glutamate transporter VGLUT1 in central terminals of myelinated afferent fibres in the rat spinal cord. Brain Res. 1017, 69-76. Hur, E.E., Zaborszky, L., 2005. Vglut2 afferents to the medial prefrontal and primary somatosensory cortices: a combined

CC E

retrograde tracing in situ hybridization study [corrected]. J. Comp. Neurol. 483, 351-373. Hwang, S.J., Burette, A., Rustioni, A., Valtschanoff, J.G., 2004. Vanilloid receptor VR1-positive primary afferents are glutamatergic and contact spinal neurons that co-express neurokinin receptor NK1 and glutamate receptors. J. Neurocytol. 33, 321-329.

A

Inta, D., Vogt, M.A., Perreau-Lenz, S., Schneider, M., Pfeiffer, N., Wojcik, S.M., Spanagel, R., Gass, P., 2012. Sensorimotor gating, working and social memory deficits in mice with reduced expression of the vesicular glutamate transporter VGLUT1. Behav. Brain Res. 228, 328-332.

Izumi, Y., Sasaki, M., Hashimoto, S., Sawa, T., Amaya, F., 2015. mTOR signaling controls VGLUT2 expression to maintain pain hypersensitivity after tissue injury. Neuroscience 308, 169-179. Jacquin, M.F., Renehan, W.E., Rhoades, R.W., Panneton, W.M., 1993. Morphology and topography of identified primary afferents in trigeminal subnuclei principalis and oralis. J. Neurophysiol. 70, 1911-1936. Jacquin, M.F., Stennett, R.A., Renehan, W.E., Rhoades, R.W., 1988. Structure-function relationships in the rat brainstem subnucleus interpolaris: II. Low and high threshold trigeminal primary afferents. J. Comp. Neurol. 267, 107-130. 39

Jean, Y.H., Wen, Z.H., Chang, Y.C., Lee, H.S., Hsieh, S.P., Wu, C.T., Yeh, C.C., Wong, C.S., 2006. Hyaluronic acid attenuates osteoarthritis development in the anterior cruciate ligament-transected knee: Association with excitatory amino acid release in the joint dialysate. J. Orthop. Res. 24, 1052-1061. Jean, Y.H., Wen, Z.H., Chang, Y.C., Hsieh, S.P., Tang, C.C., Wang, Y.H., Wong, C.S., 2007. Intra-articular injection of the cyclooxygenase-2 inhibitor parecoxib attenuates osteoarthritis progression in anterior cruciate ligament-transected knee in rats: role of excitatory amino acids. Osteoarthritis Cartilage. 15, 638-645. Juge, N., Gray, J.A., Omote, H., Miyaji, T., Inoue, T., Hara, C., Uneyama, H., Edwards, R.H., Nicoll, R.A., Moriyama, Y., 2010. Metabolic control of vesicular glutamate transport and release. Neuron 68, 99-112. Juge, N., Yoshida, Y., Yatsushiro, S., Omote, H., Moriyama, Y., 2006. Vesicular glutamate transporter contains two independent transport machineries. J. Biol. Chem. 281, 39499-39506. Neurosci. Res. 42, 243-250.

IP T

Kaneko, T., Fujiyama, F., 2002. Complementary distribution of vesicular glutamate transporters in the central nervous system. Kaneko, T., Mizuno, N., 1988. Immunohistochemical study of glutaminase-containing neurons in the cerebral cortex and thalamus of the rat. J. Comp. Neurol. 267, 590-602.

Kaneko, T., Urade, Y., Watanabe, Y., Mizuno, N., 1987. Production, characterization, and immunohistochemical application of

SC R

monoclonal antibodies to glutaminase purified from rat brain. J. Neurosci. 7, 302-309.

Kehrl, J., Althaus, J.C., Showalter, H.D., Rudzinski, D.M., Sutton, M.A., Ueda, T., 2017. Vesicular glutamate transporter inhibitors: Structurally modified brilliant yellow analogs. Neurochem. Res. 42, 1823-1832.

Kemplay, S., Webster, K.E., 1989. A quantitative study of the projections of the gracile, cuneate and trigeminal nuclei and of the

U

medullary reticular formation to the thalamus in the rat. Neuroscience 32, 153-167.

Khodorova, A., Richter, J., Vasko, M.R., Strichartz, G., 2009. Early and late contributions of glutamate and CGRP to mechanical

N

sensitization by endothelin-1. J. Pain. 10, 740-749.

Kim, Y.S., Kim, T.H., McKemy, D.D., Bae, Y.C., 2015. Expression of vesicular glutamate transporters in transient receptor

A

potential melastatin 8 (TRPM8)-positive dental afferents in the mouse. Neuroscience 303, 378-388. Koga, K., Chen, T., Li, X.Y, Descalzi. G., Ling, J., Gu, J., Zhuo, M., 2011. Glutamate acts as a neurotransmitter for gastrin

M

releasing peptide-sensitive and insensitive itch-related synaptic transmission in mammalian spinal cord. Mol. Pain. 7, 47. Lagerstrom, M.C., Rogoz, K., Abrahamsen, B., Lind, A.L., Olund, C., Smith, C., Mendez, J.A., Wallen-Mackenzie, A., Wood, J.N.,

ED

Kullander, K., 2011. A sensory subpopulation depends on vesicular glutamate transporter 2 for mechanical pain, and together with substance P, inflammatory pain. Proc. Natl. Acad. Sci. U S A 108, 5789-5794. Lagerstrom, M.C., Rogoz, K., Abrahamsen, B., Persson, E., Reinius, B., Nordenankar, K., Olund, C., Smith, C., Mendez, J.A., Chen, Z.F., Wood, J.N., Wallen-Mackenzie, A., Kullander, K., 2010. VGLUT2-dependent sensory neurons in the TRPV1

PT

population regulate pain and itch. Neuron 68, 529-542. Landry, M., Bouali-Benazzouz, R., El Mestikawy, S., Ravassard, P., Nagy, F., 2004. Expression of vesicular glutamate transporters in rat lumbar spinal cord, with a note on dorsal root ganglia. J. Comp. Neurol. 468, 380-394.

CC E

Laursen, J.C., Cairns, B.E., Dong, X.D., Kumar, U., Somvanshi, R.K., Arendt-Nielsen, L., Gazerani, P., 2014. Glutamate dysregulation in the trigeminal ganglion: a novel mechanism for peripheral sensitization of the craniofacial region. Neuroscience 256, 23-35.

Lechner, S.G., Siemens, J., 2011. Sensory transduction, the gateway to perception: mechanisms and pathology. EMBO Rep. 12,

A

292-295.

Leo, S., Moechars, D., Callaerts-Vegh, Z., D'Hooge, R., Meert, T., 2009. Impairment of VGLUT2 but not VGLUT1 signaling reduces neuropathy-induced hypersensitivity. Eur. J. Pain 13, 1008-1017.

Li. C.L., Li, K.C., Wu, D., Chen, Y., Luo, H., Zhao, J.R., Wang, S.S., Sun, M.M., Lu, Y.J., Zhong, Y.Q., Hu, X.Y., Hou, R., Zhou, B.B.,

Bao,

L.,

Xiao,

H.S.,

Zhang,

X.,

2016.

Somatosensory neuron types identified by high-coverage single-cell RNA-sequencing and functional heterogeneity. Cell Res. 26, 83-102. Li, J.L., Fujiyama, F., Kaneko, T., Mizuno, N., 2003a. Expression of vesicular glutamate transporters, VGluT1 and VGluT2, in axon terminals of nociceptive primary afferent fibers in the superficial layers of the medullary and spinal dorsal horns of 40

the rat. J. Comp. Neurol. 457, 236-249. Li, J.L., Xiong, K.H., Dong, Y.L., Fujiyama, F., Kaneko, T., Mizuno, N., 2003b. Vesicular glutamate transporters, VGluT1 and VGluT2, in the trigeminal ganglion neurons of the rat, with special reference to coexpression. J. Comp. Neurol. 463, 212-220. Li, X., Ge, S.N., Li, Y., Wang, H.T., 2017. Neurokinin-1 receptor-immunopositive neurons in the medullary dorsal horn provide collateral axons to both the thalamus and parabrachial nucleus in rats. Neurochem. Res. 42, 375-388. Li, Z., Ge, S., Zhang, F., Zhang, T., Mizuno, N., Hioki, H., Kaneko, T., Gao, G., Li, J., 2012. Distribution of gephyrin-immunoreactivity in the trigeminal motor nucleus: an immunohistochemical study in rats. Anat. Rec. (Hoboken) 295, 641-651. Liguz-Lecznar, M., Skangiel-Kramska, J., 2007a. Vesicular glutamate transporters (VGLUTs): the three musketeers of glutamatergic system. Acta. Neurobiol. Exp. (Wars) 67, 207-218. mouse barrel cortex. Int. J. Dev. Neurosci. 25, 107-114.

IP T

Liguz-Lecznar, M., Skangiel-Kramska, J., 2007b. Vesicular glutamate transporters VGLUT1 and VGLUT2 in the developing Liu, Q., Tang, Z., Surdenikova, L., Kim, S., Patel, K.N., Kim, A., Ru, F., Guan, Y., Weng, H.J., Geng, Y., Undem, B.J., Kollarik, M.,

Chen,

Z.F.,

Anderson,

D.J.,

Dong,

X.,

2009.

SC R

Sensory neuron-specific GPCR Mrgprs are itch receptors mediating chloroquine-induced pruritus. Cell.139,1353-1365. Liu, T.T., Bannatyne, B.A., Jankowska, E., Maxwell, D.J., 2010a. Properties of axon terminals contacting intermediate zone excitatory and inhibitory premotor interneurons with monosynaptic input from group I and II muscle afferents. J. Physiol. 588, 4217-4233.

U

Liu, Y., Abdel Samad, O., Zhang, L., Duan, B., Tong, Q., Lopes, C., Ji, R.R., Lowell, B.B., Ma, Q., 2010b. VGLUT2-dependent glutamate release from nociceptors is required to sense pain and suppress itch. Neuron 68, 543-556.

N

Lopes, D.M., Denk, F., McMahon, S.B., 2017. The molecular fingerprint of dorsal root and trigeminal ganglion neurons. Front. Mol. Neurosci. 10, 304.

A

Lou, S., Duan, B., Vong, L., Lowell, B.B., Ma, Q., 2013. Runx1 controls terminal morphology and mechanosensitivity of VGLUT3-expressing C-mechanoreceptors. J. Neurosci. 33, 870-882.

M

Lou, S., Pan, X., Huang, T., Duan, B., Yang, F.C., Yang, J., Xiong, M., Liu, Y., Ma, Q., 2015. Incoherent feed-forward regulatory loops control segregation of C-mechanoreceptors, nociceptors, and pruriceptors. J. Neurosci. 35, 5317-5329.

ED

Lund, J.P., Sadeghi, S., Athanassiadis, T., Caram Salas, N., Auclair, F., Thivierge, B., Arsenault, I., Rompre, P., Westberg, K.G., Kolta, A., 2010. Assessment of the potential role of muscle spindle mechanoreceptor afferents in chronic muscle pain in the rat masseter muscle. PLoS One 5, e11131.

Luo, P., Dessem, D., 1995. Inputs from identified jaw-muscle spindle afferents to trigeminothalamic neurons in the rat: a

PT

double-labeling study using retrograde HRP and intracellular biotinamide. J. Comp. Neurol. 353, 50-66. Luo, P.F., Wang, B.R., Peng, Z.Z., Li, J.S., 1991. Morphological characteristics and terminating patterns of masseteric neurons of the mesencephalic trigeminal nucleus in the rat: an intracellular horseradish peroxidase labeling study. J. Comp. Neurol.

CC E

303, 286-299.

Ma, Q., 2014. Itch Modulation by VGLUT2-Dependent Glutamate Release from Somatic Sensory Neurons. In: Carstens E, Akiyama T, editors. Itch: Mechanisms and Treatment. Boca Raton (FL): CRC Press/Taylor & Francis, Chapter 21. Mackenzie, B., Illing, A.C., Morris, M.E., Varoqui, H., Erickson, J.D., 2008. Analysis of a vesicular glutamate transporter

A

(VGLUT2) supports a cell-leakage mode in addition to vesicular packaging. Neurochem. Res. 33, 238-247. Magnusson, K.R., Clements, J.R., Larson, A.A., Madl, J.E., Beitz, A.J., 1987. Localization of glutamate in trigeminothalamic projection neurons: a combined retrograde transport-immunohistochemical study. Somatosens. Res. 4, 177-190.

Magnusson, K.R., Larson, A.A., Madl, J.E., Altschuler, R.A., Beitz, A.J., 1986. Co-localization of fixative-modified glutamate and glutaminase in neurons of the spinal trigeminal nucleus of the rat: an immunohistochemical and immunoradiochemical analysis. J. Comp. Neurol. 247, 477-490. Maksimovic, S., Baba, Y., Lumpkin, E.A., 2013. Neurotransmitters and synaptic components in the Merkel cell-neurite complex, a gentle-touch receptor. Ann. N. Y. Acad. Sci.1279,13-21. Maksimovic, S., Nakatani, M., Baba, Y., Nelson, A.M., Marshall, K.L., Wellnitz, S.A., Firozi, P., Woo, S.H., Ranade, S., 41

Patapoutian, A., Lumpkin, E.A., 2014. Epidermal Merkel cells are mechanosensory cells that tune mammalian touch receptors. Nature. 509, 617-621. Malet, M., Brumovsky, P.R., 2015. VGLUTs and glutamate synthesis-focus on DRG neurons and pain. Biomolecules 5, 3416-3437. Malet, M., Vieytes, C.A., Lundgren, K.H., Seal, R.P., Tomasella, E., Seroogy, K.B., Hokfelt, T., Gebhart, G.F., Brumovsky, P.R., 2013. Transcript expression of vesicular glutamate transporters in lumbar dorsal root ganglia and the spinal cord of mice - effects of peripheral axotomy or hindpaw inflammation. Neuroscience 248, 95-111. Manteniotis, S., Lehmann, R., Flegel, C., Vogel, F., Hofreuter, A., Schreiner, B.S., Altmüller, J., Becker, C., Schöbel, N., Hatt, H., Gisselmann, G., 2013. Comprehensive RNA-Seq expression analysis of sensory ganglia with a focus on ion channels and GPCRs in trigeminal ganglia. PLoS One. 8, e79523. Mantle-St John, L.A., Tracey, D.J., 1987. Somatosensory nuclei in the brainstem of the rat: independent projections to the

IP T

thalamus and cerebellum. J. Comp. Neurol. 255, 259-271.

Matsuo, K., Ban, R., Hama, Y., Yuzuriha, S., 2015. Eyelid opening with trigeminal proprioceptive activation regulates a brainstem arousal mechanism. PLoS One 10, e0134659. in superficial laminae of the rat dorsal horn. J. Physiol. 584, 521-533.

SC R

Maxwell, D.J., Belle, M.D., Cheunsuang, O., Stewart, A., Morris, R., 2007. Morphology of inhibitory and excitatory interneurons McCarter, G.C., Reichling, D.B., Levine, J.D., 1999. Mechanical transduction by rat dorsal root ganglion neurons in vitro. Neurosci. Lett. 273, 179-182.

McCarthy, C.J., Tomasella, E., Malet, M., Seroogy, K.B., Hökfelt, T., Villar, M.J., Gebhart, G.F., Brumovsky, P.R., 2016. Axotomy neurons and the spinal cord. Brain Struct. Funct. 221, 1985-2004.

U

of tributaries of the pelvic and pudendal nerves induces changes in the neurochemistry of mouse dorsal root ganglion

N

McMahon, S.B., Koltzenburg, M., 1992. Itching for an explanation.Trends Neurosci. 15, 497-501. microscopic study. J. Invest. Dermatol. 73, 325-334.

A

Mihara, M., Hashimoto, K., Ueda, K., Kumakiri, M., 1979. The specialized junctions between Merkel cell and neurite: an electron Miller, K.E., Hoffman, E.M., Sutharshan, M., Schechter, R., 2011. Glutamate pharmacology and metabolism in peripheral primary

M

afferents: physiological and pathophysiological mechanisms. Pharmacol. Ther. 130, 283-309. Minnery, B.S., Simons, D.J., 2003. Response properties of whisker-associated trigeminothalamic neurons in rat nucleus principalis.

ED

J. Neurophysiol. 89, 40-56.

Miyata, M., 2007. Distinct properties of corticothalamic and primary sensory synapses to thalamic neurons. Neurosci. Res. 59, 377-382.

Moechars, D., Weston, M.C., Leo, S., Callaerts-Vegh, Z., Goris, I., Daneels, G., Buist, A., Cik, M., van der Spek, P., Kass, S.,

PT

Meert, T., D'Hooge, R., Rosenmund, C., Hampson, R.M., 2006. Vesicular glutamate transporter VGLUT2 expression levels control quantal size and neuropathic pain. J. Neurosci. 26, 12055-12066. Morris, J.L., Konig, P., Shimizu, T., Jobling, P., Gibbins, I.L., 2005. Most peptide-containing sensory neurons lack proteins for

CC E

exocytotic release and vesicular transport of glutamate. J. Comp. Neurol. 483, 1-16. Mu, D., Deng, J., Liu, K.F., Wu, Z.Y., Shi, Y.F., Guo, W.M., Mao, Q.Q., Liu, X.J., Li, H., Sun, Y.G., 2017. A central neural circuit for itch sensation. Science. 357, 695-699. Nahmani, M., Erisir, A., 2005. VGluT2 immunochemistry identifies thalamocortical terminals in layer 4 of adult and developing

A

visual cortex. J. Comp. Neurol. 484, 458-473. Nakamura, F., Strittmatter, S.M., 1996. P2Y1 purinergic receptors in sensory neurons: contribution to touch-induced impulse generation. Proc. Natl. Acad. Sci. U S A. 93, 10465-10470.

Nakamura, K., Hioki, H., Fujiyama, F., Kaneko, T., 2005. Postnatal changes of vesicular glutamate transporter (VGluT)1 and VGluT2 immunoreactivities and their colocalization in the mouse forebrain. J. Comp. Neurol. 492, 263-288. Nakatani, M., Maksimovic, S., Baba, Y., Lumpkin, E.A., 2015. Mechanotransduction in epidermal Merkel cells. Pflugers Arch. 467, 101-108. Nash, P.G., Macefield, V.G., Klineberg, I.J., Gustin, S.M., Murray, G.M., Henderson, L.A., 2010. Bilateral activation of the trigeminothalamic tract by acute orofacial cutaneous and muscle pain in humans. Pain 151, 384-393. 42

Nathan, P.W., Smith, M., Deacon, P., 2001. The crossing of the spinothalamic tract. Brain 124, 793-803. Ni, B., Du, Y., Wu, X., DeHoff, B.S., Rosteck, P.R., Jr., Paul, S.M., 1996. Molecular cloning, expression, and chromosomal localization of a human brain-specific Na(+)-dependent inorganic phosphate cotransporter. J. Neurochem. 66, 2227-2238. Ni, B., Rosteck, P.R., Jr., Nadi, N.S., Paul, S.M., 1994. Cloning and expression of a cDNA encoding a brain-specific Na(+)-dependent inorganic phosphate cotransporter. Proc. Natl. Acad. Sci. U S A 91, 5607-5611. Ni, B., Wu, X., Yan, G.M., Wang, J., Paul, S.M., 1995. Regional expression and cellular localization of the Na(+)-dependent inorganic phosphate cotransporter of rat brain. J. Neurosci. 15, 5789-5799. Nunzi, M.G., Pisarek, A., Mugnaini, E., 2004. Merkel cells, corpuscular nerve endings and free nerve endings in the mouse palatine mucosa express three subtypes of vesicular glutamate transporters. J. Neurocytol. 33, 359-376. Oliveira, A.L., Hydling, F., Olsson, E., Shi, T., Edwards, R.H., Fujiyama, F., Kaneko, T., Hokfelt, T., Cullheim, S., Meister, B., 2003. Cellular localization of three vesicular glutamate transporter mRNAs and proteins in rat spinal cord and dorsal root

IP T

ganglia. Synapse 50, 117-129.

Omote, K., Kawamata, T., Kawamata, M., Namiki, A., 1998. Formalin-induced release of excitatory amino acids in the skin of the rat hindpaw. Brain Res. 787, 161-164. glutamate transport. Biochemistry 50, 5558-5565. Otis, T.S., 2001. Vesicular glutamate transporters in cognito. Neuron 29, 11-14.

SC R

Omote, H., Miyaji, T., Juge, N., Moriyama, Y., 2011. Vesicular neurotransmitter transporter: bioenergetics and regulation of

Ozkan, E.D., Ueda, T., 1998. Glutamate transport and storage in synaptic vesicles. Jpn. J. Pharmacol. 77, 1-10. Park, S.K., Ko, S.J., Paik, S.K., Rah, J.C., Lee, K.J., Bae, Y.C., 2018. Vesicular glutamate transporter 1 (VGLUT1)- and

U

VGLUT2-immunopositive axon terminals on the rat jaw-closing and jaw-opening motoneurons. Brain Struct. Funct. 223, 2323-2334.

N

Paik, S.K., Kim, S.K., Choi, S.J., Yang, E.S., Ahn, S.H., Bae, Y.C., 2012. Vesicular glutamate transporters in axons that innervate the human dental pulp. J. Endod. 38, 470-474. glutamate in brain. Front. Biosci. 3, d701-718.

A

Palmada, M., Centelles, J.J., 1998. Excitatory amino acid neurotransmission. Pathways for metabolism, storage and reuptake of

M

Pang, Y.W., Ge, S.N., Nakamura, K.C., Li, J.L., Xiong, K.H., Kaneko, T., Mizuno, N., 2009. Axon terminals expressing vesicular glutamate transporter VGLUT1 or VGLUT2 within the trigeminal motor nucleus of the rat: origins and distribution

ED

patterns. J. Comp. Neurol. 512, 595-612.

Pang, Y.W., Li, J.L., Nakamura, K., Wu, S., Kaneko, T., Mizuno, N., 2006. Expression of vesicular glutamate transporter 1 immunoreactivity in peripheral and central endings of trigeminal mesencephalic nucleus neurons in the rat. J. Comp. Neurol. 498, 129-141.

PT

Peirs, C., Williams, S.P., Zhao, X., Walsh, C.E., Gedeon, J.Y., Cagle, N.E., Goldring, A.C., Hioki, H., Liu, Z., Marell, P.S., Seal, R.P., 2015. Dorsal horn circuits for persistent mechanical pain. Neuron. 87, 797-812. Persson, S., Boulland, J.L., Aspling, M., Larsson, M., Fremeau, R.T., Jr., Edwards, R.H., Storm-Mathisen, J., Chaudhry, F.A.,

CC E

Broman, J., 2006. Distribution of vesicular glutamate transporters 1 and 2 in the rat spinal cord, with a note on the spinocervical tract. J. Comp. Neurol. 497, 683-701. Pierret, T., Lavallee, P., Deschenes, M., 2000. Parallel streams for the relay of vibrissal information through thalamic barreloids. J. Neurosci. 20, 7455-7462.

A

Pintelon, I., Brouns, I., De Proost, I., Van Meir, F., Timmermans, J.P., Adriaensen, D., 2007. Sensory receptors in the visceral pleura: neurochemical coding and live staining in whole mounts. Am. J. Respir. Cell Mol. Biol. 36, 541-551.

Preobraschenski, J., Zander, J.F., Suzuki, T., Ahnert-Hilger, G., Jahn, R., 2014. Vesicular glutamate transporters use flexible anion and cation binding sites for efficient accumulation of neurotransmitter. Neuron 84, 1287-1301. Renehan, W.E., Jacquin, M.F., Mooney, R.D., Rhoades, R.W., 1986. Structure-function relationships in rat medullary and cervical dorsal horns. II. Medullary dorsal horn cells. J. Neurophysiol. 55, 1187-1201. Restrepo, C.E., Lundfald, L., Szabo, G., Erdelyi, F., Zeilhofer, H.U., Glover, J.C., Kiehn, O., 2009. Transmitter-phenotypes of commissural interneurons in the lumbar spinal cord of newborn mice. J. Comp. Neurol. 517, 177-192. Rethelyi, M., Horvath-Oszwald, E., Boros, C., 2008. Caudal end of the rat spinal dorsal horn. Neurosci. Lett. 445, 153-157. 43

Rogoz, K., Andersen, H.H., Kullander, K., Lagerstrom, M.C., 2014a. Glutamate, substance P, and calcitonin gene-related peptide cooperate in inflammation-induced heat hyperalgesia. Mol. Pharmacol. 85, 322-334. Rogoz, K., Andersen, H.H., Lagerstrom, M.C., Kullander, K., 2014b. Multimodal use of calcitonin gene-related peptide and substance P in itch and acute pain uncovered by the elimination of vesicular glutamate transporter 2 from transient receptor potential cation channel subfamily V member 1 neurons. J. Neurosci. 34, 14055-14068. Rogoz, K., Lagerstrom, M.C., Dufour, S., Kullander, K., 2012. VGLUT2-dependent glutamatergic transmission in primary afferents is required for intact nociception in both acute and persistent pain modalities. Pain 153, 1525-1536. Rogoz, K., Stjarne, L., Kullander, K., Lagerstrom, M.C., 2015. VGLUT2 controls heat and punctuate hyperalgesia associated with nerve injury via TRPV1-Cre primary afferents. PLoS One 10, e0116568. Rokx, J.T., van Willigen, J.D., 1988. Organization of neuronal clusters in the mesencephalic trigeminal nucleus of the rat: fluorescent tracing of temporalis and masseteric primary afferents. Neurosci. Lett. 86, 21-26.

IP T

Ross, S. E., Mardinly, A. R., McCord, A. E., Zurawski, J., Cohen, S., Jung, C., Hu, L., Mok, S.I., Shah, A., Savner, E.M., Tolias, C., Corfas, R., Chen, S., Inquimbert, P., Xu, Y., McInnes, R.R,, Rice, F.L., Corfas, G., Ma, Q., Woolf, C.J., Greenberg, M.E., 2010. Loss of inhibitory interneurons in the dorsal spinal cord and elevated itch in Bhlhb5 mutant mice. Neuron. 65, 886–898

SC R

Ross, S.E., Hachisuka, J., Todd, A.J., 2014. Spinal Microcircuits and the Regulation of Itch. In: Carstens E, Akiyama T, editors. Itch: Mechanisms and Treatment. Boca Raton (FL): CRC Press/Taylor & Francis, Chapter 20. Rossano, A.J., Kato, A., Minard, K.I., Romero, M.F., Macleod, G.T., 2017. Na(+) /H(+) exchange via the Drosophila vesicular glutamate transporter mediates activity-induced acid efflux from presynaptic terminals. J. Physiol. 595, 805-824.

U

Rotterman, T.M., Nardelli, P., Cope, T.C., Alvarez, F.J., 2014. Normal distribution of VGLUT1 synapses on spinal motoneuron dendrites and their reorganization after nerve injury. J. Neurosci. 34, 3475-3492.

N

Sakata-Haga, H., Kanemoto, M., Maruyama, D., Hoshi, K., Mogi, K., Narita, M., Okado, N., Ikeda, Y., Nogami, H., Fukui, Y., Kojima, I., Takeda, J., Hisano, S., 2001. Differential localization and colocalization of two neuron-types of

A

sodium-dependent inorganic phosphate cotransporters in rat forebrain. Brain Res. 902, 143-155. Saporta, S., Kruger, L., 1977. The organization of thalamocortical relay neurons in the rat ventrobasal complex studied by the

M

retrograde transport of horseradish peroxidase. J. Comp. Neurol. 174, 187-208. Sathyamurthy, A., Johnson, K.R., Matson, K.J.E., Dobrott, C.I., Li, L., Ryba, A.R., Bergman, T.B., Kelly, M.C., Kelley, M.W.,

ED

Levine, A.J., 2018. Massively parallel single nucleus transcriptional profiling defines spinal cord neurons and their activity during behavior. Cell Rep. 22, 2216-2225. Scarfone, E., Demêmes, D., Jahn, R., De Camilli, P., Sans, A., 1988. Secretory function of the vestibular nerve calyx suggested by presence of vesicles, synapsin I, and synaptophysin. J. Neurosci. 8, 4640-4645.

PT

Schafer, M.K., Varoqui, H., Defamie, N., Weihe, E., Erickson, J.D., 2002. Molecular cloning and functional identification of mouse vesicular glutamate transporter 3 and its expression in subsets of novel excitatory neurons. J. Biol. Chem. 277, 50734-50748.

CC E

Schenck, S., Wojcik, S.M., Brose, N., Takamori, S., 2009. A chloride conductance in VGLUT1 underlies maximal glutamate loading into synaptic vesicles. Nat. Neurosci. 12, 156-162. Scherrer, G., Low, S.A., Wang, X., Zhang, J., Yamanaka, H., Urban, R., Solorzano, C., Harper, B., Hnasko, T.S., Edwards, R.H., Basbaum, A.I., 2010. VGLUT2 expression in primary afferent neurons is essential for normal acute pain and

A

injury-induced heat hypersensitivity. Proc. Natl. Acad. Sci. U S A 107, 22296-22301. Schneider, S.P., Walker, T.M., 2007. Morphology and electrophysiological properties of hamster spinal dorsal horn neurons that express VGLUT2 and enkephalin. J. Comp. Neurol. 501, 790-809.

Scott, A., Alfredson, H., Forsgren, S., 2008. VGluT2 expression in painful Achilles and patellar tendinosis: evidence of local glutamate release by tenocytes. J. Orthop. Res. 26, 685-692. Seal, R.P., 2016. Do the distinct synaptic properties of VGLUTs shape pain? Neurochem. Int. 98, 82-88. Seal, R.P., Wang, X., Guan, Y., Raja, S.N., Woodbury, C.J., Basbaum, A.I., Edwards, R.H., 2009. Injury-induced mechanical hypersensitivity requires C-low threshold mechanoreceptors. Nature 462, 651-655. Shigeri, Y., Seal, R.P., Shimamoto, K., 2004. Molecular pharmacology of glutamate transporters, EAATs and VGLUTs. Brain Res. 44

Brain Res. Rev. 45, 250-265. Shrestha, S.S., Bannatyne, B.A., Jankowska, E., Hammar, I., Nilsson, E., Maxwell, D.J., 2012. Excitatory inputs to four types of spinocerebellar tract neurons in the cat and the rat thoraco-lumbar spinal cord. J. Physiol. 590, 1737-1755. Singh, R.P., Cheng, Y.H., Nelson, P., Zhou, F.C., 2009. Retentive multipotency of adult dorsal root ganglia stem cells. Cell Transplant. 18, 55-68. Sreedharan, S., Shaik, J.H., Olszewski, P.K., Levine, A.S., Schioth, H.B., Fredriksson, R., 2010. Glutamate, aspartate and nucleotide transporters in the SLC17 family form four main phylogenetic clusters: evolution and tissue expression. BMC Genomics. 11, 17. Sun, Y.G., Chen, Z.F., 2007. A gastrin-releasing peptide receptor mediates the itch sensation in the spinal cord. Nature. 448, 700-703. 1531-1534.

IP T

Sun, Y. G., Zhao, Z. Q., Meng. X. L., Yin, J., Liu, X. Y., Chen, Z. F., 2009. Cellular basis of itch sensation. Science. 325, Tachibana, T., Endoh, M., Kumakami, R., Nawa, T., 2003. Immunohistochemical expressions of mGluR5, P2Y2 receptor, PLC-beta1, and IP3R-I and -II in Merkel cells in rat sinus hair follicles. Histochem. Cell Biol. 120, 13-21.

Tachibana, T., Nawa, T., 2005. Immunohistochemical reactions of receptors to met-enkephalin, VIP, substance P, and CGRP

SC R

located on Merkel cells in the rat sinus hair follicle. Arch. Histol. Cytol. 68:383-389.

Takamori, S., 2006. VGLUTs: 'exciting' times for glutamatergic research? Neurosci Res 55, 343-351.

Takamori, S., 2016a. Presynaptic molecular determinants of quantal size. Front. Synaptic Neurosci. 8, 2. Takamori, S., 2016b. Vesicular glutamate transporters as anion channels? Pflugers Arch 468, 513-518.

U

Takamori, S., Rhee, J.S., Rosenmund, C., Jahn, R., 2000. Identification of a vesicular glutamate transporter that defines a glutamatergic phenotype in neurons. Nature 407, 189-194.

N

Takamori, S., Rhee, J.S., Rosenmund, C., Jahn, R., 2001. Identification of differentiation-associated brain-specific phosphate transporter as a second vesicular glutamate transporter (VGLUT2). J. Neurosci. 21, RC182.

A

Thalakoti, S., Patil, V.V., Damodaram, S., Vause, C.V., Langford, L.E., Freeman, S.E., Durham, P.L., 2007. Neuron-glia signaling in trigeminal ganglion: implications for migraine pathology. Headache 47, 1008-1023; discussion 1024-1005.

M

Todd, A.J., Hughes, D.I., Polgar, E., Nagy, G.G., Mackie, M., Ottersen, O.P., Maxwell, D.J., 2003. The expression of vesicular glutamate transporters VGLUT1 and VGLUT2 in neurochemically defined axonal populations in the rat spinal cord with

ED

emphasis on the dorsal horn. Eur. J. Neurosci. 17, 13-27.

Tordera, R.M., Totterdell, S., Wojcik, S.M., Brose, N., Elizalde, N., Lasheras, B., Del Rio, J., 2007. Enhanced anxiety, depressive-like behaviour and impaired recognition memory in mice with reduced expression of the vesicular glutamate transporter 1 (VGLUT1). Eur. J. Neurosci. 25, 281-290.

PT

Tracey, D. 2004. CHAPTER 25 - Somatosensory System A2 - Paxinos, George. In: The Rat Nervous System (THIRD EDITION). pp. 797-815. Academic Press: Burlington. Ueda, T., 2016. Vesicular glutamate uptake. Adv. Neurobiol. 13, 173-221.

CC E

Uezato, A., Meador-Woodruff, J.H., McCullumsmith, R.E., 2009. Vesicular glutamate transporter mRNA expression in the medial temporal lobe in major depressive disorder, bipolar disorder, and schizophrenia. Bipolar Disord. 11, 711-725. Ulrich-Lai, Y.M., Flores, C.M., Harding-Rose, C.A., Goodis, H.E., Hargreaves, K.M., 2001. Capsaicin-evoked release of immunoreactive calcitonin gene-related peptide from rat trigeminal ganglion: evidence for intraganglionic

A

neurotransmission. Pain 91, 219-226. Usoskin, D., Furlan, A., Islam, S., Abdo, H., Lönnerberg, P., Lou, D., Hjerling-Leffler, J., Haeggström, J., Kharchenko, O., Kharchenko,

P.

V.,

Linnarsson,

S.,

Ernfors,

P.,

2015.

Unbiased classification of sensory neuron types by large-scale single-cell RNA sequencing. Nat. Neurosci. 18, 145-153. Varoqui, H., Schafer, M.K., Zhu, H., Weihe, E., Erickson, J.D., 2002. Identification of the differentiation-associated Na+/PI transporter as a novel vesicular glutamate transporter expressed in a distinct set of glutamatergic synapses. J. Neurosci. 22, 142-155. Vigneault, E., Poirel, O., Riad, M., Prud'homme, J., Dumas, S., Turecki, G., Fasano, C., Mechawar, N., El Mestikawy, S., 2015. Distribution of vesicular glutamate transporters in the human brain. Front. Neuroanat. 9, 23. 45

Vilimas, P.I., Yuan, S.Y., Haberberger, R.V., Gibbins, I.L., 2011. Sensory innervation of the external genital tract of female guinea pigs and mice. J. Sex. Med. 8, 1985-1995. Voglmaier, S.M., Kam, K., Yang, H., Fortin, D.L., Hua, Z., Nicoll, R.A., Edwards, R.H., 2006. Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling. Neuron 51, 71-84. Waite, P.M.E. 2004. CHAPTER 26 - Trigeminal Sensory System A2 - Paxinos, George. In: The Rat Nervous System (THIRD EDITION). pp. 817-851. Academic Press: Burlington. Wallen-Mackenzie, A., Wootz, H., Englund, H., 2010. Genetic inactivation of the vesicular glutamate transporter 2 (VGLUT2) in the mouse: what have we learnt about functional glutamatergic neurotransmission? Ups. J. Med. Sci. 115, 11-20. Wang, H.S., Yu, G., Wang, Z.T., Yi, S.P., Su, R.B., Gong, Z.H., 2016. Changes in VGLUT1 and VGLUT2 expression in rat dorsal root ganglia and spinal cord following spared nerve injury. Neurochem. Int. 99, 9-15. Wang, Z.T., Yu, G., Wang, H.S., Yi, S.P., Su, R.B., Gong, Z.H., 2015. Changes in VGLUT2 expression and function in pain-related

IP T

supraspinal regions correlate with the pathogenesis of neuropathic pain in a mouse spared nerve injury model. Brain Res. 1624, 515-524.

Weng, H.R., Chen, J.H., Cata, J.P., 2006. Inhibition of glutamate uptake in the spinal cord induces hyperalgesia and increased responses of spinal dorsal horn neurons to peripheral afferent stimulation. Neuroscience 138, 1351-1360.

SC R

Wiedey, J., Alexander, M.S., Marson, L., 2008. Spinal neurons activated in response to pudendal or pelvic nerve stimulation in female rats. Brain Res. 1197, 106-114.

Willis, W.D., Jr., 2007. The somatosensory system, with emphasis on structures important for pain. Brain Res. Rev. 55, 297-313. Wilson, N.R., Kang, J., Hueske, E.V., Leung, T., Varoqui, H., Murnick, J.G., Erickson, J.D., Liu, G., 2005. Presynaptic regulation

U

of quantal size by the vesicular glutamate transporter VGLUT1. J. Neurosci. 25, 6221-6234.

Wilson, P., Kitchener, P.D., 1996. Plasticity of cutaneous primary afferent projections to the spinal dorsal horn. Prog. Neurobiol.

N

48, 105-129.

Wojcik, S.M., Rhee, J.S., Herzog, E., Sigler, A., Jahn, R., Takamori, S., Brose, N., Rosenmund, C., 2004. An essential role for

A

vesicular glutamate transporter 1 (VGLUT1) in postnatal development and control of quantal size. Proc. Natl.Acad. Sci. U S A 101, 7158-7163. gradient. J. Biol. Chem. 271, 11726-11731.

M

Wolosker, H., de Souza, D.O., de Meis, L., 1996. Regulation of glutamate transport into synaptic vesicles by chloride and proton

ED

Woo, S.H., Baba, Y., Franco, A.M., Lumpkin, E.A., Owens, D.M., 2012. Excitatory glutamate is essential for development and maintenance of the piloneural mechanoreceptor. Development 139, 740-748. Woolf, C.J., King, A.E., 1987. Physiology and morphology of multireceptive neurons with C-afferent fiber inputs in the deep dorsal horn of the rat lumbar spinal cord. J. Neurophysiol. 58, 460-479.

PT

Wu, L., Wu, J., Chang, H.H., Havton, L.A., 2012. Selective plasticity of primary afferent innervation to the dorsal horn and autonomic nuclei following lumbosacral ventral root avulsion and reimplantation in long term studies. Exp. Neurol. 233, 758-766.

CC E

Wu, S.X., Koshimizu, Y., Feng, Y.P., Okamoto, K., Fujiyama, F., Hioki, H., Li, Y.Q., Kaneko, T., Mizuno, N., 2004. Vesicular glutamate transporter immunoreactivity in the central and peripheral endings of muscle-spindle afferents. Brain Res. 1011, 247-251.

Xiang, C.X., Zhang, K.H., Johnson, R.L., Jacquin, M.F., Chen, Z.F., 2012. The transcription factor, Lmx1b, promotes a neuronal

A

glutamate phenotype and suppresses a GABA one in the embryonic trigeminal brainstem complex. Somatosens. Mot. Res. 29, 1-12.

Yang, E.S., Jin, M.U., Hong, J.H., Kim, Y.S., Choi, S.Y., Kim, T.H., Cho, Y.S., Bae, Y.C., 2014. Expression of vesicular glutamate transporters VGLUT1 and VGLUT2 in the rat dental pulp and trigeminal ganglion following inflammation. PLoS One 9, e109723. Ye, M.X., Ge, S.N., Li, J.L., 2009. Projection of vesicular glutamate transporter II-like immunoreactive termina is from the spinal trigeminal nucleus to lateral parabrachial nucleus in rats (in Chinese). Chin. J. Neuroanat. 25, 255-260. Yezierski, R.P., Kaneko, T., Miller, K.E., 1993. Glutaminase-like immunoreactivity in rat spinomesencephalic tract cells. Brain Res. 624, 304-308. 46

Zeng, C., Shroff, H., Shore, S.E., 2011. Cuneate and spinal trigeminal nucleus projections to the cochlear nucleus are differentially associated with vesicular glutamate transporter-2. Neuroscience 176, 142-151. Zerari-Mailly, F., Braud, A., Davido, N., Toure, B., Azerad, J., Boucher, Y., 2012. Glutamate control of pulpal blood flow in the incisor dental pulp of the rat. Eur. J. Oral. Sci. 120, 402-407. Zhang, F.X., Pang, Y.W., Zhang, M.M., Zhang, T., Dong, Y.L., Lai, C.H., Shum, D.K., Chan, Y.S., Li, J.L., Li, Y.Q., 2011. Expression of vesicular glutamate transporters in peripheral vestibular structures and vestibular nuclear complex of rat. Neuroscience. 173, 179-189. Zhang, X., Chen, Y., Wang, C., Huang, L.Y., 2007. Neuronal somatic ATP release triggers neuron-satellite glial cell communication in dorsal root ganglia. Proc. Natl. Acad. Sci. U S A 104, 9864-9869. Zhang, Y., Wesolowski, M., Karakatsani, A., Witzemann, V., Kröger, S., 2014. Formation of cholinergic synapse-like specializations at developing murine muscle spindles. Dev. Biol. 393, 227-235. horn. J. Neurochem. 108, 305-318.

IP T

Zhou, H.Y., Chen, S.R., Chen, H., Pan, H.L., 2009. The glutamatergic nature of TRPV1-expressing neurons in the spinal dorsal Zhou, J., Nannapaneni, N., Shore, S., 2007. Vessicular glutamate transporters 1 and 2 are differentially associated with auditory nerve and spinal trigeminal inputs to the cochlear nucleus. J. Comp.Neurol. 500, 777-787.

SC R

Zink, M., Vollmayr, B., Gebicke-Haerter, P.J., Henn, F.A., 2010. Reduced expression of glutamate transporters vGluT1, EAAT2

A

CC E

PT

ED

M

A

N

U

and EAAT4 in learned helpless rats, an animal model of depression. Neuropharmacology 58, 465-473.

47

Figure 1．A scheme epitomizing the topography of VGLUT1 and VGLUT2 in somatosensory pathways. A: Skin VGLUT1 and VGLUT2 are mainly represented by the myelinated and unmyelinated afferent endings of DRG neurons, respectively. Accordingly, large-sized DRG neurons mainly express VGLUT1, while medium to small-sized ones favor VGLUT2 or co-expression of VGLUT1 and VGLUT2. VGLUT2-immunolabeled central terminals of

IP T

DRG neurons mainly locate in superficial layer of spinal dorsal horn (SDH), as compared with the VGLUT1-immunoreactive (-IR) ones in deep layer. B: The second-order neurons of

SC R

the spinothalamic pathway projecting to contralateral VPL exclusively express VGLUT2 mRNA and protein. C: The proprioceptive endings of muscle spindles of trunk and limbs

U

only express VGLUT1, implicating the VGLUT1 identity of their parental DRG neurons and

N

the associated central terminals projecting to gracile (Cr) and cuneate (Cu) nuclei. In spinal

A

cord VGLUT2 mRNA preponderates in SDH, with few cells expressing VGLUT1 mRNA or

M

co-expressing VGLUT1 and VGLUT2 mRNAs. D: The second-order neurons in Cr and Cu

ED

where VGLUT2 mRNA preponderates, relay proprioceptive signals to contralateral VPL through VGLUT2-IR axons composing medial lemniscus in the brainstem. E: The orofacial

PT

skin nerve endings of TG neurons tend to be VGLUT1- and VGLUT2-IR in the myelinated

CC E

and unmyelinated terminals respectively. Large-sized TG neurons mainly express VGLUT1, while medium- to small-sized ones express VGLUT2 or co-express VGLUT1 and VGLUT2. The central terminals of TG neurons in superfacial layer of MDH (Vc) express primarily

A

VGLUT2, and those located in all area of TSNC except superfacial layer of Vc are mainly VGLUT1-IR. F: VPM-/Po-projections from TSNC where VGLUT2 mRNA predominates to contralateral VPM/Po are exclusively VGLUT2-IR, while the Vp-VPM projections co-express VGLUT1 and VGLUT2. G: The proprioceptive endings of masseter muscle 48

spindles only express VGLUT1 protein, and the Vme neurons and the large neurons in Vpdm express VGLUT1 mRNA. H. Trigemio-cerebellum projections only expressed VGLUT1. In all subnuclei of TSNC, VGLUT2 mRNA signals take preponderance, being numerous and evenly distributed; in contrast, VGLUT1 mRNAs are heterogeneous, hardly seen in Vc, evenly distributed in moderate density in Vi, and represented by a small number restricted to

IP T

dorsal division of Vo. Vpdm contains many large-sized VGLUT1 mRNA-positive neurons, while the middle and ventral portions of Vp host numerous medium- to small-sized neurons

SC R

co-expressing VGLUT1 and VGLUT2 mRNAs. I: The three somatosensory thalamic nuclei contain low to moderately expressed VGLUT1 mRNA and significant VGLUT2 mRNA

U

signals. Po shows the low and preponderant levels of VGLUT1 mRNA and VGLUT2 mRNA,

N

respectively; VPL and VPM display more neurons with co-expression of VGLUT1 and

A

VGLUT2 mRNAs. Accordingly, the thalamocortical projections from VPM/VPL to

M

somatosensory cortex, mainly those terminating in the layer Ⅳ, assume the phenotype of

ED

VGLUT2 or co-express VGLUT1/VGLUT2; while projections from Po, mainly those to layers I and Va, mainly express VGLUT2. In somatosensory cortex VGLUT1 mRNA signal

PT

is strong, except in layers I and IV where the signal is weak; and VGLUT2 mRNA well

CC E

delineates the cortical strata, being void in layers I/II, abundant in layer III and scattered in

A

layers IV to VI . For abbreviations, see the text.

49

50

A ED

PT

CC E

IP T

SC R

U

N

A

M

Table 1. The expression of VGLUT1/VGLUT2 mRNA/proteins in somatic sensory structures and the literature cited. . Proteins Perikarya Terminals

mRNAs

Spinal Pathway Periphery Skin Muscle spindles DRG#

VGLUT1, 2

SDH

VGLUT1, 2*

VGLUT1, 2* VGLUT1

Brumovsky, 2007; Haeberle, 2004; Hitchcock, 2004; Vilimas, 2011;Woo, 2012 Wu, 2004 Akiyama, 2014; Atoji, 2009; Brumovsky, 2007, 2011, 2013; Hwang, 2004; Landry, 2004; Malet, 2013; McCarthy, 2016; Oliveira, 2003; Woo, 2012 Alvarez, 2004, 2011; Bannatyne, 2006; Brooke, 2006; Brumovsky, 2007; Clarke, 2011; Hughes, 2004; Landry, 2004; Liu, 2010; Malet, 2013; Maxwell, 2007; Oliveira, 2003; Persson, 2006; Restrepo, 2009; Rethelyi, 2008; Rotterman, 2014; Schneider, 2007; Shrestha, 2012; Todd, 2003; Wiedey, 2008; Wu, 2012; Zhou, 2009 Hioki, 2003; Hisano, 2002; Pang, 2009

SC R

VGLUT1, 2

References

IP T

Nuclei

U

VGLUT1, 2

A

CC

EP

TE

D

M

A

N

DCN VGLUT1, 2* VGLUT1, 2 Trigeminal pathway Periphery Braud, 2010; Honma, 2012; Nunzi, 2004; Paik, 2012; Yang, 2014; Zerari-Mailly, 2012 Skin VGLUT1, 2 Lund, 2010; Pang, 2006 Muscle spindles VGLUT1 # Chen, 2014; Li, 2003;Yang, 2014 TG VGLUT1, 2 VGLUT1, 2 TSNC Ge, 2010, 2014; Hegarty, 2010; Hisano, 2002; Li, 2003; Li, 2017; Pang, 2009 Vc VGLUT1, 2* VGLUT1, 2 Ge, 2010, 2014; Hegarty, 2010; Hisano, 2002; Pang, 2009 Vi VGLUT1, 2* VGLUT1, 2 Ge, 2010, 2014; Hisano, 2002; Pang, 2009 Vo VGLUT1, 2* VGLUT1, 2 # Dong, 2007; Ge, 2010, 2014; Hisano, 2002; Pang, 2009; Xiang, 2012 Vp VGLUT1, 2* VGLUT1, 2 Pang, 2006 Vme VGLUT1 VGLUT1 Barroso-Chinea, 2007, 2008; Graziano, 2008 VGLUT1, 2* VGLUT1, 2 Somatosensory # thalamus Bai, 2001; Conti, 2005; Fujiyama, 2001; Graziano, 2008; Herzog, 2001; Hisano, 2000; VGLUT1*, 2 VGLUT1*, 2 Somatosensory # Hur, 2005; Liguz-Lecznar, 2007; Ni, 1994, 1995; Vigneault, 2015 cortex # Structures contain neurons frequently colabeled by VGLUT1 and VGLUT2 proteins/mRNAs. *The VGLUT isoform taking preponderance over the other.

51

Table 2. A literature based summary of the alteration in response by different types of stimuli to provoke distinct sensory modalities, and hence the animal nociceptive or itch related- behavior following full or cell type-specific ablation of VGLUT2

Acute nociception Mechanical

-/

VGLUT2f/f;

v

/ v, RS -/ v

VGLUT2f/f; Per-Cre Nav1.8-Cre

/ -/ v, RS

/ H / H

RS

VGLUT2f/f; Ht-Pa-Cre

-/ v, RS

VGLUT2f/f; Trpv1-Cre f/f

VGLUT2 ; Nav1.8-Cre

-/ v

U

VGLUT2f/f; Nav1.8-Cre

Chemical

/ H / H / H -/ H -/ h,H

Spontaneous

Mechanical

Neuropathic pain Thermal

f, c

-/2p

Mechanical

c

Capsaicin/

c,CFA

Capsaicin/

CFA

/-/ v

/-/

H

c,CFA

//H CFA //H

//v SNI //v CCI v /-/ CCI v /-/

Thermal

SNI

/f /-

SC R

VGLUT2f/f; Th IRES-Cre

Cold

-/ h,t,H -/ h,t

VGLUT2+/VGLUT2+/-

Thermal

Inflammatory pain

IP T

Mutant

Visceral Cold

//a SNI //a CCI //a

pain

Itch

SNI

Author Moechars, 2006

aip/-

Leo, 2009

CCI

// H

Scherrer, 2010



Liu, 2010

f

/



Lagerström,2010

f/-

 -

//

-/ a

f/

/ e -/ e

f/

v

CCI

//

v

NGF//H

PSNL/-/ v

PSNL/-/ H

PSNL/-/ a

c,NGF//H

PSNL// v

PSNL// H

PSNL// a

PSNL// v

PSNL// H

PSNL/-/ a

Lagerström,2011

/RS

VGLUT2f/f;

Trpv1-Cre Trpv1-Cre

/ H / H

c,NGF//v

f/-

c,NGF/-/v

c,NGF/-/H

 

Rogoz, 2012 Rogoz, 2014b Rogoz, 2015 Rogoz, 2014a

D

M

VGLUT2f/f;

Trpv1-Cre

A

VGLUT2f/f;

/ v, RS

N

VGLUT2f/f; Ht-Pa-Cre

A

CC

EP

TE

The trend of alteration in behavioral response to stimuli ： -“ indicating no change. Abbreviations for neuropathic pain models: SNI, spared nerve injury; CCI, chronic constriction injury; PSNL, partial sciatic nerve ligation. Abbreviations for chemicals used to construct inflammatory pain model: f, formalin; c, carrageenan; CFA, complete Freund’s adjuvant. NGF, Nerve growth factor. Abbreviations for methods for pain test: Thermal pain test: h, hot plate test; t, tail withdrawal test; H, Hargreaves’ test; Mechanical pain test; v, von Frey test; RS, Randall–Selitto test; Cold allodynia test: 2p, two-platform preference assay; a, acetone test; e, tail withdrawal test from a -15℃ ethanol solution. Visceral pain model: aip, 1% acetic acid ip injection. VGLUT2f/f; Per-Cre: VGLUT2 conditionally knocked-out in most nociceptive DRG cells. VGLUT2f/f; Nav1.8-Cre, VGLUT2 conditionally knocked-out in Nav1.8 expressing neurons. VGLUT2f/f; Th IRES-Cre, VGLUT2 conditionally knocked-out in neurons expressing tyrosine hydroxylase (Th). VGLUT2f/f; Ht-Pa-Cre: VGLUT2 conditionally knocked-out in most DRG cells in which Ht-Pa-Cre was active.

52