Human Dorsal Root Ganglia

1Sensory neurons relay information about a variety of intrinsic and environmental cues such as temperature, touch, muscle length, organ volume or actual or potential harm to the body. They also contribute to regulation of blood supply and change neuronal sensitivity and other functions by ortho- and antidromic release of molecules. The cell bodies of sensory neurons are located primarily in dorsal root ganglia (DRG) or trigeminal ganglia (TG; see reviews;;; ). The last three decades have seen significant advances in understanding the electrochemical, cellular and molecular characteristics of sensory neurons found in DRG, primarily stemming from animal studies. These studies have focused heavily on understanding mechanisms underlying the development and pathophysiology of chronic and/or neuropathic pain. Far less, however, is known about the cellular and molecular characteristics of human DRG. The emergence of recent comparative genetic and proteomic studies between animal and human models has highlighted critical differences and similarities in molecular and cellular characteristics of DRG. These may have profound implications for translating data from rodent models to human pathologies, and subsequent therapeutic developments. In view of the large-scale failure of clinical trials based on animal models, the success of new drugs to treat pain in the clinic will likely require studies of human cells and tissues. The emergence of researchers with the capacity to acquire and study native human sensory neurons in the DRG through organ-donor networks, in conjunction with data gained from clinical trials of DRG stimulation for treatment of chronic neuropathic pain, will be critical to validate important pain mechanisms discovered in animal models. There are numerous comprehensive reviews summarizing advances in the understanding of rodent DRG, however, to our knowledge, no reviews focused on collating information on human DRG have been published to date. This review aims to encapsulate existing information about human DRG neurons in relation to their size, location, blood supply, and neurochemical content under non-pathological conditions.

2Dorsal root ganglia do not only contain the cell bodies of primary sensory neurons but also a variety of other cell types such as a specific form of glia, called satellite cells, that form a layer (envelope) around neuronal cell bodies. Neurons and satellite cells form a functionally close relationship (Figure 1). Studies on cat DRG demonstrate the presence of microvilli as extensions of the neuronal cell surface, in close contact with surrounding satellite cells. Satellite cells express a characteristic pattern of surface receptors, transporters, and enzymes. Glutamine synthetase and proteins of the S100 family can be used to neurochemically identify these cells. Satellite cells are able to modify the microenvironment of neurons by uptake and release of molecules, but interestingly seem not to have a barrier function. In addition to neurons and satellite cells, DRG contain small blood vessels, thus endothelial and smooth muscle cells, delivering blood to satisfy the extensive energy and therefore oxygen demand of sensory neurons. With neuronal processes as long as a meter, ongoing synthesis and transport of proteins over hundreds of millimeters is critical for normal neuronal function. The blood vessels build an extensive network of arterioles and capillaries within DRG. The interface between accumulations of sensory neurons and blood vessels in DRG is unique. Capillaries in DRG are fenestrated and in the absence of a blood–brain barrier, many blood borne molecules can directly enter the DRG and interact with neuronal and non-neuronal cells. Non-neuronal target cells include a group of immune cells contained within DRG that consist mainly of macrophages and T-lymphocytes and a lower number of B-lymphocytes (Figure 2).

3Compared to rodents, human DRG are larger and contain more cells with different proportions of sensory neuron subtypes and substantially more connective tissue between neurons. Recent studies have showed that compared to classical laboratory animals, human sensory neurons contain similar sets of receptor and channel proteins but their expression levels and function of key components relevant to mechanisms underlying chronic pain, such as sodium channels, can differ. Given that sodium channel blockers are currently in clinical trials for analgesic drug development, this further demonstrates the importance of comparative human data. Additional differences are demonstrated by functional studies examining non-neuronal cells in the DRG. Recent animal studies suggest an important function for satellite glia and immune cells in the development of pain. However, only one publication to date addresses the presence of immune cells such as macrophages and lymphocytes in human DRG under non-pathological conditions. Shifting focus to the functional characteristics of mast cells in the DRG, there are no human studies. Considering that a small number of recent comparative studies have demonstrated critical rodent and human inter-species differences between the cellular machinery associated with the development of pathological pain conditions, it is fundamentally important to investigate the cellular and molecular components of human DRG to advance our understanding of these diseases. As a first step toward the objective of investigating human DRG, we believe it is pivotal to summarize current existing knowledge in this field. Here, we focus on describing the location and structure of human DRG, and neurochemical characteristics of satellite glia cells (SGCs) and DRG neurons with emphasis on the nociceptor-related neurochemistry.

4Information about the location and size of human DRG is mainly based on investigations using cadaveric material or studies using magnetic resonance imaging (MRI).

5Humans possess 31 pairs of spinal nerves containing, inter alia, sensory nerve fibers with cell bodies in DRG. The number of DRG often equals the number of spinal nerves. Nevertheless, the first cervical (C1) DRG has been shown to be smaller compared to DRG at other vertebral levels, in addition to being present in only about a quarter of investigated bodies (28.5%). The size of human DRG depends on the vertebral level. Even though there might be size differences between ganglia in individuals, on average, no differences exist between ganglia on the left and right side of the body and no age-dependent differences have been reported. In the cervical region, DRG size increases from the very small and sometimes absent DRG at the level C1 to larger DRG at the C8 level. The C8 DRG also has a larger volume (177 ± 27.5 mm3) than the adjacent T1 DRG (144 ± 30.8 mm3). No studies to date have measured human thoracic DRG at lower levels (T3–T12), whereas several studies provide data for DRG at lumbar and sacral levels. Table 1 summarizes studies that have identified various human DRG dimensions: width; width and length; or volume. The size of lumbar DRG increases from vertebral levels L1 to L5, from approximately 3–5 mm × 4–5 mm at L1 to 5–6 mm × 9–11 mm at L5. Conversely, the size of sacral DRG decreases from S1 to S4 with a very large ganglion at level S1 (6–7 mm × 11–12 mm) (Table 1). The correlation between DRG size and the number of neurons contained within has also been demonstrated.

6Considering that human DRG are susceptible to damage by compression, e.g., by disc herniation, the location of DRG in relation to adjacent structures is important. Compared to other DRG, the second cervical (C2) DRG has an unusual relationship to unique adjacent structures such as atlas and axis or the non-bony posterior border created by the ligamentum flavum. Substantial mobility at this intervertebral level might contribute to vulnerability of the ganglion. Besides mobility, the location of blood vessels can also influence DRG function. described the close location to and compression of the C5 cervical ganglion and ventral roots via the vertebral artery which normally lie anterior or lateral to the cervical DRG.

7Dorsal root ganglia are normally localized within, or close to, intervertebral foramina, the openings between the pedicles of vertebrae that allow peripheral structures to connect with the vertebral canal. Within the intervertebral foramen, DRG are normally localized superolaterally, but at lower vertebral levels they tend to be positioned more centrally within the foramen. classified DRG location as intraspinal (in the vertebral canal, proximal to the vertebral foramina), intraforaminal (in the intervertebral foramina) or extraforaminal (distal to the intervertebral foramina). A study investigating C6 and C7 DRG showed that about half were situated extraforaminally. Lumbar DRG are predominantly intraforaminal. Sacral DRG are localized more centrally, with DRG at levels S1 and S2 localized intraforaminal and S3 and S4 in the vertebral canal (intraspinal). No differences have been observed in the location of DRG with respect to gender, age, height, and weight.

8Interestingly in humans, individual DRG can occasionally consist of one, two or three smaller and distinctly sheathed ganglia. DRG containing two or even three ganglia are predominantly present at vertebral levels L3 and L4. Whether this is of functional significance remains unknown.

9Human DRG normally consist of a peripheral region that contains the somata of primary sensory neurons and a central region that predominantly contains bundles of nerve fibers (Figure 1). They are encased by meninges including a thick layer of dura mater, comprised of collagenous connective tissue. Based on light microscopy and routine hematoxylin and eosin (HE) staining, neuronal cell bodies can be subdivided into small phase-dark neurons and large phase-light neurons, with distinct types of neurons already present at gestational week 6. Fluorescence and light microscopy show human DRG neurons often contain highly autofluorescent accumulations of material (fluorescence microscopy) or accumulations of brownish material. This is the pigment lipofuscin which consists of a mixture of lipids, misfolded proteins and sugar molecules (Figure 1,2). Human DRG neurons show age-dependent inclusions of lipofuscin, which are accompanied by accumulations of melanin. The nuclei of human DRG neurons normally contain one nucleolus that is relative in size to the cell body, and possess Cajal bodies, suborganelles that are in involved in RNA processing (Figure 1, 2).

10It is difficult to determine the exact number of primary sensory neurons within DRG as the ganglia are not round but rather ovoid and elongated. Furthermore, DRG contain not only neurons but also non-neuronal cells, connective tissue, blood vessels and bundles of nerve fibers. Stereological quantification was used to determine the number of DRG neurons that project with the brachial plexus to peripheral targets. The study determined that about 60,000 neurons are present in DRG at the level of C5 and about 100,000 at the C7 level.

11Developmentally, all DRG neurons are initially similar in size, and differences in cell size start to appear from week 6 of gestation on. The size of human DRG neurons identified in cryostat sections ranges from approximately 20 to 100 μm in diameter. Human neurons are larger compared to rodent DRG but the distribution of small and large neurons is similar. Small to medium sized human neurons are considered those with somata of <60 μm. In contrast to human DRG neurons in situ, neurons of small to medium sizes dominate in neuronal cultures of dissociated ganglia [36 ± 2.6 μm in diameter] as the conditions of isolation probably destroy larger neurons, similar to laboratory animals. In cultured, dissociated human DRG neurons ranged from 28 to 56 μm in diameter.

12Neurons in DRG possess a T-shaped pseudo-unipolar process that originates from the cell body via an initial segment (Figure 2). Animal studies have shown that it extends with a shorter “axonal” central process connected to and arborizing within the spinal cord dorsal horn (see review, ) and a peripheral “axonal” process innervating target tissues. Data in relation to the central sensory fiber arborization in human spinal cord do not exist. Peripheral processes have been described in different human tissues. Some processes are only one micrometer in diameter but travel large distances. The peripheral process from a lumbar DRG neuron that innervates for example the big toe, can exceed 1 m = 1,000,000 μm. This presents a massive extension of the cell, restricting only 1–2% of the cytosol to the cell body with the majority of cytosol and cytoskeletal elements present in pseudo-unipolar processes (see review, ).

13The initial segment of the pseudo-unipolar process of human DRG neurons is elongated and it forms a glomerulus-like structure (Figure 2). With age, the structure is increasingly surrounded by glial fibrillary acidic protein (GFAP)-positive SGCs but neurons and satellite glia are still separated by a basement membrane approximately 100 nm thick.

14Dorsal root ganglia are situated outside of the blood brain barrier and animal studies clearly show the presence of fenestrated capillaries (Figure 2). Hence, molecules circulating in the vascular system can directly access to the DRG. This vascular organization provides the human DRG with a robust blood supply, serving neurons that have long processes with the required high-energy demand critical for maintaining the production and transport of receptors, ion channels, cytoskeletal and transport proteins. Two interconnected arterial plexuses, situated superficially and deep, supply human DRG. These plexuses originate from arteries that derive from the radiculomedullary branches of segmental arteries. Peri-ganglionic venous plexuses drain predominantly from the dorsal side of DRG into intervertebral veins. Recently,, ) used dynamic-contrast-enhanced MRI perfusion to investigate the dynamics of blood supply of human DRG. The blood supply in vivo, expressed as perfusion of DRG determined by blood-tissue permeability and interstitial leakage fraction, was higher in DRG compared to spinal nerves. Interestingly the perfusion of DRG was significantly higher in women compared to men.

15This specialized group of DRG and TG-specific glia cells not only surround the initial segment, but build an envelope around the somata of nerve cells (Figure 1, 2) and are able to modulate neuronal function. Animal studies to date have revealed a substantial amount of receptors, transporters and ion channels are expressed in SGC (see review, ). Unfortunately, only a small number of studies have investigated the neurochemical characteristic of human SGC (Table 2). Those studies show that, similar to those of laboratory animals, human glia cells exhibit characteristic immunoreactivities for S100 beta protein and glutamine synthetase. performed a carefully controlled immunohistochemical analysis of characteristic molecules in human SGCs, and showed the presence of the metabotropic glutamate receptor 2/3, (mGlu2/3), the ATP-sensitive inward rectifying potassium channel 1.4 (Kir 1.4) and the excitatory amino acid transporter 1 (EAAT1) in human SGCs (Table 2). Since the primary transmitter of nociceptive DRG neurons is glutamate, the presence of mGlu2/3 and EAAT1 suggest active involvement of SGC in the glutamate turnover of human DRG neurons. Animal studies showing inter-SGC connections and the presence of connexin 43, a major component of gap-junctions, suggest SGC involvement in cells-to-cell communication. Recently, demonstrated immunoreactivity for NaV1.7 in GFAP-positive putative SGCs within DRG removed from patients with cancer-related neuropathic pain. It remains unknown if this “nociceptive” ion channel is present in SGC under normal conditions. Nevertheless, immunoreactivity for glial-derived neurotrophic factor (GDNF), another molecule relevant for both DRG development and pain signaling, is present in SGC.

16Evidence indicates satellite cells recognize foreign molecules and participate in immune-mediated processes. Earlier studies showed that SGC in human DRG express class I and II Major Histocompatibility Complex. More recently, studies in human SGC obtained from TG have demonstrated a class of pathogen- and damage-associated pattern recognition receptors, the Toll-like receptors. Studies characterizing subpopulations of DRG neurons through the differential expression of surface carbohydrate antigens discovered that SGC express gangliosides such as the GD1b ganglioside and fucosyl GM1. Similarly, interest in expression of molecules in DRG neurons demonstrated the expression of a variety of molecules in SGC, such as the EGF receptor and the Bcl-2 homologous antagonist Bak. SGCs also express the olfactory receptor 6B2 protein. In summary, human SGCs share many features with SGCs of small laboratory animals, such as the expression of glia- and neuron-related molecules. But compared to results of animal experiments human data is limited and taking into account the established close relationship between sensory neurons and DRG-specific glia, a lot of work needs to be done to further characterize this important cell type in human DRGs.

17Dorsal root ganglia contain a complex array of sensory neuron cell bodies that have different functions and innervate different targets. Many proteins such as channel proteins are highly related to neuron function. The method capable of identifying protein localization in complex tissues with high resolution is immunohistochemistry. This section aims to provide data for normal, uninjured human DRG immunohistochemistry, and does not focus on molecules related to any form of pathology. Only data obtained from studies of normal human DRG and from studies using normal human DRG as control tissues are discussed in this segment.

18As noted previously, human DRG neurons measure between about 20 and 100 μm in diameter and occupy areas between 1,500 and 5,000 μm2. There are no reliable and distinct morphological characteristics that allow the functional subdivision of sensory neuron subpopulations based on neuronal soma size or shape. However, neurochemical characteristics represent the most commonly used method to describe aspects of neuronal function and classification in the DRG. The emergence of genomic and transcriptomic studies has recently opened a window for researchers to re-evaluate the classification of DRG neuron subtypes based on mRNA expression patterns in individual rodent neurons. However, further validation of these groundbreaking studies will be required before a more advanced classification systems is established and widely accepted. This section will focus on neurochemical characteristics of human DRG neurons, starting with the description of common neuronal markers with an overarching focus on neurochemical characteristics associated with nociceptive function. Given the relative wealth of data included in this section, Supplementary Table 1 provides an appended overview of critical variables, including the number of participants or DRG donors, and more importantly the presence or absence of controls used for the specificity of the primary antisera/antibodies.

19Neurofilaments (NFs) are cytoskeletal intermediate filaments of varying molecular weights ranging from ∼56 to 200 kDa. They provide structural support and regulate axonal diameter, and are present in all neurons, including the DRG. In rodents, NF200, a neurofilament of 200 kDa, labels a population of larger, myelinated A-fiber neurons. Human studies have shown the presence of neurofilaments in DRG neurons early in development. Immunoreactivity for a neurofilament 150 kDa was present in fetal DRG neurons at week 6. Neurofilament 200 immunoreactivity was detected in few cells in week 10 but in all neurons at weeks 17–18 of gestation. This is also true for adult human DRG where, in contrast to rodents, NF200 immunoreactivity is not restricted to larger neurons but present in virtually all DRG neurons. Similarly, most or all adult human DRG neurons show immunoreactivity for other cytoskeletal molecules such as α- and β-tubulin, microtubule-associated proteins 1 and 5, and neurofilaments 68 and 160.

20PGP9.5 (UCHL1), a ubiquitin C-terminal hydrolase is present in all neurons and is a pan-neuronal marker. This protein has emerged as one of the key neuronal markers in rodents and humans, and is used to differentiate between neuronal and non-neuronal structures in DRG.

21Tuj1 (beta3 tubulin, TUBB3) is part of microtubule element within the tubulin family found predominantly in neurons and testes in healthy tissues. Detection of this protein is widely used as a pan-neuronal marker in immunohistochemical studies, as it readily differentiates neuronal from glial cells, which do not express beta3 tubulin. It is primarily detected in somata and processes of human DRG neurons in situ and under culture conditions.

22Peripherin (PRPH) is an intermediate filament protein that is expressed in neurons of the peripheral nervous system such as small DRG neurons and in central neurons that innervate peripheral targets such as motor neurons.

23Brn3a (Pou4f1), a sensory neuron marker, is a POU homeodomain transcription factor that regulates gene expression and differentiation of sensory neurons. In animal studies it has been shown to be expressed from nearly all sensory DRG neurons. It has been found present in all human DRG neurons of adults and gestational weeks 9–11, and has been found colocalized with NF200.

24NeuN (RBFOX3) is a RNA binding protein found predominantly in the neuronal nuclei, and another common neuronal biomarker found in the vast majority of postmitotic (mature) neurons. In the limited studies performed to date, NeuN is expressed in all adult human DRG neurons but was not detected at gestational week 10.

25Ion channels are essential for the regulation of neuronal excitability leading to the generation and conduction of action potentials and are therefore critical to sensory neuron function. Important channel proteins for the generation of inward membrane currents in nociceptors belong to the groups of voltage-gated sodium (NaV) and calcium (CaV) channels as well as transient receptor potential (TRP) channels. Given the sensory nature of the DRG, and their role in the development of chronic pain conditions, it’s not surprising that in relation to the detection of channel proteins in individual neurons, human DRG studies have predominantly focused on channels related to nociception.

26Voltage-activated sodium channels are key components of action potential generation in DRG neurons. Out of the nine NaV subtypes, the NaV 1.7, 1.8, and 1.9 are of particular interest as they have established roles as key components of pain signaling events. Studies using multiple labeling immunohistochemistry have demonstrated that immunoreactivity for the voltage-gated sodium channel NaV1.7 is present in about half of all DRG neurons. In contrast to animal studies, in humans the NaV1.7 immunoreactivity is not restricted to small neurons but present in neurons of all sizes. Immunoreactivities for NaV1.7 (PN1), NaV1.8 (SNS/PN3) and NaV 1.9 (NaN/SNS2) have been detected in all small (<30 μm), medium-sized (<50–55 μm) and large (>55 μm) neurons, which also suggests the presence of voltage-gated sodium channels NaV 1.7, 1.8, and 1.9 in neurons of all sizes. Nevertheless, the strongest immunoreactivity was detected in small neurons.

27A recent study investigating the presence of NaV 1.6, 1.7, 1.8, and 1.9 in human DRG using in situ hybridization, has shown that all NaV channels were present in human DRG and expressed in presumably nerve growth factor-dependent, TrkA expressing neurons. The NaV 1.9 showed the lowest level of colocalization with TrkA (present in about 25% of neurons), the NaV1.8 the highest (about 70% of neurons). Interestingly, the study directly compared proportions of positive cells in human and mouse DRG and showed significant differences between mouse and humans for NaV1.8 and NaV1.9.

28These findings are supported by RT-PCR and RNAseq studies that demonstrated the expression of mRNA for NaV subunits in human DRG explants. Interestingly, qRT-PCR showed that the expression levels and proportions of NaV channel subtypes were different between humans and mice. The expression of the NaV 1.8 channel subtype gene SCN10A, was much lower in human DRG, when compared to gene expression levels reported in mouse studies. In contrast, the expression of the NaV1.7 channel subtype gene SCN9A, was much higher in human than mouse samples. These findings are supported by an RNAseq study that found channel subunit SCN9A expression levels are more abundant in human DRG compared to mouse data. The sequencing data support qRT-PCR data and show differences in expression levels between mice and human DRG.

29In addition to transcriptional and translational data, electrophysiological studies confirm the functional presence of NaV1.7 and NaV1.8 channels in human DRG neurons. Sensitivity to the puffer fish toxin tetrodotoxin (TTX) selectively differentiates between channel subtypes, where NaV1.8 and 1.9 are TTX-resistant, NaV1.1, 1.2, 1.3, 1.6, and 1.7 are TTX-sensitive. Human DRG neurons possess TTX-sensitive and TTX-resistant channels, but in contrast to rodents, where TTX-resistant currents are mainly restricted to small diameter neurons, in humans they are present in small and large diameter neurons. Nevertheless, the NaV1.7 channel is the major TTX-sensitive sodium channel in DRGs in both humans and mice. However, in humans, NaV1.8 channels exhibit different functional properties and the density of TTX-sensitive and -resistant channels is much higher than that detected in animal studies.

30Voltage-gated calcium channels (CaV) are essential components of sensory neuron function as activation of these channels contributes to exocytosis of transmitter-filled vesicles at synaptic endings. The channels can be subdivided based on different criteria such as high-voltage activated and low voltage activated (HVA, LVA), similarity of the α1-subunit (CaV1, CaV2, CaV3) or sensitivity to pharmacological inhibitors (L, N, P/Q, R, T). To date no studies have reported the cellular location of CaV proteins in human DRG neurons, however, the expression of CaV-mRNA has been confirmed by RT-PCR as well as single cell PCR. Interestingly, the CaV2.2 channel was expressed in 56.4% of cultured human DRG neurons whereas the CaV2.3 was found to be expressed only in 5% of neurons.

31Calcium-activated potassium channels (KCa) are important contributors to the after hyper-polarization of neurons which can be modulated by NMDA-type glutamate receptor activation and nerve ligation, and therefore contribute to nociceptive signaling. Immunoreactivities for voltage-independent human KCa2.1 (SK1) and KCa3.1 (IK1) channels were shown in almost all (between 87% and 95%) sensory DRG neurons independent of size.

32Purinergic receptor (P2X) subunits, P2X2 and P2X3, can build homo- or heterotrimeric ligand-gated ion channels that are activated by ATP. The channels are part of a variety of neuronal signaling pathways including nociception. Studies in rodents have established that P2X2 and P2X3 channels are expressed in DRG neurons with the P2X3 subunit predominantly expressed in non-peptidergic, GDNF-dependent but NGF-independent nociceptors.

33Interestingly, mRNA for the P2X2 subunit was reported to be absent in human DRG whereas P2X3 mRNA and protein were clearly detected in human DRG. Using a carefully tested antiserum, confirmed the presence of P2X3-immunoreactivity in virtually all DRG neurons, independent of size. and demonstrated the presence of strong P2X3-immunoreactivity only in small to medium sized neurons which, similar to rodents, usually do not express the nociceptor subtype-defining NGF receptor TrkA.

34The functional validation of P2X channels in human DRG neurons was also supported by electrophysiological studies and Ca2+-imaging. However, in this instance only a subgroup of small-sized (30–60 μm in diameter) isolated human DRG neurons responded to ATP with action potential discharges, and increase in intracellular Ca2+ levels.

35Transient receptor potential cation channel subfamily V member 1 (TRPV1) is a channel protein that is activated by the vanilloid capsaicin, an ingredient of hot chili peppers, by low pH and noxious heat. Endogenous agonists are endocannabinoids such as anandamide and N-arachidonoyl-dopamine. TRPV1 is a non-selective cation channel that has been shown to be an important component of nociceptive signaling. Capsaicin induces pain in humans, but also induces desensitization of TRPV1 channels and modulates nociceptor function. Consequently, topical capsaicin is currently being successfully used in treatment of pain conditions such as postherpetic neuralgia.

36As has been done in animal studies, immunohistochemical studies have confirmed the presence of the TRPV1 protein in human DRG neurons. Interestingly, most studies describe the presence of immunoreactivity not only in small-sized neurons but also in medium and some studies in large-sized somata. The average diameter of TRPV1-immunoreactive neurons was reported as 44 ± 7 μm. TRPV1 expression was also validated in cultured human DRG neurons, with a high proportion of neurons showing TRPV1-immunoreactivity.

37TRPV1 mRNA expression was detected in human DRG explants and cultured human DRG. Functional evidence for the presence of TRPV1 channels in human DRG neurons has been provided using capsaicin, a TRPV1 agonist. Capsaicin activation of cultured human DRG neurons induced action potential discharge and increased intracellular Ca2+ levels.

38Transient receptor potential cation channel ankyrin 1 (TRPA1) is a channel protein activated by mustard oil and cinnamaldehyde, and plays an important role as an irritant sensor of a vast amount of compounds in nociceptive signaling, with its expression confirmed in animal DRG neurons. Immunohistochemistry showed the presence of TRPA1 immunoreactivity in 20% of human DRG neurons, predominantly in small-medium sized (<50 μm) neurons with some staining in larger neurons. Most of the TRPA1 positive cells were also immunoreactive for TRPV1. Cultured human DRG neurons obtained from patients with avulsion injury, responded to cinnamaldehyde with an increase in intracellular Ca2+ levels. Additionally, the TRPA1 channel has also been detected in human DRG via in situ hybridization, which demonstrated that TRPA1 mRNA expressing cells also express TRPV1 mRNA.

39Neuropeptides such as CGRP, SP and galanin are neuromodulators that are co-released with transmitters at the central and peripheral terminals of sensory neurons. In addition to being important cellular markers used in identifying subpopulations of sensory neurons, they are also fundamental contributors to nociceptor function.

40Calcitonin-gene-related-peptide (CGRP) is a neuropeptide composed of 37 amino acids. Two isoforms of the peptide exist (α-CGRP and β-CGRP) encoded from two separate genes. CGRP interacts with heteromeric receptors consisting of the calcitonin-receptor-like-receptor (CRLR) and receptor activity-modifying proteins (RAMPs). Although the peptide is a strong arterial vasodilator, it also plays a major role in nociception. Furthermore, CGRP is widely used to define a subpopulation of nociceptive DRG neurons.

41Immunoreactivity for CGRP is present in human DRG neurons from early fetal life, initially appearing at weeks 14–16, with staining intensity in DRG neurons increasing between 5 months and adulthood. Subpopulations of CGRP-containing neurons possess immunoreactivity for other peptides such as substance P or signaling molecules such as TRPV1 and phospholipase C beta 3 (PLCβ3) or angiotensin II.

42In situ hybridization confirmed the presence of CGRP mRNA in 50–70% of DRG neurons, predominantly but not exclusively in small-medium sized neurons. Some studies have identified only about 20% of DRG neurons showing CGRP-immunoreactivity, although others placed the reported proportion as high as 60%.

43Substance P (SP) is a neuropeptide composed of 11 amino acids. It is synthesized by alternative splicing from a larger precursor mRNA, preprotachykinin-A, coded by the TAC1 gene. It selectively binds to the neurokinin 1 receptor present on nociceptive projection neurons in the rat spinal cord dorsal horn and causes enhanced synaptic activity. Animal studies demonstrate a clear involvement of SP in nociceptive signaling, however, in humans, the evidence to date is not as convincing. SP is present in human DRG, with pre-protachykinin mRNA detected in small-sized neurons representing 10% of all DRG, whereas the proportion of human DRG neurons reported to show immunoreactivity for the peptide varies from 5 to 60%. Similarly, SP immunoreactivity is present early in development in neuronal cell bodies of fetal DRG but reports vary. detected immunoreactivity from week 24 onward, whereas reported the presence of SP immunoreactivity from week 11 onward. Despite the apparent similarities between humans and rodents, with both having a subpopulation of nociceptive DRG neurons containing SP, blockade of the action of SP via inhibition of neurokinin 1 receptors is effective in relieving pain in mice, but has failed to generate analgesia in human clinical trials.

44Galanin is a peptide consisting of 29/30 amino acids. Galanin modulates the excitability of dorsal horn neurons and the presynaptic release of glutamate from primary afferents (see review, ). It is present in DRG of laboratory animals. Galanin mRNA has been detected in human DRG via in situ hybridization in a small subpopulation (12.5% ± 1.4) of small-sized (<1550 μm2) neurons. The majority of galanin mRNA-expressing neurons also contained CGRP-mRNA.

45Somatostatin is a neuropeptide of either 14 or 28 amino acids in length, generated from a precursor peptide and is involved in pain processing via interaction with its cognate receptors producing inhibitory, analgesic effects. More recent studies suggest that somatostatin is also involved in the signaling of itch.

46Dorsal root ganglia neurons with immunoreactivity for somatostatin are present by gestational weeks 9 and 10, and a small population of immunoreactive cells are detectable throughout all fetal stages, with enduring expression within cells present in DRG of 4-month-old infants. Furthermore, somatostatin immunoreactivity is also present in a subpopulation (17%) of adult human DRG neurons whereas the somatostatin2A receptor is present only in few neurons in human DRG.

47Endothelin-1 (ET1) is one of three peptide isoforms, 21 amino acids in length, which act as vasoconstrictors but also induce pruritus and pain. The ET1 peptide is elevated in patients suffering from sickle cell disease, which is associated with episodes of severe pain and animal studies showed that absence of the ETA receptor subtype blocked sickle cells disease-related pain behavior.

48In human DRG 30% of neurons show ET1 immunoreactivity. In situ hybridization shows the presence of ET1 in 75% of large and small DRG neurons. The mRNA for ET1 has been reported to be often colocalized with mRNAs for preprotachykinin or CGRP, where all preprotachykinin mRNA expressing DRG neurons contained mRNA for ET1.

49The eight amino acids long peptide angiotensin II is part of the renin–angiotensin–aldosterone system (RAAS) that controls water and electrolyte balance and therefore blood pressure. This peptide also contributes to the regulation of nociception. Animal studies show intrathecally applied angiotensin II elicits nociceptive behavioral responses, suggesting that angiotensin II released by central projections of DRG neurons may contribute to nociception. In human DRG, the presence of angiotensin II has to date been demonstrated by radio-immuno-assay, HPLC and immunolabeling. Angiotensin II immunoreactivity was observed in 75% of small and medium-sized human DRG neurons. Double-labeling of human DRG showed angiotensin II in neurons expressing CGRP, synaptophysin and cathepsin D.

50There is also evidence that human DRG neurons themselves respond to angiotensin II. The angiotensin II type 2 receptor (AT2R) has been identified in cultured human DRG and in 60% of small and medium diameter neurons in immunolabeled sections of human DRG. Multiple labeling studies affirm the expression of angiotensin II, AT2R and TRPV1 in the same neurons, where over 40% of TRPV1-positive neurons expressed AT2R. Direct involvement of angiotensin II in pain signaling pathways, was supported by experiments where angiotensin II treatment of cultured human DRG neurons increased their response to capsaicin, whereas treatment with an AT2R antagonist reduced capsaicin responses. However, more recently the presence of AT2R in human and rodent DRG has been disputed, by studies finding no evidence of mRNA for AT2R genes in mouse DRG. Instead, these reports that mechanical pain hypersensitivity induced by angiotensin II is mediated by macrophage AT2R, leading to production of reactive nitrogen/oxygen species that in turn activate neuronal TRPA1. Whether this occurs via a direct action on neurons, or via effects on immune cell-neuron interactions remains to be determined. Despite the uncertainty surrounding their mechanism of action, AT2R antagonists are being used as effective analgesics in humans and laboratory animals.

51Isolectin B4 (I-B4) is a plant lectin isolated from Griffonia simplicifolia, which labels a subpopulation of nociceptive DRG neurons. In mice, these neurons have been shown represent the group of GDNF-dependent, non-peptidergic nociceptors. Although noted that they could not detect I-B4 staining in cultured human DRG neurons, others have readily demonstrated I-B4 staining in sections of human DRG. However, it is known that control for the specificity of lectin binding in human sections is difficult, and this is further highlighted in these studies reporting varying detection between membrane and cytosolic I-B4 staining identified by different research groups. On the other hand, mRNA for the gene of the binding partner of I-B4, versican, is expressed in human DRG explants, but whether the versican V2 isoform that binds I-B4 is produced in human DRG remains to be confirmed.

52Neurotrophins are a family of neurotrophic factors that includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5). Receptors for these factors include the tropomyosin receptor kinases (Trk) A, B, and C, and the low affinity receptor p75. NGF and the activation of its cognate receptor TrkA, is a key factor in the development of DRG neurons, but also critical for the induction of hyperalgesia and pain via modulation of signaling events in adult DRG neurons. Neurotrophin receptors are present in DRG from early in development through to adulthood, however, the dynamics of receptor expression patterns from development to adulthood remain to be studied. Immunoreactivity for TrkB and TrkC is present in human fetal DRG at gestational weeks 9–11, however, the confirmation of immunoreactivity for their respective ligands BDNF and NT-3 is still lacking.

53Glial-derived neurotrophic factor is another neurotrophic factor which, after interaction with its receptors RET proto-oncogene tyrosine kinase (RET) and co-receptor GFRalpha1, modulates a subpopulation of nociceptive, I-B4 binding neurons. The effect of GDNF is complex. Similar to NGF, GDNF is a key factor in the development of DRG neurons, in particular nociceptive neurons but it also impacts adult DRG neurons. In neuropathic pain animal models, intrathecal GDNF reversed pain behavior, but GDNF injected into rat muscle induced prolonged hyperalgesia.

54Nerve growth factor and its receptors have been described in human DRG. Immunohistochemistry shows NGF-like immunoreactivity in small cells, whereas TrkA was present in 65% of DRG neurons with the expression of the receptor distributed between both small and medium-sized cells. More recently, the presence of TrkA, B, and C proteins in human DRG has been described, with almost no overlap of immunoreactivities between TrkA and TrkB, or between TrkA and TrkC-positive cells. TrkA immunoreactivity was localized to small cells with cell bodies measuring between 400 and 800 μm2, whereas TrkB and TrkC immunoreactivity was detected in cells with areas much larger cell bodies (the majority around 1,400 and 1,600 μm2). As with animal studies, a substantial proportion (about 50%) of human TrkA positive cells express TRPV1. In addition to NGF and its receptors, immunoreactivity for both GDNF protein and one of its receptors, RET, has been detected at 25% and 37% respectively in cell bodies of all sizes.

55The validation of protein expression is further supported by the detection of mRNA in numerous studies reporting high levels of mRNAs for neurotrophins and their receptors, with NGF, BDNF, NT-3 and GDNF, p75 and TrkA, TrkB and TrkC having been validated to date. Detection of mRNA via in situ hybridization for the GDNF receptors RET and GFRalpha 1–3 in human DRG neurons has been also reported in multiple studies with RET present in about 70% of investigated neurons.

56The size of DRG neurons immunoreactive for NGF and GDNF receptors, compared to BDNF receptors was not different. The response to NGF and GDNF determines the fate of nociceptors, at least in laboratory animals, where neurons develop into NGF- or GDNF-dependent subtypes with different neuropeptide expression patterns. Therefore, it is of fundamental interest to determine factors that drive neurotrophin receptor mRNA expression in development. To date, receptor mRNAs for p75, TrkA-C, as well as the GDNF receptors RET and GFRalpha3 have been validated in fetal DRG at gestational age weeks 9–11 with additional presence of TrkA and TrkB. In contrast to adult DRG, BDNF and neurotrophin-3 (NT-3) mRNAs were detected in fetal DRG.

57The roles of NGF and GDNF in rodent DRG neurons have become more clearly defined, especially in driving cell differentiation, survival and target innervation. However, in humans, NGF and GDNF appear to also modulate the size of cultured human DRG neurons obtained from patients with brachial plexus avulsion. Presence of these growth factors increased the mean diameter of cultured human cervical DRG neurons significantly from 42 ± 4 μm to 62 ± 5 μm and, in line with animal studies, the presence of NGF and GDNF also increased the percentage of TRPV1 immunoreactive neurons coupled with an increased response to the TRPV1 agonist capsaicin.

58Nitric oxide synthase (NOS) isoforms 1–3 are present in DRG and its product nitric oxide (NO) is involved in nociceptive signaling with evidence supporting analgesic and algesic actions. In particular NOS1 (neuronal NOS) has been shown to be upregulated in DRG neurons in animal models of neuropathic and inflammatory pain. NOS1 has been described in small-medium sized human DRG neurons and was present in 40–50% of all neurons in the investigated ganglia.

59Gamma amino butyric acid (GABA) and its receptors are the main inhibitors in the nervous system. GABAA-receptors are ligand-gated chloride channels whereas GABAB receptors are G-protein coupled receptors. Both are expressed in DRG neurons. Activation of GABAA leads to conformational change in GABAB, inhibiting the excitability of neurons via blockade of CaV channels. Animal models showed that GABAA-receptors have a role in the pathophysiology of neuropathic pain.

60Pharmacological evidence for the presence of GABAA receptors in human DRG neurons, has been provided by experiments that completely blocked GABA-induced currents by the GABAA receptor antagonists, bicuculline and picrotoxin. Interestingly, electrophysiological properties of GABAA-mediated current were different between human and mouse DRG neurons.

61Additional evidence for the presence and involvement of GABAB receptors in the excitability of human DRG neurons has been provided from experiments investigating the inhibitory action of a cone-snail venom VC1.1. Those experiments showed the expression of GABAB and demonstrated the absence of an inhibitory effect VC1.1 when GABAB could not be activated.

62Phospholipases (PLC) are present in DRG neurons and participate in pain signaling. Phospolipases β, γ, δ, and 𝜀 are part of a magnitude of different signaling pathways with the PLC β3 isoform implicated in nociceptive signaling. This isoform is present in small human DRG neurons and colocalises with CGRP immunoreactive and I-B4 positive neurons.

63In summary, only a small population of molecules that have been described to be involved in the function of DRG neurons in laboratory animals have so far been investigated in humans. It is evident from existing studies that expression patterns and functions of molecules in DRG do not perfectly match between human and laboratory animal. Important differences exist between human DRG compared to laboratory animals and careful conducted future studies will be essential to reconcile and validate these to appropriately translate animal data into human context. At the physiological level, the longer peripheral processes and associated soma size of human DRG is likely to account for some of these differences, such as immunoreactivity for neurofilament 200 in all human DRG neurons including those classified as large. Similarly, many molecules characteristic of nociceptors including TRPV1, CGRP and P2X3 and voltage-gated sodium channels are restricted to small and medium sized neurons in mice but in not in humans where nociception-related proteins (immunohistochemistry) and mRNAs (in situ hybridization) are present in neurons of all sizes. Furthermore, ion channel proteins such as TRPV1, NaV1.8, NaV1.9, and nicotinic receptor subtypes seem to be expressed in larger proportions of human DRG nociceptors compared to mice. In addition, the separation of nociceptive DRG neurons into NGF- and GDNF-dependent populations might also be questioned in relation to human DRG as the GDNF receptor protein RET is present in neurons that express the receptor for NGF, TrkA.

64By all means, these discrepancies do not completely invalidate results from animal studies in a human translational context. Indeed, most human DRG neurons show remarkably similar patterns in respect to immunoreactivities for pain-related molecules being detected in smaller sized neurons characteristic for nociceptors. But the diversity across sizes combined with differences in electrophysiological properties suggests a more complex array of human DRG neuronal subtypes, that may differ in detecting and conveying nociceptive information when compared to laboratory animals such as rats and mice.

65Regardless of species, DRG contain multiple types of neurons and multiple types of other cells including satellite cells and cells associated with immune and vascular function. Dissociation of ganglia and culture of primary sensory neurons is useful to identify neuronal characteristics, but inferences from these studies must recognize that some types of neurons, specifically those with larger size, will likely not survive mechanical isolation and subsequent culture conditions. More importantly, critical issues related to antibody specificity highlight challenges relevant to data collections from both human and animal tissues, including the ability to compare neuronal subpopulations across species.

66An emerging and clinically significant area for further investigation is the interaction of neuronal and non-neuronal cells within DRG, and certain neuron-immune cell interactions involved in pain sensitivity have been shown to be consistent in humans and in laboratory animals. Sensory neuron-immune cell interactions are increasingly recognized as important mechanisms that contribute to chronic pain, yet there is surprisingly sparse investigative reporting of cells such as macrophages and satellite cells in human DRG. In the next few decades, researchers will hopefully find increasing opportunities to investigate and validate molecular and cellular characteristics of human DRG tissues. The failure of swathes of clinical trials based on animal model data in the past few decades reinforces the importance of human studies in clinical translation and therapeutic development, especially in very complex conditions such as chronic pain. Early insights from a handful of comparative studies suggest fundamental differences in molecular characteristics of rodent and human DRG nociceptive neurons, as well as other cell types in the DRG, and may provide key pieces of information to select optimal targets and aid more effective drug design strategies.

67RH planned the manuscript. RH, DM, CB, and ND wrote the manuscript.

68The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.