Unexplained peculiarities of the dorsal root ganglion

PAIN Pain Supplement 6 (1999) S27-S35

Unexplained peculiarities of the dorsal root ganglion Marshall Devor” Department

of Cell and Animal Biology, Institute of Life Sciences.

Hebrew

Universit?, of Jerusalem,

Jerusalem

91904.

Israel

Abstract The cell soma of primary afferent neurons in the dorsal root ganglion (DRG) is assigned by classical neurophysiology the role of a metabolic depot, charged with supporting the peripheral sensory ending, the conducting axon, and the central synaptic terminals. However, certain peculiarities of DRG morphology and physiology do not sit well with this being its only role. For example, why are DRG cell somata electrically excitable, why are some able to fire repetitively on sustained depolarization, and why does the DRG lack a blood-nerve barrier? Consideration of these and related questions leads to several intriguing hypotheses: (1) Electrical excitability of the soma may be required to insure the reliable propagation of impulses past the DRG T-junction and into the spinal cord. (2) Invasion of the afferent spike into the cell soma may provide an essential feedback signal necessary for the cell soma to regulate the excitability of the sensory ending. 3) The subpopulation of DRG neurons that have repetitive firing capability may be responsible for generating the background sensation that we feel as our body schema. Moreover, these neurons may be chemical sensors that provide essential information about our body’s internal

milieu. 0 1999 International Association for the Study of Pain. F’ublished by Elsevier Science B.V. Keywords:

Branch-point:

Dorsal root ganglion;

Spike invasion

1. Introduction The dorsal root ganglion (DRG) is an odder beast than most of us realize. Superficially, its function is obvious. The DRG contains most of the body’s primary sensory neurons, the cells that are responsible for transducing stimulus energies in peripheral tissues and passing the resulting sensory signal on to the central nervous system (CNS). Sensory signaling is the job of the axon. The cell soma is thought to play a strictly supportive role, providing metabolic support for the axon. DRG cells are among the few neuronal types without dendrites or afferent synapses; the soma has no known integrative functions in its own right (Lieberman, 1976). However, there are a number of peculiarities associated with DRG cell bodies that do not sit well with this simple, traditional view.. .enough so that the skeptic might reasonably suspect a ‘hidden agenda’, some significant functional role(s) that has not yet been identified. This article is about possible hidden agendas of the DRG. It is written as a tribute to Patrick D. Wall, a mentor and a friend, on the 25th anniversary of the founding of the journal Pain. Pat Wall, of course, has made numerous contributions to the pain field. But to my mind the richest of them all have

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been his contributions as a detective, probing through layers of academic myth and dogma to find buried treasure lying just beneath the surface. What is there about the DRG that makes me suspect hidden agendas ? Here are some ‘clues’ (see references below): 1. The DRG resides midway between peripheral innervated tissue and the spinal cord. Why this odd location? In invertebrates, for example, sensory neurons are located either within the CNS, or distally near the peripheral sensory apparatus. 2. Why do DRG neurons have their peculiar ‘pseudounipolar’ morphology, practically unique in the nervous system? 3. The DRG cell soma sits off the main conducting axon, at the end of a T-stem axon. Nonetheless, afferent spikes invade the cell soma. For what purpose? 4. DRG cells are separated from one another in the ganglion; each is wrapped in a layer of ensheathing satellite glial cells, and they do not interact synaptically. Yet despite these indicators of functional isolation, nearly all undergo (subthreshold) excitation during the course of spike activity in their neighbors. 5. Somata of many DRG neurons have the specialized membrane characteristics necessary for spike initiation, and some are even capable of repetitive firing in the

for the Study of Pain. Published by Elsevier Science B.V

Fig.

I. Largeandsmall DRG cell somata, the T-stem axon with its glomerulus. and the T-junction (from Cajal, 191 I. p. 411X)

presence of a sustained depolarization. Classically. spike initiation in these neurons always occurs at the peripheral sensory ending, not within the ganglion. Why is the cell soma adapted for spike initiation? 6. Somata of DRG neurons are richly endowed with receptor molecules to a wide variety of neurotransmitters and other chemical agonists. What do they sense? 7. The entire central and peripheral nervous system (CNS, PNS) has evolved a unique structural specialization designed to control its internal milieu, to isolate neural tissue from all but selected plasma molecules. This is the blood-brain barrier (BBB) and the blood-nerve barrier (BNB). However, the DRG is not protected by a BNB. Why? My attention was drawn to these and other anomalies of the DRG during the course of studies, begun together with Pat Wall, on chronic pain states associated with nerve pathology. We found that in the event of nerve injury, somata of primary sensory neurons begin to generate ectopit neural discharge, activity which almost certainly contributes to neuropathic paraesthesias and pain (Wall and Devor, 1983). However, the electrical excitability and membrane receptors involved in this abnormal discharge are also present, if to a lesser degree, in intact DRGs. The present article is not about neuropathy, but about normal DRG functions. Why do healthy DRG cell bodies look as if they were designed to generate impulses?

2. The primary sensory neuron 2.1. Varieties of sensor_v neurons DRGs. together with the nearly identical cranial nerve ganglia, contain the bulk of the primary afferent neurons of the vertebrate body. Exceptions are sensory neurons in enteric ganglia and in the CNS. Within a typical DRG there are many thousands of sensory neurons, with a great deal of heterogeneity among them. The most important type of variability is in the sensory information transduced and conveyed centrally. Somatovisceral information is traditionally divided into three modalities, mechanical, thermal and chemical, where many C-neurons combine two or three of these modalities (e.g. Koerber and Mendell, 1992). Each modality may be represented by a range of sensitivities and have diverse kinetics (e.g. adaptation rates). Heterogeneity is also reflected in myelination (and hence conduction velocity and frequency following capability), and in the variety of neurotransmitter, neuromodulator. transduction, receptor and channel molecules that they express. Many of these properties correlate with one another. It is, after all, the complement of molecules expressed by DRG neurons that determines their functional properties. 2.2. Basics of structure and,function DRG

neurons

are large,

roughly

round

cells.

with

M. Decor/ Pain Supplement 6 (I 999) S27-S35

+

f

Cell body

Fig. 2. Geometry of the DRG neuron. (A) A typical textbook sketch of a pseudounipolar DRG neuron showing the cell soma (cell body), the dorsal root axon branch (left) and the peripheral nerve axon branch (right). The direction of spike propagation is indicated by an arrow. The sketch is not incorrect, but it provides a fundamentally misleading picture of the metabolic challenge facing the cell soma. (B) The actual size relations of the various parts of a typical DRG neurons innervating hindlimb skin in man. The cell body, and the T-stem, dorsal root and peripheral nerve axons are drawn in their true proportions. For example. the thickness of the line representing the axon is l/lOth the diameter of the cell soma. The drawing is based on a soma diameter of 50 pm, axon diameter 5 pm, T-stem axon length 500 pm, dorsal root axon (upper) length 30 cm, and peripheral nerve axon length 120 cm (lower).

diameters ranging from about 20-150 pm in humans. They fall into two overlapping classes based on diameter and on the density of cytoplasmic neurofilamentous domains, ‘large-light’ neurons and ‘small-dark’ neurons. Since these two classes tend to correlate with sensory transduction properties, and with axonal conduction velocity (Lieberman, 1976; Scott, 1992), they have come in recent years to be called DRG A- and C-neurons, respectively, terms that I will use here. Early in development DRG neurons are in direct contact with one another, and many are coupled with gap junctions (Fulton, 1995). Prenatally, specialized

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glial cells that resemble Schwann cells insinuate themselves between the neurons and then proceed to envelope them in a thin satellite cell sheath. As a result, most adult DRG neurons are separated from one another by two layers of satellite cell cytoplasm, although some pairs are separated by only one layer, and rare pairs are in direct apposition (Pannese, 1981; Shinder et al., 1998). The surface of many DRG neurons, particularly the larger ones, is densely invested with microvilli (‘perikaryal projections’). These microvilli, which are embedded within the lamella of ensheathing satellite cells, greatly increase the membrane surface area of the neuron (Lieberman, 1976; Pannese, 1981; Pannese et al., 1990). DRG neurons are ‘pseudounipolar’. That is, each emits a single stem axon from its axon hillock-initial segment pole. A few tens or hundreds of microns from the soma this stem axon divides in two like a T or a Y. One branch proceeds from the T-junction into the spinal nerve and from there to a sensory ending in skin, muscle, viscera etc. The second branch enters the dorsal root and spinal cord (or brainstem). With rare exceptions, both axon branches are either myelinated or unmyelinated in any given neuron. Very few axons branch within the nerve or dorsal root (Devor et al., 1984) although branching is extensive in both the peripheral and the central terminal fields. In some neurons the stem axon follows a tortuous, coiled path before extending to the Tjunction, sometimes spiraling around the cell soma. This socalled ‘glomerulus’ greatly lengthens the stem axon (Fig. 1). The ‘special senses’ such as hearing and balance use specialized non-neural cells (e.g. cochlear hair cells) to transduce sensory energies. However, such cells are absent in skin and deep tissue. Instead, the transducer is the terminal membrane of the sensory axon itself. Therefore, the mechano-, thermo- and chemo- sensing molecules responsible for generating the sensory signal must be synthesized in the DRG cell soma and transported down to the axon end by axoplasmic flow. The same is true of the voltage sensitive ion channels responsible for encoding the generator potential into a train of action potentials, and for the molecules needed by the cell’s spinal synaptic apparatus. The traditional textbook sketch of the DRG neuron, the sketch which defines the way we usually think about DRG neurons, successfully illustrates the cell’s role in sensory communication. However, it is grossly misleading with regard to the cell soma’s metabolic task. A typical sketch of a DRG neuron is shown in Fig. 2a. The impression given is that much of the cell’s cytoplasmic mass is contained in the cell soma. This is simply not true. Consider a typical 50 km diameter neuron in the human L4DRG that has a 5 Frn axon innervating the foot. The volume of the cell soma is 65 x lo3 km” (4/3 n-R’). The volume of 1.5 meter of axon, a conservative length estimate given that many such axons make synaptic endings in the dorsal horn and then proceed up the spinal cord to the brainstem, is 29 X lo6 km” (TR’ X L). That is, 0.2% of the cell’s cytoplasm is in the soma, and 99.8% is in the axon (Fig. 2b).

WI

M. Dmw / Pain Supplenwnt 6

Since virtually all of the cell’s protein synthesis, and much of its energy metabolism, occurs in the soma, the soma should be seen as an incredibly busy little factory struggling full time to provide for the huge metabolic load of its enormous axon.

3. Electrical propagation

excitability of DRG neurons, and spike into the cell soma

Afferent impulses running centrally from the peripheral sensory ending pass the T-junction and continue straight into the dorsal root and spinal cord (arrow in Fig. 2a). Sensory communication between periphery and center does not require the spike to invade the cell soma. However, in most DRG neurons, the afferent spike does propagate along the T-stem axon and invade the soma. For what purpose? It is possible, of course, that electrical excitability of the stem axon and soma is meaningless and does not reflect any particular functional utility. Since the axon must be excitable, perhaps the voltage sensitive channels that subserve electrical excitability are inserted promiscuously into the soma membrane due to sloppy targeting. While this possibility cannot be ruled out, most systems appear to adhere to the biological principle that the expression and targeting of gene products is tightly regulated. Many cell types, including nerve cells, have a complex and elegant machinery for vectorially transporting membrane proteins from their sites of synthesis and inserting them with great regional specificity and precision at functionally appropriate targets (e.g. Rodriguez-Boulan and Powell, 1992). If we adopt as a working hypothesis that soma excitability is not a result of sloppy targeting, but rather a specific design feature of DRG neurons, what might its evolutionary purpose be‘? 3.1. Hypothesis Tyjunction

I: protection from conduction

block at the

Some years ago I proposed a hypothesis concerning the role of spike invasion of the soma (Devor and Obermeyer, 1984). Imagine that the DRG neuron were bipolar, with the peripheral axon emerging from one end of the soma and the dorsal root axon emerging from the other end. As a spike propagating centrally along the peripheral axon approached the cell body a problem would arise. As the axon diameter expands into the much larger soma, much of the longitudinal current needed to keep the spike moving would be dissipated in charging the capacitance of the soma membrane (capacitative load), or be shunted through soma membrane conductances (resistive load). Either way, the current would be unavailable to excite the next nodes of Ranvier along the axon. Both factors, capacitative and resistive load. threaten to abort the forward propagation of the spike especially at high firing frequencies (Ito and Takahashi, 1960; Luscher et al., 1994;Rall, 1970). A potential solution to this problem is to move the cell soma off the line of spike propagation, e.g.

(1999)S27-X3.5

to the end of a fine T-stem axon, just as nature has done. Assuming that the soma must reside somewhere in midaxon (a non-trivial assumption, see below) this solution to the problem of conduction failure provides an explanation of the pseudounipolar structure of DRG neurons. Even with pseudounipolar T-stem morphology, a spike propagating centrally towards the T-junction would still see an excess resistive and capacitative load ahead of it. That is, some of the longitudinal current intended to insure throughconduction of the spike would be drawn (shunted) into the stem axon. The larger the soma, the larger the load. Shunting is minimized by reducing the caliber of the stem axon and increasing its length, thus increasing its internal resistance. This may explain the stem axon glomerulus present in larger DRG cells (Fig. 1). Another potential solution to the problem of conduction block at the T-junction is to enhance the electrical excitability of the axon in the region of the Tjunction. This would increase the longitudinal current available for forward spike propagation, helping to overcome the tendency for conduction block. Indeed, nature has adopted this solution by spacing nodes of Ranvier more closely than normal in the region of the T-junction, both on the nerve and on the dorsal root axons (e.g.Ito and Takahashi, 1960; Spencer et al., 1973). A third potential solution is to make the Tstem axon, and cell soma itself, electrically excitable. The inward ion flow generated by the evolving spike in these structures would contribute to the longitudinal current propagating past the T-junction into the dorsal root, partly compensating for the loss they imposed by shunting current away from the T-junction (Devor and Obermeyer, 1984). Under this hypothesis, cell soma excitability emerges as a design compromise that serves to insure reliable spike transmission past the T-junction and toward the spinal cord. We are currently in the process of evaluating these explanations of afferent neuron geometry and excitability using a computational model of the DRG (Amir, unpublished). This model permits us to examine the reliability of propagation past the T-junction in the presence or absence of soma excitability. 3.2. Hypothesis metabolism

2: coupling between spike activity and cell

Spike invasion of the soma may also have a specific positive role in the physiology of the DRG neuron. In the intact DRG, primary sensory neurons need to closely regulate the sensitivity of their peripheral sensory ending. A-neurons must maintain stable low-threshold mechanoreceptor properties, and most C-neurons must maintain the properties of nociceptors. These transduction and impulse encoding properties depend on precise control of the type and density of specific macromolecules present in the neuronal membrane at the sensory ending. The macromolecules involved (transducer proteins, receptors, and voltage-sensitive ion channels) are all large polypeptides that are synthesized in the DRG cell soma and transported down to the end of the axon in vesicular vectors in the axoplasmic freight. Moreover, the

M. Decor / Pain

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creation of the transduction/encoding apparatus at the sensory axon end is not a one-time event. Each of the proteins involved has a limited half-life, and undergoes continual turnover. Membrane Na+ channels, for example, are thought to be replaced about every 3 days, on average (Schmidt and Catterall, 1986). This means that their rate of synthesis, transport and incorporation into the membrane at the sensory ending, and their ultimate reuptake, must be precisely regulated on an ongoing basis. An excess or deficit in the supply of one of these molecules would alter the sensory coding properties of the afferent, and hence alter sensory signaling. It is unlikely that the regulation of sensory ending excitability is accomplished in an open-loop mode, that is, without feedback about the ongoing level of excitability. The reason is that stability of the transduction/encoding process must be maintained over very long periods of time (a lifetime) under conditions where numerous associated factors are in constant flux. For example, minor inflammation in the skin triggers increased expression and retrograde transport of neurotrophic factors including nerve growth factor (NGF). NGF, in turn, is known to upregulate the synthesis of Na+ channels in DRG neurons (Gould et al., 1998; Tanaka et al., 1998). In all instances where biological parameters are homeostatically regulated some sort of feedback signal is available that tells the synthetic machinery when its product is needed in greater abundance and when delivery can be reduced. I suggest that for afferent A-neurons, a regulatory feedback signal might be provided by ongoing sensory impulse traffic. For this to work, however, the spike would have to propagate along the T-stem axon and into the cell soma. If a neuron fired more over a particular integration epoch than mandated by an intrinsic set-point, for example, the sensitivity of the sensory ending would be appropriately reduced by modulation of the synthesis and transport of the transducer and voltage-sensitive channel proteins that determine afferent excitability. Likewise, if spike activity integrated over this time window were low, the cell would respond by increasing the excitability of the sensory ending. There are a number of examples in which impulse traffic during development has been shown to exert metabolic control on neurons, including sensory afferents (Fields, 1996; Hyman, 1996). I am now proposing that this principle may extend to the ongoing regulation of afferent excitability in adulthood.

4. Spiking as a means of functional among DRG neurons 4.1. DRG cross-depolarization

communication

and cross-excitation

The main role of DRG neurons is to convey to the CNS information on stimulus events that occur at discrete loci in the peripheral tissue, the cell’s receptive field. Correspond-

S31

ingly, each axon in the nerve and root is individually wrapped by Schwann cells in a myelin or mesaxonal sheath, and each DRG soma is wrapped in a satellite cell sheath (Lieberman, 1976; Pannese, 1981). Synapses are so rare in DRGs as to be virtually nonexistent. The whole structure appears purpose-built to avoid interactions among adjacent afferent neurons. This is why it is so surprising that in fact, nearly all neurons in the DRG are functionally linked to one another by a novel, non-synaptic coupling mechanism. Pat Wall and I first became aware of functional crossexcitation among DRG neurons during experiments aimed at characterizing the discharge that originates ectopically in DRGs following nerve injury (Devor and Wall, 1990). While recording ongoing activity in ectopically firing DRG neurons we applied tetanic stimulation to the axons of neighboring neurons that share the ganglion. In most cases this transiently increased the firing rate of the recorded neuron. Subsequent studies, mostly carried out in vitro, showed that roughly 90% of all neurons in the DRG, neurons with A-fibers as well as neurons with C-fibers, undergo several millivolts of depolarization (and are functionally excited) by impulse traffic carried in neighboring DRG neurons (Amir and Devor, 1996, 1999; Liu et al., 1998; Utzschneider et al., 1992). We call this phenomenon DRG ‘cross-depolarization’ (and ‘cross-excitation’). The ability of the soma to support spike discharge is essential for this mutual crosstalk to occur. In terms of neural mechanism, K+ release during spike activity, with consequent depolarization due to elevated [K],,, probably plays a role (Utzschneider et al., 1992). K+ cannot be the whole story, however, as cross-depolarization is associated with a change (net decrease) in membrane conductance and has a reversal potential (at about - 20 mV). At least part of the effect appears to be due to a neurotransmitter(s) released non-synaptically from the activated neurons, that diffuses in the extracellular space, and accesses receptors on neighboring neurons (Amir and Devor, 1996). The release of neurotransmitters from the cell soma, in the absence of presynaptic specializations, is in itself a notable peculiarity on DRG neurons that demands explanation (Haung and Neher, 1996). Non-synaptic ‘volume transmission’ has been discussed recently as an alternative mode of cellular interaction in the brain (Bachy-Rita, 1996). The DRG may be an advantageous venue for studying this fascinating process. But in the present context the question is not how DRG cross-excitation occurs, but why? In the intact nervous system the amplitude of DRG cross-depolarization is rarely enough to reach threshold and trigger spikes; the mutual interaction among DRG neurons is subthreshold. For example, stroking the palm of your hand probably induces crossdepolarization in DRG neurons innervating your fingertips, but it does not induce spikes in these neurons. If it did, the stroking would be felt both on the palm and in the fingertips. However, spikes can be evoked by cross-depolarization if the neuron is artificially depolarized, or if its threshold is

lowered, e.g. due to nerve injury. Indeed. in neuropathy, cross-excitation might enter a positive feedback mode triggering painful electric shock-like paroxysms and hyperpathia (Rappaport and Devor, 1994). Be that as it may, there seems no p~irna facie role for DRG cross-depolarization in the intact nervous system.

4.2. A functional depolarizution?

role

,for subthreshold cross-

Being unwilling to conclude that the ubiquitous crosstalk in the DRG is an epiphenomenon, I offer the following speculation. In the section on electrical excitability of the DRG above (Section 3.2) I discussed the need for primary sensory neurons to receive a use-related feedback signal in order to regulate the synthesis and export of the macromolecules involved in transduction and encoding at the peripheral sensory ending. For A-neurons such a signal is readily provided by afferent impulse traffic, so long as the spike is able to invade the cell soma. In contrast nociceptive Cneurons do not, in general, have access to such a feedback signal. Most fire only on the rare occasion of a nociceptive stimulus, and many never fire at all in the normal course of events (e.g. nociceptors innervating internal organs such as the ureter. or the pericardium). Nonetheless, when called upon for the first time. perhaps after decades of silence. they must respond at precisely predetermined stimulus thresholds. It is unlikely that open-loop regulation, without feedback, could meet this need. DRG cross- depolarization provides a potential solution. Like A-neurons. the bulk of C-neurons are cross-depolarized by spike activity in neighbors. including neighboring A-neurons (Amir and Devor, 1999). In principle, such A- to C-neuron cross-depolarization could provide a feedback signal sufficient to permit ongoing regulation of the sensitivity of nociceptor endings, in the absence of frank peripheral nociceptor activation. Specifically, I suggest that the integrated spike activity of A-neurons is sensed by Cneurons in the DRG by means of A- to C-neuron crossdepolarization. This signal is then used by the C-neurons to regulate their excitability within functionally appropriate bounds informed by the integrated activity of the Aneurons. If the system detected a drop in the aggregate activity of the A-neurons, it would respond by increasing the sensitivity of both A- and C-neurons. The reverse would happen if an increase in A-fiber activity were detected. By this hypothesis, it is the ratio of C- to A- fiber excitability that is regulated. I am not aware of any specific examples in which it has been demonstrated that the metabolism or gene expression of primary sensory neurons is regulated by (subthreshold) membrane potential. However, it has been shown recently in cortical and in cerebellar neurons that alteration of resting membrane potential can modulate cellular gene expression (Scherer et al., 1992; Hombourg et al., 1997).

5. Repetitive firing capability (rhythmogenesis)

in DRG neurons

The processes just discussed provide a rationale for the DRG cell soma being able to support spike invasion from the axonal T-junction. However, somata of a particular subset of DRG neurons exhibit repetitive firing capability. which is quite another matter. Repetitive firing in the presence of a sustained depolarization requires a much higher level of electrical excitability than is required to generate a single spike on pulse stimulation. or afferent spike invasion (Matzner and Devor, 1992). In neurons designed to fire repetitively, this capability is usually provided by a specialized patch of membrane, the axon hillock-initial segment in cell somata, or the spike encoding region in slowly adapting sensory endings. These regions are typically very rich in voltage sensitive Na’ channels. There is an even more striking indicator that repetitive firing capability is an evolutionarily designed feature of DRG neurons. We have recently found that at least a subset of DRG neurons possess an atypical, special-purpose repetitive firing mechanism. In most neurons rhythmogenesis is dependent on the classical Hodgkin-Huxley process whereby a sustained inward current repeatedly draws the membrane potential towards firing threshold after each spike in a train. However, we now have evidence that DRG neurons capable of repetitive firing are intrinsically resonant, and display subthreshold sinusoidal oscillations of their resting membrane potential (Amir et al.. 1998). On depolarization the peaks of the oscillations trigger trains of spikes. DRG neurons without subthreshold oscillations appear to be incapable of generating repetitive spike trains even when deeply depolarized, although they do fire briefly on step depolarization. It is hard to imagine that the precise combination of ionic conductances responsible for membrane potential oscillations in DRG neurons occurred by chance rather than as an endpoint of adaptive evolutionary pressure. On the other hand, it is hard to imagine what purpose repetitive firing capability might serve in intact DRG neurons! After all, DRGs appear to be carefully protected from sustained depolarizations. those evoked synaptically as well as those evoked by sensory-generator processes. What factor(s) in vivo could provide the sustained depolarization required to trigger repetitive firing?

6. A chemosensory

role for the DRG?

6. I. Numerous chemoreceptor DRG neuron somata

molecules

ure present

on

An important subclass of primary afferent C-neurons, the polymodal nociceptors. have chemosensitive sensory endings. Ever since the discovery of the first intrinsic mediators of inflammation (bradykinin, serotonin, various prostanoids, hydrogen ions (pH) etc.), it has been assumed that

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chemosensitivity is an adaptation designed to detect tissue damage and inflammation, and to trigger protective nocifensive responses. The presence of receptors for plant-derived toxins such as capsaicin (Caterina et al., 1997) suggests that this principle might even extend to extrinsic agonists, although the possibility of intrinsic capsaicin (vanilloid) receptor ligands cannot be ruled out. Chemosensitivity is also a relevant property for the intraspinal ending of primary afferents. In many systems neurotransmitter release is modulated by presynaptic autoreceptors. Since receptors in the sensory and spinal ends of afferent axons are manufactured in the cell soma, it is no surprise that mRNAs for many different kinds of receptors can be detected in the DRG using hybridization techniques. However, it is now known that receptors for inflammatory mediators and for capsaicin are expressed not only at the endings of afferent axons, but also on the cell soma within the DRG. Why? Moreover, this dilemma is quite general. Based on a range of techniques, primarily immunocytochemistry and electrophysiological recording during drug application, it has been shown that a large number of different chemoreceptor molecules are expressed on DRG cell somata, of A-neurons as well as C-neurons (examples cited in Shinder and Devor, 1994). A directed screen using probes against consensus sequences of transmembrane receptors is likely to yield many more examples. Why should the cell soma be chemosensitive? Are these receptors intended by the cell for export to the peripheral or central endings. but instead promiscuously placed in the cell soma? Is the metabolic machinery of the DRG neuron incapable of selectively targeting receptor molecules to the places where they are needed? If not, then it is hard to avoid the conclusion that some form of chemoreception is accomplished within the DRG. One form was already noted, chemically mediated DRG cross-excitation. The absence of a blood-nerve barrier in DRGs suggests another. 6.2. DRGs are located outside of the blood-nerve

barrier

(BNB) The entire CNS and PNS has evolved a unique structural specialization designed to isolate neural tissue from the systemic circulation, and to regulate which plasma molecules can gain access to the neural tissue and which will be excluded. This is the blood-brain barrier (BBB) and the blood-nerve barrier (BNB). Physically, the barrier is located at the lines of endothelial tight junctions that seal most CNS and PNS blood capillaries, and the astrocytic endfeet that surround CNS capillaries. The presence of these barriers forces blood-borne molecules to pass through the endothelial cell itself (and perhaps also through pericytic and astrocytic processes), where selective control can be exercised. In the brain, only a few spots are devoid of a BBB. These include the so-called circumventricular organs, the median eminence, and the area postrema (Gross, 1987). Each of these locations is presumed to play a central chemosensory

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role, a conclusion based on the absence of the BBB itself, on the presence of homeostatic regulatory nuclei nearby, and on direct electrophysiological recordings of neuronal responses to relevant blood-borne substances. The axons of DRG neurons in the dorsal root and the central terminals in the spinal cord and brainstem, are protected by the BBB. Afferent axons in the peripheral nerve are protected by the BNB. However, the DRG itself has no BNB. This unexpected observation has been confirmed repeatedly over the years in studies in which various types of BBB/BNB impenetrant tracer molecules are injected into the systemic circulation, and are later detected in the DRG parenchyma, and even within the narrow cleft that separates the neuronal soma from its satellite cell sheath (e.g. Jacobs et al., 1976; Allen and Kieman, 1994; Wadhwani and Rapoport, 1987). Extrapolating from the chemosensory function of the circumventricular organs etc. (Gross, 1987) might DRGs be carrying out some as-yet unidentified chemosensory function associated with the body’s internal milieu? This speculation, radical as it may seem, has several charms in addition to explaining the presence of somatic chemoreceptors. First, it accounts for the peculiar location of DRGs. If they resided in the peripherally innervated tissue they might be subject to the dangers of trauma, and to exogenous stimulus energies and chemicals. On the other hand, if they resided within the spinal cord, they would be within the BBB. In fact, the DRGs reside in the bony confines of the intervertebral foramen, well protected from external stimuli. and yet accessible to the blood-borne chemical milieu. A second paradox resolved by the chemosensory hypothesis is repetitive firing capability. In the discussion of excitability above (Section 5), we were faced with a process specially adapted for generating spike trains (resonance), but with no obvious source of a sustained generator depolarization adequate to bring the neuron to repetitive spiking threshold. However, if DRG neurons indeed respond to blood-borne chemicals, these might induce the necessary depolarization. Finally, a chemosensory role is consistent with the abundant microvilli that decorate the surface of many DRG neurons. Microvilli expand the membrane surface area available for exchange of metabolites with the immediate micro-environment. In addition, however, they are typical of chemosensory neurons (e.g. in the olfactory epithelium). We have previously shown that despite the satellite cell sheath, maker molecules in the extracellular space have diffusional access to the soma membrane of DRG neurons, including the microvilli (Shinder and Devor, 1994). 6.3. Evidence animals

qf impulse initiation in the DRG in intact

Following nerve injury DRG neurons are known to respond to blood-borne chemicals, circulating adrenalin for example (Burchiel, 1984; Devor et al., 1994). But is there any evidence for such responses in intact DRGs?

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In 1983 Pat Wall and I reported electrophysiological observations of ongoing discharge originating in the DRG of intact, unoperated rats (Wall and Devor, 1983). The prevalence of such cells was relatively low, but not insignificant (4-5% of the A- and C-neurons sampled). Special efforts were devoted to insuring that this activity was not an artifact of mechanical stimulation applied unintentionally while setting up the electrophysiological preparation, or a result of trauma from exposure of the DRG. We know that hypoxia and/or hypotension, and adrenalin. can accelerate the ongoing firing in the DRG, but we have not otherwise explored blood borne factors that might initiate or modulate it. The inability at present to point to a specific chemical agonist in the intact animal is the weakest link in the chemosensory hypothesis. Indeed, one could argue that there is positive evidence against the chemosensory hypothesis. Each of us is a sensitive detector of electrical impulses generated in our own DRGs. If such impulses were generated we should feel them, just as we feel sensory impulses generated in the skin. But could it be that we do feel impulses originating in the DRG, but that we are so used to the resulting sensation that it has become part of an unappreciated sensory background? One obvious way to bring any such ongoing background sensation to the level of conscious awareness is to eliminate input originating in the periphery. This can be done simply using local anaesthetic nerve blocks in intact, healthy subjects. In fact, during nerve block, the affected arm is not felt as being absent. Rather, subjects report the sensation of a phantom arm (Melzack and Bromage, 1973: Wall, 198 1; Gandevia and Phegan, 1999). Indeed, many of us have experienced such ‘normal phantoms’ (i.e. phantoms in the absence of amputation) in the dentist’s office. Local anaesthetic block of nerves serving the jaw, tongue and teeth does not, as one might expect, yield the sensation of a hole in one’s face. Rather, the sensation is of a swollen lip, a phantom lip. Such anomalous sensations might also be provoked actively. Possible examples are the unpleasant aching feeling of being ill, potentially associated with primary afferent responses to circulating cytokines (Watkins et al., 1995). or the quasi-somatic shuddering sensation sometimes felt when we hear the squeak of chalk on the blackboard. At present there is no direct evidence that such everyday sensations are in fact related to impulse discharge originating in the DRG. An equally likely source is the CNS. In principle, this distinction could be made experimentally using microneurographic recordings from peripheral nerves central to a nerve block. Another approach would be to see if normal phantoms evoked by nerve block vanish during spinal block.

hitherto unsuspected sensory roles of primary sensory neuron somata. These clues, and others, have been interpreted within the framework of two key questions: ( I ) Wh?! is the DRG cell smna electrically excitable ut all?. and 2) Why are some DRG cells able to jre repetitively? In answer to the first question two hypotheses were offered. The first hypothesis was that electrical excitability is required to insure reliable propagation of impulses past the T-junction and into the spinal cord. The second hypothesis was that invasion of the afferent spike into the cell soma provides a feedback signal useful for regulating the metabolism of the sensory neuron. The answer offered to the second question was even more unconventional. The proposal is that a subpopulation of DRG neurons is responsible for generating the background sensation that we feel as our body schema. In addition, chemosensitivity of DRG neurons, and consequent responsiveness to still-undefined chemical moieties in the blood, may provide essential information about our internal milieu. Within the framework of these hypotheses all of the peculiarities listed in the Section 1 have been provided with a rational interpretation. Specifically: The midnerve location of the DRG provides physical protection without denying it essential access to bloodborne chemical stimuli. The ‘pseudounipolar’ morphology of DRG neurons, and glomerulus on the initial segment stem axon, protects against the risk of conduction block at the T-junction. Spike invasion of the cell soma has a similar role, and also provides the feedback necessary to match cell metabolism with functional requirements of the sensory ending. Subthreshold crosstalk among DRG neurons permits metabolic regulation of receptor excitability in neurons that do not fire during normal everyday activity (esp. nociceptors). The DRG serves not only as a metabolic depot, but also as a source of afferent input. Consequently, some DRG neurons are endowed with repetitive firing capability. Membrane receptors of DRG neurons permit subthreshold crosstalk, and responsiveness of DRG neurons to blood-borne chemical signals. The hypothesis that the DRG acts, in part, as a chemoreceptor organ requires that the as- yet unidentified bloodborne signals have direct access to neurons within the ganglion. Hence the absence of a blood-nerve barrier (BNB). Do these speculations capture biological truths about the somatosensory system? My hope is that, at a minimum, the illumination of these issues has provoked some thought.

7. Summary Acknowledgements I have assembled a collection of peculiarities about DRG structure and function that may constitute ‘clues’ pointing to

I wish to thank Ron Amir for helpful comments

on the

M. Decor/

Pain Supplement 6 (1999) S27-35

manuscript. The author’s work on DRGs is supported by grants from the United States-Israel Binational Science Foundation (BSF), the German-Israel Foundation for Research and Development (GIF), and the Hebrew University Center for Research on Pain.

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