**1. Introduction**

166 Neuroscience – Dealing with Frontiers

Yeh TK, Wu JP, Chang LW, Tsai MH, Chang WH, Tsai HT, Yang CS & Lin P (2011)

Zhao X, Kuja-Panula J, Rouhiainen A, Chen YC, Panula P & Rauvala H (2011) High mobility

*Biol Chem*, Vol. 286, No. 26, pp. 23200-23213, ISSN 0021-9258

5, No. 1, pp. 91-97, ISSN 1743-5390

Comparative tissue distributions of cadmium chloride and cadmium-based quantum dot 705 in mice: Safety implications and applications. *Nanotoxicology*, Vol.

group box-1 (HMGB1; amphoterin) is required for zebrafish brain development. *J* 

Somatic sensations of the head and orofacial region are transmitted by trigeminal primary afferent neurons, a group of neural-crest derived sensory neurons. Most of their cell bodies are located outside the central nervous system, residing in the trigeminal ganglion (TG) but some of them lie centrally within the brainstem, in the mesencephalic trigeminal nucleus (MTN).

The TG represents a cranial analog of the dorsal root ganglia in the peripheral nervous system (Darian-Smith, 1973). TG neurons have a unique morphology and are classified as pseudounipolar (Krastev, 2009). Their centripetal processes, usually called trigeminal primary afferents, carry somatosensory information from mechanoreceptors, thermoreceptors and nociceptors in the face, the oral and nasal cavities, and through the portio major of the trigeminal nerve reach their main target neurons in the trigeminal sensory nuclei (Fig. 1), where they establish synaptic contacts with their perikarya (for reviews, see Darian-Smith, 1973; Dubner et al., 1978; Kruger & Young, 1981).

Mesencephalic trigeminal neurons are considered centrally displaced ganglion cells but in spite of their curious central location they maintain some characteristics of neural crest cell derivates. The great majority of MTN cells are large pseudounipolar neurons which provide the innervation of the masticatory muscle spindles and periodontal ligament pressoreceptors. Their central branches enter the trigeminal motor nucleus and several other brainstem nuclei around it (Fig. 1), where they make excitatory synaptic connections with jaw-closing motor or premotor (last-order interneurons) neurons, respectively (see Capra & Dessem, 1992 for a review). Unlike the TG cells however, MTN neurons receive synaptic inputs that potentially modify their output (reviewed in Lazarov, 2000). MTN neurons are also remarkable insofar as they, without an exception, constitute one distinct functional class of trigeminal sensory neurons, i.e. proprioceptive neurons (Jerge, 1963; Cody et al., 1972). Due to their ectopic location within the brain, in addition to this classical function, some mesencephalic trigeminal neurons may act as interneurons capable of integrating peripheral and central information prior to reaching the trigeminal motor nucleus (Kolta et al., 1995). The functional segregation between peripheral and central primary afferent neurons is a further striking feature of the mammalian trigeminal sensory system (reviewed in Waite, 2004).

The Neurochemical Anatomy of Trigeminal Primary Afferent Neurons 169

A commonly held hypothesis in neurobiology is that neuronal morphology frequently mirrors chemical neuroanatomy and also neurons in different functional pathways, within which they lie, can be characterized by their neurochemical profiles. In this respect, the various neuronal populations that constitute the TG and MTN can be identified not only on the basis of their morphological characteristics and electrophysiological properties but also by their neurochemical content. This issue is of key importance since the transmitter content of different neuronal populations often correlates well with their target projections. It is assumed that trigeminal primary afferent neurons exhibit pathway-specific patterns of neurochemical expression, a concept that has been called chemical coding (Costa et al., 1986). It has also been proposed that the differently fated embryonic migration, synaptogenesis, and peripheral and central target field innervation could affect the individual neurochemical phenotype of TG and MTN neurons. Trigeminal primary afferent neurons utilize a wide variety of chemical neuromessengers for synaptic transmission and possess the ability to produce relevant adaptive changes in their neurochemical phenotype

in response to environmental cues (for recent reviews, see Lazarov, 2002, 2007).

implications.

**neurons** 

neurotransmitters and neuroactive substances.

**2.1 Classical transmitters**

This chapter therefore focuses on the chemical neuroanatomy of the TG and MTN neuronal populations under normal conditions, the role and relationship of neurotransmitters and their corresponding receptors in relaying orofacial sensations and also refers to the interactions with other atypical neuromessengers and neurotrophic factors. We have also surveyed the chemical plasticity of developing and mature TG and MTN neurons to gain insight into their structural and functional properties in an altered neurochemical balance, with special reference to trigeminal nerve degeneration and regeneration, and clinical

**2. Neurotransmitters and their known receptors in trigeminal primary afferent** 

Using immunohistochemistry and *in situ* hybridization histochemistry we have identified distinct neuronal, partly chemically coded, subpopulations in the intact TG and MTN. Our findings suggest that trigeminal primary afferent neurons are chemically heterogeneous and appear to use various chemical neuromediator candidates for synaptic transmitters. These include classical and peptide transmitters, calcium-binding proteins as neuronal markers and other neuroactive molecules (Table 1). In addition, we have demonstrated that TG and MTN neurons receive inputs from different groups of neurons that contain multiple transmitter substances. Indeed, both the TG and MTN receive catecholaminergic, nitrergic and peptidergic innervation in the form of perineuronal arborizations encircling in a basketlike manner the perikarya of large unstained neurons. It is assumed that the pericellular baskets can function as a key communication medium between immunopositive projections and immunonegative neuronal somata in the orofacial somatosensory information processing. Last but not least, it is now well established that the cell bodies of TG and MTN neurons are richly endowed with postsynaptic receptors for a huge array of

In recent years, a variety of 'classical' transmitter substances and their receptors have been associated with subsets of trigeminal primary afferent neurons. Among them are amino acids (both excitatory and inhibitory) and monoamines. On the other hand, our studies

Fig. 1. Schematic illustration of the trigeminal pathways for orofacial somatic sensation. Tactile (touch and pressure) sensibility of the face and mouth is relayed through the large diameter Aβ axons of the trigeminal ganglion (TG) neurons to the principal sensory trigeminal nucleus (PrTN) and rostral part (subnucleus oralis and interpolaris) of the spinal trigeminal nucleus (SpTN), while pain and thermal sensations from the orofacial structures are conveyed by the thin A and C trigeminal primary afferents to the subnucleus caudalis of the SpTN. Proprioceptive sensation from the face and oral cavity is transmitted directly or through premotor neurons in the supratrigeminal nucleus (SuTN) to the motor trigeminal nucleus (MoTN) via the central processes of primary afferent neurons whose pseudounipolar cell bodies are mainly located in the mesencephalic trigeminal nucleus (MTN). The efferent limb (depicted in red) of the reflex arc producing jaw closure (jaw jerk or masseteric reflex) is formed by the axons of trigeminal motoneurons traveling through the motor root of the trigeminal nerve to the muscles of mastication. V1, V2, V3, ophthalmic, maxillary and mandibular divisions of the trigeminal nerve, respectively.

Fig. 1. Schematic illustration of the trigeminal pathways for orofacial somatic sensation. Tactile (touch and pressure) sensibility of the face and mouth is relayed through the large diameter Aβ axons of the trigeminal ganglion (TG) neurons to the principal sensory

pseudounipolar cell bodies are mainly located in the mesencephalic trigeminal nucleus (MTN). The efferent limb (depicted in red) of the reflex arc producing jaw closure (jaw jerk or masseteric reflex) is formed by the axons of trigeminal motoneurons traveling through the motor root of the trigeminal nerve to the muscles of mastication. V1, V2, V3, ophthalmic,

nucleus (MoTN) via the central processes of primary afferent neurons whose

maxillary and mandibular divisions of the trigeminal nerve, respectively.

trigeminal nucleus (PrTN) and rostral part (subnucleus oralis and interpolaris) of the spinal trigeminal nucleus (SpTN), while pain and thermal sensations from the orofacial structures are conveyed by the thin A and C trigeminal primary afferents to the subnucleus caudalis of the SpTN. Proprioceptive sensation from the face and oral cavity is transmitted directly or through premotor neurons in the supratrigeminal nucleus (SuTN) to the motor trigeminal

A commonly held hypothesis in neurobiology is that neuronal morphology frequently mirrors chemical neuroanatomy and also neurons in different functional pathways, within which they lie, can be characterized by their neurochemical profiles. In this respect, the various neuronal populations that constitute the TG and MTN can be identified not only on the basis of their morphological characteristics and electrophysiological properties but also by their neurochemical content. This issue is of key importance since the transmitter content of different neuronal populations often correlates well with their target projections. It is assumed that trigeminal primary afferent neurons exhibit pathway-specific patterns of neurochemical expression, a concept that has been called chemical coding (Costa et al., 1986). It has also been proposed that the differently fated embryonic migration, synaptogenesis, and peripheral and central target field innervation could affect the individual neurochemical phenotype of TG and MTN neurons. Trigeminal primary afferent neurons utilize a wide variety of chemical neuromessengers for synaptic transmission and possess the ability to produce relevant adaptive changes in their neurochemical phenotype in response to environmental cues (for recent reviews, see Lazarov, 2002, 2007).

This chapter therefore focuses on the chemical neuroanatomy of the TG and MTN neuronal populations under normal conditions, the role and relationship of neurotransmitters and their corresponding receptors in relaying orofacial sensations and also refers to the interactions with other atypical neuromessengers and neurotrophic factors. We have also surveyed the chemical plasticity of developing and mature TG and MTN neurons to gain insight into their structural and functional properties in an altered neurochemical balance, with special reference to trigeminal nerve degeneration and regeneration, and clinical implications.

#### **2. Neurotransmitters and their known receptors in trigeminal primary afferent neurons**

Using immunohistochemistry and *in situ* hybridization histochemistry we have identified distinct neuronal, partly chemically coded, subpopulations in the intact TG and MTN. Our findings suggest that trigeminal primary afferent neurons are chemically heterogeneous and appear to use various chemical neuromediator candidates for synaptic transmitters. These include classical and peptide transmitters, calcium-binding proteins as neuronal markers and other neuroactive molecules (Table 1). In addition, we have demonstrated that TG and MTN neurons receive inputs from different groups of neurons that contain multiple transmitter substances. Indeed, both the TG and MTN receive catecholaminergic, nitrergic and peptidergic innervation in the form of perineuronal arborizations encircling in a basketlike manner the perikarya of large unstained neurons. It is assumed that the pericellular baskets can function as a key communication medium between immunopositive projections and immunonegative neuronal somata in the orofacial somatosensory information processing. Last but not least, it is now well established that the cell bodies of TG and MTN neurons are richly endowed with postsynaptic receptors for a huge array of neurotransmitters and neuroactive substances.

#### **2.1 Classical transmitters**

In recent years, a variety of 'classical' transmitter substances and their receptors have been associated with subsets of trigeminal primary afferent neurons. Among them are amino acids (both excitatory and inhibitory) and monoamines. On the other hand, our studies

The Neurochemical Anatomy of Trigeminal Primary Afferent Neurons 171

5-HT3 receptor ND +IC Postsynaptic slow

P2X2 receptor + + Postsynaptic fast

Substance P (SP) + - Neuromodulation SP receptor - - Neuromodulation

Enkephalin (ENK) + - Neuromodulation

Orexin (hypocretin) A - +F/T Neuromodulation

P2X3 receptor - + Presynaptic

H1 receptor + +IC Postsynaptic excitation H3 receptor - +P Presynaptic inhibition

Histamine (HIS) ND +F/T Membrane

trigeminal nucleus Effect of activation

Intracellular

activation

excitation

depolarization

Membrane depolarization Facilitation of neuronal

discharge

excitation

release

Membrane depolarization: promotion of intracellular cGMP

synthesis;

stimulation

Induction of calcium

currents

+ + tonic background

modulation of

neurotransmitter

transduction pathways

Neuroactive substance Trigeminal ganglion Mesencephalic

5-HT1D receptor + ND 5-HT1F receptor + ND

5-HT2 receptor ND +IC

5-HT7 receptor + ND

triphosphate (ATP) ND +F/T

P2X4 receptor + +

P2X5 receptor + + P2X6 receptor + + P2Y receptor - -

Nitric oxide (NO) + +IC

SOM ss2(b) receptor - + Neuropeptide Y (NPY) + - NPY Y1 receptor + - NPY Y2 receptor + - NPY Y5 receptor - +

Preprodynorphin + ND µ-opioid receptor + - -opioid receptor + κ-opioid receptor + -

Orexin receptor-1 ND +

Adenosine 5I

Carbon monoxide (CO) Neuropeptides and their known receptors



AMPA receptors - +IC Postsynaptic rapid

Aspartate + ±IC, F/T Membrane

GABAA receptor + (γ1, γ2 subunits) +IC Postsynaptic fast

Dopamine (DA) - +F/T Membrane

trigeminal nucleus Effect of activation

Membrane depolarization: fast and/or slow excitation

excitation

Presynaptic modulation of transmitter release

Presynaptic auto- or postsynaptic heteroreceptors

Heteroreceptors and/or autoreceptors: neuronal excitability and transmission regulation

depolarization

Membrane hyperpolarization: inhibition Membrane depolarization: excitation

inhibition

Postsynaptic facilitation of ATP, SER and DA release

hyperpolarization

Periodontal ligament proprioceptive processing

Masseter muscle proprioceptive processing

Membrane depolarization: modulation of sodium

currents

Presynaptic regulation of K+, Ca2+channels: long-term inhibition of synaptic transmission

Neuroactive substance Trigeminal ganglion Mesencephalic

Glutamate (GLU) + +IC, F/T

KA receptors + +IC

NMDA receptors - +IC

metabotropic receptors mGluR5 mGluR1α, mGluR2/3

acid (GABA) + ±IC, F/T

GABAB receptor + (GABAB2 subunit) +IC

Glycine - -

D1 receptor - +IC

D2 receptor + +P

Serotonin (SER) - +F/T

5-HT1B receptor + ND

Neurotransmitters and their known receptors

Gamma aminobutyric


The Neurochemical Anatomy of Trigeminal Primary Afferent Neurons 173

clearly show that other classical neurotransmitters, such as acetylcholine, and purines like adenosine 5I-triphosphate (ATP) and its metabolite adenosine are not present in TG and

There are three major amino acid neurotransmitters in the nervous system: glutamic acid (Lglutamate), gamma-amino butyric acid (GABA) and glycine. Glutamate (Glu) is considered a promising excitatory transmitter of the trigeminal primary afferent neurons in rats and cats. It is stored in both the large and small TG cells (Wanaka et al., 1987; Azérad et al., 1992; Stoyanova et al., 1998), and, in addition, all five known kainate (KA) ionotropic receptor subtypes are expressed in a majority of them, occasionally combined with metabotropic mGluR5 subunits (Sahara et al., 1997). Experimental results indicate that the metabotropic Glu receptors play an important role in the somatic sensation of TG neurons together with the ionotropic ones (Araki et al., 1993). Functional contribution of peripherally localized Glu receptors in acute and chronic pain processing is amply documented (Carlton, 2001) and further discussed in Section 5. Similarly, most mammalian MTN neurons contain glutaminase, a major enzyme involved in the biosynthesis of Glu (Kaneko et al., 1989; Turman & Chandler, 1994b) and receive glutamatergic synaptic input (Chandler, 1989; Copray et al., 1990). Further, recent research has revealed that vesicular glutamate transporter 1 is expressed in the cell bodies as well as both in the central axon terminals and peripheral sensory endings of MTN neurons in newborn and adult rats (Pang et al., 2006). Finally, all ionotropic receptor subtypes, AMPA (Mineff et al., 1998; Pelkey & Marshall, 1998; Petralia & Wenthold, 1992; Turman et al., 2000), KA and NMDA (Pelkey & Marshall, 1998; Petralia et al., 1994a,b,c; Turman et al., 2002) and some metabotropic subtypes, mGluR1α, mGluR5 and mGluR2/3 (Turman et al., 2001) have been localized on mesencephalic trigeminal neurons. Iontophoretic studies have also suggested that monosynaptic transmission between jaw-closing primary afferents and jaw-closing motoneurons is mediated primarily by non-NMDA receptors (Chandler, 1989), whereas both NMDA and non-NMDA receptors have been involved in the transmission from premotoneurons to jaw-opening motoneurons (Katakura & Chandler, 1990). The identification of mGluR subunits in mesencephalic trigeminal neurons which receive, as already noted, axosomatic input and/or synthesize mGluRs in the soma and then translocate the proteins to central terminals suggests that these, along with NMDA but not AMPA receptors, may function as either auto- or heteroreceptors in central MTN terminals

GABA and glycine are both known to participate in the control of masticatory rhythms (Chandler et al., 1985). The presence of glycine, however, has been reported neither in TG nor in MTN neurons (Copray et al., 1990; Lazarov, 2002). On the other hand, GABA is localized in a substantial number of TG cells in rats (Szabat et al., 1992) and cats (Stoyanova et al., 1998). In addition, TG cells express two distinct GABA receptors, ionotropic GABAA γ1 and γ2 subunits, mostly co-localized in the same neuron (Kondo et al., 1994), and metabotropic GABAB2 (Durkin et al., 1999). Our experiments have also pointed out that a subpopulation of smaller MTN cells, presumably interneurons, which are apposed to large mesencephalic trigeminal neurons, may be of a GABAergic nature (Lazarov & Chouchkov,

MTN neurons.

**2.1.1 Amino acids** 

(Turman et al., 2001).


MTN

Table 1. Overview of the established and putative neuromessengers, specific markers, neurotrophic factors and their known receptors and major functions in orofacial somatosensory signaling under normal conditions

clearly show that other classical neurotransmitters, such as acetylcholine, and purines like adenosine 5I-triphosphate (ATP) and its metabolite adenosine are not present in TG and MTN neurons.

#### **2.1.1 Amino acids**

172 Neuroscience – Dealing with Frontiers

Orexin (hypocretin) B - +F/T Neuromodulation

Parvalbumin (PV) + +IC Intracellular Ca

Calbindin D-28 (CB) + +IC Selective neuronal

Osteocalcin + +IC Selective marker Osteopontin + +IC Selective marker Peptide 19 + +IC Selective marker

receptor P75NTR + +IC Increase in mature

(NGF) + +IC,F Trophic support

TrkB + +IC Neuronal survival

TrkC + +IC Neuronal survival

IC, intracellular; F/T, fibers and/or terminals; ND, no data; NA, not analyzed; P, pontine portion of

Table 1. Overview of the established and putative neuromessengers, specific markers, neurotrophic factors and their known receptors and major functions in orofacial

+ +IC

TrkA + +IC NA

4/5) + - NA

GFRalpha-1 receptor + +IC NA Ret receptor + +IC NA

somatosensory signaling under normal conditions

Neurotrophin 3 (NT-3) + +IC Trophic support

S-100 + - Selective glial marker

Calretinin (CR) + +IC NA

trigeminal nucleus Effect of activation

Induction of calcium

Selective marker for

neuronal excitability

Trophic support Neuronal phenotype maintenance

Modulation of neuronal electric

Trophic support

activity

currents

buffering

orofacial proprioceptors

marker

Neuroactive substance Trigeminal ganglion Mesencephalic

Orexin receptor-2 ND +

Neurocalcin + -

Calcium-binding proteins

Nerotrophic factors and their receptors Pan-neurotrophin

Nerve growth factor

Brain derived neurotrophin factor (BDNF)

Neurotrophin 4-5 (NT-

Glial cell line-derived neurotrophic factor (GDNF)

MTN

There are three major amino acid neurotransmitters in the nervous system: glutamic acid (Lglutamate), gamma-amino butyric acid (GABA) and glycine. Glutamate (Glu) is considered a promising excitatory transmitter of the trigeminal primary afferent neurons in rats and cats. It is stored in both the large and small TG cells (Wanaka et al., 1987; Azérad et al., 1992; Stoyanova et al., 1998), and, in addition, all five known kainate (KA) ionotropic receptor subtypes are expressed in a majority of them, occasionally combined with metabotropic mGluR5 subunits (Sahara et al., 1997). Experimental results indicate that the metabotropic Glu receptors play an important role in the somatic sensation of TG neurons together with the ionotropic ones (Araki et al., 1993). Functional contribution of peripherally localized Glu receptors in acute and chronic pain processing is amply documented (Carlton, 2001) and further discussed in Section 5. Similarly, most mammalian MTN neurons contain glutaminase, a major enzyme involved in the biosynthesis of Glu (Kaneko et al., 1989; Turman & Chandler, 1994b) and receive glutamatergic synaptic input (Chandler, 1989; Copray et al., 1990). Further, recent research has revealed that vesicular glutamate transporter 1 is expressed in the cell bodies as well as both in the central axon terminals and peripheral sensory endings of MTN neurons in newborn and adult rats (Pang et al., 2006). Finally, all ionotropic receptor subtypes, AMPA (Mineff et al., 1998; Pelkey & Marshall, 1998; Petralia & Wenthold, 1992; Turman et al., 2000), KA and NMDA (Pelkey & Marshall, 1998; Petralia et al., 1994a,b,c; Turman et al., 2002) and some metabotropic subtypes, mGluR1α, mGluR5 and mGluR2/3 (Turman et al., 2001) have been localized on mesencephalic trigeminal neurons. Iontophoretic studies have also suggested that monosynaptic transmission between jaw-closing primary afferents and jaw-closing motoneurons is mediated primarily by non-NMDA receptors (Chandler, 1989), whereas both NMDA and non-NMDA receptors have been involved in the transmission from premotoneurons to jaw-opening motoneurons (Katakura & Chandler, 1990). The identification of mGluR subunits in mesencephalic trigeminal neurons which receive, as already noted, axosomatic input and/or synthesize mGluRs in the soma and then translocate the proteins to central terminals suggests that these, along with NMDA but not AMPA receptors, may function as either auto- or heteroreceptors in central MTN terminals (Turman et al., 2001).

GABA and glycine are both known to participate in the control of masticatory rhythms (Chandler et al., 1985). The presence of glycine, however, has been reported neither in TG nor in MTN neurons (Copray et al., 1990; Lazarov, 2002). On the other hand, GABA is localized in a substantial number of TG cells in rats (Szabat et al., 1992) and cats (Stoyanova et al., 1998). In addition, TG cells express two distinct GABA receptors, ionotropic GABAA γ1 and γ2 subunits, mostly co-localized in the same neuron (Kondo et al., 1994), and metabotropic GABAB2 (Durkin et al., 1999). Our experiments have also pointed out that a subpopulation of smaller MTN cells, presumably interneurons, which are apposed to large mesencephalic trigeminal neurons, may be of a GABAergic nature (Lazarov & Chouchkov,

The Neurochemical Anatomy of Trigeminal Primary Afferent Neurons 175

and NAergic baskets originating from the A9 and A10 cell groups, the substantia nigra and ventral tegmental area, and the neighboring locus coeruleus, respectively. Our immunohistochemical and *in situ* hybridization experiments have further shown that MTN neurons express the two principal subtypes of DA receptors, though they are unequally distributed within the nucleus: as in muscle spindle afferents, D1 receptors are found throughout the MTN of the rat, whereas D2 receptors and periodontal afferent neurons are confined to the caudal part of the nucleus (Lazarov & Pilgrim, 1997). This suggests that the two types of primary afferents may be modulated differentially by DA. The DA input to the MTN may modulate neuronal excitability, rates of transmitter synthesis, transport and

release, as well as the number of pre- and postsynaptic receptors (Liem et al., 1997).

(Tanaka & Chandler, 2006).

In addition to the catecholaminergic input, the TG and MTN are under the influence of another monoaminergic system, namely the serotoninergic system, one of the oldest amine systems in the brain. Indeed, several immunohistochemical studies have revealed the serotoninergic supply of the mammalian TG and MTN. In fact, we have found that the TG lacks intrinsic serotonin (SER)-containing cells but a plexus of varicose nerve fibers of extraganglionic origin covers the immunonegative neurons in a basket-like manner (Chouchkov et al., 1988). Likewise, SER is present neither in the cell bodies nor in neuronal processes of MTN neurons (Lazarov & Chouchkov, 1995a). However, SERergic axonal varicosities reaching the MTN from the mesopontine and medullary raphe nuclei form a pericellular basket-like network around immunonegative mesencephalic trigeminal neurons (Tashiro et al., 1989). Electron microscopic studies show direct synaptic contacts between SER-containing terminals and MTN perikarya in rats (Copray et al., 1991; Liem et al., 1993; Liem & Copray, 1996; Li et al., 2000), cats (Lazarov & Chouchkov, 1995a) and rabbits (Kolta et al., 1993). Out of the large group of the serotonin receptors, also known as 5 hydroxytryptamine receptors or 5-HT receptors, the presence of high affinity 5-HT1B and 5- HT1D receptors is demonstrated at protein and mRNA levels in TG neurons of the rat (Bruinvels et al., 1992; Wotherspoon & Priestley, 2000), guinea pig (Bonaventure et al., 1998) and human (Longmore et al., 1997). The mRNA encoding the 5-HT7 receptor is also found to be expressed in the human TG (Terrón et al., 2001). At the same time, in the rat MTN an abundant number of the 5-HT2 (Cornea-Hébert et al., 1999), 5-HT3 (Morales et al., 1998) and 5-HT4 receptor (Lazarov, 2007) have been established. SER has been hypothesized to be involved in trigeminal pain (Moskowitz et al., 1979) and SER antagonists have important clinical implications for antimigraine drug development (discussed later in Section 5). Recent data indicate that SER plays a significant role in the control of oral-motor activity as well (Li et al., 2000) and various oral-motor disorders, either drug induced or occurring as a consequence of injury, might result from altered modulation of sodium channels by SER

As in the case of catecholamines, both peripheral and central trigeminal primary afferent neurons do not contain histamine in their cell bodies but MTN neuronal perikarya receive a direct histaminergic input by hypothalamic descending fibers (Inagaki et al., 1987). In particular, neuronal somata throughout the whole rostrocaudal length of the nucleus are encircled by histaminergic fibers and their terminals, many of the latter forming axo-somatic synapses on them. Our laboratory has provided immunohistochemical evidence for the presence of two distinct histamine receptor subtypes, H1 and H3, in the rat MTN, albeit in

1995b; Lazarov, 2000, 2002). Besides, molecular biological studies have shown that pseudounipolar MTN neurons respond to GABA and accordingly express GABAA receptor α2, β2 and γ2 subunit mRNAs (Hayar et al., 1997; Ishii & Kang, 2002), and GABAB1 (Margeta-Mitrovic et al., 1999) and GABAB2 (Li et al., 2001) receptor proteins. In line with this evidence, several research groups have consequently reported GABAergic innervation of the MTN in rats (Copray et al., 1990; Ginestal & Matute, 1993), guinea pigs (Turman & Chandler, 1994a), rabbits (Kolta et al., 1991a,b) and cats (Lazarov & Chouchkov, 1995b; Lazarov, 2000, 2002). Ultrastructural and confocal laser-scanning studies have additionally revealed the existence of GABAergic synapses upon the cell bodies of MTN neurons in the rat (Chen et al., 2001). Taken together, these results suggest that excitability of jaw muscle spindle afferents is presynaptically controlled by interneurons containing GABA and these play an important role in modulating the jaw-jerk reflex.

Our findings now permit definitive conclusions that both TG and MTN neurons contain a stable concentration of Glu and GABA as possible transmitters. It still remains to clarify the possible synaptic relationships between Glu- and GABA-immunoreactive profiles in the TG and MTN, and their related functional implications. We have observed that both amino acid neurotransmitters are present in separate subpopulations of trigeminal neurons, e.g. most large neurons are glutamatergic while certain small neurons are GABAergic (Lazarov, 2002). Thus, it seems likely that excitatory amino acid(s) may be the transmitter(s) of large myelinated non-nociceptive primary afferents whereas GABA is probably the mediator of smaller trigeminal neurons, as suggested by Salt & Hill (1983).

#### **2.1.2 Monoamines**

Out of the six different types of monoamines, catecholamines [dopamine (DA), noradrenaline (NA) and adrenaline (A)] are the most important group. The earliest evidence for the catecholaminergic innervation of the TG sprang from the works of Santini (1966) and Lukás et al. (1970). In these initial studies by using immunocytochemistry with antiserum against tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine synthesis, the immunostained neurons within the ganglion were proposed to be DAergic. Applying an antibody against the DA molecule itself, it is now inferred that TH-containing neurons in the TG do not synthesize DA but they are enveloped by DAergic pericellular arborizations (Kummer et al., 1990). A similar pattern has been observed for NA within the ganglion: TG cells are immunonegative to the NA-synthesizing enzyme, dopamine-β-hydroxylase (Katz et al., 1983) but receive dense NAergic innervation from postganglionic sympathetic neurons (Kummer et al., 1990). Hence, immunohistochemical evidence has suggested that TG neurons do not utilize catecholamines as possible transmitters but are under the influence of catecholaminergic afferent fibers of presumable sympathetic origin. Clinical observations imply that primary afferent neurons whose cell bodies reside in the TG express receptors for DA of the D2 subtype, although these receptors do not function as autoreceptors but rather have a role in pain syndromes involving the head and the neck (Peterfreund et al., 1995).

In the same way, none of the MTN neurons in rats (Copray et al., 1990; Liem et al., 1997), cats (Lazarov & Chouchkov, 1995b) and humans (Usunoff et al., 1997) exhibits immunoreactivity for DA or NA but their perikarya are closely surrounded by fine DAergic

1995b; Lazarov, 2000, 2002). Besides, molecular biological studies have shown that pseudounipolar MTN neurons respond to GABA and accordingly express GABAA receptor α2, β2 and γ2 subunit mRNAs (Hayar et al., 1997; Ishii & Kang, 2002), and GABAB1 (Margeta-Mitrovic et al., 1999) and GABAB2 (Li et al., 2001) receptor proteins. In line with this evidence, several research groups have consequently reported GABAergic innervation of the MTN in rats (Copray et al., 1990; Ginestal & Matute, 1993), guinea pigs (Turman & Chandler, 1994a), rabbits (Kolta et al., 1991a,b) and cats (Lazarov & Chouchkov, 1995b; Lazarov, 2000, 2002). Ultrastructural and confocal laser-scanning studies have additionally revealed the existence of GABAergic synapses upon the cell bodies of MTN neurons in the rat (Chen et al., 2001). Taken together, these results suggest that excitability of jaw muscle spindle afferents is presynaptically controlled by interneurons containing GABA and these

Our findings now permit definitive conclusions that both TG and MTN neurons contain a stable concentration of Glu and GABA as possible transmitters. It still remains to clarify the possible synaptic relationships between Glu- and GABA-immunoreactive profiles in the TG and MTN, and their related functional implications. We have observed that both amino acid neurotransmitters are present in separate subpopulations of trigeminal neurons, e.g. most large neurons are glutamatergic while certain small neurons are GABAergic (Lazarov, 2002). Thus, it seems likely that excitatory amino acid(s) may be the transmitter(s) of large myelinated non-nociceptive primary afferents whereas GABA is probably the mediator of

Out of the six different types of monoamines, catecholamines [dopamine (DA), noradrenaline (NA) and adrenaline (A)] are the most important group. The earliest evidence for the catecholaminergic innervation of the TG sprang from the works of Santini (1966) and Lukás et al. (1970). In these initial studies by using immunocytochemistry with antiserum against tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine synthesis, the immunostained neurons within the ganglion were proposed to be DAergic. Applying an antibody against the DA molecule itself, it is now inferred that TH-containing neurons in the TG do not synthesize DA but they are enveloped by DAergic pericellular arborizations (Kummer et al., 1990). A similar pattern has been observed for NA within the ganglion: TG cells are immunonegative to the NA-synthesizing enzyme, dopamine-β-hydroxylase (Katz et al., 1983) but receive dense NAergic innervation from postganglionic sympathetic neurons (Kummer et al., 1990). Hence, immunohistochemical evidence has suggested that TG neurons do not utilize catecholamines as possible transmitters but are under the influence of catecholaminergic afferent fibers of presumable sympathetic origin. Clinical observations imply that primary afferent neurons whose cell bodies reside in the TG express receptors for DA of the D2 subtype, although these receptors do not function as autoreceptors but rather have a role in pain syndromes involving the head and the neck

In the same way, none of the MTN neurons in rats (Copray et al., 1990; Liem et al., 1997), cats (Lazarov & Chouchkov, 1995b) and humans (Usunoff et al., 1997) exhibits immunoreactivity for DA or NA but their perikarya are closely surrounded by fine DAergic

play an important role in modulating the jaw-jerk reflex.

smaller trigeminal neurons, as suggested by Salt & Hill (1983).

**2.1.2 Monoamines** 

(Peterfreund et al., 1995).

and NAergic baskets originating from the A9 and A10 cell groups, the substantia nigra and ventral tegmental area, and the neighboring locus coeruleus, respectively. Our immunohistochemical and *in situ* hybridization experiments have further shown that MTN neurons express the two principal subtypes of DA receptors, though they are unequally distributed within the nucleus: as in muscle spindle afferents, D1 receptors are found throughout the MTN of the rat, whereas D2 receptors and periodontal afferent neurons are confined to the caudal part of the nucleus (Lazarov & Pilgrim, 1997). This suggests that the two types of primary afferents may be modulated differentially by DA. The DA input to the MTN may modulate neuronal excitability, rates of transmitter synthesis, transport and release, as well as the number of pre- and postsynaptic receptors (Liem et al., 1997).

In addition to the catecholaminergic input, the TG and MTN are under the influence of another monoaminergic system, namely the serotoninergic system, one of the oldest amine systems in the brain. Indeed, several immunohistochemical studies have revealed the serotoninergic supply of the mammalian TG and MTN. In fact, we have found that the TG lacks intrinsic serotonin (SER)-containing cells but a plexus of varicose nerve fibers of extraganglionic origin covers the immunonegative neurons in a basket-like manner (Chouchkov et al., 1988). Likewise, SER is present neither in the cell bodies nor in neuronal processes of MTN neurons (Lazarov & Chouchkov, 1995a). However, SERergic axonal varicosities reaching the MTN from the mesopontine and medullary raphe nuclei form a pericellular basket-like network around immunonegative mesencephalic trigeminal neurons (Tashiro et al., 1989). Electron microscopic studies show direct synaptic contacts between SER-containing terminals and MTN perikarya in rats (Copray et al., 1991; Liem et al., 1993; Liem & Copray, 1996; Li et al., 2000), cats (Lazarov & Chouchkov, 1995a) and rabbits (Kolta et al., 1993). Out of the large group of the serotonin receptors, also known as 5 hydroxytryptamine receptors or 5-HT receptors, the presence of high affinity 5-HT1B and 5- HT1D receptors is demonstrated at protein and mRNA levels in TG neurons of the rat (Bruinvels et al., 1992; Wotherspoon & Priestley, 2000), guinea pig (Bonaventure et al., 1998) and human (Longmore et al., 1997). The mRNA encoding the 5-HT7 receptor is also found to be expressed in the human TG (Terrón et al., 2001). At the same time, in the rat MTN an abundant number of the 5-HT2 (Cornea-Hébert et al., 1999), 5-HT3 (Morales et al., 1998) and 5-HT4 receptor (Lazarov, 2007) have been established. SER has been hypothesized to be involved in trigeminal pain (Moskowitz et al., 1979) and SER antagonists have important clinical implications for antimigraine drug development (discussed later in Section 5). Recent data indicate that SER plays a significant role in the control of oral-motor activity as well (Li et al., 2000) and various oral-motor disorders, either drug induced or occurring as a consequence of injury, might result from altered modulation of sodium channels by SER (Tanaka & Chandler, 2006).

As in the case of catecholamines, both peripheral and central trigeminal primary afferent neurons do not contain histamine in their cell bodies but MTN neuronal perikarya receive a direct histaminergic input by hypothalamic descending fibers (Inagaki et al., 1987). In particular, neuronal somata throughout the whole rostrocaudal length of the nucleus are encircled by histaminergic fibers and their terminals, many of the latter forming axo-somatic synapses on them. Our laboratory has provided immunohistochemical evidence for the presence of two distinct histamine receptor subtypes, H1 and H3, in the rat MTN, albeit in

The Neurochemical Anatomy of Trigeminal Primary Afferent Neurons 177

the C-fiber range, PEP 19 and NPY are usually expressed in the large ones, and opioid peptides, CCK, VIP and pituitary adenylate cyclase activating polypeptide (PACAP) are observed in both small and large trigeminal neurons (Kummer & Heym, 1986; Weihe, 1990). Our previous studies have also revealed that although devoid of synaptic contacts, TG neurons express an array of peptide receptors for SP, CGRP, CCK, opioid peptides and NPY (see Lazarov, 2002, and references therein). The occurrence of peptidergic arborizations of extrinsic origin around the perikarya of some TG neurons suggests that these are under the

Several lines of physiological evidence indicate that SP and CGRP have excitatory effects and depolarize TG neurons (Otsuka & Konishi, 1976; Spigelman & Puil, 1991) while SOM and opioids appear to be inhibitory in nature (Randic & Miletic, 1978). Considering the important functional segregation of TG cells into large (mostly mechanoreceptive) and small (mainly nociceptive) neurons, it is not surprising that the neuropeptides SP and CGRP have been associated with the transmission of nociceptive impulses. However, small-diameter primary afferent neurons not only transmit noxious messages to central neurons but are also active in the periphery in mediating axon-reflex mechanisms and an inflammation response (Couture & Cuello, 1984; Foreman, 1987). Therefore, it is more reasonable to consider the role of SP and CGRP both in the transmission of sensory information from the periphery and in the peripheral effector functions such as neurogenic vasodilatation (McCarthy & Lawson, 1989, 1990), thus implicating them into the pathophysiology of migraine. Moreover, SOM can interact with SP causing inhibition of its release and consequent neurogenic vasodilatation (Brodin et al., 1981). Several lines of evidence indicate that sensory opioids could act synergistically with SP to induce histamine release (Foreman, 1987) and GAL may have an inhibitory effect on the nociceptive transmission (see Xu et al., 1990, and references therein). Therefore, all these peptides play a co-transmitter role and may have significant functions in disease mechanisms associated with head pain in humans (for a recent review,

In view of the presence of a large number of neuropeptides in the TG cells, their absence in the morphologically homologous MTN neurons is rather surprising. Obviously, the absence of a peptide in two distinct populations of trigeminal primary afferent neurons indicates that different peptides subserve diverse sensory modalities, at least under normal conditions. In extension of previous inferences, we are confident that there exists a complex coexistent relationship between the chemoanatomical constellation of trigeminal primary afferent neurons and sensory modality transmission (Lazarov, 2002). Notwithstanding that intact mesencephalic trigeminal neurons do not express neuropeptides in their perikarya they are largely influenced by various synaptic inputs. Relevant to the dense peptidergic innervation of the nucleus, receptors for certain peptides have been localized on MTN neuronal somata (reviewed in Lazarov, 2002, 2007). According to Copray et al. (1990) it is likely that the synaptic input on MTN cells only affects the neuronal activity expressed at the central and peripheral terminals, in a more indirect mode and after a longer interval. The authors claim that this points to the presence and involvement of receptor systems that are not directly linked to ion channels but to a much slower secondary-messenger-induced biochemical effector cascade. As argued in the Introduction section, MTN neurons may operate under some circumstances like traditional "integrate and fire" neurons in addition

influence of multiple biologically active peptides.

see Edvinsson & Uddman, 2005).

different neuronal subpopulations (Lazarov & Gratzl, 2006). Overlapping with muscle spindle afferents, histamine H1 receptors are scattered throughout the full extent of the MTN, whereas H3 receptors and periodontal afferent neurons are restricted to its caudal region. Since H1 receptors are excitatory, histamine may act in this way on MTN neurons via the axo-somatic synapses, as suggested by Inagaki et al. (1987). Conversely, the H3 receptors do not function only as autoreceptors but also as heteroreceptors, modulating MTN neuronal activity and release of other neurotransmitters from them. Therefore, it seems that the majority of MTN neurons respond to central histamine via the activation of H1 and inhibition of H3 receptors, thus participating in the control of feeding behavior.

The purine nucleosides adenosine and ATP can function as neurotransmitters or neuromodulators in both the CNS and PNS by signaling through specific receptors termed adenosine (also known as P1) and P2 receptors, respectively. Our attempts fail to identify purines in the TG and MTN neurons, though the latter are innervated by adenosine deaminase-containing projections from the hypothalamus (Nagy et al., 1986). Inasmuch as their terminals contacting MTN perikarya also express immunoreactivity to histamine, it seems that the two substances may coexist there, as noted by Yamamoto et al. (1988). In the TG, an abundant expression of the adenosine A1 receptor protein (Schindler et al., 2001) and the six P2X receptor subtypes (Xiang et al., 1998; Dunn et al., 2001) has been shown in a large number of small nociceptive neurons, which may be suggestive of a role of these receptors in analgesia. The existence of excitatory adenosine A2A receptors (Rosin et al., 1998) and P2X2, P2X3, P2X4, P2X5 and P2X6 purinoceptors (Khakh et al., 1997; Patel et al., 2001; Lazarov, 2007) has been shown in populations of large proprioceptive mesencephalic trigeminal neurons. P2X receptors, which are ATPgated cation channels, have been shown to be responsible for mediating both fast excitatory responses in central and peripheral neurons and the presynaptic modulation of neurotransmitter release (reviewed by Ralevic & Burnstock, 1998). It is likely that certain ionotropic P2X purinoceptors may be involved in the processing of proprioceptive information, thus suggesting a potentially important physiological role of ATP at sites where it is released extracellularly (Khakh et al., 1997).

#### **2.2 Neuropeptides**

Neuropeptides are a heterogeneous group of several hundred biologically active peptides, present in neurons of both the mammalian CNS and PNS, and involved in the transmission of signals as pure neuromediators or neuromodulators. In general, a large number of putative peptide transmitters have been identified in neurons and/or neuronal processes in the TG, but none of them has been found in mammalian MTN neuronal somata under normal conditions (see reviews by Lazarov, 1994, 2002, 2007). In particular, two subpopulations of primary afferent neurons, containing neuroactive peptides are distinguished in the TG: a number of substance P (SP)-, neurokinin A (NKA)-, calcitonin gene-related peptide (CGRP)-, cholecystokinin (CCK)-, somatostatin (SOM)-, vasoactive intestinal polypeptide (VIP)- and galanin (GAL)-immunoreactive ganglion cells with smalland medium-sized somata, and relatively fewer in number larger-sized neuropeptide Y (NPY)- and peptide 19 (PEP 19)-immunoreactive trigeminal neurons. It is noteworthy that SP, CGRP, SOM and GAL are found in small-diameter TG cells with conduction velocities in

different neuronal subpopulations (Lazarov & Gratzl, 2006). Overlapping with muscle spindle afferents, histamine H1 receptors are scattered throughout the full extent of the MTN, whereas H3 receptors and periodontal afferent neurons are restricted to its caudal region. Since H1 receptors are excitatory, histamine may act in this way on MTN neurons via the axo-somatic synapses, as suggested by Inagaki et al. (1987). Conversely, the H3 receptors do not function only as autoreceptors but also as heteroreceptors, modulating MTN neuronal activity and release of other neurotransmitters from them. Therefore, it seems that the majority of MTN neurons respond to central histamine via the activation of H1 and

The purine nucleosides adenosine and ATP can function as neurotransmitters or neuromodulators in both the CNS and PNS by signaling through specific receptors termed adenosine (also known as P1) and P2 receptors, respectively. Our attempts fail to identify purines in the TG and MTN neurons, though the latter are innervated by adenosine deaminase-containing projections from the hypothalamus (Nagy et al., 1986). Inasmuch as their terminals contacting MTN perikarya also express immunoreactivity to histamine, it seems that the two substances may coexist there, as noted by Yamamoto et al. (1988). In the TG, an abundant expression of the adenosine A1 receptor protein (Schindler et al., 2001) and the six P2X receptor subtypes (Xiang et al., 1998; Dunn et al., 2001) has been shown in a large number of small nociceptive neurons, which may be suggestive of a role of these receptors in analgesia. The existence of excitatory adenosine A2A receptors (Rosin et al., 1998) and P2X2, P2X3, P2X4, P2X5 and P2X6 purinoceptors (Khakh et al., 1997; Patel et al., 2001; Lazarov, 2007) has been shown in populations of large proprioceptive mesencephalic trigeminal neurons. P2X receptors, which are ATPgated cation channels, have been shown to be responsible for mediating both fast excitatory responses in central and peripheral neurons and the presynaptic modulation of neurotransmitter release (reviewed by Ralevic & Burnstock, 1998). It is likely that certain ionotropic P2X purinoceptors may be involved in the processing of proprioceptive information, thus suggesting a potentially important physiological role of ATP at sites

Neuropeptides are a heterogeneous group of several hundred biologically active peptides, present in neurons of both the mammalian CNS and PNS, and involved in the transmission of signals as pure neuromediators or neuromodulators. In general, a large number of putative peptide transmitters have been identified in neurons and/or neuronal processes in the TG, but none of them has been found in mammalian MTN neuronal somata under normal conditions (see reviews by Lazarov, 1994, 2002, 2007). In particular, two subpopulations of primary afferent neurons, containing neuroactive peptides are distinguished in the TG: a number of substance P (SP)-, neurokinin A (NKA)-, calcitonin gene-related peptide (CGRP)-, cholecystokinin (CCK)-, somatostatin (SOM)-, vasoactive intestinal polypeptide (VIP)- and galanin (GAL)-immunoreactive ganglion cells with smalland medium-sized somata, and relatively fewer in number larger-sized neuropeptide Y (NPY)- and peptide 19 (PEP 19)-immunoreactive trigeminal neurons. It is noteworthy that SP, CGRP, SOM and GAL are found in small-diameter TG cells with conduction velocities in

inhibition of H3 receptors, thus participating in the control of feeding behavior.

where it is released extracellularly (Khakh et al., 1997).

**2.2 Neuropeptides**

the C-fiber range, PEP 19 and NPY are usually expressed in the large ones, and opioid peptides, CCK, VIP and pituitary adenylate cyclase activating polypeptide (PACAP) are observed in both small and large trigeminal neurons (Kummer & Heym, 1986; Weihe, 1990). Our previous studies have also revealed that although devoid of synaptic contacts, TG neurons express an array of peptide receptors for SP, CGRP, CCK, opioid peptides and NPY (see Lazarov, 2002, and references therein). The occurrence of peptidergic arborizations of extrinsic origin around the perikarya of some TG neurons suggests that these are under the influence of multiple biologically active peptides.

Several lines of physiological evidence indicate that SP and CGRP have excitatory effects and depolarize TG neurons (Otsuka & Konishi, 1976; Spigelman & Puil, 1991) while SOM and opioids appear to be inhibitory in nature (Randic & Miletic, 1978). Considering the important functional segregation of TG cells into large (mostly mechanoreceptive) and small (mainly nociceptive) neurons, it is not surprising that the neuropeptides SP and CGRP have been associated with the transmission of nociceptive impulses. However, small-diameter primary afferent neurons not only transmit noxious messages to central neurons but are also active in the periphery in mediating axon-reflex mechanisms and an inflammation response (Couture & Cuello, 1984; Foreman, 1987). Therefore, it is more reasonable to consider the role of SP and CGRP both in the transmission of sensory information from the periphery and in the peripheral effector functions such as neurogenic vasodilatation (McCarthy & Lawson, 1989, 1990), thus implicating them into the pathophysiology of migraine. Moreover, SOM can interact with SP causing inhibition of its release and consequent neurogenic vasodilatation (Brodin et al., 1981). Several lines of evidence indicate that sensory opioids could act synergistically with SP to induce histamine release (Foreman, 1987) and GAL may have an inhibitory effect on the nociceptive transmission (see Xu et al., 1990, and references therein). Therefore, all these peptides play a co-transmitter role and may have significant functions in disease mechanisms associated with head pain in humans (for a recent review, see Edvinsson & Uddman, 2005).

In view of the presence of a large number of neuropeptides in the TG cells, their absence in the morphologically homologous MTN neurons is rather surprising. Obviously, the absence of a peptide in two distinct populations of trigeminal primary afferent neurons indicates that different peptides subserve diverse sensory modalities, at least under normal conditions. In extension of previous inferences, we are confident that there exists a complex coexistent relationship between the chemoanatomical constellation of trigeminal primary afferent neurons and sensory modality transmission (Lazarov, 2002). Notwithstanding that intact mesencephalic trigeminal neurons do not express neuropeptides in their perikarya they are largely influenced by various synaptic inputs. Relevant to the dense peptidergic innervation of the nucleus, receptors for certain peptides have been localized on MTN neuronal somata (reviewed in Lazarov, 2002, 2007). According to Copray et al. (1990) it is likely that the synaptic input on MTN cells only affects the neuronal activity expressed at the central and peripheral terminals, in a more indirect mode and after a longer interval. The authors claim that this points to the presence and involvement of receptor systems that are not directly linked to ion channels but to a much slower secondary-messenger-induced biochemical effector cascade. As argued in the Introduction section, MTN neurons may operate under some circumstances like traditional "integrate and fire" neurons in addition

The Neurochemical Anatomy of Trigeminal Primary Afferent Neurons 179

MTN neurons (Ichikawa et al., 1999; 2000). Data from ongoing experiments have provided compelling evidence that CaBPs might function as intracellular calcium transporters or as a buffering system for cell protection during neuronal activity in normal circumstances. They may also affect calcium-dependent neuronal properties such as excitability, release of neurotransmitters and resistance to excitotoxicity in mammals (see Baimbridge et al., 1992). Additionally to intracellular roles, S-100 may be involved in the neurotrophic functions of

Neurotrophic factors or neurotrophins are a family of structurally and functionally related polypeptides that promote neuronal differentiation, survival and neurochemical plasticity during development by signaling via both a low-affinity p75 receptor and high-affinity transmembrane receptors belonging to the Trk proto-oncogene family. It has been proposed that neurotrophins and their receptors play an essential role in long-term neural trophics and trophic mechanisms during adult life, and may also regulate phenotypic expression. Recent advances in neuroscience have shown that in addition to their trophic support actions, neurotrophins share many functional properties with classical neurotransmitters as

In fact, the concepts of neurotrophin-dependent survival, neurotrophin switching and neurotrophin co-operativity have largely arisen from works on the trigeminal system (Davies, 1997). Indeed, it is now well documented that developing trigeminal afferent neurons respond to all four known neurotropic factors, albeit in different capacities. Specifically, the localization of the nerve growth factor (NGF), brain-derived neurotrophin factor (BDNF), neurotrophin-3 (NT-3) and NT-4/5 has been demonstrated in discrete neuronal subsets of the human TG at an age ranging from 23 weeks of gestation to adulthood (Quartu et al., 1997). In early development, embryonic TG neurons depend for their survival on the action of the BDNF, NT-3 or NT-4/5 but not on NGF (Buchmann & Davies, 1993). Accordingly, within the developing TG neurotrophin receptor expression is high around the time of target innervation (Ernfors et al., 1992) and, moreover, two or more Trk receptor isoforms are co-expressed in embryonic rat TG neurons (Moshnyakov et al., 1996). Similarly, MTN neurons display modality specific neurotrophin dependence in their development (Davies, 1997). For instance, during the earliest stages of neurogenesis the developing MTN neurons are transiently supported by BDNF and NT-3, but not by NGF (Copray & Liem, 1993; Davies et al., 1987). Recent work on the human MTN also suggests an active role for the glial cell line-derived neurotrophic factor and possibly other cognate ligands in the trophism from prenatal life to adulthood of the cells subserving the proprioceptive sensory transmission (Quartu et al., 2006). Studies on the neurotrophin receptor expression indicate the presence of p75 (Henry et al., 1993) and all the Trk receptor types within the adult (Yamuy et al., 2000) and developing MTN (Williams et al., 1995). Besides, most MTN neurons express multiple Trk isoforms (Jacobs & Miller, 1999). These results conclusively support the notion that MTN neurons are sensitive to the direct effects of more than one neurotrophin. Recent studies provide some of the initial evidence that the neurotrophin requirements of trigeminal primary afferent neurons are related to a specific sensory modality. For example, the high-affinity NGF receptor, TrkA, is considered a

trigeminal primary afferent neurons.

**3. Neurotrophic factors and neurotrophin receptors**

well and may function as neuromodulators in neuronal signaling.

to typically sensory neurons, which generally do not receive synaptic contacts at their somata and do not discharge repetitively (Del Negro & Chandler, 1997).

#### **2.3 Gaseous neuromessengers**

In addition to the classical and peptide transmitters, several second messenger systems may be involved in orofacial signal processing. During the last decade the free radical gases nitric oxide (NO) and carbon monoxide (CO) have been found to function as putative messenger molecules both in central and peripheral trigeminal primary afferent neurons. Indeed, recent research in animals and humans has shown that the neurons in the MTN, along with TG cells, contain heme oxygenase, the CO-synthesizing enzyme (Uddman et al., 2004; Fan et al., 2008). The enzyme responsible for the synthesis of NO, nitric oxide synthase (NOS), and its histochemical marker, NADPH-diaphorase are expressed both in TG and MTN neurons in rats (Stoyanova & Lazarov, 2005), rabbits (Kolesar et al., 2006) and cats (Lazarov & Dandov, 1998) as well. The nerve cell bodies containing NOS are predominantly of small to medium size and they also express SP and CGRP (Edvinsson et al., 1998). Moreover, all these studies demonstrate that large unstained trigeminal neurons are innervated by nitrergic fibers and that NO increases the excitability and modifies other electrophysiological properties of these cells. Rather than acting via traditional receptors on the postsynaptic membrane, NO exerts its effect by diffusion into the adjacent neurons to activate soluble guanylyl cyclase, leading to an increase in intracellular cGMP. Furthermore, NO possibly produces an increase in MTN neuronal electrotonic coupling and therefore is involved in the synchronization of their activity too. Finally, there is functional evidence to support the involvement of NO in the development and maintenance of inflammation and pain (Yun et al., 1996). It has been suggested that as in other regions, both gaseous messengers possibly interact in a complex, dynamic way in the orofacial sensory processing where CO, being a more stable gas, may be responsible for basal activity and provide tonic background stimulation, whereas surges of NO transiently amplify or deliver phasic signaling (Fan et al., 2008).

#### **2.4 Calcium-binding proteins**

The calcium-binding proteins (CaBPs) represent one of the physiological systems for maintaining calcium ion intracellular homeostasis (reviewed in Baimbridge et al., 1992). In the last two decades they have received increased attention due to their implementation as specific markers for large-sized primary afferent subpopulations and their involvement in many calcium-dependent phenomena in the nervous system both under normal and abnormal conditions (Anderssen et al., 1993). Neuron-specific CaBPs, parvalbumin (PV), calbindin D-28k (CB) and calretinin (CR), are observed to be expressed predominantly in the large-sized TG and MTN neurons (Ichikawa et al., 1994; Lazarov et al., 1998), a subpopulation with inward calcium current. Interestingly, a typical glial cell-specific protein, S-100, is also localized, mostly co-expressed with CB, in TG neurons of epibranchial placode origin (Ichikawa et al., 1997). In addition, immunoreactivity to neurocalcin, a newly identified member of the neuronal CaBP family, has been shown in large or medium in size TG cells (Iino et al., 1998). Two additional newly discovered bone matrix CaBPs, osteocalcin and osteopontin, have recently been co-localized with PV in the cell bodies of both TG and

to typically sensory neurons, which generally do not receive synaptic contacts at their

In addition to the classical and peptide transmitters, several second messenger systems may be involved in orofacial signal processing. During the last decade the free radical gases nitric oxide (NO) and carbon monoxide (CO) have been found to function as putative messenger molecules both in central and peripheral trigeminal primary afferent neurons. Indeed, recent research in animals and humans has shown that the neurons in the MTN, along with TG cells, contain heme oxygenase, the CO-synthesizing enzyme (Uddman et al., 2004; Fan et al., 2008). The enzyme responsible for the synthesis of NO, nitric oxide synthase (NOS), and its histochemical marker, NADPH-diaphorase are expressed both in TG and MTN neurons in rats (Stoyanova & Lazarov, 2005), rabbits (Kolesar et al., 2006) and cats (Lazarov & Dandov, 1998) as well. The nerve cell bodies containing NOS are predominantly of small to medium size and they also express SP and CGRP (Edvinsson et al., 1998). Moreover, all these studies demonstrate that large unstained trigeminal neurons are innervated by nitrergic fibers and that NO increases the excitability and modifies other electrophysiological properties of these cells. Rather than acting via traditional receptors on the postsynaptic membrane, NO exerts its effect by diffusion into the adjacent neurons to activate soluble guanylyl cyclase, leading to an increase in intracellular cGMP. Furthermore, NO possibly produces an increase in MTN neuronal electrotonic coupling and therefore is involved in the synchronization of their activity too. Finally, there is functional evidence to support the involvement of NO in the development and maintenance of inflammation and pain (Yun et al., 1996). It has been suggested that as in other regions, both gaseous messengers possibly interact in a complex, dynamic way in the orofacial sensory processing where CO, being a more stable gas, may be responsible for basal activity and provide tonic background stimulation, whereas surges of NO transiently amplify or deliver phasic

The calcium-binding proteins (CaBPs) represent one of the physiological systems for maintaining calcium ion intracellular homeostasis (reviewed in Baimbridge et al., 1992). In the last two decades they have received increased attention due to their implementation as specific markers for large-sized primary afferent subpopulations and their involvement in many calcium-dependent phenomena in the nervous system both under normal and abnormal conditions (Anderssen et al., 1993). Neuron-specific CaBPs, parvalbumin (PV), calbindin D-28k (CB) and calretinin (CR), are observed to be expressed predominantly in the large-sized TG and MTN neurons (Ichikawa et al., 1994; Lazarov et al., 1998), a subpopulation with inward calcium current. Interestingly, a typical glial cell-specific protein, S-100, is also localized, mostly co-expressed with CB, in TG neurons of epibranchial placode origin (Ichikawa et al., 1997). In addition, immunoreactivity to neurocalcin, a newly identified member of the neuronal CaBP family, has been shown in large or medium in size TG cells (Iino et al., 1998). Two additional newly discovered bone matrix CaBPs, osteocalcin and osteopontin, have recently been co-localized with PV in the cell bodies of both TG and

somata and do not discharge repetitively (Del Negro & Chandler, 1997).

**2.3 Gaseous neuromessengers**

signaling (Fan et al., 2008).

**2.4 Calcium-binding proteins**

MTN neurons (Ichikawa et al., 1999; 2000). Data from ongoing experiments have provided compelling evidence that CaBPs might function as intracellular calcium transporters or as a buffering system for cell protection during neuronal activity in normal circumstances. They may also affect calcium-dependent neuronal properties such as excitability, release of neurotransmitters and resistance to excitotoxicity in mammals (see Baimbridge et al., 1992). Additionally to intracellular roles, S-100 may be involved in the neurotrophic functions of trigeminal primary afferent neurons.
