**3. Neurotrophic factors and neurotrophin receptors**

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 well and may function as neuromodulators in neuronal signaling.

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

The Neurochemical Anatomy of Trigeminal Primary Afferent Neurons 181

the proximal stump of the transected masseteric nerve delays the loss of MTN neurons after the cut (Ichikawa et al., 2007). On the other hand, altered levels of CaBPs may be related to adequate cell body response since the sensitivity of damaged neurons to the intracellular

NO Upregulation Upregulation Cell death or defenc

synthesis

synthesis

synthesis

synthesis Increase in neurotrophin responsiveness

Downregulation Downregulation Cell survival

NGF Upregulation Upregulation Peptide synthesis induction

TrkA Upregulation Upregulation Enhanced neuronal survival

calcium concentration is different from that of intact ones. A logical explanation for the reported down-regulation in CaBP expression may be the actual reduction of the MTN cell number following periphery axotomy (Ichikawa et al., 2007). This would be in line with the suggestion that persistently increased levels of NOS in mesencephalic trigeminal neurons may be involved in slowly progressive nerve cell death following nerve damage because they may lead to an augmented vulnerability of the neurons to calcium-mediated

Parvalbumin (PV) Downregulation Downregulation Cell defence

BDNF Upregulation Upregulation Cell protection Table 2. Summary of the injury-induced alterations in the expression of neuroactive substances and their effects on mammalian trigeminal primary afferent neurons

Mesencephalic trigeminal nucleus

Effect

Supportive role in

Cell protection

Cell regeneration

responsiveness

Increase in neurotrophin

Enhancement membrane Enhancement of membrane potential oscillations

and maintenance

Trigeminal ganglion

Injury consequence

Neuroactive substance

Neurotransmitters

SP Downregulation

CGRP Downregulation De novo

NPY Upregulation De novo

GAL Upregulation De novo

PACAP Upregulation De novo

Neurotrophic factors and their receptors

Neuropeptides

Calcium-binding

Calbindin D-28

proteins

(CB)

marker for peptide-containing nociceptors. Interestingly, Trk receptors appear to be coexpressed with SP and CGRP in a population of small trigeminal primary afferent neurons (Quartu et al., 1996).

#### **4. Trigeminal response to injury and neurochemical plasticity of trigeminal primary afferent neurons**

Research on putative neuromessengers in the trigeminal sensory system has reached another peak of interest over the last decade since it was shown that primary afferent neurons possess the ability to make adaptive changes in their transmitter phenotype in response to environmental cues (Table 2). It is now well known that injury to the peripheral trigeminal nerve results in nerve cell degeneration and that peripheral nerve damage evokes dynamic alterations in the levels of expressions of neurotrophins, neuropeptides and their receptors in the projection areas of injured axons, a phenomenon called chemical plasticity (Hökfelt et al., 1994). Indeed, after axotomy of the inferior alveolar nerve, there is a marked reduction in the total SP and CGRP expression in TG neurons (Elcock et al., 2001) and their release is largely enhanced by peripheral inflammation (Neubert et al., 2000). Conversely, the NPY and VIP levels are dramatically increased in axotomized TG neurons (Sasaki et al., 1994; Fristad et al., 1998). It is generally presumed that neuropeptides repressed after axotomy participate normally in sensory transmission while those induced may function as neurotrophic factors involved in the response to injury and in axonal regeneration (Nielsch & Keen, 1989). The axonal signals induced in response to nerve injury activate several signaling pathways of genes in the neuronal cell bodies that may lead to two opposing outcomes: cell death or regenerative response. In effect, peripheral nerve injury causes the surviving neurons to shift their activity away from normal maintenance and neurotransmission toward a regenerative state (Navarro, 2009). It is thought that changes in gene expression after axonal injury are due to a blockage of NGF retrograde axonal flow from the periphery to the cell body. This may explain why both high (TrkA) and low (p75) affinity neurotrophin receptor transcripts in the TG neurons increase after tooth injury (Wheeler et al., 1998)

As it can be inferred from Table 2, axotomy-induced alterations in the expression of neuroactive substances in MTN neurons include a long-lasting decrease (down-regulation) in the content of CaBPs, up-regulation of NO and some neurotrophins, and a *de novo* synthesis of certain neuropeptides, such as GAL, NPY and CGRP (see Lazarov, 2002, 2007, and references therein). A commonly shared view is that a characteristic of neuropeptides is the plasticity in their expression, reflecting the fact that release has to be compensated by *de novo* synthesis in the neuronal body (Navarro et al., 2007). It may be postulated that the newly synthesized neuropeptides can enhance MTN neuronal survival and neurite regeneration in the adaptive processes following nerve injury. Therefore, a peptide involvement in the proprioceptive function develops mainly in abnormal conditions. Navarro and co-workers (2007) also state that injured neurons respond by up-regulation of neurotrophins, either by autocrine or paracrine sources, and that additional exogenous supply of neurotrophic factors may further enhance the regenerative response of peripherally axotomized neurons. It should be noted that the survival of proprioceptors during the early postnatal period is probably dependent upon BDNF since its application to

marker for peptide-containing nociceptors. Interestingly, Trk receptors appear to be coexpressed with SP and CGRP in a population of small trigeminal primary afferent neurons

**4. Trigeminal response to injury and neurochemical plasticity of trigeminal** 

Research on putative neuromessengers in the trigeminal sensory system has reached another peak of interest over the last decade since it was shown that primary afferent neurons possess the ability to make adaptive changes in their transmitter phenotype in response to environmental cues (Table 2). It is now well known that injury to the peripheral trigeminal nerve results in nerve cell degeneration and that peripheral nerve damage evokes dynamic alterations in the levels of expressions of neurotrophins, neuropeptides and their receptors in the projection areas of injured axons, a phenomenon called chemical plasticity (Hökfelt et al., 1994). Indeed, after axotomy of the inferior alveolar nerve, there is a marked reduction in the total SP and CGRP expression in TG neurons (Elcock et al., 2001) and their release is largely enhanced by peripheral inflammation (Neubert et al., 2000). Conversely, the NPY and VIP levels are dramatically increased in axotomized TG neurons (Sasaki et al., 1994; Fristad et al., 1998). It is generally presumed that neuropeptides repressed after axotomy participate normally in sensory transmission while those induced may function as neurotrophic factors involved in the response to injury and in axonal regeneration (Nielsch & Keen, 1989). The axonal signals induced in response to nerve injury activate several signaling pathways of genes in the neuronal cell bodies that may lead to two opposing outcomes: cell death or regenerative response. In effect, peripheral nerve injury causes the surviving neurons to shift their activity away from normal maintenance and neurotransmission toward a regenerative state (Navarro, 2009). It is thought that changes in gene expression after axonal injury are due to a blockage of NGF retrograde axonal flow from the periphery to the cell body. This may explain why both high (TrkA) and low (p75) affinity neurotrophin receptor transcripts in the TG neurons increase after tooth injury

As it can be inferred from Table 2, axotomy-induced alterations in the expression of neuroactive substances in MTN neurons include a long-lasting decrease (down-regulation) in the content of CaBPs, up-regulation of NO and some neurotrophins, and a *de novo* synthesis of certain neuropeptides, such as GAL, NPY and CGRP (see Lazarov, 2002, 2007, and references therein). A commonly shared view is that a characteristic of neuropeptides is the plasticity in their expression, reflecting the fact that release has to be compensated by *de novo* synthesis in the neuronal body (Navarro et al., 2007). It may be postulated that the newly synthesized neuropeptides can enhance MTN neuronal survival and neurite regeneration in the adaptive processes following nerve injury. Therefore, a peptide involvement in the proprioceptive function develops mainly in abnormal conditions. Navarro and co-workers (2007) also state that injured neurons respond by up-regulation of neurotrophins, either by autocrine or paracrine sources, and that additional exogenous supply of neurotrophic factors may further enhance the regenerative response of peripherally axotomized neurons. It should be noted that the survival of proprioceptors during the early postnatal period is probably dependent upon BDNF since its application to

(Quartu et al., 1996).

(Wheeler et al., 1998)

**primary afferent neurons** 

Neuroactive substance Trigeminal ganglion Mesencephalic trigeminal nucleus Effect Injury consequence Neurotransmitters NO Upregulation Upregulation Cell death or defenc Neuropeptides SP Downregulation CGRP Downregulation De novo synthesis Supportive role in NPY Upregulation De novo synthesis Cell protection GAL Upregulation De novo synthesis Cell regeneration PACAP Upregulation De novo synthesis Increase in neurotrophin responsiveness Increase in neurotrophin responsiveness Calcium-binding proteins Parvalbumin (PV) Downregulation Downregulation Cell defence Calbindin D-28 (CB) Downregulation Downregulation Cell survival Neurotrophic factors and their receptors NGF Upregulation Upregulation Peptide synthesis induction Enhancement membrane Enhancement of membrane potential oscillations TrkA Upregulation Upregulation Enhanced neuronal survival and maintenance BDNF Upregulation Upregulation Cell protection

the proximal stump of the transected masseteric nerve delays the loss of MTN neurons after the cut (Ichikawa et al., 2007). On the other hand, altered levels of CaBPs may be related to adequate cell body response since the sensitivity of damaged neurons to the intracellular

Table 2. Summary of the injury-induced alterations in the expression of neuroactive substances and their effects on mammalian trigeminal primary afferent neurons

calcium concentration is different from that of intact ones. A logical explanation for the reported down-regulation in CaBP expression may be the actual reduction of the MTN cell number following periphery axotomy (Ichikawa et al., 2007). This would be in line with the suggestion that persistently increased levels of NOS in mesencephalic trigeminal neurons may be involved in slowly progressive nerve cell death following nerve damage because they may lead to an augmented vulnerability of the neurons to calcium-mediated

The Neurochemical Anatomy of Trigeminal Primary Afferent Neurons 183

(Holthusen et al., 1997). It has recently been demonstrated that SP release in the TG is predominantly increased after orofacial inflammation (Neubert et al. 2000) and such a release may play an important role in determining the trigeminal inflammatory alloying concerning the temporomandibular joint disorder (Takeda et al., 2005). The authors point out that NK1 receptor antagonists may be useful as therapeutic agents to prevent the mechanical allodynia. P2X3 receptors may be another therapeutic target for treating

Another common clinical concern regarding the trigeminal nerve is trigeminal neuralgia. Evidence for the role of SP and CGRP in trigeminal neuralgia pain is clearly apparent (Stoyanova & Lazarov, 2001). Inhibitory neurotransmitters, such as GABA, are thought to have a role in analgesia and many GABAergic drugs, acting through metabotropic GABAB receptors, are useful in the treatment of migraine and trigeminal neuralgia. With regard to the latter, a GABA analogue, gabapentin, has been reported to be effective in the management of migraine and trigeminal neuralgia, and also displays anti-nociceptive activity in various animal pain models. In addition, a selective GABAB receptor agonist, baclofen, has been shown to elicit pain relief and, thus, it might play a therapeutic role in the

Clinically relevant is also pain, caused by a central or peripheral nerve lesion which is commonly termed neuropathic pain, and the concomitant neurogenic inflammation. Orofacial neuropathic pain, like anywhere in the body, may occur as a result of tissue damage and the activation of nociceptors, which transmit a noxious stimulus to the brain (Vickers & Cousins, 2000). The abnormal facial pain involves regeneration of damaged nerve fibers and may account for chemical changes in injured neuronal cell bodies. As mentioned above, a variety of neuropeptides, such as SP, CGRP, GAL and NPY, are upregulated following peripheral axotomy (see Table 2) and craniofacial muscle inflammation (Ambalavanar et al., 2006). Results from studies on animal pain models have suggested that NPY and its receptors are potential targets for treatment of pain, especially neuropathic pain (Silva et al., 2002). The efficacy of opioid receptor agonists in modulation of nociceptive inputs in a wide range of orofacial pain models, including neuropathic pain (Catheline et al., 1998) and inflammatory pain (Ko et al., 1998) is also acknowledged. Given that NGF is responsible for the increased expression of SP and CGRP during neurogenic inflammation (Lundy & Linden, 2004), it is not much surprising that the systemic administration of anti-NGF neutralizing antibodies prevents the up-regulation of neuropeptides in primary afferent neurons innervating the inflamed skin (Woolf et al., 1994). Changes in the injured neurons can also influence the ability of the surrounding glial cells to release neuromodulators such as NO and ATP, thus implicating satellite glial cells in the TG as a determinant of orofacial neuropathic pain. This fits well with the notion that the P2X3 receptor is transiently up-regulated and anterogradely transported in trigeminal primary afferent neurons after neuropathic injury (Eriksson et al., 1998). Purinergic receptors on TG neurons are thus likely to be a legitimate target for therapeutic intervention in neuropathic pain and orofacial inflammation (Ambalavanar et al., 2005). Recent findings further demonstrate that masseter inflammation differentially modulates Glu receptor subunits and that the induced changes in them may contribute to functionally different aspects of craniofacial muscle pain processing under inflammatory conditions (Lee and Ro, 2007).

inhibition of nociceptive hypersensitivity in trigeminal neuralgia (Fromm, 1994).

temporomandibular joint disorder pain (Shinoda et al., 2005).

neurotoxicity. Alternatively, it is reasonable to speculate that the possible endogenous production of NO might underlie a defense mechanism of the neurons against nerve injury and, thus, improve survival and active regeneration of MTN neurons.

In summary, these findings provide compelling evidence that the content of the neurochemicals in both central and peripheral trigeminal primary afferent neurons is not static and their level may vary in case of marked changes in the environmental conditions, thus implying neuroplasticity as another major attribute of theirs.
