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This study has revealed direct projections from the dorsal peduncular cortex (DP) in the medial prefrontal cortex (mPfC) to the trigeminal brainstem sensory nuclear complex and other lower brainstem areas in rats. We first examined the distribution of mPfC neurons projecting directly to the medullary dorsal horn (trigeminal subnucleus caudalis [Vc]) and trigeminal subnucleus oralis (Vo) which are known to receive direct projections from the lateral prefrontal cortex (insular cortex). After injections of the retrograde tracer Fluorogold (FG) into the rostro-dorsomedial part of laminae I/II of Vc (rdm-I/II-Vc), many neurons were labeled bilaterally (with an ipsilateral predominance) in the rostrocaudal middle level of DP (mid-DP) and not in other mPfC areas. After FG injections into the lateral and caudal parts of laminae I/II of Vc, or the Vo, no neurons were labeled in the mPfC. We then examined projections from the mid-DP by using the anterograde tracer biotinylated dextranamine (BDA). After BDA injections into the mid-DP, many axons and terminals were labeled bilaterally (with an ipsilateral predominance) in the rdm-I/II-Vc, periaqueductal gray and solitary tract nucleus, and ipsilaterally in the parabrachial nucleus and trigeminal mesencephalic nucleus. In addition, the connections of the mid-DP with the insular cortex were examined. Many BDA-labeled axons and terminals from the mid-DP were also found ipsilaterally in the caudalmost level of the granular and dysgranular insular cortex (GI/DI). After BDA injections into the caudalmost GI/DI, many axons and terminals were labeled ipsilaterally in the mid-DP. The projections from the mid-DP to the rdm-I/II-Vc and other brainstem nuclei suggest that mid-DP neurons may regulate intraoral and perioral sensory processing (including nociceptive processing) of rdm-I/II-Vc neurons directly or indirectly through the brainstem nuclei. The reciprocal connections between the mid-DP and caudalmost GI/DI suggest that this regulation may involve mid-DP interactions with the caudalmost GI/DI neurons., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: To evaluate whether P2X receptors are involved in responses to noxious pulp stimulation, the P2X3 and P2X2/3 receptor agonist α,β-methyleneATP (α,β-meATP) was applied to the molar tooth pulp and nocifensive behavior and extracellular-signal regulated kinase (ERK) phosphorylation in trigeminal spinal subnucleus caudalis (Vc), trigeminal spinal subnucleus interpolaris (Vi), upper cervical spinal cord (C1/C2) and paratrigeminal nucleus (Pa5) neurons were analyzed in rats., Results: Genioglossus (GG) muscle activity was evoked by pulpal application of 100 mM α,β-meATP and was significantly larger than GG activity following vehicle (phosphate-buffered saline PBS) application (p < 0.01). The enhanced GG muscle activity following 100 mM α,β-meATP was significantly reduced (p < 0.05) by co-application of 1 mM TNP-ATP (P2X1, P2X3 and, P2X2/3 antagonist). A large number of pERK-LI cells were expressed in the Vc, Vi/Vc, C1/C2 and Pa5 at 5 min following pulpal application of 100 mM α,β-meATP compared to PBS application to the pulp (p < 0.05). The pERK-LI cell expression and GG muscle activity induced by 100 mM α,β-meATP pulpal application were significantly reduced after intrathecal injection of the MAPK/ERK kinase (MEK) inhibitor PD 98059 and by pulpal co-application of 1 mM TNP-ATP (p < 0.05)., Conclusions: The present findings suggest that activation of P2X3 and P2X2/3 receptors in the tooth pulp is sufficient to elicit nociceptive behavioral responses and trigeminal brainstem neuronal activity.

In order to evaluate the neuronal mechanisms underlying functional abnormalities of swallowing in orofacial pain patients, this study investigated the effects of noxious orofacial stimulation on the swallowing reflex, phosphorylated extracellular signal-regulated kinase (pERK) and gamma-aminobutyric acid (GABA) immunohistochemical features in brainstem neurons, and also analysed the effects of brainstem lesioning and of microinjection of GABA receptor agonist or antagonist into the nucleus tractus solitarii (NTS) on the swallowing reflex in anaesthetized rats. The swallowing reflex elicited by topical administration of distilled water to the pharyngolaryngeal region was inhibited after capsaicin injection into the facial (whisker pad) skin or lingual muscle. The capsaicin-induced inhibitory effect on the swallowing reflex was itself depressed after the intrathecal administration of MAPK kinase (MEK) inhibitor. No change in the capsaicin-induced inhibitory effect was observed after trigeminal spinal subnucleus caudalis lesioning, but the inhibitory effect was diminished by paratrigeminal nucleus (Pa5) lesioning. Many pERK-like immunoreactive neurons in the NTS showed GABA immunoreactivity. The local microinjection of the GABA(A) receptor agonist muscimol into the NTS produced a significant reduction in swallowing reflex, and the capsaicin-induced depression of the swallowing reflex was abolished by microinjection of the GABA(A) receptor antagonist bicuculline into the NTS. The present findings suggest that facial skin-NTS, lingual muscle-NTS and lingual muscle-Pa5-NTS pathways are involved in the modulation of swallowing reflex by facial and lingual pain, respectively, and that the activation of GABAergic NTS neurons is involved in the inhibition of the swallowing reflex following noxious stimulation of facial and intraoral structures.

This article briefly highlights some of the recent advances in knowledge of the neural processes underlying pain in the face and mouth. It particularly focuses on those mechanisms operating in the brainstem that are involved in the transmission and modulation of nociceptive signals and that reveal a remarkable degree of plasticity following injury or inflammation of craniofacial tissues. Insights into these processes hold promise of the development of new or improved therapeutic procedures for the relief of pain.

This paper reviews the recent advances in knowledge of brainstem mechanisms related to craniofacial pain. It also draws attention to their clinical implications, and concludes with a brief overview and suggestions for future research directions. It first describes the general organizational features of the trigeminal brainstem sensory nuclear complex (VBSNC), including its input and output properties and intrinsic characteristics that are commensurate with its strategic role as the major brainstem relay of many types of somatosensory information derived from the face and mouth. The VBSNC plays a crucial role in craniofacial nociceptive transmission, as evidenced by clinical, behavioral, morphological, and electrophysiological data that have been especially derived from studies of the relay of cutaneous nociceptive afferent inputs through the subnucleus caudalis of the VBSNC. The recent literature, however, indicates that some fundamental differences exist in the processing of cutaneous vs. other craniofacial nociceptive inputs to the VBSNC, and that rostral components of the VBSNC may also play important roles in some of these processes. Modulatory mechanisms are also highlighted, including the neurochemical substrate by which nociceptive transmission in the VBSNC can be modulated. In addition, the long-term consequences of peripheral injury and inflammation and, in particular, the neuroplastic changes that can be induced in the VBSNC are emphasized in view of the likely role that central sensitization, as well as peripheral sensitization, can play in acute and chronic pain. The recent findings also provide new insights into craniofacial pain behavior and are particularly relevant to many approaches currently in use for the management of pain and to the development of new diagnostic and therapeutic procedures aimed at manipulating peripheral inputs and central processes underlying nociceptive transmission and its control within the VBSNC.

1. The subcutaneous injection of Formalin (5%, 50 microliters) into the receptive field of convergent (wide dynamic range) nociceptive neurons in the spinal dorsal horn has previously been reported to produce a prolonged biphasic response with a time course similar to the observed in behavioral experiments. However, conflicting data in other studies led us to examine the effects of Formalin on the activity of convergent nociceptive neurons at two levels of the trigeminal (V) brain stem complex, namely V subnuclei oralis (Sp5O) and caudalis (Sp5C). 2. Single neuron activity was extracellularly recorded in anesthetized rats. Each neuron was classified as convergent on the basis of its responses to both mechanical and transcutaneous electrical stimuli applied to its mechanoreceptive field (RF). All neurons responded to innocuous and noxious mechanical stimuli and had electrically evoked responses corresponding to both A and C fiber afferent inputs. Seventeen Sp5O and 32 Sp5C convergent neurons received an injection (50 microliters sc) of 5% Formalin into the center of their RF. 3. Three groups of neurons were distinguished: one group that was not activated by the Formalin injection (Sp5O, n = 1; Sp5C, n = 2), another group that responded only with an early and short-lasting response [monophasic neurons: Sp5O, n = 11 (65%); Sp5C, n = 10 (31%)], and a third group that responded with two phases separated by a short period of quiescence [biphasic neurons: Sp5O, n = 5 (29%); Sp5C, n = 20 (62.5%)]. The proportion of biphasic neurons was significantly higher in Sp5C than in Sp5O. 4. The first phases of the Sp5O neurons were not significantly different in terms of duration and frequency from those of the Sp5C neurons. However, duration and discharge frequency of the first phase of biphasic neurons were significantly greater than for monophasic neurons in Sp5C (nonsignificant in Sp5O). The second tonic period of excitation of all biphasic neurons was gradual in outset and offset, and long in duration (23-39 min). The duration of the second phase was significantly longer for the biphasic neurons in Sp5C than for those in Sp5O. 5. Our findings suggest that the mono- and biphasic neurons may have different roles in the transmission of nociceptive information induced by the peripheral injection of Formalin.(ABSTRACT TRUNCATED AT 400 WORDS)

It is known that removal of the tooth pulp from mandibular molar teeth in adult rats alters the mechanoreceptive field properties of many low-threshold mechanoreceptive neurons in the trigeminal brainstem nuclear complex. The present study investigates one possible way that such deafferentation-induced receptive field changes could occur: altered central projections of uninjured trigeminal low-threshold mechanoreceptive primary afferent fibers. Intra-axonal injection of horseradish peroxidase (n = 22) or neurobiotin (n = 44) into characterized fibers was performed ipsilateral to, and 10-32 days after, removal of the coronal pulp from the left mandibular molars in adult rats. Collaterals were reconstructed, quantified, and compared by means of multivariate analyses of variance to equivalent fibers stained in normal adult rats. Stained mechanosensitive fibers from experimental animals were rapidly conducting and responded to light mechanical stimulation of one vibrissa, one tooth, oral mucosa, facial hairy skin, or guard hairs. Their central projections were indistinguishable from those of control axons in all four trigeminal subnuclei. The numbers of collaterals, areas subtended by collateral arbors, numbers of boutons per collateral, and arbor circularity did not differ from those of control afferents. Collateral somatotopy was also unaffected. These data suggest that following pulpotomy, the central collaterals of uninjured trigeminal afferents display normal morphologies and maintain normal somatotopy. Changes in the morphology of low-threshold primary afferents cannot account for the changes that occur in the receptive field properties of trigeminal brainstem neurons after pulp deafferentation.

1. The aim of this study was to determine whether there are differential neuroplastic effects of cutaneous and deep stimuli on the mechanoreceptive field (RF) properties of nociceptive brain stem neurons recorded in trigeminal (V) subnucleus caudalis. 2. A total of 31 caudalis neurons were functionally identified in anesthetized rats as nociceptive neurons receiving convergent cutaneous and deep afferent inputs, and the effects of the small-fiber excitant and inflammatory irritant mustard oil applied to either tongue muscle or facial skin were tested on their cutaneous and deep RFs. 3. The expression of neuroplasticity on the cutaneous and deep RFs of the neurons was found to depend on the site of application of this irritant. Mustard oil injected into the tongue muscle produced a significant and reversible expansion of both the cutaneous and deep RFs. In contrast, application of mustard oil to facial skin resulted in a significant and reversible expansion of the cutaneous RF, but no deep RF expansion occurred. 4. These findings suggest that considerable functional neuroplasticity of the cutaneous and deep RF properties of V nociceptive neurons can be induced by a peripheral inflammatory irritant and that deep inputs are especially effective in evoking these neuroplastic changes.

Previous studies have demonstrated that tooth pulp deafferentation is associated with statistically significant alterations in the low-threshold facial mechanoreceptive field properties of brain stem neurons in trigeminal (V) subnucleus oralis. A loss of spinal afferent-induced presynaptic inhibition as a consequence of a decrease in primary afferent depolarization (PAD) following spinal nerve deafferentation has been invoked as a mechanism underlying deafferentation-induced somatosensory neuroplasticity. Therefore, this study was initiated to determine if these pulp deafferentation-induced neuroplastic changes could be accounted for by an alteration in PAD of low-threshold facial afferent endings in subnucleus oralis of anesthetized rats. In control (unoperated) rats (n = 7) and rats (n = 7) that had undergone mandibular pulp deafferentation 6-10 days previously, antidromic compound action potentials evoked by test stimulation in V subnucleus oralis were recorded in branches of the infraorbital (IO) and supraorbital (SO) nerves, and conditioning stimuli were applied to some of the same nerves. PAD of the afferent endings in oralis of these nerve branches was documented in all animals, and there was no significant difference between the two groups in the incidence or any of the other features of PAD. The features of the PAD were consistent with those described in several previous studies of normal animals. These findings indicate that the reported deafferentation-induced loss of spinal presynaptic regulatory mechanisms cannot be entrapolated to all forms of deafferentation injury and that the mechanoreceptive field changes that can occur in central V somatosensory neurons as a result of tooth pulp deafferentation may not reflect an alteration in PAD.

The effects of tooth pulp deafferentation on brainstem neuronal properties were examined in the rat trigeminal (V) subnucleus oralis. Deafferentation was produced by removal of the coronal tooth pulp of all left mandibular molars. Neurons in the subnucleus oralis were then electrophysiologically characterized in chloralose/urethane-anesthetized rats at a single postoperative time. The mechanoreceptive field and response properties of low-threshold mechanoreceptive (LTM) neurons in rats studied at postoperative times of 3-4 days, 7-13 days, and 28-40 days were compared to those in control (unoperated) rats. Functional changes in oralis LTM neurons were observed in all tooth-pulp-deafferented rats, but most statistically significant changes were apparent only at the 7- to 13-day postoperative period. In 7- to 13-day pulp-deafferented rats, there was a significant increase in the incidence of neurons with a two-divisional mechanoreceptive field, accompanied by a significant decrease in the incidence of neurons with a maxillary mechanoreceptive field. This group of rats also showed a significant increase in the incidence of neurons with a mechanoreceptive field involving both mandibular and maxillary divisions. For neurons that could be activated by light mechanical stimulation of one or more mandibular or maxillary teeth, the 7- to 13-day pulp-deafferented rats showed a significant increase in the incidence of neurons with such periodontal mechanosensitive inputs involving both mandibular and maxillary divisions. There was also a significant increase in the incidence of spontaneously active neurons in this group of rats. For neurons with a mechanoreceptive field involving mystacial vibrissae, there was a significant increase in all three groups of pulp-deafferented rats in the maximal width of the vibrissal row (i.e., the number of vibrissae in the longest horizontal row of vibrissae, stimulation of which was effective in activating a given neuron). No significant differences were found between groups in the incidence of neurons with a mandibular mechanoreceptive field or in the proportions of neurons with a mechanoreceptive field located in each of several defined orofacial regions. There was also no significant difference between groups in the mean latency to electrical stimulation of the neuronal mechanoreceptive field, or in the proportions of rapidly adapting (RA) and slowly adapting (SA) neurons. These results show many similarities with the functional changes of oralis LTM neurons in the cat following tooth pulp deafferentation, and indicate that the rat may serve as a very useful model for V brainstem neuroplasticity induced by tooth pulp deafferentation.

In the young cat, the natural replacement of deciduous teeth with a permanent dentition may be accompanied by extensive peripheral nerve degeneration and reinnervation. Since we have previously reported significant physiological effects of tooth pulp deafferentation on V brainstem neurons, we wished to determine if deafferentation associated with deciduous tooth exfoliation was accompanied by comparable neuroplastic changes in the brainstem of kittens. We have examined the pulpal afferent terminating pattern within the brainstem as well as the electrophysiological properties of V brainstem neurons during this naturally occurring deafferentation phenomenon. The pattern of the HRP-labelled terminals in different parts of V brainstem subnuclei in kittens was similar to that in adult cats. The highest density of labelled terminals was found in the subnucleus oralis. The properties of subnucleus oralis neurons also showed many similarities between kittens at age of 3-4 months old and adult cats, e.g., (1) in the proportions of neurons activated by tactile stimulation of 1-4 teeth (mandibular and maxillary canines and premolars), (2) the ratio of neurons showing fast adapting or slow adapting responses to dental tactile stimulation, (3) the proportions of neurons activated by electrical pulp stimulation of 1-4 teeth, and (4) the minimal latencies of pulp-evoked responses. However, the incidences of pulpal inputs as well as the convergent pattern to oralis neurons of afferent inputs evoked by periodontal stimulation and electrical pulpal stimulation were less common in kittens than adult cats.

Responses evoked in anaesthetized or decerebrate cats by stimulation of afferents supplying the face, mouth, pharynx, larynx, tooth pulp and jaw muscles were recorded from single neurones located in the trigeminal (V) main sensory nucleus, V nucleus oralis, and adjacent regions. Many cells (both V-thalamic relay and non-relay with localized V mechanoreceptive cutaneous fields could be activated by stimulation of a number of these afferents. A particularly prominent short-latency (often monosynaptic) input was noted from the canine tooth pulp, stimulation of which is generally considered to elicit only responses of pain in man. Control experiments showed that pulp-evoked responses were not the result of stimulus spread to tissues outside the pulp. The interaction of these various inputs to neurones at this level of the V brain stem complex typically resulted in a prolonged period of inhibition that was sometimes preceded by a short-lasting facilitatory phase. This inhibitory effect was also apparent in neurones located outside the complex, although a late facilitatory phase was frequently also noted. Our findings indicate a significant nociceptive input to V main sensory-oralis neurones, a proportion of which relay directly to the ventrobasal thalamus. The interactions described may be involved in perceptual and reflex aspects of responses to noxious and innocuous V stimuli.

The activity of 221 single primary afferent units was recorded extracellularly in the trigeminal (V) ganglion of chloralose-anaesthetized cats to examine the receptive field properties of nonnociceptive and nociceptive cutaneous afferents and the effect of conditioning stimulation of the raphe system and orofacial afferents on the antidromic excitability of their brain stem endings in V subnucleus caudalis. In addition to slowly adapting and rapidly adapting low-threshold mechanosensitive afferents, we functionally identified three classes of cutaneous nociceptive afferents: these included A-delta high-threshold mechanoreceptive afferents (A-delta HTMs), C-fiber high-threshold mechanoreceptive afferents (C-HTMs), and C-polymodal nociceptive afferents (CPNs). Most of the CPNs could be activated by light tactile stimuli as well as by heavy pressure and pinch and noxious radiant heat applied to their mechanoreceptive field which usually involved a localized spot (approximately 1 mm in diameter) of skin. In contrast, the C-HTMs and A-delta HTMs could not be activated by radiant heat stimuli although some did show sensitization which was also a feature of the CPNs; they did respond to noxious mechanical stimulation of a localized area of skin. We noted that orofacial conditioning stimulation could produce an increase in antidromic excitability which was considered a reflection of primary afferent depolarization (PAD) in both nociceptive and nonnociceptive afferents innervating the cat's facial skin; nonnoxious mechanical stimuli and electrical stimuli were particularly effective in the low-threshold mechanosensitive afferents and noxious mechanical and high-intensity electrical stimuli were especially effective in the cutaneous nociceptive afferents. Raphe conditioning stimulation also was very effective in inducing PAD in these nociceptive afferents; however, the raphe conditioning effects were not limited to these nociceptive afferents since PAD was also frequently demonstrated in the low-threshold mechanosensitive afferents.

Single relay (to thalamus) and nonrelay neurones that responded to innocuous and/or noxious oral-facial stimuli were located in trigeminal brain stem nuclei oralis and caudalis. The responses of the cells and the digastric muscle to these stimuli were tested with conditioning stimulation of the periaqueductal gray matter (PGM) and somatosensory cerebral cortex in cats. A greater suppression of nociceptive responses with PGM stimulation was noted, and this effect may contribute to the profound analgesic action that has been reported to occur with PGM stimulation.

To determine if the nucleus raphe magnus (NRM) and locus coeruleus (LC) might exert presynaptic modulatory influences on the endings in the medullary solitary tract nucleus of laryngeal afferents, multi-unit and single unit laryngeal afferent activity was recorded in the nodose ganglion in chloralose-anaesthetized cats. The afferents could be orthodromically activated by superior laryngeal nerve stimulation and antidromically activated from the solitary tract nucleus. An increase in antidromic excitability of laryngeal afferents, which was considered a reflection of primary afferent depolarization, could be induced by either NRM or LC conditioning stimuli and lasted for 300-400 msec. The findings suggest that presynaptic mechanisms may contribute to the suppression of solitary tract neuron or reflex activities induced by NRM or LC stimulation.

Trigeminal (V) tractotomy and cold block of synaptic transmission in V nucleus caudalis were used to show that caudalis modulates the responses to innocuous and noxious stimuli of single V main sensory-oralis neurones recorded in anaesthetized or decerebrate cats. Cold block caused a reversible depression of mechanosensitivity of 91 of 105 V-thalamic relay and non-relay cells tested; V tractotomy also decreased sensitivity. The possibliity that the effects observed with cold block of caudalis were caused by direct spread of cooling to the main sensory-oralis region, and not by depression of a tonic, net facilitatory influence of caudalis on main sensory-oralis cells, was ruled out by several controls. With cold block of caudalis there also occurred a reversible shrinkage in mechanoreceptive field size and reversible reduction in sensitivity of rapidly adapting and slowly adapting mechanoreceptive neurones. Occasionally no change or an increase in sensitivity occurred, the latter suggesting the liklihood of an inhibitory influence from caudalis as well as the facilitatory influence. The effects of interactions of innocuous and noxious V stimuli were likewise subject to ascending influences from caudalis. Cold block also reversibly depressed responses to tooth pulp and V cutaneous noxious stimuli, although pulp-evoked responses were depressed less than mechanical or infraorbital nerve-elicited responses. Our results indicate that caudalis, as well as acting as a relay site to thalamus, also exerts a predominantly facilitatory influence on the relay to the thalamus and local reflex centres of mechanoreceptive and nociceptive information through the V main sensory-oralis region. The findings also might in part explain the analgesia, partial loss of tactile sensibility, and relief from V neuralgia reported after V tractotomy.

Possible opiate-related descending influences from the periaqueductal gray matter (PAG) and nucleus raphe magnus (NRM) were tested on the activity of neural systems involved in respiration and related reflex functions in cats. Stimulation of PAG and NRM could powerfully suppress the simple buccopharyngeal reflexes of jaw-opening and tongue-protrusion and the more complex reflexes of coughing and swallowing; respiration in contrast appeared to be only weakly influenced. The reflexly induced responses of 57 single reflex interneurons recorded in the solitary tract nucleus (NST) could also be markedly suppressed by PAG and NRM conditioning stimulation. In contrast, the rhythmic activity of 30 respiratory neurons in NST was not abolished by PAG and NRM stimuli but most did show a decrease in the peak firing frequency of each rhythmic burst. The suppressive effect of PAG and NRM stimulation on the reflexes and NST reflex interneurons could be reduced by the intravenous administration of naloxone. These studies indicate that neuronal functions associated with respiration and respiratory-related activities can be suppressed by descending influences from PAG and NRM that are in part opiate-related. The observations add to the accumulating evidence that the raphe system is implicated in functions other than pain and its control, and they may also be relevant to clinical observations of opiate-induced effects on respiration and the cough reflex.