David A. Bereiter

Excitatory amino release within spinal trigeminal nucleus after mustard oil injection into the temporomandibular joint region of the rat

&NA; Inflammation of the temporomandibular joint (TMJ) region evokes pain and hyperalgesia as well as causing persistent changes in the response properties of central trigeminal neurons. To determine if excitatory amino acids have a role in TMJ‐induced responses, extracellular concentrations were measured in microdialysate samples from probes positioned in the spinal trigeminal nucleus (Vsp) near the transition region between subnucleus interpolaris and subnucleus caudalis (Vi/Vc) in chloralose‐anesthetized rats. Injection of the selective small fiber excitant, mustard oil (20 &mgr;l, 20% solution), into the ipsilateral TMJ region caused a transient (by 10 min) increase in glutamate (from 0.48 ± 0.16 to 1.94 ± 0.78 &mgr;M, P < 0.005) and aspartate (from 0.29 ± 0.11 to 1.78 ± 0.82 &mgr;M, P < 0.025) among sites located at the ventrolateral pole of the Vi/Vc transition region (n = 6). Samples from probes located within the ventral Vsp, but outside this Vi/Vc transition region (n = 9), did not show significant changes in amino acid concentrations. Glutamate and aspartate also increased after mustard oil injections into the contralateral TMJ region. Dialysate concentrations of serine and taurine did not change significantly after mustard oil injections. Addition of high potassium (150 mM) to the perfusate solution caused increases in glutamate and aspartate regardless of probe location. The transient and selective release of glutamate and aspartate within the Vi/Vc transition after acute irritation of the TMJ region is consistent with a proposed role for excitatory amino acids in mediating noxious sensory input from deep orofacial structures. Together with previous reports of c‐fos expression, these results suggest that neurons within the ventrolateral portion of the Vi/Vc transition may serve as a relay site for the integration of sensory or reflex responses to acute inflammation of the TMJ region.

Influence of sex on reflex jaw muscle activity evoked from the rat temporomandibular joint.

Injection of glutamate into the rat temporomandibular joint (TMJ) evoked a concentration-dependent increase in jaw muscle activity. We investigated whether there are sex-related differences in glutamate-evoked jaw muscle activity that are mediated by sex hormones and whether prior injection of glutamate into the TMJ alters the magnitude of jaw muscle activity evoked by a subsequent injection of the algesic and inflammatory compound mustard oil (MO) into the TMJ. The magnitude of glutamate-evoked digastric and masseter muscle activity was significantly greater in female than male rats when 1000 mM glutamate was injected into the TMJ. Gonadectomy significantly reduced the magnitude of glutamate-evoked digastric muscle activity in female rats. Treatment of gonadectomized female rats with estrogen (20 microg/day) increased the magnitude of glutamate-evoked digastric muscle activity. Glutamate-evoked jaw muscle activity in gonadectomized and estrogen-treated gonadectomized males was not significantly different from intact males. Prior injection of glutamate over a concentration range of 10-1000 mM significantly increased digastric muscle activity evoked by MO injection into the TMJ 30 min later. In contrast, MO-evoked masseter muscle activity was significantly increased by prior injection of 250 mM glutamate only. There were, however, no sex-related differences in the enhancement of MO-evoked jaw muscle activity by prior injection of glutamate. These findings indicate that there are sex-related differences in glutamate-evoked jaw muscle activity that are dependent on female sex hormones, and increased glutamate concentrations sensitize the TMJ to noxious chemical stimuli.

Caudal portions of the spinal trigeminal complex are necessary for autonomic responses and display Fos-like immunoreactivity after corneal stimulation in the cat

Corneal input to the spinal trigeminal nucleus (Vsp) was assessed by examining Fos-like immunoreactivity (Fos-LI) after chemical irritant stimulation by mustard oil in chloralose-anesthetized cats. The distribution of Fos-LI within the ipsilateral Vsp was bimodal: a dominant group of cells within the superficial laminae at caudal levels of subnucleus caudalis and a second group of cells within the ventrolateral pole of Vsp at obex levels and within the adjacent interstitial islands. Few Fos-positive cells were seen within the Vsp rostral to the mid-portion of subnucleus interpolaris or within the contralateral Vsp. To assess the involvement of caudal portions of the Vsp in mediating the adrenal and autonomic responses to corneal stimulation, mustard oil was applied before and after lidocaine blockade of the Vsp at obex levels in a second group of cats. Corneal stimulation alone increased significantly (P < 0.001) the adrenal secretion of catecholamines, adrenal blood flow, mean arterial pressure and heart rate. With the exception of heart rate, the adrenal and autonomic responses to mustard oil were greatly attenuated or abolished by lidocaine blockade of the ipsilateral Vsp at the level of the obex, a region that displayed a high number of Fos-positive cells after corneal stimulation. These results indicate that neurons within the Vsp at or more caudal than the level of the obex process chemical irritant input from the cornea and are necessary for corneal-evoked changes in adrenal and autonomic function.

The NMDA receptor antagonist MK-801 reduces Fos-like immunoreactivity in central trigeminal neurons and blocks select endocrine and autonomic responses to corneal stimulation in the rat

&NA; The N‐methyl‐D‐aspartate (NMDA) receptor is implicated in multiple aspects of pain processing by the central nervous system. However, the role of NMDA receptors in the endocrine and autonomic aspects of nociception remains uncertain. The present study examined the influence of the NMDA receptor antagonist, MK‐801 (0.02–2.0 mg/kg, intracarotid), on the adrenal and autonomic responses to corneal stimulation (mustard oil, 20% sol.) in barbiturate‐anesthetized rats. Fos‐like immunoreactivity (Fos‐LI) evoked by corneal stimulation was quantified within the spinal trigeminal nucleus (Vsp) of MK‐801 pretreated animals to assess activation of central trigeminal neurons. Corneal stimulation‐evoked increases in the plasma concentrations of adrenocorticotropin (ACTH), epinephrine and norepinephrine were reduced dose‐dependently by MK‐801. Plasma ACTH also increased after moderate hemorrhage, a response that was not affected by MK‐801. MK‐801 did not reduce the magnitude of corneal stimulation‐evoked increases in arterial pressure and heart rate; however, prestimulus arterial pressure was reduced by drug treatment. Fos‐LI was distributed bimodally within the ipsilateral caudal Vsp: one peak of Fos‐LI in the subnucleus interpolaris/caudalis transition region and a second peak within the superficial laminae of the subnucleus caudalis/upper cervical cord transition region. The magnitude of both peaks of Fos‐LI was reduced dose‐dependently by MK‐801. These results indicate a significant contribution from NMDA receptors in control of select endocrine and autonomic responses that accompany trigeminal nociception and in activation of central trigeminal neurons that process corneal nociceptive input.

Parabrachial area and nucleus raphe magnus inhibition of corneal units in rostral and caudal portions of trigeminal subnucleus caudalis in the rat.

&NA; The cornea has been used extensively as a means to selectively stimulate trigeminal nociceptive neurons. The aim of this study was to determine the effects of descending modulatory control pathways on corneal unit activity by comparing the effects of conditioning stimulation of the pontine parabrachial area (PBA CS) and nucleus raphe magnus (NRM CS). Electrical stimulation of the cornea at A‐ and C‐fiber intensities was used to activate neurons in two regions of the trigeminal spinal nucleus, the subnucleus interpolaris/caudalis transition (Vi/Vc, ‘rostral units’) and laminae I–II at the subnucleus caudalis/cervical cord transition (Vc/C1, ‘caudal units’), in chloralose‐anesthetized rats. Corneal units were further classified according to convergent cutaneous receptive field properties and PBA projection status. None of 48 rostral and 23/28 caudal units projected to the ipsilateral or contralateral PBA. PBA CS inhibited the cornea‐evoked responses (<75% change from control) of approximately 65% of rostral and caudal units regardless of neuronal class. For rostral corneal units, PBA CS inhibited A‐ and C‐fiber input equally (15±3 and 18±14% of control, respectively), whereas among caudal units, A‐fiber input was inhibited more than C‐fiber input (26±5 and 64±12% of control, respectively, P<0.01). The magnitude of NRM CS inhibition on cornea‐evoked activity of both rostral and caudal units was not different from that seen after PBA CS. Glutamate microinjections into PBA also inhibited rostral and caudal corneal units (6/9 tested). These results indicate that corneal input to rostral and caudal units is modified by activation of descending controls from the PBA and NRM. The significance for processing corneal sensory information is discussed in terms of functional differences between rostral and caudal neurons.

Glutamate activation of neurons within trigeminal nucleus caudalis increases adrenocorticotropin in the cat

&NA; The role of trigeminal nucleus caudalis (Vc) in control of the autonomic and endocrine correlates of nociception was assessed in chloralose‐anesthetized cats. Microinjections of the neuroexcitatory agent, L‐glutamate (0.5 M), were directed at the marginal layers, at the central magnocellular portion, and at the deep magnocellular portion of Vc. Changes in the plasma concentration of adrenocorticotropin (ACTH), in mean arterial pressure, and in heart rate were examined. Glutamate excitation of neurons within the marginal layers of Vc evoked a significant (+143 ± 52 pg/ml, P < 0.01) increase in plasma ACTH during the 10 min postinjection sampling period. Glutamate injections into the deep magnocellular portion of Vc also increased plasma ACTH (+97 ± 28 pg/ml, P < 0.05), whereas activation of neurons in the central magnocellular portion of Vc had no consistent effect on plasma ACTH (−25 ± 29 pg/ml, P > 0.10). Arterial pressure increased transiently after glutamate injections into the marginal layers or central magnocellular portion of Vc, whereas injections into the deep magnocellular portion of Vc did not affect arterial pressure. Heart rate increased transiently regardless of the laminar site of injection within Vc. These data indicate that activation of neurons in laminar regions of Vc that process nociceptive information cause an increase in plasma ACTH, whereas activation of neurons in laminae of Vc that process mainly non‐nociceptive input have no significant influence on plasma ACTH.

Comparison of the influence of rostral and caudal raphe neurons on the adrenal secretion of catecholamines and on the release of adrenocorticotropin in the cat

&NA; Neuroendocrine and autonomic responses were assessed in chloralose‐anesthetized cats after chemical stimulation of medial brain‐stem regions, including those that influence nociceptive input to the medullary or spinal dorsal horn. Microinjections of l‐glutamate (0.5 M, 160 nl) were directed at the following rostral and caudal raphe nuclei: the periaqueductal gray (PAG), the dorsal raphe nucleus (DR), the raphe magnus (RM), and the raphe obscurus/raphe pallidus (Ro/Rpa). Activation of DR neurons evoked a significant increase in the adrenal secretion of epinephrine (+ 2.6 ± 1.1 ng/min, P < 0.01) that returned towards prestimulus values by 6 min, whereas microinjections into other raphe nuclei had no consistent effect. Activation of Ro/Rpa neurons evoked an increase in the plasma concentration of adrenocorticotropin (ACTH, + 47.9 ± 12.3 pg/ml, P < 0.01), whereas microinjections into other raphe nuclei did not affect ACTH. Arterial pressure increased significantly after activation of PAG (+ 7.5 ± 2.1 mm Hg, P < 0.01) or of DR (+ 4.8 ± 2.0 mm Hg, P < 0.05) neurons, whereas heart rate increased significantly (P < 0.05) after stimulation of cells wi the Ro/Rpa. Glutamate microinjections within the RM, a raphe nucleus that exerts a significant descending influence on nociceptive input to the medullary and to the spinal dorsal horns, had no consistent effect on any measured variable. No evidence was seen to suggest that chemical activation of neurons within raphe nuclei inhibited the adrenal secretion of catecholamines or inhibited the release of ACTH. The results indicated that glutamate activation of neurons within different raphe nuclei evoked non‐uniform effects on neuroendocrine and autonomic function. Further, these data suggested that the neural substrate underlying the control of the adrenal secretion of catecholamines and of the release of ACTH in response to activation of raphe neurons is likely distinct from that which contributes to the descending influence on nociceptive input to the medullary and spinal dorsal horn.

Substance P and GABAergic effects on adrenal and autonomic function evoked by microinjections into trigeminal subnucleus caudalis in the cat

To assess the contribution of putative neurotransmitters in mediating changes in adrenal and autonomic function evoked by activation of medullary dorsal horn neurons, microinjections of substance P, bicuculline methiodide, or muscimol were directed at various laminac of trigeminal subnucleus caudalis in the anesthetized cat. Injections of substance P (35.6 pmol) into the superficial layers (lamina I-II) of subnucleus caudalis increased the adrenal secretion of epinephrine (+8.3 +/- 2.3 ng/min, P less than 0.01), arterial pressure (+11 +/- 5.3 mm Hg, P less than 0.01), and heart rate (+19.4 +/- 4.9 beats/min, P less than 0.01) by 1 min, and increased the plasma concentration of adrenocorticotropin (+26 +/- 10 pg/ml, P less than 0.01) by 3 min. Substance P injections into the magnocellular layers (lamina III-IV) or deep magnocellular layers (lamina V-VI) had no significant effects. Microinjections of the GABAA antagonist, bicuculline methiodide (62.4 pmol), into the superficial layers of subnucleus caudalis increased the adrenal secretion of epinephrine (+4.5 +/- 3.2 ng/min, P less than 0.01) by 1 min, whereas injections of the GABAA agonist, muscimol (280 pmol), decreased the secretion (-5.8 +/- 2.8 ng/min, P less than 0.05) by 6 min. Arterial pressure increased after bicuculline (+17.8 +/- 8.2 mm Hg, P less than 0.01) and decreased after muscimol (-6.3 +/- 2.9 mm Hg, P less than 0.01) injections into the superficial layers. Injections of bicuculline or muscimol into the magnocellular layers or into the deep magnocellular layers had no effect on adrenal secretion of catecholamines or on systemic cardiovascular function. Peripheral venous concentrations of adrenocorticotropin were not affected significantly by microinjections of GABAergic agents regardless of the laminar site of injection within subnucleus caudalis. Equivalent volume injections of artificial cerebrospinal fluid into the superficial laminae of subnucleus caudalis had no significant influence on any measured variable. Substance P-evoked changes in the adrenal secretion of epinephrine were not correlated with changes in adrenal venous blood flow, whereas bicuculline- and muscimol-evoked changes in adrenal secretion of catecholamines were positively correlated with changes in adrenal blood flow (P less than 0.01). The results indicate that substance P and GABA contribute significantly to the trigeminal control of adrenal and autonomic function by acting on neurons in the superficial layers of subnucleus caudalis, a brainstem region that processes nociceptive sensory information.

Microinjections of glutamate within trigeminal subnucleus interpolaris alters adrenal and autonomic function in the cat

The influence of rostral portions of the trigeminal sensory complex on adrenal and autonomic function was assessed by microinjections of L-glutamate (500 or 5 mM, 100 nl) directed at subnucleus interpolaris (Vi) or at the nucleus principalis/subnucleus oralis level (Vp/Vo) in chloralose-anesthetized cats. Microinjections of glutamate (500 mM) within Vi evoked prompt (by +1 min) dose-related increases in the adrenal secretion of epinephrine (+11.4 +/- 2.5 ng/min, P < 0.001), adrenal blood flow (+0.19 +/- 0.06 ml/min, P < 0.05), mean arterial pressure (+6.6 +/- 3.0 mmHg, P < 0.025) and heart rate (+8.0 +/- 2.7 beats/min, P < 0.01, n = 16). Microinjections of lower doses of L-glutamate (5 mM, n = 7) within Vi had no effect. Microinjections of 500 mM glutamate within VP/Vo (n = 15) or within the spinal trigeminal tract (n = 13) had no consistent effect on adrenal or autonomic function. Plasma concentrations of ACTH were not altered significantly by glutamate regardless of dose or of the site of injection. The results suggest that local release of glutamate within Vi, but not within Vp/Vo, influences adrenal and autonomic function. Together with previous results obtained after injections of glutamate within subnucleus caudalis, these data indicate that glutaminergic input to both Vi and to more caudal portions of the spinal trigeminal nucleus contribute to the control of autonomic function such as that which often accompanies trigeminal nociception.

Adrenalectomy increases reduced nicotinamide adenine dinucleotide phosphate-diaphorase activity in the rat spinal trigeminal subnucleus caudalis

Neurons exhibiting reduced nicotinamide adenine dinucleotide phosphate-diaphorase activity (NADPHd) were quantified at 500 microns rostrocaudal intervals in spinal trigeminal nucleus (Vsp) of adenalectomized (ADX), ADX + corticosterone, and sham-ADX rats 6-12 days after surgery. NADPHd neurons were found predominantly in Vsp subnucleus caudalis (Vc) and in dorsomedial subnucleus oralis. ADX significantly increased the number of NADPHd neurons in superficial laminae of Vc, an effect reversed by chronic corticosterone replacement. ADX effects on NADPHd in superficial laminae of Vc but not in deep laminae of Vc or in the periobex region of Vsp paralleled previously observed sites of ADX enhancement of noxious stimulus-induced Fos-like immunoreactivity. The results indicate that chronic changes in adrenal steroid status regulate NADPHd, a mechanism that may both derive from changes in nitric oxide synthase expression and influence the processing of nociceptive information by central trigeminal neurons.