Organization of the efferent projections from the spinal cervical enlargement to the parabrachial area and periaqueductal gray: a PHA-L study in the rat.

The organization of efferent projections from the spinal cervical enlargement to the parabrachial (PB) area and the periaqueductal gray (PAG) was studied in the rat by using microinjections of Phaseolus vulgaris-leucoagglutinin (PHA-L) into different laminae around the C7 level. The results demonstrated two areas of cervical enlargement which project in different ways to the PB area and PAG. First, the superficial laminae (I, II) showed a very dense projection, with a clear contralateral dominance at the coronal level where the inferior colliculus merges with the pons, to a restricted "superficial" portion of the PB area, namely the lateral crescent area, the dorsal lateral, the superior lateral (PBsl), and the outer portion of the external lateral PB subnuclei. Less dense projections were observed in the Kölliker-Fuse nucleus (KF) and in the ventrolateral/lateral quadrant of the caudal and mid PAG. By contrast, the labeling was weak or absent in the other PB subnuclei and the outer adjacent regions; in particular, no, or very little, labeling was found in the cuneiform nucleus. The PB area appeared to be the supraspinal target that received the densest projection from laminae I and II. Projections were less dense in the PAG and the thalamus and markedly less in other sites such as the ventrolateral medulla, the subnucleus reticularis dorsalis, and the nucleus of the solitary tract. Second, the reticular portion of lamina V, the medial portion of laminae IV-VI up to X and lamina VIII, showed bilateral projections with a weak ipsilateral dominance and a high to medium density on a very restricted portion of the PB area, namely the internal lateral PB subnucleus. A lesser projection was also observed in the adjacent portion of the PBsl, the KF, and the lateral quadrant of the PAG. These results suggest that signals carried by neurons from lamina I-II converge on a restricted superficial portion of the PB area and the ventral part of the lateral quadrant of the PAG. These results are discussed in the context of the role of the spino-PB and spino-PAG pathways in nociception.

Organization of efferent projections from the spinal cervical enlargement to the medullary subnucleus reticularis dorsalis and the adjacent cuneate nucleus: a PHA-L study in the rat.

The distribution and organization of projections from the spinal cervical enlargement to subnucleus reticularis dorsalis (SRD) and the neighbouring Cuneate nucleus (Cu) area was studied in the rat by using microinjections of Phaseolus vulgaris leucoagglutinin (PHA-L) into different laminae around the C7 level. The Cu received very dense projections from the dorsal horn, with the highest density being observed following injections into the medial part of laminae III-IV. The SRD received dense projections from laminae V-VII of the cervical enlargement, particularly from the reticular and medial aspects of lamina V, lamina VI, and the dorsal part of lamina VII. By contrast, the superficial part of the dorsal horn (laminae I to IV) and the dorsal part of lamina X provided only sparse projections to the SRD. Clusters of labelled terminals and boutons were observed mainly in the SRD areas subjacent to the Cu. In the caudorostral axis, labelled terminals were spread along the whole SRD from the cervicomedullary junction up to the caudal-most part of the area postrema. Contralateral projections to the SRD were scarce and were observed mainly after injections into the medial part of laminae VI-VII. These data give further support to the proposal that there are two parallel systems in neighbouring structures of the caudal medulla, viz. the Cu and the SRD, which, respectively, relay lemniscal and nociceptive information from the spinal cord to the thalamus.

Stimulation of craniofacial muscle afferents induces prolonged facilitatory effects in trigeminal nociceptive brain-stem neurones.

Stimulation of small-diameter afferents supplying deep tissues has been shown to increase the excitability of spinal cord neurones responding to cutaneous afferent inputs. This facilitation has been implicated as integral central mechanisms of deep pain that may contribute to the tenderness and spread and/or referral of pain following injury of deep tissues. In view of the recent documentation of deep craniofacial afferent inputs, as well as cutaneous afferent inputs to the trigeminal (V) spinal tract nucleus, we wished to determine the effects of deep inputs excited by the small-fibre irritant mustard oil on trigeminal nociceptive neurones. The extracellular activity of single brain-stem neurones was recorded in subnuclei caudalis and oralis of the V spinal tract nucleus of anaesthetized rats. The neurones were classified as low-threshold mechanosensitive (LTM), wide dynamic range (WDR) and nociceptive specific (NS) on the basis of their cutaneous mechanoreceptive field properties and their responses evoked by electrical stimulation of their cutaneous afferent inputs. Injection of 5% mustard oil (2-5 microliters) into the deep masseter muscle produced a facilitatory effect in 12 of 27 nociceptive neurones tested in caudalis and in 5 of 12 nociceptive neurones in oralis. This effect was reflected in an expansion of the cutaneous mechanoreceptive field, an increase in spontaneous activity or an increase in responsivity to electrical stimulation of cutaneous afferent inputs to the neurones. The facilitation was reversible and typically became apparent within 3-5 min of the injection, reached its peak at 5-10 min, and lasted for 20-30 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoaryl- and bisaryldihydroxytropolones as potent inhibitors of inositol monophosphatase.

The first successful preparation of mono- and disubstituted 3,7-dihydroxytropolone involves a four-step synthetic scheme. Thus, bromination of 3,7-dihydroxytropolone (8) followed by permethylation of the resultant products furnished gram quantities of intermediates 13-18. Single or double Suzuki coupling reactions between these permethylated monobromo- and dibromodihydroxytropolone derivatives and a variety of boronic acids delivered the expected products whose deprotection yielded the desired compounds 1a-u and 26a-n, usually in fair to good yields. Tropolones 1 and 26 were found to be potent inhibitors of inositol monophosphatase with IC50 values in the low-micromolar range. The results are discussed in the context of the recently described novel mode of inhibition of the enzyme by 3,7-dihydroxytropolones.

Organization of diencephalic projections from the spinal trigeminal nucleus oralis: an anterograde tracing study in the rat.

The organization of the efferent projections from the spinal trigeminal nucleus oralis (Sp5O) to the diencephalon was studied in the rat using the anterograde tracer Phaseolus vulgaris leucoagglutinin. The present study confirms the existence of trigemino-thalamic pathways originating from the Sp5O and details their distribution. The main diencephalic targets of the Sp5O are the ventral posteromedial thalamic nucleus (VPM), the posterior thalamic nuclei (Po) and the ventral part of the zona incerta (ZIv), contralaterally, and the parvicellular part of the ventral posterior thalamic nucleus (VPpc), bilaterally. The distribution of these projections varies according to the dorso-ventral location of the injection sites: the dorsal part of the Sp5O projects to the medial part of the VPM and the Po, and to the caudal part of the ZIv, as well as to the VPpc. The ventral part of the Sp5O projects to the lateral part of the VPM and the Po and to the rostral part of the ZIv. These results suggest that the trigemino-diencephalic pathways originating from the Sp5O are involved in the processing of gustatory and somatosensory information.

Systemic morphine reduces the wind-up of trigeminal nociceptive neurons.

We assessed the effects of intravenous morphine on the wind-up of nociceptive neurons of the spinal trigeminal nucleus oralis (Sp5O). Extracellular recordings of Sp5O nociceptive convergent neurons were performed in intact halothane-anesthetized rats. Wind-up of C-fiber-evoked responses was elicited by repetitive electrical stimulation (train of 16 shocks, 0.66 Hz) of their receptive field at C-fiber intensity (3 times the threshold). Wind-up was tested for its sensitivity to morphine (6 mg/kg,i.v.), and the specificity of the effects was verified with naloxone (0.4 mg/kg, i.v.). Nineteen convergent neurons displaying wind-up were recorded. Morphine reduced the wind-up of all but one. In five cases, notwithstanding a reduced wind-up, the neuronal response evoked by the first stimulus in the train (initial input) was unexpectedly increased. Naloxone always antagonized morphine inhibitory effects on the wind-up. When administered systemically, morphine reduced the wind-up of trigeminal nociceptive neurons. This inhibitory effect occurred independently of morphine's ability to affect the initial C-fiber-evoked input. Our findings support the idea that systemic morphine probably blocks wind-up by acting at opioid receptors located postsynaptically to nociceptive primary afferents.

The rostral part of the trigeminal sensory complex is involved in orofacial nociception.

Single units responsive to noxious mechanical stimulation of orofacial receptive fields were recorded within the ventrobasal complex of the rat thalamus. The induced activities were compared before and after deafferentation of the subnucleus caudalis by a trigeminal tractotomy performed at the obex level. The receptive fields activated by noxious stimulation were classified as 'oral' when included in the oral, perioral or paranasal areas, and as 'facial' when included in facial regions distant from the oral cavity. After tractotomy, the unit responses to noxious stimulation of an oral field remained unchanged in 8 cases, decreased in 3 cases, and were suppressed in 4 cases. For units responding to noxious stimulation of a facial field, the responses were suppressed in 8 cases, decreased in two cases and remained unchanged in two other cases. So it appears that the rostral part of the trigeminal sensory complex (1) receives nociceptive afferents mainly from the oral and perioral areas and (2) is a relay in ascending pathways which convey painful sensations.

Differential effects of trigeminal tractotomy on Adelta- and C-fiber-mediated nociceptive responses.

In this study we have tested in the rat, whether trigeminal tractotomy, which deprives the spinal trigeminal nucleus caudalis (Sp5C) of its trigeminal inputs, affected differentially nociceptive responses mediated by C- vs. Adelta-nociceptors from oral and perioral regions. Tractotomy had no effect on the threshold of the jaw opening reflex, induced by incisive pulp stimulation (Adelta-fiber-mediated), but blocked the formalin response (mainly C-fiber-mediated). These results suggest that nociceptive responses mediated by trigeminal C-fibers completely depend on the integrity of the Sp5C, while intraoral sensations triggered Adelta-fibers (especially of dental origin) are primarily processed in the rostral part of the spinal trigeminal nucleus.

Properties of nociceptive and non-nociceptive neurons in trigeminal subnucleus oralis of the rat.

Recent studies have provided evidence suggesting the involvement of rostral components of the V brainstem complex such as trigeminal (V) subnucleus oralis in orofacial pain mechanisms. Since there has been no detailed investigation of the possible existence of nociceptive oralis neurons in the rat to substantiate this recent evidence, the present study was initiated to determine if neurons responsive to noxious orofacial stimuli were present in subnucleus oralis and to characterize their functional properties. In anesthetized rats, recordings were made of the extracellular activity of single neurons functionally characterized as low-threshold mechanoreceptive (LTM), wide dynamic range (WDR) or nociceptive-specific (NS) neurons. The 342 LTM neurons responded only to light mechanical stimulation of orofacial tissues. The mechanoreceptive field of the LTM neurons included the intraoral region in 28% and was localized to the adjacent perioral area in 65%. For 95% the field was localized within one V division. Responses evoked in LTM neurons by electrical stimulation of the orofacial mechanoreceptive field revealed A fiber afferent inputs but no activity that could be attributed to C fiber afferent inputs. The 72 nociceptive neurons included 52 WDR neurons which responded to light (e.g. tactile) as well as noxious (e.g. heavy pressure; pinch) mechanical stimulation of perioral cutaneous and intraoral structures, and 20 NS neurons which responded exclusively to noxious mechanical stimuli. They also differed from the LTM neurons in that 36% of the WDR and 20% of the NS neurons had a mechanoreceptive field involving more than one V division. However, in accordance with our findings for the LTM neurons, the majority of WDR and NS neurons had a mechanoreceptive field involving the intraoral and perioral representations of the mandibular and/or maxillary divisions; those neurons having a mandibular field which especially included intraoral structures predominated in the dorsomedial zone of subnucleus oralis whereas those with a perioral mechanoreceptive field which particularly involved the maxillary division were concentrated in the ventrolateral zone of oralis. In contrast to the LTM neurons, 57% of the WDR and 67% of the NS neurons showed evidence of electrically evoked C fiber as well as A fiber afferent inputs from their mechanoreceptive field. We also noted suppression of the electrically evoked responses by heating of the tail or pinching of the paw. This effect was considered to be a reflection of diffuse noxious inhibitory controls, and was seen in NS as well as WDR neurons; most, but not all, of these neurons received A fiber as well as C fiber orofacial afferent inputs.(ABSTRACT TRUNCATED AT 400 WORDS)

Localisation of the first-order neurone of the jaw opening reflex elicited by periodontal stimulation.

The localisation of the first-order neurone of the jaw-opening reflex (JOR), provoked by periodontal stimulation, was investigated in the rat. A section of the mandibular part of the trigeminal ganglion was carried out without impairing the motor root. It suppressed the reflex triggered by the stimulation of the lower incisor. Bilateral destruction of the mesencephalic nucleus and tract does not modify the JOR. These results suggest that the first-order neurone of the reflex is located in the trigeminal ganglion.

Inhibition of platelet function by administration of MRS2179, a P2Y1 receptor antagonist.

The effects of a potent P2Y1 receptor antagonist, N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179) on adenosine-5'-diphosphate (ADP)-induced platelet aggregation in vitro, ex vivo and on the bleeding time in vivo were determined. In suspensions of washed platelets, MRS2179 inhibited ADP-induced platelet shape change, aggregation and Ca2+ rise but had no effect on ADP-induced inhibition of adenylyl cyclase. Binding studies using the new radioligand [33P]MRS2179 showed that washed human platelets displayed 134+/-8 binding sites per platelet with an affinity (Kd) of 109+/-18 nM. Finally, intravenous injection of MRS2179 resulted in inhibition of rat platelet aggregation in response to ADP and prolonged the bleeding time, in rats or mice, as compared to controls. These results suggest this potent P2Y1 receptor antagonist to be a promising tool to evaluate the in vivo effects of pharmacologically targeting the P2Y1 receptor with a view to antithrombotic therapy.

Application of the formalin test to the study of orofacial pain in the rat.

A modification of the formalin test for assessing pain and analgesia in the orofacial region of the rat is described. A formalin solution (5%) was subcutaneously injected into the upper lip, then the length of time the animal spent rubbing the injected zone was recorded. Two distinct periods of intensive rubbing activity were identified: an early phase between 0 and 3 min after the injection and a late phase between 18 and 42 min after the injection. Acetylsalicylic acid, paracetamol and morphine all had an antinociceptive effect during the two phases although incomplete during the early phase. Our results indicate that this orofacial formalin test is a valid technique for the study of orofacial pain.

Responses of trigeminal subnucleus oralis nociceptive neurones to subcutaneous formalin in the rat.

Extracellular recordings of 33 single nociceptive neurones of the trigeminal subnucleus oralis (SNO) were made in rats under halothane nitrous oxide anaesthesia. These neurones were tested for their responses to a s.c. injection of formalin in their receptive field. Such a chemical noxious stimulation is known to induce a biphasic response of nociceptive dorsal horn neurones, the second period of which would be due to inflammation. Twenty-three neurones were characterized as nociceptive non-specific (NnS) and 10 as nociceptive specific neurones (NS). Formalin activated both SNO NS and NnS neurones, but, when they responded, NS neurones (n = 5) showed only the first phase of activity while NnS neurones showed either one (n = 13) or two phases (n = 6). Biphasic responses were most often observed for NnS neurones with A delta- and C-fibre inputs. These results indicate that the time-course similarity between the behavioural and the neuronal responses to formalin exists only for some SNO convergent neurones and that therefore the SNO does not seem to be very involved in the inflammatory component of the pain caused by formalin.

Responses of neurones in the ventrobasal complex of the thalamus to orofacial noxious stimulation after large trigeminal tractotomy.

Single units were recorded, using extra-cellular glass microelectrodes, in the ventrobasal complex of the thalamus of rats under halothanenitrous oxide anaesthesia. The animals had previously undergone a large bilateral section of the trigeminal sensory complex just above the obex to deprive the caudal part of the trigeminal sensory complex (subnucleus caudalis) of its trigeminal afferents. As observed on frontal slices our lesions impaired the whole descending tract and, in most cases, the intratrigeminal pathways between the rostral and the caudal part of the complex. Forty-seven units responding to a somatic mechanical noxious stimulation applied to the trigeminal area were recorded in these conditions. Forty-two of these had a receptive field (or at least a part of it) in or around the oral and nasal cavities, and 5 in the peripheral part of the face. These data confirm the hypothesis that the rostral part of the trigeminal sensory complex participates in pain sensory pathways, as a first relay site between nociceptive primary afferents coming from oral, perioral and perinasal areas, and the ventrobasal complex of the thalamus. In addition, they suggest that the intratrigeminal pathways are not essential for the transmission of these nociceptive inputs, to the lateral thalamus.

MK-801 neurotoxicity in the guinea pig cerebral cortex: susceptibility and regional differences compared with the rat.

N-methyl-D-aspartate (NMDA) receptor antagonists induce transient vacuole formation in neurons of the retrosplenial cortex and, after higher doses, necrosis in the same region. To our knowledge, all studies demonstrating these effects have been carried out in rats or mice. The present study investigated whether vacuolization occurs in the guinea pig, rats being used as controls. Female Dunkin-Hartley guinea pigs (age 15-18 weeks) were given a single subcutaneous injection of saline or the non-competitive NMDA antagonist dizocilpine maleate [(+)-MK-801; 1, 4, or 12 mg/kg]. Female Sprague-Dawley rats (age 16 weeks) received saline or MK-801 (1 mg/kg). Whatever the dose of MK-801, guinea pigs showed only occasional vacuolated neurons in the retrosplenial cortex. However, affected neurons (mainly large pyramidal cells of layer V) were found in the frontoparietal neocortex. The reaction was limited after 1 mg/kg, and seemed to reach a maximum at 4 mg/kg. Rats injected with 1 mg/kg MK-801 showed an intense vacuole reaction in neurons from layers III-IV of the retrosplenial cortex, but no affected neurons were noted in neocortical areas. We conclude that there are significant species differences in susceptibility to, and location of, vacuolization induced by NMDA receptor antagonists.

[Participation of the rostral portion of the spinal nucleus of the trigeminal sensory complex in nociception]. / Participation de la partie rostrale du noyau spinal du complexe sensitif du trijumeau à la nociception.

Single unit responses elicited through noxious mechanical stimulation of orofacial receptive fields were recorded, with glass micro-electrodes, within the rat ventrobasal complex of the thalamus. The evoked activities were compared before and after de-afferentation of the caudal sub-nucleus by trigeminal tractotomy at the level of the obex. Only units responding to noxious stimulation of oral receptive fields were unaffected by tractotomy. These results provide evidence that the rostral part of the spinal nucleus is involved in trigeminal painful sensation.

Effects of subcutaneous formalin on the activity of trigeminal brain stem nociceptive neurones in the rat.

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)

Anti-TNF-alpha properties of new 9-benzyladenine derivatives with selective phosphodiesterase-4- inhibiting properties.

In inflammatory cells, intracellular cAMP concentration is regulated by cyclic nucleotide phosphodiesterases 4. Therefore, PDE4 inhibition appears as a rational goal for treating acute or chronic inflammatory diseases. Selective PDE4 inhibitors have been developed, but due to unwanted side effects, search for new selective PDE4-inhibitors had to be pursued. Recently, Boichot et al. (J. Pharmacol. Exp. Ther. (2000) 292, 647-653) showed that 9-benzyladenine derivatives are selective PDE4 inhibitors. In vivo data in animals suggested that they may induce fewer side effects (emesis). We examined the effects of new 9-benzyladenines on TNF-alpha, interleukin (IL)-1beta, IL-6 and IL-8 production by lipopolysaccharide-activated peripheral blood mononuclear cells, and compared them to other PDEs inhibitors. Selected potent 9-benzyladenines, strongly inhibited TNF-alpha production. Interleukin-1beta, IL-6, and IL-8 production was not significantly affected. Our results suggest that some of these new adenines (i.e., NCS 675 and NCS 700), may be potential therapeutic candidates for the treatment of inflammatory diseases.