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Sensory sciatic nerve afferent inputs to the dorsal lateral medulla in the rat
Autonomic Neuroscience: Basic and Clinical 140 (2008) 80 – 87 www.elsevier.com/locate/autneu
Sensory sciatic nerve afferent inputs to the dorsal lateral medulla in the rat☆ Olavo Egídio Alioto a , Charles Julian Lindsey a , Janice Koepp a,b , Cristofer André Caous a,c,⁎ a
b
Department of Biophysics, Universidade Federal de São Paulo, Brazil Department of Pharmacology, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil c Instituto Israelita de Ensino e Pesquisa Albert Einstein, São Paulo, SP, Brazil Received 21 November 2007; received in revised form 11 April 2008; accepted 15 April 2008
Abstract Investigations show the paratrigeminal nucleus (Pa5) as an input site for sensory information from the sciatic nerve field. Functional or physical disruption of the Pa5 alters behavioral and somatosensory responses to nociceptive hindpaw stimulation or sciatic nerve electrostimulation (SNS), both contralateral to the affected structure. The nucleus, an input site for cranial and spinal nerves, known for orofacial nociceptive sensory processing, has efferent connections to structures associated with nociception and cardiorespiratory functions. This study aimed at determining the afferent sciatic pathway to dorsal lateral medulla by means of a neuronal tract-tracer (biocytin) injected in the iliac segment of the sciatic nerve. Spinal cord samples revealed bilateral labeling in the gracile and pyramidal or cuneate tracts from survival day 2 (lumbar L1/L2) to day 8 (cervical C2/C3 segments) following biocytin application. From day 10 to day 20 medulla samples showed labeling of the contralateral Pa5 to the injection site. The ipsilateral paratrigeminal nucleus showed labeling on day 10 only. The lateral reticular nucleus (LRt) showed fluorescent labeled terminal fibers on day 12 and 14, after tracer injection to contralateral sciatic nerve. Neurotracer injection into the LRt of sciatic nerve-biocytin-treated rats produced retrograde labeled neurons soma in the Pa5 in the vicinity of biocytin labeled nerve terminals. Therefore, Pa5 may be considered one of the first sites in the brain for sensory/nociceptive inputs from the sciatic nerve. Also, the findings include Pa5 and LRt in the neural pathway of the somatosympathetic pressor response to SNS and nocifensive responses to hindpaw stimulation. © 2008 Elsevier B.V. All rights reserved. Keywords: Nociception; Sciatic innnervation field; Primary afferent fibers; Spinal trigeminal tract; Neuronal tract-tracing; Lateral reticular nucleus
1. Introduction Cardiovascular modulatory mechanisms linked to baroreflex (Balan et al., 2004; Caous et al., 2004; Possas et al., 2001; Reis et al., 1989) and somatosensory responses to nociceptive sciatic nerve electrostimulation in the dorsallateral medulla (Yu et al., 2002) have been localized to a ☆
small collection of neurons (Phelan and Falls, 1989; ChanPalay, 1978a,b) immersed in a neuropil area of the spinal trigeminal tract, the paratrigeminal nucleus (Pa5). Briefly, Pa5 receives primary afferents from spinal, trigeminal, vagus and glossopharyngeal nerves, and presents neuroanatomical efferent projections to other medullary and diencephalic nuclei (Buck et al., 2001; Caous et al., 2001) related to
Grant sponsors: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). Dr. Charles Lindsey is a recipient of a “Bolsa de Produtividade em Pesquisa 1” from the Conselho Nacional de Pesquisa e Desenvolvimento Tecnológico (CNPq). Dr. Cristofer Caous is a neuroscientist researcher at Albert Einstein Hospital and Mr. Alioto is a FAPESP PhD degree student fellow. ⁎ Corresponding author. Universidade Federal de São Paulo, Departamento de Biofísica, Rua Botucatu, 862 7° andar, 04023-062 São Paulo, SP, Brazil. Tel.: +55 11 5576 4530x206; fax: +55 11 5571 5780. E-mail address: [email protected] (C.A. Caous). 1566-0702/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2008.04.006
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cardiovascular functions and or nociception as the lateral reticular nucleus, nucleus of the solitary tract (NTS), the rostral ventral lateral reticular nucleus (RVL), parabrachial nuclei and the ventral posterior medial thalamic nucleus (VPM). This connectivity, suggesting an important role of the paratrigeminal nucleus in pain and mediation of cardiovascular reflexes (Yu and Lindsey, 2003), called for further investigations. Other findings provide substantial evidence for a role of the paratrigeminal nucleus in mediation of nociceptive inputs from the innervation area of the sciatic nerve. Bradykinin and opioids in the paratrigeminal nucleus, but not tachykinins, were shown to play a role in the mediation or modulation of the somatosensory response to sciatic nerve stimulation (Koepp et al., 2005; Lindsey et al., 1997; Lopes and Couture, 1997). Kinins (Corrêa and Graeff, 1975; Davis and Dostrovsky, 1988; Fior et al., 1993; Couture and Lindsey, 2000) and opioids (Arvidsson et al., 1995a,b; Mansour et al., 1996) are known peptide mediators of central or peripheral nociceptive pathways. Furthermore, neurochemical lesioning of the nucleus selectively alters behavioral responses to nociceptive stimulation to the contralateral hindpaw (Koepp et al., 2006) while the same lesions impair the somatosensory response to contralateral sciatic nerve (Caous et al., accepted for publication). Peripheral nociceptive stimulation elicits profound effects on somatic and autonomic activity including adjustments of both ventilation and circulation in mammalian and other species. Associated with somatic motor behaviors such as withdrawal, fight or flight reactions, all important for survival, the visceral reflexes apparently represent early stages in preparation for the appropriate behavioral responses. These, moment to moment, fast visceral responses are mostly mediated by spinal or medullary reflex arcs. The orofacial-derived nociceptors evoked cardiovascular and respiratory responses are of large intensity and foremost behavioral importance and, in some cases, are clinically significant (Andersen, 1996; Dellow and Morgan, 1969; Angell-James and Daly, 1972; Kumada et al., 1977; Mochizuki et al., 1989; Allen and Pronych 1997; Boscan and Paton, 2001). Accumulated evidence suggest that paratrigeminal nucleus, located in the dorsal lateral medulla is implicated in the processing of orofacial nociceptive information (Marfurt, 1981; Salt and Hill, 1983; Arvidsson and Thomander, 1984; Marfurt and Turner, 1984; Cechetto et al., 1985; Panneton and Burton, 1981, 1985). Although these studies mostly focused on neuroanatomical aspects, they provide important evidence suggesting that Pa5 acts as a primary termination site for orofacial nociception afferents. The Pa5 presents neuroanatomical efferent projections to other medullary, pontine and diencephalic nuclei associated with nociception, thermoregulation and cardiorespiratory responses (Buck et al., 2001; Caous et al., 2001). Many experiments provide functional evidence for a role of the Pa5 in nociception. Chemical or mechanical stimulation of tongue nociceptors (Carstens, 1995; Strass-
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man et al., 1993), laryngopharyngeal mucosa (Boucher et al., 2003) or hypoglossal nerve (Bereiter et al., 2000) upregulates c-Fos expression in Pa5 neurons. However the phenomenon is not restricted to orofacial nociceptive stimulation, since infusion of cyclophosphamide into the urinary bladder, an experimental model of visceral pain, also upregulates c-Fos in the Pa5 (Bon et al., 1997, 1998). Recent evidence suggests that the Pa5 may also represent an input site for sensory afferents of the sciatic nerve innervation field since physical or functional disruption of the Pa5 alters behavioral and somatosensory responses to nociceptive hindpaw stimulation or sciatic nerve electrostimulation (Koepp et al., 2005, 2006; Yu et al., 2002; Caous et al., accepted for publication). In establishing the pathway taken by the nociceptorsevoked cardiorespiratory or behavioral responses, the location of the first brain synapse is of particular importance in that the entire reflex arc may be deciphered. Although the collected evidence suggesting that the Pa5 may represent one of the first input sites of the brain for ascending nociceptive information is more than compelling the neuroanatomical substrates for such an assumption was lacking. Despite the excellent match among physiological, pharmacological and behavioral data regarding hind paw nociceptive mechanisms the neuroanatomical pathway had yet to be complemented or supplemented. Aiming at a comprehensive description of the sciatic medullary pathway a double label anterograde and retrograde tract tracer approach was used to: 1) identify the medullary input site to the medulla, the first brain synapse of the pathway; 2) follow the ascending spinal route of the sciatic afferent input to the medulla and 3) identify the projection site (second synapse) of the first order paratrigeminal nucleus neuron in the reflex arc. 2. Methods 2.1. Animal care and handling The care and handling, housing, surgical procedures and research protocols were conformed to the guidelines principles for animal experimentation as enunciated by the International Association for the Study of Pain and also approved by the internal Ethics Committee of the Universidade Federal de São Paulo, UNIFESP. All animals received food and water ad libitum and after surgery care was taken to ensure that animals did not suffer at any stage in the experimental procedure. 2.2. General procedures Three-month-old male normotensive Wistar (Wistar EPM-1 strain) rats weighing 280–300 g were anaesthetized with thiopental 20 mg kg− 1 and chloral hydrate 300 mg kg− 1, i.p. Animals that did not attain an adequate anaesthesia level within the first 7–10 min after anesthetic administration
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were not used. The level of anaesthesia was verified before and during surgery by evaluating vibrissa movement, tail tonus, breathing rate and response to external stimuli. This
protocol ensured that animals remained under complete anaesthesia for at least 60 min. They received an analgesic (sodium diclofenac, 3 mg/kg/day; Medley S/A, Campinas,
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SP, Brazil), and local anesthetic (2% xylocain solution; Astra-Zeneca, Cotia, SP, Brazil) was applied to the incision surfaces before suture and a wide spectrum of veterinary antibiotic was administered by intramuscular route. All animals were individually housed in 30 × 20 × 20 cm plastic cages for recovery. 2.3. Nomenclature and coordinates Stereotaxic coordinates and nomenclature for structures used in this paper refer to The Rat Brain Stereotaxic Atlas published by Paxinos and Watson (1986, 1997). The stereotaxic coordinates adapted for the lateral reticular nucleus, referenced to stereotaxic zero: − 4.3 mm anterior– posterior, ± 1.8 mm lateral and 0.4 mm vertical.
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(model 900, David Kopf, Tujunga, CA, USA), the neck was flexed to − 11 mm in the incisor bar. The occipital bone and the atlanto-occipital membrane were exposed by incision and dissection of the skin and along the dorsal midline muscles of the neck. The medulla oblongata was exposed after the removal of the occipital bone over the posterior cerebellum, and the meninges cut to expose the dorsal and dorsal lateral surfaces of the caudal medulla. Five µl of 2% w/v of fluorogold were delivered to the LRt with a glass micropipette connected to a 1.0-µl precision syringe (Hamilton, Reno, NE, USA) l. The glass micropipette was lowered to the target of interest and injection volume was delivered over a one minute period with the pipette kept in place for 5 min to avoid tracer spread. After surgery the animals, individually housed in plastic cages, were kept for an additional 4 day survival period.
2.4. Surgery for neurotracer injection in the sciatic nerve 2.6. Histological procedures The anterograde transsynaptically transport (Izzo, 1991; King, 1989) tract-tracer biocytin (Sigma, St. Louis, MO, USA) was applied to the sciatic nerve of anaesthetized animals. The iliac segments of the sciatic nerves of 36 anaesthetized animals (A05-A37) were accessed on the lateral aspect of the limb and isolated from surrounding tissue. With a 15 µm (tip diameter) glass micropipette, connected to a 1.0-µl precision syringe (Hamilton, Reno, NE, USA), 5 µl of 10% w/v biocytin, suspended in 0.05 M Tris buffer (pH = 7.5), was delivered below the sciatic nerve sheath. Five minutes was acceptable for absorption into the nerve helped by carefully flexing and extending the limb. The dissection and biocytin injection was done with the help of a surgical microscope (Carl Zeiss, Germany), and the preparation was observed to ensure that no leakage occurred. The animals were then split in distinct survival time groups, from 0 to 20 days at 48 hour intervals.
Following planned survival periods all animals were deeply anaesthetized with 80 mg kg− 1 i.p., of sodium pentobarbital and perfused transcardially with 200 ml of 0.9% saline and 200 ml of cold 4% paraformaldehyde solution in 0.1 M phosphate-buffered saline (PBS). Brains were removed and overnight cryoprotected in a 4% paraformaldehyde with 30% sucrose solution. Coronal and serial sections were cut at 40 µm on a HM 500 cryostat (Microm, Waldorf, Germany). Biocytin experimental slices were incubated for 24 h in a 1:1500 streptavidin-FITC conjugate (Sigma) in 0.2 M PBS. Histological slices were treated in PBS (0.1 M) three times for 10 min and mounted on gelatin-coated slides. The slides were coverslipped with Fluoromount G (Electron Microscopy Science, Washington, USA) for analysis. 2.7. Data analysis
2.5. Double-labeling experiments The neuronal fluorescent retrograde transport (Schmued and Fallon, 1986) tracer Fluoro-gold (Lumafluor, Englewood, CO, USA), dissolved in distilled water (2% w/v) was microinjected (0.5 µl) in the medial lateral reticular nucleus of two anaesthetized 8 day sciatic nerve-biocytin-injected animals. With the rat proned on the stereotaxic apparatus
Animals with injection sites centered in sciatic nerve and/ or LRt were included in analysis. Images were captured on an Axiovert 135 microscope (Carl Zeiss, Germany) equipped with fluorescent epi-illumination coupled to a Metamorph digitalize system (Universal Imaging, West Chester, PA, USA). Double-labeling experiments were also analyzed under a confocal laser scanning microscope.
Fig. 1. Time course of spinal cord and medullary labeling after application of an anterograde neuronal tract-tracer to the iliac segment of the sciatic nerve. Panel A shows labeling of the paratrigeminal nucleus on days 10 to 20 following application of the contralateral sciatic nerve and bilateral labeling of the nucleus on day 10. The lateral reticular nucleus was labeled on days 12 to 14 contralateral to the side of the tract-tracer application to the sciatic nerve. The photomicrographs insets in the columns at the far left show (clockwise from the bottom): (i) transynaptic fluorescent labeling of synaptic buttons, nerve terminals and varicosities in the lateral reticular nucleus resulting from tract-tracer application to the sciatic nerve; (ii) confocal laser microscopy imaging of Pa5 neuron cell bodies labeled either by biocytin injected in the sciatic nerve (green) or fluoro-gold microinjected in the lateral reticular nucleus; (iii) injection site of the retrograde neurotracer fluoro-gold in the lateral reticular nucleus. Panel B shows labeling of the spinal cord at different survival periods following tract-tracer microinjection in the sciatic nerve. Labeling of the lateral spinal nucleus at lumbar spinal cord L3, ipsilateral to the sciatic nerve tracer injection; bilateral labeling of the gracile fascicule from lumbar to cervical spinal cord (L1 to C3) from days 2 to 8; pyramidal tract (L1 to T10) from days 2 to 6 and the cuneiform fascicule in the cervical spinal cord (C3) on day 8. Abbreviations: cc, central channel; Ecu, external cuneate nucleus; cu, fascicule cuneiform; gr, gracile fascicule; Lsp, lateral spinal nucleus; py, pyramidal tract; Sp5I, spinal trigeminal nucleus interpolar; sp5, spinal trigeminal tract; LRt, lateral reticular nucleus; Pa5, paratrigeminal nucleus; Fg, fluoro-gold; Bct, biocytin. Bars: 100 μm in medulla and spinal cord photomicrographs; 25 μm in i and ii; 200 μm in iii and 50 μm in iv-vi. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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3. Results
3.2. Medullary labeling
3.1. Spinal cord labeling
Transverse medullary sections showed labeling of the Pa5 and the lateral reticular nucleus. The labeled structures were nerve terminals and neuron cell bodies in the paratrigeminal nucleus nerve terminals exclusively in the lateral reticular nucleus. The most intense labeling was observed in the paratrigeminal nucleus 10 to 18 days after application of the neuronal tract-tracer to the contralateral sciatic nerve. The ipsilateral Pa5 showed faint labeling only on sections taken from animals 10 days after biocytin application to the sciatic nerve and by day 20 only faint fluorescent labeling was discernable in the contralateral Paratrigeminal nucleus. Labeling was also observed in the lateral reticular nucleus on days 12 and 14 following tract-tracer-injection to the contralateral sciatic nerve (Fig. 1A and Table 1).
Biocytin, applied to the iliac segment of the sciatic nerve produced ascending labeling of the lateral spinal nucleus, the gracile fascicule, the pyramidal tract and the cuneiform fascicule. The labeled spinal structures were mostly fiber clusters with the exception of the lateral spinal nucleus contained labeled neuron cell bodies. Labeling of the spinal cord time related. From 48 h of tracer administration florescent labeling was observed in the spinal lateral nucleus at the level of lumbar L3 vertebra, ipsilateral to the tract-tracer injection side, and in both ipsi- and contralateral the gracile fascicule and pyramidal tract at L2 and L1 vertebrae levels (Fig. 1B). The pyramidal tract showed symmetrical ipsilateral labeling at L1/L2 to T8 but not at T5. The gracile fasciculus showed bilateral asymmetric labeling from L1 to the cervical spinal cord segments C2/C3 on survival days 2 to 8 (Table 1). In the cervical spinal cord labeled fibers were also present in the cuneiform fasciculus disposed as a lamina on the medial aspect of the posterior intermediate septum. Tract-tracer transport distance rate was measured and the tracer traveled from lumbar segments L1 to cervical segment C2 after the injection into the sciatic nerve. The conduction speed was estimated in approximately 0.5 mm hr− 1 (0.49± 0.2 mm hr− 1) for both the gracile fascicule and the pyramidal tract independent of the laterality relative to the injection site.
3.3. Double-labeling of the paratrigeminal nucleus The microinjection of a retrograde transport tracer, fluorogold, in the medial reticular nucleus (LRt) of sciatic nerve biocytin-treated rats produced double labeling of the caudal paratrigeminal nucleus. Confocal laser microscopy imaging (Figs. 1A and 2) showed retrogradely labeled neuron soma in the caudal region of the Pa5 in the immediate vicinity of biocytin-labeled terminal axons suggesting the presence of synaptic vesicles in varicosities. Some neurons soma showed double labeling with fluoro-gold and biocytin (Fig. 2). 4. Discussion
Table 1 Survival period, labeled structures and density measurement of transverse spinal cord and medullary brainstem histological sections after biocytin microinjections in the sciatic nerve (n = 31) Survival Labeled structures period (days) Spinal cord
0 2 2 2 4 4 6 6 8 8 Medulla 10 12 12 14 14 16 18 20
None lateral spinal nucleus (L3) gracile fasciculus (L2/L1) pyramidal tract (L2/L1) gracile fasciculus (T10/T9) pyramidal tract (T10/T9) gracile fasciculus (T5/T4) pyramidal tract (T5/T4) gracile fasciculus (C3/C2) cuneate fasciculus (C3/C2) paratrigeminal nucleus paratrigeminal nucleus lateral reticular nucleus paratrigeminal nucleus lateral reticular nucleus paratrigeminal nucleus paratrigeminal nucleus paratrigeminal nucleus
Density of fluorescent labeling (relative units) Ipsilateral Contralateral 0 136 ± 7* 78 ± 14 140 ± 7 128 ± 6 120 ± 13 79 ± 6 89 ± 3 52 ± 4 44 ± 2 53 ± 12 0 0 0 0 0 0 0
0 0 142 ± 3 79 ± 14 157 ± 2* 124 ± 14 100 ± 6* 100 ± 3* 119 ± 10* 49 ± 1 133 ± 1* 122 ± 1* 136 ± 4* 107 ± 1* 118 ± 3* 93 ± 4* 77 ± 2* 21 ± 2*
Values are given as means ± standard error. Variance Analysis with comparison between means by Newman–Keuls posthoc test. *Differs statistically, p b 0.05.
The present investigation identified sensory sciatic nerve input to the paratrigeminal nucleus, in the dorsal lateral medulla, and a relay to the lateral reticular nucleus (LRt) in the caudal ventral lateral medulla. These findings provide data on the anatomical substrate of the nocifensive and somatosensory responses to nociceptive stimuli to the rat hindpaw or sciatic nerve. Further than furnishing a striking parallel to the behavioral and physiological data, the impact of these findings bring to light an undisclosed view on the brain handling of reflex responses to nociceptive input from the sciatic nerve innervation field. Firstly, the sciatic input to the medulla is directed to the caudal segment of the paratrigeminal nucleus that in turn contains neurons projecting to the LRt (Caous et al., 2001). The paratrigeminal nucleus, located in the dorsal lateral medulla, is commonly known as an input site for orofacial nociceptive inputs of the trigeminal nerve (Boscan and Paton, 2001). Thus, this manuscript and related publications (Koepp et al., 2006; Caous et al., accepted for publication) bring the focus to the dorsal lateral medulla in the handling of nociceptive input. Another interesting finding is that the LRt receives input from high order neurons of paratrigeminal nucleus, suggesting that the reflex arc handling behavioral and cardiovascular responses to nociceptive stimulation from the hindpaw differs from that of the baroreflex arc that comprises the paratrigeminal nucleus/rostral ventral lateral reticular nucleus (RVL) pathway
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Fig. 2. Confocal microscopy imaging (A–D) from 4 adjacent rat transverse medullary sections (40 µm — animal A37) of biocytin-labeled nerve terminals, synaptic buttons (yellow arrows) and varicosities (blue arrows) in the paratrigeminal nucleus. Biocytin tract-tracer microinjection site was the contralateral sciatic nerve (all in green pseudo color). In red, labeled neuron soma ensuing from retrograde transport of fluoro-gold microinjected in the lateral reticular nucleus and in yellow, a double-labeled (biocytin and fluoro-gold) neuron soma (white arrows) is shown. Bars: 25 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
(Caous et al., 2004; Balan et al., 2004; Dampney et al., 1982). This finding also contradicts earlier reporting (Stornetta et al., 1989) suggesting the RVL as an exclusive component of the somatosensory reflex arc. The lateral reticular nucleus is related to the processing of nociception (Janss and Gehart, 1988; Liu et al., 1989; Sotgiu, 1990; Robbins et al., 2005; Leite-Almeida et al., 2006) and homoeostatic cardiovascular reflexes (Tian and Hartle, 1994) and thus may well be involved in the somatosympathetic response to sciatic nerve stimulation. Also, the present data imply that the Pa5/lateral reticular nucleus pathway may mediate the descending modulatory influences on nocifensive responses to hindpaw stimulation (Koepp et al., 2006) although the participation pontine or thalamic structures that receive paratrigeminal nucleus inputs (Caous et al., 2001). Regarding the spinal cord, labeling from the tract-tracer injection to the iliac portion of the sciatic nerve was apparent 48 h later in the lateral spinal nucleus, and in lumbar L3, L2 and L1 segments in the gracile fasciculus and pyramidal tract. The labeling of the gracile fasciculus apparent from the lumbar to cervical spinal cord, always asymmetric, showed higher density on the contralateral side to that of the injected nerve and thus may represent the main input to the paratrigeminal nucleus from the sciatic nerve. The gracile fasciculus that conveys sensory inputs from limbs is more effective than the spinothalamic tract in activating thalamic neurons (Willis and Westlund, 2001). The pyramidal tract on
the other hand was symmetrically labeled from the upper lumbar level to the higher thoracic level (T5/T4). At the cervical spinal cord the pyramidal tract did not show labeling. Instead, bilateral symmetric labeling was observed in the cuneate fasciculus as in laminae I next to the posterior intermedial septum. The asymmetry of the gracile fascicule and Pa5 labeling most probably reflects the larger number of labeled fibers in the contralateral spinal cord and the larger amount of nerve terminals in the contralateral Pa5. The transport rate of the tract-tracer, estimated at approximately 0.5 mm per hour in the spinal cord, was similar for all tracts independently of the laterality. Considering these kinetics, it would be expected that the tract-tracer would reach the Pa5 in about 230 h after the injection according to the measured distances and that the lateral reticular nucleus be reached 6 to 7 h later and thus be perceptible in medullae sections of 240 hour (10 days) survival group animals. In fact the delay of the lateral reticular nucleus labeling may be explained by the delay imposed by the transsynaptically labeling of the Pa5 neurons projecting to the lateral reticular nucleus. Double-labeled neurons soma by biocytin injected in the sciatic nerve and fluoro-gold in the lateral reticular nucleus assure a monosynaptic connection based on the rate of dye transport between the sciatic nerve inputs to the Pa5 and the lateral reticular nucleus. The assumption that the LRt is labeled via
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a synaptic connection in the Pa5 is strengthened by the fact that biocytin is known to be transported transsynaptically (Izzo, 1991; King, 1989) and may label neuron soma by uptake from extra cellular space (McDonald, 1992). The transsynaptic labeling raises the question as to whether the sciatic-paratrigeminal input is mono or poly synaptic. Labeled cell bodies in the lateral spinal nucleus suggest that the first synapse of the sciatic fibers occur in the lower spinal cord. Along the gracile, pyramidal or cuneiform tracts there was no clear evidence of labeled cell bodies or nerve terminals and thus it is possible that there are only two synapses in this neuronal pathway. The labeling patterns in the spinal cord and medulla, as a result of tract-tracer application to the sciatic nerve, bring to view a different pattern from other models of nociceptive input pathways involving the dorsal horn and especially laminae I (Mouton and Holstege, 2000; Ren et al., 1994) comprising three neuronal systems with projections to lateral reticular nucleus: subnucleus reticularis dorsalis and lateral gigantocelullar and paragigantocellular reticular nuclei from the deep laminae; ventral posterior lateral and ventral medial thalamic nuclei and to the parabrachial nuclei also from lamina I (Gauriau and Bernard, 2004). Interestingly, in the present experimental model, no labeling of dorsal horn laminae was observed, notwithstanding their importance as nociceptive input pathways. This fact, most probably relates to the selectivity of the tract-tracer biocytin used in the present investigation. Phaseolus vulgaris is the choice tracttracer for C and A-delta fibers in the dorsal horn and in special laminae I. However, our results with biocytin reveal a nociceptive circuit that is independent of capsaicin sensitive pain conveying fiber pathways of the deep and superficial dorsal horn laminae. Insomuch, the somatosensory response to sciatic nerve stimulation was potentiated, not abolished, in neonate or adult rats with capsaicin-induced C and A-delta fiber denervation (Caous et al., accepted for publication). Thus, in accordance to the present findings and cited functional studies, the gracile fasciculus/Pa5/lateral reticular nucleus arc would mainly be related to visceral and behavioral reflex responses to noxious stimulation while the lamina I/thalamic and parabrachial circuits would mainly be related to the perceived motivational and affective component of pain and pain related to the homoeostatic processing (Gauriau and Bernard, 2004). Although apparently independent, interactions in the handling of nociceptive information these pain processing systems may interact, at several brain levels, represented by structures such as the, lateral reticular nucleus, ventral posterior medial thalamic nucleus and parabrachial nuclei that receive inputs from the Pa5. From the clinical aspect the nociception-related reflex circuit here exposed may relate to the plantar extensor reflex (Babinsky sign) and other related reflexes that depend on the functional integrity of the pyramidal tract (Maranhão-Filho et al., 2005) and supra-spinal structures (de Freitas and André, 2005). In summary, the findings do indicate that the paratrigeminal nucleus is a primary site in the brain that
receives sensory/nociceptive input from the sciatic nerve and also strongly suggest that lateral reticular nucleus receives inputs from the sciatic nerve that are relayed through the paratrigeminal nucleus. References Allen, G.V., Pronych, S.P., 1997. Trigeminal-autonomic pathways involved in nociception-induced reflex cardiovascular responses. Brain Res. 754, 269–278. Andersen, H.T., 1996. Physiological adaptation in diving vertebrates. Physiol. Rev. 46, 212–234. Angell-James, J.E., Daly, M. de B., 1972. Reflex respiratory and cardiovascular effects of stimulation of receptors in the nose of the dog. J. Physiol. 220, 673–696. Arvidsson, J., Thomander, L., 1984. An HRP study of the central course of sensory intermediate and vagal fibers in peripheral facial nerve branches in the cat. J. Comp. Neurol. 223, 35–45. Arvidsson, U., Riedl, M., Chakrabarti, S., Vulchanova, L., Lee, J.H., Nakano, A.H., Lin, X., Loh, H.H., Law, P.Y., Wessendorf, M.W., et al., 1995a. The kappa-opioid receptor is primarily postsynaptic: combined immunohistochemical localization of the receptor and endogenous opioids. Proc. Natl. Sci. USA 92, 5062–5066. Arvidsson, J., Riedl, M., Chakrabarti, S., Lee, J.H., Law, P.Y., Loh, H.H., Wessendorf, M.W., 1995b. delta-Opioid receptor immunoreactivity: distribution in brainstem and spinal cord, and relationship to biogenic amines and enkephalin. J. Neurosci. 15, 1215–1235. Balan, A.C., Caous, C.A., Yu, Y.G., Lindsey, C.J., 2004. Barosensitive neurons in the rat tractus solitarius and paratrigeminal nucleus: a new model for medullary, cardiovascular reflex regulation. Can. J. Physiol. Pham. 82, 474–484. Bereiter, D.A., Hirata, H., Hu, J.W., 2000. Trigeminal subnucleus caudalis: beyond homologies with the spinal dorsal horn. Pain 88, 221–224. Bon, K., Lanteri-Minet, M., de Pommery, J., Michiels, J.F., Menetrey, D., 1997. Cyclophosphamide cystitis as a model of visceral pain in rats: minor effects at mesodiencephalic levels as revealed by expression of cFos, with a note on Krox-24. Exp. Brain Res. 113, 249–264. Bon, K., Lanteri-Minet, M., Michiels, J.F., Menetrey, D., 1998. Cyclophosphamide cystitis as a model of visceral pain in rats: a c-Fos and Krox-24 study at telencephalic levels, with a note on pituitary adenylate cyclase activating polypeptide (PACAP). Exp. Brain Res. 122, 165–174. Boscan, P., Paton, J.F., 2001. Role of the solitary tract nucleus in mediating nociceptive evoked cardiorespiratory responses. Auton. Neurosci. 86, 170–182. Boucher, Y., Simons, C.T., Cuellar, J.M., Jung, S.W., Carstens, M.I., Carstens, E., 2003. Activation of brain stem neurons by irritant chemical stimulation of the throat assessed by c-Fos immunohistochemistry. Exp. Brain Res. 8, 211–218. Buck, H.S., Caous, C.A., Lindsey, C.J., 2001. Projections of the paratrigeminal nucleus to the ambiguus, rostroventrolateral and lateral reticular nuclei, and the solitary tract. Auton. Neurosci. 87, 187–200. Caous, C.A., Buck, H.S., Lindsey, C.J., 2001. Neuronal connections of the paratrigeminal nucleus: a topographic analysis of neurons projecting to bulbar, pontine e thalamic nuclei related to cardiovascular, respiratory and sensory functions. Auton. Neurosci. 94, 14–24. Caous, C.A., Balan, A., Lindsey, C.J., 2004. Bradykinin microinjection in the paratrigeminal nucleus triggers neuronal discharge in the rat rostroventrolateral reticular nucleus. Can. J. Physiol. Pham. 82, 485–492. Caous, C.A., Koepp, J., Couture, R., Balan, A.C., Lindsey, C.J., accepted for publication. The role of the paratrigeminal nucleus in the pressor response to sciatic nerve stimulation in the rat. Auton. Neurosci. Carstens, E., 1995. Neural mechanisms of hyperalgesia: peripheral or central sensitization. News Physiol. Sci. 77, 2499–2514. Cechetto, D.F., Standaert, D.G., Saper, C.B., 1985. Spinal and trigeminal dorsal horn projections to the parabrachial nucleus in the rat. J. Comp. Neurol. 240, 153–160.
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McDonald, A.J., 1992. Neuroanatomical labeling with biocytin: a review. NeuroReport 3, 821–827. Mochizuki, M., Yokota, S.Y., Murata, H., Watanabe, M., Nishibori, N., Kubota, Y., 1989. Changes in heart rate and blood pressure during dental procedures with local anesthesia. Anesth. Prog. 36, 234–235. Mouton, L.J., Holstege, G., 2000. Segmental and laminar organization of the spinal neurons projecting to the periaqueductal gray (PAG) in the cat suggests the existence of at least five separate clusters of spino-PAG neurons. J. Comp. Neurol. 428, 389–410. Panneton, W.M., Burton, H., 1981. Projections from the paratrigeminal nucleus and the medullary and spinal dorsal horns to the peribrachial area in the cat. Aut. Neuroscience. 15, 779–797. Panneton, W.M., Burton, H., 1985. Projections from the paratrigeminal nucleus and the medullary and spinal dorsal horns to the peribrachial area in the cat. Aut. Neuroscience. 15, 779–797. Paxinos, G., Watson, C., 1986. The Rat Brain in Stereotaxic Coordinates. Academic Press, New York. Paxinos, G., Watson, C., 1997. The Rat Brain in Stereotaxic Coordinates. Academic Press, New York. Phelan, K.D., Falls, W.M., 1989. The interstitial system of the spinal trigeminal tract in the rat: anatomical evidence for morphological and functional heterogeneity. Somatosens. Motor Res. 6 (4), 367–399. Possas, O.S., Lopes, O.U., Cravo, S.L., 2001. Glutamatergic and GABAergic inputs to the RVL mediate cardiovascular adjustments to noxious stimulation. Am. J. Physiol., Regul. Integr. Comp. Physiol. 280, 434–440. Reis, D.J., Ruggiero, D.A., Morrison, S.F., 1989. The C1 area of the rostral ventrolateral medulla oblongata. A critical brainstem region for control of resting and reflex integration of arterial pressure. Am. J. Hypertens. 2, 363S–374S. Ren, K., Williams, G.M., Ruda, M.A., Dubner, R., 1994. Inflammation and hyperalgesia in rats neonatally treated with capsaicin: effects on two classes of nociceptive neurons in the superficial dorsal horn. Pain 59, 287–300. Robbins, M.T., Uzzel, T.W., Aly, S., Ness, T.J., 2005. Visceral nociceptive input to the area of the medullary lateral reticular nucleus ascends in the lateral spinal cord. Neurosci. Lett. 381, 329–333. Salt, T.E., Hill, R.G., 1983. Excitation of single sensory neurones in the rat caudal trigeminal nucleus by iontophoretically applied adenosine 5'triphosphate. Neurosci. Lett. 35, 53–57. Schmued, L.C., Fallon, J.H., 1986. Fluoro-Gold: a new fluorescent retrograde axonal tracer with numerous unique properties. Brain Res. 377, 147–154. Sotgiu, M.L., 1990. Bulbar influences on the periaqueductal gray. Potential role in nociception. Neuroreport 1, 5–8. Stornetta, R.L., Morrison, S.F., Ruggiero, D.A., Reis, D.J., 1989. Neurons of the rostral ventrolateral medulla mediate somatic pressor reflex. Am. J. Physiol. 256, 448–462. Strassman, A.M., Vos, B.P., Mineta, Y., Naderi, S., Borsook, D., Burstein, R., 1993. Fos-like immunoreactivity in the superficial medullary dorsal horn induced by noxious and innocuous thermal stimulation of facial skin in the rat. J. Neurophysiol. 70, 1811–1821. Tian, B., Hartle, D.K., 1994. Cardiovascular effects of NMDA and MK-801 infusion at area postrema and mNTS in rat. Pharmacol. Biochem. Behav. 49, 489–495. Willis Jr., W.D., Westlund, K.N., 2001. The role of the dorsal column pathway in visceral nociception. Curr. Pain Headache Rep. 58, 20–26. Yu, Y., Lindsey, C.J., 2003. Baroreceptor sensitive neurons in the rat paratrigeminal nucleus. Auton. Neurosci. Basic Clin. 98, 70–74. Yu, Y.G., Caous, C.A., Balan, A.C., Rae, G.A., Lindsey, C.J., 2002. Cardiovascular responses to sciatic nerve stimulation are blocked by paratrigeminal nucleus lesion. Auton. Neurosci. 98, 70–74.
Sensory sciatic nerve afferent inputs to the dorsal lateral medulla in the rat☆ Olavo Egídio Alioto a , Charles Julian Lindsey a , Janice Koepp a,b , Cristofer André Caous a,c,⁎ a
b
Department of Biophysics, Universidade Federal de São Paulo, Brazil Department of Pharmacology, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil c Instituto Israelita de Ensino e Pesquisa Albert Einstein, São Paulo, SP, Brazil Received 21 November 2007; received in revised form 11 April 2008; accepted 15 April 2008
Abstract Investigations show the paratrigeminal nucleus (Pa5) as an input site for sensory information from the sciatic nerve field. Functional or physical disruption of the Pa5 alters behavioral and somatosensory responses to nociceptive hindpaw stimulation or sciatic nerve electrostimulation (SNS), both contralateral to the affected structure. The nucleus, an input site for cranial and spinal nerves, known for orofacial nociceptive sensory processing, has efferent connections to structures associated with nociception and cardiorespiratory functions. This study aimed at determining the afferent sciatic pathway to dorsal lateral medulla by means of a neuronal tract-tracer (biocytin) injected in the iliac segment of the sciatic nerve. Spinal cord samples revealed bilateral labeling in the gracile and pyramidal or cuneate tracts from survival day 2 (lumbar L1/L2) to day 8 (cervical C2/C3 segments) following biocytin application. From day 10 to day 20 medulla samples showed labeling of the contralateral Pa5 to the injection site. The ipsilateral paratrigeminal nucleus showed labeling on day 10 only. The lateral reticular nucleus (LRt) showed fluorescent labeled terminal fibers on day 12 and 14, after tracer injection to contralateral sciatic nerve. Neurotracer injection into the LRt of sciatic nerve-biocytin-treated rats produced retrograde labeled neurons soma in the Pa5 in the vicinity of biocytin labeled nerve terminals. Therefore, Pa5 may be considered one of the first sites in the brain for sensory/nociceptive inputs from the sciatic nerve. Also, the findings include Pa5 and LRt in the neural pathway of the somatosympathetic pressor response to SNS and nocifensive responses to hindpaw stimulation. © 2008 Elsevier B.V. All rights reserved. Keywords: Nociception; Sciatic innnervation field; Primary afferent fibers; Spinal trigeminal tract; Neuronal tract-tracing; Lateral reticular nucleus
1. Introduction Cardiovascular modulatory mechanisms linked to baroreflex (Balan et al., 2004; Caous et al., 2004; Possas et al., 2001; Reis et al., 1989) and somatosensory responses to nociceptive sciatic nerve electrostimulation in the dorsallateral medulla (Yu et al., 2002) have been localized to a ☆
small collection of neurons (Phelan and Falls, 1989; ChanPalay, 1978a,b) immersed in a neuropil area of the spinal trigeminal tract, the paratrigeminal nucleus (Pa5). Briefly, Pa5 receives primary afferents from spinal, trigeminal, vagus and glossopharyngeal nerves, and presents neuroanatomical efferent projections to other medullary and diencephalic nuclei (Buck et al., 2001; Caous et al., 2001) related to
Grant sponsors: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). Dr. Charles Lindsey is a recipient of a “Bolsa de Produtividade em Pesquisa 1” from the Conselho Nacional de Pesquisa e Desenvolvimento Tecnológico (CNPq). Dr. Cristofer Caous is a neuroscientist researcher at Albert Einstein Hospital and Mr. Alioto is a FAPESP PhD degree student fellow. ⁎ Corresponding author. Universidade Federal de São Paulo, Departamento de Biofísica, Rua Botucatu, 862 7° andar, 04023-062 São Paulo, SP, Brazil. Tel.: +55 11 5576 4530x206; fax: +55 11 5571 5780. E-mail address: [email protected] (C.A. Caous). 1566-0702/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2008.04.006
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cardiovascular functions and or nociception as the lateral reticular nucleus, nucleus of the solitary tract (NTS), the rostral ventral lateral reticular nucleus (RVL), parabrachial nuclei and the ventral posterior medial thalamic nucleus (VPM). This connectivity, suggesting an important role of the paratrigeminal nucleus in pain and mediation of cardiovascular reflexes (Yu and Lindsey, 2003), called for further investigations. Other findings provide substantial evidence for a role of the paratrigeminal nucleus in mediation of nociceptive inputs from the innervation area of the sciatic nerve. Bradykinin and opioids in the paratrigeminal nucleus, but not tachykinins, were shown to play a role in the mediation or modulation of the somatosensory response to sciatic nerve stimulation (Koepp et al., 2005; Lindsey et al., 1997; Lopes and Couture, 1997). Kinins (Corrêa and Graeff, 1975; Davis and Dostrovsky, 1988; Fior et al., 1993; Couture and Lindsey, 2000) and opioids (Arvidsson et al., 1995a,b; Mansour et al., 1996) are known peptide mediators of central or peripheral nociceptive pathways. Furthermore, neurochemical lesioning of the nucleus selectively alters behavioral responses to nociceptive stimulation to the contralateral hindpaw (Koepp et al., 2006) while the same lesions impair the somatosensory response to contralateral sciatic nerve (Caous et al., accepted for publication). Peripheral nociceptive stimulation elicits profound effects on somatic and autonomic activity including adjustments of both ventilation and circulation in mammalian and other species. Associated with somatic motor behaviors such as withdrawal, fight or flight reactions, all important for survival, the visceral reflexes apparently represent early stages in preparation for the appropriate behavioral responses. These, moment to moment, fast visceral responses are mostly mediated by spinal or medullary reflex arcs. The orofacial-derived nociceptors evoked cardiovascular and respiratory responses are of large intensity and foremost behavioral importance and, in some cases, are clinically significant (Andersen, 1996; Dellow and Morgan, 1969; Angell-James and Daly, 1972; Kumada et al., 1977; Mochizuki et al., 1989; Allen and Pronych 1997; Boscan and Paton, 2001). Accumulated evidence suggest that paratrigeminal nucleus, located in the dorsal lateral medulla is implicated in the processing of orofacial nociceptive information (Marfurt, 1981; Salt and Hill, 1983; Arvidsson and Thomander, 1984; Marfurt and Turner, 1984; Cechetto et al., 1985; Panneton and Burton, 1981, 1985). Although these studies mostly focused on neuroanatomical aspects, they provide important evidence suggesting that Pa5 acts as a primary termination site for orofacial nociception afferents. The Pa5 presents neuroanatomical efferent projections to other medullary, pontine and diencephalic nuclei associated with nociception, thermoregulation and cardiorespiratory responses (Buck et al., 2001; Caous et al., 2001). Many experiments provide functional evidence for a role of the Pa5 in nociception. Chemical or mechanical stimulation of tongue nociceptors (Carstens, 1995; Strass-
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man et al., 1993), laryngopharyngeal mucosa (Boucher et al., 2003) or hypoglossal nerve (Bereiter et al., 2000) upregulates c-Fos expression in Pa5 neurons. However the phenomenon is not restricted to orofacial nociceptive stimulation, since infusion of cyclophosphamide into the urinary bladder, an experimental model of visceral pain, also upregulates c-Fos in the Pa5 (Bon et al., 1997, 1998). Recent evidence suggests that the Pa5 may also represent an input site for sensory afferents of the sciatic nerve innervation field since physical or functional disruption of the Pa5 alters behavioral and somatosensory responses to nociceptive hindpaw stimulation or sciatic nerve electrostimulation (Koepp et al., 2005, 2006; Yu et al., 2002; Caous et al., accepted for publication). In establishing the pathway taken by the nociceptorsevoked cardiorespiratory or behavioral responses, the location of the first brain synapse is of particular importance in that the entire reflex arc may be deciphered. Although the collected evidence suggesting that the Pa5 may represent one of the first input sites of the brain for ascending nociceptive information is more than compelling the neuroanatomical substrates for such an assumption was lacking. Despite the excellent match among physiological, pharmacological and behavioral data regarding hind paw nociceptive mechanisms the neuroanatomical pathway had yet to be complemented or supplemented. Aiming at a comprehensive description of the sciatic medullary pathway a double label anterograde and retrograde tract tracer approach was used to: 1) identify the medullary input site to the medulla, the first brain synapse of the pathway; 2) follow the ascending spinal route of the sciatic afferent input to the medulla and 3) identify the projection site (second synapse) of the first order paratrigeminal nucleus neuron in the reflex arc. 2. Methods 2.1. Animal care and handling The care and handling, housing, surgical procedures and research protocols were conformed to the guidelines principles for animal experimentation as enunciated by the International Association for the Study of Pain and also approved by the internal Ethics Committee of the Universidade Federal de São Paulo, UNIFESP. All animals received food and water ad libitum and after surgery care was taken to ensure that animals did not suffer at any stage in the experimental procedure. 2.2. General procedures Three-month-old male normotensive Wistar (Wistar EPM-1 strain) rats weighing 280–300 g were anaesthetized with thiopental 20 mg kg− 1 and chloral hydrate 300 mg kg− 1, i.p. Animals that did not attain an adequate anaesthesia level within the first 7–10 min after anesthetic administration
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were not used. The level of anaesthesia was verified before and during surgery by evaluating vibrissa movement, tail tonus, breathing rate and response to external stimuli. This
protocol ensured that animals remained under complete anaesthesia for at least 60 min. They received an analgesic (sodium diclofenac, 3 mg/kg/day; Medley S/A, Campinas,
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SP, Brazil), and local anesthetic (2% xylocain solution; Astra-Zeneca, Cotia, SP, Brazil) was applied to the incision surfaces before suture and a wide spectrum of veterinary antibiotic was administered by intramuscular route. All animals were individually housed in 30 × 20 × 20 cm plastic cages for recovery. 2.3. Nomenclature and coordinates Stereotaxic coordinates and nomenclature for structures used in this paper refer to The Rat Brain Stereotaxic Atlas published by Paxinos and Watson (1986, 1997). The stereotaxic coordinates adapted for the lateral reticular nucleus, referenced to stereotaxic zero: − 4.3 mm anterior– posterior, ± 1.8 mm lateral and 0.4 mm vertical.
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(model 900, David Kopf, Tujunga, CA, USA), the neck was flexed to − 11 mm in the incisor bar. The occipital bone and the atlanto-occipital membrane were exposed by incision and dissection of the skin and along the dorsal midline muscles of the neck. The medulla oblongata was exposed after the removal of the occipital bone over the posterior cerebellum, and the meninges cut to expose the dorsal and dorsal lateral surfaces of the caudal medulla. Five µl of 2% w/v of fluorogold were delivered to the LRt with a glass micropipette connected to a 1.0-µl precision syringe (Hamilton, Reno, NE, USA) l. The glass micropipette was lowered to the target of interest and injection volume was delivered over a one minute period with the pipette kept in place for 5 min to avoid tracer spread. After surgery the animals, individually housed in plastic cages, were kept for an additional 4 day survival period.
2.4. Surgery for neurotracer injection in the sciatic nerve 2.6. Histological procedures The anterograde transsynaptically transport (Izzo, 1991; King, 1989) tract-tracer biocytin (Sigma, St. Louis, MO, USA) was applied to the sciatic nerve of anaesthetized animals. The iliac segments of the sciatic nerves of 36 anaesthetized animals (A05-A37) were accessed on the lateral aspect of the limb and isolated from surrounding tissue. With a 15 µm (tip diameter) glass micropipette, connected to a 1.0-µl precision syringe (Hamilton, Reno, NE, USA), 5 µl of 10% w/v biocytin, suspended in 0.05 M Tris buffer (pH = 7.5), was delivered below the sciatic nerve sheath. Five minutes was acceptable for absorption into the nerve helped by carefully flexing and extending the limb. The dissection and biocytin injection was done with the help of a surgical microscope (Carl Zeiss, Germany), and the preparation was observed to ensure that no leakage occurred. The animals were then split in distinct survival time groups, from 0 to 20 days at 48 hour intervals.
Following planned survival periods all animals were deeply anaesthetized with 80 mg kg− 1 i.p., of sodium pentobarbital and perfused transcardially with 200 ml of 0.9% saline and 200 ml of cold 4% paraformaldehyde solution in 0.1 M phosphate-buffered saline (PBS). Brains were removed and overnight cryoprotected in a 4% paraformaldehyde with 30% sucrose solution. Coronal and serial sections were cut at 40 µm on a HM 500 cryostat (Microm, Waldorf, Germany). Biocytin experimental slices were incubated for 24 h in a 1:1500 streptavidin-FITC conjugate (Sigma) in 0.2 M PBS. Histological slices were treated in PBS (0.1 M) three times for 10 min and mounted on gelatin-coated slides. The slides were coverslipped with Fluoromount G (Electron Microscopy Science, Washington, USA) for analysis. 2.7. Data analysis
2.5. Double-labeling experiments The neuronal fluorescent retrograde transport (Schmued and Fallon, 1986) tracer Fluoro-gold (Lumafluor, Englewood, CO, USA), dissolved in distilled water (2% w/v) was microinjected (0.5 µl) in the medial lateral reticular nucleus of two anaesthetized 8 day sciatic nerve-biocytin-injected animals. With the rat proned on the stereotaxic apparatus
Animals with injection sites centered in sciatic nerve and/ or LRt were included in analysis. Images were captured on an Axiovert 135 microscope (Carl Zeiss, Germany) equipped with fluorescent epi-illumination coupled to a Metamorph digitalize system (Universal Imaging, West Chester, PA, USA). Double-labeling experiments were also analyzed under a confocal laser scanning microscope.
Fig. 1. Time course of spinal cord and medullary labeling after application of an anterograde neuronal tract-tracer to the iliac segment of the sciatic nerve. Panel A shows labeling of the paratrigeminal nucleus on days 10 to 20 following application of the contralateral sciatic nerve and bilateral labeling of the nucleus on day 10. The lateral reticular nucleus was labeled on days 12 to 14 contralateral to the side of the tract-tracer application to the sciatic nerve. The photomicrographs insets in the columns at the far left show (clockwise from the bottom): (i) transynaptic fluorescent labeling of synaptic buttons, nerve terminals and varicosities in the lateral reticular nucleus resulting from tract-tracer application to the sciatic nerve; (ii) confocal laser microscopy imaging of Pa5 neuron cell bodies labeled either by biocytin injected in the sciatic nerve (green) or fluoro-gold microinjected in the lateral reticular nucleus; (iii) injection site of the retrograde neurotracer fluoro-gold in the lateral reticular nucleus. Panel B shows labeling of the spinal cord at different survival periods following tract-tracer microinjection in the sciatic nerve. Labeling of the lateral spinal nucleus at lumbar spinal cord L3, ipsilateral to the sciatic nerve tracer injection; bilateral labeling of the gracile fascicule from lumbar to cervical spinal cord (L1 to C3) from days 2 to 8; pyramidal tract (L1 to T10) from days 2 to 6 and the cuneiform fascicule in the cervical spinal cord (C3) on day 8. Abbreviations: cc, central channel; Ecu, external cuneate nucleus; cu, fascicule cuneiform; gr, gracile fascicule; Lsp, lateral spinal nucleus; py, pyramidal tract; Sp5I, spinal trigeminal nucleus interpolar; sp5, spinal trigeminal tract; LRt, lateral reticular nucleus; Pa5, paratrigeminal nucleus; Fg, fluoro-gold; Bct, biocytin. Bars: 100 μm in medulla and spinal cord photomicrographs; 25 μm in i and ii; 200 μm in iii and 50 μm in iv-vi. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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3. Results
3.2. Medullary labeling
3.1. Spinal cord labeling
Transverse medullary sections showed labeling of the Pa5 and the lateral reticular nucleus. The labeled structures were nerve terminals and neuron cell bodies in the paratrigeminal nucleus nerve terminals exclusively in the lateral reticular nucleus. The most intense labeling was observed in the paratrigeminal nucleus 10 to 18 days after application of the neuronal tract-tracer to the contralateral sciatic nerve. The ipsilateral Pa5 showed faint labeling only on sections taken from animals 10 days after biocytin application to the sciatic nerve and by day 20 only faint fluorescent labeling was discernable in the contralateral Paratrigeminal nucleus. Labeling was also observed in the lateral reticular nucleus on days 12 and 14 following tract-tracer-injection to the contralateral sciatic nerve (Fig. 1A and Table 1).
Biocytin, applied to the iliac segment of the sciatic nerve produced ascending labeling of the lateral spinal nucleus, the gracile fascicule, the pyramidal tract and the cuneiform fascicule. The labeled spinal structures were mostly fiber clusters with the exception of the lateral spinal nucleus contained labeled neuron cell bodies. Labeling of the spinal cord time related. From 48 h of tracer administration florescent labeling was observed in the spinal lateral nucleus at the level of lumbar L3 vertebra, ipsilateral to the tract-tracer injection side, and in both ipsi- and contralateral the gracile fascicule and pyramidal tract at L2 and L1 vertebrae levels (Fig. 1B). The pyramidal tract showed symmetrical ipsilateral labeling at L1/L2 to T8 but not at T5. The gracile fasciculus showed bilateral asymmetric labeling from L1 to the cervical spinal cord segments C2/C3 on survival days 2 to 8 (Table 1). In the cervical spinal cord labeled fibers were also present in the cuneiform fasciculus disposed as a lamina on the medial aspect of the posterior intermediate septum. Tract-tracer transport distance rate was measured and the tracer traveled from lumbar segments L1 to cervical segment C2 after the injection into the sciatic nerve. The conduction speed was estimated in approximately 0.5 mm hr− 1 (0.49± 0.2 mm hr− 1) for both the gracile fascicule and the pyramidal tract independent of the laterality relative to the injection site.
3.3. Double-labeling of the paratrigeminal nucleus The microinjection of a retrograde transport tracer, fluorogold, in the medial reticular nucleus (LRt) of sciatic nerve biocytin-treated rats produced double labeling of the caudal paratrigeminal nucleus. Confocal laser microscopy imaging (Figs. 1A and 2) showed retrogradely labeled neuron soma in the caudal region of the Pa5 in the immediate vicinity of biocytin-labeled terminal axons suggesting the presence of synaptic vesicles in varicosities. Some neurons soma showed double labeling with fluoro-gold and biocytin (Fig. 2). 4. Discussion
Table 1 Survival period, labeled structures and density measurement of transverse spinal cord and medullary brainstem histological sections after biocytin microinjections in the sciatic nerve (n = 31) Survival Labeled structures period (days) Spinal cord
0 2 2 2 4 4 6 6 8 8 Medulla 10 12 12 14 14 16 18 20
None lateral spinal nucleus (L3) gracile fasciculus (L2/L1) pyramidal tract (L2/L1) gracile fasciculus (T10/T9) pyramidal tract (T10/T9) gracile fasciculus (T5/T4) pyramidal tract (T5/T4) gracile fasciculus (C3/C2) cuneate fasciculus (C3/C2) paratrigeminal nucleus paratrigeminal nucleus lateral reticular nucleus paratrigeminal nucleus lateral reticular nucleus paratrigeminal nucleus paratrigeminal nucleus paratrigeminal nucleus
Density of fluorescent labeling (relative units) Ipsilateral Contralateral 0 136 ± 7* 78 ± 14 140 ± 7 128 ± 6 120 ± 13 79 ± 6 89 ± 3 52 ± 4 44 ± 2 53 ± 12 0 0 0 0 0 0 0
0 0 142 ± 3 79 ± 14 157 ± 2* 124 ± 14 100 ± 6* 100 ± 3* 119 ± 10* 49 ± 1 133 ± 1* 122 ± 1* 136 ± 4* 107 ± 1* 118 ± 3* 93 ± 4* 77 ± 2* 21 ± 2*
Values are given as means ± standard error. Variance Analysis with comparison between means by Newman–Keuls posthoc test. *Differs statistically, p b 0.05.
The present investigation identified sensory sciatic nerve input to the paratrigeminal nucleus, in the dorsal lateral medulla, and a relay to the lateral reticular nucleus (LRt) in the caudal ventral lateral medulla. These findings provide data on the anatomical substrate of the nocifensive and somatosensory responses to nociceptive stimuli to the rat hindpaw or sciatic nerve. Further than furnishing a striking parallel to the behavioral and physiological data, the impact of these findings bring to light an undisclosed view on the brain handling of reflex responses to nociceptive input from the sciatic nerve innervation field. Firstly, the sciatic input to the medulla is directed to the caudal segment of the paratrigeminal nucleus that in turn contains neurons projecting to the LRt (Caous et al., 2001). The paratrigeminal nucleus, located in the dorsal lateral medulla, is commonly known as an input site for orofacial nociceptive inputs of the trigeminal nerve (Boscan and Paton, 2001). Thus, this manuscript and related publications (Koepp et al., 2006; Caous et al., accepted for publication) bring the focus to the dorsal lateral medulla in the handling of nociceptive input. Another interesting finding is that the LRt receives input from high order neurons of paratrigeminal nucleus, suggesting that the reflex arc handling behavioral and cardiovascular responses to nociceptive stimulation from the hindpaw differs from that of the baroreflex arc that comprises the paratrigeminal nucleus/rostral ventral lateral reticular nucleus (RVL) pathway
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Fig. 2. Confocal microscopy imaging (A–D) from 4 adjacent rat transverse medullary sections (40 µm — animal A37) of biocytin-labeled nerve terminals, synaptic buttons (yellow arrows) and varicosities (blue arrows) in the paratrigeminal nucleus. Biocytin tract-tracer microinjection site was the contralateral sciatic nerve (all in green pseudo color). In red, labeled neuron soma ensuing from retrograde transport of fluoro-gold microinjected in the lateral reticular nucleus and in yellow, a double-labeled (biocytin and fluoro-gold) neuron soma (white arrows) is shown. Bars: 25 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
(Caous et al., 2004; Balan et al., 2004; Dampney et al., 1982). This finding also contradicts earlier reporting (Stornetta et al., 1989) suggesting the RVL as an exclusive component of the somatosensory reflex arc. The lateral reticular nucleus is related to the processing of nociception (Janss and Gehart, 1988; Liu et al., 1989; Sotgiu, 1990; Robbins et al., 2005; Leite-Almeida et al., 2006) and homoeostatic cardiovascular reflexes (Tian and Hartle, 1994) and thus may well be involved in the somatosympathetic response to sciatic nerve stimulation. Also, the present data imply that the Pa5/lateral reticular nucleus pathway may mediate the descending modulatory influences on nocifensive responses to hindpaw stimulation (Koepp et al., 2006) although the participation pontine or thalamic structures that receive paratrigeminal nucleus inputs (Caous et al., 2001). Regarding the spinal cord, labeling from the tract-tracer injection to the iliac portion of the sciatic nerve was apparent 48 h later in the lateral spinal nucleus, and in lumbar L3, L2 and L1 segments in the gracile fasciculus and pyramidal tract. The labeling of the gracile fasciculus apparent from the lumbar to cervical spinal cord, always asymmetric, showed higher density on the contralateral side to that of the injected nerve and thus may represent the main input to the paratrigeminal nucleus from the sciatic nerve. The gracile fasciculus that conveys sensory inputs from limbs is more effective than the spinothalamic tract in activating thalamic neurons (Willis and Westlund, 2001). The pyramidal tract on
the other hand was symmetrically labeled from the upper lumbar level to the higher thoracic level (T5/T4). At the cervical spinal cord the pyramidal tract did not show labeling. Instead, bilateral symmetric labeling was observed in the cuneate fasciculus as in laminae I next to the posterior intermedial septum. The asymmetry of the gracile fascicule and Pa5 labeling most probably reflects the larger number of labeled fibers in the contralateral spinal cord and the larger amount of nerve terminals in the contralateral Pa5. The transport rate of the tract-tracer, estimated at approximately 0.5 mm per hour in the spinal cord, was similar for all tracts independently of the laterality. Considering these kinetics, it would be expected that the tract-tracer would reach the Pa5 in about 230 h after the injection according to the measured distances and that the lateral reticular nucleus be reached 6 to 7 h later and thus be perceptible in medullae sections of 240 hour (10 days) survival group animals. In fact the delay of the lateral reticular nucleus labeling may be explained by the delay imposed by the transsynaptically labeling of the Pa5 neurons projecting to the lateral reticular nucleus. Double-labeled neurons soma by biocytin injected in the sciatic nerve and fluoro-gold in the lateral reticular nucleus assure a monosynaptic connection based on the rate of dye transport between the sciatic nerve inputs to the Pa5 and the lateral reticular nucleus. The assumption that the LRt is labeled via
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a synaptic connection in the Pa5 is strengthened by the fact that biocytin is known to be transported transsynaptically (Izzo, 1991; King, 1989) and may label neuron soma by uptake from extra cellular space (McDonald, 1992). The transsynaptic labeling raises the question as to whether the sciatic-paratrigeminal input is mono or poly synaptic. Labeled cell bodies in the lateral spinal nucleus suggest that the first synapse of the sciatic fibers occur in the lower spinal cord. Along the gracile, pyramidal or cuneiform tracts there was no clear evidence of labeled cell bodies or nerve terminals and thus it is possible that there are only two synapses in this neuronal pathway. The labeling patterns in the spinal cord and medulla, as a result of tract-tracer application to the sciatic nerve, bring to view a different pattern from other models of nociceptive input pathways involving the dorsal horn and especially laminae I (Mouton and Holstege, 2000; Ren et al., 1994) comprising three neuronal systems with projections to lateral reticular nucleus: subnucleus reticularis dorsalis and lateral gigantocelullar and paragigantocellular reticular nuclei from the deep laminae; ventral posterior lateral and ventral medial thalamic nuclei and to the parabrachial nuclei also from lamina I (Gauriau and Bernard, 2004). Interestingly, in the present experimental model, no labeling of dorsal horn laminae was observed, notwithstanding their importance as nociceptive input pathways. This fact, most probably relates to the selectivity of the tract-tracer biocytin used in the present investigation. Phaseolus vulgaris is the choice tracttracer for C and A-delta fibers in the dorsal horn and in special laminae I. However, our results with biocytin reveal a nociceptive circuit that is independent of capsaicin sensitive pain conveying fiber pathways of the deep and superficial dorsal horn laminae. Insomuch, the somatosensory response to sciatic nerve stimulation was potentiated, not abolished, in neonate or adult rats with capsaicin-induced C and A-delta fiber denervation (Caous et al., accepted for publication). Thus, in accordance to the present findings and cited functional studies, the gracile fasciculus/Pa5/lateral reticular nucleus arc would mainly be related to visceral and behavioral reflex responses to noxious stimulation while the lamina I/thalamic and parabrachial circuits would mainly be related to the perceived motivational and affective component of pain and pain related to the homoeostatic processing (Gauriau and Bernard, 2004). Although apparently independent, interactions in the handling of nociceptive information these pain processing systems may interact, at several brain levels, represented by structures such as the, lateral reticular nucleus, ventral posterior medial thalamic nucleus and parabrachial nuclei that receive inputs from the Pa5. From the clinical aspect the nociception-related reflex circuit here exposed may relate to the plantar extensor reflex (Babinsky sign) and other related reflexes that depend on the functional integrity of the pyramidal tract (Maranhão-Filho et al., 2005) and supra-spinal structures (de Freitas and André, 2005). In summary, the findings do indicate that the paratrigeminal nucleus is a primary site in the brain that
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