Central projections of primary afferent fibers from the rat trigeminal nerve labeled with isolectin B4-HRP

doi:10.1016/0304-3940(96)13056-1

Neuroscience Letters

Volume 217, Issues 2–3, 18 October 1996, Pages 89-92

https://doi.org/10.1016/0304-3940(96)13056-1 Get rights and content

Abstract

The central projections of unmyelinated primary afferent fibers from the rat trigeminal nerve were investigated using retrograde and transganglionic transport of isolectin V4-horseradish peroxidase (B4-HRP). After the injection of a small amount of the tracer into various areas of the facial dermis and oral mucosa, small neuronal somata were retrogradely labeled in the trigeminal ganglion. Clearly delineated transganglionic labeling was observed exclusively in lamina II of the medullary and upper cervical dorsal horn. Its distribution showed a somatotopic organization. Fibers from the oral mucosa terminated in the region 0–1.7 mm caudal to the obex, whereas those from the facial skin terminated in the region from 1.3 mm caudal to the obex through the second cervical segment. In each of these two terminal regions, the rostral areas in the periphery were represented more rostrally than the caudal areas, and the dorsal areas in the periphery were represented more ventrolaterally than the ventral areas.

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Facial i.d. injection might deliver drugs to the human brain via the trigeminal nerve. Accumulating evidences indicates that facial intradermal injection might deliver lectins and HRP to the trigeminal nerve [13–16]. However, currently, facial intradermal injection of drugs to treat human CNS diseases has not been reported.
Although intranasal delivery bypasses the blood-brain barrier (BBB), the anatomical location of the olfactory mucosa and respiratory airflow interference lead to less brain-targeted drug delivery. In addition to intranasal delivery, evidence indicates that facial intradermal injection might be a novel strategy for bypassing the BBB via the trigeminal nerve (TN). The hypothesis was verified by pharmacokinetic evaluation, nasal injury, lymphatic vessels inhibition and immunohistochemistry. Intradermal injection into the rat mystacial pad (i.d.) elevated the brain sub-areas and trigeminal Evans Blue (EB) concentrations, C_max and AUC_(0 − t). I.d. also increased them in brain sub-areas beyond those of intranasal (i.n.) and intravenous injection (i.v.), especially the pons varolii and the medulla oblongata (sub-areas associated with TN). I.d. injection increased the brain drug targeting efficiency, brain direct transport percentage and brain bioavailability of EB while i.n. injection altered them slightly. Trigeminal transection and nasal injury reduced trigeminal EB with i.d. administration. Trigeminal perineurium, epineurium, perivascular spaces, neurons and Schwann cells were involved in the EB brain-targeted delivery. The lymphatic system mediated EB diffusion from the mystacial pad to the nasal mucosa and the brain. Thus, facial intradermal injection might be a promising strategy for brain-targeting delivery, bypassing the BBB via the trigeminal substructures.
Local group I mGluR antagonists reduce TMJ-evoked activity of trigeminal subnucleus caudalis neurons in female rats
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Group I metabotropic glutamate receptors (mGluR1 and mGluR5) are functionally linked to estrogen receptors and play a key role in the plasticity of central neurons. Estrogen status strongly influences sensory input from the temporomandibular joint (TMJ) to neurons at the spinomedullary (Vc/C_1–2) region. This study tested the hypothesis that TMJ input to trigeminal subnucleus caudalis/upper cervical cord (Vc/C_1–2) neurons involved group I mGluR activation and depended on estrogen status. TMJ-responsive neurons were recorded in superficial laminae at the Vc/C_1–2 region in ovariectomized (OvX) female rats treated with low-dose estradiol (2 μg/day, LE) or high-dose estradiol (20 μg/day, HE) for 2 days. TMJ-responsive units were activated by adenosine triphosphate (ATP, 1 mM) injected into the joint space. Receptor antagonists selective for mGluR1 (CPCCOEt) or mGluR5 (MPEP) were applied topically to the Vc/C_1–2 surface at the site of recording 10 min prior to the intra-TMJ ATP stimulus. In HE rats, CPCCOEt (50 and 500 μM) markedly reduced ATP-evoked unit activity. By contrast, in LE rats, a small but significant increase in neural activity was seen after 50 μM CPCCOEt, while 500 μM caused a large reduction in activity that was similar in magnitude as that seen in HE rats. Local application of MPEP produced a significant inhibition of TMJ-evoked unit activity independent of estrogen status. Neither mGluR1 nor mGluR5 antagonism altered the spontaneous activity of TMJ units in HE or LE rats. High-dose MPEP caused a small reduction in the size of the convergent cutaneous receptive field in HE rats, while CPCCOEt had no effect. These data suggest that group I mGluRs play a key role in sensory integration of TMJ nociceptive input to the Vc/C_1–2 region and are largely independent of estrogen status.
Gabaergic influence on temporomandibular joint-responsive spinomedullary neurons depends on estrogen status
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Citation Excerpt :
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Sensory input from the temporomandibular joint (TMJ) to neurons in superficial laminae at the spinomedullary (Vc/C_1–2) region is strongly influenced by estrogen status. This study determined if GABAergic mechanisms play a role in estrogen modulation of TMJ nociceptive processing in ovariectomized female rats treated with high- (HE) or low-dose (LE) estradiol (E2) for 2 days. Superficial laminae neurons were activated by ATP (1 mM) injections into the joint space. The selective GABA_A receptor antagonist, bicuculline methiodide (BMI, 5 or 50 μM, 30 μl), applied at the site of recording greatly enhanced the magnitude and duration of ATP-evoked responses in LE rats, but not in units from HE rats. The convergent cutaneous receptive field (RF) area of TMJ neurons was enlarged after BMI in LE but not HE rats, while resting discharge rates were increased after BMI independent of estrogen status. By contrast, the selective GABA_A receptor agonist, muscimol (50 μM, 30 μl), significantly reduced the magnitude and duration of ATP-evoked activity, resting discharge rate, and cutaneous RF area of TMJ neurons in LE and HE rats, whereas lower doses (5 μM) affected only units from LE rats. Protein levels of GABA_A receptor β3 isoform at the Vc/C_1–2 region were similar for HE and LE rats. These results suggest that GABAergic mechanisms contribute significantly to background discharge rates and TMJ-evoked input to superficial laminae neurons at the Vc/C_1–2 region. Estrogen status may gate the magnitude of GABAergic influence on TMJ neurons at the earliest stages of nociceptive processing at the spinomedullary region.
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Citation Excerpt :
A similar laminar pattern is seen for TMJ and jaw muscles afferents at the Vc/C1–2 region [5,21,63]. The TMJ and jaw muscles are supplied mainly by small diameter sensory fibers [40,68] that express TRPV1 [18,35] or isolectin B4 (IB4) [3], whereas the expression of TRPV1 [6] and IB4 [41,65] at the Vc/C1–2 region is restricted to the superficial laminae. Thus, a peripheral basis for estrogen status and stress induced lamina-specificity in medullary dorsal horn could derive from preferential actions on unmyelinated C fibers.
Estrogen status and psychological stress contribute to the expression of several chronic pain conditions including temporomandibular muscle and joint disorders (TMJD). Sensory neurons that supply the temporomandibular joint (TMJ) region terminate in laminae I and V of the spinal trigeminal nucleus (Vc/C_1–2 region); however, little is known about lamina-specificity and environmental influences on the encoding properties of TMJ brainstem neurons. To test the hypothesis that Vc/C_1–2 neurons integrate both interoceptive and exteroceptive signals relevant for TMJ nociception, we recorded TMJ-evoked activity in superficial and deep laminae of ovariectomized rats under high and low estradiol (E2) and stress conditions. Rats received daily injections of low (LE) or high (HE) dose E2 and were subjected to forced swim (FS) or sham swim conditioning for 3 days. The results revealed marked lamina-specificity in that HE rats displayed enhanced TMJ-evoked activity in superficial, but not deep, laminae independent of stress conditioning. By contrast, FS conditioned rats displayed increased background firing and TMJ-evoked activity of neurons in deep, but not superficial, laminae independent of E2 status. FS also enhanced TMJ-evoked masseter muscle activity and suggested the importance of deep dorsal horn neurons in mediating evoked jaw muscle activity. In conclusion, E2 status and psychophysical stress play a significant role in modifying the encoding properties of TMJ-responsive medullary dorsal horn neurons with a marked lamina-specificity.
Distinct time courses of microglial and astrocytic hyperactivation and the glial contribution to pain hypersensitivity in a facial cancer model
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Forty micrometer-thick serial sections were cut using a freezing microtome (WS-112-N, Yamatokohki, Saitama, Japan). An earlier study reported that the vibrissal pad- and infraorbital area-innervated regions in the trigeminocervical complex are located 2–3 and 4–5 mm caudal to the obex, respectively (Kobayashi and Matsumura, 1996); therefore, the sections from these two respective regions were taken to be the MDH and UCDH. The free-floating sections were incubated in 3% normal goat serum in phosphate-buffered saline for 1 h at room temperature and then incubated with anti-Iba1 rabbit polyclonal antibody (diluted 1:300, 019-19741, Wako) or anti-GFAP antibody (diluted 1:200) overnight at 4 °C.
Although recent evidence suggests that central glial hyperactivation is involved in cancer-induced persistent pain, the time course of this hyperactivation and the glial contribution to pain hypersensitivity remain unclear. The present study investigated the time-dependent spatial changes of microglial and astrocytic hyperactivation in the trigeminocervical complex, which consists of the medullary (MDH) and upper cervical (UCDH) dorsal horns, and pain-related behaviors in a rat facial cancer model in which Walker 256B-cells are inoculated into the vibrissal pad. In this model, the tumors grew within the vibrissal pad, from which sensory nerve fibers project into the MDH, but did not expand into the infraorbital region, from which fibers project into the UCDH. Nevertheless, mechanical allodynia and thermal hyperalgesia were observed not only in the vibrissal pad but also in the infraorbital region. Western blotting and immunofluorescence studies indicated that microglia were widely activated in the trigeminocervical complex on day 4 and gradually inactivated by day 11. In contrast, astrocytes were only activated in the MDH on day 4; the hyperactivation later expanded into the UCDH. Daily administration of the glial hyperactivation inhibitor propentofylline beginning on day 4 suppressed the glial hyperactivation on later days. Propentofylline treatment largely prevented allodynia/hyperalgesia in the infraorbital region beginning on day 5, although established allodynia/hyperalgesia in the vibrissal pad was less sensitive to the treatment. These results suggest that central glial hyperactivation, transient microglial hyperactivation and persistent astrocytic hyperactivation, contributes to the development of pain hypersensitivity but not to the maintenance of pain in this model.
Unmyelinated fibers of the anterior ethmoidal nerve in the rat co-localize with neurons in the medullary dorsal horn and ventrolateral medulla activated by nasal stimulation
2009, Brain Research
Citation Excerpt :
Presumably this is because WGA has high affinity for N-acetyl-d-glucosamine, a carbohydrate found on the plasma membrane of small diameter afferent fibers (Nagata and Burger, 1974). In contrast, HRP conjugated with Isolectin-B4 from Bandeiraea simplicifolia (IB4-HRP) has been used to selectively trace unmyelinated C-fibers (Kitchener et al., 1993; Kobayashi and Matsumura, 1996; Wang et al., 1998). IB4 binds to terminal α-d-galactose groups of the plasma membrane of nervous tissue (Ambalavanar and Morris, 1992; Ambalavanar and Morris, 1993), and so IB4-HRP labels only unmyelinated neurons because the myelin sheathes of myelinated axons do not contain galactose glycoconjugates (Streit et al., 1985).
The anterior ethmoidal nerve (AEN) innervates the nasal passages and external nares, and serves as the afferent limb of the nasopharyngeal and diving responses. However, although 65% of the AEN is composed of unmyelinated fibers, it has not been determined whether this afferent signal is carried by unmyelinated or myelinated fibers. We used the transganglionic tracers WGA-HRP, IB4-HRP, and CTB-HRP to trace the central projections of the AEN of the rat. Interpretation of the labeling patterns suggests that AEN unmyelinated fibers project primarily to the ventral tip of the ipsilateral medullary dorsal horn (MDH) at the level of the area postrema. Other unmyelinated projections were to the ventral paratrigeminal nucleus and ventrolateral medulla, specifically the Bötzinger and RVLM/C1 regions. Myelinated AEN fibers projected to the ventral paratrigeminal and mesencephalic trigeminal nuclei. Stimulating the nasal passages of urethane-anesthetized rats with ammonia vapors produced the nasopharyngeal response that included apnea, bradycardia and an increase in arterial blood pressure. Central projections of the AEN co-localized with neurons within both MDH and RVLM/C1 that were activated by nasal stimulation. Within the ventral MDH the density of AEN terminal projections positively correlated with the rostral–caudal location of activated neurons, especially at and just caudal to the obex. We conclude that unmyelinated AEN terminal projections are involved in the activation of neurons in the MDH and ventrolateral medulla that participate in the nasopharyngeal response in the rat. We also found that IB4-HRP was a much less robust tracer than WGA-HRP.

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Central projections of primary afferent fibers from the rat trigeminal nerve labeled with isolectin B4-HRP

Abstract

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