Morphological Studies Of Premotor Neurons In The Brainstem Local Circuits Involving In The Orofacial Nociceptive Reflex Of The Rat

Premotor neurons play critical role in carrying out motor activity and maintenance of body homeostasis. The previous studies have proven that a number of premotor neurons involved in the local circuits of reflex. Nociceptive reflex (NR) is one of the basic protective reflexes, and we learned a lot about central transmission of nociception, central sensitization, the mechanism of hyperalgesia, allodynia and referred pain by applying these reflex models. Many structural abnormalities, such as tumor, haematoma or infarcts at the level of the brainstem, may cause abolition or turbulence of these reflexes. So, the recording of different reflexes is helpful to exclude structural lesions or, conversely, to localize more accurately lesions within the central nerve system. However, to date, the studies about nociceptive reflexes are focus on the spinal level, such as nocicptive flexion reflex (NFR), and little information is far available about the orofacial nociceptive reflexes, as jaw reflex, corneal reflex, especially the premotor neurons located in these arcs due to the complication of the structure and function of the trigeminal nervous system contributing to the transmission of the orofacial sensory information and the motor nuclei which innervate the orofacial muscle activity. On the basis of above reason, the present study used morphological methods to examine the distribution and branched projection of premotor neurons involving in orofacial nociceptive information transmission in the brainstem, and the neurochemical characters of these premotor neurons as well as the axonal terminals originating from the caudal spinal trigeminal nucleus (Vc). The contents of this paper are as follows:1. The distribution of premotor neurons involving in the local circuits of the orofacial nociceptive reflexes in the rat brainstemIn the first part of our experiments, we examined the distribution of the premotor neurons projecting to VII or XII in the brainstem under an epifluorescence microscope by using tracer-tracing methods containing retrograde tracer Fluoro-gold (FG) or horseradish peroxide (HRP) injected into facial (VII) or hypoglossal (XII) nucleus iontophoretically (FG) or by pressure (HRP) and anterogradely transported tracers biotinylated dextran amine (BDA, 10000MW) into Vc ipsilateral side. Then we examined if axon terminals originating from Vc might make synapses on the premotor neurons projecting to VII or XII under the confocal laser-scanning microscope (CLSM) and electron microscope (EM). Subsequently, as a marker of nociceptive neurons, the Fos-immunoreactivity (IR) was observed after formalin injected into the lips to make sure the existence of local circuits involving in the orofacial nociceptive reflex in the brainstem. The results are follows:(1) The FG retrogradely labeled premotor neurons projecting to VII or XII were mainly distributed bilaterally with ipsilateral predominance in the pontomedullary reticular formation (RF), including parvicellular reticular formation (PCRt), dorsal or ventral medullary reticular formation (MdD, MdV), gigantocellular reticular formation (Gi), intermediated reticular formation (IRt); peri-motor nuclei of brainstem, such as supratrigeminal nucleus (Vsup), intertrigeminal nucleus (IntV), medial and lateral parabrachial nuclei (PBN), Ko lliker-Fuse nucleus (K-F nucleus); interpolar spinal trigeminal nucleus (Vi), oral spinal trigrminal nucleus (Vo) and the principal sensory trigeminal nucleus(Vp).(2) Under the CLSM, FG retrogradelly labeled neurons (green colour) were observed to make close appositions upon BDA anterogradelly labeled fibers or axonal terminals (red colour). And these close appositions were found mainlyipsilaterally in PCRt, MdD, MdV, Vsup, Vp, Vo, Vi, PBN.(3) A few FG retrogradelly labeled premotor neurons which received projections coming from Vc in the brainstem showed Fos-IRs after formalin injected into the lips of the rats.(4) Under electron microscope (EM), some BDA-labeled axon terminals were observed to make synaptic contact with dendritic and somatic profiles of the HRP-labeled neurons. In all of the double-labeled synapses, 73% of BDA-labeled boutons (88/120) synapsed on dendrites of HRP-labeled premotor neurons, while 27% of BDA-labeled boutons (32/120) made synapses with somata of HRP-labeled premotor neurons. The majority of axo-dendritic or axo-somatic synapses were asymmetric.The results aforementioned elucidated that the premotor neurons, which were distributed widely in the brainstem reticular formation, could integrate the orofacial nociceptive information and then transfer it to the brainstem motoneurons, by which mediated to bring the brainstem reflexes.2. Premotor neurons which involved in the orofacial nociceptive reflexes projected simultaneously to W and XII by sending the branched axons.After retrograde tracers FQ TMR or anterogradely transported tracers BDA were injected into VD, XII or Vc on the same side by iontophoretically (FG) or by pressure (TMR and BDA), the distribution and axon branched projection of single premotor neurons projecting into VII and XII in the brainstem were examined under the epifluorescence microscope. Then we further examined whether the FG/TMR-double labeled premotor neurons make close appositions with BDA-labeled axonal terminals originating from Vc under the CLSM. And finally, following formalin injected into the lips, the Fos-IRs were observed in the FG/TMR-double labeled neurons to make sure that single premotor neuron involves simultaneously in different orofacial nociceptive reflexes mediated bydifferent orofacial motonuclei. The results are follows:(1) Under the epifluorescence microscope, FG/TMR double-labeded neurons were found bilaterally in the brainstems with ipsilateral predominance. The distribution was abundance in the dorsal part of PCRt, MdD, dorsal part of MdV, IRt, peri-motor nucleus regions, such as Vsup and reticular formation ventral to trigeminal motor nucleus.(2) A few BDA anterogradely labeled fibers originating from Vc in the areas above mentioned made the close contacts with the somatic and dendritic profiles of FG/TMR-double labeled neurons.(3) After formalin injected into the lips, Fos-IR could be found in some of FG/TMR double-labeled neurons.Above results implicated that single premotor neurons could transmit orofacial nociceptive information simultaneously to different brainstem motonuclei by axonal collateral, which probably related with in the complicated and coordinated oral reflexes.3. Chemical neuroanatomy studies of premotor neurons and axonal terminals originating from Vc in the orofacial nociceptive reflex pathwaysThe chemical neuroanatomy characters of premotor neurons and axonal terminals originating from Vc in the orofacial nociceptive reflex pathways were investigated after injections of retrogradely transported tracer FG or HRP into VD or XH, and anterograde tracer BDA into Vc combined with triple-labeled imrnuno-fluorescence histochemical and immuno-electron microscopic techniques. The results are follows:(1) A lot of FG-labeled premotor neurons, which distributed in the PCRt, MdD, Vsup and were apposed upon Vc projecting fibers or terminals, showed y-aminobutyric acid (GABA)-IR under CLSM.(2) Anterogradely BDA-labeled fibers or boutons made the synaptic contacts...