Quantitative analysis of cervical musculature in rats: histochemical composition and motor pool organization. I. Muscles of the spinal accessory complex

In this paper we characterize the architecture and segmental innervation, histochemical composition, muscle spindle populations, and motor pool organization of rat spinal accessory (SA) muscles: sternomastoid (SM), cleidomastoid (CM), cleidotrapezius (CT), and acromiotrapezius (AT). We also consider whether individual rat neck muscles are supplied by more than one population of motor neurons as they are in turtles and cats and whether in SA muscles motor neuron size scales with target muscle fiber type. SM, CM, and CT are ventral, parallel strap muscles. Each can be divided into grossly visible white and red compartments. AT is a dorsolateral sheetlike muscle that shows no gross compartmentalization. All four muscles are dominated by fast-twitch glycolytic (FG) and fast oxidative glycolytic (FOG) fibers, and FG fibers are significantly more numerous than the FOG type in three out of four muscles. Thus SA muscles in rats appear to be specialized for rapid, phasic head movements. Topographical analyses revealed that there is a striking compartmentalization of fiber types in the ventral muscles that corresponds to the red and white segments seen grossly. Spindles are found only in regions containing slow-twitch oxidative (SO) fibers. Cross-muscle comparisons indicate that there are significant differences between SA muscles in their fiber type composition. The motor pools of SA muscles form a single column from lower medulla to C5. Rostral cells lie dorsomedially in the ventral horn and, at the C1/C2 junction, the column shifts ventrolaterally. Within this column, each motor pool occupies a characteristic rostrocaudal position in the order SM:CM:CT:AT. Thus SM and (in part) CM motor neurons lie more medially than cells supplying the trapezius complex, suggesting that they may be under different patterns of synaptic drive. We saw no evidence that rat SA muscles are supplied by more than one population of motor neurons. Direct comparisons between the soma sizes of motor neurons that supply muscles or parts of muscles with significantly different histochemical compositions indicate that these size differences are in the direction predicted from their histochemical profiles, thus suggesting that in these muscles motor neuron soma size may scale with muscle fiber type.     

Neuroprotective effects of recombinant human erythropoietin on facial motoneurons after facial nerve injury in rats

BACKGROUND: Erythropoietin and recombinant human erythropoietin (rhEPO) inhibit apoptosis of motor neurons caused by spinal cord injury and brain damage in rats. However, it still remains to be shown whether rhEPO can protect facial motoneurons (FMNs) as well.OBJECTIVE: To test the neuroprotective effects of rhEPO on injured FMNs, as well as the influence on Caspase-3 expression.DESIGN, TIME AND SETTING: Randomized, controlled, animal experiment. This study was performed at the Central Laboratory of Basic Medical College, Chongqing Medical University from January to October 2007.MATERIALS: Seventy-five female SD rats, weighing 210-230g. rhEPO injection was provided by Sansheng pharmaceuticals company, Shenyang City, Liaoning Province, China, and the License number was HMLN S20010001.METHODS: A total of 75 female rats were randomly divided into rhEPO treatment, control, and sham operation groups, with 25 rats in each group. Rat models of facial nerve injury were established in the rhEPO treatment group and the control group by crushing the main trunk of the left facial nerve. Surgical microscopic observation of the facial nerve damage displayed perineurial disruption. The left stylomastoid foramen of the sham operation group were only exposed, but without nerve injury. The rhEPO treatment group was treated with rhEPO (5000U/kg, i. p.) once following injury and once a day for two weeks. The control and sham operation groups were treated with the same dose of normal saline (i. p.), once following injury and once a day for two weeks.MAIN OUTCOME MEASURES: Rats were sacrificed 3, 7, 14, 21, and 28 days after injury, FMN survival after facial nerve injury was analyzed by Toluidine blue staining, and then survival ratios (L/R) were calculated. The number of apoptotic profiles in the injured FMNs were evaluated by TUNEL staining. Expression of Caspase-3 in the facial nucleus was detected by immunohistochemistry methods.RESULTS: A total of 75 rats were included in the final analysis. FMN survival ratios, both in rhEPO treatment group and control group, decreased gradually between seven and 28 days; however, FMN survival ratios were significantly greater in the rhEPO treatment group compared to the control group (P<0.05). No TUNEL-positive cells were observed three days after injury in the rhEPO treatment and control groups; however, by seven days after injury, apoptotic cells were observed and peaked by 14 days in the control group. Between seven and 21 days, apoptotic cell numbers were significantly lower in the rhEPO treatment group compared to the control group (P<0.05). The expression of Caspase-3 increased three days after injury and peaked at 14 days in the control group. Nevertheless, Caspase-3 expression was significantly lower in the rhEPO treatment group compared to the control group at each time point (P<0.05).CONCLUSION: Treatment with rhEPO can effectively protect facial motoneurons by reducing expression of Caspase-3 and inhibiting apoptosis.     

Morphological relationships among extensor digitorum longus, tibialis anterior, and semitendinosus motor nuclei of the cat: an investigation employing the retrograde transport of multiple fluorescent tracers.

To determine the morphological relationships among extensor digitorum longus (EDL), tibialis anterior (TA), and semitendinosus (St) motor nuclei in the spinal cord of the cat, these nuclei were retrogradely labeled with three different fluorescent tracers. The fluorochromes--bisbenzimide, nuclear yellow, and propidium iodide--were applied by intramuscular injection or soaking the muscle nerve. The positions of the labeled motor nuclei were bilaterally symmetrical. The EDL and TA motoneurons were located in close proximity to one another, in the lateral regions of lamina IX in spinal segments L6 and L7. Although the boundaries of each nucleus were tightly opposed, the EDL and TA motor nuclei overlapped minimally, with the somata of EDL motoneurons positioned dorsal to those of TA. The St motor nucleus was located ventromedial to that of EDL and extended from the caudal portion of L6 through S1. Supplemental studies of the reflex effects evoked in EDL, TA, and St muscles by cutaneous nerve stimulation provided physiological observations that may be related to these anatomical results.     

In vivo requirement of TGF-β/GDNF cooperativity in mouse development: focus on the neurotrophic hypothesis

Neurotrophic factors are well-recognized extracellular signaling molecules that regulate neuron development including neurite growth, survival and maturation of neuronal phenotypes in the central and peripheral nervous system. Previous studies have suggested that TGF-β plays a key role in the regulation of neuron survival and death and potentiates the neurotrophic activity of several neurotrophic factors, most strikingly of GDNF. To test the physiological relevance of this finding, TGF-β2/GDNF double mutant (d-ko) mice were generated. Double mutant mice die at birth like single mutants due to kidney agenesis (GDNF−/−) and congential cyanosis (TGF-β2−/−), respectively. To test for the in vivo relevance of TGF-β2/GDNF cooperativity to regulate neuron survival, mesencephalic dopaminergic neurons, lumbar motoneurons, as well as neurons of the lumbar dorsal root ganglion and the superior cervical ganglion were investigated. No loss of mesencephalic dopaminergic neurons was observed in double mutant mice at E18.5. A partial reduction in neuron numbers was observed in lumbar motoneurons, sensory and sympathetic neurons in GDNF single mutants, which was further reduced in TGF-β2/GDNF double mutant mice at E18.5. However, TGF-β2 single mutant mice showed no loss of neurons. These data point towards a cooperative role of TGF-β2 and GDNF with regard to promotion of survival within the peripheral motor and sensory systems investigated.     

Safety factor for neuromuscular transmission at type-identified diaphragm fibers

The safety factor (SF) for neuromuscular transmission varies across limb muscles of different fiber-type composition. Using intracellular recordings in rat diaphragm fibers, we found that SF varies across muscle fiber types (even within a single muscle), being larger for type IIx or IIb fibers than for type I or IIa fibers. Fiber-type differences in activation history or mechanical load may contribute to differences in SF and are important determinants of neuromuscular plasticity.     

In vivo evidence for serotonin 5-HT2C receptor-mediated long-lasting excitability of lumbar spinal reflex and its functional interaction with 5-HT1A receptor in the mammalian spinal cord

The purpose of the present study was to investigate the 5-HT(2C) receptor-mediated effects on the spinal monosynaptic mass reflex activities and also its functional interactions with 5-HT(1A) receptors in anesthetized, acutely spinalized mammalian adult spinal cord in vivo. Intravenous administration of (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) (0.1 mg/kg), an agonist of 5-HT(2A/2C) receptors, significantly increased the excitability of spinal motoneurons as reflected by an increase in the spinal monosynaptic mass reflex amplitude to 150-200% of the control. 5-HT(2A/2C) receptor-induced motoneuron excitability was slow, persistent and long-lasting for more than 2h that was significantly inhibited by 5-HT(2C) receptor specific antagonist SB 242084 administered 10 min prior to DOI. Simultaneous administration of DOI (0.1 mg/kg, i.v.) along with (+/-)-8-hydroxy dipropylaminotetraline hydrobromide (8-OH-DPAT) (0.1 mg/kg, i.v.) completely inhibited DOI-induced spinal monosynaptic mass reflex facilitation. In another separate study, administration of 8-OH-DPAT (0.1 mg/kg, i.v.) at the maximum response of DOI also inhibited the motoneuron's excitability; however, the inhibition lasted only for a period of 40-60 min after administration of 8-OH-DPAT, after which the spinal monosynaptic mass reflex amplitude reached its maximum level. These findings suggest that the 5-HT(2C) receptor is primarily involved in the mediation of the long-lasting excitability of spinal motoneurons and possibly interacts with its functional counterpart, 5-HT(1A) receptors in the mammalian adult spinal cord.     

Neuroendocrine secretory protein 55 (NESP55) in the spinal cord of rat: an immunocytochemical study

The immunohistochemical expression of a novel chromogranin-like protein, neuroendocrine secretory protein 55 (NESP55), in the rat spinal cord was investigated. NESP55-immunoreactive cells were detected in the ventral horn, intermediate laminae, and deep dorsal horn, comprising motoneurons, autonomic neurons, and interneurons throughout all spinal segments. Within laminae I-II of the dorsal horn, one or two NESP55-positive cells were often seen. Nerve fibers also contained NESP55 immunoreactivity (IR) and were particularly prominent in the ventral horn. No nerve terminals/varicosities appeared to contain NESP55 in any spinal lamina. Double-staining experiments revealed that a high proportion of the NESP55-positive neurons were cholinergic. Moreover, NESP55-IR in the motoneurons was evenly distributed in the whole cytoplasm with a finely granular appearance. In contrast, the fluorescent material in the preganglionic neurons was concentrated in the perinuclear region and largely overlapped with the trans-Golgi network marker TGN38. Our data provide detailed morphological information on the distribution of NESP55-IR in the rat spinal cord. Also, the differential intracellular expression of NESP55-IR in the spinal motoneurons and autonomic neurons suggests that NESP55 may be processed into different secretory granules and may be involved in both constitutive and regulated pathways in these neurons.     

Axon terminals expressing vesicular glutamate transporter VGLUT1 or VGLUT2 within the trigeminal motor nucleus of the rat: Origins and distribution patterns

Little is known about the significance of the two types of glutamatergic neurons (those expressing vesicular glutamate transporter VGLUT1 or VGLUT2) in the control of jaw movements. We thus examined the origin and distribution of axon terminals with VGLUT1 or VGLUT2 immunoreactivity within the trigeminal motor nucleus (Vm) in the rat. The Vm was divided into the dorsolateral division (Vm.dl; jaw‐closing motoneuron pool) and the ventromedial division (Vm.vm; jaw‐opening motoneuron pool). VGLUT1‐immunopositive terminals were seen within the Vm.dl only, whereas VGLUT2‐immunopositive ones were distributed to both the Vm.dl and the Vm.vm. Transection of the motor root eliminated almost all VGLUT1‐immunopositive axons in the Vm.dl, with no changes of VGLUT2 immunoreactivity in the two divisions, indicating that the VGLUT1‐ and VGLUT2‐immunopositive axons came from primary afferents in the mesencephalic trigeminal nucleus and premotor neurons for the Vm, respectively. In situ hybridization histochemistry revealed that VGLUT2 neurons were much more numerous than VGLUT1 neurons in the regions corresponding to the reported premotoneuron pool for the Vm. The results of immunofluorescence labeling combined with anterograde tract tracing further indicated that premotor neurons with VGLUT2 in the trigeminal sensory nuclei, the supratrigeminal region, and the reticular region ventral to the Vm sent axon terminals contacting trigeminal motoneurons and that some of the VGLUT1‐expressing premotor neurons in the reticular region ventral to the Vm sent axon terminals to jaw‐closing motoneurons. The present results suggested that the roles played by glutamatergic neurons in controlling jaw movements might be different between VGLUT1‐ and VGLUT2‐expressing neurons. J. Comp. Neurol. 512:595–612, 2009. © 2008 Wiley‐Liss, Inc.     

Detailed mapping of CGRP mRNA expression in the rat central nervous system: Comparison with previous immunocytochemical findings

The localization of CGRP mRNA in neurons of the rat brain and spinal cord was assessed by in situ hybridization histochemistry (ISH) using a radiolabeled synthetic 57-mer oiigodeoxynucleotide probe complementary to the rat prepro CGRP mRNA. Results were compared with previously published findings of CGRP-immunoreactive (CGRP-IR) cell bodies revealed by an indirect immunofluorescence technique. The highest numbers of CGRP mRNA expressing neurons as well as the greatest intensity of staining were found in the lateral hypothalamic area, the parabrachial nuclei, and among the cranial motor nuclei, especially in the nuclei of the 7th and 12th nerve and the ambiguus nucleus, which is generally in good agreement with findings assessed by immunocytochemistry (ICH). However, some mismatches between the localizaton of the peptide by ICH and the localization of the CGRP mRNA were also observed. Thus, ISH was not able to confirm CGRP-IR in cells of the amygdaloid complex and parts of the medial hypothalamus, the central gray, and the inferior colliculus, but ISH revealed considerably more CGRP mRNA expressing cells in the lateral hypothalamic area, arcuate nucleus, posterior and peripeduncular thalamic nuclei, and all cranial motor nuclei than CGRP-IR containing cells found by ICH. Moreover, ISH also revealed CGRP mRNA synthesis in the nucleus of the lateral olfactory tract and in the perihypoglossal nuclei that were devoid of CGRP-IR. The reasons for the observed mismatches still remain to be elucidated; however, intracerebroventricular colchicine pretreatment used to increase immunocytochemical signals also might have induced or suppressed gene expression in certain brain regions in an unpredictable matter. On the other hand, detection of only the mRNA in a certain region does not necessarily mean that also the active peptide is synthesized there.