Reduced cholinergic and glutamatergic synaptic input to regenerated motoneurons after facial nerve repair in rats: potential implications for recovery of motor function

Deafferentation of motoneurons after facial nerve injury is a well-documented phenomenon but whether synaptic inputs to facial motoneurons are completely restored after reinnervation is unknown. Here, we tested the hypothesis that deficits in motor performance after transection/suture of the facial nerve (facial–facial anastomosis, FFA) in adult rats are associated with incomplete recovery of synaptic inputs. At 2 months after FFA, we found, in congruence with previous results, that the amplitude of whisking had recovered to only 31 % of control (sham operation). In the same FFA-treated rats, estimates of number of chemically defined synaptic terminals in the facial nucleus by immunohistochemistry and stereology showed a significant loss, compared with sham controls, of glutamatergic terminals (?26 %) and cholinergic perisomatic boutons (?31 %), but not inhibitory (GABA/glycinergic) terminals (?14 %). Synaptic deficits were accompanied by persistent microgliosis in the facial nucleus but soma area, dendritic arbor volume, and total number of motoneurons were normal. Correlation analyses revealed significant co-variations of whisking amplitude with number of glutamatergic and cholinergic synapses. Compared with 2 months, analyses of animals at 4 months after FFA showed no attenuation of the functional deficit and structural aberrations with one exception, increase of inhibitory terminal numbers beyond control level (+11 %) leading to further reduction of the excitatory/inhibitory terminal ratio. We suggest that deficits in motoneuron innervation in the regenerated facial nucleus—reduced glutamatergic and cholinergic input and reduced excitatory/inhibitory terminal ratio—could attenuate the motor output and, thus, negatively impact the functional performance after facial nerve regeneration.     

Synaptic remodeling in the rat arcuate nucleus during the estrous cycle

Adult female rats showing regular vaginal cycles were studied in order to determine the number of axosomatic synapses in thin sections of the arcuate nucleus. The number of synapses per length of perikaryal membrane was significantly decreased in estrus, compared to other days of the estrous cycle (P less than 0.05). The reduction in the number of synapses in estrus was accompanied by a decrease in the percentage of the average length of perikaryal membrane covered by presynaptic terminals and by an increase in the percentage of membrane in close apposition of glial processes. Since the average perikaryal perimeter was not significantly changed during the estrous cycle, these results indicate a net decrease in the number of arcuate nucleus axosomatic synapses between proestrus and estrus, with a reinnervation of arcuate neurons between estrus and metestrus. These results suggest that there is a physiological synaptic turnover in the arcuate nucleus of the rat during the estrous cycle.     

Synaptic organization of damaged infraorbital nerve axons in perinatal rats: demonstration by galanin immunocytochemistry

Neonatal transection of the infraorbital nerve (ION; the trigeminal, V, branch that supplies the mystacial vibrissae follicles) results in an upregulation of galanin in the central arbors of primary afferent axons. The present study was undertaken to evaluate the synaptic organization of these galanin-positive primary afferents and compare it with that of normal neurobiotin/biocytin-labeled primary afferent axons from animals of the same age. Examination of 1200 neurobiotin/biocytin-labeled profiles in V nucleus principalis (PrV) of rats killed on postnatal day (P-) 7 indicated that 23.3% (n=279) of these profiles made synaptic contacts: 87.4% were axodendritic, 8.9% were axoaxonic, 2.8% were axosomatic, and 0.7% were axospinous. Evaluation of 1200 galanin-positive profiles in PrV from rats that sustained transection of the ION on P-0 and were killed on P-7 indicated that only 64 (5.3%) of these profiles made synaptic contacts (P<0.05 compared with the intact animals). Of the galanin-positive profiles that did make synapses in PrV, 81.2% (n=52) were axodendritic and 18.8% (n=12) were axoaxonic. These results indicate that galanin released by damaged ION primary afferents in PrV is likely to affect the activity of second-order V neurons by a paracrine action rather than by acting at specific synapses.     

Manual stimulation of forearm muscles does not improve recovery of motor function after injury to a mixed peripheral nerve

Transection and re-anastomosis of the purely motor facial nerve leads to poor functional recovery. However, we have recently shown in rat that manual stimulation (MS) of denervated vibrissal muscles reduces the number of polyinnervated motor endplates and promotes full recovery of whisking. Here, we examined whether MS of denervated rat forearm muscles would also improve recovery following transection and suture of the mixed (sensory and motor) median nerve (median–median anastomosis, MMA). Following MMA of the right median nerve, animals received no postoperative treatment, daily MS of the forearm muscles or handling only. An almost identical level of functional recovery, measured by the force of grip in grams, was reached in all animals by the sixth postoperative week and maintained till 3 months following surgery regardless of the postoperative treatment. Also, we found no differences among the groups in the degree of axonal sprouting, the extent of motor endplate polyinnervation and in the soma size of regenerated motoneurons. Taken together, we show that while MS is beneficial following motor nerve injury, combined strategies will be required for functional recovery following mixed nerve injury. S. A. Dunlop and D. N. Angelov contributed equally and share last authorship.     

Effects of testosterone on hippocampal CA1 spine synaptic density in the male rat are inhibited by fimbria/fornix transection

This study investigated the contribution of sub-cortical afferent input to the effects of testosterone (T) on spine synapse density in the CA1 subfield of the hippocampus, in adult male rats. Gonadectomized (GDX) male rats exhibited a considerably lower density of spine synapses in the CA1 region than control, intact males. The effects of GDX were reversed by treatment with testosterone propionate (TP; 500 microg/day, for 2 days). Transection of the fimbria/fornix (FF) had no significant effect on the synaptic density in non-GDX males. However, FF transection partially inhibited the responses to TP in GDX animals. These data suggest that the effects of T on spine synapse density in the CA1 region of the male rat hippocampus are partially, but not completely, dependent on afferent sub-cortical input.     

大鼠面神经切断及修复后面神经核内胆碱乙酰转移酶的变化

为了研究面神经核胆碱乙酰转移酶在神经损伤及修复后的动态变化 ,用免疫组织化学方法 ,观察了成年大鼠面神经外周切断和即刻端端吻合后面神经核胆碱乙酰转移酶阳性神经元的数量和免疫反应强度的时程变化。结果证明 ,面神经切断后 ,损伤侧面神经核胆碱乙酰转移酶阳性神经元的数量和免疫反应强度均明显下降 ,术后第 7d下降至最低。面神经切断后立即行端端吻合 ,术后 7d内手术侧面神经核胆碱乙酰转移酶阳性神经元的数量和免疫反应强度的下降规律与面神经单纯切断组相同。面神经吻合后 ,术侧面神经核胆碱乙酰转移酶阳性神经元的数量和免疫反应强度的回升最早出现在吻合后第 14 d,术后 3 5 d手术侧面神经核胆碱乙酰转移酶阳性神经元的数量恢复至正常。上述结果提示 ,面神经吻合并不能阻止损伤侧面神经核胆碱乙酰转移酶免疫阳性产物的下降 ;伴随着面神经再支配的发生 ,面神经核胆碱乙酰转移酶免疫阳性产物逐渐恢复正常     

Transformation of synaptic vesicle phenotype in the intramedullary axonal arbors of cat spinal motoneurons following peripheral nerve injury

Permanent transection of a peripheral motor nerve induces a gradual elimination of whole axon collateral systems in the axotomized spinal motoneurons. There is also an initial concurrent decrease in the amount of recurrent inhibition exerted by these arbors in the spinal cord for up to 6 weeks after the injury, whereas the same reflex action returns to normal by the 12-week postoperative state. The aim of the present investigation was to study the fine structure of the intramedullary axonal arbors of axotomized α-motoneurons in the adult cat spinal cord following a permanent peripheral motor nerve lesion. For this purpose, single axotomized α-motoneurons were labeled intracellularly with horseradish peroxidase at 12 weeks after permanent transection of their peripheral motor nerve. The intramedullary portions of their motor axon and axon collateral arbors were first reconstructed at the light microscopic level and subsequently studied ultrastructurally. This study shows that the synaptic contacts made by the intramedullary axon collateral arbors of axotomized motoneurons have undergone a change in synaptic vesicle ultrastructure from spherical and clear vesicles to spherical and dense-cored vesicles at 12 weeks after the transection of their peripheral axons. We suggest that the present transformation in synaptic vesicle fine structure may also correspond to a change in the contents of these boutons. This may, in turn, be responsible for the strengthening and recovery of the recurrent inhibitory reflex action exerted by the axotomized spinal motoneurons following a prolonged permanent motor nerve injury. Electronic Publication     

Synaptic loss following depletion of noradrenaline and/or serotonin in the rat visual cortex: a quantitative electron microscopic study

Biogenic amines have a trophic-like role for the formation and the maintenance of synapses in the CNS. We examined the changes in the number of synaptic profiles in the developing and adult rat visual cortex following selective depletion of noradrenaline and/or serotonin. By the drug-induced decreases in levels of noradrenaline or serotonin between 1 and 2 weeks after birth, the number of synaptic profiles was decreased by 29-55% compared with that of control animals. The magnitude of reduction in the number of synaptic profiles was virtually the same following simultaneous depletion of both noradrenaline and serotonin compared with the depletion of noradrenaline or serotonin alone. Later in the developmental period, the function of noradrenaline and serotonin in facilitating synapse formation and maintenance became less prominent than that in younger animals. In the control animals, the number of axosomatic synapses was the highest at around 2 weeks after birth, and decreased with development. The number of axodendritic synapses was the highest between 2 and 7 weeks after birth, and decreased to 50% at 11 weeks after birth. These data demonstrate that synapses in the rat visual cortex are overproduced during the early developmental period. We suggest that both serotonin and noradrenaline are necessary for synapse formation during the early stages of development of the rat visual cortex.     

Testosterone-induced acceleration of recovery from facial paralysis in male hamsters: temporal requirements of hormone exposure

In this study, the temporal requirements of testosterone propionate (TP) exposure necessary for acceleration of recovery from facial paralysis to occur following facial nerve crush were examined. For each of two series of experiments, adult castrated male hamsters were subjected to crush axotomies of the facial nerve at its exit from the stylomastoid foramen. In the first experimental paradigm, one-half of the animals with facial nerve crush axotomies received subcutaneous TP capsules beginning on postoperative (PO) day 6 and continuing throughout the regeneration period, with the remainder of the animals sham implanted. The results indicate that, without the early exposure to TP, the accelerative effects of the hormone on facial nerve regeneration were abolished. In the second experimental paradigm, one-half of the animals with facial nerve crush axotomies received subcutaneous TP implants immediately after the crush surgeries and until PO day 7, with the remainder of the animals sham implanted. The results indicate that an early, discontinuous dose of TP immediately after crush surgeries was sufficient to produce a partial accelerative effect on the return of facial nerve function. It is hypothesized from these findings that there is a priming effect of TP that is exerted at the level of the neuron, temporally precedes behavioral recovery by a week or more and is critical to subsequent acceleration of recovery from facial paralysis.     

Compensatory loss of axosomatic synapses in the dentate gyrus of the senescent rat

A portentous reorganization of the dentate gyrus occurs characteristically in senescent rats. This reorganization includes atrophy of dendrites, hypertrophy of astrocytes and a 27% loss of axodendritic synapses in the molecular layer of the dentate gyrus.A coincident loss of axosomatic synapses is now reported. These synapses on granule cell somata were counted in electron micrographs of representative coronal sections through the dentate gyri of five 3-month-old and five 25-month-old Fischer-344 male rats. A 15% decrease in the number of axosomatic synapses per 100 μm length of granule cell plasma membrane and a 22% decrease in the amount of neuronal surface covered by synapses were found in the senescent, as compared with the young adult, animals. These differences were statistically significant.As synapses on granule cell somata are inhibitory terminals of GABAergic interneurons it is suggested that their loss in senescence may be compensatory for the loss of axodendritic synapses, which are excitatory. By means of a compensatory loss of synapses it may be possible for the aging animal to maintain a reasonably adaptive level of function in spite of ongoing changes in the level of granule cell excitation resulting from the reorganization of the dentate gyrus in senescence.     

Effects of age on axon terminals forming axosomatic and axodendritic inhibitory synapses in prefrontal cortex

Much of the cognitive decline shown by aging primates can be attributed to dysfunction of prefrontal cortex and, as shown previously, about 30% of asymmetric (excitatory) and symmetric (inhibitory) axodendritic synapses are lost from the neuropil of layer 2/3 in prefrontal area 46 with age [Peters A, Sethares C, Luebke JI (2008) Neuroscience 152:970–981]. Whether there is a similar loss of inhibitory axosomatic synapses from this cortex has not been determined, but a study in primate motor cortex suggests that axosomatic synapses are not lost with age [Tigges J, Herndon JG, Peters A (1992) Anat Rec 232:305–315]. The present study is focused upon whether the remaining axon terminals forming inhibitory synapses in old monkeys hypertrophy to compensate for any age-related loss. Analysis of electron micrographs show that in layer 2/3 of area 46 in both young and old monkeys, axon terminals forming axosomatic synapses are significantly larger and contain more mitochondria than those forming axodendritic synapses and both axodendritic and axosomatic terminals become larger with age. However, while mitochondria in axodendritic terminals do not change in either size or amount with age, the mitochondria in axosomatic terminals become larger. Similarly, in terminals forming axodendritic synapses, the mean numbers of synaptic vesicle profiles is the same in young and old monkeys, whereas in terminals forming axosomatic synapses there is an increase in the numbers of synaptic vesicles with age. We also show that among these age-related changes, only the numbers of synaptic vesicles in axosomatic synapses are significantly correlated with the cognitive impairment indices displayed by the same monkeys. In summary, the data provide original evidence that axosomatic axon terminals increase in size and in their content of mitochondria and synaptic vesicles. Furthermore, based on our and previously published results, we speculate that these changes are linked to age-related cognitive decline.     

大鼠下丘脑弓状核突触的衰老性变化

用透射电镜结合体视学方法，对３月龄、１０月龄和３０～３４月龄大鼠弓状核突触进行了定性和定量研究。结果显示：老龄组大鼠神经毯呈萎缩变性相，大树突内脂褐素增多，小到中等大小的树突出现空泡变性、多泡体和膜被多层体等，棘萎缩减少；轴突终末内突触囊泡减少而大颗粒囊泡积聚，部分突触前、后膜变薄、缩短或间断，突触小球少见；轴－体、轴－树和轴－棘突触数减少，突触密度、突触连接带面密度和突触膜总长度降低，ＧｒａｙⅠ型和即Ⅱ型突触间隙增宽。上述结果表明，老年弓状核突触在数量、形态和结构上发生了衰老性改变，这是导致下丘脑神经内分泌衰老障碍的主要原因之一。     

T cell memory in the injured facial motor nucleus: relation to functional recovery following facial nerve crush

T cells have the ability to mount a memory response to a previously encountered antigen such that re-exposure to the antigen results in a response that is greater in magnitude and function. Following facial nerve transection, T cells have been shown to traffic to injured motor neurons in the facial motor nucleus (FMN) and may have the ability to promote neuronal survival and functional recovery. Previously, we demonstrated that early exposure to neuronal injury on one side of the brain during young adulthood elicited a T cell response that was greater in magnitude following exposure to the same form of injury on the contralateral side later in adulthood. Whether the T cell memory response to neuronal injury influenced functional recovery following nerve crush injury was unknown. In the current study, we tested the hypotheses that (1) transection of the right facial nerve in sensitized mice would result in faster recovery of the whisker response when the contralateral facial nerve is crushed 10 weeks later, and (2) the early recovery would be associated with an increase in the magnitude of the T cell response in the contralateral FMN following crush injury in sensitized mice. The onset of modest recovery in sensitized mice occurred between 3 and 5 days following crush injury of the contralateral facial nerve, approximately 1.5 days earlier than naïve mice, and was associated with more than a two-fold increase in the magnitude of the T cell response in the contralateral FMN following crush injury. There was no difference between groups in the number of days to full recovery. Further study of how T cell memory influences neuroregeneration may have important implications for translational research.     

Synaptic relationships between induced neuropeptide Y-like immunoreactive terminals and cuneothalamic projection neurons in the rat cuneate nucleus following median nerve transection

Previous studies have demonstrated that following complete median nerve transection (CMNT), neuropeptide Y-like immunoreactive (NPY-LI) fibers were dramatically increased and predominantly expressed in the ventral portion of the middle cuneate nucleus (CN), reaching maximum numbers at four weeks. Ultrastructurally, NPY-LI terminals made axodendritic synapses, but the postsynaptic elements are unknown. In the present study, using retrograde tract-tracing of wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP) and NPY immunocytochemistry we examined the synaptic relationships between cuneothalamic projection neurons (CTNs) and NPY-LI terminals in the rat CN following CMNT. The injury-induced NPY-LI fibers were distributed throughout the rostrocaudal extent of the CN. Further, the greatest number of HRP-labeled CTNs was observed in the ventral portion of the middle CN. Ultrastructurally, the NPY-LI terminals made asymmetric axodendritic synaptic contact with the HRP-labeling CTN dendrites. These data suggest that injury-induced NPY may modulate the excitability of CTNs, and thus, play a role in the transmission of neuropathic sensation following median nerve injury.     

Effect of chronic vitamin E deficiency on the synapses of cerebellar glomeruli in young rats

A morphometric investigation has been carried out on the synaptic junctions in the cerebellar glomeruli of young-adult rats chronically deprived of vitamin E for 10 months and control animals of the same age. The following parameters were evaluated: the average length of the synapses (L), the numerical (NV) as well as the surface (SV) density of the synaptic contact zones. The results from these experimental groups were compared with data from young, adult and old rats. The results obtained show a significant decrease of the surface density of the synaptic contact zones in old and alpha-tocopherol deprived young-adult (11-month-old) rats as compared to younger and normally fed animals. This reduction of the synaptic contact area seems to be due to the marked decline in the number of synapses found in both cases. The average size (L) of the synaptic junctions, on the other hand, was increased in alpha-tocopherol deficient rats as compared to normally fed littermates. The significant reduction of the synaptic contact area in old and vitamin E deprived young rats supports the hypothesis that a common denominator may be responsible to explain this alteration. Because of the recognized protective role of alpha-tocopherol against free radical attacks on plasma membranes, the present findings support an involvement of membrane structural alterations in aging as well as in vitamin E deficiency.     

Vulnerability of cerebellar development in malnutrition-II. Intrinsic determination of total synaptic area on purkinje cell spines

In a previous investigation, we demonstrated a greater reduction in the ratio of granule cells to Purkinje cells in malnourished female rats than male rats that were reared under the same conditions (Hillman &; Chen, 1981). In this study, the synaptic relationships between these two types of cells were analyzed to determine how this difference in ratio was accommodated in cerebellar circuitry. Control diets (25% casein) and experimental diets (8% casein), having the same calories, were administered pregestationally and through lactation with continuation of the respective diets to offspring until death at 60 days. Molecular layer volume and synaptic density were determined and the relative number of synapses on each Purkinje cell was estimated from the number of synapses in the molecular layer and total number of Purkinje cells. The average length of synaptic profiles on Purkinje cell spines was measured and the average synaptic contact area on each Purkinje cell was calculated. We found that the average number of synapses on individual Purkinje cells decreased in the deficient female group but not in the deficient male group and there was an increase in the average length of synaptic profiles in the malnourished female group but not in the deficient male group.In some cerebella of females, elongated synaptic profiles on giant spines were seen on Purkinje cells. Comparison of the average number of synapses on each Purkinje cell with average length of synaptic profiles revealed an inverse relationship between size and number of synapses for two control and two experimental groups and a subgroup of females with giant spines. After conversion of average profile length to average contact area and taking into account the number of synapses on each Purkinje cell, we found that Purkinje cells for all five groups had the same total synaptic area. This inverse relationship was believed to reflect a constant total contact area for parallel fiber synapses on Purkinje cells.It is suggested that a constancy in total synaptic area means that the amounts of macromolecules that are incorporated into postsynaptic membrane specializations are determined intrinsically by the Purkinje cell. However, the distribution of these molecules to synaptic sites on dendritic trees can be shifted by interaction of parallel fiber synapses with the Purkinje cell.     

面神经损伤后降钙素基因相关肽在大鼠面神经核中的表达

目的研究降钙素基因相关肽(calcitonin gene-related peptide,CGRP)在面神经损伤后再生过程中面神经核中的表达。方法健康SD大鼠分别行左侧面神经茎乳孔切断术,术后饲养3、7、14、21、28、35d,取出脑干含面神经核团部分,用免疫组化及图像分析技术,观察面神经核中CGRP的变化。结果CGRP分布于正常SD大鼠面神经各亚核,面神经损伤后3d,损伤侧的面神经核中CGRP比对照侧增强,图像分析CGRP灰度值与对照侧比较,差异有统计学意义(P<0.05);损伤后7d达最高峰(P<0.05),以后渐减。结论损伤导致CGRP在面神经运动神经元中的表达增加,提示CGRP在面神经再生修复过程中发挥调理作用。     

The formation and regression of synapses during the re-innervation of axolotl striated muscles.

1. A study has been made of the formation and regression of synapses formed by spinal nerves 16 and 17 in axolotl hind-limb flexor muscles following the severing of nerve 16, using histological, ultrastructural and electrophysiological techniques. 2. Axolotl hind-limb flexor myofibres possessed 'en plaque' end-plates from either spinal nerve 16 or 17 or both at intervals of about 1000 micronm along their length; the myofibre's length constant was about 700 micronm allowing electrophysiological observations of at least two of these synapses during a single impalement; transmitter release at these synapses could be described by binomial statistics and in a given set of ionic conditions the binomial statistic parameter n was directly proportional to the size of the nerve terminals whilst the binomial statistic parameter p was invariant to changes in nerve terminal size. 3. The distribution of synapses formed by spinal nerves 16 and 17 in different sectors of the axolotl hind-limb flexor muscles was determined from a study of evoked end-plate potentials; the middle and proximal sectors of the flexor muscles contained myofibres which received an innervation from nerve 16 only, whereas the sectors surrounding these contained myofibres innervated either by nerve 16 or nerve 17 or by both nerves. 4. Six days following the severing of spinal nerve 16, evoked transmitter release from the synapses formed by this nerve had failed; transmission was subsequently recorded at a few synapses formed by nerve 17 in the middle and proximal sectors of the flexor muscles which are not normally innervated by this nerve and these synapses had a low n; during the succeeding four weeks the value of n at the synapses increased to a size about 70% that of the terminals normally formed by nerve 16 at these sites. 5. Four weeks after severing nerve 16, myofibres which possessed synapses formed by nerve 17 also possessed synapses from re-innervating nerve 16 and these were sometimes formed at the same synaptic sites as those occupied by nerve 17. 6. In the subsequent sixteen weeks, the n value of synapses formed by nerve 17 declined whilst the n values of synapses formed by re-innervating nerve 16 on the same myofibres matured to their control size. 7. It is suggested that on severing nerve 16 collateral sprouting of nearby intact nerve 17 occurs and these collateral sprouts innervate the denervated synaptic sites, although the sprouts arenot as well matched to the denervated synaptic sites as are the original nerve terminals; thus if nerve 16 returns it preferentially forms synapses at its original synaptic sites, and the collateral synapses formed by nerve 17 regress.     

Ultrastructural features of neurons and synaptic contacts in the posterodorsal medial amygdala of adult male rats

The aim of the present study was to describe the ultrastructure of neurons (from eight animals) and to analyse the synaptic terminal distribution (from two animals) in the posterodorsal subnucleus of the medial amygdala (MePD) of adult male rats. Using transmission electron microscopy, it was possible to identify many spiny and aspiny dendrites, unmyelinated axonal bundles, single axonal processes, a few myelinated axons, blood vessels and glial processes in the neuropil. Axodendritic synapses were the most frequently observed (67.5%), appearing to be of either the inhibitory or the excitatory types. The presynaptic region contained round or flattened vesicles that occurred either singly or with dense-cored vesicles (DCVs). The dendrites often received many synapses on a single shaft, and axon terminals displayed synaptic contacts with one or more postsynaptic structures. Dendritic spines showed different morphologies and the synapses on them (23.1%) formed a single and apparently excitatory synaptic contact with round, electron-lucid vesicles alone or, less frequently, with DCVs. Inhibitory and excitatory axosomatic synapses (8.2%) and excitatory axoaxonic synapses (1.2%) were also identified. The present report provides new findings relevant to the study of the MePD cellular organization and could be combined with other morphological data in order to reveal the functional activity of this area in male rats.