Kainic acid-mediated increase of preprotachykinin-A messenger RNA expression in the rat hippocampus and a region-selective attenuation by dexamethasone.

The hippocampus contains the highest number of glucocorticoid-sensitive neurons in the rat brain and excessive exposure to glucocorticoids can cause damage to hippocampal neurons and impair the capacity of the hippocampus to survive neuronal insults. In this study in situ hybridization combined with quantitative image analysis was used to study preprotachykinin-A mRNA levels after administration of a toxic dose of kainic acid in animals pretreated with glucocorticoids. Kainic acid was injected into dorsal hippocampus CA3 region in animals pretreated with the synthetic glucocorticoid receptor agonist dexamethasone and in control animals. Preprotachykinin-A mRNA was not detected in the hippocampus of untreated animals or in animals analysed 30 min after a kainic acid injection. However, 4 h after injection of kainic acid, the level of preprotachykinin-A mRNA increased to 20-times above the detection limit both in the dentate gyrus and the CA3 region of the hippocampus. Treatment of kainic acid-injected animals with dexamethasone 30 min before and 2 h after the injection attenuated the increase in the granule cells of the dentate gyrus by 50%. In contrast, dexamethasone pretreatment had no significant effect on the kainic acid-induced increase of preprotachykinin-A mRNA in pyramidal cells in regions CA3 or CA1. These results show that an excitatory stimulus within the hippocampus causes a substantial increase in the level of preprotachykinin-A mRNA in hippocampal granule and pyramidal cells and suggest that in granule cells of the dentate gyrus this increase can be modulated by glucocorticoids.     

Temporal changes in the level of neurotrophins in the spinal cord and associated precentral gyrus following spinal hemisection in adult Rhesus monkeys

Neurotrophins (NTs) appear to be crucial for the survival and potential regeneration of injured neurons. However, their temporal changes and remote regulations following spinal cord injury (SCI) have been only partially determined, especially in primates. In this study, ELISA was performed on the extracts of injured spinal cord and the associated precentral gyrus contralateral to the site of spinal cord hemisection to investigate the temporal changes in the levels of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) in adult rhesus monkeys subjected to T8 spinal hemisection. Animals were allowed to survive 3, 7, 14, 30 and 90 days post-operation (dpo). In the spinal cord, the levels of NGF, BDNF and NT-3 sharply decreased between 3 and 7dpo. Thereafter, the levels of NGF and BDNF were transiently elevated while NT-3 level continuously increased and recovered to normal level at 30dpo. In the contralateral precentral gyrus (cPG), only the NT-3 level was altered and in fact elevated above the normal value. No obvious changes were observed in NT-4 level in any of the regions studied. Taken together, the present findings indicated that intrinsic NGF, BDNF and NT-3 may play a local role in the responses to the SCI in primates. Especially, the increase of NT-3 level occurred continuously in both the cPG and the spinal cord pointed to a possible transportation of NT-3 to the cord following SCI.     

Temporal changes in the level of neurotrophins in the spinal cord and associated precentral gyrus following spinal hemisection in adult Rhesus monkeys

Neurotrophins (NTs) appear to be crucial for the survival and potential regeneration of injured neurons. However, their temporal changes and remote regulations following spinal cord injury (SCI) have been only partially determined, especially in primates. In this study, ELISA was performed on the extracts of injured spinal cord and the associated precentral gyrus contralateral to the site of spinal cord hemisection to investigate the temporal changes in the levels of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) in adult rhesus monkeys subjected to T8 spinal hemisection. Animals were allowed to survive 3, 7, 14, 30 and 90 days post-operation (dpo). In the spinal cord, the levels of NGF, BDNF and NT-3 sharply decreased between 3 and 7 dpo. Thereafter, the levels of NGF and BDNF were transiently elevated while NT-3 level continuously increased and recovered to normal level at 30 dpo. In the contralateral precentral gyrus (cPG), only the NT-3 level was altered and in fact elevated above the normal value. No obvious changes were observed in NT-4 level in any of the regions studied. Taken together, the present findings indicated that intrinsic NGF, BDNF and NT-3 may play a local role in the responses to the SCI in primates. Especially, the increase of NT-3 level occurred continuously in both the cPG and the spinal cord pointed to a possible transportation of NT-3 to the cord following SCI.     

Septal cholinergic afferents regulate expression of brain-derived neurotrophic factor andβ-nerve growth factor mRNA in rat hippocampus

Summary In situ hybridization was used to study the expression of members of the nerve growth factor family of trophic factors in rat hippocampus following stimulation of afferent cholinergic and glutamatergic pathways with quisqualate. A transient increase in brain-derived neurotrophic factor (BDNF) and-nerve growth factor (NGF) mRNA expression in the hippocampus was seen 4 h after a quisqualate injection into the medial septal nucleus. Both BDNF and NGF mRNA levels increased more than 4-fold in the granule layer of the dentate gyrus and for BDNF mRNA also in the pyramidal cells of CA1, while the levels of BDNF mRNA in CA3 increased 2-fold. The increase in BDNF and NGF mRNA levels were completely prevented by pretreatment with systemic injections of either scopolamine or diazepam. A quisqualate injection into the entorhinal cortex, containing glutamatergic afferents to the hippocampus, resulted in a 15-, 5- and 17-fold increase in the expression of BDNF mRNA in the ipsilateral granule cells, CA3 and CA1 pyramidal cells, respectively. Similar increases were also seen in the hippocampus contralateral to the injections. In contrast, the level of NGF mRNA did not increase significantly in any of the subfields in the hippocampus. The increase in BDNF mRNA after cortex injections was attenuated by diazepam but not by scopolamine. These findings imply that increased activity in afferent cholinergic and glutamatergic pathways to the hippocampus differentially regulate expression of the NGF family of neurotrophic factors in the hippocampus.     

A role for neurotrophin-3 in targeting developing cholinergic axon projections to cerebral cortex

This study examined the relationship between expression of neurotrophin-3 (NT-3) and the ingrowth of cholinergic axonal projections in cerebral cortex. Patterns of expression of NT-3 (defined by beta-galactosidase reporter expression in heterozygous offspring of transgenic NT-3(lacZneo/+) mice) revealed that limbic cortical regions (including frontal, cingulate, and insular cortex, as well as the dentate gyrus) express NT-3 and that these cortical regions receive early and relatively dense cholinergic axons (stained for acetylcholinesterase, AChE). Using the dentate gyrus as a model system, studies revealed that expression of the NT-3 reporter parallels, and precedes by approximately 2 days, the ingrowth of AChE positive cholinergic axons. Studies of forebrain organotypic slice cultures demonstrate that basal forebrain-derived cholinergic axons extend into cortical regions in a pattern that mimics the pattern of expression of the NT-3 reporter. Similarly, chimeric co-cultures, combining wild type septum with a slice of hippocampus from heterozygous NT-3(lacZneo/+) mice, demonstrate that cholinergic axons grow into regions of the dentate gyrus that express the NT-3 reporter. Hemisphere slice cultures made from NT-3 knockout mice reveal cholinergic axonal growth into cortex, but these axons do not form the regional pattern characteristic of slice cultures made from wild type or heterozygous NT-3(lacZneo/+) mice. Further, chimeric co-cultures made using slices of wild type septum combined with slices of hippocampus from NT-3 knockout mice demonstrate robust cholinergic axonal growth into the hippocampus, but the cholinergic axons do not form the characteristic preterminal pattern associated with the dentate gyrus. Slice cultures from limbic cortical tissue from the NT-3 null mice do not display exaggerated levels of cell death. In aggregate, these data support the hypothesis that expression of NT-3 by cortical neurons serves to attract basal forebrain cholinergic projections to their target cells in cerebral cortex.     

脊髓半横断损伤后腹角神经元神经生长因子、脑源性神经营养因子、神经营养因子-3和神经营养因子-4表达的变化

目的:探讨大鼠脊髓半横断损伤(htSCI)后脑源性神经营养因子(BDNF)、神经生长因子(NGF)、神经营养因子(NT-3、NT-4)在脊髓腹角神经元表达的早期变化。方法:免疫组织化学ABC法分别染4种神经因子并作阳性细胞计数。结果:NGF主要分布于脊髓腹角神经元的胞核,BDNF、NT-4与NT-3主要分布于胞浆。htSCI前后它们在细胞内的分布范围没有变化。BDNF、NGF与NT-3的3 d在损伤尾侧段脊髓双侧腹角阳性神经元数与对照组相比显著减少。BDNF与NGF的14 d的双侧腹角阳性神经元数量均较正常组明显增多,NT-3与NT-4的14 d~21 d的双侧腹角阳性神经元数量均较正常组明显增多,BDNF7~21 d以及NGF14 d的健侧的阳性神经元数量均分别多于相应的伤侧。结论:内源性BDNF、NGF、NT-3、NT-4增加对脊髓损伤修复具有重要作用,BDNF和NGF在健侧表达的增加说明健侧代偿功能的活跃。     

Developmental changes in concentrations and distributions of neurotrophins in the monkey cerebellar cortex

Neurotrophins are involved in the survival, differentiation, migration and neurite outgrowth of various neuronal populations. Neurotrophins and their receptors are widely expressed in the developing cerebellum of various experimental animals. To gain some insight into the possible roles played by these molecules in monkey cerebellum, we examined the protein levels of BDNF, NT-4/5 and NT-3 and distributions of those neurotrophins and TrkC, a high affinity receptor for NT-3, in the cerebellum of developing macaque monkeys using ELISAs and immunohistochemical methods. We found that the level of BDNF increased during development, while the level of NT-3 was higher during embryonic stages and decreased toward adulthood. The level of NT-4/5 increased from embryonic stages to infant stages and gradually declined with age. Among the three neurotrophins, BDNF immunoreactivity was found in all kinds of cerebellar neurons, including all inhibitory interneurons, throughout the postnatal periods examined, indicating that BDNF may be an essential factor for the maintenance of cerebellar neural functions. The Bergmann glial fibers and the internal part of the external granule cell layer were strongly NT-3 immunopositive at the early postnatal stages, and more weakly immunoreactive toward adulthood. In addition, we found that the premigratory precursors of the granule cells were TrkC immunopositive at early postnatal stages. These findings suggest that NT-3 in Bergmann glial fibers may be involved in the migration of the premigratory granule cells.     

Autoradiographic analysis of [H]ryanodine binding sites in rat brain: Regional distribution and the effects of lesions on sites in the hippocampus

Quantitative and qualitative autoradiographic methods together with lesion approaches were used to determine the distribution of [3H]ryanodine binding sites in rat brain and the neuronal localization of these sites in the hippocampus. In normal animals, levels of [3H]ryanodine binding sites ranged from a low of about 1 fmol/mg tissue in subcortical structures to a high of 12-18 fmol/mg tissue in subregions of the hippocampus and the olfactory bulb. Relatively high densities of sites (5-9 fmol/mg tissue) were also seen in the olfactory tubercle, most areas of the cerebral cortex, accumbens nucleus, striatum, lateral septal nuclei, pontine nucleus, superior colliculus and granule cell layer of the cerebellum. Specific binding was undetectable in white matter. In experimental animals, intracerebral injections of kainic acid caused neuronal degeneration and a near total depletion of [3H]ryanodine binding sites in the dentate gyrus and in fields CA1, CA2 and CA3 of the hippocampus. Injections of kainic acid that left dentate granule cells largely intact while destroying all neurons in field CA3 had no effect on binding sites in the dentate gyrus. However, these lesions substantially reduced the density of binding in field CA3, leaving a narrow band of sites outlining the position of the degenerated CA3 pyramidal cells. Mechanical knife-cut lesions that severed the granule cell mossy fiber input to field CA3 reduced the density of binding sites in the CA3 region. The results indicate that [3H]ryanodine binding sites in brain are heterogeneously distributed and suggest that a proportion of these sites in the hippocampus may be contained in mossy fiber terminals where a presumptive calcium channel/ryanodine receptor complex may be involved in the regulation of calcium mobilization and/or neurotransmitter release.     

Cellular hybridization for BDNF. trkB, and NGF mRNAs and BDNF-immunoreactivity in rat forebrain after pilocarpine-induced status epilepticus

The messenger RNAs (mRNAs) for the neurotrophins, brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF), are upregulated during epileptic seizure activity, as visualized by in situ hybridization techniques. Neurotrophins might be protective against excitotoxic cell stress, and the upregulation during seizures might provide such cell protection. In this study, a high dose of pilocarpine (300 mg/kg) was used to induce long-lasting, limbic motor status epilepticus and a selective pattern of brain damage. The regulation of BDNF, trkB, and NGF mRNA was studied by in situ hybridization at 1, 3, 6, and 24 h after induction of limbic motor status epilepticus. BDNF immunoreactivity was examined with an anti-peptide antibody and the neuropathological process studied in parallel. BDNF mRNA increased in hippocampus, neocortex, piriform cortex, striatum, and thalamus with a maximum at 3–6 h. Hybridization levels increased earlier in the resistant granule and CA1 cells as compared to the vulnerable CA3 neurons. BDNF immunoreactivity was elevated in dentate gyrus at 3–6 h. trKB mRNA increased in the entire hippocampus. NGF mRNA in hippocampus appeared in dentate gyrus at 3–6 h and declined in hilar neurons at 6–24 h. Cell damage was found in the CA3 area, entire basal cortex, and layers II/III of neocortex. Endogenous neurotrophins are upregulated during status epilepticus caused by pilocarpine, which is related to the coupling between neuronal excitation and trophic factor expression. This upregulation of neurotrophic factors may serve endogenous protective effects; however, the excessive levels of neuronal hyperexcitation resulting from pilocarpine seizures lead to cell damage which cannot be prevented by endogenous neurotrophins.     

NGF,BDNF,NT-3和GDNF在背根节不同神经元的配布的免疫组织化学证据

为了探讨神经生长因子、脑源性神经生长因子 ,神经营养素 -3,胶原细胞源性神经生长因子在成年猫背根节神经元不同大小细胞的分布。取 L6背根节制成 2 0μm厚冰冻切片 ,用上述四种因子抗血清进行免疫组化反应。计数每一种因子阳性大、中、小型 (小于 42μm者为小型神经元 ;42～ 5 7μm者为中型神经元 ;大于 5 7μm者为大型神经元 )神经元的百分数。结果证明 ,此四种因子在背根节大、中、小神经元的阳性百分数分别为 :1.2± 0 .7,2 .1± 0 .9,3.3± 1.1;1.7± 0 .4,11.2± 1.2 ,30 .0± 1.5 ;2 6 .7± 2 .2 ,6 .2± 1.2 ,13.2± 2 .9;2 7.4± 3.3,8.1± 1.7,2 0 .1± 2 .4。表明 :在成年猫 L6背根节仅存在少数神经生长因子阳性细胞 ,而脑源性神经生长因子则主要配布于中、小神经元 ,神经营养素 -3和胶质细胞源性生长因子的免疫阳性细胞主要是大神经元 ,但也包括一些中小神经元。提示此四种因子在成年猫背根节的生理功能可能与神经元的不同大小有关。     

NGF、BDNF和NT-3在正常成年大鼠主要脑区表达的免疫组织化学研究

目的研究神经生长因子(NGF)﹑脑源性神经营养因子(BDNF)﹑神经营养素-3(NT-3)在正常成年大鼠主要脑区的表达。方法将正常成年大鼠用4%多聚甲醛灌注固定后取脑制成20μm厚的冰冻切片,应用上述3种因子的抗体进行免疫组织化学染色。结果3种因子的表达既有共同点也有不同点:共同点是在主要脑区都能表达,不同点是在单个细胞中,BDNF主要表达于胞浆边缘;NGF在大部分神经元的整个细胞都表达,但少量神经元则在胞核无表达;NT-3则与BDNF相似。结论3种因子在正常成年大鼠主要脑区都能表达。     

Enhanced but fragile inhibition in the dentate gyrus in vivo in the kainic acid model of temporal lobe epilepsy: a study using current source density analysis

Temporal lobe epilepsy is related to many structural and physiological changes in the brain. We used kainic acid in rats as an animal model of temporal lobe epilepsy, and studied the neural interactions of the dentate gyrus in urethane-anesthetized rats in vivo. Our initial hypothesis was that sprouting of mossy fibers, the axons of the granule cells, increases proximal dendritic excitatory currents in the inner molecular layer of the dentate gyrus. Extracellular currents were detected in vivo using current source density analysis. Backfiring the mossy fibers in CA3 or orthodromic excitation of the granule cells through the medial perforant path induced a current sink at the inner molecular layer. However, the sink or inferred excitation at the inner molecular layer was not increased in kainic acid-treated rats and the sink actually correlated negatively with the degree of mossy fiber sprouting. It is inferred that the latter sink was mediated mainly by association fibers and not by recurrent mossy fibers. After kainic acid treatment, paired-pulse inhibition of the population spikes in the dentate gyrus was increased. In contrast, reverberant activity that involved looping around an entorhinal-hippocampal circuit was increased in kainic acid-treated rats, compared to control rats. The increase of inhibition in kainic acid-treated rats was readily blocked by a small dose of GABA(A) receptor antagonist bicuculline. The latter dose of bicuculline induced paroxsymal spike bursts in kainic acid-treated but not control rats, demonstrating that the increased inhibition in dentate gyrus was fragile.In conclusion, after kainic acid induced seizures, the dentate gyrus in vivo showed an increase in inhibition that appeared to be fragile. The hypothesized increase in proximal dendritic excitation due to mossy fiber sprouting was not detected. However, the fragile inhibition could explain the seizure susceptibility in patients with temporal lobe epilepsy.     

BDNF Up-Regulates TrkB Protein and Prevents the Death of CA1 Neurons Following Transient Forebrain Ischemia

The neurotrophin family of growth factors, which includes Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT3) and Neurotrophin-4/5 (NT4/5) bind and activate specific tyrosine kinase (Trk) receptors to promote cell survival and growth of different cell populations. For these reasons, growing attention has been paid to the use of neurotrophins as therapeutic agents in neurodegeneration, and to the regulation of the expression of their specific receptors by the ligands. BDNF expression, as revealed by immunohistochemistry, is found in the pre-subiculum, CA1, CA3, and dentate gyrus of the hippocampus. Strong TrkB immunoreactivity is present in most CA3 neurons but only in scattered neurons of the CA1 area. Weak TrkB immunoreactivity is found in the granule cell layer of the dentate gyrus. Unilateral grafting of BDNF-transfected fibroblasts into the hippocampus resulted in a marked increase in the intensity of the immunoreaction and in the number of TrkB-immunoreactive neurons in the granule cell layer of the dentate gyrus, pre-subiculum and CA1 area in the vicinity of the graft. No similar effects were produced after the injection of control mock-transfected fibroblasts. Delayed cell death in the CA1 area was produced following 5 min of forebrain ischemia in the gerbil. The majority of living cells in the CA1 area at the fourth day were BDNF/TrkB immunoreactive. Unilateral grafting of control mock-transfected or BDNF fibroblasts two days before ischemia resulted in a moderate non-specific protection of TrkB-negative, but not TrkB-positive cells, in the CA1 area of the grafted side. This finding is in line with a vascular and glial reaction, as revealed, by immunohistochemistry using astroglial and microglial cell markers. This astroglial response was higher in the grafted side than in the contralateral side in ischemic gerbils, but no differences were seen between BDNF-producing and non-BDNF-producing grafts. However, grafting of BDNF-producing fibroblasts two days before ischemia significantly and specifically prevented nerve cells from dying in the CA1 area of the ipsilateral hippocampus. Cell survival was associated with increased TrkB immunoreactivity as the majority of living cells were TrkB immunoreactive. Thus, our results show that BDNF is able to up-regulate the expression of TrkB in control and pathological states, and that BDNF prevention of neuronal death following transient forebrain ischemia is associated with increased expression of its specific receptor.     

不同年龄双峰驼海马的形态学变化及其神经生长因子的表达

目的 探讨胎驼、青年双峰驼及成年双峰驼海马不同区域内主要神经元形态特征变化及神经生长因子（NGF）的表达情况。 方法 应用常规石蜡切片Nissl染色及免疫组织化学方法,对胎驼、青年双峰驼和成年双峰驼海马CA1～CA3区锥体细胞、齿状回（DG)颗粒细胞的形态学变化进行研究,并观察了NGF的表达变化情况。 结果 Nissl染色结果显示,从胎驼到青年双峰驼再到成年双峰驼,海马各区锥体细胞和颗粒细胞的体积增大、密度降低、细胞的核体比减小。NGF免疫组织化学结果表明,成年双峰驼海马锥体细胞中NGF的表达量显著高于胎驼和青年双峰驼（P<0.01）,颗粒细胞中NGF的表达量逐渐增加。 结论 从胎驼到成年双峰驼,海马中的主要神经元不断成熟,从体积小且少有突起和分支的神经元发育成为体积较大、具有明显形态和功能的神经元。NGF在成年双峰驼海马神经元中表达量最高,表明NGF可调节已分化的神经元,使其形态和功能发生改变以促进成年双峰驼与海马相关的学习记忆功能的逐步完善。     

Differential neurotrophin levels in cerebrospinal fluid and their changes during development in newborn rat

Cerebrospinal fluid concentration of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) was measured in normal developing rat from birth to postnatal day (PND) 21 by enzyme-linked immunosorbent assay. NGF levels were significantly higher than those of BDNF and NT-3 from PND 1–21. NGF levels decreased from PND 1–3 to PND 9. At PND 15 and 17, NGF levels peaked a second time and rapidly decreased to PND 21. BDNF peaked at PND 13–15, while NT-3 levels peaked at PND 7–9. Each of the three neurotrophins has its own characteristic pattern in changes in cerebrospinal fluid levels.     

Functional changes in neuropeptide Y- and somatostatin-containing neurons induced by limbic seizures in the rat

The influence of sustained epileptic seizures evoked by intraperitoneal injection of kainic acid on the gene expression of the neuropeptides somatostatin and neuropeptide Y and on the damage of neurons containing these peptides was studied in the rat brain. Injection of kainic acid induced an extensive loss of somatostatin and, though less pronounced, of neuropeptide Y neurons in the inner part of the hilus of the dentate gyrus. Neuropeptide Y-immunoreactive neurons located in the subgranular layer of the hilus, presumably pyramidal-shaped basket cells, were spared by the treatment. Although neuropeptide Y messenger RNA was not detected in granule cells of control rats, it was found there after kainic acid seizures at all time intervals investigated (12 h to 90 days after injection of kainic acid). High concentrations of neuropeptide Y messenger RNA were especially observed 24 h after injection of kainic acid. At this time neuropeptide Y messenger RNA was also transiently observed in CA1 pyramidal cells. Neuropeptide Y synthesis in granule cells in turn gave rise to an intense immunoreactivity of the peptide in the terminal field of mossy fibers which persisted for the entire time period (90 days) investigated. In addition, neuropeptide Y messenger RNA concentrations were also drastically elevated in presumptive basket cells located at the inner surface of the granule cell layer, especially at the “late” time intervals investigated (30–90 days after kainic acid).

These data support the concept that extensive activation of granule cells by limbic seizures contributes to the observed neuronal cell death in CA3 pyramidal neurons and interneurons of the hilus. Consecutively, basket cells containing neuropeptide Y and presumably GABA might be activated and participate in recurrent inhibition of granule cells. Neuropeptide Y-immunoreactive fibers observed in the inner molecular layer at “late” time intervals after kainic acid may result either from collateral sprouting of mossy fibers or from basket cells extensively expressing the peptide.

It is speculated that neuropeptide Y synthesized and released at a high rate from granule cells and basket cells may exert a protective action against seizures.     

Neurotrophin-3 is a target-derived neurotrophic factor for penile erection-inducing neurons

The aim of this study was to determine whether the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin (NT)-3 could act as endogenous target-derived trophic factors for erection-inducing, i.e. penis-projecting major pelvic ganglion (MPG) neurons, and/or penile sensory neurons in adult rat. This was accomplished by studying the expression of NT mRNAs in the penis and their cognate receptors in the MPG and dorsal root ganglia (DRGs), and the retrograde axonal transport of radioiodinated NTs injected into the corpora cavernosa. Northern hybridization showed that NGF, BDNF, and NT-3 mRNAs are expressed in the shaft of the penis. In situ hybridization combined with usage of the retrograde tracer Fluoro-Gold showed that TrkC and p75 receptors are expressed in penis-projecting neurons of the MPG whereas the mRNAs for TrkA and TrkB receptors were undetectable. However, all the NT receptor mRNAs were expressed in penile sensory neurons of sacral level 1 (S1) DRG. (125)I-NT-3 injected into the shaft of the penis was retrogradely transported into the MPG and S1 DRG, whereas radioiodinated NGF and BDNF were transported specifically into the S1 DRG, thus confirming the existence of functional NT receptors in these penile neurons. In conclusion, these data suggest that NT-3 may act as a target-derived neurotrophic factor for both erection-inducing and penile sensory neurons, whereas NGF and BDNF may be more important for the sensory innervation of the penis.     

Expression of some neurotrophins in the spinal motoneurons after cord hemisection in adult rats

There are numerous studies reporting on the crucial roles of neurotrophins (NTFs) in neuronal survival and sprouting after spinal cord injury (SCI). But studies on endogenous changes of neurotrophins after SCI are few. In this study we explored by means of immunohistochemistry the localization of NGF, BDNF and NT-3 in the normal adult spinal cord (SC) and the changes in the expression of these chemicals in the ventral horn after right cord hemisection at T9-10. The results showed an obvious increase in the numbers of NGF, BDNF and NT-3-immunoreactive neurons in the ventral horn and also an increase in their intracellular optical density (O.D.) at 3, 7 and 21 days after cord hemisection, when compared with sham-operated rats. The expression of NGF peaked at 7 days postoperation (dpo), while BDNF and NT-3 expressions peaked at 3 dpo. Evaluation of hindlimb functions by Basso Beattie Bresnahan (BBB) scoring showed that the hindlimb support and stepping function improved very quickly at 7 dpo. This study indicated that NGF, BDNF and NT-3 could play important but different roles in the mechanisms of spinal neuroplasticity at different times after SCI.     

Investigating the synergistic effect of combined neurotrophic factor concentration gradients to guide axonal growth

Neurotrophic factors direct axonal growth toward the target tissue by a concentration gradient, which is mediated through different tyrosine kinase cell surface receptors. In this study, well-defined concentration gradients of neurotrophic factors (NFs) allowed us to study the synergistic effect of different NFs (e.g. nerve growth factor [NGF], neurotrophin-3 [NT-3] and brain-derived neurotrophic factor [BDNF]) for axonal guidance of embryonic lumbar dorsal root ganglion cells (DRGs). Effective guidance of DRG axons was achieved with a minimum NGF concentration gradient of 133 ng/ml/mm alone, or combined NGF and NT-3 concentration gradients of 80 ng/ml/mm each. Interestingly, the combined concentration gradients of NGF and BDNF did not show any significant synergism at the concentration gradients studied. The synergism observed between NGF and NT-3 indicates that axons may be guided over a 12.5 mm distance, which is significantly greater than that of 7.5 mm calculated by us for NGF alone or that of 2 mm observed by others.     

Recurrent excitatory connectivity in the dentate gyrus of kindled and kainic acid-treated rats

Repeated seizures induce mossy fiber axon sprouting, which reorganizes synaptic connectivity in the dentate gyrus. To examine the possibility that sprouted mossy fiber axons may form recurrent excitatory circuits, connectivity between granule cells in the dentate gyrus was examined in transverse hippocampal slices from normal rats and epileptic rats that experienced seizures induced by kindling and kainic acid. The experiments were designed to functionally assess seizure-induced development of recurrent circuitry by exploiting information available about the time course of seizure-induced synaptic reorganization in the kindling model and detailed anatomic characterization of sprouted fibers in the kainic acid model. When recurrent inhibitory circuits were blocked by the GABA(A) receptor antagonist bicuculline, focal application of glutamate microdrops at locations in the granule cell layer remote from the recorded granule cell evoked trains of excitatory postsynaptic potentials (EPSPs) and population burst discharges in epileptic rats, which were never observed in slices from normal rats. The EPSPs and burst discharges were blocked by bath application of 1 microM tetrodotoxin and were therefore dependent on network-driven synaptic events. Excitatory connections were detected between blades of the dentate gyrus in hippocampal slices from rats that experienced kainic acid-induced status epilepticus. Trains of EPSPs and burst discharges were also evoked in granule cells from kindled rats obtained after > or = 1 wk of kindled seizures, but were not evoked in slices examined 24 h after a single afterdischarge, before the development of sprouting. Excitatory connectivity between blades of the dentate gyrus was also assessed in slices deafferented by transection of the perforant path, and bathed in artificial cerebrospinal fluid (ACSF) containing bicuculline to block GABA(A) receptor-dependent recurrent inhibitory circuits and 10 mM [Ca(2+)](o) to suppress polysynaptic activity. Low-intensity electrical stimulation of the infrapyramidal blade under these conditions failed to evoke a response in suprapyramidal granule cells from normal rats (n = 15), but in slices from epileptic rats evoked an EPSP at a short latency (2.59 +/- 0.36 ms) in 5 of 18 suprapyramidal granule cells. The results are consistent with formation of monosynaptic excitatory connections between blades of the dentate gyrus. Recurrent excitatory circuits developed in the dentate gyrus of epileptic rats in a time course that corresponded to the development of mossy fiber sprouting and demonstrated patterns of functional connectivity corresponding to anatomic features of the sprouted mossy fiber pathway.