Effect of reserpine on 5-hydroxytryptophan (5HTP)-immunoreactive neurons in the rat brain

By immunohistochemistry of rat brain in conjunction with a specific antibody against 5-hydroxytryptophan (5HTP), we examined immunoreactivity to 5HTP in neurons, from which 5-hydroxytryptamine (5HT; serotonin) was depleted by reserpine treatment. The distribution patterns of 5HTP-positive neurons overlapped with those of 5HT neurons. Treatment with reserpine (5 mg/kg, 90 min before death) caused a complete suppression of 5HT-positive staining, but 5HTP-immunostaining remained in perikarya of the nuclei raphe dorsalis, centralis superior and obscurus. Treatment with reserpine (25 mg/kg, 90 min before death) suppressed the 5HTP-immunoreaction in certain perikarya (e.g. of the nucleus raphe dorsalis) and fibres; however, 5HTP-immunostaining remained in perikarya of the nuclei centralis superior and raphe obscurus. This suggests that these neurons synthesize more 5HTP by a process which appears to be stimulated by reserpine.     

Distribution of serotonin-immunoreactive nerve cells and fibers in the rat gastrointestinal tract

 The distribution of serotonin-immunoreactive (5HT-IR) nerve cells and fibers was thoroughly investigated immunohistochemically in the rat stomach, duodenum, jejunum, ileum, and colon. The immunoreactivity of the 5HT neurons was compared between non-treated controls and animals treated with colchicine, colchicine plus 5-hydroxytryptophan (5HTP), colchicine plus pargyline, and reserpine. The intensity of immunoreactivity in nerve fibers as well as nerve cell bodies was enhanced mostly in colchicine plus pargyline treated animals, therefore these animals were used for an observation of precise localization of 5HT in the rat gastrointestinal (GI) tract. Immunoreactivity in the nerve cell bodies and fibers was completely abolished in the GI tract of reserpine treated animals. The pattern of localization and projection of 5HT-IR neurons was similar in all segments of the rat GI tract. 5HT-IR nerve cell bodies were located in the myenteric plexus and showed the distinctive features of Dogiel type I neurons. Prominent bundles of varicose fibers traversed the myenteric ganglia and some of them surrounded the cell bodies of immunopositive and immunonegative neurons. 5HT-IR nerve fibers were located in the submucous plexus, densely entwined about the submucosal blood vessels. Most characteristically, 5HT-IR nerve fibers invaded the lamina propria of mucosa where they underlay the crypt epithelium. In conclusion, the present study showed that 5HT-IR neurons located in the myenteric plexus projected fibers widely in the rat GI tract. The localization of fibers in the lamina propria of mucosa implies that this neuron may exert an important role in the epithelial function of the GI tract. Accepted: 8 October 1996     

Protein synthesis at synapse versus cell body: Enhanced but transient expression of long‐term facilitation at isolated synapses

Protein synthesis at synaptic terminals contributes to LTP in hippocampus and to the formation of new synaptic connections by sensory neurons (SNs) of Aplysia. Here we report that after removal of the SN cell body, isolated SN synapses of Aplysia in culture express protein‐synthesis dependent long‐term facilitation (LTF) produced by 5‐HT that decays rapidly. Changes in expression of a SN‐specific neuropeptide sensorin in isolated SN varicosities parallel the changes in synaptic efficacy. At 24 h after 5‐HT the magnitude of LTF produced at isolated SN synapses was significantly greater than that produced when SN cell bodies were present. LTF was maintained at 48 h at connections with SN cell bodies, but not at isolated SN synapses. The increase in synaptic efficacy at isolated SN synapses at 24 h was blocked by the protein synthesis inhibitor anisomycin. LTF was accompanied by changes in expression of sensorin. The increase in sensorin level at isolated SN varicosities with 5‐HT was blocked by anisomycin or was reversed 48 h after 5‐HT treatment alone. The results suggest that, as is the case for initial synapse formation between SNs and L7, changes in protein synthesis at synaptic terminals may contribute directly to LTF of stable synapses. Changes in expression within the cell body provide additional contributions for long‐term maintenance of the new level of synaptic efficacy that was initiated directly by local changes in protein synthesis at or near synaptic terminals. © 2003 Wiley Periodicals, Inc. J Neurobiol 56: 275–286, 2003     

5—HT能纤维向猫脊髓膈神经核及延髓膈肌前运动神经元的投射

实验在6只成年猫上进行,将WGA－HRP微量注入C5膈神经核内,通过逆行追踪及5－HT免疫组织化学FITC荧光双重标记方法,研究了中缝核5－HT能神经元向脊髓膈神经核的投射,同时观察了延髓膈肌产运动神经元接受5－HT能纤维投射的情况,结果在中缝苍白核观察到较多的HRP－5－HT双标记神经元,在中缝大核,中缝隐核观察到少数散在的双标记神经元,在延髓疑核,孤束核腹外侧区域的HRP单核记神经元（即膈肌前运动神经元）周围观察到5－HT能轴突末梢,结构表明：发自中缝苍白核5－HT能神经元的传出纤维可投射到脊髓膈神经核,延髓膈肌前运动神经元接受5－HT能纤维的传入投射。     

Postembryonic development of serotonin-immunoreactive neurons in the central nervous system of the blowfly,Calliphora erythrocephala

Summary The postembryonic development of serotonin-immunoreactive (5-HTi) neurons was studied in the optic lobe of the blowfly. In the adult fly there are 24 5-HTi neurons invading each optic lobe. The perikarya of two of these neurons are situated in the dorso-caudal part of the protocerebrum (LBO-5HT neurons; large bilateral optic lobe 5-HTi neurons). The cell bodies of the remaining 22 neurons are located anteriorly at the medial base of the medulla (2 innervating the lobula, LO-5HT neurons; and 20 neurons innervating the medulla, ME-5HT neurons). The two central neurons (LBO-5HT neurons) are derived from metamorphosing larval neurons, while the ME- and LO-5HT neurons are imaginai optic lobe neurons differentiating during pupal development.The 5-HTi neurons of the optic lobe seem to have different ancestors. The LBO-5HT neurons are probably derived from segmental protocerebral neuroblasts, whereas the ME-and LO-5HT neurons are most likely derived from the inner optic anlage. The first 5-HTi fibers to reach the imaginal optic lobes are seen in the late third instar larva and are derived from the LBO-5HT neurons. The first ME- and LO-5HT neurons become immunoreactive at 24 h (10%) pupal development. At about 96 h (40%) of pupal development all the 5-HTi neurons of the optic lobes have differentiated and attained their basic adult morphology. The further development mainly entails increase in volume of arborizations and number of finer processes. The differentiation and outgrowth of 5-HTi processes follows that of, e.g., columnar neurons in the optic lobe neuropils. Hence, 5-HTi processes invade neuropil relatively late in the differentiation of the optic lobe.     

Purinergic mechanosensory transduction and visceral pain

Background

Little is known about whether peripheral nerve injury during the early postnatal period modulates synaptic efficacy in the immature superficial dorsal horn (SDH) of the spinal cord, or whether the neonatal SDH network is sensitive to the proinflammatory cytokine TNFα under neuropathic conditions. Thus we examined the effects of TNFα on synaptic transmission and intrinsic membrane excitability in developing rat SDH neurons in the absence or presence of sciatic nerve damage.

Results

The spared nerve injury (SNI) model of peripheral neuropathy at postnatal day (P)6 failed to significantly alter miniature excitatory (mEPSCs) or inhibitory (mIPSCs) postsynaptic currents in SDH neurons at P9-11. However, SNI did alter the sensitivity of excitatory synapses in the immature SDH to TNFα. While TNFα failed to influence mEPSCs or mIPSCs in slices from sham-operated controls, it significantly increased mEPSC frequency and amplitude following SNI without modulating synaptic inhibition onto the same neurons. This was accompanied by a significant decrease in the paired-pulse ratio of evoked EPSCs, suggesting TNFα increases the probability of glutamate release in the SDH under neuropathic conditions. Similarly, while SNI alone did not alter action potential (AP) threshold or rheobase in SDH neurons at this age, TNFα significantly decreased AP threshold and rheobase in the SNI group but not in sham-operated littermates. However, unlike the adult, the expression of TNFα in the immature dorsal horn was not significantly elevated during the first week following the SNI.

Conclusion

Developing SDH neurons become susceptible to regulation by TNFα following peripheral nerve injury in the neonate. This may include both a greater efficacy of glutamatergic synapses as well as an increase in the intrinsic excitability of immature dorsal horn neurons. However, neonatal sciatic nerve damage alone did not significantly modulate synaptic transmission or neuronal excitability in the SDH, which could reflect a relatively weak expression of TNFα in the injured spinal cord at early ages. The above data suggest that although the sensitivity of the SDH network to proinflammatory cytokines after nerve injury is present from the first days of life, the profile of spinal cytokine expression under neuropathic conditions may be highly age-dependent.     

Speaking out of turn: a role for silent synapses in pain

Severe tissue or nerve injury can result in a chronic and inappropriate sensation of pain, mediated in part by the sensitization of spinal dorsal horn neurons to input from primary afferent fibers. Synaptic transmission at primary afferent synapses is mainly glutamatergic. Although a functioning excitatory synapse contains both alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors in the postsynaptic membrane, recent evidence suggests that dorsal horn neurons contain some "silent" synapses, which exhibit purely NMDA receptor-mediated evoked postsynaptic currents and do not conduct signals at resting membrane potential. Serotonin, which is released onto dorsal horn neurons by descending fibers from the rostroventral medulla, potentiates sensory transmission by activating silent synapses on those neurons, i.e., by recruiting functional AMPA receptors to the postsynaptic membrane. This phenomenon may contribute to the hyperexcitability of dorsal horn neurons seen in chronic pain conditions.     

移植的5—羟色胺能神经元跨软脊膜迁入大鼠脊髓的研究

本文对移植的5-HT神经元从蛛网膜下腔跨软脊膜迁移进入脊髓作了初步研究。将含有5-HT细胞的胚胎中缝核组织小块或神经细胞悬浮液作为移植物,以5-HT免疫组织化学方法跟踪移植细胞,结果如下:(1)在低胸水平横切脊髓,10d后,横断脊髓内的5-HT纤维消失。(2)横切脊髓(方法同上)后,立即将中缝核组织小块移植在胸腰段脊髓的蛛网膜下腔,一月后.在横断脊髓内出现5-HT阳性神经元和纤维。5-HT纤维能在灰白质内延伸。(3)脊髓横断后,若以中缝核的细胞悬浮液代替组织小块,作上述移植,则在移植区附近的灰质内出现大量的5-HT阳性神经元。这些神经元在灰质内的分布范围与神经细胞悬浮液在蛛网膜下腔的移植范围相一致。迁入神经元能在灰质内重新形成5-HT阳性纤维网。(4)经上述移植后,灰质内出现的5-HT阳性纤维随远离细胞体而变得稀疏。白质内的5-HT阳性纤维远比灰质内稀少。本实验结果表明:移植在脊髓蛛网膜下腔的脑干5-HT细胞能跨软脊膜迁移进入脊髓。     

Ultrastructural observation of 5-hydroxytryptamine-storing granules in the domestic fowl thrombocytes

Summary Ultrastructural observations of domestic fowl thrombocytes were made with special reference to their intracellular storage site of 5HT. Using a modified method for sampling blood and isolating thrombocytes, a characteristic structure was confirmed in the cytoplasm; the structure was highly osmiophilic, rough-surfaced and quite larger than 5HT granules observed in mammalian platelets. Disappearance of the structure, associated with a marked reduction of 5HT content, was caused by in vivo treatment with reserpine; this indicated its function to store the amine. From various morphological patterns indicative of stepwise advancement of degranulation, it was concluded that the 5HT-storing organelle is the multigranular body, which contains smooth-surfaced granules aggregated with fine filamentous structures.Sponsored partly by the Ministry of Education of Japan.The authors wish to acknowledge the valuable reviews of Professors A. Inouye and H. Takagi.     

Serotonin-induced changes in the excitability of cultured antennal-lobe neurons of the sphinx moth Manduca sexta

The modulatory actions of 5-hydroxy-tryptamine (5HT or serotonin) on a morphologically identifiable class of neurons dissociated from antennal lobes of Manduca sexta at stages 9–15 of the 18 stages of metamorphic adult development were examined in vitro with whole-cell patch-clamp recording techniques. Action potentials could be elicited from approximately 20% of the cells. These cells were used to examine effects of 5HT (5 × 10^–6 to 5 × 10^–4 M) on cell excitability and action-potential waveform. 5HT increased the number of spikes elicited by a constant depolarizing current pulse and reduced the latency of responses. 5HT also led to broadening of action potentials in these neurons and increased cell input resistance. Modulation of potassium channels by 5HT is likely to contribute to these responses. 5HT causes reversible reduction of at least 3 distinct potassium currents, one of which is described for the first time in this study. Because effects of 5HT on antennal-lobe neurons in culture mimic those observed in situ in the brain of the adult moth, in vitro analysis should contribute to elucidation of the cellular mechanisms that underlie the modulatory effects of 5HT on central olfactory neurons in the moth.     

S100 is present in developing chicken neurons and schwann cell and promotes motor neuron survival in vivo

We used polyclonal antisera recognizing S100, a small acidic protein highly enriched in nervous tissue, to stain sections of embryonic chicken lumbosacral spinal cord and hindlimb. S100 immunoreactivity was detected in developing sensory neurons of the dorsal root ganglia (DRG) and motor neurons of the ventral spinal cord as early as embryonic day (E) 5, and staining persisted through hatching. In contrast, expression of S100 first became apparent in Schwann cells at E13, just before myelination, and was not detected in developing skin or muscle. Since S100β was present in motor and sensory neurons and is known to promote neuronal survival and neurite extension in vitro (Winningham-Major, Staecker, Barger, Coats, and Van Eldik, 1989), we tested the ability of S100 to promote neuron survival in an in ovo survival assay. Addition of S100 to chick embryos in ovo during the period of naturally occurring motor neuron cell death resulted in a significant increase in motor neuron survival, but had no effect on the in vivo survival of sensory neurons in the DRG. The findings that S100 is present in spinal motor neurons and that the addition of S100 enhances the survival of these cells in vivo are consistent with the possibility that S100 may act as a naturally occurring neuron survival factor during development. © 1992 John Wiley & Sons, Inc.     

离体培养的小鼠脊髓固有神经元的突触构筑

运用电子显微镜观察分析原代分离培养鼠胚脊髓的固有神经元的突触构筑。培养中主要可见中、小型神经元,彼此之间可形成大量的突触,以非对称性突触占多数,有轴-树突触和轴-体突触,树-树突触为少见。根据以前学者分类标准将终扣分成S、F、M、和G四型。超微结构有利于提示固有神经元经过简单突触从脊髓固有神经纤维接受突触传入,表示它们的冲动只是突触后机制控制信息传递。     

离体培养的小鼠脊髓固有神经元的突触构筑

An electron microscopic analysis of the synaptic architecture in propriospinal neurons of cultured fetal mouse spinal cord has been undertaken. The size of the perikarya in the cultured spinal cord represents a range from small- to medium sized neurons, which form many synapses each other. There are many axo-dendritic and axo-somatic synapses in the culture but direct dendro-dendritic apposition is rarely seen. Four morphological types of synaptic boutons, S, F, M and G are classified according to criteria used by previous investigators. The ultrastructural details available suggest that the propriospinal neurons receive synaptic input from propriospinal fibers through simple synapses. It may indicate that their impulses can be controlled only postsynaptically.     

Serotonin modulation of calcium transients in cells in the suprachiasmatic nucleus.

Information about environmental lighting conditions is conveyed to the suprachiasmatic nucleus (SCN), at least in part, via a glutamatergic fiber pathway originating in the retina, known as the retinohypothalamic tract (RHT). Previous work indicates that serotonin (5HT) can inhibit this pathway, although the underlying mechanisms are unknown. The authors became interested in the possibility that 5HT can inhibit the glutamatergic regulation of Ca2+ in SCN neurons and, by this mechanism, modulate light-induced phase shifts of the circadian system. To start to examine this hypothesis, optical techniques were used to measure Ca2+ levels in SCN cells in a brain slice preparation. First, it was found that 5HT produced a reversible and significant inhibition of Ca2+ transients evoked by synaptic stimulation. Next, it was found that 5HT did not alter the magnitude or duration of Ca2+ transients evoked by the bath application of glutamate or N-methyl-D-aspartate acid (NMDA) in the presence of tetrodotoxin (TTX). The authors feel that the simplest explanation for these results is that 5HT can act presynaptically at the RHT/SCN synaptic connection to inhibit the release of glutamate. The demonstration that 5HT can have a dramatic modulatory action on synaptic-evoked Ca2+ transients measured in SCN neurons adds support to the notion that the serotonergic innervation of the SCN may function to regulate environmental input to the circadian system. In addition, it was found that the administration of higher concentrations of 5HT can increase Ca2+ in at least a subpopulation of SCN neurons. This effect of 5HT was concentration dependent and blocked by a broad-spectrum 5HT antagonist (metergoline). In addition, both TTX and the gamma-amino-N-butyric acid (GABA) receptor blocker bicuculline inhibited the 5HT-induced Ca2+ transients. Therefore, the interpretation of this data is that 5HT can act within the SCN to alter GABAergic activity and, by this mechanism, cause changes in intracellular Ca2+. It is also suggested that this 5HT-induced Ca2+ increase might play a role in 5HT-induced phase shifts of the SCN circadian oscillator.     

Capsaicin Does Not Change Tissue Levels of Glutamic Acid, Its Uptake, or Release in the Rat Spinal Cord

Capsaicin treatment (50 mg/kg, subcutaneous) of newborn rats resulted in 1 75% decrease of substance P immunoreactivity in the dorsal spinal cord of the adult animal, but failed to affect levels of the proposed sensory neurotransmitter glutamic acid or to alter high-affinity uptake of [3H]glutamic acid into synaptosomes of the same tissue. Furthermore, capsaicin (30 microM) in vitro had no influence on the release of [3H]glutamic acid from spinal cord P2 fractions of untreated adult rats, but induced a marked release of substance P. The results suggest that, in contrast to substance P fibers, neurons containing glutamic acid are not sensitive to capsaicin. Eleven other neurochemical parameters measured in the spinal cord did not appear to be changed by the treatment with capsaicin, suggesting a considerable neurochemical selectivity of the lesion.     

猫腰髓中GABA能神经元的分布及年龄相关变化

目的观察和比较GABA能神经元在青年猫和老年猫L6段脊髓的分布,探讨GABA能神经元在脊髓中分布的年龄相关变化及意义.方法免疫组织化学ABC法.结果青年猫与老年猫L6段脊髓灰质内,GABA能神经元及神经纤维分布广泛,各个Rexed板层均可见GABA-IR细胞,其中背侧灰质阳性最强,其次是腹侧灰质.标记的GABA能神经元胞体为卵圆形、三角形、多角形和星形,可分为大、中、小三种类型.经比较,老年组GABA能神经元的数量及免疫反应性均明显低于青年组.结论老年动物脊髓调节功能的减弱可能与GABA能神经元减少有少.     

大鼠脊髓胶状质内GABA能神经元突触联系的超微结构研究

本文用免疫电镜方法对脊髓胶状质内ＧＡＢＡ能神经元的突触联系进行了超微结构研究。结果表明;脊髓胶状质内有许多ＧＡＢＡ能神经元胞体和末梢分布;标记的ＧＡＢＡ能神经末梢可作为突触前成分与未标记的ＧＡＢＡ形成输一树突触。未标记的末梢可与标记的ＧＡＢＡ末梢形成输一轴突触。此外,标记的ＧＡＢＡ能神经末梢还可作为突触前成分与标记的ＧＡＢＡ能轴突、树突或胞体形成输－轴、轴－树或轴－体突触,即自调节突触。上述结果揭示：ＧＡＢＡ能末梢可对脊髓胶状质内其它神经元产生抑制或脱抑制作用。值得注意的是胶状质内含ＧＡｎＡ的神经结构可形成各种形式的自调节突触,并借此实现其对脊髓功能的复杂调节。     

The effects of the perinatal treatment with 5-hydroxytryptophan or tranylcypromine on the peripheral and central serotonin homeostasis in adult rats

Serotonin (5HT) is a biologically active amine present in mammals in the brain and the peripheral tissues. Autism is a neurodevelopmental disorder in which 5HT homeostasis is disturbed both centrally and peripherally, but the relationship between the 5HT disturbances in the two compartments is not understood. In an attempt to explore the relationship between the disturbed peripheral 5HT homeostasis and central 5HT functioning, we exposed the developing rat brain to increased 5HT concentrations, by treatment of rats with subcutaneous injections of the immediate 5HT precursor 5-hydroxy-l-tryptophan (5HTP, 25 mg/kg), or the non-selective MAO inhibitor tranylcypromine (TCP, 2 mg/kg), during the period of the most intensive development of 5HT neurons - from gestational day 13 to post-natal day 21. The effects of the mentioned treatments on peripheral and central 5HT levels were then studied in adult rats. Platelet and plasma 5HT concentrations (measured by ELISA), as well as cortical and midbrain 5HT, tryptophan and 5-hydroxyindoleacetic acid levels (measured by HPLC) were determined in twelve 5HTP treated and eight TCP treated rats, and compared with the values measured in 10 control, saline treated rats. Treatment with 5HTP significantly raised peripheral but not central 5HT concentrations. At adult age, peripheral 5HT homeostasis was re-established, while modest decrease in 5HT concentration was observed in frontal cortex, presumably due to hyperserotonemia-induced loss of 5HT terminals during brain development. Treatment with TCP induced significant 5HT elevations in both compartments. At adult age, permanent changes in 5HT homeostasis were observed, both peripherally (as hyperserotonemia) and centrally (as altered 5HT metabolism with decreased 5HT concentrations). Further studies are planned in order to explore the nature of the different disturbances of 5HT homeostasis induced by the two compounds, and their results are expected to shed some light on the role of hyperserotonemia in autism.     

Fluorescence microscopic observations of catecholamines in cultures of the sympathetic chains

Summary A histochemical technique for the demonstration of catecholamines developed by Falck et al. has been successfully applied to the sympathetic chains of rats and mice maintained in vitro. Catecholamines were localized in the nerve fibers, showing identical green fluorescence as in tissue sections of healthy rats. The cultures 8 days in vitro exhibited positive reaction in a few terminals, whereas sister cultures 1 month in vitro showed strong fluorescence reaction in thicker proximal axons and networks of nerve fibers as well. Reactivity of neuron somas became positive after 1 month of cultivation. Application of reserpine in amount of 0.00025 mg/ml for 2 hours resulted in complete disappearance of fluorescence. Furthermore, cultures of spinal ganglia from fetal rat produced no fluorescence reaction with this technique. Therefore, the reaction is specific for sympathetic nervous tissue and reliable for the differentiation of sympathetic neurons from other types of nerve cells.This work was supported by research grant NBO 3173 from the National Institute of Neurological Diseases and Blindness, U.S. Public Health Service, and research grant No. 355 from the National Multiple Sclerosis Society, New York.