Neurokinin-1 receptor expression in dorsal root ganglion neurons of young rats

The expression of neurokinin-1 receptors was studied in the fourth lumbar dorsal root ganglia of young rats using immunohistochemical and electrophysiological techniques. Use of a specific immunoserum raised against the C-terminal fragment of rat neurokinin-1 receptor revealed immunoreactivity in 32 +/- 1.5% of dorsal root ganglion neurons. The diameter of the majority of the neurokinin-1 receptor immunostained neurons was smaller than 30 microm. Double immunohistochemical labelling using neurokinin-1 receptor and substance P antibodies revealed that about 1/3 of the neurokinin-1 receptor expressing neuron contains substance P. Likewise, about 1/3 of the substance P producing DRG cells expressed the neurokinin-1 receptor. Superfusion of substance P (1 microM) to an in vitro preparation of the fourth lumbar dorsal root ganglion induced a reversible long-lasting depolarization as measured by extracellular suction electrodes attached to the dorsal roots. This response to substance P was only partially antagonized by the selective neurokinin-1 receptor antagonist RP 67580 (1 microM). Intracellular recordings distinguished between Aalpha/beta-, Adelta- and C-sub-types of ganglion neurons. Superfusion of substance P (1 microM) evoked excitatory responses in Adelta- and C-type neurons. These results demonstrate the expression of functional neurokinin-1 receptors on a subpopulation of Adelta- and C-type sensory ganglion neurons. Our data suggest the possible physiological importance of peripheral neurokinin-1 receptors located on dorsal root ganglion neurons.     

GDNF and NGF family members and receptors in human fetal and adult spinal cord and dorsal root ganglia.

We describe the expression of mRNA encoding ligands and receptors of members of the GDNF family and members of the neurotrophin family in the adult human spinal cord and dorsal root ganglia (DRG). Fetal human spinal cord and ganglia were investigated for the presence of ligands and receptors of the neurotrophin family. Tissues were collected from human organ donors and after routine elective abortions. Messenger RNA was found encoding RET, GFR alpha-1, BDNF, trkB, and trkC in the adult human spinal cord and BDNF, NT-3, p75, trkB, and trkC in the fetal human spinal cord. The percentage of adult human DRG cells expressing p75, trkA, trkB, or trkC was 57, 46, 29, and 24%, respectively, and that of DRG cells expressing RET, GFR alpha-1, GFR alpha-2, or GFR alpha-3 was 79, 20, 51, and 32%, respectively. GFR alpha-2 was expressed selectively in small, GFR alpha-3 principally in small and GFR alpha-1 and RET in both large and small adult human DRG neurons. p75 and trkB were expressed by a wide range of DRG neurons while trkA was expressed in most small diameter and trkC primarily in large DRG neurons. Fetal DRG cells were positive for the same probes as adult DRG cells except for NT-3, which was only found in fetal DRG cells. Messenger RNA species only expressed at detectable levels in fetal but not adult spinal cord tissues included GDNF, GFR alpha-2, NT-3, and p75. Notably, GFR alpha-2, which is expressed in the adult rat spinal cord, was not found in the adult human spinal cord.     

Myelination of cultured dorsal root ganglion neurons by oligodendrocytes obtained from adult rats

Enzymatically dissociated cell suspensions from adult rat spinal cord were added at low densities (5 X 10(3) cells/culture) to cultures of pure dorsal root ganglion neurons. Oligodendrocytes, identified by immunostaining with a monoclonal antibody to galactocerebroside, began to proliferate by 4 days after their addition, forming large colonies of cells by the 14th day. Myelin formation by oligodendrocytes began 4 weeks after addition and myelin was abundant by 6 weeks. Oligodendrocyte proliferation and myelination did not require the immediate presence of astrocytes; the number of astrocytes overall remained low throughout the culture period. Preliminary studies indicated that the specific removal of galactocerebroside-positive cells from the cultures with anti-galactocerebroside antibody and complement 3 days after their addition prevented the subsequent generation of new oligodendrocytes and myelination. These preliminary results suggest that a major source of new myelinating cells in the adult central nervous system (CNS) might be already committed, galactocerebroside-positive, oligodendrocytes rather than uncommitted stem cells. The absence of cellular barriers between the myelinating cells and the medium make these cultures well suited for studies probing cellular and molecular mechanisms of myelination in the CNS.     

Hypoxia-induced apoptosis in adult rat dorsal root ganglion neurons in vitro

Following spinal root injury, dorsal root ganglia suffer mechanical trauma and compromised blood supply. Little is known about the consequences for neuronal survival. Here we used cyanide treatment in vitro to examine effects of moderate hypoxia on adult rat dorsal root ganglion cells identified by GAP-43 immunostaining. 400 microM-4 mM cyanide caused sustained increases in intracellular Ca2+. Cyanide at 2 mM led to a significant increase in apoptosis, detected using TUNEL labelling and confirmed by ultrastructural analysis, and a further increase when cultures were left overnight in fresh medium. Our study shows that dorsal root ganglion neurons die by apoptosis following hypoxia and that cell death increases over time. Cyanide response provides a simple assay for testing neuroprotective agents and examining underlying mechanisms.     

大鼠背根神经节细胞的分离及特性探讨

目的探讨大鼠背根神经节（dorsal root ganglion，DRG）细胞的分离方法以及细胞形态和电生理特征。方法采用显微外科技术获取大鼠DRG体，用双酶法急性分离大鼠DRG获得DRG细胞，全细胞膜片钳技术记录动作电位和钠电流。结果本实验能得到完整圆形或椭圆长条形的大鼠DRG体。正常的单个DRG细胞呈圆形或椭圆形，大小不等，胞膜清晰，折光性好，隐约可见细胞核。在DRG细胞上记录的动作电位都具有从0期到4期，呈正立锐角三角形，静息电位小，动作电位时程短。DRG细胞的钠通道最大电流密度在-30mV左右，几乎能被，TTX完全抑制，具有可逆性恢复。结论本实验采用分离方法简单易行，DRG细胞容易获得和辨认，适合膜片钳技术要求，电生理特征明确可靠，值得推崇。     

Three types of sodium channels in adult rat dorsal root ganglion neurons

Several types of Na+ currents have previously been demonstrated in dorsal root ganglion (DRG) neurons isolated from neonatal rats, but their expression in adult neurons has not been studied. Na+ current properties in adult dorsal root ganglion (DRG) neurons of defined size class were investigated in isolated neurons maintained in primary culture using a combination of microelectrode current clamp, patch voltage clamp and immunocytochemical techniques. Intracellular current clamp recordings identified differing relative contributions of TTX-sensitive and -resistant inward currents to action potential waveforms in DRG neuronal populations of defined size. Patch voltage clamp recordings identified three distinct kinetic types of Na+ current differentially distributed among these size classes of DRG neurons. 'Small' DRG neurons co-express two types of Na+ current: (i) a rapidly-inactivating, TTX-sensitive 'fast' current and (ii) a slowly-activating and -inactivating, TTX-resistant 'slow' current. The TTX-sensitive Na+ current in these cells was almost completely inactivated at typical resting potentials. 'Large' cells expressed a single TTX-sensitive Na+ current identified as 'intermediate' by its inactivation rate constants. 'Medium'-sized neurons either co-expressed 'fast' and 'slow' current or expressed only 'intermediate' current. Na+ channel expression in these size classes was also measured by immunocytochemical techniques. An antibody against brain-type Na+ channels (Ab7493)10 labeled small and large neurons with similar intensity. These results demonstrate that three types of Na+ currents can be detected which correlate with electrogenic properties of physiologically and anatomically distinct populations of adult rat DRG neurons.     

NGF stimulates extensive neurite outgrowth from implanted dorsal root ganglion neurons following transplantation into the adult rat inner ear

Neuronal tissue transplantation is a potential way to replace degenerated spiral ganglion neurons (SGNs) since these cells cannot regenerate in adult mammals. To investigate whether nerve growth factor (NGF) can stimulate neurite outgrowth from implanted neurons, mouse embryonic dorsal root ganglion (DRG) cells expressing enhanced green fluorescent protein (EGFP) were transplanted into the scala tympani of adult rats with a supplement of NGF or artificial perilymph. DRG neurons were observed in the cochlea for up to 6 weeks postoperatively. A significant difference was identified in the number of DRG neurons between the NGF and non-NGF groups. In the NGF group, extensive neurite projections from DRGs were found penetrating the osseous modiolus towards the spiral ganglion. These results suggest the possibility that embryonic neuronal implants may become integrated within the adult auditory nervous system. In combination with a cochlear prosthesis, a neuronal implantation strategy may provide a possibility for further treatment of profoundly deaf patients.     

Plasticity of developing and adult dorsal root ganglion neurons as revealed in vitro

We review recent data on the plasticity of dorsal root ganglion (DRG) neurons as revealed during cultivation in vitro. Some experiments on cultured developing DRG neurons and on adult DRG neurons in vivo are also mentioned. Cultured developing and adult DRG neurons can be switched from an apolar to a multipolar phenotype by fetal calf serum or fibronectin. The effect is concentration dependent and occurs through an early modification of cell-substratum interaction. Adult DRG neurons synthesize and release within hours after injury TGFβ-1, which is a mitogen and a differentiation factor for Schwann cells. Finally, adult DRG neurons express in vitro neurotransmitters that are not expressed in vivo. This neurotransmitter plasticity can be modulated in vitro by some growth factors and in vivo by distal or proximal axotomy.     

P2X1 receptor subunit immunoreactivity and ATP-evoked fast currents in adult rat dorsal root ganglion neurons

Triple fluorescent staining for P2X1 and P2X3 subunits and isolectin I-B4 (IB4) were performed on acutely dissociated rat DRG neurons. Immunoreactivity for P2X1 and P2X3 subunits was present separately or together in DRG neurons. P2X1 immunoreactivity was present in both IB4-positive and IB4-negative cells. When combining patch-clamp recordings with immunostaining for the P2X1 and P2X3 subunits on single recorded cells, ATP-evoked fast currents were shown to be present on DRG neurons that have immunoreactivity for the P2X3 subunit only, the P2X1 subunit only, or both P2X1 and P2X3 subunits. These results raised a possibility that, in addition to the P2X3 receptor subunit, the P2X1 subunit may also contribute to functional P2X receptors with fast kinetics in DRG neurons.     

Differential expression patterns of mRNAs for P2X receptor subunits in neurochemically characterized dorsal root ganglion neurons in the rat

The ionotropic purine receptors, P2X receptors, are composed of an assembly of multiple P2X subunits. At present, seven subunits have been cloned and named "P2X1-7." We examined the precise distribution of mRNAs for these subunits in the rat lumbar dorsal root ganglion (DRG) by in situ hybridization histochemistry (ISHH) using riboprobes and characterized their expression among some neuronal subpopulations by ISHH and immunohistochemistry. P2X1 was not expressed by DRG neurons. P2X2 mRNA was preferentially expressed by neurofilament (NF)-200 negative small-sized neurons expressing Ret, but not TrkA or TrkC mRNAs. P2X3 mRNA was mainly expressed by NF-200-negative neurons. Most P2X3-positive neurons had Ret mRNA, and about a half of them coexpressed TrkA and TRPV1 mRNAs. P2X4 was the most ubiquitous subunit, evenly distributing among all examined neuronal subpopulations. P2X5 and P2X6 were expressed by about half of the neurons, and most of these neurons were NF-200-positive. P2X7 mRNA-expressing neurons were quite rare. We further examined the coexpression of all pairs of P2X2-P2X6 mRNAs in DRG neurons and found that: 1) P2X4 was always present in combination with the other subunits. 2) All TrkC neurons had three subunits, P2X4, P2X5, and P2X6, and made up 32% of the total neurons. 3) 12.5% of the total neurons had both P2X2 and P2X3. 4) 12.9% of the neurons had both P2X3 and P2X5. We determined the neuronal subpopulation-specific distribution of P2X subunits in the DRG. These findings suggest possible combinations of subunits of native P2X receptor in DRG neurons.     

The maturation-dependent change in fibronectin receptor density of mouse dorsal root ganglion neurons

The maturation-dependent change in fibronectin receptor density of mouse dorsal root ganglion neurons were investigated by an immuno-cytofluorometric method. The receptor density showed a drastic decrease around birth and a smaller change after birth.     

Co-expression of heat sensitive vanilloid receptor subtypes in rat dorsal root ganglion neurons

Expression of the heat sensitive cation channels TRPV1 and TRPV2 was investigated by immunofluorescence in rat dorsal root ganglion (DRG) neurons. TRPV1-positive neurons were more frequent and had smaller diameters than TRPV2-positive neurons (35.7% vs 7.3%; 22.3 microm vs 27.6 microm), but size distributions overlapped and significant co-expression was seen in 20.7% of TRPV2-positive neurons (1.7% of all). Expression patterns did not differ between tissue sections typically used in immunocytochemistry and dissociated DRG neurons typically used in electrophysiology. Rectangular temperature pulses revealed two patterns of heat-evoked inward currents in small DRG neurons: low-threshold rapidly activating and high-threshold slowly activating. Slowly activating heat-evoked currents have not been described previously, but correspond to the type I heat responses of primary nociceptive afferents, which have been suggested to be mediated by TRPV2.     

Neurite outgrowth and GAP-43 mRNA expression in cultured adult rat dorsal root ganglion neurons: Effects of NGF or prior peripheral axotomy

Adult dorsal root ganglion (DRG) cells are capable of neurite outgrowth in vivo and in vitro after axotomy. We have investigated, in cultured adult rat DRG cells, the relative influence of nerve growth factor (NGF) or a prior peripheral nerve lesion on the capacity of these neurons to produce neurites. Since there is evidence suggesting that the growth-associated protein GAP-43 may play a crucial role in axon elongation during development and regeneration, we have also compared the effect of these treatments on GAP-43 mRNA expression. NGF increased the early neurite outgrowth in a subpopulation of DRG cells. This effect was substantially less, however, than that resulting from preaxotomy, which initiated an early and profuse neurite outgrowth in almost all cells. No difference in the expression of GAP-43 mRNA was found between neurons grown in the presence or absence of NGF over 1 week of culture, in spite of the increased growth produced by NGF. In contrast, cultures of neurons that had been preaxotomized showed substantial increase in GAP-43 mRNA and NGF had, as expected, a significant effect on substance P mRNA levels. Two forms of growth may be present in adult DRG neurons: an NGF-independent, peripheral nerve injury-provoked growth associated with substantial GAP-43 upregulation, and an NGF-dependent growth that may underlie branching or sprouting of NGF-sensitive neurons, but which is not associated with increased levels of GAP-43 mRNA. © 1994 Wiley-Liss, Inc.     

阿霉素中毒大鼠背根神经节神经元及细胞核孔的改变

目的 探讨阿霉素中毒所致神经元坏死过程中胞核,胞质及核孔的变化,是否存在凋亡。方法 ＳＤ大鼠静脉注射阿霉素８ｍｇ／ｋｇ,使其腰背根神经节细胞发生变性。利用光镜,电镜、聚焦激光扫描显微镜,ＴＵＮＥＬ染色等方法观察不同时期神经元和核孔的变化,寻找凋亡存在的证据。结果 阿霉素应用１４ｄ后,核膜切线位核孔的结构变得模糊不清,数量减少;垂直位核膜隔明显稀疏,甚至消失,神经核中度变性及胞质明显变性。结论 核孔     

Culture of dorsal root ganglion neurons from aged rats: Effects of acetyl-l-carnitine and NGF

In vitro neuronal preparations are used to study the action mechanism of substances which are active in normal and pathological brain aging. One major concern with in vitro assays is that the use of embryonic or adult neurons may hamper an appreciation of the relevance of these substances on aged nervous tissue. In the present study for the first time cultures of aged dorsal root ganglia from 24-months-old rats were maintained in vitro up to 2 weeks. This model was used to investigate the neurotrophic/neuroprotective action of nerve growth factor and acetyl-L-carnitine. A large population of aged dorsal root ganglia neurons was responsive to nerve growth factor (100 ng/ml). Nerve growth factor induced an increase of initial rate of axonal regeneration and influenced the survival time of these neurons. Acetyl-L-carnitine (250 microM) did not affect the axonal regeneration but substantially attenuated the rate of neuronal mortality. A significant difference was evident between the acetyl-L-carnitine-treated and the untreated neurons from the first cell counting (day 3 in culture). After 2 weeks the number of aged neurons treated with acetyl-L-carnitine was almost double that of the controls. The effects of acetyl-L-carnitine on aged DRG neurons potentially explain the positive effects in clinical and in vivo experimental studies.     

GDNF family ligand receptor components Ret and GFRalpha-1 in the human trigeminal ganglion and sensory nuclei

The occurrence of Ret and GFRalpha-1 receptors is shown by immunohistochemistry in the human trigeminal sensory system at pre-, postnatal and adult age. Receptor-labeled neurons occur in both trigeminal ganglion and mesencephalic nucleus. In adult trigeminal ganglion, about 75% of Ret- and 65% of GFRalpha-1-labeled neurons are small- and medium-sized. The proportion of Ret+ and GFRalpha-1+ trigeminal ganglion neurons in the adult is about 25 and 60%, respectively. The majority of Ret+ are double labeled for GFRalpha-1 and glial cell line-derived neurotrophic factor (GDNF). Most of the GFRalpha-1+ cells contain GDNF and about 50% of them contain Ret. Triple labeling shows many Ret+/GDNF+/GFRalpha-1+ neurons, but also a number of Ret-/GDNF+/GFRalpha-1+ and Ret+/GDNF-/GFRalpha-1+ cells. Both Ret+ and GFRalpha-1+ neuronal subpopulations overlap with that containing calcitonin gene-related peptide. Ret+ pericellular basket-like nerve fibers occur in the adult trigeminal ganglion. Centrally, immunoreactivity is restricted to the spinal nucleus pars caudalis and pars interpolaris and to the mesencephalic nucleus. In adult specimens, Ret+ nerve fibers and puncta gather in the inner substantia gelatinosa. Ret+ neurons occur in the spinal nucleus and are more frequent in newborn than in adult subjects. Central GFRalpha-1+-labeled neurons and punctate elements are sparse. These findings support the involvement of GDNF and possibly other cognate ligands in the trophism of human trigeminal primary sensory neurons from prenatal life to adulthood, indicating a selective commitment to cells devoted to protopathic and proprioceptive sensory transmission. They also support the possibility that receptor molecules other than Ret could be active in transducing the ligand signal.