Cloning and functional expression of the bovine GABA(C) rho2 subunit. Molecular evidence of a widespread distribution in the CNS

GABA(C) receptors were first described as a non-desensitizing, bicuculline- and baclofen-insensitive component in Xenopus oocytes expressing bovine retina mRNA. However, the expression, tissue distribution and functional properties of GABA(C) receptors from other areas of the CNS still remain controversial. In previous experiments, the injection of rat cerebellum mRNA into Xenopus oocytes induced the expression of receptors that generated currents with both GABA(A) and GABA(C) characteristics; the latter component apparently being given by the rho2 subunit, suggesting the expression of GABA(C) receptors in the CNS and the formation of homooligomeric receptors. In this study, using RT-PCR, we found that the rho1 and rho2 subunits are widely expressed in the CNS including areas where they have not been previously described such as the bulb, pons and the caudate nucleus. To determine if the GABA(C) component of the GABA-currents elicited by oocytes expressing cerebellum mRNA was caused by activation of homomeric GABA rho2 receptors, we cloned the corresponding cDNA and expressed it in Xenopus oocytes. It was found that oocytes injected with rho2 cDNA, efficiently formed GABA-gated homooligomeric receptors. The GABA-dose-current response gave an EC50=1.19muM and the currents were resistant to bicuculline and reversibly antagonized by the specific GABA(C) receptor antagonist TPMPA. Altogether, our results indicate a widespread distribution of both rho1 and rho2 subunits in the bovine CNS and show further that the rho2 subunit cDNA isolated from cerebellum, forms fully functional receptors when expressed in Xenopus oocytes.     

甘氨酸受体在大鼠心肌细胞的表达

目的：检测成年大鼠心肌组织和新生大鼠心 肌细胞是否有甘氨酸受体（GlyR）mRNA和蛋白的表达。方法：选用成年 雄性SD大鼠心肌组织和新生SD大鼠培养心肌细胞，提取总RNA和膜蛋白，用逆转录－巢式聚 合酶链反应（RT-PCR）和蛋白免疫印迹（Western blotting）检测GlyRα1和β亚基的mRNA 和蛋白表达。结果：成年大鼠心肌组织和新生大鼠培养心肌细胞均有与 脊髓组织GlyRβ亚基极为相似的mRNA和蛋白的表达；对于GlyRα1亚基，仅在新生SD大鼠培 养心肌细胞发现有mRNA的表达。结论：本研究证实在成年大鼠心肌组织 和新生大鼠心肌细胞中有与脊髓组织相似的GlyR亚基的表达，提示大鼠心肌细胞的膜上有Gl yR的存在。     

Cholecystokinin B-type receptors mediate a G-protein-dependent depolarizing action of sulphated cholecystokinin ocatapeptide (CCK-8s) on rodent neonatal spinal ventral horn neurons

Reports of cholecystokinin (CCK) binding and expression of CCK receptors in neonatal rodent spinal cord suggest that CCK may influence neuronal excitability. In patch-clamp recordings from 19/21 ventral horn motoneurons in neonatal (PN 5-12 days) rat spinal cord slices, we noted a slowly rising and prolonged membrane depolarization induced by bath-applied sulfated CCK octapeptide (CCK-8s; 1 microM), blockable by the CCK B receptor antagonist L-365,260 (1 microM). Responses to nonsulfated CCK-8 or CCK-4 were significantly weaker. Under voltage clamp (V H -65 mV), 22/24 motoneurons displayed a CCK-8s-induced tetrodotoxin-resistant inward current [peak: -136 +/- 28 pA] with a similar time course, mediated via reduction in a potassium conductance. In 29/31 unidentified neurons, CCK-8s induced a significantly smaller inward current (peak: -42.8 +/- 5.6 pA), and I-V plots revealed either membrane conductance decrease with net inward current reversal at 101.3 +/- 4.4 mV (n = 16), membrane conductance increase with net current reversing at 36.1 +/- 3.8 mV (n = 4), or parallel shift (n = 9). Intracellular GTP-gamma-S significantly prolonged the effect of CCK-8s (n = 6), whereas GDP-beta-S significantly reduced the CCK-8s response (n = 6). Peak inward currents were significantly reduced after 5-min perfusion with N-ethylmaleimide. In isolated neonatal mouse spinal cord preparations, CCK-8s (30-300 nM) increased the amplitude and discharge of spontaneous depolarizations recorded from lumbosacral ventral roots. These observations imply functional postsynaptic G-protein-coupled CCK B receptors are prevalent in neonatal rodent spinal cord.     

Silent NMDA receptor-mediated synapses are developmentally regulated in the dorsal horn of the rat spinal cord

In vitro whole cell patch-clamp recording techniques were utilized to study silent pure-N-methyl-D-aspartate (NMDA) receptor-mediated synaptic responses in lamina II (substantia gelatinosa, SG) and lamina III of the spinal dorsal horn. To clarify whether these synapses are present in the adult and contribute to neuropathic pain, transverse lumbar spinal cord slices were prepared from neonatal, naive adult and adult sciatic nerve transected rats. In neonatal rats, pure-NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) were elicited in SG neurons either by focal intraspinal stimulation (n = 15 of 20 neurons) or focal stimulation of the dorsal root (n = 2 of 7 neurons). In contrast, in slices from naive adult rats, no silent pure-NMDA EPSCs were recorded in SG neurons following focal intraspinal stimulation (n = 27), and only one pure-NMDA EPSC was observed in lamina III (n = 23). Furthermore, in rats with chronic sciatic nerve transection, pure-NMDA EPSCs were elicited by focal intraspinal stimulation in only 2 of 45 SG neurons. Although a large increase in Abeta fiber evoked mixed alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and NMDA receptor-mediated synapses was detected after sciatic nerve injury, Abeta fiber-mediated pure-NMDA EPSCs were not evoked in SG neurons by dorsal root stimulation. Pure-NMDA receptor-mediated EPSCs are therefore a transient, developmentally regulated phenomenon, and, although they may have a role in synaptic refinement in the immature dorsal horn, they are unlikely to be involved in receptive field plasticity in the adult.     

Depolarizing glycine responses in Cajal-Retzius cells of neonatal rat cerebral cortex

We investigated the properties of glycine-induced responses in Cajal-Retzius cells, a neuronal cell type essential for the establishment of neocortical lamination. Whole-cell and gramicidin-perforated patch-clamp recordings were performed on visually identified Cajal-Retzius cells in tangential slices from neonatal rat cortex (postnatal days 0-3). With a pipette Cl(-) concentration of 50 mM, bath application of 1 mM glycine induced a membrane depolarization of 32.8+/-7.4 mV and a massive decrease in membrane resistance by 88+/-1.4%. The membrane depolarization was abolished in the presence of the glycinergic antagonists strychnine (30 microM) and phenylbenzene-omega-phosphono-alpha-amino acid (100 microM), while the GABA(A) receptor antagonist bicuculline (100 microM) and the glutamatergic antagonist (+/-)-2-amino-5-phosphonopentatonic acid (60 microM) were without effect, suggesting that the glycine-induced membrane responses were mediated exclusively by the strychnine-sensitive glycine receptor. The EC(50) for activation of glycine receptors was 0.54 mM, 1.62 mM and 2.41 mM, for the glycinergic agonists glycine, beta-alanine and taurine, respectively. Since the reversal potential of the glycine-induced currents showed a strong dependency on the intracellular chloride concentration and was virtually unaffected under HCO(3)(-)-free conditions, the activation of glycine receptors was probably linked to Cl(-) fluxes with little contribution of HCO(3)(-) ions. Perforated patch recordings from Cajal-Retzius cells demonstrated that glycine elicited depolarizing responses mediated by Cl(-) currents which reversed at -41+/-3.7 mV.In summary, from these results we suggest that Cajal-Retzius cells of the neonatal rat cerebral cortex express functional strychnine-sensitive glycine receptors that mediate depolarizing membrane responses via Cl(-) efflux.     

Increased neurosteroid sensitivity of hippocampal GABAA receptors during postnatal development

To investigate developmental changes in neurosteroid modulation of GABA(A) receptors, whole-cell currents were elicited by applying GABA with allopregnanolone or pregnenolone sulfate (PS) to dentate granule cells (DGCs), acutely isolated from 7-14-day-old and adult rats. GABA evoked larger currents from dentate granule cells acutely isolated from adult rats (adult DGCs) than from neonatal DGCs, due to increased efficacy (1662+/-267 pA in adult DGCs versus 1094+/-198 pA in neonatal DGCs, P=0.004), and current density (0.072+/-0.01 pA/microm(2) in neonatal rat DGCs to 0.178+/-0.02 pA/microm(2) in adult DGCs), but unchanged potency (EC(50) was 18.5+/-2 microm in adult DGCs, and 26.6+/-7.9 microm in neonatal DGCs, P=0.21). Allopregnanolone sensitivity of GABA(A) receptor currents increased during development due to an increased potency (21.1+/-4.7 nM in adult DGCs versus 94.6+/-9 nM in neonatal DGCs, P=0.0002). The potency and efficacy of PS inhibition of GABA(A) receptor currents were remained unchanged during development (13+/-6 microm and 13.2+/-5.9 microm, P=0.71 and 85.5%+/-3.5% and 83.6%+/-0.8%, P=0.29, respectively). To investigate possible mechanism of developmental changes in GABA(A) receptor properties, in situ hybridization for alpha1, alpha4 and gamma2 subunit mRNAs was performed in dentate gyrus of the two age groups. Qualitatively, alpha1 subunit mRNA was expressed at low levels in neonatal rats while it was well expressed in adult rats. The alpha4 and gamma2 subunits were well expressed in the dentate gyrus of adult and neonatal rats. Immunohistochemical staining for alpha1 subunit in hippocampal slices from neonatal and adult rats was examined under confocal laser scanning microscope. This demonstrated that cell bodies and dendrites of granule cells are moderately positive for the alpha1 staining in adult rats but weakly so in neonatal rats. Higher-magnification images demonstrate large number of clusters of alpha1-subunit in the cell bodies of dentate granule cells of adult rat but rare clusters in granule cells of neonatal rats. Maturation of GABA(A) receptors in DGCs is characterized by increased number of GABA(A) receptors that are more sensitive to endogenous neurosteroid allopregnanolone, which might be related to increased expression of alpha1 subunit.     

NT-3 evokes an LTP-like facilitation of AMPA/kainate receptor-mediated synaptic transmission in the neonatal rat spinal cord

Neurotrophin-3 (NT-3) is a neurotrophic factor required for survival of muscle spindle afferents during prenatal development. It also acts postsynaptically to enhance the monosynaptic excitatory postsynaptic potential (EPSP) produced by these fibers in motoneurons when applied over a period of weeks to the axotomized muscle nerve in adult cats. Similar increases in the amplitude of the monosynaptic EPSP in motoneurons are observed after periodic systemic treatment of neonatal rats with NT-3. Here we show an acute action of NT-3 in enhancing the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA/kainate) receptor-mediated fast monosynaptic EPSP elicited in motoneurons by dorsal root (DR) stimulation in the in vitro hemisected neonatal rat spinal cord. The receptor tyrosine kinase inhibitor K252a blocks this action of NT-3 as does the calcium chelator bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) injected into the motoneuron. The effect of NT-3 resembles long-term potentiation (LTP) in that transient bath application of NT-3 to the isolated spinal cord produces a long-lasting increase in the amplitude of the monosynaptic EPSP. An additional similarity is that activation of N-methyl-D-aspartate (NMDA) receptors is required to initiate this increase but not to maintain it. The NMDA receptor blocker MK-801, introduced into the motoneuron through the recording microelectrode, blocks the effect of NT-3, indicating that NMDA receptors in the motoneuron membrane are crucial. The effect of NT-3 on motoneuron NMDA receptors is demonstrated by its enhancement of the depolarizing response of the motoneuron to bath-applied NMDA in the presence of tetrodotoxin (TTX). The potentiating effects of NT-3 do not persist beyond the first postnatal week. In addition, EPSPs with similar properties evoked in the same motoneurons by stimulation of descending fibers in the ventrolateral funiculus (VLF) are not modifiable by NT-3 even in the initial postnatal week. Thus, NT-3 produces synapse-specific and age-dependent LTP-like enhancement of AMPA/kainate receptor-mediated synaptic transmission in the spinal cord, and this action requires the availability of functional NMDA receptors in the motoneuron.     

Electrophysiological evidence for vasopressin V(1) receptors on neonatal motoneurons, premotor and other ventral horn neurons

Prominent arginine-vasopressin (AVP) binding and AVP V(1) type receptors are expressed early in the developing rat spinal cord. We sought to characterize their influence on neural excitability by using patch-clamp techniques to record AVP-induced responses from a population of motoneurons and interneurons in neonatal (5-18 days) rat spinal cord slices. Data were obtained from 58 thoracolumbar (T(7)-L(5)) motoneurons and 166 local interneurons. A majority (>90%) of neurons responded to bath applied AVP (10 nM to 3 microM) and (Phe(2), Orn(8))-vasotocin, a V(1) receptor agonist, but not V(2) or oxytocin receptor agonists. In voltage-clamp, postsynaptic responses in motoneurons were characterized by slowly rising, prolonged (7-10 min) and tetrodotoxin-resistant inward currents associated with a 25% reduction in a membrane potassium conductance that reversed near -100 mV. In interneurons, net AVP-induced inward currents displayed three patterns: decreasing membrane conductance with reversal near -100 mV, i.e., similar to that in motoneurons (24 cells); increasing conductance with reversal near -40 mV (21 cells); small reduction in conductance with no reversal within the current range tested (41 cells). A presynaptic component recorded in most neurons was evident as an increase in the frequency but not amplitude (in motoneurons) of inhibitory and excitatory postsynaptic currents (IPSCs and EPSCs), in large part due to AVP-induced firing in inhibitory (mainly glycinergic) and excitatory (glutamatergic) neurons synapsing on the recorded cells. An increase in frequency but not amplitude of miniature IPSCs and EPSCs also indicated an AVP enhancement of neurotransmitter release from axon terminals of inhibitory and excitatory interneurons. These observations provide support for a broad presynaptic and postsynaptic distribution of AVP V(1) type receptors and indicate that their activation can enhance the excitability of a majority of neurons in neonatal ventral spinal cord.     

Modulation of inhibitory glycine receptors in cultured embryonic mouse hippocampal neurons by zinc, thiol containing redox agents and carnosine

Modulation of inhibitory glycine receptors by zinc (Zn(2+)) and endogenous redox agents such as glutathione may alter inhibition in the mammalian brain. Despite the abundance of Zn(2+) in the hippocampus and its ability to modulate glycine receptors, few studies have examined Zn(2+) modulation of hippocampal glycine receptors. Whether redox agents modulate hippocampal glycine receptors also remains unknown. This study examined Zn(2+) and redox modulation of glycine receptor-mediated currents in cultured embryonic mouse hippocampal neurons using whole-cell recordings. Zn(2+) concentrations below 10 microM potentiated currents elicited by low glycine, beta-alanine, and taurine concentrations by 300-400%. Zn(2+) concentrations above 300 microM produced nearly complete inhibition. Potentiating Zn(2+) concentrations shifted the dose-response curves for the three agonists to the left and decreased the Hill coefficient for glycine and beta-alanine but not taurine. Inhibiting Zn(2+) concentrations shifted the dose-response curves for glycine and beta-alanine to the right but reduced the maximum taurine response. Histidine residues may participate in potentiation because diethyl pyrocarbonate and pH 5.4 diminished Zn(2+) enhancement of glycine currents. pH 5.4 diminished Zn(2+) block of glycine currents, but diethyl pyrocarbonate did not. These findings indicate that separate sites mediate Zn(2+) potentiation and inhibition. The redox agents glutathione, dithiothreitol, tris(2-carboxyethyl)phosphine, and 5,5'-dithiobis(2-nitrobenzoic acid) did not alter glycine currents by a redox mechanism. However, glutathione and dithiothreitol interfered with the effects of Zn(2+) on glycine currents by chelating it. Carnosine had similar effects. Thus, Zn(2+) and thiol containing redox agents that chelate Zn(2+) modulate hippocampal glycine receptors with the mechanism of Zn(2+) modulation being agonist dependent.     

Intracellular Ca2+-dependent and Ca2+-independent responses of rat brain serotonin receptors transplanted to Xenopus oocytes

Xenopus oocytes injected with messenger RNA extracted from rat brain are induced to acquire a variety of neurotransmitter receptors and voltage-operated membrane channels. Activation of the receptors to serotonin, acetylcholine (muscarinic) and glutamate elicits oscillatory membrane currents carried by chloride ions. These currents are not abolished by removing external calcium, but are completely abolished after EGTA is injected into the oocytes to chelate intracellular calcium. A smooth current response to serotonin remained in EGTA-loaded oocytes, indicating that this response does not require intracellular calcium. In contrast to the oscillatory chloride currents, the chloride currents activated by GABA or glycine are not abolished by intracellular injection of EGTA. Thus, there appear to be two classes of chloride channels one of which requires intracellular calcium to open.     

针刺对脊髓全横断SD大鼠BDNF基因表达的影响

目的探讨电针刺对脊髓全横断成年SD大鼠脑源性神经营养因子(brain-derived neurotrophic fac-tor,BDNF)mRNA在横断脊髓上端、下端、大脑皮质和比目鱼肌中的表达及变化的影响。方法建立40只SD大鼠脊髓全横断损伤模型,雌雄不限,随机分为针刺1d、3d、7d、14d和手术1d、3d、7d、14d组,每组5只,选"足三里 悬钟"及"三阴交 伏兔"2组穴位隔天交替针刺。采用RT-PCR技术检测各时间段脊髓横断上、下端、大脑皮质、比目鱼肌四部位中BDNF mRNA的表达。结果针刺14d组比目鱼肌中BDNF mRNA较手术14d组显著增加(P<0.05)。结论电针刺可促进BDNF在损伤部位所支配的肌肉-靶组织中的表达。     

Replacement of missing motoneurons by embryonic grafts in the rat spinal cord

The ventral quadrant of embryonic spinal cord with its motoneurons prelabelled by 5-bromo-2'-deoxyuridine was grafted into the spinal cord of adult rats. The ventral horn of the host had been previously partially depleted of its own motoneurons by a neonatal nerve lesion. To enhance the chances of survival of the transplanted embryonic motoneurons a target muscle was provided for their axons. Two to three months after the grafts were inserted into the cord nuclei containing 5'-bromo-2'-deoxyuridine were found in the graft and in the host's spinal cord. Many of the stained nuclei were much larger than those of embryonic motoneurons, and their size distribution was similar to that of nuclei from control motoneurons. Retrograde labelling with horseradish peroxidase, injected into the target muscle provided for the embryonic motoneurons, showed that some motoneurons had reached the muscle and presumably made contact with it. Physiological and histological examination of the target muscle showed that it was innervated and that it contained at least three different types of muscle fibres. Thus embryonic motoneurons can survive and develop in the adult spinal cord. Moreover, they seem to be able to make functional connections with skeletal muscle fibres. The heterogeneity of the muscle indicates that the motoneurons that supply them are able to differentiate into various types of cells.     

Central projections of extraocular muscle afferents in cat.

The extraocular muscles (EOMs) of adult cats were injected with wheat germ agglutinin-horseradish peroxidase (WGA-HRP). In addition to motoneurons, labelled cells corresponding to the sensory receptors were found in both the Gasser ganglion and the mesencephalic trigeminal nucleus. Central transganglionic terminals were observed in the pars interpolaris and caudalis of the spinal trigeminal nucleus, in the paratrigeminal nucleus, and in the dorsal horn of the cervical spinal cord. Double labelling experiments were carried out with either Fast blue or Complex gold tracer, injected in the EOM, and either Diamidino yellow or HRP tracer injected in the cervical dorsal horn. Some Gasser ganglion neurons were found to contain both tracers, providing evidence that the transganglionic terminals are localized in the cervical segments of the spinal cord.     

Patch clamp measurements onXenopus laevis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels

Functional acetylcholine receptor (AChR) and sodium channels were expressed in the membrane of Xenopus laevis oocytes following injection with poly(A)+-mRNA extracted from denervated rat leg muscle. Whole-cell currents, activated by acetylcholine or by depolarizing voltage steps had properties comparable to those observed in rat muscle. Oocytes injected with specific mRNA, transcribed from cDNA templates and coding for the AChR of Torpedo electric organ, expressed functional AChR channels at a much higher density. Single-channel currents were recorded from the oocyte plasma membrane following removal of the follicle cell layer and the vitelline membrane from the oocyte. The follicle cell layer was removed enzymatically with collagenase. The vitelline membrane was removed either mechanically after briefly exposing the oocyte to a hypertonic solution, or by enzyme treatment with pronase. Stretch activated (s.a.) currents were observed in most recordings from cell-attached patches obtained with standard patch pipettes. S.a.-currents were evoked by negative or positive pressure (greater than or equal to 5 mbar) applied to the inside of the pipette, and were observed in both normal and mRNA injected oocytes indicating that they are endogenous to the oocyte membrane. The s.a.-channels are cation selective and their conductance is 28 pS in normal frog Ringer's solution (20 +/- 1 degree C). Their gating is voltage dependent, and their open probability increases toward more positive membrane potentials. The density of s.a.-channels is estimated to be 0.5-2 channels per micron 2 of oocyte plasma membrane. In cell-attached patches s.a.-currents are observed much less frequently when current measurement is restricted to smaller patches of 3-5 micron 2 area using thick-walled pipettes with narrow tips. In outside-out patches s.a.-currents occur much less frequently than in cell-attached or inside-out patches. AChR-channel and sodium channel currents were observed only in a minority of patches from oocytes injected with poly(A)+-mRNA from rat muscle. AChR-channel currents were seen in all patches of oocytes injected with specific mRNA coding for Torpedo AChR. In normal frog Ringer's solution (20 +/- 2 degrees C) the conductance of implanted rat muscle AChR-channels was 38 pS and that of sodium channels 20 pS. The conductance of implanted Torpedo AChR channels was 40 pS. The conductance of implanted channels was similar in cell-attached and in cell-free patches.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ontogeny and characterization of [125I]Bolton Hunter-eledoisin binding sites in rat spinal cord by quantitative autoradiography.

The distribution and characteristics of [125I]Bolton Hunter-eledoisin binding sites in rat lumbar spinal cord were studied during postnatal development by in vitro receptor autoradiography. At three, six and 10 days of age, specific [125I]eledoisin binding was distributed throughout the dorsal and ventral horns of the spinal cord. In contrast, from day 24 onwards, specific binding of [125I]eledoisin was confined to superficial layers of the dorsal horn, with negligible amounts of specific binding in the ventral horn. [125I]Eledoisin binding to neonatal (three day) and adult (eight to 12 weeks) spinal cord sections was characterized using tachykinin agonists. In both dorsal and ventral horns of neonatal spinal cord, the rank order of potency of agonists indicated that the majority (64%) of specific [125I]eledoisin binding was to neurokinin-3 binding sites. The identity of the non-neurokinin-3 sites labelled by [125I]eledoisin remains to be determined. In adult rat spinal cord, [125I]eledoisin appeared to bind exclusively to neurokinin-3 binding sites. These results suggest that major changes take place in the localization of neurokinin-3 receptors during postnatal ontogeny of the rat spinal cord. These changes may reflect an important role for tachykinins in neuronal plasticity of the developing spinal cord.     

The origin of projections from the medullary reticular formation to the spinal cord, the diencephalon and the cerebellum at different stages of development in the North American opossum: studies using single and double labeling techniques

We have employed the retrograde transport of horseradish peroxidase alone or conjugated to wheat germ agglutinin, to label neurons within the medullary reticular formation which project to the spinal cord, the diencephalon and the cerebellum at different stages of development in the North American opossum. At selected ages, the fluorescent markers Fast Blue and Diamidino Yellow were also used in double-labeling experiments to determine if single neurons innervate both the spinal cord and diencephalon or the spinal cord and cerebellum, presumably via axonal collaterals. The opossum was employed because it is born in a very immature state, 12 days after conception, and is thus available for injections at early stages of development. At all ages studied, the location of retrograde labeling within the medullary reticular formation after spinal, diencephalic or cerebellar placements of horseradish peroxidase or its conjugate appeared similar to that obtained in the adult animal. Such results suggest that the origin of projections from the medullary reticular formation to the areas injected is specified early in development. At some ages, however, the labeling density appeared greater than in the adult animal. When either Fast Blue or Diamidino Yellow was injected into the spinal cord and the other marker was placed into the diencephalon at such ages, relatively few neurons of the medullary reticular formation were double-labeled. When one marker was injected into the spinal cord and the other was placed within the cerebellum, no double-labeled neurons were found. These results indicate that at the ages studied, relatively few neurons of the medullary reticular formation provide collateral innervation to either the spinal cord and diencephalon or the spinal cord and cerebellum. Similar conclusions have been reached previously for the adult opossum. We have interpreted our results to suggest that the organization of reticular projections, at least to the areas injected, may not be shaped by the selective elimination of axonal collaterals as in certain other areas of the brain.     

Neutralizing antibodies against neurite growth inhibitor NI-35/250 do not promote regeneration of sensory axons in the adult rat spinal cord

Neutralization of the myelin-associated neurite growth inhibitors NI-35 and NI-250 by IN-1 antibodies can promote axonal regeneration of several types of central nervous neurons. Here, we investigated in adult rats whether IN-1 can promote regeneration of ascending sensory axons across a peripheral nerve bridge back into the spinal cord. IN-1 was administered by hybridoma cells injected in the cerebral cortex or thoracic cord, its presence confirmed in tissue sections and cerebrospinal fluid, and its effectiveness demonstrated in co-cultures of oligodendrocytes and sensory neurons. With a two week infusion of control vehicle into the dorsal spinal cord 3 mm rostral to the nerve graft, only 3±2% of the anterogradely labeled sensory fibers present at the rostral end of the nerve graft had grown up to 0.5 mm, but not farther into the spinal cord. A similar limited extent of regeneration was seen with IN-1 or with infusion of Dantrolene, an inhibitor of NI-35/250 activity in vitro. With infusion of nerve growth factor rostral to the nerve graft, 40% of the fibers at the rostral end of the graft were found at 0.5 mm, 34% at 1 mm, 24% at 2 mm and 14% at 3 mm (the infusion site) into the spinal cord. Treatment with IN-l antibodies did not enhance the growth-promoting effects of nerve growth factor.

We suggest that the neurite growth inhibitors NI-35 or NI-250 do not play a major inhibitory role in the regeneration of the ascending sensory fibers across a nerve bridge and back into the spinal cord of the adult rat.