Spinal Cord Of Young Rats Research Articles

Aging diminishes the resistance of AO rats to EAE: putative role of enhanced generation of GM-CSF Expressing CD4+ T cells in aged rats.

BackgroundAging influences immune response and susceptibility to EAE in a strain specific manner. The study was designed to examine influence of aging on EAE induction in Albino Oxford (AO) rats.ResultsDifferently from 3-month-old (young) rats, which were resistant to EAE induction, the majority of aged (24-26-month-old) rats developed mild chronic form of EAE. On 16th day post-immunization, when in aged rats the neurological deficit reached plateau, more mononuclear cells, including CD4+ T lymphocytes was retrieved from spinal cord of aged than young rats. The frequencies of IL-17+ and GM-CSF+ cells within spinal cord infiltrating CD4+ lymphocytes were greater in aged rats. To their increased frequency contributed the expansion of GM-CSF + IL-17 + IFN-γ+ cells, which are highly pathogenic in mice. The expression of the cytokines (IL-1β and IL-23/p19) driving GM-CSF + IL-17 + IFN-γ + cell differentiation in mice was also augmented in aged rat spinal cord mononuclear cells. Additionally, in aged rat spinal cord the expansion of GM-CSF + IL-17-IFN-γ- CD4+ T lymphocytes was found. Consistently, the expression of mRNAs for IL-3, the cytokine exhibiting the same expression pattern as GM-CSF, and IL-7, the cytokine driving differentiation of GM-CSF + IL-17-IFN-γ- CD4 + lymphocytes in mice, was upregulated in aged rat spinal cord mononuclear cells, and the tissue, respectively. This was in accordance with the enhanced generation of the brain antigen-specific GM-CSF+ CD4+ lymphocytes in aged rat draining lymph nodes, as suggested by (i) the higher frequency of GM-CSF+ cells (reflecting the expansion of IL-17-IFN-γ- cells) within their CD4+ lymphocytes and (ii) the upregulated GM-CSF and IL-3 mRNA expression in fresh CD4+ lymphocytes and MBP-stimulated draining lymph node cells and IL-7 mRNA in lymph node tissue from aged rats. In agreement with the upregulated GM-CSF expression in aged rats, strikingly more CD11b + CD45int (activated microglia) and CD45hi (mainly proinflammatory dendritic cells and macrophages) cells was retrieved from aged than young rat spinal cord. Besides, expression of mRNA for SOCS1, a negative regulator of proinflammatory cytokine expression in innate immunity cells, was downregulated in aged rat spinal cord mononuclear cells.ConclusionsThe study revealed that aging may overcome genetic resistance to EAE, and indicated the cellular and molecular mechanisms contributing to this phenomenon in AO rats.Electronic supplementary materialThe online version of this article (doi:10.1186/s12979-015-0044-x) contains supplementary material, which is available to authorized users.

Local axon collaterals of lamina I projection neurons in the spinal cord of young rats

Abstractlpsilateral spinal cord areas occupied by axon collaterals of different types of ALT projection neurons. Dorsal Collateral Type projection neurons (DCT‐I and II, upper right and left corners; respectively) send axon collaterals into the dorsal laminae of the same segment and these axons may extend into the overlying white matter. The collateral area of Lateral Collateral Type (LCT) neurons (purple cell, lower left) extends beyond the segment of the neuron, includes the lateral spinal nucleus, a part of the dorsolateral funiculus and may include the lateral part of laminae I‐V. Collaterals of Ventral Collateral Type (VCT) neurons (yellow cell, lower right) are occupying an area that could be best described as a transverse slice of the grey matter ventral to lamina IV, centered on the rostrocaudal position of the neuron soma. The Journal of Comparative Neurology, Volume 518, Number 14, pages 2645–2665.

Local axon collaterals of lamina I projection neurons in the spinal cord of young rats

Large, mediolaterally oriented neurons in lamina I of the spinal cord, frequently referred to as marginal cells of Waldeyer, are known to project to supraspinal targets via the anterolateral tract (ALT). Although dendritic organization of lamina I neurons has been extensively studied, little is known about their local axonal morphology and branching. With the help of oblique illumination, we visually identified large lamina I neurons in the isolated lumbar enlargement (L1-L6) of the spinal cord of P14-P20 rats. By using intracellular and cell-attached biocytin injections, we achieved extensive axonal and dendritic labeling in 77 lamina I cells, 40 of which were identified as ALT projection neurons. In the majority of the cases (n = 28), the main axon of these projection neurons gave rise to one or more thin collaterals on the ipsilateral side. Based on their trajectory and location, these collaterals could be divided into three major categories: dorsal, lateral, and ventral. Lamina I projection neurons had dorsal (n = 5), lateral (n = 8), or ventral (n = 6) collaterals only or a combination of these collateral types (n = 9). Our results suggest that lamina I ALT projection neurons can additionally function as local-circuit and propriospinal neurons participating in intra- and intersegmental spinal cord processing.

Mexiletine and Lidocaine Suppress the Excitability of Dorsal Horn Neurons

Spinal sensitization and facilitatory processes in dorsal horn neurons after nerve injury alter spinal outflow leading to enhanced pain perception and chronic pain syndromes. Clinically used Na+ channel blockers at doses which do not block conduction can relieve such chronic pain. Although much attention has been paid to their effect upon afferents, less work has been done with their effect on the excitability of central sensory neurons. Thus, we investigated the effects of the Na+ channel blockers mexiletine and lidocaine on sensory spinal dorsal horn neurons. Patch-clamp recordings were directly performed in visualized neurons of the substantia gelatinosa in the spinal cord of young rats to investigate the effect of mexiletine and lidocaine in different types of dorsal horn neurons (tonically firing, adapting-firing, and single spike neurons). All three different types of neurons responded dose-dependently to mexiletine and lidocaine. Both local anesthetics reversibly inhibited Na+ and K+ currents. The half-maximal inhibitory concentration for Na+ conductance block was 89 +/- 2 or 54 +/- 6 microM and for delayed-rectifier K+ conductance block was 582 +/- 36 or 398 +/- 14 microM for lidocaine and mexiletine, respectively. The inhibition of Na+ and K+ currents consecutively altered the properties of single action potentials and reduced the firing rate of tonically firing and adapting-firing neurons. In clinically relevant concentrations, lidocaine and mexiletine reduced the excitability of sensory dorsal horn neurons via a blockade of Na+ and K+ channels. Our work confirms that, in addition to the peripheral effects of lidocaine and mexiletine, modulation of voltage-gated ion channels in the central nervous system contributes to the antinociceptive effects of these drugs used in pain therapy.

Network-based activity induced by 4-aminopyridine in rat dorsal horn in vitro is mediated by both chemical and electrical synapses.

This study investigated the role of electrical and chemical synapses in sustaining 4-aminopyridine (4-AP)-evoked network activity recorded extracellularly from substantia gelatinosa (SG) of young rat spinal cord in vitro. Superfusion of 4-AP (50 microM) induced two types of activity, the first was observed as large amplitude field population spiking activity and the second manifested within the inter-spike interval as low amplitude rhythmic oscillations in the 4-12 Hz frequency range (mean peak of 8.0 +/- 0.1 Hz). The AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) abolished field population spiking and disrupted 4-12 Hz rhythmic oscillatory activity whereas the NMDA receptor antagonist D-AP5 (50 microM) had no significant effect on either activity component. The glycine receptor antagonist strychnine (4 microM) and the GABA(A) receptor antagonist bicuculline (10 microM) diminished and abolished, respectively, field population spiking and both antagonists reduced the power of 4-12 Hz oscillations. The non-specific gap junction blockers carbenoxolone (100 microM) and octanol (1 mM) attenuated both types of 4-AP-induced activity. By comparison, the neuronal-specific gap junction uncouplers quinine (250 microM) and mefloquine (500 nM) both disrupted 4-12 Hz oscillations but only quinine reduced the frequency of field population spiking. These data demonstrate the existence of 4-AP-sensitive neuronal networks within SG that can generate rhythmic activity, are differentially modulated by excitatory and inhibitory ionotropic neurotransmission and are at least partly reliant on neuronal and/or glial-mediated electrical connectivity. The physiological significance of these putative intrinsic SG networks and the implications in the context of processing of nociceptive inputs are discussed.

Differential contribution of GABAergic and glycinergic components to inhibitory synaptic transmission in lamina II and laminae III–IV of the young rat spinal cord

Using whole-cell patch-clamp recordings from spinal cord slices of young (10-15 days old) rats, we have characterized and compared the properties of inhibitory synaptic transmission in lamina II and laminae III-IV of the dorsal horn, which are involved in the processing of nociceptive and non-nociceptive sensory information, respectively. All (100%) of laminae III-IV neurons, but only 55% of lamina II neurons, received both gamma-aminobutyric acid (GABA)ergic and glycinergic inputs. The remaining 45% of lamina II neurons received only GABAergic synapses. Neurons receiving only glycinergic synapses were never observed. Among the 55% of lamina II neurons receiving both GABAergic and glycinergic inputs, all displayed a small proportion (approximately 10%) of mixed miniature inhibitory postsynaptic currents (mIPSCs), indicating the presence of a functional GABA/glycine co-transmission at a subset of synapses. Such a co-transmission was never observed in laminae III-IV neurons. The presence of mixed mIPSCs and the differences in decay kinetics of GABAA-type receptor mIPSCs between lamina II and laminae III-IV were due to the endogenous tonic production of 3alpha5alpha-reduced steroids (3alpha5alpha-RS) in lamina II. Stimulation of the local production of 3alpha5alpha-RS was possible in laminae III-IV after incubation of slices with progesterone, subcutaneous injection of progesterone or induction of a peripheral inflammation. This led to the prolongation of GABAergic mIPSCs, but failed to induce the appearance of mixed mIPSCs in laminae III-IV. Our results indicate that, compared with lamina II, inhibitory synaptic transmission in laminae III-IV is characterized by a dominant role of glycinergic inhibition and the absence of a functional GABA/glycine co-transmission.

Alginate enhances elongation of early regenerating axons in spinal cord of young rats.

Freeze-dried alginate sponge cross-linked with covalent bonds has been demonstrated to enhance nerve regeneration in peripheral nerves and spinal cords. The present study examined, at early stages after surgery, the outgrowth of regenerating axons and reactions of astrocytes at the stump of transected spinal cord in young rats. Two segments (Th7-8) were resected, and alginate was implanted in the lesion. As controls, collagen gel was implanted in place of alginate or the lesion was left without implantation. Two and 4 weeks after surgery, nerve outgrowth and astrocyte reactions were examined. Many regenerating axons, some of which were accompanied by astrocytic processes, were found to extend from the stump into the alginate-implanted lesion. In the all nonimplanted animals, large cystic cavities were formed at both interfaces with no definite axonal outgrowth into the lesion. In collagen-implanted animals, cavity formation was found in some rats, and regenerating axons once formed at the stumps did not extend further into the lesion. Astrocytic processes extending into alginate-implanted lesion had no basal laminae, whereas those found in control experiments were covered by basal laminae. These findings suggest that alginate contributed to reducing the barrier composed of connective tissues and reactive astrocytic processes, and served as a scaffold for the outgrowth of regenerating axons and elongation of astrocytic processes.

Distinctive membrane and discharge properties of rat spinal lamina I projection neurones in vitro.

Most lamina I neurones with a projection to the brainstem express the neurokinin 1 receptor and thus belong to a small subgroup of lamina I neurones that are necessary for the development of hyperalgesia in rat models of persisting pain. These neurones are prone to synaptic plasticity following primary afferent stimulation in the noxious range while other nociceptive lamina I neurones are not. Here, we used whole-cell patch-clamp recordings from lamina I neurones in young rat spinal cord transverse slices to test if projection neurones possess membrane properties that set them apart from other lamina I neurones. Neurones with a projection to the parabrachial area or the periaqueductal grey (PAG) were identified by retrograde labelling with the fluorescent tracer DiI. The properties of lamina I projection neurones were found to be fundamentally different from those of unidentified, presumably propriospinal lamina I neurones. Two firing patterns, the gap and the bursting firing pattern, occurred almost exclusively in projection neurones. Most spino-parabrachial neurones showed the gap firing pattern while the bursting firing pattern was characteristic of spino-PAG neurones. The underlying membrane currents had the properties of an A-type K(+) current and a Ca(2+) current with a low activation threshold, respectively. Projection neurones, especially those of the burst firing type, were more easily excitable than unidentified neurones and received a larger proportion of monosynaptic input from primary afferent C-fibres. Intracellular labelling with Lucifer yellow showed that projection neurones had larger somata than unidentified neurones and many had a considerable extension in the mediolateral plane.

Epileptiform activity in rat spinal dorsal horn in vitro has common features with neuropathic pain

Neuropathic pain and epileptic seizures bear several similarities, among them is the response to anticonvulsant drugs. It has therefore been hypothesized that epileptiform activity of nociceptive spinal dorsal horn neurons may contribute to paroxysmal forms of neuropathic pain. We used patch-clamp and field potential recordings from young rat spinal cord slices to test if nociceptive dorsal horn structures are indeed able to sustain epileptiform activity. Application of the convulsant 4-aminopyridine (100 μM) evoked epileptiform activity that was most pronounced in superficial dorsal horn and involved nociceptive lamina I neurons with a projection to the brain. The epileptiform activity was dependent on fast excitatory and inhibitory synaptic transmission through ionotropic glutamate receptors and GABA A receptors. During epileptiform activity, previously silent polysynaptic pathways from primary afferent C-fibers to superficial dorsal horn neurons were opened. Stimulation of primary afferents at Aδ- and C-fiber intensity interfered with the epileptiform rhythm, suggesting that both affect the same dorsal horn structures. Similar to neuropathic pain, spinal dorsal horn epileptiform activity was much less reduced by classical analgesics than by anticonvulsant agents.

Involvement of intracellular calcium and protein phosphatases in long-term depression of A-fiber-mediated primary afferent neurotransmission

Long-term depression (LTD) of monosynaptic and polysynaptic excitatory postsynaptic potentials (EPSPs) in substantia gelatinosa (SG) neurons can be induced by brief high-frequency electrical stimulation (HFS, 300 pulses at 100 Hz) of primary afferent fibers in dorsal roots [57]. Here we examined the possible cellular mechanism underlying spinal LTD. Conventional intracellular recordings were made from SG neurons in a transverse slice-dorsal root preparation of the young rat lumbar spinal cord. LTD of both monosynaptic and polysynaptic EPSPs was induced in 16 of 24 SG neurons by HFS of dorsal root in either the presence or absence of the GABA A receptor antagonist bicuculline and the glycine receptor antagonist strychnine. Loading the postsynaptic cell with BAPTA, an intracellular Ca 2+ chelator, almost completely blocked the induction of LTD. Induction of LTD was abolished by bath application of calyculin A (100 nM), a potent inhibitor of protein phosphatases 1 and 2A. These results indicate that: (i) a rise in postsynaptic Ca 2+ is necessary for LTD induction, (ii) synaptic activation of protein phosphatases 1 and 2A plays an important role in the induction of LTD of primary afferent A-fiber neurotransmission in the young rat spinal cord, and (iii) the effect of LTD may be physiologically relevant for transmission and integration of sensory information, including nociception.

Migration, integration, and differentiation of hippocampus-derived neurosphere cells after transplantation into injured rat spinal cord

Hippocampus-derived neurospheres were prepared from transgenic rat fetuses expressing green fluorescent protein (GFP), and transplanted into an alginate-filled lesion of young rat spinal cord. One, two and four weeks after transplantation, a large number of grafted cells survived, many of which expressed immunoreactivity for glial fibrillary acidic protein, and a few expressed immunoreactivity for β-tubulin III. The grafted cells closely attached to the host tissue including astrocytes at the border of the lesion. It was notable that numerous GFP-positive cells had migrated within host spinal cord tissue up to 2 mm away from the implanted site 4 weeks postoperation. These results demonstrate that rat fetal hippocampus-derived neurosphere cells could survive, differentiate, extensively migrate, and integrate well into the host spinal cord tissue.

Inflammatory pain and hypersensitivity are selectively reversed by epidural bupivacaine and are developmentally regulated.

Low doses of local anesthetics applied to the young rat spinal cord in vitro have been shown to inhibit C-fiber-evoked responses. The aim of this work was to investigate whether such low doses applied epidurally selectively reduce nociceptive responses in vivo and to investigate the influence of postnatal development on such local anesthetic actions. Three groups of rat pups aged 3, 10, and 21 days were studied. The threshold of the flexion withdrawal reflex to mechanical stimulation was determined in the hind limb at each age. Inflammatory pain was induced in the right hind limb with 2% carrageenan, causing a reduction in the sensory threshold on that side. The difference in threshold between the two sides represents inflammatory hypersensitivity. The effect of low-dose epidural bupivacaine on sensory thresholds and thus the induced hypersensitivity was also determined for each age group. Inflammatory hypersensitivity was selectively attenuated by very low doses of bupivacaine (concentration range. 0.004-0.0625%), which did not affect the sensory threshold in the contralateral uninflamed limb. This effect was also age-related, with younger rats being more sensitive than older rats. The effects of epidural bupivacaine in the infant rat are developmentally regulated. Lower doses have a selective analgesic effect that decreases with increasing postnatal age.

The anticonvulsant remacemide and its metabolite AR-R12495AA attenuate spinal synaptic transmission and carrageenan-induced inflammation in the young rat

The effects of the anticonvulsants remacemide [(±)-2-amino-N-(1-methyl-1,2-diphenylethyl)-acetamide hydrochloride] and its des -glycinated metabolite AR-R12495AA [(±)-1-methyl-1,2-diphenylethylamine- monohydrochloride] on primary afferent-induced synaptic transmission and frequency-dependent summation of synaptic potentials were assessed in the young rat spinal cord in vitro. Behavioural studies in the rat determined the effects of these anticonvulsant compounds in the carrageenan model of inflammation. Recordings of the extracellular dorsal root-evoked ventral root potential (DR-VRP) revealed a significant reduction of the duration andt1 -2decay of the long latency, slow DR-VRP by remacemide (50 and 100 μM) and AR-R12495AA (25, 50 and 100 mM). The short-latency, fast monosynaptic DR-VRP peak was reduced by only the highest concentration of AR-R12495AA (100 μM). In intracellular dorsal root-evoked excitatory postsynaptic potentials (DR-EPSPs) of single ventral horn neurons, AR-R12495AA (100 μM) attenuated the time course of the long-latency (slow) EPSP. Frequency-dependent (0.5–2.0 Hz) summation of dorsal root-evoked synaptic events (recorded extracellularly as the cumulative ventral root depolarization (CVRD), and intracellularly as wind-up) was attenuated by remacemide (100 μM) and AR-R12495AA (50 and 100 μM). Pre-treatment with intra-peritoneal injection of 75 mg/kg of remacemide or AR-R12495AA caused a significant reduction of carrageenan-induced mechanical hyperalgesia and oedema. These electrophysiological and behavioural data provide evidence that remacemide and AR-R12495AA may also possess analgesic and anti-inflammatory activity. Copyright 2000 European Federation of Chapters of the International Association for the Study of Pain

Immunohistochemical localization of neurokinin-1 receptor in the lumbar spinal cord of young rats: morphology and distribution

The type and distribution of neurokinin-1 (NK-1) receptor-expressing neurones were studied in young (14-day-old) rats' lumbar spinal cord using pre-embedding immunohistochemistry. The heaviest immunoreactivity was observed in the middle part and lateral fourth of lamina I where the great majority of immunoreactive perikarya represented fusiform and multipolar cells. In lamina II the middle and medial part showed moderate immunoreactivity, most of the cells resembled stalked cells. In lamina III the labelled perikarya were evenly distributed, while those in lamina IV accumulated mainly in the lateral part. In both laminae most of the labelled neurones represented central cells, the rest of them belonged to the antenna-type cells with long dorsally directed dendrites penetrating the superficial laminae. The immunoreactivity in laminae V-VII was uniform and relatively weak. In lamina VIII the immunopositive perikarya were encountered only rarely while in lamina IX virtually all motoneurones showed weak immunoreactivity. Lamina X contained small, multipolar and fusiform labelled perikarya. In conclusion, we found that the general appearance of the NK-1 receptor immunostaining and the major type of NK-1 receptor-expressing neurones were similar to that found previously in adult spinal cord. Using the same method as Brown and colleagues the number of labelled NK-1 receptor immunoreactive cells was similar in young and adult animals except lamina I where the number of immunoreactive neurones was twice that in adults.

Serotonergic modulation of the responses to excitatory amino acids of rat dorsal horn neurons in vitro: implications for somatosensory transmission.

Serotonin (5-HT) is one of the major transmitters involved in supraspinal control of somatic sensation and nociception. The aim of the present study was to investigate if the 5-HT-induced modulation of sensory transmission in the dorsal horn could be due to regulation of neuronal responses to excitatory amino acids. Experiments were performed in an in vitro preparation of the young rat spinal cord. Responses to dorsal root stimulation (DR-EPSP) and to droplet application of N-methyl-D-aspartic acid (NMDA) and alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA) were obtained by means of intracellular recordings of dorsal horn neurons. Bath applications of 5-HT (50 microM) generally caused reductions in amplitude and integrated area of DR-EPSPs and of responses to NMDA but the responses to AMPA were unaltered. A linear correlation was found between the effects of 5-HT on the DR-EPSP and on the NMDA response measured as percentage change in amplitude (r2 = 0.45; P < or = 0.01) and integrated area (r2 = 0.77; P < or = 0.001). The NMDA receptor antagonist d-AP5 (50 microM) completely abolished NMDA responses and caused a depression of the DR-EPSP similar to that of 5-HT. The 5-HT1 receptor agonist 5-carboxamidotryptamine (5-CT; 1 microM) mimicked the depressant effects of 5-HT but had a stronger depressant action on the DR-EPSP than 5-HT. The depression of NMDA responses induced by 5-HT and 5-CT was tetrodotoxin (1 microM) resistant. It is concluded that 5-HT-induced depression of NMDA responses explains partially the depressant action of 5-HT on dorsal horn synaptic transmission activating a postsynaptic site sensitive to 5-CT. The possible activation of coadjuvant mechanisms is discussed.

Reduction of NMDA-induced Ca2+ transients by a mu-opioid receptor agonist in dorsal horn neurons.

The effect of DAMGO, a mu-opioid receptor agonist, on intracellular Ca2+ transients evoked by the application of NMDA, was studied in freshly dissociated neurons from the superficial dorsal horn in the spinal cord of young rats. DAMGO (5-10 microns) reduced the amplitude of the Ca2+ transients measured with Fura-2 to 58 +/- 17% of the controls in 41% of the neurons tested. The effect of DAMGO was dose dependent and reversible. The reduction of NMDA-induced Ca2+ transients by DAMGO was prevented by application of the opioid antagonists naloxone (0.1-5 microM) and CTAP (0.2-2 microM). DAMGO also reduced Ca2+ transients induced by high K+ in 29% of the neurons. These data suggest that mu-receptor activation regulates NMDA-induced Ca2+ transients in a complex manner, by reducing both a depolarization-induced component and the NMDA-channel component of this Ca2+ signal.

Profile of neuronal excitation following selective activation of the neurokinin-1 receptor in rat deep dorsal horn in vitro

The excitatory actions of the selective neurokinin-1 receptor (NK1R) agonist [Sar 9,Met(O 2) 11]substance P (SP) were tested on a sample ( n=50) of deep dorsal horn neurones in the isolated and hemisected young rat spinal cord. Superfusion of the NK1R agonist (2 μM) elicited a prolonged membrane depolarisation (6.6±0.5 mV) and an increase in action potential firing in 41/50 (82%) neurones. These [Sar 9,Met(O 2) 11]SP-induced depolarisations were attenuated by the selective NK1R antagonist GR82334 (1 μM). An increased neuronal excitability after [Sar 9,Met(O 2) 11]SP application was indicated by an augmented spike frequency generated in response to long duration, step depolarisations. In order to assess whether a direct excitatory action existed, [Sar 9,Met(O 2) 11]SP was re-tested on a sample of TTX-treated neurones ( n=14). The majority (9/14) retained agonist sensitivity although the amplitude of the depolarisation was reduced to 48% of the control value. A sample of neurones ( n=7) that responded to the NK1R agonist were morphologically characterised after filling with the intracellular dye, biocytin. Dorsal dendrites that clearly penetrated lamina II and that could receive a direct C-afferent input, were identified in only 2/7 neurones. These electrophysiological and neuroanatomical data demonstrate that deep dorsal horn neurones possess functional NK1Rs. The implications of the existence of these NK1Rs in the context of spinal somatosensory systems and SP is considered.

Spinal cord dysmyelination induced in vivo by IgM antibodies to three different myelin glycolipids.

It was shown previously (Rosenbluth et al.: J. Neurosci. 16:2635-2641, 1996) that implantation of hybridoma cells that produce an IgM antigalactocerebroside into the spinal cord of young rats results in the development of myelin sheaths with a repeat period approximately 2-3x normal, similar to the abnormal peripheral myelin sheaths seen in human IgM gammopathies. We now present evidence that this effect can be reproduced in the spinal cord by implanting either of two other hybridomas, O4 and A2B5, that secrete, respectively, antisulfatide and antiganglioside IgM antibodies. The formation of expanded CNS myelin thus does not depend on antibodies to galactocerebroside specifically but can be mediated by IgM antibodies that react with other myelin glycolipids as well.

Kappa-opioid receptor agonists modulate excitatory transmission in substantia gelatinosa neurons of the rat spinal cord

This study examined the effects of selective activation of kappa 1-opioid receptors on excitatory transmission in substantia gelatinosa (SG) using intracellular recordings from SG neurons in transverse slices of the young rat lumbar spinal cord. Monosynaptic and polysynaptic excitatory postsynaptic potentials (EPSPs) were evoked by orthodromic electrical stimulation of A delta or C primary afferent fibers in the dorsal root after blocking inhibitory inputs with bicuculline and strychnine, NMDA receptors with D-2-amino-5-phosphonovaleric acid and mu- and delta-opioid receptors with CTAP and ICI 174,864, respectively. Bath application of dynorphin A1-17 or U-69, 593 caused dual modulation of the peak amplitude of presumed monosynaptic AMPA receptor-mediated EPSPs, decreasing synaptic potentials at nanomolar concentrations in a majority of SG cells examined (dynorphin, 63%; U-69,593, 91%), and increasing EPSPs at micromolar concentrations. Only the inhibitory action of dynorphin A1-17 was consistently and completely blocked by norbinaltorphimine (nor-BNI). Since U-69,593 and nor-BNI are selective for the kappa 1-opioid receptors, the depression of EPSPs is likely to be mediated by the kappa1-opioid receptors. Under conditions of blockade of synaptic transmission with TTX and mu-and delta-opioid receptors, dynorphin A1-17 and U-69,593 hyperpolarize most of SG neurons and decrease their membrane input resistance, the finding suggesting that direct interaction of kappa-agonists with a postsynaptic receptor is likely explanation for the inhibition of EPSPs. However, in some SG cells, the inhibition of EPSPs appears to be of presynaptic origin since dynorphin A1-17 and U-69,593 did depress the EPSPs in the absence of changes in passive membrane properties. Rp-cAMPS, a membrane permeant potent competitive inhibitor of cAMP-activated protein kinase, prevented the depressant effect of dynorphin A 1-17. This finding suggested a possibility that dynorphin A1-17, acting through a decrease in intracellular cyclic AMP levels, can reduce the synaptic responses of SG neurons. These results provide the first electrophysiological demonstration that the activation of kappa 1-opioid receptors inhibits AMPA receptor-mediated primary afferent neurotransmission in the substantia gelatinosa of the young rat spinal cord. This effect may mediate the ability of kappa-receptor agonists to produce antinociception.

Cobalt accumulation in neurons expressing ionotropic excitatory amino acid receptors in young rat spinal cord: morphology and distribution.

Excitatory amino acids (EAA) acting on N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate receptors play an important role in synaptic transmission in the spinal cord. Quantitative autoradiography and physiological experiments suggest that NMDA receptors are localized mainly in lamina II while kainate and AMPA receptors are found on both dorsal and ventral horn neurons. However the cell types expressing EAA receptors and their laminar distribution is not known. We have used a cobalt uptake method to study the morphology and distribution of spinal cord neurons expressing AMPA, kainate, or NMDA excitatory amino acid receptors in the lumbar enlargement of the rat spinal cord. The technique involved superfusion of hemisected spinal cords of 14 day-old rat pups in vitro with excitatory amino acid receptor ligands in the presence of CoCl2. Cobalt has been shown to enter cells through ligand-gated ion channels in place of Ca2+. Cells which accumulated cobalt ions following activation by ionotropic excitatory amino acid receptors were visualized histochemically. The cobalt uptake generated receptor-specific labeling of cells, as the NMDA receptor antagonist D-(-)-2-amino-(5)-phosphonovaleric acid (D-AP-5) (20 microM) blocked the NMDA, but not kainate-induced cobalt uptake. The kainate-induced cobalt labeling was reduced by the non-selective excitatory amino acid receptor antagonist kynurenic acid (4 mM). Passive opening of the voltage-gated Ca(2+)-channels by KCl (50 mM) did not result in cobalt uptake, indicating that cobalt enters the cells through ligand-gated Ca(2+)-channels. AMPA (500 microM), kainate (500 microM), or NMDA (500 microM) each induced cobalt uptake with characteristic patterns and distributions of neuronal staining. Overall, kainate induced cobalt uptake in the greatest number of neuronal staining. Overall, kainate induced cobalt uptake in the greatest number of neuronal perikarya while NMDA-induced uptake was the lowest. AMPA and kainate, but not NMDA superfusion, resulted in cobalt labeling of glial cells. Our results show that the cobalt uptake technique is a useful way to study the morphology and distribution of cells expressing receptors with ligand-gated Ca2+ channels.