Do premotor interneurons act in parallel on spinal motoneurons and on dorsal horn spinocerebellar and spinocervical tract neurons in the cat?

It has previously been established that ventral spinocerebellar tract (VSCT) neurons and dorsal spinocerebellar tract neurons located in Clarke's column (CC DSCT neurons) forward information on actions of premotor interneurons in reflex pathways from muscle afferents on α-motoneurons. Whether DSCT neurons located in the dorsal horn (dh DSCT neurons) and spinocervical tract (SCT) neurons are involved in forwarding similar feedback information has not yet been investigated. The aim of the present study was therefore to examine the input from premotor interneurons to these neurons. Electrical stimuli were applied within major hindlimb motor nuclei to activate axon-collaterals of interneurons projecting to these nuclei, and intracellular records were obtained from dh DSCT and SCT neurons. Direct actions of the stimulated interneurons were differentiated from indirect actions by latencies of postsynaptic potentials evoked by intraspinal stimuli and by the absence or presence of temporal facilitation. Direct actions of premotor interneurons were found in a smaller proportion of dh DSCT than of CC DSCT neurons. However, they were evoked by both excitatory and inhibitory interneurons, whereas only inhibitory premotor interneurons were previously found to affect CC DSCT neurons [as indicated by monosynaptic excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs) in dh DSCT and only IPSPs in CC DSCT neurons]. No effects of premotor interneurons were found in SCT neurons, since monosynaptic EPSPs or IPSPs were only evoked in them by stimuli applied outside motor nuclei. The study thus reveals a considerable differentiation of feedback information provided by different populations of ascending tract neurons.     

Modulation of μ-opioid receptor desensitization in peripheral sensory neurons by phosphoinositide 3-kinase γ

G protein-coupled opioid receptors undergo desensitization after prolonged agonist exposure. Recent in vitro studies of μ-opioid receptor (MOR) signaling revealed an involvement of phosphoinositide 3-kinases (PI3K) in agonist-induced MOR desensitization. Here we document a specific role of the G protein-coupled class IB isoform PI3Kγ in MOR desensitization in mice and isolated sensory neurons. The tail-withdrawal nociception assay evidenced a compromised morphine-induced tolerance of PI3Kγ-deficient mice compared to wild-type animals. Consistent with a role of PI3Kγ in MOR signaling, PI3Kγ was expressed in a subgroup of small-diameter dorsal root ganglia (DRG) along with MOR and the transient receptor potential vanilloid type 1 (TRPV1) receptor. In isolated DRG acute stimulation of MOR blocked voltage-gated calcium currents (VGCC) in both wild-type and PI3Kγ-deficient DRG neurons. By contrast, following long-term opioid administration the attenuating effect of MOR was strongly compromised in wild-type DRG but not in PI3Kγ-deficient DRG. Our results uncover PI3Kγ as an essential modulator of long-term MOR desensitization and tolerance development induced by chronic opioid treatment in sensory neurons.     

Functional heterogeneity of calretinin‐expressing neurons in the mouse superficial dorsal horn: implications for spinal pain processing

Key points

• The superficial spinal dorsal horn contains a heterogeneous population of neurons that process sensory inputs.
• Information on the properties of excitatory interneurons in this region is limited. As calretinin is a protein thought to be restricted to an excitatory population in this region, the aim of this study was to characterize calretinin‐expressing neurons.
• Most calretinin cells (85%) exhibited large A‐type potassium currents and delayed firing action potential discharge, and received strong excitatory synaptic input, whereas the remainder exhibited hyperpolarization‐activated cation currents and low threshold T‐type calcium currents, and tonic‐ or initial bursting firing patterns, and received weak excitatory synaptic input. These respective features are consistent with properties of excitatory and inhibitory interneuron populations in this region of the spinal cord.
• Our findings have resolved a previously unidentified population of inhibitory interneurons. Furthermore, the contrasting excitability patterns of excitatory and inhibitory calretinin‐expressing neurons suggest that they play distinct roles in spinal sensory processing circuits.

Abstract

Neurons in the superficial dorsal horn (SDH) of the spinal cord play an important role in nociceptive, thermal, itch and light touch sensations. Excitatory interneurons comprise ∼65% of all SDH neurons but surprisingly few studies have investigated their role in spinal sensory processing. Here we use a transgenic mouse to study putative excitatory SDH neurons that express the calcium binding protein calretinin (CR). Our immunocytochemical, morphological and electrophysiological analysis identified two distinct populations of CR‐expressing neurons, which we termed ‘Typical’ and ‘Atypical’. Typical CR‐expressing neurons comprised ∼85% of the population and exhibited characteristic excitatory interneuron properties including delayed firing discharge, large rapid A‐type potassium currents, and central, radial or vertical cell morphologies. Atypical neurons exhibited properties consistent with inhibitory interneurons, including tonic firing or initial bursting discharge, I _h currents, and islet cell morphology. Although both Typical and Atypical CR‐expressing neurons responded to noxious peripheral stimulation, the excitatory drive onto Typical CR‐expressing neurons was much stronger. Furthermore, Atypical CR‐expressing cells comprise at least two functionally distinct subpopulations based on their responsiveness to noxious peripheral stimulation and neurochemical profile. Together our data suggest CR expression is not restricted to excitatory neurons in the SDH. Under normal conditions, the contribution of ‘Typical’ excitatory CR‐expressing neurons to overall SDH excitability may be limited by the presence of A‐type potassium currents, which limit the effectiveness of their strong excitatory input. Their contribution may, however, be increased in pathological situations where A‐type potassium currents are decreased. By contrast, ‘Atypical’ inhibitory neurons with their excitable phenotype but weak excitatory input may be more easily recruited during increased peripheral stimulation.

Abbreviations

AP

action potential

CR

calretinin

DH

dorsal horn

eGFP

enhanced GFP

GFP

green fluorescent protein

IR

immunoreactive

nNOS

neuronal nitric oxide synthase

NPY

neuropeptide Y

RMP

resting membrane potential

SDH

superficial dorsal horn

     

Metabotropic glutamate receptor 5 regulates excitability and Kv4.2-containing K⁺ channels primarily in excitatory neurons of the spinal dorsal horn

Metabotropic glutamate (mGlu) receptors play important roles in the modulation of nociception. Previous studies demonstrated that mGlu5 modulates nociceptive plasticity via activation of ERK signaling. We have reported recently that the Kv4.2 K(+) channel subunit underlies A-type currents in spinal cord dorsal horn neurons and that this channel is modulated by mGlu5-ERK signaling. In the present study, we tested the hypothesis that modulation of Kv4.2 by mGlu5 occurs in excitatory spinal dorsal horn neurons. With the use of a transgenic mouse strain expressing enhanced green fluorescent protein (GFP) under control of the promoter for the γ-amino butyric acid (GABA)-synthesizing enzyme, glutamic acid decarboxylase 67 (GAD67), we found that these GABAergic neurons express less Kv4.2-mediated A-type current than non-GAD67-GFP neurons. Furthermore, the mGlu1/5 agonist, (R,S)-3,5-dihydroxyphenylglycine, had no modulatory effects on A-type currents or neuronal excitability in this subgroup of GABAergic neurons but robustly modulated A-type currents and neuronal excitability in non-GFP-expressing neurons. Immunofluorescence studies revealed that Kv4.2 was highly colocalized with markers of excitatory neurons, such as vesicular glutamate transporter 1/2, PKCγ, and neurokinin 1, in cultured dorsal horn neurons. These results indicate that mGlu5-Kv4.2 signaling is associated with excitatory dorsal horn neurons and suggest that the pronociceptive effects of mGlu5 activation in the spinal cord likely involve enhanced excitability of excitatory neurons.     

Co-induction of neuronal interferon-gamma and nitric oxide synthase in rat motor neurons after axotomy: a role in nerve repair or death?

Summary Induction of an interferon-gamma-like molecule, previously isolated from neurons (N-IFN-), and of the neuronal isoform I of the synthetic enzyme of the free radical nitric oxide, nitric oxide synthase I, as well as of NADPH-diaphorase, were examined in axotomized dorsal motor vagal and hypoglossal neurons. Unilateral transection of the vagal and hypoglossal nerves was performed in the same rat and an induction of N-IFN- and nitric oxide synthase I immunostaining as well as NADPH-diaphorase histochemical positivity was observed in the ipsilateral motoneurons after 2–4 days. The immuno- and enzyme- histochemical positivities were much stronger in the dorsal motor vagal neurons than in hypoglossal neurons. Two and 4 weeks after axotomy N-IFN- immunoreactivity and NADPH-diaphorase positivity persisted in the former, but started to decrease in the latter neurons. Previous data have shown that 23 weeks after nerve transection the majority of the dorsal motor vagal neurons are lost, while the majority of the hypoglossal neurons survive. The high and persistent expression of N-IFN- and nitric oxide synthase I after axotomy in the dorsal motor vagal neurons, that are largely destined to die, indicates that the co-induction of these two molecules may be implicated in the pathogenesis of neuronal degeneration.     

Acute inflammation induces segmental,bilateral, supraspinally mediated opioid release in the rat spinal cord,as measured by μ-opioid receptor internalization

The objective of this study was to measure opioid release in the spinal cord during acute and long-term inflammation using μ-opioid receptor (MOR) internalization. In particular, we determined whether opioid release occurs in the segments receiving the noxious signals or in the entire spinal cord, and whether it involves supraspinal signals. Internalization of neurokinin 1 receptors (NK1Rs) was measured to track the intensity of the noxious stimulus. Rats received peptidase inhibitors intrathecally to protect opioids from degradation. Acute inflammation of the hind paw with formalin induced moderate MOR internalization in the L5 segment bilaterally, whereas NK1R internalization occurred only ipsilaterally. MOR internalization was restricted to the lumbar spinal cord, regardless of whether the peptidase inhibitors were injected in a lumbar or thoracic site. Formalin-induced MOR internalization was substantially reduced by isoflurane anesthesia. It was also markedly reduced by a lidocaine block of the cervical-thoracic spinal cord (which did not affect the evoked NK1R internalization) indicating that spinal opioid release is mediated supraspinally. In the absence of peptidase inhibitors, formalin and hind paw clamp induced a small amount of MOR internalization, which was significantly higher than in controls. To study spinal opioid release during chronic inflammation, we injected complete Freund's adjuvant (CFA) in the hind paw and peptidase inhibitors intrathecally. Two days later, no MOR or NK1R internalization was detected. Furthermore, CFA inflammation decreased MOR internalization induced by clamping the inflamed hind paw. These results show that acute inflammation, but not chronic inflammation, induces segmental opioid release in the spinal cord that involves supraspinal signals.     

Effects of κ- and μ-opioid agonists on cholinergic neurotransmission and contraction in isolated bovine trachealis

The effects of the selective μ-opioid agonist DAMGO and the selective κ-opioid agonist U-50488H on tritiated acetylcholine release ([³H]-ACh) and contractile responses to electrical stimulation (ES) were simultaneously determined in isolated bovine trachealis. The inhibitory effect of DAMGO 10⁻⁵ M on [³H]-ACh release was not significantly different from the effect of the non-selective muscarinic agonist pilocarpine 10⁻⁵ M, whereas the effect of U-50488H 10⁻⁵ M was significantly greater. The effects of both opioids were not significantly different when muscles were pre- or co-incubated with the unselective muscarinic antagonist atropine 10⁻⁷ M. Both DAMGO and U-50488H attenuated ES-induced contraction and this effect was significantly correlated with the inhibition of [³H]ACh-release (r² = 0.8552). These data suggest that (1) opioids are important modulators of airway smooth muscle tone, (2) their effect is not altered by the activity of muscarinic autoregulation, and (3) their inhibitory effect of airway smooth muscle contraction can be almost totally explained by inhibition of ACh release.     

Differential effect of glutamate transporter inhibition on EPSCs in the morphine naïve and morphine tolerant neonatal spinal cord slice

Opioid analgesic tolerance is a phenomenon defined as a need for increasingly higher doses of opiates to maintain suitable pain relief following repeated drug exposure. Research suggests that analgesic tolerance may result from heightened NMDA receptor (NMDAR) activity, but little is known regarding the mechanisms by which this elevated NMDAR activity develops. Recent evidence suggests that glutamate transporter down-regulation follows repeated opiate exposure and contributes to heightened pain sensitivity. Though glutamate transporter inhibition has been shown to increase activity of spinal cord neurons, it is unknown whether this increase contributes to the heightened NMDAR activity that underlies opiate tolerance. We directly tested this hypothesis by comparing the effects of glutamate transporter inhibition on excitatory post-synaptic currents (EPSCs) in the spinal cord dorsal horn of opiate naïve and opiate tolerant rats. We show that non-selective glutamate transporter inhibition increases the rate of spontaneous excitatory post-synaptic currents (sEPSCs) in the opiate naïve, but not opiate tolerant slice. This potentiation occurs in the presence of the sodium channel blocker tetrodotoxin (TTX) and is blocked by the NMDAR antagonist d-2-amino-5-phosphonovalerate (APV). The sEPSC rate is elevated at baseline in the opiate tolerant spinal cord slice compared to the opiate naïve slice, and glutamate transporter inhibition eliminates this difference. Taken together, we conclude that glutamate transporter inhibition directly contributes to heightened NMDAR activity. Furthermore, we propose that the increased neural activity observed in the opiate tolerant slice is due to a state of glutamate transporter down-regulation and resultant heightened NMDAR activity.     

Co-localization of the β-subtype of protein kinase C and phosphorylation-dependent immunoreactivity of neurofilaments in intact, decentralized and axotomized rat peripheral neurons

Summary The localizations of protein kinase C--immunoreactivity and phosphorylation-dependent immunoreactivity of neurofilaments were compared in rat dorsal root, hypogastric, and superior cervical ganglia. In all the ganglia studied, protein kinase C- and phosphorylation-dependent immunoreactivity of neurofilaments were co-localized in nerve fibres, and no fibres with only protein kinase C--immunoreactivity or phosphorylation-dependent immunoreactivity of neurofilaments were observed. Most intense perikaryal protein kinase c- and phosphorylation-dependent neurofilament-staining were seen in large dorsal root ganglion neurons, whereas in the superior cervical ganglion only very faint protein-kinase C- and no phosphorylation-dependent staining was seen in the neuronal cell bodies. Both decentralization and axotomy of the superior cervical ganglion induced an accumulation of protein-kinase C--immunoreactivity and phosphorylation-dependent immunoreactivity of neurofilaments in the majority of neuronal perikarya. The accumulation was first observed at 1–2 days postoperation and it persisted up to 6–10 days postoperation. In strongly labelled decentralized neuronal perikarya, precipitation of immunoreactivity was seen near the cell and nuclear membranes, whereas in axotomized neurons, immunoreactivity was often concentrated as a unipolar clump in the cytoplasm. The results show that protein kinase C--immunoreactivity and phosphorylation-dependent immunoreactivity of neurofilaments are colocalized in intact rat peripheral ganglia and that both accumulate transiently in cell bodies of the superior cervical ganglion after decentralization and axotomy.     

Late blocking of peripheral TNF-α is ineffective after spinal cord injury in mice

Spinal cord injury (SCI) is characterized by different phases of inflammatory responses. Increasing evidence indicates that the early chronic phase (two to three weeks after SCI) is characterized by a dramatic invasion of immune cells and a peak of pro-inflammatory cytokine levels, such as tumor necrosis factor-α (TNF-α) derived from the injured spinal cord as well as from injured skin, muscles and bones. However, there is substantial controversy whether these inflammatory processes in later phases lead to pro-regenerative or detrimental effects. In the present study, we investigated whether the inhibition of peripheral TNF-α in the early chronic phase after injury promotes functional recovery in a dorsal hemisection model of SCI. Three different approaches were used to continuously block peripheral TNF-α in vivo, starting 14 days after injury. We administered the TNF-α blocker etanercept intraperitoneally (every second day or daily) as well as continuously via osmotic minipumps. None of these administration routes for the TNF-α inhibitor influenced locomotor restoration as assessed by the Basso mouse scale (BMS), nor did they affect coordination and strength as evaluated by the Rotarod test. These data suggest that peripheral TNF-α inhibition may not be an effective therapeutic strategy in the early chronic phase after SCI.     

Morphine sensitization increases the extracellular level of glutamate in CA1 of rat hippocampus via μ-opioid receptor

Repeated administration of abuse drugs such as morphine elicits a progressive enhancement of drug-induced behavioral responses, a phenomenon termed behavioral sensitization. These changes in behavior may reflect plastic changes requiring regulation of glutamatergic system in the brain. In this study, we investigated the effect of morphine sensitization on extracellular glutamate concentration in the hippocampus, a brain region rich in glutamatergic neurons. Sensitization was induced by subcutaneous (s.c.) injection of morphine, once daily for 3 days followed by 5 days free of the opioid treatment. The results showed that extracellular glutamate concentration in the CA1 was decreased following administration of morphine in non-sensitized rats. However, morphine-induced behavioral sensitization significantly increased the extracellular glutamate concentration in this area. The enhancement of glutamate in morphine sensitized rats was prevented by administration of naloxone 30 min before each of three daily doses of morphine. These results suggest an adaptation of the glutamatergic neuronal transmission in the hippocampus after morphine sensitization and it is postulated that opioid receptors may play an important role in this effect.     

Effects of systemic 3-nitropropionic acid-induced lesions of the dorsal striatum on cannabinoid and µ-opioid receptor binding in the basal ganglia

Systemic administration of 3-nitropropionic acid (3NPA) in experimental animals produces bilateral striatal lesions similar to those seen in Huntington’s disease (HD) caudate and putamen. [³H]-CP55,940 binding to cannabinoid receptors in human basal ganglia nuclei has been shown to be highly susceptible to the earliest pathological changes in the HD brain. In this study, to assess further the suitability of 3NPA-induced striatal lesions as a model for HD neuropathology, we examined the effects of striatal lesions induced by the systemic administration of 3NPA on the binding of [³H]-CP55,940 to pre- and postsynaptic cannabinoid receptors in striatum, globus pallidus, entopeduncular nucleus and substantia nigra pars reticulata and also the effect of 3NPA-induced striatal lesions on the binding of [³H]-DAMGO to μ-opioid receptors in striatal striosomes. Systemic administration of 3NPA induced bilateral and symmetrical lesions in dorsolateral striatum. Within the lesion core, [³H]-CP55,940 and [³H]-DAMGO binding density was reduced to background levels. Beyond the immediate borders of the central core of the 3NPA-induced lesion, striatal binding density was not significantly different from that measured in unlesioned rats. [³H]-CP55,940 binding in globus pallidus, entopeduncular nucleus and substantia nigra in 3NPA-lesioned rats was significantly reduced compared to controls, and the individual decreases were similar for each site. However, these reductions were statistically marginal. These data suggest that, while producing striatal lesions which bear some similarity to those seen in HD, the consequences of 3NPA for striatopallidal and striatonigral efferent projections do not reflect the reported neurodegenerative changes seen in the HD brain. Received: 18 November 1998 / Accepted: 12 July 1999     

Effect of chronic γ-irradiation and β-carotene on lipid metabolism in presynaptic membranes in rat brain

Lysophosphatidylcholine is involved in radiation-induced modulation of presynaptic membranes in rat brain. High sensitivity of the cortical integrative functions to chronic low-dose γ-irradiation is demonstrated. β-Carotene produced a protective effect during chronic irradiation. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 129, No. 6, pp. 629–632, June, 2000     

Effect of deoxycorticosterone on 5′-nucleotidase and adenosine deaminase activity in the rat hypothalamus and hippocampus

Laboratory of Neurochemistry, Kiev Research Institute of Endocrinology and Metabolism, Ministry of Health of the Ukrainian SSR. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 110, No. 7, pp. 56–58, July, 1990.     

Effect of IFN-γ and IL-4 levels on the expression of Fas and Bcl-2 in peripheral blood lymphocytes in hemodialysis patients

To study the correlation between the levels of IFN-γ and IL-4 and the expression of Fas and Bcl-2 in peripheral blood lymphocytes (PBL) in hemodialysis patients, the indirect immune fluorescein labeling method of flow cytometry and solid sandwich enzyme-linked immunosorbent assay were performed for detecting the expression of Fas and Bcl-2 in PBL and the levels of IFN-γ and IL-4 in the serum of 30 hemodialysis patients, respectively. It was found that the expression of Fas in PBL and the level of IL-4 in the serum of hemodialysis patients were significantly higher (P < 0.01), whereas Bcl-2 in PBL and IFN-γ in the serum were significantly lower (P < 0.01) than those of the normal controls. According to statistical analysis, the expression of Fas in PBL had a negative correlation with the level of IFN-γ, but a positive correlation with IL-4 in the serum of hemodialysis patients. Contrarily, the expression of Bcl-2 had a positive correlation with IFN-γ, but a negative correlation with IL-4 in the serum of hemodialysis patients. These results suggest that hemodialysis patients have a suppressed secretion of Th1-associated cytokine IFN-γ, but an increased secretion of Th2-associated cytokines IL-4, and these two aspects may play an important role in the abnormal apoptosis of PBL and its accompanying immune deficiency.     

Effect of α1-adrenergic stimulation and lithium on calcium sensitivity of hyperpermeable rat myocardial fibers

All-Union Cardiologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. N. Smirnov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 112, No. 11, pp. 478–480, November, 1991.     

Effect of μ-Opiate Receptor Agonist Tetrapeptide A10 on DNA Synthesis and Protein Content in the Myocardium of Albino Rats

Effect of intraperitoneal injection of tetrapeptide A10 (H-Tyr-D-Orn-Phe-Gly-OH), selective -opiate receptor agonist, synthetic analog of dermorphine, in a dose of 100 g/kg on DNA synthesis and protein content in the myocardium was studied in albino rats. Five injections of tetrapeptide on days 2-6 after birth caused no changes in DNA synthesis 17 days after the last injection, i. e. in 24-day rats. The number of nucleoli and their area increased. In adult males long-term (3-week) treatment with tetrapeptide A10 increased the number of nucleoli and the mean and integral optical density of isolated cardiomyocytes stained with amido black B, which probably attested to activation of protein synthesis in the myocardium. Simultaneously, the content of catecholamines in the heart increased. These data are comparable with delayed effects of k-opiate receptor agonist dinorphine A1-13 and indicate that morphogenetic properties of opioid peptides in rat myocardium are realized via the same routes.