Pretreatment with Group I Metabotropic Glutamate Receptors Antagonists Attenuates Lethality Induced by Acute Cocaine Overdose and Expression of Sensitization to Hyperlocomotor Effect of Cocaine in Mice

Cocaine abuse and dependence is a worldwide health problem. However, there are no currently approved medications to reduce cocaine abuse/relapse and toxicity. The aim of the present study was to test, whether group I metabotropic glutamate receptors (mGluRs) antagonists (mGluR1 and mGluR5) differentially regulate toxic versus behavioral effects of cocaine, both phenomena relevant to the psychopathology of cocaine addiction in humans. In the present study, we assessed the impact of mGluR1 antagonist—EMQMCM and mGluR5 antagonist—MTEP on the cocaine-induced lethality and the expression of sensitization to hyperlocomotor effect of cocaine in mice. Our study indicated that EMQMCM and MTEP, both substances at the doses of 5 and 10 mg/kg (but not 2.5 mg/kg), decreased cocaine-induced lethality produced by 75 mg/kg of cocaine, which was given acutely. The effect of EMQMCM was dose-dependent, and this compound at the dose of 10 mg/kg almost completely abolished the lethality induced by cocaine. MTEP reduced this cocaine effect at the doses of 5 and 10 mg/kg, equally. Furthermore, EMQMCM (1.25–5 mg/kg) at the doses of 2.5 and 5.0 mg/kg, and MTEP (2.5–10 mg/kg) only at the highest dose of 10 mg/kg, significantly reduced the expression of cocaine-induced (10 mg/kg) behavioral sensitization. Our results suggest that stimulation of mGluR1 and mGluR5 is involved in lethal effect of cocaine overdose and cocaine seeking behavior evaluated in behavioral sensitization test. However, the participation of mGluR1 in these cocaine effects seems to be dominant. Therefore, antagonists showing preferences towards mGluR1 might be useful in therapy of cocaine toxicity and abuse.     

Perinatal changes of I(h) in phrenic motoneurons

The hyperpolarization-activated cationic current (I(h)) was characterized and its maturation studied on phrenic motoneurons (PMNs), from reduced preparations of foetal (E18 and E21) and newborn (P0-P3) rats, using the whole-cell patch-clamp technique. In voltage-clamp mode, 2-s hyperpolarizing steps (5-mV, -50 to -110 mV) elicited a noninactivating inward current, blocked by external application of Cs+ or ZD 7288. At -110 mV, Ih current density averaged 0.67 +/- 0.41 pA/pF at E18, reached a transient peak at E21 (1.38 +/- 0.11 pA/pF) and decreased at P0-P3 (0.77 +/- 0.22 pA/pF). V1/2 was similar at E18 and E21 (-79 mV) but was significantly hyperpolarized at P0-P3 (-90 mV). The time constant of activation was voltage-dependent, and significantly faster at E21. Reversal potential was similar at all ages when estimated by extrapolation or tail current procedures. It was positively shifted by 25 +/- 6 mV when external potassium was raised from 3 to 10 m M, suggesting a similar sensitivity to K+ from E18 to P0-3. Cs(+) or ZD 7288 applications on PMNs at rest in current-clamp mode, in a partitioned chamber, induced a 10 +/- 2 mV hyperpolarization at E18 and E21, and an 8 +/- 2 mV hyperpolarization at P0-3. The area of the central respiratory drive potential or current was increased by 33 and 31%, respectively, at E21, but was not significantly modified at E18 and P0-3. Our data suggest a critical period during the perinatal maturation of Ih during which it is transiently upregulated and attenuates the influence of the central respiratory drive on PMNs just prior to birth.     

Bilobalide Protects Mitochondrial Function in Ovariectomized Rats by Up-regulation of mRNA and Protein Expression of Cytochrome c Oxidase Subunit I

Bilobalide (BB), a sesquiterpene trilactone from Ginkgo biloba has been proposed to have protective effects on mitochondrial function. Using ovariectomized rats to mimic the post-menopausal pathophysiological changes in women, this study demonstrated that BB treatment could prevent estrogen withdrawal-induced decrease in mitochondrial adenosine triphosphate content, cytochrome c oxidase subunit I (COXI) mRNA and protein levels and COX activity in hippocampal tissues as effectively as estradiol benzoate. But neither ovariectomy nor BB treatment affected citrate synthase activity. These results suggested that BB was able to regulate COX activity via up-regulation of the gene and protein expression of its mitochondrial DNA-coded subunits, and modulation of COX activity by BB might contribute to its protective effects on mitochondrial function. Given that ovariectomy induces decrease in estrogen levels similar to that of menopause, BB may be useful in developing therapy for neurodegenerative diseases such as Alzheimer’s disease in post-menopausal females.     

Dihydroprogesterone Increases the Gene Expression of Myelin Basic Protein in Spinal Cord of Diabetic Rats

Alterations in myelin membranes, as well as in the expression of myelin proteins have been reported in experimental models of diabetes. Data here reported show for the first time that the mRNA levels of two isoforms of myelin basic protein (MBP), 18.5 and 21.5 kDa, are decreased in the spinal cord of streptozotocin-treated rats and that treatment with a neuroactive steroid, such as progesterone (P), may counteract this effect. Interestingly, metabolism of progesterone into dihydroprogesterone (DHP) by the enzyme 5α-reductase seems to exert an important role in such an effect. As here demonstrated, 5α-reductase mRNA and DHP levels are reduced by diabetes in spinal cord, but treatment with P, is able to counteract these effects. Moreover, treatment with DHP is able to mimic the effect of P on MBP gene expression. Thus, the effects of P here observed are due to its enzymatic conversion into DHP. Because DHP, like P, interacts with P receptor (PR), the present results may suggest the importance to analyze the effects of PR modulators as tools of therapeutic strategies for diabetic complications occurring in nervous system.     

Methionine-Mediated Protein Phosphatase 2A Catalytic Subunit (PP2Ac) Methylation Ameliorates the Tauopathy Induced by Manganese in Cell and Animal Models

The molecular mechanism of Alzheimer-like cognitive impairment induced by manganese (Mn) exposure has not yet been fully clarified, and there are currently no effective interventions to treat neurodegenerative lesions related to manganism. Protein phosphatase 2 A (PP2A) is a major tau phosphatase and was recently identified as a potential therapeutic target molecule for neurodegenerative diseases; its activity is directed by the methylation status of the catalytic C subunit. Methionine is an essential amino acid, and its downstream metabolite S-adenosylmethionine (SAM) participates in transmethylation pathways as a methyl donor. In this study, the neurotoxic mechanism of Mn and the protective effect of methionine were evaluated in Mn-exposed cell and rat models. We show that Mn-induced neurotoxicity is characterized by PP2Ac demethylation accompanied by abnormally decreased LCMT-1 and increased PME-1, which are associated with tau hyperphosphorylation and spatial learning and memory deficits, and that the poor availability of SAM in the hippocampus is likely to determine the loss of PP2Ac methylation. Importantly, maintenance of local SAM levels through continuous supplementation with exogenous methionine, or through specific inhibition of PP2Ac demethylation by ABL127 administration in vitro, can effectively prevent tau hyperphosphorylation to reduce cellular oxidative stress, apoptosis, damage to cell viability, and rat memory deficits in cell or animal Mn exposure models. In conclusion, our data suggest that SAM and PP2Ac methylation may be novel targets for the treatment of Mn poisoning and neurotoxic mechanism-related tauopathies.Electronic supplementary materialThe online version of this article (10.1007/s13311-020-00930-6) contains supplementary material, which is available to authorized users.Key Words: Methionine, ABL127, manganism, tau, neurodegeneration, PP2Ac methylation     

Expression of Neuronal Nicotinic Acetylcholine Receptor Subunit Genes in the Rat Autonomic Nervous System

In the autonomic nervous system efferent signals are relayed in sympathetic and parasympathetic ganglia. Fast synaptic transmission between pre- and postsynaptic neurons is achieved by neuronal nicotinic acetylcholine receptors (nAChRs). There is still little known about the subunit composition of these receptors. Establishing the subunit composition of native neuronal nAChRs is important for the understanding of their functional properties both in vivo and after expression in heterologous expression systems. We have combined in situ hybridization and autoradiography to detect the presence of mRNAs encoding subunits of neuronal nAChRs in sympathetic and parasympathetic ganglia. Inspection of the autoradiographs showed that the hybridization signal of five riboprobes (α3, 014–1, α7, β2 and β4) was significantly higher than the unspecific signal obtained with sense riboprobes. The distribution of α7 was tissue-dependent: a7 riboprobe binding was detected in the neurons of the superior cervical ganglion, adrenal medulla and ciliary ganglion. In contrast, the α7 hybridization signal was found only in a small fraction (1 -3%) of the neurons of the sphenopalatine and otic ganglia. Our results are consistent with the idea that α3 mRNA expression levels are somewhat higher than those of α7, α4 -1, β2 and β4.     

Expression of the Cyclic AMP-dependent Protein Kinase (PKA) Catalytic Subunit from a Herpes Simplex Virus Vector Extends the Survival of Rat Sympathetic Neurons in the Absence of NGF

Superior cervical ganglion neurons from neonatal rats are dependent on nerve growth factor for their survival both in vivo and in vitro. In culture this requirement can be largely replaced by cAMP or its analogues. Since activation of protein kinase A by cAMP is likely to be the pathway by which it exerts its survival-promoting effect, we have tested the feasibility of using herpes simplex virus (HSV) as a vector for expressing survival-promoting genes in neurons by cloning the catalytic subunit of the CAMP-dependent protein kinase (PKAcat) with a metallothionein gene promoter into the HSV thymidine kinase gene by homologous recombination. About 95% of the neurons became infected using 2.5 p.f.u. per cell. When this construct was used to express PKAcat in superior cervical ganglion neurons, in the presence of nerve growth factor (NGF) increases of 1.9- to 2.4-fold in PKA activity were found 8–10 h after infection; levels remained elevated (1.4- to 2.1-fold) up to 18 h, returning to basal by 24 h. After infection in the absence of NGF, cumulative activity over 24 h was ～3.5-fold lower in the first 24 h. Although the level of the inhibitory regulatory subunit type I was raised by 18 h, this is unlikely to completely explain the transient activity of PKAcat. When neurons were induced to express maximum PKAcat levels in the presence of NGF and then deprived of NGF, survival was extended by up to 2 days, demonstrating a direct role for PKA in promoting survival. By this time, some neurite degeneration was beginning which appeared to be partly due to toxic effects of the virus. However, replenishment with NGF supported further survival, showing that at this time the neurons were still viable. Similar rates of survival were obtained using a tsK-based PKAcat vector, but no significant survival was obtained with parental HSV or tsK virus strains. These data demonstrate the feasibility, and highlight some of the problems, of using HSV-based vectors as tools for expressing functional survival proteins in sympathetic neurons.     

Ultrastructural Localization of a Voltage-gated K+ Channel a Subunit (Kv1.2) in the Rat Cerebellum

A highly specific monoclonal antibody and pre-embedding immunocytochemistry were employed to examine the distribution of the K⁺ channel a subunit K_v1.2 in the rat cerebellum. At the light microscopic level, the heaviest immunoreactivity was seen in the basket cell pinceau at the base of Purkinje cells, with lighter staining of basket and Golgi cell bodies and a punctate pattern in the granule cell and molecular layers. Electron microscopy was performed to identify the ultrastructural location of K_v1.2 α subunit in these labelled structures. This revealed that the labelling of the pinceau was confined to the preterminal axonal plexus, the area immediately around the Purkinje axon initial segment being relatively devoid of staining. Basket cell parent axons were not immunostained, but gave rise to heavily stained fine processes. Immunoreactivity was also seen in myelinated axons in the granule cell layer and in the medial cerebellar nucleus, the staining being most concentrated at the juxtaparanodal regions of the axons. An unusual pattern of staining was seen in some mossy fibre terminals, with staining restricted to fine protuberances of mossy fibre glomeruli. Structures contacted by these protuberances included adjoining glial processes. Immunostaining was absent from Purkinje cell bodies, dendrites, their axon initial segments and their terminals in the medial cerebellar nucleus. In this study, the a subunit K_v1.2 was localized to a number of different cell types in the cerebellum. Each neuronal type displays a distinct subcellular distribution of the subunit.     

Expression of the Adrenoleukodystrophy Protein in the Human and Mouse Central Nervous System

The gene mutated in X-linked adrenoleukodystrophy (ALD), a progressive demyelinating disease, codes for a protein (ALDP) involved in very-long-chain fatty acid (VLCFA) transport. The expression of ALDP and of two peroxisomal enzymes involved in β-oxidation of VLCFA, acyl-CoA oxidase, and catalase was studied in human and mouse brain. The pattern of expression was similar in both species. While acyl-CoA oxidase and catalase are found in all types of CNS cells, including neurons and oligodendrocytes, ALDP expression is restricted mostly to the white matter and endothelial cells. ALDP is highly expressed in astrocytes and microglial cellsin vivoand in regenerating oligodendrocytesin vitro. In contrast,in vivo,ALDP is detected in much fewer oligodendrocytes and quantitative Western blot analysis confirmed the lower abundance of ALDP in these cells than in astrocytes. Only oligodendrocytes localized in corpus callosum, internal capsules, and anterior commissure express ALDP at levels comparable to those seen in astrocytes. In ALD, demyelination is first detected in these white matter regions, suggesting that the ALD gene mutation selectively affects those oligodendrocytes strongly expressing ALDP. Because of their failure to express ALDP, microglia and astrocytes may also contribute to demyelination in ALD patients.     

Differential modulation of I(h) by 5-HT receptors in mouse CA1 hippocampal neurons

CA1 pyramidal neurons of the hippocampus express various types of serotonin (5-HT) receptors, such as 5-HT(1A), 5-HT(4) and 5-HT(7) receptors, which couple to Galpha(i) or Galpha(s) proteins and operate on different intracellular signalling pathways. In the present paper we verify such differential serotonergic modulation for the hyperpolarization-activated current I(h). Activation of 5-HT(1A) receptors induced an augmentation of current-induced hyperpolarization responses, while the responses declined after 5-HT(4) receptors were activated. The resting potential of neurons hyperpolarized (-2.3 +/- 0.7 mV) after 5-HT(1A) receptor activation, activation of 5-HT(4) receptors depolarized neurons (+3.3 +/- 1.4 mV). Direct activation of adenylyl cyclase (AC) by forskolin also produced a depolarization. In voltage clamp, the Ih current was identified by its characteristic voltage- and time-dependency and by blockade with CsCl or ZD7288. Activation of 5-HT(1A) receptors reduced I(h) and shifted the activation curve to a more negative voltage by -5 mV at half-maximal activation. Activation of 5-HT(4) and 5-HT(7) receptors increased I(h) and shifted the activation curve to the right by +5 mV. Specific activation of 5-HT(4) receptors by BIMU8 increased membrane conductance and showed an increase in I(h) in a subset of cells, but did not induce a significant alteration in the activation curve. In order to verify spatial differences, we applied BIMU8 selectively to the soma and to the dendrites. Only somatic application induced receptor activation. These data are confirmed by immunofluorescence stainings with an antibody against the 5-HT(4) receptor, revealing receptor expression at the somata of the CA1 region. A similar expression pattern was found with a new antibody against 5-HT(7) receptors which reveals immunofluorescence staining on the cell bodies of pyramidal neurons.     

Neuromodulation of I(h) in layer II medial entorhinal cortex stellate cells: a voltage-clamp study

Stellate cells in layer II of medial entorhinal cortex (mEC) are endowed with a large hyperpolarization-activated cation current [h current (I(h))]. Recent work using in vivo recordings from awake behaving rodents demonstrate that I(h) plays a significant role in regulating the characteristic spatial periodicity of "grid cells" in mEC. A separate, yet related, line of research demonstrates that grid field spacing changes as a function of behavioral context. To understand the neural mechanism or mechanisms that could be underlying these changes in grid spacing, we have conducted voltage-clamp recordings of I(h) in layer II stellate cells. In particular, we have studied I(h) under the influence of several neuromodulators. The results demonstrate that I(h) amplitude can be both upregulated and downregulated through activation of distinct neuromodulators in mEC. Activation of muscarinic acetylcholine receptors produces a significant decrease in the I(h) tail current and a hyperpolarizing shift in the activation, whereas upregulation of cAMP through application of forskolin produces a significant increase in the I(h) amplitude and a depolarizing shift in I(h) activation curve. In addition, there was evidence of differential modulation of I(h) along the dorsal-ventral axis of mEC. Voltage-clamp protocols were also used to determine whether M current is present in stellate cells. In contrast to CA1 pyramidal neurons, which express M current, the data demonstrate that M current is not present in stellate cells. The results from this study provide key insights into a potential mechanism that could be underlying changes seen in grid field spacing during distinct behavioral contexts.     

Propofol block of I(h) contributes to the suppression of neuronal excitability and rhythmic burst firing in thalamocortical neurons

Although the depressant effects of the general anesthetic propofol on thalamocortical relay neurons clearly involve gamma-aminobutyric acid (GABA)(A) receptors, other mechanisms may be involved. The hyperpolarization-activated cation current (I(h)) regulates excitability and rhythmic firing in thalamocortical relay neurons in the ventrobasal (VB) complex of the thalamus. Here we investigated the effects of propofol on I(h)-related function in vitro and in vivo. In whole-cell current-clamp recordings from VB neurons in mouse (P23-35) brain slices, propofol markedly reduced the voltage sag and low-threshold rebound excitation that are characteristic of the activation of I(h). In whole-cell voltage-clamp recordings, propofol suppressed the I(h) conductance and slowed the kinetics of activation. The block of I(h) by propofol was associated with decreased regularity and frequency of delta-oscillations in VB neurons. The principal source of the I(h) current in these neurons is the hyperpolarization-activated cyclic nucleotide-gated (HCN) type 2 channel. In human embryonic kidney (HEK)293 cells expressing recombinant mouse HCN2 channels, propofol decreased I(h) and slowed the rate of channel activation. We also investigated whether propofol might have persistent effects on thalamic excitability in the mouse. Three hours following an injection of propofol sufficient to produce loss-of-righting reflex in mice (P35), I(h) was decreased, and this was accompanied by a corresponding decrease in HCN2 and HCN4 immunoreactivity in thalamocortical neurons in vivo. These results suggest that suppression of I(h) may contribute to the inhibition of thalamocortical activity during propofol anesthesia. Longer-term effects represent a novel form of propofol-mediated regulation of I(h).     

Characterisation of hyperpolarization-activated currents (I(h)) in the medial septum/diagonal band complex in the mouse

Hyperpolarization-activated cyclic nucleotide gated (HCN) channel subunits are distributed widely, but selectively, in the central nervous system, and underlie hyperpolarization-activated currents (I(h)) that contribute to rhythmicity in a variety of neurons. This study investigates, using current and voltage-clamp techniques in brain slices from young mice, the properties of I(h) currents in medial septum/diagonal band (MS/DB) neurons. Subsets of neurons in this complex, including GABAergic and cholinergic neurons, innervate the hippocampal formation, and play a role in modulating hippocampal theta rhythm. In support of a potential role for I(h) in regulating MS/DB firing properties and consequently hippocampal neuron rhythmicity, I(h) currents were present in around 60% of midline MS/DB complex neurons. The I(h) currents were sensitive to the selective blocker ZD7288 (10 microM). The I(h) current had a time constant of activation of around 220 ms (at -130 mV), and tail current analysis revealed a half-activation voltage of -98 mV. Notably, the amplitude and kinetics of I(h) currents in MS/DB neurons were insensitive to the cAMP membrane permeable analogue 8-bromo-cAMP (1 mM), and application of muscarine (100 microM). Immunofluoresence using antibodies against HCN1, 2 and 4 channel subunits revealed that all three HCN subunits are expressed in neurons in the MS/DB, including neurons that express the calcium binding protein parvalbumin (marker of fast spiking GABAergic septo-hippocampal projection neurons). The results demonstrate, for the first time, that specific HCN channel subunits are likely to be coexpressed in subsets of MS/DB neurons, and that the resultant I(h) currents show both similarities, and differences, to previously described I(h) currents in other CNS neurons.     

Tacrolimus (FK506) Increases Neuronal Expression of GAP-43 and Improves Functional Recovery after Spinal Cord Injury in Rats

Tacrolimus (FK506), a widely used immunosuppressant drug, has neurite-promoting activity in cultured PC12 cells and peripheral neurons. The present study investigated whether tacrolimus affects the expression of the neuronal growth-associated protein, GAP-43, as well as functional recovery after photothrombotic spinal cord injury in the rat. In injured animals receiving tacrolimus, the number of neurons expressing GAP-43 mRNA and protein approximately doubled compared to that in injured animals receiving vehicle alone. This increase in GAP-43-positive cells was paralleled by a significant improvement in neurological function evaluated by open-field and inclined plane tests. Another FKBP-12 ligand (V-10,367) had similar effects on GAP-43 expression and functional outcome, indicating that the observed effects of tacrolimus do not involve inhibition of the phosphatase calcineurin. Thus, tacrolimus, a drug which is already approved for use in humans, as well as other FKBP-12 ligands which do not inhibit calcineurin, could potentially enhance functional outcome after CNS injury in humans.