European Journal of Neuroscience

Published by Wiley

Online ISSN: 1460-9568

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Print ISSN: 0953-816X

Articles


Changes in the Expression of the Extracellular Matrix Molecules Tenascin-C and Tenascin-R after 3-Acetylpyridine-induced Lesion of the 01ivocerebe1lar System of the Adult Rat
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April 1997

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17 Reads

Eva Sabine Wintergerst

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In the central nervous system of rodents, the extracellular matrix glycoproteins tenascin-C and tenascin-R are expressed predominantly by astrocytes and oligodendrocytes respectively. Both molecules support neurite outgrowth from several neuronal cell types when presented as uniform substrates. When offered as a sharp boundary with a permissive substrate, however, both molecules prevent neurite elongation. On the basis of these observations it has been suggested that tenascin-C and tenascin-R may be relevant in determining the cellular response after injury in the adult rodent central nervous system. To investigate whether tenascin-C and tenascin-R may play important functional roles in the lesioned central nervous system, we have analysed their expression in the olivocerebellar system of the adult rat after 3-acetylpyridine-induced degeneration of nerve cells in the inferior olivary nucleus. Tenascin-C mRNA was not detectable at any time in the unlesioned or lesioned inferior olivary nucleus by in situ hybridization. In the cerebellar cortex, tenascin-C mRNA in Golgi epithelial cells was down-regulated 3 days after the lesion and returned to control values 80 days after the lesion. Tenascin-R mRNA was expressed by distinct neural cell types in the unlesioned olivocerebellar system. After a lesion, the density of cells containing tenascin-R transcripts increased significantly in the inferior olivary nucleus and in the white matter of the cerebellar cortex. Immunohistochemical and immunochemical investigations confirmed these observations at the protein level. Our data thus suggest differential functions of tenascin-C and tenascin-R in the injured central nervous system.
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Long-term reduction of brain-derived neurotrophic factor levels and signaling impairment following prenatal treatment with the cannabinoid receptor 1 receptor agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinyl-methyl) pyrrolo[1,2,3-de]-1,4-benzoxazin-

July 2007

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70 Reads

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It is well accepted that adverse life events occurring early in development may alter the correct program of brain maturation leading to enhanced vulnerability to neuropsychiatric disorders. It has recently been demonstrated that prenatal exposure to the cannabinoid receptor 1 agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinyl-methyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN 55,212-2) produces memory deficit in adulthood, an effect associated with a reduced functionality of the glutamatergic system. The aim of our study was to identify molecular changes produced by prenatal exposure to WIN 55,212-2 that might contribute to late disruption in synaptic plasticity and cognition. For this purpose, WIN 55,212-2 was injected in pregnant wistar rats from gestation day 5 to 20 and a detailed analysis of the levels of the neurotrophin brain-derived neurotrophic factor (BDNF) as well as of the signaling molecules extracellular signal-regulated kinase (ERK)1/2 and alpha-calcium/calmodulin-dependent protein kinase II (alpha-CaMKII) was carried out in adult offspring. We found that exposure to WIN 55,212-2 significantly reduced BDNF levels in hippocampus and frontal cortex. This effect was associated with decreased activation of pathways linked to neurotrophin and glutamate receptor signaling. In particular, prenatal cannabinoid treatment reduced the phosphorylated levels of ERK1/2 in selected subcellular compartments of hippocampus, frontal and prefrontal cortex, whereas no changes were observed in the total levels of these proteins. Furthermore, a robust reduction of total and phospho-alpha-CaMKII was found in the hippocampus of rats prenatally exposed to WIN 55,212-2. In summary, the present data suggest that deficits of BDNF levels and signaling through ERK1/2 and alpha-CaMKII might contribute to cognitive and neuroplastic defects associated with prenatal exposure to cannabinoids.

EGb761 protects against nigrostriatal dopaminergic neurotoxicity in 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine-induced Parkinsonism in mice: Role of oxidative stress

August 2008

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50 Reads

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes nigrostriatal dopaminergic neurotoxicity and behavioral impairment in rodents. Previous studies suggest that oxidative stress, via free radical production, is involved in MPTP-induced neurotoxicity. The MPTP-treated mouse has been the most widely used model for assessing neuroprotective agents for Parkinson's disease. It has been reported previously that EGb761 prevents dopaminergic neurotoxicity of MPTP. This compound is multifunctional via different mechanisms. Here, we report the neuroprotective effect of EGb761 against oxidative stress induced by MPTP in C57BL/6J mice. EGb761 is a patented and well-defined mixture of active compounds extracted from Ginkgo biloba leaves, with neuroprotective effects, exerted probably via its antioxidant or free radical scavenger action. MPTP administration resulted in a significant decrease in striatal dopamine levels and tyrosine hydroxylase immunostaining in the striatum and substantia nigra pars compacta. Mice receiving EGb761 had significantly attenuated MPTP-induced loss of striatal dopamine levels and tyrosine hydroxylase immunostaining in the striatum and substantia nigra pars compacta. The neuroprotective effect of EGb761 against MPTP neurotoxicity is associated with blockade of lipid peroxidation and reduction of superoxide radical production (indicated by a down-regulation of Mn-superoxide dismutase activity), both of which are indices of oxidative stress. Behavioral analyses showed that EGb761 improved MPTP-induced impairment of locomotion in a manner that correlated with enhancement of striatal dopamine levels. These findings suggest that, in mice, EGb761 attenuates MPTP-induced neurodegeneration of the nigrostriatal pathway and that an inhibitory effect against oxidative stress may be partly responsible for its observed neuroprotective effects.

Some Actions of 9-Amino-1,2,3,4-Tetrahydroacridine (THA) on Cholinergic Transmission and Membrane Currents in Rat Sympathetic Ganglia

February 1990

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12 Reads

THA (Tacrine) is an anticholinesterase drug reported to alleviate cognitive deficit in Alzheimer's disease. We have used rat isolated superior cervical sympathetic ganglia as a model mammalian cholinergic neural system to study effects of THA on cholinergic synaptic transmission and postsynaptic membrane currents. At 0.1 - 3 microM, THA augmented the postsynaptic depolarizations and inward clamp currents produced by acetylcholine but not by the cholinesterase-resistant analogue, DMPP. Higher concentrations depressed these responses to both acetylcholine and DMPP, and reduced the acetylcholine-induced increase in membrane current noise. At 1 microM, THA did not affect the amplitude or time-course of fast (nicotinic) excitatory postsynaptic currents (epscs) evoked by single orthodromic volleys, but higher concentrations induced a biphasic epsc decay. In contrast, low concentrations of THA (1 - 3 microM) greatly augmented and prolonged the muscarinic slow epsc evoked by repetitive orthodromic volleys: this effect was blocked by 1 microM atropine. Concentrations above 0.1 mM produced a membrane depolarization and inhibited a variety of membrane ionic currents, including voltage-gated Ca current and subsequent Ca-activated K currents, and voltage-gated M- and A-type K currents. It is concluded that the principal effect of THA is to inhibit cholinesterase, and that the main consequence of this is to augment and prolong the muscarinic slow epsc. In contrast, the nicotinic fast epsc is not increased but instead may be reduced through a nicotinic channel-blocking action. Although THA could also block several other ion channels the concentrations required were too high to contribute significantly to its principal pharmacological actions on ganglionic transmission.

Use of catechol- O -methyltransferase inhibition to minimize L-3,4-dihydroxyphenylalanine-induced dyskinesia in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned macaque

January 2013

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20 Reads

L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesia is a complication of dopaminergic treatment in Parkinson's disease. Lowering the L-DOPA dose reduces dyskinesia but also reduces the antiparkinsonian benefit. A therapy that could enhance the antiparkinsonian action of low-dose L-DOPA (LDl) without exacerbating dyskinesia would thus be of considerable therapeutic benefit. This study assessed whether catechol-O-methyltransferase (COMT) inhibition, as an add-on to LDl, might be a means to achieve this goal. Cynomolgus macaques were administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Dyskinesia was established by chronic treatment with L-DOPA. Two doses of L-DOPA were identified - high-dose L-DOPA (LDh), which provided good antiparkinsonian benefit but was compromised by disabling dyskinesia, and LDl, which was sub-threshold for providing significant antiparkinsonian benefit, without dyskinesia. LDh and LDl were administered in acute challenges in combination with vehicle and, for LDl, with the COMT inhibitor entacapone (5, 15 and 45 mg/kg). The duration of antiparkinsonian benefit (ON-time), parkinsonism and dyskinesia were determined. The ON-time after LDh was ∼170 min and the ON-time after LDl alone (∼98 min) was not significantly different to vehicle (∼37 min). In combination with LDl, entacapone significantly increased the ON-time (5, 15 and 45 mg/kg being ∼123, ∼148 and ∼180 min, respectively). The ON-time after LDl/entacapone 45 mg/kg was not different to that after LDh. However, whereas the percentage ON-time that was compromised by disabling dyskinesia was ∼56% with LDh, it was only ∼31% with LDl/entacapone 45 mg/kg. In addition to the well-recognized action of COMT inhibition to reduce wearing-OFF, the data presented suggest that COMT inhibition in combination with low doses of L-DOPA has potential as a strategy to alleviate dyskinesia.

Striatal leucine-rich repeat kinase 2 mRNA is increased in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned common marmosets (Callithrix jacchus) with L-3, 4-dihydroxyphenylalanine methyl ester-induced dyskinesia

August 2007

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135 Reads

The level of leucine-rich repeat kinase 2 (Lrrk2) mRNA expression was measured by reverse transcription-polymerase chain reaction in anterior striatum from normal and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated common marmosets (Callithrix jacchus) that had L-3,4-dihydroxyphenylalanine methyl ester (L-DOPA)-induced dyskinesia. The level of striatal Lrrk2 mRNA was increased in MPTP-treated common marmosets that had L-DOPA-induced dyskinesia compared with normal animals that did not receive l-DOPA. Marmosets that exhibited higher levels of dyskinesia had the greatest increase in striatal Lrrk2 mRNA. Lrrk2 mRNA expression was also measured in human striatum and substantia nigra from control subjects and patients dying with Parkinson's disease. In contrast to marmoset tissue, no alteration in Lrrk2 mRNA expression was found in parkinsonian human brain. However, the brain was from patients who had an overall low level of dyskinesia. The correlation between striatal Lrrk2 mRNA levels in MPTP-treated common marmoset striatum and L-DOPA-induced dyskinesia indicates that LRRK2 may have a role in the molecular alterations that cause L-DOPA-induced dyskinesia.

Granulocyte-macrophage colony-stimulating factor promotes survival of dopaminergic neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced murine Parkinson's disease model

March 2009

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37 Reads

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic cytokine that has the potential for clinical application. The biological effects of GM-CSF have been well characterized, and include stimulation of bone marrow hematopoietic stem cell proliferation and inhibition of apoptosis of hematopoietic cells. In contrast, the therapeutic effects of GM-CSF on the central nervous system in acute injury such as stroke and spinal cord injury have been reported only recently. To better understand the protective effect of GM-CSF on dopaminergic neurons in Parkinson's disease (PD), we investigated the effect of GM-CSF on the survival of dopamine neurons and changes in locomotor behavior in a murine PD model. We investigated the neuroprotective effects of GM-CSF in 1-methyl-4-phenylpyridinium (MPP+)-treated PC12 cells as well as in embryonic mouse primary mesencephalic neurons (PMNs) in vitro. To investigate the role of GM-CSF in vivo, we prepared a mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) PD model, and examined the effects of GM-CSF on dopaminergic neuron survival in the substantia nigra and on locomotor behavior. Treatment with GM-CSF significantly reduced MPP+-induced dopaminergic cell death in PC12 cells and PMNs in vitro. GM-CSF modulated the expression of apoptosis-related proteins, Bcl-2 and Bax, in vitro. Furthermore, administration of GM-CSF (50 microg/kg body weight/day) in vivo for 7 days protected dopaminergic neurons in the substantia nigra and improved locomotor behavior in a mouse MPTP model of PD.

Unilateral pallidotomy in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated common marmosets exhibiting levodopa-induced dyskinesia

October 2005

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18 Reads

Pallidotomy paradoxically reduces the intensity of levodopa-induced dyskinesia without worsening motor symptoms. The reasons for this are not clear and no experimental study has investigated this phenomenon. The objective of this investigation was to evaluate the effects of unilateral pallidotomy on locomotor activity, motor disability and levodopa-induced dyskinesia in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated levodopa-primed common marmosets. Animals were primed to exhibit dyskinesia by daily administration of levodopa until stable dyskinesia was evoked by each dose. Locomotor activity, motor disability and dyskinesia were assessed weekly at baseline and following an acute levodopa challenge. Prior to pallidotomies, two distinct groups of animals emerged: poor responders to levodopa with mild dyskinesia (Group 1) and those exhibiting a marked increase in motor activity and pronounced dyskinesia (Group 2). Electrolytic lesions were placed in the left internal segment of the globus pallidus. Pallidotomy had no effect on basal or levodopa-induced motor activity in either group but significantly improved basal motor disability in Group 2. Following pallidotomy, the ability of levodopa to reduce motor disability was significantly increased in both groups. Pallidotomy improved dyskinesia in both Groups 1 and 2 but it was more effective in reducing dystonia compared with chorea. The effect of pallidotomy on dyskinesia in Group 2 was transient, with the intensity of involuntary movements reverting to presurgery levels 4 weeks later. This study shows that in levodopa-primed, parkinsonian marmosets, placement of discrete globus pallidus lesions can ameliorate levodopa-induced dyskinesia but not akinesia. This model allows the evaluation of pallidotomy-induced biochemical changes in dyskinetic primates.

Adaptive acetylcholinesterase splicing patterns attenuate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism in mice

July 2006

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23 Reads

Balanced dopaminergic cholinergic interactions are crucial for proper basal ganglia function. This is dramatically demonstrated by the worsening of Parkinson's disease symptoms following acetylcholinesterase (AChE) inhibition. Typically, in the brain, the synapse-anchored synaptic AChE (AChE-S) variant is prevalent whereas the soluble readthrough AChE (AChE-R) variant is induced in response to cholinesterase inhibition or stress. Because of the known functional differences between these variants and the fact that AChE-R expression is triggered by various stimuli that themselves are often associated with Parkinson's disease risk, we hypothesized that the splice shift to AChE-R plays a functional role in Parkinsonian progression. After establishing that Paraoxon-induced AChE inhibition indeed aggravates experimental Parkinsonism triggered by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice, we tested the roles of individual AChE variants by exposing transgenic mice overexpressing either the AChE-S or AChE-R variant to MPTP. Differential reductions of tyrosine hydroxylase levels in the striatum and substantia nigra indicated that transgenic AChE-R expression confers resistance as compared with the parent FVB/N strain. In contrast, AChE-S overexpression accelerated the MPTP-induced damage. Survival, behavioral measures and plasma corticosterone levels were also compatible with the extent of the dopaminergic damage. Our findings highlight the functional differences between individual AChE variants and indicate that a naturally occurring stress or AChE inhibitor-induced splicing shift can act to minimize dopaminergic cholinergic imbalances. We propose that inherited or acquired alternative splicing deficits could accelerate Parkinsonism and that, correspondingly, adaptive alternative splicing events may attenuate disease progression.

Excitatory Actions of the Metabotropic Excitatory Amino Acid Receptor Agonist, trans-(+-)-1-amino-cyclopentane-1,3-dicarboxylate (t-ACPD), on Rat Thalamic Neurons In Vivo

November 1991

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11 Reads

The metabotropic excitatory amino acid receptor agonist trans-(+/-)-1-amino-cyclopentane-1,3-dicarboxylate (t-ACPD) was applied to rat ventrobasal thalamic neurons by iontophoresis. This agonist typically evoked an excitatory response which was slower in onset and of longer duration than responses to the other excitatory amino acid agonists, N-methyl-aspartate, kainate or (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. Responses to t-ACPD were resistant to the excitatory amino acid antagonists 6-cyano-7-nitroquinoxaline-2,3-dione, 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid and kynurenate. These results suggest that t-ACPD may exert its effects via the so-called 'metabotropic' excitatory amino acid receptor. The putative antagonists at this receptor, d-2-amino-4-phosphono-butyrate (d-AP4), l-2-amino-4-phosphono-butyrate (l-AP4) and l-2-amino-3-phosphono-propionate (l-AP3), were able to reduce responses to t-ACPD under certain circumstances. However, such antagonism was always accompanied by similar reductions in excitatory responses to other agonists. These non-selective effects would appear to limit the usefulness of AP4 and AP3 as antagonists of t-ACPD.

Expression and subcellular localization of p42IP4/centaurin-alpha, a brain-specific, high-affinity receptor for inositol 1,3,4,5-tetrakisphosphate and phosphatidylinositol 3,4,5-trisphosphate in rat brain

November 1997

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42 Reads

Recently emerging evidence suggests important roles for inositol polyphosphates and inositol phospholipids in neuronal Ca2+ signalling, membrane vesicle trafficking and cytoskeletal rearrangement. A prerequisite for a detailed physiological characterization of the signalling of both potential second messengers inositol-(1,3,4,5)-tetrakisphosphate (InsP4) and phosphatidylinositol-3,4,5-trisphosphate (PtdInsP3) in the nervous system is the precise cellular localization of their receptors. Based on the cDNA sequence of a recently cloned brain-specific receptor with high affinity for both InsP4 and PtdInsP3 (InsP4-PtdInsP3R), p42IP4/centaurin-alpha, we localized the mRNA and the protein in rat brain. In situ hybridization revealed a widespread expression of the InsP4-PtdInsP3R with prominent labelling in cerebellum, hippocampus, cortex and thalamus, which moreover is developmentally regulated. Using peptide-specific antibodies, the immunoreactivity was localized in the adult brain in the vast majority of neuronal cell types and probably also in some glial cells. Prominent immunoreactivity was found in axonal processes and in cell types characterized by extensive neurites. In the hypothalamus a subpopulation of parvocellular neurons in the peri- and paraventricular nuclei was most heavily labelled. This was confined by strong immunoreactivity in the lamina externa of the median eminence in close proximity to portal plexus blood vessels. Electron microscopy revealed that the InsP4-PtdInsP3R was frequently associated with presynaptic vesicular structures. Further studies should identify the role of the InsP4-PtdInsP3R in cellular neural processes.

FIG. 1. Respective autoradiographic distribution of [3H]1,4,5-IP3 (left column), [3H]1,3,4,5-IP, (middle column) and [3H]IP6 (right column) binding sites in coronal sections of the rat brain. The pH conditions used in this study are shown at the top while autoradiograms at the level of hippocampal formation obtained at the other pH tested are shown at the bottom. It is clear that [3H]1,4,5-IP3 binding sites are abundant at pH 8.5 while [3H]t,3,4,5-IP4 binding is more prominent at pH 5.0. Each class of specific binding site is discretely distributed; highly enriched areas especially include the hippocampal formation and the cerebellum.
FIG. 2. Quantitative analysis of the distribution of [3H]1,4,5-IP3, [3H]1 ,3,4,5-IP4 and [3H]rP6 binding in the rat brain. Filled bars represent values obtained at basic pH while shipped bars relate to data obtained at acidic pH. For [3H]IP6 binding, values obtained at both pH were significantly different (P < 0.05) only in a few regions. For 1,4,5-IP3 and 1,3,4,5-IP4 binding assays performed at the other pH investigated failed to generate reproducible specific binding. Accordingly, only results obtained with optimum pH values are presented. The ratio (% binding) of 1,4,5-1P3/1 ,3,4,5-IP4 values obtained at their optimal pH is shown for comparison.
Differential Localization and pH Dependency of Phosphoinositide 1,4,5‐IP3, 1,3,4,5‐IP4 and IP6 Receptors in Rat and Human Brains
  • Article
  • Full-text available

February 1994

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36 Reads

It is well established that the inositol lipids mediate signal transduction in several cellular populations. Many neurotransmitters, hormones and growth factors act at plasma membrane receptors to induce the hydrolysis of phosphatidylinositols and hence the generation of various inositol phosphates (IP). The best known member of this family is 1,4,5-IP3, which is associated with the release of Ca2+ from intracellular pools. It has also been proposed that two others inositides, 1,3,4,5-IP4 and IP6, may be involved in Ca2+ homeostasis. In order to study the possible relevance of these various inositides in neuronal tissues, we have localized the respective receptors in rat and human brain under both acidic and basic pH conditions. In the hippocampal formation, [3H]1,3,4,5-IP4 binding sites are concentrated in the hilus and the molecular layer while a clearly different pattern of distribution is seen for [3H]1,4,5-IP3, its highest concentration of labelling being concentrated in the oriens and radiatum laminae. This contrasting profile of distribution is also observed in other brain areas such as the caudate-putamen, the septo-hippocampal area, and the molecular and granular layers of the cerebellum. Moreover, while highest amounts of specific [3H]1,4,5-IP3 binding are obtained at pH 8.5, the opposite is found for [3H]1,3,4,5-IP4, with high binding levels seen under acidic conditions. [3H]IP6 binding sites are broadly distributed with specific labelling concentrated in areas enriched with neuronal perikarya such as the granular cell layer of the dentate gyrus, the pyramidal cell layers of the hippocampus and the granular cell layer of the cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)
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Endothelin Induces a Rise of Inositol 1,4,5-Trisphosphate, Inositol 1,3,4,5-Tetrakisphosphate Levels and of Cytosolic Ca2+ Activity in Neural Cell Lines

February 1990

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6 Reads

The mechanism of action of the vasoconstricting peptide endothelin was investigated in two neural cell lines. In rat glioma cells endothelin-1 caused a biphasic rise in cytosolic Ca2+ activity. A large peak of 40 s duration was followed by another, however smaller, transient rise of comparable duration. In the absence of extracellular Ca2+ only the first peak was detected. Pretreatment with Ca2+ ionophores suppressed the Ca2+ response to endothelin. At the concentrations used the Ca2+ ionophores primarily deplete internal Ca2+ stores and prevent their refilling. Measurements of 45Ca2+ fluxes corroborate the conclusion that in the glioma cells endothelin induces firstly a release of Ca2+ from internal stores and subsequently a stimulation of Ca2+ entry. In neuronal cells (mouse neuroblastoma x rat glioma hybrid cells), endothelin caused a monophasic rise in cytosolic Ca2+ activity, most likely due to release from internal stores. In the glioma cells the concentrations of both inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate were raised about 2.5-fold for ca. 90 s after addition of endothelin. In the neuronal cells a shorter, smaller rise in inositololigophosphate concentrations was induced. Thus, endothelin seems to act as a neuropeptide activating phospholipase C and intracellular Ca2+.

Lack of effects of GHB precursors GBL and 1,4-BD following i.c.v. administration in rats

December 2006

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61 Reads

Gamma-hydroxybutyrate (GHB) is used therapeutically and recreationally worldwide. Since the scheduling of GHB by the USA and the United Nations in 2000-2001, the recreational use of GHB precursors has reportedly increased. The aim of this study was to examine if potency differences of GHB and GHB-like compounds are due to their blood-brain barrier permeability. The effects of peripheral and central administration of GHB, GHB precursors gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD), and the gamma-aminobutyric acid (GABA)(B) receptor agonist baclofen on schedule-controlled responding were examined in rats. GHB and baclofen were 276- and 253-fold more potent, respectively, after intracerebroventricular (i.c.v.) administration than after intraperitoneal (i.p.) administration, whereas GBL and 1,4-BD, up to a dose of 1780 microg were without effect after i.c.v. administration. These data suggest that GBL and 1,4-BD are not metabolically converted to GHB in the brain, that enhanced brain penetration cannot account for potency differences between compounds, and that baclofen, like GHB, can readily cross the blood-brain barrier.

Regional Variation in Expression of Calbindin and Inositol 1,4,5‐trisphosphate Receptor Type 1 mRNAs in the Cerebellum of the Staggerer Mutant Mouse

August 1996

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13 Reads

The Purkinje cells in the staggerer mutant mouse have various cellular abnormalities, including reduced cell number, ectopia, smaller size and absence of dendritic spines. It is also know that some of these abnormalities exhibit regional variations in the cerebellum. In this paper we have investigated expression in the staggerer Purkinje cells of the calbindin and inositol 1,4, 5-trisphosphate receptor type 1 mRNAs by in situ hybridization. Although the transcription levels of both mRNAs were significantly reduced compared with the wild-type cells, the reduction among the Purkinje cell populations was not even, varying greatly from region to region. Purkinje cells with different transcription levels were distributed in discrete regions and arranged alternately in the mediolateral direction. Moreover, the cell bodies with higher transcription levels were larger in size and aligned in a monolayer between the granular and molecular layers, whereas those with lower levels were smaller in size, fewer in number and dispersed throughout the granular layer. These findings suggest that there is a distinct mediolateral heterogeneity in the staggerer cerebellum with respect to transcription levels of these Purkinje cell-specific molecules, which might correlate with some cytological phenotypes.

Figure 1: Increase in InsP3 levels 10 s after agonist addition. Cerebellar granule cells were preincubated in extracellular buffer (see Materials and methods) prior to addition of 1 mm carbachol (CCh), 1 mm histamine (His), 0.5 mm noradrenaline (Nor) or 1 mm 1S,3R ACPD (ACPD). Hatched bars represent the responses of resting cells, while solid bars show the responses of cells depolarized with 20 mm KCl 5 min before addition of the agonists. Data are expressed as mean ± SEM obtained from 106 cells. *P < 0.01, Significantly higher than control, anova.
Figure 2: Intracellular Ca2+ release in resting and depolarized CGCs. Changes in Ca2+i in resting (A and B) and 20 mm KCl-depolarized (C–F) CGCs stimulated with 1 mm carbachol (CCh) (A and C), 1 mm histamine (His) (B and D), 1 mm 1S,3R ACPD (E) or 0.5 mm noradrenaline (Nor) (F). Fura-2 loading conditions and cytoplasmic Ca2+ calibration are described in Materials and methods. Each plot contains the traces of 15–20 individual CGC somata present in the field of a representative experiment.
Figure 3: Frequency distribution of Ca2+-release responses in resting and depolarized CGCs. Changes in Ca2+i in CGCs stimulated with 1 mm carbachol (A), 1 mm histamine (B), 1 mm 1S,3R ACPD (C) or 0.5 mm noradrenaline (D). Hatched bars represent the increase in Ca2+i in resting CGCs while solid bars display the increase in Ca2+i in CGCs previously depolarized with 20 mm KCl. Data correspond to 150–200 individual CGCs in no less than six independent experiments from at least three different tissue culture preparations. KCl-depolarization caused a significant (P < 0.001, by anova) increase in the magnitude of the Ca2+-release responses to all agonists except noradrenaline.
Differential coupling of G-protein-linked receptors to Ca2+ mobilization through inositol(1,4,5)trisphosphate or ryanodine receptors in cerebellar granule cells in primary culture

October 1999

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26 Reads

Rat cerebellar granule cells in primary culture possess muscarinic, metabotropic glutamatergic, histaminergic and alpha-adrenergic receptors which couple to phosphoinositide-specific phospholipase C. We have determined the ability of these receptors to elevate inositol(1,4,5)trisphosphate and to release intracellular calcium, in order to establish the correlation between these two responses. In resting cerebellar granule cells, only the muscarinic agonist carbachol evoked significant increases in both inositol(1,4, 5)trisphosphate and cytoplasmic free Ca2+. Mild depolarization (20 mM KCl) enhanced inositol(1,4,5)trisphosphate elevation by carbachol and histamine, but not by noradrenaline or the metabotropic glutamate agonist 1S,3R ACPD. In contrast, Ca2+-release responses were modified differently by 20 mM KCl-depolarization: the responses to carbachol, histamine and 1S,3R ACPD, but not the responses to noradrenaline, were markedly enhanced. The contribution of ryanodine-sensitive Ca2+-release channels (ryanodine receptors) to the calcium release signal in depolarized cells was determined. Ryanodine (10 microM) inhibited most effectively the cytoplasmic Ca2+ elevation evoked by 1S,3R ACPD (> 90%), while Ca2+ release upon stimulation by carbachol and histamine was only inhibited by approximately 60% and remained larger than in the absence of KCl. Our data are consistent with a specific coupling between metabotropic glutamate receptors and ryanodine-sensitive Ca2+-release channels which may not require generation of inositol(1, 4,5)trisphosphate.

Inositol 1,4,5-trisphosphate activates non-selective cation conductance via intracellular Ca2+ increase in isolated frog taste cells

May 1998

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5 Reads

The effect of intracellular Ca2+ increase was analysed in isolated frog taste cells under the whole-cell patch clamp. External application of a Ca2+-ionophore, ionomycin (3 microM) induced the sustained inward current of -200+/-17 pA (mean +/- SE, n = 23) at -50 mV in taste cells. The ionomycin-induced response was observed in most of the cells exposed in the drug, but not when 10 mM BAPTA (1,2-bis (O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) was included in the pipette (eight cells). Steady-state I-V relationships of ionomycin-induced currents were almost linear and reversed at -8+/-1 mV (n = 23). The simultaneous removal of Na+ and Ca2+ from the external solution eliminated the response completely (three cells). Intracellular dialysis with 1 mM Ca2+ or 50 microM inositol 1,4,5-trisphosphate (IP3) in K+-internal solution also induced an inward current in the taste cells. The Ca2+-induced and IP3-induced responses were observed in 82% and 36% of the cells dialysed with the drugs, respectively. The Ca2+-induced and IP3-induced currents were inhibited by external Cd2+ (1-2 mM). The reversal potentials of the inward currents were -15+/-3 mV (n = 9) in Ca2+ dialysis and -11+/-3 mV (n = 13) in IP3 dialysis. The half-maximal Ca2+ concentration in the pipette to induce the inward current was approximately 170 microM. The results suggest that IP3 can depolarize the taste cell with mediation by intracellular Ca2+.

Basic properties of an inositol 1,4,5-trisphosphate-gated channel in carp olfactory cilia

September 2000

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11 Reads

In addition to the activation of cAMP-dependent pathways, odorant binding to its receptor can lead to inositol 1,4,5-trisphosphate (InsP3) production that may induce the opening of plasma membrane channels. We therefore investigated the presence and nature of such channels in carp olfactory cilia. Functional analysis was performed by reconstitution of the olfactory cilia in planar lipid bilayers (tip-dip method). In the presence of InsP3 (10 microM) and Ca2+ (100 nM), a current of 1.6 +/- 0.1 pA (mean +/- SEM, n = 4) was measured, using Ba2+ as charge carrier. The I/V curve displayed a slope conductance of 45 +/- 5 pS and a reversal potential of -29 mV indicating a higher selectivity for divalent cations. This current was characterized by two mean open times (3.0 +/- 0.4 ms and 42.0 +/- 2.6 ms, n = 4) and was strongly inhibited by ruthenium red (30 microM) or heparin (10 microg/mL). Importantly, the channel activity was closely dependent on the Ca2+ concentration, with the highest open probability (Po) at 100 nM Ca2+ (Po = 0.50 +/- 0.02, n = 4). Po is lower at both higher and lower Ca2+ concentrations. A structural identification of the channel was attempted by using a large panel of antibodies, raised against several InsP3 receptor (InsP3R)/Ca2+ release channel isoforms. The type 1 InsP3R was detected in carp cerebellum and whole brain, while a lower molecular mass InsP3R, which may correspond to type 2 or 3, was detected in heart, whole brain and the soma of the olfactory neurons. None of the antibodies, however, cross-reacted with olfactory cilia. Taken together, these results indicate that in carp olfactory cilia an InsP3-dependent channel is present, distinct from the classical InsP3Rs localized on intracellular membranes.

HAP1 facilitates effects of mutant huntingtin on inositol 1,4,5‐trisphosphate‐induced Ca2+ release in primary culture of striatal medium spiny neurons

November 2004

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68 Reads

Huntington's disease is caused by polyglutamine expansion (exp) in huntingtin (Htt). Htt-associated protein-1 (HAP1) was the first identified Htt-binding partner. The type 1 inositol (1,4,5)-trisphosphate receptor (InsP3R1) is an intracellular Ca2+ release channel that plays an important role in neuronal function. Recently, we identified a InsP3R1-HAP1A-Htt ternary complex in the brain and demonstrated that Httexp, but not normal Htt, activates InsP3R1 in bilayers and facilitates InsP3R1-mediated intracellular Ca2+ release in medium spiny striatal neurons [MSN; T.-S. Tang et al. (2003) Neuron, 39, 227-239]. Here we took advantage of mice with targeted disruption of both HAP1 alleles (HAP1 -/-) to investigate the role of HAP1 in functional interactions between Htt and InsP3R1. We determined that: (i) HAP1 is expressed in the MSN; (ii) HAP1A facilitates functional effects of Htt and Htt(exp) on InsP3R1 in planar lipid bilayers; (iii) HAP1 is required for changes in MSN basal Ca2+ levels resulting from Htt or Htt(exp) overexpression; (iv) HAP1 facilitates potentiation of InsP3R1-mediated Ca2+ release by Htt(exp) in mouse MSN. Our present results indicate that HAP1 plays an important role in functional interactions between Htt and InsP3R1.

Inositol 1,4,5‐trisphosphate and adenophostin analogues induce responses in turtle olfactory sensory neurons

March 2000

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11 Reads

Using the whole-cell mode of the patch-clamp technique, we recorded inward currents in response to inositol-1,4,5-trisphosphate (IP3) and adenophostin analogues in turtle olfactory sensory neurons. Dialysis of IP3 into the neurons induced inward currents with an increase in membrane conductance in a dose-dependent manner under the voltage-clamp conditions (holding potential -70 mV). The application of Ca2+-free Ringer solution to neurons previously dialysed with IP3 induced inward currents that were reversibly inhibited by application of Na+, Ca2+-free Ringer solution, normal Ringer solution or 10 microM ruthenium red. Dialysis of the adenophostin analogues, novel IP3 receptor ligands, also induced inward currents with an increase in membrane conductance. The magnitude of the responses to the adenophostin analogues varied among these derivatives. The application of Ca2+-free Ringer solution to neurons previously dialysed with the adenophostin analogues induced inward currents that were inhibited by the application of normal Ringer solution. The reversal potential of inward currents induced by an adenophostin analogue was similar to that induced by IP3, suggesting that inward currents induced by the adenophostin analogue were generated by a similar ionic mechanism to that induced by IP3. The present study demonstrated that IP3-mediated transduction pathways exist in turtle olfactory receptor neurons and that adenophostin analogues act as agonists of IP3.

1-Aminoindan-1,5-dicarboxylic acid and (S)-(+)-2-(3′-carboxybicyclo[1.1.1] pentyl)-glycine, two mGlu1 receptor-preferring antagonists, reduce neuronal death in in vitro and in vivo models of cerebral ischaemia

November 1999

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15 Reads

Metabotropic glutamate (mGlu) receptors have been implicated in a number of physiological and pathological responses to glutamate, but the exact role of group I mGlu receptors in causing postischaemic injury is not yet clear. In this study, we examined whether the recently-characterized and relatively selective mGlu1 receptor antagonists 1-aminoindan-1,5-dicarboxylic acid (AIDA) and (S)-(+)-2-(3'-carboxybicyclo[1.1.1]pentyl)-glycine (CBPG) could reduce neuronal death in vitro, following oxygen-glucose deprivation (OGD) in murine cortical cell and rat organotypic hippocampal cultures, and in vivo, after global ischaemia in gerbils. When present in the incubation medium during the OGD insult and the subsequent 24 h recovery period, AIDA and CBPG significantly reduced neuronal death in vitro. The extent of protection was similar to that observed with the nonselective mGlu receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine [(+)MCPG] and with typical ionotropic glutamate (iGlu) receptor antagonists. Neuroprotection was also observed when AIDA or CBPG were added only after the OGD insult was terminated. Neuronal injury was not attenuated by the inactive isomer (-)MCPG, but was significantly enhanced by the nonselective mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid [(1S,3R)-ACPD] and the group I mGlu receptor agonist 3,5-dihydroxyphenylglycine (3,5-DHPG). The antagonists (+)MCPG, AIDA and CBPG were also neuroprotective in vivo, because i. c.v. administration reduced CA1 pyramidal cell degeneration examined 7 days following transient carotid occlusion in gerbils. Our results point to a role of mGlu1 receptors in the pathological mechanisms responsible for postischaemic neuronal death and propose a new target for neuroprotection.

Episodic ataxia type 1 mutations in the KCNA1 gene impair the fast inactivation properties of the human potassium channels Kv1.4-1.1/Kvβ1.1 and Kv1.4-1.1/Kvβ1.2

January 2007

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110 Reads

Episodic ataxia type 1 (EA1) is an autosomal dominant neurological disorder characterized by constant muscle rippling movements (myokymia) and episodic attacks of ataxia. Several heterozygous point mutations have been found in the coding sequence of the voltage-gated potassium channel gene KCNA1 (hKv1.1), which alter the delayed-rectifier function of the channel. Shaker-like channels of different cell types may be formed by unique hetero-oligomeric complexes comprising Kv1.1, Kv1.4 and Kvbeta1.x subunits. Here we show that the human Kvbeta1.1 and Kvbeta1.2 subunits modulated the functional properties of tandemly linked Kv1.4-1.1 wild-type channels expressed in Xenopus laevis oocytes by (i) increasing the rate and amount of N-type inactivation, (ii) slowing the recovery rate from inactivation, (iii) accelerating the cumulative inactivation of the channel and (iv) negatively shifting the voltage dependence of inactivation. To date, the role of the human Kv1.4-1.1, Kv1.4-1.1/Kvbeta1.1 and Kv1.4-1.1/Kvbeta1.2 channels in the aetiopathogenesis of EA1 has not been investigated. Here we also show that the EA1 mutations E325D, V404I and V408A, which line the ion-conducting pore, and I177N, which resides within the S1 segment, alter the fast inactivation and repriming properties of the channels by decreasing both the rate and degree of N-type inactivation and by accelerating the recovery from fast inactivation. Furthermore, the E325D, V404I and I177N mutations shifted the voltage dependence of the steady-state inactivation to more positive potentials. The results demonstrate that the human Kvbeta1.1 and Kvbeta1.2 subunits regulate the proportion of wild-type Kv1.4-1.1 channels that are available to open. Furthermore, EA1 mutations alter heteromeric channel availability which probably modifies the integration properties and firing patterns of neurones controlling cognitive processes and body movements.

Ultrastructural localization of a voltage-gated K+ channel alpha subunit (KV 1.2) in the rat cerebellum

May 1996

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12 Reads

A highly specific monoclonal antibody and pre-embedding immunocytochemistry were employed to examine the distribution of the K+ channel, alpha subunit K(V)1.2 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(V)1.2 alpha 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 alpha subunit K(V)1.2 was localized to a number of different cell types in the cerebellum. Each neuronal type displays a distinct subcellular distribution of the subunit.

Distribution and functional characterization of human Na v 1.3 splice variants

August 2005

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33 Reads

The focus of the present study is the molecular and functional characterization of four splice variants of the human Nav1.3 alpha subunit. These subtypes arise due to the use of alternative splice donor sites of exon 12, which encodes a region of the alpha subunit that resides in the intracellular loop between domains I and II. This region contains several important phosphorylation sites that modulate Na+ channel kinetics in related sodium channels, i.e. Nav1.2. While three of the four Nav1.3 isoforms, 12v1, 12v3 and 12v4 have been previously identified in human, 12v2 has only been reported in rat. Herein, we evaluate the distribution of these splice variants in human tissues and the functional characterization of each of these subtypes. We demonstrate by reverse transcriptase-polymerase chain reaction (RT-PCR) that each subtype is expressed in the spinal cord, thalamus, amygdala, cerebellum, adult and fetal whole brain and heart. To investigate the functional properties of these different splice variants, each alpha subunit isoform was cloned by RT-PCR from human fetal brain and expressed in Xenopus oocytes. Each isoform exhibited functional voltage-dependent Na+ channels with similar sensitivities to tetrodotoxin (TTX) and comparable current amplitudes. Subtle shifts in the V 1/2 of activation and inactivation (2-3 mV) were observed among the four isoforms, although the functional significance of these differences remains unclear. This study has demonstrated that all four human splice variants of the Nav1.3 channel alpha subunit are widely expressed and generate functional TTX-sensitive Na+ channels that likely modulate cellular excitability.

Sekine-Aizawa Y, Omori A & Fujita SC. MuSC, a novel member of the immunoglobulin superfamily, is expressed in neurons of a subset of cranial sensory ganglia in the mouse embryo. Eur J Neurosci10: 2810-2824 10.1046/j.1460-9568.1998.00288.x

October 1998

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10 Reads

In contrast to the spinal sensory ganglia which reiterate a basic organizational and functional unit, each cranial ganglion mediates a distinct sensory modality and exhibits a characteristic pattern of peripheral and central neuronal connectivity. Molecules responsible for establishment and maintenance of the cranial ganglion-specific networks are not known. Our hamster monoclonal antibody 802C11 strongly stained neurons and their processes of the VIIIth cranial ganglion (hearing and equilibrium), but not of the Vth cranial (somatosensory) or spinal ganglia in the mouse embryo. The cellular staining pattern of positive neurons suggested that the antigen was associated with the cell membrane, and biochemical analyses of the antigen from adult mouse brain showed the antigen to be a glycosylated intrinsic membrane protein of approximately 100 kDa. The antigen was purified, and based on the partial amino acid sequences, its entire cDNA was cloned. A bacterially expressed polypeptide encoded by the cDNA was recognized by the antibody. The deduced amino acid sequence revealed that the antigen belongs to the immunoglobulin superfamily with a significant homology (73.5% identity) to chicken SC1 protein. Chicken SC1 has been shown to be a cell-cell adhesion molecule in vitro with a proposed role in neurite extension of spinal motor neurons. These results suggest that our murine SC1-related protein (MuSC) is involved in the pathfinding and/or fasciculation of specific cranial sensory nerve fibres.

Colussi-Mas J, Geisler S, Zimmer L, Zahm DS, Bérod A. Activation of afferents to the ventral tegmental area in response to acute amphetamine: a double labeling study. Eur J Neurosci 26: 1011-1025

September 2007

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83 Reads

The ventral tegmental area (VTA), primary source of the mesocorticolimbic dopaminergic system, is regarded as a critical site for initiation of behavioural sensitization to psychostimulants. The present study was undertaken to identify the neural pathways converging on the VTA that are potentially implicated in this process. Rats were sensitized by a single exposure to amphetamine (5 mg/kg, s.c.). The distribution of VTA-projecting neurons activated by amphetamine was examined by combining retrograde transport of the cholera toxin beta subunit (CTb), injected into the VTA, with immunodetection of Fos. The quantitative analysis of CTb-Fos double labelling demonstrates that amphetamine induced a rapid activation of Fos in a large number of brain areas projecting to the VTA. More than half of the CTb-Fos double-labelled neurons were located in the prefrontal cortex, lateral preoptic area-lateral hypothalamus, pontomesencephalic tegmentum, dorsal raphe nucleus, ventral pallidum and nucleus accumbens. In addition, scattered CTb-Fos double-labelled cells were observed in many other VTA afferent structures, such as claustrum, lateral septum, diagonal band-magnocellular preoptic nucleus, deep mesencephalic nucleus, oral part of pontine reticular nucleus and dorsomedial tegmental area. This suggests that systemic amphetamine activates a wide population of neurons projecting to the VTA that may be important for the modulation of neurobehavioural plasticity produced by this psychostimulant.

Morris RG, Schenk F, Tweedie F, Jarrard LE. Ibotenate lesions of Hippocampus and/or subiculum: dissociating components of allocentric spatial learning. Eur J Neurosci 2: 1016-1028

February 1990

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59 Reads

This study examined the effects of ibotenic acid-induced lesions of the hippocampus, subiculum and hippocampus +/- subiculum upon the capacity of rats to learn and perform a series of allocentric spatial learning tasks in an open-field water maze. The lesions were made by infusing small volumes of the neurotoxin at a total of 26 (hippocampus) or 20 (subiculum) sites intended to achieve complete target cell loss but minimal extratarget damage. The regional extent and axon-sparing nature of these lesions was evaluated using both cresyl violet and Fink - Heimer stained sections. The behavioural findings indicated that both the hippocampus and subiculum lesions caused impairment to the initial postoperative acquisition of place navigation but did not prevent eventual learning to levels of performance almost as effective as those of controls. However, overtraining of the hippocampus + subiculum lesioned rats did not result in significant place learning. Qualitative observations of the paths taken to find a hidden escape platform indicated that different strategies were deployed by hippocampal and subiculum lesioned groups. Subsequent training on a delayed matching to place task revealed a deficit in all lesioned groups across a range of sample choice intervals, but the subiculum lesioned group was less impaired than the group with the hippocampal lesion. Finally, unoperated control rats given both the initial training and overtraining were later given either a hippocampal lesion or sham surgery. The hippocampal lesioned rats were impaired during a subsequent retention/relearning phase. Together, these findings suggest that total hippocampal cell loss may cause a dual deficit: a slower rate of place learning and a separate navigational impairment. The prospect of unravelling dissociable components of allocentric spatial learning is discussed.

Pistis M, Porcu G, Melis M, Diana M, Gessa GL. Effects of cannabinoids on prefrontal neuronal responses to ventral tegmental area stimulation. Eur J Neurosci 14: 96-102

August 2001

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86 Reads

Cannabinoids activate the firing of mesoprefrontocortical dopamine neurons and release dopamine in the prefrontal cortex. This study was undertaken with the aim of clarifying the interaction between cannabinoids and mesocortical system in the prefrontal cortex. The effect of Delta9-tetrahydrocannabinol (Delta9-THC) and the synthetic CB1 agonist WIN55,212-2 (WIN) was studied by extracellular single unit recordings, in chloral hydrate anaesthetised rats, on the spontaneous activity of pyramidal neurons and on the inhibition produced on these neurons by the electrical stimulation of the ventral tegmental area (VTA). Intravenously administered Delta9-THC and WIN (1.0 and 0.5 mg/kg, respectively), increased the firing rate of pyramidal neurons projecting to the VTA. VTA stimulation produced a phasic inhibition (167 +/- 6 ms) in 79% of prefrontal cortex pyramidal neurons. Delta9-THC and WIN reverted this inhibition in 73% and 100% of the neurons tested, respectively. The subsequent administration of the selective CB1 antagonist SR141716A (1 mg/kg) readily suppressed the effects of both cannabinoids and restored the inhibitory response to VTA stimulation. Moreover, when administered alone, SR141716A prolonged the inhibition in 55.6% of the neurons tested. The results indicate that stimulation of CB1 receptors by cannabinoids results in an enhanced excitability of prefrontal cortex pyramidal neurons as indexed by the suppression of the inhibitory effect of VTA stimulation and by the increase in firing rate of antidromically identified neurons projecting to the VTA. Furthermore, our results support the view that endogenous cannabinoids exert a negative control on dopamine activity in the prefrontal cortex. This study may be relevant in helping to understand the influence of cannabinoids on cognitive processes mediated by the prefrontal cortex.

Figure 1: A) Schematic representation of location of microinjection sites described in this study. Coronal sections (left) and corresponding and enlarged details of the areas of interest (right) (from Paxinos & Watson, 1986, with modifications). Distances (mm) are from the interaural line. Microinjection sites within the dorsal raphe nucleus (DRN) (n = 7) are represented by open circles. Microinjection sites outside and rostral to the DRN (n = 7), lying in the region of the oculomotor nucleus, are represented by open triangles. For the sake of clarity of the illustration, injection sites outside the DRN between coronal planes at 1.7 mm and 1.36 mm from the interaural line (n = 6) are not represented. (B) Microphotograph and schematic camera lucida drawing of a histological section showing track and site of microinjection into the DRN. Section shown corresponds approximately to the section at 1.7 mm from the interaural line shown in A. PAG, periaqueductal grey matter. Scale bar = 500 µm.
Figure 2: Interleukin-1 (IL-1) microinjected into the dorsal raphe nucleus (DRN) increased non-rapid eye movements (NREM) sleep. Changes induced in NREM, rapid eye movements (REM) sleep, and brain cortical temperature (Tcort) by IL-1 microinjection into the DRN and outside this area (not DRN) during the first 2 h post-injection. REM sleep was inhibited when IL-1 was microinjected both into and outside the DRN. Values are means ± SEM and are expressed as differences from vehicle. *P < 0.05 vs. vehicle.
Figure 3: Electrophysiological and pharmacological identification of serotonergic dorsal raphe nucleus (DRN) neurons. Representative voltage recordings from DRN serotonergic neurons obtained under current clamp in brain slice. No direct current current was injected. (A) Slow and regular firing rate of serotonergic DRN neurons in spontaneous conditions. (B) Detail of the recording in A, showing typical serotonergic action potentials, characterized by an initiating ramp of depolarization and by a large after-hyperpolarization. (C) Intracellular recording showing the effect of 1-min bath perfusion with 5-HT 40 µm (starting at arrow) on a serotonergic DRN neuron. In this case 5-HT induced a complete cessation of firing and a hyperpolarization of about 10 mV.                             (D) In the same neuron depicted in C, 1-min bath perfusion (starting at arrow) with NA 30 µm induced a reversible increase in the firing rate. The effects in C and D were rapidly reverted after wash out.
Figure 4: Interleukin-1 (IL-1) inhibited serotonergic dorsal raphe nucleus (DRN) neurons intracellularly recorded in vitro. Intracellular recordings showing the effects of IL-1 (25 ng/mL) addition to bath perfusate for 2 min on two different representative serotonergic DRN neurons. Upper panel: IL-1 perfusion (starting at arrow) induced a decrease in spontaneous firing rate. Lower panel: in this neuron during perfusion with IL-1 the spontaneous activity first decreased and then ceased. In both neurons the effects were rapidly and completely reverted by washout.
Manfridi A, Brambilla D, Bianchi S, Mariotti M, Opp MR, Imeri L. Interleukin-1beta enhances non-rapid eye movement sleep when microinjected into the dorsal raphe nucleus and inhibits serotonergic neurons in vitro. Eur J Neurosci 18: 1041-1049

October 2003

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195 Reads

Interleukin-1 (IL-1) and IL-1 receptors are constitutively expressed in normal brain. IL-1 increases non-rapid eye movements (NREM) sleep in several animal species, an effect mediated in part by interactions with the serotonergic system. The site(s) in brain at which interactions between IL-1 and the serotonergic system increase NREM sleep remain to be identified. The dorsal raphe (DRN) is the origin of the major ascending serotonergic pathways to the forebrain, and it contains IL-1 receptors. This study examined the hypothesis that IL-1 increases NREM sleep by acting at the level of the DRN. IL-1beta (0.25 and 0.5 ng) was microinjected into the DRN of freely behaving rats and subsequent effects on sleep-wake activity were determined. IL-1beta 0.5 ng increased NREM sleep during the first 2 h post-injection from 33.5 +/- 3.7% after vehicle microinjection to 42.9 +/- 3.0% of recording time. To determine the effects of IL-1beta on electrophysiological properties of DRN serotonergic neurons, intracellular recordings were performed in a guinea-pig brain stem slice preparation. In 26 of 32 physiologically and pharmacologically identified serotonergic neurons, IL-1beta superfusion (25 ng/mL) decreased spontaneous firing rates by 50%, from 1.6 +/- 0.2 Hz (before IL-1beta superfusion) to 0.8 +/- 0.2 Hz. This effect was reversible upon washout. These results show that IL-1beta increases NREM sleep when administered directly into the DRN. Serotonin enhances wakefulness and these novel data also suggest that IL-1beta-induced enhancement of NREM sleep could be due in part to the inhibition of DRN serotonergic neurons.

Feenstra MG, Vogel M, Botterblom MH, Joosten RN, de Bruin JP. Dopamine and noradrenaline efflux in the rat prefrontal cortex after classical aversive conditioning to an auditory cue. Eur J Neurosci 13: 1051-1054

April 2001

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26 Reads

We used bilateral microdialysis in the medial prefrontal cortex (PFC) of awake, freely moving rats to study aversive conditioning to an auditory cue in the controlled environment of the Skinner box. The presentation of the explicit conditioned stimuli (CS), previously associated with foot shocks, caused increased dopamine (DA) and noradrenaline (NA) efflux. This conditioned response was dependent on the immediate pairing of the two stimuli; in the pseudoconditioned group that received an equal number of both stimuli, but in an unpaired fashion, no conditioned increases in efflux were observed.

Figure 1: Coronal sections of the chick brain showing the location of the ventral and dorsal hippocampus (vHp and dHp). AA, anterior arcopallium; AD, dorsal arcopallium; CPi, piriform cortex; GP, globus pallidus; LSt, lateral striatum; M, mesopallium; N, nidopallium; SL, lateral septal nucleus; SM, medial septal nucleus; TnA, nucleus taeniae of amygdala; black dots A14, dopaminergic neurons in the paraventricular nucleus (PVN).
Figure 2: Photomicrographs of synaptic contacts in the chick Hp. (A–D). (A) An asymmetric shaft synapse (on dendrites) in dorsal 1-day-old chick Hp indicated with a black asterisk. The presynaptic part can be clearly distinguished by the presence of vesicles (ves). (B) Representative symmetric shaft synapse marked with an arrowhead from the dorsal Hp of the MeA-trained group. Asterisk indicates spine synapses. (C) Representation of asymmetric spine synapses marked with black asterisks from the right hemisphere of the ventral Hp of the water-trained group. (D) Two dendrites in the ventral Hp of a control bird 24 h post-training receiving two asymmetric synapses from a presynaptic axon terminal (At) resulting in axo-dendritic synapses. At, axon terminal; Den, dendrite; mit, mitochondrion; sp, spine. Scale bars, 200 nm.
Figure 4: Histograms showing asymmetric synapse densities (Nv/µm3) in the dorsal (A) and ventral (B) hippocampus (Hp) of the right and left hemisphere of chicks 6 h after avoidance training, and in water-trained and naïve controls (control n = 6, water n = 5, MeA n = 5). Vertical bars on the histogram blocks represent means ± SEM.                             (A) The asterisks indicate significant reductions in asymmetric spine synapse density in the dorsal Hp of the right hemisphere of MeA-trained chicks (P = 0.0008).                             (B) Significant differences are indicated between the right and left hemisphere in the water-trained group (n = 5) (P = 0.045) for asymmetric shaft synapses (‡), whilst † and + show differences for asymmetric spine synapse densities between right and left hemisphere of water and MeA-trained groups (P = 0.017 and P = 0.04, respectively).
Figure 5: Histograms showing asymmetric synapse densities (Nv/µm3) in the dorsal (A) and ventral (B) hippocampus (Hp) in the right and left hemisphere of chicks 24 h after avoidance training, and in water-trained and naïve controls (control n = 6, water n = 6, MeA n = 6). Vertical bars on the histogram blocks represent means ± SEM.                             (A) There are significantly fewer (‡) asymmetric shaft synapses in the dorsal Hp of the left hemisphere of control animals, compared with the right hemisphere (P = 0.031). Also the MeA-trained group has significantly fewer asymmetric shaft synapses (*) in comparison to the control group (P = 0.038). (B) None of the differences in the ventral Hp between the groups or hemispheres at 24 h for either asymmetric shaft or spine synapses are significant.
Nikolakopoulou AM, Davies HA, Stewart MG. Passive avoidance training decreases synapse density in the hippocampus of the domestic chick. Eur J Neurosci 23: 1054-1062

March 2006

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267 Reads

The bird hippocampus (Hp), although lacking the cellular lamination of the mammalian Hp, possesses comparable roles in spatial orientation and is implicated in passive avoidance learning. As in rodents it can be divided into dorsal and ventral regions based on immunocytochemical, tracing and electrophysiological studies. To study the effects of passive avoidance learning on synapse morphometry in the Hp, spine and shaft synapse densities of 1-day-old domestic chicks were determined in dorsal and ventral Hp of each hemisphere by electron microscopy, 6 and 24 h following training to avoid pecking at a bead coated with a bitter-tasting substance, methyl anthranilate (MeA). The density of asymmetric spine and shaft synapses in MeA-trained birds at 6 h post-training was significantly lower in the dorsal and ventral Hp of the right hemisphere relative to control (untrained) chicks, but by 24 h this difference was absent. A hemispheric asymmetry was apparent in the ventral Hp where the water-trained group showed enhanced shaft and spine synapse density in the left hemisphere, whilst in the MeA-trained group only asymmetric shaft synapses follow the same pattern in relation to the right hemisphere. There were no differences in asymmetric shaft synapses in the dorsal Hp at 6 h post-training, but at 24 h post-training there was a reduction in the density of shaft synapses in the right hemisphere in MeA compared with control birds. These data are discussed in relation to the pruning effects of stress and learning on synapse density in chick Hp.

Patel S, Roelke CT, Rademacher DJ, Hillard CJ. Inhibition of restraint stress-induced neural and behavioural activation by endogenous cannabinoid signaling. Eur J Neurosci 21: 1057-1069

March 2005

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182 Reads

The role of endocannabinoid (eCB) signalling in restraint stress-induced neuronal activation was studied. Male mice exposed to 30 min of restraint exhibit increased Fos protein within prefrontal cortex (PFC), lateral septum (LS), nucleus accumbens (Acb) and medial amygdala. SR141716 (2 mg/kg) itself had no effect on Fos but pretreatment with SR141716 significantly potentiated restraint-induced Fos expression in cingulate, LS and Acb. SR141716 also significantly increased the time spent in active escape behaviours during the restraint. In restraint-habituated mice (mice exposed to four previous restraint episodes), the fifth restraint exposure resulted in decreased expression of active escape behaviours compared to the first exposure and only induced Fos protein in the central and medial amygdala. Administration of SR141716 prior to the fifth restraint episode resulted in greater potentiation of restraint-induced Fos induction than the first; significant increases occurred within all regions of PFC examined, LS and Acb. Brain regional eCB content was measured immediately after restraint. N-arachidonylethanolamine content within the amygdala was significantly decreased after both restraint episodes. 2-Arachidonylglycerol content was significantly increased in both the limbic forebrain and amygdala after the fifth restraint but not the first. Restraint had no effect on cerebellar eCB content. These data suggest that eCB activation of CB(1) receptors opposes the behavioural and neuronal responses to aversive stimuli. Because repeated homotypic stress increased both limbic 2-AG and resulted in a greater effect of SR141716 on limbic Fos expression, we hypothesize that increased CB(1) receptor activity contributes to the expression of habituation to homotypic stress.

Breukel AI, Besselsen E, Lopes da Silva FH, Ghijsen WE. A presynaptic N-methyl-D-aspartate autoreceptor in rat hippocampus modulating amino acid release from a cytoplasmic pool. Eur J Neurosci 10: 106-114

February 1998

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11 Reads

A possible role of the N ‐methyl‐ d ‐aspartate receptor (NMDA‐R) as a presynaptic autoreceptor was investigated using Percoll‐purified hippocampus nerve terminals (synaptosomes). This preparation contained only a neglectable amount of postsynaptic structures. Two main effects of NMDA were observed. First, NMDA dose‐dependently (10–100 μ m ) and in the absence of Mg ²⁺ , stimulated basal release of aspartate and glutamate, but not of GABA. MK801 (10 μ m ), an open NMDA‐R‐channel blocker, reduced this effect even below control levels, indicating endogenous NMDA‐R activation. By superfusing synaptosomes, which prevents a tonic receptor occupation, also basal GABA release was stimulated by NMDA. The NMDA‐induced potentiation of amino acid superfusate levels was blocked both by MK801 and Mg ²⁺ (1 m m ), was slow in onset and returned to baseline after NMDA‐removal. The NMDA‐effect was also found in the absence of extracellular Ca ²⁺ , suggesting that amino acids were released from a non‐vesicular (cytoplasmic) pool. Secondly, in KCl‐depolarized synaptosomes exposed to 1 m m Mg ²⁺ , NMDA did not affect the release of the amino acids. MK801, however, reduced the KCl‐evoked Ca ²⁺ ‐independent release of aspartate and glutamate, but not of GABA. l ‐ trans ‐PDC, the selective inhibitor of the glutamate/aspartate transporter, prevented this MK801‐effect, suggesting a coupling between NMDA‐Rs and these transporters. These data provide evidence for a presynaptic NMDA autoreceptor in rat hippocampus. We speculate on the role of this NMDA‐R to depolarize the presynaptic membrane by Na ⁺ ‐entry, which may induce reversal of amino acid transporters and thereby releasing amino acids from a cytoplasmic pool.

Hayley S, Wall P, Anisman H. Sensitization to the neuroendocrine, central monoamine and behavioural effects of murine TNF-: peripheral and central mechanisms. Eur J Neurosci 15: 1061-1076

April 2002

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16 Reads

Systemic administration of murine tumour necrosis factor-alpha (mTNF-alpha; 0.1-2.0 microg, i.p.) dose-dependently increased plasma corticosterone and augmented monoamine utilization within the paraventricular nucleus of the hypothalamus (PVN), locus coeruleus, medial prefrontal cortex (PFC), central and medial amygdala. A time-dependent sensitization was induced in mice, wherein reexposure to mTNF-alpha 28 days (but not 1 day) following the initial cytokine treatment provoked marked signs of illness (diminished activity, ptosis, piloerection) and increased plasma corticosterone levels. Serotonin (5-HT) activity was augmented upon mTNF-alpha reexposure at the 1- or 28-day intervals in the PFC and medial amygdala, respectively. Intracerebroventricular (i.c.v.; 1-500 ng) mTNF-alpha did not promote illness, but modestly increased plasma corticosterone levels. Neither the illness nor the corticosterone changes were subject to a sensitization upon i.c.v. cytokine reexposure. Acute i.c.v. mTNF-alpha increased norepinephrine (NE), 5-HT and dopamine (DA) activity within the PVN and median eminence/arcuate nucleus complex (ME/ARC), and NE utilization within the central amygdala. Subsequent i.c.v. mTNF-alpha further enhanced the hypothalamic monoamine variations. Finally, systemic (i.p.) mTNF-alpha pretreatment did not proactively influence sickness or corticosterone responses upon later i.c.v. cytokine challenge, but augmented locus coeruleus NE activity and 5-HT and DA utilization within the ME/ARC. It is suggested that the sensitization with respect to sickness and corticosterone activity in response to mTNF-alpha reflect the involvement of peripheral mechanisms. Moreover, it appears that mTNF-alpha promotes central neurochemical plasticity through independent central and peripheral mechanisms.

Rodefer JS, Murphy ER, Baxter MG. PDE10A inhibition reverses subchronic PCP-induced deficits in attentional set-shifting in rats. Eur J Neurosci 21: 1070-1076

March 2005

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47 Reads

Persistent suppression of N-methyl-d-aspartate (NMDA) receptor function produces enduring structural changes in neocortical and limbic regions in a pattern similar to changes reported in schizophrenia. This similarity suggests that chronic NMDA receptor antagonism in animals may represent a useful model of neurobiological and related cognitive deficits in schizophrenia. Schizophrenia is associated with impairments in frontal lobe-dependent cognitive functions, including working memory and attentional shifting. Deficits in attention and executive function have not been well characterized in animal models of schizophrenia using chronic NMDA receptor antagonist administration. We investigated whether subchronic systemic administration of the NMDA receptor antagonist phencyclidine (PCP) to rats followed by a drug washout period would produce enduring cognitive deficits on an attentional set-shifting task. The task is functionally analogous to a sensitive test of frontal function in humans and non-human primates. Subchronic PCP administration selectively impaired extradimensional shift learning without affecting other discrimination or reversal tasks. Moreover, acute treatment with the PDE10A inhibitor papaverine immediately prior to testing attenuated the PCP-induced deficits in extradimensional shift learning across a range of doses. These data suggest that subchronic PCP administration may model effectively some of the cognitive deficits that are observed in schizophrenia, and that PDE10A inhibition may be an effective therapeutic route to improve executive function deficits associated with schizophrenia.

Centonze D, Picconi B, Gubellini P, Bernardi G, Calabresi P. Dopaminergic control of synaptic plasticity in the dorsal striatum. Eur J Neurosci 13: 1071-1077

April 2001

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45 Reads

Cortical glutamatergic and nigral dopaminergic afferents impinge on projection spiny neurons of the striatum, providing the most significant inputs to this structure. Isolated activation of glutamate or dopamine (DA) receptors produces short-term effects on striatal neurons, whereas the combined stimulation of both glutamate and DA receptors is able to induce long-lasting modifications of synaptic excitability. Repetitive stimulation of corticostriatal fibres causes a massive release of both glutamate and DA in the striatum and, depending on the glutamate receptor subtype preferentially activated, produces either long-term depression (LTD) or long-term potentiation (LTP) of excitatory synaptic transmission. D1-like and D2-like DA receptors interact synergistically to allow LTD formation, while they operate in opposition during the induction phase of LTP. Corticostriatal synaptic plasticity is severely impaired after chronic DA denervation and requires the stimulation of DARPP-32, a small protein expressed in dopaminoceptive spiny neurons which acts as a potent inhibitor of protein phosphatase-1. In addition, the formation of LTD and LTP requires the activation of PKG and PKA, respectively, in striatal projection neurons. These kinases appear to be stimulated by the activation of D1-like receptors in distinct neuronal populations.

The alpha1I T-type calcium channel exhibits faster gating properties when overexpressed in neuroblastoma/glioma NG 108-15 cells

December 2001

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17 Reads

The recently cloned T-type calcium channel alpha1I (Cav3.3) displays atypically slow kinetics when compared to native T-channels. Possible explanations might involve alternative splicing of the alpha1I subunit, or the use of expression systems that do not provide a suitable environment (auxiliary subunit, phosphorylation, glycosylation...). In this study, two human alpha1I splice variants, the alpha1I-a and alpha1I-b isoforms that harbour distinct carboxy-terminal regions were studied using various expression systems. As the localization of the alpha1I subunit is primarily restricted to neuronal tissues, its functional expression was conducted in the neuroblastoma/glioma cell line NG 108-15, and the results compared to those obtained in HEK-293 cells and Xenopus oocytes. In Xenopus oocytes, both isoforms exhibited very slow current kinetics compared to those obtained in HEK-293 cells, but the alpha1I-b isoform generated faster currents than the alpha1I-a isoform. Both activation and inactivation kinetics of alpha1I currents were significantly faster in NG 108-15 cells, while deactivating tail currents were two times slower, compared to those obtained in HEK-293 cells. Moreover, the alpha1-b isoform showed significantly slower deactivation kinetics both in NG 1080-15 and in HEK-293 cells. Altogether, these data emphasize the advantage of combining several expression systems to reveal subtle differences in channel properties and further indicate that the major functional differences between both human alpha1I isoforms are related to current kinetics. More importantly, these data suggest that the expression of the alpha1I subunit in neuronal cells contributes to the "normalization" of current kinetics to the more classical, fast-gated T-type Ca2+ current.

Lerma J, Morales M, Ibarz JM, Somohano F. Rectification properties and Ca2+ permeability of glutamate receptor channels in hippocampal cells. Eur J Neurosci 6: 1080-1088

August 1994

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21 Reads

Excitatory amino acids exert a depolarizing action on central nervous system cells through an increase in cationic conductances. Non-NMDA receptors have been considered to be selectively permeable to Na+ and K+, while Ca2+ influx has been thought to occur through the NMDA receptor subtype. Recently, however, the expression of cloned non-NMDA receptor subunits has shown that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are permeable to Ca2+ whenever the receptor lacks a particular subunit (edited GluR-B). The behaviour of recombinant glutamate receptor channels predicts that Ca2+ would only permeate through receptors that show strong inward rectification and vice versa, i.e. AMPA receptors with linear current-voltage relationships would be impermeable to Ca2+. Using the whole-cell configuration of the patch-clamp technique, we have studied the Ca2+ permeability and the rectifying properties of AMPA receptors, when activated by kainate, in hippocampal neurons kept in culture or acutely dissociated from differentiated hippocampus. Cells were classified according to whether they showed outward rectifying (type I), inward rectifying (type II) or almost linear (type III) current-voltage relationships for kainate-activated responses. AMPA receptors of type I cells (52.2%) were mostly Ca(2+)-impermeable (PCa/PCs = 0.1), while type II cells (6.5%) expressed Ca(2+)-permeable receptors (PCa/PCs = 0.9). Type III cells (41.3%) showed responses with low but not negligible Ca2+ permeability (PCa/PCs = 0.18). The degree of Ca2+ permeability and inward rectification were well correlated in cultured cells, i.e. more inward rectification corresponded to higher Ca2+ permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutant huntingtin affects the rate of transcription of striatum-specific isoforms of phosphodiesterase 10A

January 2005

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67 Reads

Huntington's disease (HD) is caused by the inheritance of a copy of the gene encoding mutant huntingtin with an expanded CAG repeat. Phosphodiesterase 10A (PDE10A) mRNA decreases in transgenic HD mice expressing exon 1 of the human huntingtin gene (HD). The mouse PDE10A mRNA is expressed through alternative splicing and polyadenylation in a tissue-specific manner and that transcription of striatal PDE10A mRNA is driven by two promoters. PDE10A2 is the predominant isoform of the gene is expressed in the striatum. Using in situ hybridization and quantitative RT-PCR, we determined that decreased steady-state levels of PDE10A2 mRNA were caused by an altered transcription initiation rate rather than by post-transcriptional mRNA instability in HD mice. Transcription from three initiation sites located within a 50-bp region in the PDE10A2-specific promoter was differentially affected by the presence of the mutant huntingtin transgene. The mouse and human PDE10A2 promoters are highly conserved with respect to the relative position of cis-regulatory elements. Several transcription factors that have been shown to interact with mutant huntingtin, including Sp1, neuron restrictive silencing factor, TATA-binding protein and cAMP-response element binding protein, are unlikely to be involved in mutant huntingtin-induced PDE10A2 transcriptional dysregulation.

Dietrich D, Kral T, Clusmann H, Friedl M, Schramm JReduced function of L-AP4-sensitive metabotropic glutamate receptors in human epileptic sclerotic hippocampus. Eur J Neurosci 11:1109-1113

April 1999

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13 Reads

Human temporal lobe epilepsy is characterized by strong synaptic reorganization that leads to abnormal recurrent excitatory synaptic connections among hippocampal neurons. In addition, electrophysiological studies show that synaptic activity of the main afferent input to the hippocampus, the perforant path, is prolonged and amplified by changes in postsynaptic glutamate receptors. The current view is that these morphological and physiological abnormalities contribute significantly to the hyperexcitability seen in the hippocampus of temporal lobe epilepsy. Recently, it was found that presynaptic inhibitory metabotropic glutamate receptors are an important negative feedback mechanism that controls synaptic release of glutamate in the hippocampus. In this study, we assessed the functionality of this feedback system by investigating the metabotropic glutamate receptor mediated depression of excitatory synaptic transmission in surgically removed hippocampi from patients with marked synaptic reorganization (Ammon's horn sclerosis group) and from patients without detectable reorganization (lesion group). We report here that this control of synaptic transmission is lost in hippocampi from the Ammon's horn sclerosis group whereas this control is preserved in hippocampi from the lesion group. The data presented here suggest that the loss of feedback inhibition mediated by metabotropic glutamate receptors could be a further, previously not recognized, mechanism in the pathophysiology of temporal lobe epilepsy.

Neuhoff H, Roeper J, Schweizer M. Activity-dependent formation of perforated synapses in cultured hippocampal neurons. Eur J Neurosci 11: 4241-4250

January 2000

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40 Reads

The study investigated the formation of perforated synapses in rat hippocampal cell cultures. Perforated synapses are defined by their discontinuous postsynaptic densities (PSDs) and are believed to occur in parallel with changes in synaptic activity and possibly also synaptic efficacy. Several in vivo studies have demonstrated an increase in the frequency of perforated synapses induced by development and environmental stimulation as well as long-term potentiation (LTP). Also in in vitro brain slices, LTP was associated with an elevated number of perforated spine synapses. Our study demonstrated for the first time that the formation of perforated synapses can be induced by a short-term increase in spontaneous neural activity in a hippocampal cell culture model. Stimulation with the GABAA-antagonist picrotoxin (PTX) induced a significant increase in the percentage of perforated synapses. This strong increase was blocked when APV was added together with PTX, indicating that the formation of perforated synapses depended on the activation of NMDA receptors. We also showed that inhibition of the tissue type plasminogen activator (tPA-stop/PAI-1) significantly interfered with the activity-induced increase in perforated synapses. This implies that the proteolytic activities of tPA might be involved in steps which are downstream from the NMDA receptor-mediated synaptic plasticity leading to structural changes at synaptic contacts. In contrast, even long-term inhibition of electrical network activity by tetrodotoxin had no effect on the number of perforated synapses, but almost completely abolished the formation of spine synapses. These results indicate that a short-term increase in neural activity via NMDA receptors and a proteolytic cascade involving tPA lead to the formation of perforated synapses.

Bergeron M, Yu AY, Solway KE, Semenza GL, Sharp FRInduction of hypoxia-inducible factor-1 (HIF-1) and its target genes following focal ischemia in rat brain. Eur J Neurosci 11:4159-4170

January 2000

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51 Reads

HIF-1 is a heterodimeric transcription factor, induced by hypoxia, that is composed of HIF-1alpha and HIF-1beta protein subunits. It binds to promoter/enhancer elements and stimulates the transcription of hypoxia-inducible target genes, including glucose transporter-1 and the glycolytic enzymes. Because HIF-1 activation might promote cell survival in hypoxic tissues, we studied the effect of permanent middle cerebral artery occlusion on the expression of HIF-1alpha, HIF-1beta and several HIF-1 target genes in adult rat brain. After focal ischaemia, mRNAs encoding HIF-1alpha, glucose transporter-1 and several glycolytic enzymes were up-regulated in the peri-infarct penumbra. This was observed by 7.5 h after the onset of ischaemia and increased further at 19 and 24 h. Regional cerebral blood flow was moderately decreased at 1 and 24 h after the ischaemia in areas of HIF-1 and HIF-1 target gene induction. Because hypoxia induces HIF-1 in other tissues, systemic hypoxia (6% O2 for 4.5 h) was also shown to increase HIF-1alpha protein expression in the adult rat brain. It is proposed that decreased blood flow to the penumbra decreases the supply of oxygen and that this induces HIF-1 and its target genes. This is the first study to show induction of HIF-1 after focal ischaemia in brain. Increased expression of HIF-1 target genes as a result of HIF-1 activation by hypoxia may contribute to tissue viability in the hypoxic/ischaemic penumbra by increasing glucose transport and glycolysis.

Humby T, Laird FM, Davies W, Wilkinson LS. Visuo-spatial attentional functioning in mice: interactions between cholinergic manipulations and genotype. Eur J Neurosci 11: 2813-2823

September 1999

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27 Reads

Attentional functioning in mice was assessed in an analogue of the five-choice serial reaction time task in which the requirement was to detect brief visual stimuli presented across five spatial locations. Two hybrid strains of mice were assessed; F1 C57Bl/6xDBA/2 and C57Bl/6x129sv. Both strains acquired the task to high levels of performance with, in particular, no problems due to premature responding. At performance, systematic manipulation of the task parameters indicated a pattern of effects consistent with the task, taxing aspects of visuospatial attention. There were some differential effects of task manipulations at baseline across strain. However, the pattern of effects suggested these were likely to be the result of effects on factors other than attentional functioning per se, such as behavioural reactivity and inhibition. There was evidence in both strains of specific, centrally mediated effects of scopolamine on attentional functioning, with the C57Bl/6xDBA/2 hybrid showing greater sensitivity to the drug manipulation. Specific effects on discriminative accuracy were observed at doses of 0.02 and 0.2 mg/kg scopolamine. At the 2 mg/kg dose, large reductions in accuracy were associated with large effects on other measures, including omissions and response latencies, suggestive of nonspecific effects on task performance. These data indicate, for the first time, the utility of operant methods in assessing visuospatial attentional functioning in mice. They confirm the importance of cholinergic mechanisms in attentional processes across species, and suggest interactions between cholinergic mechanisms and genotype in the expression of attentional phenotypes.

Mikkonen M, Soininen H, Tapiola T, Alafuzoff I, Miettinen R. Hippocampal plasticity in Alzheimer's disease: changes in highly polysialylated NCAM immunoreactivity in the hippocampal formation. Eur J Neurosci 11: 1754-1764

June 1999

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35 Reads

The highly polysialylated neural cell adhesion molecule (PSA-NCAM) is one of the most promising molecules that contributes to plasticity in the central nervous system. We evaluated PSA-NCAM immunoreactivity in the hippocampal formation of Alzheimer's disease (AD) patients. We found significant increases over control levels in the optical density ratios of PSA-NCAM immunoreactivity in the outer molecular layer/granule cell layer (ODoml/grl) and in the inner molecular layer/granule cell layer (ODiml/grl) in the dentate gyrus of AD patients. The optical density of the granule cell layer in the dentate gyrus did not differ significantly between AD patients and control subjects. However, the number of PSA-NCAM-immunopositive infragranule cells was higher in the AD group compared with control subjects. The major finding in the CA1, subiculum and entorhinal cortex of AD patients was the disorganization of PSA-NCAM-immunoreactive fibres. These results indicate that neuronal remodelling occurs, especially in the dentate gyrus of patients with AD.

Characteristic Localization of gpl30 (the Signal‐transducing Receptor Component Used in Common for IL‐6/IL‐11/CNTF/LIF/OSM) in the Rat Brain

September 1996

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15 Reads

The localization of gp130, the signal transducing receptor component used in common for interleukin (IL)-6, IL-11, ciliary neurotrophic factor (CNTF), LIF and OSM, in the rat brain was demonstrated by immunohistochemistry using an antibody specific to gp130. The gp130 immunoreactivity was observed in both glial and neuronal cells. Two distinct neuronal staining patterns were observed. The first showed neuropil staining, observed mainly in telencephalic structures including the hippocampus, cerebral cortex and caudate-putamen. The second pattern was observed on the cytoplasmic membrane of neuronal somata and was found primarily in the lower brainstem, in the large neurons of the reticular formation, and in spinal and cranial motor neurons. Electron-microscopic analysis revealed that both types of gp130 immunoreactivity were primarily associated with the cytoplasmic membrane and were not localized exactly at synaptic sites. Further, gp130 immunoreactivity was also observed in the oligodendrocytes and subependymal zone. These widespread but characteristic patterns of gp130 immunoreactivity overlap well with those of IL-6 receptor and CNTF alpha chains, suggesting a role of cytokines and growth factors such as IL-6 and CNTF via gp130 in certain specific regions of the brain.

Schnell L, Fearn S, Klassen H, Schwab ME, Perry VHAcute inflammatory responses to mechanical lesions in the CNS: differences between brain and spinal cord. Eur J Neurosci 11:3648-3658

November 1999

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38 Reads

Lesion-induced inflammatory responses in both brain and spinal cord have recently become a topic of active investigation. Using C57BL/6J mice, we compared the tissue reaction in these two central nervous system (CNS) compartments with mechanical lesions of similar size involving both grey and white matter. This evaluation included the quantitative assessment of neutrophils, lymphocytes and activated macrophages/microglia, as well as astrocyte activation, upregulation of vascular cell adhesion molecules (ICAM-1, VCAM-1, PECAM) and the extent of blood-brain barrier (BBB) breakdown. Time points analysed post-lesioning included 1, 2, 4 and 7 days (as well as 10 and 14 days for the BBB). We found clear evidence that the acute inflammatory response to traumatic injury is significantly greater in the spinal cord than in the cerebral cortex. The numbers of both neutrophils and macrophages recruited to the lesion site were significantly higher in the spinal cord than in the brain, and the recruitment of these cells into the surrounding parenchyma was also more widespread in the cord. The area of BBB breakdown was substantially larger in the spinal cord and vascular damage persisted for a longer period. In the brain, as in spinal cord, the area to which neutrophils were recruited correlated well with the area of BBB breakdown. It will be of interest to determine the extent to which the infiltration of inflammatory cells contributes, either directly or indirectly, to the vascular permeability and secondary tissue damage or, conversely, to local tissue repair in the brain and the spinal cord.

Mahanty NK, Sah P. Excitatory synaptic inputs to pyramidal neurons of the lateral amygdala. Eur J Neurosci 11: 1217-1222

May 1999

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15 Reads

Whole-cell patch clamp recordings were made from pyramidal neurons in the rat lateral amygdala (LA). Synaptic currents were evoked by stimulating in either the external capsule (ec), internal capsule (ic) or basolateral nucleus (BLA). Stimulation of either the ic, ec or BLA evoked a glutamatergic excitatory synaptic current (EPSC) which was mediated by both non-NMDA and NMDA (N-methyl-d-aspartic acid) receptors. The ratio of the amplitude of the NMDA receptor-mediated component measured at +40 mV to the amplitude of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) component measured at -60 mV was similar regardless of whether EPSCs were evoked in the ec, ic or BLA. At resting membrane potentials, excitatory synaptic potentials evoked from either the ec or putative thalamic inputs were unaffected by application of the NMDA receptor antagonist APV. Spontaneous glutamatergic currents had two components to their decay phase. The slow component was selectively blocked by the NMDA receptor antagonist D-APV, indicating that AMPA and NMDA receptors are colocalized in spiny neurons. We conclude that pyramidal cells of the LA receive convergent inputs from the cortex, thalamus and basal nuclei. At all inputs, both AMPA/kainate and NMDA-type receptors are active and colocalized in the postsynaptic density.

Fiumelli H, Jabaudon D, Magistretti PJ, Martin JLBDNF stimulates expression, activity and release of tissue-type plasminogen activator in mouse cortical neurons. Eur J Neurosci 11:1639-1646

June 1999

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27 Reads

Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor involved in neuronal development and synaptic plasticity. Although the physiological effects of BDNF have been examined in detail, target proteins which mediate its actions remain largely unknown. Here, we report that BDNF stimulates the expression of tissue-type plasminogen activator (tPA) in primary cultures of cortical neurons in a time- and concentration-dependent manner. Among the other members of the neurotrophin family, neurotrophin-4 (NT-4) and to a lesser extent neurotrophin-3 (NT-3) also increased tPA mRNA expression, while nerve growth factor (NGF) was devoid of any effect. Induction of tPA expression by BDNF is accompanied by an increase in the proteolytic activity of tPA associated with cortical neurons and a release of tPA into the extracellular space. Release of tPA induced by BDNF depends on extracellular Ca2+ since it is markedly reduced in the presence of ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). Up-regulation of tPA expression by BDNF is followed by the induction of plasminogen activator inhibitor 2 (PAI-2), an inhibitor of tPA. Together these results suggest that activation of tPA by BDNF may contribute to structural changes associated with neuronal development or synaptic plasticity.

Rat Brain: Distribution of Immunoreactivity of PE-11, a Peptide Derived from Chromogranin B

January 1997

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13 Reads

An antiserum was raised against the peptide PE-11 whose sequence is present in the chromogranin B molecule. The antiserum reacts only with the free C-terminal end of this peptide. PE-11 immunoreactivity in brain was characterized by molecular size exclusion high performance liquid chromatography. Only the free peptide and a N-terminally elongated peptide were detected, indicating that proteolytic processing of chromagranin B in brain is quite extensive. In immunohistochemistry PE-11 immunoreactivity was found in varicosities, fibres and perikarya throughout the brain. Strong staining was detected in the shell sector of the nucleus accumbens, in the lateral septum, in subregions of the extended amygdala, in some areas of the hippocampus and of the hypothalamus, in the locus coeruleus, in the Purkinje cells of the cerebellum and in the dorsal horn of the spinal cord. Our results, which demonstrate significant processing of chromogranin B in brain and its widespread distribution, can be taken as an indication that chromogranin B represents a precursor of peptides with functional relevance for this organ.

Distinct Effects of Recombinant Tenascin-R Domains in Neuronal Cell Functions and Identification of the Domain Interacting with the Neuronal Recognition Molecule F3/11

May 1996

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14 Reads

We have identified distinct domains of the rat extracellular matrix glycoprotein tenascin-R using recombinant fragments of the molecule that confer neuronal cell functions. In short-term adhesion assays (0.5 h), cerebellar neurons adhered best to the fragment representing the fibrinogen knob (FG), but also the fibronectin type III (FN) repeats 1-2 and 6-8. FG, FN1-2 and FN3-5 were the most repellent fragments for neuronal cell bodies. Neurites and growth cones were strongly repelled from areas coated with fragments containing the cysteine-rich stretch and the EGF-like domains (EGF-L), FN1-2, FN3-5 and FG. Polarization of morphology of hippocampal neurons was exclusively associated with FG, while EGF-L prevented neurite outgrowth altogether. The binding site of the neuronal receptor for tenascin-R, the immunoglobulin superfamily adhesion molecule F3/11, was localized to EGF-L. The combined observations show distinct, but also overlapping functions for the different tenascin-R domains. They further suggest the existence of multiple neuronal tenascin-R receptors which influence the response of neurons to their extracellular matrix environment.

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