Tyrosine kinase A, C and fibroblast growth factor-2 receptors in bovine embryos cultured in vitro.

Neurotrophins and basic fibroblast growth factor are ligands of tyrosine kinase receptors, though they bind to different tyrosine kinase receptor classes. Neurotrophins bind to receptor tyrosine kinase class VII, Trk receptor family, while basic fibroblast growth factor binds to receptor tyrosine kinase class IV, FGF receptor family. The mammalian uterine tract immunolocalizes neurotrophins and bFGF; therefore their cognate receptors might exert a role during embryonic development. Using RT-PCR, we found mRNA for p75(NTR) TrkA, TrkC and FGFr2 throughout the early bovine embryonic development in vitro. Immunofluorescent staining, assessed by confocal microscopy, showed the expression of TrkA and TrkC proteins in oocytes and all embryonic stages analyzed. We have provided a novel description of TrkA and TrkC proteins, and TrkA, TrkC, p75(NTR) and FGFr2 mRNA expression throughout mammalian embryonic development. This work may help to design future research with neurotrophins in bovine embryo culture and embryonic stem cells.

Nefopam is more potent than carbamazepine for neuroprotection against veratridine in vitro and has anticonvulsant properties against both electrical and chemical stimulation.

Nefopam (NEF) is a known analgesic that has recently been shown to be effective in controlling both neuropathic pain and convulsions in rodents. In this study we compared nefopam to carbamazepine (CBZ), a reference antiepileptic drug (AED), for their ability to protect cerebellar neuronal cultures from neurodegeneration induced by veratridine (VTD). Furthermore, we tested nefopam for protection against both, maximal electroshock-induced seizures (MES), and isoniazid-induced seizures in mice. Both NEF and CBZ were effective in preventing both signs of excitotoxicity and neurodegeneration following exposure of cultures to 5 microM veratridine for 30 min and 24 h, respectively. Concentrations providing full neuroprotection were 500 microM CBZ and 50 microM NEF, while the concentration providing 50% neuroprotection was 200 microM for CBZ and 20 microM for NEF. Neither NEF nor CBZ reduced excitotoxicity following direct exposure of cultures to glutamate, but CBZ failed to reduce increases in intracellular calcium following stimulation of L-type voltage sensitive calcium channels. In vivo, NEF (20 mg/kg i.p.) significantly reduced MES and fully prevented MES-induced terminal clonus (TC). In comparison, NEF was significantly more effective than CBZ in preventing MES, although both drugs were equally effective against MES-induced TC. Furthermore, nefopam provided protection against isoniazid-induced seizures at doses similar to those protecting against MES.

Nefopam inhibits calcium influx, cGMP formation, and NMDA receptor-dependent neurotoxicity following activation of voltage sensitive calcium channels.

Nefopam hydrochloride is a potent non sedative benzoxazocine analgesic that possesses a profile distinct from that of anti-inflammatory drugs. Previous evidence suggested a central action of nefopam but the detailed mechanism remains unclear. We have investigated the actions of nefopam on voltage sensitive calcium channels and calcium-mediated pathways. We found that nefopam prevented N-methyl-D-aspartate (NMDA)-mediated excitotoxicity following stimulation of L-type voltage sensitive calcium channels by the specific agonist BayK8644. Nefopam protection was concentration-dependent. 47 muM nefopam provided 50% protection while full neuroprotection was achieved at 100 muM nefopam. Neuroprotection was associated with a 73% reduction in the BayK8644-induced increase in intracellular calcium concentration. Nefopam also inhibited intracellular cGMP formation following BayK8644 in a concentration-dependent manner, 100 muM nefopam providing full inhibition of cGMP synthesis and 58 muM allowing 50% cGMP formation. Nefopam reduced NMDA receptor-mediated cGMP formation resulting from the release of glutamate following activation of channels by BayK8644. Finally, we also showed that nefopam effectively reduced cGMP formation following stimulation of cultures with domoic acid, while not providing neuroprotection against domoic acid. Thus, the novel action of nefopam we report here may be important both for its central analgesic effects and for its potential therapeutic use in neurological and neuropsychiatric disorders involving an excessive glutamate release.

Nefopam, an analogue of orphenadrine, protects against both NMDA receptor-dependent and independent veratridine-induced neurotoxicity.

Nefopam hyghochloride is a potent analgesic compound commercialized in most Western Europe for 20 years, which possesses a profile distinct from that of opioids or anti-inflammatory drugs. Previous evidence suggested a central action of nefopam but the detailed mechanisms remain unclear. While, nefopam structure resembles that of orphenadrine, an uncompetitive NMDA receptor antagonist, here we report that differently from orphenadrine, nefopam (100 microM) failed to protect cultured cerebellar neurons from excitotoxicity following direct exposure of neurons to glutamate. Moreover, nefopam failed to displace MK-801 binding to hippocampal membranes. Nefopam effectively prevented NMDA receptor-mediated early appearance (30 min) of toxicity signs induced by the voltage sensitive sodium channel (VSSC) activator veratridine. The later phase (24 h) of neurotoxicity by veratridine occurring independently from NMDA receptor activation, was also prevented by nefopam. Nefopam effect was not mimicked by the GABA receptor agonist muscimol.

Comparison of the in vitro and in vivo neurotoxicity of three new sources of kainic acid.

Historically, all commercially available kainic acid has been derived from a single biological source using a consistent method of extraction and purification. That source became unavailable in 1995. Recently, three new commercial suppliers of kainic acid have made the product available, but the source of the material and the purification processes used differ. Our objective was to systematically compare the response produced by each of these new sources of kainic acid using three established neurobiological techniques: neuronal cell culture, hippocampal slice electrophysiology, and whole animal behavioural toxicity. Results in all three systems indicated no overall differences between the three formulations, although studies in both cerebellar neuron cultures and whole animal toxicity testing in mice, revealed some significant differences that may imply subtle differences in receptor selectivity and/or potency. We conclude that all three sources of kainic acid are viable alternatives to traditional kainate but they may not be identical. Until further information becomes available researchers may want to avoid using the three formulations interchangeably, and take note of the source of kainic acid when evaluating literature describing results from other laboratories.

Novel effect of nefopam preventing cGMP increase, oxygen radical formation and neuronal death induced by veratridine.

Nefopam hydrochloride is a potent analgesic compound that possesses a profile distinct from that of opiods or anti-inflammatory drugs. Previous evidence suggested a central action of nefopam but the detailed mechanisms remain unclear. Here we have used cultured cerebellar neurons to test the hypothesis that nefopam may modulate voltage sensitive sodium channel (VSSC) activity. Nefopam (100 microM) effectively prevented NMDA receptor-mediated early appearance (30 min) of toxicity signs induced by the VSSC activator veratridine. Delayed neurotoxicity by veratridine occurring independently from NMDA receptor activation, was also prevented by nefopam. In contrast, excitotoxicity following direct exposure of neurons to glutamate was not affected. Neuroprotection by nefopam was dose-dependent. 50% protection was obtained at 57 microM while full neuroprotection was achieved at 75 microM nefopam. Veratridine-induced sodium influx was completely abolished in nefopam-treated neurons. Intracellular cGMP and oxygen radical formation following VSSC stimulation by veratridine were also effectively prevented by nefopam. Our data are consistent with an inhibitory action of nefopam on VSSC and suggest that nefopam may modulate the release of endogenous glutamate following activation of these channels. This novel action of nefopam may be of great interest for the treatment of neurodegenerative disorders involving excessive glutamate release and neurotransmission.

Antihistamine terfenadine potentiates NMDA receptor-mediated calcium influx, oxygen radical formation, and neuronal death.

We previously reported that the histamine H1 receptor antagonist terfenadine enhances the excitotoxic response to N-methyl-D-aspartate (NMDA) receptor agonists in cerebellar neurons. Here we investigated whether this unexpected action of terfenadine relates to its antihistamine activity, and which specific events in the signal cascade coupled to NMDA receptors are affected by terfenadine. Low concentrations of NMDA (100 microM) or glutamate (15 microM) that were only slightly (<20%) toxic when added alone, caused extensive cell death in cultures pre-exposed to terfenadine (5 microM) for 5 h. Terfenadine potentiation of NMDA receptor response was mimicked by other H1 antagonists, including chlorpheniramine (25 microM), oxatomide (20 microM), and triprolidine (50 microM), was prevented by histamine (1 mM), and did not require RNA synthesis. Terfenadine increased NMDA-mediated intracellular calcium and cGMP synthesis by approximately 2.4 and 4 fold respectively. NMDA receptor-induced cell death in terfenadine-treated neurons was associated with a massive production of hydrogen peroxides, and was significantly inhibited by the application of either (+)-alpha-tocopherol (200 microM) or the endogenous antioxidant melatonin (200 microM) 15 min before or up to 30 min after receptor stimulation. This operational time window suggests that an enduring production of reactive oxygen species is critical for terfenadine-induced NMDA receptor-mediated neurodegeneration, and strengthens the importance of antioxidants for the treatment of excitotoxic injury. Our results also provide direct evidence for antihistamine drugs enhancing the transduction signaling activated by NMDA receptors in cerebellar neurons.

NMDA receptor dependent and independent components of veratridine toxicity in cultured cerebellar neurons are prevented by nanomolar concentrations of terfenadine.

Exposure of cultured neurons to nanomolar concentrations of terfenadine prevented the NMDA receptor-mediated early appearance (30min.) of toxicity signs induced by the voltage sensitive sodium channel activator veratridine. Terfenadine also provided an histamine-insensitive protection against delayed neurotoxicity by veratridine (24h), occurring independently of NMDA receptor activation, while not protecting from excitotoxicity following direct exposure of neurons to glutamate. Terfenadine reduced tetrodotoxin-sensitive inward currents, and reduced intracellular cGMP formation following veratridine exposure. Our data suggest that nanomolar concentrations of TEF may reduce excitatory aminoacid release following neuronal depolarization via a presynaptic mechanism involving voltage sensitive sodium channels, and therefore may be considered as a prototype for therapeutic drugs in the treatment of diseases that involve excitatory aminoacid neurotransmission.

Terfenadine prevents NMDA receptor-dependent and -independent toxicity following sodium channel activation.

Exposure of cultured cerebellar neurons to terfenadine prevented the N-methyl-D-aspartate (NMDA) receptor-mediated early appearance (30 min) of toxicity signs induced by the voltage sensitive sodium channel (VSSC) activator veratridine. Delayed neurotoxicity by veratridine (24 h) occurring independently from NMDA receptor activation was also prevented by terfenadine. Terfenadine did not protect from excitotoxicity following direct exposure of neurons to glutamate. Our results suggest that terfenadine may modulate endogenous glutamate release following activation of VSSCs.

Aluminum-induced degeneration of astrocytes occurs via apoptosis and results in neuronal death.

The mechanisms by which aluminum interacts with the nervous system are only partly understood. In this study, we used cultured astrocytes and neurons to investigate the effects of long exposures to aluminum (1 mM). We found that aluminum accumulated both in neurons and astrocytes. After 8-12 days exposure, aluminum caused strong changes in the morphology of astrocytes including shrinkage of cell bodies and retraction of processes. Exposures over 15-18 days reduced astrocytes viability by 50%. Aluminum-induced degeneration of astrocytes involved the DNA fragmentation characteristic of apoptosis, and staining of aluminum-treated astrocytes with the DNA-binding fluorochrome Hoeschst 33258 revealed the typical apoptotic condensation and fragmentation of chromatin. Aluminum was also found to be neurotoxic, causing first (4-6 days) abnormal clustering and aggregation, and later (8-12 days) neuronal death. Interestingly, aluminum neurotoxicity occurred in neuroglial cultures containing approximately 10% astrocytes but not in near-pure neuronal cultures containing only 1% astrocytes. Staining of co-cultured cells with Hoeschst 33258 showed apoptotic condensation and fragmentation of chromatin in aluminum-treated astrocytes but not in co-cultured neurons. Our study demonstrates that aluminum can induce the apoptotic degeneration of astrocytes, and that this toxicity is critical in determining neuronal degeneration and death. Aluminum-mediated apoptosis of cultured astrocytes may be also a valuable model system to study the mechanisms underlying apoptosis in glial cells.

Terfenadine induces toxicity in cultured cerebellar neurons: a role for glutamate receptors.

Exposure of cultured cerebellar neurons to the histamine H1 receptor antagonist terfenadine resulted in neuronal degeneration and death. Terfenadine neurotoxicity was dependent upon concentration and time of exposure. After 2 h exposure, 20 microM terfenadine reduced the number of surviving neurons by 75%, and as low as 10 nM terfenadine induced significant neurotoxicity after 5 days of exposure. Neuronal sensitivity to terfenadine changed with age in culture, and at 25 days in culture neurons appeared to be much less sensitive than at 5 or 9-17 days in culture. Neurotoxicity by terfenadine could not be prevented by high concentrations of histamine (5 mM), but it was significantly delayed by blocking NMDA or non-NMDA glutamate receptors with MK-801 or CNQX respectively, suggesting the involvement of excitatory transmission mediated by glutamate in the neurotoxicity induced by terfenadine in these neurons. We also found that the presence of terfenadine (5 microM) unveiled the potential excitotoxity of the non-NMDA receptor agonist AMPA (100 microM), and reduced the concentration of glutamate necessary to induce excitotoxicity, compared to untreated cultures. These results suggest a role for terfenadine in the modulation of the excitotoxic response mediated in cerebellar neurons through ionotropic glutamate receptors.

Inhibition of protein phosphatases induces IGF-1-blocked neurotrophin-insensitive neuronal apoptosis.

We have previously described the marine toxin okadaic acid (OKA) to be a potent neurotoxin for cultured rat cerebellar neurons. Here we show that OKA-induced neurodegeneration involves the DNA fragmentation characteristic of apoptosis and is protein synthesis-dependent. DNA fragmentation and neurotoxicity correlated with inhibition of protein phosphatase (PP) 2A rather than PP1 activity. Neurotrophins NT-3 and BDNF failed to protect from OKA-induced apoptotic neurotoxicity that was, however, totally prevented by insulin-like growth factor-1. Neuronal death by OKA was significantly reduced by protein kinase C inhibitors and by the L-type calcium channel agonist Bay K8644, while it was potentiated by the reduction of free extracellular calcium concentrations.

Beta-amyloid precursor protein in human digital skin.

The occurrence and distribution of beta-amyloid precursor protein (beta APP) and of beta-amyloid peptide (beta/A4) was investigated using immunoblotting and immunohistochemical techniques in the digital skin of healthy adult subjects. beta APP-like proteic bands with apparent molecular masses between 55-60 kDa, 100-125 kDa (corresponding to the full-length beta APP isoforms), 145-150 kDa, and 200 kDa were found in pellets and supernatants of whole skin and dermis. The same proteins, except that of approximately 200 kDa, were also found in pellets from the epidermis, whereas epidermic supernatants were unreactive. beta/A4 was not found by immunoblotting. Light microscope immunohistochemistry showed beta APP immunoreactivity (IR) in: (a) dermal nerves; (b) lamellar cells of Meissner, as well as inner-core, outer-core and capsule of Pacinian corpuscles; and (c) dermal blood vessels, sweat glands and, occasionally, epidermis. The distribution of beta/A4 IR matched that of beta APP, and no evidence of extracellular beta/A4 IR was encountered. Present results demonstrate that beta APP, but not beta/A4, is normally present in human glabrous (digital) skin. The potential clinical relevance of these findings is discussed.

Expression of beta-amyloid precursor protein (APP) in human dorsal root ganglia.

The present study reports the occurrence and localization of beta-amyloid precursor protein (APP) immunoreactivity (IR) in human lumbar dorsal root ganglia of healthy adult subjects (age range 25-43 years). To ascertain that ganglionic cells displayed APP IR, neurofilament (NFP) and S-100 proteins (S100P) were studied in parallel. Immunoblotting revealed four or five major proteins with apparent molecular masses between 100-125 kDa, which corresponded with the different full-length APP isoforms. Moreover, an additional protein of approximately 55 kDa was detected. Selective APP IR was observed restricted to the satellite glial cell cytoplasms whereas neuron cell bodies resulted unlabeled. Moreover, some intraganglionic nerve fibers also displayed APP IR, apparently labelling Schwann cells. No individual differences among subjects were observed neither in the pattern of APP IR distribution, nor in the intensity of APP IR. Although it remains to be demonstrated whether or not human primary sensory neurons express APP, present results strongly suggest that supporting glial cells may be a primary source of APP or any related peptide, at least in adult healthy people. The functional and clinical relevance of these findings, if any, remain to be clarified.

Two components in neurotoxicity by L-2-amino-3-phosphonopropionate in cultured cerebellar neurons.

Exposure of cultured cerebellar neurons to the putative metabotropic glutamate receptor antagonist L-2-amino-3-phosphonopropionate (L-AP3) for 24 h produced a neurotoxic effect which was prevented by the addition of the NMDA receptor antagonist (+)-10,11-dihydro-5-methyl-5-H-dibenzo-[a,d]-cyclohepten-5,1 0-imine hydrogen maleate (MK-801). MK-801 did also reduce neurotoxicity following 72 h exposure to L-AP3 neurotoxicity in the presence of MK-801 was antagonized by glutamate. Our results suggest that metabotropic glutamate receptors may play an important role in neuronal survival by controlling NMDA receptor-dependent as well as independent pathways.

Basic fibroblast growth factor protects cerebellar neurons in primary culture from NMDA and non-NMDA receptor mediated neurotoxicity.

We have investigated the ability of bFGF to protect cerebellar neurons from neurotoxicity by excitatory amino acids. We have found that preincubation with 1-2.5 nM bFGF for 1-6 days significantly protected neurons from excitotoxic damage via NMDA receptors as well as ionotropic non-NMDA receptors. bFGF neuroprotection appeared not to be dependent upon neuronal differentiation and was not mimicked by other neurotrophins including BDNF, NT-3 and NGF. A greater rise in extracellular calcium-dependent cGMP formation, following either depolarization or excitatory amino acid receptor activation was observed in bFGF-pretreated neurons. We suggest that neuroprotection from excitotoxicity following bFGF treatment may be associated to the modulation of neurochemical pathways dependent upon extracellular calcium influx.

Domoic acid-containing toxic mussels produce neurotoxicity in neuronal cultures through a synergism between excitatory amino acids.

In 1987, an intoxication by cultured mussels produced neurological problems, such as headache, confusion, and loss of memory, particularly severe at times. Neuronal damage was found in the hippocampus and amygdala of four patients. The intoxication was attributed to the presence in mussels of domoic acid, a rare excitatory amino acid acting at the non-NMDA receptor. We now report that a domoic acid-containing mussel extract is more neurotoxic for cultured neurons than purified domoic acid. Moreover, we show that this increase in neurotoxicity is selectively due to domoic acid potentiation of the excitotoxic effect of glutamic acid and aspartic acid present in high concentrations in mussel tissue. We also show that subtoxic concentrations of domoic acid are sufficient to potentiate glutamic acid and aspartic acid neurotoxicity, and we present evidence suggesting that the neurotoxic synergism may occur through a reduction of the voltage-dependent Mg2+ block at the NMDA receptor-associated channel, following activation of non-NMDA receptors by domoic acid. Thus, based on our results, we suggest that the contemporary presence in the brain of concentrations of domoic acid insufficient alone to be toxic, together with excitatory amino acids, of endogenous and eventually of diet-related origin, may have been relevant in the occurrence of the neurological problems reported.

The amnesic shellfish poison domoic acid enhances neurotoxicity by excitatory amino acids in cultured neurons.

A recent episode of human intoxication by cultured mussels containing a rare excitatory amino acid named domoic acid, received particular attention for its neurological implications. The intoxication produced neurological problems, such as headache, confusion, and loss of memory, particularly severe at times. Neuronal damage was found in the hippocampus and amygdala of four patients. We now report that in neuronal cultures the neurotoxicity of a domoic acid-containing mussel extract is the result of domoic acid potentiation of the excitotoxic effect of glutamic acid and aspartic acid present in high amounts in mussel tissue. Moreover, we show that subtoxic concentrations of domoic acid are sufficient to potentiate glutamic acid and aspartic acid neurotoxicity. We present evidence suggesting that the neurotoxic synergism may be due to a reduction of Mg(+ +) block at the NMDA receptor-associated channel, following activation of NON-NMDA receptors by domoic acid.