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We investigated the effect of chronically blocking NMDA receptor stimulation to examine changes in GABA(A) receptor expression and pharmacology in cerebellar granule cells at different stages of maturation. We have previously shown that NMDA-selective glutamate receptor stimulation alters GABA(A) receptor pharmacology in cerebellar granule neurons in vitro by altering the levels of selective subunits. When NMDA receptor stimulation is blocked with MK-801 during the first week in vitro, a decrease in the alpha1, gamma2S, and gamma2L receptor subunit mRNAs occurred. When present only during the second week, changes were limited to the alpha1 and gamma2L mRNAs. Finally, if MK-801 was present during the first week and removed during the second week, these changes reversed. Whole-cell voltage-clamp recordings showed that treatment with MK-801 during either the first or second week increased the EC50 of the receptors for GABA and attenuated the potentiation mediated by flunitrazepam. Last, these properties were reversed if MK-801 was removed after the first week in vitro. Our results suggest that MK-801 reversibly inhibits GABA(A) receptor maturation by modulating receptor subunit expression and that the altered pharmacological responses appear to be dominated by changes in the levels of allosteric modulation mediated by the gamma2 receptor subunit.

Immunoblot analysis, using antibodies against distinct N-methyl-D-aspartic acid (NMDA) receptor subunits, illustrated that the NR2A and NR2B subunit proteins have developmental profiles in cultured cortical neurons similar to those seen in vivo. NR1 and NR2B subunits display high levels of expression within the first week. In contrast, the NR2A subunit is barely detectable at 7 days in vitro (DIV) and then gradually increased to mature levels at DIV21. Immunocytochemical analysis indicated that NMDA receptor subunits cluster in the dendrites and soma of cortical neurons. Clusters of NR1 and NR2B subunits were observed as early as DIV3, while NR2A clusters were rarely observed before DIV10. At DIV18, NR2B clusters partially co-localize with those of NR2A subunits, but NR2B clusters always co-localize with those of NR1 subunits. Synapse formation, as indicated by the presence of presynaptic synaptophysin staining, was observed as early as 48-72 h after plating. However, in several neurons at ages less than DIV5 where synapses were scarce, NR2B and NR1 clusters were abundant. Furthermore, while NR2B subunit clusters were seen both at synaptic and extrasynaptic sites, NR2A clusters occurred almost exclusively in front of synaptophysin-labelled boutons. This result was supported by electrophysiological recording of NMDA-mediated synaptic activity [NMDA-excitatory postsynaptic currents (EPSCs)] in developing neurons. At DIV6, but not at DIV12, CP101, 606, a NR1/NR2B receptor antagonist, antagonized spontaneously occurring NMDA-EPSCs. Our data indicate that excitatory synapse formation occurs when NMDA receptors comprise NR1 and NR2B subunits, and that NR2A subunits cluster preferentially at synaptic sites.

Recordings of NMDA-activated currents from cerebellar granule neurons in culture revealed a developmental increase in current density accompanied by a slight decrease of the half-maximal effective concentration. At the same time, a decrease of NMDA receptors comprising NR2B subunits was demonstrated by the reduction in the antagonism of NMDA currents by ifenprodil. Ifenprodil antagonism increased after treatment for 24 h with KN93- and KN62-selective inhibitors of the Ca2+/calmodulin-dependent protein kinases (CaM kinases), indicating a selective increase of receptor containing NR2B subunit. This increase was observed at all ages tested: 4 days in vitro (DIV4), DIV6, and DIV13. Western blot analysis with specific NMDA receptor antibodies performed at DIV6 confirmed the electrophysiological data. At this age, the negative control KN92 was ineffective. The increasing ifenprodil antagonism after KN93 treatment was proportionally greater in cells at DIV13 than at DIV4. Treatment with NMDA (100 microM) of cerebellar cultures for 24 h produced a decrease in the NMDA-induced current density by almost 50% at all ages tested. Ifenprodil antagonism, however, was unchanged. We propose that the expression of NR2B subunits in cerebellar granule cells is selectively stimulated by the inhibition of CaM kinases.

We have shown that the vertebrate neuropeptide N-acetylaspartylglutamate (NAAG) meets the criteria for a neurotransmitter, including function as a selective metabotropic glutamate receptor (mGluR) 3 agonist. Short-term treatment of cerebellar granule cells with NAAG (30 microM) results in the transient increase in content of GABA(A) alpha6 subunit mRNA. Using quantitative PCR, this increase was determined to be up to 170% of control values. Similar effects are seen following treatment with trans-1-aminocyclopentane-1,3-dicarboxylate and glutamate and are blocked by the mGluR antagonists (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl) glycine and (2S)-alpha-ethylglutamic acid. The effect is pertussis toxin-sensitive. The increase in alpha6 subunit mRNA level can be simulated by activation of other receptors negatively linked to adenylate cyclase activity, such as adenosine A1, alpha2-adrenergic, muscarinic, and GABA(B) receptors. Forskolin stimulation of cyclic AMP (cAMP) levels abolished the effect of NAAG. The change in alpha6 levels induced by 30 microM NAAG can be inhibited in a dose-dependent manner by simultaneous application of increasing doses of the beta-adrenergic receptor agonist isoproterenol. The increase in alpha6 mRNA content is followed by a fourfold increase in alpha6 protein level 6 h posttreatment. Under voltage-clamped conditions, NAAG-treated granule cells demonstrate an increase in the furosemide-induced inhibition of GABA-gated currents in a concentration-dependent manner, indicating an increase in functional alpha6-containing GABA(A) receptors. These data support the hypothesis that NAAG, acting through mGluR3, regulates expression of the GABA(A) alpha6 subunit via a cAMP-mediated pathway and that cAMP-coupled receptors for other neurotransmitters may similarly influence GABA(A) receptor subunit composition.

A case is reported of amyotrophic lateral sclerosis with occupational exposure to solvents and metals. The environmental toxin theory of motorneuron disease, including aspects of epidemiological, clinical, experimental and individual susceptibility is discussed. An overall evaluation of animal and human data was made using a methodological approach developed by the International Agency for Research on Cancer and a Scandinavian group of experts. It is concluded that there is a probable linkage between metals/solvents exposure and motorneuron disease.

A case of rhabdomyolysis with acute renal failure associated with bezafibrate and furosemide treatment is reported. The possible interaction between these drugs in determining myopathic toxicity is underlined.

Insulin response and FFA behavior have been evaluated during an IVGTT in 63 subjects of whom 18 were normal, 31 were obese (with varying degrees of carbohydrate tolerance) and 14 were mild non insulin-dependent diabetics. The extreme reduction of insulin secretion in the early phase (delta 0-15 min) and the less severe impairment of the late phase (delta 15-60 min) have been confirmed; obese subjects showed on the average an active insulin response to venous loading; this was more marked and more consistent in the late phase. Compared to controls, FFA concentration both in basal conditions and during IVGTT was progressively higher in obese and diabetic patients. When analyzing the interplay between IRI, KG and FFA in the course of IVGTT, it was observed that: (1) a close correlation exists between IG and early insulin response (r = 0.72); (2) a correlation between delta IRI 0-15 min and percentage decrease of FFA at 45 min is found only in normal subjects; (3) a negative highly significant correlation is found between KG and mean FFA plasma level 0-60 min. This last correlation is evidence of the important role played by FFA in carbohydrate tolerance. The conflicting results reported by others have been discussed.

We have studied the correlation between NEFA and KB in 25 controls and in 24 diabetics, receiving or no insulin therapy. There is a correlation (r=0,64) between NEFA and KB in normal subjects and a more significant correlation (r=0,85) in diabetics. The "b" value of the two regression lines in the two groups is different, and this is dependent by variations in the hormonal (insulin and glucagon) and metabolic responses.

13 normal and 16 obese subjects have been chosen for a double insulin tolerance test: 0,02 U/Kg of insulin were administered i.v. with an interval of 60'. The glycemic curve of the normal subjects show an identical lay out after both pulses; on the contrary the obese subjects could be divided into two subgroups. In the first one the lowering glucose action can be compared after both pulses, while in the second one the first stimulus causes a weather lowering glucose action, than in the first group, which is furtherly reduced during the subsequent pulse. An insulin resistance in these subjects is thus stressed. In the obese subjects the NEFA have a higher concentration in both groups than in the normal ones, show a normal decrease, but a certain delay is observed in the rebound phase.