17ß-Estradiol/N-acetylcysteine interaction enhances the neuroprotective effect on dopaminergic neurons in the weaver model of dopamine deficiency.

The weaver mouse, is a phenocopy of Parkinson's disease (PD) in which dopaminergic neurons degenerate gradually during development, reaching at P21 a neurodegeneration of 55%. Thus, the weaver mouse constitutes an appropriate in vivo PD model for investigating the effect of neuroprotective agents. In the present study, long-term treatment (from P1 to P21) with 17ß-estradiol (17ß-estradiol) significantly protected the dopaminergic neurons in the substantia nigra (SN) of weaver mouse by 54%, as was detected by immunohistochemical experiments, using the specific antibody against tyrosine hydroxylase (TH). This dopaminergic neuroprotection is in line with our biochemical results showing that 17ß-estradiol treatment significantly decreased the high lipid peroxidation levels seen in the SN of weaver mouse, indicating high oxidative stress. Interestingly, co-administration of 17ß-estradiol with N-acetylcysteine (NAC, precursor molecule of glutathione (GSH)) further significantly increased the survival of dopaminergic neurons in the SN (by 85%), with a parallel further decrease of lipid peroxidation to normal levels. Our results show the in vivo neuroprotective effect of 17ß-estradiol, which is strongly enhanced by co administration of NAC, indicating a strong synergistic effect of the two drugs. Furthermore, the main mechanism underlying this neuroprotective action seems to be the reversal of the oxidative stress shown by the high peroxidation levels. These results could be of clinical relevance since both drugs are already used separately in the clinic, 17ß-estradiol for treatment of PD and NAC as a mucolytic agent and for the treatment of several disorders.

Synergistic interactions of dopamine D1 and glutamate NMDA receptors in rat hippocampus and prefrontal cortex: involvement of ERK1/2 signaling.

Interactions between dopamine and glutamate receptors are essential for the prefrontal cortical (PFC) and hippocampal cognitive functions. In order to understand the molecular basis of dopamine/glutamate interactions in rat PFC and hippocampus, we investigated (a) the effect of in vitro dopamine D1 receptor stimulation on glutamate N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits' phosphorylation and (b) the signal transduction pathway underlying these interactions, by examining the involvement of D1-extracellular regulated kinase 1/2 (ERK1/2) and D1/protein kinase A (PKA)/dopamine- and cyclic AMP-regulated phosphoprotein-32 (DARPP-32) signaling pathways. Furthermore, we compared the D1/NMDA/AMPA receptor interactions seen in PFC and hippocampus with those appearing in striatum, in which the D1 receptors' density is the highest within the mammalian brain. Our results showed that stimulation of D1 receptor by the specific agonist SKF38393 (10 microM) in PFC and hippocampal slices significantly increased the phosphorylation state of NR1ser897 and NR2Bser1303 subunits of NMDA receptor and of the GLUR1 (ser831 and ser845) subunit of AMPA receptor, as well as of ERK1/2, but not of DARPP-32. Interestingly, co-stimulation of D1 and NMDA receptors with an ineffective dose of SKF38393 (2 microM) and NMDA (5 microM) respectively, elevated further the phosphorylation level of NMDA and AMPA receptor subunits, as well as of ERK1/2, but not of DARPP-32. The D1- and D1/NMDA-induced phosphorylations were totally inhibited by SL327 (specific ERK1/2 inhibitor). Conversely, in striatal slices our data confirm that the D1-mediated phosphorylation of NMDA and AMPA receptor subunits relies on D1/PKA/DARPP-32 signaling. In conclusion, in PFC and hippocampus: (a) a strong synergistic interaction of D1 and NMDA receptors exists, which results in a significant ERK1/2 pathway activation, (b) the D1 and the D1/NMDA receptor-induced phosphorylation of NMDA and AMPA receptor subunits seems to rely on ERK1/2 signaling and could to some extent underlie the enhancement of NMDA and AMPA receptor currents mediated by D1 receptor activation.

Differential expression of NMDA and AMPA receptor subunits in rat dorsal and ventral hippocampus.

Several studies have demonstrated anatomical and functional segregation along the dorsoventral axis of the hippocampus. This study examined the possible differences in the AMPA and NMDA receptor subunit composition and receptor binding parameters between dorsal and ventral hippocampus, since several evidence suggest diversification of NMDA receptor-dependent processes between the two hippocampal poles. Three sets of rat dorsal and ventral hippocampus slices were prepared: 1) transverse slices for examining a) the expression of the AMPA (GluRA, GluRB, GluRC) and NMDA (NR1, NR2A, NR2B) subunits mRNA using in situ hybridization, b) the protein expression of NR2A and NR2B subunits using Western blotting, and c) by using quantitative autoradiography, c(1)) the specific binding of the AMPA receptor agonist [(3)H]AMPA and c(2)) the specific binding of the NMDA receptor antagonist [(3)H]MK-801, 2) longitudinal slices containing only the cornus ammonis 1 (CA1) region for performing [(3)H]MK-801 saturation experiments and 3) transverse slices for electrophysiological measures of NMDA receptor-mediated excitatory postsynaptic potentials. Ventral compared with dorsal hippocampus showed for NMDA receptors: 1) lower levels of mRNA and protein expression for NR2A and NR2B subunits in CA1 with the ratio of NR2A /NR2B differing between the two poles and 2) lower levels of [(3)H]MK-801 binding in the ventral hippocampus, with the lowest value observed in CA1, apparently resulting from a decreased receptor density since the B(max) value was lower in ventral hippocampus. For the AMPA receptors CA1 our results showed in ventral hippocampus compared with dorsal hippocampus: 1) lower levels of mRNA expression for GluRA, GluRB and GluRC subunits, which were more pronounced in CA1 and in dentate gyrus region and 2) lower levels of [(3)H]AMPA binding. Intracellular recordings obtained from pyramidal neurons in CA1 showed longer NMDA receptor-mediated excitatory postsynaptic potentials in ventral hippocampus compared with dorsal hippocampus. In conclusion, the differences in the subunit mRNA and protein expression of NMDA and AMPA receptors as well as the lower density of their binding sites observed in ventral hippocampus compared with dorsal hippocampus suggest that the glutamatergic function differs between the two hippocampal poles. Consistently, the lower value of the ratio NR2A/NR2B seen in the ventral part would imply that the ventral hippocampus NMDA receptor subtype is functionally different than the dorsal hippocampus subtype, as supported by our intracellular recordings. This could be related to the lower ability of ventral hippocampus for long-term synaptic plasticity and to the higher involvement of the NMDA receptors in the epileptiform discharges, observed in ventral hippocampus compared with dorsal hippocampus.

Stimulation of adenosine A2A receptors elicits zif/268 and NMDA epsilon2 subunit mRNA expression in cortex and striatum of the "weaver" mutant mouse, a genetic model of nigrostriatal dopamine deficiency.

Interaction between basal ganglia and cerebral cortex is critical for normal goal-directed behavior. In the present study we have used the immediate early gene zif/268, as functional marker to investigate how the stimulation of adenosine A2A receptors, i.e. of the "indirect" striatal output pathway, affects striatal and cortical function in "weaver" mouse, a genetic model of dopamine deficiency. Furthermore, we have examined the effect of A2A receptor stimulation on glutamate receptor expression in the "weaver" brain. A single injection of CGS21680 (A2A receptor agonist), induced strong expression of zif/268 mRNA, detected by in situ hybridization, not only in striatum but also in the motor cortex of the "weaver" mutant. This cortical response seems to be elicited through the basal-ganglia-thalamo-cortical circuit, rather than through a direct cortical effect, since A2A receptors are not detectable in cortex according to our autoradiographic study. Co-administration of CGS21680 and quinpirole (D2 receptor agonist) attenuated the expression of zif/268 mRNA in dorsal striatum but not in motor cortex, indicating that the cortical response is dopamine-D2-receptor-independent. However, this co-administration induced an increase in zif/268 mRNA expression in somatosensory cortex, which could rely on disinhibition of the thalamo-cortical pathway. The motor cortical response could be of clinical interest, as it would further stimulate the "indirect" striatal pathway in a feed forward circuit, thus worsening the parkinsonian symptoms. Furthermore, the up-regulation of epsilon2 subunit mRNA of the NMDA receptor, induced by CGS21680 administration, seen in striatum and cortex of the "weaver" mouse, would lead to overactivity of these receptors worsening dyskinesias. These results suggest adenosine to play a significant role in regulating striatal and cortical neurochemistry in a dopamine-depleted mouse. Blockade of these receptors by specific A2A antagonists could ameliorate parkinsonian symptoms.

Changes in AMPA receptor binding and subunit messenger RNA expression in hippocampus and cortex in the pentylenetetrazole-induced 'kindling' model of epilepsy.

'Kindling' is a phenomenon of epileptogenesis, which has been widely used as an experimental model of temporal lobe epilepsy. In the present study, we have examined the contribution of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) glutamate receptors and their subunits (GluR-A, -B, -C and -D) to the acquisition and maintenance of the kindled state in the pentylenetetrazole (PTZ)-induced 'kindling' mouse model, by using quantitative autoradiography and in situ hybridization. Region-specific increases in [3H]AMPA binding were seen in kindled animals in the CA3 region of hippocampus and in the temporal cortex 1 week after the last PTZ injection. At the same time, a significant decrease in the level of transcripts encoding the GluR-B and -C subunits was detected in the hippocampal CA1 region and dentate gyrus, suggestive of a higher proportion of Ca(2+)-permeable AMPA receptors in these neurons. These changes did not persist 1 month after establishment of kindling, indicating a transient role of AMPA receptors in the acquisition of the kindled state. At 1 month after the last PTZ injection, an upregulation in [3H]AMPA binding appeared in the motor cortex and the basal ganglia of kindled animals, which is consistent with electrophysiological data showing hyperexcitability in the cortex of the PTZ-kindled animals at that time. Interestingly, an increase in mRNA for the GluR-B subunit appeared in the outer layers of motor and somatosensory cortices of the kindled animals 1 month after acquisition of the kindled state, possibly as part of a gene-regulated, compensatory mechanism against seizure susceptibility, since this change should give rise to a higher proportion of Ca(2+)-impermeable AMPA receptors. These results support the evidence of a transient role of hippocampal AMPA receptors in the acquisition of the 'kindling' phenomenon and they also suggest an involvement of AMPA receptors in the maintenance of kindled state at least in two brain areas, cortex and basal ganglia.

Reduction of A1 adenosine receptors in rat hippocampus after kainic acid-induced limbic seizures.

In a temporal lobe epilepsy (TLE) model induced by kainic acid (KA), we examined the effect of limbic seizures on A1 adenosine receptor distribution in hippocampus and cortex. By using quantitative autoradiography, we determined a progressive decrease in A1 receptor density in CA1 and CA3 regions of hippocampus, which coincided in time with the degenerating process of hippocampal pyramidal cells. This result indicates that a great amount of A1 receptors are located postsynaptically on pyramidal cell dendrites. No difference in A1 receptor density was observed in the inner compared to the outer molecular layer of dentate gyrus, or in the infrapyramidal band compared to the outer layer of stratum oriens of CA3. This could indicate that the newly sprouted mossy fiber glutamatergic terminals do not contain A1 receptors, thus lacking a restrain in the release of glutamate.

Age-dependent changes in adenosine A1 receptor and uptake site binding in the mouse brain: an autoradiographic study.

Ageing is a multifactorial, inevitable event of life span, which affects neurotransmission in the CNS. Since adenosine is a major neuromodulator of the synaptic activity, it was of interest to investigate the possible modification of the adenosinergic system in the brain during ageing. Using "in vitro" quantitative autoradiography and the radioactive ligands [(3)H]Cyclohexyladenosine and [(3)H]Nitrobenzylthioinosine, we have studied the distribution of A1 adenosine receptors and adenosine uptake sites in the aged mice (26 months) compared to the young ones (3 months). Our results showed a widespread reduction in A1 receptor binding in the aged animals, which was brain area-specific, occurring in areas where adenosine plays a significant neuromodulatory role such as the hippocampus, cortex, basal ganglia, and thalamus. Interestingly, the significant reduction in NBI-sensitive adenosine uptake sites was restricted to few areas of the aged brain, mainly in thalamic nuclei. Since the alterations in the density of A1 receptors and adenosine uptake sites showed no regional correlation and since no significant changes in either neuronal or glial cell number are observed, at least in hippocampus and cortex in this mouse strain during ageing, our findings could be explained by a selective age-dependent reduction of these adenosinergic components rather than by a general neuronal cell degeneration. As adenosine depresses electrical activity in hippocampus, a downregulation of adenosinergic function could probably be related to enhanced excitability seen in hippocampal neurons of the CA1 subregion and dentate gyrus of aged animals.

Upregulation of NMDA receptors in hippocampus and cortex in the pentylenetetrazol-induced "kindling" model of epilepsy.

"Kindling" is a phenomenon of epileptogenesis, which has been widely used as an experimental model of temporal lobe epilepsy. At the present work we investigated the contribution of NMDA receptors in the Pentylenetetrazol-induced "kindling" model in the mouse brain, by using quantitative autoradiography and the radioactive ligands [3H]MK801 and [3H]L-glutamate (NMDA-sensitive component). One week after establishment of kindling, a small but significant increase in [3H]MK801 as well as NMDA-sensitive [3H]glutamate binding was seen, being restricted to the molecular layer (ML) of the dentate gyrus (DG) and the CA3 region of the hippocampus. These binding augmentations persisted one month after establishment of kindling. A significant increase of NMDA receptor binding was also observed in the cortex-somatosensory and temporal one week after acquisition of the kindled state. The upregulation of NMDA receptors seen in DG and CA3 region of the hippocampus could be associated with the kindling process of this model especially with its maintenance phase, since it persists at long term, is area-specific and consistent with electrophysiological data. The increase of NMDA receptors seen in the cortex of the kindled animals could underlie the hyperexcitability detected by electrophysiological studies in this area.

Time development and regional distribution of [3H]nitrobenzylthioinosine adenosine uptake site binding in the mouse brain after acute Pentylenetetrazol-induced seizures.

Adenosine has been shown to play a significant role as a modulator of neuronal activity in convulsion disorders, acting as an endogenous anticonvulsant agent. In the present study, we have investigated in mice the effect of acute tonic-clonic seizures induced by a single Pentylenetetrazol (PTZ)-injection (a) on the time development of adenosine uptake site binding after seizures in membranes of hippocampus, cortex, cerebellum, and striatum, and (b) on the regional distribution of adenosine uptake sites in the mouse brain by using "in vitro" quantitative autoradiography. As radioligand, the specific adenosine uptake blocker [3H]N-9-nitrobenzylthioinosine ([3H]NBI) was used. Acute seizures induced a rapid significant increase in [3H]NBI uptake site binding in hippocampus and cerebellum within 5 min, in cortex within 10 min after seizures, which reached a maximum level at 1 hr and reversed to control levels in about 150 min after seizures. On the contrary, in striatum a significant decrease of [3H]NBI uptake site binding was observed within 10 min after seizures, which reached its maximum at 1 hr and reversed to control levels at 150 min after seizures. With this single exception of striatum the "in vitro" quantitative autoradiography revealed a rather widespread upregulation of [3H]NBI uptake site density in the mouse brain, which was specifically enhanced in certain areas known to mediate seizure activity, such as hippocampus, specific thalamic nuclei, temporal cortex, and substantia nigra. The pattern of increases in [3H]NBI uptake site binding as they develop after acute seizures correlates well in time with the rapid enhancement of endogenous adenosine concentration released during epileptic activity. Since extracellular adenosine levels seem to be regulated by a rapid reuptake system, it seems likely that in our study, the [3H]NBI adenosine uptake system is acutely activated by seizures in order to compensate for the excess of endogenous adenosine. Furthermore, the upregulation of [3H]NBI uptake sites as revealed by the "in vitro" quantitative autoradiography seems to be organized in selective brain areas related to seizure propagation.

Lower density of A1 adenosine receptors in nucleus reticularis thalami in rats with genetic absence epilepsy.

The possible involvement of the adenosinergic modulatory system in the pathogenesis of absence seizures was investigated in genetic absence epilepsy rats from Strasbourg (GAERS). Using in vitro quantitative autoradiography, the distribution of A1 adenosine receptors and adenosine uptake sites in the brain of GAERS was studied and compared to that of control animals. An area-specific lower density of A1 receptors (15% decrease) was detected in reticular (nRT) and anterior ventral (AV) thalamic nuclei as well as basal ganglia in the brains of GAERS animals compared with control animals. Since adenosine exerts an anti-oscillatory effect on the thalamic nuclei by suppressing (via A1 receptors) excitatory as well as inhibitory neurotransmitter release, the impairment in A1 receptor density seen here, especially in nRT, could be implicated in the thalamic rhythmicity underlying spike and wave discharges present in this absence epilepsy model.

Pentylenetetrazol-induced seizures decrease gamma-aminobutyric acid-mediated recurrent inhibition and enhance adenosine-mediated depression.

To elucidate the consequences of convulsions, we examined biochemically and electrophysiologically the brains of mice that had sustained two complete tonic-clonic convulsions after administration of pentylenetetrazol (PTZ 50 mg/kg intraperitoneally, i.p.), 48 and 24 h before decapitation. Control mice were injected with saline. Input/output curves of the extracellular synaptic responses in the CA1 area of hippocampal slices showed that PTZ-induced seizures do not establish the persistent change in hippocampal excitability itself that can be detected in vitro. However, use of the paired-pulse stimulation paradigm showed that gamma-aminobutyric acid A (GABAA)-mediated recurrent inhibition was significantly weaker (by 19-25%) in the CA1 area of slices from PTZ-treated mice (PTZ slices) as compared with slices from control mice (control slices). The density of GABAA receptors (high-affinity component) was also lower in hippocampus (by 19%) and cortex (by 14%) of PTZ-treated mice. A GABA-related disinhibitory mechanism underlying PTZ seizures may thus persist for 1 day after the seizure, predisposing the brain to subsequent seizures. On the other hand, the depressant effect of a single dose of adenosine 10 microM on the CA1 synaptic response was stronger (by 35% on population spikes) and longer lasting in PTZ slices as compared with controls. This could be attributed to significantly higher adenosine A1 receptor density in hippocampus (Bmax of [3H]CHA was higher by 34%) as well as cortex and cerebellum of these animals. The phenomenon may reflect an adenosine A1-mediated adaptive mechanism that offers protection from subsequent seizures.

Upregulation of A1 adenosine receptors in human temporal lobe epilepsy: a quantitative autoradiographic study.

A significant increase of A1 adenosine receptor binding (48% increase of mean) was detected in human neocortex obtained from patients suffering from temporal lobe epilepsy as compared to control neocortex from non-epileptic patients. Such increase was equally distributed in the six cortical layers and reached similar levels in each of the five specimens tested independently of age, sex and pharmacological treatment of the patient. Since adenosine exerts a depressant effect on neocortical neurons in slices obtained from epileptic patients, this upregulation of A1 receptor binding may constitute a protective mechanism against subsequent seizures, which is exerted by elevating the depressant response of the brain to endogenous adenosine.

Effect of pentylentetrazol-induced seizures on A1 adenosine receptor regional density in the mouse brain: a quantitative autoradiographic study.

Adenosine has been shown to be a major regulator of neuronal activity in convulsive disorders, exerting its anticonvulsant effect through central A1 adenosine receptors. The aim of the present study was to investigate the effect of generalized tonic-clonic seizures induced by pentylentetrazol on regional changes in A1 adenosine receptor density and distribution in the mouse brain by in vitro quantitative autoradiography. As radioligand the specific agonist of A1 receptors [3H]cyclohexyladenosine was used. After two consecutive (once daily) pentylentetrazol-induced convulsions a widespread upregulation of A1 receptor density was detected with a marked enhancement in structures that mediate seizure activity like hippocampus, mamillary bodies, septum, substantia nigra, thalamic nuclei and cerebral cortices. On the contrary, in basal ganglia a significant downregulation of A1 receptors was observed. These results indicate that: (i) the observed increases or decreases in A1 receptor density are organized in selective anatomical structures related to seizure development rather than uniform in the brain; and (ii) since the upregulation of A1 receptors is sufficient to enhance the physiological depressive response of adenosine, the overall evoked increases seen here may lead to a stronger inhibitory tone and accordingly to a more efficient anticonvulsant effect of endogenous adenosine.

Reduction of A1 adenosine receptors in cortex, hippocampus and cerebellum in ageing mouse brain.

Age related changes in A1 adenosine receptor binding were investigated in mouse brain using the selective agonist, [3H]-cyclohexyladenosine ([3H]CHA). In the cortex, hippocampus and cerebellum of aged mice (28 months old), a significant decrease of about 44%, 50% and 12%, respectively, in [3H]CHA binding compared to young animals (3 months old) was observed. According to the Scatchard analysis of the binding data in the cortex, this decrease was due to a receptor density reduction and not to a Kd change. Since the weight and protein content of each tissue tested did not differ significantly between the old and the young animals, our findings may be partly explained by specific reductions of A1 receptors rather than a general cell degeneration in old age.

Changes in seizure latency correlate with alterations in A1 adenosine receptor binding during daily repeated pentylentetrazol-induced convulsions in different mouse brain areas.

The seizure latency changed during daily pentylentetrazol (PTZ) induced convulsions showing an increase between days 2 and 4, a rapid decrease between days 5 and 10 and a slight increase again between days 11 and 14. At the respective timepoints, [3H]CHA binding, in cortex and cerebellum of PTZ treated animals followed exactly the same pattern, suggesting that the alterations in A1 receptors in these areas may partly determine the PTZ seizure latency curve. On the contrary, the changes of [3H]CHA binding in hippocampus (sustained increase) and striatum (sustained decrease) didn't follow the latency curve pattern. These results suggest that changes in A1 receptor density in specific brain areas may be involved in the modulation of seizure susceptibility.

Alterations of A1 adenosine receptors in different mouse brain areas after pentylentetrazol-induced seizures, but not in the epileptic mutant mouse 'tottering'.

Single and repeated Pentylentetrazol (PTZ)-induced convulsions are associated with significant changes of A1 adenosine receptors (detected using the radioligand [3H]cyclohexyladenosine, [3H]CHA) in 4 different brain areas of the mouse, namely cortex, hippocampus, cerebellum and striatum. In hippocampus and cerebellum, a rapid increase in [3H]CHA binding, by 26% and 30% respectively, was observed 1 h after a single PTZ convulsion. In striatum, on the contrary, a significant decrease by 30% in [3H]CHA binding was seen, whereas in cortex no significant change could be detected. After daily repeated PTZ convulsions, a significant increase of A1 receptors by 26% appeared also in cortex, while the changes of A1 receptors observed in the other brain areas after a single PTZ convulsion were maintained in almost the same range. All the alterations observed were due to changes of the total number of A1 receptors (Bmax) without changes in receptor affinity (Kd). A significant increase in the latency of PTZ seizure (time between the PTZ-injection and the beginning of the seizure) was also observed after repeated PTZ-induced convulsions at the time when the changes in A1 adenosine receptors were noted. Considered together, these results provide further evidence for an A1 receptor-mediated modulation of seizure susceptibility and indicate that specific brain areas may play different roles in this modulation. The binding of [3H]CHA to membranes from different cortical and subcortical areas of the epileptic mutant mouse 'tottering' was not different from that in control animals.

L-aspartate and L-glutamate binding sites in developing normal and 'nervous' mutant mouse cerebellum.

This study concerns the ontogeny and the cellular localization of L-aspartate and L-glutamate binding sites in normal and 'nervous' mutant mouse cerebellar membranes. The binding kinetics revealed for L-aspartate a single binding system (Kd = 750 nM) and for L-glutamate also a single binding component of higher affinity (Kd = 344 nM). The pharmacological study, using various amino acid analogues, revealed a differential specificity for the binding sites of the two amino acids. The developmental study showed that the binding sites of both amino acids appear mainly during the second and third week of life, a period when parallel and climbing fiber synaptogenesis occurs, but they follow a slightly different developmental pattern. The study using 'nervous', mutant mouse cerebellum showed an age-dependent decrease of L-aspartate and L-glutamate binding, which coincides in time with the Purkinje cell degeneration in this mutant, indicating a cellular localization of these binding sites on the Purkinje cell membranes. These results suggest that L-aspartate and L-glutamate binding sites may be respectively associated with the postsynaptic target of climbing and parallel fibers on the Purkinje cell dendrites. However, the decrease of specific binding in 'nervous' mutant mouse cerebellum was about 50% for L-aspartate and 60% for L-glutamate, implying that a significant number of L-aspartate and L-glutamate binding sites are located on cerebellar elements other than the Purkinje cell membranes.

Absence of modification in GABA and benzodiazepine binding and in choline acetyltransferase activity in brain areas of the epileptic mutant mouse tottering.

1. In the tottering mutant mouse, which suffers from epilepsy and cerebellar ataxia, we examined whether possible changes in GABA, benzodiazepine receptors and choline acetyltransferase (ChAT) activity are implicated in the pathophysiology of these animals. 2. No alteration in GABAA and GABAB binding could be detected in cerebellar membranes of epileptic mice as compared to normal mice. 3. Benzodiazepine receptor density and affinity showed no statistical difference in cerebellar membranes of epileptic and normal mice. 4. The activity of ChAT determined in the cortices of epileptic and normal mice did not differ significantly between the two groups.

Purification and properties of two gentamicin-modifying enzymes, coded by a single plasmid pPK237 originating from Pseudomonas aeruginosa.

A broad host range multiresistance plasmid pPK237, originating from Pseudomonas aeruginosa mediates high-level resistance to gentamicin and tobramycin. It was found to code for two gentamicin modifying enzymes, which from their substrate profile by radioenzymatic assay were characterized as aminoglycoside acetyltransferase AAC(3)-I and aminoglycoside adenylyltransferase AAD(2"). The two enzymes were studied after purification from an Escherichia coli K12 host. The two gentamicin-modifying enzymes coded by PPK237 were completely separated by DEAE chromatography. The purification (126 fold) of the acetyltransferase was achieved by (NH4)2SO4 precipitation, DEAE chromatography and affinity chromatography. The purification of the adenylyltransferase was performed by affinity chromatography directly after (NH4)2SO4 precipitation. Both purified enzyme preparations showed a single protein band on disc electrophoresis. The Km for gentamicin C1 of the acetyltransferase was 0.066 mM. The amino acid analysis of the acetyltransferase coded by pPK237 showed a different aminoacid composition than that of the gentamicin acetyltransferase AAC(3)-I purified by Williams and Northrop17). The acetyltransferase after DEAE chromatography is stable for many months at -20 degrees C, while the adenylyltransferase after purification is highly unstable; it shows enzymatic activity only in the presence of Mg++.

Multiresistant plasmids from Pseudomonas aeruginosa highly resistant to either or both gentamicin and carbenicillin.

High-level resistance to gentamicin and carbenicillin was found in 30 and 10.7%, respectively, of Pseudomonas aeruginosa strains, especially in isolates from urine. In 23 out of 25 strains tested, these resistances were R mediated and linked to multiresistant plasmids, carrying genes for resistances to five other aminoglycosides, tobramycin, kanamycin, neomycin, streptomycin, and spectinomycin, and for resistances to chloramphenicol, tetracycline, sulfonamides, and mercury chloride. Carbenicillin resistance was unstable in Pseudomonas, and in its presence the multiresistant plasmids had a host range extended to the Enterobacteriaceae (group I plasmids). Otherwise they were transferable intragenerically only (group II plasmids). The extended host range plasmids were, as a rule, in fi(-) incompatibility class A-C. Segregants incompatible with both class A-C and P plasmids were detected. The beta-lactamase specified by the carbenicillin marker was of the TEM-like type. Multiple linkages of resistance determinants to the aminoglycosides were concomitantly present in most of the plasmids. Results from the bioassay indicated the presence of at least two aminoglycoside-inactivating enzymes.