Percolation of aligned rigid rods on two-dimensional triangular lattices.

The percolation behavior of aligned rigid rods of length k (k-mers) on two-dimensional triangular lattices has been studied by numerical simulations and finite-size scaling analysis. The k-mers, containing k identical units (each one occupying a lattice site), were irreversibly deposited along one of the directions of the lattice. The connectivity analysis was carried out by following the probability R_{L,k}(p) that a lattice composed of L×L sites percolates at a concentration p of sites occupied by particles of size k. The results, obtained for k ranging from 2 to 80, showed that the percolation threshold p_{c}(k) exhibits a increasing function when it is plotted as a function of the k-mer size. The dependence of p_{c}(k) was determined, being p_{c}(k)=A+B/(C+sqrt[k]), where A=p_{c}(k→∞)=0.582(9) is the value of the percolation threshold by infinitely long k-mers, B=-0.47(0.21), and C=5.79(2.18). This behavior is completely different from that observed for square lattices, where the percolation threshold decreases with k. In addition, the effect of the anisotropy on the properties of the percolating phase was investigated. The results revealed that, while for finite systems the anisotropy of the deposited layer favors the percolation along the parallel direction to the alignment axis, in the thermodynamic limit, the value of the percolation threshold is the same in both parallel and transversal directions. Finally, an exhaustive study of critical exponents and universality was carried out, showing that the phase transition occurring in the system belongs to the standard random percolation universality class regardless of the value of k considered.

MicroRNA-125b regulates microglia activation and motor neuron death in ALS.

Understanding the means by which microglia self-regulate the neuroinflammatory response helps modulating their reaction during neurodegeneration. In amyotrophic lateral sclerosis (ALS), classical NF-κB pathway is related to persistent microglia activation and motor neuron injury; however, mechanisms of negative control of NF-κB activity remain unexplored. One of the major players in the termination of classical NF-κB pathway is the ubiquitin-editing enzyme A20, which has recognized anti-inflammatory functions. Lately, microRNAs are emerging as potent fine-tuners of neuroinflammation and reported to be regulated in ALS, for instance, by purinergic P2X7 receptor activation. In this work, we uncover an interplay between miR-125b and A20 protein in the modulation of classical NF-κB signaling in microglia. In particular, we establish the existence of a pathological circuit in which termination of A20 function by miR-125b strengthens and prolongs the noxious P2X7 receptor-dependent activation of NF-κB in microglia, with deleterious consequences on motor neurons. We prove that, by restoring A20 levels, miR-125b inhibition then sustains motor neuron survival. These results introduce miR-125b as a key mediator of microglia dynamics in ALS.

Percolation of aligned rigid rods on two-dimensional square lattices.

The percolation behavior of aligned rigid rods of length k (kmers) on two-dimensional square lattices has been studied by numerical simulations and finite-size scaling analysis. The kmers, containing k identical units (each one occupying a lattice site), were irreversibly deposited along one of the directions of the lattice. The process was monitored by following the probability R(L,k)(p) that a lattice composed of L×L sites percolates at a concentration p of sites occupied by particles of size k. The results, obtained for k ranging from 1 to 14, show that (i) the percolation threshold exhibits a decreasing function when it is plotted as a function of the kmer size; (ii) for any value of k (k>1), the percolation threshold is higher for aligned rods than for rods isotropically deposited; (iii) the phase transition occurring in the system belongs to the standard random percolation universality class regardless of the value of k considered; and (iv) in the case of aligned kmers, the intersection points of the curves of R(L,k)(p) for different system sizes exhibit nonuniversal critical behavior, varying continuously with changes in the kmer size. This behavior is completely different to that observed for the isotropic case, where the crossing point of the curves of R(L,k)(p) do not modify their numerical value as k is increased.

Isotropic-nematic phase diagram for interacting rigid rods on two-dimensional lattices.

The phase behavior of interacting rigid rods of length k (k-mers) on two-dimensional square and triangular lattices has been studied by theoretical calculations in the framework of the lattice-gas model. The process was analyzed by comparing the dependence on coverage of the free energy per site of an isotropic submonolayer of interacting k-mers f(iso)(θ) with that corresponding to a fully aligned (nematic) system f(nem)(θ). The existence of an intersection point between the curves f(iso)(θ) and f(nem)(θ), which is indicative of the occurrence of an isotropic-nematic phase transition in the adlayer, allowed us to obtain the complete (temperature, coverage, k-mer size) phase diagram of the system.

The protective role of catalase against cerebral ischemia in vitro and in vivo.

The present study aims to assess the protective role of the antioxidant enzyme catalase (CAT) with relation to hydrogen peroxide (H(2)O(2)) degradation in oxygen plus water on electrophysiological and fluorescence changes induced by in vitro ischemia and on brain damage produced by transient in vivo ischemia. Neuroprotective effects of CAT were determined by means of electrophysiological recordings and confocal fluorescence microscopy in the hippocampal slice preparation. Ischemia was simulated in vitro by oxygen/glucose deprivation (OGD). In vivo ischemia was produced by transient middle cerebral artery occlusion (MCAo). A protection of the rat CA1 field excitatory postsynaptic potential (fEPSP) loss caused by a prolonged OGD (40 min) was observed after exogenous CAT (500 U/mL) bath-applied before a combined exposure to OGD and H(2)O(2) (3 mM). Of note, neither H(2)O(2) nor exogenous CAT alone had a protective action when OGD lasted for 40 min. The CAT-induced neuroprotection was confirmed in a transgenic mouse model over-expressing human CAT [Tg(CAT)]. In the presence of H(2)O(2), the hippocampus of Tg(CAT) showed an increased resistance against OGD compared to that of wild-type (WT) animals. Moreover, CAT treatment reduced for about 50 min fEPSP depression evoked by repeated applications of H(2)O(2) in normoxia. A lower sensitivity to H(2)O(2)-induced depression of fEPSPs was also indicated by the rightward shift of concentration-response curve in Tg(CAT) compared to WT mice. Noteworthy, Tg(CAT) mice had a reduced infarct size after MCAo. Our data suggest new strategies to reduce neuronal damage produced by transient brain ischemia through the manipulation of CAT enzyme.

Postsynaptic alteration of NR2A subunit and defective autophosphorylation of alphaCaMKII at threonine-286 contribute to abnormal plasticity and morphology of upper motor neurons in presymptomatic SOD1G93A mice, a murine model for amyotrophic lateral sclerosis.

Although amyotrophic lateral sclerosis (ALS) has long been considered as a lower motor neuron (MN) disease, degeneration of upper MNs arising from a combination of mechanisms including insufficient growth factor signaling and enhanced extracellular glutamate levels is now well documented. The observation that these mechanisms are altered in presymptomatic superoxide dismutase (SOD1) mice, an ALS mouse model, suggests that defective primary motor cortex (M1) synaptic activity might precede the onset of motor disturbances. To examine this point, we assessed the composition of AMPAR and NMDAR subunits and of the alphaCa²(+)/calmodulin-dependent kinase autophosphorylation at threonine-286 in the triton insoluble fraction from the M1 in postnatal P80-P85 SOD1(G93A) and wild-type mice. We show that presymptomatic SOD1(G93A) exhibit a selective decrease of NR2A subunit expression and of the alphaCa²(+)/calmodulin-dependent kinase autophosphorylation at threonine-286 in the triton insoluble fraction of upper MNs synapses. These molecular alterations are associated with synaptic plasticity defects, and a reduction in upper MN dendritic outgrowth revealing that abnormal neuronal connectivity in the M1 region precedes the onset of motor symptoms. We suggest that the progressive disruption of M1 corticocortical connections resulting from the SOD1(G93A) mutation might extend to adjacent regions and promote development of cognitive/dementia alterations frequently associated with ALS.

Abnormal medial prefrontal cortex connectivity and defective fear extinction in the presymptomatic G93A SOD1 mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal progressive neuropathy associated with the degeneration of spinal and brainstem motor neurons. Although ALS is essentially considered as a lower motor neuron disease, prefrontal cortex atrophy underlying executive function deficits have been extensively reported in ALS patients. Here, we examine whether prefrontal cortex neuronal abnormalities and related cognitive impairments are present in presymptomatic G93A Cu/Zn superoxide dismutase mice, a mouse model for familial ALS. Structural characteristics of prelimbic/infralimbic (PL/IL) medial prefrontal cortex (mPFC) neurons were studied in 3-month-old G93A and wild-type mice with the Golgi-Cox method, while mPFC-related cognitive operations were assessed using the conditioned fear extinction paradigm. Sholl analysis performed on the dendritic material showed a reduction in dendrite length and branch nodes on basal dendrites of PL/IL neurons in G93A mice. Spine density was also decreased on basal dendrite segments of branch order five. Consistent with the altered morphology of PL/IL cortical regions, G93A mice showed impaired extinction of conditioned fear. Our findings indicate that abnormal prefrontal cortex connectivity and function are appreciable before the onset of motor disturbances in this model.

Decreased plasma and cerebrospinal fluid content of neuroactive steroids in Parkinson's disease.

The levels of the neuroactive steroids allopregnanolone (THP) and 5alpha-dihydroprogesterone (DHP) were quantified in the plasma of 11 (group 1) and in the liquor of 12 (group 2) Parkinson's disease (PD) patients using a gas-chromatographic/mass-spectrometric method. When compared with controls, both groups showed a significant decrease in DHP and THP concentrations. These decreases could be a useful marker of PD. Moreover, in view of the importance of GABA-ergic transmission to substantia nigra (SN) neurons and GABA-ergic modulation exerted by the two neuroactive steroids, our data indicate a global dysregulation of the SN GABA-ergic system in PD patients. Moreover, a lack of neuroprotective factors (i. e., GDNF, BDNF), promoted by DHP, may contribute to dopaminergic cell death.

alpha-Amino-3-hydroxy-5-methyl-isoxazole-4-propionate receptors in spinal cord motor neurons are altered in transgenic mice overexpressing human Cu,Zn superoxide dismutase (Gly93-->Ala) mutation.

There are many evidences implicating glutamatergic toxicity as a contributory factor in the selective neuronal injury occurring in amyotrophic lateral sclerosis (ALS). This neurodegenerative disorder is characterized by the progressive loss of motor neurons, whose pathogenesis is thought to involve Ca(2+) influx mediated by alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate receptors (AMPARs). In the present study we report alterations in the AMPARs function in a transgenic mouse-model of the human SOD1(G93A) familial ALS. Compared with those expressed in motor neurons carrying the human wild type gene, AMPAR-gated channels expressed in motor neurons carrying the human mutant gene exhibited modified permeability, altered agonist cooperativity between the sites involved in the process of channel opening and were responsible for slower spontaneous synaptic events. These observations demonstrate that the SOD1(G93A) mutation induces changes in AMPAR functions which may underlie the increased vulnerability of motor neurons to glutamatergic excitotoxicity in ALS.

Altered long-term corticostriatal synaptic plasticity in transgenic mice overexpressing human CU/ZN superoxide dismutase (GLY(93)-->ALA) mutation.

Apart from the extensive loss of motor neurons, degeneration of midbrain dopaminergic cells has been described in both familial and sporadic forms of amyotrophic lateral sclerosis (ALS). Mice overexpressing the mutant human Cu/Zn superoxide dismutase (SOD1) show an ALS-like phenotype in that they show a progressive death of motor neurons accompanied by degeneration of dopaminergic cells. To describe the functional alterations specifically associated with this dopaminergic dysfunction, we have investigated the corticostriatal synaptic plasticity in mice overexpressing the human SOD1 (SOD1+) and the mutated (Gly(93)-->Ala) form (G93A+) of the same enzyme. We show that repetitive stimulation of the corticostriatal pathway generates long-term depression (LTD) in SOD1+ mice and in control (G93A-/SOD1-) animals, whereas in G93A+ mice the same stimulation generates an N-methyl-D-aspartic acid receptor-dependent long-term potentiation. No significant alterations were found in the intrinsic membrane properties of striatal medium spiny neurons and basal corticostriatal synaptic transmission of G93A+ mice. Bath perfusion of dopamine or the D(2) dopamine receptor agonist quinpirole restored LTD in G93A+ mice. Consistent with these in vitro results, habituation of locomotor activity and striatal-dependent active avoidance learning were impaired in G93A+ mice. Thus, degeneration of dopaminergic neurons in the substantia nigra of G93A+ mice causes substantial modifications in striatal synaptic plasticity and related behaviors, and may be a cellular substrate of the extrapyramidal motor and cognitive disorders observed in familial and sporadic ALS.

The regulation of hippocampal nicotinic acetylcholine receptors (nAChRs) after a protracted treatment with selective or nonselective nAChR agonists.

In rats, 1 mg/kg twice daily for 10 d of nicotine, a nonselective agonist of nicotinic acetylcholine receptors (nAChRs), fails to change alpha4 and beta2 nAChR subunit mRNA but significantly decreased alpha7 nAChR subunit mRNA and protein expression, which is associated with a 35-40% decrease in the number of 125I-alpha-Bgtx binding sites in hippocampus. In addition, this schedule of nicotine treatment produced a 40% increase in the number of high- (K(D) 1 nM), but decreased by 25% the number of low-affinity (K(D) 30 nM) binding sites for 3H-epibatidine in hippocampus. In contrast, repeated treatment with lobeline (2.7 mg/kg twice daily for 10 d), which selectively binds to high-affinity binding nAChRs, fails to change the expression of high- or low-affinity nAChRs. These data suggest that a simultaneous upregulation of high-affinity nAChRs and downregulation of low-affinity nAChRs is elicited by ligands that can bind to both low- and high-affinity nAChRs, but not by selective agonists of high-affinity nAChRs. One might infer that in hippocampus, high- and low-affinity nAChRs may be located in the same cells. When these two receptor types are stimulated simultaneously by nonselective ligands for high- and low-affinity nAChRs, they interact, bringing about an increase in binding site density of the high-affinity nAChRs.

Changes in AMPA receptor-spliced variant expression and shift in AMPA receptor spontaneous desensitization pharmacology during cerebellar granule cell maturation in vitro.

Using appropriate internal standards, quantitative reverse transcripase-polymerase chain reaction (RT-PCR), and cerebellar granule cell (CG) in primary cultures we have quantified the expression of mRNAs encoding for GluR1-4 DL-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunits during neuronal maturation in vitro. GluR1 is the mRNA that increases during CG maturation; the expression changes of the other GluR mRNAs are minimal and the translation products of these mRNAs change with a similar pattern. During CG maturation, there is an 8- to 10-fold increase in the GluR1 FLOP mRNA and a twofold increase in the expression of FLOP mRNA for GluR4 and GluR4C. The GluR1 FLIP mRNA increases, but by a smaller extent. We found that the GluR2 mRNA is completely edited at its Q/R site during CG maturation. The increase on the expression of GluR1 FLIP and FLOP and of GluR4 FLOP mRNA variants during development is associated with a 10-fold increase in AMPA-mediated Na+ currents and in the increased amplification of this current by 7-chloro-3-methyl-3,4 dihydro-2H-1,2,4 benzothiadiazine S-S-dioxide (IDRA21) or by 6-chloro-3,4 dihydro-3-(2-norbornen-5-yl)-7-sulfamoyl-1,2,4-benzothiadiazine 1,1 dioxide (cyclothiazide [CT]).

Aging-associated up-regulation of neuronal 5-lipoxygenase expression: putative role in neuronal vulnerability.

Aging is associated with neurodegenerative processes. 5-Lipoxygenase (5-LO), which is also expressed in neurons, is the key enzyme in the synthesis of leukotrienes, inflammatory eicosanoids that are capable of promoting neurodegeneration. We hypothesized that neuronal 5-LO expression can be up-regulated in aging and that this may increase the brain's vulnerability to neurodegeneration. We observed differences in the distribution of 5-LO-like immunoreactivity in various brain areas of adult young (2-month-old) vs. old (24-month-old) male rats. Greater 5-LO-like immunoreactivity was found in old vs. young rats, in particular in the dendrites of pyramidal neurons in limbic structures, including the hippocampus, and in layer V pyramidal cells of the frontoparietal cortex and their apical dendrites. The aging-increased expression of neuronal 5-LO protein appears to be due to increased 5-LO gene expression. Using a quantitative reverse transcription/polymerase chain reaction assay and 5-LO-specific oligonucleotide primers and their mutated internal standards, we observed about a 2.5-fold greater hippocampal 5-LO mRNA content in old rats. 5-LO-like immunoreactivity was also observed in small, nonpyramidal cells, which were positive for glutamic acid decarboxylase or glial fibrillary acid protein. This type of 5-LO immunostaining did not increase in the old rats. Hippocampal excitotoxic injury induced by systemic injection of kainate was greater in old rats. Neuroprotection was observed with the 5-LO inhibitor, caffeic acid. Together, these results suggest that aging increases both neuronal 5-LO expression and neuronal vulnerability to 5-LO inhibitor-sensitive excitotoxicity, and indicate that the 5-LO system might play a significant role in the pathobiology of aging-associated neurodegenerative diseases.

7-Chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine S,S-dioxide: a partial modulator of AMPA receptor desensitization devoid of neurotoxicity.

In cerebellar granule neurons of neonatal rats micromolar concentrations of 7-chloro-3-methyl-3,4-dihydro-2H-1,2, 4-benzothiadiazine S,S-dioxide (IDRA-21) and cyclothiazide, two negative modulators of the spontaneous agonist-dependent rapid desensitization of alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA)-gated ion channels, facilitate AMPA receptor function by increasing the content of free cytosolic Ca2+ as measured by single-cell fura-2 acetoxymethyl ester (Fura-2) Ca2+-dependent fluorescence and intracellular Na+ measured with the sodium-binding bezofuran isophthalate acetoxymethyl ester fluorescence indicator. IDRA-21 increases intracellular Na+ transient with a threshold (5 microM) that is approximately 10 times higher and has an intrinsic activity significantly lower than that of cyclothiazide. By virtue of its low intrinsic activity, IDRA-21 elicits a free cytosolic Ca2+ transient increase that is shorter lasting than that elicited by cyclothiazide even when the drug is left in contact with cultured granule cells for several minutes. Additionally, while dose dependently, 5-25 microM cyclothiazide in the presence of AMPA is highly neurotoxic, IDRA-21 (up to 100 microM) is devoid of neurotoxicity. The neurotoxicity elicited by cyclothiazide persists in the presence of dizocilpine (an antagonist of N-methyl-D-aspartate-selective glutamate receptors) but is blocked by 2,3-dihydroxy-6-nitrosulfamoylbenzo[f]quinoxaline (a competitive AMPA receptor antagonist) and the 1-(aminophenyl)-4-methyl-7, 8-methylendioxy-5H-2,3-benzodiazepine (GYKI 52466; a noncompetitive AMPA receptor antagonist). Since the doses of IDRA-21 that enhance cognitive processes in rats and monkeys are several orders of magnitude lower than those required to elicit marginal neurotoxicity in cultured neurons, it can be surmised that IDRA-21 is a potent cognition-enhancing drug virtually devoid of neurotoxic liability because it acts as a partial negative allosteric modulator of AMPA receptor desensitization.

Full-length and N-terminally truncated chicken intestinal diazepam-binding inhibitor. Purification, structural characterization and influence on insulin release.

Two forms of diazepam-binding inhibitor (DBI) have been purified from chicken intestine and identified as the intact avian polypeptide (residues 1-86) and a truncated variant (residues 35-86). At 10 nM concentration, both the intact and the truncated peptide suppress in vitro-monitored glucose-induced insulin release by 50 (p < 0.02) and 64% (p < 0.01) respectively. The truncation starts at a segment. -Thr-Val-Gly-Asp-, that is strictly conserved between characterized DBI species, indicating special restrictions on the structure. However, overall DBI conservation appears to be complex. A number of differently bioactive fragments with separate processings and tissue distributions have been observed, suggesting multiple functions of DBI and its sub-segments.

Increased hippocampal 5-lipoxygenase mRNA content in melatonin-deficient, pinealectomized rats.

Melatonin is neuroprotective because of its antioxidative action, but it also can modify neuronal vulnerability by altering gene expression. 5-Lipoxygenase (5-LO) gene expression is suppressed by the binding of melatonin to its high-affinity nuclear receptors. Recently, we reported that in rats the melatonin deficiency elicited by pinealectomy increases hippocampal susceptibility to excitotoxic injury. Here we have hypothesized that pinealectomy may increase hippocampal vulnerability by eliminating the tonic inhibitory action of melatonin on 5-LO gene expression. Sham-pinealectomized controls and pinealectomized rats were killed 15 days after surgery. Their hippocampi were dissected, and total RNA was extracted and processed for quantitative reverse transcription-polymerase chain reaction assay of 5-LO and cyclophilin mRNAs. Mutated primers were used as internal standards to assay attomole quantities of these two specific mRNAs per microgram of total RNA; the ratio 5-LO/cycophilin was used to compare samples from control and pinealectomized rats. Pinealectomy increased hippocampal 5-LO mRNA content by about threefold. These results support our hypothesis that melatonin deficiency may abate the tonic inhibition of 5-LO mRNA expression and thereby up-regulate 5-LO gene expression, which in turn would increase the brain's synthesis rate of potentially harmful eicosanoids, leukotrienes.

Modifications of gamma-aminobutyric acidA receptor subunit expression in rat neocortex during tolerance to diazepam.

We evaluated whether tolerance to the antagonism of bicuculine-induced seizures by diazepam is associated with changes (i) in the content of mRNAs encoding for gamma-aminobutyric acidA (GABAA) receptor subunits, (ii) in the expression density of these subunits, and (iii) in the 1,4-benzodiazepine binding site characteristics in discrete neocortical structures. We found that in diazepam-tolerant rats, the content of the mRNA encoding for the alpha 1 subunit of the GABAA receptor decreased in the frontoparietal motor (FrPaM) cortex and in the hippocampus (42% and 20%, respectively) but not in the frontoparietal somatosensory (FrPaSS) cortex, striatum, olfactory bulb, and cerebellum. In the FrPaM cortex, gamma 2S and gamma 2L subunit mRNA contents were also decreased (48% and 30%, respectively), whereas that of alpha 5 was increased (30%). In the FrPaM and FrPaSS cortices as well as in cerebellum of diazepam-tolerant rats, the content of alpha 2, alpha 3, alpha 6, beta 2, and delta subunit mRNA was unchanged, as was the content of alpha 2, alpha 5, gamma 1, and gamma 2S subunit mRNA in the hippocampus. Furthermore, the reduction in alpha 1 subunit mRNA content in the FrPaM cortex and the anticonvulsant tolerance to diazepam returned to control values 72 hr after termination of the protracted diazepam treatment. Rats receiving a treatment with imidazenil in doses equipotent and with a schedule identical to that of diazepam failed to exhibit tolerance to the anticonvulsant action of this drug or cross-tolerance to diazepam. In these rats, the content of mRNA encoding for alpha 1, alpha 2, alpha 3, alpha 5, alpha 6, gamma 1, gamma 2S, gamma 2L, and delta GABAA receptor subunits failed to change in the FrPaM and FrPaSS cortices, in the hippocampus, and in the other brain areas that were studied in diazepam-tolerant rats. Although the density and affinity of [3H]flumazenil and [3H]imidazenil binding failed to change in the FrPaM and FrPaSS cortices of diazepam-tolerant rats, the expression density of alpha 1 subunit immunogold labeling decreased by 37%, whereas that of alpha 5, gamma 2L/S, and beta 2/3 increased by 158%, 50%, and 47%, respectively, in the FrPaM cortex, and the density of the alpha 5 subunit selectively increased (209%) in the FrPaSS cortex. In contrast, the immunogold labeling density of the alpha 1, alpha 5, gamma 2L/S, and beta 2/3 subunits failed to change in either the FrPaM or FrPaSS cortex of rats receiving protracted imidazenil treatment.

Reversible modification of GABAA receptor subunit mRNA expression during tolerance to diazepam-induced cognition dysfunction.

Benzodiazepines (BZs) that are endowed with full positive allosteric modulatory (FAM) activity on GABAA receptors cause anterograde amnesia in both animals and humans. In rats subjected to a delayed object recognition test, diazepam, endowed with FAM activity, exerted an amnesic action, whereas BZs endowed with partial allosteric modulatory (PAM) activity on GABAA receptors, such as imidazenil, failed to induce amnesia, even if administered at doses five times higher than those equipotent to a standard anticonvulsant dose of diazepam (17.6 mumol/kg/os). After discontinuation of 14 days' treatment with vehicle, diazepam, or imidazenil (three times daily with increasing doses starting from 17.6 mumol/kg/os for diazepam and 2.5 mumol/kg/os for imidazenil), we compared the persistence of tolerance to the amnesic effect of diazepam with the persistence of the changes in the context of four (alpha 1, alpha 5, gamma 2L, gamma 2S) GABAA receptor subunit mRNAs in the fronto-parietal motor (FrPaM) cortex and the hippocampus. Rats receiving the long-term treatment with diazepam developed a tolerance to the amnesic effect of this drug and showed a decrease (30-50%) in the expression of mRNAs encoding for alpha 1 gamma 2L, gamma 2S GABAA receptor subunits, an increase, by approximately 30%, of the expression of mRNA of the alpha 5 subunit in the FrPaM cortex and a decrease, by approximately 25%, in the expression of mRNA, of the alpha 1 subunit in the hippocampus. These changes of subunit mRNA expression and the tolerance to the amnesic effect of diazepam returned to control values 72 hr after termination of the long-term treatment with diazepam. No tolerance to the amnesic effect of diazepam and no changes in GABAA receptor subunit mRNA expression were found in rats undergoing long-term treatment with imidazenil.

Identification of three transcriptional regulatory elements in the rat mitochondrial benzodiazepine receptor-encoding gene.

The sequence upstream from the first exon in the rat mitochondrial benzodiazepine receptor-encoding gene (MBR) was analyzed for transcriptional promoter activity by three techniques: promoter deletion analysis in vectors containing the gene cat encoding chloramphenicol acetyltransferase, electrophoretic mobility shift analysis (EMSA) and DNase I protection assay. All three methods are in uniformity with the identification of at least three regulatory elements corresponding to locations -51/-33, -267/-249 and -555/-526. The most distal and proximal domains are positive-acting, whereas the element at -267/-249 acts in a negative manner. The positive-acting -51/-33 element contains the middle of three consensus Sp1-recognition sequences found in this region of the gene. Binding of Y1 cell nuclear protein to a DNA fragment corresponding to this region of the gene is competed by a synthetic oligodeoxyribonucleotide bearing the consensus Sp1-binding site sequence. These studies provide the first reported functional evidence localizing transcriptional elements of MBR.