Disease mechanisms revealed by transcription profiling in SOD1-G93A transgenic mouse spinal cord.

Mutations of copper,zinc-superoxide dismutase (cu,zn SOD) are found in patients with a familial form of amyotrophic lateral sclerosis. When expressed in transgenic mice, mutant human cu,zn SOD causes progressive loss of motor neurons with consequent paralysis and death. Expression profiling of gene expression in SOD1-G93A transgenic mouse spinal cords indicates extensive glial activation coincident with the onset of paralysis at 3 months of age. This is followed by activation of genes involved in metal ion regulation (metallothionein-I, metallothionein-III, ferritin-H, and ferritin-L) at 4 months of age just prior to end-stage disease, perhaps as an adaptive response to the mitochondrial destruction caused by the mutant protein. Induction of ferritin-H and -L gene expression may also limit iron catalyzed hydroxyl radical formation and consequent oxidative damage to lipids, proteins, and nucleic acids. Thus, glial activation and adaptive responses to metal ion dysregulation are features of disease in this transgenic model of familial amyotrophic lateral sclerosis.

Enhanced oxygen radical production in a transgenic mouse model of familial amyotrophic lateral sclerosis.

Mutations of the SOD1 gene encoding copper/zinc superoxide dismutase (CuZnSOD) cause an inherited form of amyotrophic lateral sclerosis. When expressed in transgenic mice, the same SOD1 mutations cause progressive loss of spinal motor neurons with consequent paralysis and death. In vitro biochemical studies indicate that SOD1 mutations enhance free radical generation by the mutant enzyme. We investigated those findings in vivo by using a novel, brain-permeable spin trap, azulenyl nitrone. Reaction of azulenyl nitrone with a free radical forms a nitroxide adduct that then fragments to yield the corresponding azulenyl aldehyde. Transgenic mice expressing mutant SOD1-G93A show enhanced free radical content in spinal cord but not brain. This correlates with tissue-specific differences in the level of transgene expression. In spinal cord, the increase in free radical content is in direct proportion to the age-dependent increase in mutant human CuZnSOD expression. This increase precedes motor neuron degeneration. The higher level of human CuZnSOD expression seen in spinal cord compared with brain, and consequent difference in free radical generation, provides a basis for understanding the selective vulnerability of the spinal cord in this disease model.

U-95666E: a potential anti-parkinsonian drug with anxiolytic activity.

1. U-95666E, a D2 selective dopamine agonist, was investigated for its effect on rat striatal acetylcholine (ACh) concentration and the results were compared with those obtained with pergolide, pramipexole and bromocriptine under similar conditions. 2. U-95666E, pergolide, pramipexole and bromocriptine dose-dependently increased striatal ACh concentration both in the non-reserpinized and reserpinized rats. 3. Intrinsic activity of U-95666E was similar to pergolide and pramipexole in non-reserpinized rats, but significantly lower in reserpinized rats. 4. The sensitivity of these dopamine agonists for increasing ACh levels in the denervated as compared to innervated striatum were significantly (p < 0.01) higher. 5. U-95666E also has anxiolytic activity in mice. 6. In conclusion, U-95666E may have potential for the treatment of Parkinson's Disease and associated anxiety.

D2 and D1 dopaminergic activity of 7-OH-DPAT.

The D1 and D2 dopamine receptor agonist properties of 7-hydroxy-2-(N,N-di-n-propylamino) tetraline (7-OH-DPAT) was determined by investigating the effect of this compound on rat striatal acetylcholine (ACh) concentration and increase in cAMP formation in primary cerebellar granule cell cultures. 7-OH-DPAT at low doses (0.01 to 0.1 mumol/ kg) had no significant effect, and at high doses (0.3 to 30 mumol/kg) significantly (P < 0.01) increased striatal ACh levels. Likewise, quinpirole was found to significantly elevate ACh content. Pretreatment with haloperidol, a non-selective antagonist of the D2 family of receptors, significantly (P < 0.01) blocked 7-OH-DPAT- and quinpirole-induced increases in ACh. U-99194A, a D3 selective dopamine antagonist, had no significant effect on 7-OH-DPAT-induced increases in striatal ACh. However, raclopride, a D2 selective dopamine antagonist, completely blocked 7-OH-DPAT-induced elevations in ACh. 7-OH-DPAT in the mumolar range increased cAMP formation in granule cell cultures, and this effect was antagonized by SCH 23390, a D1 selective dopamine antagonist. The neurochemical study indicates that, at high doses, 7-OH-DPAT has both D1 and D2 agonist activities.

Comparative dopaminergic and muscarinic antagonist activity of clozapine and haloperidol.

Clozapine is an atypical antipsychotic drug, and its dopamine and muscarinic antagonist activity has been compared with haloperidol in rodents. Elevation in rat striatal acetylcholine (ACh) and mice cerebellar cGMP has been used as an agonist response for oxotremorine and quinpirole. Pretreatment with clozapine significantly blocked oxotremorine-induced elevation in striatal ACh (p<0.01) and cerebellar cGMP(p<0.05). At the same doses, clozapine had no significant effect on quinpirole-induced increases in ACh and cGMP levels. Pretreatment with haloperidol significantly antagonized quinpirole-induced elevation in striatal ACh (p<0.01) and cerebellar cGMP(p<0.05), and haloperidol had no significant effect on oxotremorine-induced agonist responses. Thus, clozapine is antimuscarinic at a dose level that lacks dopamine antagonist properties, whereas haloperidol is a dopamine antagonist and lacks antimuscarinic activity. The atypical neuroleptic profile of clozapine may be due to its high antimuscarinic and low antidopaminergic activity.

Regulation of muscarinic receptors by intrahippocampal injections of gallamine.

Multiple intrahippocampal injections of gallamine impair performance of a representational memory task in rats. The binding of [3H]-(-)-quinuclidinyl benzilate (QNB) to rat brain sections was measured to determine if changes in receptor binding were associated with the deleterious effects of gallamine. [3H]-(-)-QNB binding to sections taken from gallamine-injected animals was compared with binding in saline-injected control animals. Autoradiographic analyses indicated an increase in [3H]-(-)-QNB binding sites within all layers of the cerebral cortex and in the superior colliculus in gallamine-treated animals as compared to saline-injected controls. Significant increases were noted in cortical layers IV and V (P less than 0.025) in gallamine-treated animals. No significant changes (P greater than 0.05) in the number of binding sites were observed in the hippocampus, neostriatum or various thalamic nuclei. The ability of unlabeled pirenzepine, gallamine and carbamylcholine to inhibit 0.2 nM [3H]-(-)-QNB binding also was measured to determine changes in the distribution of receptor subtypes. No significant changes were observed in any brain region for the binding of the selective antagonists pirenzepine and gallamine or the agonist carbamyl-choline. Although other possibilities are considered, the data suggest that an increase in the number of muscarinic receptors may contribute to the observed behavioral deficits associated with long-term gallamine treatment.

Biochemical and behavioral responses of pilocarpine at muscarinic receptor subtypes in the CNS. Comparison with receptor binding and low-energy conformations.

Pilocarpine was tested biochemically in vitro for its ability to stimulate phosphoinositide (PI) turnover in the hippocampus (M1/M3 responses) where it displayed 35% of the maximal carbachol response with an EC50 value of 18 microM, and low-Km GTPase in the cortex (M2 response), where it had 50% of the maximal carbachol response with an EC50 value of 4.5 microM. Behaviorally, pilocarpine was able to restore deficits in a representational memory task (sensitive to M1 antagonists) produced by intrahippocampal injections of AF64A. Twenty-three low-energy conformations of protonated pilocarpine were generated using the program MacroModel. The data indicate that pilocarpine is a partial agonist at both M1 and M2 muscarinic receptors in the CNS. Behaviorally, with respect to the memory task, M1 effects of pilocarpine apparently predominate. It also is conceivable that different conformations of pilocarpine are active as agonists at different muscarinic receptor subtypes.

Biochemical and behavioral evidence for muscarinic autoreceptors in the CNS.

Muscarinic autoreceptors of the M2 subclass were examined in rat forebrain using a number of different methodologies, including receptor autoradiography and image analysis, regulation of acetylcholine release, phosphoinositide turnover, low-Km GTP hydrolysis, and behavioral analysis. The relatively minor population of M2 receptors in coronal sections was visualized by autoradiography and image analysis using [3H]quinuclidinyl benzilate in the presence of a concentration of pirenzepine that blocked most of M1 (and M4) receptors. The highest densities of M2 receptors in forebrain regions were found in the outer layers of the cortex, CA1 region of the hippocampus and striatum. The M2-, but not M1-selective antagonists were able to block the oxotremorine-induced attenuation of acetylcholine release in forebrain synaptosomes. Low concentrations of the M2-selective antagonist gallamine increased phosphoinositide turnover, which is thought to be an M1 postsynaptic response in the forebrain, in brain slices by a Ca2(+)-dependent mechanism. The M2-selective agonist oxotremorine produced a substantial stimulation of low-Km GTPase in cortical membranes, suggesting that M2 forebrain receptors are efficiently coupled to G-proteins in the cortex. Behavioral signs of cholinergic stimulation were observed after intracerebroventricular injections of M2-, but not M1-selective antagonists. It is suggested that a minor population of forebrain M2 receptors regulates acetylcholine release by a mechanism that includes coupling through G-proteins presynaptically at synapses for which the postsynaptic response involves phosphoinositide turnover. Selective blockade of these receptors produces both biochemical and behavioral signs of acetylcholine release.

Identification of four brain areas each enriched in a unique muscarinic receptor subtype.

The affinities of muscarinic agonists and antagonists were determined by autoradiography and image analysis in selected areas of the rat brain. IC50 values and Hill coefficients for the inhibition of the binding of 0.2 nM [3H]-QNB to dentate gyrus, superior colliculus, rhomboid thalamus and substantia nigra were measured in coronal sections. Pirenzepine displayed a high affinity for receptors in the dentate gyrus and AF-DX 116, the superior colliculus. Both pirenzepine and AF-DX 116 had high affinities for the substantia nigra and low affinities for the rhomboid thalamus. Gallamine displayed a 50-fold preference for superior colliculus over dentate gyrus receptors. Amitriptyline was less selective, showing a modest preference for substantia nigra receptors and 4-DAMP was essentially nonselective. Carbachol was the most selective agonist with a 4000-fold preference for superior colliculus over dentate gyrus receptors. Other agonists except RS 86 were also selective for superior colliculus receptors in the order carbachol much greater than arecoline greater than bethanechol greater than McN A343 = oxotremorine = pilocarpine.

Regional differences in the binding of selective muscarinic receptor antagonists in rat brain: comparison with minimum-energy conformations.

The binding of selective muscarinic receptor antagonists to regions of rat brain was examined through quantitative autoradiographic techniques. 5,11-Dihydro-11-[(4-methyl-1-piperazinyl)acetyl]-6H- pyrido[2,3-b][1,4]benzodiazepin-6-one [pirenzepine (compound I)] and 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro- 6H-pyrido[2,3-b][1,4]benzodiazepin-6-one [AF-DX 116 (compound II)] were chosen on the basis of their selectivity for M1 and M2 muscarinic receptors, respectively, and similarities in chemical structure. Pirenzepine displayed a higher potency than II for inhibition of [3H]-l-quinuclidinyl benzilate ([3H]-l-QNB) binding to rat brain sections. Scatchard analyses of binding to brain sections revealed heterogeneous binding profiles for both antagonists, suggesting the presence of multiple receptor binding sites. Quantitative autoradiographic techniques were utilized in regional analyses of antagonist binding. Pirenzepine displayed the highest affinity for hippocampal, striatal, and amygdaloid muscarinic receptors (IC50 values less than 0.4 microM), with a slightly lower affinity for cortical receptors (IC50 values between 0.4 and 0.8 microM). Pirenzepine displayed the lowest affinity for thalamic and brainstem regions with IC50 values generally greater than 1.0 microM. In contrast, II bound with higher affinity to muscarinic receptors in brainstem, cerebellar, and hypothalamic nuclei (IC50 values less than 0.5 microM) than to receptors in thalamic nuclei (IC50 values between 0.5 and 2.0 microM). Binding sites with the lowest affinity for II were found in cortical, striatal, and hippocampal regions (IC50 values greater than 2.0 microM). The binding profiles of the two selective muscarinic antagonists reveal the complexity and diversity of muscarinic receptor subtypes throughout the brain. The data provide a basis for identifying muscarinic receptor subtypes (as defined through cloning procedures) with selective ligands. Minimum-energy conformations of pirenzepine and II were calculated by using the program MacroModel (version 2.0). Pirenzepine displayed three energy minima, differing in the relative position of the piperazine ring with respect to the tricyclic system. In contrast, the (diethylamino)methyl substituent on the piperidine ring conferred a much larger set of minimum-energy conformations on II. It is suggested that the greater conformational flexibility of the side chain allows II to achieve a conformation inaccessible to pirenzepine, which allows it to bind preferentially to M2 receptors.