The ability of environmental context to facilitate psychomotor sensitisation to amphetamine can be dissociated from its effect on acute drug responsiveness and on conditioned responding.

Doses of amphetamine or cocaine that fail to induce psychomotor sensitization when given to a rat in its home cage can produce robust sensitization if given immediately following placement into a relatively novel, distinct environment. A drug-associated context can serve as a conditioned stimulus, and therefore may promote robust sensitization by facilitating associative learning processes. We examined this hypothesis by habituating rats to the test environment for 1 or 6-8 hr prior to each drug injection, which degrades the ability of environmental context to serve as an effective conditioned stimulus. When 0.5 mg/kg of amphetamine was administered intravenously immediately after placement into a distinct environment there was a large acute psychomotor response (rotational behavior) on the first test day, and robust sensitization developed with repeated daily injections. When the same treatment was administered in the home cage, there was a small acute response and no sensitization developed. The enhanced acute response seen in the distinct environment was significantly attenuated by 1 hr of habituation to the test environment, and completely abolished by 6-8 hr of habituation. Also, as little as 1 hr of habituation completely prevented the development of a conditioned rotational response. In contrast, neither 1 nor 6-8 hr of habituation had any effect on the ability of amphetamine to induce robust behavioral sensitization. It is concluded that the ability of a distinct environment to facilitate sensitization to amphetamine can be dissociated from its effect on acute drug responsiveness and from the ability of drug-associated environmental stimuli to elicit a conditioned response. Possible mechanisms by which a distinct environment facilitates sensitization are discussed. © 2001 American College of Neuropsychopharmacology

Neuronal Atrophy Early in Degenerative Ataxia Is a Compensatory Mechanism to Regulate Membrane Excitability.

Neuronal atrophy in neurodegenerative diseases is commonly viewed as an early event in a continuum that ultimately results in neuronal loss. In a mouse model of the polyglutamine disorder spinocerebellar ataxia type 1 (SCA1), we tested the hypothesis that cerebellar Purkinje neuron atrophy serves an adaptive role rather than being simply a nonspecific response to injury. In acute cerebellar slices from SCA1 mice, we find that Purkinje neuron pacemaker firing is initially normal but, with the onset of motor dysfunction, becomes disrupted, accompanied by abnormal depolarization. Remarkably, subsequent Purkinje cell atrophy is associated with a restoration of pacemaker firing. The early inability of Purkinje neurons to support repetitive spiking is due to unopposed calcium currents resulting from a reduction in large-conductance calcium-activated potassium (BK) and subthreshold-activated potassium channels. The subsequent restoration of SCA1 Purkinje neuron firing correlates with the recovery of the density of these potassium channels that accompanies cell atrophy. Supporting a critical role for BK channels, viral-mediated increases in BK channel expression in SCA1 Purkinje neurons improves motor dysfunction and partially restores Purkinje neuron morphology. Cerebellar perfusion of flufenamic acid, an agent that restores the depolarized membrane potential of SCA1 Purkinje neurons by activating potassium channels, prevents Purkinje neuron dendritic atrophy. These results suggest that Purkinje neuron dendritic remodeling in ataxia is an adaptive response to increases in intrinsic membrane excitability. Similar adaptive remodeling could apply to other vulnerable neuronal populations in neurodegenerative disease.Significance statementIn neurodegenerative disease, neuronal atrophy has long been assumed to be an early nonspecific event preceding neuronal loss. However, in a mouse model of spinocerebellar ataxia type 1 (SCA1), we identify a previously unappreciated compensatory role for neuronal shrinkage. Purkinje neuron firing in these mice is initially normal, but is followed by abnormal membrane depolarization resulting from a reduction in potassium channels. Subsequently, these electrophysiological effects are counteracted by cell atrophy, which by restoring normal potassium channel membrane density, re-establishes pacemaker firing. Reversing the initial membrane depolarization improved motor function and Purkinje neuron morphology in the SCA1 mice. These results suggest that Purkinje neuron remodeling in ataxia is an active compensatory response that serves to normalize intrinsic membrane excitability

Neuronal Atrophy Early in Degenerative Ataxia Is a Compensatory Mechanism to Regulate Membrane Excitability

Neuronal atrophy in neurodegenerative diseases is commonly viewed as an early event in a continuum that ultimately results in neuronal loss. In a mouse model of the polyglutamine disorder spinocerebellar ataxia type 1 (SCA1), we tested the hypothesis that cerebellar Purkinje neuron atrophy serves an adaptive role rather than being simply a nonspecific response to injury. In acute cerebellar slices from SCA1 mice, we find that Purkinje neuron pacemaker firing is initially normal but, with the onset of motor dysfunction, becomes disrupted, accompanied by abnormal depolarization. Remarkably, subsequent Purkinje cell atrophy is associated with a restoration of pacemaker firing. The early inability of Purkinje neurons to support repetitive spiking is due to unopposed calcium currents resulting from a reduction in large-conductance calcium-activated potassium (BK) and subthreshold-activated potassium channels. The subsequent restoration of SCA1 Purkinje neuron firing correlates with the recovery of the density of these potassium channels that accompanies cell atrophy. Supporting a critical role for BK channels, viral-mediated increases in BK channel expression in SCA1 Purkinje neurons improves motor dysfunction and partially restores Purkinje neuron morphology. Cerebellar perfusion of flufenamic acid, an agent that restores the depolarized membrane potential of SCA1 Purkinje neurons by activating potassium channels, prevents Purkinje neuron dendritic atrophy. These results suggest that Purkinje neuron dendritic remodeling in ataxia is an adaptive response to increases in intrinsic membrane excitability. Similar adaptive remodeling could apply to other vulnerable neuronal populations in neurodegenerative disease. SIGNIFICANCE STATEMENT In neurodegenerative disease, neuronal atrophy has long been assumed to be an early nonspecific event preceding neuronal loss. However, in a mouse model of spinocerebellar ataxia type 1 (SCA1), we identify a previously unappreciated compensatory role for neuronal shrinkage. Purkinje neuron firing in these mice is initially normal, but is followed by abnormal membrane depolarization resulting from a reduction in potassium channels. Subsequently, these electrophysiological effects are counteracted by cell atrophy, which by restoring normal potassium channel membrane density, re-establishes pacemaker firing. Reversing the initial membrane depolarization improved motor function and Purkinje neuron morphology in the SCA1 mice. These results suggest that Purkinje neuron remodeling in ataxia is an active compensatory response that serves to normalize intrinsic membrane excitability

Monomeric G protein-coupled receptor rhodopsin in solution activates its G protein transducin at the diffusion limit

G protein-coupled receptors mediate biological signals by stimulating nucleotide exchange in heterotrimeric G proteins (Gαβγ). Receptor dimers have been proposed as the functional unit responsible for catalytic interaction with Gαβγ. To investigate whether a G protein-coupled receptor monomer can activate Gαβγ, we used the retinal photoreceptor rhodopsin and its cognate G protein transducin (Gt) to determine the stoichiometry of rhodopsin/Gt binding and the rate of catalyzed nucleotide exchange in Gt. Purified rhodopsin was prepared in dodecyl maltoside detergent solution. Rhodopsin was monomeric as concluded from fluorescence resonance energy transfer, copurification studies with fluorescent labeled and unlabeled rhodopsin, size exclusion chromatography, and multiangle laser light scattering. A 1:1 complex between light-activated rhodopsin and Gt was found in the elution profiles, and one molecule of GDP was released upon complex formation. Analysis of the speed of catalytic rhodopsin/Gt interaction yielded a maximum of ≈50 Gt molecules per second and molecule of activated rhodopsin. The bimolecular rate constant is close to the diffusion limit in the diluted system. The results show that the interaction of Gt with an activated rhodopsin monomer is sufficient for fully functional Gt activation. Although the activation rate in solution is at the physically possible limit, the rate in the native membrane is still 10-fold higher. This is likely attributable to the precise orientation of the G protein to the membrane surface, which enables a fast docking process preceding the actual activation step. Whether docking in membranes involves the formation of rhodopsin dimers or oligomers remains to be elucidated

Constitutive Dimerization of the G-Protein Coupled Receptor, Neurotensin Receptor 1, Reconstituted into Phospholipid Bilayers

Neurotensin receptor 1 (NTS1), a Family A G-protein coupled receptor (GPCR), was expressed in Escherichia coli as a fusion with the fluorescent proteins eCFP or eYFP. A fluorophore-tagged receptor was used to study the multimerization of NTS1 in detergent solution and in brain polar lipid bilayers, using fluorescence resonance energy transfer (FRET). A detergent-solubilized receptor was unable to form FRET-competent complexes at concentrations of up to 200 nM, suggesting that the receptor is monomeric in this environment. When reconstituted into a model membrane system at low receptor density, the observed FRET was independent of agonist binding, suggesting constitutive multimer formation. In competition studies, decreased FRET in the presence of untagged NTS1 excludes the possibility of fluorescent protein-induced interactions. A simulation of the experimental data indicates that NTS1 exists predominantly as a homodimer, rather than as higher-order multimers. These observations suggest that, in common with several other Family A GPCRs, NTS1 forms a constitutive dimer in lipid bilayers, stabilized through receptor-receptor interactions in the absence of other cellular signaling components. Therefore, this work demonstrates that well-characterized model membrane systems are useful tools for the study of GPCR multimerization, allowing fine control over system composition and complexity, provided that rigorous control experiments are performed

Structural Insights for Activation of Retinal Guanylate Cyclase by GCAP1

Guanylyl cyclase activating protein 1 (GCAP1), a member of the neuronal calcium sensor (NCS) subclass of the calmodulin superfamily, confers Ca(2+)-sensitive activation of retinal guanylyl cyclase 1 (RetGC1) upon light activation of photoreceptor cells. Here we present NMR assignments and functional analysis to probe Ca(2+)-dependent structural changes in GCAP1 that control activation of RetGC. NMR assignments were obtained for both the Ca(2+)-saturated inhibitory state of GCAP1 versus a GCAP1 mutant (D144N/D148G, called EF4mut), which lacks Ca(2+) binding in EF-hand 4 and models the Ca(2+)-free/Mg(2+)-bound activator state of GCAP1. NMR chemical shifts of backbone resonances for Ca(2+)-saturated wild type GCAP1 are overall similar to those of EF4mut, suggesting a similar main chain structure for assigned residues in both the Ca(2+)-free activator and Ca(2+)-bound inhibitor states. This contrasts with large Ca(2+)-induced chemical shift differences and hence dramatic structural changes seen for other NCS proteins including recoverin and NCS-1. The largest chemical shift differences between GCAP1 and EF4mut are seen for residues in EF4 (S141, K142, V145, N146, G147, G149, E150, L153, E154, M157, E158, Q161, L166), but mutagenesis of EF4 residues (F140A, K142D, L153R, L166R) had little effect on RetGC1 activation. A few GCAP1 residues in EF-hand 1 (K23, T27, G32) also show large chemical shift differences, and two of the mutations (K23D and G32N) each decrease the activation of RetGC, consistent with a functional conformational change in EF1. GCAP1 residues at the domain interface (V77, A78, L82) have NMR resonances that are exchange broadened, suggesting these residues may be conformationally dynamic, consistent with previous studies showing these residues are in a region essential for activating RetGC1