Dorsolateral Prefrontal Cortex Glutamate/Gamma-Aminobutyric Acid (GABA) Alterations in Clinical High Risk and First-Episode Schizophrenia: A Preliminary 7-T Magnetic Resonance Spectroscopy Imaging Study.

Converging lines of evidence suggest that an imbalance between excitation and inhibition is present in the dorsolateral prefrontal cortex (DLPFC) of schizophrenia (SCZ). Gamma-aminobutyric-acid (GABA) and, to a lesser extent, glutamate (Glu) abnormalities were reported in the DLPFC of SCZ patients, especially on the right hemisphere, by post-mortem studies. However, in vivo evidence of GABA, Glu, and Glu/GABA DLPFC abnormalities, particularly on the right side and the early stages of illness, is limited. In this preliminary study, we utilized 7-Tesla magnetic resonance spectroscopic imaging (MRSI) to investigate bilateral Glu/Creatine (Cre), GABA/Cre, and Glu/GABA in the DLPFC of sixteen first episode schizophrenia (FES), seventeen clinical high risk (CHR), and twenty-six healthy comparison (HC) subjects. FES and CHR had abnormal GABA/Cre and Glu/GABA in the right DLPFC (rDLPFC) compared with HC participants, while no differences were observed in the left DLPFC (lDLPFC) among the three groups. Furthermore, HC had higher Glu/GABA in rDLPFC compared to lDLPFC (R > L), whereas the opposite relationship (R < L) was observed in the DLPFC Glu/GABA of FES patients. Altogether, these findings indicate that GABA/Cre and Glu/GABA DLPFC alterations are present before illness manifestation and worsen in FES patients, thus representing a putative early pathophysiological biomarker for SCZ and related psychotic disorders.

Fast 3D rosette spectroscopic imaging of neocortical abnormalities at 3 T: Assessment of spectral quality.

PURPOSE: To use a fast 3D rosette spectroscopic imaging acquisition to quantitatively evaluate how spectral quality influences detection of the endogenous variation of gray and white matter metabolite differences in controls, and demonstrate how rosette spectroscopic imaging can detect metabolic dysfunction in patients with neocortical abnormalities. METHODS: Data were acquired on a 3T MR scanner and 32-channel head coil, with rosette spectroscopic imaging covering a 4-cm slab of fronto-parietal-temporal lobes. The influence of acquisition parameters and filtering on spectral quality and sensitivity to tissue composition was assessed by LCModel analysis, the Cramer-Rao lower bound, and the standard errors from regression analyses. The optimized protocol was used to generate normative white and gray matter regressions and evaluate three patients with neocortical abnormalities. RESULTS: As a measure of the sensitivity to detect abnormalities, the standard errors of regression for Cr/NAA and Ch/NAA were significantly correlated with the Cramer-Rao lower bound values (R = 0.89 and 0.92, respectively, both with P < 0.001). The rosette acquisition with a duration of 9.6 min, produces a mean Cramer-Rao lower bound (%) over the entire slab of 4.6 ± 2.6 and 5.8 ± 2.3 for NAA and Cr, respectively. This enables a Cr/NAA standard error of 0.08 (i.e., detection sensitivity of 25% for a 50/50 mixed gray and white matter voxel). In healthy controls, the regression of Cr/NAA versus fraction gray matter in the cingulate differs from frontal and parietal regions. CONCLUSIONS: Fast rosette spectroscopic imaging acquisitions with regression analyses are able to identify metabolic differences across 4-cm slabs of the brain centrally and over the cortical periphery with high efficiency, generating results that are consistent with clinical findings. Magn Reson Med 79:2470-2480, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Development of frontal GABA and glutamate supports excitation/inhibition balance from adolescence into adulthood.

Animal and human postmortem studies provide evidence for changes in gamma-aminobutyric acid (GABA) and glutamate in prefrontal cortex (PFC) during adolescence, suggesting shifts in excitation and inhibition balance consistent with critical period plasticity. However, how GABA and glutamate change through adolescence and how the balance of these inhibitory and excitatory neurotransmitters changes is not well understood in vivo in humans. High field (7 Tesla) Magnetic Resonance Spectroscopic Imaging was used to investigate age-related changes in the balance of GABA/creatine (Cr) and glutamate/Cr in multiple developmentally-relevant regions of frontal cortex in 144 10-30-year-olds. Results indicated a homogenous pattern of age-related Glu/Cr decreases across regions, while age-related changes in GABA/Cr were heterogenous, with a mix of stable and decreasing age effects. Importantly, balance between glutamate/Cr and GABA/Cr in areas of frontal cortex increased through adolescence, suggesting the presence of critical period plasticity in frontal cortex at this significant time of development when adult trajectories are established.

A longitudinal investigation of GABA, glutamate, and glutamine across the insula during antipsychotic treatment of first-episode schizophrenia.

Individuals with first-episode schizophrenia (FES) typically present with acute psychotic symptoms. Though antipsychotic drugs are the mainstay for treatment, the neurobiology underlying successful treatment remains largely elusive. Recent evidence from functional connectivity studies highlights the insula as a key structure in the neural mechanism of response. However, molecular contributions to response across insular regions remain largely unknown. We used 7-Tesla magnetic resonance spectroscopic imaging (MRSI) to measure glutamate (Glu), Glutamine (Gln), and GABA from anterior and posterior regions of the insula across antipsychotic treatment. A total of 36 participants were examined, including 15 individuals with FES and moderate to severe psychosis who were scanned at two time points, while starting and after 6 weeks of antipsychotic treatment. Symptoms were carefully monitored across the study period to characterize treatment response. GABA, Glu, and Gln levels were calculated relative to creatine in anterior and posterior insular regions, bilaterally. In relation to psychotic symptom reduction, we observed a significant increase in Glu across all insular regions with (p < 0.001), but no corresponding changes in Gln or GABA. In group analyses, the FES cohort showed lower levels of Glu (p < 0.001) and GABA (p = 0.02) at baseline. Finally, in exploratory analyses, treatment remitters demonstrated a normalization of lower insular Glu levels across treatment, unlike non-remitters. Overall, these findings contribute to our understating of molecular changes associated with antipsychotic response and demonstrate abnormalities specific to the insula in FES.

K⁺ dynamics in ischemic rat brain in vivo by 87Rb MRI at 7 T.

The aims of the present study were as follows: (i) to perform the first (87)Rb MRI in live rats with focal ischemic stroke; and (ii) to test the hypothesis that K(+) egress from the brain in this model is quantifiable in individual animals by high-field (7-T) K/Rb substitution MRI. Rats preloaded with dietary Rb(+) (resulting in Rb/(K + Rb) replacement ratios of 0.1-0.2 in the brain) were subjected to permanent occlusion of the middle cerebral artery, and (87)Rb MRI was implemented with 13-min temporal resolution using a dedicated RF coil and a spiral ultrashort-TE sequence (TR/TE = 3/0.07 ms). The ischemic core was localized by apparent diffusion coefficient mapping, by microtubule-associated protein-2 immunohistochemistry and by changes in surface reflectivity. [K], [Na] and [Rb] were determined independently in the micropunched samples by post-mortem flame photometry. Both techniques were generally in agreement in the nonischemic cortex; however, the MRI-assessed [K(+) + Rb(+)] drop in ischemic brain was less pronounced (average efflux rate of 4.8 ± 0.2 nEq/mm(3) /h versus 10 ± 1 nEq/mm(3)/h by flame photometry; p < 0.0001). The use of higher field gradients for better spatial resolution, and hence more accurate quantification, is suggested.

Reduced GABA/glutamate in the thalamus of individuals at clinical high risk for psychosis.

Youth at clinical high risk (CHR) are a unique population enriched for precursors of major psychiatric disorders, especially schizophrenia (SCZ). Recent neuroimaging findings point to abnormalities in the thalamus of patients with SCZ, including chronic and early course patients, as well as in CHR individuals relative to healthy comparison groups, thus suggesting that thalamic dysfunctions are present even before illness onset. Furthermore, modeling data indicate that alteration between excitatory and inhibitory control, as reflected by alteration in GABAergic and glutamatergic balance (i.e., GABA/Glu), may underlie thalamic deficits linked to the risk and development of psychosis. There is, however, a lack of in vivo evidence of GABA/Glu thalamic abnormalities in the CHR state. Magnetic resonance spectroscopic imaging (MRSI) 7 Tesla (7 T) provides enhanced resolution to quantify GABA and Glu levels in the thalamus of CHR individuals. In this study, we performed 7 T MRSI in 15 CHR and 20 healthy control (HC) participants. We found that GABA/Glu was significantly reduced in the right medial anterior and right medial posterior thalamus of CHR relative to HC groups. The GABA/Glu reduction was negatively correlated with general symptoms in the right medial anterior thalamus, as well as with disorganization symptoms in the right medial posterior thalamus. Altogether, these findings indicate that GABA/Glu abnormalities are present in the thalamus before the onset of full-blown psychosis and are associated with symptom severity, thus providing putative molecular and neuronal targets for early interventions in youth at CHR.

NMR study of general anesthetic interaction with nAChR beta2 subunit.

The molecular basis of anesthetic interaction with membrane proteins has been explored via determination of anesthetic effects on the structure and dynamics of the extended second transmembrane domain (TM2e) of the human neuronal nicotinic acetylcholine receptor (nAChR) beta(2) subunit in dodecylphosphocholine (DPC) micelles by (1)H and (15)N solution-state NMR. Both 1-chloro-1,2,2-trifluorocyclobutane (F3) and isoflurane, two volatile general anesthetics, induced nonuniform changes in chemical shifts among residues in TM2e. Saturation transfer difference NMR experiments further confirmed the direct anesthetic interaction with TM2e. A significant and more specific anesthetic interaction was observed on three leucine residues at the helix C-terminus. Although the TM2e helical structure remained after addition of anesthetics, plausible shortening and lengthening of helix hydrogen bonds were evidenced by periodic changes in backbone amide chemical shifts. The TM2e backbone dynamics were determined on the basis of the (15)N relaxation rate constants, R(1) and R(2), and the (15)N-[(1)H] NOE using the model-free approach. The global tumbling time (11.7 ns) of TM2e in micelles slightly increased ( approximately 12.3-12.5 ns) in the presence of anesthetics. The order parameter, S(2), exceeded 0.9 for all (15)N-labeled residues, showing a restricted internal motion. Anesthetics appear to have minor effect on the TM2e's internal motion. This study provided the basis for subsequent more comprehensive studies of anesthetic effects on the transmembrane domain complex of neuronal nAChR.

Stroke onset time using sodium MRI in rat focal cerebral ischemia.

BACKGROUND AND PURPOSE: Thrombolytic therapy with intravenous tPA must be administered within 3 hours after stroke onset. However, stroke onset time cannot be established in 20% to 45% of potential patients. We propose that the rate of increase of the brain concentration of sodium ([Na+]br) after stroke, monitored using sodium MRI in a rat model of cortical ischemia, is linear in each individual animal, can locate the ischemic region, and can be used to estimate onset time. METHODS: After induction of focal cortical ischemia in rats under isoflurane anesthesia, [Na+]br time course maps were acquired continuously on a 3 T whole body scanner from 2 to 7 hours after occlusion followed by T2-weighted proton images. Microtubule-associated protein-2 immunostained brain sections were used to verify the location of the infarct. RESULTS: The ischemic region identified with microtubule-associated protein-2 corresponded to the region of maximum [Na+]br increase (P<0.001; n=5), and all of the animals demonstrated high linearity. [Na+]br increased at a mean rate of 25+/-4.7%/h in ischemic tissue (P=0.013) but not in normal cortex (1.0+/-1.1%/h; P=0.42). The mean onset time error was 1+/-4 minutes (n=4). CONCLUSIONS: These results of sodium MRI show that the region of maximum [Na+]br increase corresponds to the ischemic region. Although [Na+]br increases at a different rate in each animal, the increase is linear, and, therefore, onset time can be estimated. These findings suggest that this method can be used as a ticking clock to estimate time elapsed after vascular occlusion.

Differences between arterial occlusive and cortical photothrombosis stroke models with magnetic resonance imaging and microtubule-associated protein-2 immunoreactivity.

The differences between two models of cerebral ischemia [middle cerebral arterial transection (MCAT) and cortical photothrombosis (PT)] were explored with multiparametric MRI of apparent diffusion coefficient trace (ADCtr), cerebral blood flow (CBF) and T1. Microtubule-associated protein-2 (MAP2) immunoreactivity sections aligned with the MR images in the same coronal plane were used to map the infarct and to guide region-of-interest selection. In ischemic cortex, the larger T1 increase in PT versus MCAT (42+/-7% vs. 16+/-5%) is related to the different character of edema between these models; yet, neither CBF nor ADCtr discriminated between them at 3.5 h, suggesting that different mechanisms of ischemic damage to the brain cells resulted in the same ADCtr value. CBF and ADCtr were depressed in immediately adjacent ischemic border by 27+/-7% and 47+/-10%, respectively, in MCAT but not in PT, suggesting marginal perfusion in MCAT. CBF in homotopic normal cortex in the opposite hemisphere was higher for PT compared with MCAT (199+/-20 and 134+/-10 ml/100 g/min, respectively). Different pathological processes in the two models affect CBF, ADCtr and T1 in a unique, regionally specific manner. The PT model differs substantially from the MCAT and is not a model of cortical ischemia with an appreciable border zone.

Late reversal of cerebral perfusion and water diffusion after transient focal ischemia in rats.

Region-specific cerebral blood flow (CBF) and the apparent diffusion coefficient (ADC) of tissue water in the rat brain were quantified by high-field magnetic resonance imaging at 9.4 T in the rat suture occlusion model. Cerebral blood flow and ADC were compared during the short- (4.5 hours) and long-term (up to 6 days) reperfusion after 80 minutes of transient middle cerebral artery occlusion, and correlated with the histology analysis. On occlusion, average CBF fell from approximately 100 to less than 50 mL x 100 g(-1) x min(-1) in the cortex, and to less than 20 mL x 100 g(-1) x min(-1) in the caudate putamen (CP). Corresponding ADC values decreased from (6.98 +/- 0.82) x 10(-4) to (5.49 +/- 0.54) x 10(-4) mm2/s in the cortex, and from (7.16 +/- 0.58) x 10(-4) to (4.86 +/- 0.62) x 10(-4) mm2/s in the CP. On average, CBF recovered to approximately 50% of baseline in the first 24 hours of reperfusion. After 2 to 4 days, a strong hyperperfusion in the ipsilateral cortex and CP, up to approximately 300 mL x 100 g(-1) x min(-1), was observed. The ADC ratio in the ipsilateral and contralateral CP was also inverted in the late reperfusion period. Histology revealed more severe tissue damage at the late stage of reperfusion than at 4.5 hours. Significant reversal of CBF and ADC during the late reperfusion period may reflect the impairment of autoregulation in the ischemic regions. Vascular factors may play an important role in the infarct development after 80-minute focal ischemia.

Aggregation of Fe(III)TPPS(4) on Biological Structures Is pH-Dependent, Suggesting Oxo-Bridging in the Aggregates.

Interaction of the Fe(III) derivative of tetra(4-sulfonatophenyl)porphyrin (TPPS(4)), and diamagnetic ZnTPPS(4) and metal-free TPPS(4), with the simplest models for membranes and protein reaction centers, aqueous (AM) and reversed (RM) ionic micelles, was studied by high-resolution (1)H NMR and proton magnetic relaxation measurements. AM were much more sensitive than RM to the bulky porphyrins, seemingly, because of the more restricted motion of surfactant chains in AM. TPPS(4) and its derivatives were incorporated into the AM of cationic cetyltrimethylammonium chloride (CTAC) or zwitterionic lysophosphatidylcholine (LPC) near the terminal part of their hydrocarbon chains, as evidenced by a strong upfield shift of the corresponding peaks. At a FeTPPS(4)/CTAC molar ratio greater than 0.05 and a pH > 4, FeTPPS(4) partly formed nonparamagnetic aggregates, which dissociated into monomers at pH < 4. In CTAC RM, FeTPPS(4) was predominantly aggregated, the transition to the monomer form occurring upon acidification of the water RM interior to about pH -1. No similar pH dependencies were found for ZnTPPS(4) and TPPS(4). It is supposed that charged porphyrins may interact with cellular membranes. Characteristic pH dependence of the FeTPPS(4) aggregation in micelles suggests that aggregated units are bound through a &mgr;-oxo-bridge. Similar mechanisms may be operative in other systems, such as porphyrin-protein.

NMR studies of drug interaction with membranes and membrane-associated proteins.

This review focuses on the recent developments in the study of drug interactions with biological membranes and membrane-associated proteins using nuclear magnetic resonance (NMR) spectroscopy and other spectroscopic techniques. Emphasis is placed on a class of low-affinity neurological agents as exemplified by volatile general anesthetics and structurally related compounds. The technical aspects are reviewed of how to prepare membrane-mimetic systems and of NMR approaches that are either in current use or opening new prospects. A brief literature survey covers studies ranging from drug distribution in simplified lipid matrix to specific drug interaction with neuronal receptors reconstituted in complicated synthetic membrane systems.

Correlated sodium and potassium imbalances within the ischemic core in experimental stroke: a 23Na MRI and histochemical imaging study.

This study addresses the spatial relation between local Na(+) and K(+) imbalances in the ischemic core in a rat model of focal ischemic stroke. Quantitative [Na(+)] and [K(+)] brain maps were obtained by (23)Na MRI and histochemical K(+) staining, respectively, and calibrated by emission flame photometry of the micropunch brain samples. Stroke location was verified by diffusion MRI, by changes in tissue surface reflectivity and by immunohistochemistry with microtubule-associated protein 2 antibody. Na(+) and K(+) distribution within the ischemic core was inhomogeneous, with the maximum [Na(+)] increase and [K(+)] decrease typically observed in peripheral regions of the ischemic core. The pattern of the [K(+)] decrease matched the maximum rate of [Na(+)] increase ('slope'). Some residual mismatch between the sites of maximum Na(+) and K(+) imbalances was attributed to the different channels and pathways involved in transport of the two ions. A linear regression of the [Na(+)]br vs. [K(+)]br in the samples of ischemic brain indicates that for each K(+) equivalent leaving ischemic tissue, 0.8±0.1 Eq, on average, of Na(+) enter the tissue. Better understanding of the mechanistic link between the Na(+) influx and K(+) egress would validate the (23)Na MRI slope as a candidate biomarker and a complementary tool for assessing ischemic damage and treatment planning.

Sodium mapping in focal cerebral ischemia in the rat by quantitative (23)Na MRI.

PURPOSE: To validate (23)Na twisted projection magnetic resonance imaging (MRI) as a quantitative technique to assess local brain sodium concentration ([Na(+)](br)) during rat focal ischemia every 5.3 minutes. MATERIALS AND METHODS: The MRI protocol included an ultrashort echo-time (0.4 msec), a correction of radiofrequency (RF) inhomogeneities by B(1) mapping, and the use of 0-154 mM NaCl calibration standards. To compare MRI [Na(+)](br) values with those obtained by emission flame photometry in precision-punched brain samples of about 0.5 mm(3) size, MR images were aligned with a histological three-dimensional reconstruction of the punched brain and regions of interest (ROIs) were placed precisely over the punch voids. RESULTS: The Bland-Altman analysis of [Na(+)](br) in normal and ischemic cortex and caudate putamen of seven rats quantitated by (23)Na MRI and flame photometry yielded a mean bias and limits of agreement (at +/-1.96 SD) of 2% and 43% of average, respectively. A linear increase in [Na(+)](br) was observed between 1 and 6 hours after middle cerebral artery occlusion. CONCLUSION: (23)Na MRI provides accurate and reliable results within the whole range of [Na(+)](br) in ischemia with a temporal resolution of 5.3 minutes and precisely targeted submicroliter ROIs in selected brain structures.

Inhomogeneous sodium accumulation in the ischemic core in rat focal cerebral ischemia by 23Na MRI.

PURPOSE: To test the hypotheses that (i) the regional heterogeneity of brain sodium concentration ([Na(+)](br)) provides a parameter for ischemic progression not available from apparent diffusion coefficient (ADC) data, and (ii) [Na(+)](br) increases more in ischemic cortex than in the caudate putamen (CP) with its lesser collateral circulation after middle cerebral artery occlusion in the rat. MATERIALS AND METHODS: (23)Na twisted projection MRI was performed at 3 Tesla. [Na(+)](br) was independently determined by flame photometry. The ischemic core was localized by ADC, by microtubule-associated protein-2 immunohistochemistry, and by changes in surface reflectivity. RESULTS: Within the ischemic core, the ADC ratio relative to the contralateral tissue was homogeneous (0.63 +/- 0.07), whereas the rate of [Na(+)](br) increase (slope) was heterogeneous (P < 0.005): 22 +/- 4%/h in the sites of maximum slope versus 14 +/- 1%/h elsewhere (here 100% is [Na(+)](br) in the contralateral brain). Maximum slopes in the cortex were higher than in CP (P < 0.05). In the ischemic regions, there was no slope/ADC correlation between animals and within the same brain (P > 0.1). Maximum slope was located at the periphery of ischemic core in 8/10 animals. CONCLUSION: Unlike ADC, (23)Na MRI detected within-core ischemic lesion heterogeneity.

MAP2 immunostaining in thick sections for early ischemic stroke infarct volume in non-human primate brain.

The delineation of early infarction in large gyrencephalic brain cannot be accomplished with triphenyl-tetrazolium chloride (TTC) due to its limitations in the early phase, nor can it be identified with microtubule-associated protein 2 (MAP2) immunohistochemistry, due to the fragility of large thin sections. We hypothesize that MAP2 immunostaining of thick brain sections can accurately identify early ischemia in the entire monkey brain. Using ischemic brains of one rat and three monkeys, a thick-section MAP2 immunostaining protocol was developed to outline the infarct region over the entire non-human primate brain. Comparison of adjacent thick and thin sections in a rat brain indicated complete correspondence between ischemic regions (100.4mm(3)+/-1.2%, n=7, p=0.44). Thick sections in monkey brain possessed the increased structural stability necessary for the extensive MAP2 immunostaining procedure permitting quantification of the ischemic region as a percent of total monkey brain, giving infarct volumes of 11.4, 16.3, and 19.0% of total brain. Stacked 2D images of the intact thick brain tissue sections provided a 3D representation for comparison to MRI images. The infarct volume of 16.1cm(3) from the MAP2 sections registered with MRI images agreed well with the volume calculated directly from the stained sections of 16.6 cm(3). Thick brain tissue section MAP2 immunostaining provides a new method for determining infarct volume over the entire brain at early time points in a non-human primate model of ischemic stroke.

Monitoring of brain potassium with rubidium flame photometry and MRI.

An animal model was developed to monitor [K(+)] in the brain using partial K(+) replacement with Rb(+) and (87)Rb MRI. Fifty-one rats were given 0-80 mM of RbCl in the drinking water for up to 90 days. Focal cerebral ischemia was produced in 15 of the animals. Na, K, and Rb content in precision-guided submilligram samples of cortical brain were determined by emission flame photometry. Multinuclear (87)Rb/(23)Na/(1)H MRI was performed on phantoms and rats at 3T using a twisted projection imaging (TPI) scheme for (87)Rb/(23)Na, and custom-built surface or parallel cosine transmit/receive coils. Brain [Rb(+)] was safely brought up to 17-25 mEq/kg within 2-3 weeks of feeding. The characteristic patterns of [K(+)] decrease (with a sharp drop at 3-4 hr of ischemia) and [Na(+)] increase (at a rate of 31%/hr) observed previously in animals without Rb/K substitution were reproduced in ischemic cortex. The Rb/(Rb+K) ratio increased over time in ischemic areas (R = 0.91, P < 0.001), suggesting an additional index of ischemia progression. Preliminary (87)Rb MRI gave an estimate of 20-25 mEq Rb/kg brain weight (N = 2). In conclusion, brain Rb(+) is detectable by (87)Rb MRI and does not significantly interfere with ion dynamics in ischemic brain, which enables (87)Rb MRI studies of K(+) in ischemia.

NMR structure and dynamics of the second transmembrane domain of the neuronal acetylcholine receptor beta 2 subunit.

Structure and backbone dynamics of a selectively [(15)N]Leu-labeled 28-residue segment of the extended second transmembrane domain (TM2e) of the human neuronal nicotinic acetylcholine receptor (nAChR) beta(2) subunit were studied by (1)H and (15)N solution-state NMR in dodecylphosphocholine micelles. The TM2e structure was determined on the basis of the nuclear Overhauser effects (NOEs) and the hydrogen bond restraints, which were inferred from the presence of H(alpha)(i)-H(N)(i+3), H(alpha)(i)-H(beta)(i+3), and H(alpha)(i)-H(N)(i+4) NOE connectivity and from the slow amide hydrogen exchange with D(2)O. The TM2e structure of the nAChR beta(2) subunit contains a helical region between T4 and K22. Backbone dynamics were calculated using the model-free approach based on the (15)N relaxation rate constants, R(1) and R(2), and on the (15)N-[(1)H] NOE. The data acquired at 9.4 and 14.1 T and calculations using different dynamic models demonstrated no conformational exchange and internal motions on the nanosecond time scale. The global tumbling time of TM2e in micelles was 14.4 +/- 0.2 ns; the NOE values were greater than 0.63 at 9.4 T, and the order parameter, S(2), was 0.83-0.96 for all (15)N-labeled leucine residues, suggesting a restricted internal motion. This is the first report of NMR structure and backbone dynamics of the second transmembrane domain of the human nAChR beta(2) subunit in a membrane-mimetic environment, providing the basis for subsequent studies of subunit interactions in the transmembrane domain complex of the neuronal nAChR.

NMR structure and backbone dynamics of the extended second transmembrane domain of the human neuronal glycine receptor alpha1 subunit.

The structure and backbone dynamics of an extended second transmembrane segment (TM2e) of the human neuronal glycine receptor alpha(1) subunit in sodium dodecyl sulfate micelles were studied by (1)H and (15)N solution-state NMR. The 28-amino acid segment contained the consensus TM2 domain plus part of the linker between the second and third transmembrane domains. The presence of a well-structured helical region of at least 13 amino acids long and an unstructured region near the linker was evident from the proton chemical shifts and the pattern of midrange nuclear Overhauser effects (NOE). (15)N relaxation rate constants, R(1) and R(2), and (15)N-[(1)H] NOE indicated restricted internal motions in the helical region with NOE values between 0.6 and 0.8. The squared order parameter (S(2)), the effective correlation time for fast internal motions (tau(e)), and the global rotational correlation time (tau(m)) were calculated for all TM2e backbone N-H bonds using the model-free approach. The S(2) values ranged about 0.75-0.86, and the tau(e) values were below 100 ps for most of the residues in the helical region. The tau(m) value, calculated from the dynamics of the helical region, was 5.1 ns. The S(2) values decreased to 0.1, and the tau(e) values sharply increased up to 1.2 ns at the linker near the C-terminus, indicating that the motion of this region is unrestricted. The results suggest a relatively high degree of motional freedom of TM2e in micelles and different propensities of the N- and C-terminal moieties of the transmembrane domain to assume stable helical structures.

ADC characterization of region-specific response to cerebral perfusion deficit in rats by MRI at 9.4 T.

Region-specific cerebral blood flow (CBF) and apparent diffusion coefficient (ADC) of water in the rat brain were quantified in vivo by high-field MRI (9.4 T) for 6-7 h after middle cerebral artery occlusion (MCAO). Upon occlusion, average CBF fell from about 1.5-2 ml/g/min to below 0.5 ml/g/min in cortical areas and the amygdala, and below 0.2 ml/g/min in the caudate putamen. CBF in some of the homologous contralateral areas also decreased by 20-30%. Average ADC decreased from about 8 center dot 10(-4) to 5 center dot 10(-4) mm(2)/s in the caudate putamen and parietal cortex. Corresponding changes in ADC were lower in the frontal cortex and negligible in the piriform cortex, suggesting that the perfusion threshold for ADC decrease may be different for different brain regions in the same animal. The area of decreased ADC correlated well with the infarction area revealed by 2,3,5-triphenyltetrazolium chloride (TTC) staining of brain slices in vitro. A better understanding of the mechanisms linking ADC and CBF changes to ischemic cell disorders may prove useful in characterizing the degree of tissue damage, and in developing and evaluating treatment strategies.