Florence D. Morgenthaler

Preliminary evidence for a fronto-parietal dysfunction in able-bodied participants with a desire for limb amputation

BACKGROUND Reports of able-bodied participants with the persisting desire for limb amputation raise legal and ethical questions that are partly due to insufficient empirical knowledge about the condition. Here, we searched for potential neurological mechanisms in participants with desire for limb amputation in order to help develop adequate nosological classifications, diagnosis, and treatment. METHODS Semi-structured interviews were carried out with 20 participants who self-identified themselves as able-bodied individuals desiring amputation of a limb. RESULTS The results suggest that amputation desire is not unspecific, but in most cases specific for a circumscribed part of the body. Most frequently affected was the leg, mostly on the left, non-dominant side. Left-sidedness and limb specificity was associated with elementary and complex somatosensory disturbances of the affected limb akin to those reported by neurological patients. The most frequent neurological co-morbidity was migraine headache. CONCLUSIONS These results document the existence of an unusual condition in able-bodied participants characterized by a persons desire for the amputation of one or more particular limbs. Left-sidedness, limb specificity and somatosensory disturbances of the affected limb are suggestive of abnormal brain mechanisms in right fronto-parietal cortex. Based on this association we suggest that desire for limb amputation may be conceptualized as asomatognosia due to disturbed integration of multi-sensory information of the affected body parts into a coherent cerebral representation of the own body. This suggestion has to be regarded with caution as we did not perform any neurological examination.

Biochemical quantification of total brain glycogen concentration in rats under different glycemic states

All (13)C NMR studies of brain glycogen to date relied on observing the incorporation of (13)C label into glycogen, and thus interpretation was potentially affected by changes in (13)C label turnover rates. The goal of this study was to quantify total brain glycogen concentration under conditions of hypoglycemia or normoglycemia using biochemical methods. Rats were sacrificed using a focused microwave fixation device. The results showed that metabolism of brain glycogen was Glc- and insulin-sensitive and that insulin-induced hypoglycemia promoted a gradual glycogenolysis. Moreover, we show that there are very mild effects of isoflurane and alpha-chloralose anesthesia on brain glycogen concentration. Altogether these results show that total brain glycogen serves as a substantial source of glucosyl units during insulin-induced moderate hypoglycemia and therefore may be neuroprotective. Finally we also conclude that previous interpretation of (13)C NMR spectroscopy data accurately reflected the changes in total brain glycogen content.

Cerebral glutamine metabolism under hyperammonemia determined in vivo by localized 1H and 15N NMR spectroscopy

Brain glutamine synthetase (GS) is an integral part of the glutamate—glutamine cycle and occurs in the glial compartment. In vivo Magnetic Resonance Spectroscopy (MRS) allows noninvasive measurements of the concentrations and synthesis rates of metabolites. 15N MRS is an alternative approach to 13C MRS. Incorporation of labeled 15N from ammonia in cerebral glutamine allows to measure several metabolic reactions related to nitrogen metabolism, including the glutamate—glutamine cycle. To measure 15N incorporation into the position 5N of glutamine and position 2N of glutamate and glutamine, we developed a novel 15N pulse sequence to simultaneously detect, for the first time, [5-15N]Gln and [2-15N]Gln + Glu in vivo in the rat brain. In addition, we also measured for the first time in the same experiment localized 1H spectra for a direct measurement of the net glutamine accumulation. Mathematical modeling of 1H and 15N MRS data allowed to reduce the number of assumptions and provided reliable determination of GS (0.30 ± 0.050 μmol/g per minute), apparent neurotransmission (0.26 ± 0.030 μmol/g per minute), glutamate dehydrogenase (0.029 ± 0.002 μmol/g per minute), and net glutamine accumulation (0.033 ± 0.001 μmol/g per minute). These results showed an increase of GS and net glutamine accumulation under hyperammonemia, supporting the concept of their implication in cerebral ammonia detoxification.

Morphological and molecular heterogeneity in release sites of single neurons

We have previously shown that labelling intensities for synaptic proteins vary strongly among synaptic boutons. Here we addressed the questions as to whether there are heterogeneous levels of integral membrane synaptic vesicle proteins at distinct active release sites of single neurons and if these sites possess the ultrastructural features of synapses. By double‐immunostaining with specific antibodies against synaptophysin, synaptotagmin I, VAMP1 and VAMP2, we identified different relative levels of these integral membrane proteins of synaptic vesicles in comparison to boutons of the same rat cortical neuron. This heterogeneity could also be observed between the two isoforms VAMP1 and VAMP2. By studying pairs of these proteins implicated in neurotransmitter release, including both VAMP isoforms, we also show that the sites that contained predominantly one protein were nevertheless functional, as they internalized and released FM1‐43 upon potassium stimulation. Using electron microscopy, we show that these active sites could have either synaptic specializations, or the features of vesicle‐containing varicosities without a postsynaptic target. Different varicosities of the same neuron showed different intensities for synaptic vesicle proteins; some varicosities were capable of internalizing and releasing FM1‐43, while others were silent. These results show that integral membrane synaptic vesicle proteins are differentially distributed among functional release sites of the same neuron.

Non-invasive quantification of brain glycogen absolute concentration

The only currently available method to measure brain glycogen in vivo is 13C NMR spectroscopy. Incorporation of 13C‐labeled glucose (Glc) is necessary to allow glycogen measurement, but might be affected by turnover changes. Our aim was to measure glycogen absolute concentration in the rat brain by eliminating label turnover as variable. The approach is based on establishing an increased, constant 13C isotopic enrichment (IE). 13C‐Glc infusion is then performed at the IE of brain glycogen. As glycogen IE cannot be assessed in vivo, we validated that it can be inferred from that of N‐acetyl‐aspartate IE in vivo: After [1‐13C]‐Glc ingestion, glycogen IE was 2.2 ± 0.1 fold that of N‐acetyl‐aspartate (n = 11, R2 = 0.77). After subsequent Glc infusion, glycogen IE equaled brain Glc IE (n = 6, paired t‐test, p = 0.37), implying isotopic steady‐state achievement and complete turnover of the glycogen molecule. Glycogen concentration measured in vivo by 13C NMR (mean ± SD: 5.8 ± 0.7 μmol/g) was in excellent agreement with that in vitro (6.4 ± 0.6 μmol/g, n = 5). When insulin was administered, the stability of glycogen concentration was analogous to previous biochemical measurements implying that glycogen turnover is activated by insulin. We conclude that the entire glycogen molecule is turned over and that insulin activates glycogen turnover.

Glucose and lactate are equally effective in energizing activity-dependent synaptic vesicle turnover in purified cortical neurons

This study examines the role of glucose and lactate as energy substrates to sustain synaptic vesicle cycling. Synaptic vesicle turnover was assessed in a quantitative manner by fluorescence microscopy in primary cultures of mouse cortical neurons. An electrode-equipped perfusion chamber was used to stimulate cells both by electrical field and potassium depolarization during image acquisition. An image analysis procedure was elaborated to select in an unbiased manner synaptic boutons loaded with the fluorescent dye N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM1-43). Whereas a minority of the sites fully released their dye content following electrical stimulation, others needed subsequent K(+) depolarization to achieve full release. This functional heterogeneity was not significantly altered by the nature of metabolic substrates. Repetitive stimulation sequences of FM1-43 uptake and release were then performed in the absence of any metabolic substrate and showed that the number of active sites dramatically decreased after the first cycle of loading/unloading. The presence of 1 mM glucose or lactate was sufficient to sustain synaptic vesicle cycling under these conditions. Moreover, both substrates were equivalent for recovery of function after a phase of decreased metabolic substrate availability. Thus, lactate appears to be equivalent to glucose for sustaining synaptic vesicle turnover in cultured cortical neurons during activity.

Direct Validation of In Vivo Localized 13C MRS Measurements of Brain Glycogen

With the use of localized 13C MRS in conjunction with [1‐13C]‐D‐glucose infusion, it is possible to study brain glycogen metabolism in vivo. The purpose of this study was to validate in vivo 13C MRS measurements by comparing them with results from a standard biochemical assay. To increase the [1–13C] glycogen concentration, 11 rats were subjected to an episode of acute hypoglycemia followed by a mild hyperglycemic recovery period during which [1‐13C]‐D‐glucose was infused. The total brain [1‐13C] glycogen of the same animal was determined from the enzymatically determined total brain glycogen content, which was fixed by focused microwave irradiation (4 kW in 1.4 s) immediately after the end of the in vivo NMR measurements. The corresponding isotopic enrichment (IE) of glycogen was measured by in vitro 1H MRS of protons bound to glucose C1‐α. The in vivo [1‐13C] glycogen concentration was strongly correlated to the in vitro [1‐13C] glycogen content determined by biochemical measurement in a linear manner (R = 0.79). The results are consistent with the notion that localized 13C MRS measurements closely reflect 13C glycogen content in the brain. Magn Reson Med 57:243–248, 2007.

Quantification of brain glycogen concentration and turnover through localized 13C NMR of both the C1 and C6 resonances

We have recently shown that at isotopic steady state 13C NMR can provide a direct measurement of glycogen concentration changes, but that the turnover of glycogen was not accessible with this protocol. The aim of the present study was to design, implement and apply a novel dual‐tracer infusion protocol to simultaneously measure glycogen concentration and turnover. After reaching isotopic steady state for glycogen C1 using [1‐13C] glucose administration, [1,6‐13C2] glucose was infused such that isotopic steady state was maintained at the C1 position, but the C6 position reflected 13C label incorporation. To overcome the large chemical shift displacement error between the C1 and C6 resonances of glycogen, we implemented 2D gradient based localization using the Fourier series window approach, in conjunction with time‐domain analysis of the resulting FIDs using jMRUI. The glycogen concentration of 5.1 ± 1.6 mM measured from the C1 position was in excellent agreement with concomitant biochemical determinations. Glycogen turnover measured from the rate of label incorporation into the C6 position of glycogen in the α‐chloralose anesthetized rat was 0.7 µmol/g/h. Copyright

Steady-state brain glucose transport kinetics re-evaluated with a four-state conformational model

Glucose supply from blood to brain occurs through facilitative transporter proteins. A near linear relation between brain and plasma glucose has been experimentally determined and described by a reversible model of enzyme kinetics. A conformational four-state exchange model accounting for trans-acceleration and asymmetry of the carrier was included in a recently developed multi-compartmental model of glucose transport. Based on this model, we demonstrate that brain glucose (Gbrain) as function of plasma glucose (Gplasma) can be described by a single analytical equation namely comprising three kinetic compartments: blood, endothelial cells and brain. Transport was described by four parameters: apparent half saturation constant Kt, apparent maximum rate constant Tmax, glucose consumption rate CMRglc, and the iso-inhibition constant Kii that suggests Gbrain as inhibitor of the isomerisation of the unloaded carrier. Previous published data, where Gbrain was quantified as a function of plasma glucose by either biochemical methods or NMR spectroscopy, were used to determine the aforementioned kinetic parameters. Glucose transport was characterized by Kt ranging from 1.5 to 3.5 mM, Tmax/CMRglc from 4.6 to 5.6, and Kii from 51 to 149 mM. It was noteworthy that Kt was on the order of a few mM, as previously determined from the reversible model. The conformational four-state exchange model of glucose transport into the brain includes both efflux and transport inhibition by Gbrain, predicting that Gbrain eventually approaches a maximum concentration. However, since Kii largely exceeds Gplasma, iso-inhibition is unlikely to be of substantial importance for plasma glucose below 25 mM. As a consequence, the reversible model can account for most experimental observations under euglycaemia and moderate cases of hypo- and hyperglycaemia.

A comparison of in vivo 13C MR brain glycogen quantification at 9.4 and 14.1 T

The high molecular weight and low concentration of brain glycogen render its noninvasive quantification challenging. Therefore, the precision increase of the quantification by localized 13C MR at 9.4 to 14.1 T was investigated. Signal‐to‐noise ratio increased by 66%, slightly offset by a T1 increase of 332 ± 15 to 521 ± 34 ms. Isotopic enrichment after long‐term 13C administration was comparable (∼40%) as was the nominal linewidth of glycogen C1 (∼50 Hz). Among the factors that contributed to the 66% observed increase in signal‐to‐noise ratio, the T1 relaxation time impacted the effective signal‐to‐noise ratio by only 10% at a repetition time = 1 s. The signal‐to‐noise ratio increase together with the larger spectral dispersion at 14.1 T resulted in a better defined baseline, which allowed for more accurate fitting. Quantified glycogen concentrations were 5.8 ± 0.9 mM at 9.4 T and 6.0 ± 0.4 mM at 14.1 T; the decreased standard deviation demonstrates the compounded effect of increased magnetization and improved baseline on the precision of glycogen quantification. Magn Reson Med, 2011.