Leif Oltedal

Intramolecular protein-protein and protein-lipid interactions control the conformation and subcellular targeting of neuronal Ykt6

Although the membrane-trafficking functions of most SNAREs are conserved from yeast to humans, some mammalian SNAREs have evolved specialized functions unique to multicellular life. The mammalian homolog of the prenylated yeast SNARE Ykt6p might be one such example, because rat Ykt6 is highly expressed only in brain neurons. Furthermore, neuronal Ykt6 displayed a remarkably specialized, punctate localization that did not overlap appreciably with conventional compartments of the endomembrane system, suggesting that Ykt6 might be involved in a pathway unique to or specifically modified for neuronal function. Targeting of Ykt6 to its unique subcellular location was directed by its profilin-like longin domain. We have taken advantage of high-resolution structural data available for the yeast Ykt6p longin domain to examine mechanisms by which the mammalian longin domain controls Ykt6 conformation and subcellular targeting. We found that the overall tertiary structure of the longin domain, not sequence-specific surface features, drives direct targeting to the Ykt6 punctate structures. However, several sequence-specific surface features of the longin domain indirectly regulate Ykt6 localization through intramolecular interactions that mask otherwise-dominant targeting signals on the SNARE motif and lipid groups. Specifically, two hydrophobic binding pockets, one on each face of the longin domain, and one mixed hydrophobic/charged surface, participate in protein-protein interactions with the SNARE motif and protein-lipid interactions with the lipid group(s) at the molecules C-terminus. One of the hydrophobic pockets suppresses protein-palmitoylation-dependent mislocalization of Ykt6 to the plasma membrane. The Ykt6 intramolecular interactions would be predicted to create a compact, closed conformation of the SNARE that prevents promiscuous targeting interactions and premature insertion into membranes. Interestingly, both protein-protein and protein-lipid interactions are required for a tightly closed conformation and normal targeting.

Sec6 is localized to the plasma membrane of mature synaptic terminals and is transported with secretogranin II-containing vesicles

The sec6/8 (exocyst) complex is implicated in targeting of vesicles for regulated exocytosis in various cell types and is believed to play a role in synaptogenesis and brain development. We show that the subunits sec6 and sec8 are present at significant levels in neurons of adult rat brain, and that immunoreactivity for the two subunits has a differential subcellular distribution. We show that in developing as well as mature neurons sec6 is concentrated at the inside of the presynaptic plasma membrane, while sec8 immunoreactivity shows a diffuse cytoplasmic distribution. Among established, strongly synaptophysin-positive neuronal boutons, sec6 displays highly differential concentrations, indicating a role for the complex independent of the ongoing synaptic-vesicle release activity. Sec6 is transported along neurites on secretogranin II-positive vesicles, while sec6-negative/secretogranin II-positive vesicles stay in the cell body. In PC12 cells, sec6-positive vesicles accumulate at the plasma membrane at sites of cell-cell contact. Neuronal induction of the PC12 cells with nerve growth factor shows that sec8 is not freely soluble, but may probably interact with cytoskeletal elements. The complex may facilitate the targeting of membrane material to presynaptic sites and may possibly shuttle vesicles from the cytoskeletal transport machinery to presynaptic membrane sites. Thus, we suggest that the exocyst complex serves to modulate exocytotic activity, by targeting membrane material to its presynaptic destination.

Passive membrane properties and electrotonic signal processing in retinal rod bipolar cells

Rod bipolar cells transmit visual signals from their dendrites, where they receive input from rod photoreceptors, to their axon terminals, where they synapse onto amacrine cells. Little is known, however, about the transmission and possible transformation of these signals. We have combined axon terminal recording in retinal slices, quantitative, light‐microscopic morphological reconstruction and computer modelling to obtain detailed compartmental models of rat rod bipolar cells. Passive cable properties were estimated by directly fitting the current responses of the models evoked by voltage pulses to the physiologically recorded responses. At a holding potential of −60 mV, the average best‐fit parameters were 1.1 μF cm−2 for specific membrane capacitance (Cm), 130 Ω cm for cytoplasmic resistivity (Ri), and 24 kΩ cm2 for specific membrane resistance (Rm). The passive integration of excitatory and inhibitory synaptic inputs was examined by computer modelling with physiologically realistic synaptic conductance waveforms. For both transient and steady‐state synaptic inhibition, the inhibitory effect was relatively insensitive to the location of the inhibition. For transient synaptic inhibition, the time window of effective inhibition depended critically on the relative timing of inhibition and excitation. The passive signal transmission between soma and axon terminal was examined by the electrotonic transform and quantified as the frequency‐dependent voltage attenuation of sinusoidal voltage waveforms. For the range of parameters explored (axon diameter and length, Ri), the lowest cutoff frequency observed was ∼300 Hz, suggesting that realistic scotopic visual signals will be faithfully transmitted from soma to axon terminal, with minimal passive attenuation along the axon.

Electrical Synapses Between AII Amacrine Cells: Dynamic Range and Functional Consequences of Variation in Junctional Conductance

AII amacrine cells form a network of electrically coupled interneurons in the mammalian retina and tracer coupling studies suggest that the junctional conductance (G(j)) can be modulated. However, the dynamic range of G(j) and the functional consequences of varying G(j) over the dynamic range are unknown. Here we use whole cell recordings from pairs of coupled AII amacrine cells in rat retinal slices to provide direct evidence for physiological modulation of G(j), appearing as a time-dependent increase from about 500 pS to a maximum of about 3,000 pS after 30-90 min of recording. The increase occurred in recordings with low- but not high-resistance pipettes, suggesting that it was related to intracellular washout and perturbation of a modulatory system. Computer simulations of a network of electrically coupled cells verified that our recordings were able to detect and quantify changes in G(j) over a large range. Dynamic-clamp electrophysiology, with insertion of electrical synapses between AII amacrine cells, allowed us to finely and reversibly control G(j) within the same range observed for physiologically coupled cells and to examine the quantitative relationship between G(j) and steady-state coupling coefficient, synchronization of subthreshold membrane potential fluctuations, synchronization and transmission of action potentials, and low-pass filter characteristics. The range of G(j) values over which signal transmission was modulated depended strongly on the specific functional parameter examined, with the largest range observed for action potential transmission and synchronization, suggesting that the full range of G(j) values observed during spontaneous run-up of coupling could represent a physiologically relevant dynamic range.

Electrical coupling and passive membrane properties of AII amacrine cells.

AII amacrine cells in the mammalian retina are connected via electrical synapses to on-cone bipolar cells and to other AII amacrine cells. To understand synaptic integration in these interneurons, we need information about the junctional conductance (g(j)), the membrane resistance (r(m)), the membrane capacitance (C(m)), and the cytoplasmic resistivity (R(i)). Due to the extensive electrical coupling, it is difficult to obtain estimates of r(m), as well as the relative contribution of the junctional and nonjunctional conductances to the total input resistance of an AII amacrine cell. Here we used dual voltage-clamp recording of pairs of electrically coupled AII amacrine cells in an in vitro slice preparation from rat retina and applied meclofenamic acid (MFA) to block the electrical coupling and isolate single AII amacrines electrically. In the control condition, the input resistance (R(in)) was approximately 620 Mohms and the apparent r(m) was approximately 760 Mohms. After block of electrical coupling, determined by estimating g(j) in the dual recordings, R(in) and r(m) were approximately 4,400 Mohms, suggesting that the nongap junctional conductance of an AII amacrine cell is approximately 16% of the total input conductance. Control experiments with nucleated patches from AII amacrine cells suggested that MFA had no effect on the nongap junctional membrane of these cells. From morphological reconstructions of AII amacrine cells filled with biocytin, we obtained a surface area of approximately 900 microm(2) which, with a standard value for C(m) of 0.01 pF/microm(2), corresponds to an average capacitance of approximately 9 pF and a specific membrane resistance of approximately 41 kohms cm(2). Together with information concerning synaptic connectivity, these data will be important for developing realistic compartmental models of the network of AII amacrine cells.

Antibodies to CRMP3-4 associated with limbic encephalitis and thymoma.

We present a case with subacute limbic encephalitis (LE) and thymoma. Neither classical onconeural antibodies nor antibodies to voltage gated potassium channels (VGKC) were detected, but the serum was positive for anti‐glutamic acid decarboxylase (GAD). The patient serum also stained synaptic boutons of pyramidal cells and nuclei of granule cells of rat hippocampus. The objective of the study was to identify new antibodies associated with LE. Screening a cDNA expression library identified collapsin response mediator protein 3 (CRMP3), a protein involved in neurite outgrowth. The serum also reacted with both CRMP3 and CRMP4 by Western blot. Similar binding pattern of hippocampal granule cells was obtained with the patient serum and rabbit anti‐serum against CRMP1–4. The CRMP1–4 antibodies stained neuronal nuclei of a biopsy from the patients temporal lobe, but CRMP1–4 expression in thymoma could only be detected by immunoblotting. Absorption studies with recombinant GAD failed to abolish the staining of the hippocampal granule cells. Our findings illustrate that CRMP3–4 antibodies can be associated with LE and thymoma. This has previously been associated with CRMP5.

Transient release kinetics of rod bipolar cells revealed by capacitance measurement of exocytosis from axon terminals in rat retinal slices

Presynaptic transmitter release has mostly been studied through measurements of postsynaptic responses, but a few synapses offer direct access to the presynaptic terminal, thereby allowing capacitance measurements of exocytosis. For mammalian rod bipolar cells, synaptic transmission has been investigated in great detail by recording postsynaptic currents in AII amacrine cells. Presynaptic measurements of the dynamics of vesicular cycling have so far been limited to isolated rod bipolar cells in dissociated preparations. Here, we first used computer simulations of compartmental models of morphologically reconstructed rod bipolar cells to adapt the ‘Sine + DC’ technique for capacitance measurements of exocytosis at axon terminals of intact rod bipolar cells in retinal slices. In subsequent physiological recordings, voltage pulses that triggered presynaptic Ca2+ influx evoked capacitance increases that were proportional to the pulse duration. With pulse durations ≤100 ms, the increase saturated at ∼10 fF, corresponding to the size of a readily releasable pool of vesicles. Pulse durations ≥400 ms evoked additional capacitance increases, probably reflecting recruitment from additional pools of vesicles. By using Ca2+ tail current stimuli, we separated Ca2+ influx from Ca2+ channel activation kinetics, allowing us to estimate the intrinsic release kinetics of the readily releasable pool, yielding a time constant of ∼1.1 ms and a maximum release rate of 2–3 vesicles (release site)−1 ms−1. Following exocytosis, we observed endocytosis with time constants ranging from 0.7 to 17 s. Under physiological conditions, it is likely that release will be transient, with the kinetics limited by the activation kinetics of the voltage‐gated Ca2+ channels.

Vesicular release of glutamate from hippocampal neurons in culture: an immunocytochemical assay

Glutamate, the main excitatory neurotransmitter in the brain, may cause excitotoxic damage through excessive release during a number of pathological conditions. We have developed an immunocytochemical assay to investigate the mechanisms and regulation of glutamate release from intact, cultured neurons. Our results indicate that cultured hippocampal neurons have a large surplus of glutamate available for release upon chemically induced depolarization. Long incubations with high K+-concentrations, and induction of repetitive action potentials with the K+-channel blocker 4-aminopyridine (4-AP), caused a significant reduction in glutamate labeling in a subset of boutons, demonstrating that transmitter release exceeded the capacity for replenishment. The number of boutons where release exceeded replenishment increased continuously with time of stimulation. This depletion was Ca2+-dependent and sensitive to bafilomycin A1 (baf), indicating that it was dominated by vesicular release mechanisms. The depletion of glutamate from cell bodies and dendrites was also Ca2+-dependent. Thus, under the present conditions, cytosolic glutamate is taken up in vesicles prior to release, and the main escape route for the amino acid is through vesicular exocytosis. Depolarization with lower concentrations of K+ caused sustainable release of glutamate, i.e., without full depletion.

The Global ECT-MRI Research Collaboration (GEMRIC): Establishing a multi-site investigation of the neural mechanisms underlying response to electroconvulsive therapy

Major depression, currently the worlds primary cause of disability, leads to profound personal suffering and increased risk of suicide. Unfortunately, the success of antidepressant treatment varies amongst individuals and can take weeks to months in those who respond. Electroconvulsive therapy (ECT), generally prescribed for the most severely depressed and when standard treatments fail, produces a more rapid response and remains the most effective intervention for severe depression. Exploring the neurobiological effects of ECT is thus an ideal approach to better understand the mechanisms of successful therapeutic response. Though several recent neuroimaging studies show structural and functional changes associated with ECT, not all brain changes associate with clinical outcome. Larger studies that can address individual differences in clinical and treatment parameters may better target biological factors relating to or predictive of ECT-related therapeutic response. We have thus formed the Global ECT-MRI Research Collaboration (GEMRIC) that aims to combine longitudinal neuroimaging as well as clinical, behavioral and other physiological data across multiple independent sites. Here, we summarize the ECT sample characteristics from currently participating sites, and the common data-repository and standardized image analysis pipeline developed for this initiative. This includes data harmonization across sites and MRI platforms, and a method for obtaining unbiased estimates of structural change based on longitudinal measurements with serial MRI scans. The optimized analysis pipeline, together with the large and heterogeneous combined GEMRIC dataset, will provide new opportunities to elucidate the mechanisms of ECT response and the factors mediating and predictive of clinical outcomes, which may ultimately lead to more effective personalized treatment approaches.

Brain morphology in school-aged children with prenatal opioid exposure: A structural MRI study

BACKGROUND Both animal and human studies have suggested that prenatal opioid exposure may be detrimental to the developing fetal brain. However, results are somewhat conflicting. Structural brain changes in children with prenatal opioid exposure have been reported in a few studies, and such changes may contribute to neuropsychological impairments observed in exposed children. AIM To investigate the association between prenatal opioid exposure and brain morphology in school-aged children. STUDY DESIGN A cross-sectional magnetic resonance imaging (MRI) study of prenatally opioid-exposed children and matched controls. SUBJECTS A hospital-based sample (n=16) of children aged 10-14years with prenatal exposure to opioids and 1:1 sex- and age-matched unexposed controls. OUTCOME MEASURES Automated brain volume measures obtained from T1-weighted MRI scans using FreeSurfer. RESULTS Volumes of the basal ganglia, thalamus, and cerebellar white matter were reduced in the opioid-exposed group, whereas there were no statistically significant differences in global brain measures (total brain, cerebral cortex, and cerebral white matter volumes). CONCLUSIONS In line with the limited findings reported in the literature to date, our study showed an association between prenatal opioid exposure and reduced regional brain volumes. Adverse effects of opioids on the developing fetal brain may explain this association. However, further research is needed to explore the causal nature and functional consequences of these findings.