Molecular analysis of Sigma-1 receptor modulation of the dopamine transporter

Sigma (σ) receptors are well established as a non-opioid, non-phencyclidine, and haloperidol-sensitive receptor family with its own binding profile and a characteristic distribution in the central nervous system (CNS) as well as in endocrine, immune, and some peripheral tissues. Two σ receptors subtypes, termed σ1 and σ2, have been pharmacologically characterized, but, to date, only the σ1 has also been cloned. Activation of σ1 receptors alter several neurotransmitter systems and dopamine (DA) neurotrasmission has been often shown to constitute an important target of σ receptors in different experimental models; however the exact role of σ1 receptor in dopaminergic neurotransmission remains unclear. The DA transporter (DAT) modulates the spatial and temporal aspects of dopaminergic synaptic transmission and interprer the primary mechanism by wich dopaminergic neurons terminate the signal transmission. For this reason present studies have been focused in understanding whether, in cell models, the human subtype of σ1 (hσ1) receptor is able to directly modulate the human DA transporter (hDAT). In the first part of this thesis, HEK-293 and SH-SY5Y cells were permanently transfected with the hσ1 receptor. Subsequently, they were transfected with another plasmid for transiently expressing the hDAT. The hDAT activity was estimated using the described [3H]DA uptake assay and the effects of σ ligands were evaluated by measuring the uptaken [3H]DA after treating the cells with known σ agonists and antagonists. Results illustrated in this thesis demonstrate that activation of overexpressed hσ1 receptors by (+)-pentazocine, the σ1 agonist prototype, determines an increase of 40% of the extracellular [3H]DA uptake, in comparison to non-treated controls and the σ1 antagonists BD-1047 and NE-100 prevent the positive effect of (+)-pentazocine on DA reuptake DA is likely to be considered a neurotoxic molecule. In fact, when levels of intracellular DA abnormally invrease, vescicles can’t sequester the DA which is metabolized by MAO (A and B) and COMT with consequent overproduction of oxygen reactive species and toxic catabolites. Stress induced by these molecules leads cells to death. Thus, for the second part of this thesis, experiments have been performed in order to investigate functional alterations caused by the (+)-pentazocine-mediated increase of DA uptake; particularly it has been investigated if the increase of intracellular [DA] could affect cells viability. Results obtained from this study demonstrate that (+)-pentazocine alone increases DA cell toxicity in a concentration-dependent manner only in cells co-expressing hσ1 and hDAT and σ1 antagonists are able to revert the (+)-pentazocine-induced increase of cell susceptibility to DA toxicity. In the last part of this thesis, the functional cross-talking between hσ1 receptor and hDAT has been further investigated using confocal microscopy. From the acquired data it could be suggested that, following exposure to (+)-pentazocine, the hσ1 receptors massively translocate towards the plasma membrane and colocalize with the hDATs. However, any physical interaction between the two proteins remains to be proved. In conclusion, the presented study shows for the first time that, in cell models, hσ1 receptors directly modulate the hDAT activity. Facilitation of DA uptake induced by (+)-pentazocine is reflected on the increased cell susceptibility to DA toxicity; these effects are prevented by σ1 selective antagonists. Since numerous compounds, including several drugs of abuse, bind to σ1 receptors and activating them could facilitate the damage of dopaminergic neurons, the reported protective effect showed by σ1 antagonists would represent the pharmacological basis to test these compounds in experimental models of dopaminergic neurodegenerative diseases (i.e. Parkinson’s Disease).

Cognitive binding in schizophrenia: weakened integration of temporal intersensory information

Cognitive functioning is based on binding processes, by which different features and elements of neurocognition are integrated and coordinated. Binding is an essential ingredient of, for instance, Gestalt perception. We have implemented a paradigm of causality perception based on the work of Albert Michotte, in which 2 identical discs move from opposite sides of a monitor, steadily toward, and then past one another. Their coincidence generates an ambiguous percept of either "streaming" or "bouncing," which the subjects (34 schizophrenia spectrum patients and 34 controls with mean age 27.9 y) were instructed to report. The latter perception is a marker of the binding processes underlying perceived causality (type I binding). In addition to this visual task, acoustic stimuli were presented at different times during the task (150 ms before and after visual coincidence), which can modulate perceived causality. This modulation by intersensory and temporally delayed stimuli is viewed as a different type of binding (type II). We show here, using a mixed-effects hierarchical analysis, that type II binding distinguishes schizophrenia spectrum patients from healthy controls, whereas type I binding does not. Type I binding may even be excessive in some patients, especially those with positive symptoms; Type II binding, however, was generally attenuated in patients. The present findings point to ways in which the disconnection (or Gestalt) hypothesis of schizophrenia can be refined, suggesting more specific markers of neurocognitive functioning and potential targets of treatment.

Cortical and subcortical correlates of electroencephalographic alpha rhythm modulation

Neural correlates of electroencephalographic (EEG) alpha rhythm are poorly understood. Here, we related EEG alpha rhythm in awake humans to blood-oxygen-level-dependent (BOLD) signal change determined by functional magnetic resonance imaging (fMRI). Topographical EEG was recorded simultaneously with fMRI during an open versus closed eyes and an auditory stimulation versus silence condition. EEG was separated into spatial components of maximal temporal independence using independent component analysis. Alpha component amplitudes and stimulus conditions served as general linear model regressors of the fMRI signal time course. In both paradigms, EEG alpha component amplitudes were associated with BOLD signal decreases in occipital areas, but not in thalamus, when a standard BOLD response curve (maximum effect at approximately 6 s) was assumed. The part of the alpha regressor independent of the protocol condition, however, revealed significant positive thalamic and mesencephalic correlations with a mean time delay of approximately 2.5 s between EEG and BOLD signals. The inverse relationship between EEG alpha amplitude and BOLD signals in primary and secondary visual areas suggests that widespread thalamocortical synchronization is associated with decreased brain metabolism. While the temporal relationship of this association is consistent with metabolic changes occurring simultaneously with changes in the alpha rhythm, sites in the medial thalamus and in the anterior midbrain were found to correlate with short time lag. Assuming a canonical hemodynamic response function, this finding is indicative of activity preceding the actual EEG change by some seconds.

Modulation of nociceptive withdrawal reflexes evoked by single and repeated nociceptive stimuli in conscious dogs by low-dose acepromazine

OBJECTIVES: To investigate the modulation of the nociceptive withdrawal reflex (NWR) and temporal summation (TS) by low-dose acepromazine (ACP) in conscious dogs. To assess the short- and long-term stability of the reflex thresholds. STUDY DESIGN: Randomized, blinded, placebo-controlled cross-over experimental study. ANIMALS: Eight adult male Beagles. METHODS: The NWR was elicited using single transcutaneous electrical stimulation of the ulnar nerve. Repeated stimuli (10 pulses, 5 Hz) were applied to evoke TS. The responses of the deltoideus muscle were recorded and quantified by surface electromyography and the behavioural reactions were scored. Each dog received 0.01 mg kg(-1) ACP or an equal volume saline intravenously (IV) at 1 week intervals. Measurements were performed before (baseline) and 20, 60 and 100 minutes after drug administration. Sedation was scored before drug administration and then at 10 minutes intervals. Data were analyzed with Friedman repeated measures analysis of variance on ranks and Wilcoxon signed rank tests. RESULTS: Acepromazine resulted in a mild tranquilization becoming obvious at 20 minutes and peaking 30 minutes after injection. Single (I(t)) and repeated stimuli (TS(t)) threshold intensities, NWR and TS characteristics and behavioural responses were not affected by the ACP at any time point. Both I(t) and TS(t) were stable over time. CONCLUSIONS AND CLINICAL RELEVANCE: In dogs, 0.01 mg kg(-1) ACP IV had no modulatory action on the NWR evoked by single or repeated stimuli, suggesting no antinociceptive activity on phasic nociceptive stimuli. The evidence of the stability of the NWR thresholds supports the use of the model as an objective tool to investigate nociception in conscious dogs. A low dose of ACP administered as the sole drug, can be used to facilitate the recordings in anxious subjects without altering the validity of this model.

The subjective present and its modulation in clinical contexts

Time is a basic dimension in psychology, underlying behavior and experience. Timing and time perception constitute implicit processes that are often inaccessible to the individual person. Research in this field has shown that timing is involved in many areas of clinical significance. In the projects presented here, we combine timing with seemingly different fields of research, such as psychopathology, perceptual grouping, and embodied cognition. Focusing on the time scale of the subjective present, we report findings from three different clinical studies: (1) We studied perceived causality in schizophrenia patients, finding that perceptual grouping (‘binding’, ‘Gestalt formation’), which leads to visual causality perceptions, did not distinguish between patients and healthy controls. Patients however did integrate context (provided by the temporal distribution of auditory context stimuli) less into perceptions, in significant contrast to controls. This is consistent with reports of higher inaccuracy in schizophrenia patients’ temporal processing. (2) In a project on auditory Gestalt perception we investigated auditory perceptual grouping in schizophrenia patients. The mean dwell time was positively related to how much patients were prone to auditory hallucinations. Dwell times of auditory Gestalts may be regarded as operationalizations of the subjective present; findings thus suggested that patients with hallucinations had a shorter present. (3) The movement correlations of interacting individuals were used to study the non-verbal synchrony between therapist and patient in psychotherapy sessions. We operationalized the duration of an embodied ‘social present’ by the statistical significance of such associations, finding a window of roughly 5.7 seconds in conversing dyads.We discuss that temporal scales of nowness may be modifiable, e.g., by mindfulness. This yields promising goals for future research on timing in the clinical context: psychotherapeutic techniques may alter binding processes, hence the subjective present of individuals, and may affect the social present in therapeutic interactions.

Spatial modulation of primate inferotemporal responses by eye position.

BACKGROUND: A key aspect of representations for object recognition and scene analysis in the ventral visual stream is the spatial frame of reference, be it a viewer-centered, object-centered, or scene-based coordinate system. Coordinate transforms from retinocentric space to other reference frames involve combining neural visual responses with extraretinal postural information. METHODOLOGY/PRINCIPAL FINDINGS: We examined whether such spatial information is available to anterior inferotemporal (AIT) neurons in the macaque monkey by measuring the effect of eye position on responses to a set of simple 2D shapes. We report, for the first time, a significant eye position effect in over 40% of recorded neurons with small gaze angle shifts from central fixation. Although eye position modulates responses, it does not change shape selectivity. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that spatial information is available in AIT for the representation of objects and scenes within a non-retinocentric frame of reference. More generally, the availability of spatial information in AIT calls into questions the classic dichotomy in visual processing that associates object shape processing with ventral structures such as AIT but places spatial processing in a separate anatomical stream projecting to dorsal structures.

Dopaminergic modulation of the reward system in schizophrenia: A placebo-controlled dopamine depletion fMRI study

Background The brain reward circuitry innervated by dopamine is critically disturbed in schizophrenia. This study aims to investigate the role of dopamine-related brain activity during prediction of monetary reward and loss in first episode schizophrenia patients. Methods We measured blood–oxygen-level dependent (BOLD) activity in 10 patients with schizophrenia (SCH) and 12 healthy controls during dopamine depletion with α-methylparatyrosine (AMPT) and during a placebo condition (PLA). Results AMPT reduced the activation of striatal and cortical brain regions in SCH. In SCH vs. controls reduced activation was found in the AMPT condition in several regions during anticipation of reward and loss, including areas of the striatum and frontal cortex. In SCH vs. controls reduced activation of the superior temporal gyrus and posterior cingulate was observed in PLA during anticipation of rewarding stimuli. PLA patients had reduced activation in the ventral striatum, frontal and cingulate cortex in anticipation of loss. The findings of reduced dopamine-related brain activity during AMPT were verified by reduced levels of dopamine in urine, homovanillic-acid in plasma and increased prolactin levels. Conclusions Our results indicate that dopamine depletion affects functioning of the cortico-striatal reward circuitry in SCH. The findings also suggest that neuronal functions associated with dopamine neurotransmission and attribution of salience to reward predicting stimuli are altered in schizophrenia.

Periodic time-domain modulation for the electrically tunable control of optical pulse train envelope and repetition rate multiplication

An electrically tunable system for the control of optical pulse sequences is proposed and demonstrated. It is based on the use of an electrooptic modulator for periodic phase modulation followed by a dispersive device to obtain the temporal Talbot effect. The proposed configuration allows for repetition rate multiplication with different multiplication factors and with the simultaneous control of the pulse train envelope by simply changing the electrical signal driving the modulator. Simulated and experimental results for an input optical pulse train of 10 GHz are shown for different multiplication factors and envelope shapes.

Modulation of cognition-specific cortical activity by gonadal steroids: A positron-emission tomography study in women

There is considerable evidence from animal studies that gonadal steroid hormones modulate neuronal activity and affect behavior. To study this in humans directly, we used H215O positron-emission tomography to measure regional cerebral blood flow (rCBF) in young women during three pharmacologically controlled hormonal conditions spanning 4–5 months: ovarian suppression induced by the gonadotropin-releasing hormone agonist leuprolide acetate (Lupron), Lupron plus estradiol replacement, and Lupron plus progesterone replacement. Estradiol and progesterone were administered in a double-blind cross-over design. On each occasion positron-emission tomography scans were performed during (i) the Wisconsin Card Sorting Test, a neuropsychological test that physiologically activates prefrontal cortex (PFC) and an associated cortical network including inferior parietal lobule and posterior inferolateral temporal gyrus, and (ii) a no-delay matching-to-sample sensorimotor control task. During treatment with Lupron alone (i.e., with virtual absence of gonadal steroid hormones), there was marked attenuation of the typical Wisconsin Card Sorting Test activation pattern even though task performance did not change. Most strikingly, there was no rCBF increase in PFC. When either progesterone or estrogen was added to the Lupron regimen, there was normalization of the rCBF activation pattern with augmentation of the parietal and temporal foci and return of the dorsolateral PFC activation. These data directly demonstrate that the hormonal milieu modulates cognition-related neural activity in humans.

A long marine history of carbon cycle modulation by orbital-climatic changes

Pacing of the marine carbon cycle by orbital forcing during the Pliocene and Pleistocene Ice Ages [past 2.5 million years (Myr)] is well known. As older deep-sea sediment records are being studied at greater temporal resolution, it is becoming clear that similar fluctuations in the marine carbon system have occurred throughout the late Mesozoic and Tertiary, despite the absence of large continental ice sheets over much of this time. Variations in both the organic and the calcium carbonate components of the marine carbon system seem to have varied cyclically in response to climate forcing, and carbon and carbonate time series appear to accurately characterize the frequency spectrum of ancient climatic change. For the past 35 Myr, much of the variance in carbonate content carries the “polar” signal of obliquity [41,000 years (41 kyr)] forcing. Over the past 125 Myr, there is evidence from marine sediments of the continued role of precessional (≈21 kyr) climatic cycles. Repeat patterns of sedimentation at about 100, 400, and 2,400 kyr, the modulation periods of precession, persistently enter into marine carbon cycle records as well. These patterns suggest a nonlinear response of climate and/or the sedimentation of organic carbon and carbonates to precessional orbital perturbations. Nonlinear responses of the carbon system may help to amplify relatively weak orbital insolation anomalies into more significant climatic perturbations through positive feedback effects. Nonlinearities in the carbon cycle may have transformed orbital-climatic cycles into long-wavelength features on time scales comparable to the residence times of carbon and nutrient elements in the ocean.

Characterising the central mechanisms of sensory modulation in human swallowing motor cortex

Objective: Pharyngeal stimulation can induce remarkable increases in the excitability of swallowing motor cortex, which is associated with short-term improvements in swallowing behaviour in dysphagic stroke patients. However, the mechanism by which this input induces cortical change remains unclear. Our aims were to explore the stimulus-induced facilitation of the cortico-bulbar projections to swallowing musculature and examine how input from the pharynx interacts with swallowing motor cortex. Methods: In 8 healthy subjects, a transcranial magnetic stimulation (TMS) paired-pulse investigation was performed comprising a single conditioning electrical pharyngeal stimulus (pulse width 0.2 ms, 240 V) followed by cortical TMS at inter-stimulus intervals (ISI) of 10-100 ms. Pharyngeal sensory evoked potentials (PSEP) were also measured over the vertex. In 6 subjects whole-brain magnetoencephalography (MEG) was further acquired following pharyngeal stimulation. Results: TMS evoked pharyngeal motor evoked potentials were facilitated by the pharyngeal stimulus at ISI between 50 and 80 ms (Δ mean increase: 47±6%, P<0.05). This correlated with the peak latency of the P1 component of the PSEP (mean 79.6±8.5 ms). MEG confirmed that the equivalent P1 peak activities were localised to caudolateral sensory and motor cortices (BA 4, 1, 2). Conclusions: Facilitation of the cortico-bulbar pathway to pharyngeal stimulation relates to coincident afferent input to sensorimotor cortex. Significance: These findings have mechanistic importance on how pharyngeal stimulation may increase motor excitability and provide guidance on temporal windows for future manipulations of swallowing motor cortex. © 2004 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.

Numerical modelling of advanced modulation formats and WDM transmission

This thesis presents a theoretical investigation of the application of advanced modelling formats in high-speed fibre lightwave systems. The first part of this work focuses on numerical optimisation of dense wavelength division multiplexing (DWDM) system design. We employ advanced spectral domain filtering techniques and carrier pulse reshaping. We then apply these optimisation methods to investigate spectral and temporal domain characteristics of advanced modulation formats in fibre optic telecommunication systems. Next we investigate numerical methods used in detecting and measuring the system performance of advanced modulation formats. We then numerically study the combination of return-to-zero differential phase-shift keying (RZ-DPSK) with advanced photonic devices. Finally we analyse the dispersion management of Nx40 Gbit/s RZ-DPSK transmission applied to a commercial terrestrial lightwave system.

Spatial and temporal dependencies of cross-orientation suppression in human vision.

A well-known property of orientation-tuned neurons in the visual cortex is that they are suppressed by the superposition of an orthogonal mask. This phenomenon has been explained in terms of physiological constraints (synaptic depression), engineering solutions for components with poor dynamic range (contrast normalization) and fundamental coding strategies for natural images (redundancy reduction). A common but often tacit assumption is that the suppressive process is equally potent at different spatial and temporal scales of analysis. To determine whether it is so, we measured psychophysical cross-orientation masking (XOM) functions for flickering horizontal Gabor stimuli over wide ranges of spatio-temporal frequency and contrast. We found that orthogonal masks raised contrast detection thresholds substantially at low spatial frequencies and high temporal frequencies (high speeds), and that small and unexpected levels of facilitation were evident elsewhere. The data were well fit by a functional model of contrast gain control, where (i) the weight of suppression increased with the ratio of temporal to spatial frequency and (ii) the weight of facilitatory modulation was the same for all conditions, but outcompeted by suppression at higher contrasts. These results (i) provide new constraints for models of primary visual cortex, (ii) associate XOM and facilitation with the transient magno- and sustained parvostreams, respectively, and (iii) reconcile earlier conflicting psychophysical reports on XOM.

Neuronal network pharmacodynamics of GABAergic modulation in the human cortex determined using pharmaco-magnetoencephalography

Neuronal network oscillations are a unifying phenomenon in neuroscience research, with comparable measurements across scales and species. Cortical oscillations are of central importance in the characterization of neuronal network function in health and disease and are influential in effective drug development. Whilst animal in vitro and in vivo electrophysiology is able to characterize pharmacologically induced modulations in neuronal activity, present human counterparts have spatial and temporal limitations. Consequently, the potential applications for a human equivalent are extensive. Here, we demonstrate a novel implementation of contemporary neuroimaging methods called pharmaco-magnetoencephalography. This approach determines the spatial profile of neuronal network oscillatory power change across the cortex following drug administration and reconstructs the time course of these modulations at focal regions of interest. As a proof of concept, we characterize the nonspecific GABAergic modulator diazepam, which has a broad range of therapeutic applications. We demonstrate that diazepam variously modulates ? (4–7 Hz), a (7–14 Hz), ß (15–25 Hz), and ? (30–80 Hz) frequency oscillations in specific regions of the cortex, with a pharmacodynamic profile consistent with that of drug uptake. We examine the relevance of these results with regard to the spatial and temporal observations from other modalities and the various therapeutic consequences of diazepam and discuss the potential applications of such an approach in terms of drug development and translational neuroscience.

Clinical studies of spatial and temporal aspects of vision: an investigation using psychophysical and electrophysiological techniques

Separate physiological mechanisms which respond to spatial and temporal stimulation have been identified in the visual system. Some pathological conditions may selectively affect these mechanisms, offering a unique opportunity to investigate how psychophysical and electrophysiological tests reflect these visual processes, and thus enhance the use of the tests in clinical diagnosis. Amblyopia and optical blur were studied, representing spatial visual defects of neural and optical origin, respectively. Selective defects of the visual pathways were also studied - optic neuritis which affects the optic nerve, and dementia of the Alzheimer type in which the higher association areas are believed to be affected, but the primary projections spared. Seventy control subjects from 10 to 79 years of age were investigated. This provided material for an additional study of the effect of age on the psychophysical and electrophysiological responses. Spatial processing was measured by visual acuity, the contrast sensitivity function, or spatial modulation transfer function (MTF), and the pattern reversal and pattern onset-offset visual evoked potential (VEP). Temporal, or luminance, processing was measured by the de Lange curve, or temporal MTF, and the flash VEP. The pattern VEP was shown to reflect the integrity of the optic nerve, geniculo striate pathway and primary projections, and was related to high temporal frequency processing. The individual components of the flash VEP differed in their characteristics. The results suggested that the P2 component reflects the function of the higher association areas and is related to low temporal frequency processing, while the Pl component reflects the primary projection areas. The combination of a delayed flash P2 component and a normal latency pattern VEP appears to be specific to dementia of the Alzheimer type and represents an important diagnostic test for this condition.