Quantitative pharmacologic MRI: Mapping the cerebral blood volume response to cocaine in dopamine transporter knockout mice

The use of pharmacologic MRI (phMRI) in mouse models of brain disorders allows noninvasive in vivo assessment of drug-modulated local cerebral blood volume changes (ΔCBV) as one correlate of neuronal and neurovascular activities. In this report, we employed CBV-weighted phMRI to compare cocaine-modulated neuronal activity in dopamine transporter (DAT) knockout (KO) and wild-typemice. Cocaine acts to block the dopamine, norepinephrine, and serotonin transporters (DAT, NET, and SERT) that clear their respective neurotransmitters from the synapses, helping to terminate cognate neurotransmission. Cocaine consistently reduced CBV, with a similar pattern of regional ΔCBV in brain structures involved inmediating reward in both DAT genotypes. The largest effects (−20% to −30% ΔCBV) were seen in the nucleus accumbens and several cortical regions. Decreasing response amplitudes to cocaine were noted in more posterior components of the cortico-mesolimbic circuit. DAT KO mice had significantly attenuated ΔCBV amplitudes, shortened times to peak response, and reduced response duration in most regions. This study demonstrates that DAT knockout does not abolish the phMRI responses to cocaine, suggesting that adaptations to loss of DAT and/or retained cocaine activity in other monoamine neurotransmitter systems underlie these responses in DAT KO mice

Quantitative pharmacologic MRI in mice

Pharmacologic MRI (phMRI) uses functional MRI techniques to provide a noninvasive in vivo measurement of the hemodynamic effects of drugs. The cerebral blood volume change (ΔCBV) serves as a surrogate for neuronal activity via neurovascular coupling mechanisms. By assessing the location and time course of brain activity in mouse mutant studies, phMRI can provide valuable insights into how different behavioral phenotypes are expressed in deferring brain activity response to drug challenge. In this report, we evaluate the utility of three different intravascular ultrasmall superparamagnetic iron oxide (USPIO) contrast agents for phMRI using a gradient-echo technique, with temporal resolution of one min at high magnetic field. The tissue half-life of the USPIOs was studied using a nonlinear detrending model. The three USPIOs are candidates for CBV weighted phMRI experiments, with r_2/r_1 ratios ≥ 20 and apparent half-lives ≥ 1.5 h at the described doses. An echo-time of about 10 ms or longer results in a functional contrast to noise ratio (fCNR) > 75 after USPIO injection, with negligible decrease between 1.5-2 h. phMRI experiments were conducted at 7 T using cocaine as a psychotropic substance and acetazolamide, a global vasodilator, as a positive control. Cocaine acts as a dopamine-serotonin-norepinephrine reuptake inhibitor, increasing extracellular concentrations of these neurotransmitters, and thus increasing dopaminergic, serotonergic and noradrenergic neurotransmission. phMRI results showed that CBV was reduced in the normal mouse brain after cocaine challenge, with the largest effects in the nucleus accumbens, whereas after acetazolamide, blood volume was increased in both cerebral and extracerebral tissue

Rapid quantitative pharmacodynamic imaging by a novel method: theory, simulation testing and proof of principle

Pharmacological challenge imaging has mapped, but rarely quantified, the sensitivity of a biological system to a given drug. We describe a novel method called rapid quantitative pharmacodynamic imaging. This method combines pharmacokinetic-pharmacodynamic modeling, repeated small doses of a challenge drug over a short time scale, and functional imaging to rapidly provide quantitative estimates of drug sensitivity including EC50 (the concentration of drug that produces half the maximum possible effect). We first test the method with simulated data, assuming a typical sigmoidal dose-response curve and assuming imperfect imaging that includes artifactual baseline signal drift and random error. With these few assumptions, rapid quantitative pharmacodynamic imaging reliably estimates EC50 from the simulated data, except when noise overwhelms the drug effect or when the effect occurs only at high doses. In preliminary fMRI studies of primate brain using a dopamine agonist, the observed noise level is modest compared with observed drug effects, and a quantitative EC50 can be obtained from some regional time-signal curves. Taken together, these results suggest that research and clinical applications for rapid quantitative pharmacodynamic imaging are realistic.Comment: 26 pages total, 4 tables, 10 figures. The original PDF file at https://peerj.com/articles/117/ includes active hyperlinks. This version is the final published version. (Differs from v2 only in that I corrected the abstract on the arXiv.org page.

Hypoinsulinemia Regulates Amphetamine-Induced Reverse Transport of Dopamine

The behavioral effects of psychomotor stimulants such as amphetamine (AMPH) arise from their ability to elicit increases in extracellular dopamine (DA). These AMPH-induced increases are achieved by DA transporter (DAT)-mediated transmitter efflux. Recently, we have shown that AMPH self-administration is reduced in rats that have been depleted of insulin with the diabetogenic agent streptozotocin (STZ). In vitro studies suggest that hypoinsulinemia may regulate the actions of AMPH by inhibiting the insulin downstream effectors phosphotidylinositol 3-kinase (PI3K) and protein kinase B (PKB, or Akt), which we have previously shown are able to fine-tune DAT cell-surface expression. Here, we demonstrate that striatal Akt function, as well as DAT cell-surface expression, are significantly reduced by STZ. In addition, our data show that the release of DA, determined by high-speed chronoamperometry (HSCA) in the striatum, in response to AMPH, is severely impaired in these insulin-deficient rats. Importantly, selective inhibition of PI3K with LY294002 within the striatum results in a profound reduction in the subsequent potential for AMPH to evoke DA efflux. Consistent with our biochemical and in vivo electrochemical data, findings from functional magnetic resonance imaging experiments reveal that the ability of AMPH to elicit positive blood oxygen level–dependent signal changes in the striatum is significantly blunted in STZ-treated rats. Finally, local infusion of insulin into the striatum of STZ-treated animals significantly recovers the ability of AMPH to stimulate DA release as measured by high-speed chronoamperometry. The present studies establish that PI3K signaling regulates the neurochemical actions of AMPH-like psychomotor stimulants. These data suggest that insulin signaling pathways may represent a novel mechanism for regulating DA transmission, one which may be targeted for the treatment of AMPH abuse and potentially other dopaminergic disorders

Peripheral Blood Pressure Changes Induced by Dobutamine Do Not Alter BOLD Signals in The Human Brain

In extending the use of functional MRI to neuropharmacology, a primary area of concern is that peripheral blood pressure changes induced by pharmacological agents could independently produce a change in the blood oxygenation level-dependent (BOLD) signal, resulting in difficulties distinguishing or interpreting drug-induced neural activations. In the present study, we utilized intravenous dobutamine, a beta-adrenergic receptor agonist, to increase the mean arterial blood pressure (MABP), while examining the effects of MABP changes on the BOLD signal in cocaine-dependent participants. Dobutamine infusion significantly increased the MABP from 93 ± 8 mm Hg to 106 ± 12 mm Hg (P \u3c 0.0005), but did not produce a significant global BOLD signal. Yet, a few voxels in the anterior cingulate showed BOLD signal changes that paralleled the changes in blood pressure (BP). Our observations support the conclusion that following the infusion of psychoactive agents, brain BOLD signals accurately reflect neuronal activity, even in the face of relatively large peripheral cardiovascular effects that transiently increase systemic BP

In vivo imaging of dopamine and serotonin release: response to psychopharmacological challenges

De neurotransmitters dopamine en serotonine zijn betrokken bij vele processen waaronder de regulatie van beloningsmechanismen (dopamine) en de stemming (serotonine). Ook is gevonden dat deze neurotransmitters betrokken zijn bij het ontstaan en de behandeling van verschillende psychiatrische en neurologische ziektebeelden. Het is echter nog steeds niet bekend wat de precieze rol van deze neurotransmitters hierbij is. Met behulp van neuroimaging technieken zoals PET (positron emission tomography) is het mogelijk om hierin meer inzicht te krijgen. In dit proefschrift werden een aantal methoden onderzocht die gericht zijn op het meten van de serotonerge en dopaminerge transmissie. In de eerste studies van dit proefschrift werd onderzocht of PET gebruikt kan worden om veranderingen in serotonine afgifte te meten. Hierbij werd gebruik gemaakt van de serotonine-1A ligand 18F-MPPF. Het principe van deze methode berust op het gegeven dat de binding van een radioligand wordt beïnvloed door veranderingen in de neurotransmitterconcentratie. Deze studies werden uitgevoerd bij gezonde vrijwilligers en proefdieren. Helaas bleek de binding van 18F-MPPF niet gevoelig voor veranderingen in de serotonineconcentratie. In een volgende studie werd het effect van een verhoging van de dopamineconcentratie onderzocht met behulp van de dopamine D2 ligand 11C-raclopride, bij gezonde vrijwilligers. Hierbij werd ondermeer een verband gevonden tussen de mate van dopamineafgifte en emoties zoals angst en euforie. In de laatste studie werd het effect van een dopamine toename op de hersenactiviteit gemeten met behulp van 15O-H2O PET. In deze studie werd een relatie gevonden tussen de dopamine-geïnduceerde toename in activiteit in de “anterior cingulate cortex” en de mate van euforie. Deze laatste twee methoden kunnen mogelijk worden toegepast bij patiëntenonderzoek en onderzoek naar het werkingsmechanisme van geneesmiddelen.

Probing brain function with pharmacological MRI

Lo sviluppo di tecniche di risonanza magnetica funzionale (fMRI) ha rivoluzionato le ricerca neuroscientifica clinica, determinando la possibilit\ue0 di investigare le dinamiche spazio-temporali dell\u2019attivit\ue0 cerebrale in maniera non invasiva e con grande accuratezza. Sebbene la tecnica sia stata originariamente sviluppata in ambito clinico, essa ha il potenziale di poter essere utilizzata in ambito preclinico come efficace strumento investigativo e traslazionale. Tuttavia, l\u2019implementazione preclinica di questi metodi \ue8 complicata da una serie di costrizioni sperimentali, in primis l\u2019utilizzo di anestetici, che minano fortemente il potenziale traslazionale di queste tecniche. Il recente sviluppo di tecniche di "MRI farmacologico" (phMRI) offre la possibilit\ue0 di superare alcune delle limitazioni sperimentali correlate all\u2019implementazione di approcci fMRI classici in animali da laboratorio. La tecnica si basa sull'utilizzo di metodi fMRI per mappare alterazioni di attivit\ue0 cerebrale prodotte dalla somministrazione di sostanze psicoattive. Studi preliminari hanno evidenziato la capacit\ue0 di generare robusti e specifici segnali phMRI anche in condizioni di anestesia, ed ha dimostrato la possibilit\ue0 di stimolare selettivamente diversi sistemi di neurotrasmettitori. Sfruttando la conservazione di circuiti cerebrali tra specie, tecniche phMRI offrono quindi l\u2019opportunit\ue0 di ampliare in maniera significativa il repertorio di stimolazione neuronale a disposizione in ambito preclinico, consentendo di indagare selettivamente specifici aspetti della funzione cerebrale in diversi stati di precondizionamento neuronale. In tale contesto, le attivit\ue0 di ricerca di questa tesi sono state finalizzate ad ampliare il campo di applicazione di metodi phMRI preclinici in due diversi ambiti sperimentali: a) come modalit\ue0 di indagine traslazionale, qualora applicata a modelli di malattia clinicamente rilevanti, b) pi\uf9 in generale come piattaforma investigativa per l'indagine della funzione cerebrale e della sua topologia funzionale in contesti sperimentali diversi. In un primo gruppo di studi, tecniche phMRI sono state impiegate per mappare i circuiti neuronali attivati da antagonisti del recettore del glutammato NMDA nel cervello del ratto (Sezione 4.1). Tali composti, grazie alle loro propriet\ue0 psicotogeniche, sono ampiamente sfruttati come modelli sperimentali di schizofrenia in animali ed in volontari allo scopo di valutare e validare nuovi trattamenti per la malattia. I risultati di questa ricerca hanno evidenziato uno specifico circuito corticolimbo- talamico che risulta essere attivato da antagonisti NMDAR sia nell'uomo che in Riassunto XII specie precliniche, e che \ue8 risultato essere modulabile da meccanismi antipsicotici diversi (Sezione 4.2). Il potenziale traslazionale dei metodi phMRI \ue8 stato ulteriormente avvalorato da un secondo gruppo di studi, in cui un approccio multi-parametrico \u201cphMRI-based\u201d \ue8 stato impiegato per indagare molteplici aspetti della funzione cerebrale in un modello murino di dipendenza da cocaina. Questa linea di investigazione ha evidenziato multiple alterazioni della funzione cerebrale basale e reattiva nel cervello di roditori esposti alla cocaina strettamente connesse a quelle osservate in analoghi studi di imaging su pazienti cocaina-dipendenti (Sezione 4.2). In una terza linea d\u2019 investigazione, l'uso combinato di avanzate strategie di targeting neuro-genetico (pharmaco-genetic silencing) e phMRI si \ue8 dimostrato efficace nello stabilire correlazioni dirette tra cellule, circuito e comportamento in linee di topo geneticamente modificate. Questi studi hanno portato all\u2019identificazione di una nuova e circoscritta popolazione neuroni nell'amigdala, in grado di controllare qualitativamente la risposta comportamentale alla paura attraverso il reclutamento di circuiti colinergici corticali (Sezione 4.3) Infine, l'approccio phMRI si \ue8 dimostrato uno strumento potente e versatile per l\u2019implementazione di misure di connettivit\ue0 funzionale nel cervello di roditori. Questo aspetto ha permesso l\u2019esplorazione di nuovi approcci statistici per l\u2019analisi della topologia funzionale del cervello basati sulla rappresentazione di misure di connettivit\ue0 in termini di reti complesse (Sezione 4.4). Complessivamente, i risultati di questo lavoro avvalorano il potenziale traslazionale di metodi phMRI nell\u2019ambito di diverse aree delle neuroscienze e della psicofarmacologia. La combinazione di phMRI e tecniche di manipolazione genetica avanzate definisce una nuova, potente piattaforma tecnologica per lo studio delle basi circuitali del comportamento in animali da laboratorio.The development of functional Magnetic Resonance Imaging (fMRI) has heralded a revolution in neuroscience, providing clinicians with a method to non-invasively investigate the spatio-temporal patterns of neuro-functional activity. Although primarily developed for human investigations, there exists significant scope for the application of fMRI in pre-clinical species as a translational and investigational platform across different areas of neuroscience and psychiatry research. However, the realization of this potential is hampered by a number of experimental constraints which make the application of fMRI methods to animal models less than straightforward. As a result, most fMRI research in laboratory species has been reduced to the employment of basic somato-sensory stimulation paradigms, thus greatly limiting the translational potential of the technique. An interesting approach to overcome some of these limitations has been dubbed \u201cpharmacological MRI\u201d (phMRI) and relies on the use of fMRI to map patterns of brain activity induced by psychoactive drugs. The approach has demonstrated the ability to elicit reliable fMRI signals even under anaesthesia, and to enable selective stimulation of different neurotransmitter systems. Building upon the homology between brain circuits in humans and laboratory animals, phMRI techniques thus offer the opportunity of significantly expanding the stimulation repertoire available to preclinical fMRI research, by allowing to selectively probe specific aspects of brain function under different preconditioning states. Within this framework, the research presented herein was aimed to broaden the scope of application of preclinical phMRI both as a translational technique, when applied to clinically-relevant disease models, and more generally as a versatile platform for the pre-clinical investigation of brain activity and its functional topology as a function of behavioural, pharmacological or genetic preconditioning. In a first group of studies, we developed a phMRI assay to map the circuitry activated by NMDAR antagonists in the rat brain. These psychotogenic compounds are widely exploited to model schizophrenia symptoms and to provide experimental models that may prove useful in the development of novel treatments for the disorder. The results of this research highlighted a conserved cortico-limbo-thalamic circuit that is activated by NMDAR antagonists both in humans and preclinical species, which can be modulated by existing and novel antipsychotic drugs (Section 4.1). The translational potential of phMRI measurements was further corroborated by a second group of studies, where a multi-parametric phMRI-based approach was applied to investigate multiple facets of brain function in a rodent cocaine selfSummary X administration model, a behavioural paradigm of established construct-validity for research of drug addiction. This line of investigation revealed specific basal and reactive functional alterations in the brain of cocaine-exposed rodents closely related to those observed in analogous neuroimaging studies in humans (Section 4.2). In a third line of investigation, the combined use of advanced neuro-genetic targeting strategies (i.e. pharmacogenetic silencing) and phMRI has proven successful in establishing direct correlations between cells, circuit and complex behaviours in genetically engineered mouse lines. These studies (Section 4.3) have led to the identification of a novel cell population in the amygdala that controls the behavioural response to fear through the recruitment of cholinergic circuits. Finally, the phMRI approach has proven a powerful tool to explore functional connectivity in rodents, and to map a variety of different neurotransmitter pathways by performing measures of correlated responses in spatially remote brain areas. This has provided a useful playground to explore novel statistical methods of analysis of functional connectivity represented in terms of complex networks (Section 4.4). Collectively, the results of this work strongly corroborate the translational use of phMRI approaches, and pave the way to the integrated implementation of phMRI and advance genetic manipulation as a novel powerful platform for basic neurobiological research

Experimental magnetic resonance imaging modalities as tools to evaluate brain function, structure and networks : exercise and cocaine induced adaptations

Psychiatric disease is common, costly and globally spread. Currently there are few efficient treatments that help patients to be remitted to a high degree. There is a need for improvement of robust objective diagnostic markers that can help the physicians to diagnose, follow-up or monitor treatment schedules. In the field of depression, only about 50% of patients experience medical relief by pharmacological treatment, and with psychostimulant use disorders there are no routinely used pharmacological treatments available. Therefore, it would be valuable to have better robust tools to be used for evaluation of new putative treatments as well as gaining a more comprehensive understanding of the etiology and pathophysiology of psychiatric diseases. The aim of this study was to further evaluate the translational potential of resting state functional connectivity (rsfMRI) brain networks by Magnetic Resonance Imaging (MRI) with special focus on rodent models for psychiatric disease. MRI can be utilized to analyze both structure and function of the brain. The images obtained by an MRI scan with EPI (Echo Planar Imaging) pulse sequences have the right contrast to enable analysis of different functional brain networks. In the case of rsfMRI, subjects are asked to close their eyes, not think about any specific and lay still inside an MRI scanner. For rodents, this technique can be utilized on lightly anaesthetized animals. This study demonstrates how wheel running induces enlargement of hippocampus in rats. Also, it shows how remarkably similar resting state functional connectivity networks are in rats and humans. It was further discovered that 14 days cocaine administration to rats induced alterations of functional brain networks. The data obtained was processed with the novel method Quantitative Data-driven Analysis (QDA), where connection strength and numbers of connections in the whole brain was measured. This was the first time QDA was applied in rodent rsfMRI. Finally, we used QDA to show that functional brain network connections involving insular cortex were correlated to the behavioral response of rats in a novel environment

Stress, Monoamines, and Cognitive Flexibility

Stress has been implicated in psychiatric disorders that are characterized by impaired executive function, which is mediated by the prefrontal cortex (PFC). The stress-related neuropeptide, corticotropin-releasing factor (CRF) regulates monoamine systems that project to the PFC, including the locus coeruleus norepinephrine (LC-NE) system and the dorsal raphe-serotonin (DRN-5-HT) system. CRF actions on these systems may underlie cognitive symptoms of stress-related disorders. The age at which stress occurs can determine its impact, and adolescent stress has been linked to adult psychopathology. This dissertation explores the role of CRF in stress-induced modulation of the LC-NE and DRN-5-HT systems and the developmental time course of the impact of stress on PFC-dependent cognitive function using attentional set-shifting tasks, microdialysis, and immunohistochemistry. CRF microinfusion into the LC and DRN produced dose-dependent effects on distinct cognitive functions. Low doses CRF in the LC facilitated set-shifting and increased c-fos expression in the PFC. In contrast, high doses of CRF in the LC facilitated reversal learning, suggesting that mild and severe stress affect different cognitive processes through LC-PFC projections. In the DRN, CRF facilitated set-shifting at a dosage that decreased 5-HT levels in the PFC. This effect switched to facilitation of reversal learning in a defeat-resistant subpopulation of rats exposed to social stress, underscoring the importance of stress history and coping strategy in determining the impact of stress. Finally, adolescent social stress produced an enduring impairment of cognitive flexibility that was seen in adulthood and occurred selectively in rats that resisted social defeat, further reinforcing the importance of coping style in the consequences of stress. Together these studies demonstrate how CRF modulation of monoamine systems can affect cognitive flexibility in ways that are adaptive for dealing with acute stress. They also show the importance of stress history, coping style, and age at which stress occurs as determinants of the impact of stress on cognition. This research may lead to the development of novel, individualized monoamine-targeted treatments for individuals suffering from stress-related cognitive impairments that may be related to the etiology of a diverse range of psychiatric disorders