Quantification of Retrograde Axonal Transport in the Rat Optic Nerve by Fluorogold Spectrometry

PURPOSE: Disturbed axonal transport is an important pathogenic factor in many neurodegenerative diseases, such as glaucoma, an eye disease characterised by progressive atrophy of the optic nerve. Quantification of retrograde axonal transport in the optic nerve usually requires labour intensive histochemical techniques or expensive equipment for in vivo imaging. Here, we report on a robust alternative method using Fluorogold (FG) as tracer, which is spectrometrically quantified in retinal tissue lysate. METHODS: To determine parameters reflecting the relative FG content of a sample FG was dissolved in retinal lysates at different concentrations and spectra were obtained. For validation in vivo FG was injected uni- or bilaterally into the superior colliculus (SC) of Sprague Dawley rats. The retinal lysate was analysed after 3, 5 and 7 days to determine the time course of FG accumulation in the retina (n = 15). In subsequent experiments axona transport was impaired by optic nerve crush (n = 3), laser-induced ocular hypertension (n = 5) or colchicine treatment to the SC (n = 10). RESULTS: Spectrometry at 370 nm excitation revealed two emission peaks at 430 and 610 nm. We devised a formula to calculate the relative FG content (c(FG)), from the emission spectrum. c(FG) is proportional to the real FG concentration as it corrects for variations of retinal protein concentration in the lysate. After SC injection, c(FG) monotonously increases with time (p = 0.002). Optic nerve axonal damage caused a significant decrease of c(FG) (crush p = 0.029; hypertension p = 0.025; colchicine p = 0.006). Lysates are amenable to subsequent protein analysis. CONCLUSIONS: Spectrometrical FG detection in retinal lysates allows for quantitative assessment of retrograde axonal transport using standard laboratory equipment. It is faster than histochemical techniques and may also complement morphological in vivo analyses

Decoupling the Effects of the Amyloid Precursor Protein From Amyloid-β Plaques on Axonal Transport Dynamics in the Living Brain

Amyloid precursor protein (APP) is the precursor to Aβ plaques. The cytoplasmic domain of APP mediates attachment of vesicles to molecular motors for axonal transport. In APP-KO mice, transport of Mn²⁺ is decreased. In old transgenic mice expressing mutated human (APP^(SwInd)) linked to Familial Alzheimer’s Disease, with both expression of APP^(SwInd) and plaques, the rate and destination of Mn²⁺ axonal transport is altered, as detected by time-lapse manganese-enhanced magnetic resonance imaging (MEMRI) of the brain in living mice. To determine the relative contribution of expression of APP^(SwInd) versus plaque on transport dynamics, we developed a Tet-off system to decouple expression of APP^(SwInd) from plaque, and then studied hippocampal to forebrain transport by MEMRI. Three groups of mice were compared to wild-type (WT): Mice with plaque and APP^(SwInd) expression; mice with plaque but suppression of APP^(SwInd) expression; and mice with APP^(SwInd) suppressed from mating until 2 weeks before imaging with no plaque. MR images were captured before at successive time points after stereotactic injection of Mn²⁺ (3–5 nL) into CA3 of the hippocampus. Mice were returned to their home cage between imaging sessions so that transport would occur in the awake freely moving animal. Images of multiple mice from the three groups (suppressed or expressed) together with C57/B6J WT were aligned and processed with our automated computational pipeline, and voxel-wise statistical parametric mapping (SPM) performed. At the conclusion of MR imaging, brains were harvested for biochemistry or histopathology. Paired T-tests within-group between time points (p = 0.01 FDR corrected) support the impression that both plaque alone and APP^(SwInd) expression alone alter transport rates and destination of Mn²⁺ accumulation. Expression of APP^(SwInd) in the absence of plaque or detectable Aβ also resulted in transport defects as well as pathology of hippocampus and medial septum, suggesting two sources of pathology occur in familial Alzheimer’s disease, from toxic mutant protein as well as plaque. Alternatively mice with plaque without APP^(SwInd) expression resemble the human condition of sporadic Alzheimer’s, and had better transport. Thus, these mice with APP^(SwInd) expression suppressed after plaque formation will be most useful in preclinical trials

Decoupling the Effects of the Amyloid Precursor Protein From Amyloid-β Plaques on Axonal Transport Dynamics in the Living Brain

Amyloid precursor protein (APP) is the precursor to Aβ plaques. The cytoplasmic domain of APP mediates attachment of vesicles to molecular motors for axonal transport. In APP-KO mice, transport of Mn²⁺ is decreased. In old transgenic mice expressing mutated human (APP^(SwInd)) linked to Familial Alzheimer’s Disease, with both expression of APP^(SwInd) and plaques, the rate and destination of Mn²⁺ axonal transport is altered, as detected by time-lapse manganese-enhanced magnetic resonance imaging (MEMRI) of the brain in living mice. To determine the relative contribution of expression of APP^(SwInd) versus plaque on transport dynamics, we developed a Tet-off system to decouple expression of APP^(SwInd) from plaque, and then studied hippocampal to forebrain transport by MEMRI. Three groups of mice were compared to wild-type (WT): Mice with plaque and APP^(SwInd) expression; mice with plaque but suppression of APP^(SwInd) expression; and mice with APP^(SwInd) suppressed from mating until 2 weeks before imaging with no plaque. MR images were captured before at successive time points after stereotactic injection of Mn²⁺ (3–5 nL) into CA3 of the hippocampus. Mice were returned to their home cage between imaging sessions so that transport would occur in the awake freely moving animal. Images of multiple mice from the three groups (suppressed or expressed) together with C57/B6J WT were aligned and processed with our automated computational pipeline, and voxel-wise statistical parametric mapping (SPM) performed. At the conclusion of MR imaging, brains were harvested for biochemistry or histopathology. Paired T-tests within-group between time points (p = 0.01 FDR corrected) support the impression that both plaque alone and APP^(SwInd) expression alone alter transport rates and destination of Mn²⁺ accumulation. Expression of APP^(SwInd) in the absence of plaque or detectable Aβ also resulted in transport defects as well as pathology of hippocampus and medial septum, suggesting two sources of pathology occur in familial Alzheimer’s disease, from toxic mutant protein as well as plaque. Alternatively mice with plaque without APP^(SwInd) expression resemble the human condition of sporadic Alzheimer’s, and had better transport. Thus, these mice with APP^(SwInd) expression suppressed after plaque formation will be most useful in preclinical trials

Activity Dependent Changes In Functional And Morphological Characteristics Among Presympathetic Neurons Of The Rostral Ventrolateral Medulla

A sedentary lifestyle is a major risk factor for the development of cardiovascular disease (CVD), the leading cause of death among Americans. Increasing evidence implicates increased sympathetic nerve activity (SNA) as the link between a sedentary lifestyle and CVD. The research presented in this dissertation examines the region of the brainstem known as the rostral ventrolateral medulla (RVLM) and how its regulation of SNA changes as a result of sedentary conditions. Our group has previously reported that sedentary conditions enhance splanchnic SNA in response to pharmacologically induced decreases in blood pressure or by direct activation of the RVLM via microinjection of the amino acid glutamate. More recently, our group has published the first evidence of overt structural differences in phenotypically identified RVLM neurons from sedentary versus physically active rats. Although collectively these studies suggest that a sedentary lifestyle results in increased activity and sensitivity of presympathetic RVLM neurons involved in blood pressure regulation, direct evidence of this proposed mechanism for the observed increased splanchnic SNA is lacking. The studies presented in this dissertation use in vivo characterization and juxtacellular labeling of RVLM neurons to examine the potential mechanistic connection and physiological relevance of overt changes in their structure and function and how they relate to enhanced SNA in sedentary versus physically active rats. These cross sectional studies are complemented by longitudinally based studies of in vivo neuronal activity in the RVLM utilizing manganese-enhanced magnetic resonance imaging (MEMRI). The information gained from these studies will contribute to our understanding of how a sedentary lifestyle contributes to the development of CVD and may provide information on new therapeutic targets in the brain to prevent or slow the progression of CVD

Contribution of ipsilesional versus contralesional pyramidal tract plasticity

Schlaganfall trägt zu erhöhter Mortalität und Morbidität trotz akut intensivmedizinischer Behandlung. Trotz vielversprechenden Ergebnisse von Seite der Akutbehandlung, die preklinische und klinische Versuche, die die Morbidität und Mortalität beim Schlaganfall in die postakute bis chronische Phase zu reduzieren versuchen, bleiben frustrierend. Nach dem Schlaganfall, das Hirnparenchym ist einem Reorganisationsprozess ausgesetzt indem vertiefte interzelluläre und zellulär-extrazelluläre Interaktionen eine wichtige Rolle spielen. Das Ontogenese Software wird „restarted“ und damit die endogene Plastizität nach dem Schlaganfall steigt. In wie fern wir diese Eigenschaft für therapeutische Zwecke „ausbeuten“ können bleibt bis jetzt unklar, allerdings man beobachtet bereits in viele Studien eine breitere Therapiefenster mit verlängertem Effekt und Erholungspotenzial nach dem Schlaganfall. In unserem Projekt haben wir mittels mehreren Readouts die Effekten von Erythropoietin (EPO) und Vascular endothelial growth factor (VEGF) auf die Neuroplastizität der langen Bahnen nach dem Schlaganfall untersucht. VEGF ist ein Wachstumsfaktor mit pleiotropen Effekte nach dem Schlaganfall und ist vor allem in die Astroglia und Mikroglia zusammen mit dem Rezeptoren VEGFR1 und VEGFR2 zu finden. In die akute Schlaganfall Phase VEGF ist für verschiedene Effekten in die Penumbra Region zuständig wie Förderung des neuronalen Überlebens, der Angiogenese Prozessen, der Proliferation von neuralen Stamzellen, deren Migration und Differenzierung. Anhalt neuen Studien wurde den VEGFR2 in den kontralesionalen Motorkortex nach dem Schlaganfall identifiziert . Diese Entdeckung weisst indirekt auf einer funktionelle Rolle der VEGF auf die kontralesionale Hemisphäre. Bis jetzt bleibt allerdings die Rolle der VEGF Therapie in die axonale Plastizität der langen Bahnen nach dem Schlaganfall unerforscht und dadurch auch seiner möglichen therapeutischen Rolle in die funktionelle Erholung. EPO ist ein Wachstumsfaktor mit einem breiten Spektrum der schon für klinische Verwendung zugelassen ist und damit zu einer schnelleren Translationsphase von preklinischen zu klinischen Experimenten geeignet ist. Die erste klinische Studie in der Erythropoietin nach dem Schlaganfall untersucht worden ist, zeigte eine Verbesserung der neurologischen Outcome mit Verkleinerung der ischämischen Schaden. In eine weitere Studie wurde dann der Effekt von EPO in 522 Patienten untersucht und zeigte dass die Verabreichung des EPO zusammen mit recombinant tissue-plasminogen activator (rt-PA) akut nach dem Schlaganfall zu einer signifikanten Nachblutung, Hirnödem und thromboembolische Ereignisse führte. Eine Falschinterpretation dieser Studie wird in die Zukunft für eine Ablehnung der EPO Therapie in Schlaganfall führen, obwohl eine alleinige Verabreichung des Epo eine Verbesserung zeigte. Unsere Studie untersucht die Effekte der Epo Therapie verabreicht drei Tage nach dem Schlaganfall mit Fokus auf die axonale Plastizität und funktionelle Erholung. Als Schlaganfall Model wurde eine 30 minutige Okklusion der A. cerebri media (MCAO) linkshemisphärisch durchgeführt. Die Verabreichung der Wachstumsfaktoren erfolgte am dritten postoperativen Tag mittels Alzet Pumpen die intraventrikulär implantiert wurden und eine kontinuierliche Verabreichung für verschiedene Zeitspannen gewährleistete. Als Kontrolle wurden die Pumpen mit NaCl 0.9% gefüllt und gemäss dem gleichen Protokoll intraventrikulär verabreicht. Sechs Wochen nach dem Schlaganfall und Therapie wurden die motorische Kortexanteile ipsi- und kontralesional mittels anterograden Tracttracers [Biotinyliertes Dextrane amid (BDA) in den kontralesionalen Motorkortex und Cascade Blue (CB) in den ipsilesionalen Motorkortex] markiert. Zwei Monate Postischämie wurden die Mäuse in Narkose getötet und das Gewebe für weitere molekularbiologische, biochemische, enzymologische, zytologische und genetische Untersuchungen verwendet. Die funktionelle Erholung als Korrelat zu Neurorehabilitation nach dem Schlaganfall wurde mittels RotaRod test, Grips strength test und Anxiety Test untersucht. Gemäss unseren Ergebnisse, VEGF und EPO Therapie verabreicht drei Tage nach dem Schlaganfall unterstützen die funktionelle Erholung durch koordinierte jedoch unterschiedliche Mechanismen die die Plastizität der langen Bahnen in der kontralesionalen Hemisphäre stimulieren. EPO zeigte vor allem eine vermehrte Translation der Plastizitätsgenen in der kontralesionalen Hemisphäre nach dem Schlaganfall die für eine erhöhte axonale Plastizität kontralesional verantwortlich waren. VEGF therapierten Tieren zeigten auch Erhöhung der axonalen Plastizität nach dem Schlaganfall kontralesional aber auf molekularer Ebene war das Effekt am besten durch eine Unterregulation von Plastizität hemmenden Substanzen in der Extrazellulären Matrix zu erklären.Stroke incidence is increasing due to the rapidly aging population in developed countries. Whereas untreated acute middle cerebral artery occlusion (MCAO) causes death in 20% of patients and long-term disability in more than 70% of patients, acute stroke therapy with rapid vessel recanalization significantly reduces mortality without influencing functional recovery beyond the acute stroke phase. This lack of functional recovery suggests a need for innovative therapies that can restore function after stroke. The purpose of these studies was to examine the effects of delayed administration of the growth factors erythropoietin (Epo) and vascular endogenous growth factor (VEGF) on functional neurological recovery and pyramidal tract plasticity in mice. The first study investigated how subacute delivery of Epo, starting at 3 days after stroke onset and continuing for 30 days (1 I.U. /day or 10 I.U. /day; via mini osmotic pump), influenced neuronal survival, axonal sprouting and neurological function recovery in C57Bl6/j mice submitted to 30-min MCAO. Epo administered in a 10 I.U. /day dose, in contrast to the 1 I.U. /day administration, showed a significant increase in neuronal survival and CD31 + newly-formed capillaries. This vascular growth enabled further neuroregeneration processes. Functional behavioral tests showed a significant improvement of motor coordination (RotaRod test) and grip strength (Grips strength test) among mice with 10 I.U. /day Epo administration, with no improvement for the low dose group. To investigate the neurological changes underlying these results, two anterograde tract tracers (dextrane amines) were injected in the motor cortex ipsilateral and contralateral to the ischemic lesion, in mice treated with the higher Epo dose. Histological evaluation of the tracers, both at the level of rubral and facial nucleus, showed that functional recovery in these animals was due to an increase of contralateral projections, accompanied by a compensatory decrease of ipsilateral projections. In the second study, VEGF was investigated due to its dual actions on vessels and neurons, which have potential for promoting long distance axonal plasticity in the ischemic brain. Mice were submitted to 30 minutes MCAO, followed by the intraventricular delivery of normal saline or VEGF (0.004 or 0.02 µg/day) starting 3 days post-ischemia. The outcome parameters were functional neurological recovery, long distance axonal plasticity by anterograde tract tracing and cellular and molecular responses examined by histochemistry, RT-PCR and Western blots. VEGF promoted neurological recovery when administered at the higher dosage, by stimulating long distance axonal plasticity in the contralesional but not ipsilesional pyramidal tract system. This observation was accompanied by deactivation of matrix metalloproteinase-9 (MMP9) in the ipsilesional brain tissue and downregulation of axonal growth inhibitors and guidance molecules in the contralesional brain tissue. The results support the concept that brain plasticity is consistent with coordinated axonal growth responses both ipsilateral and contralateral to the site of stroke. Considering that Epo is well tolerated in humans, clinical studies are now conceivable in which Epo is applied in patients in the post-acute stroke phase

Development of a peripheral nerve specific extracellular matrix-based hydrogel and its use in augmenting peripheral nerve injury and repair

Peripheral nerve injury commonly results in loss of neuromuscular function, often resulting in significant impact upon both quality of life and cost of care for patients. One promising target for improving patient outcomes is the use of a peripheral nerve specific extracellular matrix hydrogel (PNM) as an injectable, regenerative support. It has been long understood that the extracellular matrix (ECM) not only provides structural support but also regulates cell growth, survival, maturation, differentiation, and development of resident cells. The objective of this dissertation was to develop and characterize a decellularized peripheral nerve hydrogel, investigate its effect on key properties of peripheral nerve regeneration, and finally assess its ability to enhance return to function in several peripheral nerve injury models. We found that PNM provides a tissue-specific microenvironment which is conducive to nerve repair, including: nerve specific growth factors that are chemotactic signals for Schwann cells, promote neurite outgrowth, as well as factors that modulate the macrophage inflammatory response to injury. When employed as a lumen filler for conduit repair of peripheral nerve defects, a switch in the ratio of M1:M2 phenotype macrophages was observed, a phenomenon associated with improved nerve growth and promotion of Schwann cell migration across a gap defect. This was associated with improved function over time in non-critical common peroneal and sciatic nerve defects. Furthermore, we provided proof-of-concept for the use of PNM in treating nerve crush injuries. The injection of the PNM hydrogel directly into the nerve injury was found to be safe with no impact on downstream function. The application of PNM to the crush injury resulted in enhanced return to function and a more robust axon regrowth across the injury. In conclusion, we developed an injectable material that provides a regeneration promoting, tissue-specific microenvironment at the site of injury. The material has shown the ability to promotes recruitment of alternately activated, M2 macrophages, enhance Schwann cell migration, and axon extension. Finally, the use of PNM has enhanced recovery and return to function in numerous peripheral nerve injury models. PNM shows promise in augmenting current surgical practices for peripheral nerve injury and repair and has the potential to significantly improve quality of life for affected patients

Volumetric Manganese Enhanced Magnetic Resonance Imaging in mice (mus musculus)

The present doctoral thesis introduces a method for semi-automatic volumetric analysis of the hippocampus and other distinct brain regions in laboratory mice. The method of volumetric manganese enhanced magnetic resonance imaging (vMEMRI) makes use of the paramagnetic property of the manganese ion, Mn2+, which results in a positive contrast enhancement of specific brain areas on the MR image and enables a more detailed image of brain morphology. The chemical similarity of Mn2+ to Calcium leads to an accumulation of Mn2+ in excited cells and consequentially an enhanced signal in certain brain regions in an activity dependent manner. However, one major drawback for vMEMRI is the toxicity of Mn2+. Therefore, the aims of the thesis have been: (1) Establishment of a MEMRI protocol in mice (2) Optimization of a Mn2+ application procedure to reduce toxic side effects (3) Development of an automatized method to determine hippocampal volume (4) Validation of vMEMRI analysis (5) Application of volumetric analysis in mouse models of psychopathology This thesis splits into 3 studies. Study 1 deals with Mn2+ toxicity and introduces an application method that considerably reduces the toxic side effects of Mn2+. Study 2 validates vMEMRI as a method to reliably determine hippocampal volume and explores its utilization it in animals with genetically and chemically modified hippocampi. Study 3 displays the application vMEMRI in a mouse model of a psychiatric disorder. Study 1 shows that a single application of Mn2+ in dosages used in current MEMRI studies leads to considerable toxic side effects measurable with physiological, behavioral and endocrine markers. In contrast, a fractionated application of a low dose of Mn2+ is proposed as an alternative to a single injection of a high dose. Repeated application of low dosages of 30 mg/kg Mn2+ showed less toxic side effects compared to the application schemes with higher dosages of 60 mg/kg. Additionally, the best vMEMRI signal contrast was seen for an injection protocol of 30 mg/kg 8 times with an inter-injection interval of 24 h (8x30/24 protocol). The impact of the 8x30/24 application protocol on longitudinal studies was tested by determining whether learning processes are disturbed. Mice were injected with the 8x30/24 protocol 2 weeks prior to receiving a single footshock. Manganese injected mice showed less contextual freezing to the shock context and a shock context reminder one month after shock application. Furthermore, mice showed increased hyperarousal and no avoidance of shock context related odors. This impairment in fear conditioning indicates a disturbed associative learning of Mn2+ injected mice. Therefore, it was investigated whether Mn2+ application shows a specific disturbance of hippocampus dependent learning. Mice were subjected to habitual and spatial learning protocols 12 h after each injection in a water cross-maze. There was no impairment in learning protocols which allowed for hippocampus-independent habitual learning. However, Mn2+ injected mice were specifically impaired in the hippocampus-dependent spatial learning protocol. Furthermore, it was shown that only mice with higher Mn2+ accumulation showed this impairment. Altogether, the results of this chapter argue for a fractionated application scheme such as 30 mg/kg every 24 h for 8 days to provide sufficient MEMRI signal contrast while minimizing toxic side effects. However, the treatment procedure has to be further improved to allow for an analysis of hippocampus-dependent learning processes as well. Because of the potential side effects, the vMEMRI method was applied as a final experiment in study 2 and 3. Study 2 introduces the method of vMEMRI, which allows, for the first time, an in vivo semi-automatic detection of hippocampal volume. Hippocampal volume of mice with genetically altered adult neurogenesis and those with chemically lesioned hippocampi could be analyzed with vMEMRI. Even the highly variable differences in hippocampal volume of these animals could be detected with vMEMRI. vMEMRI data correlated with manually obtained volumes and are in agreement with previously reported histological findings, indicating the high reliability of this method. Study 3 investigates the ability of vMEMRI to detect even small differences in brain morphology by examining volumetric changes of the hippocampus and other brain structures in a mouse model of PTSD supplemented with enriched housing conditions. It was shown, that exposure to a brief inescapable foot shock led to a volume reduction in both the left hippocampus and right central amygdala two months later. Enriched housing decreased the intensity of trauma-associated contextual fear independently of whether it was provided before or after the shock. vMEMRI analysis revealed that enriched housing led to an increase in whole brain volume, including the lateral ventricles and the hippocampus. Furthermore, the enhancement of hippocampal volume through enriched housing was accompanied by the amelioration of trauma-associated PTSD-like symptoms. Hippocampal volume gain and loss was mirrored by ex vivo ultramicroscopic measurements of the hippocampus. Together, these data demonstrate that vMEMRI is able to detect small changes in hippocampal and central amygdalar volumes induced by a traumatic experience in mice. In conclusion, vMEMRI proves to be very reliable and able to detect small volumetric differences in various brain regions in living mice. vMEMRI opens up a great number possibilities for future research determining neuroanatomical structure, volumes and activity in vivo as well as the ability to repeatedly determine such characteristics within each subject, given an improvement of the Mn2+ treatment protocols to minimize potential toxic side effects

Alkoholin palkitseviin ominaisuuksiin ja amfetamiinin myrkkyvaikutuksiin liittyvien hermostollisten järjestelmien funktionaalinen kuvantaminen

Alcohol addiction is one of the most prevalent brain disorders in the world. A major hurdle for reducing alcohol-related harms and developing effective treatments is the poor understanding of neural processes responsible for the development of dependence and addiction. Alcohol has been shown to affect various neurotransmitter systems; however, the mesolimbic dopamine (DA) system, which projects from the ventral tegmental area (VTA) to the nucleus accumbens (NAc), has been thought to play a key role in producing the reinforcing effects of alcohol. The VTA region has also been suggested to be the anatomical site for the interaction of the dopaminergic system with the opioidergic and γ-aminobutyric acid (GABA) systems. Here, manganese-enhanced magnetic resonance imaging (MEMRI) and behavioral tests were used to study drug-induced alterations in brain activity of the alcohol-preferring AA (Alko Alcohol) and heterogeneous Wistar rats. MEMRI is based on the ability of paramagnetic Mn2+ ions to accumulate in excitable neurons, thus enhancing the T1-weighted signal in activated brain regions. Mn2+ ions can also be transported anterogradely and retrogradely in neurons, released to the synaptic cleft, and taken up by other neurons. These properties allow MEMRI to measure long-term changes in brain activity, as well as map neural pathways involved in acute and long-term drug actions, including drug reward and toxicity. The AA rats exposed to alcohol compared to water controls displayed a widespread and persistent activation in brain regions that have been previously linked with alcohol reinforcement. Similarly, activity in neural pathways originating in the NAc and projecting caudally to the midbrain was enhanced in alcohol drinking rats. Moreover, this alcohol-induced activation was blocked by systemic naltrexone (NLX) administration. Comparison of naïve AA and Wistar rats revealed a lowered basal activity in the caudal linear nucleus (CLi) of AA rats, which was restored by voluntary alcohol drinking. The intra-CLi γ-aminobutyric acid type A receptor (GABAA) agonist muscimol produced a dose-dependent increase in alcohol drinking, blocked by co-administration of the GABAA antagonist bicuculline, suggesting that the CLi GABAergic system is involved in the regulation of alcohol reward. MEMRI was also employed for assessing stimulant-induced toxicity. Methamphetamine and mephedron displayed disparate effects on brain activity, as methamphetamine produced widespread decreases in activity, whereas mephedron increased activity in limited brain areas. Taken together, the use of MEMRI for mapping alcohol- and stimulant-induced alterations in functional brain activity revealed networks and specific pathways that have potential for guiding further translational efforts to develop medications for drug abuse disorders, as well as for evaluating drug-induced toxicity.Alkoholismi on maailman yleisimpiä aivosairauksia. Alkoholin aiheuttamien haittojen vähentämistä ja tehokkaiden hoitomuotojen kehittämistä haittaa se, että päihderiippuvuuden kehittymiseen vaikuttavat aivojen toiminnan muutokset ymmärretään yhä huonosti. Alkoholin vaikutukset syntyvät monien hermoston välittäjäaineiden toiminnan kautta, ja etenkin nk. mesolimbisellä dopamiinijärjestelmällä on arveltu olevan keskeinen rooli alkoholin tuottamassa mielihyvässä. Töissämme käytettiin mangaanitehosteista magneettiresonanssikuvantamista (MRI) selvitettäessä alkoholin ja stimulanttien vaikutuksia alkoholiin mieltyneiden AA-rottien ja normaalien Wistar-rottien aivoissa. Kyseinen kuvantamismenetelmä perustuu siihen, että magneettisia ominaisuuksia omaavat mangaani-ionit kulkeutuvat hermopäätteiden kalsiumkanavien kautta hermosoluihin niiden aktivoituessa. Mangaani-ioneja sisältävien hermosolujen muodostamat radat ja aivoalueet tuottavat mitattavissa olevan signaalin. Mangaani voi myös siirtyä synapsiraon ylitse viereiseen hermosoluun ja tuottaa siten MRI-kuvan aktiivisista hermoverkoista. Viikkoja kestänyt alkoholin juominen johti aivojen aktiivisuusmuutoksiin sellaisilla aivojen alueilla, joiden on aikaisemmin oletettu liittyvän alkoholin positiivisiin vaikutuksiin. Selvitettäessä tarkemmin yksittäisten hermoratojen merkitystä havaittiin, että etuaivojen accumbens-tumakkeen keskiaivoihin lähettävä rata aktivoitui alkoholin juomisen seurauksena, kun taas alkoholismin hoidossa käytettävä lääkeaine, naltreksoni, vähensi alkoholin aiheuttamaa aktivaatiota. Verrattaessa alkoholiin mieltyneitä rottia tavalliseen rottakantaan löydettiin keskiaivoista tumake (kaudaalinen lineaaritumake), jonka aktiivisuus oli ennen alkoholin juomista tavallista alhaisempi, mutta jota alkoholi aktivoi. Kun tähän tumakkeeseen annettiin ainetta, joka salpasi gamma-aminovoihapon (GABA) vastaanottokohdat, alkoholin kulutus lisääntyi huomattavasti. Tämä löydös viittasi keskiaivojen GABA-järjestelmän merkitykseen alkoholin kulutuksen säätelyssä. Kaikkiaan kehittämämme mangaanitehosteinen magneettiresonanssikuvantaminen tuotti uutta tietoa alkoholin juomista säätelevistä hermoradoista ja yksittäisistä aivojen alueista. Tätä tietoa voidaan käyttää hyväksi, kun suunnitellaan ja testataan alkoholismin hoitoon tarkoitettuja lääkeaineita