Reward circuitry is perturbed in the absence of the serotonin transporter

The serotonin transporter (SERT) modulates the entire serotonergic system in the brain and influences both the dopaminergic and norepinephrinergic systems. These three systems are intimately involved in normal physiological functioning of the brain and implicated in numerous pathological conditions. Here we use high-resolution magnetic resonance imaging (MRI) and spectroscopy to elucidate the effects of disruption of the serotonin transporter in an animal model system: the SERT knock-out mouse. Employing manganese-enhanced MRI, we injected Mn^(2+) into the prefrontal cortex and obtained 3D MR images at specific time points in cohorts of SERT and normal mice. Statistical analysis of co-registered datasets demonstrated that active circuitry originating in the prefrontal cortex in the SERT knock-out is dramatically altered, with a bias towards more posterior areas (substantia nigra, ventral tegmental area, and Raphé nuclei) directly involved in the reward circuit. Injection site and tracing were confirmed with traditional track tracers by optical microscopy. In contrast, metabolite levels were essentially normal in the SERT knock-out by in vivo magnetic resonance spectroscopy and little or no anatomical differences between SERT knock-out and normal mice were detected by MRI. These findings point to modulation of the limbic cortical–ventral striatopallidal by disruption of SERT function. Thus, molecular disruptions of SERT that produce behavioral changes also alter the functional anatomy of the reward circuitry in which all the monoamine systems are involved

Live imaging of neuronal connections by magnetic resonance: Robust transport in the hippocampal–septal memory circuit in a mouse model of Down syndrome

Connections from hippocampus to septal nuclei have been implicated in memory loss and the cognitive impairment in Down syndrome (DS). We trace these connections in living mice by Mn^(2+) enhanced 3D MRI and compare normal with a trisomic mouse model of DS, Ts65Dn. After injection of 4 nl of 200 mM Mn^(2+) into the right hippocampus, Mn^(2+) enhanced circuitry was imaged at 0.5, 6, and 24 h in each of 13 different mice by high resolution MRI to detect dynamic changes in signal over time. The pattern of Mn^(2+) enhanced signal in vivo correlated with the histologic pattern in fixed brains of co-injected 3kD rhodamine–dextran–amine, a classic tracer. Statistical parametric mapping comparing intensity changes between different time points revealed that the dynamics of Mn2+ transport in this pathway were surprisingly more robust in DS mice than in littermate controls, with statistically significant intensity changes in DS appearing at earlier time points along expected pathways. This supports reciprocal alterations of transport in the hippocampal-forebrain circuit as being implicated in DS and argues against a general failure of transport. This is the first examination of in vivo transport dynamics in this pathway and the first report of elevated transport in DS

Decoupling the effect of mutant amyloid precursor protein (APP) from the effect of plaque on axonal transport dynamics in the living mouse brain: A correlation MRI-microscopy study

The parent protein for amyloid plaques, amyloid precursor protein (APP), mediates cargo‐motor attachments for intracellular transport. Axonal transport is decreased and the distal location of accumulation is altered in transgenic mice expressing human APP with the Swedish and Indiana mutations (APPSwInd) linked to Familial Alzheimer’s Disease, as detected by time‐lapse magnetic resonance imaging (MRI) of transport in living mouse brains (Bearer et al. 2017). Transport is also altered in brains of Down syndrome mice with 3 copies of APP gene. Questions now become whether expression of mutated APP effects transport dynamics independent of plaque, and do plaques alone contribute to transport defects? To address these we used the Tet‐Off system to decouple expression of APPSwInd from presence of plaques, and then studied transport using our MRI technique in three experimental groups of transgenic mice in which the timing and duration of APPSwInd expression, and thereby plaque formation, was altered with doxycycline: Group A (+ plaques, + APPSwInd); Group B (+ plaques, no APPSwInd), and group C (no plaques, + APPSwInd). Manganese‐enhanced MRI (MEMRI) allows us to perform cell biological experiments in live animals with T1‐weighted MRI in a Bruker 11.7T scanner (Medina et al 2016). Time‐lapse MR images were captured before and after stereotactic injection of Mn2+ (3‐5nL) into CA3 of the hippocampus at successive time‐points. Images of multiple individuals were aligned and processed with our automated computational pipeline (Medina et al. 2017) and statistical parametric mapping (SPM) performed. After MRI brains were harvested for histopathology or biochemistry. Results show that within group between time‐point have altered transport locations as well as diminished transport in all groups compared to wildtype (p<0.05 FDR n= 36). Preliminary ANOVA between‐group comparisons both by SPM and by region of interest measurements of images support the visual impression that APPSwInd expression alone may compromise transport. Groups A and B displayed plaques, but not C, and Western blots showed APPSwInd expressed 3.2‐fold over normal at sacrifice in Groups A and C but not B, with Aβ detected only in Groups A and B, where phospho‐tau was also present in dystrophic neurites surrounding plaques. Cholinergic neurons that project to hippocampus from the medial septal nucleus were decreased in Group C (p=0.0006 by ANOVA, n=15). Isolated hippocampal vesicles contained Mn2+, as well as Trk (NGF receptor), Rab 5 and 7 (associated with transport vesicles), suggesting a distinct vesicle population is affected by these APP mutations. These surprising results implicate mutated APPSwInd in transport defects, separable from the effect of plaque

Developing a Model for Slow Hypoxic Injury and Vascular Degeneration in Amyloid Burdened Brains

The breakdown of neurovascular systems may play a crucial role in the pathogenesis of Alzheimer’s disease. However whether this breakdown initiates a degenerative mechanism or is the consequence of some other deleterious process remains unknown. We examined hippocampal pathology in double transgenic mice overexpressing a human mutant gene encoding the amyloid precursor protein (APPSwe/Ind) using a combination of histochemistry and stereologic techniques. Expression of APPSwe/Ind in these mice is driven by a tetracycline-sensitive promoter. Tetracycline transcriptional activator (tTA), the second transgene, is driven in turn by a CAM KIIa promoter that is only active in neurons. Thus this double transgenic construct allows us to control expression of APPSwe/Ind with doxycycline. Utilizing this characteristic, we created three distinct experimental groups: A, display abeta plaque pathology and express APPSwe/Ind at time of sacrifice; B, display abeta plaque pathology but do not express APPSwe/Ind at time of sacrifice; and C, do not display abeta plaque pathology but do express APPSwe/Ind at time of sacrifice. Stereologic investigation revealed decreased hippocampal volume in groups A(n=5) and B(n=5) when compared to group C(n=5) and age-matched wildtype (n=9)

Fusion of phospholipid vesicles arrested by quick-freezing. The question of lipidic particles as intermediates in membrane fusion

We have examined the early events in Ca2+-induced fusion of large (0.2 μm diameter) unilamellar cardiolipin/phosphatidylcholine and phosphatidylserine/phosphatidylethanolamine vesicles by quick-freezing freeze-fracture electron microscopy, eliminating the necessity of using glycerol as a cryoprotectant. Freeze-fracture replicas of vesicle suspensions frozen after 1-2 s of stimulation revealed that the majority of vesicles had already undergone membrane fusion, as evidenced by dumbbell-shaped structures and large vesicles. In the absence of glycerol, lipidic particles or the hexagonal HII phase, which have been proposed to be intermediate structures in membrane fusion, were not observed at the sites of fusion. Lipidic particles were evident in less than 5% of the cardiolipin/phosphatidylcholine vesicles after long-term incubation with Ca2+, and the addition of glycerol produced more vesicles displaying the particles. We have also shown that rapid fusion occurred within seconds of Ca2+ addition by the time-course of fluorescence emission produced by the intermixing of aqueous contents of two separate vesicle populations. These studies therefore have produced no evidence that lipidic particles are necessary intermediates for membrane fusion. On the contrary, they indicate that lipidic particles are structures obtained at equilibrium long after fusion has occurred and they become particularly prevalent in the presence of glycerol. © 1982

Witnessing microtubule-based transport in the living brain: Impact of the cargomotor receptor, amyloid precursor protein, and Alzheimer’s plaques

Most amyloid precursor protein (APP)-based Alzheimer’s models overexpress mutant human APP resulting in Abeta plaques. Yet the relative contribution of this elevated APP and the presence of plaques to neurodegeneration remains a big question. APP’s role as a cargo-motor receptor for axonal transport suggests that overexpression might lead to increased transport. Indeed we showed that transport is increased in Down’s syndrome and decreased in APP knockout mice. Hence transport may be elevated in APP overexpressors and lead to either beneficial or deleterious consequences. Here we use high field microMRI with Mn2+, an MR contrast agent useful as a track-tracer, to pose this cell biological quest ion within the whole living brains of wildtype and Alzheimer’s model mice. Injection of Mn2+ into the CA3 region of the hippocampus results in measurable transport over time. Application of 3D unbiased whole brain image analysis detects all circuitry emanating from the hippocampus. By driving APP Swe/Ind transgene expression with a tetracycline-sensitive promoter, APPSwe/Ind expression can be decoupled from the presence of plaques with doxycycline (doxy). Three groups of mice were studied: group ‘A’ (no doxy, +plaques, +APP); group ‘B’ (doxy at 8 days before sacrifice, +plaques, no APP), and group ‘C’ (doxy prior to conception, and stopped 8 days before sacrifice, no plaques, +APP). Images were captured before and sequentionally after Mn2+ injection into CA 3 (1, 7, 25 hr). Images were aligned and analyzed by statistical parametric mapping to identify differential accumulation within the hippocampal projections. Histopathology revealed well-developed plaques in A and B, and Western blots showed human APP expressed five-fold over WT in in A and C. Our preliminary results show increased transport in A and C, with APP Swe/Ind expression when compared with B, where expression is suppressed. Cholinergic neurons in the medial septal nucleus were decreased as determined by anti-ChAT staining in Group C (p=0.0006 by one-way ANOVA, n=15). In conclusion, the effects of elevated APP expression are separable from consequences of plaque, and each may

Increased anatomical detail by in vitro MR microscopy with a modified Golgi impregnation method

Golgi impregnation is unique in its ability to display the dendritic trees and axons of large numbers of individual neurons by histology. Here we apply magnetic resonance microscopy to visualize the neuroanatomy of animal models by combining histologic fixation chemistry with paramagnetic contrast agents. Although there is some differential uptake of the standard small-molecular-weight contrast agents by different tissue types, detailed discrimination of tissue architecture in MR images does not approach that of standard histology. Our modified Golgi impregnation method significantly increases anatomic detail in magnetic resonance microscopy images. Fixed mouse brains were treated with a solution containing a paramagnetic contrast agent (gadoteridol) and potassium dichromate. Results demonstrate a specific contrast enhancement likely due to diamagnetic hexavalent chromium undergoing tissue specific reduction to paramagnetic trivalent chromium. This new method dramatically improves neuroanatomical contrast compared to conventional fixation, displaying detail approximating that of histologic specimens at low (4×) magnification

Role of neuronal activity and kinesin on tract tracing by manganese-enhanced MRI (MEMRI)

MEMRI offers the exciting possibility of tracing neuronal circuits in living animals by MRI. Here we use the power of mouse genetics and the simplicity of the visual system to test rigorously the parameters affecting Mn^(2+) uptake, transport and trans-synaptic tracing. By measuring electrical response to light before and after injection of Mn^(2+) into the eye, we determine the dose of Mn^(2+) with the least toxicity that can still be imaged by MR at 11.7 T. Using mice with genetic retinal blindness, we discover that electrical activity is not necessary for uptake and transport of Mn^(2+) in the optic nerve but is required for trans-synaptic transmission of this tracer to distal neurons in this pathway. Finally, using a kinesin light chain 1 knockout mouse, we find that conventional kinesin is a participant but not essential to neuronal transport of Mn^(2+) in the optic tract. This work provides a molecular and physiological framework for interpreting data acquired by MEMRI of circuitry in the brain