The impact of selective and non-selective medial septum stimulation on hippocampal neuronal oscillations: A study based on modeling and experiments.

Alzheimer's disease (AD) is a neurodegenerative disorder with a rising socioeconomic impact on societies. The hippocampus (HPC), which plays an important role in AD, is affected in the early stages. The medial septum (MS) in the forebrain provides major cholinergic input to the HPC and has been shown to play a significant role in generating oscillations in hippocampal neurons. Cholinergic neurons in the basal forebrain are particularly vulnerable to neurodegeneration in AD. To better understand the role of MS neurons including the cholinergic, glutamatergic, and GABAergic subpopulations in generating the well-known brain rhythms in HPC including delta, theta, slow gamma, and fast gamma oscillations, we designed a detailed computational model of the septohippocampal pathway. We validated the results of our model, using electrophysiological recordings in HPC with and without stimulation of the cholinergic neurons in MS using designer receptors exclusively activated by designer drugs (DREADDs) in healthy male ChAT-cre rats. Then, we eliminated 75% of the MS cholinergic neurons in the model to simulate degeneration in AD. A series of selective and non-selective stimulations of the remaining MS neurons were performed to understand the dynamics of oscillation regulation in the HPC during the degenerated state. In this way, appropriate stimulation strategies able to normalize the aberrant oscillations are proposed. We found that selectively stimulating the remaining healthy cholinergic neurons was sufficient for network recovery and compare this to stimulating other subpopulations and a non-selective stimulation of all MS neurons. Our data provide valuable information for the development of new therapeutic strategies in AD and a tool to test and predict the outcome of potential theranostic manipulations.

Altered basal forebrain function during whole-brain network activity at pre- and early-plaque stages of Alzheimer's disease in TgF344-AD rats.

BACKGROUND: Imbalanced synaptic transmission appears to be an early driver in Alzheimer's disease (AD) leading to brain network alterations. Early detection of altered synaptic transmission and insight into mechanisms causing early synaptic alterations would be valuable treatment strategies. This study aimed to investigate how whole-brain networks are influenced at pre- and early-plague stages of AD and if these manifestations are associated with concomitant cellular and synaptic deficits.  METHODS: To this end, we used an established AD rat model (TgF344-AD) and employed resting state functional MRI and quasi-periodic pattern (QPP) analysis, a method to detect recurrent spatiotemporal motifs of brain activity, in parallel with state-of-the-art immunohistochemistry in selected brain regions. RESULTS: At the pre-plaque stage, QPPs in TgF344-AD rats showed decreased activity of the basal forebrain (BFB) and the default mode-like network. Histological analyses revealed increased astrocyte abundance restricted to the BFB, in the absence of amyloid plaques, tauopathy, and alterations in a number of cholinergic, gaba-ergic, and glutamatergic synapses. During the early-plaque stage, when mild amyloid-beta (Aß) accumulation was observed in the cortex and hippocampus, QPPs in the TgF344-AD rats normalized suggesting the activation of compensatory mechanisms during this early disease progression period. Interestingly, astrogliosis observed in the BFB at the pre-plaque stage was absent at the early-plaque stage. Moreover, altered excitatory/inhibitory balance was observed in cortical regions belonging to the default mode-like network. In wild-type rats, at both time points, peak activity in the BFB preceded peak activity in other brain regions-indicating its modulatory role during QPPs. However, this pattern was eliminated in TgF344-AD suggesting that alterations in BFB-directed neuromodulation have a pronounced impact in network function in AD. CONCLUSIONS: This study demonstrates the value of rsfMRI and advanced network analysis methods to detect early alterations in BFB function in AD, which could aid early diagnosis and intervention in AD. Restoring the global synaptic transmission, possibly by modulating astrogliosis in the BFB, might be a promising therapeutic strategy to restore brain network function and delay the onset of symptoms in AD.

Combining magnetic resonance imaging with readout and/or perturbation of neural activity in animal models: Advantages and pitfalls.

One of the main challenges in brain research is to link all aspects of brain function: on a cellular, systemic, and functional level. Multimodal neuroimaging methodology provides a continuously evolving platform. Being able to combine calcium imaging, optogenetics, electrophysiology, chemogenetics, and functional magnetic resonance imaging (fMRI) as part of the numerous efforts on brain functional mapping, we have a unique opportunity to better understand brain function. This review will focus on the developments in application of these tools within fMRI studies and highlight the challenges and choices neurosciences face when designing multimodal experiments.

Chemogenetic silencing of neurons in the mouse anterior cingulate area modulates neuronal activity and functional connectivity.

The anterior cingulate area (ACC) is an integral part of the prefrontal cortex in mice and supports cognitive functions, including attentional processes, motion planning and execution as well as remote memory, fear and pain. Previous anatomical and functional imaging studies demonstrated that the ACC is interconnected with numerous brain regions, such as motor and sensory cortices, amygdala and limbic areas, suggesting it serves as a hub in functional networks. However, the exact role of the ACC in regulating functional network activity and connectivity remains to be elucidated. Recently developed neuromodulatory techniques, such as Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) allow for precise control of neuronal activity. In this study, we used an inhibitory kappa-opioid receptor DREADD (KORD) to temporally inhibit neuronal firing in the right ACC of mice and assessed functional network activity and connectivity using non-invasive functional magnetic resonance imaging (MRI). We demonstrated that KORD-induced inhibition of the right ACC induced blood oxygenation-level dependent (BOLD) signal decreases and increases in connected brain regions of both hemispheres. More specifically, altered neuronal activity could be observed in functional brain networks including connections with sensory cortex, thalamus, basolateral amygdala and ventral pallidum, areas involved in attention processes, working memory, fear behavior and reward respectively. Furthermore, these modulations in neuronal activity were associated with decreased intra- and interhemispheric functional connectivity. Our results consolidate the hub role of the mouse ACC in functional networks and further demonstrate that the combination of the DREADD technology and non-invasive functional imaging methods is a valuable tool for unraveling mechanisms of network function and dysfunction by reversible inactivation of selected targets.

Combining designer receptors exclusively activated by designer drugs and neuroimaging in experimental models: A powerful approach towards neurotheranostic applications.

The combination of chemogenetics targeting specific brain cell populations with in vivo imaging techniques provides scientists with a powerful new tool to study functional neural networks at the whole-brain scale. A number of recent studies indicate the potential of this approach to increase our understanding of brain function in health and disease. In this review, we discuss the employment of a specific chemogenetic tool, designer receptors exclusively activated by designer drugs, in conjunction with non-invasive neuroimaging techniques such as PET and MRI. We highlight the utility of using this multiscale approach in longitudinal studies and its ability to identify novel brain circuits relevant to behaviour that can be monitored in parallel. In addition, some identified shortcomings in this technique and more recent efforts to overcome them are also presented. Finally, we discuss the translational potential of designer receptors exclusively activated by designer drugs in neuroimaging and the promise it holds for future neurotheranostic applications.

The effect of pharmacological inhibition of Serine Proteases on neuronal networks in vitro.

Neurons are embedded in an extracellular matrix (ECM), which functions both as a scaffold and as a regulator of neuronal function. The ECM is in turn dynamically altered through the action of serine proteases, which break down its constituents. This pathway has been implicated in the regulation of synaptic plasticity and of neuronal intrinsic excitability. In this study, we determined the short-term effects of interfering with proteolytic processes in the ECM, with a newly developed serine protease inhibitor. We monitored the spontaneous electrophysiological activity of in vitro primary rat cortical cultures, using microelectrode arrays. While pharmacological inhibition at a low dosage had no significant effect, at elevated concentrations it altered significantly network synchronization and functional connectivity but left unaltered single-cell electrical properties. These results suggest that serine protease inhibition affects synaptic properties, likely through its actions on the ECM.

Bottom-up sensory processing can induce negative BOLD responses and reduce functional connectivity in nodes of the default mode-like network in rats.

The default mode network is a large-scale brain network that is active during rest and internally focused states and deactivates as well as desynchronizes during externally oriented (top-down) attention demanding cognitive tasks. However, it is not sufficiently understood if salient stimuli, able to trigger bottom-up attentional processes, could also result in similar reduction of activity and functional connectivity in the DMN. In this study, we investigated whether bottom-up sensory processing could influence the default mode-like network (DMLN) in rats. DMLN activity was examined using block-design visual functional magnetic resonance imaging (fMRI) while its synchronization was investigated by comparing functional connectivity during a resting versus a continuously stimulated brain state by unpredicted light flashes. We demonstrated that the BOLD response in DMLN regions was decreased during visual stimulus blocks and increased during blanks. Furthermore, decreased inter-network functional connectivity between the DMLN and visual networks as well as decreased intra-network functional connectivity within the DMLN was observed during the continuous visual stimulation. These results suggest that triggering of bottom-up attention mechanisms in sedated rats can lead to a cascade similar to top-down orienting of attention in humans and is able to deactivate and desynchronize the DMLN.

Hypersynchronicity in the default mode-like network in a neurodevelopmental animal model with relevance for schizophrenia.

BACKGROUND: Immune activation during pregnancy is an important risk factor for schizophrenia. Brain dysconnectivity and NMDA receptor (NMDAR) hypofunction have been postulated to be central to schizophrenia pathophysiology. The aim of this study was to investigate resting-state functional connectivity (resting-state functional MRI-rsfMRI), microstructure (diffusion tension imaging-DTI) and response to NMDAR antagonist (pharmacological fMRI-phMRI) using multimodal MRI in offspring of pregnant dams exposed to immune challenge (maternal immune activation-MIA model), and determine whether these neuroimaging readouts correlate with schizophrenia-related behaviour. METHODS: Pregnant rats were injected with Poly I:C or saline on gestational day 15. The maternal weight response was assessed. Since previous research has shown behavioural deficits can differ between MIA offspring dependent on the maternal response to immune stimulus, offspring were divided into three groups: controls (saline, n = 11), offspring of dams that gained weight (Poly I:C WG, n = 12) and offspring of dams that lost weight post-MIA (Poly I:C WL, n = 16). Male adult offspring were subjected to rsfMRI, DTI, phMRI with NMDAR antagonist, behavioural testing and histological assessment. RESULTS: Poly I:C WL offspring exhibited increased functional connectivity in default mode-like network (DMN). Poly I:C WG offspring showed the most pronounced attenuation in NMDAR antagonist response versus controls. DTI revealed no differences in Poly I:C offspring versus controls. Poly I:C offspring exhibited anxiety. CONCLUSIONS: MIA offspring displayed a differential pathophysiology depending on the maternal response to immune challenge. While Poly I:C WL offspring displayed hypersynchronicity in the DMN, altered NMDAR antagonist response was most pronounced in Poly I:C WG offspring.

Neuroimaging of Subacute Brain Inflammation and Microstructural Changes Predicts Long-Term Functional Outcome after Experimental Traumatic Brain Injury.

There is currently a lack of prognostic biomarkers to predict the different sequelae following traumatic brain injury (TBI). The present study investigated the hypothesis that subacute neuroinflammation and microstructural changes correlate with chronic TBI deficits. Rats were subjected to controlled cortical impact (CCI) injury, sham surgery, or skin incision (naïve). CCI-injured (n = 18) and sham-operated rats (n = 6) underwent positron emission tomography (PET) imaging with the translocator protein 18 kDa (TSPO) radioligand [18F]PBR111 and diffusion tensor imaging (DTI) in the subacute phase (≤3 weeks post-injury) to quantify inflammation and microstructural alterations. CCI-injured, sham-operated, and naïve rats (n = 8) underwent behavioral testing in the chronic phase (5.5-10 months post-injury): open field and sucrose preference tests, two one-week video-electroencephalogram (vEEG) monitoring periods, pentylenetetrazole (PTZ) seizure susceptibility tests, and a Morris water maze (MWM) test. In vivo imaging revealed pronounced neuroinflammation, decreased fractional anisotropy, and increased diffusivity in perilesional cortex and ipsilesional hippocampus of CCI-injured rats. Behavioral analysis revealed disinhibition, anhedonia, increased seizure susceptibility, and impaired learning in CCI-injured rats. Subacute TSPO expression and changes in DTI metrics significantly correlated with several chronic deficits (Pearson's |r| = 0.50-0.90). Certain specific PET and DTI parameters had good sensitivity and specificity (area under the receiver operator characteristic [ROC] curve = 0.85-1.00) to distinguish between TBI animals with and without particular behavioral deficits. Depending on the investigated behavioral deficit, PET or DTI data alone, or the combination, could very well predict the variability in functional outcome data (adjusted R2 = 0.54-1.00). Taken together, both TSPO PET and DTI seem promising prognostic biomarkers to predict different chronic TBI sequelae.

Decreased levels of active uPA and KLK8 assessed by [111 In]MICA-401 binding correlate with the seizure burden in an animal model of temporal lobe epilepsy.

OBJECTIVE: Urokinase-type plasminogen activator (uPA) and kallikrein-related peptidase 8 (KLK8) are serine proteases that contribute to extracellular matrix (ECM) remodeling after brain injury. They can be labelled with the novel radiotracer [111 In]MICA-401. As the first step in exploring the applicability of [111 In]MICA-401 in tracing the mechanisms of postinjury ECM reorganization in vivo, we performed in vitro and ex vivo studies, assessing [111 In]MICA-401 distribution in the brain in two animal models: kainic acid-induced status epilepticus (KASE) and controlled cortical impact (CCI)-induced traumatic brain injury (TBI). METHODS: In the KASE model, in vitro autoradiography with [111 In]MICA-401 was performed at 7 days and 12 weeks post-SE. To assess seizure burden, rats were monitored using video-electroencephalography (EEG) for 1 month before the 12-week time point. In the CCI model, in vitro autoradiography was performed at 4 days and ex vivo autoradiography at 7 days post-TBI. RESULTS: At 7 days post-SE, in vitro autoradiography revealed significantly decreased [111 In]MICA-401 binding in hippocampal CA3 subfield and extrahippocampal temporal lobe (ETL). In the chronic phase, when animals had developed spontaneous seizures, specific binding was decreased in CA3 and CA1/CA2 subfields of hippocampus, dentate gyrus, ETL, and parietal cortex. Of interest, KASE rats with the highest frequency of seizures had the lowest hippocampal [111 In]MICA-401 binding (r = -0.76, p ≤ 0.05). Similarly, at 4 days post-TBI, in vitro [111 In]MICA-401 binding was significantly decreased in medial and lateral perilesional cortex and ipsilateral dentate gyrus. Ex vivo autoradiography at 7 days post-TBI, however, revealed increased tracer uptake in perilesional cortex and hippocampus, which was likely related to tracer leakage due to blood-brain barrier (BBB) disruption. SIGNIFICANCE: Strong association of reduced [111 In]MICA-401 binding with seizure burden in the KASE model suggests that analysis of reduced levels of active uPA/KLK8 represents a novel biomarker candidate to be explored as a biomarker for epilepsy severity. However, limited BBB permeability of [111 In]MICA-401 currently limits its application in vivo.

Evaluation of [18 F]BR420 and [18 F]BR351 as radiotracers for MMP-9 imaging in colorectal cancer.

MMP-9 is a zinc-dependent endopeptidase that is involved in the proteolytic degradation of the extracellular matrix and plays an important role in cancer migration, invasion, and metastasis. The aim of this study was to evaluate the potential of MMP-tracers [18 F]BR420 and [18 F]BR351 for MMP-9 imaging in a colorectal cancer xenograft model. [18 F]BR420 and [18 F]BR351 were synthesized using an automated synthesis module. For [18 F]BR420, a novel and improved radiosynthesis was developed. Plasma stability and MMP-9-targeting capacity of both radiotracers was compared in the Colo205 colorectal cancer model. MMP-9 and MMP-2 expression levels in the tumors were evaluated by immunohistochemistry and in situ zymography. µPET imaging as well as ex vivo biodistribution revealed a higher tumor uptake for [18 F]BR420 (3.15% ± 0.03% ID/g vs 0.94% ± 0.18% ID/g for [18 F]BR351 at 2 hours pi) but slower blood clearance compared with [18 F]BR351. [18 F]BR351 was quickly metabolized in plasma with 20.28% ± 5.41% of intact tracer remaining at 15 minutes postinjection (PI). By contrast, [18 F]BR420 displayed a higher metabolic stability with >86% intact tracer remaining at 2 hours PI. Immunohistochemistry revealed the presence of MMP-9 and MMP-2 in the tumor tissue, which was confirmed by in situ zymography. However, an autoradiography analysis of tracer distribution in the tumors did not correlate with MMP-9 expression. [18 F]BR420 displayed a higher tumor uptake and higher stability compared with [18 F]BR351 but a low tumor-to-blood ratio and discrepancy between tracer distribution and MMP-9 immunohistochemistry. Therefore, both tracers will not be usefulness for MMP-9 imaging in colorectal cancer.

P2X7 receptor antagonism reduces the severity of spontaneous seizures in a chronic model of temporal lobe epilepsy.

BACKGROUND: The available pharmacotherapy for patients with epilepsy primarily address the symptoms and are ineffective in about 40% of patients. Brain inflammation gained support as potential target for developing new therapies, especially the P2X7 receptor (P2X7R), involved in processing of IL-1ß, might be an interesting candidate. This study was designed to investigate the effect of a novel P2X7R antagonist on the severity and on the number of chronic spontaneous recurrent seizures (SRS), which was unexplored until now. METHODS: After one-week of vehicle treatment (20% HP-ß-cyclodextrin), JNJ-42253432 was administered subcutaneously for another week under continuous video-electroencephalography monitoring (n = 17) in Sprague Dawley rats 3 months after kainic acid-induced status epilepticus. The proportion of different seizure classes, as well as the number of SRS/day were calculated for the vehicle and treatment period. In addition, post-mortem microglial activation and astrogliosis were assessed. RESULTS: A significant decrease of the proportion of type 4-5 SRS (p < 0.05), while an increase of type 1-3 was demonstrated (p < 0.05) from the vehicle to the treatment period. There was no effect of the P2X7R antagonist on the number of SRS/day or the glial markers. CONCLUSIONS: The P2X7R antagonist gave rise to a less severe profile of the chronic seizure burden without suppressing the SRS frequency. More studies are needed to unravel the underlying mechanisms of the beneficial effect on seizure severity and whether the administration of the compound during early epileptogenesis could induce long-term disease-modifying effects.

Hypolocomotive behaviour associated with increased microglia in a prenatal immune activation model with relevance to schizophrenia.

Over the past decade a neurodevelopmental animal model with high validity for schizophrenia has been developed based on the environmental risk factor known as maternal immune activation (MIA). The immunological basis of this model, together with extensive data from clinical and preclinical context, suggests the involvement of an aberrant neuro-immune system in the pathophysiology of schizophrenia. The goal of this study was to examine microglia activation in adult behaviourally phenotyped MIA offspring. MIA was induced in pregnant rats using viral mimetic Poly I:C at gestational day 15. Adult offspring were behaviourally phenotyped at postnatal days (PND) 56, 90 and 180 through the evaluation of prepulse inhibition (PPI) of the acoustic startle and spontaneous locomotion. Finally, the presence of activated microglia in brain regions associated with schizophrenia was evaluated using post-mortem immunohistochemistry against OX-42 (CD11b) and ED-1 (CD68). Although a deficit in PPI could not be replicated despite the high number of animals tested, we found an overall decrease in basal startle response and spontaneous locomotion in offspring born to Poly I:C- compared to saline-treated dams, accompanied by increased microglial density with characteristics of non-reactive activation in the chronic stage of the model. These findings provide additional evidence for a role played by microglial activation in schizophrenia-related pathology in general and psychomotor slowing in particular, and warrant extensive research on the underlying mechanism in order to establish new drug targets for the treatment of schizophrenia patients with an inflammatory component.