Mapping brain activity in vivo during spatial learning in mice

Deficits in the encoding, retrieval and manipulation of sensory or memory information in the brain contribute to a number of neuro- and psychopathologies in humans. To better understand the underlying principles of memory processes, efforts still have to rely on animal research. The laboratory mouse as a model organism provides many advantages to study the underlying mechanisms as one can directly interfere with brain functions via a number of tools including genetic manipulation. For this reason, it is very important to have memory tasks that match human memory testings and suits the characteristics of mice. One cognitive capability that is highly preserved across species is spatial learning, what enables the daily required relocation of certain positions in space. Mice and men have developed two different strategies to do so, which rely on different types of reference information. During place learning, environmental cues are adopted and incorporated into a cognitive map. In contrast, response learning is based on directed movements along a specific route. Place and response learning can also be dissected in terms of their biological substrate. While place learning requires the hippocampus (HPC), response learning depends on the striatum. Most behavioral tests, that can clearly distinguish between the two strategies were originally designed for rats. Since the behavior of mice and rats can be considerably different, it is necessary to adapt these tasks to the requirements of mice. In order to improve then the translation of structural and functional results from rodents to humans, methods must be applied that match the coarse in vivo imaging typically used in humans. Manganese-enhanced magnetic resonance imaging (MEMRI) may therefore be useful, as it provides three-dimensional maps of the living mouse brain. Since manganese increases the brain contrast in magnetic resonance (MR) images, MEMRI is regularly applied to depict the volume of specific brain structures in vivo. A second feature of manganese is, that it enters neurons through L-type voltage-dependent calcium channels (LTCCs) and therefore might be an indicator for neuronal action. However, ithas never been shown that LTCCs directly regulate the MEMRI contrast in vivo, which would be essential to establish it as a functional tool in order to measure brain activity in the living mouse brain. The application of MEMRI to cognitive tasks might then be helpful to identify underlying brain circuits and in combination with other techniques also essentially involved neurobiological mechanisms. Finally, it may also increase the comparability of human and rodent research. Therefore, I wanted to establish the water cross maze (WCM) as a suitable tool to study different learning strategies in mice and relate them to HPC functioning. Next, I aimed to dissect the influence of LTCCs on MEMRI contrast (specifically Cav1.2 and Cav1.3 as the two major LTCCs in the brain) in order to justify a functional application of MEMRI. At last, MEMRI should be implemented to depict learning processes in the WCM before I wanted to interfere with LTCC functioning to further explore their role in spatial learning. I had been able to demonstrate that theWCMwas particularly suitable for mice because it prevented most unwanted strategies that mice often adopt during the Morris water maze task. Further the test clearly dissected response from place strategies, which were both successfully acquired by C57Bl/6N mice. However, mice failed to relearn under response training independent of the original navigation strategy that was adopted within the week before. These results suggested, that not only place learning but also relearning is predicated on the HPC. Accordingly, HPC-lesioned mice were unable to acquire a place strategy, however, they adopted a response strategy instead. Further, relearning was blocked by the lesion, if less than 40% of the entire HPC remained. The inability to relearn was best reflected in the residual volume of the left ventral HPC. Second, I investigated the contribution of Cav1.2 and Cav1.3 on MEMRI contrast with the help of corresponding knockout mice. I was able to demonstrate that the Cav1.2 knockout affected at least 50% of the manganese-dependent contrast increase seen in MR images, whereas Cav1.3 knockouts caused no significant alterations. In addition, a locally defined knockout of Cav1.2 induced contrast differences in a projection region far away from the knockout side suggesting a bias in contrast differences away from the soma towards the axon terminals. Overall, this indicated a voltage-dependent manganese displacement in the brain and therefore suggested the functional application of MEMRI. For this reason, I combined place training in the WCM with manganese injections to map brain activity in vivo. On the one hand, the accuracy score was related to a fear associated network comprising the basolateral amygdala (BLA) and ventral HPC. On the other hand, the latency correlated with the dorsal HPC, specifically the left CA3 and the right CA1 region. First, this was in line with functional magnetic resonance imaging (fMRI) results obtained in humans, where the left HPC indicated response navigation and the right place memory formation. Second, the associations indicate the integration of emotional information into cognitive processing. At last, learning and relearning capabilities of Cav1.2 knockout mice were explored. Despite reduced MEMRI intensities in learning associated regions, knockout mice successfully acquire place and response memories and were also capable to revert the place memory afterwards. However, animals exhibited a significant retardation during place learning ,which can be attributed to impairments of late long-term potentiation (LTP) in the CA1 region of the HPC. Overall, the WCM suits the characteristics of mice and allows the distinction of different learning strategies. Further, mice similar to rats require an intact HPC to use place strategies in the WCM and at least 40% of the total HPC volume is necessary to accomplish relearning. Since I could demonstrate for the first time that MEMRI contrast largely depends on Cav1.2, MEMRI was employed to map brain activity in freely moving mice. I could identify brain regions most in the HPC that correlate with place learning parameters in the WCM for the first time in vivo. These results further match findings in humans, where place and response learning occur in parallel during place navigation in the left and right HPC, respectively. In addition, they suggest the integration of emotional information into cognitive precessing. At last, Cav1.2 is involved but not essential for place learning in the WCM. Future investigations with temporary knockouts might be useful to further elaborate the role of Cav1.2 in learning and memory functions

Tau protein is essential for stress-induced brain pathology

Exposure to chronic stress is frequently accompanied by cognitive and affective disorders in association with neurostructural adaptations. Chronic stress was previously shown to trigger Alzheimer's-like neuropathology, which is characterized by Tau hyper-phosphorylation and missorting into dendritic spines followed by memory deficits. Here, we demonstrate that stress-driven hippocampal deficits in wild-type mice are accompanied by synaptic missorting of Tau and enhanced Fyn/GluN2B-driven synaptic signaling. In contrast, mice lacking Tau [Tau knockout (Tau-KO) mice] do not exhibit stress-induced pathological behaviors and atrophy of hippocampal dendrites or deficits of hippocampal connectivity. These findings implicate Tau as an essential mediator of the adverse effects of stress on brain structure and function.We thank Dr. Peter Davies (Albert Einstein College) for the PHF1 antibody. This work was funded by Portuguese Foundation for Science & Technology (FCT) Grants PTDC/SAU-NMC/113934/2009 (to I.S.); the European Union FP7 Project SwitchBox (N.S. and O.F.X.A.); the Portuguese North Regional Operational Program (ON.2-O Novo Norte) under the National Strategic Reference Framework (QREN) through the European Regional Development Fund (FEDER); and the Education and Lifelong Learning, Supporting Postdoctoral Researchers and Large Scale Cooperative Project, cofinanced by the European Social Fund and the Greek General Secretariat for Research and Technology. J.V.-S. is a recipient of a PhD fellowship (PD/BD/105938/2014) of the University of Minho MD/PhD Program funded by the FCT

Mapping brain activity in vivo during spatial learning in mice

Deficits in the encoding, retrieval and manipulation of sensory or memory information in the brain contribute to a number of neuro- and psychopathologies in humans. To better understand the underlying principles of memory processes, efforts still have to rely on animal research. The laboratory mouse as a model organism provides many advantages to study the underlying mechanisms as one can directly interfere with brain functions via a number of tools including genetic manipulation. For this reason, it is very important to have memory tasks that match human memory testings and suits the characteristics of mice. One cognitive capability that is highly preserved across species is spatial learning, what enables the daily required relocation of certain positions in space. Mice and men have developed two different strategies to do so, which rely on different types of reference information. During place learning, environmental cues are adopted and incorporated into a cognitive map. In contrast, response learning is based on directed movements along a specific route. Place and response learning can also be dissected in terms of their biological substrate. While place learning requires the hippocampus (HPC), response learning depends on the striatum. Most behavioral tests, that can clearly distinguish between the two strategies were originally designed for rats. Since the behavior of mice and rats can be considerably different, it is necessary to adapt these tasks to the requirements of mice. In order to improve then the translation of structural and functional results from rodents to humans, methods must be applied that match the coarse in vivo imaging typically used in humans. Manganese-enhanced magnetic resonance imaging (MEMRI) may therefore be useful, as it provides three-dimensional maps of the living mouse brain. Since manganese increases the brain contrast in magnetic resonance (MR) images, MEMRI is regularly applied to depict the volume of specific brain structures in vivo. A second feature of manganese is, that it enters neurons through L-type voltage-dependent calcium channels (LTCCs) and therefore might be an indicator for neuronal action. However, ithas never been shown that LTCCs directly regulate the MEMRI contrast in vivo, which would be essential to establish it as a functional tool in order to measure brain activity in the living mouse brain. The application of MEMRI to cognitive tasks might then be helpful to identify underlying brain circuits and in combination with other techniques also essentially involved neurobiological mechanisms. Finally, it may also increase the comparability of human and rodent research. Therefore, I wanted to establish the water cross maze (WCM) as a suitable tool to study different learning strategies in mice and relate them to HPC functioning. Next, I aimed to dissect the influence of LTCCs on MEMRI contrast (specifically Cav1.2 and Cav1.3 as the two major LTCCs in the brain) in order to justify a functional application of MEMRI. At last, MEMRI should be implemented to depict learning processes in the WCM before I wanted to interfere with LTCC functioning to further explore their role in spatial learning. I had been able to demonstrate that theWCMwas particularly suitable for mice because it prevented most unwanted strategies that mice often adopt during the Morris water maze task. Further the test clearly dissected response from place strategies, which were both successfully acquired by C57Bl/6N mice. However, mice failed to relearn under response training independent of the original navigation strategy that was adopted within the week before. These results suggested, that not only place learning but also relearning is predicated on the HPC. Accordingly, HPC-lesioned mice were unable to acquire a place strategy, however, they adopted a response strategy instead. Further, relearning was blocked by the lesion, if less than 40% of the entire HPC remained. The inability to relearn was best reflected in the residual volume of the left ventral HPC. Second, I investigated the contribution of Cav1.2 and Cav1.3 on MEMRI contrast with the help of corresponding knockout mice. I was able to demonstrate that the Cav1.2 knockout affected at least 50% of the manganese-dependent contrast increase seen in MR images, whereas Cav1.3 knockouts caused no significant alterations. In addition, a locally defined knockout of Cav1.2 induced contrast differences in a projection region far away from the knockout side suggesting a bias in contrast differences away from the soma towards the axon terminals. Overall, this indicated a voltage-dependent manganese displacement in the brain and therefore suggested the functional application of MEMRI. For this reason, I combined place training in the WCM with manganese injections to map brain activity in vivo. On the one hand, the accuracy score was related to a fear associated network comprising the basolateral amygdala (BLA) and ventral HPC. On the other hand, the latency correlated with the dorsal HPC, specifically the left CA3 and the right CA1 region. First, this was in line with functional magnetic resonance imaging (fMRI) results obtained in humans, where the left HPC indicated response navigation and the right place memory formation. Second, the associations indicate the integration of emotional information into cognitive processing. At last, learning and relearning capabilities of Cav1.2 knockout mice were explored. Despite reduced MEMRI intensities in learning associated regions, knockout mice successfully acquire place and response memories and were also capable to revert the place memory afterwards. However, animals exhibited a significant retardation during place learning ,which can be attributed to impairments of late long-term potentiation (LTP) in the CA1 region of the HPC. Overall, the WCM suits the characteristics of mice and allows the distinction of different learning strategies. Further, mice similar to rats require an intact HPC to use place strategies in the WCM and at least 40% of the total HPC volume is necessary to accomplish relearning. Since I could demonstrate for the first time that MEMRI contrast largely depends on Cav1.2, MEMRI was employed to map brain activity in freely moving mice. I could identify brain regions most in the HPC that correlate with place learning parameters in the WCM for the first time in vivo. These results further match findings in humans, where place and response learning occur in parallel during place navigation in the left and right HPC, respectively. In addition, they suggest the integration of emotional information into cognitive precessing. At last, Cav1.2 is involved but not essential for place learning in the WCM. Future investigations with temporary knockouts might be useful to further elaborate the role of Cav1.2 in learning and memory functions

Hippocampus-dependent place learning enables spatial flexibility in mice

Spatial navigation is a fundamental capability necessary in everyday life to locate food, social partners and shelter. It results from two very different strategies: (i) place learning which enables for flexible way finding and (ii) response learning that leads to a more rigid ‘route following’. Despite the importance of knockout techniques that are only available in mice, little is known about mice’ flexibility in spatial navigation tasks.Here we demonstrate for C57BL6/N mice in a water-cross maze that only place learning enables spatial flexibility and relearning of a platform position, whereas response learning does not. This capability depends on an intact hippocampal formation, since hippocampus lesions by ibotenic acid disrupted relearning. In vivo manganese-enhanced magnetic resonance imaging revealed a volume loss of ≥ 60% of the hippocampus as a critical threshold for relearning impairments. In particular the changes in the left ventral hippocampus were indicative of relearning deficits.In summary, our findings establish the importance of hippocampus-dependent place learning for spatial flexibility and provide a first systematic analysis on spatial flexibility in mice

Tau protein is essential for stress-induced brain pathology

Exposure to chronic stress is frequently accompanied by cognitive and affective disorders in association with neurostructural adaptations. Chronic stress was previously shown to trigger Alzheimer’s-like neuropathology, which is characterized by Tau hyper-phosphorylation and missorting into dendritic spines followed by memory deficits. Here, we demonstrate that stress-driven hippocampal deficits in wild-type mice are accompanied by synaptic missorting of Tau and enhanced Fyn/GluN2B-driven synaptic signaling. In contrast, mice lacking Tau [Tau knockout (Tau-KO) mice] do not exhibit stress-induced pathological behaviors and atrophy of hippocampal dendrites or deficits of hippocampal connectivity. These findings implicate Tau as an essential mediator of the adverse effects of stress on brain structure and function

Distinct behavioral consequences of short-term and prolonged GABAergic depletion in prefrontal cortex and dorsal hippocampus

GABAergic interneurons are essential for a functional equilibrium between excitatory and inhibitory impulses throughout the CNS. Disruption of this equilibrium can lead to various neurological or neuropsychiatric disorders such as epileptic seizures or schizophrenia. Schizophrenia itself is clinically defined by negative- (e.g. depression) and positive- (e.g. hallucinations) symptoms as well as cognitive dysfunction. GABAergic interneurons are proposed to play a central role in the etiology and progression of schizophrenia; however, the specific mechanisms and the time-line of symptom development as well as the distinct involvement of cortical and hippocampal GABAergic interneurons in the etiology of schizophrenia-related symptoms are still not conclusively resolved.Previous work demonstrated that GABAergic interneurons can be selectively depleted in adult mice by means of saporin-conjugated anti-vesicular GABA transporter antibodies (SAVAs) in vitro and in vivo. Given their involvement in Schizophrenia-related disease etiology, we ablated GABAergic interneurons in the medial prefrontal cortex (mPFC) and dorsal hippocampus (dHPC) in adult male C57BL/6N mice. Subsequently we assessed alterations in anxiety, sensory processing, hyperactivity and cognition after long-term (>14 days) and short-term (< 14 days) GABAergic depletion. Long-term GABAergic depletion in the mPFC resulted in a decrease in sensorimotor-gating and impairments in cognitive flexibility. Notably, the same treatment at the level of the dHPC completely abolished spatial learning capabilities. Short-term GABAergic depletion in the dHPC revealed a transient hyperactive phenotype as well as marked impairments regarding the acquisition of a spatial memory. In contrast, recall of a spatial memory was not affected by the same intervention. These findings emphasize the importance of functional local GABAergic networks for the encoding but not the recall of hippocampus-dependent spatial memories