Intercellular Signaling Pathways as Therapeutic Targets for Vascular Dementia Repair.

Vascular dementia (VaD) is a white matter ischemic disease and the second-leading cause of dementia, with no direct therapy. Within the lesion site, cell-cell interactions dictate the trajectory towards disease progression or repair. To elucidate the underlying intercellular signaling pathways, a VaD mouse model was developed for transcriptomic and functional studies. The mouse VaD transcriptome was integrated with a human VaD snRNA-Seq dataset. A custom-made database encompassing 4053 human and 2032 mouse ligand-receptor (L-R) interactions identified significantly altered pathways shared between human and mouse VaD. Two intercellular L-R systems, Serpine2-Lrp1 and CD39-A3AR, were selected for mechanistic study as both the ligand and receptor were dysregulated in VaD. Decreased Seprine2 expression enhances OPC differentiation in VaD repair. A clinically relevant drug that reverses the loss of CD39-A3AR function promotes tissue and behavioral recovery in the VaD model. This study presents novel intercellular signaling targets and may open new avenues for VaD therapies.

Control of feeding by a bottom-up midbrain-subthalamic pathway.

Investigative exploration and foraging leading to food consumption have vital importance, but are not well-understood. Since GABAergic inputs to the lateral and ventrolateral periaqueductal gray (l/vlPAG) control such behaviors, we dissected the role of vgat-expressing GABAergic l/vlPAG cells in exploration, foraging and hunting. Here, we show that in mice vgat l/vlPAG cells encode approach to food and consumption of both live prey and non-prey foods. The activity of these cells is necessary and sufficient for inducing food-seeking leading to subsequent consumption. Activation of vgat l/vlPAG cells produces exploratory foraging and compulsive eating without altering defensive behaviors. Moreover, l/vlPAG vgat cells are bidirectionally interconnected to several feeding, exploration and investigation nodes, including the zona incerta. Remarkably, the vgat l/vlPAG projection to the zona incerta bidirectionally controls approach towards food leading to consumption. These data indicate the PAG is not only a final downstream target of top-down exploration and foraging-related inputs, but that it also influences these behaviors through a bottom-up pathway.

Early Post-Natal Immune Activation Leads to Object Memory Deficits in Female Tsc2+/- Mice: The Importance of Including Both Sexes in Neuroscience Research.

There is evidence that viral infections during pre-natal development constitute a risk factor for neuropsychiatric disorders and lead to learning and memory deficits. However, little is known about why viral infections during early post-natal development have a different impact on learning and memory depending on the sex of the subject. We previously showed that early post-natal immune activation induces hippocampal-dependent social memory deficits in a male, but not in a female, mouse model of tuberous sclerosis complex (TSC; Tsc2+/- mice). Here, we explored the impact of a viral-like immune challenge in object memory. We demonstrate that early post-natal immune activation (during the first 2 weeks of life) leads to object memory deficits in female, but not male, mice that are heterozygous for a gene responsible for tuberous sclerosis complex (Tsc2+/- mice), while no effect was observed in wild type (WT) mice. Moreover, we found that the same immune activation in Tsc2+/- adult mice was not able to cause object memory deficits in females, which suggests that the early post-natal development stage constitutes a critical window for the effects of immune challenge on adult memory. Also, our results suggest that mTOR plays a critical role in the observed deficit in object memory in female Tsc2+/- mice. These results, together with previous results published by our laboratory, showing sex-specific memory deficits due to early post-natal immune activation, reinforce the necessity of using both males and females for research studies. This is especially true for studies related to immune activation, since the higher levels of estrogens in females are known to affect inflammation and to provide neuroprotection.

A dual-pathway architecture enables chronic stress to promote habit formation.

Chronic stress can change how we learn and, thus, how we make decisions by promoting the formation of inflexible, potentially maladaptive, habits. Here we investigated the neuronal circuit mechanisms that enable this. Using a multifaceted approach in male and female mice, we reveal a dual pathway, amygdala-striatal, neuronal circuit architecture by which a recent history of chronic stress shapes learning to disrupt flexible goal-directed behavior in favor of inflexible habits. Chronic stress inhibits activity of basolateral amygdala projections to the dorsomedial striatum to impede the action-outcome learning that supports flexible, goal-directed decisions. Stress also increases activity in direct central amygdala projections to the dorsomedial striatum to promote the formation of rigid, inflexible habits. Thus, stress exerts opposing effects on two amygdala-striatal pathways to promote premature habit formation. These data provide neuronal circuit insights into how chronic stress shapes learning and decision making, and help understand how stress can lead to the disrupted decision making and pathological habits that characterize substance use disorders and other psychiatric conditions.

Role of Type I Interferon Signaling and Microglia in the Abnormal Long-term Potentiation and Object Place Recognition Deficits of Male Mice With a Mutation of the Tuberous Sclerosis 2 Gene.

Background: Tuberous sclerosis complex is a genetic disorder associated with high rates of intellectual disability and autism. Mice with a heterozygous null mutation of the Tsc2 gene (Tsc2+/-) show deficits in hippocampal-dependent tasks and abnormal long-term potentiation (LTP) in the hippocampal CA1 region. Although previous studies focused on the role of neuronal deficits in the memory phenotypes of rodent models of tuberous sclerosis complex, the results presented here demonstrate a role for microglia in these deficits. Methods: To test the possible role of microglia and type I interferon in abnormal hippocampal-dependent memory and LTP of Tsc2+/- mice, we used field recordings in CA1 and the object place recognition (OPR) task. We used the colony stimulating factor 1 receptor inhibitor PLX5622 to deplete microglia in Tsc2+/- mice and interferon alpha/beta receptor alpha chain null mutation (Ifnar1-/-) to manipulate a signaling pathway known to modulate microglia function. Results: Unexpectedly, we demonstrate that male, but not female, Tsc2+/- mice show OPR deficits. These deficits can be rescued by depletion of microglia and by the Ifnar1-/- mutation. In addition to rescuing OPR deficits, depletion of microglia also reversed abnormal LTP of the Tsc2+/- mice. Altogether, our results suggest that altered IFNAR1 signaling in microglia causes the abnormal LTP and OPR deficits of male Tsc2+/- mice. Conclusions: Microglia and IFNAR1 signaling have a key role in the hippocampal-dependent memory deficits and abnormal hippocampal LTP of Tsc2+/- male mice.

Miniscope-LFOV: A large-field-of-view, single-cell-resolution, miniature microscope for wired and wire-free imaging of neural dynamics in freely behaving animals.

Imaging large-population, single-cell fluorescent dynamics in freely behaving animals larger than mice remains a key endeavor of neuroscience. We present a large-field-of-view open-source miniature microscope (MiniLFOV) designed for large-scale (3.6 mm × 2.7 mm), cellular resolution neural imaging in freely behaving rats. It has an electrically adjustable working distance of up to 3.5 mm ± 100 µm, incorporates an absolute head orientation sensor, and weighs only 13.9 g. The MiniLFOV is capable of both deep brain and cortical imaging and has been validated in freely behaving rats by simultaneously imaging >1000 GCaMP7s-expressing neurons in the hippocampal CA1 layer and in head-fixed mice by simultaneously imaging ~2000 neurons in the dorsal cortex through a cranial window. The MiniLFOV also supports optional wire-free operation using a novel, wire-free data acquisition expansion board. We expect that this new open-source implementation of the UCLA Miniscope platform will enable researchers to address novel hypotheses concerning brain function in freely behaving animals.

Aversive experience drives offline ensemble reactivation to link memories across days.

Memories are encoded in neural ensembles during learning and stabilized by post-learning reactivation. Integrating recent experiences into existing memories ensures that memories contain the most recently available information, but how the brain accomplishes this critical process remains unknown. Here we show that in mice, a strong aversive experience drives the offline ensemble reactivation of not only the recent aversive memory but also a neutral memory formed two days prior, linking the fear from the recent aversive memory to the previous neutral memory. We find that fear specifically links retrospectively, but not prospectively, to neutral memories across days. Consistent with prior studies, we find reactivation of the recent aversive memory ensemble during the offline period following learning. However, a strong aversive experience also increases co-reactivation of the aversive and neutral memory ensembles during the offline period. Finally, the expression of fear in the neutral context is associated with reactivation of the shared ensemble between the aversive and neutral memories. Taken together, these results demonstrate that strong aversive experience can drive retrospective memory-linking through the offline co-reactivation of recent memory ensembles with memory ensembles formed days prior, providing a neural mechanism by which memories can be integrated across days.

Local memory allocation recruits memory ensembles across brain regions.

Memories are thought to be stored in ensembles of neurons across multiple brain regions. However, whether and how these ensembles are coordinated at the time of learning remains largely unknown. Here, we combined CREB-mediated memory allocation with transsynaptic retrograde tracing to demonstrate that the allocation of aversive memories to a group of neurons in one brain region directly affects the allocation of interconnected neurons in upstream brain regions in a behavioral- and brain region-specific manner in mice. Our analysis suggests that this cross-regional recruitment of presynaptic neurons is initiated by downstream memory neurons through a retrograde mechanism. Together with statistical modeling, our results indicate that in addition to the anterograde flow of information between brain regions, the establishment of interconnected, brain-wide memory traces relies on a retrograde mechanism that coordinates memory ensembles at the time of learning.

New research tools suggest a "levels-less" image of the behaving organism and dissolution of the reduction vs. anti-reduction dispute.

A kind of "ruthless reductionism" characterized the experimental practices of the first two decades of molecular and cellular cognition (MCC). More recently, new research tools have expanded experimental practices in this field, enabling researchers to image and manipulate individual molecular mechanisms in behaving organisms with an unprecedented temporal, sub-cellular, cellular, and even circuit-wide specificity. These tools dramatically expand the range and reach of experiments in MCC, and in doing so they may help us transcend the worn-out and counterproductive debates about "reductionism" and "emergence" that divide neuroscientists and philosophers alike. We describe examples of these new tools and illustrate their practical power by presenting an exemplary recent case of MCC research using them. From these tools and results, we provide an initial sketch of a new image of the behaving organism in its full causal-interactive complexity, with its molecules, cells, and circuits combined within the single system that it is. This new image stands in opposition to the traditional "levels" image of the behaving organism, and even the initial sketch we provide of it here offers hope for avoiding the dreary metaphysical debates about "emergence" and "downward causation," and even the reduction vs. anti-reduction dispute, all dependent upon the familiar "levels" image.

A locus coeruleus-dorsal CA1 dopaminergic circuit modulates memory linking.

Individual memories are often linked so that the recall of one triggers the recall of another. For example, contextual memories acquired close in time can be linked, and this is known to depend on a temporary increase in excitability that drives the overlap between dorsal CA1 (dCA1) hippocampal ensembles that encode the linked memories. Here, we show that locus coeruleus (LC) cells projecting to dCA1 have a key permissive role in contextual memory linking, without affecting contextual memory formation, and that this effect is mediated by dopamine. Additionally, we found that LC-to-dCA1-projecting neurons modulate the excitability of dCA1 neurons and the extent of overlap between dCA1 memory ensembles as well as the stability of coactivity patterns within these ensembles. This discovery of a neuromodulatory system that specifically affects memory linking without affecting memory formation reveals a fundamental separation between the brain mechanisms modulating these two distinct processes.

CCR5 closes the temporal window for memory linking.

Real-world memories are formed in a particular context and are often not acquired or recalled in isolation1-5. Time is a key variable in the organization of memories, as events that are experienced close in time are more likely to be meaningfully associated, whereas those that are experienced with a longer interval are not1-4. How the brain segregates events that are temporally distinct is unclear. Here we show that a delayed (12-24 h) increase in the expression of C-C chemokine receptor type 5 (CCR5)-an immune receptor that is well known as a co-receptor for HIV infection6,7-after the formation of a contextual memory determines the duration of the temporal window for associating or linking that memory with subsequent memories. This delayed expression of CCR5 in mouse dorsal CA1 neurons results in a decrease in neuronal excitability, which in turn negatively regulates neuronal memory allocation, thus reducing the overlap between dorsal CA1 memory ensembles. Lowering this overlap affects the ability of one memory to trigger the recall of the other, and therefore closes the temporal window for memory linking. Our findings also show that an age-related increase in the neuronal expression of CCR5 and its ligand CCL5 leads to impairments in memory linking in aged mice, which could be reversed with a Ccr5 knockout and a drug approved by the US Food and Drug Administration (FDA) that inhibits this receptor, a result with clinical implications. Altogether, the findings reported here provide insights into the molecular and cellular mechanisms that shape the temporal window for memory linking.

The emergence of molecular systems neuroscience.

Systems neuroscience is focused on how ensemble properties in the brain, such as the activity of neuronal circuits, gives rise to internal brain states and behavior. Many of the studies in this field have traditionally involved electrophysiological recordings and computational approaches that attempt to decode how the brain transforms inputs into functional outputs. More recently, systems neuroscience has received an infusion of approaches and techniques that allow the manipulation (e.g., optogenetics, chemogenetics) and imaging (e.g., two-photon imaging, head mounted fluorescent microscopes) of neurons, neurocircuits, their inputs and outputs. Here, we will review novel approaches that allow the manipulation and imaging of specific molecular mechanisms in specific cells (not just neurons), cell ensembles and brain regions. These molecular approaches, with the specificity and temporal resolution appropriate for systems studies, promise to infuse the field with novel ideas, emphases and directions, and are motivating the emergence of a molecularly oriented systems neuroscience, a new discipline that studies how the spatial and temporal patterns of molecular systems modulate circuits and brain networks, and consequently shape the properties of brain states and behavior.

Experiment Selection in Meta-Analytic Piecemeal Causal Discovery.

Scientists try to design experiments that will yield maximal information. For instance, given the available evidence and a limitation on the number of variables that can be observed simultaneously, it may be more informative to intervene on variable X and observe the response of variable Y than to intervene on X and observe Z; in other situations, the opposite may be true. Scientists must often make these decisions without primary data. To address this problem, in previous work, we created software for annotating aggregate statistics in the literature and deriving consistent causal explanations, expressed as causal graphs. This meta-analytic pipeline is useful not only for synthesizing evidence but also for planning experiments: one can use it strategically to select experiments that could further eliminate causal graphs from consideration. In this paper, we introduce interpretable policies for selecting experiments in the context of piecemeal causal discovery, a common setting in biological sciences in which each experiment can measure not an entire system but rather a strict subset of its variables. The limits of this piecemeal approach are only beginning to be fully characterized, with crucial theoretical work published recently. With simulations, we show that our experiment-selection policies identify causal structures more efficiently than random experiment selection. Unlike methods that require primary data, our meta-analytic approach offers a flexible alternative for those seeking to incorporate qualitative domain knowledge into their search for causal mechanisms. We also present a method that categorizes hypotheses with respect to their utility for identifying a system's causal structure. Although this categorization is usually infeasible to perform manually, it is critical for conducting research efficiently.

Postnatal immune activation causes social deficits in a mouse model of tuberous sclerosis: Role of microglia and clinical implications.

There is growing evidence that prenatal immune activation contributes to neuropsychiatric disorders. Here, we show that early postnatal immune activation resulted in profound impairments in social behavior, including in social memory in adult male mice heterozygous for a gene responsible for tuberous sclerosis complex (Tsc2+/−), a genetic disorder with high prevalence of autism. Early postnatal immune activation did not affect either wild-type or female Tsc2+/− mice. We demonstrate that these memory deficits are caused by abnormal mammalian target of rapamycin­dependent interferon signaling and impairments in microglia function. By mining the medical records of more than 3 million children followed from birth, we show that the prevalence of hospitalizations due to infections in males (but not in females) is associated with future development of autism spectrum disorders (ASD). Together, our results suggest the importance of synergistic interactions between strong early postnatal immune activation and mutations associated with ASD.

Dorsal premammillary projection to periaqueductal gray controls escape vigor from innate and conditioned threats.

Escape from threats has paramount importance for survival. However, it is unknown if a single circuit controls escape vigor from innate and conditioned threats. Cholecystokinin (cck)-expressing cells in the hypothalamic dorsal premammillary nucleus (PMd) are necessary for initiating escape from innate threats via a projection to the dorsolateral periaqueductal gray (dlPAG). We now show that in mice PMd-cck cells are activated during escape, but not other defensive behaviors. PMd-cck ensemble activity can also predict future escape. Furthermore, PMd inhibition decreases escape speed from both innate and conditioned threats. Inhibition of the PMd-cck projection to the dlPAG also decreased escape speed. Intriguingly, PMd-cck and dlPAG activity in mice showed higher mutual information during exposure to innate and conditioned threats. In parallel, human functional magnetic resonance imaging data show that a posterior hypothalamic-to-dlPAG pathway increased activity during exposure to aversive images, indicating that a similar pathway may possibly have a related role in humans. Our data identify the PMd-dlPAG circuit as a central node, controlling escape vigor elicited by both innate and conditioned threats.

Dimensions and mechanisms of memory organization.

Memory formation is dynamic in nature, and acquisition of new information is often influenced by previous experiences. Memories sharing certain attributes are known to interact so that retrieval of one increases the likelihood of retrieving the other, raising the possibility that related memories are organized into associative mnemonic structures of interconnected representations. Although the formation and retrieval of single memories have been studied extensively, very little is known about the brain mechanisms that organize and link related memories. Here we review studies that suggest the existence of mnemonic structures in humans and animal models. These studies suggest three main dimensions of experience that can serve to organize related memories: time, space, and perceptual/conceptual similarities. We propose potential molecular, cellular, and systems mechanisms that might support organization of memories according to these dimensions.

Pharmacological blockers of CCR5 and CXCR4 improve recovery after traumatic brain injury.

CCR5 and CXCR4 are structurally related chemokine receptors that belong to the superfamily of G-protein coupled receptors through which the HIV virus enters and infects cells. Both receptors are also related to HIV-associated neurocognitive disorders that include difficulties in concentration and memory, impaired executive functions, psychomotor slowing, depression and irritability, which are also hallmarks of the long-term sequelae of TBI. Moreover, A growing body of evidence attributes negative influences to CCR5 activation on cognition, particularly after stroke and traumatic brain injury (TBI). Here we investigated the effect of their blockage on motor and cognitive functions, on brain tissue loss and preservation and on some of the biochemical pathways involved. We examined the effect of maraviroc, a CCR5 antagonist used in HIV patients as a viral entry inhibitor, and of plerixafor (AMD3100), a CXCR4 antagonist used in cancer patients as an immune-modulator, on mice subjected to closed head injury (CHI). Mice were treated with maraviroc or plerixafor after CHI for the following 4 or 5 days, respectively. Neurobehavior was assessed according to the Neurological Severity Score; cognitive tests were performed by using the Y-maze, Barnes maze and the novel object recognition test; anxiety was evaluated with the open field test. The mice were sacrificed and brain tissues were collected for Western blot, pathological and immunohistochemical analyses. Both drugs enhanced tissue preservation in the cortex, hippocampus, periventricular areas, corpus callosum and striatum, and reduced astrogliosis)GFAP expression). They also increased the levels of synaptic cognition-related signaling molecules such as phosphorylated NR1 and CREB, and the synaptic plasticity protein PSD95. Both treatments also enhanced the expression of CCR5 and CXCR4 on different brain cell types. In summary, the beneficial effects of blocking CCR5 and CXCR4 after CHI suggest that the drugs used in this study, both FDA approved and in clinical use, should be considered for translational research in TBI patients.

Chemokine Receptors CC Chemokine Receptor 5 and C-X-C Motif Chemokine Receptor 4 Are New Therapeutic Targets for Brain Recovery after Traumatic Brain Injury.

Recently, chemokine receptor CC chemokine receptor 5 (CCR5) was found to be a negative modulator of learning and memory. Its inhibition improved outcome after stroke and traumatic brain injury (TBI). To better understand its role after TBI and establish therapeutic strategies, we investigated the effect of reduced CCR5 signaling as a neuroprotective strategy and of the temporal changes of CCR5 expression after TBI in different brain cell types. To silence CCR5 expression, ccr5 short hairpin RNA (shRNA) or dsred shRNA (control) was injected into the cornu ammonis (CA) 1 and CA3 regions of the hippocampus 2 weeks before induction of closed-head injury in mice. Animals were then monitored for 32 days and euthanized at different time points to assess lesion area, inflammatory components of the glial response (immunohistochemistry; IHC), cytokine levels (enzyme-linked immunosorbent array), and extracellular signal-regulated kinase (ERK) phosphorylation (western blot). Fluorescence-activated cell sorting (FACS) analysis was performed to study post-injury temporal changes of CCR5 and C-X-C motif chemokine receptor 4 (CXCR4) expression in cortical and hippocampal cell populations (neurons, astrocytes, and microglia). Phosphorylation of the N-methyl-d-aspartate subunit 1 (NR1) subunit of N-methyl-d-aspartate (western blot) and cAMP-response-element-binding protein (CREB; IHC) were also assessed. The ccr5 shRNA mice displayed reduced lesion area, dynamic alterations in levels of inflammation-related CCR5 ligands and cytokines, and higher levels of phosphorylated ERK. The ccr5 shRNA also reduced astrocytosis in the lesioned and sublesioned cortex. FACS analysis revealed increased cortical CCR5 and CXCR4 expression in CD11b-positive cells, astrocytes, and neurons, which was most evident in cells expressing both receptors, at 3 and 11 days post-injury. The lowest levels of phosphorylated NR1 and phosphorylated CREB were found at day 3 post-injury, suggesting that this is the critical time point for therapeutic intervention.

Breakdown of spatial coding and interneuron synchronization in epileptic mice.

Temporal lobe epilepsy causes severe cognitive deficits, but the circuit mechanisms remain unknown. Interneuron death and reorganization during epileptogenesis may disrupt the synchrony of hippocampal inhibition. To test this, we simultaneously recorded from the CA1 and dentate gyrus in pilocarpine-treated epileptic mice with silicon probes during head-fixed virtual navigation. We found desynchronized interneuron firing between the CA1 and dentate gyrus in epileptic mice. Since hippocampal interneurons control information processing, we tested whether CA1 spatial coding was altered in this desynchronized circuit, using a novel wire-free miniscope. We found that CA1 place cells in epileptic mice were unstable and completely remapped across a week. This spatial instability emerged around 6 weeks after status epilepticus, well after the onset of chronic seizures and interneuron death. Finally, CA1 network modeling showed that desynchronized inputs can impair the precision and stability of CA1 place cells. Together, these results demonstrate that temporally precise intrahippocampal communication is critical for spatial processing.

Human Memories Can Be Linked by Temporal Proximity.

Real-world memories involve the integration of multiple events across time, yet the mechanisms underlying this integration is unknown. Recent rodent studies show that distinct memories encoded within a few hours, but not several days, share a common neural ensemble, and a common fate whereby later fear conditioning can transfer from one memory to the other. Here, we tested if distinct memories could be linked by temporal proximity in humans. 74 young adults encoded two memories (A and B) close (3-h) or far apart (7-day) in time. One day after encoding the second memory (B), Memory A was updated by pairing it with electric shock (i.e., fear conditioning). We tested whether the memory and fear associated with Memory B would be stronger in the 3-h, compared with the 7-day condition. Results were generally consistent with rodent studies, where we found heightened Memory B fear expression when the two memories were encoded close, but not far apart, in time. Furthermore, there was less forgetting of Memory B in the 3-h compared to 7-day condition. Our results suggest that temporally proximal memories may be linked, such that updating one experience updates the other.