bioRxiv Subject Collection: Neuroscience's Journal

Long-Term Reactivation of Multiple Sub-Assemblies in the Hippocampus and Prefrontal Cortex
In resting or sleep periods following a task, neurons in various parts of the brain become reactivated, with firing patterns often similar to that during the task. This reactivation plays an essential role in memory consolidation. However, detecting these reactivating episodes has been challenging because not all neurons recorded may be directly relevant to the memory being consolidated. Here, we propose a novel spike train clustering (STC) method for detecting groups of neurons (clusters) with partial synchronous firing. From a mechanistic standpoint, the correlated activity of an ensemble can arise from neuronal interactions within the recorded local ensemble, common external inputs, or both. To quantify these contributions, we propose to use information geometry (IG) by taking advantage of its capacity for orthogonal decomposition of neural interactions. We analyzed simultaneous single unit activity from rat medial prefrontal cortex (mPFC) and area CA1 of the hippocampus when animals explored novel objects and when they slept before and after the exploration. We demonstrate that multiple reactivations by different subsets of neurons (clusters) occurred. Those reactivations could extend over 11 hours, the entire recording duration of post-task rest after the exploration epoch. Long-lasting reactivation was not detected when all neurons were included as a single cluster in the analysis. We also showed that pairwise interactions in reactivating clusters tended to be more strongly modified than in non-reactivating ones. In addition, the pairwise interactions of the reactivating clusters in CA1 were strongly modulated by the task experience but not in the mPFC. These results indicate that hippocampal reactivation following novel experience is likely to be induced within the hippocampal circuits. In contrast, mPFC reactivation is likely to be driven by external inputs, possibly in part from the hippocampus.

A vestibulospinal pathway for context-dependent motor control of the mouse tail
The tail movement is critical for maintaining balance during locomotion in many animal species, yet its underlying neuro-muscular control remains poorly understood. In this study we investigated what are the neuronal substrates responsible for tail control in mice. Using high-resolution microCT scans and retrograde labeling, we lay out the neuro-muscular organization of the tail and identified distinct pools of motoneurons in the spinal cord that innervate proximal and distal muscles. We further show that the spinal vestibular nucleus (SpVN) in the brainstem sends direct projections to the same spinal cord segments where tail motoneurons are located. The activation of these vestibulospinal neurons using optogenetics results in more accurate tail movements during challenging balance tasks. Our results reveal that the vestibular systems influence on tail control is context-dependent, enhancing balance performance under uncertain sensory conditions. These findings provide novel insights into the neural circuits responsible for maintaining balance, highlighting the role of the vestibulospinal pathway in context-dependent modulation of tail movement to maintain stability during complex locomotor tasks.

A novel nitric oxide (NO)-dependent 'molecular switch' mediates LTP in the Octopus vulgaris brain through persistent activation of nitric oxide synthase (NOS)
Cephalopods are a renowned example of the independent evolution of complex behavior in invertebrates. The octopus's outstanding learning capability is a prominent feature that depends on the vertical lobe (VL). Previously, we found that the synaptic input into the VL exhibits robust activity-dependent long-term potentiation (LTP) mediated by molecular processes that are only partially understood. Here, we reveal that the VL LTP is mediated by nitric oxide (NO). In contrast to the prevailing dogma, in the octopus VL, NO does not mediate LTP induction, as tetanization-induced LTP occurs even in the presence of NO-synthase (NOS) inhibitors. Remarkably, however, NOS inhibitors block the long-term presynaptic expression of LTP, and high doses of NO donor induce short-term synaptic potentiation, suggesting that a persistent elevation of NO concentration mediates LTP expression. Moreover, in a distinct group of synapses, NOS inhibitors also disrupted LTP maintenance, as following drug washout, a high-frequency stimulation reinstated full LTP, suggesting that NO is also involved in maintaining LTP. We propose a novel molecular-switch mechanism whereby a positive feedback loop of NO-dependent NOS reactivation mediates persistent NOS activation, thus providing an LTP maintenance mechanism. Subsequently, retrograde NO diffusion facilitates presynaptic transmitter release, driving LTP expression. These findings demonstrate how evolutionary adaptation of molluscan molecular mechanisms has contributed to the emergence of the advanced cognitive abilities observed in octopuses.

p75NTR modulation by LM11A-31 counteracts oxidative stress and cholesterol dysmetabolism in a rotenone-induced cell model of Parkinson's disease
Neurotrophins play pivotal roles in the development and proper functioning of the nervous system. The effects of these growth factors are mediated through the binding of high-affinity receptors, known as Trks, as well as the low-affinity receptor p75NTR. The latter has the capacity to induce complex signal pathways, as it favors both pro-survival and pro-apoptotic cascades depending on the physiopathological condition. Recent findings have indicated that p75NTR expression is increased in post-mortem Parkinsons disease (PD) brains, and this upregulation is associated with a significant reduction in neuroprotection. Given its double-edged sword nature, p75NTR has recently been identified as a promising therapeutic target to counteract neurodegenerative events. The present study aims to assess the neuroprotective effects of p75NTR modulation in a rotenone-induced neuronal model of PD. To this end, differentiated SH-SY5Y cells were exposed to rotenone to mimic the PD phenotype, and the small molecule LM11A-31 was used to modulate p75NTR activity. The main results revealed that LM11A-31 significantly mitigated the hallmarks of PD, including cell death, neuromorphological aberrations, and -synuclein accumulation. Pharmacological manipulation of p75NTR also reduced oxidative damage by increasing the expression of transcriptional factors that regulate the antioxidant response and by decreasing the expression of the pro-oxidant NADPH-oxidase modulatory subunits. Furthermore, LM11A-31 hampered cholesterol buildup induced by rotenone, normalizing the expression of proteins involved in cholesterol biosynthesis, uptake and intracellular trafficking. Taken together, these findings suggest that p75NTR modulation may represent a novel approach to counteracting PD abnormalities of redox and cholesterol metabolism.

Inhibition of PDE5 induces rapid non-genomic increase in hippocampal dendritic spines via specific kinases
Cyclic guanosine monophosphate (cGMP) is a typical neuromodulator that is used in neuronal synapses. Inhibitor of phosphodiesterase 5 (PDE5) could regulates signaling pathways by elevating cGMP levels.

In the current study, we found that the treatments with tadalafil, a PDE5 inhibitor, for 2 h rapidly and non-genomically increased the total density of dendritic spines, using confocal imaging of Lucifer Yellow-injected hippocampal CA1 pyramidal neurons. Since inhibition of PDE5 elevates cGMP levels at synapses, we analyzed downstream nongenomic signaling. We analyzed the involvement of kinase signaling, because rapid postsynaptic modulation involves kinase networks observed from many investigations including our works.

Upon co-treatments of PKG inhibitor (KT5823) with tadalafil, the spine increase was considerably blocked. In addition, co-treatments of GSK-3 inhibitor (I8) also blocked the spine increase induced by tadalafil. However, inhibitors of other representative synaptic kinases, including LIMK, Erk/MAPK, PKA, PKC and PI3K, did not suppress the tadalafil-induced spine increases.

PDE5 inhibitors have attracted attention on their recovery effects from pathological damages, including memory improvement from cognitive impairment by Stroke, Amyloid {beta} (A{beta}) accumulation and tau phosphorylation. The current finding of tadalafils function about rapid modulation of neural plasticity may add new elemental steps of anti-aging capacity against cognitive decline.

Dissecting the evolving cellular landscape of a remyelinating microenvironment
Demyelination, or the loss of myelin in the central nervous system (CNS) is a hallmark of multiple sclerosis (MS) and occurs in various forms of CNS injury and neurodegenerative diseases. The regeneration of myelin, or remyelination, occurs spontaneously following demyelination. The lysophosphatidylcholine (LPC)-induced focal demyelination model enables investigations into the mechanisms of remyelination, providing insight into the molecular basis underlying an evolving remyelinating microenvironment over a tractable time course. Here, we present a detailed analysis using high-resolution single nucleus RNA sequencing to investigate gene expression dynamics across multiple cell populations involved in the remyelination process. We examine three specific time points following focal demyelinating injury in mice, and by delineating activation states within the heterogeneous cell populations of demyelinated lesions, we highlight changes in gene expression within subclusters of each cell population from the early stages of injury response to the initiation and maintenance of remyelination. Our findings reveal how shifts in microglial, astrocytic and fibroblast activities within lesions are associated with efficient oligodendrocyte differentiation during remyelination.

LPS-induced sepsis disrupts brain activity in a region- and vigilance-state specific manner
Sepsis-associated encephalopathy (SAE) is a common complication of sepsis and the systemic inflammatory response syndrome that leads to lasting consequences in survivors. It manifests as early EEG changes, that are region-, time- and state-specific, possibly reflecting distinct mechanisms of injury.

Here, we investigated the effects of 5mg/kg lipopolysaccharide (LPS) on hippocampal and cortical sleep-wake states, oscillatory and non-oscillatory neuronal activity, as well as on within and between state dynamics using state-space analysis.

LPS induced rapid-onset severe temporal and spatial vigilance state fragmentation, which preceded all other spectral changes by [~]90 minutes. Thereafter, LPS led to specific destabilization and increased delta oscillatory activity in wakefulness, but not NREM sleep, although state transitions remained largely normal. Instead, reduced NREM delta power resulted from aperiodic spectrum changes. LPS specifically reduced higher frequency hippocampal gamma oscillations (60-80Hz peak) in wakefulness, but not cortical high gamma or lower frequency gamma oscillations.

These results suggest that disruption of sleep-wake patterns could serve as an early indicator of sepsis and associated encephalopathy, independent of spectral changes. Moreover, treatment aimed at stabilizing vigilance states in early stages of sepsis might prove to be a novel option preventing the development of further pathological neurophysiology, as well as limiting inflammation-related brain damage.

Contribution of glutamatergic projections to neurons in the nonhuman primate lateral substantia nigra pars reticulata for the reactive inhibition
The basal ganglia play a crucial role in action selection by facilitating desired movements and suppressing unwanted ones. The substantia nigra pars reticulata (SNr), a key output nucleus, facilitates movement through disinhibition of the superior colliculus (SC). However, its role in action suppression, particularly in primates, remains less clear. We investigated whether individual SNr neurons in three male macaque monkeys bidirectionally modulate their activity to both facilitate and suppress actions and examined the role of glutamatergic inputs in suppression. Monkeys performed a sequential choice task, selecting or rejecting visually presented targets. Electrophysiological recordings showed SNr neurons decreased firing rates during target selection and increased firing rates during rejection, demonstrating bidirectional modulation. Pharmacological blockade of glutamatergic inputs to the lateral SNr disrupted saccadic control and impaired suppression of reflexive saccades, providing causal evidence for the role of excitatory input in behavioral inhibition. These findings suggest that glutamatergic projections, most likely from the subthalamic nucleus, drive the increased SNr activity during action suppression. Our results highlight conserved basal ganglia mechanisms across species and offer insights into the neural substrates of action selection and suppression in primates, with implications for understanding disorders such as Parkinsons disease.

Significance StatementUnderstanding how the basal ganglia facilitate desired actions while suppressing unwanted ones is fundamental to neuroscience. This study shows that neurons in the primate substantia nigra pars reticulata (SNr) bidirectionally modulate activity to control action, decreasing firing rates to facilitate movements and increasing rates to suppress them. Importantly, we provide causal evidence that glutamatergic inputs to the lateral SNr mediate action suppression. These findings reveal a conserved mechanism of action control in primates and highlight the role of excitatory inputs in behavioral inhibition. This advances our understanding of basal ganglia function and has significant implications for treating movement disorders like Parkinsons disease.

Longitudinal assessment of DREADD expression and efficacy in the monkey brain
Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) offer a powerful means for reversible control of neuronal activity through systemic administration of inert actuators. Because chemogenetic control relies on DREADD expression levels, understanding and quantifying the temporal dynamics of their expression is crucial for planning long-term experiments in monkeys. In this study, we longitudinally quantified in vivo DREADD expression in macaque monkeys using positron emission tomography with the DREADD-selective tracer [11C]deschloroclozapine (DCZ), complemented by functional studies. Twenty macaque monkeys were evaluated after being injected with adeno-associated virus vectors expressing the DREADDs hM4Di or hM3Dq, whose expression was quantified as changes in [11C]DCZ binding potential from baseline levels. Expression levels of both hM4Di and hM3Dq peaked around 60 days post-injection, remained stable for about 1.5 years, and declined gradually after two years. Significant chemogenetic control of neural activity and behavior persisted for about two years. Virus titer and the presence of protein tags significantly influenced expression levels, with co-expressed protein tags reducing overall expression levels. These findings provide valuable insights and guidelines for optimizing the use of DREADDs in long-term primate studies and potential therapeutic applications.

Modulation of hippocampal sharp-wave ripples by behavioral states and body movements in head-fixed rodents
Hippocampal sharp-wave ripples (SWRs) are critical events implicated in memory consolidation, planning, and the reactivation of recent experiences. Under freely moving conditions, a well-established dichotomy exists: hippocampal networks predominantly generate theta oscillations during periods of reward pursuit (preparatory behaviors) and exhibit pronounced SWR activity once the reward is achieved (consummatory behaviors). Here, we examined how SWRs are modulated by reward delivery and subtle movements in head-fixed rats. Contrary to the canonical view established in freely moving settings, we found that the dominant and more enduring effect was a sustained suppression of SWR activity immediately following water delivery. Moreover, even minor, localized movements (such as whisking or body adjustments) decreased SWR occurrence, demonstrating that hippocampal ripple generation is highly sensitive to motor engagement, irrespective of reward timing. Such movement-induced suppression of ripples persisted during both sleep-like states and quiet wakefulness, suggesting that while large-scale brain states modulate the overall likelihood of SWR generation, local motor-related influences exert a state-independent inhibitory effect on hippocampal ripples. Our results show that SWR modulation by behavioral states and body movements is more context-dependent than previously appreciated.

Hippocampal sharp-wave ripples decrease during physical actions including consummatory behavior in immobile rodents
Hippocampal sharp-wave ripples (SWRs) are intermittent, fast synchronous oscillations that play a pivotal role in memory formation. It is well known that SWRs occur during "consummatory behaviors", e.g., eating or drinking a reward for correct action. However, most of typical behavioral experiments using freely-moving rodents have not rigorously distinguished eating/drinking itself (regardless of whether it means consummation or consumption) from stopping locomotion (immobility). In this study, therefore, we examined the occurrence of SWRs during a reward-seeking action and subsequent consummatory reward licking in constantly immobile rats under head fixation and body restriction. The immobile rats performed a pedal hold-release action that was rewarded with water every other time for their correct action (false and true consummation). Unexpectedly, the SWRs remarkably decreased during reward licking as well as pedal release action. Unlearned rats also showed a similar SWR decrease during water licking. On the other hand, the SWRs increased gradually during pedal hold period, which was enhanced by reward expectation. A cluster of hippocampal neurons responded to Cue/pedal release and reward as known previously. Some other clusters exhibited spike activity changes similar to the SWR occurrence, i.e., decreasing during the pedal release action and reward licking, and enhanced by reward expectation during pedal hold period. These task event-responsive neurons and SWR-like neurons displayed stronger spiking synchrony with the SWRs than task-unrelated neurons. These findings suggest that the hippocampus generates the SWRs, which may associate action with outcome, in animals "relative immobility" (action pauses) rather than specific consummation or consumption.

Significance StatementTo clarify the nature of sharp-wave ripples (SWRs) in the hippocampus, we analyzed the SWRs occurring during operant task performance by immobile rats under head-fixation and body restriction. First, we found the SWRs decreased when they licked and drank water, which conflicts with the theory that the SWRs occur in consummatory behavior. Second, hippocampal neurons showed different task-related activities, particularly those that resemble the SWR occurrence and those that conveyed specific signals on task events. Third, these task-related neurons displayed strong synchronous discharges during the SWRs in task-engaged periods. These findings may explain the neuronal mechanism underlying the association between an action and its outcome.

Hindering memory suppression by perturbing the right dorsolateral prefrontal cortex
A reminder of the past can trigger the involuntary retrieval of an unwanted memory. Yet, we can intentionally stop this process and thus prevent the memory from entering awareness. Such suppression not only transiently hinders the retrieval of the memory, it can also induce forgetting. Neuroimaging has implicated the right dorsolateral prefrontal cortex (dlPFC) in initiating this process. Specifically, this region seems to downregulate activity in brain systems that would otherwise support the reinstatement of the memory. We here probed the causal contribution of the right dlPFC to suppression by combining the Think/No-Think task with repetitive transcranial magnetic stimulation (rTMS). Participants first learned pairs of cue and target words, and then repeatedly recalled some of the targets (think condition) and suppressed others (no-think condition). We applied 10 Hz rTMS bursts to the right dlPFC during the suppression of half the no-think items, and to the contra-lateral primary motor area (M1) as an active control site during the other half. As hypothesized, participants experienced less success at keeping the memories out of awareness with concurrent dlPFC than M1 stimulation. Similarly, a memory test yielded evidence for suppression-induced forgetting (SIF) following M1 but not dlPFC stimulation. However, the difference in forgetting between the stimulation conditions was not significant. The study thus provides causal evidence for the role of the dlPFC in preventing retrieval. Future work will need to conclusively establish the relationship between this transient effect and suppression-induced forgetting.

Dorsolateral prefrontal cortex TMS evokes responses in the subgenual anterior cingulate cortex: Evidence from human intracranial EEG
Transcranial magnetic stimulation combined with intracranial local field potential recordings in humans (TMS-iEEG) represents a new method for investigating electrophysiologic effects of TMS with spatiotemporal precision. We applied TMS-iEEG to the dorsolateral prefrontal cortex (dlPFC) in two subjects and demonstrate evoked activity in the subgenual anterior cingulate cortex (sgACC). This study provides direct electrophysiologic evidence that dlPFC TMS, as targeted for depression treatment, can modulate brain activity in the sgACC.

Motor sequence learning involves better prediction of the next action andoptimization of movement trajectories
Learning new sequential movements is a fundamental skill for many animals. Although the behavioral manifestations of sequence learning are clear, the underlying mechanisms remain poorly understood. Motor sequence learning may arise from three distinct processes: (1) improved execution of individual movements independent of their sequential context; (2) enhanced anticipation of "what" movement should be executed next, enabling faster initiation; and (3) the development of motoric sequence-specific representations that encode "how" movements should be optimally performed within a sequence. However, many existing paradigms conflate the "what" and "how" components of learning, as participants often acquire both the sequence content (what to do) and its execution (how to do it). This overlap obscures the distinct contributions of each mechanism to motor sequence learning. In this study, we disentangled these mechanisms using a continuous reaching task. Performance in trained sequences was compared to random sequences to rule out improvements attributable solely to isolated movement execution. By also varying how many upcoming targets were visible we assessed the role of anticipation in learning. When participants could only see one future target, improvements were mostly due to them learning which target would come next. When they could see four future targets, participants immediately demonstrated fast movement times and increased movement smoothness, surpassing late-stage performance in the one target condition. Crucially, even with full visibility of future targets, participants showed further sequence-specific learning caused by a continuous optimization of movement trajectories. Follow-up experiments revealed that the learned sequence representations were effector-specific and encoded contextual information of four movements or longer. Our paradigm enables a clear dissociation between the "what" and "how" components of motor sequence learning and provides compelling evidence for the development of effector-specific sequence representations that guide optimal movement execution.

Neurodevelopmental deviations in schizophrenia: Evidences from multimodal connectome-based brain ages
BackgroundPathologic schizophrenia process originate early in brain development, leading to detectable brain alterations via structural and functional magnetic resonance imaging (MRI). Recent MRI studies have sought to characterize disease effects from a brain age perspective, but developmental deviations from the typical brain age trajectory in youths with schizophrenia remain unestablished.

AimThis study investigated brain development deviations in early-onset schizophrenia (EOS) patients by applying machine learning algorithms to structural and functional MRI data.

MethodsMultimodal MRI data, including T1-weighted MRI (T1w-MRI), diffusion MRI, and resting-state functional MRI (rs-fMRI) data, were collected from 80 antipsychotic-naive first-episode EOS patients and 91 typically developing (TD) controls. The morphometric similarity connectome (MSC), structural connectome (SC), and functional connectome (FC) were separately constructed by using these three modalities. According to these connectivity features, eight brain age estimation models were first trained with the TD group, the best of which was then used to predict brain ages in patients. Individual brain age gaps (BAGs) were assessed as brain ages minus chronological ages.

ResultsBoth the SC and MSC features performed well in brain age estimation, whereas the FC features did not. Compared with the TD controls, the EOS patients presented widened structural BAGs, with opposite trends between childhood and adolescence. These increased absolute BAG scores for EOS patients were positively correlated with the severity of their clinical symptoms.

ConclusionThese findings from a multimodal brain age perspective suggest that advanced BAGs exist early in youths with schizophrenia.

Universality of representation in biological and artificial neural networks
Many artificial neural networks (ANNs) trained with ecologically plausible objectives on naturalistic data align with behavior and neural representations in biological systems. Here, we show that this alignment is a consequence of convergence onto the same representations by high-performing ANNs and by brains. We developed a method to identify stimuli that systematically vary the degree of inter-model representation agreement. Across language and vision, we then showed that stimuli from high-and low-agreement sets predictably modulated model-to-brain alignment. We also examined which stimulus features distinguish high-from low-agreement sentences and images. Our results establish representation universality as a core component in the model-to-brain alignment and provide a new approach for using ANNs to uncover the structure of biological representations and computations.

Visual circuitry for distance estimation in Drosophila
Animals must infer the three-dimensional structure of their environment from two-dimensional images on their retinas. In particular, visual cues like motion parallax and binocular disparity can be used to judge distances to objects. Studies across several animal models have found neural signals that correlate with visual distance, but the causal role of these neurons in distance estimation as well as the range of possible neural properties that can inform distance estimation have remained poorly understood. Here, we developed a novel high-throughput behavioral assay to identify neurons in the Drosophila visual system that are involved in distance estimation during free locomotion. We found that silencing the primary motion detectors in the fly visual system eliminated their ability to perceive distance, consistent with a reliance on motion parallax to judge distance. Through a targeted silencing screen of visual neurons during behavior and through in vivo two-photon microscopy, we identified a visual projection neuron that encodes the parallax signal in the relative motion of foreground and background. Interestingly, it differs from previously identified parallax-tuned neurons in its lack of direction selectivity both to moving bars and to moving backgrounds. This non-canonical tuning is interpretable in the context of parallax signals that the fly would likely encounter during naturalistic walking behavior. Our results demonstrate how both direction selective and non-direction selective feature-detecting neurons can contribute to distance estimation using parallax cues, providing a framework for considering broader classes of parallax-encoding neurons in distance estimation across visual systems.