bioRxiv Subject Collection: Neuroscience's Journal

Mechanism, and treatment of anti-CV2/CRMP5 autoimmune pain
Paraneoplastic neurological syndromes arise from autoimmune reactions against nervous system antigens due to a maladaptive immune response to a peripheral cancer. Patients with small cell lung carcinoma or malignant thymoma can develop an autoimmune response against the CV2/collapsin response mediator protein 5 (CRMP5) antigen. For reasons that are not understood, approximately 80% of patients experience painful neuropathies. Here, we investigated the mechanisms underlying anti-CV2/CRMP5 autoantibodies (CV2/CRMP5-Abs)-related pain. We found that patient-derived CV2/CRMP5-Abs can bind to their target in rodent dorsal root ganglia (DRG) and superficial laminae of the spinal cord. CV2/CRMP5-Abs induced DRG neuron hyperexcitability and mechanical hypersensitivity in rats that were abolished by preventing binding to their cognate autoantigen CRMP5. The effect of CV2/CRMP5-Abs on sensory neuron hyperexcitability and mechanical hypersensitivity observed in patients was recapitulated in rats using genetic immunization providing an approach to rapidly identify possible therapeutic choices for treating autoantibody-induced pain including the repurposing of a monoclonal anti-CD20 antibody that selectively deplete B-lymphocytes. These data reveal a previously unknown neuronal mechanism of neuropathic pain in patients with paraneoplastic neurological syndromes resulting directly from CV2/CRMP5-Abs-induced nociceptor excitability. CV2/CRMP5-Abs directly sensitize pain responses by increasing sensory neuron excitability and strategies aiming at either blocking or reducing CV2/CRMP5-Abs can treat pain as a comorbidity in patients with paraneoplastic neurological syndromes.

Differential effects of PDE4A5 on cAMP-dependent forms of long-term potentiation
cAMP signaling is critical for memory consolidation and certain of forms long-term potentiation (LTP). Phosphodiesterases (PDEs), enzymes that degrade the second messenger cAMP and cGMP, are highly conserved during evolution and represent a unique set of drug targets, given the involvement of these enzymes in several pathophysiological states including brain disorders. The PDE4 family of cAMP selective PDEs, exert regulatory roles in memory and synaptic plasticity, but the specific roles of distinct PDE4 isoforms in these processes are poorly understood. Building on our previous work demonstrating that spatial and contextual memory deficits were caused by expressing selectively the long isoform of the PDE4A subfamily, PDE4A5, in hippocampal excitatory neurons, we now investigate the effects of PDE4A isoforms on different cAMP-dependent forms of LTP. We find that PDE4A5 impairs long-lasting LTP induced by theta burst stimulation (TBS) while sparing long-lasting LTP induced by spaced 4-train stimulation (4X100Hz). This effect requires the unique N-terminus of PDE4A5 and is specific to this long isoform. Targeted overexpression of PDE4A5 in area CA1 is sufficient to impair TBS-LTP, suggesting that cAMP levels in the postsynaptic neuron are critical for TBS-LTP. Our results shed light on the inherent differences among the PDE4A subfamily isoforms, emphasizing the importance of the long isoforms, which have a unique N-terminal region. Advancing our understanding of the function of specific PDE isoforms will pave the way for developing isoform-selective approaches to treat the cognitive deficits that are debilitating aspects of psychiatric, neurodevelopmental, and neurodegenerative disorders.

State- and circuit-dependent opponent-processing of fear
The presence of valence coding neurons in the basolateral amygdala (BLA) that form distinct projections to other brain regions implies functional opposition between aversion and reward during learning. However, evidence for opponent interactions in fear learning is sparse and may only be apparent under certain conditions. Here we test this possibility by studying the roles of the BLA-central amygdala (CeA) and BLA-nucleus accumbens (Acb) pathways in fear learning in male rats. First, we assessed the organisation of these pathways in the rat brain. BLA-CeA and BLA-Acb pathways were largely segregated in the BLA but shared overlapping molecular profiles. Then we assessed activity of the BLA-CeA and BLA-Acb pathways during two different forms of fear learning - fear learning in a neutral context and fear learning in a reward context. BLA-CeA neurons were robustly recruited by footshock regardless of where fear learning occurred whereas recruitment of BLA-Acb neurons was state-dependent because footshock only recruited this pathway in a reward context. Finally, we assessed the causal roles of activity in these pathways in fear learning. Photoinhibition of the BLA-CeA pathway during the footshock US impaired fear learning, regardless of where fear learning occurred. In contrast, photoinhibition of the BLA-Acb pathway augmented fear learning, but only in the reward context. Taken together, our findings show circuit- and state-dependent opponent processing of fear. Footshock-driven activity in the BLA-Acb pathway can functionally oppose the BLA-CeA pathway to limit how much fear is learned.

Scheduled feeding improves sleep in a mouse model of Huntington's disease.
Sleep disturbances are common features of neurodegenerative disorders including Huntington's disease (HD). The sleep and circadian disruptions are recapitulated in animal models, and these models provide the opportunity to evaluate whether circadian interventions can be effective countermeasures for neurodegenerative disease. Time restricted feeding (TRF) interventions successfully improve activity rhythms, sleep behavior and motor performance in mouse models of HD. Seeking to determine if these benefits of scheduled feeding extend to physiological measures of sleep, electroencephalography (EEG) was used to measure sleep/wake states and polysomnographic patterns in adult mice (six month-old) under TRF and ad lib feeding (ALF). With each diet, both male and female wild-type (WT) and bacterial artificial chromosome transgenic (BACHD) mice were evaluated. Our findings show that male, but not female, BACHD mice exhibited significant changes in the temporal patterning of wake and non-rapid eye movement (NREM) sleep. The TRF intervention reduced the inappropriate early morning activity by increasing NREM sleep in the male BACHD mice. In addition, the scheduled feeding reduced sleep fragmentation (# bouts) in the male BACHD mice. The phase of the rhythm in rapid-eye movement (REM) sleep was significantly altered by the scheduled feeding. The treatment did impact the power spectral curves during the day in male but not female mice. Sleep homeostasis, as measured by the response to six hours of gentle handling, was not altered by the diet. Thus, TRF improves the temporal patterning and fragmentation of NREM sleep without impacting sleep homeostasis. This work adds critical support to the view that sleep is a modifiable risk factor in neurodegenerative diseases.

An ultra-short-acting benzodiazepine in thalamic nucleus reuniens undermines fear extinction via intermediation of hippocamposeptal circuits
Benzodiazepines, commonly used for anxiolytics, hinder conditioned fear extinction, and the underlying circuit mechanisms are unclear. Utilizing remimazolam, an ultra-short-acting benzodiazepine, we reveal its impact on the thalamic nucleus reuniens (RE) and interconnected hippocamposeptal circuits during fear extinction. Systemic or RE-specific administration of remimazolam impedes fear extinction by reducing RE activation through A type GABA receptors. Remimazolam enhances long-range GABAergic inhibition from lateral septum (LS) to RE, underlying the compromised fear extinction. RE projects to ventral hippocampus (vHPC), which in turn sends projections characterized by feed-forward inhibition to the GABAergic neurons of the LS. This is coupled with long-range GABAergic projections from the LS to RE, collectively constituting an overall positive feedback circuit construct that promotes fear extinction. RE-specific remimazolam negates the facilitation of fear extinction by disrupting this circuit. Thus, remimazolam in RE disrupts fear extinction caused by hippocamposeptal intermediation, offering mechanistic insights for the dilemma of combining anxiolytics with extinction-based exposure therapy.

Type-I interferons drive the gastrointestinal inflammatory response in a mouse model of Parkinsons disease
Background and Aims Parkinsons disease (PD) is an age-related neurodegenerative disorder characterised by classical motor symptoms due to a loss of dopaminergic neurons in the substantia nigra pars compacta. The type-I interferons (IFNs) are elevated in the aging brain and we have implicated them in the neuroinflammatory response in PD. With increasing evidence of gastrointestinal (GI) dysfunction in PD patients, this study explored the contribution of the type-I IFNs to the transmission of pathology from the brain to the gut in PD. Methods Young (10-12 weeks) and aged (40-50 weeks) wildtype and IFNAR1-/- mice received an intrastriatal injection of human alpha-synuclein (-Syn) pre-formed fibrils (PFF) (8ug) with gut tissue analysed 6-months post-injection (p.i). A mouse intestinal organoid culture model was established to further characterise the -Syn induced inflammatory response in the gut. Results An intrastriatal injection of human -Syn PFFs was shown to initiate a type-I IFN-dependent neuroinflammatory response in the GI tract of wildtype mice at 6-months p.i. This response was attributed to an elevation in type-I IFN signalling in aged mice that was absent in the IFNAR1-/- mice. Mouse intestinal organoid cultures confirmed -Syn was taken up by the enteroendocrine cells (EECs) to induce a type-I IFN mediated pro-inflammatory response that was attenuated in IFNAR1-/- cultures. Conclusion This study has confirmed the type-I IFNs modulate the -Syn PFF induced inflammatory response within the gut potentiating pathology progression along the gut-brain axis. Early intervention of this type-I IFN response may be a potential therapeutic target to limit the progression of PD. Keywords Parkinsons disease, type-I interferons, alpha-synuclein, gut-brain axis

Velocity-selective arterial spin labelling bolus duration measurements: Implications for consensus recommendations
Velocity-selective arterial spin labelling (VSASL) MRI is insensitive to arterial transit time. This is an advantage over other perfusion measurements, where long arterial transit times can lead to bias. Therefore, VSASL can be used to study perfusion in the presence of long arterial transit times, such as in the ageing brain, in vascular pathologies, and cancer, or where arterial transit time changes, such as during measurement of cerebrovascular reactivity (CVR). However, when calculating perfusion (cerebral blood flow, CBF, in the brain) from VSASL signal, it is assumed that images are acquired before the trailing edge of the labelled blood has arrived in the imaging slice. The arrival of the trailing edge of the labelled bolus of blood will cause an underestimation of perfusion. Here we measure bolus duration in young, healthy human brains, both at rest and during elevated CBF. Grey matter bolus duration was 1.61 +/- 0.31 s, but there was a large spatial heterogeneity, with bolus duration being lower in anterior brain regions, with some areas having bolus duration < 1.2 s. We place these results in context of recommendations from a recent consensus paper, which recommends imaging 1.4 s after the label, potentially underestimating CBF in anterior regions. Further, we observed a 0.23 +/- 0.12 s reduction in grey matter bolus duration with 5% CO2 inhalation. These results can be used to inform the experimental design of future VSASL studies, to avoid underestimating perfusion by imaging after the arrival of the trailing edge of the labelled bolus.

Cdc25A phosphatase is activated and mediates neuronal cell death by PUMA via pRB/E2F1 pathway in a model of Parkinson's disease
Parkinson's disease (PD) is a predominant movement disorder caused mainly due to selective loss of the dopaminergic neurons in the substantia nigra pars compacta of the mid brain. There is currently no cure for PD barring treatments to manage symptoms. The reasons might be due to lack of precise understanding of molecular mechanisms leading to neurodegeneration. Aberrant cell cycle activation has been implicated in neuronal death pathways of various neurodegenerative diseases including PD. This study investigates the role of cell cycle regulator Cell division cycle 25A (Cdc25A) in a PD-relevant neuron death model induced by 6-OHDA treatment. We find Cdc25A is rapidly elevated, activated and is playing a key role in neuron death by regulating Rb phosphorylation and E2F1 activity. Knockdown of Cdc25A via shRNA downregulates the levels of pro-apoptotic PUMA, an E2F1 target and cleaved Caspase-3 levels, suggesting Cdc25A may regulate neuronal apoptosis through these effectors. Our work sheds light on the intricate signaling networks involved in neurodegeneration and highlights Cdc25A as a potential therapeutic target for mitigating aberrant cell cycle re-entry underlying PD pathogenesis. These novel insights into molecular mechanisms provide a foundation for future development of neuroprotective strategies to slow or prevent progression of this debilitating disease.

Ih Block Reveals Separation of Timescales in Pyloric Rhythm Response to Temperature Changes in Cancer borealis
Motor systems operate over a range of frequencies and relative timing (phase). We studied the contribution of the hyperpolarization-activated inward current (Ih) to frequency and phase in the pyloric rhythm of the stomatogastric ganglion (STG) of the crab, Cancer borealis as temperature was altered from 11 degrees C to 21 degrees C. Under control conditions, the frequency of the rhythm increased monotonically with temperature, while the phases of the pyloric dilator (PD), lateral pyloric (LP), and pyloric (PY) neurons remained constant. When we blocked Ih> with cesium (Cs+) PD offset, LP onset, and LP offset were all phase advanced in Cs+ at 11 degrees C, and the latter two further advanced as temperature increased. In Cs+ the steady state increase in pyloric frequency with temperature diminished and the Q10 of the pyloric frequency dropped from ~1.75 to ~1.35. Unexpectedly in Cs+, the frequency displayed non-monotonic dynamics during temperature transitions; the frequency initially dropped as temperature increased, then rose once temperature stabilized, creating a characteristic jag. Interestingly, these jags were still present during temperature transitions in Cs+ when the pacemaker was isolated by picrotoxin, although the temperature-induced change in frequency recovered to control levels. Overall, these data suggest that Ih plays an important role in the ability of this circuit to produces smooth transitory responses and persistent frequency increases by different mechanisms during temperature fluctuations.

Assessing the subarachnoid space anatomy on clinical imaging: utilizing normal and pathology to understand compartmentalization of the subarachnoid space
BACKGROUND: The goal of the study is to use CT imaging in patients with aSAH to evaluate the anatomic distribution of hemorrhage and compartmentalization of subarachnoid space to investigate potential in vivo visualization of recently discovered layer named subarachnoid lymphatic like membrane (SLYM). METHODS: We conducted a retrospective cohort study of cases with aneurysmal SAH (aSAH) at our institution between January 2015 and June 2022. Subarachnoid hemorrhage distribution into superficial and deep subarachnoid spaces was classified based on proximity to the dural or pial surfaces, respectively, as seen on multiplanar CT head. RESULTS: A total of 97 patients with aSAH were included. Patients with lower modified Fisher score (MFS) of 1-2 were more likely to have SAH compartmentalizing in the (deep) pial-adjacent subarachnoid space. Patients with higher MFS of 3-4 were more likely to have SAH in both (superficial) and (deep) compartments along the brainstem. There is a significant association between the severity of aSAH - quantified by the MFS - and the distribution of the blood. Patients with higher MFS scores were roughly 7.6 times (p-value = 0.049) more likely to have hemorrhage at the (Superficial) juxta-dural subarachnoid compartment when compared to those with lower MFS scores. CONCLUSION: This study suggests an imaging correlate to the recently discovered SLYM, potentially influencing aSAH compartmentalization, particularly in low-grade bleeds. While compartmentalization is limited in high-grade cases, these findings warrant further investigation with advanced imaging techniques to validate this membrane's role and potential impact on CSF flow and aSAH pathophysiology.

Afferent projections to the Calca/CGRP-expressing parabrachial neurons in mice
The parabrachial nucleus (PB), located in the dorsolateral pons, contains primarily glutamatergic neurons which regulate responses to a variety of interoceptive and cutaneous sensory signals. The lateral PB subpopulation expressing the Calca gene which produces the neuropeptide calcitonin gene-related peptide (CGRP) relays signals related to threatening stimuli such as hypercarbia, pain, and nausea, yet the afferents to these neurons are only partially understood. We mapped the afferent projections to the lateral part of the PB in mice using conventional cholera toxin B subunit (CTb) retrograde tracing, and then used conditional rabies virus retrograde tracing to map monosynaptic inputs specifically targeting the PBCalca/CGRP neurons. Using vesicular GABA (vGAT) and glutamate (vGLUT2) transporter reporter mice, we found that lateral PB neurons receive GABAergic afferents from regions such as the lateral part of the central nucleus of the amygdala, lateral dorsal subnucleus of the bed nucleus of the stria terminalis, substantia innominata, and the ventrolateral periaqueductal gray. Additionally, they receive glutamatergic afferents from the infralimbic and insular cortex, paraventricular nucleus, parasubthalamic nucleus, trigeminal complex, medullary reticular nucleus, and nucleus of the solitary tract. Using anterograde tracing and confocal microscopy, we then identified close axonal appositions between these afferents and PBCalca/CGRP neurons. Finally, we used channelrhodopsin-assisted circuit mapping to test whether some of these inputs directly synapse upon the PBCalca/CGRP neurons. These findings provide a comprehensive neuroanatomical framework for understanding the afferent projections regulating the PBCalca/CGRP neurons.

In utero adeno-associated virus (AAV)-mediated gene delivery targeting sensory and supporting cells in the embryonic mouse inner ear
In vivo gene delivery to tissues using adeno-associated vector (AAVs) has revolutionized the field of gene therapy. Yet, while sensorineural hearing loss is one of the most common sensory disorders worldwide, gene therapy applied to the human inner ear is still in its infancy. Recent advances in the development recombinant AAVs have significantly improved their cell tropism and transduction efficiency across diverse inner ear cell types to a level that renders this tool valuable for conditionally manipulating gene expression in the context of developmental biology studies of the mouse inner ear. Here, we describe a protocol for in utero micro-injection of AAVs into the embryonic inner ear, using the AAV-PHP.eB and AAV-DJ serotypes that respectively target the sensory hair cells and the supporting cells of the auditory sensory epithelium. We also aimed to standardize procedures for imaging acquisition and image analysis to foster research reproducibility and allow accurate comparisons between studies. We find that AAV-PHP.eB and AAV-DJ provide efficient and reliable tools for conditional gene expression targeting cochlear sensory and supporting cells in the mouse inner ear, from late embryonic stages on.

Concurrent maintenance of visual imagery and short-term memory provides evidence for their distinct representations
Recent research indicates there is overlap in the neural resources used during imagery and visual short-term memory. But do visual short-term memory and visual imagery operate on similar representations during recall? Here we investigated this question by asking participants to perform a delayed match to sample task for the contrast of visual gratings as cues. In the Imagery condition, participants were asked to form an accurate mental image of the visual cue, and at the end of the trial, perform a matching task on the mental image contrast. In the Memory condition, participants were not required to perform visual imagery but merely instructed to perform the delayed contrast-matching task. In Experiment 1, participants were told at the beginning each block whether to engage in memory or imagery. The results showed that for the relevant contrast feature, matching judgments were more accurately in the Memory than in the Imagery condition. Thus imagery did not maintain an accurate representation of the encoded image, even when the visual features could still be maintained in visual short-term memory. In Experiment 2, participants were required to engage in memory and imagery simultaneously, and were told which to base their judgment on after each trial. The key finding was that the superior accuracy for memory over imagery remained, indicating that the contents of VSTM and imagery are based on distinct representations.

HisCl1 regulates gustatory habituation in sweet taste neurons and mediates sugar ingestion in Drosophila
Similar to other animals, the fly, Drosophila melanogaster, reduces its responsiveness to tastants with repeated exposure, a phenomenon called gustatory habituation. Previous studies have focused on the circuit basis of gustatory habituation in the fly chemosensory system 1,2. However, gustatory neurons reduce their firing rate during repeated stimulation 3, suggesting that cell-autonomous mechanisms also contribute to habituation. Here, we used deep learning-based pose estimation and optogenetic stimulation to demonstrate that continuous activation of sweet taste neurons causes gustatory habituation in flies. We conducted a transgenic RNAi screen to identify genes involved in this process and found that knocking down Histamine-gated chloride channel subunit 1 (HisCl1) in the sweet taste neurons significantly reduced gustatory habituation. Anatomical analysis showed that HisCl1 is expressed in the sweet taste neurons of various chemosensory organs. Using single sensilla electrophysiology, we showed that sweet taste neurons reduced their firing rate with prolonged exposure to sucrose. Knocking down HisCl1 in sweet taste neurons suppressed gustatory habituation by reducing the spike frequency adaptation observed in these neurons during high-concentration sucrose stimulation. Finally, we showed that flies lacking HisCl1 in sweet taste neurons increased their consumption of high-concentration sucrose solution at their first meal bout compared to control flies. Together, our results demonstrate that HisCl1 tunes spike frequency adaptation in sweet taste neurons and contributes to gustatory habituation and food intake regulation in flies. Since HisCl1 is highly conserved across many dipteran and hymenopteran species, our findings open a new direction in studying insect gustatory habituation.

The dorsal thalamic lateral geniculate nucleus is required for visual control of head direction cell firing direction in rats
Head direction (HD) neurons, signalling facing direction, generate a signal that is primarily anchored to the outside world by visual inputs. We investigated the route for visual landmark information into the HD system in rats. There are two candidates: an evolutionarily older, larger subcortical (retino-tectal) pathway specialised for coarse vision, and a smaller cortical (retino-geniculo-striate) pathway for higher acuity vision. We disrupted the cortical pathway by lesioning the dorsal lateral geniculate (dLGN) thalamic nuclei bilaterally, and recorded HD cells in postsubicular (PoS) cortex as rats foraged in a visual-cue-controlled enclosure. In dLGN-lesioned rats we found the expected number of PoS HD cells. Although directional tuning curves were broader across a trial, this was due to increased instability of otherwise normal-width tuning curves. Tuning curves were also poorly responsive to polarizing visual landmarks, and did not distinguish cues based on their visual pattern. Thus, the retino-geniculo-striate pathway is not critical for generation of an underlying, tightly-tuned directional signal, but does provide the main route for vision-based anchoring of the signal to the outside world, even when visual cues are high-contrast and low in detail.

NEK1 modulates neurite outgrowth in motor neurons through coordinating retromer formation
Loss of function of a cell cycle-associated gene NEK1 causes amyotrophic lateral sclerosis (ALS), but how this leads to motor neuron degeneration is unknown. We studied the function of NEK1 in human stem cell-derived motor neurons and found that loss of NEK1 causes decreased neurite length accompanied by transcriptional alterations. We also found that NEK1 interacts with and modulates the formation of the retromer, and that impaired retromer function contributes to neurite outgrowth deficits. We identified SMC3, which interacts with NEK1 during the cell cycle, as a kinase substrate of NEK1 in motor neurons. Notably, loss of SMC3 not only recapitulates the decreased neurite outgrowth, but also affects retromer formation. We suggest that NEK1 interacts with multiple proteins in postmitotic neurons to coordinate retromer formation, and that loss of this leads to impaired neurite outgrowth.

Evidence of optimal control theory over active inference in corticospinal excitability modulations
Two theories, optimal control theory and active inference, dominate the motor control field. We use transcranial magnetic stimulation (TMS) in force and angle tasks to examine whether corticospinal excitability represents a motor command, as proposed by the optimal control theory, or a proprioceptive prediction, as proposed by active inference. Our results strongly support optimal control theory. We encourage comparisons of the theories against each other based on empirically testable predictions.