Long COVID: From olfactory dysfunctions to viral Parkinsonism.

Neurological and psychiatric complications continue to be a public health concern in long coronavirus disease 2019 (COVID-19). This varies from olfactory dysfunctions such as parosmia to cognitive and emotional challenges. Historically, the surge of neurological disorders followed the viral pandemics, for example, the emergence of Encephalitis Lethargica after the outbreak of Spanish Influenza. During and after COVID-19 infection, the problems associated with the sense of smell and the reports of affected olfactory and limbic brain areas are leading to a growing concern about the similarity with the symptoms and the pattern of degeneration observed at the onset of Parkinson's disease and Alzheimer's disease. These reports reveal the essentiality of long-term studies of olfactory and cognitive functions in the post-COVID era and the experiments using animal models to dissect the neural basis of these complications. In this manuscript, we summarize the research reporting the potential correlation between neurological disorders and viral pandemic outbreaks with a historical perspective. Further, we discuss the studies providing evidence of neurodegeneration due to severe acute respiratory syndrome coronavirus 2 infection by focusing on viral Parkinsonism.

Perceptual learning deficits mediated by somatostatin releasing inhibitory interneurons of olfactory bulb in an early life stress mouse model.

Early life adversity (ELA) causes aberrant functioning of neural circuits affecting the health of an individual. While ELA-induced behavioural disorders resulting from sensory and cognitive disabilities can be assessed clinically, the neural mechanisms need to be probed using animal models by employing multi-pronged experimental approaches. As ELA can alter sensory perception, we investigated the effect of early weaning on murine olfaction. By implementing go/no-go odour discrimination paradigm, we observed olfactory learning and memory impairments in early life stressed (ELS) male mice. As olfactory bulb (OB) circuitry plays a critical role in odour learning, we studied the plausible changes in the OB of ELS mice. Lowered c-Fos activity in the external plexiform layer and a reduction in the number of dendritic processes of somatostatin-releasing, GABAergic interneurons (SOM-INs) in the ELS mice led us to hypothesise the underlying circuit. We recorded reduced synaptic inhibitory feedback on mitral/tufted (M/T) cells, in the OB slices from ELS mice, explaining the learning deficiency caused by compromised refinement of OB output. The reduction in synaptic inhibition was nullified by the photo-activation of ChR2-expressing SOM-INs in ELS mice. The role of SOM-INs was revealed by learning-dependent refinement of Ca2+dynamics quantified by GCaMP6f signals, which was absent in ELS mice. Further, the causal role of SOM-INs involving circuitry was investigated by optogenetic modulation during the odour discrimination learning. Photo-activating these neurons rescued the ELA-induced learning deficits. Conversely, photo-inhibition caused learning deficiency in control animals, while it completely abolished the learning in ELS mice, confirming the adverse effects mediated by SOM-INs. Our results thus establish the role of specific inhibitory circuit in pre-cortical sensory area in orchestrating ELA-dependent changes.

Knockout of angiotensin converting enzyme-2 receptor leads to morphological aberrations in rodent olfactory centers and dysfunctions associated with sense of smell.

Neuronal morphological characterization and behavioral phenotyping in mouse models help dissecting neural mechanisms of brain disorders. Olfactory dysfunctions and other cognitive problems were widely reported in asymptomatic carriers and symptomatic patients infected with Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). This led us to generate the knockout mouse model for Angiotensin Converting Enzyme-2 (ACE2) receptor, one of the molecular factors mediating SARS-CoV-2 entry to the central nervous system, using CRISPR-Cas9 based genome editing tools. ACE2 receptors and Transmembrane Serine Protease-2 (TMPRSS2) are widely expressed in the supporting (sustentacular) cells of human and rodent olfactory epithelium, however, not in the olfactory sensory neurons (OSNs). Hence, acute inflammation induced changes due to viral infection in the olfactory epithelium may explain transient changes in olfactory detectabilities. As ACE2 receptors are expressed in different olfactory centers and higher brain areas, we studied the morphological changes in the olfactory epithelium (OE) and olfactory bulb (OB) of ACE2 KO mice in comparison with wild type animals. Our results showed reduced thickness of OSN layer in the OE, and a decrease in cross-sectional area of glomeruli in the OB. Aberrations in the olfactory circuits were revealed by lowered immunoreactivity toward microtubule associated protein 2 (MAP2) in the glomerular layer of ACE2 KO mice. Further, to understand if these morphological alterations lead to compromised sensory and cognitive abilities, we performed an array of behavioral assays probing their olfactory subsystems' performances. ACE2 KO mice exhibited slower learning of odor discriminations at the threshold levels and novel odor identification impairments. Further, ACE2 KO mice failed to memorize the pheromonal locations while trained on a multimodal task implying the aberrations of neural circuits involved in higher cognitive functions. Our results thus provide the morphological basis for the sensory and cognitive disabilities caused by the deletion of ACE2 receptors and offer a potential experimental approach to study the neural circuit mechanisms of cognitive impairments observed in long COVID.

Persistent olfactory learning deficits during and post-COVID-19 infection.

Quantifying olfactory impairments can facilitate early detection of Coronavirus disease 2019 (COVID-19). Despite being a debated topic, many reports provide evidence for the neurotropism of SARS-CoV-2. However, a sensitive, specific, and accurate non-invasive method for quantifying persistent neurological impairments is missing to date. To quantify olfactory detectabilities and neurocognitive impairments in symptomatic COVID-19 patients during and post-infection periods, we used a custom-built olfactory-action meter (OAM) providing accurate behavioral readouts. Ten monomolecular odors were used for quantifying olfactory detectabilities and two pairs of odors were employed for olfactory matching tests. We followed cohorts of healthy subjects, symptomatic patients, and recovered subjects for probing olfactory learning deficits, before the Coronavirus Omicron variant was reported in India. Our method identifies severe and persistent olfactory dysfunctions in symptomatic patients during COVID-19 infection. Symptomatic patients and recovered subjects showed significant olfactory learning deficits during and post-infection periods, 4-18 months, in comparison to healthy subjects. On comparing olfactory fitness, we found differential odor detectabilities and olfactory function scores in symptomatic patients and asymptomatic carriers. Our results indicate probable long-term neurocognitive deficits in COVID-19 patients imploring the necessity of long-term tracking during post-infection period. Differential olfactory fitness observed in symptomatic patients and asymptomatic carriers demand probing mechanisms of potentially distinct infection routes.

Sulfation of Heparan and Chondroitin Sulfate Ligands Enables Cell-Specific Homing of Nanoprobes.

Demystifying the sulfation code of glycosaminoglycans (GAGs) to induce precise homing of nanoparticles in tumor cells or neurons influences the development of a potential drug- or gene-delivery system. However, GAGs, particularly heparan sulfate (HS) and chondroitin sulfate (CS), are structurally highly heterogeneous, and synthesizing well-defined HS/CS composed nanoparticles is challenging. Here, we decipher how specific sulfation patterns on HS and CS regulate receptor-mediated homing of nanoprobes in primary and secondary cells. We discovered that aggressive cancer cells such as MDA-MB-231 displayed a strong uptake of GAG-nanoprobes compared to mild or moderately aggressive cancer cells. However, there was no selectivity towards the GAG sequences, thus indicating the presence of more than one form of receptor-mediated uptake. However, U87 cells, olfactory bulb, and hippocampal primary neurons showed selective or preferential uptake of CS-E-coated nanoprobes compared to other GAG-nanoprobes. Furthermore, mechanistic studies revealed that the 4,6-O-disulfated-CS nanoprobe used the CD44 and caveolin-dependent endocytosis pathway for uptake. These results could lead to new opportunities to use GAG nanoprobes in nanomedicine.

Multimodal learning of pheromone locations.

Memorizing pheromonal locations is critical for many mammalian species as it involves finding mates and avoiding competitors. In rodents, pheromonal information is perceived by the main and accessory olfactory systems. However, the role of somatosensation in context-dependent learning and memorizing of pheromone locations remains unexplored. We addressed this problem by training female mice on a multimodal task to locate pheromones by sampling volatiles emanating from male urine through the orifices of varying dimensions or shapes that are sensed by their vibrissae. In this novel pheromone location assay, female mice' preference toward male urine scent decayed over time when they were permitted to explore pheromones vs neutral stimuli, water. On training them for the associations involving olfactory and whisker systems, it was established that they were able to memorize the location of opposite sex pheromones, when tested 15 days later. This memory was not formed either when the somatosensory inputs through whisker pad were blocked or when the pheromonal cues were replaced with that of same sex. The association between olfactory and somatosensory systems was further confirmed by the enhanced expression of the activity-regulated cytoskeleton protein. Furthermore, the activation of main olfactory bulb circuitry by pheromone volatiles did not cause any modulation in learning and memorizing non-pheromonal volatiles. Our study thus provides the evidence for associations formed between different sensory modalities facilitating the long-term memory formation relevant to social and reproductive behaviors.

Local Postsynaptic Signaling on Slow Time Scales in Reciprocal Olfactory Bulb Granule Cell Spines Matches Asynchronous Release.

In the vertebrate olfactory bulb (OB), axonless granule cells (GC) mediate self- and lateral inhibitory interactions between mitral/tufted cells via reciprocal dendrodendritic synapses. Locally triggered release of GABA from the large reciprocal GC spines occurs on both fast and slow time scales, possibly enabling parallel processing during olfactory perception. Here we investigate local mechanisms for asynchronous spine output. To reveal the temporal and spatial characteristics of postsynaptic ion transients, we imaged spine and adjacent dendrite Ca2 +- and Na+-signals with minimal exogenous buffering by the respective fluorescent indicator dyes upon two-photon uncaging of DNI-glutamate in OB slices from juvenile rats. Both postsynaptic fluorescence signals decayed slowly, with average half durations in the spine head of t1 / 2_Δ[Ca2 +]i ∼500 ms and t1 / 2_Δ[Na+]i ∼1,000 ms. We also analyzed the kinetics of already existing data of postsynaptic spine Ca2 +-signals in response to glomerular stimulation in OB slices from adult mice, either WT or animals with partial GC glutamate receptor deletions (NMDAR: GluN1 subunit; AMPAR: GluA2 subunit). In a large subset of spines the fluorescence signal had a protracted rise time (average time to peak ∼400 ms, range 20 to >1,000 ms). This slow rise was independent of Ca2 + entry via NMDARs, since similarly slow signals occurred in ΔGluN1 GCs. Additional Ca2 + entry in ΔGluA2 GCs (with AMPARs rendered Ca2 +-permeable), however, resulted in larger ΔF/Fs that rose yet more slowly. Thus GC spines appear to dispose of several local mechanisms to promote asynchronous GABA release, which are reflected in the time course of mitral/tufted cell recurrent inhibition.

Quantitative assessment of olfactory dysfunction accurately detects asymptomatic COVID-19 carriers.

BACKGROUND: COVID-19 threatens the global community because a large fraction of infected people are asymptomatic, yet can effectively transmit SARS-CoV-2. Finding and isolating these silent carriers is a crucial step in confining the spread of the disease. A sudden loss of the sense of smell has been self-reported by COVID-19 patients across different countries, consistent with expression of the molecular factors mediating SARS-CoV-2 uptake into human olfactory epithelial supporting cells. However, precise quantification of olfactory loss in asymptomatic COVID-19 carriers is missing to date. METHODS: To quantify olfactory functions in asymptomatic COVID-19 patients, we designed an olfactory-action meter that determines detectability indices at different odor concentrations and an olfactory matching accuracy score using monomolecular odors. The optimization of test parameters allowed us to reliably and accurately assess olfactory deficits in a patient within 20 minutes. FINDINGS: Measurement of detection indices at low concentrations revealed a 50% reduction in asymptomatic COVID-19 carriers. Further, patients with better detection scores showed significantly reduced olfactory matching accuracies compared to normal healthy subjects. Our quantification of olfactory loss, considering all parameters, identified 82% of the asymptomatic SARS-CoV-2 carriers with olfactory deficits. However, on subjective evaluation, only 15% of the patients noticed a compromised ability to smell. INTERPRETATION: Compromised olfactory fitness can serve as a strong basis for identifying asymptomatic COVID-19 patients. Detailed design specifications and protocols provided here should enable the development of a sensitive, fast, and economical screening strategy that can be administered to large populations to prevent the rapid spread of COVID-19. FUNDING: This work was supported by the DBT - Wellcome Trust India Alliance intermediate grant (IA/I/14/1/501,306 to N.A.) and UGC NET Fellowship (A.B.). All the funding sources played no roles in the study.

Similarity and Strength of Glomerular Odor Representations Define a Neural Metric of Sniff-Invariant Discrimination Time.

The olfactory environment is first represented by glomerular activity patterns in the olfactory bulb. It remains unclear how these representations intersect with sampling behavior to account for the time required to discriminate odors. Using different chemical classes, we investigate glomerular representations and sniffing behavior during olfactory decision-making. Mice rapidly discriminate odorants and learn to increase sniffing frequency at a fixed latency after trial initiation, independent of odor identity. Relative to the increase in sniffing frequency, monomolecular odorants are discriminated within 10-40 ms, while binary mixtures require an additional 60-70 ms. Intrinsic imaging of glomerular activity in anesthetized and awake mice reveals that Euclidean distance between activity patterns and the time needed for discriminations are anti-correlated. Therefore, the similarity of glomerular patterns and their activation strengths, rather than sampling behavior, define the extent of neuronal processing required for odor discrimination, establishing a neural metric to predict olfactory discrimination time.

Neuronal pattern separation in the olfactory bulb improves odor discrimination learning.

Neuronal pattern separation is thought to enable the brain to disambiguate sensory stimuli with overlapping features, thereby extracting valuable information. In the olfactory system, it remains unknown whether pattern separation acts as a driving force for sensory discrimination and the learning thereof. We found that overlapping odor-evoked input patterns to the mouse olfactory bulb (OB) were dynamically reformatted in the network on the timescale of a single breath, giving rise to separated patterns of activity in an ensemble of output neurons, mitral/tufted (M/T) cells. Notably, the extent of pattern separation in M/T assemblies predicted behavioral discrimination performance during the learning phase. Furthermore, exciting or inhibiting GABAergic OB interneurons, using optogenetics or pharmacogenetics, altered pattern separation and thereby odor discrimination learning in a bidirectional way. In conclusion, we propose that the OB network can act as a pattern separator facilitating olfactory stimulus distinction, a process that is sculpted by synaptic inhibition.