Faculty Bibliography

Digital spatial profiling (DSP) facilitates RNA sequencing (RNA-seq) of small populations of cells, specifically ~20 pyramidal neurons in postmortem formalin-fixed paraffin-embedded (FFPE) human brain tissue. This approach enables precise expression profiling via RNA-seq data linked to spatially characterized samples utilizing tissue-bound probes. We present a comprehensive protocol for manual slide preparation using the GeoMx DSP system as preliminary steps to spatial characterization of lamina-specific pyramidal neurons in human postmortem Brodmann area 9 (BA9) frontal cortex brain tissue.

Oral cancer patients suffer pain that is patient-specific and heterogeneous at the site of the tumor. Protease-activated receptor 2 (PAR₂), a G-protein coupled receptor and an oral cancer mediator, is activated by protease cleavage of an N-terminal ligand. Once cleaved, the receptor is retained at the plasma membrane or re-distributed (trafficking) to internal sites followed by downstream protease-specific signaling. The oral cancer tumor microenvironment (TME) is replete with protases that activate PAR₂, potentially evoking PAR₂ trafficking and signaling that is unique to each cancer and reflecting the protease composition of the individual TMEs. To measure PAR₂ cleavage activity in oral cancer samples, an array of overlapping peptides spanning the PAR₂ N-terminus was assembled. In this feasibility study, we assayed the cleavage activity of individual PAR₂-activating proteases and mixtures of proteases modeling the oral cancer TME. Additionally, we confirmed that PAR₂-cleaving proteases are active in oral cancer patient saliva, a sample that can be collected non-invasively to measure PAR₂ cleavage in patients when they are experiencing pain prior to surgery. Further studies should elucidate the utility of the peptide array to measure PAR₂-activating proteolysis in cancer samples.

Astrocyte contributions to CNS antigen presentation remain poorly defined. We show that interferon gamma (IFN-γ, the sole type II interferon), with proinflammatory cytokines, induces astrocytes to express components mediating attraction of and antigen presentation to CD4⁺ T cells. Re-analysis of astrocyte-enriched single-cell sequencing from bacterial mimetic-injected mice revealed a transcriptionally distinct, IFN-responsive astrocyte population enriched for antigen-presentation genes 24 h after peripheral immune challenge. In vivo, type II major histocompatibility complex (MHC-II⁺)GFAP⁺ astrocytes localize to the brain surface across multiple mouse models of neuroinflammation and in human neurological disease tissue. In vitro, purified astrocytes stimulated with IFN-γ and proinflammatory cytokines expressed MHC-II, costimulatory molecules, and lymphocyte-recruiting chemokines, and in co-culture supported rapid antigen-dependent expansion of previously activated CD4⁺ T cells. Human iPSC-derived CNS cultures showed equivalent induction of antigen-presentation machinery in human astrocytes following inflammatory cytokine stimulation. These findings define a cytokine-induced astrocyte state expressing antigen-presentation machinery at neuroimmune interfaces that can support local CD4⁺ T cell responses.

Functional abnormalities of the globus pallidus interna (GPi) are linked to psychosis severity and treatment response in schizophrenia. Transcranial ultrasound stimulation (TUS) is a noninvasive method for precisely targeting deep brain structures. The effects of GPi-targeted TUS on psychosis had not been examined. We conducted a random-order, sham-controlled, crossover pilot study assessing safety, tolerability, and preliminary efficacy of right GPi TUS for psychotic symptoms. Ten outpatients (mean age 37.5; 50% female) with schizophrenia or schizoaffective disorder and persistent psychosis received one TUS and one sham session, one week apart. TUS was MRI-guided, targeting right GPi with a 350 kHz transducer (100 Hz pulse repetition frequency, 5% duty cycle, in situ I _spta 400 mW/cm²). Outcomes-delusion experience sampling assessment (DESA) and auditory hallucinations rating scale (AHRS)-were measured before, during, and immediately, 1 h, 1, and 7 days after stimulation. DESA scores decreased significantly more after TUS than sham (p = 0.002, Cohen's d = 0.68); AHRS and cognition changes were not significant. Mild transient adverse effects occurred in both conditions; none were serious. GPi-targeted TUS was safe, well-tolerated, and associated with a preliminary reduction in delusional severity, warranting further study in larger, randomized trials. ClinicalTrials.gov: NCT05643196, registered 21 November 2022.

Conscious object recognition is a critical brain function, key to making sense of the plethora of visual inputs and guiding adaptive behavior. Yet, the sequence of neural processing underlying conscious object recognition remains unclear. Existing proposals alternatively suggest that conscious object recognition proceeds in a "coarse-to-fine" sequence or a "fine-to-coarse" sequence. Here, we recorded high-density EEG while participants of both sexes performed a threshold-level object recognition task under different viewing conditions. Both superordinate and basic-level category information emerged early in neural activity, followed by a late superordinate category representation in an abstract format that is disentangled from low-level object features. Strikingly, in the early period, while basic-level category information positively correlated with perceptual awareness, superordinate category information negatively predicted perceptual awareness. By contrast, both superordinate and basic-level category information positively correlated with objective categorization accuracy in the early period. In the late period, both levels of category information positively correlated with awareness. These results suggest that neural processing underlying conscious object perception proceeds from the basic to the superordinate category level.Significance statement Despite significant interests, the sequence of neural processing underlying conscious perception remains unclear. Here, we investigated the unfolding of neural events in time that support perceptual awareness of visual objects. Using a threshold-level object perception task involving three image conditions (normal, low-pass filtered, masked) and high-density EEG, we reveal that basic and superordinate category information both emerge early in the neural activity (at ∼100 ms), but only the basic-level category representation positively contributes to awareness. By contrast, the early superordinate category representation negatively correlates with perceptual awareness. At ∼300 ms, an abstract superordinate category representation emerges, which also positively correlates with perceptual awareness. Therefore, category-level neural processing underlying conscious object perception proceeds from the basic to the superordinate level.

The locus coeruleus (LC) brainstem cell group is evolutionarily conserved across vertebrates and influences both mind (cognition) and body (cardiorespiratory, metabolic, and immune functions) via two-putatively distinct-spontaneous activity patterns. Yet, the degree to which spontaneous LC firing rates and patterns are homogeneous across species has never been assessed due to inherently limited sample sizes. To remedy this, we pooled cross-species data from 20 laboratories to show that firing rates differ across species and are modulated by sex, age, and type of in vitro or in vivo preparation. Contrary to the prevailing dual-mode firing pattern schema, we observed patterns spread across a low-dimensional manifold, with subregions enriched for specific biological factors and neurodegenerative disease models. Our findings show considerable diversity in an ancestral vertebrate neuromodulatory system.

The extent to which conserved neural circuit architectures depend on shared molecular specification programs remains unclear. Here, we address this question by examining the somatosensory system of the little skate, Leucoraja erinacea, an early-diverging vertebrate that retains ancestral features of both finned and limb-based body plans. We show that core features of somatosensory circuit organization, including laminar organization of the spinal cord and dorsally restricted targeting of sensory afferents, are deeply conserved. Unexpectedly, the molecular programs specifying dorsal root ganglion (DRG) sensory subtypes diverge extensively from those of mammals. Although DRG neuron subtype specification and spinal connectivity rely on target-derived cues, skates employ distinct neurotrophin receptor and transcription factor identity codes. These findings support a model in which conserved spinal circuit architectures provide a stable scaffold that leverages flexible sensory neuron specification programs, enabling the evolutionary diversification of vertebrate somatosensory systems.

Polycomb Repressive Complexes PRC1 and PRC2 establish and maintain chromatin architectures critical for cell-type specific gene expression. In the developing spinal cord, PRC1 is essential for implementing Hox-dependent transcriptional programs that diversify motor neuron identities. Although PRC1 can assemble into subnuclear condensates, whether these structures contribute to the specification and maintenance of neuronal subtype fates remains unclear. Using chick and mouse models, we show that canonical PRC1 components - BMI1, PHC2, CBX2, RING1B - coalesce into subnuclear assemblies as neural progenitors differentiate into motor neurons. Disrupting PHC2 polymerization in either neural progenitors or postmitotic neurons dismantles BMI1-containing PRC1 assemblies, triggering derepression of caudal Hox genes and a cell autonomous loss of segment-specific motor neuron fates. Mouse mutants lacking the core PRC1 subunit Ring1 fail to form BMI1-containing assemblies, despite intact PHC2 and CBX2 polymers. In contrast, motor neuron-restricted deletion of core PRC2 subunits (Ezh1/2) depletes H3K27me3 histone marks but retains PRC1 assemblies and Hox boundaries. Together, these findings indicate that PHC2-dependent PRC1 assemblies stabilize Hox transcriptional boundaries and motor neuron segmental identities.

Can the transcriptomic profile of a neuron predict its physiological properties? Using a Patch-seq dataset of the primary visual cortex, we addressed this question by focusing on spike rate adaptation (SRA), a well-known phenomenon that depends on small conductance calcium (Ca)-dependent potassium (SK) channels. We first show that in parvalbumin-expressing (PV) and somatostatin-expressing (SST) interneurons, expression levels of genes encoding the ion channels underlying action potential generation are correlated with the half-width (HW) of spikes. Surprisingly, the SK encoding genes are not correlated with the degree of SRA (dAdap). Instead, genes that encode proteins upstream from the SK current, as well as the low-voltage-activated calcium channel, are correlated with dAdap. This general principle is supported by datasets from the mouse primary motor cortex and multiple macaque brain regions. Finally, we generate testable predictions by constructing a minimal computational model.

Cochlear implants (CIs) stimulate auditory neurons with electric pulse trains whose amplitudes are modulated using envelopes extracted from different frequency bands, conveying essential speech cues. This study investigated how the rate of stimulation pulses affects envelope processing and speech perception in ten post-lingually deaf CI users. Amplitude modulation (AM) detection and consonant identification were examined using acoustic stimuli processed by multi-channel and single-channel strategies, with pulse rates ranging from 125 to 2000 pulses per second (pps) for multi-channel stimulation and from 125 to 4000pps for single-channel stimulation. The single-channel conditions encoded the overall broad-band speech envelope by a single electrode and were used to isolate within-electrode temporal modulation coding. For AM detection, the modulation frequency was 25 Hz ensuring sufficient modulation sampling across all carrier rates tested. Even at 125pps, the 25-Hz modulation was represented by five pulses per modulation cycle. Results revealed a significant decline in both AM detection and consonant identification at 125pps, suggesting that poorer speech recognition at low rates may be partly attributable to limitations in temporal modulation processing rather than solely to a restricted range of representable envelope frequencies. Consonant recognition performance remained stable across clinically relevant pulse rates (250-2000pps), though significant listener- and feature-specific effects were observed. Notably, while AM detection and consonant recognition were not significantly correlated within this small cohort, significant correlations were found between single-channel and multi-channel performance in both tasks. These findings support an important role for within-electrode temporal coding in CI speech perception and highlight the clinical relevance of individual variability in pulse rate effects.