Kristen R. Maynard

The habenula is a critical regulator of affect, negative reinforcement, avoidance, and coping behavior. It is thus unsurprising that accumulating evidence supports pivotal roles for habenular circuits in the expression of major psychiatric illnesses including depression and addiction. Though high levels of mu-opioid receptors (MORs) were identified in the habenula more than a generation ago, the functional effects of MORs on habenular circuits remained largely unexplored due to challenges of selectively targeting this small and heterogeneous brain structure. In this SfN mini-symposium report, we highlight studies that resolve how MOR is distributed across medial and lateral habenular subnuclei and how MOR expression maps onto molecularly defined habenular cell types. We further describe some of the habenula-intersecting circuits that express MORs and how MOR signaling modulates neural activity and neurotransmission within these unique habenular circuits and cell types. Going beyond cellular-level effects of MOR signaling, we illustrate how neural activity in the habenula can influence affect and behaviors associated with opioid analgesia, opioid seeking, opioid dependence, and opioid withdrawal. Drawing on works outside the opioid field, we suggest commonalities through which drugs of abuse may influence habenula circuits to induce states of negative affect that contribute to continued drug use or relapse. Finally, we discuss how better understanding the molecular and functional properties of habenular cell types and circuits can identify new druggable targets, such as the orphan receptors GPR151 and GPR139, for the treatment of pain, mood disorders, or opioid and other substance use disorders.

Major depressive disorder (MDD) and bipolar disorder (BD) exhibit both overlapping and distinct clinical features, including sex differences in prevalence and symptomatology, but how these similarities and differences are reflected at the molecular and cellular level in the human brain remains poorly understood. Here, we applied spatial transcriptomics to the human dorsolateral prefrontal cortex from individuals with MDD, BD, and neurotypical controls to resolve molecular programs associated with diagnosis and biological sex. Our analysis revealed sex- and diagnosis-specific differences across white matter, the neurovascular unit (NVU), and neuronal compartments. Within white matter, we identified female-specific survival-response programs shared in both MDD and BD, and divergent myelin-related gene expression patterns across disorders ( CLDN11 , CNDP1 ). Within the NVU, transcriptional evidence of impaired vascular integrity was accompanied by astrocytic response profiles in females with MDD and males with BD, with inflammatory responses further evident in females with MDD ( IFITM3 , CEBPD , IL1R1 ). In donors with BD of both sexes and in females with MDD, inhibitory neurons exhibited impaired neuropeptide expression ( SST , CORT , CRH ). Integration with donor genotype information highlighted that genetic risk for mood disorders influences MAPK3 expression, implicating altered ERK signaling, which was further supported by disorder-associated changes in Ras/MEK/ERK pathway components in neurons ( MAPK3, DUSP6 , RASD1 , ELK1 ). Together, these findings reveal coordinated, cell type- and spatial domain-specific molecular programs that distinguish shared and disorder-specific transcriptomic changes across mood disorders in a sex-dependent manner.

The habenula (Hb) is a small epithalamic midline structure critical for reward processing and mood regulation that is implicated in many complex brain disorders, including depression and addiction. The molecular anatomy of the Hb has been well-characterized in model organisms, but little is known about its organization in the human brain, including the transcriptomic and epigenetic signatures of spatially-organized lateral (LHb) and medial (MHb) cell types. Here, we generated a high resolution spatiomolecular map of the human Hb using both multiomic single nucleus sequencing (snMultiome) and spatially-resolved transcriptomics. We measured gene expression and chromatin accessibility across 4 MHb and 5 LHb neuronal cell types as defined by transcriptomic conservation with rodents and zebrafish. We identified cell type-specific molecular specializations related to GABAergic and glutamatergic signaling as well as regulatory relationships between open chromatin regions, transcription factors, and target genes associated with depression and addiction. High resolution spatial transcriptomics identified the anatomical location of heterogeneous LHb subpopulations, including two putative inhibitory populations in the LHb. We further characterized cell-type relationships between Hb neurons and glial subtypes and predicted ligand-receptor interactions between LHb and MHb neurons. Finally, we delineated Hb cell types and spatial domains enriched in genes associated with genetic risk for psychiatric disorders, identifying the strongest enrichment in the MHb. Collectively, the generated data and analyses resolve the spatiomolecular organization of the human Hb and identify molecular-genetic associations with neuropsychiatric disease.

Spatial transcriptomics has transformed molecular characterization of the human brain, but most studies profile individual two-dimensional tissue sections and therefore cannot capture molecular organization across 3D neuroanatomical axes. Here, we developed a spatial genomics framework to reconstruct the 3D cellular architecture of the human nucleus accumbens (NAc) by densely sampling and aligning serial sections across its anterior-posterior (AP) extent. Combining single-cell Xenium profiling with near-transcriptome-wide VisiumHD, we mapped cell type composition and topography across AP, mediolateral and dorsoventral axes. Neuronal populations varied in relative abundance across the AP axis while maintaining characteristic spatial topographies. These differences were particularly pronounced among D1-islands, a topographically organized structure within the NAc in which transcriptionally distinct subpopulations of DRD1 -expressing medium spiny neurons (MSNs) aggregate into discrete cellular formations. D1-islands comprised two molecularly distinct spatial domains with conserved counterparts across species. Both domains varied in relative abundance across the AP axis, but demonstrated distinct 3D distribution; one was present throughout the NAc, whereas the other, corresponding to islands of Calleja (ICj), was more restricted to intermediate-to-posterior levels. Local spatial analyses revealed region- and AP-dependent cellular relationships, including segregation of the two island populations despite their anatomical proximity. Together, these findings establish a framework for three dimensional spatial genomics of human brain tissue and reveal molecular and cellular organization not captured by individual tissue sections.

The amygdala is a central hub for emotional learning that is dysregulated across numerous psychiatric disorders, yet its molecular architecture in humans remains poorly defined. We here present a spatial transcriptomic atlas of the human amygdala from nine neurotypical donors. Integrating Visium, Xenium, and VisiumHD technologies, we profiled over 1.1 million cells across 13 spatial domains spanning the basolateral complex, central, medial, and cortical nuclei, as well as the intercalated islands, each defined by distinct marker genes and gene co-expression networks. By integrating snRNA-seq reference atlases, we found that each subdivision of the basolateral subnucleus contains distinct excitatory neuron classes. We additionally found three transcriptionally distinct populations of intercalated neuron types, two of which form spatially distinct islands neighboring the basolateral complex, and were able to refine cell type diversity across the central nucleus and related amygdalostriatal transition areas. Finally, we localized psychiatric genetic risk across the amygdala, revealing both broad neuronal enrichment and subnuclear specificity. Together, this atlas provides a foundational resource and framework for accelerating cross-species comparisons and human disease-focused investigations.

Fentanyl is a potent synthetic opioid associated with overdose. However, little is known about fentanyl‐induced molecular adaptations in the habenula and amygdala, two brain regions implicated in opioid use and withdrawal. We performed bulk RNA‐sequencing in the rat habenula and amygdala to identify transcriptomic changes associated with fentanyl intake. Male rats self‐administered intravenous saline or fentanyl over 22–24 days. Ninety minutes following the final session, brains were collected for transcriptomic profiling. In Hb, we identified 453 differentially expressed genes (DEGs) between saline and fentanyl rats, with upregulated genes associated with synaptic transmission and ionic conductance. In the amygdala, we identified 3041 fentanyl‐associated DEGs with upregulated genes implicated in metabolic and vesicular functions. Downregulated genes in both regions were enriched for extracellular matrix functions. Integration of DEGs with single‐cell RNA‐sequencing data from rodents and humans revealed that fentanyl DEGs were enriched in specific habenula and amygdala cell type markers. Furthermore, fentanyl downregulated DEGs in the amygdala were enriched in genes associated with the risk for substance use disorders. Together, we define how fentanyl intake alters transcriptional programs in the rat habenula and amygdala, and we link these changes to specific human cell types and risk genes for neuropsychiatric disorders and addiction.

Divergent transcription from bidirectional promoters is frequently observed in eukaryotic genomes, but the biological relevance of divergent RNA transcripts (DT) is unknown. We identified and characterized BDNF-DT, a novel DT gene, and BDNF-AS-DT, a novel readthrough gene, in the locus containing BDNF, a gene with key roles in neuronal development, differentiation, and synaptic plasticity. BDNF-DT is independent from the known BDNF antisense (BDNF-AS), and its expression is developmentally regulated and positively correlated with BDNF in human postmortem dorsolateral prefrontal cortex (DLPFC). BDNF-DT and BDNF-AS-DT expression increase after induced depolarization, but the temporal dynamics follow expression of BDNF, suggesting a regulatory role. Moreover, CRISPR-mediated upregulation of BDNF in human neural progenitor cells drives BDNF-DT expression. Finally, BDNF-DT shows higher expression in DLPFC from patients diagnosed with schizophrenia compared to neurotypical controls, and genetically predicted lower expression of the BDNF-AS-DT readthrough transcript is associated with schizophrenia and with the schizophrenia-associated C allele of the rs6265 single-nucleotide polymorphism. These findings identify BDNF-DT and BDNF-AS-DT as novel, low-abundance genes that show coordinated expression with BDNF and association with schizophrenia risk, though their biological significance requires further validation given detection limitations and the need to establish causal roles.