Sex-differences in the ventral subiculum’s microcircuitry and function

Abstract

The ventral subiculum has long been appreciated as the major output structure of the ventral hippocampus but remains greatly understudied relative to the other hippocampal subregions. Over the last decade, investigation into ventral subiculum synapses and connectivity have revealed a critical role for this brain region in context-induced drug seeking, stress integration, and psychiatric disorders. While the majority of literature has historically focused on male rodent models, recent works including sex as a biological variable uncover that the ventral subiculum may have complex cell-type-, synapse-, and sex-specific roles in its functions. In this review, we discuss the primary functions of the ventral subiculum, emphasize works that uncover sex-differences, and highlight avenues for future research.

Part I: Introduction

The hippocampal formation is a heavily investigated structure that participates in diverse functions due to its functional heterogeneity across the dorsal-ventral (D-V) axis and its five functionally distinct subdivisions: dentate gyrus, CA3, CA2, CA1, and subiculum. The subiculum serves as the major output structure of the hippocampus but is understudied relative to other hippocampal subregions. Additionally, examination of the hippocampus often groups the subiculum and CA1 together, which can obscure our understanding of the subiculum’s unique contributions to different functions and disease states. The subiculum contains two distinct subclasses of pyramidal cells that are distinguished based on their action potential firing pattern. Subicular pyramidal cells exhibit either regular or burst action potential firing patterns (O’Mara, 2005), and these cell subtypes are differentially implicated in disease states (Lee, Lee, Woo, Kang, & Shin, 2019b). To add further complexity to this hippocampal subregion, the subiculum has documented sex-differences in clinical and preclinical models (Emma E. Boxer et al., 2021; Teicher et al., 2012) and has the densest expression of estrogen receptors (ER) in the hippocampus (Blurton-Jones & Tuszynski, 2002).

The subiculum’s role in neural processes and overall size notably changes across the D-V axis (Fig. 1). While the dorsal subiculum (dSUB) is relatively smaller and involved in spatial memory (Yanakieva, Frost, Amin, Nelson, & Aggleton, 2024) and object recognition(Chang & Huerta, 2012), the ventral subiculum (vSUB) is larger and implicated in stress, drug seeking, and emotional responses (O’Mara, 2005). Thus, vSUB is a sexually dimorphic brain region and regulates behaviors that model psychiatric diseases with well-known sex-differences in incidence and symptomology. Despite this, sex-differences in vSUB have been underexamined due to studies exclusively using male rodents, not directly examining sex-differences, or not studying the hippocampus with subregion specificity. The focus of this review is to discuss the major functions of the vSUB, highlight works that uncovered sex-differences in the function of vSUB, underscore importance of differentiating vSUB from other hippocampal subregions, and highlight areas of future research.

Figure 1.

The function and size of the subiculum changes over the dorsal-ventral axis.

Part II: Microcircuitry

Excitatory pyramidal neurons

The principal cell types within the vSUB are excitatory pyramidal cells that possess either a regular or burst action potential firing pattern in the subiculum (Graves et al., 2012). Within the subiculum, regular- and burst-spiking cells exist in roughly equal proportions regardless of sex (Graves et al., 2012; Staff, Jung, Thiagarajan, Yao, & Spruston, 2000). The expression of burst spiking is caused by Cav3.1 T-type calcium channels and is thought to enhance the fidelity of neurotransmission to downstream targets (Joksimovic et al., 2017). These pyramidal subtypes are understood to have distinct organization, connectivity, and function within the vSUB. Thus, the purpose of having two unique principal cells in the vSUB may allow for greater versatility or more complex signaling relative to other subregions in the ventral hippocampus (vHIPP).

Organizationally, regular-spiking cells in the subiculum are generally more abundant close to CA1 (proximal subiculum) whereas burst-spiking cells are generally found close to the presubiculum (distal subiculum) (Böhm et al., 2015; Cembrowski, Phillips, et al., 2018; Staff et al., 2000). Consistent with this gradient of regular- to burst-spiking predominance, projection patterns and gene expression vary markedly across the proximal-distal axis. For example, proximal vSUB preferentially innervates prefrontal cortex (PFC), medial-proximal vSUB innervates nucleus accumbens (NAc), and distal vSUB preferentially innervates the lateral hypothalamus (LH) (Kim & Spruston, 2012; Wee & MacAskill, 2020). The organizational segregation of gene expression and downstream projection targets likely underlie the distinct connectivity patterns and involvement in disease states of regular- versus burst-spiking cells. For instance, our lab and others found that regular-spiking cells predominantly (~70%) project to the nucleus accumbens medial shell (NAcMS), a projection critical to substance use disorders (SUDs) and schizophrenia behaviors (Emma E. Boxer, Kim, Dunn, & Aoto, 2023; Kim & Spruston, 2012; Lee, Lee, Woo, Kang, & Shin, 2019a). Importantly, biases observed for the projections of regular- and burst-spiking cells to the NAcMS from vSUB do not appear to be impacted by biological sex (Emma E. Boxer et al., 2023). Thus, when examining vSUB functions, it is essential to consider the functional heterogeneity of vSUB along the proximal-distal axis in terms of projections, gene expression, and action potential firing profiles.

Despite the functional and physiological differences between regular- and burst-spiking cells, efforts to identify molecular markers to reliably distinguish them are ongoing. Transcriptomic approaches to delineate regular- versus burst-spiking cells along their proximal-distal axis are limited by the graded changes in gene expression across this axis. The graded changes result in a transition zone with a mix of regular- and burst-spiking cells possessing seemingly undistinguishable gene expression profiles (Cembrowski, Phillips, et al., 2018). Despite this, vesicular glutamate transporter 2 (vGluT2) has gained attention as a marker for burst-spiking cells due to its preferential expression in the distal subiculum (Cembrowski, Wang, et al., 2018). vGluT2 was examined as a molecular marker for burst-spiking cells using a combination of whole cell patch clamp electrophysiology and vGluT2-Cre transgenic mice (Wozny et al., 2018). The expression of channelrhodopsin-2 (ChR2) in vGluT2-expressing subicular neurons reliably elicited action potentials in 36/39 burst-spiking cells; however, in regular-spiking cells, photoactivation of ChR2 was sufficient to depolarize but insufficient to drive action potential firing. Thus, while vGluT2 is highly expressed in burst-spiking cells, regular-spiking cells may also express vGluT2, albeit at lower levels. A limitation to this study and others seeking genetic markers for regular versus burst-spiking cells is the inherent low throughput of whole cell patch clamp electrophysiology, which is currently required to distinguish them. Until there is a greater molecular handle on differentiating regular- versus burst-spiking cells, we lack the ability to examine these cells with methods outside of electrophysiology which greatly limits our understanding of how these subtypes may contribute to physiology in vivo.

In addition to segregating pyramidal cells by their firing patterns, subiculum pyramidal cells can be further divided into 4 distinct subclasses based on scRNA-seq data. dSUB expresses Col5a2, Tpbg, and Ly6g6e genes in a laminar fashion from superficial to deep, respectively. By contrast, vSUB expresses Cbln4, Dlk1, Tpbg, and Ly6g6e genes spanning the superficial to deep layers, respectively (Cembrowski, Wang, et al., 2018). Thus, Tbpg and Ly6g6e represent the middle-deeper layers throughout the subiculum, but Clbn4 and Dlk1 are specific to the superficial layers of vSUB. Consistent with a non-specific role for Tbpg across the subiculum, Tpbg encodes trophoblast glycoprotein which is specifically critical for overall hippocampal structural organization (Park et al., 2021). Ly6g6e encodes lymphocyte antigen 6 family member G6E which may represent a marker of excitatory “non-pyramidal ovoid neurons” that control non-spatial learning in dSUB (Kinman et al., 2025). Learning and memory are more strongly associated with dSUB than vSUB and perhaps Ly6g6e expression serves different functions in vSUB, but this is untested. Although Clbn4 and Dlk1 expression are unique to vSUB, their functional significance in vSUB has not been directly tested. Clbn4 encodes cerebellin 4 precursor protein which primarily interacts with neurexins to influence synaptic plasticity in the entorhinal cortex-DG pathway of the hippocampus (Liakath-Ali et al., 2022), but, given the pleiotropy of neurexins, it is unknown if Clbn4 shares a similar functionality in vSUB. DLK1 encodes delta like non-canonical Notch ligand 1 which is implicated in adult neurogenesis in DG, anxiety-like behavior, and spatial navigation (Montalbán-Loro et al., 2021). While neurogenesis is exclusive to DG and spatial navigation is associated with dSUB, anxiety-like behavior is regulated by vSUB activity and DLK1 may represent a population of cells involved in this function. Consistent with Cembrowski et al., another group found that the cellular expression of Clbn4 is restricted to the ventral portion of the subiculum, however, the cells that express Clbn4 are largely restricted to the stratum radiatum layer, which suggests that these cells are primarily inhibitory (Seigneur & Südhof, 2017). Taken together, defining vSUB cell groups by mRNA and laminar organization may be informative for inferring functionality and represent distinct cell-types such as ovoid neurons (Ly6g6e) or inhibitory interneurons (Clbn4).

Along with revealing vSUB neuronal subpopulations and lamina, mRNA identity can also co-vary with downstream projection targets. For example, Gpc3 positive neurons mostly project to the amygdala whereas Dlk1 positive neurons largely project to the NAc and PFC (Cembrowski, Wang, et al., 2018). Based on the previous studies that identified the cell-type-specific output to these brain regions (Emma E. Boxer et al., 2023; Kim & Spruston, 2012; Lee et al., 2019a), we can infer that Gpc3 and Dlk1 might identify different subclasses of regular-spiking cells in vSUB, however, this remains untested. Future consideration of the projection specific scRNA-seq data together with the projection specific vSUB spiking data may help better inform the classification of vSUB neuron subtypes. Further, while highly informative, the transcriptomic profiles were determined for cells that projected to three downstream targets (PFC, NAc, and amygdala), thus, while covariance was observed, it is unclear whether Gpc3 and Dlk1 can specifically identify subicular neurons with a unique output target or whether these markers are shared more broadly with the multiple known output targets of vSUB (Tang et al., 2016). Taken together, principal excitatory pyramidal cells may exhibit heterogeneity based on location along the proximal-distal axis, action potential firing pattern, and gene expression. Although basal sex-differences are not noted when examining vSUB cell types by firing pattern, it would be advantageous to examine whether there are sex-differences in gene expression by including females in future studies. Finally, gaining a firmer molecular handle on these subtypes would allow greater investigation of their functional relevance.

Inhibitory interneurons

Unlike the hippocampus proper, our understanding of the subtypes of GABAergic interneurons is limited in the subiculum (Böhm et al., 2018). Within the subiculum, the activity of pyramidal cells is modulated by local inhibition from several interneuron classes including: parvalbumin (PV) expressing, somatostatin (SST) expressing, and cholecystokinin (CCK) expressing. All subicular interneurons express the inhibitory marker GAD67 and, interestingly, males have marginally more GAD67 immunoreactive subicular neurons than females (Urien et al., 2022). This finding suggests that there may be basal sex-differences in the abundance of inhibitory neurons in vSUB. However, differences in expression levels of GAD67 in female and male interneurons, and not the abundance of inhibitory neurons, may account for the marginal differences in GAD67 immunoreactivity in subiculum. It will be important to further validate this interesting finding. Of these interneuron classes identified in subiculum, subicular PV interneurons are the most examined, provide the greatest inhibitory control, and striking sexual dimorphism has been reported in local PV connectivity and PV abundance throughout neurodevelopment.

PV interneurons express P/Q -type calcium channels to allow for their characteristic fast-spiking action potential profile and they exert formidable perisomatic inhibition over pyramidal cells (Freund & Katona, 2007; Pelkey et al., 2017; Zaitsev, Povysheva, Lewis, & Krimer, 2007). Dysfunction of PV interneurons in vHIPP is a feature in several psychiatric diseases such as schizophrenia and stress-disorders (Konradi et al., 2011; Nguyen et al., 2014; Tiwari et al., 2024). In both sexes, subicular PV interneurons have the densest estrogen receptor (ER) expression in the entire rodent hippocampus, propounding that estrogen may have profound influences on inhibitory tone in the subiculum (Blurton-Jones & Tuszynski, 2002). Both ER-alpha and –beta receptor subtypes are expressed on PV cells and, interestingly, neither their expression nor overall PV cell abundance are altered by adolescent ovariectomy or castration in vHIPP (Blurton-Jones & Tuszynski, 2002; Wu, Du, Buuse, & Hill, 2014). However, there may be sex-differences in the adolescent developmental time-course of PV cells, with evidence that females increase PV cell numbers in vHIPP from 3–12 weeks of age whereas male PV cell numbers remain constant (Wu et al., 2014). Interestingly, this developmental sex difference in PV cell numbers was again unaffected by adolescent ovariectomy or castration. Unlike PV expression, GAD67 expression remained constant throughout development in both sexes, suggesting that other interneuron subtypes may compensate for the changes in PV abundance across development in females. PV dysfunction in vHIPP is a notable feature in schizophrenia (Konradi et al., 2011), a disease strongly linked to adverse experiences during adolescence (Gomes & Grace, 2017). Schizophrenia has notable sex-differences in disease course, which raises the intriguing possibility that sex-differences in PV expression across development may underlie the sex-differences in schizophrenia, but more work is necessary to determine this. Taken together, the differences observed in PV cell abundance in vSUB across neurodevelopment are not likely due to circulating gonadal hormones and may reflect an organizational effect of sex hormones that precedes puberty and influence sex-specific vulnerabilities to disease. Future works should characterize postnatal development of the other interneuron subtypes and determine the physiological significance of the female specific trajectory for PV cell abundance during postnatal development.

In vSUB, our lab reported sex-differences in transcriptomes of vSUB PV interneurons. Compared to females, males have 6 upregulated protein coding genes (Eif2s3y, Ddx3y, Dkk3, Vip, Penk, Plp1) (Emma E. Boxer et al., 2021). Eif2s3y is located on the Y chromosome and overexpression increases synaptic transmission and causes autism-like behaviors in male mice (Zhang et al., 2021). Ddx3y and Dkk3 are important for neural cell differentiation in males (Fukusumi et al., 2015; Vakilian et al., 2015). The remaining genes encode for proteins that contribute to proteolipids (Plp1) and that function in neuromodulation (Vip, Penk). Compared to males, females have 1 differentially upregulated gene (Gstm1), which contributes to neuroprotection against oxidative stress (Gravina et al., 2011). These differentially expressed genes in PV interneurons based on sex represent rich areas for future exploration. In males, enhanced Eif2s3y expression in PV interneurons should be further examined as a genetic contributor to sex-differences in autism spectrum disorder which predominantly effects males and has sex-specific symptoms (Napolitano et al., 2022). Differential expression of Ddx3y and Dkk3 may represent a fruitful avenue to pursue as the source of sexual dimorphic development of vSUB PV interneurons. In females, it is intriguing that vSUB PV interneurons have elevated Gtsm1, a gene encoding glutathione S-transferase, because reduced activity of glutathione S-transferases and increased oxidative stress-driven PV impairments are risk factors for schizophrenia, a disease that disproportionately impacts males (Giordano, Bucci, Mucci, Pezzella, & Galderisi, 2021). Future studies should examine whether upregulated Gstm1 is a neuroprotective factor in females against schizophrenia.

Sex-differences in the basal connectivity of PV interneurons with regular- and burst-spiking principal neurons have been described in vSUB by our lab (Fig. 2; Boxer et al., 2021). Within the vSUB of female mice, PV interneurons have greater inhibitory control over burst-spiking cells compared to regular-spiking cells, which is driven by enhanced inhibitory postsynaptic strength in burst-spiking cells. By contrast, in male mice, PV interneurons exert more inhibition onto regular-spiking cells relative to burst-spiking cells, which is facilitated through increased PV-regular-spiking synapse numbers. However, another study using male rats did not report a bias of vSUB PV interneuron-mediated inhibition onto regular-spiking cells (Böhm et al., 2015). Our lab identified that neurexin-3, an evolutionarily conserved presynaptic adhesion molecule which is highly expressed in hippocampal interneurons (Fuccillo et al., 2015; Ullrich, Ushkaryov, & Sudhof, 1995), is critical for inhibitory synapse function and contributes to sex-differences in vSUB PV inhibition (Fig. 2). Independent of sex, the genetic ablation of neurexin-3 from PV interneurons selectively impacted PV-regular-spiking synapses, however, the resulting phenotypes were sexually dimorphic. In females, neurexin-3 knockout enhanced presynaptic release at PV–regular-spiking synapses, indicating that the primary role of neurexin-3 at these synapses is to suppress presynaptic release, thereby contributing to the PV inhibitory bias onto burst-spiking cells observed in females. By contrast, in males, the deletion of neurexin-3 revealed that neurexin-3 functions to positively influence the postsynaptic strength and number PV-regular-spiking synapses, suggesting that neurexin-3 maintains stronger inhibition at these synapses. Curiously, these sex-differences are not explained by differences in neurexin-3 isoform expression nor alternative splicing in vSUB PV cells. Further, the sex specific role of neurexin-3 at synapses only partially explains the basal sex-differences of PV inhibition in the vSUB, begging for further examination into other proteins or factors that may be responsible. One possibility could be the differential localization or usage of postsynaptic neurexin-3 ligands, such as neuroligin-2 and neuroligin-3, at synapses formed by PV interneurons (E. E. Boxer & Aoto, 2022). Another avenue for further investigation is the phosphorylation status of gephyrin which controls sex-differences in PV density and electrophysiological properties in dorsal CA1. Of note, neuroligin-2 is critical for the accumulation of gephyrin in inhibitory synapses and can indirectly affect gephyrin phosphorylation states (Poulopoulos et al., 2009). Our lab did not suspect circulating sex hormones as drivers of these sex-differences because we observed similar variability in data between sexes, however we did not directly test this. Taken together, there is profound basal sexual dimorphism in PV postnatal neurodevelopment, local connectivity, and function in the vSUB. The literature suggests these changes are due to the organizational effects of sex steroid hormones rather than circulating gonadal hormones, but more work is necessary to determine this.

Figure 2.

PV neurexin-3 contributes to the basal sex-differences in vSUB microcircuitry. Adapted with permission from Boxer et al. (2021). Red principal cells are regular-spiking and blue principal cells are burst-spiking.

The remaining subclasses of vSUB interneurons are grossly understudied. Histological studies indicate the presence of CCK and SST interneurons in the subiculum. However, to the best of our knowledge, there are no studies characterizing their connectivity or physiology in either sex. A brief overview of their function and directions for future research will be covered. In CA1 of the hippocampus, the CCK subclass directly exerts powerful perisomatic inhibition over pyramidal cells and exhibits regular-spiking activity (Pawelzik, Hughes, & Thomson, 2002). While vastly understudied in vSUB, CCK dysfunction in other parts of the brain is linked to dementia, Alzheimer’s disease, and schizophrenia (Asim, Wang, & Chen, 2024). In other brain regions, CCK has basal sex-differences in abundance, augmented release of CCK in the presence of estradiol-17beta (Micevych 1988), and sex-specific C1BR signaling effects (Ferraro, Wig, Boscarino, & Reich, 2020), suggesting an important sex-specific role for this interneuron class throughout the brain although never directly tested in the vSUB. CCK neurons have historically been challenging to study due to a lack of specific markers, as pyramidal cells also express CCK mRNA (Taniguchi et al., 2011). This challenge was recently overcome by using the Dlx 5/6 enhancer to induce expression of Cre-dependent viruses in CCK-Cre mice (Liu, Dimidschstein, Fishell, & Carter, 2020) and should facilitate more research into the role of these interneurons in vSUB.

Unlike PV and CCK neurons, SST interneurons appear to exert somatic and dendritic inhibitory regulation over vSUB pyramidal cells (Aylward & Totterdell, 1993). SST cells possess a regular-spiking pattern and have high levels of spontaneous activity (Urban-Ciecko, Barth, Urban-Ciecko, & Barth, 2016), although SST cells have never been functionally characterized within vSUB to our knowledge. In vHipp, CCK dysfunction is implicated in fear and anxiety which are behaviors that have sex-differences and the vSUB is involved in regulating (Rehfeld, 2021). Past work reveals basal sex-differences in the transcription profiles in PFC SST interneurons (Girgenti et al., 2019), although sex-differences have not been examined in vSUB SST cells. SST driver lines are commercially available through the Jackson Laboratory and will be useful to further characterize this understudied vSUB interneuron subclass. In summary, PV cells are the most robustly examined interneurons in vSUB and exhibit remarkable sex-differences in neurodevelopment, gene expression, and roles in disease. Future works should continue examining the involvement of vSUB PV interneurons in psychiatric diseases with special attention for potential sex-specific roles in pathophysiology. The remaining vSUB interneuron cell types are incredibly understudied, and future research should examine their role in vSUB functions while considering biological sex as a variable.

Part III: vSUB Functions

Substance use disorders: vSUB-NAc

The vSUB has an instrumental role in SUDs by integrating contextual information relevant to drug seeking (Sesack & Grace, 2009) and controlling the activity of dopamine neurons (Todd & Grace, 1999). One key feature of substances of abuse is their ability to produce highly reinforcing effects through increased dopamine release in the brain (Pierce & Kumaresan, 2006), which is easily monitored behaviorally through measuring drug-induced locomotor activation (Beninger, 1983). vSUB modulates the activity of the locomotor response to psychostimulants by increasing dopamine in NAc (Burns, Robbins, & Everitt, 1993), partly via a glutamatergic relay that causes hyperactivity of VTA dopamine neurons (Glangetas et al., 2015). For example, high frequency stimulation of vSUB increases the locomotor activating effects of acute cocaine administration, without impacting basal locomotion (Glangetas et al., 2015). Complementary inactivation work shows that excitotoxic lesions of vSUB decrease the locomotor-stimulating effects of systemic amphetamine (Caine, Humby, Robbins, & Everitt, 2001). Together, vSUB activity has a positive relationship with locomotion psychostimulatory effects and dopamine release in NAc in response to drugs of abuse (Table 1).

Table 1.

Summary of drug-related behavioral and synaptic phenotypes following manipulations of vSUB or vSUB-NAcS.

Brain region	Manipulation	Sex	Model	Direction	Phenotype	References
vSUB	High frequency stimulation	Not specified	Sprague Dawley rats	Increase	Locomotor response to cocaine	Glangetas 2015
vSUB	Excitotoxic lesion	Male	Lister Hooded rats	Decrease	Locomotor response to amphetamine	Caine 2001
vSUB	Muscimol-baclofen inactivation	Male & Female	Sprague Dawley Rats	Decrease	Context-induced reinstatement of oxycodone seeking	Fredriksson 2023
vSUB	Muscimol-baclofen inactivation	Male	Long-Evans rats, Sprague Dawley rats	Decrease	Context-induced reinstatement of heroin seeking	Bossert 2014, Bossert 2016
vSUB	Muscimol-baclofen inactivation	Male	Alcohol Preferring Rats	Decrease	Context-induced reinstatement of alcohol seeking	Marchant 2016
vSUB	Lidocaine inactivation	Male	Sprague Dawley Rats	Decrease	Context-induced reinstatement of cocaine seeking	Sun 2003
vSUB-NAcS	Chemogenetic inactivation	Male	Alcohol Preferring Rats	Decrease	Context-induced reinstatement of alcohol seeking	Marchant 2016
vSUB-NAcMS	Chronic cocaine exposure	Male & Female	C57 mice	Increase	Synaptic plasticity at D2R MSN synapses	Boxer 2023

Another hallmark of SUD is the propensity for individuals to relapse drug use when re-exposed to an environment previously associated with drug use and this is modeled preclinically with context-induced reinstatement of drug seeking. Pharmacological inactivation of vSUB reduced context-induced reinstatement of alcohol, heroin, cocaine, and oxycodone seeking (Bossert & Stern, 2014; Fredriksson et al., 2023; Marchant et al., 2016; Sun & Rebec, 2003). The expression of designer receptor exclusively activated by designer drugs (DREADDs) in nucleus accumbens shell (NAcS)-projecting vSUB neurons revealed that the vSUB to NAcS glutamatergic projection specifically drives this effect on context-induced drug reinstatement (Marchant et al., 2016). While in humans, females report greater drug craving and rates of relapse during abstinence when compared to males (Becker & Koob, 2016), however, the aforementioned studies only used male rodents or included females without examining sex as a biological variable (Table 1). Using both sexes in future models will be informative for whether this pathway contributes to sex-differences observed in drug relapse.

Given the crucial role for the vSUB-NAcS projection for context-induced drug reinstatement, this pathway has been heavily scrutinized. Early work characterizing the subiculum-NAc projection in males uncovered that the majority of NAc afferents originate in the proximal subiculum and are regular-spiking pyramidal cells (Kim & Spruston, 2012; Lee et al., 2019a). Recent work uncovered that the ventral portion of the subiculum provides the densest glutamatergic input to the NAcMS (Jin, Maddern, Campbell, & Lawrence, 2022), so the vSUB-NAcMS connectivity has recently gained attention and was examined with granularity. Excitatory pyramidal vSUB neurons express two types of strictly presynaptic vesicular glutamate transporters, vGluT1 and/or vGluT2, that may correspond to spiking and/or projection patterns. Recent work in male rats characterized the vSUB-NAcMS projection by vesicular glutamate transporter expression and uncovered that vSUB-NAcMS afferents express vGluT1 (68%), vGluT2 (8%), or both vGluT1and vGluT2 (22%) (Jin et al., 2022). Although these data indicate that vSUB to NAcMS afferents might utilize different vesicular glutamate transporters, the functional consequences of the differential expression of vGluT1 and vGluT2 related to reward, depression, and SUD in male mice remains unexplored. vGluT1 is typically expressed at synapses with low release probability and a higher potential for plasticity (Weston, Nehring, Wojcik, & Rosenmund, 2011). By contrast, vGluT2 is found at synapses with higher release probability and higher fidelity of transmission (Fremeau et al., 2001; Johnson et al., 2004; Li, Santos, Park, Dobry, & Voglmaier, 2017). Thus, vGluT1-positive vSUB synapses may be poised to undergo synaptic plasticity in response to rewarding stimuli while vGluT2-positive synapses may maintain a constant basal excitatory tone. Consistent with previous reports (Kim & Spruston, 2012; Lee et al., 2019a), work from our laboratory recently characterized the vSUB-NAcMS circuit by the spiking identity of vSUB pyramidal projecting cell in both sexes of mice, which is normally equal proportions of regular-spiking and burst-spiking cells. We found that ~70% of NAcMS-projecting vSUB cells are regular-spiking and ~30% are burst-spiking, regardless of sex (Emma E. Boxer et al., 2023), which closely aligns with the proportion of vSUB cells positive for vGluT1 and vGluT2 in this projection pathway. Taken together, while there is no evidence for sexual dimorphism in the connectivity of the vSUB-NAcMS circuit, most (~70%) of vSUB-NAcMS afferents are regular-spiking and may utilize vGluT1. Given that regular-spiking cells disproportionately project to NAcMS, it will be critical to assess the contribution of the synaptic input from regular-spiking cells in reward-seeking behavior. Furthermore, a functional dissection of vGluT1, vGluT2, and vGluT1/2 boutons might provide needed granularity into how basal synaptic strength and activity-dependent plasticity are established at vSUB-NAcMS synapses.

Within the NAcMS, vSUB afferents are thought to primarily make excitatory synapses onto medium spiny neurons (MSNs) that either express dopamine receptor type 1 or 2 (D1R or D2R). D1R MSNs are understood to mediate reward/reinforcement while D2R MSNs mediate aversion (Soares-Cunha, Coimbra, Sousa, & Rodrigues, 2016). Classically, vHIPP-NAcMS undergoes cocaine-induced plasticity selectively at vHIPP-D1R synapses, which represents enhanced reinforcement by cocaine. However recent work from our lab reveals that vSUB-NAcMS is strikingly distinct from vHIPP-NAcMS in terms of basal connectivity and adaptations to cocaine exposure. In drug naive animals, functional examination of the vSUB-NAcMS projection reveals that vSUB has a basal bias of excitatory synaptic strength at D2R MSN synapses relative to D1R MSN synapses that is due to an increased number of functional synapses in both sexes (Fig. 3) (Emma E. Boxer et al., 2023). This is distinct from vHIPP-NAcMS, which includes projections from both vCA1 and vSUB, which biases D1R MSNs and suggests that while vSUB exhibits biased innervation of D2R MSNS, vCA1 preferentially targets D1R MSNs (Emma E. Boxer et al., 2023). Another unique feature of the vSUB-NAcMS projection is that the postsynaptic D1R and D2R MSNs basally express calcium-permeable AMPARs (CP-AMPARs) (Emma E. Boxer et al., 2023), which are not detected in the vHIPP-NAcMS pathway and are typically only present after chronic drug exposure (Conrad et al., 2008). Similar to projection specificity differences between vCA1 and vSUB, the lack of detectable CP-AMPARs in the vHIPP-NAcMS circuit suggests that vCA1-NAcMS synapses contain calcium-impermeable AMPARs. While the functional significance of this is undetermined, it is possible that CP-AMPAR are basally expressed to promote plasticity of this vSUB-NAcMS. Intriguingly, upon chronic cocaine exposure, vSUB-D2R MSNs are specifically potentiated (Emma E. Boxer et al., 2023), which again contrasts vHIPP-NAcMS findings and further highlights the utility of studying vSUB distinctly from other vHIPP subregions (Fig. 3). These striking differences in basal functional connectivity and plasticity highlight the functional distinction between vSUB and vCA1 and emphasize the need to study vHIPP with subregion specificity.

Figure 3.

The NAcMS-projecting vSUB cells preferentially innervate D2R MSNs and are 70% regular-spiking. vSUB-NAcMS D2R MSN synapses are potentiated after cocaine exposure.

The activation of D2R MSNs is commonly associated with encoding aversion, and the biased innervation and cocaine-induced potentiation of D2R MSNs by vSUB is seemingly at odds with the behavioral studies detailed above. If activating vSUB or the vSUB-NAcMS pathway specifically increases context-induced drug reinstatement and the activating effects of drugs, how is this driven by D2R MSN signaling? There is evidence for a more nuanced role for D2R MSNs in aversion and reward. Consistent with vSUB-NAcMS activation behavioral studies, systemic D2R agonism alone can strongly reinstate cocaine-seeking behavior (De Vries et al., 1999; Dias et al., 2004; Koob et al., 1996; Self, Barnhart, Lehman, & Nestler, 1996). Recent work showed NAcMS D2R MSNs project to excitatory ventral pallidum neurons to promote reward, which is unique from other NAc regions (Yao et al., 2021) and provides a potential mechanism by which D2R activation from vSUB-NAcMS promotes context-induced drug relapse. In sum, the vSUB sends projections to NAcMS that are distinct from other vHIPP projections to promote context-induced drug relapse in both sexes and future works should further examine the role of D2Rs and CP-AMPAR in this process.

In addition to innervating MSNs, vHIPP projections also innervate fast-spiking PV interneurons in NAcMS to induce feedforward inhibition. While elevated PV-mediated feed-forward inhibition in NAcMS promotes cocaine self-administration, it is thought that the basolateral amygdala, not the vHIPP, is responsible (J. Yu et al., 2017). Indeed, cocaine exposure did not increase the excitatory strength of vHIPP inputs onto PV interneurons in male or female NAcMS (Scudder, Baimel, Macdonald, & Carter, 2018). Given the significant role ascribed to vSUB in SUDs due to its projections to NAcMS and the intriguing role of PV-mediated feed-forward inhibition in NAcMS, it is surprising that, to our knowledge, the cell-type-specific connectivity, synaptic strengths, and plasticity of vSUB synapses made onto PV cells in NAcMS are unexamined.

In vivo fiber photometry of the vSUB-NAc circuit has begun to provide insight into how this circuit processes different information relevant to reward. NAc-projecting vSUB neurons exhibit sexually dimorphic changes in activity in response to Pavlovian threat conditioning – in males, the activity of vSUB neurons only increase during threat cues, while in females, vSUB neurons exhibit generalized encoding by responding to both threat and non-threat cues. The threat discrimination observed in males reduces reward seeking. However, despite sexually dimorphic changes in the activity of NAc-projecting vSUB neurons, threat conditioning did not alter the strength of vSUB-NAc synapses in either sex (Muir et al., 2024). A separate study using operant reward tasks found that the vSUB-NAc circuit encodes outcomes following unrewarded outcomes (Iyer et al., 2025). The encoding of unrewarded outcomes occurred independent of sex. The authors of this study postulate that the encoding of unrewarded outcomes by the vSUB-NAc circuit may increase engagement when rewards are encountered infrequently. The observations that threat discrimination, but not unrewarded outcomes, elicits sexually dimorphic neural activity suggest distinct engagement of upstream circuits of vSUB and/or plasticity in vSUB microcircuitry that may differentially regulate vSUB principal neuron activity, which underscores the need to systematically interrogate vSUB circuitry with sex-, cell-, and synapse-specificity.

Stress disorders and vSUB:

The vSUB is a major site of stress integration and regulates behaviors that model stress-related disorders (Herman & Mueller, 2006). The vSUB integrates two main signaling pathways that physiologically process stress, the hypothalamus-pituitary-adrenal axis (HPA) and the sympathetic nervous system (SNS) (Herman & Mueller, 2006). The HPA regulates the systemic release of stress hormones (e.g., corticosterone in rodents, cortisol in humans), which act on glucocorticoid receptors in vSUB while the activation of the SNS causes dense noradrenergic release from the locus coeruleus into vSUB. Early lesion studies revealed that vSUB regulates corticosterone release, and this function is exclusive to the vSUB within the vHIPP (Herman, Cullinan, Morano, Akil, & Watson, 1995; Herman, Dolgas, & Carlson, 1998). Interestingly, the regulatory role for vSUB over corticosterone release appears to be stressor specific. For example, vSUB lesions reduce corticosterone in response to hypoxia stress (Mueller, Dolgas, & Herman, 2004), do not change corticosterone in response to ether stress (Herman et al., 1998), and increase corticosterone in response to the elevated plus maze, open field test, or restraint stress (Herman et al., 1995; Herman et al., 1998; Mueller et al., 2004). These studies indicate that vSUB provides negative feedback to the HPA axis specifically in response to psychogenic but not systemic stressors (Herman & Mueller, 2006). Thus, vSUB is hypothesized to be critical for context-dependent processing of threatening or aversive contexts to guide appropriate responses. Prolonged corticosterone release after psychogenic stress is observed after vSUB lesions and is strongly linked to the development and exacerbation of stress-related disorders preclinically and clinically (cortisol) (Lenze et al., 2011; Vreeburg et al., 2010), which suggests that vSUB dysregulation may be a key feature in stress disorder pathogenesis. Generalized anxiety disorders in human patients are closely related to prior stress exposure and anxiety-like behaviors in rodents are commonly measured after stress exposure by using the open field test (OFT), elevated plus maze (EPM), acoustic startle response (ASR), and elevated zero maze (EZM) (Lezak, Missig, & Jr, 2017). These tests rely on the preference of mice to walk closely to walls (i.e., thigmotaxis) and/or their aversion to open spaces to avoid predators. Researchers interpret increased thigmotaxis or aversion to open space as increased anxiety-like behaviors. Consistent with the notion that prolonged corticosterone release corresponds to increased risk for stress disorders, vSUB lesions increase anxiety-like behavior in the OFT and ASR (Herman et al., 1998; Nettles, Pesold, & Goldman, 2000). Taken together, vSUB is crucial for contextually processing stressors, modulates anxiety-like behaviors, and negatively regulates corticosterone release.

Impacts of stress on vSUB microcircuitry:

vSUB has been heavily studied for its function in stress integration and stress-related disorders (Herman & Mueller, 2006), however these studies have been conducted primarily in male rodent models and lack cell-type specificity. Recent works reinforce the imperative to consider cell-type specificity when examining vSUB activity after stress, highlighting burst-spiking cells as uniquely stress susceptible. Chronic social defeat stress increases the total number of burst-spiking cells in the proximal vSUB and the proportion of burst-spiking cells that project to the NAc (Lee et al., 2019a, 2019b). This cellular conversion of regular to burst-spiking properties is driven by enhanced T-type calcium currents and increased expression of Cav3.1 channels (Lee et al., 2019b). This intriguing increase in the number of burst-spiking cells was monitored 1-day after the termination of chronic social defeat stress, however, it remains to be tested whether this cellular conversion represents a transient or persistent adaptation to chronic stress. Stress not only impacts the prevalence of vSUB burst-spiking cells, but also their synaptic properties. A two-way active avoidance conditioning stressor enhanced vCA1-vSUB burst-spiking long term potentiation (LTP) and reduced the threshold for late-onset LTP at these synapses (Bartsch, von Cramon, Gruber, Heinemann, & Behr, 2021). Further, the lowered induction threshold for vCA1-vSUB burst-spiking LTP required beta-adrenergic, NMDA, and DR1/DR5 receptor activation (Bartsch et al., 2021). While these studies suggest that burst-spiking is a physiological consequence of stress and that burst-spiking cells are susceptible to stress perturbations, it is important to note that these studies were only conducted using males and the behavioral significance of the changes to burst-spiking cells in vSUB is undetermined. When considering recent work that demonstrates vSUB has basal microcircuit sexual dimorphism and that stress-related diseases have well-documented sex-differences (Bangasser & Valentino, 2014), how does stress impact vSUB in females?

Recent work from our laboratory found significant sexually dimorphic synaptic adaptations in the vSUB microcircuit manifested after 1-hour acute restraint stress (Miller, Li, Beier, & Aoto, 2025). Importantly, these adaptations primarily impacted burst-spiking cells. In females, acute stress decreased the strength of excitatory input from vCA1 and increased the strength of PV-mediated synaptic inhibition via postsynaptic mechanisms. By contrast in males, the same stressor did not alter excitatory input but decreased PV-mediated inhibition onto burst-spiking cells. Further, acute restraint significantly impaired vCA1-burst-spiking cell LTP in females but not males. Thus, acute restraint stress likely drives a net reduction in the activity of burst-spiking cell in females, but a net increase in activity in males. Importantly, the sexually dimorphic adaptations in PV-mediated inhibition onto burst-spiking cells is driven by distinct signaling pathways, as adrenergic receptor signaling is necessary in females while corticosterone signaling is required in males. It will be critical to continue examining the vSUB with cell-type resolution and by including females and behavioral readouts to provide context for these electrophysiological findings.

In addition to burst-spiking vSUB cells, recent work revealed a role for vSUB cells that express D1R or D2R in anxiogenic exploration (Godino et al., 2025). Likely in concert with other stress signaling, dopamine is released in the vSUB in response to anxiogenic environments and acts through D1R or D2R expressing vSUB cells to regulate exploratory behaviors. In vivo calcium imaging during the EPM revealed that both D1R and D2R expressing vSUB cells are activated during open arm entries and in head dip behaviors preceding exploration. Interestingly, only D1R cells increased activity in response to head dipping if the subsequent behavior was avoidance and only D2R cellular activity is required for anxiogenic exploration. Together, D1R and D2R cells in vSUB may be important for coordinating and balancing motivated exploration in anxiogenic environments. Interestingly, the cell populations within vSUB that express D1R or D2R are about half glutamatergic and half GABAergic. More work is essential to examine further into other neuronal properties of the D1R and D2R cells in vSUB. For example, it will be important to know whether D1R and D2R expression correlates with spiking pattern in pyramidal cells or interneuron subtypes and whether they have unique projection patterns. Finally, it will be critical to determine whether to determine whether this remains true for females as this study exclusively used males.

Stress integration: vSUB-BNST

Despite the well-documented role for vSUB to inhibit corticosterone release, the vSUB provides only sparse direct innervation to the paraventricular nucleus (PVN) of the hypothalamus to control HPA function (Herman & Mueller, 2006). Thus, it has long been hypothesized that the excitatory afferents from vSUB indirectly regulates the PVN through an inhibitory relay structure. The bed nucleus of the stria terminalis (BNST), a largely inhibitory nucleus, has been historically appreciated as well-poised to serve as an inhibitory relay center between vSUB and PVN (Cullinan, Herman, & Watson, 1993). Recent work strengthened this hypothesis by showing that photo-stimulation of vSUB inhibited CRF+ neurons in the PVN via an inhibitory relay through the BNST (Cole, Montgomery, Bale, & Thompson, 2022). Further, activating this trisynaptic circuit in vivo inhibited corticosterone release caused by acute restraint stress (Cole et al., 2022). In sum, manipulation of the vSUB-BNST-PVN circuit reveals that BNST serves as an integral inhibitory relay center for vSUB to exert control over corticosterone release by HPA. Reciprocal to these findings, others found that in vivo potentiation of the vSUB to anteromedial area of the BNST produced anxiolysis as indicated by the light-dark box and EPM tests (Glangetas et al., 2017). While these studies imply that vSUB-BNST activity is anxiolytic, they do not examine the circuit after physiological stress nor consider sex as a biological variable despite that vSUB, BNST, and stress disorders all exhibit profound sexual dimorphism.

To this end, we recently assessed the functional connectivity between vSUB and anterior BNST (aBNST) in male and female mice (Miller et al., 2025). Using retrograde adeno associated virus, we unexpectedly found that within the vHIPP 88% of projections to aBNST originate from vSUB (Fig. 4) and that ~80% of the output from vSUB to aBNST originates from burst-spiking cells. These findings underscore the distinct role of vSUB within vHIPP to convey information to the aBNST and is in stark contrast to the over-representation of regular-spiking cells in the vSUB-NAcMS circuit. Additionally, sexually dimorphic synaptic adaptations manifest after acute restraint stress. In females, vSUB-aBNST synapses are weakened, while in males, these synapses are strengthened (Fig. 5) (Miller et al., 2025). Given the known cellular heterogeneity of the aBNST, it will be critical to assess the impact of stress on the cell-type-specific synaptic properties of the vSUB-aBNST circuit.

Figure 4.

The majority of aBNST projections in vHIPP originate from vSUB. (Left) Image depicting proper targeting of aBNST with retrograde AAV₂mRuby. VL: lateral ventricle, LS: lateral septal nucleus, HY: hypothalamus, V3: third ventricle, NDB: diagonal band nucleus, MA: magnocellular nucleus, OT: olfactory tubercle, SI: substantia inominata, FS: fundus of striatum, CP: caudoputamen. (Right) Diagram including the horizontal plane representing the vHIPP section examined from the Allen Brain Explorer. Hippocampal subregions are identified based on transcriptomic profiles (Ding et al., 2020) integrated into the Allen Brain Institute’s Brain Explorer. Adapted with permission from Miller et al., (2025).

Figure 5.

Summary of vSUB-BNST pathway functions. The majority of BNST-projecting vSUB cells are burst-spiking, a vSUB cell type that is vulnerable to acute stress induced changes.

Complementary to this work, freezing is a form of passive coping to stress and excessive freezing in anxiogenic environments is considered dysfunctional and associated with increased anxiety (Godoy, Medeiros, Souza, & Martynhak, 2022; Sartori et al., 2011; Wood & Bhatnagar, 2015). The vSUB-BNST pathway shows reduced activity after freezing in contextual fear conditioning as indexed by FOS expression (Urien et al., 2022). Consistently, chemogenetic activation of the vSUB-BNST reduces freezing behavior in contextual fear conditioning, although both studies only examined male rodents (Kopaeva, Yakimov, Urien, & Bauer, 2023). Complementary to these studies, our lab found that aBNST-projecting vSUB cells increase activity during struggling bouts, an active coping mechanism, in both sexes as measured by in vivo calcium activity (Miller et al., 2025). Together, these findings raise the enticing possibility that vSUB-BNST activity shapes coping behaviors and potentiation of this pathway could represent enhanced employment of active coping behaviors when facing future stressors like the EZM (Glangetas et al., 2017). Similarly, weakening the vSUB-BNST projection may promote passive coping behaviors like freezing which increases susceptibility to exhibiting more anxiety-like behaviors (Kopaeva et al., 2023). It would be highly informative to examine the vSUB-BNST pathway in vivo to determine whether activity directly corresponds to active versus coping behaviors. Further, given the known sex-differences in stress coping styles and prevalence of stress-related disorders (Cholankeril, Xiang, & Badr, 2023), including both sexes in future works would be highly informative.

Major depressive disorder

Loss of hippocampal volume is common in patients with major depressive disorder (MDD) (Cole, Costafreda, McGuffin, & Fu, 2011) and this loss can be reversed with antidepressants (Arnone et al., 2012), highlighting this brain region as crucial in MDD pathophysiology. Within the vHIPP, clinical fMRI studies reveal disrupted connectivity in the subiculum of MDD patients (Hao et al., 2020; Shengli et al., 2022), but see (Jacob et al., 2022). Preclinically, there is also evidence that vSUB may be involved in depression-like behaviors. MDD is a psychiatric disorder defined by anhedonia and helplessness which is modeled preclinically through the sucrose preference test (SPT) and forced swim test (FST) or tail suspension test (TST), respectively. Disrupted excitatory-inhibitory balance and intrinsic excitability of vSUB glutamatergic neurons is observed after a 14-day chronic mild unpredictable mild stress (CMUS) induced depression model which produces depression-like behaviors on the FST, TST, and SPT (Yu et al., 2022). Specifically, CMUS shifted vSUB E-I balance towards excitation and increased excitability of burst-spiking cells. Stimulating acetylcholine release from the medial septum and diagonal band of Broca (MSDB) into vSUB recapitulates increased vSUB burst-spiking cell excitability and depressive-like behaviors observed after CMUS, but a causal relationship between acetylcholine and CMUS phenotypes will need to be directly examined in the future (Yu et al., 2022). Increased excitatory tone on vSUB glutamate neurons after CMUS-induced depression may be a mechanism to compensate for reduced vHIPP volume observed in humans and rodents after stress-induced depression, although Yu et al. did not assess changes in vSUB volume in their study. Further, while other preclinical studies demonstrate hippocampal volume loss in stress-induced models of depression, the volume loss does not correlate with the onset of depression-like symptoms (Delgado y Palacios et al., 2011; Jayatissa, Henningsen, Nikolajsen, West, & Wiborg, 2010). Additionally, hippocampal volume loss is observed after models of chronic stress regardless of the expression of depression-like behaviors (Delgado y Palacios et al., 2011; Jayatissa et al., 2010), which suggests that hippocampal volume loss is specific to chronic stress rather than depression pathophysiology. Taken together, the cause of increased vSUB excitability after CMUS and relationship with loss of hippocampal volume is incompletely understood and could be explained by a number of possibilities. First, hyperactivation of vSUB glutamate neurons from chronic stress causes excitotoxicity and thus volume loss. Second, vSUB hyperactivation is a compensatory mechanism after volume loss. Third, vSUB hyperactivation occurs independently of hippocampal volume status, potentially through increased acetylcholine input from MSDB, to contribute to depression-like phenotypes.

While the above studies examining the role of vSUB in stress-induced depression are exclusively done in male rodent models or did not consider sex as a biological variable, females are more likely to develop depressive symptoms after encountering significant stress (Albert & Newhouse, 2019). Preclinical evidence has identified sex-differences in vSUB-NAc circuit activity after chronic variable stress, which is a model for depression. Using in vivo fiber photometry of NAc-projecting vSUB neurons, chronic variable stress increased the activity of NAc-projecting vSUB neurons in both sexes when mice were in the anxiogenic context of the OFT (Muir et al., 2020). However, only females exhibited increased in vivo vSUB activity during social interaction, accompanied by an increase in the frequency of spontaneous synaptic activity ex vivo. However, whether the stress-induced increase in the frequency of spontaneous activity selectively manifests in regular- or burst-spiking cells or occurs more broadly in both cell types remains to be tested. Further, although females are initially more vulnerable to stress-induced anxiety-like and depressive behavior, once these behaviors manifest, both sexes exhibit increased activity of the vSUB-NAc circuit. Given the role for vSUB in regulating outcomes with known sex-differences, it is critical to continue to include sex as a biological variable in future studies. The increased vulnerability of women to MDD lasts between puberty and menopause, strongly suggesting a role for circulating estrogens in these sex-differences (Albert & Newhouse, 2019). vSUB has both sexually dimorphic basal microcircuitry and estrogen receptor expression (Blurton-Jones & Tuszynski, 2002; Wu et al., 2014), representing a promising candidate to underlie sex-differences in depressive disorders and symptomology. Future studies should expand upon the current literature while including female subjects and considering the possible influences of estrogen cycle on outcomes.

Part IV: Conclusions

The literature solidifies vSUB as a distinct vHIPP subregion deserving special consideration when studying the pathogenesis of psychiatric diseases such as anxiety, MDD, and SUD. vSUB lesion studies produce a variety of outcomes including reduced context-induced reinstatement of drug seeking, increased anxiety-like behavior, and increased corticosterone release. These results are seemingly at odds with each other since we know that increased stress and anxiety are strongly associated with increased drug relapse. Thus, it is important to examine vSUB function in terms of physiological disease models and with projection and cell-type specificity. For example, vSUB-NAcS activation drives context-induced drug relapse, is potentiated after chronic cocaine exposure, and the majority of vSUB afferents are regular-spiking cells (Emma E. Boxer et al., 2023; Marchant et al., 2016). By contrast, vSUB-aBNST activation drives anxiolysis and the majority of vSUB afferents are burst-spiking cells (Glangetas et al., 2017; Miller et al., 2025). To add complexity to the cell-type, synapse, and projection specific role of vSUB in diseases, there are also remarkable sex-differences reported in some of these works. Taken together, vSUB is a complex structure that regulates fundamental biological responses like stress responsivity, appetite, and contextual processing and increased investigation into this brain region with projection, cell-type, and sex specificity is warranted.

Current limitations to our understanding of vSUB is the historic exclusion of females in these studies and the lack of molecular tools to examine burst and regular-spiking cells. While the literature has historically almost exclusively used male rodent models to investigate the role of vSUB in these diseases, emerging works are beginning to include sex as a biological variable which will greatly expand our understanding of vSUB function. vSUB participates in the pathogenesis of psychiatric diseases such as anxiety, MDD, and SUDs with well-documented sex-differences in onset, symptomology, and outcomes and represents an appealing brain region to underlie these sexually dimorphic features. There is ample evidence that vSUB burst-spiking and regular-spiking cells participate in different projection pathways and disease processes, but we currently lack genetic markers to distinguish them reliably. A potential unexplored genetic marker for regular-spiking cells is Dlk1+. Parallel observations reveal that NAc-projecting vSUB cells biasedly express Dlk1+ and are 70% regular-spiking (Emma E. Boxer et al., 2023; Kim & Spruston, 2012), which supports further investigation into Dlk1+ as a possible regular-spiking cell marker. While establishing molecular signatures for each cell type will be invaluable, ways around identifying these is to capitalize on cell biases in certain projections. For example, manipulating NAcMS-projecting vSUB cells will capture mostly regular-spiking cells while manipulating aBNST-projecting vSUB cells will capture mostly burst-spiking cells (Miller et al., 2025). In sum, continued investigation into vSUB should focus on the inclusion of females and use of cell-type specificity to elucidate its roles in numerous disease states and regulating biological responses.