Id2 GABAergic interneurons comprise a neglected fourth major group of cortical inhibitory cells.

Cortical GABAergic interneurons (INs) represent a diverse population of mainly locally projecting cells that provide specialized forms of inhibition to pyramidal neurons and other INs. Most recent work on INs has focused on subtypes distinguished by expression of Parvalbumin (PV), Somatostatin (SST), or Vasoactive Intestinal Peptide (VIP). However, a fourth group that includes neurogliaform cells (NGFCs) has been less well characterized due to a lack of genetic tools. Here, we show that these INs can be accessed experimentally using intersectional genetics with the gene Id2. We find that outside of layer 1 (L1), the majority of Id2 INs are NGFCs that express high levels of neuropeptide Y (NPY) and exhibit a late-spiking firing pattern, with extensive local connectivity. While much sparser, non-NGFC Id2 INs had more variable properties, with most cells corresponding to a diverse group of INs that strongly expresses the neuropeptide CCK. In vivo, using silicon probe recordings, we observed several distinguishing aspects of NGFC activity, including a strong rebound in activity immediately following the cortical down state during NREM sleep. Our study provides insights into IN diversity and NGFC distribution and properties, and outlines an intersectional genetics approach for further study of this underappreciated group of INs.

A comparative study between a transscleral sustained-release device and eyedrops on intraocular distribution of carteolol hydrochloride.

The objectives of this study were to develop a sustained-release device for carteolol hydrochloride (CH) and investigate any potential difference in the intraocular distribution of this agent between the transscleral administration of the device and treatment with eyedrops. The device was formulated with photocurable resin, poly (ethyleneglycol) dimethacrylate, to fit within the curve of the rabbit eyeball. In vitro study showed that CH was released in a sustained-release manner for 2 weeks. The concentration of CH in the retina, choroid/retinal pigment epithelium, sclera, iris, and aqueous humor was determined by high-performance liquid chromatography. Transscleral administration was able to deliver CH to the posterior segment (i.e., retina and choroid/retinal pigment epithelium) rather than the anterior segment (i.e., aqueous humor), while eyedrops delivered CH only to the anterior segment. Transscleral administration could deliver CH to aqueous humor at half the concentration versus treatment with eyedrops and reduced intraocular pressure (IOP) at 1 day after implantation; however, the IOP-lowering effect was not sustained thereafter. In conclusion, transscleral drug delivery may be a useful method for the reduction of IOP. Notably, the aqueous concentration must be equal to that delivered by the eyedrops, and this approach might be preferable for drug delivery to the posterior segment of the eye.

VMHvllCckar cells dynamically control female sexual behaviors over the reproductive cycle.

Sexual behavior is fundamental for the survival of mammalian species and thus supported by dedicated neural substrates. The ventrolateral part of ventromedial hypothalamus (VMHvl) is an essential locus for controlling female sexual behaviors, but recent studies revealed the molecular complexity and functional heterogeneity of VMHvl cells. Here, we identify the cholecystokinin A receptor (Cckar)-expressing cells in the lateral VMHvl (VMHvllCckar) as the key controllers of female sexual behaviors. The inactivation of VMHvllCckar cells in female mice diminishes their interest in males and sexual receptivity, whereas activating these cells has the opposite effects. Female sexual behaviors vary drastically over the reproductive cycle. In vivo recordings reveal reproductive-state-dependent changes in VMHvllCckar cell spontaneous activity and responsivity, with the highest activity occurring during estrus. These in vivo response changes coincide with robust alternation in VMHvllCckar cell excitability and synaptic inputs. Altogether, VMHvllCckar cells represent a key neural population dynamically controlling female sexual behaviors over the reproductive cycle.

Long-term survival in non-human primates of stem cell-derived, MHC-unmatched corneal epithelial cell sheets.

When corneal epithelial stem cells residing in the corneal limbus become dysfunctional, called a limbal stem cell deficiency (LSCD), corneal transparency is decreased, causing severe vision loss. Transplantation of corneal epithelial cell sheets (CEPS) derived from stem cells, including induced pluripotent stem cells, is a promising treatment for LSCD. However, the potential effect of human leukocyte antigen (HLA) concordance on CEPS transplantation has not been addressed. Here, we show that there is no difference in the immune response to CEPS between HLA-matched and -unmatched peripheral blood mononuclear cells in mixed lymphocyte reactions. CEPS transplantation in cynomolgus monkeys revealed that the immune response to major histocompatibility-unmatched CEPS was not strong and could be controlled by local steroid administration. Furthermore, programmed death ligand 1 was identified as an immunosuppressive molecule in CEPS under inflammatory conditions in vitro. Our results indicate that corneal epithelium has low immunogenicity and allogeneic CEPS transplantation requires mild immunosuppression.

Transcriptional and functional divergence in lateral hypothalamic glutamate neurons projecting to the lateral habenula and ventral tegmental area.

The lateral hypothalamic area (LHA) regulates feeding- and reward-related behavior, but because of its molecular and anatomical heterogeneity, the functions of defined neuronal populations are largely unclear. Glutamatergic neurons within the LHA (LHAVglut2) negatively regulate feeding and appetitive behavior. However, this population comprises transcriptionally distinct and functionally diverse neurons that project to diverse brain regions, including the lateral habenula (LHb) and ventral tegmental area (VTA). To resolve the function of distinct LHAVglut2 populations, we systematically compared projections to the LHb and VTA using viral tracing, single-cell sequencing, electrophysiology, and in vivo calcium imaging. LHAVglut2 neurons projecting to the LHb or VTA are anatomically, transcriptionally, electrophysiologically, and functionally distinct. While both populations encode appetitive and aversive stimuli, LHb projecting neurons are especially sensitive to satiety state and feeding hormones. These data illuminate the functional heterogeneity of LHAVglut2 neurons, suggesting that reward and aversion are differentially processed in divergent efferent pathways.

Generation of knockout rabbits with X-linked severe combined immunodeficiency (X-SCID) using CRISPR/Cas9.

Severe immunodeficient mice are widely used to examine human and animal cells behaviour in vivo. However, mice are short-lived and small in size; while large animals require specific large-scale equipment. Rabbits are also commonly employed as experimental models and are larger than mice or rats, easy to handle, and suitable for long-term observational and pre-clinical studies. Herein, we sought to develop and maintain stable strains of rabbits with X-linked severe combined immunodeficiency (X-SCID) via the CRISPR/Cas9 system targeting Il2rg. Consequently, X-SCID rabbits presented immunodeficient phenotypes including the loss of T and B cells and hypoplasia of the thymus. Further, these rabbits exhibited a higher success rate with engraftments upon allogeneic transplantation of skin tissue than did wild type controls. X-SCID rabbits could be stably maintained for a minimum of four generations. These results indicate that X-SCID rabbits are effective animals for use in a non-rodent model of severe immunodeficiency.

Transcriptional and Spatial Resolution of Cell Types in the Mammalian Habenula.

The habenula complex is appreciated as a critical regulator of motivated and pathological behavioral states via its output to midbrain nuclei. Despite this, transcriptional definition of cell populations that comprise both the medial habenular (MHb) and lateral habenular (LHb) subregions in mammals remain undefined. To resolve this, we performed single-cell transcriptional profiling and highly multiplexed in situ hybridization experiments of the mouse habenula complex in naive mice and those exposed to an acute aversive stimulus. Transcriptionally distinct neuronal cell types identified within the MHb and LHb, were spatially defined, differentially engaged by aversive stimuli, and had distinct electrophysiological properties. Cell types identified in mice also displayed a high degree of transcriptional similarity to those previously described in zebrafish, highlighting the well-conserved nature of habenular cell types across the phylum. These data identify key molecular targets within habenular cell types and provide a critical resource for future studies.

Paraventricular Thalamus Projection Neurons Integrate Cortical and Hypothalamic Signals for Cue-Reward Processing.

The paraventricular thalamus (PVT) is an interface for brain reward circuits, with input signals arising from structures, such as prefrontal cortex and hypothalamus, that are broadcast to downstream limbic targets. However, the precise synaptic connectivity, activity, and function of PVT circuitry for reward processing are unclear. Here, using in vivo two-photon calcium imaging, we find that PVT neurons projecting to the nucleus accumbens (PVT-NAc) develop inhibitory responses to reward-predictive cues coding for both cue-reward associative information and behavior. The multiplexed activity in PVT-NAc neurons is directed by opposing activity patterns in prefrontal and lateral hypothalamic afferent axons. Further, we find that prefrontal cue encoding may maintain accurate cue-reward processing, as optogenetic disruption of this encoding induced long-lasting effects on downstream PVT-NAc cue responses and behavioral cue discrimination. Together, these data reveal that PVT-NAc neurons act as an interface for reward processing by integrating relevant inputs to accurately inform reward-seeking behavior.

Four Unique Interneuron Populations Reside in Neocortical Layer 1.

Sensory perception depends on neocortical computations that contextually adjust sensory signals in different internal and environmental contexts. Neocortical layer 1 (L1) is the main target of cortical and subcortical inputs that provide "top-down" information for context-dependent sensory processing. Although L1 is devoid of excitatory cells, it contains the distal "tuft" dendrites of pyramidal cells (PCs) located in deeper layers. L1 also contains a poorly characterized population of GABAergic interneurons (INs), which regulate the impact that different top-down inputs have on PCs. A poor comprehension of L1 IN subtypes and how they affect PC activity has hampered our understanding of the mechanisms that underlie contextual modulation of sensory processing. We used novel genetic strategies in male and female mice combined with electrophysiological and morphological methods to help resolve differences that were unclear when using only electrophysiological and/or morphological approaches. We discovered that L1 contains four distinct populations of INs, each with a unique molecular profile, morphology, and electrophysiology, including a previously overlooked IN population (named here "canopy cells") representing 40% of L1 INs. In contrast to what is observed in other layers, most L1 neurons appear to be unique to the layer, highlighting the specialized character of the signal processing that takes place in L1. This new understanding of INs in L1, as well as the application of genetic methods based on the markers described here, will enable investigation of the cellular and circuit mechanisms of top-down processing in L1 with unprecedented detail.SIGNIFICANCE STATEMENT Neocortical layer 1 (L1) is the main target of corticocortical and subcortical projections that mediate top-down or context-dependent sensory perception. However, this unique layer is often referred to as "enigmatic" because its neuronal composition has been difficult to determine. Using a combination of genetic, electrophysiological, and morphological approaches that helped to resolve differences that were unclear when using a single approach, we were able to decipher the neuronal composition of L1. We identified markers that distinguish L1 neurons and found that the layer contains four populations of GABAergic interneurons, each with unique molecular profiles, morphologies, and electrophysiological properties. These findings provide a new framework for studying the circuit mechanisms underlying the processing of top-down inputs in neocortical L1.

The Neural Mechanisms of Sexually Dimorphic Aggressive Behaviors.

Aggression is a fundamental social behavior that is essential for competing for resources and protecting oneself and families in both males and females. As a result of natural selection, aggression is often displayed differentially between the sexes, typically at a higher level in males than females. Here, we highlight the behavioral differences between male and female aggression in rodents. We further outline the aggression circuits in males and females, and compare their differences at each circuit node. Lastly, we summarize our current understanding regarding the generation of sexually dimorphic aggression circuits during development and their maintenance during adulthood. In both cases, gonadal steroid hormones appear to play crucial roles in differentiating the circuits by impacting on the survival, morphology, and intrinsic properties of relevant cells. Many other factors, such as environment and experience, may also contribute to sex differences in aggression and remain to be investigated in future studies.

Esr1+ cells in the ventromedial hypothalamus control female aggression.

As an essential means of resolving conflicts, aggression is expressed by both sexes but often at a higher level in males than in females. Recent studies suggest that cells in the ventrolateral part of the ventromedial hypothalamus (VMHvl) that express estrogen receptor-α (Esr1) and progesterone receptor are essential for male but not female mouse aggression. In contrast, here we show that VMHvlEsr1+ cells are indispensable for female aggression. This population was active when females attacked naturally. Inactivation of these cells reduced female aggression whereas their activation elicited attack. Additionally, we found that female VMHvl contains two anatomically distinguishable subdivisions that showed differential gene expression, projection and activation patterns after mating and fighting. These results support an essential role of the VMHvl in both male and female aggression and reveal the existence of two previously unappreciated subdivisions in the female VMHvl that are involved in distinct social behaviors.

Ventromedial Hypothalamus and the Generation of Aggression.

Aggression is a costly behavior, sometimes with severe consequences including death. Yet aggression is prevalent across animal species ranging from insects to humans, demonstrating its essential role in the survival of individuals and groups. The question of how the brain decides when to generate this costly behavior has intrigued neuroscientists for over a century and has led to the identification of relevant neural substrates. Various lesion and electric stimulation experiments have revealed that the hypothalamus, an ancient structure situated deep in the brain, is essential for expressing aggressive behaviors. More recently, studies using precise circuit manipulation tools have identified a small subnucleus in the medial hypothalamus, the ventrolateral part of the ventromedial hypothalamus (VMHvl), as a key structure for driving both aggression and aggression-seeking behaviors. Here, we provide an updated summary of the evidence that supports a role of the VMHvl in aggressive behaviors. We will consider our recent findings detailing the physiological response properties of populations of VMHvl cells during aggressive behaviors and provide new understanding regarding the role of the VMHvl embedded within the larger whole-brain circuit for social sensation and action.

The neural circuits of mating and fighting in male mice.

Tinbergen proposed that instinctive behaviors can be divided into appetitive and consummatory phases. During mating and aggression, the appetitive phase contains various actions to bring an animal to a social target and the consummatory phase allows stereotyped actions to take place. Here, we summarize recent advances in elucidating the neural circuits underlying the appetitive and consummatory phases of sexual and aggressive behaviors with a focus on male mice. We outline the role of the main olfactory inputs in the initiation of social approach; the engagement of the accessory olfactory system during social investigation, and the role of the hypothalamus and its downstream pathways in orchestrating social behaviors through a suite of motor actions.

Inhibiting the Activity of CA1 Hippocampal Neurons Prevents the Recall of Contextual Fear Memory in Inducible ArchT Transgenic Mice.

The optogenetic manipulation of light-activated ion-channels/pumps (i.e., opsins) can reversibly activate or suppress neuronal activity with precise temporal control. Therefore, optogenetic techniques hold great potential to establish causal relationships between specific neuronal circuits and their function in freely moving animals. Due to the critical role of the hippocampal CA1 region in memory function, we explored the possibility of targeting an inhibitory opsin, ArchT, to CA1 pyramidal neurons in mice. We established a transgenic mouse line in which tetracycline trans-activator induces ArchT expression. By crossing this line with a CaMKIIα-tTA transgenic line, the delivery of light via an implanted optrode inhibits the activity of excitatory CA1 neurons. We found that light delivery to the hippocampus inhibited the recall of a contextual fear memory. Our results demonstrate that this optogenetic mouse line can be used to investigate the neuronal circuits underlying behavior.