Total Number and Ratio of GABAergic Neuron Types in the Mouse Lateral and Basal Amygdala.

GABAergic neurons are key circuit elements in cortical networks. Despite growing evidence showing that inhibitory cells play a critical role in the lateral (LA) and basal (BA) amygdala functions, neither the number of GABAergic neurons nor the ratio of their distinct types has been determined in these amygdalar nuclei. Using unbiased stereology, we found that the ratio of GABAergic neurons in the BA (22%) is significantly higher than in the LA (16%) in both male and female mice. No difference was observed between the right and left hemispheres in either sex. In addition, we assessed the ratio of the major inhibitory cell types in both amygdalar nuclei. Using transgenic mice and a viral strategy for visualizing inhibitory cells combined with immunocytochemistry, we estimated that the following cell types together compose the vast majority of GABAergic cells in the LA and BA: axo-axonic cells (5.5%-6%), basket cells expressing parvalbumin (17%-20%) or cholecystokinin (7%-9%), dendrite-targeting inhibitory cells expressing somatostatin (10%-16%), NPY-containing neurogliaform cells (14%-15%), VIP and/or calretinin-expressing interneuron-selective interneurons (29%-38%), and GABAergic projection neurons expressing somatostatin and neuronal nitric oxide synthase (5.5%-8%). Our results show that these amygdalar nuclei contain all major GABAergic neuron types as found in other cortical regions. Furthermore, our data offer an essential reference for future studies aiming to reveal changes in GABAergic cell number and in inhibitory cell types typically observed under different pathologic conditions, and to model functioning amygdalar networks in health and disease.SIGNIFICANCE STATEMENT GABAergic cells in cortical structures, as in the lateral and basal nucleus of the amygdala, have a determinant role in controlling circuit operation. In this study, we provide the first estimate for the total number of inhibitory cells in these two amygdalar nuclei. In addition, our study is the first to define the ratio of the major GABAergic cell types present in these cortical networks. Taking into account that hyperexcitability in the amygdala, arising from the imbalance between excitation and inhibition typifies many altered brain functions, including anxiety, post-traumatic stress disorder, schizophrenia, and autism, uncovering the number and ratio of distinct amygdalar inhibitory cell types offers a solid base for comparing the changes in inhibition in pathologic brain states.

Physiological sharp wave-ripples and interictal events in vitro: what's the difference?

Sharp wave-ripples and interictal events are physiological and pathological forms of transient high activity in the hippocampus with similar features. Sharp wave-ripples have been shown to be essential in memory consolidation, whereas epileptiform (interictal) events are thought to be damaging. It is essential to grasp the difference between physiological sharp wave-ripples and pathological interictal events to understand the failure of control mechanisms in the latter case. We investigated the dynamics of activity generated intrinsically in the Cornu Ammonis region 3 of the mouse hippocampus in vitro, using four different types of intervention to induce epileptiform activity. As a result, sharp wave-ripples spontaneously occurring in Cornu Ammonis region 3 disappeared, and following an asynchronous transitory phase, activity reorganized into a new form of pathological synchrony. During epileptiform events, all neurons increased their firing rate compared to sharp wave-ripples. Different cell types showed complementary firing: parvalbumin-positive basket cells and some axo-axonic cells stopped firing as a result of a depolarization block at the climax of the events in high potassium, 4-aminopyridine and zero magnesium models, but not in the gabazine model. In contrast, pyramidal cells began firing maximally at this stage. To understand the underlying mechanism we measured changes of intrinsic neuronal and transmission parameters in the high potassium model. We found that the cellular excitability increased and excitatory transmission was enhanced, whereas inhibitory transmission was compromised. We observed a strong short-term depression in parvalbumin-positive basket cell to pyramidal cell transmission. Thus, the collapse of pyramidal cell perisomatic inhibition appears to be a crucial factor in the emergence of epileptiform events.

Role of sanguinarine in regulating immunosuppression in a Lewis lung cancer mouse model.

Myeloid-derived suppressor cells (MDSCs) play an important role in the tumor-induced immunosuppressive microenvironment and have been linked with tumor development, proliferation, and resistance to treatment. Therefore, therapies that target MDSCs, such as sanguinarine (SNG), are now being considered potential treatments for lung cancer. However, the role of SNG in regulating the immune response in lung cancer is still not clear. In view of this, we evaluated the mechanism involved in the antitumor and immunoregulatory response to SNG therapy in a Lewis lung cancer (LLC) mouse model. The tumor mass and volume in the SNG treated LLC mouse model were significantly lower when compared with the control group (p < 0.05), indicating a good response to SNG. SNG also reduced the damage to the spleen, decreased the proportion of MDSCs, and increased the production of T helper 1 (Th1), T helper 2 (Th2), cytotoxic T-lymphocyte (CTL), macrophages, dendritic cells (DC) within the spleen. However, it did not affect the proportion of T helper 17 (Th17) and regulatory T cells (Treg). SNG also down-regulated the proportion of MDSCs in vitro and promoted their apoptosis, differentiation, and maturation. SNG was found to induce the differentiation of MDSCs into macrophages and DC through the nuclear factor kappa-B (NF-κB) pathway in vitro, while it also decreased the expression of arginase-1 (Arg-1) anti-inducible nitric oxide synthase (iNOS) and reactive oxygen species (ROS) in MDSCs.SNG also reduced the inhibitory effect on the proliferation of CD8+T cells. SNG may reduce the immunosuppressive state induced by lung cancer by promoting cell differentiation and by inhibiting the immunosuppressive activity of MDSCs.

Excitation of Diverse Classes of Cholecystokinin Interneurons in the Basal Amygdala Facilitates Fear Extinction.

There is growing evidence that interneurons (INs) orchestrate neural activity and plasticity in corticoamygdala circuits to regulate fear behaviors. However, defining the precise role of cholecystokinin-expressing INs (CCK INs) remains elusive due to the technical challenge of parsing this population from CCK-expressing principal neurons (CCK PNs). Here, we used an intersectional genetic strategy in CCK-Cre;Dlx5/6-Flpe double-transgenic mice to study the anatomical, molecular and electrophysiological properties of CCK INs in the basal amygdala (BA) and optogenetically manipulate these cells during fear extinction. Electrophysiological recordings confirmed that this strategy targeted GABAergic cells and that a significant proportion expressed functional cannabinoid CB1 receptors; a defining characteristic of CCK-expressing basket cells. However, immunostaining showed that subsets of the genetically-targeted cells expressed either neuropeptide Y (NPY; 29%) or parvalbumin (PV; 17%), but not somatostatin (SOM) or Ca2+/calmodulin-dependent protein kinase II (CaMKII)-α. Further morphological and electrophysiological analyses showed that four IN types could be identified among the EYFP-expressing cells: CCK/cannabinoid receptor type 1 (CB1R)-expressing basket cells, neurogliaform cells, PV+ basket cells, and PV+ axo-axonic cells. At the behavioral level, in vivo optogenetic photostimulation of the targeted population during extinction acquisition led to reduced freezing on a light-free extinction retrieval test, indicating extinction memory facilitation; whereas photosilencing was without effect. Conversely, non-selective (i.e., inclusive of INs and PNs) photostimulation or photosilencing of CCK-targeted cells, using CCK-Cre single-transgenic mice, impaired extinction. These data reveal an unexpectedly high degree of phenotypic complexity in a unique population of extinction-modulating BA INs.

Cell Type Specific Representation of Vibro-tactile Stimuli in the Mouse Primary Somatosensory Cortex.

Although the processing of whisker deflections in the barrel area of the rodent primary somatosensory cortex (S1) has been studied extensively, how cutaneous vibro-tactile stimuli are processed in the rodent S1 outside the barrel area has not been fully examined. Particularly, the cell-type specific representation of multiple vibration frequencies in genetically identified inhibitory cells in the S1 has not been examined. Using two-photon calcium imaging, we examined the responses to vibration stimuli of excitatory and inhibitory neurons in the S1 hind limb area of male and female mice. The excitatory cells showed relatively sharp selectivity to vibration stimuli, whereas the inhibitory cells exhibited less selectivity. The excitatory and inhibitory cells with different preferred stimuli were intermingled in a "salt and pepper" manner. Furthermore, the noise correlation tended to be especially strong in excitatory-inhibitory and inhibitory-inhibitory cell pairs that have similar stimulus selectivity. These results suggest that excitatory cells tend to represent specific stimulus information and work together with similarly tuned inhibitory cells as a functionally connected network.