Vasoactive Intestinal Polypeptide-Immunoreactive Interneurons within Circuits of the Mouse Basolateral Amygdala.

In cortical structures, principal cell activity is tightly regulated by different GABAergic interneurons (INs). Among these INs are vasoactive intestinal polypeptide-expressing (VIP+) INs, which innervate preferentially other INs, providing a structural basis for temporal disinhibition of principal cells. However, relatively little is known about VIP+ INs in the amygdaloid basolateral complex (BLA). In this study, we report that VIP+ INs have a variable density in the distinct subdivisions of the mouse BLA. Based on different anatomical, neurochemical, and electrophysiological criteria, VIP+ INs could be identified as IN-selective INs (IS-INs) and basket cells expressing CB1 cannabinoid receptors. Whole-cell recordings of VIP+ IS-INs revealed three different spiking patterns, none of which was associated with the expression of calretinin. Genetic targeting combined with optogenetics and in vitro recordings enabled us to identify several types of BLA INs innervated by VIP+ INs, including other IS-INs, basket and neurogliaform cells. Moreover, light stimulation of VIP+ basket cell axon terminals, characterized by CB1 sensitivity, evoked IPSPs in ∼20% of principal neurons. Finally, we show that VIP+ INs receive a dense innervation from both GABAergic inputs (although only 10% from other VIP+ INs) and distinct glutamatergic inputs, identified by their expression of different vesicular glutamate transporters.In conclusion, our study provides a wide-range analysis of single-cell properties of VIP+ INs in the mouse BLA and of their intrinsic and extrinsic connectivity. Our results reinforce the evidence that VIP+ INs are structurally and functionally heterogeneous and that this heterogeneity could mediate different roles in amygdala-dependent functions.SIGNIFICANCE STATEMENT We provide the first comprehensive analysis of the distribution of vasoactive intestinal polypeptide-expressing (VIP+) interneurons (INs) across the entire mouse amygdaloid basolateral complex (BLA), as well as of their morphological and physiological properties. VIP+ INs in the neocortex preferentially target other INs to form a disinhibitory network that facilitates principal cell firing. Our study is the first to demonstrate the presence of such a disinhibitory circuitry in the BLA. We observed structural and functional heterogeneity of these INs and characterized their input/output connectivity. We also identified several types of BLA INs that, when inhibited, may provide a temporal window for principal cell firing and facilitate associative plasticity, e.g., in fear learning.

Anatomically heterogeneous populations of CB1 cannabinoid receptor-expressing interneurons in the CA3 region of the hippocampus show homogeneous input-output characteristics.

A subpopulation of GABAergic cells in cortical structures expresses CB1 cannabinoid receptors (CB1 ) on their axon terminals. To understand the function of these interneurons in information processing, it is necessary to uncover how they are embedded into neuronal circuits. Therefore, the proportion of GABAergic terminals expressing CB1 and the morphological and electrophysiological properties of CB1 -immunoreactive interneurons should be revealed. We investigated the ratio and the origin of CB1 -expressing inhibitory boutons in the CA3 region of the hippocampus. Using immunocytochemical techniques, we estimated that ∼40% of GABAergic axon terminals in different layers of CA3 also expressed CB1 . To identify the inhibitory cell types expressing CB1 in this region, we recorded and intracellularly labeled interneurons in hippocampal slices. CB1 -expressing interneurons showed distinct axonal arborization, and were classified as basket cells, mossy-fiber-associated cells, dendritic-layer-innervating cells or perforant-path-associated cells. In each morphological category, a substantial variability in axonal projection was observed. In contrast to the diverse morphology, the active and passive membrane properties were found to be rather similar. Using paired recordings, we found that pyramidal cells displayed large and fast unitary postsynaptic currents in response to activating basket and mossy-fiber-associated cells, while they showed slower and smaller synaptic events in pairs originating from interneurons that innervate the dendritic layer, which may be due to dendritic filtering. In addition, CB1 activation significantly reduced the amplitude of the postsynaptic currents in each cell pair tested. Our data suggest that CB1 -expressing interneurons with different axonal projections have comparable physiological characteristics, contributing to a similar proportion of GABAergic inputs along the somato-dendritic axis of CA3 pyramidal cells.

Functionally linked amygdala and prefrontal cortical regions are innervated by both single and double projecting cholinergic neurons.

Cholinergic cells have been proposed to innervate simultaneously those cortical areas that are mutually interconnected with each other. To test this hypothesis, we investigated the cholinergic innervation of functionally linked amygdala and prefrontal cortical regions. First, using tracing experiments, we determined that cholinergic cells located in distinct basal forebrain (BF) areas projected to the different nuclei of the basolateral amygdala (BLA). Specifically, cholinergic cells in the ventral pallidum/substantia innominata (VP/SI) innervated the basal nucleus (BA), while the horizontal limb of the diagonal band of Broca (HDB) projected to its basomedial nucleus (BMA). In addition, cholinergic neurons in these two BF areas gave rise to overlapping innervation in the medial prefrontal cortex (mPFC), yet their axons segregated in the dorsal and ventral regions of the PFC. Using retrograde-anterograde viral tracing, we demonstrated that a portion of mPFC-projecting cholinergic neurons also innervated the BLA, especially the BA. By injecting retrograde tracers into the mPFC and BA, we found that 28% of retrogradely labeled cholinergic cells were double labeled, which typically located in the VP/SI. In addition, we found that vesicular glutamate transporter type 3 (VGLUT3)-expressing neurons within the VP/SI were also cholinergic and projected to the mPFC and BA, implicating that a part of the cholinergic afferents may release glutamate. In contrast, we uncovered that GABA is unlikely to be a co-transmitter molecule in HDB and VP/SI cholinergic neurons in adult mice. The dual innervation strategy, i.e., the existence of cholinergic cell populations with single as well as simultaneous projections to the BLA and mPFC, provides the possibility for both synchronous and independent control of the operation in these cortical areas, a structural arrangement that may maximize computational support for functionally linked regions. The presence of VGLUT3 in a portion of cholinergic afferents suggests more complex functional effects of cholinergic system in cortical structures.

Network-driven cancer cell avatars for combination discovery and biomarker identification for DNA damage response inhibitors.

Combination therapy is well established as a key intervention strategy for cancer treatment, with the potential to overcome monotherapy resistance and deliver a more durable efficacy. However, given the scale of unexplored potential target space and the resulting combinatorial explosion, identifying efficacious drug combinations is a critical unmet need that is still evolving. In this paper, we demonstrate a network biology-driven, simulation-based solution, the Simulated Cell™. Integration of omics data with a curated signaling network enables the accurate and interpretable prediction of 66,348 combination-cell line pairs obtained from a large-scale combinatorial drug sensitivity screen of 684 combinations across 97 cancer cell lines (BAC = 0.62, AUC = 0.7). We highlight drug combination pairs that interact with DNA Damage Response pathways and are predicted to be synergistic, and deep network insight to identify biomarkers driving combination synergy. We demonstrate that the cancer cell 'avatars' capture the biological complexity of their in vitro counterparts, enabling the identification of pathway-level mechanisms of combination benefit to guide clinical translatability.

Endocannabinoid-mediated long-term depression of afferent excitatory synapses in hippocampal pyramidal cells and GABAergic interneurons.

Although endocannabinoids have emerged as essential retrograde messengers in several forms of synaptic plasticity, it remains controversial whether they mediate long-term depression (LTD) of glutamatergic synapses onto excitatory and inhibitory neurons in the hippocampus. Here, we show that parvalbumin- and somatostatin/metabotropic glutamate receptor 1(a) (mGlu(1a))-positive GABAergic interneurons express diacylglycerol lipase-α (DGL-α), a synthesizing enzyme of the endocannabinoid 2-arachidonoylglycerol (2-AG), albeit at lower levels than principal cells. Moreover, this lipase accumulates postsynaptically around afferent excitatory synapses in all three cell types. To address the role of retrograde 2-AG signaling in LTD, we investigated two forms: (1) produced by postsynaptic spiking paired with subsequent presynaptic stimulation or (2) induced by group I mGlu activation by (S)-3,5-dihydroxyphenylglycine (DHPG). Neither form of LTD was evoked in the presence of the mGlu(5) antagonist MPEP [2-methyl-6-(phenylethynyl)-pyridine], the DGL inhibitor THL [N-formyl-l-leucine (1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]methyl]dodecyl ester], or the intracellularly applied Ca(2+) chelator BAPTA in CA1 pyramidal cells, fast-spiking interneurons (representing parvalbumin-containing cells) and interneurons projecting to stratum lacunosum-moleculare (representing somatostatin/mGlu(1a)-expressing interneurons). Both forms of LTD were completely absent in CB(1) cannabinoid receptor knock-out mice, whereas pharmacological blockade of CB(1) led to inconsistent results. Notably, in accordance with their lower DGL-α level, a higher stimulation frequency or higher DHPG concentration was required for LTD induction in interneurons compared with pyramidal cells. These findings demonstrate that hippocampal principal cells and interneurons produce endocannabinoids to mediate LTD in a qualitatively similar, but quantitatively different manner. The shifted induction threshold implies that endocannabinoid-LTD contributes to cortical information processing during distinct network activity patterns in a cell type-specific manner.

Different input and output properties characterize parvalbumin-positive basket and Axo-axonic cells in the hippocampal CA3 subfield.

In the hippocampus, parvalbumin-expressing basket (BC) and axo-axonic cells (AAC) show different discharge patterns during distinct network states, but the cellular mechanisms underlying these differences are not well understood. Using whole-cell patch-clamp techniques, we investigated the single-cell properties and excitatory synaptic features of anatomically identified BCs and AACs in the CA3 region of mouse hippocampal slices. The results showed that BCs had lower threshold for action potential (AP) generation and lower input resistance, narrower AP and afterhyperpolarization than AACs. In addition, BCs fired with higher frequencies and with more modest accommodation compared with AACs. The kinetic properties of excitatory postsynaptic currents (EPSC), the rectification of AMPA receptor-mediated currents, the fraction of the NMDA receptor-mediated component in EPSCs, and the EPSC magnitude necessary to evoke an AP were similar in both cell types. However, smaller excitatory postsynaptic potential and lower intensity fiber stimulation in stratum oriens was necessary to drive firing in BCs. Moreover, the rate of spontaneous EPSCs in BCs was higher than in AACs. Neurolucida analysis revealed that the dendrites of BCs in strata radiatum and oriens were longer and more extensively ramified. Since the density of the excitatory synapses was estimated to be comparable in both cell types, we conclude that the more elaborated dendritic arbor of BCs ensures that they receive a larger number of proximal excitatory inputs. Thus, CA3 pyramidal cells more profoundly innervate BCs than AACs, which could explain, at least in part, their distinct spiking behavior under different hippocampal network activities.

Fear learning and aversive stimuli differentially change excitatory synaptic transmission in perisomatic inhibitory cells of the basal amygdala.

Inhibitory circuits in the basal amygdala (BA) have been shown to play a crucial role in associative fear learning. How the excitatory synaptic inputs received by BA GABAergic interneurons are influenced by memory formation, a network parameter that may contribute to learning processes, is still largely unknown. Here, we investigated the features of excitatory synaptic transmission received by the three types of perisomatic inhibitory interneurons upon cue-dependent fear conditioning and aversive stimulus and tone presentations without association. Acute slices were prepared from transgenic mice: one group received tone presentation only (conditioned stimulus, CS group), the second group was challenged by mild electrical shocks unpaired with the CS (unsigned unconditioned stimulus, unsigned US group) and the third group was presented with the CS paired with the US (signed US group). We found that excitatory synaptic inputs (miniature excitatory postsynaptic currents, mEPSCs) recorded in distinct interneuron types in the BA showed plastic changes with different patterns. Parvalbumin (PV) basket cells in the unsigned US and signed US group received mEPSCs with reduced amplitude and rate in comparison to the only CS group. Coupling the US and CS in the signed US group caused a slight increase in the amplitude of the events in comparison to the unsigned US group, where the association of CS and US does not take place. Excitatory synaptic inputs onto cholecystokinin (CCK) basket cells showed a markedly different change from PV basket cells in these behavioral paradigms: only the decay time was significantly faster in the unsigned US group compared to the only CS group, whereas the amplitude of mEPSCs increased in the signed US group compared to the only CS group. Excitatory synaptic inputs received by PV axo-axonic cells showed the least difference in the three behavioral paradigm: the only significant change was that the rate of mEPSCs increased in the signed US group when compared to the only CS group. These results collectively show that associative learning and aversive stimuli unpaired with CS cause different changes in excitatory synaptic transmission in BA perisomatic interneuron types, supporting the hypothesis that they play distinct roles in the BA network operations upon pain information processing and fear memory formation.

The effects of an Echinacea preparation on synaptic transmission and the firing properties of CA1 pyramidal cells in the hippocampus.

Traditionally, Echinacea preparations are used as antiinflammatory agents and immune-enhancers. In addition to these effects, their anxiolytic potency has been recognized recently in laboratory tests. Our aim in this study was to uncover the potential effects of an Echinacea preparation on neuronal operations in the hippocampus, a brain region that is involved in anxiety and anxiety-related behaviors. Using in vitro electrophysiological techniques, we observed that excitatory synaptic transmission in hippocampal slices was significantly suppressed by an Echinacea extract found to be effective in anxiety tests. In contrast, no change in inhibitory synaptic transmission could be detected upon application of this extract. In addition, our experiments revealed that at low concentration the Echinacea extract reduced the spiking activity of CA1 pyramidal cells, while at high concentration increased it. This latter observation was parallel to the reduction in the magnitude of the h-current-mediated voltage responses in pyramidal cells. At any concentrations, the passive membrane properties of CA1 pyramidal cells were found to be unaltered by the Echinacea extract. In summary, the Echinacea extract can significantly regulate excitatory, but not inhibitory, synaptic transmission in the hippocampus, and this action might be involved in its anxiolytic effects observed in behaviour tests.

Hippocampal sharp wave-ripples and the associated sequence replay emerge from structured synaptic interactions in a network model of area CA3.

Hippocampal place cells are activated sequentially as an animal explores its environment. These activity sequences are internally recreated ('replayed'), either in the same or reversed order, during bursts of activity (sharp wave-ripples [SWRs]) that occur in sleep and awake rest. SWR-associated replay is thought to be critical for the creation and maintenance of long-term memory. In order to identify the cellular and network mechanisms of SWRs and replay, we constructed and simulated a data-driven model of area CA3 of the hippocampus. Our results show that the chain-like structure of recurrent excitatory interactions established during learning not only determines the content of replay, but is essential for the generation of the SWRs as well. We find that bidirectional replay requires the interplay of the experimentally confirmed, temporally symmetric plasticity rule, and cellular adaptation. Our model provides a unifying framework for diverse phenomena involving hippocampal plasticity, representations, and dynamics, and suggests that the structured neural codes induced by learning may have greater influence over cortical network states than previously appreciated.

Cannabinoids attenuate hippocampal γ oscillations by suppressing excitatory synaptic input onto CA3 pyramidal neurons and fast spiking basket cells.

CB(1) cannabinoid receptor (CB(1)R) activation by exogenous ligands can impair memory processes, which critically depend on synchronous neuronal activities that are temporarily structured by oscillations. In this study, we aimed to reveal the mechanisms underlying the cannabinoid-induced decrease in gamma oscillations. We first verified that cannabinoids (CP55,940 and WIN55,212-2) readily suppressed carbachol-induced gamma oscillations in the CA3 region of hippocampal slices via activation of CB(1)Rs. The cannabinoid-induced decrease in the peak power of oscillations was accompanied by reduced and less precise firing activity in CA3 pyramidal cells and fast spiking basket cells. By examining the cannabinoid sensitivity of synaptic inputs we found that the amplitude of evoked excitatory postsynaptic currents was significantly suppressed upon CB(1)R activation in both CA3 pyramidal cells and fast spiking basket cells. In contrast, evoked inhibitory postsynaptic currents in CA3 pyramidal cells were unaltered. Furthermore, we observed that a CB(1)R agonist-induced decrease in the oscillation power at the beginning of the drug application was accompanied primarily by the reduced discharge of fast spiking basket cells, while pyramidal cell firing was unaltered. This result implies that the dampening of cholinergically induced gamma oscillations in the hippocampus by cannabinoids can be explained by a reduced excitatory input predominantly onto fast spiking basket cells, which leads to a reduction in neuronal firing frequency and precision, and thus to smaller field potentials. In addition, we uncovered that the spontaneously occurring sharp wave-ripple activities in hippocampal slices could also be suppressed by CB(1)R activation suggesting that cannabinoids profoundly reduce the intrinsically generated oscillatory activities at distinct frequencies in CA3 networks by reducing synaptic neurotransmission.

Fruit juices are effective anti-amyloidogenic agents.

Amyloid fibril formation has been associated with a great variety of human diseases. Fruits contain different important bioactive molecules without causing various undesirable side effects, which are necessary for disease prevention and treatment. Here we report that various fruit juices inhibited the amyloid formation by α-chymotrypsin in aqueous ethanol at pH 7.0. Turbidity measurements, total phenolic content determination, as well as Congo red binding assay were used to analyse the inhibition of amyloid fibril formation. We showed that the black currant juice possessed the strongest inhibitory potential against protein aggregation because it contains the most polyphenolic compounds too and its effect was concentration dependent. Interestingly, white grapes, figs and bananas are relatively effective although they are not high in polyphenols. These fruits are typically sweet. The sugars in them also contribute to their effectiveness. Eating black currant can reduce the likelihood of formation of amyloid fibrils.

Organ Specific Copy Number Variations in Visceral Metastases of Human Melanoma.

Malignant melanoma is one of the most aggressive skin cancers with high potential of visceral dissemination. Since the information about melanoma genomics is mainly based on primary tumors and lymphatic or skin metastases, an autopsy-based visceral metastasis biobank was established. We used copy number variation arrays (N = 38 samples) to reveal organ specific alterations. Results were partly completed by proteomic analysis. A significant increase of high-copy number gains was found in an organ-specific manner, whereas copy number losses were predominant in brain metastases, including the loss of numerous DNA damage response genes. Amplification of many immune genes was also observed, several of them are novel in melanoma, suggesting that their ectopic expression is possibly underestimated. This "immunogenic mimicry" was exclusive for lung metastasis. We also provided evidence for the possible autocrine activation of c-MET, especially in brain and lung metastases. Furthermore, frequent loss of 9p21 locus in brain metastases may predict higher metastatic potential to this organ. Finally, a significant correlation was observed between BRAF gene copy number and mutant allele frequency, mainly in lung metastases. All of these events may influence therapy efficacy in an organ specific manner, which knowledge may help in alleviating difficulties caused by resistance.

Co-Inoculation of Organic Potato with Fungi and Bacteria at High Disease Severity of Rhizoctonia solani and Streptomyces spp. Increases Beneficial Effects.

Rhizobacteria-based technologies may constitute a viable option for biological fertilization and crop protection. The effects of two microbial inoculants (1) PPS: Pseudomonas protegens, P. jessenii and Stenotrophomonas maltophilia biocontrol bacterium strains and (2) TPB: Trichoderma atroviride, Pseudomonas putida, and Bacillus subtilis fungi, bacteria biocontrol, and biofertilizer combinations were examined on potato (Solanum tuberosum L. var. Demon) in three consecutive years in irrigated organic conditions. The number of tubers showing symptoms of Streptomyces sp. and Rhizoctonia sp. was recorded. The severity of symptoms was evaluated based on the damaged tuber surface. There was a large annual variability in both the symptoms caused by soil-borne pathogens, and the effect of bio-inoculants. In the first and second year, with a stronger Rhizoctonia and Streptomyces spp. incidence, the bacterial and fungal combination of TPB inoculums with both the potential plant nutrition and biocontrol ability of the strains seemed to have a better efficiency to control the diseases. This tendency was not supported in the third year, and this may be attributed to the relatively high natural precipitation. Further studies are required to investigate the agronomic benefits of these inoculants and to tailor their application to the soil microbial characteristics and weather conditions.

LAPTM4B gene copy number gain is associated with inferior response to anthracycline-based chemotherapy in hormone receptor negative breast carcinomas.

PURPOSE: To determine the associations between lysosomal-associated transmembrane protein 4b (LAPTM4B) gene copy number and response to different chemotherapy regimens in hormone receptor negative (HR-) primary breast carcinomas. PATIENTS AND METHODS: Two cohorts were analyzed: (1) 69 core biopsies from HR-breast carcinomas treated with neoadjuvant chemotherapy (anthracycline based in 72.5% of patients and non-anthracycline based in 27.5% of patients). (2) Tissue microarray (TMA) of 74 HR-breast carcinomas treated with adjuvant therapy (77.0% of the patients received anthracycline, 17.6% of the patients non-anthracycline-based therapy, and in 5.4% of the cases, no treatment data are available). Interphase FISH technique was applied on pretreatment core biopsies (cohort I) and on TMAs (cohort II) using custom-made dual-labelled FISH probes (LAPTM4B/CEN8q FISH probe Abnova Corp.). RESULTS: In the neoadjuvant cohort in the anthracycline-treated group, we observed a significant difference (p = 0.029) of average LAPTM4B copy number between the non-responder and pathological complete responder groups (4.1 ± 1.1 vs. 2.6 ± 0.1). In the adjuvant setting, the anthracycline-treated group of metastatic breast carcinomas was characterized by higher LAPTM4B copy number comparing to the non-metastatic ones (p = 0.046). In contrast, in the non-anthracycline-treated group of patients, we did not find any LAPTM4B gene copy number differences between responder vs. non-responder groups or between metastatic vs. non-metastatic groups. CONCLUSION: Our results confirm the possible role of the LAPTM4B gene in anthracycline resistance in HR- breast cancer. Analyzing LAPTM4B copy number pattern may support future treatment decision.

Altered integrin expression patterns shown by microarray in human cutaneous melanoma.

A large variety of molecular pathways in melanoma progression suggests that no individual molecular alteration is crucial in itself. Our aim was to define the molecular alterations underlying metastasis formation. Gene expression profiling was performed using microarray and qRT-PCR to define alterations between matched primary and metastatic melanoma cell lines. These data were integrated with publicly available unmatched tissue data. The invasiveness of cell lines was determined by Matrigel invasion assays and invasive clones from primary melanoma-derived cell lines were also selected. Two metastatic cell line models were created: the regional lymph node WM983A-WM983A-WM983B and the distant lung WM793B-WM793B-1205Lu metastatic models. The majority of metastasis genes were downregulated and enriched in adhesion and ITGA6-B4 pathways. Upregulation of immune pathways was characteristic of distant metastases, whereas increased Rap1 signaling was specific for regional (sub)cutaneous metastases. qRT-PCR analysis of selected integrins (A2, A3, A4, A9, B5, B8, A6, B1, and B3) highlighted the possible importance of ITGA3/4 and B8 in the metastatic process, distinguishing regional and distant metastases. We identified functionally relevant gene clusters that influenced metastasis formation. Our data provide further evidence that integrin expression patterns may be important in distant metastasis formation.

Genomic profiling of invasive melanoma cell lines by array comparative genomic hybridization.

Malignant melanoma is one of the most aggressive human cancers. Invasion of cells is the first step in metastasis, resulting in cell migration through tissue compartments. We aimed to evaluate genomic alterations specifically associated with the invasive characteristics of melanoma cells. Matrigel invasion assays were used to determine the invasive properties of cell lines that originated from primary melanomas. Array comparative genomic hybridization analyses were carried out to define the chromosome copy number alterations (CNAs). Several recurrent CNAs were identified by array comparative genomic hybridization that affected melanoma-related genes. Invasive primary cell lines showed high frequencies of CNAs, including the loss of 7q and gain of 12q chromosomal regions targeting PTPN12, ADAM22, FZD1, TFPI2, GNG11, COL1A2, SMURF1, VGF, RELN and GLIPR1 genes. Gain of the GDNF (5p13.1), GPAA1, PLEC and SHARPIN (8q24.3) genes was significantly more frequent in invasive cell lines compared with the noninvasive ones. Importantly, copy number gains of these genes were also found in cell lines that originated from metastases, suggesting their role in melanoma metastasis formation. The present study describes genomic differences between invasive and noninvasive melanoma cell lines that may contribute toward the aggressive phenotype of human melanoma cells.