Correction: Brüning-Richardson et al. GSK-3 Inhibition Is Cytotoxic in Glioma Stem Cells through Centrosome Destabilization and Enhances the Effect of Radiotherapy in Orthotopic Models. Cancers 2021, 13, 5939.

The authors wish to make the following corrections to this paper [1][...].

GSK-3 Inhibition Is Cytotoxic in Glioma Stem Cells through Centrosome Destabilization and Enhances the Effect of Radiotherapy in Orthotopic Models.

BACKGROUND: Previous data on glycogen synthase kinase 3 (GSK-3) inhibition in cancer models support a cytotoxic effect with selectivity for tumor cells compared to normal tissue but the effect of these inhibitors in glioma has not been widely studied. Here, we investigate their potential as cytotoxics in glioma. METHODS: We assessed the effect of pharmacologic GSK-3 inhibition on established (U87, U251) and patient-derived (GBM1, GBM4) glioblastoma (GBM) cell lines using cytotoxicity assays as well as undertaking a detailed investigation of the effect on cell cycle, mitosis, and centrosome biology. We also assessed drug uptake and efficacy of GSK-3 inhibition alone and in combination with radiation in xenograft models. RESULTS: Using the selective GSK-3 inhibitor AZD2858, we demonstrated single agent cytotoxicity in two patient-derived glioma cell lines (GBM1, GBM4) and two established cell lines (U251 and U87) with IC50 in the low micromolar range promoting centrosome disruption, failed mitosis, and S-phase arrest. Glioma xenografts exposed to AZD2858 also showed growth delay compared to untreated controls. Combined treatment with radiation increased the cytotoxic effect of clinical radiation doses in vitro and in orthotopic glioma xenografts. CONCLUSIONS: These data suggest that GSK-3 inhibition promotes cell death in glioma through disrupting centrosome function and promoting mitotic failure and that AZD2858 is an effective adjuvant to radiation at clinical doses.

Drug Resistance in Glioma Cells Induced by a Mesenchymal-Amoeboid Migratory Switch.

Cancer cell invasion is a precondition for tumour metastasis and represents one of the most devastating characteristics of cancer. The development of drugs targeting cell migration, known as migrastatics, may improve the treatment of highly invasive tumours such as glioblastoma (GBM). In this study, investigations into the role of the cell adhesion protein Cellular communication network factor 1 (CCN1, also known as CYR61) in GBM cell migration uncovered a drug resistance mechanism adopted by cells when treated with the small molecule inhibitor CCG-1423. This inhibitor binds to importin α/ß inhibiting the nuclear translocation of the transcriptional co-activator MKL1, thus preventing downstream effects including migration. Despite this reported role as an inhibitor of cell migration, we found that CCG-1423 treatment did not inhibit GBM cell migration. However, we could observe cells now migrating by mesenchymal-amoeboid transition (MAT). Furthermore, we present evidence that CCN1 plays a critical role in the progression of GBM with increased expression in higher-grade tumours and matched blood samples. These findings support a potential role for CCN1 as a biomarker for the monitoring and potentially early prediction of GBM recurrence, therefore as such could help to improve treatment of and increase survival rates of this devastating disease.

Ex-vivo recellularisation and stem cell differentiation of a decellularised rat dental pulp matrix.

Implementing the principles of tissue engineering within the clinical management of non-vital immature permanent teeth is of clinical interest. However, the ideal scaffold remains elusive. The aim of this work was to assess the feasibility of decellularising rat dental pulp tissue and evaluate the ability of such scaffold to support stem cell repopulation. Rat dental pulps were retrieved and divided into control and decellularised groups. The decellularisation protocol incorporated a low detergent concentration and hypotonic buffers. After decellularisation, the scaffolds were characterised histologically, immunohistochemistry and the residual DNA content quantified. Surface topography was also viewed under scanning electron microscopy. Biocompatibility was evaluated using cytotoxicity assays utilising L-929 cell line. Decellularised scaffolds were recellularised with human dental pulp stem cells up to 14 days in vitro. Cellular viability was assessed using LIVE/DEAD stain kit and the recellularised scaffolds were further assessed histologically and immunolabelled using makers for odontoblastic differentiation, cytoskeleton components and growth factors. Analysis of the decellularised scaffolds revealed an acellular matrix with histological preservation of structural components. Decellularised scaffolds were biocompatible and able to support stem cell survival following recellularisation. Immunolabelling of the recellularised scaffolds demonstrated positive cellular expression against the tested markers in culture. This study has demonstrated the feasibility of developing a biocompatible decellularised dental pulp scaffold, which is able to support dental pulp stem cell repopulation. Clinically, decellularised pulp tissue could possibly be a suitable scaffold for use within regenerative (reparative) endodontic techniques.

A novel workflow for three-dimensional analysis of tumour cell migration.

The limitations of two-dimensional analysis in three-dimensional (3D) cellular imaging impair the accuracy of research findings in biological studies. Here, we report a novel 3D approach to acquisition, analysis and interpretation of tumour spheroid images. Our research interest in mesenchymal-amoeboid transition led to the development of a workflow incorporating the generation and analysis of 3D data with instant structured illumination microscopy and a new ImageJ plugin.

Fibroblast-specific deletion of interleukin-1 receptor-1 reduces adverse cardiac remodeling following myocardial infarction.

It has been hypothesized that interleukin-1alpha (IL-1α) is released from damaged cardiomyocytes following myocardial infarction (MI) and activates cardiac fibroblasts via its receptor (IL-1R1) to drive the early stages of cardiac remodeling. This study aimed to definitively test this hypothesis using cell type-specific IL-1α and IL-1R1 knockout (KO) mouse models. A floxed Il1α mouse was created and used to generate a cardiomyocyte-specific IL-1α KO mouse line (MIL1AKO). A tamoxifen-inducible fibroblast-specific IL-1R1 hemizygous KO mouse line (FIL1R1KO) was also generated. Mice underwent experimental MI (permanent left anterior descending coronary artery ligation) and cardiac function was determined 4 weeks later by conductance pressure-volume catheter analysis. Molecular markers of remodeling were evaluated at various time points by real-time RT-PCR and histology. MIL1AKO mice showed no difference in cardiac function or molecular markers of remodeling post-MI compared with littermate controls. In contrast, FIL1R1KO mice showed improved cardiac function and reduced remodeling markers post-MI compared with littermate controls. In conclusion, these data highlight a key role for the IL-1R1/cardiac fibroblast signaling axis in regulating post-MI remodeling and provide support for the continued development of anti-IL-1 therapies for improving cardiac function after MI. Cardiomyocyte-derived IL-1α was not an important contributor to post-MI remodeling in this model.

FGFR1 Expression and Role in Migration in Low and High Grade Pediatric Gliomas.

The heterogeneous and invasive nature of pediatric gliomas poses significant treatment challenges, highlighting the importance of identifying novel chemotherapeutic targets. Recently, recurrent Fibroblast growth factor receptor 1 (FGFR1) mutations in pediatric gliomas have been reported. Here, we explored the clinical relevance of FGFR1 expression, cell migration in low and high grade pediatric gliomas and the role of FGFR1 in cell migration/invasion as a potential chemotherapeutic target. A high density tissue microarray (TMA) was used to investigate associations between FGFR1 and activated phosphorylated FGFR1 (pFGFR1) expression and various clinicopathologic parameters. Expression of FGFR1 and pFGFR1 were measured by immunofluorescence and by immunohistochemistry (IHC) in 3D spheroids in five rare patient-derived pediatric low-grade glioma (pLGG) and two established high-grade glioma (pHGG) cell lines. Two-dimensional (2D) and three-dimensional (3D) migration assays were performed for migration and inhibitor studies with three FGFR1 inhibitors. High FGFR1 expression was associated with age, malignancy, tumor location and tumor grade among astrocytomas. Membranous pFGFR1 was associated with malignancy and tumor grade. All glioma cell lines exhibited varying levels of FGFR1 and pFGFR1 expression and migratory phenotypes. There were significant anti-migratory effects on the pHGG cell lines with inhibitor treatment and anti-migratory or pro-migratory responses to FGFR1 inhibition in the pLGGs. Our findings support further research to target FGFR1 signaling in pediatric gliomas.

Epidermal Growth Factor Like-domain 7 and miR-126 are abnormally expressed in diffuse Systemic Sclerosis fibroblasts.

Systemic sclerosis (SSc) is characterized by microangiopathy with impaired reparative angiogenesis and fibrosis. Epidermal Growth Factor Like-domain 7 (EGFL7), firstly described in endothelial cells plays a pivotal role in angiogenesis. Fibroblasts (FBs) are involved in vascular remodeling, under physiological and pathological conditions. In this study, we investigated: (i) the expression of EGFL7 and its miR-126 in patients affected by diffuse cutaneous SSc (dcSSc); (ii) the ability of Transforming Growth Factor-beta (TGF-ß) to modulate EGFL7 expression; (iii) the ability of EGFL7 to modulate COL1A1 expression and proliferation/migration, and (iv) the functional role of EGFL7 on angiogenesis. Patients were divided in 2 subsets: patients fulfilling the classification criteria in less than one year from Raynaud's Phenomenon onset (Early Onset Subset-EOS), and all the others (Long Standing Subset-LSS). We show that EGFL7 expression is increased in EOS dcSSc skin and cultured FBs. EGFL7 is inducible by TGF-ß on Healthy Controls (HC) FBs but not in SSc-FBs. EGFL7 decreases COL1A1 expression in EOS SSc-FBs while EGFL7 silencing up-regulates COL1A1 expression. EGFL7 promotes migration/invasion of EOS SSc-FBs but not proliferation. Finally, SSc-FBs, partially inhibit angiogenesis in organotypic coculture assays, and this is reversed by treatment with human recombinant (rh)EGFL7. We conclude that EGFL7 and its specific microRNA miR-126 may be involved in the pathogenesis of SSc vasculopathy and fibrosis.

Transforming Growth Factor ß Activation Primes Canonical Wnt Signaling Through Down-Regulation of Axin-2.

OBJECTIVE: Aberrant activation of Wnt signaling has been observed in tissues from patients with systemic sclerosis (SSc). This study aimed to determine the role of transforming growth factor ß (TGFß) in driving the increased Wnt signaling, through modulation of axis inhibition protein 2 (Axin-2), a critical regulator of the Wnt canonical pathway. METHODS: Canonical Wnt signaling activation was analyzed by TOPflash T cell factor/lymphoid enhancer factor promoter assays. Axin-2 was evaluated in vitro by analysis of Axin-2 primary/mature transcript expression and decay, TGFß receptor type I (TGFßRI) blockade, small interfering RNA-mediated depletion of tristetraprolin 1, and XAV-939-mediated Axin-2 stabilization. In vivo, Axin-2 messenger RNA (mRNA) and protein expression was determined in skin and lung biopsy samples from mice that express a kinase-deficient TGFßRII specifically on fibroblasts (TßRIIΔk-fib-transgenic mice) and from littermate controls. RESULTS: SSc fibroblasts displayed an increased response to canonical Wnt ligands despite basal levels of Wnt signaling that were comparable to those in healthy control fibroblasts in vitro. Notably, we showed that SSc fibroblasts had reduced basal expression of Axin-2, which was caused by an endogenous TGFß-dependent increase in Axin-2 mRNA decay. Accordingly, we observed that TGFß decreased Axin-2 expression both in vitro in healthy control fibroblasts and in vivo in TßRIIΔk-fib-transgenic mice. Additionally, using Axin-2 gain- and loss-of-function experiments, we demonstrated that the TGFß-induced increased response to Wnt activation characteristic of SSc fibroblasts depended on reduced bioavailability of Axin-2. CONCLUSION: This study highlights the importance of reduced bioavailability of Axin-2 in mediating the increased canonical Wnt response observed in SSc fibroblasts. This novel mechanism extends our understanding of the processes involved in Wnt/ß-catenin-driven pathology and supports the rationale for targeting the TGFß pathway to regulate the aberrant Wnt signaling observed during fibrosis.

Scleroderma fibroblasts suppress angiogenesis via TGF-ß/caveolin-1 dependent secretion of pigment epithelium-derived factor.

OBJECTIVES: Systemic sclerosis (SSc) is characterised by tissue fibrosis and vasculopathy with defective angiogenesis. Transforming growth factor beta (TGF-ß) plays a major role in tissue fibrosis, including downregulation of caveolin-1 (Cav-1); however, its role in defective angiogenesis is less clear. Pigment epithelium-derived factor (PEDF), a major antiangiogenic factor, is abundantly secreted by SSc fibroblasts. Here, we investigated the effect of TGF-ß and Cav-1 on PEDF expression and the role of PEDF in the ability of SSc fibroblasts to modulate angiogenesis. METHODS: PEDF and Cav-1 expression in fibroblasts and endothelial cells were evaluated by means of immunohistochemistry on human and mouse skin biopsies. PEDF and Cav-1 were silenced in cultured SSc and control fibroblasts using lentiviral short-hairpin RNAs. Organotypic fibroblast-endothelial cell co-cultures and matrigel assays were employed to assess angiogenesis. RESULTS: PEDF is highly expressed in myofibroblasts and reticular fibroblasts with low Cav-1 expression in SSc skin biopsies, and it is induced by TGF-ß in vitro. SSc fibroblasts suppress angiogenesis in an organotypic model. This model is reproduced by silencing Cav-1 in normal dermal fibroblasts. Conversely, silencing PEDF in SSc fibroblasts rescues their antiangiogenic phenotype. Consistently, transgenic mice with TGF-ß receptor hyperactivation show lower Cav-1 and higher PEDF expression levels in skin biopsies accompanied by reduced blood vessel density. CONCLUSIONS: Our data reveal a new pathway by which TGF-ß suppresses angiogenesis in SSc, through decreased fibroblast Cav-1 expression and subsequent PEDF secretion. This pathway may present a promising target for new therapeutic interventions in SSc.

Affimer proteins are versatile and renewable affinity reagents.

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications.

Tenascin C upregulates interleukin-6 expression in human cardiac myofibroblasts via toll-like receptor 4.

AIM: To investigate the effect of Tenascin C (TNC) on the expression of pro-inflammatory cytokines and matrix metalloproteinases in human cardiac myofibroblasts (CMF). METHODS: CMF were isolated and cultured from patients undergoing coronary artery bypass grafting. Cultured cells were treated with either TNC (0.1 µmol/L, 24 h) or a recombinant protein corresponding to different domains of the TNC protein; fibrinogen-like globe (FBG) and fibronectin type III-like repeats (TNIII 5-7) (both 1 µmol/L, 24 h). The expression of the pro-inflammatory cytokines; interleukin (IL)-6, IL-1ß, TNFα and the matrix metalloproteinases; MMPs (MMP1, 2, 3, 9, 10, MT1-MMP) was assessed using real time RT-PCR and western blot analysis. RESULTS: TNC increased both IL-6 and MMP3 (P < 0.01) mRNA levels in cultured human CMF but had no significant effect on the other markers studied. The increase in IL-6 mRNA expression was mirrored by an increase in protein secretion as assessed by enzyme-linked immunosorbant assay (P < 0.01). Treating CMF with the recombinant protein FBG increased IL-6 mRNA and protein (P < 0.01) whereas the recombinant protein TNIII 5-7 had no effect. Neither FBG nor TNIII 5-7 had any significant effect on MMP3 expression. The expression of toll-like receptor 4 (TLR4) in human CMF was confirmed by real time RT-PCR, western blot and immunohistochemistry. Pre-incubation of cells with TLR4 neutralising antisera attenuated the effect of both TNC and FBG on IL-6 mRNA and protein expression. CONCLUSION: TNC up-regulates IL-6 expression in human CMF, an effect mediated through the FBG domain of TNC and via the TLR4 receptor.

High-content analysis of tumour cell invasion in three-dimensional spheroid assays.

Targeting infiltrating tumour cells is an attractive way of combating cancer invasion and metastasis. Here we describe a novel and reproducible method for high content analysis of invading cells using multicellular tumour spheroid assays in a high grade glioma model. Live cell imaging of spheroids generated from glioma cell lines, U87 and U251, gave insight into migration dynamics and cell morphology in response to anti-migratory drugs. Immunofluorescence imaging confirmed cytoskeletal rearrangements in the treated cells indicating a direct effect on cell morphology. Effect on migration was determined by a Migration Index (MI) from brightfield images which confirmed anti-migratory activity of the drugs. A marked effect on the core with treatment suggestive of disordered proliferation was also observed. A newly developed technique to prepare the spheroids and migratory cells for immunohistochemistry allowed an assessment of response to drug treatment with a selection of markers. A difference in protein expression was noted between cells maintained within the core and migratory cells indicative of the presence of cell subpopulations within the spheroid core. We conclude that this high content analysis allows researchers to perform screening of anti-tumour invasion compounds and study their effects on cellular dynamics, particularly in relation to protein expression, for the first time.

A Rac/Cdc42 exchange factor complex promotes formation of lateral filopodia and blood vessel lumen morphogenesis.

During angiogenesis, Rho-GTPases influence endothelial cell migration and cell-cell adhesion; however it is not known whether they control formation of vessel lumens, which are essential for blood flow. Here, using an organotypic system that recapitulates distinct stages of VEGF-dependent angiogenesis, we show that lumen formation requires early cytoskeletal remodelling and lateral cell-cell contacts, mediated through the RAC1 guanine nucleotide exchange factor (GEF) DOCK4 (dedicator of cytokinesis 4). DOCK4 signalling is necessary for lateral filopodial protrusions and tubule remodelling prior to lumen formation, whereas proximal, tip filopodia persist in the absence of DOCK4. VEGF-dependent Rac activation via DOCK4 is necessary for CDC42 activation to signal filopodia formation and depends on the activation of RHOG through the RHOG GEF, SGEF. VEGF promotes interaction of DOCK4 with the CDC42 GEF DOCK9. These studies identify a novel Rho-family GTPase activation cascade for the formation of endothelial cell filopodial protrusions necessary for tubule remodelling, thereby influencing subsequent stages of lumen morphogenesis.

Differential distribution of 5-HT 1A and 5-HT 1B-like immunoreactivities in rat central nucleus of the amygdala neurones projecting to the caudal dorsomedial medulla oblongata.

Neurones in the central nucleus of the amygdala (CeA) projecting to the caudal dorsomedial medulla oblongata play a key role in the autonomic expression of emotional behaviour. We have earlier shown that these projections from the CeA contain gamma-aminobutyric acid. The CeA receives a dense serotonergic innervation from the raphé nuclei and expresses several serotonin (5-hydroxytryptamine, 5-HT) receptor subtypes, including the 5-HT(1A) and 5-HT(1B) subtypes. However, there have been no reports on the cellular distribution of these receptors in CeA projection neurones. This study was therefore designed to investigate the localisation of 5-HT(1A) and 5-HT(1B) receptors in CeA projection neurones identified by microinjection of a retrograde tracer, cholera toxin B-subunit (CTb) into the caudal dorsomedial medulla, targeting projections to the nucleus of the solitary tract. A large proportion (approximately 60%) of amygdala neurones retrogradely-labelled with CTb expressed 5-HT(1B) receptor-like immunoreactivity, whereas fewer (approximately 30%) expressed 5-HT(1A) receptor-like immunoreactivity. The retrogradely-labelled neurones positive for 5-HT(1B) receptor were present in both lateral and medial parts of the CeA whereas 5-HT(1A) receptor positive neurones were located mainly in the lateral part of the CeA. Since 5-HT plays an important role in controlling emotional behaviour and the 5-HT(1A) and 5-HT(1B) receptors have been shown to have distinct roles in the regulation of anxiety and depression, the differential expression of these receptors in CeA neurones projecting to the caudal dorsomedial medulla suggests that these projection neurones may act differentially in controlling autonomic expression of emotional behaviour.

White matter lesions in an unselected cohort of the elderly: molecular pathology suggests origin from chronic hypoperfusion injury.

BACKGROUND AND PURPOSE: "Incidental" MRI white matter (WM) lesions, comprising periventricular lesions (PVLs) and deep subcortical lesions (DSCLs), are common in the aging brain. Direct evidence of ischemia associated with incidental WM lesions (WMLs) has been lacking, and their pathogenesis is unresolved. METHODS: A population-based, postmortem cohort (n=456) of donated brains was examined by MRI and pathology. In a subsample of the whole cohort, magnetic resonance images were used to sample and compare WMLs and nonlesional WM for molecular markers of hypoxic injury. RESULTS: PVL severity was associated with loss of ventricular ependyma (P=0.004). For DSCLs, there was arteriolar sclerosis compared with normal WM (vessel wall thickness and perivascular enlargement; both P<0.001). Capillary endothelial activation (ratio of intercellular adhesion molecule to basement membrane collagen IV; P<0.001) and microglial activation (CD68 expression; P=0.002) were elevated in WMLs. Immunoreactivity for hypoxia-inducible factors (HIFs) HIF1alpha and HIF2alpha was elevated in DSCLs (P=0.003 and P=0.005). Other hypoxia-regulated proteins were also increased in WMLs: matrix metalloproteinase-7 (PVLs P<0.001; DSCLs P=0.009) and the number of neuroglobin-positive cells (WMLs P=0.02) reaching statistical significance. The severity of congophilic amyloid angiopathy was associated with increased HIF1alpha expression in DSCLs (P=0.04). CONCLUSIONS: The data support a hypoxic environment within MRI WMLs. Persistent HIF expression may result from failure of normal adaptive mechanisms. WM ischemia appears to be a common feature of the aging brain.

Substance P (NK1) and somatostatin (sst2A) receptor immunoreactivity in NTS-projecting rat dorsal horn neurones activated by nociceptive afferent input.

Spinal neurones that receive inputs from primary afferent fibres and have axons projecting supraspinally to the medulla oblongata may represent a pathway through which nociceptive and non-nociceptive peripheral stimuli are able to modulate cardiorespiratory reflexes. Expression of the neurokinin-1 (NK1) receptor is believed to be an indicator of lamina I cells that receive nociceptive inputs from substance P releasing afferents, and similarly, sst2A receptor expression may be a marker for neurones receiving somatostatinergic inputs. In this study, immunoreactivity for these two receptors was investigated in rat spinal neurones retrogradely labelled by injections of cholera toxin B or Fluorogold into the nucleus of the solitary tract (NTS). In addition, nociceptive activation of these labelled cells was studied by immunodetection of Fos protein in response to cutaneous and visceral noxious chemical stimuli. NK1 and sst2A receptors in lamina I were localised to mainly separate populations of retrogradely labelled cells with fusiform, flattened and pyramidal morphologies. Examples of projection neurones expressing both receptors were, however observed. With visceral stimulation, many retrogradely labelled cells expressing c-fos were immunoreactive for the NK1 receptor, and a smaller population was sst2A positive. In contrast, with cutaneous stimulation, only NK1 positive retrogradely labelled cells showed c-fos expression. These data provide evidence that lamina I neurones receiving noxious cutaneous and visceral stimuli via NK1 receptor activation project to NTS and so may be involved in coordinating nociceptive and cardiorespiratory responses. Moreover, a subpopulation of projection neurones that respond to visceral stimuli may receive somatostatinergic inputs of peripheral, local or supraspinal origins.

Evidence for peptide co-transmission in retrograde- and anterograde-labelled central nucleus of amygdala neurones projecting to NTS.

Synaptic terminals in the nucleus of the solitary tract (NTS) from axons originating in the central nucleus of the amygdala (CeA) are known to contain gamma-aminobutyric acid (GABA) immunoreactivity. Here, we have investigated whether such projections contain neuropeptides as putative co-transmitters. Somata in the medial and lateral CeA that were retrogradely labelled with cholera toxin B (CTb) injected into the commissural NTS were found to be immunoreactive for GABA, somatostatin (SOM), neurotensin (NT), vasoactive intestinal polypeptide (VIP) and nitric oxide synthase (NOS). Subpopulations of fibres in the NTS that were anterogradely labelled with biotin dextran amine (BDA) injected into the CeA and examined using both fluorescence and electron microscopy appeared to colocalise somatostatin, but not other neuropeptides. Their varicosities were observed in proximity to NTS neurones that were immunoreactive for the somatostatin receptor sst2A subtype, substance P (SP) NK1 receptor, and the GABAA receptor alpha3, beta1 and gamma2 subunits. This morphological evidence is consistent with the possibility of GABA-somatostatin co-transmission at synapses of some of the CeA projection neurones to NTS that might inhibit cardiovascular reflex responses in response to fear or emotion-related stimuli.