Combined treatment with exercise and α5GABAAR inhibitor promotes motor function recovery after intracerebral hemorrhage.

The present study aimed to examine the synergistic effects of exercise and pharmacological inhibition of the α5 subunit-containing gamma-aminobutyric acid (GABA)A receptors (α5GABAAR) on motor function recovery after intracerebral hemorrhage (ICH). Wistar rats were divided into five groups (n = 8 per group): SHAM, ICH, ICH + exercise (ICH + EX), ICH + L-655,708 (ICH + L6), and ICH + L-655,708 and exercise (ICH + L6EX) groups. ICH was induced by microinjection of a collagenase solution. The ICH + EX and ICH + L6EX groups exercised on a treadmill (12 m/min for 30 min/day). L-655,708 (0.5 mg/kg), a negative allosteric modulator of α5GABAAR, was administered intraperitoneally to the ICH + L6 and ICH + L6EX groups. Each intervention was initiated 1 week after the ICH surgery and was performed for 3 weeks, followed by tissue collection, including the motor cortex and spinal cord. At 4 weeks after ICH, significant motor recovery was found in the ICH + L6EX group compared to the ICH group. L-655,708 administration increased brain-derived neurotrophic factor (BDNF) expression in the cortex. Regarding neuroplastic changes in the spinal cord, rats in the ICH + L6EX group showed a significant increase in several neuroplastic markers: 1) BDNF, 2) growth-associated protein 43 as an axonal sprouting marker, 3) synaptophysin as a synaptic marker, and 4) Nogo-A as an axonal growth inhibitor. This study is the first to demonstrate that combined treatment with exercise and α5GABAAR inhibitor effectively promoted motor function recovery after ICH. Regarding the underlying mechanism of post-ICH recovery with the combined treatment, the present study highlights the importance of both growth and inhibitory modification of axonal sprouting in the spinal cord.

Rosmarinic Acid, a Bioactive Phenolic Compound, Inhibits Glutamate Release from Rat Cerebrocortical Synaptosomes through GABAA Receptor Activation.

Rosmarinic acid, a major component of rosemary, is a polyphenolic compound with potential neuroprotective effects. Asreducing the synaptic release of glutamate is crucial to achieving neuroprotectant's pharmacotherapeutic effects, the effect of rosmarinic acid on glutamate release was investigated in rat cerebrocortical nerve terminals (synaptosomes). Rosmarinic acid depressed the 4-aminopyridine (4-AP)-induced glutamate release in a concentration-dependent manner. The removal of extracellular calcium and the blockade of vesicular transporters prevented the inhibition of glutamate release by rosmarinic acid. Rosmarinic acid reduced 4-AP-induced intrasynaptosomal Ca2+ elevation. The inhibition of N-, P/Q-type Ca2+ channels and the calcium/calmodulin-dependent kinase II (CaMKII) prevented rosmarinic acid from having effects on glutamate release. Rosmarinic acid also reduced the 4-AP-induced activation of CaMKII and the subsequent phosphorylation of synapsin I, the main presynaptic target of CaMKII. In addition, immunocytochemistry confirmed the presence of GABAA receptors. GABAA receptor agonist and antagonist blocked the inhibitory effect of rosmarinic acid on 4-AP-evoked glutamate release. Docking data also revealed that rosmarinic acid formed a hydrogen bond with the amino acid residues of GABAA receptor. These results suggested that rosmarinic acid activates GABAA receptors in cerebrocortical synaptosomes to decrease Ca2+ influx and CaMKII/synapsin I pathway to inhibit the evoked glutamate release.

Somatostatin contributes to long-term potentiation at excitatory synapses onto hippocampal somatostatinergic interneurons.

Somatostatin-expressing interneurons (SOM-INs) are a major subpopulation of GABAergic cells in CA1 hippocampus that receive excitation from pyramidal cells (PCs), and, in turn, provide feedback inhibition onto PC dendrites. Excitatory synapses onto SOM-INs show a Hebbian long-term potentiation (LTP) mediated by type 1a metabotropic glutamate receptors (mGluR1a) that is implicated in hippocampus-dependent learning. The neuropeptide somatostatin (SST) is also critical for hippocampal long-term synaptic plasticity, as well as learning and memory. SST effects on hippocampal PCs are well documented, but its actions on inhibitory interneurons remain largely undetermined. In the present work, we investigate the involvement of SST in long-term potentiation of CA1 SOM-IN excitatory synapses using pharmacological approaches targeting the somatostatinergic system and whole cell recordings in slices from transgenic mice expressing eYFP in SOM-INs. We report that application of exogenous SST14 induces long-term potentiation of excitatory postsynaptic potentials in SOM-INs via somatostatin type 1-5 receptors (SST1-5Rs) but does not affect synapses of PC or parvalbumin-expressing interneurons. Hebbian LTP in SOM-INs was prevented by inhibition of SSTRs and by depletion of SST by cysteamine treatment, suggesting a critical role of endogenous SST in LTP. LTP of SOM-IN excitatory synapses induced by SST14 was independent of NMDAR and mGluR1a, activity-dependent, and prevented by blocking GABAA receptor function. Our results indicate that endogenous SST may contribute to Hebbian LTP at excitatory synapses of SOM-INs by controlling GABAA inhibition, uncovering a novel role for SST in regulating long-term synaptic plasticity in somatostatinergic cells that may be important for hippocampus-dependent memory processes.

The GABAA Receptor Influences Pressure Overload-Induced Heart Failure by Modulating Macrophages in Mice.

Background: Myocardial macrophages have key roles in cardiac remodeling and dysfunction. The gamma-aminobutyric acid subtype A (GABAA) receptor was recently found to be distributed in macrophages, allowing regulation of inflammatory responses to various diseases. This study aimed to clarify the role of GABAA receptor-mediated macrophage responses in pressure overload-induced heart failure. Methods and Results: C57BL/6J mice underwent transverse aortic constriction for pressure-overload hypertrophy (POH) and were intraperitoneally treated with a specific GABAA receptor agonist (topiramate) or antagonist (bicuculline). Echocardiography, histology, and flow cytometry were performed to evaluate the causes and effects of myocardial hypertrophy and fibrosis. Activation of the GABAA receptor by topiramate reduced ejection fraction and fractional shortening, enlarged the end-diastolic and end-systolic left ventricular internal diameter, aggravated myocardial hypertrophy and fibrosis, and accelerated heart failure in response to pressure overload. Mechanistically, topiramate increased the number of Ly6Clow macrophages in the heart during POH and circulating Ly6Chigh classic monocyte infiltration in late-phase POH. Further, topiramate drove Ly6Clow macrophages toward MHCIIhigh macrophage polarization. As a result, Ly6Clow macrophages activated the amphiregulin-induced AKT/mTOR signaling pathway, and Ly6ClowMHCIIhigh macrophage polarization increased expression levels of osteopontin and TGF-ß, which led to myocardial hypertrophy and fibrosis. Conversely, GABAA receptor blockage with bicuculline reversed these effects. Conclusions: Control of the GABAA receptor activity in monocytes/macrophages plays an important role in myocardial hypertrophy and fibrosis after POH. Blockade of the GABAA receptor has the potential to improve pressure overload-induced heart failure.

Bumetanide prevents diazepam-modified anxiety-like behavior in lipopolysaccharide-treated mice.

Benzodiazepine receptor agonists are widely prescribed therapeutic agents that alter gamma-aminobutyric acid (GABA)A receptor activity and have anxiolytic effects. Post-operative use of benzodiazepines is a risk factor of delirium. Inflammatory conditions alter the anxiolytic effects of benzodiazepine. We investigated the effect of diazepam, a typical benzodiazepine anxiolytic, on changes in the emotional behavior of mice in a hole-board test after lipopolysaccharide (LPS) treatment. Diazepam dose-dependently increased the number of head-dips at doses that did not alter locomotor activity; however, diazepam dose-dependently significantly decreased the number of head-dips at doses that did not alter locomotor activity in LPS-treated mice. Flumazenil, a benzodiazepine receptor antagonist, normalized the decrease in head-dipping behavior caused by diazepam treatment in normal and LPS-treated mice. The decrease of the head-dipping effect caused by diazepam was attenuated by minocycline in LPS-treated mice. We further found that the decrease in head-dipping behavior caused by diazepam was blocked by bumetanide, a Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) antagonist, in LPS-treated mice. These findings suggest that diazepam induces the anxiety-like behavior under inflammation conditions, and may cause the GABAA receptor dysfunction associated with the chloride plasticity mediated by NKCC1, which contributes to benzodiazepine-induced delirium after surgery.

N-Demethylsinomenine, an active metabolite of sinomenine, attenuates chronic neuropathic and inflammatory pain in mice.

Chronic pain is a significant public health problem that afflicts nearly 30% of the global population, but current pharmacotherapies are insufficient. Previous report indicated that N-demethylsinomenine, an active metabolite of sinomenine, is efficacious against postoperative pain. The present study investigated whether N-demethylsinomenine is effective for chronic painful conditions or whether repeated treatment alters its effect. Both chronic constriction injury (CCI) surgery and complete Freund's adjuvant (CFA) intraplantar injection induced significant and reliable mechanical allodynia at least for 7 days. Acute treatment with N-demethylsinomenine (10-40 mg/kg, i.p.) dose-dependently attenuated the mechanical allodynia both in CCI-induced neuropathic pain and CFA-induced inflammatory pain in mice. The potency of N-demethylsinomenine for reducing CFA-induced mechanical allodynia was slightly higher than sinomenine. During the period of repeated treatment, N-demethylsinomenine maintained its anti-allodynic effect against both neuropathic and inflammatory pain without producing carry-over effect. Pretreatment with bicuculline, a selective γ-aminobutyric acid type A (GABAA) receptor antagonist, almost completely blocked the anti-allodynia of N-demethylsinomenine (40 mg/kg) both in CCI and CFA-treated mice. Our findings indicated that N-demethylsinomenine exhibits GABAA receptor-mediated anti-allodynic effects in mouse models of neuropathic and inflammatory pain, suggesting it may be a useful novel pharmacotherapy for the control of chronic pain.

Novel Three-Finger Neurotoxins from Naja melanoleuca Cobra Venom Interact with GABAA and Nicotinic Acetylcholine Receptors.

Cobra venoms contain three-finger toxins (TFT) including α-neurotoxins efficiently binding nicotinic acetylcholine receptors (nAChRs). As shown recently, several TFTs block GABAA receptors (GABAARs) with different efficacy, an important role of the TFTs central loop in binding to these receptors being demonstrated. We supposed that the positive charge (Arg36) in this loop of α-cobratoxin may explain its high affinity to GABAAR and here studied α-neurotoxins from African cobra N. melanoleuca venom for their ability to interact with GABAARs and nAChRs. Three α-neurotoxins, close homologues of the known N. melanoleuca long neurotoxins 1 and 2, were isolated and sequenced. Their analysis on Torpedocalifornica and α7 nAChRs, as well as on acetylcholine binding proteins and on several subtypes of GABAARs, showed that all toxins interacted with the GABAAR much weaker than with the nAChR: one neurotoxin was almost as active as α-cobratoxin, while others manifested lower activity. The earlier hypothesis about the essential role of Arg36 as the determinant of high affinity to GABAAR was not confirmed, but the results obtained suggest that the toxin loop III may contribute to the efficient interaction of some long-chain neurotoxins with GABAAR. One of isolated toxins manifested different affinity to two binding sites on Torpedo nAChR.

Progesterone modulates neuronal excitability bidirectionally.

Progesterone acts on neurons directly by activating its receptor and through metabolic conversion to neurosteroids. There is emerging evidence that progesterone exerts excitatory effects by activating its cognate receptors (progesterone receptors, PRs) through enhanced expression of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs). Progesterone metabolite 5α,3α-tetrahydro-progesterone (allopregnanolone, THP) mediates its anxiolytic and sedative actions through the potentiation of synaptic and extrasynaptic γ-aminobutyric acid type-A receptors (GABAARs). Here, we review progesterone's neuromodulatory actions exerted through PRs and THP and their opposing role in regulating seizures, catamenial epilepsy, and seizure exacerbation associated with progesterone withdrawal.

Bicuculline regulated protein synthesis is dependent on Homer1 and promotes its interaction with eEF2K through mTORC1-dependent phosphorylation.

The regulation of protein synthesis is a vital and finely tuned process in cellular physiology. In neurons, this process is very precisely regulated, as which mRNAs undergo translation is highly dependent on context. One of the most prominent regulators of protein synthesis is the enzyme eukaryotic elongation factor kinase 2 (eEF2K) that regulates the elongation stage of protein synthesis. This kinase and its substrate, eukaryotic elongation factor 2 (eEF2) are important in processes such as neuronal development and synaptic plasticity. eEF2K is regulated by multiple mechanisms including Ca2+ -ions and the mTORC1 signaling pathway, both of which play key roles in neurological processes such as learning and memory. In such settings, the localized control of protein synthesis is of crucial importance. In this work, we sought to investigate how the localization of eEF2K is controlled and the impact of this on protein synthesis in neuronal cells. In this study, we used both SH-SY5Y neuroblastoma cells and mouse cortical neurons, and pharmacologically and/or genetic approaches to modify eEF2K function. We show that eEF2K activity and localization can be regulated by its binding partner Homer1b/c, a scaffolding protein known for its participation in calcium-regulated signaling pathways. Furthermore, our results indicate that this interaction is regulated by the mTORC1 pathway, through a known phosphorylation site in eEF2K (S396), and that it affects rates of localized protein synthesis at synapses depending on the presence or absence of this scaffolding protein.

Low-Level Inhibition of GABAergic Synapses Enhances Gene Expressions Crucial for Neuronal Plasticity in the Hippocampus After Ischemic Stroke.

OBJECTIVE: Pharmacological inhibition of GABAergic synapses could represent a potent neuromodulation strategy to activate hippocampal neurons and increase neurotrophic factor gene expression, thus exerting a beneficial effect on post-stroke cognitive impairment (PSCI). The objective of this study was to assess the effects of low-level inhibition of GABAergic synapses on hippocampal gene expressions related to neuroplasticity using the middle cerebral artery occlusion surgery (MCAO) ischemic stroke rat model. METHODS: The animals were randomly assigned to three experimental groups-(1) a sham operated group (SHAM), (2) a control group (CON), and (3) a bicuculline group (BIC). MCAO was performed in the CON and BIC groups. A non-epileptic dose of bicuculline (0.25 mg/kg) was intraperitoneally administered every day for two weeks, starting three days after surgery, to the rats in the BIC group. The mRNA expression of brain-derived neurotrophic factor (BDNF), tropomyosin-related kinase B (TrkB), in relation to neurotrophic intracellular signal, p75, in relation to apoptosis, and synaptophysin (SYP) and PSD-95, synaptic markers, were assessed in the hippocampus ipsilateral to the ischemic site. RESULTS: MCAO increased the gene expression of TrkB. Furthermore, MCAO plus bicuculline administration increased the expression ratio of TrkB to p75 and SYP gene expression. CONCLUSION: Therefore, this study showed that administration of bicuculline after stroke beneficially modulated the expression of crucial genes for neuroplasticity, including BDNF receptors and SYP, in the ipsilateral hippocampus, suggesting that low-level inhibition of GABAergic synapses could lead to beneficial neuromodulation in the hippocampus after stroke.

The role of hippocampal GABAA receptors on anxiolytic effects of Echium amoenum extract in a mice model of restraint stress.

Echium amoenum (EA), a popular medicinal plant in Persian medicine, has anxiolytic, antioxidant, sedative, and anti-inflammatory effects. This study examined whether GABA-ergic signaling is involved in the anxiolytic effects of EA in mice. Sixty BALB/c mice (25-30 g) were divided into six groups (n = 10) as follows: the (I) control group received 10 ml/kg normal saline (NS). In the stress groups, the animals underwent 14 consecutive days of restraint stress (RS), and received following treatments simultaneously; (II) RS + NS; (III) RS + Diaz (Diazepam); (IV) RS + EA; (V) RS + Flu (Flumazenil) + EA; (VI) RS + Flu + Diaz. Behavioral tests including the open field test (OFT) and elevated plus maze (EPM) were performed to evaluate anxiety-like behaviors and the effects of the regimens. The plasma level of corticosterone and the hippocampal protein expressions of IL-1ß, TNF-α, CREB, and BDNF, as well as p-GABAA/GABAA ratio, were also assessed. The findings revealed that chronic administration of EA alone produced anxiolytic effects in both behavioral tests, while diazepam alone or in combination with Flu failed to decrease the anxiety-like behaviors. Furthermore, the p-GABAA/GABAA and p-CREB/CREB ratios, and protein levels of BDNF were significantly increased in the EA-received group. On the other hand, plasma corticosterone levels and the hippocampal IL-1ß and TNF-α levels were significantly decreased by EA. However, pre-treatment with GABAA receptors (GABAA Rs) antagonist, Flu, reversed the anxiolytic and molecular effects of EA in the RS-subjected animals. Our findings confirmed that alternation of GABAAR is involved in the effects of EA against RS-induced anxiety-like behaviors, HPA axis activation, and neuroinflammation.

Inactivation of the Ventral Pallidum by GABAA Receptor Agonist Promotes Non-rapid Eye Movement Sleep in Rats.

GABA, the most abundant inhibitory neurotransmitter in the brain, is closely linked with sleep and wakefulness. As the largest area input to the ventral pallidum (VP), the nucleus accumbens (NAc) has been confirmed to play a pivotal role in promoting non-rapid eye movement (NREM) sleep through inhibitory projections from NAc adenosine A2A receptor-expressing neurons to VP GABAergic neurons which mostly express GABAA receptors. Although these studies demonstrate the possible role of VP GABAergic neurons in sleep-wake regulation, whether and how its modulate sleep-wake cycle is not completely clear. In our study, pharmacological manipulations were implemented in freely moving rats and then the EEG and the EMG were recorded to monitor the sleep-wake states. We found that microinjection of muscimol, a GABAA receptor agonist, into the VP increased NREM sleep in both light and dark period. Microinjection of bicuculline, a GABAA receptor antagonist, into the VP increased wakefulness in the light period. Collectively, our data identify the important role of VP GABAA receptor-expressing neurons in NREM sleep of rats which may help improve the understanding of the pathological sleep disorders.

GluN2A-selective positive allosteric modulator-nalmefene-flumazenil reverses ketamine-fentanyl-dexmedetomidine-induced anesthesia and analgesia in rats.

Anesthetics are used to produce hypnosis and analgesic effects during surgery, but anesthesia for a long time after the operation is not conducive to the recovery of animals or patients. Therefore, finding appropriate treatments to counter the effects of anesthetics could enhance postoperative recovery. In the current study, we discovered the novel role of a GluN2A-selective positive allosteric modulator (PAM) in ketamine-induced anesthesia and investigated the effects of the PAM combined with nalmefene and flumazenil (PNF) in reversing the actions of an anesthetic combination (ketamine-fentanyl-dexmedetomidine, KFD). PAM treatment dose-dependently decreased the duration of the ketamine-induced loss of righting reflex (LORR). Compared with those in the KFD group, the duration of LORR and the analgesic effect of the KFD + PNF group were obviously decreased. Meanwhile, successive administration of PNF and KFD had no adverse effects on the cardiovascular and respiratory systems. Both the KFD group and the KFD + PNF group showed no changes in hepatic and renal function or cognitive function in rats. Moreover, the recovery of motor coordination of the KFD + PNF group was faster than that of the KFD group. In summary, our results suggest the potential application of the PNF combination as an antagonistic treatment strategy for anesthesia.

Output from VIP cells of the mammalian central clock regulates daily physiological rhythms.

The suprachiasmatic nucleus (SCN) circadian clock is critical for optimising daily cycles in mammalian physiology and behaviour. The roles of the various SCN cell types in communicating timing information to downstream physiological systems remain incompletely understood, however. In particular, while vasoactive intestinal polypeptide (VIP) signalling is essential for SCN function and whole animal circadian rhythmicity, the specific contributions of VIP cell output to physiological control remains uncertain. Here we reveal a key role for SCN VIP cells in central clock output. Using multielectrode recording and optogenetic manipulations, we show that VIP neurons provide coordinated daily waves of GABAergic input to target cells across the paraventricular hypothalamus and ventral thalamus, supressing their activity during the mid to late day. Using chemogenetic manipulation, we further demonstrate specific roles for this circuitry in the daily control of heart rate and corticosterone secretion, collectively establishing SCN VIP cells as influential regulators of physiological timing.

Slowly-Conducting Pyramidal Tract Neurons in Macaque and Rat.

Anatomical studies report a large proportion of fine myelinated fibers in the primate pyramidal tract (PT), while very few PT neurons (PTNs) with slow conduction velocities (CV) (<~10 m/s) are reported electrophysiologically. This discrepancy might reflect recording bias toward fast PTNs or prevention of antidromic invasion by recurrent inhibition (RI) of slow PTNs from faster axons. We investigated these factors in recordings made with a polyprobe (32 closely-spaced contacts) from motor cortex of anesthetized rats (n = 2) and macaques (n = 3), concentrating our search on PTNs with long antidromic latencies (ADLs). We identified 21 rat PTNs with ADLs >2.6 ms and estimated CV 3-8 m/s, and 67 macaque PTNs (>3.9 ms, CV 6-12 m/s). Spikes of most slow PTNs were small and present on only some recording contacts, while spikes from simultaneously recorded fast-conducting PTNs were large and appeared on all contacts. Antidromic thresholds were similar for fast and slow PTNS, while spike duration was considerably longer in slow PTNs. Most slow PTNs showed no signs of failure to respond antidromically. A number of tests, including intracortical microinjection of bicuculline (GABAA antagonist), failed to provide any evidence that RI prevented antidromic invasion of slow PTNs. Our results suggest that recording bias is the main reason why previous studies were dominated by fast PTNs.

GABA-A receptor and mitochondrial TSPO signaling act in parallel to regulate melanocyte stem cell quiescence in larval zebrafish.

Tissue regeneration and homeostasis often require recruitment of undifferentiated precursors (adult stem cells; ASCs). While many ASCs continuously proliferate throughout the lifetime of an organism, others are recruited from a quiescent state to replenish their target tissue. A long-standing question in stem cell biology concerns how long-lived, non-dividing ASCs regulate the transition between quiescence and proliferation. We study the melanocyte stem cell (MSC) to investigate the molecular pathways that regulate ASC quiescence. Our prior work indicated that GABA-A receptor activation promotes MSC quiescence in larval zebrafish. Here, through pharmacological and genetic approaches we show that GABA-A acts through calcium signaling to maintain MSC quiescence. Unexpectedly, we identified translocator protein (TSPO), a mitochondrial membrane-associated protein that regulates mitochondrial function and metabolic homeostasis, as a parallel regulator of MSC quiescence. We found that both TSPO-specific ligands and induction of gluconeogenesis likely act in the same pathway to promote MSC activation and melanocyte production in larval zebrafish. In contrast, TSPO and gluconeogenesis appear to act in parallel to GABA-A receptor signaling to regulate MSC quiescence and vertebrate pigment patterning.

Korean meditation music suppressed relapse to methamphetamine in rats.

OBJECTIVE: Methamphetamine is a drug abused worldwide. Even though its abuse is a serious problem in many countries, there are few safe and effective therapies to treat addiction. In a previous study, music therapy attenuated relapse to morphine. Based on the study, we investigated whether music therapy suppresses the reinstatement of methamphetamine self-administration behavior. METHODS: Male Sprague-Dawley rats were trained to intravenously self-administer methamphetamine (0.1 mg/kg) using a fixed ratio 1 schedule in a daily 2 h session. Following 3 weeks of training, rats who had established a stable daily intake were subjected to extinction for 1 week. On the next day, priming injection was performed to induce reinstatement. Music therapy was played twice daily during the extinction period and immediately before the test session. In the second experiment, the selective GABAA and GABAB receptor antagonists were treated prior to the last music therapy to investigate a possible neuronal mechanism. Immunofluorescence was performed to immunohistochemically examine the behavioral effects. RESULTS: The meditation music by Young-Dong Kim but not the control music by Jeff Beck reduced active lever responding during the reinstatement session. And the effects of music therapy were blocked by GABA receptors antagonists. Also, immunofluorescence showed the pattern of c-Fos expression in the nucleus accumbens paralleled the behavioral results. CONCLUSION: Results of the present study suggest that meditation music by Young-Dong Kim can be a useful therapy to prevent the reinstatement of methamphetamine addiction during abstinence.

Identification and Validation of a Novel Biologics Target in Triple Negative Breast Cancer.

The goal of this study was to identify a novel target for antibody-drug conjugate (ADC) development in triple negative breast cancer (TNBC), which has limited treatment options, using gene expression datasets and in vitro siRNA/CRISPR and in vivo functional assays. We analyzed 4467 breast cancers and identified GABRP as top expressed gene in TNBC with low expression in most normal tissues. GABRP protein was localized to cell membrane with broad range of receptors/cell (815-53,714) and expressed by nearly half of breast cancers tissues. GABRP gene knockdown inhibited TNBC cell growth and colony formation in vitro and growth of MDA-MB-468 xenografts in nude mice. Commercially available anti-GABRP antibody (5-100 µg/ml) or de novo generated Fabs (20 µg/ml) inhibited TNBC cell growth in vitro. The same antibody conjugated to mertansine (DM1) also showed significant anticancer activity at nanomolar concentrations. Our results indicate that GABRP is a potential novel therapeutic target for ADC development.

Does kisspeptin participate in GABA-mediated modulation of GnRH and GnRH receptor biosynthesis in the hypothalamic-pituitary unit of follicular-phase ewes?

BACKGROUND: The inverse relationship between GnRH transcript level and GABA neurons activity has suggested that GABA at the hypothalamic level may exert a suppressive effect on subsequent steps of the GnRH biosynthesis. In the present study, we analyzed the effects of GABA type A receptor agonist (muscimol) or antagonist (bicuculline) on molecular mechanisms governing GnRH/LH secretion in follicular-phase sheep. METHODS: ELISA technique was used to investigate the effects of muscimol and/or bicuculline on levels of post-translational products of genes encoding GnRH ligand and GnRH receptor (GnRHR) in the preoptic area (POA), anterior (AH) and ventromedial (VMH) hypothalamus, stalk/median eminence (SME), and GnRHR in the anterior pituitary (AP). Real-time PCR was chosen for determination of the effect of drugs on kisspeptin (Kiss 1) mRNA level in POA and VMH including arcuate nucleus (VMH/ARC), and on Kiss1 receptor (Kiss1r) mRNA abundance in POA-hypothalamic structures. These analyses were supplemented by RIA method for measurement of plasma LH concentration. RESULTS: The study demonstrated that muscimol and bicuculline significantly decreased or increased GnRH biosynthesis in all analyzed structures, respectively, and led to analogous changes in plasma LH concentration. Similar muscimol- and bicuculline-related alterations were observed in levels of GnRHR. However, the expression of Kiss 1 and Kiss1r mRNAs in selected POA-hypothalamic areas of either muscimol- and bicuculline-treated animals remained unaltered. CONCLUSIONS: Our data suggest that GABAergic neurotransmission is involved in the regulatory pathways of GnRH/GnRHR biosynthesis and then GnRH/LH release in follicular-phase sheep conceivably via indirect mechanisms that exclude involvement of Kiss 1 neurons.

Involvement of oxytocin and GABA in consolation behavior elicited by socially defeated individuals in mandarin voles.

Consolation, which entails comforting contact directed toward a distressed party, is a common empathetic response in humans and other species with advanced cognition. Here, using the social defeat paradigm, we provide empirical evidence that highly social and monogamous mandarin voles (Microtus mandarinus) increased grooming toward a socially defeated partner but not toward a partner who underwent only separation. This selective behavioral response existed in both males and females. Accompanied with these behavioral changes, c-Fos expression was elevated in many of the brain regions relevant for emotional processing, such as the anterior cingulate cortex (ACC), bed nucleus of the stria terminalis, paraventricular nucleus (PVN), basal/basolateral and central nucleus of the amygdala, and lateral habenular nucleus in both sexes; in the medial preoptic area, the increase in c-Fos expression was found only in females, whereas in the medial nucleus of the amygdala, this increase was found only in males. In particular, the GAD67/c-Fos and oxytocin (OT)/c-Fos colocalization rates were elevated in the ACC and PVN, indicating selective activation of GABA and OT neurons in these regions. The "stressed" pairs matched their anxiety-like behaviors in the open-field test, and their plasma corticosterone levels correlated well with each other, suggesting an empathy-based mechanism. This partner-directed grooming was blocked by pretreatment with an OT receptor antagonist or a GABAA receptor antagonist in the ACC but not by a V1a subtype vasopressin receptor antagonist. We conclude that consolation behavior can be elicited by the social defeat paradigm in mandarin voles, and this behavior may be involved in a coordinated network of emotion-related brain structures, which differs slightly between the sexes. We also found that the endogenous OT and the GABA systems within the ACC are essential for consolation behavior in mandarin voles.