GABA Cells Research Articles

Laminar specificity and coverage of viral-mediated gene expression restricted to GABAergic interneurons and their parvalbumin subclass in marmoset primary visual cortex.

In the mammalian neocortex, inhibition is important for dynamically balancing excitation and shaping the response properties of cells and circuits. The various computational functions of inhibition are thought to be mediated by different inhibitory neuron types, of which a large diversity exists in several species. Current understanding of the function and connectivity of distinct inhibitory neuron types has mainly derived from studies in transgenic mice. However, it is unknown whether knowledge gained from mouse studies applies to the non-human primate, the model system closest to humans. The lack of viral tools to selectively access inhibitory neuron types has been a major impediment to studying their function in the primate. Here, we have thoroughly validated and characterized several recently developed viral vectors designed to restrict transgene expression to GABAergic cells or their parvalbumin (PV) subtype, and identified two types that show high specificity and efficiency in marmoset V1. We show that in marmoset V1, AAV-h56D induces transgene expression in GABAergic cells with up to 91-94% specificity and 79% efficiency, but this depends on viral serotype and cortical layer. AAV-PHP.eB-S5E2 induces transgene expression in PV cells across all cortical layers with up to 98% specificity and 86-90% efficiency, depending on layer. Thus, these viral vectors are promising tools for studying GABA and PV cell function and connectivity in the primate cortex.

Laminar specificity and coverage of viral-mediated gene expression restricted to GABAergic interneurons and their parvalbumin subclass in marmoset primary visual cortex

In the mammalian neocortex, inhibition is important for dynamically balancing excitation and shaping the response properties of cells and circuits. The various computational functions of inhibition are thought to be mediated by different inhibitory neuron types, of which a large diversity exists in several species. Current understanding of the function and connectivity of distinct inhibitory neuron types has mainly derived from studies in transgenic mice. However, it is unknown whether knowledge gained from mouse studies applies to the non-human primate, the model system closest to humans. The lack of viral tools to selectively access inhibitory neuron types has been a major impediment to studying their function in the primate. Here, we have thoroughly validated and characterized several recently developed viral vectors designed to restrict transgene expression to GABAergic cells or their parvalbumin (PV) subtype, and identified two types that show high specificity and efficiency in marmoset V1. We show that in marmoset V1, AAV-h56D induces transgene expression in GABAergic cells with up to 91–94% specificity and 79% efficiency, but this depends on viral serotype and cortical layer. AAV-PHP.eB-S5E2 induces transgene expression in PV cells across all cortical layers with up to 98% specificity and 86–90% efficiency, depending on layer. Thus, these viral vectors are promising tools for studying GABA and PV cell function and connectivity in the primate cortex.

Medium Formulation and Optimisation of Fermentation Condition Enhancing γ-aminobutyric Acid (GABA) Biosynthesis by Lactiplantibacillus plantarum B7

Extensive studies on γ-aminobutyric acid (GABA) over decades highlight its significant physiological and pharmacological effects on humans. GABA produced using microbe is favoured compared to enzymatic and chemical methods due to operational ease and reduced harmful pollutant formation. This study focused on increasing γ-aminobutyric acid (GABA) biosynthesis from Lactiplantibacillus plantarum B7, employing a multi-step optimisation strategy. An unoptimised cultivation approach yielded a maximum GABA of 11.68 ± 0.04 g/L and viable cell count of 10.47 ± 0.01 log CFU/mL at 48 h. A nutrient-rich medium was developed through single-parameter optimisation, comprising 1%, 2.5% and 0.0002% of glucose, yeast extract and each trace element (CaCO3, KI, and Tween 80) respectively. Temperature, pH, incubation period, initial concentration of monosodium glutamate (MSG) and pyridoxal-5’-phosphate (PLP) demonstrated significant contributions towards GABA production and cell growth as determined using a two-level factorial design. Steepest ascent identified optimal conditions (36°C, pH 5.5, 370 mM MSG, and 0.7 mM PLP), resulting in 30.50 g/L GABA and 11.51 log CFU/mL at 60 h. Further refinement via a central composite experiment yielded optimal conditions (temperature-35.6°C, pH-5.66, initial MSG concentration-335.61 mM, PLP concentration-0.723 mM) with improved GABA production (32.18 g/L) and cell growth (11.52 log CFU/mL) over 63.66 h. Therefore, this approach utilising lactic acid bacteria capable of GABA synthesis holds promise for mass-produced, enhanced-functional foods.

Neurexin 3 Regulates Synaptic Connections Between Central Amygdala Neurons and Excitable Cells of the Lateral Parabrachial Nucleus in Rats with Varicella Zoster Induced Orofacial Pain.

Herpes Zoster in humans is the result of varicella zoster virus (VZV) infection. Injecting rats with varicella zoster virus produces pain similar to herpes zoster "shingles" pain in humans. . In a previous study, orofacial pain was induced by injecting the whisker pad of male rats with VZV and the pain response increased after attenuating neurexin 3 (Nrxn3) expression in the central amygdala. Neurons descend from the central amygdala to the lateral parabrachial nucleus and orofacial pain signals ascend to the lateral parabrachial nucleus. GABAergic neurons within the central amygdala regulate pain by inhibiting activity within the lateral parabrachial nucleus. Attenuating Nrxn3 expression in the central amygdala increased GABA release in the lateral parabrachial nucleus suggesting Nrxn3 controls pain by regulating GABA release. Nrxn3 can also control synaptic connections between neurons, and we hypothesized that Nrxn3 knockdown in the central amygdala would reduce the number of GABAergic synaptic connections in the lateral parabrachial nucleus and increase VZV associated pain. To test this idea, the number of synaptic connections between GABAergic cells of the central amygdala and excitatory or dynorphin positive neurons within the lateral parabrachial nucleus were quantitated after infusion of a virus expressing synaptophysin. Synaptophysin is a synaptic vesicle protein that labels neuronal synaptic connections. These connections were measured in rats with and without whisker pad injection of VZV and knockdown of Nrxn3 within the central amygdala. Orofacial pain was measured using a place escape avoidance paradigm. GABAergic synaptic connections were reduced in the lateral parabrachial nucleus after Nrxn3 knockdown. Rats with a reduction in the number of connections had an increase in VZV associated orofacial pain. Immunostaining with the pain marker prodynorphin indicated that the reduction in GABAergic connections was primarily associated with prodynorphin positive neurons. The results suggest Nrxn3 reduces VZV associated orofacial pain, in part, by enhancing synaptic connections between GABA cells of the central amygdala and pain neurons within the lateral parabrachial nucleus.

Laminar specificity and coverage of viral-mediated gene expression restricted to GABAergic interneurons and their parvalbumin subclass in marmoset primary visual cortex.

In the mammalian neocortex, inhibition is important for dynamically balancing excitation and shaping the response properties of cells and circuits. The various computational functions of inhibition are thought to be mediated by different inhibitory neuron types of which a large diversity exists in several species. Current understanding of the function and connectivity of distinct inhibitory neuron types has mainly derived from studies in transgenic mice. However, it is unknown whether knowledge gained from mouse studies applies to the non-human primate, the model system closest to humans. The lack of viral tools to selectively access inhibitory neuron types has been a major impediment to studying their function in the primate. Here, we have thoroughly validated and characterized several recently-developed viral vectors designed to restrict transgene expression to GABAergic cells or their parvalbumin (PV) subtype, and identified two types that show high specificity and efficiency in marmoset V1. We show that in marmoset V1 AAV-h56D induces transgene expression in GABAergic cells with up to 91-94% specificity and 79% efficiency, but this depends on viral serotype and cortical layer. AAV-PHP.eB-S5E2 induces transgene expression in PV cells across all cortical layers with up to 98% specificity and 86-90% efficiency, depending on layer. Thus, these viral vectors are promising tools for studying GABA and PV cell function and connectivity in the primate cortex.

Optimization of fermentation conditions for the production of γ-aminobutyric acid by Lactobacillus hilgardii GZ2 from traditional Chinese fermented beverage system.

γ-Aminobutyric acid (GABA) is a crucial neurotransmitter with wide application prospects. In this study, we focused on a GABA-producing strain from a traditional Chinese fermented beverage system. Among the six isolates, Lactobacillus hilgardii GZ2 exhibited the greatest ability to produce GABA in the traditional Chinese fermented beverage system. To increase GABA production, we optimized carbon sources, nitrogen sources, temperature, pH, and monosodium glutamate and glucose concentrations and conducted fed-batch fermentation. The best carbon and nitrogen sources for GABA production and cell growth were glucose, yeast extract and tryptone. Gradual increases in GABA were observed as the glucose and monosodium glutamate concentrations increased from 10g/L to 50g/L. During fed-batch fermentation, lactic acid was used to maintain the pH at 5.56, and after feeding with 0.03g/mL glucose and 0.4g/mL sodium glutamate for 72h, the GABA yield reached 239g/L. This novel high-GABA-producing strain holds great potential for the industrial production of GABA, as well as the development of health-promoting functional foods and medical fields.

Pentylenetetrazole kindling induces dynamic changes in GAD65 expression in hippocampal somatostatin interneurons

IntroductionOne of the mechanisms of epileptgenesis is impairment of inhibitory neural circuits. Several studies have compared neural changes among subtypes of gamma-aminobutyric acid-related (GABAergic) neurons after acquired epileptic seizure. However, it is unclear that GABAergic neural modifications that occur during acquisition process of epileptic seizure. MethodsMale rats were injected with pentylenetetrazole (PTZ kindling: n = 30) or saline (control: n = 15) every other day to observe the development of epileptic seizure stages. Two time points were identified: the point at which seizures were most difficult to induce, and the point at which seizures were most easy to induce. The expression of GABAergic neuron-related proteins in the hippocampus was immunohistochemically compared among GABAergic subtypes at each of these time points. ResultsBimodal changes in seizure stages were observed in response to PTZ kindling. The increase of seizure stage was transiently suppressed after 8 or 10 injections, and then progressed again by the 16th injection. Based on these results, we defined 10 injections as a short-term injection period during which seizures are less likely to occur, and 20 injections as a long-term injection period during which continuous seizures are likely to occur. The immunohistochemical analysis showed that hippocampal glutamic acid decarboxylase 65 (GAD65) expression was increased after short-term kindling but unchanged after long-term kindling. Increased GAD65 expression was limited to somatostatin-positive (SOM+) cells among several GABAergic subtypes. By contrast, GAD, GABA, GABAAR α1, GABABR1, and VGAT cells showed no change following short- or long-term PTZ kindling. ConclusionPTZ kindling induces bimodal changes in the epileptic seizure stage. Seizure stage is transiently suppressed after short-term PTZ injection with GAD65 upregulation in SOM+ cells. The seizure stage is progressed again after long-term PTZ injection with GAD65 reduction to baseline level.

Defining potential targets of prenatal androgen excess: Expression analysis of androgen receptor on hypothalamic neurons in the fetal female mouse brain.

Polycystic ovary syndrome (PCOS) is a female endocrine disorder that is associated with prenatal exposure to excess androgens. In prenatally androgenized (PNA) mice that model PCOS, GABAergic neural transmission to and innervation of GnRH neurons is increased. Evidence suggests that elevated GABAergic innervation originates in the arcuate nucleus (ARC). We hypothesized that GABA-GnRH circuit abnormalities are a direct consequence of PNA, resulting from DHT binding to androgen receptor (AR) in the prenatal brain. However, whether prenatal ARC neurons express AR at the time of PNA treatment is presently unknown. We used RNAScope in situ hybridization to localize AR mRNA (Ar)-expressing cells in healthy gestational day (GD) 17.5 female mouse brains and to assess coexpression levels in specific neuronal phenotypes. Our study revealed that less than 10% of ARC GABA cells expressed Ar. In contrast, we found that ARC kisspeptin neurons, critical regulators of GnRH neurons, were highly colocalized with Ar. Approximately 75% of ARC Kiss1-expressing cells also expressed Ar at GD17.5, suggesting that ARC kisspeptin neurons are potential targets of PNA. Investigating other neuronal populations in the ARC we found that ~50% of pro-opiomelanocortin (Pomc) cells, 22% of tyrosine hydroxylase (Th) cells, 8% of agouti-related protein (Agrp) cells and 8% of somatostatin (Sst) cells express Ar. Lastly, RNAscope in coronal sections showed Ar expression in the medial preoptic area (mPOA), and the ventral part of the lateral septum (vLS). These Ar-expressing regions were highly GABAergic, and 22% of GABA cells in the mPOA and 25% of GABA cells in the vLS also expressed Ar. Our findings identify specific neuronal phenotypes in the ARC, mPOA, and vLS that are androgen sensitive in late gestation. PNA-induced functional changes in these neurons may be related to the development of impaired central mechanisms associated with PCOS-like features.

Ethanol blocks a novel form of iLTD, but not iLTP of inhibitory inputs to VTA GABA neurons.

The ventral tegmental area (VTA) is an essential component of the mesocorticolimbic dopamine (DA) circuit that processes reward and motivated behaviors. The VTA contains DA neurons essential in this process, as well as GABAergic inhibitory cells that regulate DA cell activity. In response to drug exposure, synaptic connections of the VTA circuit can be rewired via synaptic plasticity-a phenomenon thought to be responsible for the pathology of drug dependence. While synaptic plasticity to VTA DA neurons as well as prefrontal cortex to nucleus accumbens GABA neurons are well studied, VTA GABA cell plasticity, specifically inhibitory inputs to VTA GABA neurons, is less understood. Therefore, we investigated the plasticity of these inhibitory inputs. Using whole cell electrophysiology in GAD67-GFP mice to identify GABA cells, we observed that these VTA GABA cells experience either inhibitory GABAergic long-term potentiation (iLTP) or inhibitory long-term depression (iLTD) in response to a 5 Hz stimulus. Paired pulse ratios, coefficient of variance, and failure rates suggest a presynaptic mechanism for both plasticity types, where iLTP is NMDA receptor-dependent and iLTD is GABAB receptor-dependent-this being the first report of iLTD onto VTA GABA cells. As illicit drug exposure can alter VTA plasticity, we employed chronic intermittent exposure (CIE) to ethanol (EtOH) vapor in male and female mice to examine its potential impact on VTA GABA input plasticity. Chronic EtOH vapor exposure produced measurable behavioral changes illustrating dependence and concomitantly prevented previously observed iLTD, which continued in air-exposed controls, illustrating the impact of EtOH on VTA neurocircuitry and suggesting physiologic mechanisms at play in alcohol use disorder and withdrawal states. Taken together, these novel findings of unique GABAergic synapses exhibiting either iLTP or iLTD within the mesolimbic circuit, and EtOH blockade specifically of iLTD, characterize inhibitory VTA plasticity as a malleable, experience-dependent system modified by EtOH.

Chronic Δ9-tetrahydrocannabinol impact on plasticity, and differential activation requirement for CB1-dependent long-term depression in ventral tegmental area GABA neurons in adult versus young mice.

The ventral tegmental area (VTA) mediates incentive salience and reward prediction error through dopamine (DA) neurons that are regulated by local VTA GABA neurons. In young mice, VTA GABA cells exhibit a form of synaptic plasticity known as long-term depression (LTD) that is dependent on cannabinoid 1 (CB1) receptors preceded by metabotropic glutamate receptor 5 (mGluR5) signaling to induce endocannabinoid production. This LTD was eliminated following chronic (7-10 consecutive days) exposure to the marijuana derived cannabinoid Δ9 -tetrahydrocannabinol (THC). We now examine the mechanism behind THC-induced elimination of LTD in adolescents as well as plasticity induction ability in adult versus young male and female mice using whole-cell electrophysiology experiments of VTA GABA cells. Chronic THC injections in adolescents resulted in a loss of CB1 agonist-mediated depression, illustrating chronic THC likely desensitizes or removes synaptic CB1. We noted that seven days withdrawal from chronic THC restored LTD and CB1 agonist-induced depression, suggesting reversibility of THC-induced changes. Adult mice continue to express functional mGluR5 and CB1, but require a doubling of the synaptic stimulation compared to young mice to induce LTD, suggesting a quantitative difference in CB1-dependent plasticity between young and adult mice. One potential rationale for this difference is changes in AMPA and NMDA glutamate receptors. Indeed, AMPA/NMDA ratios were increased in in adults compared to young mice. Lastly, we performed quantitative reverse-transcription PCR and identified that CB1, DAGLα, and GluA1 levels increased following chronic THC exposure. Collectively, our data demonstrate the first age-dependent GABA neuron plasticity in the VTA, which could have implications for decreased THC dependence capacity in adults, as well as the mechanism behind chronic THC-induced synaptic alterations in young mice.

The Production of γ-Aminobutyric Acid from Free and Immobilized Cells of Levilactobacillus brevis Cultivated in Anaerobic and Aerobic Conditions.

γ-aminobutyric acid (GABA) has several beneficial effects on human health. GABA may be produced via chemical synthesis or through microbial metabolism, and Levilactobacillus brevis is recognized as a GABA-producing species. In this study, 11 Lvb. brevis strains were screened for GABA production, and the best producers were selected to verify the effect of aerobic (AE) and respiratory (RS) cultivations on growth parameters, biomass, and GABA accumulation. Lvb. brevis LB12 was then used to evaluate the combined effect of the incubation atmosphere (anaerobiosis vs. aerobiosis), cell protection (free vs. immobilized cells), and cell recycling (fresh vs. starved cells) on GABA production. Glutamate (GLU) consumption and GABA accumulation were detected by Thin-layer Chromatography (TLC) and RP-HPLC analyses. The ability to produce GABA was widespread among the strains. AE and RS growth improved biomass production, but oxygen availability impaired GLU to GABA conversion, and the anaerobically growing cells had the highest GABA productivity. Immobilized strains had lower efficiency in both GLU uptake and conversion compared to free cells, probably due to the poor diffusion in alginate beads. The use of resting cells allowed further GABA production without the cultivation step, but cell activity was exhausted after three cycles of reutilization. Lvb. brevis LB12 is an excellent GABA producer, and AE cultivation can be exploited to improve the final cell density; however, the conditions for boosting GLU to GABA conversion and cell regeneration need to be further investigated.

Evaluation of Prefrontal γ-Aminobutyric Acid and Glutamate Levels in Individuals With Major Depressive Disorder Using Proton Magnetic Resonance Spectroscopy

Major depressive disorder (MDD) is one of the most prevalent illnesses worldwide. Perturbations of the major inhibitory and excitatory neurotransmitters, γ-aminobutyric acid (GABA) and glutamate (Glu), respectively, as well as Glx (Glu or glutamine [Gln]) have been extensively reported in a multitude of brain areas of individuals with depression, but few studies have examined changes in Gln, the metabolic counterpart of synaptic Glu. To investigate changes in GABA, Glx, Glu, and Gln levels in a voxel in the left dorsolateral prefrontal cortex of participants with no, past, and current MDD using proton magnetic resonance spectroscopy (1H-MRS). This community-based study used a cross-sectional design using 3-T 1H-MRS in participants not taking MDD medication recruited from the community. The sample consisted of 251 healthy controls, 98 participants with a history of past MDD, and 47 participants who met the diagnostic criteria for current MDD. Diagnostic groups were comparable regarding age, education, income, and diet. Data were collected from March 2014 to October 2021, and data were analyzed from October 2021 to June 2022. GABA, Glx, Glu, and Gln concentrations in the left dorsolateral prefrontal cortex. Of 396 included participants, 258 (65.2%) were female, and the mean (SD) age was 25.0 (4.7) years. Compared with healthy controls, those with past MDD and current MDD had lower GABA concentrations (mean [SEM] concentration: healthy controls, 2.70 [0.03] mmol/L; past MDD, 2.49 [0.05] mmol/L; current MDD, 2.54 [0.07] mmol/L; 92 with past MDD vs 236 healthy controls: r = 0.18; P = .002; 44 with current MDD vs 236 healthy controls: r = 0.13; P = .04). Compared with healthy controls, those with past MDD also had lower Glu concentrations (mean [SEM] concentration: healthy controls, 7.52 [0.06] mmol/L; past MDD, 7.23 [0.11] mmol/L; 93 with past MDD vs 234 healthy controls: r = 0.16; P = .01) and higher Gln concentrations (mean [SEM] concentration: healthy controls, 1.63 [0.04] mmol/L; past MDD, 1.84 [0.07] mmol/L; 66 with past MDD 153 healthy controls: r = 0.17; P = .04). In a large, mostly medication-free community sample, reduced prefrontal GABA concentrations were associated with past MDD, consistent with histopathologic studies reporting reduced glial cell and GABA cell density in the prefrontal cortex in individuals with depression. Patients with MDD also demonstrated increased Gln levels, indicative of increased synaptic Glu release, adding to previous evidence for the Glu hypothesis of MDD.

Characteristics and Impact of the rNST GABA Network on Neural and Behavioral Taste Responses.

The rostral nucleus of the solitary tract (rNST), the initial CNS site for processing gustatory information, is comprised of two major cell types, glutamatergic excitatory and GABAergic inhibitory neurons. Although many investigators have described taste responses of rNST neurons, the phenotypes of these cells were unknown. To directly compare the response characteristics of both inhibitory and noninhibitory neurons, we recorded from mice expressing Channelrhodopsin-2 (ChR2) under the control of GAD65, a synthetic enzyme for GABA. We observed that chemosensitive profiles of GABAergic taste neurons (G+TASTE) were similar to non-GABA taste neurons (G-TASTE) but had much lower response rates. We further observed a novel subpopulation of GABA cells located more ventrally in the nucleus that were unresponsive to taste stimulation (G+UNR), suggesting pathways for inhibition initiated by centrifugal sources. This preparation also allowed us to determine how optogenetic activation of the rNST GABA network impacted the taste responses of G-TASTE neurons. Activating rNST inhibitory circuitry suppressed gustatory responses of G-TASTE neurons across all qualities and chemosensitive types of neurons. Although the tuning curves of identified G-TASTE were modestly sharpened, the overall shape of response profiles and the ensemble pattern remained highly stable. These neurophysiological effects were consistent with the behavioral consequences of activating GAD65-expressing inhibitory neurons using DREADDs. In a brief-access licking task, concentration-response curves to both palatable (sucrose, maltrin) and unpalatable (quinine) stimuli were shifted to the right when GABA neurons were activated. Thus, the rNST GABAergic network is poised to modulate taste intensity across the qualitative and hedonic spectrum.

Abnormal mitochondria in Down syndrome iPSC-derived GABAergic interneurons and organoids

Down syndrome (DS) is caused by trisomy 21, and it is characterized by developmental brain disorders and neurological dysfunction. Clinical studies and basic research have revealed that defects in mitochondrial function contribute to the pathogenesis of DS. However, the underlying mechanisms of mitochondrial dysfunction in DS remain unclear. In this study, we first generated GABAergic interneurons and medial ganglionic eminence (MGE) organoids from DS patients and control induced pluripotent stem cells. The mitochondria were abnormally clustered in the perinuclear region of GABA neurons and cell in MGE organoids from DS patients, which exhibited impaired mitochondrial function as assessed by seahorse oxidative phosphorylation assay. Inhibition of the DSCAM-PAK1 pathway by gene editing or treatment with a small molecule corrected mitochondrial perinuclear aggregation in cells from DS patients. Therefore, our study provides insight into the potential mechanism of mitochondrial dysfunction in DS.

Abstract PD6-06: Comprehensive characterization of neurotransmitters and neuronal signaling gene alterations in invasive breast cancers

Abstract BACKGROUNDIt has been reported that the sympathetic nervous system and associated neurotransmitters (NTs) play a pivotal role in driving breast cancer (BC) tumorigenesis and metastasis, however, comprehensive characterization of these pathways in BC is lacking. The purpose of this study was to retrospectively characterize NTs and neuronal signaling (NTNS) gene alterations in a large real-world BC cohort. METHODS A total of 6464 BC tumors were analyzed by next generation sequencing (NextSeq, 592 genes and WES, NovaSEQ, 720 genes) and whole transcriptome sequencing (WTS, NovaSeq) at Caris Life Sciences. Gene set variation analysis (GSVA) scores were calculated (positive: higher gene expressions in a selected gene set compared to genes outside that gene set in each tumor specimen, vice versa for negative) to assess expression of major NTNS genes, including GABA, nicotinic (NIC), muscarinic (MUS), dopamine (DA), reelin (RELN), and glial cell line-derived neurotrophic factor (GDNF). GSVA scores were compared by histologic subtype, primary or metastatic site, and hormone receptor (HR) and HER2 status with corrected Wilcoxon-Mann-Whitney testing. All significance levels were p<0.01. RESULTS The 6464 BC specimens in this cohort included 2520 primary sites and 3944 metastasis (mets) (liver: 1012; lymph node: 714; bone: 575; lung: 420; brain: 196). Predictive biomarker status in this cohort was HR+/HER2-: 3705; HR+/HER2+: 238; HR-/HER2+: 189; TNBC: 1654. Invasive ductal carcinomas (IDC) were the most common histologic subtype and demonstrated significantly higher GSVA scores for RELN and NIC pathways with respect to invasive lobular carcinomas (ILC) (Table). TNBC tumors had significantly higher enrichment overall (GABA, -0.04 vs -0.14; RELN, -0.05 vs -0.31; DA, -0.03 vs -0.08; MUS, 0.13 vs -0.16; NIC, 0.01 vs -0.12; and GDNF, 0.04 vs -0.04). HR-/HER2+ had significantly higher scores in GABA, -0.04 vs -0.14; RELN, -0.03 vs -0.31; MUS, 0.12 vs -0.16; and NIC, -0.01 vs -0.12 genes. Brain mets had significantly enriched pathway scores for GABA, 0.30 vs -0.13; MUS, 0.15 vs -0.08; and NIC, 0.13 vs -0.09 compared to primary tumors. Similarly, GABA,0.09 vs -0.13; DA, 0.07 vs -0.05; MUS, 0.17 vs -0.08; and NIC, 0.02 vs -0.09 pathways were enriched in bone mets compared to those from primary tumors. CONCLUSION Our results demonstrate that NTNS pathways are significantly enriched in IDC, TNBC tumors, and particularly in brain and bone mets. Our data advance the current understanding of the role of NTNS pathways in BC tumorigenesis and metastasis. Further investigation on genetic. determinants and signaling alternations associated with the observed NTNS pathway deregulation is warranted and could inform the development of novel therapeutic strategies. Significant comparisons with Bonferroni corrected p values are shown with an asterisk. Citation Format: Irene Kang, Krutika Deshpande, Sarah Persing, Jun Yin, Joanne Xiu, Wolfgang Michael Korn, Jia Zeng, Evanthia T Roussos-Torres, Janice Lu, Darcy Spicer, Stephen F Sener, Antoinette R Tan, Ashley Sumrall, David SB Hoon, Cynthia X Ma, Carey K Anders, Heather L McArthur, Reva Basho, Heinz-Josef Lenz, Josh Neman. Comprehensive characterization of neurotransmitters and neuronal signaling gene alterations in invasive breast cancers [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD6-06.

Neonatal ethanol causes profound reduction of cholinergic cell number in the basal forebrain of adult animals.

In animal models that mimic human third-trimester fetal development, ethanol causes substantial cellular apoptosis in the brain, but for most brain structures, the extent of permanent neuron loss that persists into adulthood is unknown. We injected ethanol into C57BL/6J mouse pups at postnatal day 7 (P7) to model human late-gestation ethanol toxicity, and then used stereological methods to investigate adult cell numbers in several subcortical neurotransmitter systems that project extensively in the forebrain to regulate arousal states. Ethanol treatment caused especially large reductions (34-42%) in the cholinergic cells of the basal forebrain, including cholinergic cells in the medial septal/vertical diagonal band nuclei (Ch1/Ch2) and in the horizontal diagonal band/substantia innominata/nucleus basalis nuclei (Ch3/Ch4). Cell loss was also present in non-cholinergic basal forebrain cells, as demonstrated by 34% reduction of parvalbumin-immunolabeled GABA cells and 25% reduction of total Nissl-stained neurons in the Ch1/Ch2 region. In contrast, cholinergic cells in the striatum were reduced only 12% by ethanol, and those of the brainstem pedunculopontine/lateral dorsal tegmental nuclei (Ch5/Ch6) were not significantly reduced. Similarly, ethanol did not significantly reduce dopamine cells of the ventral tegmental area/substantia nigra or serotonin cells in the dorsal raphe nucleus. Orexin (hypocretin) cells in the hypothalamus showed a modest reduction (14%). Our findings indicate that the basal forebrain is especially vulnerable to alcohol exposure in the late gestational period. Reduction of cholinergic and GABAergic projection neurons from the basal forebrain that regulate forebrain arousal may contribute to the behavioral and cognitive deficits associated with neonatal ethanol exposure.

Acute cocaine exposure occludes long-term depression in ventral tegmental area GABA neurons

The ventral tegmental area (VTA) in the midbrain is essential in incentive salience of reward behavior. Drugs of abuse increase midbrain dopamine cell activity and/or dopamine levels, and can alter endogenous VTA glutamate plasticity, leading to addiction or dependence. VTA dopamine cells are regulated by local inhibitory GABA cells, which exhibit a form of pre-synaptic cannabinoid receptor 1-dependent long-term depression of their glutamatergic inputs. Our current aim was to determine cocaine's influence on VTA GABA cell glutamate plasticity and circuity. Using whole cell voltage-clamp electrophysiology in VTA slices of GAD67-GFP knock-in mice, we recorded excitatory inputs on VTA GABA cells. Acute and chronic injections of cocaine were sufficient to occlude long-term depression. The plasticity could be reversed to the naïve state however, as long-term depression was again observed following a 7-day abstinence from acute cocaine exposure. Furthermore, chronic cocaine decreased AMPA/NMDA ratios at glutamate synapses onto VTA GABA cells, compared to vehicle injection controls, the opposite change noted in dopamine cells. Collectively, our data suggest the cellular mechanism of cocaine-mediated synaptic modification that may result in dependence/withdrawal could involve changes in glutamate input to VTA GABA circuitry in addition to VTA dopamine cells. Therefore VTA GABA cells may also play a role, possibly in a synergistic manner with the dopamine circuit, in cocaine-induced changes to the VTA reward pathway than previously known.

Behavioral Deficits Induced by Somatostatin-Positive GABA Neuron Silencing Are Rescued by Alpha 5 GABA-A Receptor Potentiation.

IntroductionDeficits in somatostatin-positive gamma-aminobutyric acid interneurons (SST+ GABA cells) are commonly reported in human studies of mood and anxiety disorder patients. A causal link between SST+ cell dysfunction and symptom-related behaviors has been proposed based on rodent studies showing that chronic stress, a major risk factor for mood and anxiety disorders, induces a low SST+ GABA cellular phenotype across corticolimbic brain regions; that lowering Sst, SST+ cell, or GABA functions induces depressive-/anxiety-like behaviors (a rodent behavioral construct collectively defined as “behavioral emotionality”); and that disinhibiting SST+ cells has antidepressant-like effects. Recent studies found that compounds preferentially potentiating receptors mediating SST+ cell functions, α5-GABAA receptor positive allosteric modulators (α5-PAMs), achieved antidepressant-like effects. Together, the evidence suggests that SST+ cells regulate mood and cognitive functions that are disrupted in mood disorders and that rescuing SST+ cell function via α5-PAM may represent a targeted therapeutic strategy.MethodsWe developed a mouse model allowing chemogenetic manipulation of brain-wide SST+ cells and employed behavioral characterization 30 minutes after repeated acute silencing to identify contributions to symptom-related behaviors. We then assessed whether an α5-PAM, GL-II-73, could rescue behavioral deficits.ResultsBrain-wide SST+ cell silencing induced features of stress-related illnesses, including elevated neuronal activity and plasma corticosterone levels, increased anxiety- and anhedonia-like behaviors, and impaired short-term memory. GL-II-73 led to antidepressant- and anxiolytic-like improvements among behavioral deficits induced by brain-wide SST+ cell silencing.ConclusionOur data validate SST+ cells as regulators of mood and cognitive functions and demonstrate that bypassing low SST+ cell function via α5-PAM represents a targeted therapeutic strategy.

Lentivirus-Mediated Expression of Human Secreted Amyloid Precursor Protein-Alpha Promotes Long-Term Induction of Neuroprotective Genes and Pathways in a Mouse Model of Alzheimer's Disease.

Secreted amyloid precursor protein-alpha (sAPPα) can enhance memory and is neurotrophic and neuroprotective across a range of disease-associated insults, including amyloid-β toxicity. In a significant step toward validating sAPPα as a therapeutic for Alzheimer's disease (AD), we demonstrated that long-term overexpression of human sAPPα (for 8 months) in a mouse model of amyloidosis (APP/PS1) could prevent the behavioral and electrophysiological deficits that develop in these mice. To explore the underlying molecular mechanisms responsible for the significant physiological and behavioral improvements observed in sAPPα-treated APP/PS1 mice. We assessed the long-term effects on the hippocampal transcriptome following continuous lentiviral delivery of sAPPα or empty-vector to male APP/PS1 mice and wild-type controls using Affymetrix Mouse Transcriptome Assays. Data analysis was carried out within the Affymetrix Transcriptome Analysis Console and an integrated analysis of the resulting transcriptomic data was performed with Ingenuity Pathway analysis (IPA). Mouse transcriptome assays revealed expected AD-associated gene expression changes in empty-vector APP/PS1 mice, providing validation of the assays used for the analysis. By contrast, there were specific sAPPα-associated gene expression profiles which included increases in key neuroprotective genes such as Decorin, betaine-GABA transporter and protocadherin beta-5, subsequently validated by qRT-PCR. An integrated biological pathways analysis highlighted regulation of GABA receptor signaling, cell survival and inflammatory responses. Furthermore, upstream gene regulatory analysis implicated sAPPα activation of Interleukin-4, which can counteract inflammatory changes in AD. This study identified key molecular processes that likely underpin the long-term neuroprotective and therapeutic effects of increasing sAPPα levels in vivo.

Contribution of serotonin receptor subtypes to hallucinogenic activity of 25I-NBOMe and to its effect on neurotransmission

Background4-Iodo-2,5-dimethoxy-N-(2-methoxybenzyl)phenethylamine (25I-NBOMe) is a potent serotonin (5-HT) receptor agonist with hallucinogenic properties. The aim of our research was to examine the role of the 5-HT2A, 5-HT2C and 5-HT1A serotonin receptor subtypes in 25I-NBOMe hallucinogenic activity and its effect on dopamine (DA), 5-HT and glutamate release in the rat frontal cortex.MethodsHallucinogenic activity was investigated using the wet dog shake (WDS) test. The release of DA, 5-HT and glutamate in the rat frontal cortex was studied using a microdialysis in freely moving rats. Neurotransmitter levels were analyzed by HPLC with electrochemical detection. The selective antagonists of the 5-HT2A, 5-HT2C and 5-HT1A serotonin receptor subtypes: M100907, SB242084 and WAY100635, respectively were applied through a microdialysis probe.ResultsThe WDS response to 25I-NBOMe (1 and 3 mg/kg) was significantly reduced by local administration of M100907 and SB242084 (100 nM). The 25I-NBOMe-induced increase in glutamate, DA and 5-HT release was inhibited by M100907 and SB242084. WAY100635 had no effect on 25I-NBOMe-induced WDS and glutamate release, while it decreased DA and 5-HT release from cortical neuronal terminals.ConclusionThe obtained results suggest that 5-HT2A and 5-HT2C receptors play a role in 25I-NBOMe-induced hallucinogenic activity and in glutamate, DA and 5-HT release in the rat frontal cortex as their respective antagonists attenuated the effect of this hallucinogen. The disinhibition of GABA cells by the 5-HT1A receptor antagonist seems to underlie the mechanism of decreased DA and 5-HT release from neuronal terminals in the frontal cortex.