Dopamine Modulates Excitability of Basolateral Amygdala Neurons In Vitro

2005 ◽  
Vol 93 (3) ◽  
pp. 1598-1610 ◽  
Author(s):  
Sven Kröner ◽  
J. Amiel Rosenkranz ◽  
Anthony A. Grace ◽  
German Barrionuevo
Keyword(s):  
Basolateral Amygdala ◽  
Neuronal Response ◽  
Brain Slices ◽  
Receptor Activation ◽  
D1 Receptor ◽  
In Vivo Studies ◽  
Projection Neurons ◽  
Direct Effects

The amygdala plays a role in affective behaviors, which are modulated by the dopamine (DA) innervation of the basolateral amygdala complex (BLA). Although in vivo studies indicate that activation of DA receptors alters BLA neuronal activity, it is unclear whether DA exerts direct effects on BLA neurons or whether it acts via indirect effects on BLA afferents. Using whole cell patch-clamp recordings in rat brain slices, we investigated the site and mechanisms through which DA regulates the excitability of BLA neurons. Dopamine enhanced the excitability of BLA projection neurons in response to somatic current injections via a postsynaptic effect. Dopamine D1 receptor activation increased excitability and evoked firing, whereas D2 receptor activation increased input resistance. Current- and voltage-clamp experiments in projection neurons showed that D1 receptor activation enhanced excitability by modulating a 4-aminopyridine- and α-dendrotoxin-sensitive, slowly inactivating K+ current. Furthermore, DA and D1 receptor activation increased evoked firing in fast-spiking BLA interneurons. Consistent with a postsynaptic modulation of interneuron excitability, DA also increased the frequency of spontaneous inhibitory postsynaptic currents recorded in projection neurons without changing release of GABA. These data demonstrate that DA exerts direct effects on BLA projection neurons and indirect actions via modulation of interneurons that may work in concert to enhance the neuronal response to large, suprathreshold inputs, while suppressing weaker inputs.

scholarly journals Hyperoxia, reactive oxygen species, and hyperventilation: oxygen sensitivity of brain stem neurons

2004 ◽  
Vol 96 (2) ◽  
pp. 784-791 ◽  
Author(s):  
Jay B. Dean ◽  
Daniel K. Mulkey ◽  
Richard A. Henderson ◽  
Stephanie J. Potter ◽  
Robert W. Putnam
Keyword(s):  
Oxidative Stress ◽  
Brain Stem ◽  
Brain Slices ◽  
Respiratory Control ◽  
In Vivo Studies ◽  
Future Research ◽  
Direct Effects ◽  
Underlying Assumption

Hyperoxia is a popular model of oxidative stress. However, hyperoxic gas mixtures are routinely used for chemical denervation of peripheral O2 receptors in in vivo studies of respiratory control. The underlying assumption whenever using hyperoxia is that there are no direct effects of molecular O2 and reactive O2 species (ROS) on brain stem function. In addition, control superfusates used routinely for in vitro studies of neurons in brain slices are, in fact, hyperoxic. Again, the assumption is that there are no direct effects of O2 and ROS on neuronal activity. Research contradicts this assumption by demonstrating that O2 has central effects on the brain stem respiratory centers and several effects on neurons in respiratory control areas; these need to be considered whenever hyperoxia is used. This mini-review summarizes the long-recognized, but seldom acknowledged, paradox of respiratory control known as hyperoxic hyperventilation. Several proposed mechanisms are discussed, including the recent hypothesis that hyperoxic hyperventilation is initiated by increased production of ROS during hyperoxia, which directly stimulates central CO2 chemoreceptors in the solitary complex. Hyperoxic hyperventilation may provide clues into the fundamental role of redox signaling and ROS in central control of breathing; moreover, oxidative stress may play a role in respiratory control dysfunction. The practical implications of brain stem O2 and ROS sensitivity are also considered relative to the present uses of hyperoxia in respiratory control research in humans, animals, and brain stem tissues. Recommendations for future research are also proposed.

scholarly journals Excitation of medium spiny neurons by ‘inhibitory’ ultrapotent chemogenetics via shifts in chloride reversal potential

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Stephanie C Gantz ◽  
Maria M Ortiz ◽  
Andrew J Belilos ◽  
Khaled Moussawi
Keyword(s):  
Brain Slices ◽  
Reversal Potential ◽  
Cell Types ◽  
Receptor Activation ◽  
D1 Receptor ◽  
Medium Spiny Neurons ◽  
Inhibitory Function ◽  
Spiny Neurons

Ultrapotent chemogenetics, including the chloride-permeable inhibitory PSAM4-GlyR receptor, were recently proposed as a powerful strategy to selectively control neuronal activity in awake, behaving animals. We aimed to validate the inhibitory function of PSAM4-GlyR in dopamine D1 receptor-expressing medium spiny neurons (D1-MSNs) in the ventral striatum. Activation of PSAM4-GlyR with the uPSEM792 ligand enhanced rather than suppressed the activity of D1-MSNs in vivo as indicated by increased c-fos expression in D1-MSNs and in vitro as indicated by cell-attached recordings from D1-MSNs in mouse brain slices. Whole-cell recordings showed that activation of PSAM4-GlyR depolarized D1-MSNs, attenuated GABAergic inhibition, and shifted the reversal potential of PSAM4-GlyR current to more depolarized potentials, perpetuating the depolarizing effect of receptor activation. These data show that ‘inhibitory’ PSAM4-GlyR chemogenetics may activate certain cell types and highlight the pitfalls of utilizing chloride conductances to inhibit neurons.

Physiology, Pharmacology, and Topography of Cholinergic Neocortical Oscillations In Vitro

1997 ◽  
Vol 77 (5) ◽  
pp. 2427-2445 ◽  
Author(s):  
Heath S. Lukatch ◽  
M. Bruce Maciver
Keyword(s):  
Current Source ◽  
Brain Slices ◽  
Receptor Activation ◽  
Brain Regions ◽  
Oscillatory Activity ◽  
Cortical Layers ◽  
Eeg Activity ◽  
Layer 2

Lukatch, Heath S. and M. Bruce MacIver. Physiology, pharmacology, and topography of cholinergic neocortical oscillations in vitro. J. Neurophysiol. 77: 2427–2445, 1997. Rat neocortical brain slices generated rhythmic extracellular field [microelectroencephalogram (micro-EEG)] oscillations at theta frequencies (3–12 Hz) when exposed to pharmacological conditions that mimicked endogenous ascending cholinergic and GABAergic inputs. Use of the specific receptor agonist and antagonist carbachol and bicuculline revealed that simultaneous muscarinic receptor activation and γ-aminobutyric acid-A (GABAA)-mediated disinhibition werenecessary to elicit neocortical oscillations. Rhythmic activity was independent of GABAB receptor activation, but required intact glutamatergic transmission, evidenced by blockade or disruption of oscillations by 6-cyano-7-nitroquinoxaline-2,3-dione and (±)-2-amino-5-phosphonovaleric acid, respectively. Multisite mapping studies showed that oscillations were localized to areas 29d and 18b (Oc2MM) and parts of areas 18a and 17. Peak oscillation amplitudes occurred in layer 2/3, and phase reversals were observed in layers 1 and 5. Current source density analysis revealed large-amplitude current sinks and sources in layers 2/3 and 5, respectively. An initial shift in peak inward current density from layer 1 to layer 2/3 indicated that two processes underlie an initial depolarization followed by oscillatory activity. Laminar transections localized oscillation-generating circuitry to superficial cortical layers and sharp-spike-generating circuitry to deep cortical layers. Whole cell recordings identified three distinct cell types based on response properties during rhythmic micro-EEG activity: oscillation-on (theta-on) and -off (theta-off) neurons, and transiently depolarizing glial cells. Theta-on neurons displayed membrane potential oscillations that increased in amplitude with hyperpolarization (from −30 to −90 mV). This, taken together with a glutamate antagonist-induced depression of rhythmic micro-EEG activity, indicated that cholinergically driven neocortical oscillations require excitatory synaptic transmission. We conclude that under the appropriate pharmacological conditions, neocortical brain slices were capable of producing localized theta frequency oscillations. Experiments examining oscillation physiology, pharmacology, and topography demonstrated that neocortical brain slice oscillations share many similarities with the in vivo and in vitro theta EEG activity recorded in other brain regions.

scholarly journals SAT-742 Characterising the Metabolism, Glucuronidation and Sulfation of C11-oxy C19 Steroids

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Therina Du Toit ◽  
Amanda C Swart
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Receptor Activation ◽  
In Vitro Models ◽  
In Vivo Studies ◽  
Cancer Tissue ◽  
Mcf 7

Abstract The metabolism of 11β-hydroxyandrostenedione (11OHA4), a major adrenal C19 steroid, was first characterised in our in vitro prostate models showing that 11OHA4, catalysed by 11βHSDs, 17βHSDs and 5α-reductases, yields potent androgens, 11keto-testosterone (11KT) and 11keto-dihydrotestosterone (11KDHT) in the 11OHA4-pathway [1]. Findings have since led to the analysis of C11-oxy steroids in PCOS, CAH and 21OHD. However, the only circulating C11-oxy steroids included to date have been 11OHA4, 11keto-androstenedione (11KA4), 11β-hydroxytestosterone (11OHT) and 11KT, with 11KT reported as the only potent androgen produced from 11OHA4. We have identified higher levels of 11KDHT compared to 11KT in prostate cancer tissue and benign prostatic hyperplasia tissue and serum, with data suggesting impeded glucuronidation of the C11-oxy androgens [2,3]. The assessment of 11KDHT and the inactivation/conjugation of the C11-oxy steroids in clinical conditions is therefore crucial. We investigated the metabolism of testosterone, 11KT, 11OHT, dihydrotestosterone, 11KDHT and 11OHDHT in JEG-3 placenta choriocarcinoma, MCF-7 BUS and T-47D breast cancer cells, focusing on glucuronidation and sulfation. Steroids were assayed at 1 µM and metabolites were quantified using UPC2-MS/MS. Conjugated steroids were not detected in JEG-3 cells with DHT (0.6 µM remaining) metabolised to 5α-androstane-3α,17β-diol and androsterone (AST), and 11KDHT (0.9 µM remaining) to 11OHAST and 11KAST. 11OHA4 was converted to 11KA4 (12%) and 11KT (2.5%); and 11KT to 11KDHT (14%). In MCF-7 BUS cells, DHT was significantly glucuronidated, whereas 11KDHT was not. 11KAST was the only steroid in the MCF-7 BUS and T-47D cells that was significantly sulfated (p<0.05). In parallel we investigated sulfation in the LNCaP prostate model. Comparing sulfated to glucuronidated levels, only DHT was sulfated, 26%. Analysis showed that C19 steroids were significantly conjugated (glucuronidated + sulfated) compared to the C11-oxy C19 steroids. As there exists an intricate interplay between steroid production and inactivation, impacting pre- and post-receptor activation, efficient conjugation would limit adverse downstream effects. Our data demonstrates the production and impeded conjugation of active C11-oxy C19 steroids, allowing the prolonged presence of androgenic steroids in the cellular microenvironment. Identified for the first time is the 11OHA4-pathway in placenta and breast cancer cells, and the sulfation of 11KAST. Characterising steroidogenic pathways in in vitro models paves the direction for in vivo studies associated with characterising clinical disorders and disease, which the C11-oxy C19 steroids and their intermediates, including inactivated and conjugated end-products, have highlighted. [1] Bloem, et al. JSBMB 2015, 153; [2] Du Toit & Swart. MCE 2018, 461; [3] Du Toit & Swart, JSBMB 2020, 105497.

scholarly journals Inhibitory ultrapotent chemogenetics activate dopamine D1 receptor-expressing medium spiny neurons

2020 ◽  
Author(s):  
Stephanie C. Gantz ◽  
Maria M. Ortiz ◽  
Andrew J. Belilos ◽  
Khaled Moussawi
Keyword(s):  
Brain Slices ◽  
Reversal Potential ◽  
Cell Types ◽  
Receptor Activation ◽  
Dopamine D1 Receptor ◽  
D1 Receptor ◽  
Medium Spiny Neurons ◽  
Inhibitory Function ◽  
Spiny Neurons

SUMMARYUltrapotent chemogenetics, including the chloride-permeable inhibitory PSAM4-GlyR receptor, were recently proposed as a powerful strategy to selectively control neuronal activity in awake, behaving animals. We aimed to validate the inhibitory function of PSAM4-GlyR in dopamine D1 receptor-expressing medium spiny neurons (D1-MSNs) in the ventral striatum. Activation of PSAM4-GlyR with the uPSEM792 ligand enhanced rather than suppressed the activity of D1-MSNs in vivo as indicated by increased c-fos expression in D1-MSNs. Whole-cell recordings in mouse brain slices showed that activation of PSAM4-GlyR did not inhibit firing of action potentials in D1-MSNs. Activation of PSAM4-GlyR depolarized D1-MSNs, attenuated GABAergic inhibition, and shifted the reversal potential of PSAM4-GlyR current to more depolarized potentials, perpetuating the depolarizing effect of receptor activation. The data show that ‘inhibitory’ PSAM4-GlyR chemogenetics may actually activate certain cell types, and highlight the pitfalls of utilizing chloride conductances to inhibit neurons.

scholarly journals A fluorescent sensor for spatiotemporally resolved endocannabinoid dynamics in vitro and in vivo

2020 ◽  
Author(s):  
Ao Dong ◽  
Kaikai He ◽  
Barna Dudok ◽  
Jordan S Farrell ◽  
Wuqiang Guan ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Basolateral Amygdala ◽  
Fluorescent Sensor ◽  
Brain Slices ◽  
High Specificity ◽  
Cultured Neurons ◽  
Spatiotemporal Resolution ◽  
Wide Range

Endocannabinoids (eCBs) are retrograde neuromodulators that play an important role in a wide range of physiological processes; however, the release and in vivo dynamics of eCBs remain largely unknown, due in part to a lack of suitable probes capable of detecting eCBs with sufficient spatiotemporal resolution. Here, we developed a new eCB sensor called GRABeCB2.0. This genetically encoded sensor consists of the human CB1 cannabinoid receptor fused to circular-permutated EGFP, providing cell membrane trafficking, second-resolution kinetics, high specificity for eCBs, and a robust fluorescence response at physiological eCB concentrations. Using the GRABeCB2.0 sensor, we monitored evoked changes in eCB dynamics in both cultured neurons and acute brain slices. Interestingly, in cultured neurons we also observed spontaneous compartmental eCB transients that spanned a distance of approximately 11 μm, suggesting constrained, localized eCB signaling. Moreover, by expressing GRABeCB2.0 in the mouse brain, we readily observed foot shock-elicited and running-triggered eCB transients in the basolateral amygdala and hippocampus, respectively. Lastly, we used GRABeCB2.0 in a mouse seizure model and observed a spreading wave of eCB release that followed a Ca2+ wave through the hippocampus. Thus, GRABeCB2.0 is a robust new probe for measuring the dynamics of eCB release under both physiological and pathological conditions.

Cholecystokinin activates area postrema neurons in rat brain slices

1997 ◽  
Vol 272 (5) ◽  
pp. R1625-R1630 ◽  
Author(s):  
K. Sun ◽  
A. V. Ferguson
Keyword(s):  
Food Intake ◽  
Receptor Antagonist ◽  
Area Postrema ◽  
Brain Slices ◽  
In Vivo Studies ◽  
Unit Recording ◽  
Dose Dependent ◽  
Cckb Receptor

Peripheral cholecystokinin (CCK) reduces food intake and triggers the secretion of both oxytocin and corticotropin-releasing hormone. These responses are partially initiated by activation of receptors in the peripheral endings of the vagus nerve. However, in vivo studies showing that after vagotomy systemic CCK induces fos activation of neurons in the area postrema (AP) suggest that circulating CCK may directly influence the activity of neurons in this structure. The present study was therefore designed to investigate the responsiveness of AP neurons to CCK using in vitro extracellular single-unit recording techniques. Bath application of 100 nM CCK for 200 s resulted in excitatory responses in 41% and inhibitory effects in 6% of 143 AP neurons tested. Application of multiple doses of CCK (1-100 nM) to single neurons demonstrated that CCK effects were dose dependent. The firing rate of tested neurons increased by 48 +/- 15% in response to 1 nM, by 89 +/- 22% in response to 10 nM, and by 242 +/- 77% in response to 100 nM CCK. After we blockaded synaptic transmission with a low-Ca2+/high-Mg2+ artificial cerebrospinal fluid, the excitatory effects of CCK remained in all nine neurons tested. The CCK-receptor antagonist L-364,718 had no significant effect on the responses to CCK (P > 0.1, n = 4), whereas, after perfusion of slices with the CCKB-receptor antagonist L-365,260, mean responses to CCK were significantly reduced to 12.6 +/- 4.7% of the control value (P < 0.001, n = 4). These results demonstrate a direct and dose-dependent excitatory action of CCK on AP neurons that is abolished by CCKB-receptor antagonists. These data emphasize the potential role of AP in processing afferent information derived from circulating peptide concentrations that could be involved in the regulation of food intake.

scholarly journals Dopamine D1 receptor activation drives plasticity in the songbird auditory pallium

2020 ◽  
Author(s):  
Matheus Macedo-Lima ◽  
Hannah M. Boyd ◽  
Luke Remage-Healey
Keyword(s):  
Vocal Learning ◽  
Receptor Activation ◽  
D1 Receptor ◽  
Auditory Neurons ◽  
Production Variability ◽  
Neuronal Types ◽  
Dopamine Signaling ◽  
Motor Production

AbstractVocal learning species must form and extensively hone associations between sounds and social contingencies. In songbirds, dopamine signaling guides song motor-production, variability, and motivation, but it is unclear how dopamine regulates fundamental auditory associations for learning new sounds. We hypothesized that dopamine regulates learning in the auditory pallium, in part by interacting with local neuroestradiol signaling. Here, we show that zebra finch auditory neurons frequently coexpress D1 receptor (D1R) protein, neuroestradiol-synthase, GABA, and parvalbumin. Auditory classical conditioning increased neuroplasticity gene induction in D1R-positive neurons. In vitro, D1R pharmacological activation reduced the amplitude of GABAergic and glutamatergic currents, and increased the latter’s frequency. In vivo, D1R activation reduced the firing of putative interneurons, increased the firing of putative excitatory neurons, and made both neuronal types unable to adapt to novel stimuli. Together, these data support the hypothesis that dopamine acting via D1Rs modulates learning and memory in the songbird sensory cortex.

Abstract 235: Sonic Hedgehog Induces Angiogenesis via Rho Kinase-dependent MMP-9 and Osteopontin Expression

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Marie-Ange Renault ◽  
Jerome Roncalli ◽  
Joern Tongers ◽  
Tina Thorne ◽  
Sol Misener ◽  
...  
Keyword(s):  
Sonic Hedgehog ◽  
Rho Kinase ◽  
Dominant Negative ◽  
In Vivo Studies ◽  
Tube Length ◽  
Classical Pathway ◽  
Direct Effects ◽  
Angiogenic Activity

The morphogen Sonic Hedgehog (Shh) is known to promote neovascularization in adults via indirect induction of pro-angiogenic cytokine expression by fibroblasts. Direct effects of Shh on endothelial cell (EC) function and angiogenesis, however, have not been characterized. Accordingly, we performed a series of in vitro and in vivo studies to evaluate the direct effects of Shh on EC and to investigate certain mechanisms by which Shh modulates angiogenesis. Our data disclose that Shh promotes capillary morphogenesis (tube length on Matrigel ™ increased to 271±50% of the length in untreated cells), induces EC migration (191±35%) and increases EC expression of matrix metalloproteinase 9 (MMP-9) and osteopontin (OPN), which are shown to be essential for Shh-induced angiogenesis both in vitro and in vivo . Shh effects in ECs, however, occur while Gli-dependent transcription is not modulated. Furthemore, our studies show that changes in gene expression and EC migration are mediated by Shh induction of Rho. The Rho dependence of Shh-induced EC angiogenic activity is documented in vitro using dominant negative constructs for RhoA and Rho kinase (ROCK), showing that RhoA and ROCK blockade attenuates Shh induced migration and tube formation, as well as the Shh induced expression of MMP-9 and OPN. In vivo in the mouse corneal angiogenesis model pharmacologic inhibition of ROCK blocks Shh induced angiogenesis, confirming the ROCK dependence of this process. Furthermore, in MMP-9 and OPN null mice Shh induced angiogenesis also blocked, indicating that Shh induced angiogenesis is also dependent on MMP-9 and OPN expression. These data elucidate an entirely novel, “non-classical” pathway by which Shh directly modulates EC phenotype and angiogenic activity

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