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1.
Brain Res ; 1837: 148955, 2024 Aug 15.
Article in English | MEDLINE | ID: mdl-38679314

ABSTRACT

Swallowing is induced by a central pattern generator in the nucleus tractus solitarius (NTS). We aimed to create a medullary slice preparation to elucidate the neural architecture of the central pattern generator of swallowing (Sw-CPG) and record its neural activities. Experiments were conducted on 2-day-old Sprague-Dawley rats (n = 46). The brainstem-spinal cord was transected at the pontomedullary and cervicothoracic junctions; the medulla was sliced transversely at thicknesses of 600, 700, or 800 µm. The rostral end of the slice was 100 µm rostral to the vagus nerve. We recorded hypoglossal nerve activity and electrically stimulated the vagus nerve or microinjected bicuculline methiodide (BIC) into the NTS. The 800-µm slices generated both rhythmic respiratory activity and electrically elicited neural activity. The 700-µm slices generated only respiratory activity, while the 600-µm slices did not generate any neural activity. BIC microinjection into the NTS in 800-µm slices resulted in the typical activity that closely resembled the swallowing activity reported in other experiments. This swallowing-like activity consistently lengthened the respiratory interval. Despite complete inhibition of respiratory activity, weak swallowing-like activity was observed under bath application of a non-NMDA receptor antagonist. Contrastingly, bath application of NMDA receptor antagonists resulted in a complete loss of swallowing-like activity and no change in respiratory activity. These results suggest that the 800-µm medullary slice preparation contains both afferent and efferent neural circuits and pattern generators of swallowing activity. Additionally, NMDA receptors may be necessary for generating swallowing activity. This medullary slice preparation can therefore elucidate Sw-CPG neural networks.


Subject(s)
Animals, Newborn , Bicuculline , Central Pattern Generators , Deglutition , Hypoglossal Nerve , Medulla Oblongata , Rats, Sprague-Dawley , Vagus Nerve , Animals , Deglutition/physiology , Deglutition/drug effects , Medulla Oblongata/physiology , Medulla Oblongata/drug effects , Bicuculline/pharmacology , Bicuculline/analogs & derivatives , Rats , Vagus Nerve/physiology , Vagus Nerve/drug effects , Central Pattern Generators/physiology , Central Pattern Generators/drug effects , Hypoglossal Nerve/physiology , Hypoglossal Nerve/drug effects , Electric Stimulation , Solitary Nucleus/drug effects , Solitary Nucleus/physiology
2.
Brain Res ; 1763: 147451, 2021 07 15.
Article in English | MEDLINE | ID: mdl-33773979

ABSTRACT

Neurons in the subfornical organ (SFO) sense both neurotransmitters and circulating humoral factors such as angiotensin II (AII) and atrial natriuretic peptide (ANP), and regulate multiple physiological functions including drinking behavior. We recently reported that AII at nanomolar concentrations induced a persistent [Ca2+]i increase in acutely dissociated SFO neurons and that this effect of AII was reversibly inhibited by GABA. In the present study, we studied the inhibitory mechanism of GABA using Ca2+ imaging and patch-clamp electrophysiology. The AII-induced persistent [Ca2+]i increase was inhibited by GABA in more than 90% of AII-responsive neurons and by other two SFO inhibitory ligands, ANP and galanin, in about 60 and 30% of neurons respectively. The inhibition by GABA was mimicked by the GABAA and GABAB receptor agonists muscimol and baclofen. The involvement of both GABA receptor subtypes was confirmed by reversal of the GABA-mediated inhibition only when the GABAA and GABAB receptors antagonists bicuculline methiodide and CGP55845 were both present. The GABAB agonist baclofen rapidly and reversibly inhibited voltage-gated Ca2+ channel (VGCC) currents recorded in response to depolarizing pulses in voltage-clamp electrophysiology using Ba2+ as a charge carrier (IBa). Baclofen inhibition of IBa was antagonized by CGP55845, confirming GABAB receptor involvement; was reduced by N-ethylmaleimide, suggesting downstream Gi-mediated actions; and was partially removed by a large prepulse, indicating voltage-dependency. The magnitude of IBa inhibition by baclofen was reduced by the application of selective blockers for N-, P/Q-, and L-type VGCCs (ω-conotoxin GVIA, ω-agatoxin IVA, and nifedipine respectively). Overall, our study indicates that GABA inhibition of the AII-induced [Ca2+]i increase is mediated by both GABAA and GABAB receptors, and that GABAB receptors associated with Gi proteins suppress Ca2+ entry through VGCCs in SFO neurons.


Subject(s)
Angiotensin II/metabolism , Bicuculline/analogs & derivatives , Calcium/metabolism , GABA-A Receptor Agonists/pharmacology , GABA-B Receptor Agonists/pharmacology , Subfornical Organ/drug effects , Animals , Baclofen/metabolism , Bicuculline/pharmacology , Calcium Channels/metabolism , Ethylamines/pharmacology , Male , Neurons/drug effects , Neurons/metabolism , Rats , Rats, Wistar , Receptors, GABA-B/metabolism , Subfornical Organ/metabolism
3.
Pflugers Arch ; 472(8): 1051-1063, 2020 08.
Article in English | MEDLINE | ID: mdl-32617654

ABSTRACT

The Kölliker-Fuse (KF) nucleus is a part of the parabrachial complex, located in the dorsolateral pons. It is involved in the chemoreflex-evoked cardiovascular and respiratory changes, but the role of GABA and glutamate in cardiovascular chemoreflex has not been shown yet. This study was performed to determine the role of GABA, glutamate, and their interaction in the KF, in cardiovascular chemoreflex in anesthetized rat. The antagonists were microinjected into the KF, and arterial pressure, heart rate, and single-unit responses were recorded simultaneously. The chemoreflex was evoked by i.v. injection of KCN, consisted of a short pressor followed by long bradycardia responses. Both responses were significantly attenuated by injection of a synaptic blocker (CoCl2) into the KF, confirming involvement of the KF in generating the reflex. Microinjection of AP5, an NMDA receptor antagonist, into the KF significantly attenuated the pressor and bradycardia responses, while blocking the AMPA receptors by CNQX had no significant effect. Blockade of GABAA receptors by bicuculline methiodide (BMI) potentiated both responses. Co-injection of BMI and CNQX potentiated the responses too. Co-injection of BMI and AP5 had no significant effect on the pressor response but significantly attenuated the bradycardia response. In conclusion, the KF plays a role in generating cardiovascular chemoreflex via its glutamate NMDA but not AMPA receptors. GABA inhibits both components of this reflex through GABAA receptors. There is an interaction between GABAA and NMDA receptors in regulating the bradycardia response of the reflex. Single-unit results were also presented which were correlated with and supported the homodynamic findings.


Subject(s)
Cardiovascular System/metabolism , Chemoreceptor Cells/metabolism , Glutamic Acid/metabolism , Kolliker-Fuse Nucleus/metabolism , Reflex/physiology , gamma-Aminobutyric Acid/metabolism , Animals , Bicuculline/analogs & derivatives , Bicuculline/pharmacology , Blood Pressure/drug effects , Blood Pressure/physiology , Chemoreceptor Cells/drug effects , Heart Rate/drug effects , Heart Rate/physiology , Kolliker-Fuse Nucleus/drug effects , Male , Pons/drug effects , Pons/physiology , Rats , Rats, Sprague-Dawley , Receptors, AMPA/metabolism , Receptors, GABA-A/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Reflex/drug effects , Respiration/drug effects
4.
J Neurosci ; 40(7): 1527-1537, 2020 02 12.
Article in English | MEDLINE | ID: mdl-31911457

ABSTRACT

The ongoing activity of prefrontal neurons after a stimulus has disappeared is considered a neuronal correlate of working memory. It depends on the delicate but poorly understood interplay between excitatory glutamatergic and inhibitory GABAergic receptor effects. We administered the NMDA receptor antagonist MK-801 and the GABA(A) receptor antagonist bicuculline methiodide while recording cellular activity in PFC of male rhesus monkeys performing a delayed decision task requiring working memory. The blockade of GABA(A) receptors strongly improved the selectivity of the neurons' delay activity, causing an increase in signal-to-noise ratio during working memory periods as well as an enhancement of the neurons' coding selectivity. The blockade of NMDA receptors resulted in a slight enhancement of selectivity and encoding capacity of the neurons. Our findings emphasize the delicate and more complex than expected interplay of excitatory and inhibitory transmitter systems in modulating working memory coding in prefrontal circuits.SIGNIFICANCE STATEMENT Ongoing delay activity of prefrontal neurons constitutes a neuronal correlate of working memory. However, how this delay activity is generated by the delicate interplay of synaptic excitation and inhibition is unknown. We probed the effects of excitatory neurotransmitter glutamate and inhibitory neurotransmitter GABA in regulating delay activity in rhesus monkeys performing a delayed decision task requiring working memory. Surprisingly, the blockade of both glutamatergic NMDA and GABA(A) receptors improved neuronal selectivity of delay activity, causing an increase in neuronal signal-to-noise ratio. Moreover, individual neurons were similarly affected by blockade of both receptors. This emphasizes the delicate and more complex than expected interplay of excitatory and inhibitory transmitter systems in modulating working memory coding in prefrontal circuits.


Subject(s)
Memory, Short-Term/physiology , Neurons/drug effects , Prefrontal Cortex/physiology , Action Potentials/drug effects , Animals , Bicuculline/analogs & derivatives , Bicuculline/pharmacology , Dizocilpine Maleate , Excitatory Amino Acid Antagonists , GABA-A Receptor Antagonists , GABAergic Neurons/drug effects , GABAergic Neurons/physiology , Glutamic Acid/physiology , Macaca mulatta , Male , Memory, Short-Term/drug effects , Mental Recall , Neurons/physiology , Pattern Recognition, Visual/drug effects , Pattern Recognition, Visual/physiology , Photic Stimulation , Prefrontal Cortex/drug effects , Psychomotor Performance/drug effects , Receptors, GABA-A , Receptors, N-Methyl-D-Aspartate
5.
Chembiochem ; 21(10): 1526-1533, 2020 05 15.
Article in English | MEDLINE | ID: mdl-31859406

ABSTRACT

Bicuculline, a valued chemical tool in neurosciences research, is a competitive antagonist of specific GABAA receptors and affects other pentameric ligand-gated ion channels including the glycine, nicotinic acetylcholine and 5-hydroxytryptamine type 3 receptors. We used a fluorescence-quenching assay and isothermal titration calorimetry to record low-micromolar dissociation constants for N-methylbicuculline interacting with acetylcholine-binding protein and an engineered version called glycine-binding protein (GBP), which provides a surrogate for the heteromeric interface of the extracellular domain of the glycine receptor (GlyR). The 2.4 Šresolution crystal structure of the GBP:N-methylbicuculline complex, sequence and structural alignments reveal similarities and differences between GlyR and the GABAA receptor-bicuculline interactions. N-methylbicuculline displays a similar conformation in different structures, but adopts distinct orientations enforced by interactions and steric blocks with key residues and plasticity in the binding sites. These features explain the promiscuous activity of bicuculline against the principal inhibitory pentameric ligand-gated ion channels in the CNS.


Subject(s)
Bicuculline/analogs & derivatives , Ion Channel Gating , Receptors, GABA-A/chemistry , Receptors, Glycine/antagonists & inhibitors , Amino Acid Sequence , Bicuculline/chemistry , Bicuculline/pharmacology , Binding Sites , GABA Antagonists/chemistry , GABA Antagonists/pharmacology , Humans , Models, Molecular , Protein Conformation , Sequence Homology
6.
Neuron ; 102(2): 420-434.e8, 2019 04 17.
Article in English | MEDLINE | ID: mdl-30826183

ABSTRACT

Presynaptic inhibition (PSI) of primary sensory neurons is implicated in controlling gain and acuity in sensory systems. Here, we define circuit mechanisms and functions of PSI of cutaneous somatosensory neuron inputs to the spinal cord. We observed that PSI can be evoked by different sensory neuron populations and mediated through at least two distinct dorsal horn circuit mechanisms. Low-threshold cutaneous afferents evoke a GABAA-receptor-dependent form of PSI that inhibits similar afferent subtypes, whereas small-diameter afferents predominantly evoke an NMDA-receptor-dependent form of PSI that inhibits large-diameter fibers. Behaviorally, loss of either GABAA receptors (GABAARs) or NMDA receptors (NMDARs) in primary afferents leads to tactile hypersensitivity across skin types, and loss of GABAARs, but not NMDARs, leads to impaired texture discrimination. Post-weaning age loss of either GABAARs or NMDARs in somatosensory neurons causes systemic behavioral abnormalities, revealing critical roles of two distinct modes of PSI of somatosensory afferents in adolescence and throughout adulthood.


Subject(s)
Behavior, Animal , Nerve Tissue Proteins/genetics , Neural Inhibition , Presynaptic Terminals/metabolism , Receptors, GABA-A/genetics , Receptors, N-Methyl-D-Aspartate/genetics , Sensory Receptor Cells/metabolism , Skin/innervation , Touch , Animals , Bicuculline/analogs & derivatives , Bicuculline/pharmacology , Discrimination, Psychological , GABA-A Receptor Antagonists/pharmacology , Hyperesthesia/genetics , Mice , Nerve Tissue Proteins/metabolism , Neurons, Afferent/metabolism , Pyridazines/pharmacology , Receptors, GABA-A/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Spinal Cord Dorsal Horn
7.
Brain Struct Funct ; 224(3): 1245-1265, 2019 Apr.
Article in English | MEDLINE | ID: mdl-30680454

ABSTRACT

While recently completing a study of the effects of stimulating the lateral preoptic area (LPO) and ventral pallidum (VP) on locomotion and other movements, we also noticed LPO and VP effects on motivational drive and threat tolerance. Here, we have investigated these latter effects by testing conditioned place preference (CPP), behavior on the elevated plus maze (EPM) and the willingness of sated rats to occupy a harshly lit open field center to acquire sweet pellets, a measure of threat tolerance, following infusions of vehicle or bicuculline (bic) into the LPO and VP. LPO-bic infusions robustly increased total locomotion, and, in direct proportion, occupancy of both the harshly lit field center and open arms of the EPM. LPO bic also generated CPP, but did not increase sweet pellet ingestion. These effects were attenuated by dopamine D1 and D2 receptor antagonists, whether given individually or as a cocktail and systemically or infused bilaterally into the nucleus accumbens. VP-bic infusions did not increase total locomotion, but preferentially increased field center occupancy. VP-bic-infused rats compulsively ingested sweet pellets and did so even under the spotlight, whereas harsh illumination suppressed pellet ingestion in the control groups. VP bic produced CPP and increased open arm occupancy on the EPM. These effects were attenuated by pretreatment with dopamine receptor antagonists given systemically or as bilateral infusions into the VP, except for % distance in the field center (by D1 or D2 antagonists) and pellet ingestion (by D1 antagonist). Thus, boldness generated in association with LPO activation is tightly tied to locomotor activation and, as is locomotion itself, strongly DA dependent, whereas that accompanying stimulation of the VP is independent of locomotor activation and, at least in part, DA signaling. Furthermore, respective emboldened behaviors elicited from neither LPO nor VP could clearly be attributed to goal pursuit. Rather, emboldening of behavior seems more to be a fixed action response not fundamentally different than previously for reported locomotion, pivoting, backing, gnawing, and eating elicited by basal forebrain stimulation.


Subject(s)
Basal Forebrain/physiology , Conditioning, Operant/physiology , Exploratory Behavior/physiology , Locomotion/physiology , Preoptic Area/physiology , Animals , Basal Forebrain/drug effects , Bicuculline/analogs & derivatives , Bicuculline/pharmacology , Conditioning, Operant/drug effects , Dopamine Agents/pharmacology , Exploratory Behavior/drug effects , GABA-A Receptor Antagonists/pharmacology , Locomotion/drug effects , Male , Maze Learning/drug effects , Maze Learning/physiology , Preoptic Area/drug effects , Rats
8.
Brain Struct Funct ; 224(1): 171-190, 2019 Jan.
Article in English | MEDLINE | ID: mdl-30298290

ABSTRACT

Deficient prefrontal cortex (PFC) GABA function is hypothesized to play a role in schizophrenia and other psychiatric disorders. In rodents, PFC GABAA receptor antagonism produces cognitive and behavioral changes relevant to these disorders, including impaired spatial memory assessed with the traditional working/reference memory radial maze task. This aspect of spatial memory does not depend on PFC, suggesting that deficient PFC GABAergic transmission may interfere with non-PFC-dependent cognitive functions via aberrant increases in PFC output. To test this, we assessed whether PFC GABAA antagonism (50 ng bicuculline methbromide) alters neuronal activation in PFC terminal regions, including the striatum, thalamus, hippocampus, amygdala, and cortical regions, of adult male rats using the immediate early gene, c-Fos, as an activity marker. A subset of these animals were also trained and/or tested on the working/reference memory radial maze task. These treatments caused widespread increases in neuronal activation in animals under baseline conditions, with notable exception of the hippocampus. Furthermore, PFC GABAA antagonism impaired task performance. In most instances, training and/or testing on the radial maze had no additional effects on neuronal activation. However, in both the hippocampus and rhomboid thalamic nucleus, PFC GABAA antagonism caused a selective increase in neuronal activation in animals trained on the maze. These results indicate that deficiencies in PFC GABAergic transmission may have widespread impacts on neuronal activity that may interfere with certain PFC-independent cognitive functions. Furthermore, these alterations in activity are modulated by plasticity induced by spatial learning in the hippocampus and rhomboid thalamic nucleus.


Subject(s)
Behavior, Animal , Maze Learning , Neuronal Plasticity , Prefrontal Cortex/metabolism , Receptors, GABA-A/metabolism , Synaptic Transmission , gamma-Aminobutyric Acid/metabolism , Animals , Behavior, Animal/drug effects , Bicuculline/analogs & derivatives , Bicuculline/pharmacology , GABA-A Receptor Antagonists/pharmacology , Male , Maze Learning/drug effects , Neural Pathways/metabolism , Neuronal Plasticity/drug effects , Prefrontal Cortex/drug effects , Proto-Oncogene Proteins c-fos/metabolism , Rats, Long-Evans , Receptors, GABA-A/drug effects , Synaptic Transmission/drug effects
9.
Int J Dev Neurosci ; 71: 1-9, 2018 Dec.
Article in English | MEDLINE | ID: mdl-30055229

ABSTRACT

The morphological and functional development of inhibitory circuit in the anterior piriform cortex (aPC) during the first three postnatal weeks may be crucial for the development of odor preference learning in infant rodents. As first step toward testing this hypothesis, we examined the normal development of GABAergic synaptic transmission in the aPC of rat pups during the postnatal days (P) 5-8 and 14-17. Whole cell patch-clamp recordings of layer 2/3 (L2/3) aPC pyramidal cells revealed a significant increase in spontaneous (sIPSC) and miniature (mIPSC) inhibitory postsynaptic current frequencies and a decrease in mIPSC rise and decay-time constant at P14-P17. Moreover, as the development of neocortical inhibitory circuit can be driven by sensory experience, we recorded sIPSC and mIPSC onto L2/3 aPC pyramidal cells from unilateral naris-occluded animals. Early partial olfactory deprivation caused by naris occlusion do not affected the course of age-dependent increase IPSC frequency onto L2/3 aPC pyramidal cell. However, this age-dependent increase of sIPSC and mIPSC frequencies were lower on aPC pyramidal cells ipsilateral to the occlusion side. In addition, the age-dependent increase in sIPSC frequency and amplitude were more pronounced on aPC pyramidal cells contralateral to the occlusion. While mIPSC kinetics were not affected by age or olfactory deprivation, at P5-P8, the sIPSC decay-time constant on aPC pyramidal cells of both hemispheres of naris-occluded animals were significantly higher when compared to sham. These results demonstrated that the GABAergic synaptic transmission on the aPC changed during postnatal development by increasing inhibitory inputs on L2/3 pyramidal cells, with increment in frequency of both sIPSC and mIPSC and faster kinetics of mIPSC. Our data suggested that the maturation of GABAergic synaptic transmission was little affected by early partial olfactory deprivation. These results could contribute to unravel the mechanisms underlying the development of odor processing and olfactory preference learning.


Subject(s)
Inhibitory Postsynaptic Potentials/physiology , Piriform Cortex/cytology , Piriform Cortex/growth & development , Synaptic Transmission/physiology , Age Factors , Analysis of Variance , Animals , Animals, Newborn , Bicuculline/analogs & derivatives , Bicuculline/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Female , GABA-A Receptor Antagonists/pharmacology , In Vitro Techniques , Inhibitory Postsynaptic Potentials/drug effects , Kynurenic Acid/pharmacology , Male , Patch-Clamp Techniques , Piriform Cortex/drug effects , Pyramidal Cells/drug effects , Pyramidal Cells/radiation effects , Rats , Rats, Wistar , Sensory Deprivation , Sodium Channel Blockers/pharmacology , Synaptic Transmission/drug effects , Tetrodotoxin/pharmacology
10.
Mol Pain ; 14: 1744806918783478, 2018.
Article in English | MEDLINE | ID: mdl-29956582

ABSTRACT

Background Chronic pain is a persistent unpleasant sensation that produces pathological synaptic plasticity in the central nervous system. Both human imaging study and animal studies consistently demonstrate that the anterior cingulate cortex is a critical cortical area for nociceptive and chronic pain processing. Thus far, the mechanisms of excitatory synaptic transmission and plasticity have been well characterized in the anterior cingulate cortex for various models of chronic pain. By contrast, the potential contribution of inhibitory synaptic transmission in the anterior cingulate cortex, in models of chronic pain, is not fully understood. Methods Chronic inflammation was induced by complete Freund adjuvant into the adult mice left hindpaw. We performed in vitro whole-cell patch-clamp recordings from layer II/III pyramidal neurons in two to three days after the complete Freund adjuvant injection and examined if the model could cause plastic changes, including transient and tonic type A γ-aminobutyric acid (GABAA) receptor-mediated inhibitory synaptic transmission, in the anterior cingulate cortex. We analyzed miniature/spontaneous inhibitory postsynaptic currents, GABAA receptor-mediated tonic currents, and evoked inhibitory postsynaptic currents. Finally, we studied if GABAergic transmission-related proteins in the presynapse and postsynapse of the anterior cingulate cortex were altered. Results The complete Freund adjuvant model reduced the frequency of both miniature and spontaneous inhibitory postsynaptic currents compared with control group. By contrast, the average amplitude of these currents was not changed between two groups. Additionally, the complete Freund adjuvant model did not change GABAA receptor-mediated tonic currents nor the set of evoked inhibitory postsynaptic currents when compared with control group. Importantly, protein expression of vesicular GABA transporter was reduced within the presynpase of the anterior cingulate cortex in complete Freund adjuvant model. In contrast, the complete Freund adjuvant model did not change the protein levels of GABAA receptors subunits such as α1, α5, ß2, γ2, and δ. Conclusion Our results suggest that the induction phase of inflammatory pain involves spontaneous GABAergic plasticity at presynaptic terminals of the anterior cingulate cortex.


Subject(s)
Chronic Pain/complications , Chronic Pain/pathology , Gyrus Cinguli/pathology , Inflammation/etiology , Neuronal Plasticity/physiology , Pain Threshold/physiology , gamma-Aminobutyric Acid/metabolism , Anesthetics, Local/pharmacology , Anesthetics, Local/therapeutic use , Animals , Bicuculline/analogs & derivatives , Bicuculline/pharmacology , Chronic Pain/chemically induced , Chronic Pain/drug therapy , Freund's Adjuvant/toxicity , GABA-A Receptor Antagonists/pharmacology , Gyrus Cinguli/cytology , In Vitro Techniques , Inflammation/chemically induced , Male , Mice , Mice, Inbred C57BL , Microscopy, Confocal , Physical Stimulation/adverse effects , Synaptic Potentials/drug effects , Synaptic Potentials/physiology , Tetrodotoxin/pharmacology , Vesicular Inhibitory Amino Acid Transport Proteins/metabolism
11.
Biochem Biophys Res Commun ; 501(1): 152-157, 2018 06 18.
Article in English | MEDLINE | ID: mdl-29723524

ABSTRACT

The balance between glutamate-mediated excitation and GABA-mediated inhibition is critical to cortical functioning. However, the contribution of network structure consisting of the both neurons to cortical functioning has not been elucidated. We aimed to evaluate the relationship between the network structure and functional activity patterns in vitro. We used mouse induced pluripotent stem cells (iPSCs) to construct three types of neuronal populations; excitatory-rich (Exc), inhibitory-rich (Inh), and control (Cont). Then, we analyzed the activity patterns of these neuronal populations using microelectrode arrays (MEAs). Inhibitory synaptic densities differed between the three types of iPSC-derived neuronal populations, and the neurons showed spontaneously synchronized bursting activity with functional maturation for one month. Moreover, different firing patterns were observed between the three populations; Exc demonstrated the highest firing rates, including frequent, long, and dominant bursts. In contrast, Inh demonstrated the lowest firing rates and the least dominant bursts. Synchronized bursts were enhanced by disinhibition via GABAA receptor blockade. The present study, using iPSC-derived neurons and MEAs, for the first time show that synchronized bursting of cortical networks in vitro depends on the network structure consisting of excitatory and inhibitory neurons.


Subject(s)
Cerebral Cortex/cytology , Cerebral Cortex/physiology , Induced Pluripotent Stem Cells/physiology , Neurons/physiology , Action Potentials , Animals , Bicuculline/analogs & derivatives , Bicuculline/pharmacology , Cell Differentiation , Cell Line , GABA-A Receptor Antagonists/pharmacology , GABAergic Neurons/cytology , GABAergic Neurons/drug effects , GABAergic Neurons/physiology , Glutamic Acid/physiology , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/drug effects , Mice , Microelectrodes , Nerve Net/cytology , Nerve Net/physiology , Neurons/cytology , Neurons/drug effects , Receptors, GABA-A/physiology
12.
PLoS One ; 13(3): e0194578, 2018.
Article in English | MEDLINE | ID: mdl-29561904

ABSTRACT

Investigating cerebral metabolism in vivo at a microscopic level is essential for understanding brain function and its pathological alterations. The intricate signaling and metabolic dynamics between neurons, glia, and microvasculature requires much more detailed understanding to better comprehend the mechanisms governing brain function and its disease-related changes. We recently demonstrated that pharmacologically-induced alterations to different steps of cerebral metabolism can be distinguished utilizing 2-photon fluorescence lifetime imaging of endogenous reduced nicotinamide adenine dinucleotide (NADH) fluorescence in vivo. Here, we evaluate the ability of the phasor analysis method to identify these pharmacological metabolic alterations and compare the method's performance with more conventional nonlinear curve-fitting analysis. Visualization of phasor data, both at the fundamental laser repetition frequency and its second harmonic, enables resolution of pharmacologically-induced alterations to mitochondrial metabolic processes from baseline cerebral metabolism. Compared to our previous classification models based on nonlinear curve-fitting, phasor-based models required fewer parameters and yielded comparable or improved classification accuracy. Fluorescence lifetime imaging of NADH and phasor analysis shows utility for detecting metabolic alterations and will lead to a deeper understanding of cerebral energetics and its pathological changes.


Subject(s)
Cerebral Cortex/metabolism , Mitochondria/metabolism , NAD/metabolism , Rodentia/physiology , Seizures/diagnostic imaging , Animals , Bicuculline/analogs & derivatives , Bicuculline/pharmacology , Biomarkers/metabolism , Cerebral Cortex/drug effects , Disease Models, Animal , Humans , Intravital Microscopy/methods , Microscopy, Fluorescence, Multiphoton/methods , Mitochondria/drug effects , Models, Biological , Nonlinear Dynamics , Rats, Sprague-Dawley , Seizures/chemically induced , Seizures/metabolism
13.
Neurotoxicology ; 65: 1-8, 2018 03.
Article in English | MEDLINE | ID: mdl-29309796

ABSTRACT

Prenatal valproic acid (VPA) exposure is a well-known animal model of autism spectrum disorder (ASD) that produces alterations in embryonic and adult neurogenesis as well as adolescent/adulthood neurobehavioral phenotypes. However, the effects of prenatal VPA exposure on neural network excitability, especially during the synaptogenic period around eye opening, are not fully understood. In this study, we orally administered VPA (300 mg/kg) to pregnant Wistar rats on gestation day 15 and subsequently performed field potential recording in the CA1 area of hippocampal slices obtained from control (saline-exposed) and VPA-exposed rat pups between postnatal day (PND) 13 and PND18. In control slices, we observed an abrupt enhancement of stimulation-dependent responses including population spike (PS) amplitudes and field excitatory postsynaptic potential (fEPSP) slopes at PND16, which coincided with the average day of eye opening. In contrast, VPA-exposed pups exhibited delayed eye opening (PND17) and gradual rather than abrupt increases in PS amplitudes and fEPSP slopes over the duration of the synaptogenic period. We next investigated the involvement of ambient GABA (γ-aminobutyric acid) in PS generation using bicuculline methiodide (BMI), a GABA type A (GABAA) receptor antagonist. In control slices, BMI enhanced PS amplitudes during PND14-15 (before eye opening) and had little effect thereafter during PND16-17; a subsequent regression model analysis of BMI ratios (the ratio of PS amplitudes in the presence and absence of BMI) indicated a possible developmental change between these periods. In contrast, almost identical regression models were obtained for BMI ratios during PND14-15 and PND16-17 in the VPA-exposed group, indicating the absence of a developmental change. Our results suggest that prenatal VPA exposure accelerates the development of hippocampal excitability before eye opening. Moreover, our experimental model can be used as a novel approach for the evaluation of developmental neurotoxicity.


Subject(s)
CA1 Region, Hippocampal/drug effects , CA1 Region, Hippocampal/physiopathology , Prenatal Exposure Delayed Effects/chemically induced , Prenatal Exposure Delayed Effects/physiopathology , Valproic Acid/toxicity , Action Potentials/drug effects , Aging/drug effects , Animals , Bicuculline/analogs & derivatives , Bicuculline/pharmacology , Excitatory Postsynaptic Potentials/drug effects , Eye/drug effects , Eye/growth & development , Female , Male , Pregnancy , Prenatal Exposure Delayed Effects/psychology , Rats
14.
Eur Neuropsychopharmacol ; 27(11): 1120-1131, 2017 11.
Article in English | MEDLINE | ID: mdl-28939165

ABSTRACT

Acute γ-aminobutyric acid (GABA) disinhibition in the posterior hypothalamus (PH) elicits defensive reactions that are considered anxiety- and panic attack-like behaviour, and these defensive reactions are followed by antinociception. Evidence indicates that the PH connects with the medial prefrontal cortex, particularly the anterior cingulate cortex (ACC), which seems to regulate these unconditioned fear-induced defensive responses. However, few studies have shown the participation of cortical regions in the control of behavioural and antinociceptive responses organised by diencephalic structures. It has been suggested that the glutamatergic system can mediate this cortical influence, as excitatory imbalance is believed to play a role in both defensive mechanisms. Thus, the aim of the present study was to investigate the involvement of ACC glutamatergic connections via blockade of local N-methyl-D-aspartate (NMDA) receptors to elaborate panic-like defensive behaviours and unconditioned fear-induced antinociception organised by PH neurons. Wistar rats were treated with microinjections of 0.9% NaCl or LY235959 (a selective NMDA receptor antagonist) in the ACC at different concentrations (2, 4 and 8 nmol/0.2µL), followed by GABAA receptor blockade in the PH. Defensive reactions were analysed for 20min, and the nociceptive threshold was then measured at 10-min intervals for 60min. Pretreatment of the ACC with LY235959 reduced both panic-like defensive behaviour and fear-induced antinociception evoked by PH GABAergic disinhibition. Our findings suggest that ACC NMDA receptor-signalled glutamatergic inputs play a relevant role in the organisation of anxiety- and panic attack-like behaviours and in fear-induced antinociception.


Subject(s)
Escape Reaction/physiology , Fear/physiology , Gyrus Cinguli/metabolism , Hypothalamus, Posterior/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Signal Transduction/physiology , Analysis of Variance , Animals , Bicuculline/analogs & derivatives , Bicuculline/pharmacology , Dose-Response Relationship, Drug , Escape Reaction/drug effects , Excitatory Amino Acid Antagonists/pharmacology , Fear/drug effects , GABA-A Receptor Antagonists/pharmacology , Gyrus Cinguli/drug effects , Hypothalamus, Posterior/drug effects , Isoquinolines/pharmacology , Male , Microinjections , Pain Measurement , Panic/drug effects , Panic/physiology , Rats , Rats, Wistar , Signal Transduction/drug effects
15.
Brain Struct Funct ; 222(9): 4219-4237, 2017 Dec.
Article in English | MEDLINE | ID: mdl-28656372

ABSTRACT

A remarkable example of maladaptive plasticity is the development of epilepsy after a brain insult or injury to a normal animal or human. A structure that is considered central to the development of this type of epilepsy is the dentate gyrus (DG), because it is normally a relatively inhibited structure and its quiescence is thought to reduce hippocampal seizure activity. This characteristic of the DG is also considered to be important for normal hippocampal-dependent cognitive functions. It has been suggested that the brain insults which cause epilepsy do so because they cause the DG to be more easily activated. One type of brain insult that is commonly used is induction of severe seizures (status epilepticus; SE) by systemic injection of a convulsant drug. Here we describe an alteration in the DG after this type of experimental SE that may contribute to chronic seizures that has not been described before: large folds or gyri that develop in the DG by 1 month after SE. Large gyri appeared to increase network excitability because epileptiform discharges recorded in hippocampal slices after SE were longer in duration when recorded inside gyri relative to locations outside gyri. Large gyri may also increase excitability because immature adult-born neurons accumulated at the base of gyri with time after SE, and previous studies have suggested that abnormalities in adult-born DG neurons promote seizures after SE. In summary, large gyri after SE are a common finding in adult rats, show increased excitability, and are associated with the development of an abnormal spatial distribution of adult-born neurons. Together these alterations may contribute to chronic seizures and associated cognitive comorbidities after SE.


Subject(s)
Dentate Gyrus/physiology , Neurogenesis/physiology , Neuronal Plasticity/physiology , Neurons/physiology , Status Epilepticus/pathology , Analysis of Variance , Animals , Atropine Derivatives/pharmacology , Bicuculline/analogs & derivatives , Bicuculline/pharmacology , Bromodeoxyuridine/metabolism , Dentate Gyrus/drug effects , Excitatory Amino Acid Agonists/toxicity , Female , GABA-A Receptor Antagonists/pharmacology , In Vitro Techniques , Kainic Acid/toxicity , Male , Muscarinic Agonists/toxicity , Neurogenesis/drug effects , Neuronal Plasticity/drug effects , Neurons/drug effects , Parasympatholytics/pharmacology , Phosphopyruvate Hydratase/metabolism , Pilocarpine/toxicity , Rats , Rats, Sprague-Dawley , Status Epilepticus/chemically induced , Time Factors
16.
Brain Topogr ; 30(3): 390-407, 2017 May.
Article in English | MEDLINE | ID: mdl-28176165

ABSTRACT

Interictal spikes can be generated by blocking GABAA receptor-mediated inhibition. The nature of the hemodynamic activities associated with interictal spikes in acute models of focal epilepsy based on GABA deactivation has not been determined. We analyzed systemic changes in hemodynamic signals associated with interictal spikes generated by acute models of focal epilepsy. Simultaneous ElectroCorticoGraphy (ECoG) and Near-InfraRed Spectroscopy (NIRS) recordings were obtained in vivo from adult Sprague-Dawley rat brain during semi-periodic focal interictal spikes induced by local cortical application of low doses of Penicillin G (PG) and Bicuculline Methiodide (BM) as GABA deactivation agents. The Finite Impulse Response deconvolution technique was used to estimate the profile of hemodynamic changes in oxyhemoglobin (HbO) and deoxyhemoglobin (HbR) concentrations associated with interictal ECoG spikes in each rat. Our results show that, in both acute models of focal epilepsy, the hemodynamic changes associated with interictal spikes were characterized by pre-spike and post-spike primary NIRS responses, and recovery periods with slight differences in amplitude and latency. The pre-spike period starting at least 2 s prior to the onset of ECoG spikes was characterized by a significant decrease in HbO concomitant with an increase in HbR with respect to baseline. The post-spike primary NIRS response exhibited the expected changes described according to the classical view of neurovascular coupling, i.e., a significant increase in HbO and a significant decrease in HbR in response to interictal spikes. The recovery period was characterized by a decreased HbO signal and an increased HbR signal, followed by a return to baseline. Compared to the BM epilepsy model, the PG model was more stable and showed lower variability in the shape, amplitude and latency of the components of spike-related hemodynamic changes. Our findings support a prominent role for pre-spike hemodynamic changes in the initiation of interictal spikes. The mechanism of interactions between neuronal and vascular networks during the pre-spike period constitutes a complex process, resulting in increased sensitivity of the epileptogenic focus to induce neuronal spiking.


Subject(s)
Brain/drug effects , Epilepsies, Partial/physiopathology , GABA-A Receptor Antagonists/pharmacology , Hemodynamics/drug effects , Hemoglobins/drug effects , Neurovascular Coupling/physiology , Oxyhemoglobins/drug effects , Animals , Bicuculline/analogs & derivatives , Bicuculline/pharmacology , Brain/metabolism , Brain/physiopathology , Disease Models, Animal , Electrocorticography , Electroencephalography , Epilepsies, Partial/diagnostic imaging , Hemodynamics/physiology , Hemoglobins/metabolism , Male , Oxyhemoglobins/metabolism , Penicillin G/pharmacology , Rats , Rats, Sprague-Dawley , Spectroscopy, Near-Infrared
17.
Epilepsy Res ; 130: 21-26, 2017 02.
Article in English | MEDLINE | ID: mdl-28107659

ABSTRACT

We analyzed the patterns of seizure-like activity and associated high-frequency oscillations (HFOs) induced by the K+ channel blocker 4-aminopyridine (4AP, 50µM) or the GABAA receptor antagonist bicuculline methiodide (BMI, 50µM) in the in vitro isolated guinea pig brain preparation. Extracellular field recordings were obtained from the medial entorhinal cortex (EC) using glass pipettes or silicon probes; 4AP or BMI were applied through the basilar artery. Ripples (80-200Hz) or fast ripples (250-500Hz) occurred at higher rates shortly before ictal events induced by 4AP or BMI, respectively. In addition, during the ictal period, ripples were mostly associated with 4AP-induced ictal events whereas fast ripples predominated during ictal discharges induced by BMI. Finally, ripples occurred at higher rates during the clonic phase of 4AP-induced ictal events compared to the tonic phase, while higher rates of fast ripples characterized the clonic phase in both 4AP- and BMI-induced ictal discharges. These differences in HFO occurrence presumably reflect the diverse action of these two convulsants on GABAA receptor signaling.


Subject(s)
Entorhinal Cortex/physiopathology , Seizures/physiopathology , 4-Aminopyridine/pharmacology , Animals , Bicuculline/analogs & derivatives , Bicuculline/pharmacology , Convulsants/pharmacology , Entorhinal Cortex/drug effects , Female , Guinea Pigs , In Vitro Techniques , Microelectrodes , Periodicity , Seizures/chemically induced
18.
Behav Brain Res ; 320: 21-29, 2017 03 01.
Article in English | MEDLINE | ID: mdl-27908749

ABSTRACT

GABAergic transmission in the ventral tegmental area (VTA) exerts a tonic inhibitory influence on mesolimbic dopaminergic neurons' activity. Blockade of VTA GABAA receptors increases dopamine release in the nucleus accumbens (NAcc). Increases in NAcc dopamine levels typically accompany sexual behavior display. Copulation to satiety is characterized by the instatement of a long lasting (72h) sexual behavior inhibition and the mesolimbic system appears to be involved in this phenomenon. GABAergic transmission in the VTA might play a role in the maintenance of this long lasting sexual inhibitory state. To test this hypothesis, in the present work we investigated the effect of GABAA receptor blockade in sexually exhausted males 24h after copulation to satiety, once the sexual inhibitory state is established, and compared it with its effect in sexually experienced rats. Results showed that low doses of systemically administered bicuculline induced sexual behavior expression in sexually exhausted rats, but lacked an effect on copulation of sexually experienced animals. Intra-VTA bilateral infusion of bicuculline did not modify sexual behavior of sexually experienced rats, but induced sexual behavior expression in all the sexually exhausted males. Hence, GABA plays a role in the control of sexual behavior expression at the VTA. The role played by GABAergic transmission in male sexual behavior expression of animals with distinct sexual behavior conditions is discussed.


Subject(s)
Sexual Behavior, Animal/physiology , Synaptic Transmission/physiology , gamma-Aminobutyric Acid/metabolism , Animals , Bicuculline/analogs & derivatives , Bicuculline/pharmacology , Dose-Response Relationship, Drug , GABA-A Receptor Antagonists/pharmacology , Male , Rats , Rats, Wistar , Reaction Time/drug effects , Sexual Behavior, Animal/drug effects , Statistics, Nonparametric , Synaptic Transmission/drug effects , Ventral Tegmental Area/drug effects , Ventral Tegmental Area/physiology
19.
Behav Brain Res ; 316: 38-46, 2017 01 01.
Article in English | MEDLINE | ID: mdl-27566182

ABSTRACT

Overweight and obesity are conditions associated with an overall range of clinical health consequences, and they could be involved with the development of neuropsychiatric diseases, such as generalized anxiety disorder (GAD) and panic disorder (PD). A crucial brain nuclei involved on the physiological functions and behavioral responses, especially fear, anxiety and panic, is the dorsomedial hypothalamus (DMH). However, the mechanisms underlying the process whereby the DMH is involved in behavioral changes in obese rats still remains unclear. The current study further investigates the relation between obesity and generalized anxiety, by investigating the GABAA sensitivity to pharmacological manipulation within the DMH in obese rats during anxiety conditions. Male Wistar rats were divided in two experimental groups: the first was fed a control diet (CD; 11% w/w) and second was fed a high fat diet (HFD; 45% w/w). Animals were randomly treated with muscimol, a GABAA agonist and bicuculline methiodide (BMI), a GABAA antagonist. Inhibitory avoidance and escape behaviors were investigated using the Elevated T-Maze (ETM) apparatus. Our results revealed that the obesity facilitated inhibitory avoidance acquisition, suggesting a positive relation between obesity and the development of an anxiety-like state. The injection of muscimol (an anxiolytic drug), within the DMH, increased the inhibitory avoidance latency in obese animals (featuring an anxiogenic state). Besides, muscimol prolonged the escape latency and controlling the possible panic-like behavior in these animals. Injection of BMI into the DMH was ineffective to produce an anxiety-like effect in obese animals opposing the results observed in lean animals. These findings support the hypotheses that obese animals are susceptible to develop anxiety-like behaviors, probably through changes in the GABAergic neurotransmission within the DMH.


Subject(s)
Anxiety/etiology , Anxiety/pathology , Diet, High-Fat/adverse effects , Dorsomedial Hypothalamic Nucleus/metabolism , Obesity/etiology , gamma-Aminobutyric Acid/metabolism , Animals , Anxiety/drug therapy , Bicuculline/analogs & derivatives , Bicuculline/pharmacology , Escape Reaction/drug effects , GABA-A Receptor Agonists/pharmacology , GABA-A Receptor Antagonists/pharmacology , Male , Maze Learning/drug effects , Muscimol/pharmacology , Muscimol/therapeutic use , Obesity/complications , Obesity/psychology , Rats , Rats, Wistar , Reaction Time/drug effects
20.
Behav Brain Res ; 317: 542-552, 2017 01 15.
Article in English | MEDLINE | ID: mdl-27732892

ABSTRACT

INTRODUCTION: Decreased sociability is a symptom of psychiatric conditions including autism-spectrum disorder and schizophrenia. Both of these conditions are associated with decreases in GABA function, particularly in the medial prefrontal cortex (PFC) and the basolateral amygdala (BLA); structures that are components of the social brain. Here, we determined if decreasing GABA transmission within either the PFC or the BLA decreases social behavior. METHODS: Rats were implanted with cannulae aimed at either the medial PFC or the BLA and then were tested on up to 4 behavioral tests following bilateral infusions of 0.5µl bicuculline methiodide (BMI, a GABAA receptor antagonist) at doses of 0, 25, or 50ng/µl. Rats were tested in the social interaction test, the social preference test, the sucrose preference test and for locomotor activity (BLA infusions only). RESULTS: Intra-BLA or PFC BMI infusions decreased the amount of time and the number of social interactions in the social interaction test. Further, in the social preference test, rats infused with 50ng BMI no longer exhibited a preference to explore a social over a non-social stimulus. The change in sociability was not due to a change in reward processing or locomotor behavior. DISCUSSION: Decreasing GABA transmission in either the medial PFC or BLA decreased sociability. Thus, changes in GABA signaling observed in conditions such as autism or schizophrenia may mediate the social withdrawal characteristic of these conditions. Moreover, they suggest that social withdrawal may be treated by drugs that potentiate GABA transmission.


Subject(s)
Basolateral Nuclear Complex/metabolism , Interpersonal Relations , Prefrontal Cortex/metabolism , gamma-Aminobutyric Acid/metabolism , Animals , Basolateral Nuclear Complex/drug effects , Bicuculline/analogs & derivatives , Bicuculline/pharmacology , Dose-Response Relationship, Drug , Food Preferences/psychology , GABA-A Receptor Antagonists/pharmacology , Male , Prefrontal Cortex/drug effects , Rats , Rats, Sprague-Dawley , Sucrose/administration & dosage
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