6-cyano-7-nitroquinoxaline-2-3-dione and biocytin

Development of inhibition onto pyramidal cells may be crucial for the emergence of cortical network activity, including gamma oscillations. In primate dorsolateral prefrontal cortex (DLPFC), inhibitory synaptogenesis starts in utero and inhibitory synapse density reaches adult levels before birth. However, in DLPFC, the expression levels of γ-aminobutyric acid (GABA) synapse-related gene products changes markedly during development until young adult age, suggesting a highly protracted maturation of GABA synapse function. Therefore, we examined the development of GABA synapses by recording GABAAR-mediated inhibitory postsynaptic currents (GABAAR-IPSCs) from pyramidal cells in the DLPFC of neonatal, prepubertal, peripubertal, and adult macaque monkeys. We found that the decay of GABAAR-IPSCs, possibly including those from parvalbumin-positive GABA neurons, shortened by prepubertal age, while their amplitude increased until the peripubertal period. Interestingly, both GABAAR-mediated quantal response size, estimated by miniature GABAAR-IPSCs, and the density of GABAAR synaptic appositions, measured with immunofluorescence microscopy, were stable with age. Simulations in a computational model network with constant GABA synapse density showed that the developmental changes in GABAAR-IPSC properties had a significant impact on oscillatory activity and predicted that, whereas DLPFC circuits can generate gamma frequency oscillations by prepubertal age, mature levels of gamma band power are attained at late stages of development.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Age Factors; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Animals, Newborn; Calcium Channel Blockers; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Female; GABA Antagonists; gamma-Aminobutyric Acid; Inhibitory Postsynaptic Potentials; Lysine; Macaca mulatta; Models, Neurological; Neurons; omega-Agatoxin IVA; Prefrontal Cortex; Pyridazines; Synapses

Neurons coexpressing neuropeptide Y, agouti-related peptide, and GABA (NAG) play an important role in ingestive behavior and are located in the arcuate nucleus of the hypothalamus. NAG neurons receive both GABAergic and glutamatergic synaptic inputs, however, the developmental time course of synaptic input organization of NAG neurons in mice is unknown. In this study, we show that these neurons have low numbers of GABAergic synapses and that GABA is inhibitory to NAG neurons during early postnatal period. In contrast, glutamatergic inputs onto NAG neurons are relatively abundant by P13 and are comparatively similar to the levels observed in the adult. As mice reach adulthood (9-10 weeks), GABAergic tone onto NAG neurons increases. At this age, NAG neurons received similar numbers of inhibitory and EPSCs. To further differentiate age-associated changes in synaptic distribution, 17- to 18-week-old lean and diet-induced obesity (DIO) mice were studied. Surprisingly, NAG neurons from lean adult mice exhibit a reduction in the GABAergic synapses compared with younger adults. Conversely, DIO mice display reductions in the number of GABAergic and glutamatergic inputs onto NAG neurons. Based on these experiments, we propose that synaptic distribution in NAG neurons is continuously restructuring throughout development to accommodate the animals' energy requirements.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Age Factors; Animals; Animals, Newborn; Arcuate Nucleus of Hypothalamus; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Female; gamma-Aminobutyric Acid; Inhibitory Postsynaptic Potentials; Lysine; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neurons; Neuropeptide Y; Sodium Channel Blockers; Synapses; Tetrodotoxin; Vesicular Inhibitory Amino Acid Transport Proteins

Theta-gamma network oscillations are thought to represent key reference signals for information processing in neuronal ensembles, but the underlying synaptic mechanisms remain unclear. To address this question, we performed whole-cell (WC) patch-clamp recordings from mature hippocampal granule cells (GCs) in vivo in the dentate gyrus of anesthetized and awake rats. GCs in vivo fired action potentials at low frequency, consistent with sparse coding in the dentate gyrus. GCs were exposed to barrages of fast AMPAR-mediated excitatory postsynaptic currents (EPSCs), primarily relayed from the entorhinal cortex, and inhibitory postsynaptic currents (IPSCs), presumably generated by local interneurons. EPSCs exhibited coherence with the field potential predominantly in the theta frequency band, whereas IPSCs showed coherence primarily in the gamma range. Action potentials in GCs were phase locked to network oscillations. Thus, theta-gamma-modulated synaptic currents may provide a framework for sparse temporal coding of information in the dentate gyrus.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Biological Clocks; Biophysics; Dentate Gyrus; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Female; Inhibitory Postsynaptic Potentials; Lysine; Male; Nerve Net; Neurons; Patch-Clamp Techniques; Rats; Rats, Wistar; Synapses

Inhibitory connections among striatal projection neurons (SPNs) called "feedback inhibition," have been proposed to endow the striatal microcircuit with computational capabilities, such as motor sequence selection, filtering, and the emergence of alternating network states. These properties are disrupted in models of Parkinsonism. However, the impact of feedback inhibition in the striatal network has remained under debate. Here, we test this inhibition at the microcircuit level. We used optical and electrophysiological recordings in mice and rats to demonstrate the action of striatal feedback transmission in normal and pathological conditions. Dynamic calcium imaging with single-cell resolution revealed the synchronous activation of a pool of identified SPNs by antidromic stimulation. Using bacterial artificial chromosome-transgenic mice, we demonstrate that the activated neuron pool equally possessed cells from the direct and indirect basal ganglia pathways. This pool inhibits itself because of its own GABA release when stimuli are frequent enough, demonstrating functional and significant inhibition. Blockade of GABAA receptors doubled the number of responsive neurons to the same stimulus, revealing a second postsynaptic neuron pool whose firing was being arrested by the first pool. Stronger connections arise from indirect SPNs. Dopamine deprivation impaired striatal feedback transmission disrupting the ability of a neuronal pool to arrest the firing of another neuronal pool. We demonstrate that feedback inhibition among SPNs is strong enough to control the firing of cell ensembles in the striatal microcircuit. However, to be effective, feedback inhibition should arise from synchronized pools of SPNs whose targets are other SPNs pools.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Adrenergic Agents; Anesthetics, Local; Animals; Animals, Newborn; Bicuculline; Biophysics; Calcium; Disease Models, Animal; Electric Stimulation; Excitatory Amino Acid Antagonists; Feedback, Physiological; GABA Antagonists; gamma-Aminobutyric Acid; Green Fluorescent Proteins; In Vitro Techniques; Lidocaine; Lysine; Male; Mice; Mice, Transgenic; Monte Carlo Method; Neostriatum; Neural Inhibition; Neural Pathways; Neurons; Oxidopamine; Parkinsonian Disorders; Patch-Clamp Techniques; Pyridazines; Rats; Rats, Wistar; Reaction Time; Receptors, Dopamine D1; Receptors, Dopamine D2; Synaptic Transmission; Time Factors; Valine

Fast-spiking parvalbumin (PV)-positive interneurons in layers 2/3 of the visual cortex regulate gain control and tuning of visual processing. Synapse-associated protein 97 (SAP97) belongs to a family of proteins that have been implicated in regulating glutamatergic synaptic transmission at pyramidal-to-pyramidal connections in the nervous system. For PV interneurons in mouse visual cortex, the expression of SAP97 is developmentally regulated, being expressed in almost all juvenile but only a fraction, ~40%, of adult PV interneurons. Using whole-cell patch-clamping, single-cell RT-PCR to assay endogenous expression of SAP97 and exogenous expression of SAP97, we investigated the functional significance of SAP97 in PV interneurons in layers 2/3 of the visual cortex. PV interneurons expressing SAP97, either endogenously or via exogenous expression, showed distinct membrane properties from those not expressing SAP97. This included an overall decrease in membrane excitability, as indexed by a decrease in membrane resistance and an increase in the stimulus threshold for the first action potential firing. Additionally, SAP97-expressing PV interneurons fired action potentials more frequently and, at moderate stimulus intensities, showed irregular or stuttering firing patterns. Furthermore, SAP97-expressing PV interneurons showed increased glutamatergic input and more extensive dendritic branching when compared with non-expressing PV interneurons. These differences in membrane and synaptic properties would significantly alter how PV interneurons expressing SAP97 compared with those not expressing SAP97 would function in local networks. Thus, our results indicate that the scaffolding protein SAP97 is a critical molecular factor regulating the input-output relationships of cortical PV interneurons.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Adaptor Proteins, Signal Transducing; Age Factors; Animals; Animals, Newborn; Discs Large Homolog 1 Protein; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; Glutamate Decarboxylase; In Vitro Techniques; Interneurons; Lysine; Membrane Proteins; Mice; Mice, Transgenic; Parvalbumins; Picrotoxin; Plant Lectins; Sodium Channel Blockers; Tetrodotoxin; Transduction, Genetic; Visual Cortex

Epileptic encephalopathies comprise a heterogeneous group of severe infantile disorders for which the pathophysiological basis of epilepsy is inaccurately clarified by genotype-phenotype analysis. Because a deficit of GABA neurons has been found in some of these syndromes, notably in patients with X-linked lissencephaly with abnormal genitalia, epilepsy was suggested to result from an imbalance in GABAergic inhibition, and the notion of "interneuronopathy" was proposed. Here, we studied the impact of a polyalanine expansion of aristaless-related homeobox (ARX) gene, a mutation notably found in West and Ohtahara syndromes. Analysis of Arx((GCG)7/Y) knock-in mice revealed that GABA neuron development is not affected. Moreover, pyramidal cell migration and cortical layering are unaltered in these mice. Interestingly, electrophysiological recordings show that hippocampal pyramidal neurons displayed a frequency of inhibitory postsynaptic currents similar to wild-type (WT) mice. However, these neurons show a dramatic increase in the frequency of excitatory inputs associated with a remodeling of their axonal arborization, suggesting that epilepsy in Arx((GCG)7/Y)mice would result from a glutamate network remodeling. We therefore propose that secondary alterations are instrumental for the development of disease-specific phenotypes and should be considered to explain the phenotypic diversity associated with epileptogenic mutations.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Age Factors; Animals; Animals, Newborn; Cell Movement; Doublecortin Protein; Electroporation; Embryo, Mammalian; Excitatory Amino Acid Antagonists; Female; GABAergic Neurons; gamma-Aminobutyric Acid; Gene Expression Regulation, Developmental; Glutamate Decarboxylase; Glutamates; Green Fluorescent Proteins; HEK293 Cells; Homeodomain Proteins; Humans; Lysine; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nerve Tissue Proteins; Organ Culture Techniques; Patch-Clamp Techniques; Peptides; RNA, Small Interfering; Statistics, Nonparametric; Synaptic Potentials; Transcription Factors; Transfection

It is a matter of ongoing debate whether newly generated granule cells contribute to epileptic activity in the hippocampus. To address this question, we investigated neurogenesis and epileptiform activity (EA) along the hippocampal septotemporal axis in the intrahippocampal kainate (KA) mouse model for temporal lobe epilepsy. Multisite intrahippocampal in vivo recordings and immunolabeling for c-Fos showed that the KA-induced status epilepticus (SE) extended along the septotemporal axis of both hippocampi with stronger intensity at ipsilateral temporal and contralateral sites. Accordingly, we found a position-dependent increase in proliferation (incorporation of bromodeoxyuridine) and neurogenesis (immunolabeling for doublecortin): Both were selectively increased in the ipsilateral temporal and entire contralateral subgranular zone, sparing the septal region close to the injection site. The newborn neurons were hyperexcitable and functionally integrated into the hippocampal network as revealed by patch-clamp recordings. Analysis of chronic EA also showed a differential intensity pattern along the hippocampal axis: EA was low in the septal portion with prominent sclerosis and granule cell dispersion but most pronounced in the transition zone where neurogenesis reappeared. In conclusion, SE stimulates neurogenesis in a position-dependent manner and coincidence of neurogenesis and stronger EA distal to the injection site suggests a proepileptogenic effect of increased neurogenesis.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Analysis of Variance; Animals; Bromodeoxyuridine; Cell Count; Cell Proliferation; Convulsants; Disease Models, Animal; Doublecortin Domain Proteins; Electric Stimulation; Electroencephalography; Epilepsy, Temporal Lobe; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Functional Laterality; Hippocampus; Kainic Acid; Luminescent Proteins; Lysine; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microtubule-Associated Proteins; Motor Activity; Neurogenesis; Neuropeptides; Patch-Clamp Techniques; Picrotoxin

Unilateral damage to the peripheral vestibular receptors precipitates a debilitating syndrome of oculomotor and balance deficits at rest, which extensively normalize during the first week after the lesion due to vestibular compensation. In vivo studies suggest that GABA(B) receptor activation facilitates recovery. However, the presynaptic or postsynaptic sites of action of GABA(B) receptors in vestibular nuclei neurons after lesions have not been determined. Accordingly, here presynaptic and postsynaptic GABA(B) receptor activity in principal cells of the tangential nucleus, a major avian vestibular nucleus, was investigated using patch-clamp recordings correlated with immunolabeling and confocal imaging of the GABA(B) receptor subunit-2 (GABA(B)R2) in controls and operated chickens shortly after unilateral vestibular ganglionectomy (UVG). Baclofen, a GABA(B) agonist, generated no postsynaptic currents in principal cells in controls, which correlated with weak GABA(B)R2 immunolabeling on principal cell surfaces. However, baclofen decreased miniature excitatory postsynaptic current (mEPSC) and GABAergic miniature inhibitory postsynaptic current (mIPSC) events in principal cells in controls, compensating and uncompensated chickens three days after UVG, indicating the presence of functional GABA(B) receptors on presynaptic terminals. Baclofen decreased GABAergic mIPSC frequency to the greatest extent in principal cells on the intact side of compensating chickens, with concurrent increases in GABA(B)R2 pixel brightness and percentage overlap in synaptotagmin 2-labeled terminals. In uncompensated chickens, baclofen decreased mEPSC frequency to the greatest extent in principal cells on the intact side, with concurrent increases in GABA(B)R2 pixel brightness and percentage overlap in synaptotagmin 1-labeled terminals. Altogether, these results revealed changes in presynaptic GABA(B) receptor function and expression which differed in compensating and uncompensated chickens shortly after UVG. This work supports an important role for GABA(B) autoreceptor-mediated inhibition in vestibular nuclei neurons on the intact side during early stages of vestibular compensation, and a role for GABA(B) heteroreceptor-mediated inhibition of glutamatergic terminals on the intact side in the failure to recover function.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Baclofen; Chick Embryo; Excitatory Amino Acid Antagonists; GABA Agents; In Vitro Techniques; Lysine; Microscopy, Confocal; Microtubule-Associated Proteins; Neurons; Neurotransmitter Agents; Patch-Clamp Techniques; Presynaptic Terminals; Receptors, GABA-B; Sodium Channel Blockers; Statistics, Nonparametric; Synaptic Potentials; Synaptotagmin I; Synaptotagmin II; Tetrodotoxin; Time Factors; Valine; Vestibular Diseases; Vestibular Nuclei

Relating the function of neuronal cell types to information processing and behavior is a central goal of neuroscience. In the hippocampus, pyramidal cells in CA1 and the subiculum process sensory and motor cues to form a cognitive map encoding spatial, contextual, and emotional information, which they transmit throughout the brain. Do these cells constitute a single class or are there multiple cell types with specialized functions? Using unbiased cluster analysis, we show that there are two morphologically and electrophysiologically distinct principal cell types that carry hippocampal output. We show further that these two cell types are inversely modulated by the synergistic action of glutamate and acetylcholine acting on metabotropic receptors that are central to hippocampal function. Combined with prior connectivity studies, our results support a model of hippocampal processing in which the two pyramidal cell types are predominantly segregated into two parallel pathways that process distinct modalities of information.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Analysis of Variance; Animals; Animals, Newborn; Benzylamines; Biophysics; CA1 Region, Hippocampal; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Amino Acid Transporter 3; GABA Antagonists; Glutamate Decarboxylase; In Vitro Techniques; Lysine; Male; Membrane Potentials; Neurons; Patch-Clamp Techniques; Phosphinic Acids; Pyridazines; Rats; Receptors, Metabotropic Glutamate; Time Factors

Unitary excitatory (EPSP) and inhibitory (IPSP) postsynaptic potentials (PSPs) were evoked between neurons in Rexed's laminae (L)II-V of spinal slices from young hamsters (7-24 days old) at 27°C using paired whole cell recordings. Laminar differences in synaptic efficacy were observed: excitatory connections were more secure than inhibitory connections in LII and inhibitory linkages in LII were less reliable than those in LIII-V. A majority of connections displayed paired-pulse facilitation or depression. Depression was observed for both EPSPs and IPSPs, but facilitation was seen almost exclusively for IPSPs. There were no frequency-dependent shifts between facilitation and depression. Synaptic depression was associated with an increased failure rate and decreased PSP half-width for a majority of connections. However, there were no consistent changes in failure rate or PSP time course at facilitating connections. IPSPs evoked at high-failure synapses had consistently smaller amplitude and showed greater facilitation than low-failure connections. Facilitation at inhibitory connections was positively correlated with synaptic jitter and associated with a decrease in latency. At many connections, the paired-pulse ratio varied from trial to trial and depended on the amplitude of the first PSP; dependence was greater for inhibitory synapses than excitatory synapses. Paired-pulse ratios for connections onto neurons with rapidly adapting, "phasic" discharge to depolarizing current injection were significantly greater than for connections onto neurons with tonic discharge properties. These results are evidence of diversity in synaptic transmission between dorsal horn neurons, the nature of which may depend on the types of linkage, laminar location, and intrinsic firing properties of postsynaptic cells.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Bicuculline; Cricetinae; Electric Stimulation; Excitatory Amino Acid Antagonists; Female; GABA-A Receptor Antagonists; Glycine Agents; In Vitro Techniques; Lysine; Male; Mesocricetus; Neural Pathways; Patch-Clamp Techniques; Posterior Horn Cells; Reaction Time; Spinal Cord; Strychnine; Synapses; Synaptic Transmission; Time Factors

By regulating the neocortical excitability, nicotinic acetylcholine receptors (nAChRs) control vigilance and cognition and are implicated in epileptogenesis. Modulation of gamma-aminobutyric acid (GABA) release often accompanies these processes. We studied how nAChRs regulate GABAergic transmission in the murine neocortex with immunocytochemical and patch-clamp methods. The cholinergic fibers densely innervated the somatosensory, visual, motor, and prefrontal cortices (PFC). Laminar distribution was broadly homogeneous, especially in the PFC. The cholinergic terminals were often adjacent to the soma and dendrites of GABAergic interneurons, but well-differentiated synapses were rare. Tonically applied nicotine (1-100 microM) increased the frequency of spontaneous GABAergic inhibitory postsynaptic currents (IPSCs) on pyramidal neurons in PFC layer V. The contribution of nAChR types was assessed by using 1 microM dihydro-beta-erythroidine (DHbetaE), to block heteromeric nAChRs, and 10 nM methyllycaconitine (MLA), to block homomeric nAChRs. Both inhibitors antagonized the effect of nicotine on IPSCs, suggesting that mixed nAChR types control pyramidal neuron inhibition in layer V. To determine whether nAChRs are expressed on basket cells' terminals, we studied miniature IPSCs (mIPSCs). These were revealed using 0.5 microM tetrodotoxin and 50 microM Cd(2+) to isolate the GABAergic terminals from the action potential drive. The nicotinic stimulation of mIPSCs was antagonized by DHbetaE, but not MLA, indicating that heteromeric nAChRs prevail in GABAergic terminals. Immunocytochemistry confirmed the expression of nAChRs on basket cells' somata and terminals. Finally, when the ionotropic glutamatergic transmission was blocked, nicotine partially inhibited the IPSCs, an effect counteracted by both DHbetaE and MLA. Therefore, a fraction of nAChRs are capable of activating GABAergic interneurons that in turn inhibit other GABAergic interneurons, thereby reducing the IPSCs. We conclude that heteromeric nAChRs control GABA release presynaptically, whereas mixed nAChRs regulate both excitation and inhibition of interneurons, the balance depending on the overall glutamatergic drive.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Biophysics; Choline O-Acetyltransferase; Electric Stimulation; Excitatory Amino Acid Antagonists; gamma-Aminobutyric Acid; Glutamate Decarboxylase; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Lysine; Mice; Microscopy, Confocal; Microscopy, Electron, Transmission; Neurons; Patch-Clamp Techniques; Prefrontal Cortex; Receptors, Nicotinic; Valine; Vesicular Acetylcholine Transport Proteins

Whole-cell recordings were obtained from tyrosine hydroxylase-expressing (TH(+)) neurons in striatal slices from bacterial artificial chromosome transgenic mice that synthesize enhanced green fluorescent protein (EGFP) selectively in neurons expressing TH transcriptional regulatory sequences. Stereological cell counting indicated that there were approximately 2700 EGFP-TH(+) neurons/striatum. Whole-cell recordings in striatal slices demonstrated that EGFP-TH(+) neurons comprise four electrophysiologically distinct neuron types whose electrophysiological properties have not been reported previously in striatum. EGFP-TH(+) neurons were identified in retrograde tracing studies as interneurons. Recordings from synaptically connected pairs of EGFP-TH(+) interneurons and spiny neurons showed that the interneurons elicited GABAergic IPSPs/IPSCs in spiny neurons powerful enough to significantly delay evoked spiking. EGFP-TH(+) interneurons responded to local or cortical stimulation with glutamatergic EPSPs. Local stimulation also elicited GABA(A) IPSPs, at least some of which arose from identified spiny neurons. Single-cell reverse transcription-PCR showed expression of VMAT1 in EGFP-TH(+) interneurons, consistent with previous suggestions that these interneurons may be dopaminergic as well as GABAergic. All four classes of interneurons were medium sized with modestly branching, varicose dendrites, and dense, highly varicose axon collateral fields. These data show for the first time that there exists in the normal rodent striatum a substantial population of TH(+)/GABAergic interneurons comprising four electrophysiologically distinct subtypes whose electrophysiological properties differ significantly from those of previously described striatal GABAergic interneurons. These interneurons are likely to play an important role in striatal function through fast GABAergic synaptic transmission in addition to, and independent of, their potential role in compensation for dopamine loss in experimental or idiopathic Parkinson's disease.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Analysis of Variance; Animals; Anti-Inflammatory Agents; Bicuculline; Calcium Channel Blockers; Cardiovascular Agents; Cell Count; Colchicine; Corpus Striatum; Electric Stimulation; Excitatory Amino Acid Antagonists; Flufenamic Acid; GABA Antagonists; Green Fluorescent Proteins; In Vitro Techniques; Lysine; Mice; Mice, Transgenic; Neural Pathways; Neurons; Nimodipine; Patch-Clamp Techniques; Pyrimidines; Synapses; Synaptic Transmission; Time Factors; Tubulin Modulators; Tyrosine 3-Monooxygenase; Vesicular Monoamine Transport Proteins

We have examined the basic electrophysiology and pharmacology of cells in the anterior caudal lobe (CLa) of the mormyrid cerebellum. Intracellular recordings were performed in an in vitro slice preparation using the whole-cell patch recording method. The responses of cells to parallel fiber (PF) and climbing fiber (CF) stimulation and to somatic current injection were recorded, and then characterized by bath application of receptor and ion channel blockers. Using biocytin or neurobiotin, these cells were also morphologically identified after recording to ensure their classification. Efferent cells and two subtypes of Purkinje cells were identified on the basis of their physiology and morphology. While the majority of Purkinje cells fire a single type of spike that is mediated by Na(+), some fire a large broad spike mediated by Ca(2+) and a narrow spike mediated by Na(+) at resting potential levels. By patching one recording electrode to the soma and another to one of the proximal dendrites of the same cell simultaneously, it was found that the Na(+) spike has an axonal origin and the Ca(2+) spike is generated in the soma-dendritic region of Purkinje cells. Efferent cells fire a single type of Na(+) spike only. Despite variations in their physiology and morphology, all cell types responded to PF stimulation with graded excitatory postsynaptic potentials (EPSPs) mediated by AMPA receptors. However, none of the efferent cells and only some of the Purkinje cells responded to CF activation with a large, AMPA receptor-mediated all-or-none EPSPs. We conclude that the functional circuitry of the CLa resembles that of other regions of the mormyrid cerebellum and is largely similar to that of the mammalian cerebellum.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Axons; Biophysics; Cerebellum; Dendrites; Dose-Response Relationship, Drug; Electric Fish; Electric Stimulation; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; In Vitro Techniques; Lysine; Membrane Potentials; Mice; N-Methylaspartate; Nerve Fibers; Patch-Clamp Techniques; Purkinje Cells; Sodium Channel Blockers; Tetrodotoxin

Proprioceptive sensory signals inform the CNS of the consequences of motor acts, but effective motor planning involves internal neural systems capable of anticipating actual sensory feedback. Just where and how predictive systems exert their influence remains poorly understood. We explored the possibility that spinocerebellar neurons that convey proprioceptive sensory information also integrate information from cortical command systems. Analysis of the circuitry and physiology of identified dorsal spinocerebellar tract neurons in mouse spinal cord revealed distinct populations of Clarke's column neurons that received direct excitatory and/or indirect inhibitory inputs from descending corticospinal axons. The convergence of these descending inhibitory and excitatory inputs to Clarke's column neurons established local spinal circuits with the capacity to mark or modulate incoming proprioceptive input. Together, our genetic, anatomical and physiological results indicate that Clarke's column spinocerebellar neurons nucleate local spinal corollary circuits that are relevant to motor planning and evaluation.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; beta-Galactosidase; Bicuculline; Cerebellum; Cholera Toxin; Electric Stimulation; Estrogen Antagonists; Excitatory Amino Acid Antagonists; Feedback, Sensory; GABA Antagonists; Gene Expression Regulation; Glial Cell Line-Derived Neurotrophic Factor; Glycine Agents; Glycine Plasma Membrane Transport Proteins; Green Fluorescent Proteins; Homeodomain Proteins; Lysine; Membrane Potentials; Mice; Mice, Transgenic; Models, Neurological; Nerve Net; Neural Inhibition; Neural Pathways; Parvalbumins; Patch-Clamp Techniques; Protein Kinase C; RNA, Messenger; Sensory Receptor Cells; Spinal Cord; Stilbamidines; Strychnine; Tamoxifen; tau Proteins; Transcription Factors; Vesicular Glutamate Transport Protein 1

Ligands for the epidermal growth factor receptor ErbB1, such as epidermal growth factor (EGF) and transforming growth factor alpha (TGFalpha), negatively regulate synaptic maturation of GABAergic neurons in the developing neocortex. Here, we evaluated the effects of these ligands in vivo on developing inhibitory neurons in the dentate gyrus. Hippocampal slices were prepared from postnatal mice repeatedly challenged with EGF or from transgenic mice overexpressing TGFalpha. We monitored paired pulse depression of field population spikes evoked by perforant path stimulation to estimate the strength of local inhibition. Administration of EGF increased the paired pulse ratio, suggesting a reduction of inhibitory strength. A similar reduction was observed in TGFalpha transgenic mice. Monitoring miniature and evoked synaptic currents, we estimated EGF effects on synaptic input and output of GABAergic neurons. EGF treatment diminished the amplitude of excitatory postsynaptic currents (EPSCs) in the GABAergic neurons without affecting their miniature EPSCs. EGF also affected output strength of the GABAergic neurons: The frequency of miniature inhibitory postsynaptic currents (IPSCs) and the evoked IPSC/evoked EPSC ratio were decreased in granule cells. In parallel, EGF down-regulated the protein level of vesicular GABA transporter. Thus, ErbB1 ligands influence GABAergic inhibitory synaptic transmission in the developing dentate gyrus.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Biophysics; Dentate Gyrus; Electric Stimulation; Epidermal Growth Factor; ErbB Receptors; Evoked Potentials; Excitatory Amino Acid Antagonists; Gene Expression Regulation; Glutamate Decarboxylase; Humans; In Vitro Techniques; Lysine; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neural Inhibition; Neurons; Parvalbumins; Patch-Clamp Techniques; Synapses; Synaptic Potentials; Transforming Growth Factor alpha

Striatal GABAergic microcircuits modulate cortical responses and movement execution in part by controlling the activity of medium spiny neurons (MSNs). How this is altered by chronic dopamine depletion, such as in Parkinson's disease, is not presently understood. We now report that, in dopamine-depleted slices of the striatum, MSNs generate giant spontaneous postsynaptic GABAergic currents (single or in bursts at 60 Hz) interspersed with silent episodes, rather than the continuous, low-frequency GABAergic drive (5 Hz) observed in control MSNs. This shift was observed in one-half of the MSN population, including both "D(1)-negative" and "D(1)-positive" MSNs. Single GABA and NMDA channel recordings revealed that the resting membrane potential and reversal potential of GABA were similar in control and dopamine-depleted MSNs, and depolarizing, but not excitatory, actions of GABA were observed. Glutamatergic and cholinergic antagonists did not block the GABAergic oscillations, suggesting that they were generated by GABAergic neurons. In support of this, cell-attached recordings revealed that a subpopulation of intrastriatal GABAergic interneurons generated bursts of spikes in dopamine-deprived conditions. This subpopulation included low-threshold spike interneurons but not fast-spiking interneurons, cholinergic interneurons, or MSNs. Therefore, a population of local GABAergic interneurons shifts from tonic to oscillatory mode when dopamine deprived and gives rise to spontaneous repetitive giant GABAergic currents in one-half the MSNs. We suggest that this may in turn alter integration of cortical signals by MSNs.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Adrenergic Agents; Animals; Biological Clocks; Biophysics; Corpus Striatum; Dopamine; Dose-Response Relationship, Drug; Electric Stimulation; Excitatory Amino Acid Antagonists; GABA Agents; gamma-Aminobutyric Acid; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Interneurons; Lysine; Mice; Nicotine; Nicotinic Agonists; Oxidopamine; Patch-Clamp Techniques; Spectrum Analysis; Tyrosine 3-Monooxygenase; Valine

The current dominant concept for the control of saccadic eye movements by the basal ganglia is that release from tonic GABAergic inhibition by the substantia nigra pars reticulata (SNr) triggers burst firings of intermediate gray layer (SGI) neurons in the superior colliculus (SC) to allow saccade initiation. This hypothesis is based on the assumption that SNr cells inhibit excitatory projection neurons in the SGI. Here we show that nigrotectal fibers are connected to local GABAergic neurons in the SGI with a similar frequency to non-GABAergic neurons. This was accomplished by applying neuroanatomical tracing and slice electrophysiological experiments in GAD67-green fluorescent protein (GFP) knock-in mice, in which GABAergic neurons specifically express GFP. We also found that GABA(A), but not GABA(B), receptors subserve nigrotectal transmission. The present results revealed a novel aspect on the role of the basal ganglia in the control of saccades, e.g., the SNr not only regulates burst initiation but also modulates the spatiotemporal properties of premotor neurons via connections to local GABAergic neurons in the SC.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Animals, Newborn; Biotin; Dextrans; Electric Stimulation; Excitatory Amino Acid Antagonists; gamma-Aminobutyric Acid; Glutamate Decarboxylase; Green Fluorescent Proteins; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Lysine; Membrane Potentials; Mice; Mice, Transgenic; Nerve Fibers; Neural Inhibition; Neural Pathways; Neurons; Patch-Clamp Techniques; Substantia Nigra; Superior Colliculi

Responses of on-center starburst amacrine cells to steady light stimuli were recorded in the dark-adapted mouse retina. The response to spots of dim white light appear to show two components, an initial peak that correspond to the onset of the light stimulus and a series of oscillations that ride on top of the initial peak relaxation. The frequency of oscillations during light stimulation was three time higher than the frequency of spontaneous oscillations recorded in the dark. The light-evoked responses in starburst cells were exclusively dependent on the release of glutamate likely from presynaptic bipolar axon terminals and the binding of glutamate to AMPA/kainate receptors because they were blocked by 6-cyano-7-nitroquinoxalene-2,3-dione. The synaptic pathway responsible for the light responses was blocked by AP4, an agonist of metabotropic glutamate receptors that hyperpolarize on-center bipolar cells on activation. Light responses were inhibited by the calcium channel blockers cadmium ions and nifedipine, suggesting that the release of glutamate was calcium dependent. The oscillatory component of the response was specifically inhibited by blocking the glutamate transporter with d-threo-beta-benzyloxyaspartic acid, suggesting that glutamate reuptake is necessary for the oscillatory release. GABAergic antagonists bicuculline, SR 95531, and picrotoxin increased the amplitude of the initial peak while they inhibit the frequency of oscillations. TTX had a similar effect. Strychnine, the blocker of glycine receptors did not affect the initial peak but strongly decreased the oscillations frequency. These inhibitory inputs onto the bipolar axon terminals shape and synchronize the oscillatory component.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Amacrine Cells; Animals; Aspartic Acid; Biological Clocks; Cadmium Chloride; Calcium Channel Blockers; Electric Stimulation; Excitatory Amino Acid Antagonists; Glycine Agents; In Vitro Techniques; Light; Lysine; Membrane Potentials; Mice; Mice, Inbred C57BL; Nifedipine; Patch-Clamp Techniques; Photic Stimulation; Retina; Strychnine; Synapses

Although in situ hybridization studies have revealed the presence of kainate receptor (KAR) mRNA in neurons of the rat medial entorhinal cortex (mEC), the functional presence and roles of these receptors are only beginning to be examined. To address this deficiency, whole cell voltage clamp recordings of locally evoked excitatory postsynaptic currents (EPSCs) were made from mEC layer II and III neurons in combined entorhinal cortex-hippocampal brain slices. Three types of neurons were identified by their electroresponsive membrane properties, locations, and morphologies: stellate-like "Sag" neurons in layer II (S), pyramidal-like "No Sag" neurons in layer III (NS), and "Intermediate Sag" neurons with varied morphologies and locations (IS). Non-NMDA EPSCs in these neurons were composed of two components, and the slow decay component in NS neurons had larger amplitudes and contributed more to the combined EPSC than did those observed in S and IS neurons. This slow component was mediated by KARs and was characterized by its resistance to either 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466, 100 microM) or 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[lsqb]f[rsqb]quinoxaline-7-sulfonamide (NBQX, 1 microM), relatively slow decay kinetics, and sensitivity to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10-50 microM). KAR-mediated EPSCs in pyramidal-like NS neurons contributed significantly more to the combined non-NMDA EPSC than did those from S and IS neurons. Layer III neurons of the mEC are selectively susceptible to degeneration in human temporal lobe epilepsy (TLE) and animal models of TLE such as kainate-induced status epilepticus. Characterizing differences in the complement of postsynaptic receptors expressed in injury prone versus injury resistant mEC neurons represents an important step toward understanding the vulnerability of layer III neurons seen in TLE.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Benzodiazepines; Cell Death; Electrophysiology; Entorhinal Cortex; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Hippocampus; In Vitro Techniques; Lysine; Male; Neurons; Patch-Clamp Techniques; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Receptors, Kainic Acid

The principal cells of the chick tangential nucleus are vestibular nucleus neurons participating in the vestibuloocular and vestibulocollic reflexes. In birds and mammals, spontaneous and stimulus-evoked firing of action potentials is essential for vestibular nucleus neurons to generate mature vestibular reflex activity. The emergence of spike-firing pattern and the underlying ion channels were studied in morphologically-identified principal cells using whole-cell patch-clamp recordings from brain slices of late-term embryos (embryonic day 16) and hatchling chickens (hatching day 1 and hatching day 5). Spontaneous spike activity emerged around the perinatal period, since at embryonic day 16 none of the principal cells generated spontaneous action potentials. However, at hatching day 1, 50% of the cells fired spontaneously (range, 3 to 32 spikes/s), which depended on synaptic transmission in most cells. By hatching day 5, 80% of the principal cells could fire action potentials spontaneously (range, 5 to 80 spikes/s), and this activity was independent of synaptic transmission and showed faster kinetics than at hatching day 1. Repetitive firing in response to depolarizing pulses appeared in the principal cells starting around embryonic day 16, when <20% of the neurons fired repetitively. However, almost 90% of the principal cells exhibited repetitive firing on depolarization at hatching day 1, and 100% by hatching day 5. From embryonic day 16 to hatching day 5, the gain for evoked spike firing increased almost 10-fold. At hatching day 5, a persistent sodium channel was essential for the generation of spontaneous spike activity, while a small conductance, calcium-dependent potassium current modulated both the spontaneous and evoked spike firing activity. Altogether, these in vitro studies showed that during the perinatal period, the principal cells switched from displaying no spontaneous spike activity at resting membrane potential and generating one spike on depolarization to the tonic firing of spontaneous and evoked action potentials.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Apamin; Bicuculline; Cesium; Chick Embryo; Chlorides; Dose-Response Relationship, Radiation; Drug Combinations; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; In Vitro Techniques; Lysine; Neurons; Sodium Channel Blockers; Strychnine; Tetrodotoxin; Vestibular Nuclei

Neural circuitry of the spinal superficial dorsal horn (SDH) (laminae I and II) and its relationship to pain and other somatosensory phenomena remain poorly understood. To gain information on this issue, synaptic connections between identified SDH neurons were studied in rat spinal cord slices by simultaneous whole-cell recordings from pairs of cells. Both excitatory and inhibitory connections were noted. This report focuses on the observed excitatory linkages. Synaptic excitatory connections between SDH neurons proved highly selective and consistently were unidirectional. Two patterns repeatedly appeared (for neuron classification, see Materials and Methods) (Grudt and Perl, 2002). Lamina II central neurons, with dorsal root (DR) C-fiber input, monosynaptically excited lamina II vertical neurons with DR Adelta input. Lamina II outer vertical neurons with DR Adelta input monosynaptically excited lamina I neurons. Some of the postsynaptic lamina I cells were shown to project rostrally. In contrast to the usual case for connected neurons, in unconnected pairs, primary afferent input to the same type of neuron proved closely similar. Together, these observations indicate that the neural circuitry in the SDH, including its substantia gelatinosa (lamina II), has an explicit organization in which particular combinations of neurons comprise modules arranged to modify and transmit sensory information arriving from Adelta and C primary afferent fibers.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; In Vitro Techniques; Lysine; Models, Neurological; Nerve Net; Neurons; Rats; Rats, Sprague-Dawley; Reaction Time; Stilbamidines; Substance P; Substantia Gelatinosa; Synaptic Transmission; Tetrodotoxin

Using patch-clamp techniques, we investigated the characteristics of the spontaneous oscillatory activity displayed by starburst amacrine cells in the mouse retina. At a holding potential of -70 mV, oscillations appeared as spontaneous, rhythmic inward currents with a frequency of approximately 3.5 Hz and an average maximal amplitude of approximately 120 pA. Application of TEA, a potassium channel blocker, increased the amplitude of oscillatory currents by >70% but reduced their frequency by approximately 17%. The TEA effects did not appear to result from direct actions on starburst cells, but rather a modulation of their synaptic inputs. Oscillatory currents were inhibited by 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX), an antagonist of AMPA/kainate receptors, indicating that they were dependent on a periodic glutamatergic input likely from presynaptic bipolar cells. The oscillations were also inhibited by the calcium channel blockers cadmium and nifedipine, suggesting that the glutamate release was calcium dependent. Application of AP4, an agonist of mGluR6 receptors on on-center bipolar cells, blocked the oscillatory currents in starburst cells. However, application of TEA overcame the AP4 blockade, suggesting that the periodic glutamate release from bipolar cells is intrinsic to the inner plexiform layer in that, under experimental conditions, it can occur independent of photoreceptor input. The GABA receptor antagonists picrotoxin and bicuculline enhanced the amplitude of oscillations in starburst cells prestimulated with TEA. Our results suggest that this enhancement was due to a reduction of a GABAergic feedback inhibition from amacrine cells to bipolar cells and the resultant increased glutamate release. Finally, we found that some ganglion cells and other types of amacrine cell also displayed rhythmic activity, suggesting that oscillatory behavior is expressed by a number of inner retinal neurons.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Amacrine Cells; Aminobutyrates; Animals; Bicuculline; Biological Clocks; Cadmium Chloride; Dose-Response Relationship, Radiation; Drug Interactions; Electric Stimulation; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; GABA Antagonists; In Vitro Techniques; Lysine; Membrane Potentials; Mice; Mice, Inbred ICR; Mice, Knockout; Patch-Clamp Techniques; Picrotoxin; Potassium Channel Blockers; Potassium Channels, Voltage-Gated; Retina; Retinal Ganglion Cells; Shaw Potassium Channels; Tetraethylammonium; Time Factors

In accordance with its central role in basal ganglia circuitry, changes in the rate of action potential firing and pattern of activity in the globus pallidus (GP)-subthalamic nucleus (STN) network are apparent in movement disorders. In this study we have developed a mouse brain slice preparation that maintains the functional connectivity between the GP and STN in order to assess its role in shaping and modulating bursting activity promoted by pharmacological manipulations. Fibre-tract tracing studies indicated that a parasagittal slice cut 20 deg to the midline best preserved connectivity between the GP and the STN. IPSCs and EPSCs elicited by electrical stimulation confirmed connectivity from GP to STN in 44/59 slices and from STN to GP in 22/33 slices, respectively. In control slices, 74/76 (97%) of STN cells fired tonically at a rate of 10.3 +/- 1.3 Hz. This rate and pattern of single spiking activity was unaffected by bath application of the GABA(A) antagonist picrotoxin (50 microM, n = 9) or the glutamate receptor antagonist (6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) 10 microM, n = 8). Bursting activity in STN neurones could be induced pharmacologically by application of NMDA alone (20 microm, 3/18 cells, 17%) but was more robust if NMDA was applied in conjunction with apamin (20-100 nM, 34/77 cells, 44%). Once again, neither picrotoxin (50 microM, n = 5) nor CNQX (10 microM, n = 5) had any effect on the frequency or pattern of the STN neurone activity while paired STN and GP recordings of tonic and bursting activity show no evidence of coherent activity. Thus, in a mouse brain slice preparation where functional GP-STN connectivity is preserved, no regenerative synaptically mediated activity indicative of a dynamic network is evident, either in the resting state or when neuronal bursting in both the GP and STN is generated by application of NMDA/apamin. This difference from the brain in Parkinson's disease may be attributed either to insufficient preservation of cortico-striato-pallidal or cortico-subthalamic circuitry, and/or an essential requirement for adaptive changes resulting from dopamine depletion for the expression of network activity within this tissue complex.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Apamin; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; gamma-Aminobutyric Acid; Globus Pallidus; Glutamic Acid; In Vitro Techniques; Lysine; Male; Mice; Mice, Inbred Strains; N-Methylaspartate; Nerve Net; Neurons; Picrotoxin; Subthalamic Nucleus

Recordings of synaptic responses of pyramidal neurons to feedback (FB) inputs from higher to lower areas of visual cortex show that excitatory synaptic responses are only weakly opposed by disynaptic inhibition. Whether weak inhibition is preserved at high frequencies remains unknown. Whole-cell recordings were performed in pyramidal cells of mouse visual cortex to study the frequency dependence of excitatory and inhibitory postsynaptic currents (EPSCs, IPSCs) elicited by feedforward (FF) input from the primary visual cortex (V1) to the higher lateromedial area (LM) and by FB input from the LM to V1. EPSCs showed similar frequency dependencies in FF and FB pathways; the amplitudes decreased during stimulus trains, and the depression was larger at higher frequencies. IPSCs decreased during repetitive stimulation, and the depression increased at higher frequencies. At >20 Hz, the depression of IPSCs in the FB pathway was greater than in the FF pathway. Thus, unlike FF circuits, FB circuits provide balanced excitatory and inhibitory inputs across a wide range of frequencies. This property was shown to be critically important in cortical circuits that modulate the gain of pyramidal cell firing (Chance et al. [2002] Neuron 35:773-782).

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Bicuculline; Cell Size; Electric Stimulation; Evoked Potentials; Excitatory Amino Acid Antagonists; Feedback; GABA Antagonists; In Vitro Techniques; Lysine; Membrane Potentials; Mice; Mice, Inbred C57BL; Neural Inhibition; Neural Pathways; Patch-Clamp Techniques; Pyramidal Cells; Reaction Time; Synapses; Synaptic Transmission; Visual Cortex

Brain-derived neurotrophic factor (BDNF) acutely modulates synaptic transmission to excitatory neurons in hippocampus and neocortex. The question of whether BDNF acts similarly on excitatory synaptic transmission to GABAergic neurons was eluded in previous studies using cortical slices. To address this question, we used transgenic mice in which expression of green fluorescence protein (GFP) is regulated by glutamic acid decarboxylase 67 (GAD67) promoter. In cortical slices prepared from these GAD67-GFP knock-in mice, we could detect GABAergic neurons under a fluorescent microscope. An application of BDNF rapidly depressed excitatory postsynaptic currents (EPSCs) evoked by layer IV stimulation in most GFP-positive neurons in layer II/III of the cortex. This effect was seen at synapses activated during the BDNF application and blocked by anti-TrkB IgG, indicating that the acute inhibitory action of BDNF is activity-dependent and mediated through TrkB. Paired-pulse ratios of the amplitude of EPSCs to paired stimulation at intervals of 10-100 ms were not significantly changed after BDNF application, suggesting that the site of depression may be postsynaptic. Responses to directly applied glutamate were also depressed by BDNF in most of neurons, being consistent with the interpretation of postsynaptic action of BDNF. The depressive action of BDNF was blocked by an intracellular injection of a Ca(2+) chelator, suggesting that a rise in Ca(2+) is involved in the acute depression of EPSCs. This action of BDNF was seen in 67% of parvalbumin (PV)-positive neurons, but in only 19% of PV-negative neurons, indicating that the depressive action is biased to PV-positive GABAergic neurons.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Brain-Derived Neurotrophic Factor; Calcium; Chelating Agents; Dose-Response Relationship, Drug; Drug Interactions; Egtazic Acid; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; gamma-Aminobutyric Acid; Glutamate Decarboxylase; Green Fluorescent Proteins; Immunoglobulin G; In Vitro Techniques; Isoenzymes; Luminescent Proteins; Lysine; Mice; Mice, Transgenic; Neural Inhibition; Neurons; Parvalbumins; Receptor, trkB; Synaptic Transmission; Valine; Visual Cortex

During suckling, oxytocin (OT) neurons display a bursting electrical activity, consisting of a brief burst of action potentials which is synchronized throughout the OT neuron population and which periodically occurs just before each milk ejection in the lactating rat. To investigate the basis of such synchronization, we performed simultaneous intracellular recordings from pairs of OT neurons identified retrospectively by intracellular fluorescent labelling and immunocytochemistry in organotypic slice cultures derived from postnatal rat hypothalamus. A spontaneous bursting activity was recorded in 65% of OT neurons; the remaining showed only a slow, irregular activity. Application of OT triggered bursts in nonbursting neurons and accelerated bursting activity in spontaneously bursting cells. These cultures included rare vasopressinergic neurons showing no bursting activity and no reaction to OT. Bursts occurred simultaneously in all pairs of bursting OT neurons but, as in vivo, there were differences in burst onset, amplitude and duration. Coordination of firing was not due to electrotonic coupling because depolarizing one neuron in a pair had no effect on the membrane potential of its partner and halothane and proprionate did not desynchronize activity. On the other hand, bursting activity was superimposed on volleys of excitatory postsynaptic potentials (EPSPs) which occurred simultaneously in pairs of neurons. EPSPs, and consequently action potentials, were reversibly blocked by the non-NMDA glutamatergic receptor antagonist CNQX. Taken together, these data, obtained from organotypic cultures, strongly suggest that a local hypothalamic network governs synchronization of bursting firing in OT neurons through synchronous afferent volleys of EPSPs originating from intrahypothalamic glutamatergic inputs.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Anesthetics, Inhalation; Animals; Animals, Newborn; Bicuculline; Biotin; Calcium; Dose-Response Relationship, Drug; Drug Interactions; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; Glutamic Acid; Halothane; Hypothalamus; Lysine; Neurons; Neurophysins; Organ Culture Techniques; Oxytocin; Periodicity; Propionates; Rats; Rats, Wistar; Vasopressins

Neuronal activity is thought to play an important role in refining patterns of synaptic connectivity during development and in the molecular maturation of synapses. In experiments reported here, a 2-week infusion of tetrodotoxin (TTX) into rat hippocampus beginning on postnatal day 12 produced abnormal synchronized network discharges in in vitro slices. Discharges recorded upon TTX washout were called 'minibursts', owing to their small amplitude. They were routinely recorded in area CA3 and abolished by CNQX, an AMPA receptor antagonist. Because recurrent excitatory axon collaterals remodel and glutamate receptor subunit composition changes after postnatal day 12, experiments examined possible TTX-induced alterations in recurrent excitation that could be responsible for network hyperexcitability. In biocytin-labelled pyramidal cells, recurrent axon arbors were neither longer nor more highly branched in the TTX infusion site compared with saline-infused controls. However, varicosity size and density were increased. Whereas most varicosities contained synaptophysin and synaptic vesicles, many were not adjacent to postsynaptic specializations, and thus failed to form anatomically identifiable synapses. An increased pattern of excitatory connectivity does not appear to explain network hyperexcitability. Quantitative immunoblots also indicated that presynaptic markers were unaltered in the TTX infusion site. However, the postsynaptic AMPA and NMDA receptor subunits, GluR1, NR1 and NR2B, were increased. In electrophysiological studies EPSPs recorded in slices from TTX-infused hippocampus had an enhanced sensitivity to the NR2B containing NMDA receptor antagonist, ifenprodil. Thus, increases in subunit protein result in alterations in the composition of synaptic NMDA receptors. Postsynaptic changes are likely to be the major contributors to the hippocampal network hyperexcitability and should enhance both excitatory synaptic efficacy and plasticity.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Anesthetics, Local; Animals; Animals, Newborn; Axons; Disease Models, Animal; Epilepsy; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Hippocampus; Immunoblotting; Immunohistochemistry; In Vitro Techniques; Lysine; Membrane Potentials; Microscopy, Confocal; Microscopy, Electron; Nerve Net; Patch-Clamp Techniques; Piperidines; Pyramidal Cells; Rats; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Synapses; Synaptophysin; Tetrodotoxin; Time Factors

Activity-dependent synaptic plasticity triggered by N-methyl-d-aspartate (NMDA) receptor activation is a fundamental property of many glutamatergic synapses and may be critical for the shaping and refinement of the structural and functional properties of neuronal circuits during early postnatal development. Using a combined morphological and electrophysiological approach, we showed that chronic blockade of NMDA receptors in hippocampal slice cultures during the first two weeks of postnatal development leads to a substantial increase in synapse number and results in a more complex dendritic arborization of CA1 pyramidal cells. Thus, the development of excitatory circuitry in the hippocampus is determined by two opposing processes: NMDA receptor-independent synapse formation and NMDA receptor-dependent attenuation of synaptogenesis.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Cell Surface Extensions; Cells, Cultured; Dendrites; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Hippocampus; Histocytochemistry; In Vitro Techniques; Ion Channels; Lysine; Microscopy, Confocal; Patch-Clamp Techniques; Piperazines; Pyramidal Cells; Rats; Rats, Wistar; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Synapses

The distribution of functional neurotransmitter receptors is an important determinant of neuronal information processing. To map the location of functional glutamate and GABA receptors on individual hippocampal neurons, we photolyzed "caged" glutamate and GABA while measuring the electrical currents resulting from activation of these receptors. Responses to uncaged neurotransmitters were spatially nonuniform and varied according to the type of receptor and type of neuron. Every region of CA1 pyramidal cells responded to glutamate and GABA, but glutamate and GABA receptors increased in density along the length of their distal dendrites. Similar gradients of glutamate receptors were found in stratum radiatum interneurons, while GABA responses were detectable only in the perisomatic region of these interneurons. These regional variations in receptor distribution indicate the selective targeting of receptors on central neurons and may reflect a mechanism for local regulation of synaptic efficacy.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Cell Polarity; Dendrites; Excitatory Amino Acid Antagonists; Fluorescent Dyes; GABA Antagonists; gamma-Aminobutyric Acid; Glutamic Acid; Hippocampus; In Vitro Techniques; Interneurons; Lysine; Membrane Potentials; Patch-Clamp Techniques; Picrotoxin; Pyramidal Cells; Rats; Receptors, GABA; Receptors, Glutamate; Synapses

Paired intracellular recordings were used to investigate recurrent excitatory transmission in layers II, III and V of the rat entorhinal cortex in vitro. There was a relatively high probability of finding a recurrent connection between pairs of pyramidal neurons in both layer V (around 12%) and layer III (around 9%). In complete contrast, we have failed to find any recurrent synaptic connections between principal neurons in layer II, and this may be an important factor in the relative resistance of this layer in generating synchronized epileptiform activity. In general, recurrent excitatory postsynaptic potentials in layers III and V of the entorhinal cortex had similar properties to those recorded in other cortical areas, although the probabilities of connection are among the highest reported. Recurrent excitatory postsynaptic potentials recorded in layer V were smaller with faster rise times than those recorded in layer III. In both layers, the recurrent potentials were mediated by glutamate primarily acting at alpha-amino-3-hydroxy-5-methyl-4-isoxazole receptors, although there appeared to be a slow component mediated by N-methyl-D-aspartate receptors. In layer III, recurrent transmission failed on about 30% of presynaptic action potentials evoked at 0.2Hz. This failure rate increased markedly with increasing (2, 3Hz) frequency of activation. In layer V the failure rate at low frequency was less (19%), and although it increased at higher frequencies this effect was less pronounced than in layer III. Finally, in layer III, there was evidence for a relatively high probability of electrical coupling between pyramidal neurons. We have previously suggested that layers IV/V of the entorhinal cortex readily generate synchronized epileptiform discharges, whereas layer II is relatively resistant to seizure generation. The present demonstration that recurrent excitatory connections are widespread in layer V but not layer II could support this proposal. The relatively high degree of recurrent connections and electrical coupling between layer III cells may be a factor in it's susceptibility to neurodegeneration during chronic epileptic conditions.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Entorhinal Cortex; Epilepsy; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; In Vitro Techniques; Lysine; Male; Neural Pathways; Pyramidal Cells; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Synaptic Transmission

Morphological and electrophysiological properties of calbindin D-28k-, GABA- and dopamine-beta-hydroxylase-immunopositive neurons were investigated in the caudal nucleus of tractus solitarius of rats, using a patch-clamp whole-cell recording combined with intracellular staining and immunocytochemistry. Calbindin D-28K- and GABA-positive neurons had a small cell body (10.9+/-0.3 microm in diameter) and were distributed throughout the caudal nucleus of tractus solitarius. Double fluorescence immunocytochemistry revealed that calbindin- and GABA-positive neurons formed distinct subpopulations. Calbindin- and GABA-positive neurons double stained for biocytin showed extensive axon collaterals within the nucleus of tractus solitarius and some calbindin-positive, but not GABA-positive neurons, had also projection axons leaving the nucleus of tractus solitarius. Dopamine-beta-hydroxylase-immunopositive neurons had a small (10.8+/-0.3 microm) or large (17.2+/-0.4 microm) cell body. Neurons with a small cell body were observed in the dorsomedial nucleus at the level of the area postrema, and in the area postrema, while neurons with a large cell body were observed in the medial nucleus throughout the caudal nucleus of tractus solitarius. Double fluorescence immunocytochemistry revealed that almost all small dopamine-beta-hydroxylase-positive neurons were also immunoreactive for calbindin, while large dopamine-beta-hydroxylase-positive neurons were not. Double staining for dopamine-beta-hydroxylase and biocytin showed that neurons with a small cell body had moderate axon collaterals. On the contrary, neurons with a large cell body had few, if any, axon collaterals and a projection axon which could leave the nucleus of tractus solitarius. Following stimulation of the tractus solitarius, all neurons with a small cell body exhibited a polysynaptic excitatory response (type I neurons), while dopamine-beta-hydroxylase-immunopositive neurons with a large cell body exhibited a monosynaptic excitatory response (type II neurons) or an excitatory followed by an inhibitory response (type III neurons). Spontaneous and evoked excitatory postsynaptic currents of (type I neurons) calbindin- or GABA-positive neurons were reversibly blocked by 6-cyano-7-nitroquinoxaline-2,3-dione. Spontaneous and evoked inhibitory postsynaptic currents of type III neurons were reversibly blocked by bicuculline. Type II neurons showed no spontaneous excitatory nor inhibitory postsynaptic currents. It

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Axons; Bicuculline; Calbindins; Dopamine beta-Hydroxylase; Electric Stimulation; Excitatory Postsynaptic Potentials; gamma-Aminobutyric Acid; Immunohistochemistry; Lysine; Male; Neurons; Rats; Rats, Sprague-Dawley; S100 Calcium Binding Protein G; Solitary Nucleus

Studies in slices suggest that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated synaptic currents are not present in CA1 (Cornu ammonis) pyramidal neurons at birth (P0). We have re-examined this issue in the rat intact hippocampal formation (IHF) in vitro. Injections of biocytin or carbocyanine show that the temporo-ammonic, commissural and Schaffer collateral pathways are present at birth in the marginal zone of CA1. Electrical stimulation of these pathways evoked field excitatory postsynaptic potentials (fEPSPs) in the marginal zone of CA1 from embryonic day 19 (E19) to postnatal day 9 (P9). These fEPSPs are mediated by synaptic AMPA receptors as they are reduced or completely blocked by: (i) tetrodotoxin; (ii) high divalent cation concentrations; (iii) the adenosine A1 receptor agonist CPA; (iv) anoxic episodes; (v) the selective AMPA receptor antagonist 1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-7, 8-methylenedioxy-3,4-dihydro-5H-2,3-benzodiazepine (GYKI-53655) or the mixed AMPA-kainate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX). The amplitude of the fEPSPs is also reduced by D(-)-2-amino-5-phosphonopentanoic acid (D-APV) and its duration is increased by bicuculline suggesting the participation of N-methyl-D-aspartate (NMDA) and GABAA (gamma-aminobutyric acid) receptors. Finally, AMPA receptor-mediated fEPSPs are also recorded in P0 slices, but they are smaller and more labile than in the IHF. Our results suggest that in embryonic CA1 neurons, glutamate acting on AMPA receptors already provides a substantial part of the excitatory drive and may play an important role in the activity-dependent development of the hippocampus. Furthermore, the IHF may be a convenient preparation to investigate the properties of the developing hippocampus.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Afferent Pathways; Aging; Animals; Animals, Newborn; Benzodiazepines; Bicuculline; Cations, Divalent; Electric Stimulation; Embryonic and Fetal Development; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Fluorescent Dyes; Hippocampus; Hypoxia; Lysine; Pyramidal Cells; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Synapses; Tetrodotoxin

Serotonin (1-40 microM) reduced input resistance by 20.6 +/- 6% and hyperpolarized stellate and pyramidal neurons of layers two and three of the lateral entorhinal cortex. 5-Carboxamidotryptamine, a 5-HT1 agonist, and the selective 5-HT1A agonist 8-hydroxy-dipropylaminotetralin mimicked the action of serotonin. The reversal potential of 5-HT-mediated hyperpolarizations was sensitive to the extracellular K+ concentration, indicating a potassium conductance change. Serotonin treatment suppressed excitatory amino acid-mediated synaptic potentials (by 48%, Kd = 6.9 microM) and responses to exogenously applied glutamate (70.1 +/- 17% of control, n = 7), but did not alter paired-pulse facilitation, indicating a postsynaptic site of action. Intracellular application of QX-314, a blocker of potassium conductance, significantly reduced depression of synaptic potentials by 5-HT agonists. In cells filled with QX-314, responses to exogenously applied glutamate were not reduced by serotonin or 5-carboxamidotryptamine application. These results indicate that the observed conductance increase associated with 5-HT application accounts for most if not all of the observed depressant effects of 5-HT1A agonists on excitatory amino acid-mediated neurotransmission.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; 8-Hydroxy-2-(di-n-propylamino)tetralin; Anesthetics, Local; Animals; Electrophysiology; Entorhinal Cortex; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glutamic Acid; Lidocaine; Lysine; Male; Neural Inhibition; Neurons; Perforant Pathway; Potassium; Potassium Channels; Propranolol; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Receptors, Serotonin; Receptors, Serotonin, 5-HT1; Serotonin; Serotonin Receptor Agonists; Sympatholytics

Intracellular recordings were used to characterize the inhibitory synapses formed by Purkinje cells on neurons in the deep cerebellar nuclei of the rat. This work was performed on organotypic cerebellar cultures where functional connections between Purkinje cells and deep cerebellar neurons are formed de novo. After blocking ionotropic excitatory amino acid, and GABAA receptors with 6-cyano-7-nitro-quinoxaline-2,3-dione,D-2-amino-5-phosphonovalerate and bicuculline, respectively, the majority of deep cerebellar neurons fired spontaneously without accommodation. This tonic firing was linearly dependent on membrane potential and was abolished with hyperpolarization. Bath application of muscimol and baclofen reversibly hyperpolarized deep cerebellar nuclei cells. In the presence of excitatory amino acid receptor antagonists, field stimulation within the Purkinje cell layer induced monosynaptic inhibitory potentials in deep cerebellar neurons that were graded and completely blocked by bicuculline. Inhibitory potential amplitudes were not markedly reduced during fast repetitive stimulation of Purkinje cells, and the resulting hyperpolarization was not affected by the competitive GABAB receptor antagonist CGP 35348. A single inhibitory potential temporarily interrupted trains of action potentials induced in deep cerebellar cells by short depolarizing pulses. Trains of five inhibitory postsynaptic potentials, evoked at 20 Hz, induced a hyperpolarization which transiently blocked the spontaneous firing of deep cerebellar cells. The efficiency to block action potential discharges depended on the frequency of evoked inhibitory potentials. Bath application of bicuculline induced burst discharges in the control solution. When the excitatory amino acid receptors were pharmacologically blocked, bicuculline depolarized deep cerebellar neurons inducing sustained action potential discharges. In the presence of tetrodotoxin, bicuculline abolished miniature inhibitory postsynaptic potentials and resulted in a membrane depolarization of deep cerebellar cells. We conclude that deep cerebellar neurons isolated from synaptic inputs display a pacemaker-like activity. Although these neurons possess GABAA and GABAB receptors, we confirm that only GABAA receptors were involved in the generation of inhibitory postsynaptic potentials, even with high frequency stimulation. The amplitude of evoked inhibitory potentials was weakly frequency-dependent, thus allowing a powerful inhibition

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Baclofen; Bicuculline; Cerebellar Nuclei; Cerebral Cortex; Electrophysiology; GABA Agonists; gamma-Aminobutyric Acid; In Vitro Techniques; Lysine; Muscimol; Neural Pathways; Purkinje Cells; Rats; Rats, Wistar; Receptors, GABA-A; Receptors, GABA-B; Synaptic Transmission; Tetrodotoxin; Vestibular Nucleus, Lateral

In the present study, the nature of the synaptic transmission responsible for a monophasic potential generated by vestibular nerve stimulation of the principal cells in the chick tangential nucleus was established. This work was performed in slice preparations at the critical embryonic age of 15-16 days, the time of first observation of morphologically mixed (chemical and electrical) synapses at the axosomatic endings called spoon endings. The spoon endings are formed by the primary vestibular fibers with the largest diameters, the colossal vestibular fibers. This monophasic potential fits the criteria for chemical rather than electrical transmission due to the following responses in most cases: (i) the absence of collision between a direct spike initiated by depolarization in the principal cell and a vestibular-evoked action potential; (ii) failure to follow high frequency stimulation (up to 50 Hz); (iii) sensitivity to low calcium solution (0.1 mM). These tests indicate that strong electrical coupling between spoon endings and principal cells does not prevail at this stage. The recordings were obtained from principal cells injected intracellularly with biocytin, allowing their identification by morphological criteria. The lack of tracer coupling between the stained principal cells and their innervating vestibular fibers (n = 17) is consistent with the absence of electrical coupling. Identification of the neurotransmitter involved in this vestibular response was achieved by bath application of glutamate receptor antagonists, DL-2-amino-5-phosphonovaleric acid (40 microM) and 6-cyano-7-nitro-quinoxaline-2,3-dione (10 microM), which blocked transmission reversibly. These results suggest that at the onset of formation of these "mixed" vestibular synapses, the gap junctions identified morphologically are likely not functional, and that the main response of the principal cells to vestibular nerve stimulation is mediated by glutamate.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Chick Embryo; Electrophysiology; Excitatory Amino Acid Antagonists; Glutamates; Glutamic Acid; Intercellular Junctions; Lysine; Neurons, Afferent; Quinoxalines; Reflex, Vestibulo-Ocular; Synapses; Synaptic Transmission; Vestibular Nerve

The principal aim of this study was to characterize the transmitter mechanisms mediating fast postsynaptic potentials in identified neurons of the rat nucleus accumbens. Using the biocytin-avidin labeling technique, impaled neurons were identified as medium spiny neurons. The basic membrane characteristics of these neurons were determined. Local electrical stimulation or stimulation of the corpus callosum elicited a depolarizing postsynaptic potential consisting of an EPSP often followed by an IPSP. The quisqualate/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (4 microM) abolished most of the depolarizing postsynaptic potential. The N-methyl-D-aspartate receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid depressed a small part of the decay phase of the depolarizing postsynaptic potential. Paired-pulse facilitation of postsynaptic potentials was found using interstimulus-intervals between 10 and 150 ms. N-methyl-D-aspartate receptors were found to contribute only slightly to the facilitation of the decay phase of the depolarizing postsynaptic potential, but not to its rising phase. This contribution was particularly clear under conditions of reduced GABAA receptor mediated inhibition. The present study indicates that postsynaptic responses of medium spiny neurons in the nucleus accumbens to local stimulation or stimulation of neocortical afferents are primarily mediated by quisqualate/kainate receptors. The contribution of NMDA receptors is normally limited to a portion of the decay phase of these responses, but is enlarged in the absence of GABAergic inhibition and following paired-pulse stimulation.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Electric Stimulation; Evoked Potentials; In Vitro Techniques; Lysine; Male; Neurons; Neurons, Afferent; Nucleus Accumbens; Quinoxalines; Rats; Rats, Inbred Strains; Receptors, AMPA; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter