piperidines and biocytin

The basal forebrain (BF) controls sleep-wake cycles, attention and reward processing. Compared to cholinergic and GABAergic neurons, BF glutamatergic neurons are less well understood, due to difficulties in identification. Here, we use vesicular glutamate transporter 2 (vGluT2)-tdTomato mice, expressing a red fluorescent protein (tdTomato) in the major group of BF glutamatergic neurons (vGluT2+) to characterize their intrinsic electrical properties and cholinergic modulation. Whole-cell, patch-clamp recordings were made from vGluT2+ neurons in coronal BF slices. Most BF vGluT2+ neurons were small/medium sized (<20µm), exhibited moderately sized H-currents and had a maximal firing frequency of ∼50Hz. However, vGluT2+ neurons in dorsal BF (ventral pallidum) had larger H-currents and a higher maximal firing rate (83Hz). A subset of BF vGluT2+ neurons exhibited burst/cluster firing. Most vGluT2+ neurons had low-threshold calcium spikes/currents. vGluT2+ neurons located in ventromedial regions of BF (in or adjacent to the horizontal limb of the diagonal band) were strongly hyperpolarized by the cholinergic agonist, carbachol, a finding apparently in conflict with their increased discharge during wakefulness/REM sleep and hypothesized role in wake-promotion. In contrast, most vGluT2+ neurons located in lateral BF (magnocellular preoptic area) or dorsal BF did not respond to carbachol. Our results suggest that BF glutamatergic neurons are heterogeneous and have morphological, electrical and pharmacological properties which distinguish them from BF cholinergic and GABAergic neurons. A subset of vGluT2+ neurons, possibly those neurons which project to reward-related areas such as the habenula, are hyperpolarized by cholinergic inputs, which may cause phasic inhibition during reward-related events.

Topics: Action Potentials; Animals; Animals, Genetically Modified; Animals, Newborn; Basal Forebrain; Benzamides; Calcium Channel Blockers; Carbachol; Cholinergic Agents; Electric Stimulation; Glutamates; Luminescent Proteins; Lysine; Mice; Neurons; Patch-Clamp Techniques; Piperidines; Pyrimidines; Red Fluorescent Protein; Sodium Channel Blockers; Tetrodotoxin; Vesicular Glutamate Transport Protein 2

Activity of the dentate gyrus, which gates information flow to the hippocampus, is under tight inhibitory regulation by interneurons with distinctive axonal projections, intrinsic and synaptic characteristics and neurochemical identities. Total molecular layer cells (TML-Cs), a class of morphologically distinct GABAergic neurons with axonal projections across the molecular layer, are among the most frequent interneuronal type in the dentate subgranular region. However, little is known about their synaptic and neurochemical properties. We demonstrate that synapses from morphologically identified TML-Cs to dentate interneurons are characterized by low release probability, facilitating short-term dynamics and asynchronous release. TML-Cs consistently show somatic and axonal labeling for the cannabinoid receptor type 1 (CB1 R) yet fail to express cholecystokinin (CCK) indicating their distinctive neurochemical identity. In paired recordings, the release probability at synapses between TML-Cs was increased by the CB1 R antagonist AM251, demonstrating baseline endocannabinoid regulation of TML-C synapses. Apart from defining the synaptic and neurochemical features of TML-Cs, our findings reveal the morphological identity of a class of dentate CB1 R-positive neurons that do not express CCK. Our findings indicate that TML-Cs can mediate cannabinoid sensitive feed-forward and feedback inhibition of dentate perforant path inputs.

Topics: Action Potentials; Animals; Biophysics; Cannabinoid Receptor Modulators; Cannabinoids; Cholecystokinin; Dentate Gyrus; Electric Stimulation; In Vitro Techniques; Interneurons; Lysine; Male; Neural Inhibition; Parvalbumins; Patch-Clamp Techniques; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Statistics, Nonparametric; Synapses

The influence of local circuit interneurons is thought to play an important role in adjusting synaptic strength via endogenous cannabinoid type 1 (CB1) receptors. Using paired whole-cell recordings, combined with double immunofluorescence and biocytin labelling in acute slices of rat CA1 at postnatal day 18-23, we investigated the properties of Cholecystokinin (CCK)-positive stratum radiatum local circuit interneuron connections that utilised CB1 receptors. Three types of synaptic connections were studied, lacunosum-moleculare-radiatum perforant path-associated (LM-R PPA) to Shaffer collateral-associated (SCA) interneurons, SCA-SCA interneurons and SCA-pyramidal cells. These three synapses were differentially under tonic reduction of inhibition that was blocked by the CB1 receptor inverse agonist AM-251 (10 microM), which enhanced IPSPs. The strength of tonic reduction of inhibition was correlated with asynchronous release which was apparent at connections among interneurons. AM-251 increased the ratio of synchronous to asynchronous release (synchronicity ratio), while the CB receptor agonist anandamide (14 microM) decreased the synchronicity ratio. Fast and slow calcium chelators (BAPTA-AM and EGTA-AM) also increased the synchronicity ratio, accelerated inhibitory time courses and reduced IPSP amplitudes. These data suggest that CB1 receptors at connections among interneuron synapses play a role in tonic suppression of inhibition and govern the asynchronous release of GABA, modulating the time windows of inhibition. Effects of calcium chelators suggest that asynchronous release is a result of a long-lasting presynaptic calcium transients and/or a large distance between calcium source and sensor of exocytosis. These properties of specialised inhibitory neurons may have important modulatory roles in controlling spike timing among local circuit interneurons.

Topics: Animals; Animals, Newborn; Arachidonic Acids; CA1 Region, Hippocampal; Cannabinoid Receptor Modulators; Chelating Agents; Cholecystokinin; Dendrites; Egtazic Acid; Endocannabinoids; gamma-Aminobutyric Acid; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Interneurons; Lysine; Male; Neural Inhibition; Neural Pathways; Patch-Clamp Techniques; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Synapses

Odor coding in mammals is widely believed to involve synchronized gamma frequency (30-70 Hz) oscillations in the first processing structure, the olfactory bulb. How such inputs are read in downstream cortical structures however is not known. Here we used patch-clamp recordings in rat piriform cortex slices to examine cellular mechanisms that shape how the cortex integrates inputs from bulb mitral cells. Electrical stimulation of mitral cell axons in the lateral olfactory tract (LOT) resulted in excitation of pyramidal cells (PCs), which was followed approximately 10 ms later by inhibition that was highly reproducible between trials in its onset time. This inhibition was somatic in origin and appeared to be driven through a feedforward mechanism, wherein GABAergic interneurons were directly excited by mitral cell axons. The precise inhibition affected action potential firing in PCs in two distinct ways. First, by abruptly terminating PC excitation, it limited the PC response to each EPSP to exactly one, precisely timed action potential. In addition, inhibition limited the summation of EPSPs across time, such that PCs fired action potentials in strong preference for synchronized inputs arriving in a time window of <5 ms. Both mechanisms would help ensure that PCs respond faithfully and selectively to mitral cell inputs arriving as a synchronized gamma frequency pattern.

Topics: Action Potentials; Animals; Animals, Newborn; Bicuculline; Cerebral Cortex; Dose-Response Relationship, Radiation; Electric Stimulation; Excitatory Amino Acid Antagonists; GABA Antagonists; gamma-Aminobutyric Acid; In Vitro Techniques; Lysine; Models, Neurological; Nerve Net; Neurons; Olfactory Pathways; Patch-Clamp Techniques; Piperidines; Quinoxalines; Rats; Rats, Sprague-Dawley; Triazines

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

Cajal-Retzius (CR) cells are among the earliest generated neurons and are thought to play a role in corticogenesis and early neuronal migration. However, the role of CR cells in an early cortical microcircuit is still rather unclear. We therefore have investigated the morphology and physiology of CR cells by using whole-cell patch-clamp recordings combined with intracellular biocytin filling in acute brain slices of postnatal day 5-11 rats. CR cells are characterized by a long horizontally oriented dendrite; the axonal collaterals form a dense horizontally oriented plexus in layer 1 and to a certain extent in layer 2/3, projecting over >2 mm of cortical surface. The bouton density is relatively high, and synaptic contacts are established preferentially with dendritic spines or shafts of excitatory neurons, presumably terminal tuft dendrites of pyramidal neurons. In turn, CR cells receive dense GABAergic and non-GABAergic input on somata, dendritic shafts, and spine-like appendages. Extracellular stimulation in layer 1 could activate both GABAergic and glutamatergic synaptic inputs. The GABAergic response was blocked by the GABA(A) receptor antagonist bicuculline. The glutamatergic response was mediated solely by NMDA receptors and was highly sensitive to ifenprodil, indicating that it was mediated mainly via NR1/NR2B subunit-containing receptors. NMDA EPSPs were apparent in 1 mm extracellular Mg2+, suggesting that this pure NMDA synapse is not silent functionally. Together, the long-range horizontal projection of the axon, the high density of synaptic boutons, and the functional synaptic input of CR cells suggest that they are an integral part of an early cortical network.

Topics: Animals; Axons; Dendrites; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; In Vitro Techniques; Lysine; Neocortex; Nerve Net; Neurons; Patch-Clamp Techniques; Piperidines; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Synapses; Synaptic Transmission