N. Traverse Slater

The developmental onset of NMDA receptor-channel activity during neuronal migration

Patch-clamp recordings of granule cells in thin slices of developing rat cerebellum maintained in vitro displayed spontaneous single-channel activity mediated via activation of N-methyl-D-aspartate (NMDA) receptors. The frequency of tonic single-channel activity was reversibly inhibited by the NMDA receptor/channel antagonists D-2-amino-5-phosphonovalerate (D-AP5), 7-chloro-kynurenate (7-Cl-Kynu) and MgCl2, potentiated by glycine, and unaffected by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or tetrodotoxin (TTX). Tonic channel activity was also reversibly inhibited by enzymatic degradation of endogenous glutamate by glutamate pyruvate transaminase, which did not affect the NMDA sensitivity of granule cells. Both the frequency of spontaneous channel activity and the NMDA sensitivity were low in premigratory cells of the external germinal layer (EGL), with large increases observed in migrating cells in the molecular layer (ML) and in postmigratory cells within the internal granule cell layer (GCL). Tonic channel activity was enhanced by the glutamate uptake inhibitor L-alpha-aminoadipate (L-alpha-AA), the degree of enhancement being greater in the EGL than the GCL. The results demonstrate that a dramatic increase in the tonic NMDA receptor-channel activity occurs during the stages of granule cell differentiation, migration and synaptogenesis, which is driven by endogenous glutamate release and regulated by NMDA receptor density and local glutamate uptake.

Modification of NMDA Receptor Channels and Synaptic Transmission by Targeted Disruption of the NR2C Gene

A novel strain of mutant mouse has been generated with a deletion of the gene encoding the NR2C subunit of the NMDA receptor, which is primarily expressed in cerebellar granule cells. Patch-clamp recordings from granule cells in thin cerebellar slices were used to assess the consequences of the gene deletion. In granule cells of wild-type animals, a wide range of single-channel conductances were observed (19–60 pS). The disruption of the NR2C gene results in the disappearance of low-conductance NMDA receptor channels (<37 pS) normally expressed in granule cells during developmental maturation. The NMDA receptor-mediated synaptic current is markedly potentiated in amplitude, but abbreviated in duration (with no net difference in total charge), and the non-NMDA component of the synaptic current was reduced. We conclude that the NR2C subunit contributes to functional heteromeric NMDA receptor-subunit assemblies at the mossy fiber synapse and extrasynaptic sites during maturation, and the conductance level exhibited by a given receptor macromolecule may reflect the stochiometry of subunit composition.

Unipolar brush cells form a glutamatergic projection system within the mouse cerebellar cortex

Unipolar brush cells (UBCs) of the mammalian vestibulocerebellum receive mossy fiber projections primarily from the vestibular ganglion and vestibular nuclei. Recently, the axons of UBCs have been shown to generate an extensive system of cortex‐intrinsic mossy fibers, which resemble traditional cerebellar mossy fiber afferents and synapse with granule cell dendrites and other UBCs. However, the neurotransmitter used by the UBC axon is still unknown. In this study, we used long‐term organotypic slice cultures of the isolated nodulus (lobule X) from postnatal day 8 mouse cerebella to identify the neurotransmitter and receptors at synapses of the UBC axon terminals, relying on the notion that, in these cultures, all of the cortex‐extrinsic fibers had degenerated during the first few days in vitro. Quantification of glutamate immunogold labeling showed that the UBC axon terminals have the same high gold‐particle density as the glutamatergic parallel fiber varicosities. Furthermore, UBCs identified by calretinin immunoreactivity expressed the glutamate receptor subunits GluR2/3, NMDAR1, and mGluR2/3, like they do in the mature mouse cerebellum in situ. Evoked excitatory postsynaptic currents (EPSCs), spontaneous EPSCs, and burst discharges were demonstrated in UBCs and granule cells by patch‐clamp recording. Both the evoked and spontaneous EPSCs were blocked by ionotropic glutamate receptor antagonists CNQX and D‐AP5. We conclude that neurotransmission at the UBC axon terminals is glutamatergic. Thus, UBCs provide a powerful network of feedforward excitation within the granular layer, which may amplify vestibular signals and synchronize activity in clusters of functionally related granule cells which project vertically to patches of Purkinje cells. J. Comp. Neurol. 434:329–341, 2001.

Bicuculline and picrotoxin block phase advances induced by GABA agonists in the circadian rhythm of locomotor activity in the golden hamster by a phaclofen-insensitive mechanism

Permanent phase shifts in the free-running rhythm of locomotor activity of the golden hamster were induced with microinjections of the gamma-aminobutyric acid (GABA) agonists muscimol or baclofen in the hypothalamic suprachiasmatic nuclei. Muscimol and baclofen exhibit relatively high binding affinities for GABAA and GABAB receptors, respectively. Microinjections of the GABA antagonists, bicuculline methobromide or picrotoxinin, thought to block the actions of GABA at GABAA receptors, could block phase shifts induced by muscimol but not the benzodiazepine, triazolam. Microinjections of the postsynaptic GABAB receptor antagonist phaclofen, which blocks the actions of GABA at postsynaptic but not at presynaptic GABAB receptor sites, did not block the phase-shifting actions of either muscimol or baclofen. GABAergic antagonists when given alone did not induce phase shifts. Collectively, these studies indicate that when activated by exogenous GABAergic agents, a GABAergic system associated with both GABAA and GABAB receptors exists as a neural regulatory mechanism that can reset the mammalian circadian clock. However, GABAergic synaptic pathways may not be normally involved in the circadian timing system.

Excitatory amino acid receptors mediate slow synaptic transmission in turtle cerebellum

In the isolated turtle cerebellum intracellular recordings from Purkinje cell dendrites and somata reveal novel slow excitatory synaptic potentials evoked by activation of climbing fiber (CF) or parallel fiber (PF) inputs. Classical fast excitatory synaptic responses to CF and PF stimulation are followed by large, slow excitatory postsynaptic potentials (sEPSPs) which are associated with an increase in conductance and are enhanced by hyperpolarization. Both sEPSPs are blocked by the excitatory amino acid (EAA) antagonist kynurenate, but not by DL-2-amino-5-phosphonovalerate (AP-5). The EAA receptor antagonist L-amino-4-phosphonobutyric acid (L-AP-4) reversibly blocked the PF-sEPSP without affecting the CF-sEPSP. Two novel slow synaptic potentials mediated by excitatory amino acid receptors can therefore be observed in turtle cerebellum which may play an important role in synaptic integration.

GABAergic inhibition and epileptiform discharges in the turtle hippocampus in vitro.

The effects of excitatory amino acid (EAA) receptor antagonists were examined on intracellularly recorded epileptiform discharges in turtle hippocampal (ventromedial cortical) pyramidal neurons in vitro. Afferent synaptic activation of turtle hippocampal neurons evoked monophasic or biphasic GABAergic inhibitory postsynaptic potentials (IPSPs). In the presence of bicuculline (5 microM) or picrotoxin (100 microM) IPSPs were reduced, and long-lasting ictal-like discharges were transiently observed prior to the establishment of a regular rhythm of discharge of spontaneous paroxysmal depolarization shifts (PDSs). Bicuculline-induced PDSs were reversibly reduced in amplitude and duration, but not abolished by the EAA receptor antagonists kynurenic acid (1 mM), cis-2,3-piperidine dicarboxylic acid (cis-2,3-PDA) (1 mM), or DL-2-amino-5-phosphonovalerate (DL-AP-5) (100 microM), revealing a long-lasting hyperpolarizing afterpotential. These results indicate that the blockade of GABAergic inhibition leads to the genesis of epileptiform discharges, and EAA receptor antagonists (particularly those of the N-methyl-D-aspartate (NMDA) receptor subtype) block the maintained depolarization underlying PDSs, but do not prevent their spontaneous discharge in turtle hippocampus.

Whole-Cell Patch Recording with Simultaneous Measurement of Intracellular Calcium Concentration in Mammalian Brain Slices in Vitro

Publisher Summary This chapter describes the use of whole-cell patch-clamp recording methods in brain slices combined with the photometric method to monitor neurons dialyzed with fura-2 and imaging methods using fluo-3. The primary advantage of examining intracellular Ca 2+ concentration ([Ca 2+ ] i ) in brain slices by the photometric method is that it provides a rapid temporal means of resolving changes in [Ca 2+ ] i . The method maintains the advantages of the slice preparation, such as the avoidance of culture or dissociation artifacts, the preservation of a degree of endogenous connectivity, and control of the extracellular milieu. The photometric method provides a high temporal means of simultaneously monitoring the fluorescence of intracellular ion-sensitive dyes and the transmembrane voltage or current in neurons in slices. The key advantage of the method is speed. The chief disadvantage is the limited region of the cell that can be accurately examined and the absence of spatial resolution. Imaging methods are needed where the spatial resolution of the calcium signal is of interest, and speed can be compromised to some extent.

Cellular and Network Determinants of Visual Motion Properties in Cortical Neurons: Studies with an In Vitro Preparation of Visual Cortex

The cerebral cortex of fresh water turtles(Pseudemys scriptaandChrysemys picta)contains a visual area (review: Ulinski, 1990) that receives direct input from the dorsal lateral geniculate complex (Hall and Ebner, 1970; Heller and Ulinski, 1987). The projection of the retina to the geniculate complex is bilateral and topographically organized (Ulinski and Nautiyal, 1987), but the subsequent projection of the geniculate to the visual cortex is not topographically organized. Instead, there is a convergence of all points of visual space onto each cortical cell as a result of the spatial organization of the geniculocortical and intracortical projections (Mulligan and Ulinski, 1990; Cosans and Ulinski, 1990). The anatomy of the geniculocortical pathway is consistent with extracellular recordings in alert, paralyzed animals presented with visual stimuli (Mazurskaya, 1974). Each cortical cell in such experiments responds to moving stimuli at all points in binocular visual space. They also respond well to two stimuli presented in succession at disjunct points in visual space in an “apparent motion” paradigm and have temporal tuning curves with two facilitatory peaks, one quite constant peak at 50–75 msec and a second, more variable peak occuring between 250 and 600 msec. These neuronal properties suggest that visual cortex plays a role in visually guided orienting behaviors in turtles (Ulinski et al., 1991a).