AMPA receptor subunits expressed by single Purkinje cells.

Several subunits of the glutamate receptor of the AMPA subtype have been cloned recently. These subunits, named GluR1, GluR2, GluR3, and GluR4, exist as two splicing variants (flip and flop). We have determined the subset of AMPA receptor subunits expressed by single cerebellar Purkinje cells in culture. This was achieved by combining whole-cell patch-clamp recordings and a molecular analysis, based on the polymerase chain reaction, of the messenger RNAs harvested into the patch pipette at the end of each recording. We found that each single cell expresses the messenger RNAs encoding the following five subunits: the flip and flop versions of GluR1 and GluR2 as well as GluR3flip, GluR2 being the most abundant. In addition, GluR3flop and GluR4flip were scarcely expressed in half of these neurons, and GluR4flop was never detected.

Subunit composition at the single-cell level explains functional properties of a glutamate-gated channel.

The diversity of known glutamate-gated channels has been markedly increased by the discovery of multiple subunits and their spliced and edited variants. These subunits can potentially form different oligomeric complexes with diverging properties. A crucial question is therefore to determine the actual subunit composition of naturally occurring glutamate receptors. We have coupled patch-clamp recordings and reverse transcription followed by PCR amplification to correlate the presence of mRNAs for each subunit and the functional properties of native glutamate receptors at the single-cell level. In a homogeneous population of functionally identified hippocampal neurons (type II) in culture bearing a glutamate receptor of the AMPA subtype with a high calcium permeability, we found that, among the multiple subunits, only two, the flop forms of GluR1 and GluR4, were expressed. In particular, GluR2 was never detected. This composition explains the uncommon properties of AMPA receptors in type II neurons.

Er81 is expressed in a subpopulation of layer 5 neurons in rodent and primate neocortices.

Laminar organization is a fundamental cytoarchitecture in mammalian CNS and a striking feature of the neocortex. ER81, a transcription factor, has recently been utilized as a marker of cells in the layer 5 of the neocortex. We further pursued the distribution of ER81 to investigate the identity of the ER81-expressing cells in the brain. Er81 transcript was expressed in a subset of pyramidal cells that were scattered throughout the entire width of layer 5. In the rat cortex, Er81 transcripts were first detected in the ventricular zone at E15, remained expressed in putative prospective layer 5 neurons during infant and juvenile stages. The ER81-expressing subpopulation in adult layer 5 neurons did not segregate with the phenotypes of the projection targets. By retrograde labeling combined with immunohistochemistry or reverse transcription-polymerase chain reaction analysis, we found ER81 expression in nearly all of the layer 5 neurons projecting to the spinal cord or to the superior colliculus, while in only one-third of the layer 5 neurons projecting to the contralateral cortex. Er81 was also detected in layer 5 neurons in a P2 Japanese macaque monkey but not in adult monkey cortices. These findings suggest that a neuron class defined by a molecular criterion does not necessarily segregate with that defined by an anatomical criterion, that ER81 is involved in cell differentiation of a subset of layer 5 projection neurons and that this mechanism is conserved among rodents and primates.

Action potential propagation in dendrites of rat mitral cells in vivo.

Odors evoke beta-gamma frequency field potential oscillations in the olfactory systems of awake and anesthetized vertebrates. In the rat olfactory bulb, these oscillations reflect the synchronous discharges of mitral cells that result from both their intrinsic membrane properties and their dendrodendritic interactions with local inhibitory interneurons. Activation of dendrodendritic synapses is purportedly involved in odor memory and odor contrast enhancement. Here we investigate in vivo to what extent action potentials propagate to remote dendrodendritic sites in the entire dendritic tree and if this propagation is changed during discharges at 40 Hz. By combining intracellular recording and two-photon microscopy imaging of intracellular calcium ([Ca2+]i), we show that in remote branches of the apical tuft and basal dendrites, transient Ca2+ changes are triggered by single sodium action potentials. Neither the amplitude of these Ca2+ transients nor that of action potentials obtained from intradendritic recordings showed a significant attenuation as a function of the distance from the soma. Calcium channel density seemed homogeneous; however, propagating action potentials occasionally failed to trigger a Ca2+ transient at a site closer to the soma whereas it did farther. This suggests that measurements of calcium transients underestimate the occurrence of sodium action potentials. During 40 Hz bursts of action potentials, [Ca2+]i increases with the number of action potentials in all dendritic compartments. These results suggest that the presence of release sites in dendrites is accompanied by an "axonal-like behavior" of the entire dendritic tree of mitral cells, including their most distal dendritic branches.

Kainate receptors regulate unitary IPSCs elicited in pyramidal cells by fast-spiking interneurons in the neocortex.

Unitary IPSCs elicited by fast-spiking (FS) interneurons in layer V pyramidal cells of the neocortex were studied by means of dual whole-cell recordings in acute slices. FS to pyramidal cell unitary IPSCs were depressed by (RS)-S-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) (ATPA), a kainate (KA) receptor agonist, and by the endogenous agonist l-glutamate in the presence of AMPA, NMDA, mGluR, and GABA(B) receptor antagonists. This effect was accompanied by an increase in failure rate of synaptic transmission, in the coefficient of variation, and in the paired pulse ratio, indicating a presynaptic origin of the IPSC depression. Pairing the activation of the presynaptic neuron with a depolarization of the postsynaptic cell mimicked the decrease of unitary IPSCs, and this effect persisted when postsynaptic sodium action potentials were blocked with the local anesthetic QX314. The effects of ATPA, glutamate, and of the pairing protocol were almost totally blocked by CNQX. These data suggest that KA receptors located on presynaptic FS cell terminals decrease the release of GABA and can be activated by glutamate released from the somatodendritic compartment of the postsynaptic pyramidal cells.

Climbing Fibre Responses in Olivo-cerebellar Slice Cultures. I. Microelectrode Recordings from Purkinje Cells.

Cerebellar slices prepared from newborn rats were co-cultured with slices derived from the inferior olive of 4-day-old rats. After several weeks in vitro olivary fibres projecting into the cerebellar tissue could be assessed by anterograde labelling with the fluorescent dye 1,1-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanine perchlorate (Dil). Following electrical field stimulation of the olivary tissue, all-or-nothing complex spikes were generated in Purkinje cells, which closely resembled climbing fibre responses as seen in situ. These responses were completely and reversibly abolished by 6-cyano-7-nitroquinoxaline-2-3-dione (CNQX, 5 microM), an antagonist of non-N-methyl-d-aspartate excitatory amino acid receptors. Wash in of smaller concentrations of CNQX (0.5 - 2 microM) resulted in a graded dose-dependent depression of the climbing fibre-induced postsynaptic potentials and in a consecutive failure of distinct active components of the complex spikes. With climbing fibre synaptic transmission blocked by CNQX, complex spike-like potentials could, however, still be evoked by intrasomatic injection of depolarizing current pulses. Increasing the concentration of Mg2+ in the bathing solution from 0.5 to up to 8 mM depressed regenerative complex-spike components. Olivary stimulation elicited only monophasic postsynaptic potentials in Purkinje cells under these conditions. These observations indicate that voltage-gated conductances which are substantially involved in the generation of the complex spike, are gated by the climbing fibre synaptic depolarization rather than directly by the climbing fibre transmitter.

Afferent connections of the medial frontal cortex of the rat. II. Cortical and subcortical afferents.

In order to compare the frontal cortex of rat and macaque monkey, cortical and subcortical afferents to subdivisions of the medial frontal cortex (MFC) in the rat were analyzed with fluorescent retrograde tracers. In addition to afferent inputs common to the whole MFC, each subdivision of the MFC has a specific pattern of afferent connections. The dorsally situated precentral medial area (PrCm) was the only area to receive inputs from the somatosensory cortex. The specific pattern of afferents common to the ventrally situated prelimbic (PL) and infralimbic (IL) areas included projections from the agranular insular cortex, the entorhinal and piriform cortices, the CA1-CA2 fields of the hippocampus, the subiculum, the endopiriform nucleus, the amygdalopiriform transition, the amygdalohippocampal area, the lateral tegmentum, and the parabrachial nucleus. In all these structures, the number of retrogradely labeled cells was larger when the injection site was located in area IL. The dorsal part of the anterior cingulate area (ACd) seemed to be connectionally intermediate between the adjacent areas PrCm and PL; it receives neither the somatosensory inputs characteristic of area PrCm nor the afferents characteristic of areas PL and IL, with the exception of the afferents from the caudal part of the retrosplenial cortex. A comparison of the pattern of afferent and efferent connections of the rat MFC with the pattern of macaque prefrontal cortex suggests that PrCm and ACd areas share some properties with the macaque premotor cortex, whereas PL and IL areas may have characteristics in common with the cingulate or with medial areas 24, 25, and 32 and with orbital areas 12, 13, and 14 of macaques.

Evidence for two types of non-NMDA receptors in rat cerebellar Purkinje cells maintained in slice cultures.

Pharmacological properties of non-NMDA receptors were investigated in Purkinje cells grown in rat cerebellar slice cultures and recorded in the whole-cell configuration of the patch-clamp technique. Dose-response curves for AMPA and domoate suggest that AMPA, in the concentration range tested, activated only AMPA receptors whereas, domoate activated two types of receptors, probably AMPA and kainate receptors, with EC50 values of 8 and 0.5 microM, respectively. The Scatchard analysis of the dose-response relationship for domoate also suggest that both kainate and AMPA receptors were activated by domoate with approximate affinities of 5 and 0.07 microM-1, respectively. The non-competitive non-NMDA receptors antagonist, GYKI 52466, reduced the amplitude of both AMPA- and domoate-activated currents, with a greater potency in reducing currents evoked by AMPA (IC50 = 10 microM) than those induced by domoate (IC50 = 105 microM). These results suggest that, in addition to AMPA receptors, Purkinje cells express kainate receptors and that these two types of non-NMDA receptors can be distinguished from each other on the basis of several pharmacological properties, including affinity for AMPA, domoate and GYKI 52466.

Cellular locus of the nitric oxide-synthase involved in cerebellar long-term depression induced by high external potassium concentration.

The cellular location of the NO-synthase involved in long-term depression (LTD) of parallel fiber (PF)-mediated EPSCs induced by raising the external potassium (K) concentration has been investigated by using both whole-cell patch-clamp recordings (WCR) of Purkinje cells (PCs) in thin slices in vitro, and reverse transcription followed by polymerase chain reaction (PCR) applied to mRNAs harvested from these single PCs during WCR. In all tested cells in the control group, a large LTD of PF-mediated EPSCs was induced by perfusing the slices for 3 min with a high (30 mM) K perfusing medium. In a second group of cells for which the protein kinase C (PKC) inhibitor peptide 19-36 was added to the intrapipette solution at a concentration of 10 microM, the LTD following complete wash out of the high K solution was significantly less prominent than in the control group. Very similar results were also obtained when 30 microM NG-methyl-L-arginine (L-NMMA) was added to the perfusing medium. In contrast, when both the PKC inhibitor peptide 19-36 and L-NMMA were added to the intrapipette solution at a concentration of 10 and 30 microM respectively, no LTD was revealed following wash out of the high K solution. Finally, the PCR amplification of mRNAs harvested from these single PCs during WCR, as well as from granule cells from the same slices, confirms that mRNAs encoding the NO-synthase are expressed by granule cells, whereas they are not detected in PCs.

Electrophysiological properties of neurons recorded intracellularly in slices of the pigeon optic tectum.

The electrical properties of pigeon's optic tectum neurons located in the non-retinorecipient region of layer II have been studied in vitro slice preparations by using intracellular recordings. As judged from the somatodendritic characteristics of cells intracellularly labeled with horseradish peroxidase recordings were obtained from pyramidal neurons, the main morphological type, as well as from ganglion cells. When stimulated with depolarizing current pulses of 300-500 ms duration, three distinct modes of firing were observed. Most neurons (Type I) responded with a continuous firing of fast action potentials whose frequency rate increased regularly when current strength was raised. Another group of cells (Type II) also exhibited sustained firing. However, in Type II cells, grouped discharges formed by 2-6 fast action potentials per group fired in rapid succession were elicited within a certain range of current intensity. Finally, another group of cells (Type III) responded at all intensities tested by a short train of fast action potentials only at the onset of the current step. At current strength close to threshold the spike undershoot of type I neurons was followed by a slow hyperpolarizing afterpotential while the spike undershoot of Type II cells was followed by a hump-like depolarization and a slow hyperpolarizing afterpotential. In Type II cells, we have also observed a pronounced increase of the hyperpolarizing afterpotential after a grouped discharge. Type III cells were characterized by a small amplitude and short duration hyperpolarizing afterpotential, barely visible in most of them. In Type I and II cells the slow hyperpolarizing afterpotential was blocked by replacing Ca2+ with Mg2+ or Cd2+ in the saline. These results support the idea that in these two types of neurons the slow hyperpolarizing afterpotential is primarily caused by a Ca2+-dependent K+ conductance. Furthermore, blocking the slow hyperpolarizing afterpotential provoked a pronounced increase of the firing frequency of Type I cells. In Type II cells blockade of the slow hyperpolarizing afterpotential had a greater effect on firing behavior: i.e. when Ca2+ was replaced with Mg2+ or Cd2+, Type II neurons exhibited repetitively fired action potentials at high frequency but were incapable of discharging repetitive grouped discharges. These observations indicate that the Ca2+-dependent K+ conductance involved in the generation of the slow hyperpolarizing afterpotential is the main modulator of the firing behavior of both types of cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Excitatory synaptic potentials in neurons of the deep nuclei in olivo-cerebellar slice cultures.

Excitatory postsynaptic potentials evoked in neurons of the deep cerebellar nuclei, either by electrical stimulation within the nuclei in cerebellar slice cultures or by electrical stimulation of olivary explants in olivo-cerebellar co-cultures, were investigated in the rat by means of intracellular recordings. In neurons of the deep cerebellar nuclei, stimulation of the nuclear tissue, as well as stimulation of the olivary tissue, induced a fast rising excitatory postsynaptic potential, followed by an inhibitory postsynaptic potential and a long-lasting excitation. The fast rising excitatory postsynaptic potential and the following inhibitory postsynaptic potential were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione. The remaining depolarization was abolished by D-(-)-2-amino-5-phosphonovalerate, suggesting that this potential was mediated by N-methyl-D-aspartate receptors. With only D-(-)-2-amino-5-phosphonovalerate added to the bath, the slow excitation was depressed, whereas the fast excitatory and inhibitory postsynaptic potentials were not affected. In the presence of bicuculline, the 6-cyano-7-nitroquinoxaline-2,3-dione- and the D-(-)-2-amino-5-phosphonovalerate-sensitive excitatory postsynaptic potentials elicited by stimulation of the olivary tissue had the same latency, and were both graded with stimulation strength. The time-to-peak and the duration of the D-(-)-2-amino-5-phosphonovalerate-sensitive excitatory postsynaptic potentials were considerably longer than those of the 6-cyano-7-nitroquinoxaline-2,3-dione-sensitive excitatory postsynaptic potentials.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional and molecular analysis of glutamate-gated channels by patch-clamp and RT-PCR at the single cell level.

In the central nervous system (CNS) rapid excitatory neurotransmission is mainly mediated by ligand gated, cationic channels activated by glutamate. Three main subtypes of glutamate-gated channels have been characterized by pharmacological studies. They have been named according to their preferred agonist, N-methyl-D-aspartate (NMDA), high affinity kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). Furthermore, a large diversity within each class of glutamate-gated channels has been revealed by the molecular cloning of multiple subunits and their spliced and edited variants (for review see Wisden and Seeburg, 1993). These subunits can potentially form different oligomeric complexes with diverging properties. A crucial question is therefore to determine the actual subunit composition of naturally occurring glutamate receptors. We have combined patch-clamp recording, reverse transcription (RT) and PCR to correlate, at the single cell level, the pattern of subunits expression with the functional properties of native glutamate receptors. We describe here results obtained on the AMPA receptors of hippocampal neurones and on the NMDA receptors of cerebellar granule cells which show that the subunit composition of these two types of receptors explains some of their functional properties. Furthermore, our data also indicate that the expression of NMDA receptor subunits during the postnatal development of cerebellar granule cells is regulated by an activity-dependent mechanism.

Single cell RT-PCR proceeds without the risk of genomic DNA amplification.

We have previously described a method for detection of mRNAs expressed in single cells after patch-clamp recordings. The method, termed single cell RT-PCR, involves aspiration of the cell content, a reverse transcription (RT) step, and a polymerase chain reaction (PCR) using specific primers. Since the nucleus is frequently harvested together with the cytosol, genomic DNA may generate false positive results. Thus, we demonstrated that dilutions containing a few copies of plasmid could be detected by PCR in a range which, according to the Poisson law, suggests that the PCR method can amplify from the two genomic alleles. We performed single cell RT-PCR of intronless GluR2 or GluR5 fragments by comparing cerebellar cell types where these mRNAs are known to be present or absent. For each cell the nucleus was harvested together with the cytosol. Following RT-PCR with GluR5 primers, all Purkinje cells (n = 6) yielded the expected PCR product, whereas it was not generated from any of the granule cells (n = 5). In corresponding experiments with GluR2 primers, we obtained the GluR2 product from all Purkinje cells (n = 5), but not from any of the glial cells (n = 5). These results are in agreement with the known cellular expression of GluR2 and GluR5 mRNAs. We conclude that the single cell RT-PCR method does not amplify the genomic DNA when the nucleus is aspirated together with the cytosol. We suggest that genomic DNA amplification is avoided, because the genomic alleles are not exposed during the procedure.

Diversity of glutamate receptors in neocortical neurons: implications for synaptic plasticity.

The biochemical and functional characteristics of the AMPA subtype of the glutamate receptors expressed by pyramidal and non-pyramidal neurons of the neocortex have been studied in acute slices by means of single-cell RT-PCR and fast applications of glutamate on outside-out patches. Our results suggest that the predominant expression of the flop splice variants of the GluR1-4 AMPA subunits contributes to the faster desensitization of these receptors in non-pyramidal neurons compared to pyramidal cells where flip variants of GluR1-4 are dominant. Alternative splicing of AMPA receptors may therefore play an important role in regulating synaptic function in a cell-type specific manner.

Excitation of rat prefrontal cortical neurons by dopamine: an in vitro electrophysiological study.

The effects of dopamine (DA) on prefrontal pyramidal neurons were studied in vitro on rat cerebral cortex slices using intracellular recordings. Pyramidal neurons were first identified by Lucifer yellow and some of their basic bioelectrical properties were analysed. At resting potential, white matter stimulation mainly evoked depolarizing inhibitory postsynaptic potentials (IPSPs) which reversed between -60 and -50 mV and were almost totally abolished by bicuculline. Furthermore, pyramidal cells often exhibited spontaneous depolarizing IPSPs abolished by bicuculline. Under tetrodotoxin (TTX) this synaptic noise was partly blocked suggesting that it was due both to the spontaneous firing of presynaptic gamma-aminobutyric acid (GABA)ergic neurons and to a spontaneous quantal release from these afferent fibers. In pyramidal cells, DA enhanced the number of spikes evoked by depolarizing current pulses, and the input resistance was increased by 10-20%. DA also clearly increased the inhibitory synaptic noise. This effect was blocked by fluphenazine. In contrast, evoked IPSPs were not consistently affected by DA. Taken altogether, these results suggest, that in the prefrontal cortex, dopamine has a mild excitatory effect on both pyramidal cells and GABAergic interneurons impinging on them.

Synaptic transmission of excitation from the retina to cells in the pigeon's optic tectum.

Intracellular recordings were used to study the synaptic excitation of optic tectum neurons in the pigeon. Electrical stimulation of both contralateral optic nerve and ipsilateral optic tract evoked in the tectal neurons EPSPs which in most cases were followed by an IPSP. An extrapolation procedure based on response latency was used to reveal that the EPSPs were mediated by way of mono-, di- and polysynaptic connections with the retinal endings. The laminar location of the recorded cells was estimated according to the field potential and the recording depth with the exception of one cell which was intracellularly stained with HRP. Monosynaptic EPSPs were recorded from cells in the retinorecipient region (sublayers IIa-f) as well as in the non-retinorecipient region (sublayers IIg-j and layer III) of the tectum, while di- and polysynaptic EPSPs were never recorded from the input layers. Tectofugal projections arise largely from layer III neurons. Thus, these results indicate that retinal excitation is transmitted to the output tectal cells by way of mono-, di- or polysynaptic pathways. The conduction velocities of most retinal fibers mediating the EPSP ranged from 4 to 22 m/s (average 12 m/s). However, in a number of retinal fibers the conduction velocities were in a faster range, up to 36 m/s.

Synaptic organization of inhibitory circuits in the pigeon's optic tectum.

The synaptic organization of inhibitory systems in the pigeon's optic tectum was studied with intracellular recording techniques. An extrapolation procedure based on response latency was used to determine the synaptic delay of the postsynaptic potentials (PSPs) and the velocity of conduction of the associated retinal axons. Tectal cells receive mostly disynaptic, trisynaptic or polysynaptic inhibition from retinal ganglion cells. However, evidence was found which together with previous studies raised the possibility of the existence of a direct inhibitory retino-tectal path. Our present results also suggest that inhibition is transmitted from the retina to the tectal cells by way of both, feedforward and feedback pathways.

Afferent connections of the medial frontal cortex of the rat. A study using retrograde transport of fluorescent dyes. I. Thalamic afferents.

The present study of the medial frontal cortex of the rat was undertaken with two objectives. First, to compare the pattern of afferent thalamic neurons for each of the three subdivisions of the medial frontal cortex: the medial precentral (PrCm), dorsal anterior cingulate (ACd) and prelimbic (PL) areas. Second, to provide a firmer basis for anatomical comparisons of cortical regions between rat and monkey. Focal injections of retrogradely transported fluorescent tracers, true blue and diamidino yellow, were placed in different regions of the medial frontal cortex, to reveal the organization of afferent thalamic neurons. The PL area can be readily distinguished from PrCm and ACd areas because it receives afferents from a large number of neurons from both the medial and the lateral parts of the mediodorsal nucleus (MD) whereas only a few neurons, from the lateral MD exclusively, project to PrCm and ACd areas. Moreover, the paratenial and the paraventricular thalamic nuclei project only to the PL area, and the central medial nucleus projects mostly to the PL area. The ventrolateral nucleus projects only to the dorsal part of the medial frontal cortex. The rhomboid, reuniens, ventromedial, intralaminar, posterior and laterodorsal nuclei project to the whole medial frontal cortex. On the basis of these findings, the pattern of thalamic afferents to the PL area was compared to the pattern of thalamic afferents to cingulate and retrosplenial cortices in rat. The conclusion is that the PL area has a pattern of thalamic afferents which is different not only from those of PrCm and ACd areas but also from those of cingulate and retrosplenial cortices. On the basis of its rich innervation from the mediodorsal nucleus, the prelimbic area could very likely be a part of the prefrontal cortex of rat.

Potent and multiple regulatory actions of microglial glucocorticoid receptors during CNS inflammation.

In CNS, glucocorticoids (GCs) activate both GC receptor (GR) and mineralocorticoid receptor (MR), whereas GR is widely expressed, the expression of MR is restricted. However, both are present in the microglia, the resident macrophages of the brain and their activation can lead to pro- or anti-inflammatory effects. We have therefore addressed the specific functions of GR in microglia. In mice lacking GR in macrophages/microglia and in the absence of modifications in MR expression, intraparenchymal injection of lipopolysaccharide (LPS) activating Toll-like receptor 4 signaling pathway resulted in exacerbated cellular lesion, neuronal and axonal damage. Global inhibition of GR by RU486 pre-treatment revealed that microglial GR is the principal mediator preventing neuronal degeneration triggered by lipopolysaccharide (LPS) and contributes with GRs of other cell types to the protection of non-neuronal cells. In vivo and in vitro data show GR functions in microglial differentiation, proliferation and motility. Interestingly, microglial GR also abolishes the LPS-induced delayed outward rectifier currents by downregulating Kv1.3 expression known to control microglia proliferation and oxygen radical production. Analysis of GR transcriptional function revealed its powerful negative control of pro-inflammatory effectors as well as upstream inflammatory activators. Finally, we analyzed the role of GR in chronic unpredictable mild stress and aging, both known to prime or sensitize microglia in vivo. We found that microglial GR suppresses rather than mediates the deleterious effects of stress or aging on neuronal survival. Overall, the results show that microglial GR acts on several key processes limiting pro-inflammatory actions of activated microglia.