Dirk Feldmeyer

Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex.

Neuroscience produces a vast amount of data from an enormous diversity of neurons. A neuronal classification system is essential to organize such data and the knowledge that is derived from them. Classification depends on the unequivocal identification of the features that distinguish one type of neuron from another. The problems inherent in this are particularly acute when studying cortical interneurons. To tackle this, we convened a representative group of researchers to agree on a set of terms to describe the anatomical, physiological and molecular features of GABAergic interneurons of the cerebral cortex. The resulting terminology might provide a stepping stone towards a future classification of these complex and heterogeneous cells. Consistent adoption will be important for the success of such an initiative, and we also encourage the active involvement of the broader scientific community in the dynamic evolution of this project.

Point mutation in an AMPA receptor gene rescues lethality in mice deficient in the RNA-editing enzyme ADAR2

RNA editing by site-selective deamination of adenosine to inosine alters codons and splicing in nuclear transcripts, and therefore protein function. ADAR2 (refs 7, 8) is a candidate mammalian editing enzyme that is widely expressed in brain and other tissues, but its RNA substrates are unknown. Here we have studied ADAR2-mediated RNA editing by generating mice that are homozygous for a targeted functional null allele. Editing in ADAR2-/- mice was substantially reduced at most of 25 positions in diverse transcripts; the mutant mice became prone to seizures and died young. The impaired phenotype appeared to result entirely from a single underedited position, as it reverted to normal when both alleles for the underedited transcript were substituted with alleles encoding the edited version exonically. The critical position specifies an ion channel determinant, the Q/R site, in AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptor GluR-B pre-messenger RNA. We conclude that this transcript is the physiologically most important substrate of ADAR2.

Early-onset epilepsy and postnatal lethality associated with an editing-deficient GluR-B allele in mice.

The arginine residue at position 586 of the GluR-B subunit renders heteromeric α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-sensitive glutamate receptor channels impermeable to calcium. The codon for this arginine is introduced at the precursor messenger RNA (pre-mRNA) stage by site-selective adenosine editing of a glutamine codon. Heterozygous mice engineered by gene targeting to harbor an editing-incompetent GluR-B allele synthesized unedited GluR-B subunits and, in principal neurons and interneurons, expressed AMPA receptors with increased calcium permeability. These mice developed seizures and died by 3 weeks of age, showing that GluR-B pre-mRNA editing is essential for brain function.

New insights into the classification and nomenclature of cortical GABAergic interneurons

A systematic classification and accepted nomenclature of neuron types is much needed but is currently lacking. This article describes a possible taxonomical solution for classifying GABAergic interneurons of the cerebral cortex based on a novel, web-based interactive system that allows experts to classify neurons with pre-determined criteria. Using Bayesian analysis and clustering algorithms on the resulting data, we investigated the suitability of several anatomical terms and neuron names for cortical GABAergic interneurons. Moreover, we show that supervised classification models could automatically categorize interneurons in agreement with experts assignments. These results demonstrate a practical and objective approach to the naming, characterization and classification of neurons based on community consensus.

Coincidence detection and changes of synaptic efficacy in spiny stellate neurons in rat barrel cortex.

Paired whole-cell voltage recordings were made from synaptically connected spiny stellate neurons in layer 4 of the barrel field in young (P14) rat somatosensory cortex. When postsynaptic action potentials (APs) followed each of 5 presynaptic APs in a 10- or 20-Hz train by less than 25 ms, subsequent unitary EPSP amplitudes were persistently reduced. Induction of long-term depression (LTD) depended on activation of group II metabotropic glutamate receptors, but not on NMDA or AMPA receptors. Reducing postsynaptic increases in intracellular calcium ([Ca2+]i) by intracellular loading with a fast- (BAPTA) or a slow- (EGTA) acting Ca2+ buffer blocked synaptic depression. Analysis of EPSP failures suggested mediation of LTD by a reduction in release probability. We propose a mechanism by which coincident activity results in long-lasting reduction of synaptic efficacy between synaptically connected neurons.

Synaptic connections between layer 4 spiny neurone- layer 2/3 pyramidal cell pairs in juvenile rat barrel cortex: physiology and anatomy of interlaminar signalling within a cortical column

Whole‐cell voltage recordings were obtained from 64 synaptically coupled excitatory layer 4 (L4) spiny neurones and L2/3 pyramidal cells in acute slices of the somatosensory cortex (‘barrel’ cortex) of 17‐ to 23‐days‐old rats. Single action potentials (APs) in the L4 spiny neurone evoked single unitary EPSPs in the L2/3 pyramidal cell with a peak amplitude of 0.7 ± 0.6 mV. The average latency was 2.1 ± 0.6 ms, the rise time was 0.8 ± 0.3 ms and the decay time constant was 12.7 ± 3.5 ms. The percentage of failures of an AP in a L4 spiny neurone to evoke a unitary EPSP in the L2/3 pyramidal cell was 4.9 ± 8.8 % and the coefficient of variation (c.v.) of the unitary EPSP amplitude was 0.27 ± 0.13. Both c.v. and percentage of failures decreased with increased average EPSP amplitude. Postsynaptic glutamate receptors (GluRs) in L2/3 pyramidal cells were of the N‐methyl‐d‐aspartate (NMDA) receptor (NMDAR) and the non‐NMDAR type. At −60 mV in the presence of extracellular Mg2+ (1 mm), 29 ± 15 % of the EPSP voltage‐time integral was blocked by NMDAR antagonists. In 0 Mg2+, the NMDAR/AMPAR ratio of the EPSC was 0.50 ± 0.29, about half the value obtained for L4 spiny neurone connections. Burst stimulation of L4 spiny neurones showed that EPSPs in L2/3 pyramidal cells depressed over a wide range of frequencies (1–100 s−1). However, at higher frequencies (30 s−1) EPSP summation overcame synaptic depression so that the summed EPSP was larger than the first EPSP amplitude in the train. The number of putative synaptic contacts established by the axonal collaterals of the L4 projection neurone with the target neurone in layer 2/3 varied between 4 and 5, with an average of 4.5 ± 0.5 (n= 13 pairs). Synapses were established on basal dendrites of the pyramidal cell. Their mean geometric distance from the pyramidal cell soma was 67 ± 34 μm (range, 16–196 μm). The results suggest that each connected L4 spiny neurone produces a weak but reliable EPSP in the pyramidal cell. Therefore transmission of signals to layer 2/3 is likely to have a high threshold requiring simultaneous activation of many L4 neurons, implying that L4 spiny neurone to L2/3 pyramidal cell synapses act as a gate for the lateral spread of excitation in layer 2/3.

Reliable synaptic connections between pairs of excitatory layer 4 neurones within a single ‘barrel’ of developing rat somatosensory cortex

1 Dual whole‐cell recordings were made from pairs of synaptically coupled excitatory neurones in the ‘barrel field’ in layer (L) 4 in slices of young (postnatal day 12–15) rat somatosensory cortex. The majority of interconnected excitatory neurones were spiny stellate cells with an asymmetrical dendritic arborisation largely confined to a single barrel. The remainder were star pyramidal cells with a prominent apical dendrite terminating in L2/3 without forming a tuft. 2 Excitatory synaptic connections were examined between 131 pairs of spiny L4 neurones. Single presynaptic action potentials evoked unitary EPSPs with a peak amplitude of 1·59 ± 1·51 mV (mean ± s.d.), a latency of 0·92 ± 0·35 ms, a rise time of 1·53 ± 0·46 ms and a decay time constant of 17·8 ± 6·3 ms. 3 At 34–36 °C, the coefficient of variation (c.v.) of the unitary EPSP amplitude was 0·37 ± 0·16 and the percentage of failures to evoke an EPSP was 5·3 ± 7·8%. The c.v. and failure rate decreased with increasing amplitude of the unitary EPSP. 4 Postsynaptic glutamate receptors in spiny L4 neurones were of the AMPA and NMDA type. At −60 mV in the presence of 1 mM Mg2+, NMDA receptors contributed 39·3 ± 12·5% to the EPSP integral. In Mg2+‐free solution, the NMDA receptor/AMPA receptor ratio of the EPSC was 0·86 ± 0·64. 5 The number of putative synaptic contacts established by the projection neurone with the target neurone varied between two and five with a mean of 3·4 ± 1·0 (n= 11). Synaptic contacts were exclusively found in the barrel in which the cell pair was located and were preferentially located on secondary to quarternary dendritic branches. Their mean geometric distance from the soma was 68·8 ± 37·4 μm (range, 33·4‐168·0 μm). The number of synaptic contacts and mean EPSP amplitude showed no significant correlation. 6 The results suggest that in L4 of the barrel cortex synaptic transmission between spiny neurones is largely restricted to a single barrel. The connections are very reliable, probably due to a high release probability, and have a high efficacy because of the compact structure of the dendrites and axons of spiny neurones. Intrabarrel connections thus function to amplify and distribute the afferent thalamic activity in the vertical directions of a cortical column.

Neurological dysfunctions in mice expressing different levels of the Q/R site–unedited AMPAR subunit GluR–B

We generated mouse mutants with targeted AMPA receptor (AMPAR) GluR–B subunit alleles, functionally expressed at different levels and deficient in Q/R–site editing. All mutant lines had increased AMPAR calcium permeabilities in pyramidal neurons, and one showed elevated macroscopic conductances of these channels. The AMPAR–mediated calcium influx induced NMDA–receptor–independent long–term potentiation (LTP) in hippocampal pyramidal cell connections. Calcium–triggered neuronal death was not observed, but mutants had mild to severe neurological dysfunctions, including epilepsy and deficits in dendritic architecture. The seizure–prone phenotype correlated with an increase in the macroscopic conductance, as independently revealed by the effect of a transgene for a Q/R–site–altered GluR–B subunit. Thus, changes in GluR–B gene expression and Q/R site editing can affect critical architectural and functional aspects of excitatory principal neurons.

Efficacy and connectivity of intracolumnar pairs of layer 2/3 pyramidal cells in the barrel cortex of juvenile rats

Synaptically coupled layer 2/3 (L2/3) pyramidal neurones located above the same layer 4 barrel (‘barrel‐related’) were investigated using dual whole‐cell voltage recordings in acute slices of rat somatosensory cortex. Recordings were followed by reconstructions of biocytin‐filled neurones. The onset latency of unitary EPSPs was 1.1 ± 0.4 ms, the 20–80% rise time was 0.7 ± 0.2 ms, the average amplitude was 1.0 ± 0.7 mV and the decay time constant was 15.7 ± 4.5 ms. The coefficient of variation (c.v.) of unitary EPSP amplitudes decreased with increasing EPSP peak and was 0.33 ± 0.18. Bursts of APs in the presynaptic pyramidal cell resulted in EPSPs that, over a wide range of frequencies (5–100 Hz), displayed amplitude depression. Anatomically the barrel‐related pyramidal cells in the lower half of layer 2/3 have a long apical dendrite with a small terminal tuft, while pyramidal cells in the upper half of layer 2/3 have shorter and often more ‘irregularly’ shaped apical dendrites that branch profusely in layer 1. The number of putative excitatory synaptic contacts established by the axonal collaterals of a L2/3 pyramidal cell with a postsynaptic pyramidal cell in the same column varied between 2 and 4, with an average of 2.8 ± 0.7 (n= 8 pairs). Synaptic contacts were established predominantly on the basal dendrites at a mean geometric distance of 91 ± 47 μm from the pyramidal cell soma. L2/3‐to‐L2/3 connections formed a blob‐like innervation domain containing 2.8 mm of the presynaptic axon collaterals with a bouton density of 0.3 boutons per μm axon. Within the supragranular layers of its home column a single L2/3 pyramidal cell established about 900 boutons suggesting that 270 pyramidal cells in layer 2/3 are innervated by an individual pyramidal cell. In turn, a single pyramidal cell received synaptic inputs from 270 other L2/3 pyramidal cells. The innervation domain of L2/3‐to‐L2/3 connections superimposes almost exactly with that of L4‐to‐L2/3 connections. This suggests that synchronous feed‐forward excitation of L2/3 pyramidal cells arriving from layer 4 could be potentially amplified in layer 2/3 by feedback excitation within a column and then relayed to the neighbouring columns.

Effect of RNA editing and subunit co‐assembly single‐channel properties of recombinant kainate receptors.

1. Patch‐clamp methods have been used to examine single‐channel properties of recombinant GluR5 and GluR6 kainate‐preferring glutamate receptors which differ in a single amino acid residue as a result of RNA editing at the Q/R (glutamine/arginine) site. Subunits were expressed alone or in combination with the high‐affinity kainate receptor subunit KA ‐ 2 in transfected human embryonic kidney (HEK‐293) cells. 2. In outside‐out patches, unedited homomeric GluR6(Q) receptors exhibited directly resolved domoate‐activated single‐channel conductances of 8, 15 and 25 pS. Variance analysis of GluR6(Q) responses gave a mean conductance of 5.4 pS, while the edited isoform GluR6(R) had an unusually low channel conductance (225 fS). 3. Homomeric channels composed of GluR5(Q) subunits exhibited three conductance states of 5, 9 and 14 pS characterized by prolonged burst activations in the presence of domoate. In contrast, the GluR5(R) subunit, which has not previously been reported to form functional homomeric receptors, had an extremely low conductance (< 200 fS). 4. Heteromeric GluR6(Q)/KA‐2 kainate receptors gave single‐channel events indistinguishible from homomeric GluR6(Q) channels. Conversely, openings produced by GluR5(Q)KA‐2 and GluR5(Q) receptors differed from each other in their kinetic properties. The primary effect of co‐expression of KA‐2 with GluR5(Q) was a dramatic shortening in channel burst length. 5. Spectral and variance analyses were used to estimate mean single‐channel conductances of heteromeric edited receptor‐channels; channel conductances were 950 fS for GluR5(R)KA‐2 receptors and 700 fS for GluR6(R)/KA‐2 receptors. Both receptor types had significantly higher conductances than the respective homomeric channels, GluR5(R) and GluR6(R). 6. We conclude that Q/R site editing dramatically reduces single‐channel conductance. Furthermore, we find similarity between the kainate receptor‐channels described in sensory neurones and the recombinant GluR5(Q) homomeric channel. Characterization of recombinant single‐channel properties could therefore aid identification of the native kainate receptors.