A gradual depth-dependent change of connectivity features of supragranular pyramidal cells in rat barrel cortex

Recent experimental evidence suggests a finer genetic, structural and functional subdivision of the layers which form a cortical column. The classical layer II/III (LII/III) of rodent neocortex integrates ascending sensory information with contextual cortical information for behavioral read-out. We systematically investigated to which extent regular-spiking supragranular pyramidal neurons, located at different depths within the cortex, show different input-output connectivity patterns. Combining glutamate-uncaging with whole-cell recordings and biocytin filling, we revealed a novel cellular organization of LII/III: (i) “Lower LII/III” pyramidal cells receive a very strong excitatory input from lemniscal LIV and much fewer inputs from paralemniscal LVa. They project to all layers of the home column, including a feedback projection to LIV whereas transcolumnar projections are relatively sparse. (ii) “Upper LII/III” pyramidal cells also receive their strongest input from LIV, but in addition, a very strong and dense excitatory input from LVa. They project extensively to LII/III as well as LVa and Vb of their home and neighboring columns, (iii) “Middle LII/III” pyramidal cell show an intermediate connectivity phenotype that stands in many ways in-between the features described for lower versus upper LII/III. “Lower LII/III” intracolumnarly segregates and transcolumnarly integrates lemniscal information whereas “upper LII/III” seems to integrate lemniscal with paralemniscal information. This suggests a finegrained functional subdivision of the supragranular compartment containing multiple circuits without any obvious cytoarchitectonic, other structural or functional correlate of a laminar border in rodent barrel cortex

Glutamate uncaging responses were unchanged in DCG-IV.

<p>(A) A representative experiment for a burst firing neuron is shown. Glutamate was uncaged using a brief laser pulse (indicated with an open arrow; open circle), following synaptic stimulation (closed circle). DCG-IV reduced the synaptic response, the ‘uncaged’ response, however, was unchanged in the presence of DCG-IV. (B) Summary of all experiments (uncaging responses: n = 6; synaptic responses: n = 3). In those experiments, in which extracellular-evoked synaptic and uncaging responses were recorded simultaneously (n = 3), APV (50 µM) was added to the bath solution to block NMDA receptors. Please note that no obvious difference in synaptic depression by DCG-IV was found compared to control conditions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045039#pone-0045039-g003" target="_blank">Figure 3</a>).</p

DCG-IV acts presynaptically onto burst firing neurons.

<p>(A1 and A2) EPSCs recorded in bursting neurons in response to paired-pulse stimulation before and in the presence application of DCG-IV. Representative example is shown in A1 and A2. The paired-pulse ratio is significantly increased after chemical activation of group II mGlu receptors. A summary of seven experiments is shown in (B). (C) Analysis of the squared coefficient of variation indicates a presynaptic mechanism by which DCG-IV exerts its action.</p

The group II agonist L-CCG-1 differently affects synaptic transmission in areas CA1 and SUB.

<p>(A, B) Field potential recordings were performed and the group II agonist L-CCG 1 was bath-applied in various concentrations. Field potentials were depressed in a concentration-dependent manner (SUB: n = 4; CA1: n = 5). (C) DCG-IV (1 µM) suppressed fEPSP in the SUB to a similar extent as observed with 10 µM L-CCG-1 (SUB: n = 5; all in presence of the NMDA receptor antagonist APV, 50 µM).</p

Frequency facilitation is not limited by activation of mGluRs.

<p>(A) Bursting and regular firing cells exhibited frequency facilitation. Changes in stimulation frequency from 0.05 Hz to 1 Hz (20 stimuli) resulted in a reversible facilitation of EPSCs. (B1) A typical experiment illustrating that the group II mGluR antagonist LY341495 (10 µM) did not have an effect on frequency facilitation in bursting cells. The results for nine such experiments (ACSF and LY341495) are summarized in (B2).</p

Summary of the applied stimulation protocols to activate mGluRs.

<p>PPD: paired-pulse depression.</p>*<p>paired t-test, one-tailed.</p

DCG-IV increases failure ratio during minimal stimulation.

<p>(A) Representative experiment for a burst firing neuron specific minimal stimulation. Overlays of 10 individual sweeps each are shown for control (ACSF) and 5 min after wash-in of DCG-IV (1 µM). (B) Minimal stimulation strength was achieved by stepwise increase of extracellular current injection by 0.5 nA. After establishing a stable EPSC/failure ratio for at least 10 min DCG-IV was applied. (C) Summary of all experiments showed a significant increase in the percentage of failure in the presence of DCG-IV (n = 6). Open circles represent experiments in the presence of the NMDA receptor antagonist APV (50 µM; for details see text).</p

L-CCG-1 differentially depresses glutamatergic transmission in three different brain regions of the hippocampal formation.

<p>(A)Summary bar diagram of the effects of different concentrations of L-CCG-1 in areas CA1, CA3 (MF) and SUB (SUB: n = 4; CA1: n = 5; MF-CA3: n = 6). (B) Data were fitted to a sigmoidal function and a dose-response curve is given for the SUB, MF-CA3 and CA1. EC50 values were estimated to 7 µM, 3 µM and 28 µM for the SUB, MF-CA3 and CA1, respectively. Error bars are not shown for clarity.</p

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