Morphology and physiology of cortical neurons in layer I

Abstract

The electrophysiological and morphological properties of layer I neurons were studied in visual cortex slices from 7- to 19-d-old rats using whole-cell recording and biocytin labeling. A heterogeneous population of small, nonpyramidal neurons was found. Approximately one third of the cells we recorded were neurogliaform cells; another third were multipolar neurons with axons descending out of layer I. The remaining cells were heterogeneous and were not classified. In slices from 7- to 10-d-old animals only, we identified Cajal-Retzius cells. Neurogliaform neurons had a very dense local axonal field, which was largely contained within layer I. Cells with descending axons had a relatively sparse local axonal arbor and projected at least to layer II and sometimes deeper. Spiking in neurogliaform neurons was followed by an afterdepolarizing potential, whereas spiking in cells with descending axons was followed by a slow after-hyperpolarizing potential (AHP). In addition, neurogliaform cells exhibited less spike broadening and a larger fast AHP after single spikes than did cells with descending axons. Generally, cells in layer I received synaptic inputs characterized as either GABA- or glutamate-mediated, suggesting the presence of excitatory and inhibitory inputs. With their output largely limited to layer I, neurogliaform cells could synapse with other layer I neurons, the most distal dendritic branches of pyramidal cells, or the dendrites of layer II/III interneurons, which invade layer I. Cells with descending axons could contact a wide variety of cortical cells throughout their vertical projection.

Fig. 1.

Photomicrographs of layer I biocytin-stained neurons. A, Low-power (A1) and high-power (A2) micrograph of a neurogliaform cell located in the central portion of layer I. The axonal and dendritic arbors are contained within layer I. Note the extensive axonal arborization of this cell type in A2. The arrow points to a varicosity along the axon. B, Low-power (B1) and high-power (B2) micrograph of cell with descending axon (arrowhead) located near the border of layers I and II. Vertical bar delimits layer I. Both dendrites and axons are present in layers I and II. This neuron is drawn in Figure 5. C, Low-power (C1) and high-power (C2) micrograph of a CR cell found close to the pial surface of layer I (9-d-old rat). Both the axon (arrowhead) and the dendrite run parallel to the pial surface. This neuron is drawn in Figure 2. Scale bar inB1 applies to lower-power micrographs; Scale bar inB2 applies to high-power micrographs.

Fig. 2.

The projection tube drawing of a biocytin-stained CR cell (same cell as in Fig. 1) from a 9-d-old animal. Thearrowhead indicates the possible site of axon origin. Note the location of the cell in the upper portion of layer I (indicated by the bracket at right of the drawing). Note also the thick single dendrite with small finger-like appendages. Inset, A response to an injection of depolarizing current. Note slow action-potential time course and progressive increase in spike width. Resting potential was −50.5 mV.

Fig. 3.

A projection tube drawing of a biocytin-stained neurogliaform cell in layer I. Borders of layer I are indicated atleft of the drawing. The extensive axonal arbor extends close to the border of layer I but does not enter layer II. The central location of the somata is typical of these cells. Inset, A single action potential initiated in this cell by current injection (300 pA, 10 msec) is followed by an afterdepolarization (ADP). Resting potential: −56 mV.

Fig. 4.

Action-potential characteristics of a neurogliaform cell. All records are from the same cell.A, Steps (900 msec) of current injection (−30, −10, +10, +50, and +70 pA) from resting potential (−65 mV). Note that the response to hyperpolarizing current injection (−30 pA) suggests the presence of an inward rectification, as indicated by the sag (open circle). At threshold, a single spike was evoked with a large fAHP. Note the PSPs (arrows) indicated by the fast rise and slow decay with a peak of ∼1 mV. B, A depolarizing current injection above threshold evoked a single spike followed by a quiescent period preceding further spikes. Note the prolonged depolarization after current cessation. C, Suprathreshold current injection evokes a train of spikes with little frequency adaptation. Note the ADP after current injection (filled circle). D, ADP (filled circle) initiated by short-duration current injection. Resting potential: −65 mV.

Fig. 8.

AHP and ADP. A, AHP after a single action potential in a cell with a descending axon. Note the PSPs at the end of the voltage trace. Resting potential: −65 mV; current injection: +200 pA, 10 msec. B, ADP in a neurogliaform cell induced by a short train of spikes. Note the small PSP occurring at the decay phase of ADP. Resting potential: −62 mV; current injection: +200 pA, 50 msec. Current injection: C andC1, AHP and I-AHP in a single cell. I-AHP is induced by a brief (10 msec) depolarizing pulse of membrane potential to +20 mV from a holding potential of −60 mV. Note that the I-AHP (C1) has a time course similar to the AHP (C). D and D1, ADP and I-ADP in a single neurogliaform cell. Under current clamp (D), depolarizing current (+300 pA, 10 msec) induced a single spike from a resting potential of −61 mV. I-ADP was induced by a 2 msec voltage step to 20 mV from a holding potential of −65 mV (D1). Note the slow rising phase of the response under voltage clamp.

Fig. 5.

Cell with a descending axon. Soma is located close to layer I–II border (indicated at left of drawing). The axon, whose origin is indicated by arrowhead, descends well into lower layers but gives off a local arbor.Inset, A train of spikes is followed by an afterhyperpolarization (AHP). Resting potential: −59 mV; current injection: 150 pA, 200 msec.

Fig. 6.

Action potential characteristics of cells with descending axon. A, Depolarization induced a train of action potentials illustrating spike frequency adaptation. Note the spike broadening during the train. After the train there is an AHP (indicated by filled circle). Resting potential: −59 mV; current injection: +100 pA, 700 msec. Inset, Superimposition of the first (arrowhead) and the twelfth (arrow) spikes of the train. Note the broadening of the action potential. Spike width at half amplitude was 1.7 msec (first) and 3.4 msec (twelfth). The spikes were aligned at the threshold. Scale is 2 msec, 10 mV. B1, B2, A different cell exhibiting an AHP after a single spike (B1) or a short train of spikes (B2). Resting potential: −62 mV; current injection: 220 pA, 10 msec (B1) and 120 pA, 100 msec (B2). The AHPs are indicated by filled circles.

Fig. 7.

Action potential parameters in neurogliaform cells and cells with descending axons. The fAHP plotted against the half-width of the second spike in a train of spikes. ThefAHP of neurogliaform cells (filled symbols) is larger in amplitude than that of cells with descending axons (open symbols). The second spike half-width of the neurogliaform cell is shorter in duration compared with that of cells with descending axons. Inset, Superimposition of the first and second spikes in a train of action potentials. Inset left, Neurogliaform cell; inset right, cell with descending axon.

Fig. 9.

Conductance mechanism of the I-ADP.A, The membrane potential was −65 mV. A brief (2 msec) voltage pulse to −20 mV initiated the I-ADP. At the peak of the I-ADP, the membrane potential was stepped to different voltages: −75, −65, −60, −55, −50, and −45 as shown diagrammatically beneath the current traces. Note that the tail current after a step to −75 mV was larger than that observed at more depolarized voltages, indicating that the reversal potential was more depolarized than −75 mV.B, The current–voltage relationship of the tail currents was obtained by subtracting the current (averaged over 5 msec) measured at the time indicated by the right open arrowfrom the current just after the voltage step at the time indicated by the left open arrow. Note that the tail current reversed polarity at approximately −50 mV. Same cell as that shown in Figure8D.

Fig. 10.

mEPSCs and mIPSCs. A1–A3, mEPSCs were detected using a threshold amplitude of −6 pA (see Materials and Methods). TTX (0.5 μm) and picrotoxin (100 μm) were present. The membrane potential was −70 mV.A1, Average of 266 aligned mEPSCs with a rise time (20–80%) of <0.3 msec. The amplitude for the threshold detection was set at −6 pA. A2, The distribution of the mEPSC amplitudes (n = 318). A3, Distribution of decay time constants fitted to individual mEPSCs.B1–B3, mIPSCs recorded at a membrane potential of −70 mV in the presence of TTX (0.5 μm) and CNQX (10 μm). Chloride-rich internal solution was used (see Materials and Methods). B1, Average of 450 mIPSCs with a rise time <0.6 msec. The amplitude for the threshold detection was set at −8 pA. Note the different time scale and the relatively slow decay of the mIPSCs compared with that of the mEPSCs. B2, Distribution of the mIPSCs amplitudes. B3, Histogram of the decay time constants obtained by fitting individual mIPSCs with a single exponential function.