Synaptic interactions of late-spiking neocortical neurons in layer 1

Abstract

Layer 1 of the neocortex is an important zone in which synaptic integration of inputs originating from a variety of cerebral regions is thought to take place. Layer 1 does not contain pyramidal cells, and several histochemical studies have suggested that most layer 1 neurons are GABAergic. However, although layer 1 neurons could be an important source of inhibition in this layer, the synaptic action of these neurons and the identity of their postsynaptic targets are unknown. We studied the physiological properties and synaptic interactions of a class of cells within layer 1 called late-spiking (LS) cells. The dendrites and axons of layer 1 LS cells were confined primarily to layer 1. Using paired recording, we showed that LS cells formed GABAergic connections with other LS cells as well as with non-LS cells in layer 1 and with pyramidal cells in layer 2/3. We also found that layer 2/3 pyramidal neurons provide excitatory inputs to LS cells. It has been suggested previously that GABAergic neurons belonging to the same class in the cortex are electrically coupled. In agreement with that hypothesis, we found that LS cells were interconnected by electrical coupling (83%), whereas electrical coupling between LS cells and non-LS cells was infrequent (2%). Thus, we provide evidence showing that a group of GABAergic neurons within layer 1 are specifically interconnected by electrical coupling and can provide significant inhibitory inputs to neurons in layer 1 and to distal dendrites of pyramidal cells.

Fig. 1.

Electrophysiological properties of LS cells in layer 1. A, Top, DIC–infrared video microscopy image of an LS cell. Scale bar, 10 μm.Bottom, Firing pattern of the same cell in response to the injection of depolarizing current pulses. The two top traces were produced with the same magnitude of current injection. Note the delayed firing during the current injection, the single-component fast AHP (arrow), and the lack of spike frequency adaptation in the near-threshold discharge (middle trace). Resting V_m, −62 mV.B, DIC–infrared video microscopy image (top) and pattern of firing in response to current injection (bottom) of a non-LS cell. RestingV_m, −81 mV. Calibration as inA. C, Current-clamp recording from an LS cell in response to two near-threshold current injections. Note the slow depolarizing ramp commonly observed in LS cells under these conditions. An action potential was produced at the end of the pulse in one of the traces. Resting V_m, −69 mV. D, Recording from an LS cell. A single action potential induced by a brief current injection (300 pA, 5 msec) was followed by an afterdepolarization (ADP). The action potential has been truncated. RestingV_m, −72 mV.

Fig. 2.

Morphology of nonpyramidal cells in layer 1.A, Neurolucida reconstruction of three LS cells (A₁–A₃) filled with biocytin. Dendrites are illustrated with thick traces, and axons are illustrated with thin traces. Note the extensive axonal arborizations distributed around the soma and extending horizontally within layer 1. Insets, Pattern of firing of the reconstructed neurons in response to near-threshold current injections. B, Neurolucida reconstruction of a non-LS cell. The axon, whose origin is indicated by thearrowhead, extends into lower layers of the neocortex.Inset, Pattern of firing in response to a depolarizing current injection.

Fig. 3.

Synaptic connectivity between layer 1 LS neurons and layer 2/3 pyramidal (Pyr) cells. A, Paired recording between a presynaptic LS cell in layer 1 (Pre) and a postsynaptic pyramidal cell in layer 2/3 (Post). Top, Schematic drawing of the pair. Top trace, Presynaptic action potential induced by a brief current injection. Middle trace, Unitary IPSP recorded at a V_m of −48 mV. Dotted line, Single-exponential fit (τ = 36.3 msec).Bottom trace, unitary IPSP recorded at aV_m of −93 mV (low internal chloride). Note that the unitary IPSP reversed its polarity and became depolarizing. The capacitative surge in the postsynaptic cell was blanked.B, Paired recording between a presynaptic layer 2/3 pyramidal neuron (bottom trace, Pre) and a postsynaptic layer 1 LS cell. Top, Schematic drawing of the pair. Top trace, Unitary EPSP recorded at aV_m of −60 mV. C-Clamp, Current clamp; V-Clamp, voltage clamp. Dotted line, Single-exponential fit (τ = 7.1 msec).Middle trace, Unitary EPSC recorded under voltage-clamp mode (holding potential, −45 mV). Traces inA and B are the average of >100 trials (stimulation frequency, 0.25 Hz).

Fig. 4.

LS cells make inhibitory GABAergic chemical synapses to other interneurons in layer 1. A,B, Paired recordings from two pairs consisting of a presynaptic LS cell (Pre) and a postsynaptic non-LS cell (Post) in layer 1. A, A hyperpolarizing unitary IPSP was recorded when the postsynaptic cell was kept at aV_m of −54 mV and a low-chloride internal solution was used (E_Cl of −76 mV). B, The polarity of the unitary IPSP changed when the postsynaptic cell was filled with a high-chloride internal solution (E_Cl of −30 mV).

Fig. 5.

Paired recording between a pair of LS cells connected via both electrical and chemical synapses. A, At a V_m of −50 mV, the postsynaptic potential (bottom traces, Post) was biphasic, with a depolarizing component mediated by the electrical connection and a hyperpolarizing component reflecting the IPSP. When the postsynaptic response was recorded at −92 mV, the unitary IPSP was reduced dramatically, whereas the electrically mediated depolarization remained unmodified (low-chloride internal postsynaptic solution).Top trace, Presynaptic action potential (Pre) in response to a brief depolarizing current injection. B, Paired recording from a different pair of LS cells connected by electrical and chemical synapses. A depolarizing unitary IPSP was recorded when the postsynaptic cell was kept at a V_m of −59 mV and a high-chloride internal solution was used. C, Paired recording from a pair of LS cells connected only chemically. The depolarizing IPSP recorded at a V_m of −91 mV (Control) was blocked in the presence of bicuculline (BIC) at 20 μm(high-chloride internal solution). Traces in Aare the average of 100 trials. Traces in B andC are the average of 16–24 trials. The coupling coefficient of the pair shown in A was 14%, and that shown inB was 8.3%. The pair shown in C was not electrically coupled.

Fig. 6.

Electrical coupling among layer 1 LS neurons.A, Neurolucida reconstruction of a pair of LS cells in layer 1. Dendrites are illustrated with thick traces, and axons are illustrated with thin traces.Left, Photograph illustrating the same pair of cells filled with biocytin. Scale bar (in photomicrograph), 10 μm.Top insets, Firing pattern of the two LS cells in response to a pulse of depolarizing current. B,Left, Injecting a pulse of depolarizing or hyperpolarizing current in LS₁ affected the membrane of both LS₁ and LS₂. Similarly, injecting current in LS₂ depolarized or hyperpolarized the membrane of LS₁ (right). Data from the pair of cells shown in A. Step coupling coefficients were as follows: 6.65% in LS₁-to-LS₂ and 8.1% in LS₂-to-LS₁. Traces are the average of >130 trials.

Fig. 7.

Electrical coupling among nonpyramidal cells in layer 1 is cell type specific. A, Photograph of an LS cell and a non-LS (nLS) cell simultaneously recorded in layer 1. Scale bar (in photomicrograph), 10 μm. Theinsets show their distinct patterns of firing in response to current injection. B, Injecting a pulse of depolarizing current in the LS (left) or the non-LS cell (right) did not affect the membrane potential of the noninjected cell. C, Histogram illustrating the percentage of pairs electrically coupled. Note the high rate of electrical coupling among LS cells (83%; 49 of 59 pairs) in contrast with the very low rate among the pairs consisting of an LS and a non-LS cell (2%; 2 of 93 pairs).

Fig. 8.

Properties of electrical coupling among layer 1 LS cells. A, Electrical coupling was bidirectional. Comparison of step coupling coefficient when current was injected in LS₁ versus LS₂. B, Step coupling coefficient as a function of the distance between the somata of the two electrically coupled LS cells (n = 31 pairs).C, Example of spike transmission in a pair of LS neurons that were connected only via electrical synapses (see Materials and Methods). A prolonged depolarization of the presynaptic cell produced spontaneous action potentials that were transmitted to a postsynaptic neuron as a brief depolarization, followed by a slow hyperpolarization. The holding potential of postsynaptic cell was −75 mV.Traces were aligned to the peak of the presynaptic spike and represent the average of 102 trials. The step coupling coefficient was 16.9%, and the spike coupling coefficient was 1.4%.D, Superimposition of the presynaptic spike and the corresponding response in the coupled postsynaptic neuron. The latency between the peak of presynaptic spike (Pre) and the peak of the spikelet or postsynaptic response (Post) was 0.7 msec. Data from the same pair as in C.