Cell type-specific circuits of cortical layer IV spiny neurons

Abstract

Sensory signal processing in cortical layer IV involves two major morphological classes of excitatory neurons: spiny stellate and pyramidal cells. It is essentially unknown how these two cell types are integrated into intracortical networks and whether they play different roles in cortical signal processing. We mapped their cell-specific intracortical afferents in rat somatosensory cortex through a combination of whole-cell patch-clamp recordings and caged glutamate photolysis. Spiny stellate cells received monosynaptic excitation and inhibition originating almost exclusively from neurons located within the same barrel. Pyramidal cells, by contrast, displayed additional excitatory inputs from nongranular layers and from neighboring barrels. Their inhibitory inputs originated, as for spiny stellate cells, mainly from neurons located in the same barrel. These results indicate that spiny stellate cells act predominantly as local signal processors within a single barrel, whereas pyramidal cells globally integrate horizontal and top-down information within a functional column and between neighboring barrels.

Fig. 1.

Experimental setup for whole-cell recording in layer IV and local caged glutamate photolysis. Photomicrographs of a coronal slice of the rat somatosensory cortex taken directly after an experiment with recording and stimulation electrodes (a, c) and after histological processing (b).a, Barrel field in the living, unstained slice at low magnification and the same section after staining for biocytin and cytochrome oxidase (b). The biocytin-labeled layer IV spiny stellate cell is shown enlarged in theinset. c, Position of the recorded layer IV spiny stellate cell (white dot) and extent of the region stimulated by local photolysis of caged glutamate (grid). At 10 sec intervals, 450 fields of 50 × 50 μm in size were stimulated in sequence covering all cortical layers and at least two barrel-related columns. White frames highlight the layer IV barrels located within and near the investigated area. Roman numerals indicate cortical layers. Scale bars: a–c, 200 μm;b, inset, 100 μm.

Fig. 2.

Morphology and electrophysiology of layer IV spiny neurons. a, b, Photomicrographs (scale bar, top panel, 25 μm) and somatodendritic reconstructions (scale bar,bottom panel, 100 μm) of biocytin-stained layer IV spiny neurons. The lighter shaded areas mark the dimensions of the respective barrels.a, Spiny stellate cell with dendrites showing a spherical organization restricted to the barrel. b, Pyramidal cell with apical dendrite reaching layer I and basal dendrites forming a skirt-like pattern. Action potential firing pattern (c, d) and correlation of first-ISI versus second-ISI (e). f, Synaptic responses of layer IV spiny neurons. Spiny stellate as well as star pyramidal and pyramidal cells revealed one of the following firing patterns on injection of a suprathreshold depolarizing current pulse at resting membrane potential. In regular spiking cells, depolarizing current evoked a low-frequency train of single action potentials (c). Intrinsically bursting neurons revealed an initial high-frequency burst consisting of an action potential followed by a depolarizing afterpotential with at least two spikes of decreasing amplitude (d). The initial burst was followed by a sequence of single action potentials. e, Correlation of first-ISI versus second-ISI revealed two clusters representing the two types of action potential firing pattern: IB firing (small cluster at bottom left) and RS firing (larger cluster attop right). Data are shown for 46 spiny neurons (28 spiny stellate cells and 18 pyramidal cells). f, Postsynaptic responses of a spiny neuron to orthodromic electrical stimulation at different membrane potentials. The stimulus elicited an action potential and an EPSP followed by a fast (asterisk) and slow (double asterisk) IPSP.

Fig. 3.

Nonsynaptic and synaptic responses elicited by focal caged glutamate photolysis. a, Direct postsynaptic responses recorded at resting membrane potential in ACSF containing 0.2 mm Ca²⁺/4 mmMg²⁺ to block synaptic transmission.a1 shows suprathreshold response to perisomatic stimulation, and a2 illustrates response to stimulation of distal dendrites. b, Histogram showing delay to onset of glutamate-induced responses (n = 3 neurons) recorded in low Ca²⁺/high Mg²⁺bathing solution. Note that all direct postsynaptic events appeared within the first 3 msec after stimulus. c, Synaptic responses recorded in ASCF at a holding potential of −60 mV after local glutamate photolysis in a perisomatic field (c1), in a remote field containing one or more synaptically connected excitatory neurons (c2), and in a field containing at least one synaptically connected inhibitory cell (c3).d, Delay-to-onset distribution of glutamate-induced responses (n = 24 neurons) recorded in normal ACSF. Although direct nonsynaptic responses appeared with a delay ≤5 msec, all synaptically mediated PSPs showed delay-to-onset latencies >5 msec.

Fig. 4.

Topographic maps of functional connectivity of layer IV spiny neurons. The somatodendritic reconstruction of a recorded spiny stellate (a) and a pyramidal cell (b) as well as the respective topographic maps of their synaptic inputs were superimposed on the photomicrographs of the native slices. The topographic maps illustrate integrals of recorded EPSPs within 150 msec after stimulus (green to red) and fields of origin for IPSPs (blue). Note that for simplification, in fields where stimulation evoked EPSPs as well as IPSPs, only the IPSP is represented (blue). Fields given ingray were excluded from analysis because of a strong temporal interaction among direct postsynaptic activation, action potential generation, and synaptic events. Stimulated fields that did not induce any response are transparent. The outer black frames indicate the extent of the investigated area; therounded black rectangles highlight the dimensions of the relevant barrels. Insets show the enlarged cortical area within the slice (white frame). Action potential firing patterns on injection of a suprathreshold depolarizing current pulse atV_rmp are illustrated in a1and b1. Scale bar, 200 μm.

Fig. 5.

Spatial distribution of synaptic inputs onto layer IV spiny neurons. a, Percentages of stimulated presynaptic fields delivering intracolumnar and transcolumnar excitatory synaptic inputs onto layer IV spiny stellate cells (black bars, n = 11) and pyramidal neurons (shaded bars, n = 8).b, Mean strength of excitatory intracolumnar and transcolumnar synaptic inputs. c, Percentages of stimulated presynaptic fields delivering inhibitory synaptic inputs. Note that in a and c scales for transcolumnar inputs are enlarged. Data are means ± SD.Asterisks indicate significant differences between the two cell groups: *p < 0.05 and **p < 0.01.

Fig. 6.

Layer IV spiny neurons show direct barrel-to-barrel interactions. Topographic maps of excitatory and inhibitory synaptic inputs onto a layer IV spiny stellate cell (a) and a pyramidal cell (b). Insets show the enlarged cortical area within the slice (white frame). Action potential firing patterns on injection of a suprathreshold depolarizing current pulse at V_rmp are illustrated ina1 and b1. c, EPSPs recorded at V_hold = −60 mV on stimulation of fields in the neighboring barrel at positions indicated in a. Scale bar, 200 μm.

Fig. 7.

Schematic illustration of excitatory and inhibitory intracolumnar and transcolumnar synaptic inputs onto layer IV spiny neurons in rodent barrel cortex. Excitatory synaptic inputs are shown in gray; inhibitory inputs are shown inwhite. The density of synaptic inputs is represented by the number of arrows (1 gray arrow: 10% of fields generated EPSPs; 1 white arrow: 5% of fields generated IPSPs). The average strength of excitatory synaptic inputs is represented by the thickness of the arrows (thin arrow: <0.05 mV∗sec; medium arrow: 0.05–0.1 mV∗sec; thick arrow: >0.1 mV∗sec). Although spiny stellate cells receive predominantly intrabarrel synaptic inputs (a), layer IV pyramidal neurons are additionally innervated from supragranular and infragranular layers and from the neighboring barrel (b).