Laminar pattern of synaptic activity in rat primary visual cortex: comparison of in vivo and in vitro studies employing the current source density analysis.

In the present study we employed current source density analysis to study the major excitatory/inhibitory pathways in rat primary visual cortex in vivo and in vitro. A natural photic stimulus was used in vivo and served as a baseline for understanding the results obtained from in vivo and in vitro studies employing electrical stimulation of the white matter. The temporal pattern of synaptic activity in the cortex revealed an early excitation, characterized by sinks of short duration and high amplitude, that was followed by inhibition, characterized by long lasting, low amplitude active sources. The spatial pattern of this synaptic activity displayed early excitatory inputs to layer IV and lower layer III. Supragranular layers exhibited synaptic activity of longer latency at more superficial layers. The excitatory activity of the infragranular layers was delayed relative to that in layer IV. This spatial and temporal pattern of synaptic activity supports the model of sequential information processing in visual cortex. Based on the results of electrical and photic stimulations in vivo we conclude that electrical stimulation of white matter activate the thalamo-cortical input which results in a similar laminar pattern of postsynaptic activity evoked by photic stimulation. Electrical stimulation revealed additional antidromic and anti-orthodromic activity (collaterals of descending axons to white matter), resulting in the early fast components and the additional activity in layer VI. The major differences between in vivo and in vitro laminar pattern of synaptic activity (applying electrical stimulation) were reduced synaptic activity in layer IV and increased synaptic activity in the infragranular layers in the in vitro preparation. We concluded that the visual cortex slice preparation preserves the major pathways and electrophysiological function of this area. The technical advantages of the cortical slice preparation will facilitate studies and provide additional insight into this complex cortical network.

Organization of layers II-III connections in human visual cortex revealed by in vitro injections of biocytin.

In the search for cortical mechanisms subserving psychological phenomena, a better understanding of human cortical circuitry is crucial. In this report we describe aspects of intrinsic connectivity of supragranular layers in human visual cortex, revealed by extracellular injections of the anterograde tracer biocytin in vitro. Human cortical slices were obtained from visual association cortex in the posterior-medial portion of the dorsal bank of the occipital lobe, removed during neurosurgical tumor ablations. Small iontophoretic injections of biocytin into layers II-III revealed intense Golgi-like staining of axonal projections emanating from the injection sites. Vertically descending axons are grouped in bundles 20 microns in diameter which are spaced 15 microns apart. Some of these axons enter the white matter and send long oblique and horizontal collaterals. The main horizontal spread of the axons could be observed in layers II-III and V. The bulk of projections extends to a distance of 1.5 mm in layers II-III and 1.1 mm in layer V. Few individual axons could be observed at greater distances. In contrast, layer IV is almost devoid of horizontal connections, forming a clear gap between supra- and infragranular layers. Axon collaterals in the infragranular layers project mostly in a descending oblique direction with long horizontal collaterals in lower layer VI.

Excitatory inputs to layer V pyramidal cells of rat primary visual cortex revealed by acetylcholine activation.

Cells in layers II-III or VI were activated by microdrop application of acetylcholine (ACh), while monitoring the intracellular response of layer V pyramidal cells. This enabled the tracing of functional connections between the cells of layers II-III or VI with those of layer V. ACh activation of layer II-III or VI cells resulted in a small depolarization of these cells, accompanied by a burst of excitatory postsynaptic potentials (EPSPs) from layer V pyramidal cells. These effects of ACh were blocked by tetrodotoxin (TTX), suggesting the involvement of action potentials in their production. The input resistance of layer V pyramidal cells during and after the EPSP burst was not significantly different from control values, further suggesting an indirect effect of ACh on layer V pyramidal cells. Isolation of the supragranular layer, by horizontal cutting, did not prevent the EPSP burst evoked by ACh application to the lower layer VI, suggesting a direct input from layer VI to layer V pyramidal cells. ACh applied near pyramidal cells in layers II-III, V or VI caused transient hyperpolarization associated with a decrease in input resistance followed by a large depolarization, an increase in input resistance, and action potential discharges. The ACh-mediated hyperpolarization and the train of action potentials of layer II-III pyramidal cells were blocked by TTX. Thus the ACh-activated cells in layers II-III and VI make an excitatory synaptic contact with layer V pyramidal cells, producing the EPSP burst observed in layer V.

Golgi-like staining of visual cortex cells obtained by extracellular biocytin application in vitro.

We report here the application of biocytin (a biotin-lysine complex) as an extracellular tracer in vitro. Biocytin was applied extracellularly, revealing Golgi-like staining of cells in the adult in vitro rat visual cortex. Micropipettes were filled with a solution of 2.3-2.6% biocytin dissolved in 0.05 M Tris buffer, pH 7.4. Biocytin was applied by one of 3 methods: diffusion, pressure injection or drop application. Cell bodies and dendrites around the application site and their efferent axonal processes were stained; dendritic spines were often visible. The injection sites varied in size from a single cell to a diameter of 400 microns. When applied in layer I-III, few filled cells were also seen in layers IV and V, outside the application site. The drop application (5-10 microliters) of biocytin resulted in filling of cells throughout the cortex. The combination of biocytin and the slice preparation was found to be very useful in revealing cell morphology and tracing interlaminar connections in the visual cortex. The advantages of this technique are its ease of application, the precise and restricted injection sites, and Golgi-like morphological detail.