Recurrent circuits in layer II of medial entorhinal cortex in a model of temporal lobe epilepsy

Abstract

Patients and laboratory animal models of temporal lobe epilepsy display loss of layer III pyramidal neurons in medial entorhinal cortex and hyperexcitability and hypersynchrony of less vulnerable layer II stellate cells. We sought to test the hypothesis that loss of layer III pyramidal neurons triggers synaptic reorganization and formation of recurrent, excitatory synapses among layer II stellate cells in epileptic pilocarpine-treated rats. Laser-scanning photo-uncaging of glutamate focally activated neurons in layer II while excitatory synaptic responses were recorded in stellate cells. Photostimulation revealed previously unidentified, functional, recurrent, excitatory synapses between layer II stellate cells in control animals. Contrary to the hypothesis, however, control and epileptic rats displayed similar levels of recurrent excitation. Recently, hyperexcitability of layer II stellate cells has been attributed, at least in part, to loss of GABAergic interneurons and inhibitory synaptic input. To evaluate recurrent inhibitory circuits in layer II, we focally photostimulated interneurons while recording inhibitory synaptic responses in stellate cells. IPSCs were evoked more than five times more frequently in slices from control versus epileptic animals. These findings suggest that in this model of temporal lobe epilepsy, reduced recurrent inhibition contributes to layer II stellate cell hyperexcitability and hypersynchrony, but increased recurrent excitation does not.

Figure 1.

Laser-scanning photostimulation in layer II of medial entorhinal cortex in brain slices from control and epileptic rats uncages glutamate and evokes direct and synaptic responses. A, Questions addressed in this study: (1) do layer II stellate cells form recurrent excitatory synapses in control tissue? (2) do these neurons sprout axon collaterals and develop novel recurrent excitatory synapses in epileptic animals? and (3) is recurrent inhibitory synaptic input onto stellate cells from GABAergic interneurons in layer II diminished in epileptic animals? Laser-scanning photostimulation in layer II (L-II; gray area) activated stellate cells and inhibitory interneurons while responses were recorded in stellate cells. In this study, the term “recurrent inhibition” does not specify whether the activated interneurons receive synaptic input from the stellate cells in which IPSCs are recorded. B1, Overlay of typical responses recorded in a stellate cell evoked by pseudorandom and systematic uncaging of glutamate by flash photolysis in layer II (L II). In this and subsequent figures, recorded soma position is indicated by ⊙. The recorded neuron in layer II medial entorhinal cortex was visualized using a microscope equipped with infrared optics (R, recording electrode). B2, Enlargement of some traces from B1 reveals four types of photostimulation-evoked responses: a, direct; b, synaptic; c, mixed; d, no response. Direct responses recorded in voltage-clamp mode (holding voltage, −70 mV) peaked within 10 ms of photostimulation. Events that peaked during a measurement window 10–30 ms after photostimulation (between blue dotted lines) were identified as potential excitatory synaptic responses. C, Glutamate photo-uncaging maps of direct (top) and synaptic (bottom) responses of cell shown in B. Direct responses are expressed as peak amplitudes occurring within 10 ms of photostimulation. Synaptic responses are expressed as composite EPSC amplitudes occurring 10–30 ms after photostimulation.

Figure 2.

Direct responses recorded in current-clamp mode of entorhinal cortical neurons to glutamate photo-uncaging are similar in control and epileptic animals. A, Responses of layer II stellate cells in control and epileptic animals to photostimulation. B, Color-coded maps depict average number of action potentials evoked at each stimulation site in 10 cells from control and eight cells from epileptic animals (left and right panels, respectively). C, Quantitative comparison of action potential maps from control and epileptic animals. Hotspots are stimulation sites that evoke an action potential. D, Control experiment to evaluate specificity of layer II photostimulation. Current-clamp recordings were obtained from a layer III pyramidal cell (⊙). Photostimulation maps indicate number of action potentials evoked. Stimulation near the recorded soma in layer III (L III) evoked action potentials, whereas stimulation in overlying layer II (L II) did not. Overlapping stimulation maps are indicated by filled and open dotted circles. r, Recording electrode.

Figure 3.

Recurrent circuits in layer II of medial entorhinal cortex in control and epileptic animals evaluated with laser-scanning photostimulation. A, Maps depict composite amplitudes of EPSCs (holding potential, −70 mV) occurring in measurement windows 10–30 ms after photostimulation. Traces under maps are typical responses, and corresponding photostimulation sites are indicated by filled yellow circles. B, Maps from the same cells showing composite amplitudes of IPSCs (holding potential, 0 mV) occurring in measurement windows 10–110 ms after photostimulation. Traces under maps are typical responses. C, Neurons from which data in A and B were obtained (red, biocytin; green, NeuN immunoreactivity; L I-II, layers I-II). The yellow arrowhead indicates a cell from epileptic rat whose responses are shown above.

Figure 4.

Mediolateral distributions of probabilities of evoking a response (A, B) and average composite PSC amplitudes (C, D) in control and epileptic groups (soma at 0 μm) for EPSCs (left column) and IPSCs (right column). Error bars, where larger than the size of the symbols, indicate SEM.