Blockade of neuronal activity during hippocampal development produces a chronic focal epilepsy in the rat

Abstract

During brain development, neuronal activity can transform neurons characterized by widely ranging axonal projections to ones with more restricted patterns of synaptic connectivity. Previous studies have shown that an exuberant outgrowth of local recurrent excitatory axons occurs in hippocampal area CA3 during postnatal weeks 2 and 3. Axons are remodeled with maturation, and nearly half of the branches are eliminated. Postnatal weeks 2 and 3 also coincide with a "critical" period of development, when CA3 networks have a marked propensity to generate electrographic seizures. In an attempt to prevent axonal remodeling, local circuit activity was blocked unilaterally in dorsal hippocampus by continuous infusion of tetrodotoxin (TTX). Field potential recordings from behaving animals were dramatically altered when TTX infusion was initiated at the beginning of the critical period, week 2, but not later in life. Spontaneous, synchronized spikes and electrographic seizures with behavioral accompaniments were observed after 4 weeks of TTX infusion and persisted into adulthood. When recordings were made during TTX infusion, synchronized spiking was recorded in ventral hippocampus as early as 2 weeks after infusate introduction. At this same time, extracellular field recordings from in vitro slices demonstrated spontaneous network-driven "mini-bursts" arising from ventral hippocampal slices. These were abolished by glutamate receptor antagonists. Whole-cell recordings from CA3 neurons revealed bursts of excitatory synaptic potentials coincident with the network bursts recorded extracellularly. Thus, local assemblies of mutually excitatory CA3 pyramidal cells are hyperexcitable in these rats. Whether alterations in developmental axonal remodeling mediate these effects awaits further studies.

Fig. 1.

TTX produces a region of focal hypometabolism in infused dorsal hippocampus. Pseudocolored autoradiograms of 2DG-labeled coronal sections taken from a TTX-infused animal and ACSF-infused animals are shown in panels A, B,D, and E. The color patterns represent gray scale values of 2DG uptake. The color scale to the leftindicates blue as the lowest level of 2DG uptake, whereas red indicates the highest. A, A coronal section from a TTX-infused animal reveals almost complete blockade of 2DG uptake in the infused hippocampus (arrow). B is a coronal section that shows normal 2DG levels in both the contralateral and ipsilateral ACSF-infused hippocampus. D, A section taken more anterior to that in A in the TTX-infused animal shows a similar blockade of 2DG uptake in the infused hippocampus (arrow). In E, a section taken 3 mm posterior through the ventral hippocampus of a TTX-infused hippocampus shows no signs of hypometabolism and no differences in 2DG uptake between the infused ipsilateral and contralateral hippocampus.C is a Nissl-stained section taken from a control animal, which provides anatomical guides for A, B, andD. F is an image taken at higher magnification of a TTX infusion site. The cannula tract (arrow) is readily evident. However, no signs of neuronal loss or excessive reactive gliosis are apparent.

Fig. 2.

Comparison of hippocampal theta activity recorded from TTX- and ACSF-infused animals after 4 weeks of infusion starting on days 10–12. The timeline summarizes experimental design. In A, the amplitudes of the hippocampal theta rhythms are plotted that were recorded from the ACSF-infused and contralateral hippocampus. B compares the amplitude of theta rhythms recorded from TTX-infused and contralateral hippocampus.C compares representative EEG traces of TTX-infused hippocampal theta activity (traces 2) on days 2, 6, and 12 to that recorded simultaneously in contralateral hippocampus (traces 1).

Fig. 3.

EEG recordings from ACSF- and TTX-infused animals 4 d after cannula extraction. Timeline depicts experimental design. Surface electrodes were placed over the left cortex (LC) and right cortex (RC), and depth electrodes were placed in the left dorsal hippocampus (LDH), right dorsal hippocampus (RDH), left ventral hippocampus (LVH), and the right ventral hippocampus (RVH). An electrode placed over the occipital cortex midline (C) served as a reference electrode. TTX was infused into the right dorsal hippocampus. A,Representative EEG traces of normal hippocampal activity obtained from an ACSF-infused animal 4 d after cannula extraction.B shows representative EEG traces from a TTX-infused animal showing multifocal interictal spikes during non-REM sleep.Arrows denote representative spikes. C is a representative trace showing a brief electrographic seizure recorded from a TTX-infused animal during non-REM sleep. Arrowsdenote the beginning and end of the seizure.

Fig. 4.

An electrographic seizure recorded 8 months after TTX cannula extraction. Surface electrodes were placed over the left cortex (LC) and right cortex (RC), and depth electrodes were placed in the left dorsal hippocampus (LDH), right dorsal hippocampus (RDH), left ventral hippocampus (LVH), and the right ventral hippocampus (RVH). An electrode placed over the midline occipital cortex (C) served as a reference electrode. A referential montage was used. This seizure began with a run of high-frequency spikes (top panel, arrow) in right hippocampus and rhythmic theta activity in left hippocampus.Timeline illustrates experimental design. This seizure was 77 sec in duration. Segments of traces at the beginning (A), middle (B), and end (C) of the seizure are shown.

Fig. 5.

Interictal spikes recorded during TTX infusion illustrate a hyperexcitable surround in ventral hippocampus.Timeline depicts experimental design. Ashows interictal spiking (arrows) arising from the right ventral hippocampus (RVH) on postnatal day 34 (22 d of TTX infusion). B shows the time of occurrence of interictal spikes recorded by six electrodes during 15 min of recording. A single focus predominates at this time. InC, multiple independent foci were recorded 5 d later in the same animal (C).

Fig. 6.

Spontaneous network minibursts recorded in hippocampal slices taken immediately after TTX cannula extraction. In this experiment, TTX infusion began on postnatal day 12, and slices were taken on postnatal day 26 as TTX was infused. A,Extracellular recordings from ACSF and TTX-infused animals.Trace 1, A slow time base extracellular field recording that demonstrates the lack of spontaneous network bursting in the CA3 cell body layer of an ACSF-infused animal. Trace 2,Spontaneous synchronized network minibursts from the CA3 cell body layer of a TTX-infused animal. A selected event (asterisk) in the slow time-based recordings intrace 2 is expanded in time in the inset. B, Plotted are the percentage of slices from dorsal through ventral hippocampus that showed spontaneous network minibursts. Slices were taken from the TTX-infused hippocampus of five rats.

Fig. 7.

Spontaneous network discharges recorded in vitro evolve from minibursts to intense network discharges.Timelines 1 and 2 depict experimental design for traces 1 and 2 inA and B. A, Trace 1 shows a slow time base record of extracellular field recordings from a slice taken from an animal immediately after 2 weeks of TTX infusion.Trace 2 is a recording from a slice taken from an animal 2 weeks after 4 weeks of TTX infusion. Events denoted byasterisks in A are shown inB at a faster time base.

Fig. 8.

Kynurenic acid (KYN) blocks spontaneous network minibursts. Top, Spontaneous minibursts were recorded under control (CON) conditions in a slice taken from ventral hippocampus during TTX infusion of dorsal hippocampus. Middle, Bath application of KYN (500 μm) to hippocampal slices blocked the minibursts. Bottom, Spontaneous minibursts returned after washout of KYN.

Fig. 9.

Barrages of synaptic potentials and resulting action potentials are recorded in CA3 pyramidal cells coincident with extracellular minibursts. A, Representative whole-cell recordings (traces 1) and extracellular field recordings (traces 2) from hippocampal slices taken during TTX infusion. Selected events outlined by boxes are shown at a faster time base in Ba–Bd. Synaptic events (arrows) are shown at three different holding potentials in Bd–Bf. Recordings were made from slices of ventral hippocampus taken immediately after 2 weeks of TTX infusion of dorsal hippocampus. For this cell, the resting membrane potential was −65 mV, input resistance was 150 MΩ, the holding potential was −70 mV (A, Ba–Bd), and spike amplitude was 68 mV.

Fig. 10.

Comparison of spontaneous intrinsic bursts recorded in slices from control (A) and experimental (B) rats. Traces 1compare the waveform of the first action potential in the intrinsic bursts shown below in traces 2 and 3 at slower time bases. The duration of the depolarizing envelope that underlies the burst discharge was similar in the two treatment groups. Postburst afterhyperpolarization are evident in traces 3. Resting membrane potential and input resistance: A, −68 mV, 180 MΩ; B, 70 mV, 200 MΩ. Age: control, 31 d; experimental, 25 d.