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The majority of cortical and hippocampal interneurons originate in the subcortical telencephalon and migrate tangentially into pallial regions before settling in various cortical layers. The molecular cues that regulate final positioning of specific interneurons in cortical structures have not yet been identified. The positioning of radially migrating principal neurons of the cortex and hippocampus depends upon Reelin, an extracellular protein expressed near the pial surface during embryonic development that is absent in reeler mutant mice. To determine whether the layer specification of interneurons, like that of principal neurons, requires Reelin, we crossed reeler with transgenic mice that contain Green Fluorescent Protein (GFP)-expressing Inhibitory Neurons (GINs). These neurons express basal forebrain markers Dlx1/2 in normal and reeler mice. In normal mice, GINs express Reelin and are localized to specific layers of the cortex and hippocampus. In reeler mutant mice, we show that GINs migrate normally into the pallium, but fail to acquire proper layer position. Double labeling experiments indicate that the neurochemical profile of these interneurons is not generally altered in reeler mice. However, the extension of their cellular processes is abnormal. Quantitative analysis of GINs in the cortex revealed that they are hypertrophic, bearing longer neuritic branches than normal. Thus, the lack of Reelin signaling results in abnormal positioning and altered morphology of forebrain interneurons.

A new experimental model of developmental epilepsy is reported. Behavioral and EEG features of seizures produced by unilateral intrahippocampal injection of tetanus toxin in postnatal day 9-11 rats, are described. Within 24-72 h of tetanus toxin injection, rat pups developed frequent and often prolonged seizures which included combinations of repetitive wet dog shakes, and wild running-jumping seizures. Intrahippocampal and cortical surface EEG recordings showed that coincident with these behaviors, electrographic seizures occurred not only in the injected hippocampus, but also in the contralateral hippocampus and bilaterally in the neocortex. Analysis of the interictal EEG revealed multiple independent spike foci. One week following tetanus toxin injection, the number of seizures markedly decreased; however, interictal spiking persisted. After injection rats were allowed to mature some were observed to have unprovoked behavioral seizures and/or epileptiform EEG activity. Mature animals were also studied using in vitro slice techniques. Recordings from hippocampal slices demonstrated spontaneous epileptiform burst discharges in the majority of rats which had tetanus toxin induced seizures as infants. These events occurred in area CA3 and consisted of interictal spikes and intracellularly recorded paroxysmal depolarization shifts (PDSs). On rarer occasions, electrographic seizures were recorded. The use of the tetanus toxin model in developing rats may facilitate a better understanding of the unique features of epileptogenesis in the developing brain and the consequences early-life seizures have on brain maturation and the genesis of epileptic conditions in later life.

We have assessed the properties and usefulness of confocal scanning laser microscopy in the reflection mode for the study of neuronal morphology. In this mode, the confocal microscope detects the light reflected off the specimen as opposed to the light emitted by a fluorescent label. Neurons in slices of rat hippocampus were filled with biocytin and reacted sequentially with avidin-horseradish peroxidase and nickel-intensified diaminobenzidine (DAB/Ni). In all parts of the neuron the DAB/Ni reaction product produced a strong reflection signal in the confocal microscope. The stereo images revealed aspects of three-dimensional hippocampal cell morphology such as the conical shape of the dendritic fields and a characteristic branching pattern of the axon. Labelling neurons intracellularly is an established technique for identifying physiologically-characterized neurons. Recently, confocal microscopy has become a powerful method for examining the three-dimensional morphology of biological specimens. The resulting images in this paper show that reflection-mode confocal microscopy provides an excellent representation of the filled neurons in three dimensions and presents an opportunity for correlative electrophysiological and morphological studies and extension to the electron-microscopic level.

Penicillin's ability to produce epileptiform discharges in the CA3 region of hippocampus was examined both extracellularly and intracellularly in slices taken from immature rats 3-25 days of age. Comparisons were made to similar recordings from slices taken from mature rats. Between postnatal days 9 and 19 penicillin treatment resulted in spontaneous extracellular epileptiform bursts and coincident intracellular depolarization shifts. These events were more prolonged and less frequent than in slices from mature rats, and the bursts were followed by prolonged afterdischarges, often 20-30 s in duration. Intracellularly these afterdischarges consisted of large, rhythmic slow depolarizing potentials, which resulted in one or more action potentials in individual CA3 pyramidal cells. Extracellular field recordings showed these events to be simultaneous with synchronous discharges of a large population of CA3 pyramidal cells. In pups 1-2 weeks of age the ability of hippocampus to produce prolonged afterdischarges was associated with a slow depolarizing afterpotential, which followed the downstroke of the depolarization shift. Coincident with this afterpotential was a prolonged negative field in the CA3 pyramidal cell body layer. By postnatal days 24 and 25 the tendency to generate afterdischarges was greatly reduced. In addition, afterdischarges were observed infrequently in slices taken during the first postnatal week. Spike trains produced by prolonged intracellular current injection in slices taken on postnatal days 9-19 were followed by large afterhyperpolarizations and were unable to produce afterdischarges in individual CA3 pyramidal cells. Intracellular recordings from presumed glial cells suggest that extracellular K+ accumulation may play a role in the pronounced capacity of hippocampus from 1- and 2-week-old rat pups to generate prolonged afterdischarges.

(1) Synaptic transmission was studied in isolated transverse hippocampal slices from guinea pigs. Extracellular evoked potentials were recorded in the region CA1. (2) Changing the normal perfusion solution (containing 2 mM Ca2+) to calcium-free Ringer abolished synaptic transmission which was again restored by adding strontium. A synaptic efficacy of 25--50% ofn normal was obtained for 10 mM Sr2+. (3) Two different synaptic inputs to CA1 pyramidal cells were tested with respect to their ability to produce long-lasting synaptic potentiation after tetanization in strontium Ringer. Following a brief tetanus the field EPSP and, especially, the population spike were greatly enhanced. (4) The potentiation so produced was similar to the long-lasting potentiation seen in the normal slice, because it (i) had a very long duration (hours), (ii) was specific for the tetanized pathway, (iii) showed potentiation of both 'volley-EPSP' and 'EPSP-spike' relations, and (iv) was accompanied by short-lasting (less than 5 min) generalized depression.

After bath-perfusion with gamma-aminobutyric acid antagonists, slices of the rat's inferior colliculus were studied electrophysiologically. Synchronized epileptiform events were found to occur. The most prominent intracellular event was a sustained 30 mV depolarization which was pharmacologically and electrophysiologically characterized as an N-methyl-D-aspartate-mediated event. We propose that elicitation of this slow synaptic potential is the a priori basis of seizures arising in this midbrain nucleus.

Bath application of muscimol (10-20 microM) to hippocampal slices obtained from rats on postnatal days 10-15 produced epileptiform activity in the form of multiple population spikes in 20% of slices tested, concurrent with marked disinhibition. The disinhibition occurred in nearly 100% of cases tested at muscimol concentrations that produced epileptiform activity. Paired pulse analysis of GABAergic recurrent inhibition revealed a muscimol-induced disinhibitory effect involving a decrease in maximum possible inhibition. Spontaneous and antidromically elicited inhibitory postsynaptic potentials (IPSPs) recorded intracellularly were suppressed by muscimol. Current-voltage analysis of the recurrent IPSPs suggests that muscimol acted at a number of sites to produce disinhibition. The input conductance of the postsynaptic pyramidal cell increased due to muscimol, creating a current shunt which likely decreased the efficacy of synaptic currents. Muscimol also caused a decrease in the conductance due to the IPSP as well as a shift in the depolarizing direction of the equilibrium potential of the IPSP. The data indicate that muscimol, a GABAA agonist, can produce disinhibition resulting from the multiple consequences of its action. We conclude that the physiologic consequences of GABAA agonist treatment are complex. On the other hand, neurons are likely to be inhibited by a tonic increase in membrane conductance. However, since recurrent inhibition is simultaneously compromised, excitatory vollies of sufficient intensity may overcome the tonic inhibition and produce a hyperexcitable state. In some cases, this disinhibition may induce epileptiform activity. These observations are relevant in light of the proposed use of GABA agonists clinically to control seizures.

Bath application of therapeutic concentrations of the anticonvulsant carbamazepine suppressed penicillin-induced synchronized afterdischarging in immature rat CA3 hippocampal pyramidal cells. Afterdischarging was completely abolished in all preparations at a concentration of 30 microM (IC50 = 8.5 +/- 1.4 microM; mean +/- S.E.M.). The duration of the preceding epileptiform burst was not altered at this concentration and was diminished by only 24.4 +/- 1.2% at a supratherapeutic concentration of 100 microM. These results suggest that a carbamazepine-sensitive neurophysiological mechanism distinct from those responsible for epileptiform burst generation plays a key role in the generation of afterdischarges in developing hippocampus.

Ion-selective microelectrode techniques were used to study extracellular K+ changes associated with penicillin-induced epileptogenesis in the CA3 region of hippocampal slices. Recordings were made in slices taken from rats 9-16 days of age, which have a pronounced capacity to undergo prolonged synchronized afterdischarges. Direct comparisons were made to slices from hippocampus from more mature rats, 30-35 days old, which are much less prone to seizure-like events. The amplitude and time course of the K+ transients varied across the CA3 laminae. K+ signals were largest close to stratum pyramidale in stratum oriens (the infrapyramidal zone). Recordings from this site showed extracellular K+ accumulation to be unusually large in immature hippocampus. The ceiling [K+]o level recorded during seizure-like events ranged from 14.4 to 20.2 mM and averaged 16.9 mM. The peak amplitude of extracellular K+ transients following an epileptiform burst in slices from immature rats averaged 4.31 mM while the mean of similar recordings from mature rats was 0.97 mM. Detailed laminar distribution studies in developing hippocampus revealed that the K+ signals were large in the proximal two-thirds of the basilar dendrites and proximal half of the apical dendrites. K+ accumulation in stratum pyramidale was comparatively small even though at its very edge in stratum oriens large K+ transients were always recorded. The latter was also true in recordings from mature hippocampus. Other dendritic signals in mature tissue were comparatively small. Laminar analysis was performed of the field potentials recorded by the reference barrel of the K+ electrodes. Negative field potential for the epileptiform burst and subsequent slow potential correlated in space with sites of K+ accumulation in both immature and mature hippocampal slices. Interictal and prolonged ictal-like discharges, recorded in developing hippocampus, arose from the same baseline [K+]o. However, since [K+]o is excessively high during the course of these epileptiform events it most likely has a role in the unusual propensity of immature hippocampus for seizures.

The incidence and severity of audiogenic seizures in kanamycin (KM)-treated rat pups, from a Wistar strain which is inherently seizure-resistant, was analyzed as a function of (a) postnatal age at the time of KM injection (i.p.) and (b) KM dosage. The vigor of the pinna reflex response on postnatal day (PND) 28 was correlated with (a) age at the time of injection, (b) dosage and (c) individual audiogenic seizure severity scores on PND 28 or PND 32. The data indicate that PNDs 9-12 are the developmental period when the rat has its greatest sensitivity to induction of susceptibility to audiogenic seizures by KM. The pinna reflex data suggest that cochlear vulnerability to KM intoxication is also greatest during this period. The optimum dosage for the induction of susceptibility was 100 mg/kg X 4 days. Use of higher doses resulted in a reduction of both incidence and severity of audiogenic seizures. The pinna reflex generally exhibited a supranormal vigor in animals having the most severe seizures. The behavioral attributes of induced audiogenic seizures at postnatal ages of 28 and 32 days are described and discussed.