John W. Swann

Postnatal development of GABA-mediated synaptic inhibition in rat hippocampus

Developmental alterations in GABAergic synaptic transmission were examined physiologically and biochemically in hippocampus of rats from 3 days of age to adulthood. Neither antidromic nor orthodromic stimulation could elicit identifiable inhibitory postsynaptic potentials in CA1 neurons in slices from rats 5 or 6 days of age. In contrast, at this age these stimuli result in large inhibitory postsynaptic potentials in CA3 pyramidal cells. In the latter cells orthodromic stimulation produced a brief monosynaptic excitatory postsynaptic potential which was followed by a large prolonged biphasic hyperpolarization. These signals were strikingly similar to those recorded in 1-month-old rats. In addition, large recurrent inhibitory postsynaptic potentials were produced by antidromic stimulation. By postnatal day 9 similar inhibitory postsynaptic potentials could be elicited in a majority of neurons of the CA1 subfield. As in mature pyramidal cells, application of GABA antagonists, such as bicuculline, selectively eliminated the antidromic inhibitory postsynaptic potential and the first component of the biphasic inhibitory postsynaptic potential generated by stimulation of stratum radiatum. In the CA3 subfield, this blockade of GABA receptors resulted in prolonged afterdischarges in slices from immature but not month-old rats. Measurements of the equilibrium potential and the conductance of antidromic inhibitory postsynaptic potentials in CA3 neurons were very similar when made during the first postnatal week and at 1 month of age. While on days 10-11 the equilibrium potential was very similar to measurements made at these other ages, the conductance was 3-4 times greater. The activity of glutamate decarboxylase, the synthetic enzyme for GABA, was very low at 3 days in hippocampus, and increased until 30 days of age at which time adult values were obtained. By comparison, hippocampal GABA levels were high early in postnatal life. Glutamate decarboxylase activities in microdissected CA3 and CA1 subfields were similar in immature hippocampus. These results demonstrate dramatic differences in the ontogenesis of functional GABAergic inhibitory synaptic transmission in the CA1 and CA3 subfields of rat hippocampus. The late development of GABA-mediated synaptic inhibition in the CA1 subfield could play a role in the susceptibility of immature hippocampus to seizures. However, the large GABA-mediated inhibitory postsynaptic potentials present in the CA3 subfield at the same age have a critical role in dampening neuronal excitability.(ABSTRACT TRUNCATED AT 400 WORDS)

Penicillin-induced epileptogenesis in immature rat CA3 hippocampal pyramidal cells

Penicillins 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.

N-methyl-d-aspartate (NMDA) receptors are inactivated by trypsin

Acute isolation of hippocampal CA3 pyramidal cells using trypsin produces neurons which respond to kainate and quisqualate but not N-methyl-D-aspartate (NMDA). Incubation of 6- to 12-day-old cultured hippocampal neurons or slices of pyriform cortex with trypsin irreversibly removes the NMDA responses normally present without significant effect on responses to kainate or quisqualate. These data indicate that the NMDA receptor has a trypsin-sensitive component which is necessary for agonist recognition or ion channel activation.

Ketamine selectively suppresses synchronized afterdischarges in immature hippocampus.

The role of excitatory amino acid neurotransmission in epileptogenesis was investigated in the developing hippocampus. Bath application of ketamine blocked penicillin-induced, synchronized afterdischarges in immature rat CA3 hippocampal neurons. Ketamine also decreased the duration of the preceding intracellularly recorded depolarization shift but had no measurable effect on the resting membrane potential or input impedance of pyramidal cells. Concentrations of ketamine that blocked afterdischarge generation dramatically depressed intracellular depolarizations produced by iontophoretic application of N-methyl-D-aspartate (NMDA) but not quisqualate. The effects of the NMDA antagonist 2-amino-7-phosphonoheptanoic acid on epileptiform discharges were identical to those of ketamine. These results suggest that an endogenous excitatory amino acid acting on an NMDA receptor plays a key role in the pronounced capacity of immature hippocampus for seizures.

Analysis of aspartate and glutamate in human cerebrospinal fluid by high-performance liquid chromatography with automated precolumn derivatization

A method is described for the analysis of the neuroexcitatory amino acids, aspartate and glutamate, in human cerebrospinal fluid (CSF) by reverse-phase, high-performance liquid chromatography. Fluorescent isoindole derivatives of the amino acids were prepared by reacting the amino acids with ortho-phthalaldehyde in an automated, precolumn procedure. Chromatographic conditions were developed that resolve the isoindole derivatives of aspartate and glutamate from those of at least 10 unidentified components of CSF. Amino acids were reliably quantified in 5-microliter samples of CSF, and deproteinization of the specimens was not required. Furthermore, it was found that deproteinization by precipitation with strong acid can lead to artifactually high measurements of glutamate. The concentrations of free aspartate and glutamate in lumbar CSF from 15 neurologically normal children were 0.30 +/- 0.11 and 0.48 +/- 0.26 microM (mean +/- SD), respectively. The value for glutamate is considerably lower than has been reported in any previous study of human CSF.

Extracellular K+ accumulation during penicillin-induced epileptogenesis in the CA3 region of immature rat 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.

ONTOGENETIC FEATURES OF AUDIOGENIC SEIZURE SUSCEPTIBILITY INDUCED IN IMMATURE RATS BY NOISE

SUMMARY: Although numerous models are currently used for systematic study of the mechanisms of epileptogenesis in mature brain, few animal models have been developed that allow similar explorations in the developing nervous system. One experimental model of epilepsy supports a premise that perinatal experience can lead to eventual seizure susceptibility, however. Audiogenic seizure (AGS) susceptibility can be induced during a critical developmental period in normal mice by auditory deprivation and therefore by cochlear trauma. We studied the developmental parameters that affect success of both induction and testing of AGS‐susceptibility in the rat. Intense high‐frequency noise exposure was used as the traumatizing agent. The Wistar rat strain used is inherently seizure‐resistant because in >400 trials, untreated rats have never exhibited susceptibility at any age. Although single prolonged exposures to high‐intensity noise were administered to groups of rats at ages between postnatal days (PNDs) 12 and 36, PND 14 was the age when exposure was most likely to result in eventual seizure susceptibility. Furthermore, duration of initial exposure on PND 14 determined the rate of susceptibility when measured 2 weeks later. Accordingly, we noted that single noise exposures at an intensity of 125 dB and ranging between 6 and 10 min in duration induced susceptibility in 100% of rats tested on PND 28; nonetheless, seizures among the rats exposed for 8 min were the most severe. Typically, these seizures began as wild running attacks and were followed by tonic/clonic convulsions. In a third study, we noted that a rats maturation at the time of testing influenced the rate of susceptibility, regardless of whether the rat had been exposed for 2.5, 4, or 8 min on PND 14. In this regard, PND 28 ontogenetically appeared to be the age of greatest inherent susceptibility. Susceptibility decreased between PND 28 and 36 but remained stable thereafter into adulthood. The age of onset of susceptibility depended (inversely) on the duration of original exposure, not on maturation. Nonetheless, wild running seizure responses are not observed in fewer than 2 days after initial exposure, and tonic/clonic convulsions emerged in not fewer than 3 days. The data suggest there are at least two critically sensitive periods in genesis of AGS susceptibility, one that is specific for induction of susceptibility (PND 14) and one specific for its expression (PND 28). Together, these data strongly support a view that both experiential and maturational factors combine predictably to shape the individual rats prognosis at specific ages.

A slow NMDA-mediated synaptic potential underlies seizures originating from midbrain

After bath-perfusion with gamma-aminobutyric acid antagonists, slices of the rats 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.

The sensitive period and optimum dosage for induction of audiogenic seizure susceptibility by kanamycin in the Wistar rat

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.

Suppression of ictal-like activity by kynurenic acid does not correlate with its efficacy as an NMDA receptor antagonist

The involvement of excitatory amino acid (EAA) receptors in ictal-like activity was investigated using hippocampal slices from immature rats. Bath application of kynurenic acid (KYN) suppressed penicillin-induced synchronized afterdischarges. An identical concentration of D(-)2-amino-5-phosphonovaleric acid (D(-)AP5) spared the afterdischarges. Intracellular recordings confirmed D(-)AP5 to be the more potent N-methyl-D-aspartate antagonist. KYNs suppression of afterdischarges could not be clearly attributed to blockade of either kainate or quisqualate receptors. Thus, a novel kynurenate-preferring EAA receptor may play an important role in seizure generation in immature hippocampus.