Age-dependent long-term structural and functional effects of early-life seizures: evidence for a hippocampal critical period influencing plasticity in adulthood.

Neural activity promotes circuit formation in developing systems and during critical periods permanently modifies circuit organization and functional properties. These observations suggest that excessive neural activity, as occurs during seizures, might influence developing neural circuitry with long-term outcomes that depend on age at the time of seizures. We systematically examined long-term structural and functional consequences of seizures induced in rats by kainic acid, pentylenetetrazol, and hyperthermia across postnatal ages from birth through postnatal day 90 in adulthood (P90). Magnetic resonance imaging (MRI), diffusion tensor imaging (DTI), and electrophysiological methods at ⩾P95 following seizures induced from P1 to P90 demonstrated consistent patterns of gross atrophy, microstructural abnormalities in the corpus callosum (CC) and hippocampus, and functional alterations in hippocampal circuitry at ⩾P95 that were independent of the method of seizure induction and varied systematically as a function of age at the time of seizures. Three distinct epochs were observed in which seizures resulted in distinct long-term structural and functional outcomes at ⩾P95. Seizures prior to P20 resulted in DTI abnormalities in CC and hippocampus in the absence of gross cerebral atrophy, and increased paired-pulse inhibition (PPI) in the dentate gyrus (DG) at ⩾P95. Seizures after P30 induced a different pattern of DTI abnormalities in the fimbria and hippocampus accompanied by gross cerebral atrophy with increases in lateral ventricular volume, as well as increased PPI in the DG at ⩾P95. In contrast, seizures between P20 and P30 did not result in cerebral atrophy or significant imaging abnormalities in the hippocampus or white matter, but irreversibly decreased PPI in the DG compared to normal adult controls. These age-specific long-term structural and functional outcomes identify P20-30 as a potential critical period in hippocampal development defined by distinctive long-term structural and functional properties in adult hippocampal circuitry, including loss of capacity for seizure-induced plasticity in adulthood that could influence epileptogenesis and other hippocampal-dependent behaviors and functional properties.

Experimental, theoretical calculations of the vibrational spectra and conformational analysis of 2,4-di-tert-butylphenol.

In the present work, we reported a combined experimental and theoretical study on conformational stability, molecular structure and vibrational spectra of 2,4-di-tert-butylphenol (2,4-DTBP). The FT-IR (400-4000cm(-1)) and FT-Raman spectra (50-3500cm(-1)) of 2,4-DTBP were recorded. The molecular geometry, harmonic vibrational frequencies and bonding features of 2,4-DTBP in the ground-state have been calculated by using the density functional BLYP/B3LYP methods. The energy calculated by time-dependent density functional theory (TD-DFT) result complements with the experimental findings. The calculated highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) energies show that charge transfer occurs within the molecule. Finally the calculation results were compared with measured infrared and Raman spectra of the title compound which showed good agreement with observed spectra.

Gamma-vinyl GABA reduces paired pulse inhibition in the rat dentate gyrus in vivo and in vitro.

Gamma vinyl GABA (GVG), an irreversible GABA transaminase inhibitor, has anticonvulsant effects. GVG increases GABA levels in the brain by blocking its degradation, and is presumed to enhance GABAergic inhibition, however, in some cases it exacerbates seizures. We investigated the effects of GVG in vivo and in vitro on paired pulse inhibition (PPI) recorded in the rat dentate gyrus (DG) evoked by perforant path stimulation. At 2.5 h and 24 h after administration of GVG (1 g/kg, i.p.), there was a loss of PPI at both 15- and 25-ms interpulse intervals (IPI). Activation of presynaptic GABA(B) autoreceptors could explain this in vivo effect. We therefore further investigated the effects of co-application of GVG with the GABA(B) antagonists 2-OH saclofen (saclofen) or CGP 35348 (CGP) on PPI in hippocampal slices by in vitro study. Bath application of GVG (400 and 500 microM) not only resulted in a loss of perforant path evoked PPI at a 15-ms IPI, but produced facilitation of the second population spike relative to the first. Co-application of saclofen (250 microM) with GVG (500 microM) prevented facilitation of the second response of a paired-pulse. The facilitation of the second stimulation response produced by GVG (400 microM) was converted to inhibition by bath application of CGP 35348 (400 microM). These results suggest that activation of presynaptic GABA(B) receptors by increased extracellular GABA may be one of the contributing factors to the apparent paradoxical effect of GVG on PPI in the DG.

NMDA receptor-dependent plasticity of granule cell spiking in the dentate gyrus of normal and epileptic rats.

Because granule cells in the dentate gyrus provide a major synaptic input to pyramidal neurons in the CA3 region of the hippocampus, spike generation by granule cells is likely to have a significant role in hippocampal information processing. Granule cells normally fire in a single-spike mode even when inhibition is blocked and provide single-spike output to CA3 when afferent activity converging into the entorhinal cortex from neocortex, brainstem, and other limbic regions increases. The effects of enhancement of N-methyl-D-aspartate (NMDA) receptor-dependent excitatory synaptic transmission and reduction in gamma-aminobutyric acid-A (GABA(A)) receptor-dependent inhibition on spike generation were examined in granule cells of the dentate gyrus. In contrast to the single-spike mode observed in normal bathing conditions, perforant path stimulation in Mg(2+)-free bathing conditions evoked graded burst discharges in granule cells which increased in duration, amplitude, and number of spikes as a function of stimulus intensity. After burst discharges were evoked during transient exposure to bathing conditions that relieve the Mg(2+) block of the NMDA receptor, there was a marked increase in the NMDA receptor-dependent component of the EPSP, but no significant increase in the non-NMDA receptor-dependent component of the EPSP in normal bathing medium. Supramaximal perforant path stimulation still evoked only a single spike, but granule cell spike generation was immediately converted from a single-spike firing mode to a graded burst discharge mode when inhibition was then reduced. The induction of graded burst discharges in Mg(2+)-free conditions and the expression of burst discharges evoked in normal bathing medium with subsequent disinhibition were both blocked by DL-2-amino-4-phosphonovaleric acid (APV) and were therefore NMDA receptor dependent, in contrast to long-term potentiation (LTP) in the perforant path, which is induced by NMDA receptors and is also expressed by alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA) receptors. The graded burst discharge mode was also observed in granule cells when inhibition was reduced after a single epileptic afterdischarge, which enhances the NMDA receptor-dependent component of evoked synaptic response, and in the dentate gyrus reorganized by mossy fiber sprouting in kindled and kainic acid-treated rats. NMDA receptor-dependent plasticity of granule cell spike generation, which can be distinguished from LTP and induces long-term susceptibility to epileptic burst discharge under conditions of reduced inhibition, could modify information processing in the hippocampus and promote epileptic synchronization by increasing excitatory input into CA3.

Long-term consequences of early postnatal seizures on hippocampal learning and plasticity.

Neural activity influences the patterning of synaptic connections and functional organization of developing sensory and motor systems, but the long-term consequences of intense neural activity such as seizures in the developing hippocampus are not adequately understood. To evaluate the possibility that abnormal neural activity during early development may have long-term functional effects in hippocampal circuitry that plays a role in learning, memory and epilepsy, functional properties of hippocampal circuitry were assessed in adult rats that had experienced seizures induced by kainic acid on specific days during early postnatal development. Although previous studies have suggested that the immature hippocampus is relatively resistant to seizure-induced alterations compared with adults, independent behavioural and physiological experiments demonstrated that seizures evoked by kainic acid during early postnatal development induced a long-term loss of hippocampal plasticity manifesting as reduced capacity for long-term potentiation, reduced susceptibility to kindling, and impaired spatial learning, which was associated with enhanced paired-pulse inhibition in the dentate gyrus. The enhancement of inhibition and loss of plasticity were maximal when the seizures occurred on the first day of life, but were also observed when seizures were induced as late as postnatal day 14, which delimited a period of postnatal susceptibility in the developing rat hippocampus when disruption of normal neural activity by seizures produced consistent effects on a hippocampal-dependent behaviour and several forms of hippocampal plasticity implicated in learning, memory and the development of epilepsy in adulthood.

Gamma-vinyl-GABA potentiates the severity of naloxone-precipitated abstinence signs in morphine-dependent rats.

Effects of gamma-vinyl-GABA (GVG), an antiepileptic drug that inhibits GABA transaminase and increases extracellular GABA concentrations in the brain, were investigated on the morphine abstinence syndrome (AS) in male Wistar rats. Two morphine pellets (75 mg morphine base in each) were implanted subcutaneously on the back of the rats. Seventy-two hours after the morphine implantation, naloxone (NL, 2 mg kg-1) was injected intraperitoneally (i.p.) to induce precipitated morphine AS. GVG was administered at the doses of 250 mg kg-1 (n = 11) and 500 mg kg-1 (n = 11) i.p. 24 h prior to AS and at the dose of 500 mg kg-1 (n = 13) i.p. 6 h prior to AS. Immediately after NL injections, rats were observed for 5 min and AS signs (jumping, teeth chattering, wet dog shake, diarrhoea, ptosis and defecation) were assessed. The behavioural signs of GVG-treated rats were compared with the control groups (n = 10) during the AS. Jumping, wet dog shake, teeth chattering were found to be significantly increased in all of the GVG-treated groups. Ptosis was found to have increased in only 500 mg kg-1 GVG groups. GVG potentiated the severity of morphine AS signs. GVG does not seem to have any therapeutic potential for treatment of morphine abstinence unlike some other drugs that enhance GABAergic transmission.

Synaptic and axonal remodeling of mossy fibers in the hilus and supragranular region of the dentate gyrus in kainate-treated rats.

Seizures evoked by kainic acid and a variety of experimental methods induce sprouting of the mossy fiber pathway in the dentate gyrus. In this study, the morphological features and spatial distribution of sprouted mossy fiber axons in the dorsal dentate gyrus of kainate-treated rats were directly shown in granule cells filled in vitro with biocytin and in vivo with the anterograde lectin tracer Phaseolus vulgaris leucoagglutinin (PHAL). Sprouted axon collaterals of biocytin-filled granule cells projected from the hilus of the dentate gyrus into the supragranular layer in both transverse and longitudinal directions in kainate-treated rats but were not observed in normal rats. The sprouted axon collaterals projected into the supragranular region for 600-700 microm along the septotemporal axis. Collaterals from granule cells in the infrapyramidal blade crossed the hilus and sprouted into the supragranular layer of the suprapyramidal blade. Sprouted axon segments in the supragranular layer had more terminal boutons per unit length than the axon segments in the hilus of both normal and kainate-treated rats but did not form giant boutons, which are characteristic of mossy fiber axons in the hilus and CA3. Mossy fiber axons in the hilus of kainate-treated rats had more small terminal boutons, fewer giant boutons, and there was a trend toward greater axon length compared with mossy fibers in the hilus of normal rats. With the additional length of supragranular sprouted collaterals, there was an overall increase in the length of mossy fiber axons in kainate-treated rats. The synaptic and axonal remodeling of the mossy fiber pathway could alter the functional properties of hippocampal circuitry by altering synaptic connectivity in local circuits within the hilus of the dentate gyrus and by increasing the divergence of the mossy fiber terminal field along the septotemporal axis.

Effects of pilocarpine on paired pulse inhibition in the CA3 region of the rat hippocampus.

Pilocarpine (PILO), a muscarinic agonist, produces status epilepticus when administered to rats in vivo and induces interictal or ictal patterns of epileptiform activity in rat hippocampal slices. We investigated the effects of PILO (10 microM) on paired pulse inhibition (PPI) in the CA3 region of rat hippocampal slices. PPI was assessed by stimulating either the alveus or str. radiatum and recording the extracellular response from str. pyramidale of CA3. The evoked population spike following the second stimulus was compared to the first. PILO was bath applied for 1 h and then washed out to assess acute and long lasting effects. PILO decreased the amplitude of evoked population spikes measured in CA3. PPI following alveus stimulation was not affected by PILO; however, a significant loss of PPI at 15 and 30 ms interpulse intervals occurred following str. radiatum stimulation in the presence of PILO and 5 mM [K+]o artificial cerebrospinal fluid (ACSF). The decrease in PPI at the 15 ms interval persisted following wash-out of PILO. PILO in 7.5 mM [K+]o ACSF produced epileptiform activity and a resultant long lasting loss of PPI that followed str. radiatum stimulation. This effect was not observed following epileptiform activity produced by 7.5 mM [K+]o alone, suggesting that the loss of PPI was due to PILO. Because str. radiatum-evoked PPI was selectively impaired, PILO appears to preferentially decreased feed-forward inhibition. The more dramatic loss of PPI following exposure to PILO and high [K+]o may represent the first steps in the development of chronic seizures that results from PILO-induced status epilepticus in rats.

The significance of extracellular GABA in the substantia nigra of the rat during seizures and anticonvulsant treatments.

The effects of the anti-epileptic drugs valproic acid and gamma-vinyl-GABA (vigabatrin) on the extracellular content of GABA was determined by microdialysis. Probes were implanted in the substantia nigra reticulata (SNR) of rats. It was found that gamma-vinyl-GABA (1000 mg/kg) induced a 4-6-fold increase in the extracellular content of GABA. This increase lasted for at least 72 h. PTZ-induced convulsions were partly antagonized by the GVG treatment. The increase of extracellular GABA after gamma-vinyl-GABA was not affected by infusion of tetrodotoxin. In contrast valproic acid (200 mg/kg), although effective in preventing pentylenetetrazol (PTZ)-induced convulsions, did not affect extracellular GABA in the SNR. PTZ-induced convulsions did not modify extracellular GABA, neither in control rats nor in valproic acid or gamma-vinyl-GABA pretreated animals. The results do not support the idea that extracellular GABA in the SNR plays a significant role in anti-convulsive treatment. However, the present data can also be interpreted that extracellular GABA, as sampled by microdialysis, is not a reliable marker for GABA release.

Vigabatrin as an anticonvulsant against pentylenetetrazol seizures.

The anticonvulsant effects of gamma vinyl GABA (GVG) were investigated against pentylenetetrazol (PTZ) seizures, while sodium valproate (VP) was used as positive control. At 1000 and 1500 mg kg-1 GVG was found to decrease seizure intensity either in 4 or 24 h, as effectively as VP. At 2000 mg kg-1 GVG was found to be almost ineffective. At both doses and both time spans of drug action, seizure latency was prolonged, compared to controls and VP group.

Effects of MK 801 on morphine physical dependence: attenuation and intensification.

It has previously been reported that the noncompetitive NMDA receptor antagonists ketamine and dextromethorphan suppressed the naloxone-induced morphine abstinence syndrome. In addition, the previous blockade by ketamine and dextromethorphan of NMDA receptors has been shown to intensify the naloxone-elicited morphine abstinence syndrome. On the basis of this information, another noncompetitive NMDA receptor antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo-a,d-cyclohepten-5,10-imine maleate (MK 801), was administered to rats in which two morphine-containing (75 x 2 morphine base) pellets had been implanted. The naloxone-precipitated abstinence syndrome in rats injected with 0.3 mg/kg MK 801 36 h after pellet implantation was found significantly more intense than controls whereas the abstinence syndrome in rats that received 0.1 mg/kg MK 801 before naloxone injection was less intense. The intensification by MK 801 given 36 h following pellet implantation was attributed to the further increase in upregulation and supersensitivity of NMDA receptors caused by morphine. The attenuation was explained by the blockade by MK 801 of NMDA receptors as occurred in the case of ketamine and dextromethorphan.

Vigabatrin has an anxiolytic effect in the elevated plus-maze test of anxiety.

tau-Vinyl GABA (vigabatrin, GVG) is a novel antiepileptic drug that irreversibly inhibits GABA transaminase and elevates GABA levels in all parts of the brain. In the present study, we investigated the anxiolytic and behavioral effects of GVG in the elevated plus-maze and the hole board compared to diazepam. Doses of 500 and 1,000 mg/kg GVG were injected IP to different groups of male Wistar rats and animals were tested either 4 or 24 h after injection. Animals administered diazepam (1.5 mg/kg, IP) and saline (1 ml) were tested 20 min after injection. GVG and diazepam were found to decrease significantly the number of squares visited and rearing; both had a suppressant effect on locomotor activity. Neither drug had an effect on exploration (head dipping). GVG at a dose of 1,000 mg/kg was shown to have a similar anxiolytic activity either after 4 or 24 h as diazepam, while GVG at 500 mg/kg did not show any significant anxiolytic effect.

The effects of corticotrophin releasing factor (CRF) and handling stress on behavior in the elevated plus-maze test of anxiety.

The effects on rodent anxiety of corticotrophin releasing factor (CRF), common experimental stressors and the CRF receptor blocker, α-helical CRF, were measured using the hole board and elevated plus-maze tests. Centrally administered (intracerebroventricular, i.c.v.) CRF increased anxiety in an anxioselective manner. α-Helical CRF (i.c.v) antagonized the effects of CRF, implicating central CRF receptors. Common experimental stressors, such as surgical implantation of cannulas and intraperitoneal injections of saline also selectively increased anxiety in the plus maze. Endogenous CRF binding to central CRF receptors probably mediates the anxiogenic effects of stressors, since α-helical CRF reversed the increased anxiety following surgery. Finally, repeated gentle handling seemed to blunt the anxiogenic effect of CRF. Handling also altered the effect of CRF on behavior, creating an apparently CRF-mediated suppression of rearing and exploration which was not present in rats not stressed with repeated handling. Together the data suggest a modifiable modulatory role of CRF in rodent anxiety. The findings also suggest that careful attention should be paid to stress history when examining the role of CRF in rodent behavior.