Faculty Bibliography

Kynurenic acid (KYNA) is an antagonist of (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors and it blocks the glycine site of the NMDA receptor preferentially (IC50 = 7.9 microM). KYNA is produced endogenously by transamination of its precursor L-kynurenine (L-KYN). We tested the hypothesis that effects of endogenous, de novo produced KYNA, following bath-application of L-KYN to slices, would be different than effects of commercially-synthesized (exogenous) KYNA. The ability to block spontaneous epileptiform activity, induced by lowering extracellular magnesium, was examined in area CA3 of hippocampus and the entorhinal cortex. At a concentration of 200 microM L-KYN, which produced 0.89 +/- 0.20 microM KYNA, there were fewer slices with spontaneous epileptiform activity than slices exposed to 2 microM exogenous KYNA. The results indicate a more potent neuromodulatory action of endogenous KYNA than has been previously realized

This study examined the acute actions of brain-derived neurotrophic factor (BDNF) in the rat dentate gyrus after seizures, because previous studies have shown that BDNF has acute effects on dentate granule cell synaptic transmission, and other studies have demonstrated that BDNF expression increases in granule cells after seizures. Pilocarpine-treated rats were studied because they not only have seizures and increased BDNF expression in granule cells, but they also have reorganization of granule cell 'mossy fiber' axons. This reorganization, referred to as 'sprouting,' involves collaterals that grow into novel areas, i.e., the inner molecular layer, where granule cell and interneuron dendrites are located. Thus, this animal model allowed us to address the effects of BDNF in the dentate gyrus after seizures, as well as the actions of BDNF on mossy fiber transmission after reorganization. In slices with sprouting, BDNF bath application enhanced responses recorded in the inner molecular layer to mossy fiber stimulation. Spontaneous bursts of granule cells occurred, and these were apparently generated at the site of the sprouted axon plexus. These effects were not accompanied by major changes in perforant path-evoked responses or paired-pulse inhibition, occurred only after prolonged (30-60 min) exposure to BDNF, and were blocked by K252a. The results suggest a preferential action of BDNF at mossy fiber synapses, even after substantial changes in the dentate gyrus network. Moreover, the results suggest that activation of trkB receptors could contribute to the hyperexcitability observed in animals with sprouting. Because human granule cells also express increased BDNF mRNA after seizures, and sprouting can occur in temporal lobe epileptics, the results may have implications for understanding temporal lobe epilepsy

Transgenic mice overexpressing brain-derived neurotrophic factor from the beta-actin promoter were tested for behavioral, gross anatomical and physiological abnormalities. Brain-derived neurotrophic factor messenger RNA overexpression was widespread throughout brain. Overexpression declined with age, such that levels of overexpression decreased sharply by nine months. Brain-derived neurotrophic factor transgenic mice had no gross deformities or behavioral abnormalities. However, they showed a significant passive avoidance deficit. This deficit was dependent on continued overexpression, and resolved with age as brain-derived neurotrophic factor transcripts decreased. In addition, the brain-derived neurotrophic factor transgenic mice showed increased seizure severity in response to kainic acid. Hippocampal slices from brain-derived neurotrophic factor transgenic mice showed hyperexcitability in area CA3 and entorhinal cortex, but not in dentate gyrus. Finally, area CA1 long-term potentiation was disrupted, indicating abnormal plasticity. Our data suggest that overexpression of brain-derived neurotrophic factor in the brain can interfere with normal brain function by causing learning impairments and increased excitability. The results also support the hypothesis that excess brain-derived neurotrophic factor could be pro-convulsant in the limbic system

Chronic changes in synaptic responses of entorhinal and hippocampal neurons after amino-oxyacetic acid (AOAA)-induced entorhinal neuron loss. J. Neurophysiol. 80: 3031-3046, 1998. Synaptic responses of entorhinal cortical and hippocampal neurons were examined in vivo and in vitro, 1 mo to 1.5 yr after a unilateral entorhinal lesion caused by a focal injection of amino-oxyacetic acid (AOAA). It has been shown previously that injection of AOAA into the medial entorhinal cortex produces cell loss in layer III preferentially. Although behavioral seizures stopped approximately 2 h after AOAA treatment, abnormal evoked responses were recorded as long as 1.5 yr later in the entorhinal cortex and hippocampus. In the majority of slices from AOAA-treated rats, responses recorded in the superficial layers of the medial entorhinal cortex to white matter, presubiculum, or parasubiculum stimulation were abnormal. Extracellularly recorded responses to white matter stimulation were prolonged and repetitive in the superficial layers. Intracellular recordings showed that residual principal cells in superficial layers produced prolonged, repetitive excitatory postsynaptic potentials (EPSPs) and discharges in response to white matter stimulation compared with brief EPSPs and a single discharge in controls. Responses of deep layer neurons of AOAA-treated rats did not differ from controls in their initial synaptic response. However, in a some of these neurons, additional periods of excitatory activity occurred after a delay. Abnormal responses were recorded from slices ipsilateral as well as contralateral to the lesioned hemisphere. Recordings from the entorhinal cortex in vivo were abnormal also, as demonstrated by prolonged and repetitive responses to stimulation of the area CA1/subiculum border. Evoked responses of hippocampal neurons, recorded in vitro or in vivo, demonstrated abnormalities in selected pathways, such as responses of CA3 neurons to hilar stimulation in vitro. There was a deficit in the duration of potentiation of CA1 population spikes in response to repetitive CA3 stimulation in AOAA-treated rats. Theta activity was reduced in amplitude in area CA1 and the dentate gyrus of AOAA-treated rats, although evoked responses to angular bundle stimulation could not be distinguished from controls. The results demonstrate that a preferential lesion of layer III of the entorhinal cortex produces a long-lasting change in evoked and spontaneous activity in parts of the entorhinal cortex and hippocampus. Given the similarity of the lesion produced by AOAA and entorhinal lesions in temporal lobe epileptics, these data support the hypothesis that preferential damage to the entorhinal cortex contributes to long-lasting changes in excitability, which could be relevant to the etiology of temporal lobe epilepsy

Kynurenic acid is an excitatory amino acid antagonist with preferential activity at the N-methyl-D-aspartate subtype of glutamate receptors. It is produced endogenously in the brain, but is synthesized more effectively in the periphery. The influence of peripheral kynurenic acid on brain function is unclear because kynurenic acid is likely to penetrate the blood-brain barrier poorly. To determine the potential central effects of peripheral kynurenic acid, we compared its effects in the hippocampus after peripheral or direct administration. The hippocampus of the rat was chosen as a test system because this region receives glutamatergic inputs, and because responses to stimulation of these inputs can be compared after peripheral drug administration in vivo, and after direct administration of drugs in vitro. Peripherally-administered kynurenic acid was injected via a catheter in the jugular vein. Bath-application to hippocampal slices was used to test effects of direct administration. Area CA1 pyramidal cells and dentate gyrus granule cells were examined by extracellular recording and stimulation of area CA3 or the perforant path, respectively. Pairs of identical stimuli were used to assess paired-pulse inhibition and paired-pulse facilitation. Kynurenic acid decreased evoked responses in area CA1 and the dentate gyrus, both in vivo and in vitro. Effective concentrations were in the low micromolar range, and therefore were likely to be mediated by antagonism of N-methyl-D-aspartate receptors. In both preparations, area CA1 was more sensitive than the dentate gyrus, and paired-pulse facilitation was affected, but not paired-pulse inhibition. Control solutions had no effect. We conclude that kynurenic acid can enter the brain after peripheral administration, and that peripheral and direct effects in the hippocampus are qualitatively similar. Therefore, we predict that effects of endogenous kynurenic acid that was synthesized peripherally or centrally would be similar. Furthermore, the results suggest that modulation of the glycine site of the N-methyl-D-aspartate receptor, for example by kynurenic acid, may vary considerably among different brain areas

Effects of brain-derived neurotrophic factor (BDNF) in area CA3, the dentate gyrus, and medial entorhinal cortex were examined electrophysiologically by bath application of BDNF in slices containing the hippocampus and entorhinal cortex. Bath application of 25-100 ng/ml BDNF for 30-90 min increased responses to single afferent stimuli in selective pathways in the majority of slices. In area CA3, responses to mossy fiber stimulation increased in 73% of slices and entorhinal cortex responses to white matter stimulation increased in 64% of slices. After exposure to BDNF, these areas also demonstrated evidence of hyperexcitability, because responses to repetitive stimulation (1-Hz paired pulses for several s) produced multiple population spikes in response to mossy fiber stimulation in CA3 or multiple field potentials in response to white matter stimulation in the entorhinal cortex. Repetitive field potentials persisted after repetitive stimulation ended and usually were followed by spreading depression. Enhancement of responses to single stimuli and hyperexcitability were never evoked in untreated slices or after bath application of boiled BDNF or cytochrome C. The tyrosine kinase antagonist K252a (2 microM) blocked the effects of BDNF. In area CA3, both the potentiation of responses to single stimuli and hyperexcitability showed afferent specificity, because responses to mossy fiber stimulation were affected but responses to fimbria or Schaffer collateral stimulation were not. In addition, regional specificity was demonstrated in that the dentate gyrus was much less affected. The effects of BDNF in area CA3 were similar to those produced by bath application of low doses of kainic acid, which is thought to modulate glutamate release from mossy fiber terminals by a presynaptic action. These results suggest that BDNF has acute effects on excitability in different areas of the hippocampal-entorhinal circuit. These effects appear to be greatest in areas that are highly immunoreactive for BDNF, such as the mossy fibers and the entorhinal cortex. Although the present experiments do not elucidate mechanism(s) definitively, the afferent specificity, similarity to the effects of kainic acid, and block by K252a are consistent with previous hypotheses that BDNF affects acute excitability by a presynaptic action on trkB receptors that modulate excitatory amino acid transmission. However, we cannot rule out actions on inhibitory synapses or postsynaptic processes

The kynurenine pathway converts tryptophan into various compounds, including L-kynurenine, which in turn can be converted to the excitatory amino acid receptor antagonist kynurenic acid. The hypothesis that endogenously-produced kynurenic acid could have physiological effects was tested in combined entorhinal/hippocampal slices from adult rats. Specifically, perfusion with L-kynurenine (1 mM) was examined for its ability to suppress epileptiform activity produced by subsequent perfusion with buffer lacking added magnesium (nominal 0 mM magnesium buffer). Importantly, treatment with L-kynurenine did not appear to have depressant effects in itself, but it prevented spontaneous epileptiform activity in all 64 slices subsequently perfused with 0 mM magnesium buffer. In contrast, 45 slices that were not pretreated with L-kynurenine exhibited spontaneous epileptiform activity. These data support the hypothesis that endogenously-produced kynurenic acid can be produced and released in brain slices, where it can suppress excitatory activity in an 'anticonvulsant' manner. Therefore, manipulation of the kynurenine pathway might constitute a useful new direction for anticonvulsant drug development

1. Injection of aminooxyacetic acid (AOAA) into the entorhinal cortex in vivo produces acute seizures and cell loss in medial entorhinal cortex. To understand these effects, AOAA was applied directly to the medial entorhinal cortex in slices containing both the entorhinal cortex and hippocampus. Extracellular and intracellular recordings were made in both the entorhinal cortex and hippocampus to study responses to angular bundle stimulation and spontaneous activity. 2. AOAA was applied focally by leak from a micropipette or by pressure ejection. Evoked potentials increased gradually within 5 min of application, particularly the late, negative components. Evoked potentials continued to increase for up to 1 h, and these changes persisted for the remainder of the experiment (up to 5 h after drug application). 3. Paired pulse facilitation (100-ms interval) was also enhanced after AOAA application. Increasing stimulus frequency to 1-10 Hz increased evoked potentials further, and after several seconds of such stimulation multiple field potentials occurred. When stimulation was stopped at this point, repetitive field potentials occurred spontaneously for 1-2 min. These recordings, and simultaneous extracellular recordings in different layers, indicated that spontaneous synchronous activity occurred in entorhinal neurons. Intracellularly labeled cortical pyramidal cells depolarized and discharged during spontaneous and evoked field potentials. 4. The effects of AOAA were blocked reversibly by bath application of the N-methyl-D-aspartate (NMDA) receptor antagonist D-amino-5-phosphonovalerate (D-APV; 25 microM) or focal application of D-APV to the medial entorhinal cortex. 5. Simultaneous extracellular recordings from the entorhinal cortex and hippocampus demonstrated that spontaneous synchronous activity in layer III was often followed within several milliseconds by negative field potentials in the terminal zones of the perforant path (stratum moleculare of the dentate gyrus and stratum lacunosum-moleculare of area CA1). The extracellular potentials recorded in the dentate gyrus corresponded to excitatory postsynaptic potentials and action potentials in dentate granule cells. However, extracellular potentials in area CA1 were small and rarely correlated with discharge in CA1 pyramidal cells. 6. The results demonstrate that AOAA application leads to an NMDA-receptor-dependent enhancement of evoked potentials in medial entorhinal cortical neurons, which appears to be irreversible. The potentials can be facilitated by repetitive stimulation, and lead to synchronized discharges of entorhinal neurons. The discharges invade other areas such as the hippocampus, indicating how seizure activity may spread after AOAA injection in vivo. These data suggest that AOAA may be a useful tool to study longlasting changes in NMDA receptor function that lead to epileptiform activity and neurodegeneration

1. Microelectrode recording and fluorescence measurement with voltage-sensitive dyes were employed in horizontal hippocampal slices from rat to investigate responses in the dentate gyrus to molecular layer and hilar stimulation. 2. Both field potential and dye fluorescence measurement revealed that electrical stimulation of the molecular layer produced strong excitation throughout large regions of the dentate gyrus at considerable distances from the site of stimulation. 3. Treatment of slices with the excitatory amino acid receptor antagonists 6,7-dinitroquinoxaline-2,3-dione (DNQX) and (+/-)-2-amino-5-phosphonovaleric acid (APV) unmasked dye fluorescence signals in the outer and middle molecular layers corresponding to action potentials in axons, presumably belonging to the perforant path. The spread of these axonal signals away from the site of stimulation was far less extensive than the spread of control signals through the same regions before blockade of excitatory synapses. Large control responses could be seen in regions distant from the stimulation site where the axonal signals were not detectable. A lack of correlation between control signals and axonal signals revealed by DNQX and APV supports the hypothesis that responses in distal regions of the molecular layer were not dependent on perforant path axons. 4. The perforant path was cut by producing a lesion in the outer two-thirds of the molecular layer. Both dye fluorescence and microelectrode recording showed that stimulation on one side of the lesion could produce signals on the same side as well as across the lesion. The lesion did not block the spread of excitation through the molecular layer. Across the lesion from the site of stimulation, negative-going field potentials were observed to peak in the inner molecular layer, which is the major field of projection of hilar mossy cells. 5. Electrical stimulation in the hilus adjacent to the granule cell layer evoked dye fluorescence responses in the molecular layer. Stimulation at this site evoked negative-going field potentials that peaked in the inner molecular layer. These signals were sensitive to excitatory amino acid receptor antagonists but not to gamma-aminobutyric acid-A (GABAA) receptor antagonists. 6. Activation of excitatory amino acid receptors in the hilus by focal application of (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and N-methyl-D-aspartic acid (NMDA) elicited negative-going field potentials in the granule cell layer and depolarization of granule cells. Field potentials were blocked by tetrodotoxin (TTX), indicating that they were not caused by direct activation of receptors on granule cells, but rather by synapses from hilar neurons on granule cells. 7. These results taken together with previous studies of hilar mossy cells suggest a fundamental circuit consisting of granule cells exciting hilar mossy cells, which then excite more granule cells. This circuit provides positive feedback and can be considered a form of 'recurrent excitation' unique to the dentate gyrus. The robustness of this circuit in hippocampal slices under control conditions suggest that mossy cell excitation of granule cells could play an important role in the normal activity of the hippocampus, and, when inhibition is compromised, this circuit could contribute to the generation and spread of seizures