Brain-derived neurotrophic factor-estrogen interactions in the hippocampal mossy fiber pathway: implications for normal brain function and disease.

The neurotrophin brain-derived neurotrophic factor (BDNF) and the steroid hormone estrogen exhibit potent effects on hippocampal neurons during development and in adulthood. BDNF and estrogen have also been implicated in the etiology of diverse types of neurological disorders or psychiatric illnesses, or have been discussed as potentially important in treatment. Although both are typically studied independently, it has been suggested that BDNF mediates several of the effects of estrogen in the hippocampus, and that these interactions play a role in the normal brain as well as disease. Here we focus on the mossy fiber (MF) pathway of the hippocampus, a critical pathway in normal hippocampal function, and a prime example of a location where numerous studies support an interaction between BDNF and estrogen in the rodent brain. We first review the temporal and spatially regulated expression of BDNF and estrogen in the MFs, as well as their receptors. Then we consider the results of studies that suggest that 17ß-estradiol alters hippocampal function by its influence on BDNF expression in the MF pathway. We also address the hypothesis that estrogen influences the hippocampus by mechanisms related not only to the mature form of BDNF, acting at trkB receptors, but also by regulating the precursor, proBDNF, acting at p75NTR. We suggest that the interactions between BDNF and 17ß-estradiol in the MFs are potentially important in the normal function of the hippocampus, and have implications for sex differences in functions that depend on the MFs and in diseases where MF plasticity has been suggested to play an important role, Alzheimer's disease, epilepsy and addiction.

Vascular endothelial growth factor is up-regulated after status epilepticus and protects against seizure-induced neuronal loss in hippocampus.

Vascular endothelial growth factor (VEGF) is a protein factor which has been found to play a significant role in both normal and pathological states. Its role as an angiogenic factor is well-established. More recently, VEGF has been shown to protect neurons from cell death both in vivo and in vitro. While VEGF's potential as a protective factor has been demonstrated in hypoxia-ischemia, in vitro excitotoxicity, and motor neuron degeneration, its role in seizure-induced cell loss has received little attention. A potential role in seizures is suggested by Newton et al.'s [Newton SS, Collier EF, Hunsberger J, Adams D, Terwilliger R, Selvanayagam E, Duman RS (2003) Gene profile of electroconvulsive seizures: Induction of neurotrophic and angiogenic factors. J Neurosci 23:10841-10851] finding that VEGF mRNA increases in areas of the brain that are susceptible to cell loss after electroconvulsive-shock induced seizures. Because a linear relationship does not always exist between expression of mRNA and protein, we investigated whether VEGF protein expression increased after pilocarpine-induced status epilepticus. In addition, we administered exogenous VEGF in one experiment and blocked endogenous VEGF in another to determine whether VEGF exerts a neuroprotective effect against status epilepticus-induced cell loss in one vulnerable brain region, the rat hippocampus. Our data revealed that VEGF is dramatically up-regulated in neurons and glia in hippocampus, thalamus, amygdala, and neocortex 24 h after status epilepticus. VEGF induced significant preservation of hippocampal neurons, suggesting that VEGF may play a neuroprotective role following status epilepticus.

Acute and chronic responses to the convulsant pilocarpine in DBA/2J and A/J mice.

Characterizing the responses of different mouse strains to experimentally-induced seizures can provide clues to the genes that are responsible for seizure susceptibility, and factors that contribute to epilepsy. This approach is optimal when sequenced mouse strains are available. Therefore, we compared two sequenced strains, DBA/2J (DBA) and A/J. These strains were compared using the chemoconvulsant pilocarpine, because pilocarpine induces status epilepticus, a state of severe, prolonged seizures. In addition, pilocarpine-induced status is followed by changes in the brain that are associated with the pathophysiology of temporal lobe epilepsy (TLE). Therefore, pilocarpine can be used to address susceptibility to severe seizures, as well as genes that could be relevant to TLE. A/J mice had a higher incidence of status, but a longer latency to status than DBA mice. DBA mice exhibited more hippocampal pyramidal cell damage. DBA mice developed more ectopic granule cells in the hilus, a result of aberrant migration of granule cells born after status. DBA mice experienced sudden death in the weeks following status, while A/J mice exhibited the most sudden death in the initial hour after pilocarpine administration. The results support previous studies of strain differences based on responses to convulsants. They suggest caution in studies of seizure susceptibility that are based only on incidence or latency. In addition, the results provide new insight into the strain-specific characteristics of DBA and A/J mice. A/J mice provide a potential resource to examine the progression to status. The DBA mouse may be valuable to clarify genes regulating other seizure-associated phenomena, such as seizure-induced neurogenesis and sudden death.

Perforant path activation of ectopic granule cells that are born after pilocarpine-induced seizures.

Granule cells in the dentate gyrus are born throughout life, and various stimuli can affect their development in the adult brain. Following seizures, for instance, neurogenesis increases greatly, and some new cells migrate to abnormal (ectopic) locations, such as the hilus. Previous electrophysiological studies of this population have shown that they have intrinsic properties that are similar to normal granule cells, but differ in other characteristics, consistent with abnormal integration into host circuitry. To characterize the response of ectopic hilar granule cells to perforant path stimulation, intracellular recordings were made in hippocampal slices from rats that had pilocarpine-induced status epilepticus and subsequent spontaneous recurrent seizures. Comparisons were made with granule cells located in the granule cell layer of both pilocarpine- and saline-treated animals. In addition, a few ectopic hilar granule cells were sampled from saline-treated rats. Remarkably, hilar granule cells displayed robust responses, even when their dendrites were not present within the molecular layer, where perforant path axons normally terminate. The evoked responses of hilar granule cells were similar in several ways to those of normally positioned granule cells, but there were some differences. For example, there was an unusually long latency to onset of responses evoked in many hilar granule cells, especially those without molecular layer dendrites. Presumably this is due to polysynaptic activation by the perforant path. These results indicate that synaptic reorganization after seizures can lead to robust activation of newly born hilar granule cells by the perforant path, even when their dendrites are not in the terminal field of the perforant path. Additionally, the fact that these cells can be found in normal tissue and develop similar synaptic responses, suggests that seizures, while not necessary for their formation, strongly promote their generation and the development of associated circuits, potentially contributing to a lowered seizure threshold.

Spontaneous recurrent seizures after pilocarpine-induced status epilepticus activate calbindin-immunoreactive hilar cells of the rat dentate gyrus.

Although it is now established that neurogenesis of dentate gyrus granule cells increases after experimental seizures, little is currently known about the function of the new granule cells. One question is whether they become integrated into the network around them. Recent experiments that focused on the newly born granule cells in the hilus showed that indeed the new cells appear to become synchronized with host hippocampal neurons [Scharfman et al. (2000) J. Neurosci. 20, 6144-6158]. To address this issue further, we asked whether the new hilar granule cells were active during spontaneous limbic seizures that follow status epilepticus induced by pilocarpine injection. Thus, we perfused rats after spontaneous seizures and stained sections using antibodies to c-fos, a marker of neural activity, and calbindin, a marker of the newly born hilar granule cells [Scharfman et al. (2000) J. Neurosci. 20, 6144-6158]. We asked whether calbindin-immunoreactive hilar neurons were also c-fos-immunoreactive.C-fos was highly expressed in calbindin-immunoreactive hilar neurons. Approximately 23% of hilar cells that expressed c-fos were double-labeled for calbindin. In addition, other types of hilar neurons, i.e. those expressing parvalbumin or neuropeptide Y, also expressed c-fos. Yet other hippocampal neurons, including granule cells and pyramidal cells, had weak expression of c-fos at the latency after the seizure that hilar neuron expression occurred. In controls, there was very little c-fos or calbindin expression in the hilus.These results indicate that calbindin-immunoreactive hilar cells are activated by spontaneous seizures. Based on the evidence that many of these cells are likely to be newly born, the data indicate that new cells can become functionally integrated into limbic circuits involved in recurrent seizure generation. Furthermore, they appear to do so in a manner similar to many neighboring hilar neurons, apparently assimilating into the local environment. Finally, the results show that a number of hilar cell types are activated during chronic recurrent seizures in the pilocarpine model, a surprising result given that many hilar neurons are thought to be damaged soon after pilocarpine-induced status epilepticus.

Survival of dentate hilar mossy cells after pilocarpine-induced seizures and their synchronized burst discharges with area CA3 pyramidal cells.

The clinical and basic literature suggest that hilar cells of the dentate gyrus are damaged after seizures, particularly prolonged and repetitive seizures. Of the cell types within the hilus, it appears that the mossy cell is one of the most vulnerable. Nevertheless, hilar neurons which resemble mossy cells appear in some published reports of animal models of epilepsy, and in some cases of human temporal lobe epilepsy. Therefore, mossy cells may not always be killed after severe, repeated seizures. However, mossy cell survival in these studies was not completely clear because the methods did allow discrimination between mossy cells and other hilar cell types. Furthermore, whether surviving mossy cells might have altered physiology after seizures was not examined. Therefore, intracellular recording and intracellular dye injection were used to characterize hilar cells in hippocampal slices from pilocarpine-treated rats that had status epilepticus and recurrent seizures ('epileptic' rats). For comparison, mossy cells were also recorded from age-matched, saline-injected controls, and pilocarpine-treated rats that failed to develop status epilepticus. Numerous hilar cells with the morphology, axon projection, and membrane properties of mossy cells were recorded in all three experimental groups. Thus, mossy cells can survive severe seizures, and those that survive retain many of their normal characteristics. However, mossy cells from epileptic tissue were distinct from mossy cells of control rats in that they generated spontaneous and evoked epileptiform burst discharges. Area CA3 pyramidal cells also exhibited spontaneous and evoked bursts. Simultaneous intracellular recordings from mossy cells and pyramidal cells demonstrated that their burst discharges were synchronized, with pyramidal cell discharges typically beginning first. From these data we suggest that hilar mossy cells can survive status epilepticus and chronic seizures. The fact that mossy cells have epileptiform bursts, and that they are synchronized with area CA3, suggest a previously unappreciated substrate for hyperexcitability in this animal model.

BDNF and epilepsy: too much of a good thing?

Various studies have shown that brain-derived neurotrophic factor (BDNF) increases neuronal excitability and is localized and upregulated in areas implicated in epileptogenesis. Seizure activity increases the expression of BDNF mRNA and protein, and recent studies have shown that interfering with BDNF signal transduction inhibits the development of the epileptic state in vivo. These results suggest that BDNF contributes to epileptogenesis. Further analysis of the cellular and molecular mechanisms by which BDNF influences excitability and connectivity in adult brain could provide novel concepts and targets for anticonvulsant or anti-epileptogenic therapy.

Electrophysiological effects of exogenous and endogenous kynurenic acid in the rat brain: studies in vivo and in vitro.

In this review, recent studies on the electrophysiological effects of de novo synthesized ("endogenous") kynurenic acid (KYNA) are discussed. Endogenous KYNA is normally formed as a byproduct of tryptophan metabolism. Evidence for a physiological role in neuronal excitability has not been strong, in part because brain levels are much lower than the KD of KYNA at the glycine site of the NMDA receptor, where KYNA is thought to exert its most potent effect. The results suggest that, unexpectedly, even low concentrations of endogenous KYNA have physiological consequences. These levels of KYNA reduced the number of hippocampal slices with spontaneous epileptiform discharges after exposure to buffer lacking magnesium. However, effects on evoked responses to single afferent stimuli were not detected. Taken together, the data argue for a potentially important role of endogenous KYNA in suppression of seizure-like activity, and suggest a novel approach to anticonvulsant drug development that could have few side effects.

Granule-like neurons at the hilar/CA3 border after status epilepticus and their synchrony with area CA3 pyramidal cells: functional implications of seizure-induced neurogenesis.

A group of neurons with the characteristics of dentate gyrus granule cells was found at the hilar/CA3 border several weeks after pilocarpine- or kainic acid-induced status epilepticus. Intracellular recordings from pilocarpine-treated rats showed that these "granule-like" neurons were similar to normal granule cells (i. e., those in the granule cell layer) in membrane properties, firing behavior, morphology, and their mossy fiber axon. However, in contrast to normal granule cells, they were synchronized with spontaneous, rhythmic bursts of area CA3 pyramidal cells that survived status epilepticus. Saline-treated controls lacked the population of granule-like cells at the hilar/CA3 border and CA3 bursts. In rats that were injected after status epilepticus with bromodeoxyuridine (BrdU) to label newly born cells, and also labeled for calbindin D(28K) (because it normally stains granule cells), many double-labeled neurons were located at the hilar/CA3 border. Many BrdU-labeled cells at the hilar/CA3 border also were double-labeled with a neuronal marker (NeuN). Taken together with the recent evidence that granule cells that are born after seizures can migrate into the hilus, the results suggest that some newly born granule cells migrate as far as the CA3 cell layer, where they become integrated abnormally into the CA3 network, yet they retain granule cell intrinsic properties. The results provide insight into the physiological properties of newly born granule cells in the adult brain and suggest that relatively rigid developmental programs set the membrane properties of newly born cells, but substantial plasticity is present to influence their place in pre-existing circuitry.

Epileptogenesis in the parahippocampal region. Parallels with the dentate gyrus.

Limbic seizures have often been attributed to pathology in the hippocampus, such as the well described condition termed Ammon's Horn sclerosis, in which many of the hippocampal principal cells have degenerated. However, several studies in both the clinical and basic literature indicate that the parahippocampal region may also play an important role. This region sustains a characteristic pattern of damage in most animal models of epilepsy that is similar to that identified in humans with intractable temporal lobe epilepsy. Perhaps the most striking aspect of parahippocampal pathology is the marked loss of neurons in layer III of the entorhinal cortex. The similarity of cell loss in layer III and cell loss in the hilus of the dentate gyrus is compared, as is the characteristic resistance of layer II neurons and dentate granule cells. Cellular electrophysiological results are used as a basis for the hypothesis that synaptic inhibition plays a role in the relative vulnerability of these neurons. Studies of neurogenesis in both areas is also discussed. It is proposed that this may be an additional factor that influences vulnerability in these areas.

Kynurenergic manipulations influence excitatory synaptic function and excitotoxic vulnerability in the rat hippocampus in vivo.

Competing enzymatic mechanisms degrade the tryptophan metabolite L-kynurenine to kynurenate, an inhibitory and neuroprotective compound, and to the neurotoxins 3-hydroxykynurenine and quinolinate. Kynurenine 3-hydroxylase inhibitors such as PNU 156561 shift metabolism towards enhanced kynurenate production, and this effect may underlie the recently discovered anticonvulsant and neuroprotective efficacy of these drugs. Using electrophysiological and neurotoxicological endpoints, we now used PNU 156561 as a tool to examine the functional interplay of kynurenate, 3-hydroxykynurenine and quinolinate in the rat hippocampus in vivo. First, population spike amplitude in area CA1 and the extent of quinolinate-induced excitotoxic neurodegeneration were studied in animals receiving acute or prolonged intravenous infusions of L-kynurenine, PNU 156561, (L-kynurenine+PNU 156561) or kynurenate. Only the latter two treatments, but not L-kynurenine or PNU 156561 alone, caused substantial inhibition of evoked responses in area CA1, and only prolonged (3h) infusion of (L-kynurenine+PNU 156561) or kynurenate was neuroprotective. Biochemical analyses in separate animals revealed that the levels of kynurenate attained in both blood and brain (hippocampus) were essentially identical in rats receiving extended infusions of L-kynurenine alone or (L-kynurenine+PNU 156561) (4 and 7microM, respectively, after an infusion of 90 or 180min). However, addition of the kynurenine 3-hydroxylase inhibitor resulted in a significant decrement in the formation of 3-hydroxykynurenine and quinolinate in both blood and brain. These data suggest that the ratio between kynurenate and 3-hydroxykynurenine and/or quinolinate in the brain is a critical determinant of neuronal excitability and viability. The anticonvulsant and neuroprotective potency of kynurenine 3-hydroxylase inhibitors may therefore be due to the drugs' dual action on both branches of the kynurenine pathway of tryptophan degradation.

Quantitative differences in the effects of de novo produced and exogenous kynurenic acid in rat brain slices.

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.

Brain-derived neurotrophic factor transgenic mice exhibit passive avoidance deficits, increased seizure severity and in vitro hyperexcitability in the hippocampus and entorhinal cortex.

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.

Actions of brain-derived neurotrophic factor in slices from rats with spontaneous seizures and mossy fiber sprouting in the dentate gyrus.

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.

Chronic changes in synaptic responses of entorhinal and hippocampal neurons after amino-oxyacetic acid (AOAA)-induced entorhinal cortical neuron loss.

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.

Effects of central and peripheral administration of kynurenic acid on hippocampal evoked responses in vivo and in vitro.

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.

Hyperexcitability in combined entorhinal/hippocampal slices of adult rat after exposure to brain-derived neurotrophic factor.

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.