NT-3 modulates BDNF and proBDNF levels in naïve and kindled rat hippocampus.

Both mature and precursor forms of neurotrophins regulate nerve development, survival and plasticity. Brain-derived neurotrophic factor (BDNF) synthesis and secretion in turn are regulated by neuronal activity, such as epilepsy. Further, neurotrophins themselves are regulated by neurotrophin levels. Neurotrophin-3 (NT-3) and BDNF in particular can be co-expressed and each can regulate the levels of the other. This regulation is thought to be mediated through receptor tyrosine kinase (Trk) activity. It is not known whether this neurotrophin-neurotrophin interaction occurs in hippocampal tissue in vivo, or how it is influenced by neuronal activation. In this study, we explored the reciprocal influences of intraventricular infusions of NT-3 and BDNF in naïve and kindled hippocampi of rats using Western blotting. We confirm that hippocampal kindling resulted in a significant increase in levels of BDNF both in cytochrome C (control) infused and NT-3 infused kindled rats. However, NT-3 infusion significantly reduced BDNF levels in both kindled and non-kindled hippocampi compared to their cytochrome C infused counterparts. These results are consistent with our earlier studies demonstrating lowered levels of TrkA and TrkC (NGF modulates BDNF levels via TrkA) following chronic NT-3 infusion. Although kindling led to an increase in BDNF, this was not accompanied by any detectable change in the levels of proBDNF. However, there was a significant increase in proBDNF following NT-3 infusions, suggesting NT-3 may reduce proBDNF processing. In contrast, neither NT-3 nor proNT-3 levels were affected by kindling or chronic BDNF infusions, consistent with down-regulation of TrkB by chronic BDNF infusion. Thus, modulation of BDNF by NT-3, likely mediated by Trk receptors, occurs in naïve and kindled adult rat hippocampus.

Long-term depression and associativity in rat primary motor cortex following thalamic stimulation.

Associativity is an attractive property of LTP in terms of its possible mechanism as a model for memory storage. In this study, we compare the effects of homosynaptic vs. associative stimulation on the induction of LTP and LTD in the neocortex of freely behaving rats. Using a callosal input to the motor cortex as a 'strong' input (one that potentiates reliably following homosynaptic stimulation), we paired activity of this pathway with a 'weak' thalamocortical pathway (one that does not potentiate when stimulated homosynaptically). Surprisingly, homosynaptic HFS caused a lasting depression of the field EPSP in the thalamocortical pathway. Analysis of this effect revealed that it was largely polysynaptic. Associative HFS (HFS applied to both pathways) not only failed to induce an LTP effect in the thalamocortical pathway, it increased the magnitude of the depression. Associative HFS did, however, facilitate LTP induction in the 'strong' callosal pathway. When comparing the effects of homosynaptic and associative LTD induction (HFS on one pathway anticorrelated with LFS on the other), we found that both protocols induced a similar magnitude of depression. These results show that HFS applied to the thalamocortical pathway causes a depression and this depression is enhanced, not reversed, by associative pairing with a strong input.

Training-induced and electrically induced potentiation in the neocortex.

Long-term potentiation (LTP) shares many properties with memory and is currently the most popular laboratory model of memory. Although it has not been proven that memory is based on an LTP-like mechanism, there is evidence that learning a motor skill can induce LTP-like effects. This evidence was obtained in a slice-preparation experiment, which precluded within-animal comparisons before and after training. In the present experiments, Long-Evans rats were unilaterally trained to acquire a forelimb reaching and grasping skill. Evoked potentials were found to be larger in motor cortex layer II/III in the trained, compared to the untrained, hemisphere in slice, acute, and chronic preparations. Consistent with previous research, the trained hemisphere was less amenable to subsequent LTP induction. Furthermore, the application of either LTP- or LTD-inducing stimulation during the training phase of the reaching task disrupted the acquisition of the skill, providing further evidence that memory may be based on an LTP mechanism.

N-methyl-D-aspartate receptor-independent long-term depression and depotentiation in the sensorimotor cortex of the freely moving rat.

Bidirectional modifications in synaptic efficacy are central components in recent models of cortical learning and memory, and we previously demonstrated both long-term synaptic potentiation (LTP) and long-term synaptic depression (LTD) in the neocortex of the unanaesthetized adult rat. Here, we have examined the effects of N-methyl-D-aspartate receptor (NMDAR) blockade on the induction of LTD, LTP, and depotentiation of field potentials evoked in sensorimotor cortex by stimulation of the white matter in the adult, freely moving rat. High frequency (300 Hz) stimulation (HFS) was used to induce LTP and prolonged, low-frequency (1 Hz) stimulation was used to induce either depotentiation or LTD. LTD was expressed as a reduction in the amplitude of the short and long-latency field potential components, while depotentiation was expressed as a decrease in the amplitude of a previously enhanced late component. Under NMDAR blockade, HFS failed to induce LTP and instead produced a depression effect similar to LTD. Following washout of the drug, HFS induced a normal LTP effect. Unlike LTP, LTD and depotentiation were found to be NMDAR-independent in the neocortex of the freely moving rat.

Strain differences affect the induction of status epilepticus and seizure-induced morphological changes.

Genetic deficits have been discovered in human epilepsy, which lead to alteration of the balance between excitation and inhibition, and ultimately result in seizures. Rodents show similar genetic determinants of seizure induction. To test whether seizure-prone phenotypes exhibit increased seizure-related morphological changes, we compared two standard rat strains (Long-Evans hooded and Wistar) and two specially bred strains following status epilepticus. The special strains, namely the kindling-prone (FAST) and kindling-resistant (SLOW) strains, were selectively bred based on their amygdala kindling rate. Although the Wistar and Long-Evans hooded strains experienced similar amounts of seizure activity, Wistar rats showed greater mossy fiber sprouting and hilar neuronal loss than Long-Evans hooded rats. The mossy fiber system was affected differently in FAST and SLOW rats. FAST animals showed more mossy fiber granules in the naïve state, but were more resistant to seizure-induced mossy fiber sprouting than SLOW rats. These properties of the FAST strain are consistent with those observed in juvenile animals, further supporting the hypothesis that the FAST strain shares circuit properties similar to those seen in immature animals. Furthermore, the extent of mossy fiber sprouting was not well correlated with sensitivity to status epilepticus, but was positively correlated with the frequency of spontaneous recurrent seizures in the FAST rats only, suggesting a possible role for axonal sprouting in the development of spontaneous seizures in these animals. We conclude that genetic factors clearly affect seizure development and related morphological changes in both standard laboratory strains and the selectively bred seizure-prone and seizure-resistant strains.

The effects of brain-derived neurotrophic factor (BDNF) administration on kindling induction, Trk expression and seizure-related morphological changes.

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family that mediates synaptic plasticity and excitability in the CNS. Recent evidence has shown that increased BDNF levels can lead to hyperexcitability and epileptiform activities, while suppression of BDNF function in transgenic mice or by antagonist administration retards the development of seizures. However, several groups, including our own, have reported that increasing BDNF levels by continuous intrahippocampal infusion inhibits epileptogenesis. It is possible that the continuous administration of BDNF produces a down-regulation of its high-affinity TrkB receptor, leading to a decrease of neuronal responsiveness to BDNF. If so, then animals should respond differently to bolus injections of BDNF, which presumably do not alter Trk expression, compared with continuous infusion. To test this hypothesis, we compared the effects of intrahippocampal BDNF continuous infusion and bolus injections on kindling induction. We showed that continuous infusion of BDNF inhibited the development of behavioral seizures and decreased the level of phosphorylated Trks or TrkB receptors. In contrast, multiple bolus microinjections of BDNF accelerated kindling development and did not affect the level of phosphorylated Trks or TrkB receptors. Our results indicate that different administration protocols yield opposite effects of BDNF on neuronal excitability, epileptogenesis and Trk expression. Unlike nerve growth factor and neurotrophin-3, which affect mossy fiber sprouting, we found that BDNF administration had no effect on the mossy fiber system in naive or kindled rats. Such results suggest that the effects of BDNF on epileptogenesis are not modulated by its effect on sprouting, but rather by its effects on excitability.

EphA/ephrin-A interactions regulate epileptogenesis and activity-dependent axonal sprouting in adult rats.

The Eph family of tyrosine kinase receptors and their ligands, ephrins, are distributed in gradients and serve as molecular guidance cues for axonal patterning during neuronal development. Most of these molecules are also expressed in mature brain. Thus, we examine here the potential roles of such molecules in plasticity and activity-dependent mossy fiber sprouting of adult CNS. We show that the ligand ephrin-A3 and the receptor EphA5 are expressed in complementary gradients in the adult rat mossy fiber system. Using the kindling model, we demonstrate that exogenous immunoadhesins that affect the interaction of endogenous EphA receptors and ephrin-A ligands modulate the development of kindling, one type of long-term plasticity, in mature rat brain. These immunoadhesins, combined with epileptogenic stimulations, alter both the extent and the pattern of collateral axonal sprouting in the mossy fiber pathway. Our results suggest that EphA receptors and ephrin-A ligands modify neuronal plasticity and may serve as spatial cues that modulate the development and pattern of activation-dependent axonal growth in adult CNS.

A ligand of the p65/p95 receptor suppresses perforant path kindling, kindling-induced mossy fiber sprouting, and hilar area changes in adult rats.

Kindling, an animal model of epilepsy, results in an increased volume of the hilus of the dentate gyrus and sprouting of the mossy fiber pathway in the hippocampus. Our previous studies have revealed that chronic infusion of neurotrophins can regulate not only seizure development, but also these kindling-induced structural changes. Kindling, in turn, can alter the expression of neurotrophins and their receptors. We previously showed that intraventricular administration of a synthetic peptide that interferes with nerve growth factor stability and thus its binding to TrkA and p75(NTR) receptors suppressed kindling and sprouting. However, the precise involvement of TrkA, p75(NTR), and downstream signaling effectors of neurotrophins on kindling, sprouting and hilar changes are unknown. One of these downstream effectors is Ras. In the present study, we find that intraventricular infusion of the synthetic peptide Reo3Y, which binds to p65/p95 receptors and causes a rapid inactivation of Ras protein, impairs development of perforant path kindling, reduces the growth in afterdischarge duration, blocks kindling-induced mossy fiber sprouting in area CA3 of hippocampus and in inner molecular layer of the dentate gyrus, and prevents kindling-induced increases in hilar area. These results are consistent with a mediation of neurotrophin effects on kindling, hilar area, and axonal sprouting via Trk receptors, and suggest important roles for Ras in kindling and in kindling-induced structural changes.

Activity-dependent changes in synaptophysin immunoreactivity in hippocampus, piriform cortex, and entorhinal cortex of the rat.

Synaptophysin, an integral membrane glycoprotein of synaptic vesicles, has been widely used to investigate synaptogenesis in both animal models and human patients. Kindling is an experimental model of complex partial seizures with secondary generalization, and a useful model for studying activation-induced neural growth in adult systems. Many studies using Timm staining have shown that kindling promotes sprouting in the mossy fiber pathway of the dentate gyrus. In the present study, we used synaptophysin immunohistochemistry to demonstrate activation-induced neural sprouting in non-mossy fiber cortical pathways in the adult rat. We found a significant kindling-induced increase in synaptophysin immunoreactivity in the stratum radiatum of CA1 and stratum lucidum/radiatum of CA3, the hilus, the inner molecular layer of the dentate gyrus, and layer II/III of the piriform cortex, but no significant change in layer II/III of the entorhinal cortex, 4 weeks after the last kindling stimulation. We also found that synaptophysin immunoreactivity was lowest in CA3 near the hilus and increased with increasing distance from the hilus, a reverse pattern to that seen with Timm stains in stratum oriens following kindling. Furthermore, synaptophysin immunoreactivity was lowest in dorsal and greatest in ventral sections of both CA3 and dentate gyrus in both kindled and non-kindled animals. This demonstrates that different populations of sprouting axons are labeled by these two techniques, and suggests that activation-induced sprouting extends well beyond the hippocampal mossy fiber system.

Continuous infusion of neurotrophin-3 triggers sprouting, decreases the levels of TrkA and TrkC, and inhibits epileptogenesis and activity-dependent axonal growth in adult rats.

Neurotrophin-3 (NT-3), a member of the neurotrophin family of neurotrophic factors, is important for cell survival, axonal growth and neuronal plasticity. Epileptiform activation can regulate the expression of neurotrophins, and increases or decreases in neurotrophins can affect both epileptogenesis and seizure-related axonal growth. Interestingly, the expression of nerve growth factor and brain-derived neurotrophic factor is rapidly up-regulated following seizures, while NT-3 mRNA remains unchanged or undergoes a delayed down-regulation, suggesting that NT-3 might have a different function in epileptogenesis. In the present study, we demonstrate that continuous intraventricular infusion of NT-3 in the absence of kindling triggers mossy fiber sprouting in the inner molecular layer of the dentate gyrus and the stratum oriens of the CA3 region. Furthermore, despite this NT-3-related sprouting effect, continuous infusion of NT-3 retards the development of behavioral seizures and inhibits kindling-induced mossy fiber sprouting in the inner molecular layer of the dentate gyrus. We also show that prolonged infusion of NT-3 leads to a decrease in kindling-induced Trk phosphorylation and a down-regulation of the high-affinity Trk receptors, TrkA and TrkC, suggesting an involvement of both cholinergic nerve growth factor receptors and hippocampal NT-3 receptors in these effects. Our results demonstrate an important inhibitory role for NT-3 in seizure development and seizure-related synaptic reorganization.

Sequential changes in the synaptic structural profile following long-term potentiation in the rat dentate gyrus: III. Long-term maintenance phase.

LTP has been associated with changes in synaptic morphology but the nature of these changes over the time course of the enhanced electrophysiological response has not been fully determined. The current research involved an examination of synaptic structure in the rat hippocampus during the long-term maintenance phase of LTP. Synapses were examined in the middle third of the molecular layer (MML) of the rat dentate gyrus following repeated high frequency tetanization of the perforant path. Synapses from both the ipsilateral inner third of the dentate molecular layer (IML), which was not directly stimulated during the induction of LTP, as well as implanted, nonstimulated animals, served as controls. LTP was induced over a 4-h period, and the animals were sacrificed 5 days after the final stimulation of the LTP group. Ultrastructural quantification included the total number of synapses per neuron, synaptic curvature, the presence of synaptic perforations, and the maximum length of the synapses. No overall changes in the number of synapses per neuron, shape, or synaptic perforations were observed. There was, however, a significant increase in the length of synapses in the directly stimulated LTP tissue. This increase in synaptic length was particularly evident in the concave-shaped synapses which were also more perforated. These results, together with previous findings, describe a sequence of changes in synaptic morphology that accompany LTP in a structure that is associated with learning and memory.

Cholinergic modulation of neocortical long-term potentiation in the awake, freely moving rat.

The neocortex has proven resistant to LTP induction using standard in vitro and acute, in vivo preparations. Because the neocortex is widely thought to be involved in long-term information storage, this resistance raises questions about the validity of LTP as a memory model. Recently, we have shown that the neocortex of freely moving rats reliably supports LTP, provided that the stimulation is spaced and repeated over days. The following experiments were designed to evaluate the neuromodulatory role played by cholinergic systems in the induction of LTP in this preparation. Chronically implanted rats received either low- or high-intensity LTP-inducing tetani in combination with the administration of either a cholinergic agonist or antagonist injected systemically. Potentiation was evidenced as amplitude changes in both early and late components of the evoked field potential, the former including population spikes. The cholinergic agonist facilitated LTP induction in the late component of both high- and low-intensity groups. The cholinergic antagonist blocked LTP induction in the early component of the high-intensity group. The possibility that there are component-specific modulatory effects of cholinergic agents on the induction of neocortical LTP is discussed.

Morphology of layer III pyramidal neurons is altered following induction of LTP in sensorimotor cortex of the freely moving rat.

The organization of specific cortical connections can be altered by sensory and motor experience. These changes are believed to result from activity-dependent changes in synaptic connectivity, similar to those induced in the hippocampus by high-frequency stimulation in long-term potentiation (LTP) experiments. If similar mechanisms are involved, then neocortical LTP induction may induce some of the same morphological changes that are seen following learning. We induced LTP in the contralateral sensorimotor cortex by repeated, daily tetanization of the corpus callosum in chronically implanted, freely moving rats. Anatomical results showed that the LTP induction was associated with alterations in dendrite morphology and increased spine density. These changes are qualitatively and quantitatively similar to those commonly observed in studies in which rats are housed in complex environments. The similarity of results following exposure to complex environments and after LTP induction in the neocortex may indicate a reliance on the same cellular mechanisms in both situations.

Long-term potentiation in the reciprocal corticohippocampal and corticocortical pathways in the chronically implanted, freely moving rat.

The hippocampus and adjacent cortical structures, including the entorhinal, perirhinal, and parahippocampal cortices, appear to serve as an integrated memory system. This extended hippocampal system is believed to influence memory and consolidation through an extensive set of reciprocal connections with widespread areas of the neocortex. Long-term potentiation (LTP) has been well-examined in the intrinsic connections of the hippocampus and neocortex. However, LTP in the pathways and structures thought to convey information between the hippocampus and neocortex has received little attention. If these pathways and structures are involved in information storage, and if LTP reflects a general synaptic encoding mechanism, then these systems are also likely to support LTP. In this paper we discuss a series of experiments aimed at investigating LTP in the efferents between the hippocampus and neocortex in chronically implanted animals. In the first experiment, the efferents of the perirhinal cortex were stimulated. LTP in the dentate gyrus (DG) reached asymptote more slowly than is typically seen following perforant path stimulation, whereas the frontal area (M1) reached asymptote more quickly than reported following corticocortical stimulation. The DG and M1 LTP was long-lasting, but entorhinal cortex LTP had decayed to baseline levels after a week. In the second experiment, the hippocampal efferents were stimulated. The perirhinal, entorhinal, and frontal cortex showed a similar slow potentiation, with only the perirhinal cortex levels returning to baseline after a week. In the third experiment, the projections from M1 were tested. The perirhinal cortex and hippocampus showed a long-lasting LTP. Although LTP was found in all pathways examined, there were differences in the induction and decay rate, and these properties may correspond to differences in learning rate and longevity of information storage.

Sequential changes in the synaptic structural profile following long-term potentiation in the rat dentate gyrus. II. Induction/early maintenance phase.

Long-term potentiation (LTP), one of the most compelling models of learning and memory, has been associated with changes in synaptic morphology. In this study, LTP was induced and animals were sacrificed 1 h after the stimulation of the LTP group (induction / early maintenance phase). Synapses in the directly stimulated middle third of the dentate gyrus molecular layer (MML) were examined while synapses from the inner third of the dentate molecular layer (IML) of the LTP animals and both the MML and the IML of implanted animals served as controls. The total number of synapses per neuron, synaptic curvature, the presence of synaptic perforations, and the maximum length of the synaptic contact and active zone were examined. No overall change in the number of synapses per neuron was observed in the LTP tissue. LTP was associated with a significant increase in the proportion of perforated and irregular-shaped synapses compared to controls. The increase in perforated synapses was particularly apparent in the proportion of concave perforated synapses. Nonperforated concave synapses were found to be significantly larger in potentiated tissue. The total synaptic length per neuron of synapses in a concave configuration was also significantly higher following potentiation. These results suggest that the specific structural profile associated with 1-h post-LTP induction, which differed from the profile observed at 24 h post-induction, may represent a unique early phase of synaptic remodeling in a series of changes observed during LTP induction, maintenance, and decay.

Long-term depression and depotentiation in the sensorimotor cortex of the freely moving rat.

Activity-dependent reductions in synaptic efficacy are central components of recent models of cortical learning and memory. Here, we have examined long-term synaptic depression (LTD) and the reversal of long-term potentiation (depotentiation) of field potentials evoked in sensorimotor cortex by stimulation of the white matter in the adult, freely moving rat. Prolonged, low-frequency stimulation (1 Hz for 15 min) was used to induce either depotentiation or LTD. LTD was expressed as a reduction in the amplitude of both monosynaptic and polysynaptic field potential components. Both LTD and depotentiation were reliably induced by stimulation of the ipsilateral white matter. Stimulation of the contralateral neocortex induced only a depotentiation effect, which decayed more rapidly than that induced by ipsilateral stimulation (hours vs days). Although ipsilateral LTD was effectively induced by a single session of low-frequency stimulation, multiple sessions of stimulation, either massed or spaced, induced LTD effects that were larger in magnitude and longer lasting. Previously, we showed that the induction of long-term potentiation in the neocortex of chronic preparations required multiple, spaced stimulation sessions to reach asymptotic levels. Here, we report that LTD also required multiple stimulation sessions to reach asymptotic levels, but massed and spaced patterns of low-frequency stimulation were equally effective.

GABAergic modulation of neocortical long-term potentiation in the freely moving rat.

Although the neocortex has generally been considered resistant to the induction of long-term potentiation (LTP), we have recently shown that LTP can be reliably induced in the freely moving rat provided that the stimulation sessions are spaced and repeated. Here, we report that the induction of LTP in this preparation can be modulated by both GABAergic agonism and antagonism. The delivery of stimulation trains in the presence of the GABA(A) agonist diazepam blocked the induction of neocortical LTP, while the GABA(A) antagonist picrotoxin slowed the development of potentiation. When animals that had previously received high-frequency stimulation combined with diazepam were repotentiated, they showed greater resistance to LTP induction than animals that had received diazepam alone. These data suggest that the inhibitory circuits themselves may have potentiated. The demonstration that diazepam blocks neocortical LTP provides further support for the notion that LTP plays a role in memory formation.

Mossy fiber sprouting induced by repeated electroconvulsive shock seizures.

The elicitation of repeated focal seizures (kindling) induces mossy fiber sprouting in the hippocampus of the rat. The present study investigated whether repeated generalized seizures also induce mossy fiber sprouting. Human psychiatric patients receive repeated generalized seizures during electroconvulsive therapy (ECT). Male Long-Evans rats received a course of eight electroconvulsive shock (ECS) seizures administered on a 48-h schedule over a course of 2 1/2 weeks. Control subjects received matched handling, but no stimulation. Fourteen days after the last ECS trial, all subjects were sacrificed and their brains subjected to Timm staining. Cell counts and area measures were also taken in the hilus. Significant sprouting, but not significant cell loss, was seen in the fascia dentata of the subjects that had received ECS.

Multiple window time-frequency distribution and coherence of EEG using Slepian sequences and hermite functions.

Multiple window (MW) time-frequency analysis (TFA) is a newly developed technique to estimate a time-varying spectrum for random nonstationary signals with low bias and variance. In this paper, we describe the application of MW-TFA techniques to electroencephalogram (EEG) and compare the results with those of the conventional spectrogram. We find that the MW-TFA provide us with not only low bias and variance time-frequency (TF) distribution for EEG but also TF coherence estimation between a single realization of EEG recorded from two sites. We also compare the performance of the MW-TFA using two sets of windows, Slepian sequences, and Hermite functions. If care is taken in matching the two windows, we find no noticeable difference in the resulting TF representations.

Development of kindling-prone and kindling-resistant rats: selective breeding and electrophysiological studies.

Because of the growing need for an animal model of complex partial seizures based on a genetic predisposition, we combined the kindling model of epilepsy with selective-breeding procedures to develop two new lines (or strains) of rats that are kindling-prone or kindling-resistant. The selection of these strains was based on their rates of amygdala kindling. From a parent population of Long Evans hooded and Wistar rats, the males and females that showed the fastest and slowest amygdala kindling rates were selected and bred. Similar selection procedures continued through F11, although there was little or no overlap in the distribution of kindling rates for the two new strains (FAST and SLOW) by F6. Examination of both local and propagating seizure profiles of the new strains from F6 to F10 revealed that the FAST and SLOW rats had similar amygdala afterdischarge (AD) thresholds and associated AD durations. Also, the convulsion profiles of the stage-5 responses were similar, although the severity was greater in the FAST rats. Clearly the selection was not based on local mechanisms controlling the threshold for amygdala AD evocation, but rather for the spread of AD from the focus and the recruitment of other structures, ultimately triggering convulsive seizures. Although evoked potentials and potentiation effects were similar between the strains, the SLOW rats showed a greater paired-pulse depression, raising the possibility that they differ in inhibitory mechanisms. The specificity of strain differences for the amygdala and its associated networks is described in our accompanying paper (McIntyre et al., 1999. FAST and SLOW amygdala kindling rat strains: Comparison of amygdala, hippocampal, piriform and perirhinal cortex kindling. Epilepsy Res. 35, 197-209). These strains should provide many clues to the dispositional differences between individuals for the development of epilepsy originating in temporal lobe structures.