Differential upregulation of extracellular matrix molecules associated with the appearance of granule cell dispersion and mossy fiber sprouting during epileptogenesis in a murine model of temporal lobe epilepsy.

We have investigated changes in the extracellular matrix of the hippocampus associated with the early progression of epileptogenesis in a murine model of temporal lobe epilepsy using immunohistochemistry. In the first week following intrahippocampal injection of the glutamate agonist, domoate, there is a latent period at the end of which begins a sequential upregulation of extracellular matrix (ECM) molecules in the granule cell layer of the dentate gyrus, beginning with neurocan and tenascin-C. This expression precedes the characteristic dispersion of the granule cell layer which is evident at 14 days post-injection when the first recurrent seizures can be recorded. At this stage, an upregulation of the chondroitin sulfate proteoglycan, phosphacan, the DSD-1 chondroitin sulfate motif, and the HNK-1 oligosaccharide are also observed. The expression of these molecules is localized differentially in the epileptogenic dentate gyrus, especially in the sprouting molecular layer, where a strong upregulation of phosphacan, tenascin-C, and HNK-1 is observed but there is no expression of the proteoglycan, neurocan, nor of the DSD-1 chondroitin sulfate motif. Hence, it appears that granule cell layer dispersion is accompanied by a general increase in the ECM, while mossy fiber sprouting in the molecular layer is associated with a more restricted repertoire. In contrast to these changes, the expression of the ECM glycoproteins, laminin and fibronectin, both of which are frequently implicated in tissue remodelling events, showed no changes associated with either granule cell dispersion or mossy fiber sprouting, indicating that the epileptogenic plasticity of the hippocampus is accompanied by ECM interactions that are characteristic of the CNS.

Neuropeptide Y and epilepsy: varying effects according to seizure type and receptor activation.

In vitro and in vivo experiments suggest antiepileptic properties for NPY. In this study, the pharmacology of these effects was examined and compared in different rat models of seizures. Agonists for Y(1), Y(2) and Y(5) receptors reduced seizure-like activity in hippocampal cultures. Intracerebral injection of NPY or Y(5) agonists reduced the expression of focal seizures produced by a single electrical stimulation of the hippocampus. Conversely, NPY agonists increased the duration of generalized convulsive seizures induced by pentylenetetrazol. These results suggest that NPY reduces seizures of hippocampal origin through activation of Y(5) receptors. They also point to probable modulatory effects of NPY in brain structures other than the hippocampus, involved in initiation, propagation or control of seizures.

Brain-derived neurotrophic factor delays hippocampal kindling in the rat.

Epileptic seizures increase the expression of brain-derived neurotrophic factor in the hippocampus. Since this neurotrophin exerts modulatory effects on neuronal excitability in this structure, it may play an important role in hippocampal epileptogenesis. This question was addressed by studying the effects of chronic infusions of recombinant brain-derived neurotrophic factor and brain-derived neurotrophic factor antisense in the hippocampus during the first seven days of hippocampal kindling. Infusion with brain-derived neurotrophic factor (6-24 microg/day) significantly delayed the progression of standard hippocampal kindling and strongly suppressed seizures induced by rapid hippocampal kindling. These suppressive effects were dose dependent, long lasting, not secondary to neuronal toxicity and specific to this neurotrophin, as nerve growth factor accelerated hippocampal kindling progression. They also appeared to be specific to the hippocampus, as infusion of brain-derived neurotrophic factor (48 microg/day) in the amygdala only resulted in a slight and transient delay of amygdala kindling. Conversely to the protective effects of exogenous brain-derived neurotrophic factor, chronic hippocampal infusion of antisense oligodeoxynucleotides (12 nmol/day), resulting in reduced expression of endogenous brain-derived neurotrophic factor in the hippocampus, aggravated seizures during hippocampal kindling. Taken together, our results lead us to suggest that the seizure-induced increase in brain-derived neurotrophic factor expression in the hippocampus may constitute an endogenous regulatory mechanism able to restrain hippocampal epileptogenesis.

Endogenous control of hippocampal epileptogenesis: a molecular cascade involving brain-derived neurotrophic factor and neuropeptide Y.

PURPOSE: Seizures increase the expression of brain-derived neurotrophic factor (BDNF) in the hippocampus. Because this neurotrophin exerts modulatory effects on hippocampal neuronal excitability, it may play an important role in epileptogenesis initiated in this structure. Moreover BDNF is known to regulate the expression of neuropeptide Y (NPY), which displays modulatory properties on seizure activity. This suggests that the effects of BDNF on epileptogenesis may be mediated by NPY. METHODS: Adult male rats received a 7-day chronic intrahippocampal infusion of BDNF, BDNF antisense oligodeoxynucleotides, NPY, or anti-NPY immunoglobulin G during kindling of the hippocampus. The long-term regulation of NPY expression by BDNF was also studied by immunohistochemistry and radioimmunoassay. RESULTS: BDNF applied during the first week of hippocampal stimulation significantly delayed the progression of kindling, an effect that outlasted the end of the infusion by at least 7 days. Conversely, infusion of BDNF antisense oligodeoxynucleotides to reduce the expression of endogenous BDNF in the hippocampus aggravated the electroencephalographic expression of seizures. Chronic infusion of BDNF increased the expression of NPY in the hippocampus, with a time course similar to that of the protective effect of the neurotrophin on kindling. Finally, chronic infusion of NPY in the hippocampus delayed the progression of hippocampal kindling, whereas anti-NPY antibodies had an aggravating effect. CONCLUSIONS: Our results suggest that the seizure-induced increase in BDNF expression in the hippocampus may constitute an endogenous protective mechanism able to counteract hippocampal epileptogenesis. This protective effect appears to be mediated at least in part through the regulation of NPY expression.

Overexpression of neuropeptide Y induced by brain-derived neurotrophic factor in the rat hippocampus is long lasting.

Brain-derived neurotrophic factor (BDNF) plays an important role in hippocampal neuroplasticity. In particular, BDNF upregulation in the hippocampus by epileptic seizures suggests its involvement in the neuronal rearrangements accompanying epileptogenesis. We have shown previously that chronic infusion of BDNF in the hippocampus induces a long-term delay in hippocampal kindling progression. Although BDNF has been shown to enhance the excitability of this structure upon acute application, long-term transcriptional regulations leading to increased inhibition within the hippocampus may account for its suppressive effects on epileptogenesis. Therefore, the long-term consequences of a 7-day chronic intrahippocampal infusion of BDNF (12 microg/day) were investigated up to 2 weeks after the end of the infusion, on the expression of neurotransmitters contained in inhibitory hippocampal interneurons and which display anti-epileptic properties. Our results show that BDNF does not modify levels of immunostaining for glutamic acid decarboxylase, the rate-limiting enzyme for gamma-aminobutyric acid (GABA) synthesis, and somatostatin. Conversely, BDNF induces a long-lasting increase of neuropeptide Y (NPY) in the hippocampus, measured by immunohistochemistry and radioimmunoassay, outlasting the end of the infusion by at least 7 days. The distribution of BDNF-induced neuropeptide Y immunoreactivity is similar to the pattern observed in animals submitted to hippocampal kindling, with the exception of mossy fibres which only become immunoreactive following seizure activity. The enduring increase of neuropeptide Y expression induced by BDNF in the hippocampus suggests that this neurotrophin can trigger long-term genomic effects, which may contribute to the neuroplasticity of this structure, in particular during epileptogenesis.

Brain-derived neurotrophic factor exerts opposing effects on beta2-adrenergic receptor according to depolarization status of cerebellar neurons.

To investigate the molecular mechanisms underlying brain-derived neurotrophic factor (BDNF)-controlled synaptic plasticity, we studied beta2-adrenergic receptor (beta2-AR) expression in cultured cerebellar granule cells. We show that, depending on the state of depolarization, BDNF exerts opposite effects on beta2-AR expression. In neurons maintained in low K+ medium (5 mM K+) that will enter apoptosis, BDNF increases beta2-AR and beta2-AR transcripts. In contrast, in depolarized neurons (high K+ medium, 25 mM K+) BDNF represses beta2-AR expression. The use of reporter genes (driven by the beta2-AR promoter or restricted regulatory elements) revealed that BDNF exerts its opposite effects at the transcriptional level by recruiting a cyclic AMP response element (CRE) and the trans-acting factor CRE binding protein. These results provide the first evidence that a neurotrophin, e.g., BDNF, may exert an opposite effect on receptor expression and function (beta2-AR) according to the depolarization status of the neuron. Based on this finding, we propose that BDNF not only mediates neuronal survival, but is also involved in the modulation of the general sensitivity of the neuron to external signals, thus maintaining its optimal functional integration within the neuronal network.

Cultured rat sensory neurones express functional tachykinin receptor subtypes 1, 2 and 3.

The neuropeptide substance P (SP) is known to play a key role in peripheral nociceptive processes. We investigated the in vitro pharmacological characteristics of functional tachykinin receptors expressed in dorsal root ganglia (DRG) sensory neurones by analysing intracellular free calcium concentration changes induced after stimulation by SP or specific tachykinin agonists. We observed that about 37% of the tested neurones were responsive to either SP or an NK1-, NK2- or NK3-specific agonist. Tachykinin-responsive neurones had a small soma diameter (<20 microm) and were sensitive to capsaicin. These results suggest the presence of NK1, NK2 and NK3 receptors in noxious sensory neurones.

Pituitary adenylate cyclase-activating polypeptide triggers dual transduction signaling in CATH.a cells and transcriptionally activates tyrosine hydroxylase and c-fos expression.

We used a catecholaminergic neuron-like cell line (CATH.a cells) as a model system to investigate the likelihood that pituitary adenylate cyclase-activating polypeptide (PACAP) may participate in the regulation of specific gene expression in catecholaminergic neurons. Analysis by reverse transcriptase-PCR amplification revealed the presence in these cells of type I PACAP receptors, with a short isoform, together with a heavier so-called Hop splice variant. PACAP38 and PACAP27 enhanced, in a dose-dependent manner, both cyclic AMP formation and phosphoinositide breakdown, with EC50 values of, respectively, 0.6 x 10(-10) and 2 x 10(-9) M. These peptides, in addition, also elevated [Ca2+]i by mobilizing intracellular calcium pools. Vasoactive intestinal peptide (VIP) was approximately 1,000-fold less potent in stimulating cyclic AMP (with EC50 = 2 x 10(-7) M) and failed to change the turnover of phosphoinositides and to alter [Ca2+]i. Both forms of PACAP, as well as forskolin, stimulated transcriptional induction of tyrosine hydroxylase (TH) and c-fos promoters fused to a chloramphenicol acetyltransferase (CAT) reporter gene in transiently transfected cells (p < 0.01 vs. controls). Induction of CAT activity linked to both TH and c-fos promoters was obliterated upon coexpression of a dominant inhibitory mutant (Mt-RAB) of cyclic AMP-dependent protein kinase. We conclude that CATH.a cells do express functional PACAP type I receptors, the activation of which impinges on TH and c-fos transcription according to a process that is primarily dependent on the cyclic AMP-PKA pathway.

Evidence that GABAA receptor subunit mRNA expression during development is regulated by GABAA receptor stimulation.

The expression of six mRNA species (alpha 2, alpha 3, alpha 5, beta 2, beta 3, and gamma 2) encoding for GABAA receptor subunits was followed in cultured early postnatal cortical neurons by in situ hybridization histochemistry. In untreated control cultures it was found that these subunit mRNA expression profiles closely follow those seen during development in vivo. alpha 3, alpha 5, and beta 3 subunit expression declined, alpha 2 expression increased, whereas beta 2 and gamma 2 subunit mRNA expression remained relatively constant. To test the hypothesis that GABAA receptor stimulation regulates these expression profiles, we tested the effect of a GABAA receptor positive modulator, allopregnanolone, and a GABAA receptor noncompetitive antagonist, tert-butylbicyclophosphorothionate (TBPS). It was found that allopregnanolone augmented the rate at which the alpha 3, alpha 5, or beta 3 subunit mRNA expression declined and prevented the increase in alpha 2 subunit mRNA expression. As well, allopregnanolone down-regulated beta 2 subunit mRNA expression. TBPS, on the other hand, up-regulated alpha 3, alpha 5, beta 2, and beta 3 subunit mRNA expression. It also down-regulated the expression of alpha 2 subunit mRNA. Both allopregnanolone and TBPS had no effect on gamma 2 subunit mRNA expression. These results imply that the developmental switchover of GABA receptor subunit mRNA expression is regulated by GABAA receptor activity.

Brain-derived neurotrophic factor stimulates AP-1 and cyclic AMP-responsive element dependent transcriptional activity in central nervous system neurons.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, regulates survival and apoptosis of several neuronal populations. These effects are initiated by high-affinity membrane receptors displaying tyrosine kinase activity (trk). However, the intracellular pathways and genetic mechanisms associated with these receptors are largely unknown. Here we show that BDNF stimulates AP1 binding activity in primary cerebellar neurons. This binding corresponds to a functional complex as it is associated with the induction of AP1-dependent transactivation. Application of AP1 partner mRNAs shows an increase in levels of c-fos and c-jun mRNAs after BDNF treatment, resulting from an induction of their promoters. The cis-acting elements by which BDNF stimulates c-fos transcription were further studied. We show that BDNF impinges on multiple regulatory elements, including the serum-responsive element, Fos AP1-like element, and cyclic AMP (cAMP)-responsive element (CRE) sequences. The latter was stimulated without any detectable increase in cAMP or Ca2+ levels. To confirm that BDNF induces c-fos transcription independently of the protein kinase A/cAMP pathway, we transfected a dominant inhibitory mutant of the regulatory subunit of protein kinase A. The overexpression of this mutant does not affect the c-fos promoter transactivation by BDNF. In summary, we show that BDNF stimulates AP1- and CRE-dependent transcription through a mechanism that is distinct from the cAMP- and Ca(2+)-dependent pathways in CNS neurons.

Protective effects of brain-derived neurotrophic factor on the development of hippocampal kindling in the rat.

Recent data have suggested the involvement of neurotrophins in the cascade of events occurring during seizure development. In particular, expression of both brain-derived neurotrophic factor (BDNF) and its receptor mRNAs increases in different brain structures after convulsive seizures. The physiological significance of this increase was investigated by chronic intrahippocampal perfusion of BDNF in the model of dorsal hippocampal kindling in the rat. A 7 day perfusion of BDNF, in the region of the stimulating electrode, blocked the development of kindling during the perfusion period and for the following 15 days. These results provide in vivo evidence for a protective role of BDNF in the regulation of plasticity involved in epileptogenesis in adult brain.

Postnatal change of glycinergic IPSC decay in sympathetic preganglionic neurons.

The characteristics of glycinergic inhibitory postsynaptic currents (IPSCs) in sympathetic preganglionic neurons (SPNs) of neonatal rats were studied by whole-cell recordings in transverse spinal cord slices. In relation to postnatal age, the decay time constants of these currents decreased without a comparable effect on their rise time. This may result from alpha-subunit switching of the glycine receptor and/or increased glycine uptake during this period of postnatal life. The kinetics of glycinergic IPSCs were also temperature- and voltage-dependent. Whereas, compared with room temperature, rise and decay of the events were faster at more physiological temperature, only the decay increased upon depolarization. Visual identification of SPNs was confirmed by intracellular staining and comparison with retrogradely labeled SPNs.

Intracellular calcium regulates the survival of early sensory neurons before they become dependent on neurotrophic factors.

To investigate the role of intracellular Ca2+ in the survival of developing neurons before they become neurotrophic factor dependent, we have studied chick embryo nodose neurons, which have a particularly protracted period of neuorophic factor independence. Pharmacological reduction of intracellular free Ca2+ or depletion of either Ca(2+)-regulated or inositol trisphosphate-regulated intracellular Ca2+ stores kills early neurotrophic factor-independent neurons, but has a negligible effect on older neurons growing in the presence of brain-derived neutrotrophic factor. Shortly before they become dependent on brain-derived neurotrophic factor, nodose neurons express L-type Ca2+ channels and their survival can be enhanced by depolarization-induced Ca2+ influx. We conclude that intracellular Ca2+ plays a role in regulating neuronal survival both prior to and after the onset of neurotrophic factor dependence, but does not mediate the survival-promoting effects of neurotrophic factors.

Coordination of trophic interactions by separate developmental programs in sensory neurons and their target fields.

In the developing vertebrate nervous system the survival of sensory neurons becomes dependent on neurotrophic factors when their axons reach their target fields, and the synthesis of nerve growth factor (NGF) by target field cells commences with the arrival of the earliest axons. The timing of NGF synthesis and the onset of neurotrophic factor dependence are not, however, reliant on innervation. NGF synthesis occurs on time in developing target fields in which innervation is prevented, and sensory neurons cultured before innervating their targets become dependent on neurotrophic factors for survival after a certain length of time in culture. The length of time neurons survive in culture before becoming neurotrophic factor-dependent is related to the time they would normally contact their targets in vivo: populations of neurons that have nearby targets which are innervated early respond to neurotrophic factors before neurons that have more distant targets which are innervated later. The timing of target field innervation is governed not only by the distance axons have to grow but by the rate at which they grow. Axonal growth rate is also regulated in accordance with target distance: neurons with distant targets extend axons faster than neurons with nearby targets. In addition to reviewing evidence for separate developmental programs that control the timing of neurotrophic factor synthesis in the target field and the onset of neurotrophic factor dependence in early sensory neurons, we will consider the mechanisms that might play a role in regulating the survival of neurons during the phase of neurotrophic factor independence.

Large unit conductance voltage chloride channels are expressed in mouse neural crest cells and embryonic dorsal root ganglion cells.

The early expression of voltage-activated chloride channels of large unitary conductance (450 pS in symmetrical 140 mM KCl) was demonstrated using patch-clamp techniques in two preparations: (i) neural crest cells isolated from 9-day-old (E9) mouse embryos and (ii) acutely isolated dorsal root ganglion cells isolated from E12 mouse embryos. Properties of these ionic channels have been analyzed using single channel recordings and the group mean of these single channels.

Advantages of mouse models to study early steps of peripheral nervous system development.

The molecular mechanisms involved in the formation of mammalian peripheral nervous system remain largely unknown. Here we describe the new possibilities offered by mouse mutant analysis, new mouse in vitro models and the recent development of molecular genetic techniques which may permit analysis of the peripheral nervous system development at a level that was heretofore restricted to lower vertebrates.

Quantitative analysis of sodium and potassium activation delays in fresh axons of the squid: Loligo forbesi.

Activation kinetics of the sodium and potassium conductances were re-examined in fresh axons of Loligo forbesi exhibiting very little if any potassium accumulation and a very small leak conductance, special attention being paid to the initial lag phase which precedes the turning-on of the conductances. The axons were kept intact and voltage-clamped at 2-3 degrees C. In all cases, the rising phase of the currents could be fitted with very good accuracy using the Hodgkin-Huxley (1952) equations although, in most cases, the turning-on of the conductance did not coincide with the beginning of the depolarizing test pulse. The delay which separates the change in potential and the turning-on of current (the activation delay) was analyzed quantitatively for different prepulse and pulse potentials. The measured activation delay differed significantly from the delay predicted by the original HH equations. This difference (the 'non-HH delay') varied with prepulse and pulse potentials. For the potassium current, the relationship between the non-HH delay and pulse potential for a constant prepulse was bell shaped, the maximum value (0.7 ms for a prepulse to -80 mV) being reached for about 0 mV. For this same current, the relationship between the non-HH delay and the prepulse potential for a constant pulse potential was sigmoidal, starting from a minimum value of around 0.5 ms at -100 mV and rising to 5 ms at -15 mV. Essentially similar results were obtained for the sodium current although the non-HH delay was three to five times smaller and the dependency upon prepulse potential not significant.(ABSTRACT TRUNCATED AT 250 WORDS)