Eminy H.Y. Lee

Brain-derived neurotrophic factor antisense oligonucleotide impairs memory retention and inhibits long-term potentiation in rats

We have examined the relationship between brain-derived neurotrophic factor gene expression in the hippocampus and memory retention as well as long-term potentiation of rats. One-way inhibitory avoidance learning was adopted as the behavioural paradigm. Results revealed that brain-derived neurotrophic factor messenger RNA levels in the dentate gyrus of the hippocampus were markedly increased at 1 h, 3 h and 6 h post-training in rats showing good retention performance when compared with the poor retention controls. Direct injection of brain-derived neurotrophic factor antisense oligonucleotide into the dentate gyrus of the hippocampus before memory consolidation takes place markedly impaired retention performance in rats. It also significantly decreased brain-derived neurotrophic factor messenger RNA level in the dentate gyrus. The same antisense treatment also markedly reduced the amplitude and slope of excitatory postsynaptic potential as well as the brain-derived neurotrophic factor messenger RNA level in the dentate gyrus. These results suggest that hippocampal brain-derived neurotrophic factor gene expression plays an important role in the memory consolidation process and in the expression of long-term potentiation in rats. These results provide the first evidence to relate brain-derived neurotrophic factor gene expression and memory function in vertebrates. It further suggests that brain-derived neurotrophic factor gene expression is involved in behavioural plasticity.

Intra-amygdala injections of corticotropin releasing factor facilitate inhibitory avoidance learning and reduce exploratory behavior in rats

The effects of intra-amygdala injections of corticotropin-releasing factor (CRF) on memory and exploratory behavior in rats were examined in the present study. Rats with chronically implanted cannulae received intra-amygdala injections of vehicle or CRF at a dose of 0.01, 0.1 or 1.0 μg, either immediately after the inhibitory avoidance training or prior to the open field activity test. Results indicated that while CRF at low (0.01 μg) and high (1.0 μg) doses produced no significant effect on retention or exploration, immediate post-training intra-amygdala injections of CRF at the medium dose (0.1 μg) significantly improved retention of the inhibitory avoidance response. The same dose of CRF, given shortly prior to the open field activity test, decreased locomotor activity, rearing and hole-poke responses in rats. These results suggest that the amygdala is one of the anatomical loci involved in CRF modulation of memory processing and exploration in rats. The implication of CRF in mediating the influences of stress on behavior is discussed.

Corticotrophin-releasing factor produces a long-lasting enhancement of synaptic efficacy in the hippocampus

We have previously demonstrated that intra‐hippocampal injection of corticotrophin‐releasing factor improved memory retention of an inhibitory avoidance learning in rats; while the electrophysiological effects corticotrophin‐releasing factor produces on hippocampal neurons are largely uncharacterized. In the present study, we found that corticotrophin‐releasing factor injected into the dentate gyrus of hippocampus produced a dose‐dependent and long‐lasting enhancement in synaptic efficacy of these neurons, as measured by an increase in the amplitude and slope of population excitatory postsynaptic potentials, as well as the amplitude of population spike. The onset of corticotrophin‐releasing factor‐induced potentiation was slow. It was observed approximately 40–60 min after corticotrophin‐releasing factor administration and lasted for more than 5 h. This effect of corticotrophin‐releasing factor was blocked by pretreatment with the cyclase‐adenosine‐3,5‐monophosphate (cAMP) inhibitor Rp‐adenosine‐3,5‐cyclic monophosphothiolate triethylamine (Rp‐cAMPS) and partially blocked by the N‐methyl‐D‐aspartate receptor antagonist MK‐801. Further, pretreatment with corticotrophin‐releasing factor receptor antagonist dose‐dependently diminished tetanization‐induced long‐term potentiation, and corticotrophin‐releasing factor and tetanic stimuli had an additive effect on hippocampal neuron excitation. Moreover, direct injection of corticotrophin‐releasing factor increased cAMP level in the dentate gyrus. These results together suggest that corticotrophin‐releasing factor‐induced potentiation simulates the late phase of tetanization‐induced long‐term potentiation and cAMP seems to be the messenger mediating this effect. Moreover, corticotrophin‐releasing factor‐induced potentiation and long‐term potentiation may share some similar mechanisms, and corticotrophin‐releasing factor is probably involved in the neural circuits underlying long‐term potentiation. Thus, corticotrophin‐releasing factor may play an important role in modulating synaptic plasticity in the hippocampus.

The mesolimbic dopaminergic pathway is more resistant than the nigrostriatal dopaminergic pathway to MPTP and MPP+ toxicity : role of BDNF gene expression

In the present study we examined the role of BDNF gene expression involved in the differential vulnerability of the nigrostriatal and mesolimbic dopaminergic pathways to environmental damage. The toxins for dopamine (DA) neurons 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium (MPP+) were used as pharmacological tools. Results revealed that chronic MPTP treatment produced a significant and irreversible DA depletion in the striatum (ST) as well as a marked decrease in tyrosine-hydroxylase (TH) mRNA level in the substantia nigra (SN). Under these conditions, the endogenous brain-derived neurotrophic factor (BDNF) mRNA level was increased in the SN. Only acute DA reduction was found in the nucleus accumbens (NAc) and TH mRNA level was not affected in the ventral tegmental area (VTA) by MPTP treatment. Further, when MPP+ produced a similar extent of DA depletion in the ST and NAc, the TH mRNA level was also decreased while BDNF mRNA level was increased in the SN. The same alterations were not observed in the VTA. Results from the BDNF mRNA regional distribution study revealed that structures in the mesolimbic dopaminergic pathway expressed a more than 2-fold higher basal BDNF mRNA level than structures in the nigrostriatal dopaminergic pathway. Presumably, enhanced BDNF gene expression would help the survival of DA neurons and these findings suggest a better protective mechanism in the mesolimbic pathway. Lastly, direct BDNF infusions to the SN partially protected against MPTPs toxicity on DA neurons in the ST in mice. These results together suggest that a more abundant BDNF mRNA level along the mesolimbic pathway than the nigrostriatal pathway may, at least partially, explain the differential vulnerability of different DA neurons to MPTP and MPP+ toxicity.

Enhanced glial cell line-derived neurotrophic factor mRNA expression upon (−)-deprenyl and melatonin treatments

Glial cell line‐derived neurotrophic factor (GDNF) has been shown to be a preferentially selective neurotrophic factor for dopamine (DA) neurons. In the present study, we have examined the distribution of GDNF mRNA expression in several major DA‐containing cell body and terminal areas and the regulation of GDNF mRNA expression upon various pharmacological treatments. Results indicated that there is a relatively higher GDNF mRNA level in neurons of the nigrostriatal and mesolimbic dopaminergic pathways. Upon chronic 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) treatment (30 mg/kg, i.p., for 7 days), DA level was decreased, whereas GDNF mRNA expression was increased in the striatum, suggesting that more GDNF is synthesized and expressed to cope with the neurotoxin insult. Furthermore, among several DA neuron protective and/or therapeutic agents examined, both intrastriatal injections of (−)‐deprenyl (1.25 μg and 2.5 μg) and melatonin (30 μg, 60 μg, and 120 μg) significantly enhanced GDNF mRNA expression in the striatum, whereas the same concentrations of (−)‐deprenyl did not affect monoamine oxidase B (MAOB) activity, although it increased glutathione peroxidase (GPx) and/or superoxide dismutase (SOD) activities. Similarly, the same concentrations of melatonin did not alter SOD or GPx activities, except that the highest dose of melatonin (120 μg) increased lipid peroxidation in the striatum. Conversely, GM1 ganglioside injection (45 μg) lacked of an effect on GDNF mRNA expression. Together, these results suggest that both (−)‐deprenyl and melatonin up‐regulate GDNF gene expression at threshold doses lower than that needed for altering MAOB activity and/or the antioxidant enzyme systems, respectively. These results provide new information on the neuroprotective and therapeutic mechanisms of (−)‐deprenyl and melatonin on DA neurons. J. Neurosci. Res. 53:593–604, 1998.

sgk, a primary glucocorticoid-induced gene, facilitates memory consolidation of spatial learning in rats

By using differential display PCR, we have identified 98 cDNA fragments from the rat dorsal hippocampus that are expressed differentially between the fast learners and slow learners in the water maze learning task. One of these cDNA fragments encodes the rat serum- and glucocorticoid-inducible kinase (sgk) gene. Northern blot analysis revealed that the sgk mRNA level was approximately 4-fold higher in the hippocampus of fast learners than slow learners. In situ hybridization results indicated that sgk mRNA level was increased markedly in CA1, CA3, and dentate gyrus of hippocampus in fast learners. Transient transfection of the sgk mutant DNA to the CA1 area impaired, whereas transfection of the sgk wild-type DNA facilitated water maze performance in rats. These results provide direct evidence that enhanced sgk expression facilitates memory consolidation of spatial learning in rats. These results also elucidate the molecular mechanism of glucocorticoid-induced memory facilitation in mammals.

Enrichment enhances the expression of sgk, a glucocorticoid-induced gene, and facilitates spatial learning through glutamate AMPA receptor mediation.

We have previously demonstrated that the serum and glucocorticoid‐inducible kinase (sgk) gene plays a causal role in facilitating memory performance in rats. Environment enrichment is known to facilitate spatial learning. We therefore examined the effect of enrichment on sgk expression. We also examined the role of sgk in spatial and nonspatial learning and the regulation of sgk expression by activation of different glutamate receptors. Both real‐time polymerase chain reaction and Western blot analyses revealed that enrichment training preferentially increased sgk mRNA and protein levels in the hippocampus. Transfection of sgk mutant DNA to the hippocampal CA1 area markedly impaired spatial learning, fear‐conditioning learning and novel object‐recognition learning in rats, but enrichment training effectively reversed these learning deficits. Meanwhile, S422A mutant DNA transfection prevented enrichment‐induced spatial learning facilitation. In studying glutamate receptor regulation of sgk expression, we found that blockade of N‐methyl‐d‐aspartate (NMDA) receptors in general, and the NR2B subunit in particular both effectively blocked enrichment‐induced spatial learning facilitation, but they did not block enrichment‐induced sgk expression. Upon various glutamate agonist infusions, only α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) increased sgk mRNA levels significantly in the hippocampus. Furthermore, blockade of AMPA receptors effectively blocked both enrichment‐induced spatial learning facilitation and sgk expression. These results indicate that there is a dissociation between NMDA receptor activation and sgk expression. Enrichment enhanced spatial learning through both NMDA and AMPA receptor activation, whereas enrichment‐induced sgk expression is specifically mediated through AMPA receptors. These results suggest that sgk could serve as a novel molecular mechanism, in addition to the NMDA receptor NR2B, underlying enrichment‐induced learning facilitation.

Integrin αv and NCAM mediate the effects of GDNF on DA neuron survival, outgrowth, DA turnover and motor activity in rats

Glial cell line-derived neurotrophic factor (GDNF) is a specific neurotrophic factor for midbrain dopamine (DA) neurons, but the mechanism underlying the neurotrophic action of GDNF is not well known. The cell adhesion molecules integrin and Neural cell adhesion molecule (NCAM) play important roles in neurite outgrowth and fasciculation. In the present study, we found that subchronic GDNF administration to the pars compacta of substantia nigra in rats increased the expression of integrin alphav and NCAM. Immunostaining results demonstrated the wide distribution of integrin alphav and NCAM in all mesencephalic neurons. The results also demonstrated the co-expression of TH with integrin alphav and NCAM in the same neurons of mesencephalic culture. Further, GDNF significantly increased integrin alphav expression in single TH-positive neurons. Function-blocking anti-integrin alphav and anti-NCAM antibodies antagonized the effects of GDNF on DA neuron survival, outgrowth, DA turnover, and locomotor activity in rats. These results demonstrate that integrin alphav and NCAM mediate the effects of GDNF on DA neuron survival and outgrowth during development and on DA turnover and motor function during adulthood.

Neuroprotective mechanism of glial cell line-derived neurotrophic factor on dopamine neurons: role of antioxidation.

Recombinant human GDNF was infused into the rat striatum either acutely or subchronically. Its effects and its interactions with MPP+ on antioxidant enzyme activities were examined. Results indicated that acute GDNF infusion significantly increased glutathione peroxidase, superoxide dismutase and catalase activities. Subchronic GDNF treatment decreased the DA level and enhanced DA turnover. Pre-treatment with GDNF markedly protected DA neurons against MPP+-induced toxicity. These results suggest that GDNF protects DA neurons through its activation of the antioxidant enzyme systems.

Role of hippocampal nitric oxide in memory retention in rats

The present study investigated the role of hippocampal nitric oxide (NO) in memory retention of an inhibitory avoidance learning task in rats. The anatomical locus was aimed at the dentate gyrus (DG). Results indicated that intra-DG administration of a NO generator, sodium nitroprusside (SNP), at moderate doses enhanced retention performance in a dose-response fashion in rats. SNP at higher doses, on the other hand, impaired memory retention. Intra-DG injection of a NO inhibitor, L-NG-monomethylarginine (L-MeArg), impaired retention performance at moderate doses. Coadministration of a NO precursor L-arginine (2.9 and 7.2 micrograms) reversed the memory-impairing effect of L-MeArg. An in vitro ADP-ribosylation experiment showed five protein bands with molecular weights around 118, 94, 54, 43, and 39 kDa that were labeled. The labeling intensity of these proteins decreased as the concentration of in vivo SNP increased. These results suggest that hippocampal NO plays a facilitatory role in the memory process of an inhibitory avoidance learning task in rats.