Maija L. Castrén

Brain-Derived Neurotrophic Factor and Antidepressant Drugs Have Different But Coordinated Effects on Neuronal Turnover, Proliferation, and Survival in the Adult Dentate Gyrus

Antidepressants increase proliferation of neuronal progenitor cells and expression of brain-derived neurotrophic factor (BDNF) in the hippocampus. We investigated the role of BDNF signaling in antidepressant-induced neurogenesis by using transgenic mice with either reduced BDNF levels (BDNF+/-) or impaired trkB activation (trkB.T1-overexpressing mice). In both transgenic strains, chronic (21 d) imipramine treatment increased the number of bromodeoxyuridine (BrdU)-positive cells to degree similar to that seen in wild-type mice 24 h after BrdU administration, although the basal proliferation rate was increased in both transgenic strains. Three weeks after BrdU administration and the last antidepressant injection, the amount of newborn (BrdU- or TUC-4-positive) cells was significantly reduced in both BDNF+/- and trkB.T1-overexpressing mice, which suggests that normal BDNF signaling is required for the long-term survival of newborn hippocampal neurons. Moreover, the antidepressant-induced increase in the surviving BrdU-positive neurons seen in wild-type mice 3 weeks after treatment was essentially lost in mice with reduced BDNF signaling. Furthermore, we observed that chronic treatment with imipramine or fluoxetine produced a temporally similar increase in both BrdU-positive and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labeled neurons in the dentate gyrus, indicating that these drugs simultaneously increase both neurogenesis and neuronal elimination. These data suggest that antidepressants increase turnover of hippocampal neurons rather than neurogenesis per se and that BDNF signaling is required for the long-term survival of newborn neurons in mouse hippocampus.

Toxicological effects of dehydroabietic acid (DHAA) on the trout, Salmo gairdneri Richardson, in fresh water

Abstract Toxicological and physiological effects of dehydroabietic acid (DHAA), a major poison to fishes in pulp and paper mill effluents, were studied by two experiments with rainbow trout, Salmo gairdneri Richardson: in the first, fish were acutely exposed for 4 days to an average DHAA concentration of 1.2 mg l −1 (Exp. I) and in the second for 30 days to an average of 20 μg DHAA l −1 (Exp. II). Compared to the controls, fish of Exp. I displayed a decreased relative weight of liver, an increased blood haematocrit, and increased haemoglobin as well as plasma protein concentrations. The aspartate aminotransferase activity of heart muscle was significantly elevated, as was also the lactate dehydrogenase (LDH) of white muscle tissue. In the blood plasma, the proportion of muscle type LDH activity was simultaneously increased. UDP-glucuronyl-transferase activities of liver and kidney were strongly decreased. Results suggest an increased and altered use of body energy reserves, decreased plasma volume and impaired liver function. Fish of Exp. II showed an increased relative weight of spleen. In addition, liver and gill LDH shifted towards heart-type. We conclude that 20 μg l −1 is close to the “minimum effective concentration” of DHAA to rainbow trout.

Ecotoxicological aspects of pulp and paper mill effluents discharged to an inland water system: Distribution in water, and toxicant residues and physiological effects in caged fish (Salmo gairdneri)

Abstract Simultaneous studies were conducted on concentrations of chlorinated phenolics (CP) and resin acids (RA) in bleached kraft pulp mill effluents (BKME), their distribution in the receiving water, their uptake and accumulation in the blood plasma of rainbow trout caged in the recipient, as well as short-term physiological effects developed in fish. RA disappeared from the lake water much faster than most of the CP. Some of the BKME-related substances (e.g. 2,4,6-trichlorophenol, 4,5,6-trichloroguaiacol, tetrachloroguaiacol and dehydroabietic acid) were also detected upstream from the mill, thus implying a low-level contamination of this water system in general. In the blood plasma of caged trout free RA were detected up to 3 km from the sewer, but free CP were analysed as far as 11 km from the mill. During the 10-day caging period a partial inhibition of liver UDP-glucuronosyltransferase was observed even at 11 km from the BKME source. Brain acetylcholinesterase activity showed that BKME did not work like anticholinesterase agents, whereas a decrease of plasma cholinesterase activity was developed in a few days in the nearest 6 km from the sewer. Trout exposed at 6–11 km from the sewer displayed increased blood haemoglobin and decreased plasma protein concentrations. The plasma ionic picture (Na, Cl, Mg) indicated only negligible effects on fish osmoregulation.

Augmentation of Auditory N1 in Children with Fragile X Syndrome

We compared the N1 responses of the auditory event-related brain potentials (ERPs) in school-aged children with fragile X syndrome to age-matched controls in order to assess auditory processing. Event-related potentials to non-attended standard and deviant tone stimuli were recorded with EEG electrodes and here the standard tones were analysed. The amplitude of the N1 component to standard tones was significantly larger in children with fragile X syndrome than in control children. In addition, the global field power maximum of ERP corresponding to the N2 component was significantly (p<0.05) larger in fragile X children than in controls. The N2 scalp distribution in children with fragile X syndrome appeared more frontal than that in controls. Furthermore, the fragile X children exhibited no habituation of N1 and an absence of N2 sensitization for repeated tones. Increased responsiveness observed in the N1 evoked potential together with abnormal habituation of auditory responses in childhood may indicate increased sensory sensitivity for auditory stimuli in fragile X syndrome. The data, though very limited, suggest that stimulus processing in the auditory afferent pathways and/or in the corresponding cortical receiving areas is abnormal in children with fragile X syndrome.

Aberrant differentiation of glutamatergic cells in neocortex of mouse model for fragile X syndrome

The lack of fragile X mental retardation protein (FMRP) causes fragile X syndrome, a common form of inherited mental retardation. Our previous studies revealed alterations in the differentiation of FMRP-deficient neural progenitors. Here, we show abnormalities in neurogenesis in the mouse and human embryonic FMRP-deficient brain as well as after in utero transfection of I304N mutated FMRP, which acts in a dominant negative manner in the wild-type mouse brain. Progenitors accumulated abnormally in the subventricular zone of the embryonic Fmr1-knockout (Fmr1-KO) mouse neocortex. An increased density of cells expressing sequentially an intermediate progenitor marker, T-box transcription factor (Tbr2), and a postmitotic neuron marker, T-brain 1 (Tbr1), indicated that the differentiation to glutamatergic cell lineages was particularly disturbed. These abnormalities were associated with an increased density of pyramidal cells of the layer V in the early postnatal neocortex suggesting a role for FMRP in the regulation of the differentiation of neocortical glutamatergic neurons.

BDNF and TrkB in neuronal differentiation of Fmr1-knockout mouse.

Fragile X syndrome (FXS) is a common cause of inherited mental retardation and the best characterized form of autistic spectrum disorders. FXS is caused by the loss of functional fragile X mental retardation protein (FMRP), which leads to abnormalities in the differentiation of neural progenitor cells (NPCs) and in the development of dendritic spines and neuronal circuits. Brain-derived neurotrophic factor (BDNF) and its TrkB receptors play a central role in neuronal maturation and plasticity. We studied BDNF/TrkB actions in the absence of FMRP and show that an increase in catalytic TrkB expression in undifferentiated NPCs of Fmr1-knockout (KO) mice, a mouse model for FXS, is associated with changes in the differentiation and migration of neurons expressing TrkB in neurosphere cultures and in the developing cortex. Aberrant intracellular calcium responses to BDNF and ATP in subpopulations of differentiating NPCs combined with changes in the expression of BDNF and TrkB suggest cell subtype-specific alterations during early neuronal maturation in the absence of FMRP. Furthermore, we show that dendritic targeting of Bdnf mRNA was increased under basal conditions and further enhanced in cortical layer V and hippocampal CA1 neurons of Fmr1-KO mice by pilocarpine-induced neuronal activity represented by convulsive seizures, suggesting that BDNF/TrkB-mediated feedback mechanisms for strengthening the synapses were compromised in the absence of FMRP. Pilocarpine-induced seizures caused an accumulation of Bdnf mRNA transcripts in the most proximal segments of dendrites in cortical but not in hippocampal neurons of Fmr1-KO mice. In addition, BDNF protein levels were increased in the hippocampus but reduced in the cortex of Fmr1-KO mice in line with regional differences of synaptic plasticity in the brain of Fmr1-KO mice. Altogether, the present data suggest that alterations in the BDNF/TrkB signaling modulate brain development and impair synaptic plasticity in FXS.

BDNF regulates the expression of fragile X mental retardation protein mRNA in the hippocampus.

Both fragile X mental retardation protein (FMRP) and brain-derived neurotrophic factor (BDNF) are implicated in the maturation of neurons and in the higher cognitive functions. We have investigated whether FMRP and BDNF are reciprocally regulated in neurons. Exposure of cultured hippocampal neurons to BDNF, but not to NT-3, reduced FMR1 mRNA levels to 84.8% of control at 4 h and the levels were back to baseline by 24 h or 4 days. Furthermore, expression of FMR1 mRNA was reduced (82.4% of control) in vivo in the hippocampus of transgenic mice overexpressing TrkB receptors, and a small but significant (5.1%) decrease was also detected in FMRP protein levels. In contrast, the expression patterns of BDNF and TrkB mRNAs were not altered in FMRP-deficient mice compared to wild-type mice. Our data provide evidence that BDNF via TrkB signaling decreases FMRP expression and suggest a role for FMRP in BDNF-induced synaptic plasticity.

Epilepsy caused by CDKL5 mutations

Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) have been identified in female patients with early onset epileptic encephalopathy and severe mental retardation with a Rett-like phenotype. Subsequently CDKL5 mutations were shown to be associated with more diverse phenotypes including mild epilepsy and autism without epilepsy. Furthermore, CDKL5 mutations were found in patients with Angelman-like phenotype. The severity of epilepsy associated with CDKL5 mutations was recently shown to correlate with the type of CDKL5 mutations and epilepsy was identified to involve three distinct sequential stages. Here, we describe the phenotype of a severe form of neurodevelopmental disease in a female patient with a de novo nonsense mutation of the CDKL5 gene c.175C > T (p.R59X) affecting the catalytic domain of CDKL5 protein. Mutations in the CDKL5 gene are less common in males and can be associated with a genomic deletion as found in our male patient with a deletion of 0.3 Mb at Xp22.13 including the CDKL5 gene. We review phenotypes associated with CDKL5 mutations and examine putative relationships between the clinical epilepsy phenotype and the type of the mutation in the CDKL5 gene.

Overexpression of a truncated TrkB isoform increases the proliferation of neural progenitors

The truncated isoform of TrkB, TrkB.T1, has been shown to be expressed in the neurogenic region of rodent brain. TrkB.T1 lacks tyrosine kinase activity and it may modify the action of the full‐length TrkB. We show here that the full‐length TrkB and TrkB.T1 are expressed at the same relative expression levels in mouse neural progenitor cell cultures. The number of neurosphere‐forming progenitors was reduced and apoptosis increased in neurospheres generated from mice overexpressing TrkB.T1 when compared with wild‐type neurospheres. The proliferation of the transgenic neural progenitors was increased, as indicated by the larger average diameter of spheres (140% of control), the increased cell growth in an MTT assay (137% of control) and the faster rate of 3H‐thymidine incorporation (128% of control) in the transgenic cell cultures than in controls. The proliferation of neural progenitors was also increased after lentivirus‐mediated TrkB.T1 overexpression. A significant increase in 3H‐thymidine incorporation (119% of control) and the average diameter of spheres (112% of control) in the TrkB.T1‐transduced neurospheres compared with neurospheres transduced with the control vectors confirmed the role of TrkB.T1 in proliferation of neural progenitor. When induced to differentiate, progenitors overexpressing TrkB.T1 generated two to three times more neurons than did wild‐type cells. The increase in the number of neurons correlated with an increase in the number of apoptotic cells (two‐fold) at these time points. The data indicate that changes in the relative expression levels of different TrkB isoforms influence the replicative capacity of neural progenitors.

Neurotransmitter responsiveness during early maturation of neural progenitor cells.

Neurotransmitters are potential regulators of proliferation and differentiation of neural progenitor cells (NPC). To gain insight into the dynamics of neurotransmitter responsiveness, neurospheres were prepared from the lateral ventricles of postnatal day 6/7 mice. Individual NPCs migrating out from spheres were simultaneously monitored using Ca(2+) imaging, during the initial 8 days of differentiation, at an area between the inner edge of the sphere and outer periphery of the area of migration. At the first day of differentiation most cells showed metabotropic responses (Ca(2+) discharge from stores) to glutamate (pharmacologically identified as metabotropic glutamate receptor 5, mGluR 5), norepinephrine (NE), acetylcholine (Ach) and ATP, and a smaller proportion of cells also responded to substance P (SP). When outside the neurosphere, many of mGluR5 responding cells gained immunostaining for markers of neuronal lineage (Tuj-1 and NeuN). The number of cells responding through mGluR5 (and responses to Ach, NE and SP) showed during subsequent days of differentiation (day 2-3 onwards) a decline with time and progressively disappeared at the outer periphery of the area of migration. Conversely the number ionotropic glutamate responses as well as responses to depolarization increased in this area. After 5-8 days of differentiation mGluR5 responses could only be observed at the very inner edge of the neurosphere. At 8 days the migrated cells showed very robust ionotropic responses to glutamate, NMDA and depolarization comparable to mature neurons. Taken together, the data presented here suggest that differentiation of NPCs is a dynamic process triggered by cell migration, which leads to a loss of regulatory influences imposed by the inner milieu of the neurosphere. The subsequent switch or loss of metabotropic responses to glutamate, SP, NE, Ach and ATP with the gain of excitable characteristics such as ionotropic responses appears to be a key event in the final differentiation process.