Ger J. A. Ramakers

Maternal Care and Hippocampal Plasticity: Evidence for Experience-Dependent Structural Plasticity, Altered Synaptic Functioning, and Differential Responsiveness to Glucocorticoids and Stress

Maternal licking and grooming (LG) in infancy influences stress responsiveness and cognitive performance in the offspring. We examined the effects of variation in the frequency of pup LG on morphological, electrophysiological, and behavioral aspects of hippocampal synaptic plasticity under basal and stress-like conditions. We found shorter dendritic branch length and lower spine density in CA1 cells from the adult offspring of low compared with high LG offspring. We also observed dramatic effects on long-term potentiation (LTP) depending on corticosterone treatment. Low LG offspring, in contrast to those of high LG mothers, displayed significantly impaired LTP under basal conditions but surprisingly a significantly enhanced LTP in response to high corticosterone in vitro. This enhanced plasticity under conditions that mimic those of a stressful event was apparent in vivo. Adult low LG offspring displayed enhanced memory relative to high LG offspring when tested in a hippocampal-dependent, contextual fear-conditioning paradigm. Hippocampal levels of glucocorticoid and mineralocorticoid receptors were reduced in low compared with high LG offspring. Such effects, as well as the differences in dendritic morphology, likely contribute to LTP differences under resting conditions, as well as to the maternal effects on synaptic plasticity and behavior in response to elevated corticosterone levels. These results suggest that maternal effects may modulate optimal cognitive functioning in environments varying in demand in later life, with offspring of high and low LG mothers showing enhanced learning under contexts of low and high stress, respectively.

Improved Long-Term Potentiation and Memory in Young Tau-P301L Transgenic Mice before Onset of Hyperphosphorylation and Tauopathy

The microtubule binding protein tau is implicated in neurodegenerative tauopathies, including frontotemporal dementia (FTD) with Parkinsonism caused by diverse mutations in the tau gene. Hyperphosphorylation of tau is considered crucial in the age-related formation of neurofibrillary tangles (NFTs) correlating well with neurotoxicity and cognitive defects. Transgenic mice expressing FTD mutant tau-P301L recapitulate the human pathology with progressive neuronal impairment and accumulation of NFT. Here, we studied tau-P301L mice for parameters of learning and memory at a young age, before hyperphosphorylation and tauopathy were apparent. Unexpectedly, in young tau-P301L mice, increased long-term potentiation in the dentate gyrus was observed in parallel with improved cognitive performance in object recognition tests. Neither tau phosphorylation, neurogenesis, nor other morphological parameters that were analyzed could account for these cognitive changes. The data demonstrate that learning and memory processes in the hippocampus of young tau-P301L mice are not impaired and actually improved in the absence of marked phosphorylation of human tau. We conclude that protein tau plays an important beneficial role in normal neuronal processes of hippocampal memory, and conversely, that not tau mutations per se, but the ensuing hyperphosphorylation must be critical for cognitive decline in tauopathies.

Loss of X-Linked Mental Retardation Gene Oligophrenin1 in Mice Impairs Spatial Memory and Leads to Ventricular Enlargement and Dendritic Spine Immaturity

Loss of oligophrenin1 (OPHN1) function in human causes X-linked mental retardation associated with cerebellar hypoplasia and, in some cases, with lateral ventricle enlargement. In vitro studies showed that ophn1 regulates dendritic spine through the control of Rho GTPases, but its in vivo function remains unknown. We generated a mouse model of ophn1 deficiency and showed that it mimics the ventricles enlargement without affecting the cerebellum morphoanatomy. The ophn1 knock-out mice exhibit behavioral defects in spatial memory together with impairment in social behavior, lateralization, and hyperactivity. Long-term potentiation and mGluR-dependant long-term depression are normal in the CA1 hippocampal area of ophn1 mutant, whereas paired-pulse facilitation is reduced. This altered short-term plasticity that reflects changes in the release of neurotransmitters from the presynaptic processes is associated with normal synaptic density together with a reduction in mature dendritic spines. In culture, inactivation of ophn1 function increases the density and proportion of immature spines. Using a conditional model of loss of ophn1 function, we confirmed this immaturity defect and showed that ophn1 is required at all the stages of the development. These studies show that, depending of the context, ophn1 controls the maturation of dendritic spines either by maintaining the density of mature spines or by limiting the extension of new filopodia. Altogether, these observations indicate that cognitive impairment related to OPHN1 loss of function is associated with both presynaptic and postsynaptic alterations.

Dynamics and plasticity in developing neuronal networks in vitro

When dissociated cortical tissue is brought into culture, neurons readily grow out by forming axonal and dendritic arborizations and synaptic connections. These developing neuronal networks in vitro display spontaneous firing activity from about the end of the first week in vitro. When cultured on multielectrode arrays firing activity can be recorded from many neurons simultaneously over long periods of time. These experimental approaches provide valuable data for studying firing dynamics in neuronal networks in relation to an ongoing development of neurons and synaptic connectivity in the network. This chapter summarizes recent findings on the characteristics and developmental changes in the spontaneous firing dynamics. These changes include long-lasting transient periods of increased firing at individual sites on a time scale of days to weeks, and an age-specific repetitive pattern of synchronous network firing (network bursts) on a time scale of seconds. Especially the spatio-temporal organization of firing within network bursts showed great stability over many hours. In addition, a progressive day-to-day evolution was observed, with an initial broadening of the burst firing rate profile during the 3rd week in vitro (WIV) and a pattern of abrupt onset and precise spike timing from the 5th WIV onwards. These developmental changes are discussed in the light of structural changes in the network and activity-dependent plasticity mechanisms. Preliminary findings are presented on the pattern of spike sequences within network burst, as well as the effect of external stimulation on the spatio-temporal organization within network bursts.

A shape analysis framework for neuromorphometry

This paper addresses in an integrated and systematic fashion the relatively overlooked but increasingly important issue of measuring and characterizing the geometrical properties of nerve cells and structures, an area often called neuromorphology. After discussing the main motivation for such an endeavour, a comprehensive mathematical framework for characterizing neural shapes, capable of expressing variations over time, is presented and used to underline the main issues in neuromorphology. Three particularly powerful and versatile families of neuromorphological approaches, including differential measures, symmetry axes/skeletons, and complexity, are presented and their respective potentials for applications in neuroscience are identified. Examples of applications of such measures are provided based on experimental investigations related to automated dendrogram extraction, mental retardation characterization, and axon growth analysis. ‘…the functional superiority of the human brain is intimately linked up with the prodigious abundance and unaccustomed wealth of forms of the so-called neurons with short axons.’ (Recollections of My Life, Santiago Ramon y Cajal)

Rho proteins, mental retardation and the neurobiological basis of intelligence.

For several decades it has been known that mental retardation is associated with abnormalities in dendrites and dendritic spines. The recent cloning of eight genes which cause nonspecific mental retardation when mutated, provides an important insight into the cellular mechanisms that result in the dendritic abnormalities underlying mental retardation. Three of the encoded proteins, oligophrenin1, PAK3 and alphaPix, interact directly with Rho GTPases. Rho GTPases are key signaling proteins which integrate extracellular and intracellular signals to orchestrate coordinated changes in the actin cytoskeleton, essential for directed neurite outgrowth and the generation/rearrangement of synaptic connectivity. Although many details of the cell biology of Rho signaling in the CNS are as yet unclear, a picture is unfolding showing how mutations that cause abnormal Rho signaling result in abnormal neuronal connectivity which gives rise to deficient cognitive functioning in humans.

Dysregulation of Rho GTPases in the αPix/Arhgef6 mouse model of X-linked intellectual disability is paralleled by impaired structural and synaptic plasticity and cognitive deficits

Mutations in the ARHGEF6 gene, encoding the guanine nucleotide exchange factor αPIX/Cool-2 for the Rho GTPases Rac1 and Cdc42, cause X-linked intellectual disability (ID) in humans. We show here that αPix/Arhgef6 is primarily expressed in neuropil regions of the hippocampus. To study the role of αPix/Arhgef6 in neuronal development and plasticity and gain insight into the pathogenic mechanisms underlying ID, we generated αPix/Arhgef6-deficient mice. Gross brain structure in these mice appeared to be normal; however, analysis of Golgi-Cox-stained pyramidal neurons revealed an increase in both dendritic length and spine density in the hippocampus, accompanied by an overall loss in spine synapses. Early-phase long-term potentiation was reduced and long-term depression was increased in the CA1 hippocampal area of αPix/Arhgef6-deficient animals. Knockout animals exhibited impaired spatial and complex learning and less behavioral control in mildly stressful situations, suggesting that this model mimics the human ID phenotype. The structural and electrophysiological alterations in the hippocampus were accompanied by a significant reduction in active Rac1 and Cdc42, but not RhoA. In conclusion, we suggest that imbalance in activity of different Rho GTPases may underlie altered neuronal connectivity and impaired synaptic function and cognition in αPix/Arhgef6 knockout mice.

Tau-4R suppresses proliferation and promotes neuronal differentiation in the hippocampus of tau knockin/knockout mice

Differential isoform expression and phosphorylation of protein tau are believed to regulate the assembly and stabilization of microtubuli in fetal and adult neurons. To define the functions of tau in the developing and adult brain, we generated transgenic mice expressing the human tau‐4R/2N (htau‐4R) isoform on a murine tau null background, by a knockout/ knockin approach (tau‐KOKI). The main findings in these mice were the significant increases in hippocam‐pal volume and neuronal number, which were sustained throughout adult life and paralleled by improved cognitive functioning. The increase in hippocampal size was found to be due to increased neurogenesis and neuronal survival. Proliferation and neuronal differentiation were further analyzed in primary hippocampal cultures from tau‐KOKI mice, before and after htau‐4R expression onset. In absence of tau, proliferation increased and both neurite and axonal outgrowth were reduced. Htau‐4R expression suppressed proliferation, promoted neuronal differentiation, and restored neu‐rite and axonal outgrowth. We suggest that the tau‐4R isoform essentially contributes to hippocampal development by controlling proliferation and differentiation of neuronal precursors.–Sennvik K., Boekhoorn, K., Lasrado, R., Terwel, D., Verhaeghe, S., Korr, H., Schmitz, C., Tomiyama, T., Mori, H., Krugers, H., Joels, M., Ramakers, G. J. A., Lucassen, P. J., Van Leuven F. Tau‐4R suppresses proliferation and promotes neuronal differentiation in the hippocampus of tau knockin/knockout mice. FASEB J. 21, 2149–2161 (2007)

Hippocampal dysfunction in the Euchromatin histone methyltransferase 1 heterozygous knockout mouse model for Kleefstra syndrome

Euchromatin histone methyltransferase 1 (EHMT1) is a highly conserved protein that catalyzes mono- and dimethylation of histone H3 lysine 9, thereby epigenetically regulating transcription. Kleefstra syndrome (KS), is caused by haploinsufficiency of the EHMT1 gene, and is an example of an emerging group of intellectual disability (ID) disorders caused by genes encoding epigenetic regulators of neuronal gene activity. Little is known about the mechanisms underlying this disorder, prompting us to study the Euchromatin histone methyltransferase 1 heterozygous knockout (Ehmt1(+/-)) mice as a model for KS. In agreement with the cognitive disturbances observed in patients with KS, we detected deficits in fear extinction learning and both novel and spatial object recognition in Ehmt1(+/-) mice. These learning and memory deficits were associated with a significant reduction in dendritic arborization and the number of mature spines in hippocampal CA1 pyramidal neurons of Ehmt1(+/-) mice. In-depth analysis of the electrophysiological properties of CA3-CA1 synapses revealed no differences in basal synaptic transmission or theta-burst induced long-term potentiation (LTP). However, paired-pulse facilitation (PPF) was significantly increased in Ehmt1(+/-) neurons, pointing to a potential deficiency in presynaptic neurotransmitter release. Accordingly, a reduction in the frequency of miniature excitatory post-synaptic currents (mEPSCs) was observed in Ehmt1(+/-) neurons. These data demonstrate that Ehmt1 haploinsufficiency in mice leads to learning deficits and synaptic dysfunction, providing a possible mechanism for the ID phenotype in patients with KS.

Genotype-phenotype studies in three families with mutations in the polyglutamine-binding protein 1 gene (PQBP1).

Recently, the polyglutamine‐binding protein 1 (PQBP1) gene was found to be mutated in five of 29 families studied with X‐linked mental retardation (XLMR) linked to Xp. The reported mutations include duplications or deletions of AG dinucleotides in the fourth coding exon that resulted in shifts of the open reading frame. Three of the five families with mutations in this newly identified XLMR gene have been reported previously. We characterized the phenotypic and neuropsychological features in the two unpublished families with aberrations in PQBP1 and in a family reported 10 years ago. In total, seven patients diagnosed with aberrations in this gene were examined, including a newly identified patient at 18 months of age. Additionally, the features were compared to those reported in the literature of three other families, comprising MRXS3 (Sutherland–Haan syndrome) MRX55 and MRXS8 (Renpenning syndrome). Characteristics seen in these patients are microcephaly, lean body habitus, short stature, striking facial appearance with long narrow faces, upward slant of the eyes, malar hypoplasia, prognathism, high‐arched palate and nasal speech. In addition, small testes and midline defects as anal atresia or imperforate anus, clefting of palate and/or uvula, iris coloboma and Tetralogy of Fallot are seen in several patients. These observations contribute to the phenotypic knowledge of patients with PQBP1 mutations and make this XLMR syndrome well recognizable to clinicians.