Thomas Mittmann

More than a retrograde messenger: nitric oxide needs two cGMP pathways to induce hippocampal long-term potentiation.

Although nitric oxide (NO) has been implicated as a messenger molecule in hippocampal long-term potentiation (LTP) for almost 20 years, its precise function has not been elucidated because presynaptic and/or postsynaptic actions of NO have been reported. Most of the effects of NO as a signaling molecule are mediated by the NO receptor guanylyl cyclases (NO-GCs), two heme-containing enzymes with pronounced homology in which cGMP-forming activity is stimulated on NO binding. Here we report on knock-out (KO) mice in which either one of the NO-GC receptors has been genetically deleted. By measuring NO-induced cGMP levels, similar quantities of both NO-GC receptors were determined in the hippocampus. Surprisingly, hippocampal LTP was abolished in either one of the KO strains, demonstrating that both NO-GC receptors are required in the course of LTP. Expression of LTP was restored with a cGMP analog in one of the KO strains but did not recover in the other one. Moreover, single-cell recordings of paired pulse facilitation revealed a presynaptic role of one of the NO-GC isoforms in neurotransmitter release, confirming different roles of the NO-GC receptors in LTP. Because neither one of the NO/cGMP-induced responses by itself is sufficient for LTP, two divergent, possibly presynaptically and postsynaptically localized NO-stimulated cGMP pathways are apparently required for the expression of LTP. The unexpected role of cGMP at two sites of the synaptic cleft explains many of the controversial results in former NO research in LTP and demonstrates the necessity of presynaptic and postsynaptic changes for LTP expression.

BDNF contributes to the facilitation of hippocampal synaptic plasticity and learning enabled by environmental enrichment

Sensory, motor, and cognitive stimuli, resulting from interactions with the environment, play a key role in optimizing and modifying the neuronal circuitry required for normal brain function. An experimental animal model for this phenomenon comprises environmental enrichment (EE) in rodents. EE causes profound changes in neuronal and signaling levels of excitation and plasticity throughout the entire central nervous system and the hippocampus is particularly affected. The mechanisms underlying these changes are not yet fully understood. As brain‐derived neurotrophic factor (BDNF) supports hippocampal long‐term potentiation (LTP), we explored whether it participates in the facilitation of synaptic plasticity and hippocampus‐dependent learning that occurs following EE. In the absence of EE, LTP elicited by high‐frequency stimulation was equivalent in wildtype mice and heterozygous BDNF+/− siblings. LTP elicited by theta‐burst stimulation in BDNF+/− mice was less than in wildtypes. Long‐term depression (LTD) was also impaired. EE for three weeks, beginning after weaning, improved hippocampal LTP in both wildtype and transgenic animals, with LTP in transgenics achieving levels seen in wildtypes in the absence of EE. Object recognition memory was evident in wildtypes 24 h and 7 days after initial object exposure. EE improved memory performance in wildtypes 24 h but not 7 days after initial exposure. BDNF+/− mice in the absence of EE showed impaired memory 7 days after initial object exposure that was restored by EE. Western blotting revealed increased levels of BDNF, but not proBDNF, among both EE cohorts. These data support that BDNF plays an intrinsic role in improvements of synaptic plasticity and cognition that occur in EE.

Oligodendrocyte Precursor Cells Modulate the Neuronal Network by Activity-Dependent Ectodomain Cleavage of Glial NG2

This study shows that the activity of neurons can trigger shedding of a protein, NG2, from the surface of oligodendrocyte precursor cells; this protein in turn modulates synaptic transmission, revealing a two-way conversation between neurons and glia.

Neuronal activation of Ras regulates synaptic connectivity

A synRas mouse model was used expressing constitutively activated Ha‐Ras (Val12 mutation) in neurons to investigate the role of Ras‐MAPkinase signalling for neuronal connectivity in adult brain. Expression of the transgene in the cortex of these mice starts after neuronal differentiation is completed and allows to directly investigate the effects of enhanced Ras activity in differentiated neurons. Activation of Ha‐Ras induced an increase in soma size which was sensitive to MEK inhibitor in postnatal organotypic cultures. Adult cortical pyramidal neurons showed complex structural rearrangements associated with an increased size and ramification of dendritic arborization. Dendritic spine density was elevated and correlated with a twofold increase in number of synapses. In acute brain slices of the somatosensory and of the visual cortex, extracellular field potentials were recorded from layer II/III neurons. The input–output relation of synaptically evoked field potentials revealed a significantly higher basal excitability of the transgenic mice cortex compared to wild‐type animals. In whole cell patch clamp preparations, the frequency of AMPA receptor‐mediated spontaneous excitatory postsynaptic currents was increased while the ratio between NMDA and AMPA‐receptor mediated signal amplitude was unchanged. A pronounced depression of paired pulse facilitation indicated that Ras contributes to changes at the presynaptic site. Furthermore, synRas mice showed an increased synaptic long‐term potentiation, which was sensitive to blockers of NMDA‐receptors and of MEK. We conclude that neuronal Ras is a common switch of plasticity in adult mammalian brain sculpturing neuronal architecture and synaptic connectivity in concert with tuning synaptic efficacy.

Impaired GABAergic inhibition in the visual cortex of brain‐derived neurotrophic factor heterozygous knockout mice

Brain derived neurotrophic factor (BDNF) promotes the formation, maturation and stabilization of inhibitory synapses in the central nervous system. In addition, BDNF has been suggested to regulate the critical period for ocular dominance plasticity in the visual system. Here we further evaluated the role of BDNF in the visual cortex by studying the GABAergic synaptic transmission under conditions of chronically reduced levels of BDNF. Whole‐cell patch‐clamp recordings were performed from pyramidal neurons located in layers II/III of visual cortical slices in heterozygous BDNF knockout mice (BDNF (+/−)) and their wild‐type littermates at the age of 21–25 days. The BDNF (+/−) mice showed a decreased frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs) as well as a reduced amplitude and prolonged decay time constant of evoked IPSCs. Further analyses indicated an impaired presynaptic GABAergic function in BDNF (+/−) mice, as shown by the decreased release probability, steady‐state release and synchronous release of GABA. However, the number of functional release sites remained unchanged. In line with these observations, an impaired glutamate‐driven GABA release was observed in BDNF (+/−) mice. Furthermore, the overall balance in the strength of cortical excitation to inhibition shifted towards a decreased inhibition. Finally, the reversal potential for chloride‐mediated evoked IPSCs was not affected. These findings suggested that chronically reduced levels of BDNF strongly impair the GABAergic inhibitory function in visual cortex by altering postsynaptic properties and by reducing presynaptic GABA release as well as the overall strength of inhibition onto pyramidal neurons within the cortical network. These impairments of inhibitory function are compatible with a rather immature status of the GABAergic system in BDNF (+/−) mice, which supports the hypothesis that the level of expression for BDNF critically affects maturation and function of the GABAergic inhibition.

Long-Term Potentiation in the Visual Cortex Requires Both Nitric Oxide Receptor Guanylyl Cyclases

The role of nitric oxide (NO)/cGMP signaling in long-term potentiation (LTP) has been a lingering matter of debate. Within the cascade, the NO receptor guanylyl cyclase (GC), the cGMP-forming enzyme that is stimulated by NO, plays a key role. Two isoforms of GC (α2-GC, α1-GC) exist. To evaluate their contribution to synaptic plasticity, we analyzed knock-out mice lacking either one of the GC isoforms. We found that LTP induced in the visual cortex is NO dependent in the wild-type mice, absent in either of the GC isoform-deficient mice, and restored with application of a cGMP analog in both strains. The requirement of both NO receptor GCs for LTP indicates the existence of two distinct NO/cGMP-mediated pathways, which have to work in concert for expression of LTP.

Reduced presynaptic efficiency of excitatory synaptic transmission impairs LTP in the visual cortex of BDNF-heterozygous mice.

The neurotrophin brain‐derived neurotrophic factor (BDNF) plays an important role in neuronal survival, axonal and dendritic growth and synapse formation. BDNF has also been reported to mediate visual cortex plasticity. Here we studied the cellular mechanisms of BDNF‐mediated changes in synaptic plasticity, excitatory synaptic transmission and long‐term potentiation (LTP) in the visual cortex of heterozygous BDNF‐knockout mice (BDNF+/–). Patch‐clamp recordings in slices showed an ∼ 50% reduction in the frequency of miniature excitatory postsynaptic currents (mEPSCs) compared to wild‐type animals, in the absence of changes in mEPSC amplitudes. A presynaptic impairment of excitatory synapses from BDNF+/– mice was further indicated by decreased paired‐pulse ratio and faster synaptic fatigue upon prolonged repetitive stimulation at 40 Hz. In accordance, presynaptic theta‐burst stimulation (TBS) failed to induce LTP at layer IV to layers II‐III synapses during extracellular field‐potential recordings in BDNF+/– animals. Changes in postsynaptic function could not be detected, as no changes were observed in either the amplitudes of evoked EPSCs, the ratios of AMPA : NMDA currents or the kinetics of evoked AMPA and NMDA EPSCs. In line with this observation, an LTP pairing paradigm that relies on direct postsynaptic depolarization under patch‐clamp conditions could be induced successfully in BDNF+/– animals. These data suggest that a chronic reduction in the expression of BDNF to nearly 50% attenuates the efficiency of presynaptic glutamate release in response to repetitive stimulation, thereby impairing presynaptically evoked LTP in the visual cortex.

Presynaptic nitric oxide/cGMP facilitates glutamate release via hyperpolarization‐activated cyclic nucleotide‐gated channels in the hippocampus

In hippocampal neurons, synaptic transmission is affected by a variety of modulators, including nitric oxide (NO), which was proposed as a retrograde messenger as long as two decades ago. NO signals via two NO‐sensitive guanylyl cyclases (NO‐GCs) (NO‐GC1 and NO‐GC2) and the subsequent increase in cGMP. Lack of long‐term potentiation in mice deficient in either one of the two NO‐GCs demonstrates the involvement of both NO‐GCs in synaptic transmission. However, the physiological consequences of NO/cGMP and the cellular mechanisms involved are unknown. Here, we analyzed glutamatergic synaptic transmission, most likely reflecting glutamate release, in the hippocampal CA1 region of NO‐GC knockout mice by single‐cell recording, and found glutamate release to be reduced under basal and stimulated conditions in the NO‐GC1 knockout mice, but restorable to wild‐type‐like levels with a cGMP analog. Conversely, an inhibitor of NO/cGMP signaling, ODQ, reduced glutamate release in wild‐type mice to knockout‐like levels; thus, we conclude that presynaptic cGMP formed by NO‐GC1 facilitates glutamate release. In this pathway, NO is supplied by endothelial NO synthase. In search of a cGMP target, we found that two mechanistically distinct blockers of hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels (ZD7288 and DK‐AH269) abolished the cGMP‐induced increase in glutamate release, suggesting that cGMP either directly or indirectly signals via HCN channels. In summary, we unravel a presynaptic role of NO/cGMP most likely in glutamate release and propose that HCN channels act as effectors for cGMP.

Focal laser-lesions activate an endogenous population of neural stem/progenitor cells in the adult visual cortex

CNS lesions stimulate adult neurogenic niches. Endogenous neural stem/progenitor cells represent a potential resource for CNS regeneration. Here, we investigate the response to unilateral focal laser-lesions applied to the visual cortex of juvenile rats. Within 3 days post-lesion, an ipsilateral increase of actively cycling cells was observed in cortical layer one and in the callosal white matter within the lesion penumbra. The cells expressed the neural stem/progenitor cell marker Nestin and the 473HD-epitope. Tissue prepared from the lesion area by micro-dissection generated self-renewing, multipotent neurospheres, while cells from the contralateral visual cortex did not. The newly formed neural stem/progenitor cells in the lesion zone might support neurogenesis, as suggested by the expression of Pax6 and Doublecortin, a marker of newborn neurons. We propose that focal laser-lesions may induce the emergence of stem/progenitor cells with neurogenic potential. This could underlie the beneficial effects of laser application in neurosurgery.

Increased synaptic plasticity in the surround of visual cortex lesions in rats

Lesion-induced functional loss is reduced when new synaptic connections are established in the surround of a cortical lesion. For this, long-term synaptic plasticity can play a key role. We studied long-term potentiation (LTP) and long-term depression (LTD) in slices of rat visual cortex with small cortical lesions. Surprisingly, the normal balance between LTP and LTD was significantly altered in the first week following cortical injury. Theta-burst induced LTP was increased, whereas LTD evoked by low frequency stimulation was not affected. The increased potentiation of subcortical inputs after cortical lesions opens a window for facilitated early functional reorganization by repetitive visual training.