Johannes C. Lodder

9-Amino-1,2,3,4-tetrahydroacridine (THA), an alleged drug for the treatment of Alzheimer's disease, inhibits acetylcholinesterase activity and slow outward K+ current

The in vitro release of acetylcholine in rat brain tissue was inhibited by 9-amino-1,2,3,4-tetrahydroacridine (THA). Atropine antagonized this effect of THA. As THA does not display an affinity for muscarinic receptors, we conclude that THA inhibits acetylcholinesterase activity. In electrophysiological studies with neurons of Lymnaea stagnalis, THA inhibited the slow outward K+ current and consequently increased the duration of the action potentials. It is discussed that both effects of THA possibly contribute to its reported effect in the treatment of patients with Alzheimers disease.

Progesterone-metabolite prevents protein kinase C-dependent modulation of gamma-aminobutyric acid type A receptors in oxytocin neurons.

Gonadal steroid feedback to oxytocin neurons during pregnancy is in part mediated via the neurosteroid allopregnanolone (3alpha-OH-DHP), acting as allosteric modulator of postsynaptic gamma-aminobutyric acid type A (GABA(A)) receptors. We describe here a form of nongenomic progesterone signaling by showing that 3alpha-OH-DHP not only potentiates GABA(A) receptor-channel activity but also prevents its modulation by protein kinase C (PKC). Application of oxytocin or stimulation of PKC suppressed the postsynaptic GABA responses of oxytocin neurons in the absence, but not in the presence of 3alpha-OH-DHP. This finding was true at the juvenile stage and during late pregnancy, when the GABA(A) receptor is sensitive to 3alpha-OH-DHP. In contrast, after parturition, when the GABA(A) receptors expressed by oxytocin neurons are less sensitive to 3alpha-OH-DHP, this neurosteroid no longer counteracts PKC. The change in GABA(A)-receptor responsiveness to 3alpha-OH-DHP helps to explain the onset of firing activity and thus the induction of oxytocin release at parturition.

Induction of egg-laying in the pond snail Lymnaea stagnalis by environmental stimulation of the release of ovulation hormone from the Caudo—Dorsal Cells

Ovipository activity decreases when specimens of the pond snail, Lymnaea stagnalis, are kept in closed jars that are not cleaned regularly. When the snails are transferred from dirty to clean water, egg-laying occurs within 3 h in over 90% of the animals. The number of eggs per egg mass laid after water change is directly related to the length of the period between the induced and the previous oviposition. The relationship is similar to that found when snails are injected with a very high dose (20-times threshold) of egg-laying hormone. The latency of oviposition, which depends on the dose of ovulation hormone, is not affected by the oviposition interval. The size of the egg capsule depends on the number of ripe eggs in the ovotestis and the packaging capacity of the female accessory sex organs. The role of a number of component factors of the water-change stimulus was studied. Elevated O2-content of the water, a clear jar and clean water all have a positive effect on egg-laying. Clean water is the most e...

Cholinergic modulation of nucleus accumbens medium spiny neurons

The rat nucleus accumbens contains acetylcholine‐releasing interneurons, presumed to play a regulatory role in the electrical activity of medium spiny output neurons. In order to examine this issue in detail, we made electrophysiological recordings in rat nucleus accumbens slices. These experiments showed that γ‐aminobutyric acid‐mediated inhibition of the output neurons might be facilitated by activation of nicotinic acetylcholine receptors, in addition to being suppressed via activation of muscarinic acetylcholine receptors. In contrast, glutamatergic excitation of output neurons appeared to be inhibited by activation of muscarinic acetylcholine receptors and to be insensitive to activation of nicotinic acetylcholine receptors. The spontaneous firing frequency of cholinergic neurons appeared to be under control of both a muscarinic and a nicotinic pathway in a bi‐directional manner. Finally, we made paired recordings in which the functional connection between cholinergic neurons and output neurons was monitored. Driving the cholinergic neurons at physiological firing frequencies stimulated γ‐aminobutyric acid‐mediated inhibition of the output neurons, via activation of nicotinic acetylcholine receptors. The onset of this effect was slow and lacked a fixed delay. These data indicate that activation of nicotinic acetylcholine receptors in rat nucleus accumbens may mediate the facilitation of γ‐aminobutyric acid‐mediated inhibition of medium spiny output neurons. Possible mechanisms of neurotransmission, mediating this cholinergic modulation are discussed.

Label-free live brain imaging and targeted patching with third-harmonic generation microscopy

The ability to visualize neurons inside living brain tissue is a fundamental requirement in neuroscience and neurosurgery. Especially the development of a noninvasive probe of brain morphology with micrometer-scale resolution is highly desirable, as it would provide a noninvasive approach to optical biopsies in diagnostic medicine. Two-photon laser-scanning microscopy (2PLSM) is a powerful tool in this regard, and has become the standard for minimally invasive high-resolution imaging of living biological samples. However, while 2PLSM-based optical methods provide sufficient resolution, they have been hampered by the requirement for fluorescent dyes to provide image contrast. Here we demonstrate high-contrast imaging of live brain tissue at cellular resolution, without the need for fluorescent probes, using optical third-harmonic generation (THG). We exploit the specific geometry and lipid content of brain tissue at the cellular level to achieve partial phase matching of THG, providing an alternative contrast mechanism to fluorescence. We find that THG brain imaging allows rapid, noninvasive label-free imaging of neurons, white-matter structures, and blood vessels simultaneously. Furthermore, we exploit THG-based imaging to guide micropipettes towards designated neurons inside live tissue. This work is a major step towards label-free microscopic live brain imaging, and opens up possibilities for the development of laser-guided microsurgery techniques in the living brain.

Oxytocin and estrogen promote rapid formation of functional GABA synapses in the adult supraoptic nucleus

We here investigated inhibitory synapse turnover in the adult brain using the hypothalamic supraoptic nucleus where new synapses form during different physiological conditions, in particular on oxytocin neurons largely controlled by GABAergic inputs and locally released oxytocin. Patch clamp recordings and ultrastructural analysis of the nucleus in acute slices from late gestating rats showed that oxytocin and estrogen promoted rapid formation of inhibitory synapses. Thus, after 2-h exposure to a combination of oxytocin and 17-beta estradiol, the frequency of miniature inhibitory postsynaptic currents was significantly enhanced. Since their amplitude and presynaptic GABA release probability were unmodified, this indicated an increased number of synapses. Electron microscopy confirmed increased densities of symmetric, putative GABAergic synapses within 2-h exposure to the peptide or steroid, effects which were reversible and oxytocin receptor mediated. Our observations thus offer direct evidence that hypothalamic GABAergic microcircuitries can undergo rapid and functional remodeling under changing neuroendocrine conditions.

Suppression of egg-laying during starvation in the snailLymnaea stagnalis by inhibition of the ovulation hormone producing Caudo-Dorsal cells

Starvation inhibits egg-laying in the snail, Lymnaea stagnalis. In starved animals the neurosecretory Caudo-Dorsal Cells (CDC), which produce the egg-laying hormone, are hyperpolarized as compared to the CDC of controls. However, they are more responsive to repetitive intracellular stimulation, which induces the hormone releasing discharge. Hyperpolarization is not found in the non-neurosecretory Cerebral Giant Cells, which indicates that the effect is specific for the CDC. It is also a characteristic effect of starvation as compared to another treatment (dirty water) inhibiting egg-laying.

NMDA receptors induce somatodendritic secretion in hypothalamic neurones of lactating female rats

Many neurones in the mammalian brain are known to release the content of their vesicles from somatodendritic locations. These vesicles usually contain retrograde messengers that modulate network properties. The back‐propagating action potential is thought to be the principal physiological stimulus that evokes somatodendritic release. In contrast, here we show that calcium influx through NMDA receptor (NMDAR) channels, in the absence of postsynaptic cell firing, is also able to induce vesicle fusion from non‐synaptic sites in nucleated outside‐out patches of dorsomedial supraoptic nucleus (SON) neurones of adult female rats, in particular during their reproductive stages. The physiological significance of this mechanism was characterized in intact brain slices, where NMDAR‐mediated release of oxytocin was shown to retrogradely inhibit presynaptic GABA release, in the absence of postsynaptic cell firing. This implies that glutamatergic synaptic input in itself is sufficient to elicit the release of oxytocin, which in turn acts as a retrograde messenger leading to the depression of nearby GABA synapses. In addition, we found that during lactation, when oxytocin demand is high, NMDA‐induced oxytocin release is up‐regulated compared to that in non‐reproductive rats. Thus, in the hypothalamus, local signalling back and forth between pre‐ and postsynaptic compartments and between different synapses may occur independently of the firing activity of the postsynaptic neurone.

NMDA receptors trigger neurosecretion of 5-HT within dorsal raphe nucleus of the rat in the absence of action potential firing.

Activity and calcium‐dependent release of neurotransmitters from the somatodendritic compartment is an important signalling mechanism between neurones throughout the brain. NMDA receptors and vesicles filled with neurotransmitters occur in close proximity in many brain areas. It is unknown whether calcium influx through these receptors can trigger the release of somatodendritic vesicles directly, or whether postsynaptic action potential firing is necessary for release of these vesicles. Here we addressed this question by studying local release of serotonin (5‐HT) from dorsal raphé nucleus (DRN) neurones. We performed capacitance measurements to monitor the secretion of vesicles in giant soma patches, in response to short depolarizations and action potential waveforms. Amperometric measurements confirmed that secreted vesicles contained 5‐HT. Surprisingly, two‐photon imaging of DRN neurones in slices revealed that dendritic calcium concentration changes in response to somatic firing were restricted to proximal dendritic areas. This implied that alternative calcium entry pathways may dominate the induction of vesicle secretion from distal dendrites. In line with this, transient NMDA receptor activation, in the absence of action potential firing, was sufficient to induce capacitance changes. By monitoring GABAergic transmission onto DRN 5‐HT neurones in slices, we show that endogenous NMDA receptor activation, in the absence of postsynaptic firing, induced release of 5‐HT, which in turn increased the frequency of GABAergic inputs through activation of 5‐HT2 receptors. We propose here that calcium influx through NMDA receptors can directly induce postsynaptic 5‐HT release from DRN neurones, which in turn may facilitate GABAergic input onto these cells.

Megalencephalic leucoencephalopathy with cysts: defect in chloride currents and cell volume regulation

Megalencephalic leucoencephalopathy with subcortical cysts is a genetic brain disorder with onset in early childhood. Affected infants develop macrocephaly within the first year of life, after several years followed by slowly progressive, incapacitating cerebellar ataxia and spasticity. From early on, magnetic resonance imaging shows diffuse signal abnormality and swelling of the cerebral white matter, with evidence of highly increased white matter water content. In most patients, the disease is caused by mutations in the gene MLC1, which encodes a plasma membrane protein almost exclusively expressed in brain and at lower levels in leucocytes. Within the brain, MLC1 is mainly located in astrocyte-astrocyte junctions adjacent to the blood-brain and cereborspinal fluid-brain barriers. Thus far, the function of MLC1 has remained unknown. We tested the hypothesis that MLC1 mutations cause a defect in ion currents involved in water and ion homeostasis, resulting in cerebral white matter oedema. Using whole-cell patch clamp studies we demonstrated an association between MLC1 expression and anion channel activity in different cell types, most importantly astrocytes. The currents were absent in chloride-free medium and in cells with disease-causing MLC1 mutations. MLC1-dependent currents were greatly enhanced by hypotonic pretreatment causing cell swelling, while ion channel blockers, including Tamoxifen, abolished the currents. Down regulation of endogenous MLC1 expression in astrocytes by small interfering RNA greatly reduced the activity of this channel, which was rescued by overexpression of normal MLC1. The current-voltage relationship and the pharmacological profiles of the currents indicated that the channel activated by MLC1 expression is a volume-regulated anion channel. Such channels are involved in regulatory volume decrease. We showed that regulatory volume decrease was hampered in lymphoblasts from patients with megalencephalic leucoencephalopathy. A similar trend was observed in astrocytes with decreased MLC1 expression; this effect was rescued by overexpression of normal MLC1. In the present study, we show that absence or mutations of the MLC1 protein negatively impact both volume-regulated anion channel activity and regulatory volume decrease, indicating that megalencephalic leucoencephalopathy is caused by a disturbance of cell volume regulation mediated by chloride transport.