Márton B. Dobszay

Alzheimer's disease is associated with an increased risk of unprovoked seizures. However, the underlying mechanisms of seizure induction remain elusive. Here, we performed video-EEG recordings in mice carrying mutant human APPswe and PS1dE9 genes (APdE9 mice) and their wild-type littermates to determine the prevalence of unprovoked seizures. In two recording episodes at the onset of amyloid beta (Abeta) pathogenesis (3 and 4.5 months of age), at least one unprovoked seizure was detected in 65% of APdE9 mice, of which 46% had multiple seizures and 38% had a generalized seizure. None of the wild-type mice had seizures. In a subset of APdE9 mice, seizure phenotype was associated with a loss of calbindin-D28k immunoreactivity in dentate granular cells and ectopic expression of neuropeptide Y in mossy fibers. In APdE9 mice, persistently decreased resting membrane potential in neocortical layer 2/3 pyramidal cells and dentate granule cells underpinned increased network excitability as identified by patch-clamp electrophysiology. At stimulus strengths evoking single-component EPSPs in wild-type littermates, APdE9 mice exhibited decreased action potential threshold and burst firing of pyramidal cells. Bath application (1 h) of Abeta1-42 or Abeta25-35 (proto-)fibrils but not oligomers induced significant membrane depolarization of pyramidal cells and increased the activity of excitatory cell populations as measured by extracellular field recordings in the juvenile rodent brain, confirming the pathogenic significance of bath-applied Abeta (proto-)fibrils. Overall, these data identify fibrillar Abeta as a pathogenic entity powerfully altering neuronal membrane properties such that hyperexcitability of pyramidal cells culminates in epileptiform activity.

In utero exposure to Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the active component from marijuana, induces cognitive deficits enduring into adulthood. Although changes in synaptic structure and plasticity may underlie Delta(9)-THC-induced cognitive impairments, the neuronal basis of Delta(9)-THC-related developmental deficits remains unknown. Using a Boyden chamber assay, we show that agonist stimulation of the CB(1) cannabinoid receptor (CB(1)R) on cholecystokinin-expressing interneurons induces chemotaxis that is additive with brain-derived neurotrophic factor (BDNF)-induced interneuron migration. We find that Src kinase-dependent TrkB receptor transactivation mediates endocannabinoid (eCB)-induced chemotaxis in the absence of BDNF. Simultaneously, eCBs suppress the BDNF-dependent morphogenesis of interneurons, and this suppression is abolished by Src kinase inhibition in vitro. Because sustained prenatal Delta(9)-THC stimulation of CB(1)Rs selectively increases the density of cholecystokinin-expressing interneurons in the hippocampus in vivo, we conclude that prenatal CB(1)R activity governs proper interneuron placement and integration during corticogenesis. Moreover, eCBs use TrkB receptor-dependent signaling pathways to regulate subtype-selective interneuron migration and specification.

GABAergic interneurons with high-frequency firing, fast-spiking (FS) cells, form synapses on perisomatic regions of principal cells in the neocortex and hippocampus to control the excitability of cortical networks. Brain-derived neurotrophic factor (BDNF) is essential for the differentiation of multiple interneuron subtypes and the formation of their synaptic contacts. Here, we examined whether BDNF, alone or in conjunction with sustained KCl-induced depolarization, drives functional FS cell differentiation and the formation of inhibitory microcircuits. Homogeneous FS cell cultures were established by target-specific isolation using the voltage-gated potassium channel 3.1b subunit as the selection marker. Isolated FS cells expressed parvalbumin, were surrounded by perineuronal nets, formed immature inhibitory connections and generated slow action potentials at 12 days in vitro. Brain-derived neurotrophic factor (BDNF) promoted FS cell differentiation by increasing the somatic diameter, dendritic branching and the frequency of action potential firing. In addition, BDNF treatment led to a significant up-regulation of synaptophysin and vesicular GABA transporter expression, components of the synaptic machinery critical for GABA release, which was paralleled by an increase in synaptic strength. Long-term membrane depolarization alone was detrimental to dendritic branching. However, we observed that BDNF and KCl exerted additive effects, as reflected by the significantly accelerated maturation of synaptic contacts and high discharge frequencies, and was required for the formation of reciprocal connections between FS cells. Our results show that BDNF, along with membrane depolarization, is critical for FS cells to establish inhibitory circuitries during corticogenesis.

Recent studies implicate dendritic endocannabinoid release from subsynaptic dendrites and subsequent inhibition of neurotransmitter release from nerve terminals as a means of retrograde signaling in multiple brain regions. Here we show that type 1 cannabinoid receptor-mediated endocannabinoid signaling is not involved in the retrograde control of synaptic efficacy at inhibitory synapses between fast-spiking interneurons and pyramidal cells in layer 2/3 of the neocortex. Vesicular neurotransmitter transporters, such as vesicular glutamate transporters (VGLUTs) 1 and 2, are localized to presynaptic terminals and accumulate neurotransmitters into synaptic vesicles. A third subtype of VGLUTs (VGLUT3) was recently identified and found localized to dendrites of various cell types. We demonstrate, using multiple immunofluorescence labeling and confocal laser-scanning microscopy, that VGLUT3-like immunoreactivity is present in dendrites of layer 2/3 pyramidal neurons in the rat neocortex. Electron microscopy analysis confirmed that VGLUT3-like labeling is localized to vesicular structures, which show a tendency to accumulate in close proximity to postsynaptic specializations in dendritic shafts of pyramidal cells. Dual whole-cell recordings revealed that retrograde signaling between fast-spiking interneurons and pyramidal cells was enhanced under conditions of maximal efficacy of VGLUT3-mediated glutamate uptake, whereas it was reduced when glutamate uptake was inhibited by incrementing concentrations of the nonselective VGLUT inhibitor Evans blue (0.5-5.0 microm) or intracellular Cl- concentrations (4-145 mm). Our results present further evidence that dendritic vesicular glutamate release, controlled by novel VGLUT isoforms, provides fast negative feedback at inhibitory neocortical synapses, and demonstrate that glutamate can act as a retrograde messenger in the CNS.

Basal forebrain cholinergic neurons project to diverse cortical and hippocampal areas and receive reciprocal projections therefrom. Maintenance of a fine-tuned synaptic communication between pre- and postsynaptic cells in neuronal circuitries also requires feedback mechanisms to control the probability of neurotransmitter release from the presynaptic terminal. Release of endocannabinoids or glutamate from a postsynaptic neuron has been identified as a means of retrograde synaptic signalling. Presynaptic action of endocannabinoids is largely mediated by type 1 cannabinoid (CB1) receptors, while fatty-acid amide hydrolase (FAAH) is involved in inactivating some endocannabinoids postsynaptically. Alternatively, vesicular glutamate transporter 3 (VGLUT3) controls release of glutamate from postsynaptic cells. Here, we studied the distribution of CB1 receptors, FAAH and VGLUT3 in cholinergic basal forebrain nuclei of mouse and rat. Cholinergic neurons were devoid of CB1 receptor immunoreactivity. A fine CB1 receptor-immunoreactive (ir) fibre meshwork was present in medial septum, diagonal bands and nucleus basalis. In contrast, the ventral pallidum and substantia innominata received dense CB1 receptor-ir innervation and cholinergic neurons received CB1 receptor-ir presumed synaptic contacts. Consistent with CB1 receptor distribution, FAAH-ir somata were abundant in basal forebrain and appeared in contact with CB1 receptor-containing terminals. Virtually all cholinergic neurons were immunoreactive for FAAH. A significant proportion of cholinergic cells exhibited VGLUT3 immunoreactivity in medial septum, diagonal bands and nucleus basalis, and were in close apposition to VGLUT3-ir terminals. VGLUT3 immunoreactivity was largely absent in ventral pallidum and substantia innominata. We propose that specific subsets of cholinergic neurons may utilize endocannabinoids or glutamate for retrograde control of the efficacy of input synapses, and the mutually exclusive complementary distribution pattern of CB1 receptor-ir and VGLUT3-ir fibres in basal forebrain suggests segregated input-specific signalling mechanisms by cholinergic neurons.